An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Lung cancer is one of the cancers with highest morbidity and mortality rates and the metastasis of lung cancer is a leading cause of death. Mechanisms of lung cancer metastasis are yet to be fully understood. Herein, we demonstrate that mice deficient for REGγ, a proteasome activator, exhibited a significant reduction in tumor size, numbers, and metastatic rate with prolonged survival in a conditional Kras/p53 mutant lung cancer model. REGγ enhanced the TGFβ-Smad signaling pathway by ubiquitin-ATP-independent degradation of Smad7, an inhibitor of the TGFβ pathway. Activated TGFβ signaling in REGγ-positive lung cancer cells led to diminished expression of E-cadherin, a biomarker of epithelial–mesenchymal transitions (EMT), and elevated mesenchymal markers compared with REGγ-deficient lung cancer cells. REGγ overexpression was found in lung cancer patients with metastasis, correlating with the reduction of E-Cadherin/Smad7 and a poor prognosis. Overall, our study indicates that REGγ promotes lung cancer metastasis by activating TGF-β signaling via degradation of Smad7. Thus, REGγ may serve as a novel therapeutic target for lung cancers with poor prognosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

In Drosophila, the fat body undergoes a massive burst of autophagy at the end of larval development in preparation for the pupal transition. To identify genes involved in this process, we carried out a microarray analysis. We found that mRNA levels of the homologs of Atg8, the coat protein of early autophagic structures, and lysosomal hydrolases were upregulated, consistent with previous results. Genes encoding mitochondrial proteins and many chaperones were downregulated, including the inhibitor of eIF2alpha kinases and the peptidyl-prolyl cis-trans isomerase FK506-binding protein of 39 kDa (FKBP39). Genetic manipulation of FKBP39 expression had a significant effect on autophagy, potentially through modulation of the transcription factor Foxo. Accordingly, we found that Foxo mutants cannot properly undergo autophagy in response to starvation, and that overexpression of Foxo induces autophagy.

The Bcl-2 protein Bax normally resides in the cytosol, but during apoptosis it translocates to mitochondria where it is responsible for releasing apoptogenic factors. Using anoikis as a model, we have shown that Bax translocation does not commit cells to apoptosis, and they can be rescued by reattachment to extracellular matrix within a specific time. Bax undergoes an N-terminal conformational change during apoptosis that has been suggested to regulate conversion from its benign, cytosolic form to the active, membrane bound pore. We now show that the Bax N-terminus regulates commitment and mitochondrial permeabilisation, but not the translocation to mitochondria. We identify Proline 13 within the N-terminus of Bax as critical for this regulation. The subcellular distribution of Proline 13 mutant Bax was identical to wild-type Bax in both healthy and apoptotic cells. However, Proline 13 mutant Bax induced rapid progression to commitment, mitochondrial permeabilisation and death. Our data identify changes in Bax controlling commitment to apoptosis that are mechanistically distinct from those controlling its subcellular localisation. Together, they indicate that multiple regulatory steps are required to activate the proapoptotic function of Bax.

Expression of Bcl-x(L) correlates with the clinical outcomes of patients with cancer. While the role of Bcl-2 in angiogenesis is becoming increasingly evident, the function of Bcl-x(L) in angiogenesis is unclear. Here, we showed that epidermal growth factor (EGF) induces in vitro capillary sprouting and Bcl-x(L) expression in primary endothelial cells. Bcl-x(L)-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-x(L)), but not empty vector control cells, spontaneously organize into capillary-like sprouts. Searching for a mechanism to explain these responses, we observed that Bcl-x(L) induced expression of the pro-angiogenic chemokines CXC ligand-1 (CXCL1) and CXC ligand-8 (CXCL8), and that blockade of CXC receptor-2 (CXCR2) signaling inhibited spontaneous sprouting of HDMEC-Bcl-x(L). Bcl-x(L) led to Bcl-2 upregulation, but Bcl-2 did not upregulate Bcl-x(L), suggesting the existence of a unidirectional crosstalk from Bcl-x(L) to Bcl-2. EGF and Bcl-x(L) activate the mitogen-activated protein kinase/ERK pathway resulting in upregulation of vascular endothelial growth factor (VEGF), a known inducer of Bcl-2 in endothelial cells. Inhibition of VEGF receptor signaling in HDMEC-Bcl-x(L) prevented Bcl-2 upregulation and demonstrated the function of a VEGF-mediated autocrine loop. Bcl-2 downregulation by RNAi blocked CXCL1 and CXCL8 expression downstream of Bcl-x(L), and markedly decreased angiogenesis in vivo. We conclude that Bcl-x(L) functions as a pro-angiogenic signaling molecule controlling Bcl-2 and VEGF expression. These results emphasize a complex interplay between Bcl-2 family members beyond their classical roles in apoptosis.

The present study was undertaken to determine the significance of histone acetylation versus DNA damage in drug-induced irreversible growth arrest (senescence) and apoptosis. Cellular treatment with the DNA-damaging drugs doxorubicin and cisplatin or with the histone deacetylase inhibitor trichostatin A, led to the finding that all the three drugs induced senescence at concentrations significantly lower than those required for apoptosis. However, only doxorubicin and cisplatin induced activation of H2AX, a marker for double-strand break formation. Interestingly, this occurred mainly at apoptosis and not senescence-inducing drug concentrations, suggesting that non-DNA-damage pathways may be implicated in induction of senescence by these drugs. In agreement with this, chromatin immunoprecipitation experiments indicated that doxorubicin was able to induce acetylation of histone H3 at the promoter of p21/WAF1 only at senescence-inducing concentrations. Collectively, these findings suggest that alteration of chromatin structure by cytotoxic drugs may represent a key mediator of senescence.

Although cyclin G1 has been implicated in certain p53-related biological phenomena, other aspects of its function remain unclear. Here we report hitherto unknown mechanism by which cyclin G1 increases radiation sensitivity by regulating the level of cyclin B1. Overexpression of cyclin G1 was observable in lung carcinoma tissues. Irradiation of human lung cells with cyclin G1 overexpression resulted in increased cell death and gamma-H2AX foci suggesting that cyclin G1 rendered the cells more susceptible to DNA damage. Enhanced radiosensitivity by cyclin G1 was correlated with increased cyclin B1, CDC2/cyclin B1 complex, and MPM2. Cell cycle synchronization clearly showed coexpression of cyclin G1 and cyclin B1 in G2/M phase. Depletion of cyclin G1 by interference RNA revealed that cyclin G1 regulated transcription of cyclin B1 in a p53-independent manner, and confirmed that the increased mitotic cells and cell death by cyclin G1 were dependent upon cyclin B1. Therefore, our data suggest that cyclin G1 enhanced radiation sensitivity by overriding radiation-induced G2 arrest through transcriptional upregulation of cyclin B1.

It is unknown whether base excision DNA repair (BER) proteins interact with mitogen-activated protein kinases (MAPK) under oxidation. Here, we explored roles of BER proteins in signaling transduction involving MAPK during hyperoxia. We demonstrated that ERK1/2 phosphorylation in A549 cells was increased in 95% O(2). p38 activity in A549 cells was also increased by exposure to 95% O(2). To evaluate regulatory roles of MAPK, we have transduced A549 cells and primary alveolar epithelial type II cells (AECII) to overexpress 8-oxoguanine DNA glycosylase (hOgg1). Overexpression of hOgg1 reduced hyperoxic toxicity in A549 and AECII cells. Furthermore, protection by BER against hyperoxia appeared to involve an upregulation of ERK1/2 and downregulation of p38. These observations demonstrate, for the first time, that reduction of hyperoxic toxicity by BER proteins may be involved with MAPK activity, thereby impacting cell survival. Furthermore, our studies suggest that modulation of MAPK may be used in combination with BER proteins to counteract hyperoxic toxicity.

The genetic tools available in Drosophila have facilitated our understanding of how apoptosis is regulated and executed in the context of the developing organism. All embryonic apoptosis is initiated by the activity of three genes, rpr, grim and hid. Each of these genes is independently regulated, allowing developmental apoptosis to be finely controlled. These initiators in turn activate the core apoptotic machinery, including the caspases. Drosophila counterparts to other conserved components of the apoptotic machinery have been recently identified, and we discuss how these may be integrated into the process of normal developmentally regulated cell death. We also outline the role that phagocytosis plays in the final stages of apoptosis and consider the molecular mechanisms guiding the elimination of apoptotic corpses.

In Caenorhabditis elegans, several distinct apoptosis pathways have been characterized in the germline. The physiological pathway is though to eliminate excess germ cells during oogenesis to maintain gonad homeostasis and it is activated by unknown mechanisms. The DNA damage-induced germ cell apoptosis occurs in response to genotoxic agents and involves the proteins EGL-1 and CED-13, and the DNA damage response protein p53. Germ cell apoptosis can also be induced in response to pathogen infection through an EGL-1 dependent pathway. To gain insight into the mechanism and functions of germ cell apoptosis, we investigated whether and how other forms of stress induce this cell death. We found that oxidative, osmotic, heat shock and starvation stresses induce germ cell apoptosis through a p53 and EGL-1 independent pathway. We also learned that the MAPK kinases MEK-1 and SEK-1, and the p53 antagonist protein ABL-1, are essential for stress-induced germ cell apoptosis. We conclude that in C. elegans responses to various stresses that do not involve genotoxicity include an increase in germ cell apoptosis through the physiological pathway.