Brian P. James

EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.

Hypoxia Triggers Hedgehog-Mediated Tumor–Stromal Interactions in Pancreatic Cancer

Pancreatic cancer is characterized by a desmoplastic reaction that creates a dense fibroinflammatory microenvironment, promoting hypoxia and limiting cancer drug delivery due to decreased blood perfusion. Here, we describe a novel tumor-stroma interaction that may help explain the prevalence of desmoplasia in this cancer. Specifically, we found that activation of hypoxia-inducible factor-1α (HIF-1α) by tumor hypoxia strongly activates secretion of the sonic hedgehog (SHH) ligand by cancer cells, which in turn causes stromal fibroblasts to increase fibrous tissue deposition. In support of this finding, elevated levels of HIF-1α and SHH in pancreatic tumors were determined to be markers of decreased patient survival. Repeated cycles of hypoxia and desmoplasia amplified each other in a feed forward loop that made tumors more aggressive and resistant to therapy. This loop could be blocked by HIF-1α inhibition, which was sufficient to block SHH production and hedgehog signaling. Taken together, our findings suggest that increased HIF-1α produced by hypoxic tumors triggers the desmoplasic reaction in pancreatic cancer, which is then amplified by a feed forward loop involving cycles of decreased blood flow and increased hypoxia. Our findings strengthen the rationale for testing HIF inhibitors and may therefore represent a novel therapeutic option for pancreatic cancer.

ETS2 Mediated Tumor Suppressive Function and MET Oncogene Inhibition in Human Non–Small Cell Lung Cancer

Purpose: The ETS2 transcription factor is an evolutionarily conserved gene that is deregulated in cancer. We analyzed the transcriptome of lung adenocarcinomas and normal lung tissue by expression profiling and found that ETS2 was significantly downregulated in adenocarcinomas. In this study, we probed the yet unknown functional role of ETS2 in lung cancer pathogenesis. Experimental Design: Lung adenocarcinomas (n = 80) and normal lung tissues (n = 30) were profiled using the Affymetrix Human Gene 1.0 ST platform. Immunohistochemical (IHC) analysis was conducted to determine ETS2 protein expression in non–small cell lung cancer (NSCLC) histologic tissue specimens (n = 201). Patient clinical outcome, based on ETS2 IHC expression, was statistically assessed using the log-rank and Kaplan–Meier tests. RNA interference and overexpression strategies were used to assess the effects of ETS2 expression on the transcriptome and on various malignant phenotypes. Results: ETS2 expression was significantly reduced in lung adenocarcinomas compared with normal lung (P < 0.001). Low ETS2 IHC expression was a significant predictor of shorter time to recurrence in NSCLC (P = 0.009, HR = 1.89) and adenocarcinoma (P = 0.03, HR = 1.86). Moreover, ETS2 was found to significantly inhibit lung cancer cell growth, migration, and invasion (P < 0.05), and microarray and pathways analysis revealed significant (P < 0.001) activation of the HGF pathway following ETS2 knockdown. In addition, ETS2 was found to suppress MET phosphorylation and knockdown of MET expression significantly attenuated (P < 0.05) cell invasion mediated by ETS2-specific siRNA. Furthermore, knockdown of ETS2 augmented HGF-induced MET phosphorylation, cell migration, and invasion. Conclusion(s): Our findings point to a tumor suppressor role for ETS2 in human NSCLC pathogenesis through inhibition of the MET proto-oncogene. Clin Cancer Res; 19(13); 3383–95. ©2013 AACR.

Identification of thioredoxin-interacting protein 1 as a hypoxia-inducible factor 1α-induced gene in pancreatic cancer

Objective: To investigate the expression of thioredoxin-interacting protein (TXNIP) during hypoxia and its dependency on hypoxia-inducible factor 1&agr; (HIF-1&agr;) in pancreatic cancer cell lines. Methods: MiaPaCa-2 pancreatic cancer cells were transiently transfected with siRNA to HIF-1&agr; and TXNIP protein measured after growth in normoxia or hypoxia. In addition, HIF-1&agr; dependency was assessed by transiently transfecting MiaPaCa-2 pancreatic cancer cells with HIF-1&agr; with a mutated oxygen degradation domain resulting in stable HIF-1&agr; expression in normoxic conditions. Panc-1 pancreatic cancer cells with low endogenous TXNIP expression were stably transfected with TXNIP, and cell survival and response to platinum cancer agents were tested. Quantitative immunohistochemistry was utilized to measure the expression of TXNIP and thioredoxin 1 in human pancreatic cancer tissues. Results: Thioredoxin-interacting protein was induced during hypoxia in pancreatic cancer cells in a HIF-1&agr;-dependent manner. Overexpression of TXNIP in the Panc-1 cells resulted in a higher basal apoptosis and increased sensitivity to cisplatin and oxaliplatin. A negative correlation was observed between TXNIP and thioredoxin 1 expression in human pancreatic cancer tissues. Conclusions: Thioredoxin-interacting protein, a putative tumor suppressor gene, is induced in response to hypoxia in a HIF-1&agr;-dependent manner in pancreatic cancer cells, resulting in increased apoptosis and increased sensitivity to platinum anticancer therapy. Increased TXNIP may be a mechanism to counterbalance the prosurvival effects of HIF-1&agr;.

Superoxide dismutase is regulated by LAMMER kinase in Drosophila and human cells

LAMMER kinases (also known as CDC-2-like or CLKs) are a family of dual specificity serine/threonine protein kinases that are found in all sequenced eukaryotic genomes. In the fission yeast, Schizosaccharomyces pombe, the LAMMER kinase gene, Lkh1, positively regulates the expression of the antioxidant defense genes, superoxide dismutase 1 (sod1+, CuZn-SOD) and catalase (ctt1+, CAT). We have shown that mutations in the Drosophila LAMMER kinase gene, Darkener of apricot (Doa), protect against the decrease in life span caused by the reactive oxygen species (ROS) generator paraquat, and at the same time show an increase in cytoplasmic (CuZn-Sod or SOD1) and mitochondrial superoxide dismutase (Mn-Sod or SOD2) protein levels and activity. The siRNA-mediated knock down of the human LAMMER kinase gene, CLK-1, in HeLa and MCF-7 human cell lines leads to an increase in both SOD1 activity and mRNA transcript levels. These data suggest that SOD1 is negatively regulated by LAMMER kinases in Drosophila and human cell lines and that this regulation may be conserved during evolution.

Expression Profiling Stratifies Mesothelioma Tumors and Signifies Deregulation of Spindle Checkpoint Pathway and Microtubule Network with Therapeutic Implications

BACKGROUND Malignant pleural mesothelioma (MPM) is a lethal neoplasm exhibiting resistance to most treatment regimens and requires effective therapeutic options. Though an effective strategy in many cancer, targeted therapy is relatively unexplored in MPM because the therapeutically important oncogenic pathways and networks in MPM are largely unknown. MATERIALS AND METHODS We carried out gene expression microarray profiling of 53 surgically resected MPMs tumors along with paired normal tissue. We also carried out whole transcriptomic sequence (RNA-seq) analysis on eight tumor specimens. Taqman-based quantitative Reverse-transcription polymerase chain reaction (qRT-PCR), western analysis and immunohistochemistry (IHC) analysis of mitotic arrest deficient-like 1 (MAD2L1) was carried out on tissue specimens. Cell viability assays of MPM cell lines were carried out to assess sensitivity to specific small molecule inhibitors. RESULTS Bioinformatics analysis of the microarray data followed by pathway analysis revealed that the mitotic spindle assembly checkpoint (MSAC) pathway was most significantly altered in MPM tumors with upregulation of 18 component genes, including MAD2L1 gene. We validated the microarray data for MAD2L1 expression using quantitative qRT-PCR and western blot analysis on tissue lysates. Additionally, we analyzed expression of the MAD2L1 protein by IHC using an independent tissue microarray set of 80 MPM tissue samples. Robust clustering of gene expression data revealed three novel subgroups of tumors, with unique expression profiles, and showed differential expression of MSAC pathway genes. Network analysis of the microarray data showed the cytoskeleton/spindle microtubules network was the second-most significantly affected network. We also demonstrate that a nontaxane small molecule inhibitor, epothilone B, targeting the microtubules have great efficacy in decreasing viability of 14 MPM cell lines. CONCLUSIONS Overall, our findings show that MPM tumors have significant deregulation of the MSAC pathway and the microtubule network, it can be classified into three novel molecular subgroups of potential therapeutic importance and epothilone B is a promising therapeutic agent for MPM.

Hedgehog signaling and desmoplasia are regulated by hypoxia in pancreatic cancer

Pancreatic cancer is one of the most aggressive solid tumors, with less than 5% of patients surviving 5 years. Chemotherapy treatments have modest improvement in survival rates and surgical resection in most cases is not an option. New strategies for therapy are needed to improve overall survival in this deadly disease. Pancreatic tumors are notoriously hypoxic and characterized by a dense fibroinflammatory stromal reaction (desmoplasia). We hypothesized that signals emanating from tumor cells exposed to hypoxia enable pancreatic tumors to thrive. To explore this we conducted a transcriptome microarray study comparing genes transcribed in normoxia and hypoxia (1% O2 for 24 hrs). We have found that hypoxia significantly increases the levels of the hedgehog (Hh) pathway ligand, sonic hedgehog (sHh), and its receptor, patched (PTCH), in two pancreatic cancer cell lines, MiaPaca 2 and Panc-1. In addition, siRNA to HIF-1α showed that this increase is HIF-1α dependent. Western blotting confirmed a hypoxia/HIF-1α-dependent increase in sHh protein levels in both MiaPaca-2 and Panc-1 cells. To further explore the hypoxia-induced increase in expression of hedgehog pathway components we used a specific pathway array profiling the expression of 84 key genes involved in the hedgehog signaling. REN, (retinoic acid, EGF and NGF induced gene), was the only other hedgehog pathway gene to increase in hypoxia. Panc-1 cells transfected with a GLI-luciferase reporter of hedgehog activation, showed no increase in GLI activity in either normoxia, hypoxia or upon addition of conditioned media containing sHh, suggesting there is not an autocrine stimulation of the cancer cells themselves by the secreted sHh. This may be explained by the increase in PTCH and REN, both inhibitors of the hedgehog pathway. sHh is a known key factor promoting stromal desmoplasia in pancreatic cancer and therefore we tested whether sHh produced by cancer cells can activate fibroblasts. Indeed, NIH-3T3 fibroblasts stably transfected with GLI-luciferase (NIH-3T3/GLI-luc) showed an increase in GLI activity when co-cultured with MiaPaca-2 cells in hypoxia, but not normoxia, or when treated with conditioned media from Panc-1 or MiaPaca-2 cells grown in hypoxia. A 3-D system simulating conditions of tumor growth and demonstrating a hypoxic core within the cancer cells mass showed GLI transactivation in NIH-3T3/GLI-luc cells only when cells were co-cultured with pancreatic cancer cells (Panc-1, MiaPaca-2, Capan-2 or Su-86), but not when cells were cultured alone. Using siRNA this GLI activation was shown to be dependent on both HIF-1α and sHh. Finally, an immunohistochemical study in pancreatic cancer tumor samples showed a significant positive correlation between tumor and stromal HIF-1α staining and stromal sHh. Thus, we propose a new mechanism of the tumor9s response to hypoxia in which pancreatic tumor cells secrete sHh that acts on stroma to increase fibrosis which in turn leads to decreased blood flow, and thus a continuous cycle of more hypoxia and Hh signaling. [Supported by CA109552-05]

Abstract 2967: Pancreatic cancer cell growth requires lipids released by tumor-induced stroma autophagy

Pancreatic ductal adenocarcinoma (PDAC) is non-resectable in the majority of patients and highly resistant to chemotherapy, resulting in a poor survival. The tumor microenvironment and hypoxia are important modifiers of cancer progression in PDAC. Understanding the metabolic vulnerabilities of PDAC in the harsh tumor microenvironment may lead to novel therapeutic approaches with improved clinical efficacy. First, we found that PDAC cells showed beneficial effects of co-cultured stroma cells, but only under lipid-free serum conditions. To study the metabolic crosstalk between cancer cells and stroma in more detail, we performed an untargeted metabolomic screen of PDAC cells and fibroblasts co-cultured in normoxia and hypoxia, and performed RNA-seq profiling in parallel. We found that stromal cells are metabolically more responsive to co-culture than cancer cells. PDAC cells induce catabolic carbohydrate and protein metabolism in stromal cells, particularly in hypoxia. In contrast, 13C-based metabolic flux assays demonstrated that stromal cells display enhanced anabolic lipid metabolism in co-culture with PDAC cells. Furthermore, de novo synthesized 13C-labeled fatty acids in stromal cells were taken up by PDAC cells. In particular, PDAC cells showed extensive scavenging of lysophospholipids (lyso-PLs) from the culture medium, which was increased in co-culture under hypoxic conditions. These data were confirmed by analyzing portal vein plasma samples isolated from pancreatic cancer patients before and after surgery. In addition, we found metabolites and expression levels of metabolic enzymes from the glycerophospholipid pathway to be enriched in PDAC cells in co-culture and hypoxia. By using fibroblasts, human pancreatic stellate cells and patient-derived cancer-associated fibroblasts (CAFs), we demonstrate direct transfer of lyso-PLs from stromal to PDAC cells via lipid droplets. The transfer of lyso-PLs was abrogated by pharmacological inhibitors of autophagy, or by siRNA-mediated knockdown of autophagy genes in stromal and tumor cells. These data suggest that PDAC cells cause stroma cells to undergo autophagy, and reprogram stroma metabolism to obtain complex lipid species for their metabolic needs in the lipid-starved tumor microenvironment. Citation Format: Petrus R. De Jong, Sean-Luc Shanahan, Morgan A. Brand, Alejandro D. Campos, Anagha Srirangam, Nikolas Marino, Claudia P. Miller, Olga Zagnitko, Adam D. Richardson, David A. Scott, Brian P. James, Andrew P. Hodges, Ally Perlina, Alexey M. Eroshin, Randall French, Malene Hansen, Sally A. Litherland, Andrew M. Lowy, J. Pablo Arnoletti, Garth Powis. Pancreatic cancer cell growth requires lipids released by tumor-induced stroma autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2967. doi:10.1158/1538-7445.AM2017-2967

Abstract 3233: Modeling evolution of resistance in patient-derived xenografts: resistance to the BRAF inhibitor PLX4720.

A major hurdle for targeted cancer therapies is the development of resistance, with many agents showing promising initial results but not providing significant overall survival benefits. To address resistance we need to know the genetic and epigenetic alterations that drive resistance. This knowledge will contribute to modified treatment protocols, new targets, and predictive biomarkers. We are working on a system to model evolution of tumor resistance in a the patient-derived xenograft (PDX) mouse system. Compared to xenografts, PDXs are closer to the tumor biology of the patient, having a higher degree of molecular subtypes and intratumor heterogeneity, and a mixture of human and mouse stroma. In this study, we implanted 1-3mm chunks from a primary colorectal cancer tumor carrying the BRAF V600>E mutation into immunosuppressed NOD/SCID CB.17 mice. Once the tumors were established (100-150mmˆ3 tumor burden), the mice were stratified into two groups, control and fed chow containing the BRAF inhibitor PLX4720, 417 mg/kg (Scientific Diets). The tumors in most of the PDXs in the group fed PLX4720 chow regressed and became very small by day 77. In several cases PDXs that were allowed to develop beyond day 77 became resistant to PLX4720 treatment. To identify mechanisms of PLX4720 resistance we sequenced DNA and RNA from two resistant PDXs, one untreated control PDX, and a sample of the original patient tumor. We used the Ion AmpliSeq Comprehensive Cancer Panel from Life Technologies to sequence exons from 409 cancer related genes on the Ion Torrent PGM. Additionally, we performed SOLiD 5500 paired-end (50x25 nt) next generation sequencing (NGS) of the whole transcriptome of these samples. Since the stromal component of the PDXs is a mixture of cells of human and mouse origin, and sequence reads of mouse origin could contribute to false positive mutation calls or skew expression analysis of the resistant data, we needed to develop a method for filtering mouse reads out of our sequencing data. To this end, we have developed and applied a read filtering protocol that sorts reads that align to the human genome into three groups, better in human, better in mouse, or ambiguous. 97.7% of the reads from human tissue were defined as better in human with the remaining 2.3% being ambiguous, or better in mouse. PDX derived reads had between 8% and 13% of reads being better in mouse. Only reads defined as better in human were used for expression analysis and SNP calling in this work. In one of the two resistant tumors analyzed this method has identified a candidate resistance mutation, NRAS G13>D. The NRAS mutation is seen in both the RNA-seq and DNA-seq data, and is not seen in the original patient tumor, the control untreated PDX, or in the second resistant tumor. Based on these findings, we feel that PDX provide a powerful model system for examining the evolution of resistance to targeted agents. Citation Format: Brian P. James, Robert J. Lemos, Feng Tian, Thomas C. Motter, Amin Momin, Nastaran D. Neishaboori, Galina M. Kiriakova, Scott Kopetz, Garth Powis. Modeling evolution of resistance in patient-derived xenografts: resistance to the BRAF inhibitor PLX4720. [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 3233. doi:10.1158/1538-7445.AM2013-3233

Abstract 2177: Tumor suppressor effects of ETS2 transcriptional factor in human non-small cell lung cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Advances in prevention and treatment of non-small cell lung cancer (NSCLC) are dependent in part on the characterization of tumor suppressor genes and oncogenes and the roles they elicit in the pathogenesis of this malignancy. Although v-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) is a canonical transcription factor that regulates various cancer-associated cellular and developmental processes including proliferation and migration, its function in lung carcinogenesis is still unknown. In this study we sought to examine the role of ETS2 in NSCLC pathogenesis. We first examined ETS2 mRNA expression in lung adenocarcinomas (n=80) and normal lung (n=30) which we profiled using microarrays, and in seven matched adenocarcinoma and normal lung pairs analyzed using next-generation sequencing technology. Both array and sequencing datasets revealed that ETS2 mRNA was significantly lower in lung adenocarcinomas relative to normal lung (p<0.001) which was confirmed by quantitative PCR analysis. Moreover and in the microaray dataset, ETS2 mRNA expression was significantly anti-correlated with that of the proliferation marker KI67 (R=−0.59, p<0.001). In addition, in silico analysis of publicly available datasets demonstrated that ETS2 mRNA was lower in NSCLC compared to normal lung (all p<0.001), and interestingly, was also lower in airways of healthy smokers relative to non-smokers (p<0.001). We next assessed ETS2 immunohistochemical protein expression using tissue microarrays comprised of 342 formalin-fixed paraffin-embedded NSCLC (adenocarcinoma, n=226; SCC, n=116) tissue specimens. There were no statistically significant differences in ETS2 expression by histology, stage or age. We then assessed the association of ETS2 protein expression with clinical outcome. Non-treated all stage (n=206) or stage-I (n=157) patients with relatively lower ETS2 protein expression exhibited significantly shortened disease-free survival compared to patients with higher expression (p=0.008 and p=0.004 of the log-rank test, respectively). In addition, patients with relatively lower ETS2 expression exhibited significantly poorer response to adjuvant therapy compared to patients with higher ETS2 expression (p=0.01). We then probed the effect of modulating ETS2 expression in NSCLC cells. Knockdown of ETS2 expression by RNA interference significantly increased anchorage-dependent colony formation (p=0.004) as well as augmented cellular migration (p=0.01) and invasion through matrigel (p=0.02) compared to cells transfected with control siRNA. Our findings provide evidence that ETS2 may function as a tumor suppressor gene in NSCLC that can aid clinically in identification of aggressive tumors and biologically in increasing our understanding of the pathogenesis of this malignancy (Supported by DoD PROSPECT W81XWH-07-1-0306 and Lung Cancer Research Foundation grants). 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 2177. doi:1538-7445.AM2012-2177