Yuxiong Feng

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.

Identification of a Selective Small Molecule Inhibitor of Breast Cancer Stem Cells

A high-throughput screen (HTS) with the National Institute of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) compound collection identified a class of acyl hydrazones to be selectively lethal to breast cancer stem cell (CSC) enriched populations. Medicinal chemistry efforts were undertaken to optimize potency and selectivity of this class of compounds. The optimized compound was declared as a probe (ML239) with the NIH Molecular Libraries Program and displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control line (HMLE_sh_GFP).

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.

Cinnamides as selective small-molecule inhibitors of a cellular model of breast cancer stem cells.

A high-throughput screen (HTS) was conducted against stably propagated cancer stem cell (CSC)-enriched populations using a library of 300,718 compounds from the National Institutes of Health (NIH) Molecular Libraries Small Molecule Repository (MLSMR). A cinnamide analog displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control cell line (HMLE_sh_eGFP). Herein, we report structure-activity relationships of this class of cinnamides for selective lethality towards CSC-enriched populations.

The endoplasmic reticulum may be an Achilles' heel of cancer cells that have undergone an epithelial-to-mesenchymal transition

In a recent report published in Cancer Discovery we identified a novel vulnerability of cancer cells that have undergone an epithelial–mesenchymal transition (EMT) and established that the PERK branch of the unfolded protein response is constitutively activated upon EMT. In this commentary, we summarize and provide context for our findings.

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 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 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.