Catherine A. Del Vecchio

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.

Targeting a Glioblastoma Cancer Stem-Cell Population Defined by EGF Receptor Variant III

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.

Breakpoint analysis of transcriptional and genomic profiles uncovers novel gene fusions spanning multiple human cancer types.

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.

Targeting EGF receptor variant III: tumor-specific peptide vaccination for malignant gliomas

Glioblastoma multiforme (GBM) is the most common and deadly of the human brain cancers. The EGF receptor is often amplified in GBM and provides a potential therapeutic target. However, targeting the normal receptor is complicated by its nearly ubiquitous and high level of expression in certain tissues. A naturally occurring deletion mutant of the EGF receptor, EGFRvIII, is a constitutively active variant originally identified in a high percentage of brain cancer cases, and more importantly is rarely found in normal tissue. A peptide vaccine, rindopepimut (CDX-110, Celldex Therapeutics), is directed against the novel exon 1–8 junction produced by the EGFRvIII deletion, and it has shown high efficacy in preclinical models. Recent Phase II clinical trials in patients with newly diagnosed GBM have shown EGFRvIII-specific immune responses and significantly increased time to progression and overall survival in those receiving vaccine therapy, as compared with published results for standard of care. Rindopepimut therefore represents a very promising therapy for patients with GBM.

Epidermal Growth Factor Receptor Variant III Contributes to Cancer Stem Cell Phenotypes in Invasive Breast Carcinoma

EGFRvIII is a tumor-specific variant of the epidermal growth factor receptor (EGFR). Although EGFRvIII is most commonly found in glioblastoma, its expression in other tumor types remains controversial. In this study, we investigated EGFRvIII expression and amplification in primary breast carcinoma. Our analyses confirmed the presence of EGFRvIII, but in the absence of amplification or rearrangement of the EGFR locus. Nested reverse transcriptase PCR and flow cytometry were used to detect a higher percentage of positive cases. EGFRvIII-positive cells showed increased expression of genes associated with self-renewal and epithelial-mesenchymal transition along with a higher percentage of stem-like cells. EGFRvIII also increased in vitro sphere formation and in vivo tumor formation. Mechanistically, EGFRvIII mediated its effects through the Wnt/β-catenin pathway, leading to increased β-catenin target gene expression. Inhibition of this pathway reversed the observed effects on cancer stem cell (CSC) phenotypes. Together, our findings show that EGFRvIII is expressed in primary breast tumors and contributes to CSC phenotypes in breast cancer cell lines through the Wnt pathway. These data suggest a novel function for EGFRvIII in breast tumorigenesis.

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.

Abstract 10: Oncogenic variant EGFRvIII defines a hierarchy in glioblastoma and expression is restricted by epigenetic mechanisms despite the presence of gene amplification

Glioblastoma multiforme (GBM) is the most common and deadly primary brain tumor. Amplification and rearrangements of the epidermal growth factor receptor (EGFR) gene are frequently found in GBM. The most common variant is EGFRvIII, an exon 2-7 deletion mutant resulting from amplification and rearrangement of the EGFR locus. We have hypothesized that EGFRvIII could mark a cancer stem cell or tumor initiating cell population. If amplification and rearrangement are early events in tumorigenesis, this implies that they should be preserved throughout the tumor. However, in primary GBM EGFRvIII expression was focal and sporadic. To understand the relationship between EGFRvIII expression and the underlying genomic alterations, we utilized manual dissection to separate EGFRvIII positive and negative cells and a quantitative PCR assay that detects independently both EGFR amplification and rearrangement. Unexpectedly, we found EGFR amplification and rearrangement throughout the tumor, including regions with no EGFRvIII expression. This supports our hypothesis and further suggests that mechanisms exist to modulate EGFRvIII expression even in the presence of high gene amplification. To study this phenomenon, we characterized three GBM cell lines with endogenous EGFRvIII expression and corresponding EGFR amplification and rearrangement, confirmed by Western blot, RT-PCR and FISH. By flow cytometry analysis EGFRvIII expression was heterogeneous, with 9-50% EGFRvIII positive cells in each cell line. Both positive and negative populations maintained the genetic alterations, recapitulating what we observed in primary GBM. Importantly, EGFRvIII defined a hierarchy where EGFRvIII-positive cells gave rise to additional positive and negative cells. However, only cells that had recently lost EGFRvIII expression could re-express EGFRvIII. Epigenetic mechanisms played a role in modulating EGFRvIII expression to maintain a heterogeneous population. Demethylation induced a 20-60% increase in the percentage of EGFRvIII-positive cells, indicating that some cells could re-express EGFRvIII. Surprisingly, inhibition of histone deacetylation resulted in a 50-80% reduction in EGFRvIII expression. Collectively, this data demonstrates that EGFRvIII does follow a stem cell model for hierarchical expression and sheds light on the existence of a transient population that is able to re-express EGFRvIII, an important buffer for maintaining EGFRvIII-positive cell numbers. Furthermore, we provide the first evidence that EGFRvIII expression can be silenced by epigenetic mechanisms, suggesting that drugs which modulate the epigenome might be used successfully in glioblastoma tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 10. doi:1538-7445.AM2012-10

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 3733: Defining and targeting a Glioblastoma cancer stem cell population with EGF Receptor Variant III.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The relationship between mutated proteins and the relatively rare cancer stem cell population is unclear since driver mutations are present throughout a tumor. Glioblastoma (GBM) tumors frequently express EGFRvIII, an EGFR variant that arises via gene rearrangement and amplification. The expression of EGFRvIII is restricted despite the prevalence of the alteration. Here we show that EGFRvIII is expressed in a population of human GBM cancer stem cells. EGFRvIII positive cancer stem cells demonstrate greater self-renewal and tumor initiation than the more abundant EGFRvIII negative cells. EGFRvIII positive cells are associated with stem/progenitor markers while markers of differentiation are found in EGFRvIII negative cells. EGFRvIII is highly co-expressed with CD133, and the EGFRvIII positive/CD133 positive sub-population defines the highest self-renewal and tumor initiating population. Surprisingly, wildtype EGFR was infrequently co-expressed with EGFRvIII but present in the majority of cells, both in primary GBM and cultured GBM neurospheres. Elimination of the EGFRvIII positive or the EGFRvIII/CD133 dual positive population with an engineered anti-EGFRvIII/CD133 bi-specific antibody reduced tumorigenicity of implanted tumor cells. This work demonstrates that a mutated oncogene can have CSC specific expression and be used to specifically target this population. Citation Format: David R. Emlet, Catherine Del Vecchio, Puja Gupta, Siddhartha Mitra, Shuang-Yin Han, Marina Holgado-Madruga, Gordon Li, Kristen Jensen, Hannes Vogel, Stephen Skirboll, Albert J. Wong. Defining and targeting a Glioblastoma cancer stem cell population with EGF Receptor Variant III. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3733. doi:10.1158/1538-7445.AM2013-3733