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

The relationship between mutated proteins and the cancer stem-cell population is unclear. Glioblastoma tumors frequently express EGFRvIII, an EGF receptor (EGFR) variant that arises via gene rearrangement and amplification. However, expression of EGFRvIII is restricted despite the prevalence of the alteration. Here, we show that EGFRvIII is highly coexpressed with CD133 and that EGFRvIII⁺/CD133⁺ defines the population of cancer stem cells (CSC) with the highest degree of self-renewal and tumor-initiating ability. EGFRvIII⁺ cells are associated with other stem/progenitor markers, whereas markers of differentiation are found in EGFRvIII⁻ cells. EGFRvIII expression is lost in standard cell culture, but its expression is maintained in tumor sphere culture, and cultured cells also retain the EGFRvIII⁺/CD133⁺ coexpression, self-renewal, and tumor initiating abilities. Elimination of the EGFRvIII⁺/CD133⁺ population using a bispecific antibody reduced tumorigenicity of implanted tumor cells better than any reagent directed against a single epitope. This work demonstrates that a mutated oncogene can have CSC-specific expression and be used to specifically target this population. Cancer Res; 74(4); 1238–49. ©2013 AACR.

Purpose: Despite generally favorable outcomes, 15% to 25% of patients with human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC) will have recurrence. Current posttreatment surveillance practices rely on physical examinations and imaging and are inconsistently applied. We assessed circulating tumor tissue modified viral (TTMV)-HPV DNA obtained during routine posttreatment surveillance among a large population of real-world patients. Experimental Design: This retrospective clinical case series included 1,076 consecutive patients across 108 U.S. sites who were ≥ 3 months posttreatment for HPV-driven OPSCC and who had one or more TTMV-HPV DNA tests (NavDx, Naveris Laboratories) obtained during surveillance between February 6, 2020, and June 29, 2021. Test results were compared with subsequent clinical evaluations. Results: Circulating TTMV-HPV DNA was positive in 80 of 1,076 (7.4%) patients, with follow-up available on all. At first positive surveillance testing, 21 of 80 (26%) patients had known recurrence while 59 of 80 (74%) patients were not known to have recurrent disease. Among these 59 patients, 55 (93%) subsequently had a confirmed recurrence, 2 patients had clinically suspicious lesions, and 2 had clinically “no evidence of disease” (NED) at last follow-up. To date, the overall positive predictive value of TTMV-HPV DNA testing for recurrent disease is 95% (N = 76/80). In addition, the point-in-time negative predictive value is 95% (N = 1,198/1,256). Conclusions: These findings highlight the clinical potential for circulating TTMV-HPV DNA testing in routine practice. As a surveillance tool, TTMV-HPV DNA positivity was the first indication of recurrence in the majority of cases, pre-dating identification by routine clinical and imaging exams. These data may inform future clinical and guideline-endorsed strategies for HPV-driven malignancy surveillance. See related commentary by Colevas, p. 4171

Malignant carcinomas that recur following therapy are typically de-differentiated and multidrug resistant (MDR). De-differentiated cancer cells acquire MDR by up-regulating reactive oxygen species (ROS)–scavenging enzymes and drug efflux pumps, but how these genes are up-regulated in response to de-differentiation is not known. Here, we examine this question by using global transcriptional profiling to identify ROS-induced genes that are already up-regulated in de-differentiated cells, even in the absence of oxidative damage. Using this approach, we found that the Nrf2 transcription factor, which is the master regulator of cellular responses to oxidative stress, is preactivated in de-differentiated cells. In de-differentiated cells, Nrf2 is not activated by oxidation but rather through a noncanonical mechanism involving its phosphorylation by the ER membrane kinase PERK. In contrast, differentiated cells require oxidative damage to activate Nrf2. Constitutive PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy by reducing ROS levels and increasing drug efflux. These findings are validated in therapy-resistant basal breast cancer cell lines and animal models, where inhibition of the PERK-Nrf2 signaling axis reversed the MDR of de-differentiated cancer cells. Additionally, analysis of patient tumor datasets showed that a PERK pathway signature correlates strongly with chemotherapy resistance, tumor grade, and overall survival. Collectively, these results indicate that de-differentiated cells up-regulate MDR genes via PERK-Nrf2 signaling and suggest that targeting this pathway could sensitize drug-resistant cells to chemotherapy. The development of multidrug resistance is the primary obstacle to treating cancers. High-grade tumors that are less differentiated typically respond poorly to therapy and carry a much worse prognosis than well-differentiated low-grade tumors. Therapy-resistant cancer cells often overexpress antioxidants or efflux proteins that pump drugs out of the cell, but how the differentiation state of cancer cells influences these resistance mechanisms is not well understood. Here we used genome-scale approaches and found that the PERK kinase and its downstream target, Nrf2—a master transcriptional regulator of the cellular antioxidant response—are key mediators of therapy resistance in poorly differentiated breast cancer cells. We show that Nrf2 is activated when cancer cells de-differentiate and that this activation requires PERK. We further show that blocking PERK-Nrf2 signaling with a small-molecule inhibitor sensitizes drug-resistant cancer cells to chemotherapy. Our results identify a novel role for PERK-Nrf2 signaling in multidrug resistance and suggest that targeting this pathway could improve the responsiveness of otherwise resistant tumors to chemotherapy.

Gene fusions, like BCR/ABL1 in chronic myelogenous leukemia, have long been recognized in hematologic and mesenchymal malignancies. The recent finding of gene fusions in prostate and lung cancers has motivated the search for pathogenic gene fusions in other malignancies. Here, we developed a “breakpoint analysis” pipeline to discover candidate gene fusions by tell-tale transcript level or genomic DNA copy number transitions occurring within genes. Mining data from 974 diverse cancer samples, we identified 198 candidate fusions involving annotated cancer genes. From these, we validated and further characterized novel gene fusions involving ROS1 tyrosine kinase in angiosarcoma (CEP85L/ROS1), SLC1A2 glutamate transporter in colon cancer (APIP/SLC1A2), RAF1 kinase in pancreatic cancer (ATG7/RAF1) and anaplastic astrocytoma (BCL6/RAF1), EWSR1 in melanoma (EWSR1/CREM), CDK6 kinase in T-cell acute lymphoblastic leukemia (FAM133B/CDK6), and CLTC in breast cancer (CLTC/VMP1). Notably, while these fusions involved known cancer genes, all occurred with novel fusion partners and in previously unreported cancer types. Moreover, several constituted druggable targets (including kinases), with therapeutic implications for their respective malignancies. Lastly, breakpoint analysis identified new cell line models for known rearrangements, including EGFRvIII and FIP1L1/PDGFRA. Taken together, we provide a robust approach for gene fusion discovery, and our results highlight a more widespread role of fusion genes in cancer pathogenesis. Gene fusions represent an important class of cancer genes, created by rearrangements of the genome that bring together two different genes. Because they are unique to cancer cells, gene fusions are ideal diagnostic markers and therapeutic targets. While gene fusions were once thought restricted mainly to blood cancers, recent discoveries suggest they are more widespread. Here, we have developed an approach for mining DNA microarray data to detect the tell-tale signatures of gene fusions, as “breakpoints” occurring within the encoding DNA or expressed transcripts. We apply this approach to a large collection of nearly 1,000 human cancer specimens. From this analysis, we discover and verify twelve new gene fusions occurring in diverse cancer types. We verify that some of these rearrangements recur in other samples of the same cancer type (supporting a causal role) and that the cancers show dependency on the fusion for cancer cell growth. Notably, some of these fusions (e.g. CEP85L/ROS1 in angiosarcoma) represent the first for that cancer type and thus provide important new biological insight. Some are also good drug targets (including rearrangements of ROS1, RAF1, and CDK6 kinases), with clear implications for therapy.