Dexter X. Jin

Epithelial-to-Mesenchymal Transition Activates PERK–eIF2α and Sensitizes Cells to Endoplasmic Reticulum Stress

UNLABELLED 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.

Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis

Despite its eponymous association with the heat shock response, yeast heat shock factor 1 (Hsf1) is essential even at low temperatures. Here we show that engineered nuclear export of Hsf1 results in cytotoxicity associated with massive protein aggregation. Genome-wide analysis revealed that Hsf1 nuclear export immediately decreased basal transcription and mRNA expression of 18 genes, which predominately encode chaperones. Strikingly, rescuing basal expression of Hsp70 and Hsp90 chaperones enabled robust cell growth in the complete absence of Hsf1. With the exception of chaperone gene induction, the vast majority of the heat shock response was Hsf1 independent. By comparative analysis of mammalian cell lines, we found that only heat shock-induced but not basal expression of chaperones is dependent on the mammalian Hsf1 homolog (HSF1). Our work reveals that yeast chaperone gene expression is an essential housekeeping mechanism and provides a roadmap for defining the function of HSF1 as a driver of oncogenesis.

Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers

Significance In previous work, we used genome-wide screening to uncover a counterintuitive mechanism by which cells can acquire resistance to inhibitors of the proteasome’s catalytic core through experimentally induced imbalances in the composition of its regulatory particle. However, in many cases, mechanisms uncovered in vitro for acquired resistance often do not translate to the context of actual clinical cancers. Here, we show that this mechanism is actually deployed spontaneously and naturally in diverse human cancer lines and is associated not only with increased resistance to proteasome inhibitors both in vitro and in the clinic but also is symptomatic of a much more broadly altered state with a unique gene signature and drug targetable vulnerabilities. The use of proteasome inhibitors to target cancer’s dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers.

Perturbation-Expression Analysis Identifies RUNX1 as a Regulator of Human Mammary Stem Cell Differentiation

The search for genes that regulate stem cell self-renewal and differentiation has been hindered by a paucity of markers that uniquely label stem cells and early progenitors. To circumvent this difficulty we have developed a method that identifies cell-state regulators without requiring any markers of differentiation, termed Perturbation-Expression Analysis of Cell States (PEACS). We have applied this marker-free approach to screen for transcription factors that regulate mammary stem cell differentiation in a 3D model of tissue morphogenesis and identified RUNX1 as a stem cell regulator. Inhibition of RUNX1 expanded bipotent stem cells and blocked their differentiation into ductal and lobular tissue rudiments. Reactivation of RUNX1 allowed exit from the bipotent state and subsequent differentiation and mammary morphogenesis. Collectively, our findings show that RUNX1 is required for mammary stem cells to exit a bipotent state, and provide a new method for discovering cell-state regulators when markers are not available.

Premature polyadenylation of MAGI3 is associated with diminished N 6 -methyladenosine in its large internal exon

In cancer, tumor suppressor genes (TSGs) are frequently truncated, causing their encoded products to be non-functional or dominant-negative. We previously showed that premature polyadenylation (pPA) of MAGI3 truncates the gene, switching its functional role from a TSG to a dominant-negative oncogene. Here we report that MAGI3 undergoes pPA at the intron immediately downstream of its large internal exon, which is normally highly modified by N6-methyladenosine (m6A). In breast cancer cells that upregulate MAGI3pPA, m6A levels in the large internal exon of MAGI3 are significantly reduced compared to cells that do not express MAGI3pPA. We further find that MAGI3pPA transcripts are significantly depleted of m6A modifications, in contrast to highly m6A-modified full-length MAGI3 mRNA. Finally, we analyze public expression data and find that other TSGs, including LATS1 and BRCA1, also undergo intronic pPA following large internal exons, and that m6A levels in these exons are reduced in pPA-activated breast cancer cells relative to untransformed mammary cells. Our study suggests that m6A may play a role in regulating intronic pPA of MAGI3 and possibly other TSGs, warranting further investigation.

SMARCE1 is required for the invasive progression of in situ cancers

Significance More than half of ductal carcinoma in situ (DCIS) lesions will never progress to invasive breast cancers. However, the factors that drive invasion are not well understood. Our findings establish SMARCE1 as a clinically relevant factor that promotes the invasive progression of early-stage breast cancers. SMARCE1 drives invasion by serving as a master regulator of genes encoding proinvasive ECM and proteases required to degrade basement membrane. In functional studies in 3D cultures and animal models, SMARCE1 is dispensable for tumor growth but is required for the invasive and metastatic progression of cancers. In patients, SMARCE1 expression specifically identifies early-stage breast, lung, and ovarian cancers that are likely to eventually progress and metastasize. Advances in mammography have sparked an exponential increase in the detection of early-stage breast lesions, most commonly ductal carcinoma in situ (DCIS). More than 50% of DCIS lesions are benign and will remain indolent, never progressing to invasive cancers. However, the factors that promote DCIS invasion remain poorly understood. Here, we show that SMARCE1 is required for the invasive progression of DCIS and other early-stage tumors. We show that SMARCE1 drives invasion by regulating the expression of secreted proteases that degrade basement membrane, an ECM barrier surrounding all epithelial tissues. In functional studies, SMARCE1 promotes invasion of in situ cancers growing within primary human mammary tissues and is also required for metastasis in vivo. Mechanistically, SMARCE1 drives invasion by forming a SWI/SNF-independent complex with the transcription factor ILF3. In patients diagnosed with early-stage cancers, SMARCE1 expression is a strong predictor of eventual relapse and metastasis. Collectively, these findings establish SMARCE1 as a key driver of invasive progression in early-stage tumors.

BCL11B Drives Human Mammary Stem Cell Self-Renewal In Vitro by Inhibiting Basal Differentiation

Summary The epithelial compartment of the mammary gland contains basal and luminal cell lineages, as well as stem and progenitor cells that reside upstream in the differentiation hierarchy. Stem and progenitor cell differentiation is regulated to maintain adult tissue and mediate expansion during pregnancy and lactation. The genetic factors that regulate the transition of cells between differentiation states remain incompletely understood. Here, we present a genome-scale method to discover genes driving cell-state specification. Applying this method, we identify a transcription factor, BCL11B, which drives stem cell self-renewal in vitro, by inhibiting differentiation into the basal lineage. To validate BCL11Bs functional role, we use two-dimensional colony-forming and three-dimensional tissue differentiation assays to assess the lineage differentiation potential and functional abilities of primary human mammary cells. These findings show that BCL11B regulates mammary cell differentiation and demonstrate the utility of our proposed genome-scale strategy for identifying lineage regulators in mammalian tissues.

Abstract A28: Targeting the epigenetic state regulating cancer cell vulnerability to proteasome inhibition

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.

Abstract 2677: Unfolded protein response is required for EMT-driven metastasis by inducing CREB3L1

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.

Abstract P2-07-01: EMT activates PERK-eIF2α signaling and sensitizes cells to perturbations in endoplasmic reticulum function

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.