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  • Alterations in signaling pathways that accompany spontaneous transition to malignancy in a mouse model of BRAF mutant microsatellite stable colorectal cancer
    Neoplasia (IF 3.837) Pub Date : 2020-01-11
    Alexandra M. Kane; Lochlan J. Fennell; Cheng Liu; Jennifer Borowsky; Diane M. McKeone; Catherine E. Bond; Stephen Kazakoff; Ann-Marie Patch; Lambros T. Koufariotis; John Pearson; Nicola Waddell; Barbara A. Leggett; Vicki L.J. Whitehall

    The serrated neoplasia pathway gives rise to a distinct subgroup of colorectal cancers distinguished by the presence of mutant BRAFV600E and the CpG Island Methylator Phenotype (CIMP). BRAF mutant CRC are commonly associated with microsatellite instability, which have an excellent clinical outcome. However, a proportion of BRAF mutant CRC retain microsatellite stability and have a dismal prognosis. The molecular drivers responsible for the development of this cancer subgroup are unknown. To address this, we established a murine model of BRAFV600E mutant microsatellite stable CRC and comprehensively investigated the exome and transcriptome to identify molecular alterations in signaling pathways that drive malignancy. Exome sequencing of murine serrated lesions (mSL) and carcinomas identified frequent hot spot mutations within the gene encoding β-catenin (Ctnnb1). Immunohistochemical staining of β-catenin indicated that these mutations led to an increase in the presence of aberrant nuclear β-catenin that resulted in gene expression changes in targets of β-catenin transcription. Gene expression profiling identified a significant enrichment for transforming growth factor-β (TGF-β) signaling that was present in mSL and carcinomas. Early activation of TGF-β suggests that this pathway may be an early cue directing mSL to microsatellite stable carcinoma. These findings in the mouse model support the importance of alterations in WNT and TGF-β signaling during the transition of human sessile serrated lesions to malignancy.

  • Androgen receptor degraders overcome common resistance mechanisms developed during prostate cancer treatment
    Neoplasia (IF 3.837) Pub Date : 2020-01-10
    Steven Kregel; Chao Wang; Xin Han; Lanbo Xiao; Ester Fernandez-Salas; Pushpinder Bawa; Brooke L. McCollum; Kari Wilder-Romans; Ingrid J. Apel; Xuhong Cao; Corey Speers; Shaomeng Wang; Arul M. Chinnaiyan

    Androgen receptor (AR) antagonists, such as enzalutamide, have had a major impact on the treatment of metastatic castration-resistant prostate cancer (CRPC). However, even with the advent of AR antagonist therapies, patients continue to develop resistance, and new strategies to combat continued AR signalling are needed. Here, we develop AR degraders using PROteolysis TArgeting Chimeric (PROTAC) technology in order to determine whether depletion of AR protein can overcome mechanisms of resistance commonly associated with current AR-targeting therapies. ARD-61 is the most potent of the AR degraders and effectively induces on-target AR degradation with a mechanism consistent with the PROTAC design. Compared to clinically-approved AR antagonists, administration of ARD-61 in vitro and in vivo results in more potent anti-proliferative, pro-apoptotic effects and attenuation of downstream AR target gene expression in prostate cancer cells. Importantly, we demonstrate that ARD-61 functions in enzalutamide-resistant model systems, characterized by diverse proposed mechanisms of resistance that include AR amplification/overexpression, AR mutation, and expression of AR splice variants, such as AR-V7. While AR degraders are unable to bind and degrade AR-V7, they continue to inhibit tumor cell growth in models overexpressing AR-V7. To further explore this, we developed several isogenic prostate cell line models in which AR-V7 is highly expressed, which also failed to influence the cell inhibitory effects of AR degraders, suggesting that AR-V7 is not a functional resistance mechanism for AR antagonism. These data provide compelling evidence that full-length AR remains a prominent oncogenic driver of prostate cancers which have developed resistance to AR antagonists and highlight the clinical potential of AR degraders for treatment of CRPC.

  • Irreversible and sustained upregulation of endothelin axis during oncogene-associated pancreatic inflammation and cancer
    Neoplasia (IF 3.837) Pub Date : 2020-01-07
    Suprit Gupta; Avi Prajapati; Mansi Gulati; Shailendra K. Gautam; Sushil Kumar; Vipin Dalal; Geoffrey A. Talmon; Satyanarayana Rachagani; Maneesh Jain

    Endothelin-1 (ET-1) and its two receptors, endothelin receptor A (ETAR) and endothelin receptor B (ETBR) exhibit deregulated overexprerssion in pancreatic ductal adenocarcinoma (PDAC) and pancreatitis. We examined the expression pattern of endothelin (ET) axis components in the murine models of chronic and acute inflammation in the presence or absence of oncogenic K-ras. While the expression of endothelin converting enzyme-1 (ECE-1), ET-1, ETAR and ETBR in the normal pancreas is restricted predominantly to the islet cells, progressive increase of ET receptors in ductal cells and stromal compartment is observed in the KC model (Pdx-1 Cre; K-rasG12D) of PDAC. In the murine pancreas harboring K-rasG12D mutation (KC mice), following acute inflammation induced by cerulein, increased ETAR and ETBR expression is observed in the amylase and CK19 double positive cells that represent cells undergoing pancreatic acinar to ductal metaplasia (ADM). As compared to the wild type (WT) mice, cerulein treatment in KC mice resulted in significantly higher levels of ECE-1, ET-1, ETAR and ETBR, transcripts in the pancreas. Similarly, in response to cigarette smoke-induced chronic inflammation, the expression of ET axis components is significantly upregulated in the pancreas of KC mice as compared to the WT mice. In addition to the expression in the precursor pancreatic intraepithelial neoplasm (PanIN lesions) in cigarette smoke-exposure model and metaplastic ducts in cerulein-treatment model, ETAR and ETBR expression is also observed in infiltrating F4/80 positive macrophages and α-SMA positive fibroblasts and high co-localization was seen in the presence of oncogenic K-ras. In conclusion, both chronic and acute pancreatic inflammation in the presence of oncogenic K-ras contribute to sustained upregulation of ET axis components in the ductal and stromal cells suggesting a potential role of ET axis in the initiation and progression of PDAC.

  • Functional Cellular Anti-Tumor Mechanisms are Augmented by Genetic Proteoglycan Targeting
    Neoplasia (IF 3.837) Pub Date : 2019-12-30
    Purva Gupta; Scott C. Johns; So Young Kim; Roland El Ghazal; Elina I. Zuniga; Mark M. Fuster

    While recent research points to the importance of glycans in cancer immunity, knowledge on functional mechanisms is lacking. In lung carcinoma among other tumors, anti-tumor immunity is suppressed; and while some recent therapies boost T-cell mediated immunity by targeting immune-checkpoint pathways, robust responses are uncommon. Augmenting tumor antigen-specific immune responses by endogenous dendritic cells (DCs) is appealing from a specificity standpoint, but challenging. Here, we show that restricting a heparan sulfate (HS) loss-of-function mutation in the HS sulfating enzyme Ndst1 to predominantly conventional DCs (Ndst1f/f CD11cCre+ mutation) results in marked inhibition of Lewis lung carcinoma growth along with increased tumor-associated CD8+ T cells. In mice deficient in a major DC HS proteoglycan (syndecan-4), splenic CD8+ T cells showed increased anti-tumor cytotoxic responses relative to controls. Studies examining Ndst1f/f CD11cCre + mutants revealed that mutation was associated with an increase in anti-tumor cytolysis using either splenic CD8+ T cells or tumor-infiltrating (TIL) CD8+ T cells purified ex-vivo, and tested in pooled effector-to-target cytolytic assays against tumor cells from respective animals. On glycan compositional analysis, HS purified from Ndst1f/f CD11cCre + mutant DCs had reduced overall sulfation, including reduced sulfation of a tri-sulfated disaccharide species that was intriguingly abundant on wildtype DC HS. Interestingly, antigen presentation in the context of major histocompatibility complex class-I (MHC-I) was enhanced in mutant DCs, with more striking effects in the setting of HS under-sulfation, pointing to a likely regulatory role by sulfated glycans at the antigen/MHC-I – T-cell interface; and possibly future opportunities to improve antigen-specific T cell responses by immunologic targeting of HS proteoglycans in cancer.

  • SHARPIN Inhibits Esophageal Squamous Cell Carcinoma Progression by Modulating Hippo Signaling
    Neoplasia (IF 3.837) Pub Date : 2019-12-26
    Aijia Zhang; Weilong Wang; Zhijun Chen; Dan Pang; Xiaofeng Zhou; Kui Lu; Jinghan Hou; Sujie Wang; Can Gao; Benjie Lv; Ziyi Yan; Zhen Chen; Jian Zhu; Lidong Wang; Ting Zhuang; Xiumin Li

    Esophageal cancer is one of the leading malignancies worldwide, while around sixty percent of newly diagnosed cases are in China. In recent years, genome-wide sequencing studies and cancer biology studies show that Hippo signaling functions a critical role in esophageal squamous cell carcinoma (ESCC) progression, which could be a promising therapeutic targets in ESCC treatment. However, the detailed mechanisms of Hippo signaling dys-regulation in ESCC remain not clear. Here we identify SHARPIN protein as an endogenous inhibitor for YAP protein. SHARPIN depletion significantly decreases cell migration and invasion capacity in ESCC, which effects could be rescued by further YAP depletion. Depletion SHARPIN increases YAP protein level and YAP/TEAD target genes, such as CTGF and CYR61 in ESCC. Immuno-precipitation assay shows that SHARPIN associates with YAP, promoting YAP degradation possibly via inducing YAP K48-dependent poly-ubiquitination. Our study reveals a novel post-translational mechanism in modulating Hippo signaling in ESCC. Overexpression or activation of SHARPIN could be a promising strategy to target Hippo signaling for ESCC patients.

  • Long interspersed element-1 ribonucleoprotein particles protect telomeric ends in alternative lengthening of telomeres dependent cells
    Neoplasia (IF 3.837) Pub Date : 2019-12-14
    Thomas Aschacher; Brigitte Wolf; Olivia Aschacher; Florian Enzmann; Viktoria Laszlo; Barbara Messner; Adrian Türkcan; Serge Weis; Sabine Spiegl-Kreinecker; Klaus Holzmann; Günther Laufer; Marek Ehrlich; Michael Bergmann

    Malignant cells ensure telomere maintenance by the alternative lengthening of telomeres (ALT) in the absence of telomerase activity (TA). The retrotransposons “long interspersed nuclear element-1” (LINE-1, L1) are expressed in malignant cells and are primarily known to contribute to complex karyotypes. Here we demonstrate that LINE-1 ribonucleoprotein particles (L1-RNPs) expression is significantly higher in ALT+- versus in TA+-human glioma. Analyzing a role of L1-RNP in ALT, we show that L1-RNPs bind to telomeric repeat containing RNA (TERRA), which is critical for telomere stabilization and which is overexpressed in ALT+ cells. In turn, L1-RNP knockdown (KD) abrogated the nuclear retention of TERRA, resulted in increased telomeric DNA damage, decreased cell growth and reduced expression of ALT characteristics such as c-circles and PML-bodies. L1-RNP KD also decreased the expression of Shelterin- and the ALT-regulating protein Topoisomerase IIIα (TopoIIIα) indicating a more general role of L1-RNPs in supporting telomeric integrity in ALT. Our findings suggest an impact of L1-RNP on telomere stability in ALT+ dependent tumor cells. As L1-RNPs are rarely expressed in normal adult human tissue those elements might serve as a novel target for tumor ablative therapy.

  • Targeting histone deacetylase SIRT1 selectively eradicates EGFR TKI-resistant cancer stem cells via regulation of mitochondrial oxidative phosphorylation in lung adenocarcinoma
    Neoplasia (IF 3.837) Pub Date : 2019-11-22
    Jiangtao Sun, Guifang Li, Yiwen Liu, Mingyang Ma, Kaifang Song, Huaxu Li, Daxing Zhu, XiaoJun Tang, Jinyu Kong, Xiang Yuan

    Lung adenocarcinoma (LAD) is a human malignancy successfully treated with the tyrosine kinase inhibitor (TKI) gefitinib; however, the enrichment of therapy resistant cancer stem cells (CSCs) in such patients is assumed to be a source of treatment failure. Evaluation of LAD cell populations treated with the TKI inhibitor gefitinib identified unique aspects of a subpopulation of tumor cells exhibiting stem-like properties and mitochondria-specific metabolic features along with their reliance on sirtuin 1 (SIRT1) for survival advantage. This addiction to bioenergetic metabolism in LAD treated with EGFR-targeted therapy suggests that mitochondrial targeting should be synthetically lethal using established cytotoxic therapies. Accordingly, loss of the phenotype present in resistant CSC clones either by targeting the energy metabolism with tigecycline, a mitochondrial DNA-translation inhibitor, or tenovin-6 (TV-6), a SIRT1 inhibitor, inhibited their dependency on mitochondrial oxidative phosphorylation (mtOXPHOS) and sensitized them for a more pronounced and long-lasting TKI therapeutic effect. The results specifically demonstrated that combined therapy with TV-6 and gefitinib resulted in tumor regression in xenograft mouse models, whereas administration of a single agent showed no such efficacy. Importantly, combined treatment with TV-6 also decreased the effective dose of gefitinib necessary for treatment response. Clinical analysis demonstrated that high-profile SIRT1 and mtOXPHOS proteins were associated with recurrence and poor prognosis in LAD patients. These observations support the CSC hypothesis for cancer relapse and advocate use of mitochondria-targeting inhibitors as part of combinatorial therapy in a variety of clinical settings, as well as for reducing first-line TKI dosage in LAD patients.

  • CDK5RAP3 is a co-factor for the oncogenic transcription factor STAT3
    Neoplasia (IF 3.837) Pub Date : 2019-11-22
    Susana P. Egusquiaguirre, Suhu Liu, Isidora Tošić, Kevin Jiang, Sarah R. Walker, Maria Nicolais, Tzuen Yih Saw, Michael Xiang, Katarina Bartel, Erik A. Nelson, David A. Frank

    The transcription factor STAT3 regulates genes governing critical cellular processes such as proliferation, survival, and self-renewal. While STAT3 transcriptional function is activated rapidly and transiently in response to physiologic signals, through a variety of mechanisms it can become constitutively activated in the pathogenesis of cancer. This leads to chronic expression of genes that underlie malignant cellular behavior. However, STAT3 is known to interact with other proteins, which may modulate its function. Understanding these interactions can provide insights into novel aspects of STAT3 function and may also suggest strategies to therapeutically target the large number of cancers driven by constitutively activated STAT3. To identify critical modulators of STAT3 transcriptional function, we performed an RNA-interference based screen in a cell-based system that allows quantitative measurement of STAT3 activity. From this approach, we identified CDK5 kinase regulatory-subunit associated protein 3 (CDK5RAP3) as an enhancer of STAT3-dependent gene expression. We found that STAT3 transcriptional function is modulated by CDK5RAP3 in cancer cells, and silencing CDK5RAP3 reduces STAT3-mediated tumorigenic phenotypes including clonogenesis and migration. Mechanistically, CDK5RAP3 binds to STAT3-regulated genomic loci, in a STAT3-dependent manner. In primary human breast cancers, the expression of CDK5RAP3 expression was associated with STAT3 gene expression signatures as well as the expression of individual STAT3 target genes. These findings reveal a novel aspect of STAT3 transcriptional function and potentially provide both a biomarker of enhanced STAT3-dependent gene expression as well as a unique mechanism to therapeutically target STAT3.

  • Glutamine deprivation counteracts hypoxia-induced chemoresistance
    Neoplasia (IF 3.837) Pub Date : 2019-11-22
    Jessica Wappler, Martijn Arts, Anjali Röth, Ron M.A. Heeren, Ulf Peter Neumann, Steven W. Olde Damink, Zita Soons, Thorsten Cramer

    The microenvironment of solid tumors is a key determinant of therapy efficacy. The co-occurrence of oxygen and nutrient deprivation is a common phenomenon of the tumor microenvironment and associated with treatment resistance. Cholangiocarcinoma (CCA) is characterized by a very poor prognosis and pronounced chemoresistance. A better understanding of the underlying molecular mechanisms is urgently needed to improve therapy strategies against CCA. We sought to investigate the importance of the conditionally essential amino acid glutamine, a centrally important nutrient for a variety of solid tumors, for CCA. Glutamine levels were strongly decreased in CCA samples and the growth of established human CCA cell lines was highly dependent on glutamine. Using gradual reduction of external glutamine, we generated derivatives of CCA cell lines which were able to grow without external glutamine (termed glutamine-depleted (GD)). To analyze the effects of coincident oxygen and glutamine deprivation, GD cells were treated with cisplatin or gemcitabine under normoxia and hypoxia. Strikingly, the well-established phenomenon of hypoxia-induced chemoresistance was completely reversed in GD cells. In order to better understand the underlying mechanisms, we focused on the oncogene c-Myc. The combination of cisplatin and hypoxia led to sustained c-Myc protein expression in wildtype cells. In contrast, c-Myc expression was reduced in response to the combinatorial treatment in GD cells, suggesting a functional importance of c-Myc in the process of hypoxia-induced chemoresistance. In summary, these findings indicate that the mechanisms driving adaption to tumor microenvironmental changes and their relevance for the response to therapy are more complex than expected.

  • Liver X Receptor Agonism Sensitizes a Subset of Hepatocellular Carcinoma to Sorafenib by Dual-Inhibiting MET and EGFR
    Neoplasia (IF 3.837) Pub Date : 2019-11-18
    Weiqing Shao, Wenwei Zhu, Jing Lin, Mengjun Luo, Zhifei Lin, Lu Lu, Huliang Jia, Lunxiu Qin, Ming Lu, Jinhong Chen

    Sorafenib is the first approved systemic therapy for advanced hepatocellular carcinoma (HCC) and is the first-line choice in clinic. Sustained activation of receptor tyrosine kinases (RTKs) is associated with low efficacy of sorafenib in HCC. Activation of liver X receptor (LXR) has been reported to inhibit some RTKs. In this study, we found that the LXR agonist enhanced the anti-tumor activity of sorafenib in a subset of HCC cells with high LXR-β/α gene expression ratio. Mechanically, the activation of LXR suppressed sorafenib dependent recruitment of MET and epidermal growth factor receptor (EGFR) in lipid rafts through cholesterol efflux. Our findings imply that LXR agonist can serve as a potential sensitizer to enhance the anti-tumor effect of sorafenib.

  • Somatic copy number alterations are associated with EGFR amplification and shortened survival in patients with primary glioblastoma
    Neoplasia (IF 3.837) Pub Date : 2019-11-18
    Lisandra Muñoz-Hidalgo, Teresa San-Miguel, Javier Megías, Daniel Monleón, Lara Navarro, Pedro Roldán, Miguel Cerdá-Nicolás, Concha López-Ginés

    Glioblastoma (GBM) is the most common malignant primary tumor of the central nervous system. With no effective therapy, the prognosis for patients is terrible poor. It is highly heterogeneous and EGFR amplification is its most frequent molecular alteration. In this light, we aimed to examine the genetic heterogeneity of GBM and to correlate it with the clinical characteristics of the patients. For that purpose, we analyzed the status of EGFR and the somatic copy number alterations (CNAs) of a set of tumor suppressor genes and oncogenes. Thus, we found GBMs with high level of EGFR amplification, low level and with no EGFR amplification. Highly amplified tumors showed histological features of aggressiveness. Interestingly, accumulation of CNAs, as a measure of tumor mutational burden, was frequent and significantly associated to shortened survival. EGFR-amplified GBMs displayed both a higher number of concrete CNAs and a higher global tumor mutational burden than their no EGFR-amplified counterparts. In addition to genetic changes previously described in GBM, we found PARK2 and LARGE1 CNAs associated to EGFR amplification. The set of genes analyzed allowed us to explore relevant signaling pathways on GBM. Both PARK2 and LARGE1 are related to receptor tyrosine kinase/PI3K/PTEN/AKT/mTOR-signaling pathway. Finally, we found an association between the molecular pathways altered, EGFR amplification and a poor outcome. Our results underline the potential interest of categorizing GBM according to their EGFR amplification level and the usefulness of assessing the tumor mutational burden. These approaches would open new knowledge possibilities related to GBM biology and therapy.

  • Prostaglandin F2α-induced Prostate Transmembrane Protein, Androgen Induced 1 mediates ovarian cancer progression increasing epithelial plasticity
    Neoplasia (IF 3.837) Pub Date : 2019-11-14
    Alba Jiménez-Segovia, Alba Mota, Alejandro Rojo-Sebastián, Beatriz Barrocal, Angela Rynne-Vidal, María-Laura García-Bermejo, Raquel Gómez-Bris, Lukas J.A.C. Hawinkels, Pilar Sandoval, Ramon Garcia-Escudero, Manuel López-Cabrera, Gema Moreno-Bueno, Manuel Fresno, Konstantinos Stamatakis

    The role of prostaglandin (PG) F2α has been scarcely studied in cancer. We have identified a new function for PGF2α in ovarian cancer, stimulating the production of Prostate Transmembrane Protein, Androgen Induced 1 (PMEPA1). We show that this induction increases cell plasticity and proliferation, enhancing tumor growth through PMEPA1. Thus, PMEPA1 overexpression in ovarian carcinoma cells, significantly increased cell proliferation rates, whereas PMEPA1 silencing decreased proliferation. In addition, PMEPA1 overexpression buffered TGFβ signaling, via reduction of SMAD-dependent signaling. PMEPA1 overexpressing cells acquired an epithelial morphology, associated with higher E-cadherin expression levels while β-catenin nuclear translocation was inhibited. Notwithstanding, high PMEPA1 levels also correlated with epithelial to mesenchymal transition markers, such as vimentin and ZEB1, allowing the cells to take advantage of both epithelial and mesenchymal characteristics, gaining in cell plasticity and adaptability. Interestingly, in mouse xenografts, PMEPA1 overexpressing ovarian cells had a clear survival and proliferative advantage, resulting in higher metastatic capacity, while PMEPA1 silencing had the opposite effect. Furthermore, high PMEPA1 expression in a cohort of advanced ovarian cancer patients was observed, correlating with E-cadherin expression. Most importantly, high PMEPA1 mRNA levels were associated with lower patient survival.

  • Extracellular acidosis differentiates pancreatitis and pancreatic cancer in mouse models using acidoCEST MRI
    Neoplasia (IF 3.837) Pub Date : 2019-11-14
    Rachel A. High, Edward A. Randtke, Kyle M. Jones, Leila R. Lindeman, Jacqueline C. Ma, Shu Zhang, Lucia G. LeRoux, Mark D. Pagel

    Differentiating pancreatitis from pancreatic cancer would improve diagnostic specificity, and prognosticating pancreatitis that progresses to pancreatic cancer would also improve diagnoses of pancreas pathology. The high glycolytic metabolism of pancreatic cancer can cause tumor acidosis, and different levels of pancreatitis may also have different levels of acidosis, so that extracellular acidosis may be a diagnostic biomarker for these pathologies. AcidoCEST MRI can noninvasively measure extracellular pH (pHe) in the pancreas and pancreatic tissue. We used acidoCEST MRI to measure pHe in a KC model treated with caerulein, which causes pancreatitis followed by development of pancreatic cancer. We also evaluated the KC model treated with PBS, and wild-type mice treated with caerulein or PBS as controls. The caerulein-treated KC cohort had lower pHe of 6.85–6.92 before and during the first 48 h after initiating treatment, relative to a pHe of 6.92 to 7.05 pHe units for the other cohorts. The pHe of the caerulein-treated KC cohort decreased to 6.79 units at 5 weeks when pancreatic tumors were detected with anatomical MRI, and sustained a pHe of 6.75 units at the 8-week time point. Histopathology was used to evaluate and validate the presence of tumors and inflammation in each cohort. These results showed that acidoCEST MRI can differentiate pancreatic cancer from pancreatitis in this mouse model, but does not appear to differentiate pancreatitis that progresses to pancreatic cancer vs. pancreatitis that does not progress to cancer.

  • Fibrin Deposit on the Peritoneal Surface Serves as a Niche for Cancer Expansion in Carcinomatosis Patients
    Neoplasia (IF 3.837) Pub Date : 2019-11-14
    Shah Shahid, Aldybiat Iman, Ullah Matti, Kaci Rachid, Alassaf Assaf, Clarisse Eveno, Pocard Marc, Mirshahi Massoud

    Peritoneal metastasis (PM) is a very serious complication of gastrointestinal and gynecological malignancies which is poorly documented. Modified mesothelial cell layer and their microenvironments can favor fibrin deposition for cancer cell adhesion. Scanning and transmission electron microscopy of peritoneal surface and cancer cell clusters from cancer patients was done. Ascites and its impact on mesothelial cells were assessed by cytokine array. Neprilysin, matrix metalloprotease, epithelial mesenchymal transition (EMT) related molecules (E-cadherin, Snail, Slug, Twist, Vimentin and Fibronectin), tissues factor (TF), endothelial protein C receptors (EPCR) were quantified by q-PCR. Fibrin in the simples were stained using anti fibrin F1E1 antibody. Migration ability was assessed by scratch assay. Cell viability and neprilysin activity were analyzed by bioluminescence. Cancer cells-fibrin interaction was investigated by scanning electron microscopy (SEM) and microcinematography (MCG). Mesothelial cells change their morphology after incubation with carcinomatosis peritoneal fluids in vitro. EMT associated with upregulation of neprilysin, matrix metalloproteinase-2, tissue factor and cytokines secretions such as interleukin-6, and 8, hepatocyte growth factor and granulocyte chemotactic protein-2 mRNA and protein were observed. EPCR expression as a natural anticoagulant was decreased. In parallel, carcinomatosis cell clusters extracted from peritoneal fluids were found to be associated with fibrin. Kinetic analysis of cancer cell-fibrin interaction in vitro studied by MCG showed that fiber filaments generated from clots inhibited cancer cell adhesion on fibrin clots. These results indicated that fibrin deposit on the peritoneal surface serve as niches for cancer expansion in carcinomatosis patients.

  • Genome-Wide Profiling of Acquired Uniparental Disomy Reveals Prognostic Factors in Head and Neck Squamous Cell Carcinoma
    Neoplasia (IF 3.837) Pub Date : 2019-11-14
    Musaffe Tuna, Wenbin Liu, Christopher I. Amos, Gordon B. Mills

    Acquired uniparental disomy (aUPD) leads to homozygosity facilitating identification of monoallelically expressed genes. We analyzed single-nucleotide polymorphism array-based genotyping data of 448 head and neck squamous cell carcinoma (HNSCC) samples from The Cancer Genome Atlas to determine the frequency and distribution of aUPD regions and their association with survival, as well as to gain a better understanding of their influence on the tumor genome. We used expression data from the same dataset to identify differentially expressed genes between groups with and without aUPD. Univariate and multivariable Cox proportional hazards models were performed for survival analysis. We found that 82.14% of HNSCC samples carried aUPD; the most common regions were in chromosome 17p (31.25%), 9p (30.13%), and 9q (27.46%). In univariate analysis, five independent aUPD regions at chromosome 9p, two regions at chromosome 9q, and the CDKN2A region were associated with poor overall survival in all groups, including training and test sets and human papillomavirus (HPV)-negative samples. Forty-three genes in areas of aUPD including PD-L1 and CDKN2A were differentially expressed in samples with aUPD compared to samples without aUPD. In multivariable analysis, aUPD at the CDKN2A region was a significant predictor of overall survival in the whole cohort and in patients with HPV-negative HNSCC. aUPD at specific regions in the genome influences clinical outcomes of HNSCC and may be beneficial for selection of personalized therapy to prolong survival in patients with this disease.

  • Targeting bromodomain-containing protein 4 (BRD4) inhibits MYC expression in colorectal cancer cells
    Neoplasia (IF 3.837) Pub Date : 2019-11-14
    C. Otto, S. Schmidt, C. Kastner, S. Denk, J. Kettler, N. Müller, C.T. Germer, E. Wolf, P. Gallant, A. Wiegering

    The transcriptional regulator BRD4 has been shown to be important for the expression of several oncogenes including MYC. Inhibiting of BRD4 has broad antiproliferative activity in different cancer cell types. The small molecule JQ1 blocks the interaction of BRD4 with acetylated histones leading to transcriptional modulation. Depleting BRD4 via engineered bifunctional small molecules named PROTACs (proteolysis targeting chimeras) represents the next-generation approach to JQ1-mediated BRD4 inhibition. PROTACs trigger BRD4 for proteasomale degradation by recruiting E3 ligases. The aim of this study was therefore to validate the importance of BRD4 as a relevant target in colorectal cancer (CRC) cells and to compare the efficacy of BRD4 inhibition with BRD4 degradation on downregulating MYC expression. JQ1 induced a downregulation of both MYC mRNA and MYC protein associated with an antiproliferative phenotype in CRC cells. dBET1 and MZ1 induced degradation of BRD4 followed by a reduction in MYC expression and CRC cell proliferation. In SW480 cells, where dBET1 failed, we found significantly lower levels of the E3 ligase cereblon, which is essential for dBET1-induced BRD4 degradation. To gain mechanistic insight into the unresponsiveness to dBET1, we generated dBET1-resistant LS174t cells and found a strong downregulation of cereblon protein. These findings suggest that inhibition of BRD4 by JQ1 and degradation of BRD4 by dBET1 and MZ1 are powerful tools for reducing MYC expression and CRC cell proliferation. In addition, downregulation of cereblon may be an important mechanism for developing dBET1 resistance, which can be evaded by incubating dBET1-resistant cells with JQ1 or MZ1.

  • Microenvironmental Factors Drive Tenascin C and Src Cooperation to Promote Invadopodia Formation in Ewing Sarcoma
    Neoplasia (IF 3.837) Pub Date : 2019-09-13
    Allegra G. Hawkins, Claire M. Julian, Sonja Konzen, Sydney Treichel, Elizabeth R. Lawlor, Kelly M. Bailey

    Ewing sarcoma is a bone tumor most commonly diagnosed in adolescents and young adults. Survival for patients with recurrent or metastatic Ewing sarcoma is dismal and there is a dire need to better understand the mechanisms of cell metastasis specific to this disease. Our recent work demonstrated that microenvironmental stress leads to increased Ewing sarcoma cell invasion through Src activation. Additionally, we have shown that the matricellular protein tenascin C (TNC) promotes metastasis in Ewing sarcoma. A major role of both TNC and Src is mediation of cell–cell and cell-matrix interactions resulting in changes in cell motility, invasion, and adhesion. However, it remains largely unknown, if and how, TNC and Src are linked in these processes. We hypothesized that TNC is a positive regulator of invadopodia formation in Ewing sarcoma through its ability to activate Src. We demonstrate here that both tumor cell endogenous and exogenous TNC can enhance Src activation and invadopodia formation in Ewing sarcoma. We found that microenvironmental stress upregulates TNC expression and this is dampened with application of the Src inhibitor dasatinib, suggesting that TNC expression and Src activation cooperate to promote the invasive phenotype. This work reports the impact of stress-induced TNC expression on enhancing cell invadopodia formation, provides evidence for a feed forward loop between TNC and Src to promote cell metastatic behavior, and highlights a pathway by which microenvironment-driven TNC expression could be therapeutically targeted in Ewing sarcoma.

  • Rectal cancer sub-clones respond differentially to neoadjuvant therapy
    Neoplasia (IF 3.837) Pub Date : 2019-09-12
    Lynn M Frydrych, Peter Ulintz, Armand Bankhead, Christopher Sifuentes, Joel Greenson, Lillias Maguire, Regina Irwin, Eric R. Fearon, Karin M Hardiman

    Treatment of locally advanced rectal cancer includes chemotherapy, radiation, and surgery but patient responses to neoadjuvant treatment are variable. We have shown that rectal tumors are comprised of multiple genetically distinct sub-clones. Unique sub-clones within tumors may harbor mutations which contribute to inter-patient variation in response to neoadjuvant chemoradiotherapy (nCRT). Analysis of the influence of nCRT on the extent and nature of intra-tumoral genetic heterogeneity in rectal cancer may provide insights into mechanisms of resistance. Locally advanced rectal cancer patients underwent pre-treatment biopsies. At the time of surgery, tissue from the treated tumor was obtained and analyzed. Pre- and post-treatment specimens were subjected to whole exome and confirmatory deep sequencing for somatic mutations. Copy number variation was assessed using OncoScan SNP arrays. Genomic data were analyzed using PyClone to identify sub-clonal tumor population following nCRT. Alterations that persisted or were enriched in the post-treatment tumor specimen following nCRT were defined for each patient. Thirty-two samples were obtained from ten patients. PyClone identified 2 to 10 genetic sub-clones per tumor. Substantial changes in the proportions of individual sub-clones in pre- versus post-treatment tumor material were found in all patients. Resistant sub-clones recurrently contained mutations in TP53, APC, ABCA13, MUC16, and THSD4. Recurrent copy number variation was observed across multiple chromosome regions after nCRT. Pathway analysis including variant alleles and copy number changes associated with resistant sub-clones revealed significantly altered pathways, especially those linked to the APC and TP53 genes, which were the two most frequently mutated genes. Intra-tumoral heterogeneity is evident in pre-treatment rectal cancer. Following treatment, sub-clonal populations are selectively modified and enrichment of a subset of pre-treatment sub-clones is seen. Further studies are needed to define recurrent alterations at diagnosis that may contribute to resistance to nCRT.

  • VCAM-1 Density and Tumor Perfusion Predict T-cell Infiltration and Treatment Response in Preclinical Models
    Neoplasia (IF 3.837) Pub Date : 2019-09-11
    Johannes Riegler, Herman Gill, Annie Ogasawara, Maj Hedehus, Vincent Javinal, Jason Oeh, Gregory Z. Ferl, Jan Marik, Simon Williams, Deepak Sampath, Jill Schartner, Richard A.D. Carano

    Cancer immunotherapies have demonstrated durable responses in a range of different cancers. However, only a subset of patients responds to these therapies. We set out to test if non-invasive imaging of tumor perfusion and vascular inflammation may be able to explain differences in T-cell infiltration in pre-clinical tumor models, relevant for treatment outcomes. Tumor perfusion and vascular cell adhesion molecule (VCAM-1) density were quantified using magnetic resonance imaging (MRI) and correlated with infiltration of adoptively transferred and endogenous T-cells. MRI biomarkers were evaluated for their ability to detect tumor rejection 3 days after T-cell transfer. Baseline levels of these markers were used to assess their ability to predict PD-L1 treatment response. We found correlations between MRI-derived VCAM-1 density and infiltration of endogenous or adoptively transferred T-cells in some preclinical tumor models. Blocking T-cell binding to endothelial cell adhesion molecules (VCAM-1/ICAM) prevented T-cell mediated tumor rejection. Tumor rejection could be detected 3 days after adoptive T-cell transfer prior to tumor volume changes by monitoring the extracellular extravascular volume fraction. Imaging tumor perfusion and VCAM-1 density before treatment initiation was able to predict the response of MC38 tumors to PD-L1 blockade. These results indicate that MRI based assessment of tumor perfusion and VCAM-1 density can inform about the permissibility of the tumor vasculature for T-cell infiltration which may explain some of the observed variance in treatment response for cancer immunotherapies.

  • MCM2, MCM4, and MCM6 in Breast Cancer: Clinical Utility in Diagnosis and Prognosis
    Neoplasia (IF 3.837) Pub Date : 2019-08-30
    Marianne Samir Makboul Issac, Einas Yousef, Muhammad Ramzan Tahir, Louis A. Gaboury

    Breast cancer is a heterogeneous disease comprising the estrogen receptor (ER)–positive luminal subtype which is subdivided into luminal A and luminal B and ER-negative breast cancer which includes the triple-negative subtype. This study has four aims: 1) to examine whether Minichromosome Maintenance (MCM)2, MCM4, and MCM6 can be used as markers to differentiate between luminal A and luminal B subtypes; 2) to study whether MCM2, MCM4, and MCM6 are highly expressed in triple-negative breast cancer, as there is an urgent need to search for surrogate markers in this aggressive subtype, for drug development purposes; 3) to compare the prognostic values of these markers in predicting relapse-free survival; and 4) to compare the three approaches used for scoring the protein expression of these markers by immunohistochemistry (IHC). MCM2, MCM4, MCM6, and MKI67 mRNA expression was first studied using in silico analysis of available breast cancer datasets. We next used IHC to evaluate their protein expression on tissue microarrays using three scoring methods. MCM2, MCM4, and MCM6 can help in distinction between luminal A and luminal B whose therapeutic management and clinical outcomes are different. MCM2, MCM4, MCM6, and Ki-67 are highly expressed in breast cancer of high histological grades that comprise clinically aggressive tumors such as luminal B, HER2-positive, and triple-negative subtypes. Low transcript expression of these markers is associated with increased probability of relapse-free survival. A positive relationship exists among the three scoring methods of each of the four markers. An independent validation cohort is needed to confirm their clinical utility.

  • Iroquois Homeobox 1 Acts as a True Tumor Suppressor in Multiple Organs by Regulating Cell Cycle Progression
    Neoplasia (IF 3.837) Pub Date : 2019-08-23
    In Hye Jung, Da-Woon Esther Jung, Yong-Yoon Chung, Kyung-Sik Kim, Seung Woo Park

    Iroquois homeobox 1 (IRX1) belongs to the Iroquois homeobox family known to play an important role during embryonic development. Interestingly, however, recent studies have suggested that IRX1 also acts as a tumor suppressor. Here, we use homozygous knockout mutants of zebrafish to demonstrate that the IRX1 gene is a true tumor suppressor gene and mechanism of the tumor suppression is mediated by repressing cell cycle progression. In this study, we found that knockout of zebrafish Irx1 gene induced hyperplasia and tumorigenesis in the multiple organs where the gene was expressed. On the other hands, overexpression of the IRX1 gene in human tumor cell lines showed delayed cell proliferation of the tumor cells. These results suggest that the IRX1 gene is truly involved in tumor suppression. In an attempt to identify the genes regulated by the transcription factor IRX1, we performed microarray assay using the cRNA obtained from the knockout mutants. Our result indicated that the highest fold change of the differential genes fell into the gene category of cell cycle regulation, suggesting that the significant canonical pathway of IRX1 in antitumorigenesis is done by regulating cell cycle. Experiment with cell cycle blockers treated to IRX1 overexpressing tumor cells showed that the IRX1 overexpression actually delayed the cell cycle. Furthermore, Western blot analysis with cyclin antibodies showed that IRX1 overexpression induced decrease of cyclin production in the cancer cells. In conclusion, our in vivo and in vitro studies revealed that IRX1 gene functionally acts as a true tumor suppressor, inhibiting tumor cell growth by regulating cell cycle.

  • Pseudogene Associated Recurrent Gene Fusion in Prostate Cancer
    Neoplasia (IF 3.837) Pub Date : 2019-08-22
    Balabhadrapatruni VSK Chakravarthi, Pavithra Dedigama-Arachchige, Shannon Carskadon, Shanker Kalyana Sundaram, Jia Li, Kuan-Han Hank Wu, Darshan Shimoga Chandrashekar, James O Peabody, Hans Stricker, Clara Hwang, Dhananjay A Chitale, Sean R Williamson, Nilesh S Gupta, Nora M Navone, Craig Rogers, Mani Menon, Sooryanarayana Varambally, Nallasivam Palanisamy

    We present the functional characterization of a pseudogene associated recurrent gene fusion in prostate cancer. The fusion gene KLK4-KLKP1 is formed by the fusion of the protein coding gene KLK4 with the noncoding pseudogene KLKP1. Screening of a cohort of 659 patients (380 Caucasian American; 250 African American, and 29 patients from other races) revealed that the KLK4-KLKP1 is expressed in about 32% of prostate cancer patients. Correlative analysis with other ETS gene fusions and SPINK1 revealed a concomitant expression pattern of KLK4-KLKP1 with ERG and a mutually exclusive expression pattern with SPINK1, ETV1, ETV4, and ETV5. Development of an antibody specific to KLK4-KLKP1 fusion protein confirmed the expression of the full-length KLK4-KLKP1 protein in prostate tissues. The in vitro and in vivo functional assays to study the oncogenic properties of KLK4-KLKP1 confirmed its role in cell proliferation, cell invasion, intravasation, and tumor formation. Presence of strong ERG and AR binding sites located at the fusion junction in KLK4-KLKP1 suggests that the fusion gene is regulated by ERG and AR. Correlative analysis of clinical data showed an association of KLK4-KLKP1 with lower preoperative PSA values and in young men (<50 years) with prostate cancer. Screening of patient urine samples showed that KLK4-KLKP1 can be detected noninvasively in urine. Taken together, we present KLK4-KLKP1 as a class of pseudogene associated fusion transcript in cancer with potential applications as a biomarker for routine screening of prostate cancer.

  • Targeting c-MYC through Interference with NAMPT and SIRT1 and Their Association to Oncogenic Drivers in Murine Serrated Intestinal Tumorigenesis
    Neoplasia (IF 3.837) Pub Date : 2019-08-20
    Lydia B., Yina Z., Nina K., Andrea S., Sophie Luise B., Peter J., Florian G., Roland R., Antje M.

    We recently described a positive feedback loop connecting c-MYC, NAMPT, DBC1 and SIRT1 that contributes to unrestricted cancer cell proliferation. Here we determine the relevance of the loop for serrated route intestinal tumorigenesis using genetically well-defined BrafV600E and K-rasG12D mouse models. In both models we show that c-MYC and SIRT1 protein expression increased through progression from hyperplasia to invasive carcinomas and metastases. It correlated with high NAMPT expression and was directly associated to activation of the oncogenic drivers. Assessing functional and molecular consequences of pharmacological interference with factors of the loop, we found that inhibition of NAMPT resulted in apoptosis and reduced clonogenic growth in human BRAF-mutant colorectal cancer cell lines and patient-derived tumoroids. Blocking SIRT1 activity was only effective when combined with a PI3K inhibitor, whereas the latter antagonized the effects of NAMPT inhibition. Interfering with the positive feedback loop was associated with down-regulation of c-MYC and temporary de-repression of TP53, explaining the anti-proliferative and pro-apoptotic effects. In conclusion we show that the c-MYC-NAMPT-DBC1-SIRT1 positive feedback loop contributes to murine serrated tumor progression. Targeting the feedback loop exerted a unique, dual therapeutic effect of oncoprotein inhibition and tumor suppressor activation. It may therefore represent a promissing target for serrated colorectal cancer, and presumably for other cancer types with deregulated c-MYC.

  • Depletion of the Transcriptional Coactivator Amplified in Breast Cancer 1 (AIB1) Uncovers Functionally Distinct Subpopulations in Triple-Negative Breast Cancer
    Neoplasia (IF 3.837) Pub Date : 2019-08-19
    FR Saenz, V Ory, MO Schmidt, BV Kallakury, SC Mueller, PA Furth, A Wellstein, AT Riegel

    The transcriptional coactivator Amplified in Breast Cancer 1 (AIB1) plays a major role in the progression of hormone and HER2-dependent breast cancers but its role in triple negative breast cancer (TNBC) is undefined. Here, we report that established TNBC cell lines, as well as cells from a TNBC patient-derived xenograft (PDX) that survive chemotherapy treatment in vitro express lower levels of AIB1 protein. The surviving cell population has an impaired tube-formation phenotype when cultured onto basement membrane, a property shared with TNBC cells that survive shRNA-mediated depletion of AIB1 (AIB1LOW cells). DNA analysis by exome sequencing revealed that AIB1LOW cells represent a distinct subpopulation. Consistent with their in vitro phenotype AIB1LOW cells implanted orthotopically generated slower growing tumors with less capacity for pulmonary metastases. Gene expression analysis of cultured cells and tumors revealed that AIB1LOW cells display a distinct expression signature of genes in pro-inflammatory pathways, cell adhesion, proteolysis and tissue remodeling. Interestingly, the presence of this AIB1LOW expression signature in breast cancer specimens is associated with shorter disease free survival of chemotherapy treated patients. We concluded that TNBC cell lines contain heterogeneous populations with differential dependence on AIB1 and that the gene expression pattern of AIB1LOW cells may represent a signature indicative of poor response to chemotherapy in TNBC patients.

  • SMAR1 favors immunosurveillance of cancer cells by modulating calnexin and MHC I expression
    Neoplasia (IF 3.837) Pub Date : 2019-08-15
    Aftab Alam, Nandaraj Taye, Sonal Patel, Milind Thube, Jayati Mullick, Vibhuti Kumar Shah, Richa Pant, Tanaya Roychowdhury, Nilanjan Banerjee, Subhrangsu Chatterjee, Rittwika Bhattacharya, Rini Roy, Ashis Mukhopadhyay, Devraj Mogare, Samit Chattopadhyay

    Down-regulation or loss of MHC class I expression is a major mechanism used by cancer cells to evade immunosurveillance and increase their oncogenic potential. MHC I mediated antigen presentation is a complex regulatory process, controlled by antigen processing machinery (APM) dictating immune response. Transcriptional regulation of the APM that can modulate gene expression profile and their correlation to MHC I mediated antigen presentation in cancer cells remain enigmatic. Here, we reveal that Scaffold/Matrix-Associated Region 1- binding protein (SMAR1), positively regulates MHC I surface expression by down-regulating calnexin, an important component of antigen processing machinery (APM) in cancer cells. SMAR1, a bonafide MAR binding protein acts as a transcriptional repressor of several oncogenes. It is down-regulated in higher grades of cancers either through proteasomal degradation or through loss of heterozygosity (LOH) at the Chr.16q24.3 locus where the human homolog of SMAR1 (BANP) has been mapped. It binds to a short MAR region of the calnexin promoter forming a repressor complex in association with GATA2 and HDAC1. A reverse correlation between SMAR1 and calnexin was thus observed in SMAR1-LOH cells and also in tissues from breast cancer patients. To further extrapolate our findings, influenza A (H1N1) virus infection assay was performed. Upon viral infection, the levels of SMAR1 significantly increased resulting in reduced calnexin expression and increased MHC I presentation. Taken together, our observations establish that increased expression of SMAR1 in cancers can positively regulate MHC I surface expression thereby leading to higher chances of tumor regression and elimination of cancer cells.

  • Dual CTLA-4 and PD-L1 Blockade Inhibits Tumor Growth and Liver Metastasis in a Highly Aggressive Orthotopic Mouse Model of Colon Cancer
    Neoplasia (IF 3.837) Pub Date : 2019-08-11
    E Fiegle, D Doleschel, S Koletnik, A Rix, R Weiskirchen, E Borkham-Kamphorst, F Kiessling, W Lederle

    Immune checkpoint inhibitors have shown clinical benefit in several cancer entities including metastatic microsatellite instable colorectal carcinomas. However, for the majority of metastatic colorectal carcinomas the potential and limitations of immune checkpoint inhibition is not fully understood. In this study, the effects of sole and dual CTLA-4 and PD-L1 blockade were investigated in a microsatellite stable highly aggressive orthotopic mouse model of colon cancer. Dual CTLA-4 and PD-L1 inhibition resulted in tumor growth stagnation and completely blocked liver metastasis. Sole CTLA-4 and PD-L1 inhibition only moderately reduced metastatic spread of the colon cancer cells, though CTLA-4 blockade being superior to PD-L1 inhibition. Dual immune checkpoint blockade and sole CTLA-4 inhibition significantly increased intratumoral CD8+ and CD4+ T cells and reduced FOXP3+/CD4+ Treg cells. This was associated with increased expression levels of the pro-inflammatory Th1/M1-related cytokines IFN-γ, IL-1α, IL-2, and IL-12. Moreover, tumors treated with combined immune checkpoint blockade showed the strongest increase in intratumoral iNOS+ macrophages, reduction of PD-L1+ and Tie2+ macrophages and the lowest expression of M2/Th2-related IL-4, TARC and COX-2. The assessment of further microenvironmental changes by DCE-MRI and immunohistology revealed no alterations in functional tumor vascularization upon combined immune checkpoint blockade, but a significant increase in intratumoral fibroblasts and collagen I deposition. Thus, the synergistic inhibitory effects of dual immune checkpoint inhibition can be explained by anti-tumorigenic T cell responses mediated by CTLA-4 inhibition and M1 macrophage polarization predominantly induced by PD-L1 blockade. This was accompanied by pronounced fibroblast activation highlighting the interconnection between immunogenicity and desmoplasia.

  • p85α Inactivates MMP-2 and Suppresses Bladder Cancer Invasion by Inhibiting MMP-14 Transcription and TIMP-2 Degradation
    Neoplasia (IF 3.837) Pub Date : 2019-08-08
    Jingjing Wang, Ning Zhang, Minggang Peng, Xiaohui Hua, Chao Huang, Zhongxian Tian, Qipeng Xie, Junlan Zhu, Jingxia Li, Haishan Huang, Chuanshu Huang

    Recent studies show p85α up-regulates epidermal growth factor (EGF) receptor, thereby promoting malignant cell transformation and migration in normal mouse embryonic fibroblasts (MEFs). However, the potential role of p85α in human bladder cancer (BC) remains unknown. Here, we show that p85α is down-regulated in BC tumor tissues. Ectopic expression of p85α inhibited cell invasion, but not migration, whereas p85α knockdown promoted invasion in BC cells, revealing that p85α inhibits BC invasion. Overexpression of kinase-deficient p110 in T24 T(p85α) cells inhibited BC cell migration, but not invasion, suggesting that the inhibition of p85α on invasion is independent of PI3K activity. The effect of p85α on inhibiting BC invasion was mediated by the inactivation of MMP-2 concomitant with the up-regulation of TIMP-2 and down-regulation of MMP-14. Mechanistic studies revealed c-Jun inactivation was associated with p85α knockdown-induced MMP-14 expression, and down-regulated miR-190, leading to ATG7 mRNA degradation. This suppressed the autophagy-dependent removal of TIMP-2 in human BC cells. The present results identify a novel function of p85α and clarify the mechanisms underlying its inhibition of BC invasion, providing insight into the role of p85α in normal and cancer cells.

  • The Adiponectin-AdipoR1 Axis Mediates Tumor Progression and Tyrosine Kinase Inhibitor Resistance in Metastatic Renal Cell Carcinoma
    Neoplasia (IF 3.837) Pub Date : 2019-08-08
    Guangxi Sun, Xingming Zhang, Zhenhua Liu, Sha Zhu, Pengfei Shen, Haoran Zhang, Mengni Zhang, Ni Chen, Jinge Zhao, Junru Chen, Jiandong Liu, Jindong Dai, Zhipeng Wang, Xudong Zhu, Yufang Wang, Hao Zeng

    The survival of patients diagnosed with metastatic renal cell carcinoma (RCC) is still limited and the current targeted therapies are only partially effective. Herein, we investigated the clinical value and functions of adiponectin receptors (AdipoR1 and AdipoR2) in metastatic renal cell carcinoma (RCC) patients treated with tyrosine kinase inhibitors (TKIs). A total of 127 mRCC patients treated with first-line TKIs between 2008 and 2017 at a single institution were collected. AdipoR1 and AdipoR2 expression was assessed by immunohistochemistry. AdipoR1 was positively expressed in 87.4% (111/127) of tumors, especially, highly expressed in pulmonary and bone lesions. Patients with low-AdipoR1 expression in primary tumor tissues were more likely to suffer from progressive disease during TKIs treatment (40.0% vs. 11.1%, P = 0 .02), and with decreased progression-free survival (PFS: 19.5 vs. 37.8 mo, P = .001) and overall survival (OS: 62.3 vs 101.1 mo, P = .004) compared to those with high-AdipoR1 expression. Moreover, low-AdipoR1 expression in metastatic tissues was also associated with poor PFS (P = .006) and OS (P = .037). In contrast, AdipoR2 expression was neither associated with sunitinib response nor patient survival. In vitro, we found that adiponectin inhibited migration, invasion and sensitized RCC cells to sunitinib though interacting with AdipoR1, but not AdipoR2. Furthermore, we demonstrated that adiponentin-AdipoR1 axis inhibits tumor cells migration and invasion by blocking the GSK3β/β-Catenin pathway and enhances sunitinib sensitivity via abrogating PI3K/AKT/NF-κB signaling. Our results suggest that adiponentin-AdipoR1 axis may serve as a predictor of TKIs response and could be a potential therapeutic target in the future treatment for metastatic RCC.

  • A Novel Pyrazolopyrimidine Ligand of Human PGK1 and Stress Sensor DJ1 Modulates the Shelterin Complex and Telomere Length Regulation
    Neoplasia (IF 3.837) Pub Date : 2019-08-08
    Alan E. Bilsland, Yu Liu, Andrew Turnbull, David Sumpton, Katrina Stevenson, Claire J. Cairney, Susan M. Boyd, Jon Roffey, David Jenkinson, W. Nicol Keith

    Telomere signaling and metabolic dysfunction are hallmarks of cell aging. New agents targeting these processes might provide therapeutic opportunities, including chemoprevention strategies against cancer predisposition. We report identification and characterization of a pyrazolopyrimidine compound series identified from screens focused on cell immortality and whose targets are glycolytic kinase PGK1 and oxidative stress sensor DJ1. We performed structure–activity studies on the series to develop a photoaffinity probe to deconvolute the cellular targets. In vitro binding and structural analyses confirmed these targets, suggesting that PGK1/DJ1 interact, which we confirmed by immunoprecipitation. Glucose homeostasis and oxidative stress are linked to telomere signaling and exemplar compound CRT0063465 blocked hypoglycemic telomere shortening. Intriguingly, PGK1 and DJ1 bind to TRF2 and telomeric DNA. Compound treatment modulates these interactions and also affects Shelterin complex composition, while conferring cellular protection from cytotoxicity due to bleomycin and desferroxamine. These results demonstrate therapeutic potential of the compound series.

  • MicroRNA-130a modulates a radiosensitivity of rectal cancer by targeting SOX4
    Neoplasia (IF 3.837) Pub Date : 2019-08-03
    Huyen Trang Ha Thi, Hye-Yeon Kim, Young-Mi Kim, Suntaek Hong

    Radioresistance poses a major challenge in the treatment of advanced rectal cancer. Therefore, understanding the detailed mechanisms of radioresistance may improve patient response to irradiation and the survival rate. To identify the novel targets that modulate the radiosensitivity of rectal cancer, we performed small RNA sequencing with human rectal cancer cell lines. Through bioinformatics analysis, we selected microRNA-310a (miR-130a) as a promising candidate to elucidate radioresistance. miR-130a was dramatically upregulated in radiosensitive rectal cancer cells and overexpression of miR-130a promotes rectal cancer cell radiosensitivity. Mechanically, miR-130a reversed the epithelial-mesenchymal transition phenotype of rectal cancer cells following inhibition of cell invasion upon irradiation. Moreover, miR-130a also inhibited the repair of irradiation-induced DNA damage followed by cell death. We identified that SOX4 was a direct target of miR-130a. Overexpression of SOX4 reversed the promotion activity of miR-130a on radiosensitivity. Together, our findings suggest that miR-130a functions as a radiosensitizer in rectal cancer and reveals a potential therapeutic target and preoperative prognostic marker for radiotherapy.

  • Down-Regulation of S100A8 is an Independent Predictor of PSA Recurrence in Prostate Cancer Treated by Radical Prostatectomy
    Neoplasia (IF 3.837) Pub Date : 2019-08-02
    Sarah Minner, Dominik Hager, Stefan Steurer, Doris Höflmayer, Maria Christina Tsourlakis, Christina Möller-Koop, Till S Clauditz, Claudia Hube-Magg, Andreas M Luebke, Ronald Simon, Guido Sauter, Cosima Göbel, Sören Weidemann, Patrick Lebok, David Dum, Christoph Fraune, Jakob Izbicki, Eike Burandt, Asmus Heumann

    Dysregulation of S100A8 is described in many different human tumor types, but its role in prostate cancer is unknown. To evaluate the clinical relevance of S100A8 expression in prostate cancer, a tissue microarray containing 13,665 tumors was analyzed by immunohistochemistry. Cytoplasmic S100A8 staining was compared to prostate cancer phenotype, patient prognosis and molecular features including TMPRSS2:ERG fusion status and deletions of PTEN, 3p, 5q and 6q. S100A8 immunostaining was typically seen in normal prostate tissue but lost in 60% of 9786 interpretable prostate cancers. In the remaining tumors, S100A8 was considered weak in 17.9%, moderate in 17.8% and strong in 5.4% of cases. Loss of S100A8 expression was linked to advanced tumor stage, high Gleason grade, positive nodal status, positive surgical margin and high preoperative PSA (P < .0001 each). In addition, loss of S100A8 expression was associated with TMPRSS2:ERG fusions (P < .0001), deletions of PTEN, 3p, and 6q (P < .005), and a high number of genomic deletions per tumor (P = .0009). Absence of S100A8 immunostaining was also linked to an elevated risk for early PSA recurrence (P < .0001). In a multivariate analysis limited to features that are preoperatively available, the prognostic impact of S100A8 expression (P < .0001) was independent of clinical stage, Gleason grade, and serum PSA level (P < .0001). Taken together, the results of our study demonstrate that complete loss of S100A8 expression is linked to adverse tumor features and predicts early biochemical recurrence in prostate cancer. S100A8 measurement, either alone or in combination might be of clinical utility in prostate cancers.

  • AsiDNA Treatment Induces Cumulative Antitumor Efficacy with a Low Probability of Acquired Resistance
    Neoplasia (IF 3.837) Pub Date : 2019-07-27
    Wael Jdey, Maria Kozlak, Sergey Alekseev, Sylvain Thierry, Pauline Lascaux, Pierre-Marie Girard, Françoise Bono, Marie Dutreix

    The Achilles heel of anticancer treatments is intrinsic or acquired resistance. Among many targeted therapies, the DNA repair inhibitors show limited efficacy due to rapid emergence of resistance. We examined evolution of cancer cells and tumors treated with AsiDNA, a new DNA repair inhibitor targeting all DNA break repair pathways. Effects of AsiDNA or Olaparib were analyzed in various cell lines. Frequency of AsiDNA- and olaparib-resistant clones was measured after 2 weeks of continuous treatment in KBM7 haploid cells. Cell survivals were also measured after one to six cycles of 1-week treatment and 1-week recovery in MDA-MB-231 and NCI-H446. Transcriptomes of cell populations recovering from cyclic treatments or mock treatment were compared. MDA-MB-231 xenografted models were treated with three cycles of AsiDNA to monitor the effects of treatment on tumor growth and transcriptional modifications. No resistant clones were selected after AsiDNA treatment (frequency < 3x10−8) in treatment conditions that generate resistance to olaparib at a frequency of 7.2x10−7 resistant clones per treated cell. Cyclic treatments promote cumulative sensitivity characterized by a higher mortality of cells having undergone previous treatment cycles. This sensitization was stable, and transcriptome analysis revealed a major gene downregulation with a specific overrepresentation of genes coding for targets of DNA-PK. Such changes were also detected in tumor models which showed impaired growth after cycles of AsiDNA treatment.

  • miR-125a-5p Functions as Tumor Suppressor microRNA And Is a Marker of Locoregional Recurrence And Poor prognosis in Head And Neck Cancer
    Neoplasia (IF 3.837) Pub Date : 2019-07-18
    Dat T. Vo, Narasimha Kumar Karanam, Lianghao Ding, Debabrata Saha, John S. Yordy, Uma Giri, John V. Heymach, Michael D. Story

    MicroRNAs (miRNAs) are short single-stranded RNAs, measuring 21 to 23 nucleotides in length and regulate gene expression at the post-transcriptional level through mRNA destabilization or repressing protein synthesis. Dysregulation of miRNAs can lead to tumorigenesis through changes in regulation of key cellular processes such as cell proliferation, cell survival, and apoptosis. miR-125a-5p has been implicated as a tumor suppressor miRNA in malignancies such as non-small cell lung cancer and colon cancer. However, the role of miR-125a-5p has not been fully investigated in head and neck squamous cell carcinoma (HNSCC). We performed microRNA microarray profiling of HNSCC tumor samples obtained from a prospective clinical trial evaluating the role of postoperative radiotherapy in head and neck cancer. We also mined through The Cancer Genome Atlas to evaluate expression and survival data. Biological experiments, including cell proliferation, flow cytometry, cell migration and invasion, clonogenic survival, and fluorescent microscopy, were conducted using HN5 and UM-SCC-22B cell lines. miR-125a-5p downregulation was associated with recurrent disease in a panel of high-risk HNSCC and then confirmed poor survival associated with low expression in HNSCC via the Cancer Genome Atlas, suggesting that miR-125a-5p acts as a tumor suppressor miRNA. We then demonstrated that miR-125a-5p regulates cell proliferation through cell cycle regulation at the G1/S transition. We also show that miR-125a-5p can alter cell migration and modulate sensitivity to ionizing radiation. Finally, we identified putative mRNA targets of miR-125a-5p, including ERBB2, EIF4EBP1, and TXNRD1, which support the tumor suppressive mechanism of miR-125a-5p. Functional validation of ERBB2 suggests that miR-125a-5p affects cell proliferation and sensitivity to ionizing radiation, in part, through ERBB2. Our data suggests that miR-125a-5p acts as a tumor suppressor miRNA, has potential as a diagnostic tool and may be a potential therapeutic target for the management and treatment of squamous cell carcinoma of the head and neck.

  • The COOH-Terminal Proline-Rich Region of GRP78 Is a Key Regulator of Its Cell Surface Expression and Viability of Tamoxifen-Resistant Breast Cancer Cells
    Neoplasia (IF 3.837) Pub Date : 2019-07-12
    Chun-Chih Tseng, Pu Zhang, Amy S. Lee

    Translocation of 78-kDa glucose-regulated protein (GRP78) from endoplasmic reticulum (ER) to plasma membrane represents a paradigm shift beyond its traditional function as an ER chaperone protein. Cell surface GRP78 (csGRP78) exerts novel signaling functions, and mechanisms underlying its cell surface expression are just emerging. Acquired tamoxifen resistance of breast cancer cells is accompanied with elevated level of csGRP78. Therefore, the tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) represents a clinically relevant model to study mechanisms of csGRP78 expression. We discovered that a proline-rich region (PRR) containing three consecutive prolines close to the COOH-terminus of GRP78 is important for its ability to form a complex with the partner protein, CD44v, as demonstrated by in vitro glutathione S-transferase pull-down assay. Proline to alanine mutations at the PRR compromised GRP78 expression level on the cell surface as evidenced by purification of biotinylated cell surface proteins. Reconstitution of MCF7-LR cells with the PRR mutant after knockdown of endogenous GRP78 diminished the capacity of GRP78 to stimulate STAT3 activation. The enforced expression of a short peptide bearing the PRR region of GRP78 led to reduction of CD44v and Cyclin D1 protein levels as well as cell viability, accompanied with increase in apoptotic signaling including cleaved Caspase-3 and PARP. These findings suggest that the COOH-terminal PRR of GRP78 is critical for its interaction with CD44v as well as its cell surface expression, and enforced expression of the short peptide bearing the PRR region may provide a new approach to lower the viability of tamoxifen-resistant breast cancer cells.

  • Engineered 3D Model of Cancer Stem Cell Enrichment and Chemoresistance
    Neoplasia (IF 3.837) Pub Date : 2019-07-09
    Maria R. Ward Rashidi, Pooja Mehta, Michael Bregenzer, Shreya Raghavan, Elyse M. Fleck, Eric N. Horst, Zainab Harissa, Visweswaran Ravikumar, Samuel Brady, Andrea Bild, Arvind Rao, Ronald J. Buckanovich, Geeta Mehta

    Intraperitoneal dissemination of ovarian cancers is preceded by the development of chemoresistant tumors with malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ. Method: We describe a highly integrated approach to establish an in vitro model of chemoresistance and stemness in ovarian cancer, using the 3D hanging drop spheroid platform. The model was established by serially passaging non-adherent spheroids. At each passage, the effectiveness of the model was evaluated via measures of proliferation, response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression and tumor initiating capacity of cancer stem-like cells (CSCs) was analyzed. RNA-seq was used to establish gene signatures associated with the evolution of tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. Results: Our serial passage model demonstrated increased cellular proliferation, enriched CSCs, and emergence of a platinum resistant phenotype. In vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSCs, compared to early passage spheroids. RNA-seq revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimentally observed data. Conclusion: Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence and development of chemoresistance in ovarian cancer in a controllable and reproducible format.

  • Overcoming Tyrosine Kinase Inhibitor Resistance in Transformed Cell Harboring SEPT9-ABL1 Chimeric Fusion Protein
    Neoplasia (IF 3.837) Pub Date : 2019-07-03
    Hidetsugu Kawai, Hiromichi Matsushita, Rikio Suzuki, Yuka Kitamura, Yoshiaki Ogawa, Hiroshi Kawada, Kiyoshi Ando

    Hematological malignancies harboring various ABL1 fusions are expected to be sensitive to tyrosine kinase inhibitors (TKIs), similar to those with BCR-ABL1. However, SEPT9-ABL1 exhibits TKI resistance both in vitro and in vivo. SEPT9-ABL1 has the same ABL1 region as seen in BCR-ABL1 but no point mutation in its kinase domain, which is one of the main mechanisms underlying TKI resistance in the leukemic cells harboring BCR-ABL1. The purpose of this study was to reveal the mechanism underlying TKI resistance induced by SEPT9-ABL1. We focused on the TP53 status because TKI-induced apoptosis in BCR-ABL1–positive cells is achieved through TP53. Mouse TP53 homologue TRP53 was downregulated and less phosphorylated in the cells expressing SEPT9-ABL1 than in those with BCR-ABL1, resulting in the prevention of apoptosis induced by TKIs. The CRM1 inhibitor KPT-330 accumulated nuclear TRP53 and NFKB1A (also known as IκBα), which is thought to capture TRP53 in the cytoplasm, and induced apoptosis in the hematopoietic cells expressing SEPT9-ABL1. In addition, the combination treatment of KPT-330 and imatinib, which induced the marked nuclear accumulation of PP2A and SET, reactivated PP2A through its dephosphorylation and inhibited SET expression, resulting in the effective induction of the apoptosis in the cells expressing SEPT9-ABL1. The combination treatment with KPT-330 and imatinib successfully reduced the subcutaneous masses expressing SEPT9-ABL1 and extended the survival of the mice intraperitoneally transplanted with SEPT9-ABL1–expressing cells. These results show that therapy with CRM1 inhibitors may be effective for overcoming TKI resistance induced by SEPT9-ABL1.

  • Circulating Tumor Cell–Based Molecular Classifier for Predicting Resistance to Abiraterone and Enzalutamide in Metastatic Castration-Resistant Prostate Cancer
    Neoplasia (IF 3.837) Pub Date : 2019-07-02
    Jae-Seung Chung, Yugang Wang, James Henderson, Udit Singhal, Yuanyuan Qiao, Alexander B. Zaslavsky, Daniel H. Hovelson, Daniel E. Spratt, Zachery Reichert, Ganesh S. Palapattu, Russell S. Taichman, Scott A. Tomlins, Todd M. Morgan

    While circulating tumor cell (CTC)–based detection of AR-V7 has been demonstrated to predict patient response to second-generation androgen receptor therapies, the rarity of AR-V7 expression in metastatic castrate-resistant prostate cancer (mCRPC) suggests that other drivers of resistance exist. We sought to use a multiplex gene expression platform to interrogate CTCs and identify potential markers of resistance to abiraterone and enzalutamide. 37 patients with mCRPC initiating treatment with enzalutamide (n = 16) or abiraterone (n = 21) were prospectively enrolled for CTC collection and gene expression analysis using a panel of 89 prostate cancer–related genes. Gene expression from CTCs was correlated with PSA response and radioclinical progression-free survival (PFS) using Kaplan-Meier and Cox regression analyses. Twenty patients (54%) had detectable CTCs. At a median follow-up of 11.3 months, increased expression of the following genes was significantly associated with shorter PSA PFS and radioclinical PFS: AR, AR-V7, PSA, PSCA, TSPAN8, NKX3.1, and WNT5B. Additionally, high SPINK1 expression was associated with increased PFS. A predictive model including all eight genes gave an area under the curve (AUC) of 0.84 for PSA PFS and 0.86 for radioclinical PFS. In comparison, the AR-V7 only model resulted in AUC values of 0.65 and 0.64.These data demonstrate that clinically relevant information regarding gene expression can be obtained from whole blood using a CTC-based approach. Multigene classifiers in this setting may allow for the development of noninvasive predictive biomarkers to guide clinical management.

  • Downregulation of Notch Signaling in Kras-Induced Gastric Metaplasia
    Neoplasia (IF 3.837) Pub Date : 2019-07-02
    Wen-Cheng Chung, Yunyun Zhou, Azeddine Atfi, Keli Xu

    Activating mutations and amplification of Kras and, more frequently, signatures for Kras activation are noted in stomach cancer. Expression of mutant KrasG12D in the mouse gastric mucosa has been shown to induce hyperplasia and metaplasia. However, the mechanisms by which Kras activation leads to gastric metaplasia are not fully understood. Here we report that KrasLSL-G12D/+;Pdx1-cre, a mouse model known for pancreatic cancer, also mediates KrasG12D expression in the stomach, causing gastric hyperplasia and metaplasia prior to the pathologic changes in the pancreas. These mice exhibit ectopic cell proliferation at the base of gastric glands, whereas wild-type mice contain proliferating cells primarily at the isthmus/neck of the gastric glands. Notch signaling is decreased in the KrasLSL-G12D/+;Pdx1-cre gastric mucosa, as shown by lower levels of cleaved Notch intracellular domains and downregulation of Notch downstream target genes. Expression of a Notch ligand Jagged1 is downregulated at the base of the mutant gland, accompanied by loss of chief cell marker Mist1. We demonstrate that exogenous Jagged1 or overexpression of Notch intracellular domain stimulates Mist1 expression in gastric cancer cell lines, suggesting positive regulation of Mist1 by Notch signaling. Finally, deletion of Jagged1 or Notch3 in KrasLSL-G12D/+;Pdx1-cre mice promoted development of squamous cell carcinoma in the forestomach, albeit short of invasive adenocarcinoma in the glandular stomach. Taken together, these results reveal downregulation of Notch signaling and Mist1 expression during the initiation of Kras-driven gastric tumorigenesis and suggest a tumor-suppressive role for Notch in this context.

  • The SOX4/miR-17-92/RB1 Axis Promotes Prostate Cancer Progression
    Neoplasia (IF 3.837) Pub Date : 2019-06-22
    Hui Liu, Zhen Wu, Haibin Zhou, Wenjie Cai, Xinjun Li, Jing Hu, Lin Gao, Tingting Feng, Lin Wang, Xijia Peng, Mei Qi, Long liu, Bo Han

    Although androgen-deprivation treatment (ADT) is the main treatment for advanced prostate cancer (PCa), it eventually fails. This failure invariably leads to castration-resistant prostate cancer (CRPC) and the development of the neuroendocrine (NE) phenotype. The molecular basis for PCa progression remains unclear. Previously, we and others have demonstrated that the sex-determining region Y-box 4 (SOX4) gene, a critical developmental transcription factor, is overexpressed and associated with poor prognosis in PCa patients. In this study, we show that SOX4 expression is associated with PCa progression and the development of the NE phenotype in androgen deprivation conditions. High-throughput microRNA profiling and bioinformatics analyses suggest that SOX4 may target the miR-17-92 cluster. SOX4 transcriptionally upregulates miR-17-92 cluster expression in PCa cells. SOX4-induced PCa cell proliferation, migration, and invasion are also mediated by miR-17-92 cluster members. Furthermore, RB1 is a target gene of miR-17-92 cluster. We found that SOX4 downregulates RB1 protein expression by upregulating the miR-17-92 expression. In addition, SOX4-knockdown restrains NE phenotype and PCa cell proliferation. Clinically, the overexpression of miR-17-92 members is shown to be positively correlated with SOX4 expression in PCa patients, whereas RB1 expression is negatively correlated with SOX4 expression in patients with the aggressive PCa phenotype. Collectively, we propose a novel model of a SOX4/miR-17-92/RB1 axis that may exist to promote PCa progression.

  • Clodronate-Liposome Mediated Macrophage Depletion Abrogates Multiple Myeloma Tumor Establishment In Vivo
    Neoplasia (IF 3.837) Pub Date : 2019-06-24
    Khatora S Opperman, Kate Vandyke, Kimberley C Clark, Elizabeth A Coulter, Duncan R Hewett, Krzysztof M Mrozik, Nisha Schwarz, Andreas Evdokiou, Peter I Croucher, Peter J Psaltis, Jacqueline E Noll, Andrew CW Zannettino

    Multiple myeloma is a fatal plasma cell malignancy that is reliant on the bone marrow microenvironment. The bone marrow is comprised of numerous cells of mesenchymal and hemopoietic origin. Of these, macrophages have been implicated to play a role in myeloma disease progression, angiogenesis, and drug resistance; however, the role of macrophages in myeloma disease establishment remains unknown. In this study, the antimyeloma efficacy of clodronate-liposome treatment, which globally and transiently depletes macrophages, was evaluated in the well-established C57BL/KaLwRijHsd murine model of myeloma. Our studies show, for the first time, that clodronate-liposome pretreatment abrogates myeloma tumor development in vivo. Clodronate-liposome administration resulted in depletion of CD169+ bone marrow–resident macrophages. Flow cytometric analysis revealed that clodronate-liposome pretreatment impaired myeloma plasma cell homing and retention within the bone marrow 24 hours postmyeloma plasma cell inoculation. This was attributed in part to decreased levels of macrophage-derived insulin-like growth factor 1. Moreover, a single dose of clodronate-liposome led to a significant reduction in myeloma tumor burden in KaLwRij mice with established disease. Collectively, these findings support a role for CD169-expressing bone marrow–resident macrophages in myeloma disease establishment and progression and demonstrate the potential of targeting macrophages as a therapy for myeloma patients.

  • Conditional Deletion of Eaf1 Induces Murine Prostatic Intraepithelial Neoplasia in Mice
    Neoplasia (IF 3.837) Pub Date : 2019-06-21
    Laura E. Pascal, Fei Su, Dan Wang, Junkui Ai, Qiong Song, Yujuan Wang, Katherine J. O'Malley, Brian Cross, Lora H. Rigatti, Anthony Green, Rajiv Dhir, Zhou Wang

    ELL-associated factor 1 is a transcription elongation factor that shares significant homology and functional similarity to the androgen-responsive prostate tumor suppressor ELL-associated factor 2. EAF2 is frequently down-regulated in advanced prostate cancer and Eaf2 deletion in the mouse induced the development of murine prostatic intraepithelial neoplasia. Here we show that similar to EAF2, EAF1 is frequently down-regulated in advanced prostate cancer. Co-downregulation of EAF1 and EAF2 occurred in 40% of clinical specimens with Gleason score >7. We developed and characterized a murine model of prostate-epithelial specific deletion of Eaf1 in the prostate and crossed it with our previously generated mouse with conventional deletion of Eaf2. The prostates of Eaf1 deletion mice displayed murine prostatic intraepithelial neoplasia lesions with increased proliferation and inflammation. Combined deletion of Eaf1 and Eaf2 in the murine model induced an increased incidence in mPIN lesions characterized by increased proliferation and CD3+ T cells and CD19+ B cells infiltration compared to individual deletion of either Eaf1 or Eaf2 in the murine prostate. These results suggest that EAF1 may play a tumor suppressive role in the prostate. Cooperation between EAF1 and EAF2 may be important for prostate maintaining prostate epithelial homeostasis, and concurrent loss of these two tumor suppressors may promote prostate tumorigenesis and progression.

  • The FUS-DDIT3 Interactome in Myxoid Liposarcoma
    Neoplasia (IF 3.837) Pub Date : 2019-06-17
    Jamie S.E. Yu, Shane Colborne, Christopher S. Hughes, Gregg B. Morin, Torsten O. Nielsen

    Myxoid liposarcoma is a malignant lipogenic tumor that develops in deep soft tissues. While local control rates are good, current chemotherapy options remain ineffective against metastatic disease. Myxoid liposarcoma is characterized by the FUS-DDIT3 fusion oncoprotein that is proposed to function as an aberrant transcription factor, but its exact mechanism of action has remained unclear. To identify the key functional interacting partners of FUS-DDIT3, this study utilized immunoprecipitation-mass spectrometry (IP-MS) to identify the FUS-DDIT3 interactome in whole cell lysates of myxoid liposarcoma cells, and results showed an enrichment of RNA processing proteins. Further quantitative MS analyses of FUS-DDIT3 complexes isolated from nuclear lysates showed that members of several chromatin regulatory complexes were present in the FUS-DDIT3 interactome, including NuRD, SWI/SNF, PRC1, PRC2, and MLL1 COMPASS-like complexes. Co-immunoprecipitation validated the associations of FUS-DDIT3 with BRG1/SMARCA4, BAF155/SMARCC1, BAF57/SMARCE1, and KDM1A. Data from this study provides candidates for functional validation as potential therapeutic targets, particularly for emerging epigenetic drugs.

  • Allosteric and ATP-Competitive Inhibitors of mTOR Effectively Suppress Tumor Progression-Associated Epithelial-Mesenchymal Transition in the Kidneys of Tsc2+/− Mice
    Neoplasia (IF 3.837) Pub Date : 2019-06-14
    Ashley T. Jones, Jian Yang, Kalin Narov, Elizabeth P Henske, Julian R. Sampson, Ming Hong Shen

    In tuberous sclerosis (TSC)–associated tumors, mutations in the TSC genes lead to aberrant activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. mTORC1 signaling impacts many biological processes including the epithelial-mesenchymal transition (EMT), which is suggested to promote tumor progression and metastasis in various types of cancer. In this study, we report hybrid cells with epithelial and mesenchymal features in angiomyolipomas and partial EMT in carcinomas from TSC patients and describe a new model of EMT activation during tumor progression from cyst to papillary adenoma to solid carcinoma in the kidneys of Tsc2+/− mice. Features of EMT occurred infrequently in TSC-associated cysts but increased as the lesions progressed through papillary adenoma to solid carcinoma where epithelial-mesenchymal hybrid cells were abundant, indicating partial EMT. We also compared the effects of the novel ATP-competitive mTOR inhibitor AZD2014 with the allosteric mTOR inhibitor rapamycin on EMT and tumor burden. Both AZD2014 and rapamycin potently suppressed EMT of renal tumors and effectively blocked tumor progression in Tsc2+/− mice. These results suggest that partial EMT is a shared feature of TSC-associated renal tumors in humans and mice and occurs during TSC-associated tumor progression. EMT-related signaling pathways may represent therapeutic targets for tumors associated with mutations in the TSC genes.

  • Induced Chromosomal Aneuploidy Results in Global and Consistent Deregulation of the Transcriptome of Cancer Cells
    Neoplasia (IF 3.837) Pub Date : 2019-06-04
    Darawalee Wangsa, Rüdiger Braun, Christina H. Stuelten, Markus Brown, Kerry M. Bauer, Georg Emons, Leigh A. Weston, Yue Hu, Howard H. Yang, Maria Vila-Casadesús, Maxwell P. Lee, Philip Brauer, Lidia Warner, Madhvi Upender, Amanda B. Hummon, Jordi Camps, Thomas Ried

    Chromosomal aneuploidy is a defining feature of epithelial cancers. The pattern of aneuploidies is cancer-type specific. For instance, the gain of chromosome 13 occurs almost exclusively in colorectal cancer. We used microcell-mediated chromosome transfer to generate gains of chromosome 13 in the diploid human colorectal cancer cell line DLD-1. Extra copies of chromosome 13 resulted in a significant and reproducible up-regulation of transcript levels of genes on chromosome 13 (P = .0004, FDR = 0.01) and a genome-wide transcriptional deregulation in all 8 independent clones generated. Genes contained in two clusters were particularly affected: the first cluster on cytoband 13q13 contained 7 highly up-regulated genes (NBEA, MAB21L1, DCLK1, SOHLH2, CCDC169, SPG20 and CCNA1, P = .0003) in all clones. A second cluster was located on 13q32.1 and contained five upregulated genes (ABCC4, CLDN10, DZIP1, DNAJC3 and UGGT2, P = .003). One gene, RASL11A, localized on chromosome band 13q12.2, escaped the copy number-induced overexpression and was reproducibly and significantly down-regulated on the mRNA and protein level (P = .0001, FDR = 0.002). RASL11A expression levels were also lower in primary colorectal tumors as compared to matched normal mucosa (P = .0001, FDR = 0.0001. Overexpression of RASL11A increases cell proliferation and anchorage independent growth while decreasing cell migration in +13 clones. In summary, we observed a strict correlation of genomic copy number and resident gene expression levels, and aneuploidy dependent consistent genome-wide transcriptional deregulation.

  • CDK9 Inhibition Induces a Metabolic Switch that Renders Prostate Cancer Cells Dependent on Fatty Acid Oxidation
    Neoplasia (IF 3.837) Pub Date : 2019-05-28
    Harri M. Itkonen, Ninu Poulose, Suzanne Walker, Ian G. Mills

    Cyclin-dependent kinase 9 (CDK9), a key regulator of RNA-polymerase II, is a candidate drug target for cancers driven by transcriptional deregulation. Here we report a multi-omics-profiling of prostate cancer cell responses to CDK9 inhibition to identify synthetic lethal interactions. These interactions were validated using live-cell imaging, mitochondrial flux-, viability- and cell death activation assays. We show that CDK9 inhibition induces acute metabolic stress in prostate cancer cells. This is manifested by a drastic down-regulation of mitochondrial oxidative phosphorylation, ATP depletion and induction of a rapid and sustained phosphorylation of AMP-activated protein kinase (AMPK), the key sensor of cellular energy homeostasis. We used metabolomics to demonstrate that inhibition of CDK9 leads to accumulation of acyl-carnitines, metabolic intermediates in fatty acid oxidation (FAO). Acyl-carnitines are produced by carnitine palmitoyltransferase enzymes 1 and 2 (CPT), and we used both genetic and pharmacological tools to show that inhibition of CPT-activity is synthetically lethal with CDK9 inhibition. To our knowledge this is the first report to show that CDK9 inhibition dramatically alters cancer cell metabolism.

  • The Deubiquitinase Inhibitor b-AP15 and Its Effect on Phenotype and Function of Monocyte-Derived Dendritic Cells
    Neoplasia (IF 3.837) Pub Date : 2019-05-25
    Moritz Schmidt, Vanessa Altdörfer, Sarah Schnitte, Alexander Rolf Fuchs, Korbinian Nepomuk Kropp, Stefanie Maurer, Martin Rudolf Müller, Helmut Rainer Salih, Susanne Malaika Rittig, Frank Grünebach, Daniela Dörfel

    The ubiquitin-proteasome system is elementary for cellular protein degradation and gained rising attention as a new target for cancer therapy due to promising clinical trials with bortezomib, the first-in class proteasome inhibitor meanwhile approved for multiple myeloma and mantle cell lymphoma. Both bortezomib and next-generation proteasome inhibitors mediate their effects by targeting the 20S core particle of the 26S proteasome. The novel small molecule inhibitor b-AP15 affects upstream elements of the ubiquitin-proteasome cascade by suppressing the deubiquitinase activity of both proteasomal regulatory 19S subunits and showed promising anticancer activity in preclinical models. Nonetheless, effects of inhibitors on the ubiquitin-proteasome system are not exclusively restricted to malignant cells: alteration of natural killer cell-mediated immune responses had already been described for drugs targeting either 19S or 20S proteasomal subunits. Moreover, it has been shown that bortezomib impairs dendritic cell (DC) phenotype and function at different levels. In the present study, we comparatively analyzed effects of bortezomib and b-AP15 on monocyte-derived DCs. In line with previous results, bortezomib exposure impaired maturation, antigen uptake, migration, cytokine secretion and immunostimulation, whereas treatment with b-AP15 had no compromising effects on these DC features. Our findings warrant the further investigation of b-AP15 as an alternative to clinically approved proteasome inhibitors in the therapy of malignancies, especially in the context of combinatorial treatment with DC-based immunotherapies.

  • Notch and mTOR Signaling Pathways Promote Human Gastric Cancer Cell Proliferation
    Neoplasia (IF 3.837) Pub Date : 2019-05-24
    Elise S. Hibdon, Nataliya Razumilava, Theresa M. Keeley, Gabriela Wong, Sumeet Solanki, Yatrik M. Shah, Linda C. Samuelson

    Notch pathway signaling is known to promote gastric stem cell proliferation, and constitutive pathway activation induces gastric tumors via mTORC1 activation in mouse genetic models. The purpose of this study was to determine whether human gastric adenocarcinomas are similarly dependent on Notch and mTORC1 signaling for growth. Gene expression profiling of 415 human gastric adenocarcinomas in The Cancer Genome Atlas, and a small set of locally obtained gastric cancers showed enhanced expression of Notch pathway components, including Notch ligands, receptors and downstream target genes. Human gastric adenocarcinoma tissues and chemically induced mouse gastric tumors both exhibited heightened Notch and mTORC1 pathway signaling activity, as evidenced by increased expression of the NOTCH1 receptor signaling fragment NICD, the Notch target HES1, and the mTORC1 target phosphorylated S6 ribosomal protein. Pharmacologic inhibition of either Notch or mTORC1 signaling reduced growth of human gastric cancer cell lines, with combined pathway inhibition causing a further reduction in growth, suggesting that both pathways are activated to promote gastric cancer cell proliferation. Further, mTORC1 signaling was reduced after Notch inhibition suggesting that mTOR is downstream of Notch in gastric cancer cells. Analysis of human gastric organoids derived from paired control and gastric cancer tissues also exhibited reduced growth in culture after Notch or mTOR inhibition. Thus, our studies demonstrate that Notch and mTOR signaling pathways are commonly activated in human gastric cancer to promote cellular proliferation. Targeting these pathways in combination might be an effective therapeutic strategy for gastric cancer treatment.

  • Telomere Trimming and DNA Damage as Signatures of High Risk Neuroblastoma
    Neoplasia (IF 3.837) Pub Date : 2019-05-23
    Eun Young Yu, Irene Y. Cheung, Yi Feng, Mohamed O. Rabie, Gail J. Roboz, Monica L. Guzman, Nai-Kong V. Cheung, Neal F. Lue

    Telomeres play important roles in genome stability and cell proliferation. High risk neuroblastoma (HRNB), an aggressive childhood cancer, is especially reliant on telomere maintenance. Three recurrent genetic aberrations in HRNB (MYCN amplification, TERT re-arrangements, and ATRX mutations) are mutually exclusive and each capable of promoting telomere maintenance mechanisms (i.e., through telomerase or ALT). We analyzed a panel of 5 representative HRNB cell lines and 30 HRNB surgical samples using assays that assess average telomere lengths, length distribution patterns, single-stranded DNA on the G- and C-strand, as well as extra-chromosomal circular telomeres. Our analysis pointed to substantial and variable degrees of telomere DNA damage in HRNB, including pervasive oxidative lesions. Moreover, unlike other cancers, neuroblastoma consistently harbored high levels of C-strand ssDNA overhangs and t-circles, which are consistent with active “telomere trimming”. This feature is observed in both telomerase- and ALT-positive tumors and irrespective of telomere length distribution. Moreover, evidence for telomere trimming was detected in normal neural tissues, raising the possibility that TMMs in HRNB evolved in the face of a canonical developmental program of telomere shortening. Telomere trimming by itself appears to distinguish neuroectodermal derived tumors from other human cancers, a distinguishing characteristic with both biologic and therapeutic implications.

  • SUMOylation of Csk Negatively Modulates its Tumor Suppressor Function
    Neoplasia (IF 3.837) Pub Date : 2019-05-21
    Nan Cui, Tianqi Liu, Yanmin Guo, Jinzhuo Dou, Qianqian Yang, Hailong Zhang, Ran Chen, Yanli Wang, Xian Zhao, Jianxiu Yu, Jian Huang

    Csk, a non-receptor tyrosine kinase, serves as an indispensable negative regulator of the Src family kinases (SFKs). However, little is known about regulation of Csk expression so far. SUMOylation, a reversible post-translational modification, has been shown to regulate many biological processes especially in tumor progression. Here we report that Csk is covalently modified by SUMO1 at lysine 53 (K53) both in vitro and in vivo. Treatment with hydrogen peroxide inhibited this modification to a certain extent, but PIAS3, identified as the main specific SUMO E3 ligase for Csk, could significantly enhance SUMO1-Csk level. In addition, phosphorylation at Ser364, the active site in Csk, had no effect on this modification. Ectopic expression of SUMO-defective mutant, Csk K53R, inhibited tumor cell growth more potentially than Csk wild-type. Consistent with the biological phenotype, the SUMO modification of Csk impaired its activity to interact with Cbp (Csk binding protein) leading to decreased c-Src phosphorylation at Y527. Our results suggest that SUMOylation of Csk mainly at lysine 53 negatively modulates its tumor suppressor function by reducing its binding with Cbp and consequently, inducing c-Src activation.

  • SIRT3 regulates cancer cell proliferation through deacetylation of PYCR1 in proline metabolism
    Neoplasia (IF 3.837) Pub Date : 2019-05-17
    Shuaiyi Chen, Xin Yang, Miao Yu, Zhe Wang, Boya Liu, Minghui Liu, Lu Liu, Mengmeng Ren, Hao Qi, Junhua Zou, Ivana Vucenik, Wei-Guo Zhu, Jianyuan Luo

    SIRT3 is a major mitochondrial deacetylase, which regulates various metabolic pathways by deacetylation; however, the effect of SIRT3 on proline metabolism is not reported. Pyrroline-5-carboxylate reductase 1 (PYCR1) participates in proline synthesis process by catalyzing the reduction of P5C to proline with concomitant generation of NAD+ and NADP+. PYCR1 is highly expressed in various cancers, and it can promote the growth of tumor cells. Here, through immunoprecipitation and mass spectrometry, we found that PYCR1 is in SIRT3’s interacting network. PYCR1 directly binds to SIRT3 both in vivo and in vitro. CBP is the acetyltransferase for PYCR1, whereas SIRT3 deacetylates PYCR1. We further identified that K228 is the major acetylation site for PYCR1. Acetylation of PYCR1 at K228 reduced its enzymatic activity by impairing the formation of the decamer of PYCR1. As a result, acetylation of PYCR1 at K228 inhibits cell proliferation, while deacetylation of PYCR1 mediated by SIRT3 increases PYCR1’s activity. Our findings on the regulation of PYCR1 linked proline metabolism with SIRT3, CBP and cell growth, thus providing a potential approach for cancer therapy.

  • Critical role of the MCAM-ETV4 axis triggered by extracellular S100A8/A9 in breast cancer aggressiveness
    Neoplasia (IF 3.837) Pub Date : 2019-05-14
    Youyi Chen, I Wayan Sumardika, Nahoko Tomonobu, Rie Kinoshita, Yusuke Inoue, Hidekazu Iioka, Yosuke Mitsui, Ken Saito, I Made Winarsa Ruma, Hiroki Sato, Akira Yamauchi, Hitoshi Murata, Ken-ichi Yamamoto, Shuta Tomida, Kazuhiko Shien, Hiromasa Yamamoto, Junichi Soh, Junichiro Futami, Masakiyo Sakaguchi

    Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes.

  • Determination of Pyruvate Metabolic Fates Modulates Head and Neck Tumorigenesis
    Neoplasia (IF 3.837) Pub Date : 2019-05-14
    Tsai-Ying Chen, Yi-Ta Hsieh, Jian-Min Huang, Chung-Ji Liu, Lu-Te Chuang, Pei-Chun Huang, Tz-Yu Kuo, Hao-Yuan Chia, Chia-Yi Chou, Ching-Wen Chang, Yi-Fen Chen, Hsin-Ming Chen, Jeng-Fan Lo, Wan-Chun Li

    Even with increasing evidence for roles of glycolytic enzymes in controlling cancerous characteristics, the best target of candidate metabolic enzymes for lessening malignancy remains under debate. Pyruvate is a main glycolytic metabolite that could be mainly converted into either lactate by Lactate Dehydrogenase A (LDHA) or acetyl-CoA by Pyruvate Dehydrogenase E1 component α subunit (PDHA1) catalytic complex. In tumor cells, accumulating lactate is produced whereas the conversion of pyruvate into mitochondrial acetyl-CoA is less active compared with their normal counterparts. This reciprocal molecular association makes pyruvate metabolism a potential choice of anti-cancer target. Cellular and molecular changes were herein assayed in Head and Neck Squamous Cell Carcinoma (HNSCC) cells in response to LDHA and PDHA1 loss in vitro, in vivo and in clinic. By using various human cancer databases and clinical samples, LDHA and PDHA1 levels exhibit reversed prognostic roles. In vitro analysis demonstrated that decreased cell growth and motility accompanied by an increased sensitivity to chemotherapeutic agents was found in cells with LDHA loss whereas PDHA1-silencing exhibited opposite phenotypes. At the molecular level, it was found that oncogenic Protein kinase B (PKB/Akt) and Extracellular signal-regulated kinase (ERK) singling pathways contribute to pyruvate metabolism mediated HNSCC cell growth. Furthermore, LDHA/PDHA1 changes in HNSCC cells resulted in a broad metabolic reprogramming while intracellular molecules including polyunsaturated fatty acids and nitrogen metabolism related metabolites underlie the malignant changes. Collectively, our findings reveal the significance of pyruvate metabolic fates in modulating HNSCC tumorigenesis and highlight the impact of metabolic plasticity in HNSCC cells.

  • Cellular Metabolic Heterogeneity In Vivo Is Recapitulated in Tumor Organoids
    Neoplasia (IF 3.837) Pub Date : 2019-05-09
    Joe T. Sharick, Justin J. Jeffery, Mohammad R. Karim, Christine M. Walsh, Karla Esbona, Rebecca S. Cook, Melissa C. Skala

    Heterogeneous populations within a tumor have varying metabolic profiles, which can muddle the interpretation of bulk tumor imaging studies of treatment response. Although methods to study tumor metabolism at the cellular level are emerging, these methods provide a single time point “snapshot” of tumor metabolism and require a significant time and animal burden while failing to capture the longitudinal metabolic response of a single tumor to treatment. Here, we investigated a novel method for longitudinal, single-cell tracking of metabolism across heterogeneous tumor cell populations using optical metabolic imaging (OMI), which measures autofluorescence of metabolic coenzymes as a report of metabolic activity. We also investigated whether in vivo cellular metabolic heterogeneity can be accurately captured using tumor-derived three-dimensional organoids in a genetically engineered mouse model of breast cancer. OMI measurements of response to paclitaxel and the phosphatidylinositol-3-kinase inhibitor XL147 in tumors and organoids taken at single cell resolution revealed parallel shifts in metaboltruic heterogeneity. Interestingly, these previously unappreciated heterogeneous metabolic responses in tumors and organoids could not be attributed to tumor cell fate or varying leukocyte content within the microenvironment, suggesting that heightened metabolic heterogeneity upon treatment is largely due to heterogeneous metabolic shifts within tumor cells. Together, these studies show that OMI revealed remarkable heterogeneity in response to treatment, which could provide a novel approach to predict the presence of potentially unresponsive tumor cell subpopulations lurking within a largely responsive bulk tumor population, which might otherwise be overlooked by traditional measurements.

  • ALDH2 Repression Promotes Lung Tumor Progression via Accumulated Acetaldehyde and DNA Damage
    Neoplasia (IF 3.837) Pub Date : 2019-05-06
    Kaimi Li, Wenzheng Guo, Zhanming Li, Yang Wang, Beibei Sun, Dongliang Xu, Jing Ling, Hongyong Song, Yueling Liao, Tong Wang, Bo Jing, Min Hu, Yanbin Kuang, Qi Wang, Feng Yao, Aijun Sun, Liang Zhu, Lishun Wang, Jiong Deng

    The major role of aldehyde dehydrogenase 2 family (ALDH2) is to detoxify acetaldehyde (ACE) to non-toxic acetic acid. Many evidences suggest that ALDH2 dysfunction contributes to a variety of human diseases including cancer. However, the biological function and molecular mechanism of ALDH2 in tumor progression remain elusive. In this study, we found that ALDH2 repression was associated with poor prognosis in lung adenocarcinoma. Overexpression of ALDH2 inhibited malignant features of lung adenocarcinoma cells, such as proliferation, stemness and migration, whereas ALDH2 knockdown increased these features. Mechanistically, ALDH2 repression led to accumulation of ACE; whereas ACE enhanced the migration features of lung adenocarcinoma cells, which was associated with increased DNA damage. Importantly, accumulated ACE and increased DNA damage were identified in Aldh2-knockout (KO) mouse lung tissues in vivo. Consistent with this concept, treatment of lung adenocarcinoma cells with ALDH2 agonist Alda-1 suppressed the proliferation, stemness and migration features of lung adenocarcinoma cells. Thus, activating ALDH2, such as via its agonist, may provide a novel strategy for treatment of lung cancer.

  • Immune Signature-Based Subtypes of Cervical Squamous Cell Carcinoma Tightly Associated with Human Papillomavirus Type 16 Expression, Molecular Features, and Clinical Outcome
    Neoplasia (IF 3.837) Pub Date : 2019-05-02
    Xiaofan Lu, Liyun Jiang, Liya Zhang, Yue Zhu, Wenjun Hu, Jiashuo Wang, Xinjia Ruan, Zhengbao Xu, Xiaowei Meng, Jun Gao, Xiaoping Su, Fangrong Yan

    Substantial heterogeneity exists within cervical cancer that is generally infected by human papillomavirus (HPV). However, the most common histological subtype of cervical cancer, cervical squamous cell carcinoma (CSCC), is poorly characterized regarding the association between its heterogeneity and HPV oncoprotein expression. We filtered out 138 CSCC samples with infection of HPV16 only as the first step; then we compressed HPV16 E6/E7 expression as HPVpca and correlated HPVpca with the immunological profiling of CSCC based on supervised clustering to discover subtypes and to characterize the differences between subgroups in terms of the HPVpca level, pathway activity, epigenetic dysregulation, somatic mutation frequencies, and likelihood of responding to chemo/immunotherapies. Supervised clustering of immune signatures revealed two HPV16 subtypes (namely, HPV16-IMM and HPV16-KRT) that correlated with HPVpca and clinical outcomes. HPV16-KRT is characterized by elevated expression of genes in keratinization, biological oxidation, and Wnt signaling, whereas HPV16-IMM has a strong immune response and mesenchymal features. HPV16-IMM exhibited much more epigenetic silencing and significant mutation at FBXW7, while MUC4 and PIK3CA were mutated frequently for HPV16-KRT. We also imputed that HPV16-IMM is much more sensitive to chemo/immunotherapy than is HPV16-KRT. Our characterization tightly links the expression of HPV16 E6/E7 with biological and clinical outcomes of CSCC, providing valuable molecular-level information that points to decoding heterogeneity. Together, these results shed light on stratifications of CSCC infected by HPV16 and shall help to guide personalized management and treatment of patients.

  • Loss of 1p36.33 Frequent in Low-Grade Serous Ovarian Cancer
    Neoplasia (IF 3.837) Pub Date : 2019-05-01
    Els Van Nieuwenhuysen, Pieter Busschaert, Annouschka Laenen, Philippe Moerman, Sileny N. Han, Patrick Neven, Diether Lambrechts, Ignace Vergote

    BACKGROUND: Low-grade serous ovarian cancer (LGSOC) is a rare subtype of epithelial ovarian carcinoma. Limited data regarding the molecular-genetic background exist beyond mutations in the RAS signaling pathway. There is a growing need to better characterize these tumors due to chemoresistance and limited therapeutic options in advanced or recurrent disease. METHODS: We performed genome-wide copy number aberration (CNA) profiles and mutation hotspot screening (KRAS, BRAF, NRAS, ERBB2, PIK3CA, TP53) in 38 LGSOC tumor samples. RESULTS: We detected mutations in the RAS-signaling pathway in 36.8% of cases, including seven KRAS, four BRAF, and three NRAS mutations. We identified two mutations in PIK3CA and one mutation in MAP3K1, EGFR, and TP53. CNAs were detected in 86.5% of cases. None of the focal aberrations was correlated with specific clinical characteristics. The most frequently detected CNA was loss of 1p36.33 in 54.1% of cases, with a trend towards lower progression-free survival and overall survival in patients with 1p36.33 loss. CONCLUSIONS: Activating RAS mutations were dominant in our series, with supplementary detection of two PIK3CA mutations which may lead to therapeutic options. Furthermore, we detected 1p36.33 deletions in half of the cases, indicating a role in tumorigenesis, and these deletions may serve as a prognostic marker.

  • Gene Expression Profiling Reveals Distinct Molecular Subtypes of Esophageal Squamous Cell Carcinoma in Asian Populations
    Neoplasia (IF 3.837) Pub Date : 2019-04-29
    Fengling Wang, Zhongyi Yan, Jiajia Lv, Junfang Xin, Yifang Dang, Xiaoxiao Sun, Yang An, Yijun Qi, Qiying Jiang, Wan Zhu, Yongqiang Li, Ao Li, Xiangqian Guo

    Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide, particularly in Asian populations, and responds poorly to conventional therapy. Subclassification of ESCCs by molecular analysis is a powerful strategy in extending conventional clinicopathologic classification, improving prognosis and therapy. Here we identified two ESCC molecular subtypes in Chinese population using gene expression profiling data and further validated the molecular subtypes in two other independent Asian populations (Japanese and Vietnamese). Subtype I ESCCs were enriched in pathways including immune response, while genes overexpressed in subtype II ESCCs were mainly involved in ectoderm development, glycolysis process, and cell proliferation. Specifically, we identified potential ESCC subtype-specific diagnostic markers (FOXA1 and EYA2 for subtype I, LAMC2 and KRT14 for subtype II) and further validated them in a fourth Asian cohort. In addition, we propose a few subtype-specific therapeutic targets for ESCC, which may guide future ESCC clinical treatment when further validated.

  • Metformin Regulates the Expression of CD133 Through the AMPK-CEBPβ Pathway in Hepatocellular Carcinoma Cell Lines
    Neoplasia (IF 3.837) Pub Date : 2019-04-28
    Osamu Maehara, Shunsuke Ohnishi, Ayaka Asano, Goki Suda, Mitsuteru Natsuizaka, Koji Nakagawa, Masanobu Kobayashi, Naoya Sakamoto, Hiroshi Takeda

    CD133 is a cellular surface protein, which has been reported to be a cancer stem cell marker, and thus is considered a potential target for cancer treatment. Metformin, one of the biguanides used for the treatment of diabetes, is also known to reduce the risk of cancer development and cancer stem-like cells (CSCs), including the expression of CD133. However, the mechanism underlying the reduction of the expression of CD133 by metformin is not yet understood. This study shows that metformin suppressed CD133 expression mainly by affecting the CD133 P1 promoter via adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling but not the mammalian target of rapamycin (mTOR). AMPK inhibition rescued the reduction of CD133 by metformin. Further experiments demonstrated that CCAAT/enhancer-binding protein beta (CEBPβ) was upregulated by metformin and that two isoforms of CEBPβ reciprocally regulated the expression of CD133. Specifically, the liver-enriched activator protein (LAP) isoform increased the expression of CD133 by directly binding to the P1 promoter region, whereas the liver-enriched inhibitory protein (LIP) isoform suppressed the expression of CD133. Consistent with these findings, a three dimensional (3D) culture assay and drug sensitivity assay demonstrated that LAP-overexpressing cells formed large spheroids and were more resistant to 5-fluorouracil (5-FU) treatment, whereas LIP-overexpressing cells were more sensitive to 5-FU and showed combined effects with metformin. Our results indicated that metformin-AMPK-CEBPβ signaling plays a crucial role in regulating the gene expression of CD133. Additionally, regulating the ratio of LAP/LIP may be a novel strategy for targeting CSCs for the treatment of cancer.

  • Epigenetically Down-Regulated Acetyltransferase PCAF Increases the Resistance of Colorectal Cancer to 5-Fluorouracil
    Neoplasia (IF 3.837) Pub Date : 2019-04-28
    Tong Liu, Xiang Wang, Wenquan Hu, Zhi Fang, Ying Jin, Xuedong Fang, Qing Robert Miao

    Only 10%–20% of colorectal cancer (CRC) patients observe effective responses to 5-fluorouracil (5-FU) based chemo-treatment. We used real-time PCR array and Western blot analysis to examine the expression alteration of acetyltransferases and deacetylases in 5-FU resistant CRC cell lines as compared to their respective parental CRC cell lines. Unlike other acetyltransferases and deacetylases, we found that the expression of acetyltransferase P300/CBP-associated factor (PCAF) is consistently decreased in three 5-FU resistant CRC cell lines. Similarly, knockdown of PCAF in HCT116 CRC parental cell line also increases the resistance to 5-FU and attenuates 5-FU-induced apoptosis. Mechanistically, we demonstrated that increased binding of trimethylated histone H3K27 in the promoter region of PCAF attenuated its transcription in 5-FU resistant HCT116/5-FU cells. Decreased PCAF impairs the acetylation of p53 and attenuates the p53-dependent transcription of p21, which results in the increased cyclin D1 and phosphorylation of Retinoblastoma 1. Conversely, overexpression of PCAF in CRC cell lines increases p21 and their susceptibility to 5-FU in vitro and in vivo. However, knockdown of p21 abolishes the beneficial effects of PCAF overexpression on increasing the sensitivity of HCT116/5-FU cells to 5-FU. Also, the reduced intensity of PCAF immunostaining was observed in the precancerous lesion, and microarray data from the public database further demonstrated the association between PCAF down-regulation and poor survival outcome. Our data suggest that PCAF-mediated p53 acetylation is an essential regulatory mechanism for increasing the susceptibility of CRC to 5-FU.

  • ATF6α Activation Enhances Survival against Chemotherapy and Serves as a Prognostic Indicator in Osteosarcoma
    Neoplasia (IF 3.837) Pub Date : 2019-04-25
    Suma Yarapureddy, Jazmine Abril, Janet Foote, Saravana Kumar, Omar Asad, Veena Sharath, Janine Faraj, Dustin Daniel, Paul Dickman, Andrea White-Collins, Pooja Hingorani, Aparna R Sertil

    Patients with metastatic or relapsed/refractory osteosarcoma (OS) have a 5-year survival rate of <30%. This has remained unchanged over several decades. One of the factors contributing to lack of improvement in survival is the development of chemoresistance. Hence, elucidating and targeting the mechanisms that promote survival against chemotherapy and lead to chemoresistance is pivotal to improving outcomes for these patients. We identified that endoplasmic reticulum (ER) stress-activated transcription factor, ATF6α, is essential for the survival of OS cells against chemotherapy induced cell death. ATF6α cleavage and activity were enhanced in OS cells compared to normal osteoblasts and knockdown of ATF6α expression enhanced sensitivity of OS cells against chemotherapy induced cell death. This was in part due to increased Bax activation. Pharmacologic inhibition or knock-down of downstream targets of ATF6α, protein disulfide isomerases (PDI) and ERO1β, a thiol oxidase that is involved in the re-oxidation of PDIs also independently induced pronounced killing of OS cells following chemotherapy. Analysis of primary tumors from OS patients reveals that patients with high levels of nuclear ATF6α: (1) also had increased expression of its downstream targets the chaperone BiP and enzyme PDI, (2) had a significant likelihood of developing metastasis at diagnosis, (3) had significantly poorer overall and progression free survival, and (4) had poorer response to chemotherapy. These findings suggest that targeting survival signaling by the ATF6α pathway in OS cells may favor eradication of refractory OS tumor cells and ATF6α could be a useful predictor for chemo-responsiveness and prognosis.

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上海纽约大学William Glover