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Epithelial-to-mesenchymal transition (EMT) plays an important role in cancer progression. By undergoing an EMT, cancer cells acquire a spectrum of malignant properties, including invasiveness, multi-drug resistance and stem-like traits. Although they play an important role in tumor progression and resistance, few vulnerabilities of EMT cancer cells have been reported to date. To identify specific vulnerabilities of EMT cells, Using small molecule and RNAi screens, we have discovered that induction of EMT greatly sensitizes cells to agents that perturb endoplasmic reticulum (ER) function. This unexpected sensitivity to ER stress is mainly due to the expression and secretion of large amount of extracellular matrix (ECM) proteins by cells that have undergone an EMT. In line with their increased secretory load, EMT cells display a branched ER morphology and constitutively activate the PERK-eIF2α branch of the unfolded protein response (UPR). Using a PERK-specific inhibitor, we found that PERK activation is also required for EMT cells to invade and metastasize. In human tumor tissues, EMT gene expression correlates strongly with both ECM and PERK-eIF2α genes. In summary, our findings identify a novel vulnerability of cells that have undergone an EMT, and demonstrate that the PERK branch of the UPR is required for their malignancy. Citation Format: Yuxiong Feng, Ethan S Sokol, Catherine A Del Vecchio, Sandhya Sanduja, Jasper HL Claessen, Theresa A Proia, Dexter X Jin, Ferenc Reinhardt, Hidde L Ploegh, Qiu Wang, Piyush B Gupta. EMT activates PERK-eIF2α signaling and sensitizes cells to perturbations in endoplasmic reticulum function [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-07-01.

Epithelial-to-mesenchymal transition (EMT) promotes both tumor progression and drug resistance, yet few vulnerabilities of this state have been identified. Using selective small molecules as cellular probes, we show that induction of EMT greatly sensitizes cells to agents that perturb endoplasmic reticulum (ER) function. This sensitivity to ER perturbations is caused by the synthesis and secretion of large quantities of extracellular matrix (ECM) proteins by EMT cells. Consistent with their increased secretory output, EMT cells display a branched ER morphology and constitutively activate the PERK–eIF2α axis of the unfolded protein response (UPR). Protein kinase RNA-like ER kinase (PERK) activation is also required for EMT cells to invade and metastasize. In human tumor tissues, EMT gene expression correlates strongly with both ECM and PERK–eIF2α genes, but not with other branches of the UPR. Taken together, our findings identify a novel vulnerability of EMT cells, and demonstrate that the PERK branch of the UPR is required for their malignancy. Significance: EMT drives tumor metastasis and drug resistance, highlighting the need for therapies that target this malignant subpopulation. Our findings identify a previously unrecognized vulnerability of cancer cells that have undergone an EMT: sensitivity to ER stress. We also find that PERK–eIF2α signaling, which is required to maintain ER homeostasis, is also indispensable for EMT cells to invade and metastasize. Cancer Discov; 4(6); 702–15. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 621

Background: Breast cancer is a heterogeneous disease encompassing multiple subtypes that differ in their molecular profiles, clinical course of progression, and response to therapeutics. Interestingly, breast cancers fall broadly into luminal and basal subtypes, which resemble the normal luminal and basal epithelial cells of the breast. This correlation suggests that the molecular mechanisms controlling epithelial cell lineage identity during normal mammary development may also influence tumor subtype during cancer formation. Since many breast cancers are driven by oncogenes that are important for normal mammary development, we hypothesize that dysregulated oncogene signaling may alter mammary epithelial cell lineage identity at an early step of cancer initiation, priming the breast tumor subtype that will be formed upon full tumorigenesis. Methods: Human breast cell lines, MCF10a and MCF10f, and primary human breast tissues were infected with virus expressing single breast cancer oncogenes. Flow cytometry and quantitative RT-PCR were performed on breast cell lines to determine changes in the balance of luminal to basal cells and expression of luminal or basal gene sets. Primary tissues were grown in hydrogels and stained for luminal and basal markers to determine the effect of oncogene expression on the growth of mammary ductal structures. Mouse tumor models, MMTV-Myc and MMTV-Wnt1, were investigated for changes in their mammary epithelial architecture by histology and flow cytometry at ages prior to tumor formation. Results: Oncogene overexpression in human breast cell lines causes shifts in basal and luminal cell balance that correlate with changes seen in mammary ductal growth in hydrogel cultures of primary human breast epithelial cells. In mouse models, individual oncogenes alter mammary epithelial architecture and cell balance at early stages prior to tumor formation. In both models, individual oncogenes are able to regulate mammary epithelial cell lineage identity in the absence of other tumor-initiating signals, indicating that oncogenes may influence breast cancer subtype through mammary epithelial cell lineage regulation at early stages of cancer progression. Citation Format: Jerrica Breindel, Felix Nampanya, Aditya Datye, Daniel H. Miller, Piyush Gupta, Charlotte Kuperwasser. Oncogenes alter mammary epithelial cell lineage identity prior to tumor formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 534.

Numerous hormonal factors contribute to the lifetime risk of breast cancer development. These include inherited genetic mutations, age of menarche, age of menopause, and parity. Inexplicably, there is evidence indicating that ovariectomy prevents the formation of both estrogen receptor (ER)–positive and ER-negative breast cancers, suggesting that ER-negative breast cancers are dependent on ovarian hormones for their formation. To examine the mechanism(s) by which this may be occurring, we investigated the hypothesis that steroid hormones promote the outgrowth of ER-negative cancers by influencing host cell types distinct from the mammary epithelial cells. We used a novel xenograft mouse model of parturition-induced breast carcinoma formation, in which the tumors that arise following pregnancy lack the expression of nuclear hormone receptors, thereby recapitulating many clinical cases of this disease. Despite lacking ER expression, the tumors arising following pregnancy in this model require circulating estrogens for their formation. Moreover, increasing the levels of circulating estrogens sufficed to promote the formation and progression of ER-negative cancers, which was accompanied by a systemic increase in host angiogenesis and was attendant with the recruitment of bone marrow–derived stromal cells. Furthermore, bone marrow cells from estrogen-treated mice were sufficient to promote tumor growth. These results reveal a novel mechanism by which estrogens promote the growth of ER-negative cancers. [Cancer Res 2007;67(5):2062–71]

The proteasome is a central regulator of protein homeostasis in all eukaryotes. Targeted pharmaceutical inhibition of the proteasome complex has been implemented as a successful therapeutic strategy to treat several cancers including multiple myeloma. Unfortunately, many of the cancers show intrinsic resistance to proteasome inhibitors, and those that are initially responsive eventually develop resistance by a poorly understood mechanism. We have recently performed genome-wide screens for mutations that allowed cells to survive in the presence of two distinct proteasome inhibitors (bortezomib and MG132). Counter to expectation, we have shown that reducing any one of the many of the subunits of the regulatory cap complex (19S) is sufficient to alter both the levels and ratio of the 26S/20S proteasome complex and induce increased resistance to proteasome inhibition. These findings motivated us to establish a cell-based screening strategy to identify molecules that specifically target the proteasome inhibitor (PI)-resistant state. We further exploited the Genomics of Drug Sensitivity in Cancer (GDSC) database to analyze the efficacy of PIs across 315 cancer cell lines for which we also had matched gene expression data. This analysis revealed that a significant decrease in the gene expression of one component of the 19S complex is both naturally occurring (in almost 6-8% of cells examined) and is sufficient to predict resistance to PIs. We termed these cells, proteasome HYDRA (Hypo expression Yielding Drug Resistance Ability) cancer cells. Further exploring the HYDRA cells utilizing the Cancer Cell Line Encyclopedia (CCLE) dataset revealed that the loss of expression of the proteasome subunit is not associated with a loss of gene copy number but rather is an epigenetic mechanism of reduced gene expression. We show here a novel cellular epigenetic switch utilized by many cancer cells that renders them resistant to proteotoxic stress. Our findings enable prediction of cancer cell vulnerability to proteasome function perturbation and also facilitate the specific targeting of this unique epigenetic state by selective drug treatment. Citation Format: Peter Tsvetkov, Ethan Sokol, Dexter Jin, Piyush Gupta, Sandro Santagata, Luke Whitesell, Susan Lindquist. Targeting the epigenetic state regulating cancer cell vulnerability to proteasome inhibition. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A28.

Cancer initiates and progresses in the presence of a diversity of stresses ranging from nutrient deficiency, low oxygen supply to pH fluctuations. A set of stress response pathways, termed unfolded protein response (UPR), was evolved to maintain homeostasis of the endoplasmic reticulum (ER) from disruption by the above stressors. While it is broadly acknowledged that the UPR is involved in tumorigenesis, as tumor cells can employ the UPR as a critical survival strategy to grow and resist chemotherapy, the role of UPR in metastasis remains largely elusive. Epithelial-to-mesenchymal transition (EMT) is a cell transdifferentiation program frequently hijacked by cancer cells to migrate and invade. Here we showed cancer cells that have undergone an EMT employ the PERK-ATF4 branch of the UPR to drive metastasis, since loss of ATF4 abrogates the cell invasion upon an EMT. By conducting gene expression profiling, we found that the PERK-ATF4 pathway mediates the expression of a subset of the essential pro-invasion EMT-signature genes. In particular, CREB3L1, an ER-associated transcription factor (TF), is highly induced upon EMT in an ATF4-dependent manner. Knockdown of CREB3L1 effectively inhibits the EMT-driven invasion, while overexpression of CREB3L1 not only promotes invasion but also rescues the decrease of invasion caused by loss of ATF4. Mechanistically, CREB3L1 facilitates invasion through an ECM-FAK cascade. In breast cancer patients, the expression of CREB3L1 is significantly upregulated in breast cancer metastases compared to primary lesions, and predicts a poor prognosis. Most importantly, unlike other EMT-TFs, CREB3L1 could be inhibited by known chemicals, since the activation of CREB3L1 is mediated by the S1P- and S2P- dependent proteolysis. In an orthotopic breast cancer model, we found that chemical inhibition of CREB3L1 drastically suppresses lung metastasis. As a summary, we discovered that an ER stress-associated transcription factor CREB3L1 is required for cancer metastasis, and the susceptibility of CREB3L1 to chemical inhibition makes it a valuable target for drug development. Citation Format: Yuxiong Feng, Dexter X. Jin, Piyush B. Gupta. Unfolded protein response is required for EMT-driven metastasis by inducing CREB3L1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2677.

Tumor recurrence and metastasis underlie the majority of cancer-related deaths. Cancer cells that recur or metastasize are often both de-differentiated and multidrug resistant, but the mechanistic basis for this has been poorly understood. We have recently shown that de-differentiation promotes multidrug resistance by activating Nrf2, which stimulates transcription of drug efflux pumps and enzymes that scavenge reactive oxygen species (ROS). De-differentiation activates Nrf2 by a non-canonical mechanism involving its phosphorylation by the ER membrane kinase PERK. PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy, and inhibiting this signaling axis re-sensitizes de-differentiated cancer cells to treatment. To further explore this pathway we profiled the effects of PERK inhibition on global gene expression in both differentiated and de-differentiated cells upon treatment with chemotherapy. This analysis showed that PERK inhibition results in an amino acid deprivation phenotype, and suggested that de-differentiated cells may be sensitive to perturbations in amino acid availability. Consistent with this, we found that the aminopeptidase inhibitor Tosedostat was selectively toxic to de-differentiated breast cancer cells when given in combination with chemotherapy. Our findings identify a novel vulnerability of therapy-resistant breast cancer cells, and suggest that targeting amino acid availability in combination with chemotherapy could be an effective treatment for aggressive breast cancers that are multidrug resistant. Citation Format: Catherine A. Del Vecchio, Yuxiong Feng, Ethan S. Sokol, Erik J. Tillman, Sandhya Sanduja, Ferenc Reinhardt, Piyush B. Gupta. Amino acid deprivation selectively targets multidrug-resistant breast cancer cells. [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 LB-244. doi:10.1158/1538-7445.AM2015-LB-244

Tumor histopathology is a strong predictor of patient mortality, although the molecular and cellular factors that are responsible for this phenotypic diversity remain poorly understood. Women with inherited mutations in the BRCA1 gene have increased risk for the development of breast cancer, but also exhibit a specific predisposition for the development of aggressive basal-like breast cancers. Here we study the interplay between inherited mutations in BRCA1 and tumor differentiation by examining the regulation of progenitor cell fate in disease-free breast tissues from BRCA1 mutation carriers. We demonstrate for the first time that cell populations derived from patients harboring mutations in BRCA1 (BRCA1mut/+) give rise to tumors with increased basal differentiation, relative to cells obtained from BRCA1+/+ patients. Molecular analysis of disease-free breast tissues from BRCA1mut/+ patients revealed significant defects in epithelial progenitor cells that are present prior to cancer incidence. Moreover, we discovered that the transcriptional repressor Slug is an important functional regulator of human breast progenitor cell lineage commitment and differentiation and that it is aberrantly expressed in BRCA1mut/+ tissues and cells prior to neoplastic transformation. In addition, Slug expression is necessary for the increased basal-like phenotypes prior to and following neoplastic transformation. These findings demonstrate that the genetic background of patient populations, in addition to affecting incidence rates, significantly impacts progenitor cell fate commitment and therefore, tumor phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 916. doi:10.1158/1538-7445.AM2011-916

Human breast cancers can be broadly classified based on their molecular and gene expression profiles into luminal and basal-like tumors. These tumor subtypes express markers corresponding to the two major differentiation states of epithelial cells in the breast: luminal cells that line the breast ducts and the outer myoepithelial/basal cells that provide contractile functions. Women with inherited mutations in the BRCA1 gene have increased breast cancer risk and also exhibit a specific predisposition to the development of aggressive basal-like breast cancers. Accumulating evidence suggests that BRCA1 has a role in breast epithelial differentiation and we wanted to further understand how this could contribute to the formation of basal-like tumors. Microarray, flow cytometry and immunohistochemical analysis of breast epithelial cells from disease-free women harboring deleterious mutations in BRCA1 (BRCA1 mut/+) compared to those from BRCA1 +/+ reduction mammoplasties showed an increase in markers of basal differentiation and a decrease in markers of luminal differentiation. We also created breast cancers from single cell suspensions of BRCA1 mut/+ and BRCA1 +/+ epithelial cells that had been transformed with identical oncogenes and injected into humanized mammary fat pads. Tumors derived from BRCA1 mut/+ cells had increased basal differentiation relative to cells obtained from BRCA1 +/+ patients, indicating that the perturbed differentiation evident prior to neoplastic transformation was mirrored in the tumors. Pathway analysis of the microarray data comparing BRCA1 mut/+ and BRCA1 +/+ cells from disease-free tissue indicated that signaling components relating to the transcriptional repressor Slug were overexpressed in BRCA1 mut/+ tissues. We confirmed that Slug protein levels were elevated in BRCA1 mut/+ tissues and in human tumors from BRCA1 mutation carriers. RNAi-mediated downregulation of slug in primary breast epithelial cells and cell lines derived from BRCA1 mut/+ tissue as well as breast cancer cell lines known to harbor BRCA1 mutations, led to a decrease in markers of basal differentiation, indicating that elevated Slug protein levels seen in BRCA1-associated tissues and tumors are contributing to the basal phenotype. Furthermore, RNAi-mediated knockdown of BRCA1 led to an increase in Slug protein expression and breast cancer cell lines harboring BRCA1 mutations showed an increase in Slug protein stability, indicating that loss of BRCA1 protein by mutation contributes to elevated Slug protein levels. These results reveal an important mechanism by which BRCA1 can regulate breast epithelial differentiation and may explain how, in addition to affecting incidence rates, the genetic background of patients could impact tumor phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-265.

Metastasis accounts for over 90% of cancer death, but its mechanisms are still poorly understood. One valuable approach that has been commonly used to study metastasis was to compare paired poorly and highly metastatic cell lines, either for roles of a few genes of interest or global gene expression differences. Our work involved in using shRNA screen to identify global key functional differences between these matched cell lines. We postulated that the distinct in vivo phenotypes of these paired cell lines are a consequence of alterations in their cellular signaling networks during tumor progression. To characterize these functional differences, we performed arrayed shRNA screens against the human kinome to examine kinase requirements for survival and proliferation on several pairs of melanoma, colon or prostate cancer cell lines. From these screens, we have identified a set of kinases that become essential for multiple highly metastatic cells, some of which have been linked to metastasis (such as c-Met) but more have not. Several of these genes were shown to be amplified or mutated in human cancers. In vivo metastatic experiments are underway to prioritize these kinases by evaluating which kinases can promote metastasis when ectopically expressed. Since inhibition of these kinases phenocopied c-Met inhibition, we also sought to test if any of these kinases are functionally linked to c-Met signaling. Indeed, several of the identified kinases became essential when immortalized melanocytes were made tumorigenic and metastatic by introduction of activated c-Met. Biochemical and genetic rescue experiments are currently ongoing to further examine the links between these genes and c-Met. These studies offer new insights into the cellular signaling pathways operating in metastatic cancer cells and also suggest potential targets to treat metastatic cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A200.