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CDK4 Regulates Lysosomal Function and Mtorc1 Activation to Promote Cancer Cell Survival

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CDK4 Regulates Lysosomal Function and Mtorc1 Activation to Promote Cancer Cell Survival Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/0008-5472.CAN-19-0708 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. CDK4 regulates lysosomal function and mTORC1 activation to promote cancer cell survival Laia Martínez-Carreres1, Julien Puyal2, Lucía C. Leal-Esteban1, Meritxell Orpinell3, Judit Castillo-Armengol1, Albert Giralt1, Oleksandr Dergai1, Catherine Moret1, Valentin Barquissau1, Anita Nasrallah1, Angélique Pabois4,5, Lianjun Zhang4,6,7, Pedro Romero4, Isabel C. Lopez-Mejia1 and Lluis Fajas1* 1Center for Integrative Genomics, University of Lausanne, 1015 Lausanne, Switzerland. 2Department of Fundamental Neurosciences, University of Lausanne, 1005 Lausanne, Switzerland. 3Department of Physiology, University of Lausanne, 1005 Lausanne, Switzerland 4Department of Fundamental Oncology, University of Lausanne, 1066 Epalinges, Switzerland. 5Ludwig Institute for Cancer Research, University of Lausanne, 1066 Epalinges, Switzerland. 6Present address: Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, 100005 Beijing, China. 7Present address: Suzhou Institute of Systems Medicine, Suzhou, Jiangsu 215123, China. *Corresponding author: Lluis Fajas, University of Lausanne, Center for Integrative Genomics, Quartier UNIL-Sorge, Bat. Genopode, CH-1015 Lausanne, Switzerland. Phone: +41.21.692.4111; E-mail: [email protected]. Declaration of interests The authors declare no competing interests. Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/0008-5472.CAN-19-0708 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Cyclin-dependent kinase 4 (CDK4) is well-known for its role in regulating the cell cycle, however, its role in cancer metabolism, especially mTOR signaling, is undefined. In this study, we established a connection between CDK4 and lysosomes, an emerging metabolic organelle crucial for mTORC1 activation. On the one hand, CDK4 phosphorylated the tumor suppressor FLCN, regulating mTORC1 recruitment to the lysosomal surface in response to amino acids. On the other hand, CDK4 directly regulated lysosomal function and was essential for lysosomal degradation, ultimately regulating mTORC1 activity. Pharmacological inhibition or genetic inactivation of CDK4, other than retaining FLCN at the lysosomal surface, led to the accumulation of undigested material inside lysosomes, which impaired the autophagic flux and induced cancer cell senescence in vitro and in xenograft models. Importantly, the use of CDK4 inhibitors in therapy is known to cause senescence but not cell death. To overcome this phenomenon and based on our findings, we increased the autophagic flux in cancer cells by using an AMPK activator in combination with a CDK4 inhibitor. The cotreatment induced autophagy (AMPK activation), and impaired lysosomal function (CDK4 inhibition), resulting in cell death and tumor regression. Altogether, we uncover a previously unknown role for CDK4 in lysosomal biology and propose a novel therapeutic strategy to target cancer cells. Statement of significance Findings uncover a novel function of CDK4 in lysosomal biology which promotes cancer progression by activating mTORC1, targeting this function offers a new therapeutic strategy for cancer treatment. 2 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/0008-5472.CAN-19-0708 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Cyclin-dependent kinase 4 (CDK4) has a well-established role in cell cycle control (1) and CDK4-cyclin complexes are commonly deregulated in tumorigenesis (2). These complexes are of great interest as therapeutic targets, and the FDA has approved the specific CDK4/6 kinase inhibitors PD0332991 (palbociclib), LEE011 (ribociclib) and LY2835219 (abemaciclib) for treating advanced or metastatic hormone receptor (HR)-positive and HER2- negative breast cancer. Clinical studies using CDK4/6 inhibitors to treat other malignancies are being conducted (3). Research from our group and others has shown that the role of CDK4 is not limited to the control of the cell cycle. Indeed CDK4 is also a major regulator of energy homeostasis (4-6) through E2F1-RB complex (7), AMPK (8) and IRS2 (9). Importantly, the CDK4 pathway has been shown to cross-talk with the mTOR pathway, which is a major regulator of cell growth and metabolism (10,11). CDK4/6 inhibition attenuates mTOR Complex 1 (mTORC1) activity in some cancer models (12,13), yet the effects of CDK4/6 inhibitors on mTORC1 seem to be cell-type specific since opposite results were observed in other cancer types (14). The exact mechanism underlying the CDK4-mTOR cross-talk in mammals is unknown, although in Drosophila it occurs via the phosphorylation of TSC2 (15). Given that mTOR activity is increased in numerous cancers and participates in the translational regulation of several oncogenic proteins, mTOR inactivation constitutes an attractive strategy for cancer treatment (16). Lysosomes, considered for years as only the digestive system of the cell, have emerged as key effectors in cell metabolism, due to their role as platforms in the activation of mTOR pathway (17-19). mTORC1 is recruited to the surface of lysosomes in a complex amino acid (AA)-dependent manner (17). Among the multiple regulators of this process, we focused on FLCN, a tumor suppressor which functions as a complex with FNIP. The FLCN- FNIP complex interacts with Rag GTPases in the absence of AAs repressing their activity. When AAs are sensed, FLCN-FNIP complexes dissociate from Rag GTPases eliciting their activation. The activation of Rag GTPases is crucial for mTORC1 recruitment to lysosomes (20). Importantly, mTORC1 activation is also triggered by the accumulation of AAs in the lysosomal lumen (21). Therefore, alterations in the lysosomal function directly impact 3 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/0008-5472.CAN-19-0708 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. mTORC1 activity (22,23). Additionally, these organelles play roles in cell survival and cell proliferation, thus becoming emerging targets for cancer therapy (24-26). In this study, we demonstrate that CDK4 is capable of modulating mTORC1 activity in a direct manner, through the phosphorylation of FLCN, and indirectly, by promoting lysosomal function. When CDK4 inhibitors are used, the lack of lysosomal function induces senescence in triple-negative breast cancer (TNBC) cells and impairs tumor growth in a mouse xenograft model. Moreover, a combination of AMPK activation and CDK4 inhibition was used in an attempt to trigger autophagy in conditions when lysosomes are dysfunctional and resulted in cell death and tumor regression. This finding is of high relevance in TNBC, a highly invasive and aggressive cancer type that does not have a clear therapeutic strategy yet (27). Materials and Methods Materials. LY2835219, PD0332991 and LEE011 were purchased from MedChem Express and used at 0.5 µM, unless otherwise indicated. R3 human IGF-1 (I11146, Sigma) was used at 30 ng/ml. Minimal Essential Media (MEM) Amino Acids Solution (50X, Gibco) was used at the indicated concentrations. Rapamycin (LC Laboratories) was kindly provided by Pedro Romero (UNIL) and used at the indicated concentrations. Bafilomycin A1 (BafA1) was purchased from Enzo Life Sciences (ALX-380-030-M001) and used at 0.3 µM. Cell permeable αKG analog DMKG (349631, Sigma) was used at 5mM. Cell culture and transfection. MDA-MB-231, CCRF-CEM, HTC116, IB115, HT29, SKOV, MCF7 and PC3 cell lines, all obtained from ATCC, were cultured in RPMI 1640 + GlutaMAX containing, 10mM HEPES, 1mM sodium pyruvate (All from Gibco) and 10% fetal bovine serum (FBS) (PAA Laboratories). Thawed cells were allowed one passage to reach exponential growth phase before being used. Cells were used during maximum of ten passages in the experiments performed in this study. PCR-based mycoplasma tests were done routinely using specific primers: 5’-TGCACCATCTGTCACTCTGTTAACCTC-3’ and 3- GGGAGCAAACAGGATTAGATACCCT-5’, the last test was performed in June 2019. MDA-MB-231 CDK4 knockout (KO), E2F1 KO and FLCN KO stable cell lines were generated with CRISPR/Cas9 technology. The lentiCRISPR v2 plasmid was a gift from Feng Zhang (MIT; Addgene plasmid 52961)(28). The pMD2.G plasmid was a gift from Didier Trono (EPFL; Addgene 12259). The pCMV-dR8.91 plasmid and the guide RNA for FLCN were 4 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/0008-5472.CAN-19-0708 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. gifts from Christian Widmann (UNIL). The target sequences for the guide RNAs are shown in Supplementary Table S1. Synthetic oligonucleotides were purchased and cloned into the digested LentiCrispR vector as described in Shalem et al (28).
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