Targeting PP2A with lomitapide suppresses colorectal tumorigenesis through the activation of AMPK/Beclin1-mediated autophagy

doi:10.1016/j.canlet.2021.09.010

Cancer Letters

Volume 521, 28 November 2021, Pages 281-293

https://doi.org/10.1016/j.canlet.2021.09.010 Get rights and content

Highlights

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Lomitapide screened from FDA-approved drugs induces CRC apoptosis and tumorigenesis inhibition.
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Lomitapide activates CRC autophagy through inducing AMPK-mediated formation of the Beclin1-Atg14-Vps34 complex.
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Lomitapide impaires the dephosphorylation of AMPK by directly targeting PP2A.

Abstract

Colorectal cancer (CRC) is one of the most common malignancies worldwide, and effective therapy remains a challenge. In this study, we take advantage of a drug repurposing strategy to screen small molecules with novel anticancer activities in a small-molecule library consisting of 1056 FDA-approved drugs. We show, for the first time, that lomitapide, a lipid-lowering agent, exhibits antitumor properties in vitro and in vivo. Activated autophagy is characterized as a key biological process in lomitapide-induced CRC repression. Mechanistically, lomitapide stimulated mitochondrial dysfunction-mediated AMPK activation, resulting in increased AMPK phosphorylation and enhanced Beclin1/Atg14/Vps34 interactions, provoking autophagy induction. Autophagy inhibition or AMPK silencing significantly abrogated lomitapide-induced cell death, indicating the significance of AMPK-regulated autophagy in the antitumor activities of lomitapide. More importantly, PP2A was identified as a direct target of lomitapide by limited proteolysis-mass spectrometry (LiP-SMap), and the bioactivity of lomitapide was attenuated in PP2A-deficient cells, suggesting that the anticancer effect of lomitapide occurs in a PP2A-dependent manner. Taken together, the results of the study reveal that lomitapide can be repositioned as a potential therapeutic drug for CRC treatment.

Introduction

Colorectal cancer (CRC) is the third leading cause of cancer mortality worldwide. With advances in diagnostic techniques and therapeutic strategies, the survival time for patients with CRC has increased in recent decades, yet the mortality rate of CRC remains high due to limited efficacy and significant side effects of treatments [1]. Hence, novel drugs for CRC treatment are urgently needed. Currently, drug repurposing has gradually emerged as an alternative approach to cancer treatment. This is a promising strategy for investigating novel antineoplastic medicines, because it capitalizes on previous investments while derisking clinical trials. In this study, a drug library consisting of 1056 U.S. Food and Drug Administration (FDA)-approved medications was tested for compounds with anticancer indications. Lomitapide, an inhibitor of mitochondrial trifunctional protein (MTP), which is usually used for the treatment of hypercholesterolemia in the clinic [2], was found to significantly suppress colorectal tumorigenesis. To the best of our knowledge, the anticancer properties of lomitapide have not been reported, and the mechanism involved is not clear.

Autophagy is an evolutionarily conserved process that maintains cellular metabolism and homeostasis [3] and is subjected to a range of cellular stresses, including oxidative stress, mitochondrial abnormalities and abnormal protein accumulation [[4], [5], [6]]. Several studies have indicated that autophagy can act to suppress tumor survival and growth in advanced cancers [7]. In addition, silencing of some key autophagy-related genes has been reported to facilitate tumor progression in colon, gastric, breast, and prostate cancers [[8], [9], [10]]. However, the regulatory role of autophagy in colorectal tumorigenesis remains ambiguous. Many studies have revealed that the modulation of autophagy by the mTOR, AMPK, and MAPK pathways plays a critical role in CRC [[11], [12], [13]]. Targeting autophagy via key signaling pathways has been recognized as an effective approach to CRC therapy. In this study, RNA sequencing was performed with lomitapide-treated CRC cells, and pathway enrichment analyses of differentially expressed genes suggested that autophagy activation may contribute to the antitumor effects of lomitapide. The direct target of lomitapide in cancer cells is important to its clinical implications but remains to be elucidated.

Quantitative proteomic analysis coupled with bioinformatics analysis has been identified as a favorable strategy for exploring the molecular mechanism underlying the actions of small molecules [14]. In previous studies, we successfully used drug affinity responsive target stability (DARTS) technology to demonstrate that ADP-ribosylation factor 1 (ARF1) acts as a direct target and mediates the anticancer effect of azelastine [15]. In contrast to DARTS technology, the optimized limited proteolysis-mass spectrometry (LiP-SMap) method takes advantage of high-resolution LC-MS/MS and high-throughput quantitative proteomic technology to identify increased peptide levels upon limited proteolysis. Moreover, LiP-SMap relies on the binding affinity between a small molecule and its target protein(s), providing more consistent peptide detection and accurate proteome quantification [16]. Here, by performing an optimized LiP-SMap, we found that protein phosphatase 2A (PP2A), which plays a crucial role in various biological processes [17], is a direct target of lomitapide in cancer cells. Although PP2A was originally characterized as a tumor suppressor, recent studies have revealed that targeting PP2A can achieve therapeutic benefits in certain malignancies via cell cycle arrest, apoptosis induction and disruption of DNA repair [[18], [19], [20]]. In the present study, a series of functional assays were performed to investigate whether lomitapide activates AMPK-regulated autophagy to inhibit the proliferation and tumorigenesis of CRC cells by directly targeting PP2A.

Section snippets

Drugs, antibodies and reagents

Lomitapide was acquired from Selleck Chemicals (Huston, TX, USA) and freshly prepared into 5 mM stock solution with DMSO. Anti-Caspase-3, anti-cleaved caspase-3, anti-cleaved PARP, anti-LC3B, anti-AMPKα, anti-phospho-AMPKα Thr172, anti-phospho-Beclin-1 Ser93, and anti-Beclin-1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Vps34, anti-Atg14 and anti-Gapdh antibodies were obtained from Proteintech (Chicago, IL, USA). The WST-1 cell proliferation and

Screening of an FDA-approved small-molecule library led to the identification of lomitapide as a novel CRC anticancer compound

To obtain novel anti-cancer agents, a drug library of 1056 FDA-approved medications was tested for antitumor effects. HCT116 cells were intervened with the 1056 compounds individually at an identical concentration of 10 μM for 48 h, and the inhibitory effects of small-molecules on cells were evaluated by WST-1 assay. As shown in Fig. 1A, lomitapide, an agent widely used for treating hypercholesterolemia, was one of the most potent compounds in suppressing the proliferation of HCT116 cells,

Discussion

Autophagy has been extensively studied in cancers, and it is now universally recognized that autophagy has dual, contradictory roles in cancer progression [35]. Intracellular autophagy can be enhanced by a series of stressors, such as oxidative stress, nutrient starvation, and misfolded protein accumulation. Once the process is activated, a portion of the cytoplasm and organelles will enter the autophagic vesicles in succession, responding to the cellular stresses [36]. Owing to sustained

Authors' contributions

Wen Wen Xu and Qian Zuo: study concept and design; manuscript writing; Qian Zuo, Long Liao, Zi-Ting Yao, Ya-Ping Liu, Ding-Kang Wang, Shu-Jun Li, Xing-Feng Yin: acquisition of data; analysis and interpretation of data; statistical analysis; Wen Wen Xu: obtained funding; study supervision.

Acknowledgments

This research was supported by National Natural Science Foundation of China of China (82073196, 81803551, 31770888), National Key R&D Program of China (2017YFA0505100), Guangdong Natural Science Research Grant International joint project (2021A0505030035), Guangdong Natural Science Research Grant (2021A1515011158, 2020A1515110760), and the Fundamental Research Funds for the Central Universities (21620429).

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Targeting PP2A with lomitapide suppresses colorectal tumorigenesis through the activation of AMPK/Beclin1-mediated autophagy

Highlights

Abstract

Introduction

Section snippets

Drugs, antibodies and reagents

Screening of an FDA-approved small-molecule library led to the identification of lomitapide as a novel CRC anticancer compound

Discussion

Authors' contributions

Acknowledgments

Free Radic. Biol. Med.

Biochim. Biophys. Acta

Semin. Cell Dev. Biol.

Biomed. Pharmacother.

Biochim. Biophys. Acta Mol. Cell Res.

FEBS Lett.

Canc. Lett.

Semin. Canc. Biol.

Curr. Biol.

J. Biol. Chem.

Cell

EBioMedicine

Exp. Cell Res.

Redox Biol

Drug Discov. Today

Cancer Treat Res Commun

Canc. Lett.

Int. J. Biochem. Cell Biol.

Cell. Signal.

J. Biol. Chem.

Colorectal cancer is a leading cause of cancer incidence and mortality among adults younger than 50 years in the USA: a SEER-based analysis with comparison to other young-onset cancers

J. Invest. Med.

Lomitapide

Br. J. Clin. Pharmacol.

Modulation of inflammation by autophagy: consequences for human disease

Autophagy

Autophagy sustains mitochondrial glutamine metabolism and growth of BrafV600E-driven lung tumors

Canc. Discov.

Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease

Nat. Genet.

Truncating mutation in the autophagy gene UVRAG confers oncogenic properties and chemosensitivity in colorectal cancers

Nat. Commun.

Tumor suppression and promotion by autophagy

BioMed Res. Int.

The role of autophagy in the treatment of BRAF mutant colorectal carcinomas differs based on microsatellite instability status

PloS One