Souvik Dey

ER stress–mediated autophagy promotes Myc-dependent transformation and tumor growth

The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc-induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/-) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc-induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.

Targeting ER stress–induced autophagy overcomes BRAF inhibitor resistance in melanoma

Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.

ATF4-dependent induction of heme oxygenase 1 prevents anoikis and promotes metastasis

The integrated stress response (ISR) is a critical mediator of cancer cell survival, and targeting the ISR inhibits tumor progression. Here, we have shown that activating transcription factor 4 (ATF4), a master transcriptional effector of the ISR, protects transformed cells against anoikis - a specialized form of apoptosis - following matrix detachment and also contributes to tumor metastatic properties. Upon loss of attachment, ATF4 activated a coordinated program of cytoprotective autophagy and antioxidant responses, including induced expression of the major antioxidant enzyme heme oxygenase 1 (HO-1). HO-1 upregulation was the result of simultaneous activation of ATF4 and the transcription factor NRF2, which converged on the HO1 promoter. Increased levels of HO-1 ameliorated oxidative stress and cell death. ATF4-deficient human fibrosarcoma cells were unable to colonize the lungs in a murine model, and reconstitution of ATF4 or HO-1 expression in ATF4-deficient cells blocked anoikis and rescued tumor lung colonization. HO-1 expression was higher in human primary and metastatic tumors compared with noncancerous tissue. Moreover, HO-1 expression correlated with reduced overall survival of patients with lung adenocarcinoma and glioblastoma. These results establish HO-1 as a mediator of ATF4-dependent anoikis resistance and tumor metastasis and suggest ATF4 and HO-1 as potential targets for therapeutic intervention in solid tumors.

PERK-ing up autophagy during MYC-induced tumorigenesis

Stress in the tumor microenvironment in the form of hypoxia and low glucose/amino acid levels activates the evolutionarily conserved cellular adaptation program called the unfolded protein response (UPR) promoting cell survival in such conditions. Our recent studies showed that cell autonomous stress such as activation of the proto-oncogene MYC/c-Myc, can also trigger the UPR and induce endoplasmic reticulum (ER) stress-mediated autophagy. Amelioration of ER stress or autophagy enhances cancer cell death in vitro and attenuates tumor growth in vivo. Here we will discuss the role of the UPR and autophagy in MYC-induced transformation. Our findings demonstrate that the EIF2AK3/PERK-EIF2S1/eIF2α-ATF4 arm of the UPR promotes tumorigenesis by activating autophagy and enhancing tumor formation. Therefore, the UPR is an attractive target in MYC-driven cancers.

The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase (PDH)-E1α Phosphorylation

Inhibition of the PI3K/Akt pathway decreases hypoxia within SQ20B human head and neck cancer xenografts. We set out to understand the molecular mechanism underlying this observation. We measured oxygen consumption using both a Clark electrode and an extracellular flux analyzer. We made these measurements after various pharmacologic and genetic manipulations. Pharmacologic inhibition of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in vitro in SQ20B and other cell lines by 30% to 40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, which inhibits activity of this critical gatekeeper of mitochondrial respiration. Expressing wild-type PTEN in a doxycycline-inducible manner in a cell line with mutant PTEN led to an increase in PDH-E1α phosphorylation and a decrease in OCR. Pretreatment of SQ20B cells with dichloroacetate (DCA), which inhibits PDH-E1α phosphorylation by inhibiting dehydrogenase kinases (PDK), reversed the decrease in OCR in response to PI3K/Akt/mTOR inhibition. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects of PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway. Mol Cancer Ther; 14(8); 1928–38. ©2015 AACR.

Radiosensitization by the PARP inhibitor olaparib in BRCA1-proficient and deficient high-grade serous ovarian carcinomas

OBJECTIVE Approximately 15-25% of high-grade serous ovarian carcinomas (HGSOC) harbor BRCA1/2 mutations. Inhibition of Poly (ADP-ribose) polymerase (PARP) is synthetically lethal to cells and tumors with BRCA1/2 mutation. Our goal was to investigate the radiosensitizing effects of PARP inhibitor olaparib in HGSOC with different BRCA1 status. METHODS The radiosensitizing effects of olaparib were tested on BRCA1-proficient and deficient HGSOC by clonogenic survival and tumor growth assays. The effects of olaparib and radiation on DNA damage, PARP activity, and apoptosis were determined. RESULTS BRCA1-deficient HGSOC cells were more sensitive to RT alone and exhibited significantly higher levels of olaparib-mediated radiosensitization compared to BRCA1-proficient cells. Furthermore, when combined with RT, olaparib inhibited DNA damage repair and PARP1 activity, increased apoptosis, decreased growth of HGSOC xenografts and increased overall host survival. The growth-inhibitory effects of the combined olaparib and RT treatment were more pronounced in mice bearing BRCA1-deficient tumors compared to BRCA1-proficient tumors. CONCLUSIONS These results provide a preclinical rationale for improved treatment modalities using olaparib as an effective radiosensitizer in HGSOC, particularly in tumors with BRCA1-deficiencies.

Abstract 5863: Identifying the radiosensitizing effects of PARP inhibitor in ovarian cancer

Ovarian cancer is the seventh-most common cancer with a high mortality rate among women. Radiation therapy (RT) is used in ovarian cancer for local relapse, but doses are limited due to concern for small bowel obstruction in patients who have had multiple surgeries and chemotherapeutic regimens. Therefore, it’s an ideal setting to improve the therapeutic window by combining RT and a radiosensitizer. Poly (ADP-ribose) polymerase-1 (PARP-1) is involved in the recognition of DNA damage and the facilitation of DNA repair by recognizing DNA single-strand breaks (SSB). Inhibition of PARP1, especially in tumors driven by BRCA1 or 2 mutations, has been shown to potentiate the DNA-damaging effects of radiation in vitro and in vivo and to increase tumor vasculature perfusion and oxygenation. Approximately half of ovarian cancers have germline or somatic BRCA gene mutation, epigenetic silencing or other mutations that affect homologous recombination (HR) competency resulting in DNA repair defects, making this tumor particularly susceptible to the radiation-sensitizing effects of PARP inhibitors. Here, we investigated the therapeutic effects of the PARP inhibitor olaparib in preclinical models of ovarian cancer. Basic BRCA1 and PARP1 protein and mRNA levels were characterized in a panel of ovarian cancer cell lines. The radiosensitizing effects of olaparib were tested on both HR-proficient and HR-deficient ovarian cancer cells by evaluating the colony formation, DNA damage, apoptosis, and PARP1 activity. Survival benefit of olaparib was also examined in xenograft models of ovarian cancer. Our results demonstrate that combination of olaparib with IR decreased colony formation, inhibited DNA damage (γH2AX) repair, and induced more apoptosis compared to olaparib or IR alone. In addition, HR-deficient cells were more sensitive to IR than HR-proficient cells in the presence of olaparib. Furthermore, olaparib inhibited PARP1 activity in vivo, significantly decreased tumor growth and increased overall survival when combined with IR in mice bearing subcutaneous xenografts of HR-deficient OVCAR8 cells while producing a relative modest effect on overall survival of mice bearing xenografts of HR-proficient SKOV3 cells. These results provide a preclinical rationale for improved treatment modalities using olaparib as an effective radiosensitizer in ovarian cancer, particularly in tumors with HR-deficiencies. Citation Format: Yue Bi, Ioannis Verginadis, Souvik Dey, linlang Guo, Lilie Lin, Yanfang Zheng, Constantinos Koumenis. Identifying the radiosensitizing effects of PARP inhibitor in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5863. doi:10.1158/1538-7445.AM2017-5863

Abstract 1247: Identifying novel regulators of the Unfolded Protein Response (UPR) by genome-scale CRISPR-Cas9 knockout screens

Development and growth of a tumor as well as its ability to metastasize involves a complex relationship with its tissue microenvironment. A proliferating tumor encounters several microenvironmental stress conditions such as hypoxia, lack of nutrients and acidosis. To cope with these conditions, cancer cells have developed elaborate cytoprotective mechanisms which provide them with distinct advantages to thrive. Thus, deciphering the signaling pathways which get activated in the tumor microenvironment has been paramount to develop new therapeutic strategies for treatment. The Unfolded Protein Response (UPR) is an adaptive prosurvival pathway elicited by stresses in the tumor microenvironment (e.g., hypoxia, low glucose) and involves translational and transcriptional activation of effector genes which act to relieve cellular stress and block cancer cell death. We developed a strategy to comprehensively analyze critical mediators of cell fate in response to UPR activation. We have delivered a lentiviral genome-scale CRISPR Cas9 knockout (GeCKOv2) library to Sq20B cells (human squamous head and neck carcinoma) and A375 (human melanoma) cells. The library is targeting 19,050 genes with 123,411 unique guide sequences and enables both negative and positive selection screening. We used the GeCKO v2 library to identify genes essential for triggering the UPR in response to thapsigargin and tunicamycin, known specific activators of ER stress. Our highest-ranking candidates include BIRC5/Survivin, a well-studied molecule that acts as an inhibitor of apoptosis and is highly expressed in cancer cells and eukaryotic translation initiation factor eIF6, whose overexpression increases motility and invasiveness of cancer cells. Our preliminary results indicate that loss of Survivin and eIF6 dramatically enhance sensitization of cells to various ER stress conditions. Moreover, this synergistic outcome is observed when cells are treated with YM155, a small molecule that selectively suppresses Survivin and is used in phase I/II clinical trials. Lastly, morphological changes like endoreduplication are observed after long term absence of Survivin indicating its endogenous ER stress inducing role. Taken together, Survivin and eIF6 are important mediators of survival following ER stress and characterizing the pathways involved can lead to the development of novel targeted agents and therapeutic approaches. Citation Format: Nektaria Maria Leli, Souvik Dey, Lauren Brady, Constantinos Koumenis. Identifying novel regulators of the Unfolded Protein Response (UPR) by genome-scale CRISPR-Cas9 knockout screens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1247. doi:10.1158/1538-7445.AM2017-1247

Abstract B05: PI3K/mTOR pathway-dependent regulation of oxygen metabolism via pyruvate dehydrogenase (PDH)-E1alpha phosphorylation

The PI3K/mTOR pathway plays a central role in coupling metabolic processes to the cellular proliferative state. In the current study we show that pharmacological inhibition of this pathway leads to a decrease in hypoxia within SQ20B human head and neck cancer xenografts. The mechanism underlying the effect appears in part to be due to reduced tumor cell oxygen consumption induced by the drug. Pharmacologic inhibitors of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in transformed cell lines in vitro by 30-40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1alpha subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, an inhibitory modification of this critical gatekeeper of mitochondrial respiration. Expressing wild type PTEN in a doxycycline-inducible manner in a glioblastoma cell line with mutant PTEN led to an increase in PDH-E1alpha phosphorylation and a decrease in OCR. The decrease in OCR with PI3K/Akt/mTOR inhibition was recapitulated by knocking down PDH-E1alpha expression using siRNA. Pre-treatment with dichloroacetate (DCA), a known inhibitor of the pyruvate dehydrogenase kinases (PDKs) that phosphorylate PDH-E1alpha prevented the upregulation in PDH-E1alpha phosphorylation, induced by inhibition of the pathway and also blunted the decrease in OCR. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption and reveal a novel link between this pathway and PDH phosphorylation. These results have important implications for understanding the effects PI3K pathway activation in tumor metabolism and also in the use of inhibitors of this pathway in cancer therapy Citation Format: George Cerniglia, Souvik Dey, Shannon M. Gallagher-Colombo, Natalie Daurio, Stephen Tuttle, Theresa M. Busch, Alexander Lin, Tatiana V. Esipova, Sergei Vinogradov, Constantinos Koumenis, Amit Maity. PI3K/mTOR pathway-dependent regulation of oxygen metabolism via pyruvate dehydrogenase (PDH)-E1alpha phosphorylation. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B05.

Abstract 1262: The transcription factor ATF4 regulates resistance to anoikis and promotes metastasis in fibrosarcoma via cooperative upregulation of Heme Oxygenase-1 with Nrf2

The Integrated Stress Response (ISR) plays a critical role in cancer cell survival, and targeting the ISR results in inhibition of tumor progression. A critical aspect of ISR involves the preferential translation of activating transcription factor 4(ATF4), a transcriptional factor regulating genes involved in metabolism, nutrient uptake, and anti-oxidant responses. We previously demonstrated that ATF4 expression is significantly increased in tumors compared to corresponding normal tissue, and that ablation of ATF4 compromised primary tumor growth in mice. Based on the central roles that ATF4 target genes play in pro-survival processes, we hypothesized that ATF4 might also play a role in tumor metastasis. Upon loss of matrix attachment, a critical step in the metastatic process, we found induction of phosphorylation of the translation factor eIF2α mediated by upstream kinase PERK in human adenocarcinoma HT1080 cells and colorectal adenocarcinoma DLD1 cells. Increased eIF2α phosphorylation resulted in translational upregulation of ATF4 and its transcriptional targets CHOP, ASNS and ATF3. Interestingly, failure to induce ISR and upregulate ATF4 resulted in increased apoptosis following matrix detachment - a process known as anoikis. Furthermore, we demonstrate that ATF4 promotes anoikis resistance by activating a coordinated program of autophagy and anti-oxidant responses. Upon detachment, ATF4 activates cytoprotective autophagy by transcriptionally upregulating several key autophagic genes (Atg5, Atg7 and Ulk1). Simultaneously, ATF4 also induces the expression of the heme oxygenase 1 (HO-1) - a major antioxidant enzyme. Activation of HO-1 following matrix detachment occurs by coordinated upregulation of ATF4 and the antioxidant PERK dependent transcription factor Nrf2, which converge to bind on antioxidant regulatory elements (ARE) in the HO-1 promoter. Failure to either initiate the autophagy response or HO-1 induction sensitizes cells to anoikis. In agreement with our in vitro observations, HT1080 cells harboring ATF4 shRNA (shATF4) injected through tail vein of nude mice fails to establish metastatic lung colonization after 4 weeks compared to the non-targeting (shNT) counterparts. Immunohistochemical analysis on the tumor bearing lungs show high and colocalized expression of ATF4 and HO-1. Reconstituting either ATF4 or HO-1 expression in ATF4-deficient cells, rescues the tumor lung colonization phenotype. Finally, higher expression of HO-1 and ATF4 was found in human primary as well as metastatic tumors compared to normal epithelium or stromal tissue and correlated with reduced overall survival of lung adenocarcinoma and glioblastoma patients. Collectively, these results establish ATF4 as a major player in tumor metastasis, and the combined activity of its downstream targets HO-1 and Nrf2 as a critical mediator in this process. Citation Format: Souvik Dey, Carly M. Sayers, Stacey L. Lehman, Yi Cheng, George J. Cerniglia, Stephen W. Tuttle, Michael D. Feldman, Paul J.L. Zhang, Serge Y. Fuchs, J. Alan Diehl, Constantinos Koumenis. The transcription factor ATF4 regulates resistance to anoikis and promotes metastasis in fibrosarcoma via cooperative upregulation of Heme Oxygenase-1 with Nrf2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1262. doi:10.1158/1538-7445.AM2015-1262