Sandhya Sanduja

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.

De-differentiation confers multidrug resistance via noncanonical PERK-Nrf2 signaling.

Upregulation of PERK-Nrf2 signaling is a key mechanism by which de-differentiated cancer cells gain multi-drug resistance.

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.

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.

Abstract LB-244: Amino acid deprivation selectively targets multidrug-resistant breast cancer cells

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

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.

Abstract 2130: The RNA-binding protein tristetraprolin controls intestinal cell differentiation and tumorigenesis through the Notch signaling pathway

Colorectal cancer (CRC) is the 2 nd leading cause of cancer death in U.S. With lifetime incidence rates as high as 1 in 20, it is critical to better define the molecular events controlling intestinal homeostasis and tumor initiation/progression. In normal intestinal homeostasis, the Notch pathway plays a fundamental role in controlling epithelial cell fate through juxtacrine signaling between membrane-bound ligands to the Notch receptor present on contacting cells. This complex promotes intestinal epithelial cell fate through transcription factors Hes1 and Atoh1/Math1, allowing for differentiation of absorptive enterocyte lineage by Hes1 or secretory (goblet, enteroendocrine and Paneth) cell lineage controlled by Atoh1. In CRC however, constitutive Notch activation is observed allowing for enhanced Hes1 expression associated with tumor progression and self-renewal of tumor-initiating cells. Yet, a clear mechanism for the constitutive activation of this pathway in CRC is not known. Here, we investigated the regulation of Notch signaling by the mRNA decay factor Tristetraprolin (TTP) in CRC and TTP knockout mice. TTP is a member of the TIS11 family of RNA-binding proteins, and the major factor promoting decay of mRNAs bearing AU-rich element (ARE) motifs in their 3′UTR. TTP plays an essential role in controlling pro-inflammatory and oncogenic gene expression in the GI tract, however during CRC progression, TTP expression is silenced in tumors. In this study, TTP-inducible CRC cells were used to show that expression of TTP suppresses proliferation and anchorage-independent growth, impacts cell cycle, and reduces xenograft tumor growth. Interestingly, TTP expression altered CRC cells such that cells now displayed a secretory cell-like morphology, consistent with studies showing effects of Notch signaling attenuation. Alcian blue staining of acidic mucins was significantly increased in TTP-expressing CRC cells and in xenograft tumors, suggesting a role for TTP in directing secretory cell differentiation. Furthermore, gene expression arrays and reporter gene assays showed that levels of Notch signaling genes were significantly impacted by TTP and TTP preferentially promoted a 3-fold downregulation of Notch1 receptor as compared to Notch2-4. The ability of TTP to target Notch1 mRNA for rapid decay occurred through its ARE-containing 3′UTR as observed using 3′UTR reporter and RNA-binding assays. To determine if these effects were specific to tumor cells, intestinal cell lineage was examined in TTP knockout mice with a remarkable reduction in goblet cell number observed as compared to wild-type mice. These findings demonstrate a novel role for TTP in controlling intestinal cell differentiation via post-transcriptional regulation of Notch1 and provide mechanistic insights into constitutive Notch activation observed in CRC occurring through loss of TTP expression. Citation Format: Shufei Zhuang, Liangyan Hu, Sandhya Sanduja, Shahid Umar, Shrikant Anant, Dan A. Dixon. The RNA-binding protein tristetraprolin controls intestinal cell differentiation and tumorigenesis through the Notch signaling pathway. [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 2130. doi:10.1158/1538-7445.AM2015-2130