Sonal Gupta

Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism

Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions. DOI: http://dx.doi.org/10.7554/eLife.10250.001

Evolution of cellular morpho-phenotypes in cancer metastasis

Intratumoral heterogeneity greatly complicates the study of molecular mechanisms driving cancer progression and our ability to predict patient outcomes. Here we have developed an automated high-throughput cell-imaging platform (htCIP) that allows us to extract high-content information about individual cells, including cell morphology, molecular content and local cell density at single-cell resolution. We further develop a comprehensive visually-aided morpho-phenotyping recognition (VAMPIRE) tool to analyze irregular cellular and nuclear shapes in both 2D and 3D microenvironments. VAMPIRE analysis of ~39,000 cells from 13 previously sequenced patient-derived pancreatic cancer samples indicate that metastasized cells present significantly lower heterogeneity than primary tumor cells. We found the same morphological signature for metastasis for a cohort of 10 breast cancer cell lines. We further decipher the relative contributions to heterogeneity from cell cycle, cell-cell contact, cell stochasticity and heritable morphological variations.

Altered hydroxymethylation is seen at regulatory regions in pancreatic cancer and regulates oncogenic pathways

Transcriptional deregulation of oncogenic pathways is a hallmark of cancer and can be due to epigenetic alterations. 5-Hydroxymethylcytosine (5-hmC) is an epigenetic modification that has not been studied in pancreatic cancer. Genome-wide analysis of 5-hmC-enriched loci with hmC-seal was conducted in a cohort of low-passage pancreatic cancer cell lines, primary patient-derived xenografts, and pancreatic controls and revealed strikingly altered patterns in neoplastic tissues. Differentially hydroxymethylated regions preferentially affected known regulatory regions of the genome, specifically overlapping with known H3K4me1 enhancers. Furthermore, base pair resolution analysis of cytosine methylation and hydroxymethylation with oxidative bisulfite sequencing was conducted and correlated with chromatin accessibility by ATAC-seq and gene expression by RNA-seq in pancreatic cancer and control samples. 5-hmC was specifically enriched at open regions of chromatin, and gain of 5-hmC was correlated with up-regulation of the cognate transcripts, including many oncogenic pathways implicated in pancreatic neoplasia, such as MYC, KRAS, VEGFA, and BRD4 Specifically, BRD4 was overexpressed and acquired 5-hmC at enhancer regions in the majority of neoplastic samples. Functionally, acquisition of 5-hmC at BRD4 promoter was associated with increase in transcript expression in reporter assays and primary samples. Furthermore, blockade of BRD4 inhibited pancreatic cancer growth in vivo. In summary, redistribution of 5-hmC and preferential enrichment at oncogenic enhancers is a novel regulatory mechanism in human pancreatic cancer.

YAP1 Oncogene is a Context-specific Driver for Pancreatic Ductal Adenocarcinoma

Transcriptomic profiling classifies pancreatic ductal adenocarcinoma (PDAC) into several molecular subtypes with distinctive histological and clinical characteristics. However, little is known about the molecular mechanisms that define each subtype and their correlation with clinical outcome. Mutant KRAS is the most prominent driver in PDAC, present in over 90% of tumors, but the dependence of tumors on oncogenic KRAS signaling varies between subtypes. In particular, squamous subtype are relatively independent of oncogenic KRAS signaling and typically display much more aggressive clinical behavior versus progenitor subtype. Here, we identified that YAP1 activation is enriched in the squamous subtype and associated with poor prognosis. Activation of YAP1 in progenitor subtype cancer cells profoundly enhanced malignant phenotypes and transformed progenitor subtype cells into squamous subtype. Conversely, depletion of YAP1 specifically suppressed tumorigenicity of squamous subtype PDAC cells. Mechanistically, we uncovered a significant positive correlation between WNT5A expression and the YAP1 activity in human PDAC, and demonstrated that WNT5A overexpression led to YAP1 activation and recapitulated YAP1-dependent but Kras-independent phenotype of tumor progression and maintenance. Thus, our study identifies YAP1 oncogene as a major driver of squamous subtype PDAC and uncovers the role of WNT5A in driving PDAC malignancy through activation of the YAP pathway.

Mitochondrial Fusion Suppresses Pancreatic Cancer Growth via Reduced Oxidative Metabolism

Pancreatic cancer is a highly lethal disease whose aggressive biology that is driven by mitochondrial oxidative metabolism. Mitochondria normally form a network of fused organelles, but we find that patient-derived and genetically engineered murine pancreatic cancer cells exhibit highly fragmented mitochondria with robust oxygen consumption rates (OCR). When mitochondrial fusion was activated by the genetic or pharmacological inhibition Drp1, the morphology and metabolism of human and murine pancreatic cancer cells more closely resembled that of normal pancreatic epithelial cells. This reduced metabolism was correlated with slower tumor growth, fewer metastases, and enhanced survival in a syngeneic orthotopic model. Similarly, directly activating mitochondrial fusion by overexpression of Mfn2 also reduced tumor growth and metastases. Mitochondrial fusion in pancreatic cancer cells was associated with reduced mitochondrial mass and Complex I expression and function. Thus, these data suggest that enhancing mitochondrial fusion through Drp1 inhibition or enhanced Mfn2 expression or function has strong tumor suppressive activity against pancreatic cancer and may thus represent a highly novel and efficacious therapeutic target.

A pipeline for rapidly generating genetically engineered mouse models of pancreatic cancer using in vivo CRISPR-Cas9 mediated somatic recombination

Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single “workhorse” mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic Kras G12D allele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic KrasG12D allele at the endogenous locus, and insertion deletion (“indel”) and frameshift mutations of targeted tumor suppressor alleles. By using a single “workhorse” mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we have created a facile autochthonous platform for interrogation of the PDAC genome.

GnasR201C Induces Murine Pancreatic Cystic Neoplasms through Suppression of YAP1 Signaling and Transcriptional Reprogramming

Background & Aims Somatic “hotspot” mutations of GNAS, which encodes for the alpha subunit of stimulatory G-protein, are present in ~60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. There are currently no cognate animal models that recapitulate the biology of mutant Gnas-induced IPMNs, and the underlying mechanisms that lead to the cystic pathway of neoplasia in the pancreas remain unknown. Methods We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras; Gnas mice) where pancreas-specific GnasR201C expression was induced by doxycycline administration. In this model, mutant Kras is constitutively expressed, and control mice were produced through absence of doxycycline. Separate cohorts of mice were utilized for timed necropsies and for Kaplan-Meier survival analysis. Isogenic cell lines (with doxycycline inducible mutant Gnas expression) were propagated from the resulting pancreatic ductal adenocarcinoma (PDAC). Results Co-expression of KrasG12D and GnasR201C resulted in the development of pancreatic cystic lesions resembling human IPMNs in 100% of mice, with higher grades of epithelial dysplasia observed over time. Approximately one-third of Kras; Gnas mice developed PDAC at a median of 38 weeks post doxycycline induction. GnasR201C did not accelerate oncogenic transformation with KrasG12D, but rather, reprogrammed Ras-induced neoplasms towards a well-differentiated phenotype. GnasR201C induction led to activation of the inhibitory Hippo kinase cascade and cytoplasmic sequestration of phosphorylated YAP1 protein, a phenomenon that was also observed in human IPMN with GNAS mutations. Conclusions GNASR201C functions not as a traditional oncogene, but rather as an “oncomodulator” of KRAS-mediated pancreatic neoplasia, through suppression of YAP1 and transcriptional reprogramming towards a differentiated (large ductal) phenotype.

Immune Cell Production of Interleukin 17 Induces Stem Cell Features of Pancreatic Intraepithelial Neoplasia Cells

BACKGROUND & AIMS Little is known about how the immune system affects stem cell features of pancreatic cancer cells. Immune cells that produce interleukin 17A (IL17A) in the chronically inflamed pancreas (chronic pancreatitis) contribute to pancreatic interepithelial neoplasia (PanIN) initiation and progression. We investigated the effects that IL17A signaling exerts on pancreatic cancer progenitor cells and the clinical relevance of this phenomena. METHODS We performed studies with Mist1Cre;LSLKras;Rosa26mTmG (KCiMist;G) and Kras(G12D);Trp53(R172H);Pdx1-Cre (KPC) mice (which upon tamoxifen induction spontaneously develop PanINs) and control littermates. Some mice were injected with neutralizing antibodies against IL17A or control antibody. Pancreata were collected, PanIN epithelial cells were isolated by flow cytometry based on lineage tracing, and gene expression profiles were compared. We collected cells from pancreatic tumors of KPC mice, incubated them with IL17 or control media, measured expression of genes regulated by IL17 signaling, injected the cancer cells into immune competent mice, and measured tumor growth. IL17A was overexpressed in pancreata of KCiMist mice from an adenoviral vector. Pancreata were collected from all mice and analyzed by histology and immunohistochemistry. Levels of DCLK1 and other proteins were knocked down in KPC pancreatic cancer cells using small interfering or short hairpin RNAs; cells were analyzed by immunoblotting. We obtained 65 pancreatic tumor specimens from patients, analyzed protein levels by immunohistochemistry, and compared results with patient survival times. We also analyzed gene expression levels and patient outcome using The Cancer Genome Atlas database. RESULTS PanIN cells from KCiMist;G mice had a gene expression pattern associated with embryonic stem cells. Mice given injections of IL17-neutralizing antibodies, or with immune cells that did not secrete IL17, lost this expression pattern and had significantly decreased expression of DCLK1 and POU2F3, which regulate tuft cell development. KCiMist mice that overexpressed IL17 formed more PanINs, with more DCLK1-positive cells, than control mice. Pancreatic tumor cells from KPC mice and human Capan-2 cells exposed to IL17A had increased activation of NF-κB and mitogen-activated protein kinase signaling and increased expression of DCLK1 and ALDH1A1 (a marker of embryonic stem cells) compared with cells in control media. These cells also formed tumors faster that cells not exposed to IL17 when they were injected into immunocompetent mice. KPC cells with knockdown of DCLK1 expressed lower levels of ALDH1A1 after incubation with IL17 than cells without knockdown. Expression of the IL17 receptor C was higher in DCLK1-positive PanIN cells from mice compared with DCLK1-negative PanIN cells. In human pancreatic tumor tissues, high levels of DCLK1 associated with a shorter median survival time of patients (17.7 months, compared with 26.6 months of patients whose tumors had low levels of DCLK1). Tumor levels of POU2F3 and LAMC2 were also associated with patient survival time. CONCLUSIONS In studies of mouse and human pancreatic tumors and precursors, we found that immune cell-derived IL17 regulated development of tuft cells and stem cell features of pancreatic cancer cells via increased expression of DCLK1, POU2F3, ALDH1A1, and IL17RC. Strategies to disrupt this pathway might be developed to prevent pancreatic tumor growth and progression.

Abstract B13: Synthetic vulnerabilities in MLL3 deficient pancreatic tumors

Myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3) is a histone 3-lysine 4 methyltransferase, frequently mutated in a variety of solid tumors including pancreatic ductal adenocarcinoma (PDAC), a nearly lethal disease due to our current inability to therapeutically target major oncogenic proteins driving this cancer. Like other tumor suppressor genes, direct targeting of MLL3 is not feasible, and thus our objective was to find biological pathways which are synthetic lethal targets in MLL3-deficient PDAC tumors. Identification of drug targets in such a specific genetic context could potentially dictate stratification of patients based on the mutational profile of their tumor. To achieve this, we generated a novel genetically engineered mouse model (GEMM) of pancreatic cancer, in which targeted mutation of Kras (G12D) and loss of functional MLL3 was driven by Cre-recombination from early pancreatic lineage-specific transcription factor, Pdx1. Our unpublished data suggests that loss of MLL3 function in exocrine pancreas cooperates with mutant Kras to accelerate the progression of invasive pancreatic neoplasia. Using cell lines generated from spontaneous tumors arising in KC (KrasG12D alone) and KMC (KrasG12D; MLL3δ/δ) mice, we performed functional genomic analyses and found several key oncogenic pathways upregulated in MLL3-deficient cells. We selected two well-known oncogenic pathways and through a series of cellular and biochemical assays, further validated their selectively activation in KMC cells, in contrast with KC cells. Next, we picked multiple pharmacological inhibitors of these pathways, which are already approved at various phases of clinical trials, and tested for their efficacy on growth of KC and KMC cells in both two- and three-dimensional growth assays. In agreement with our data showing preferential activation in KMC cells, we found KMC cells to be significantly more sensitive than KC cells to the effect of these inhibitors. Thus, identification of synthetic lethal hits in context of MLL3 functional loss could potentially enable us to design strategies to limit metastasis in patients with surgically-resectable tumors. Citation Format: Sonal Gupta, Sammy Ferri-Borgogno, Mary R. Reisenauer, Anvita k. Gupta, Anirban Maitra. Synthetic vulnerabilities in MLL3 deficient pancreatic tumors [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B13.

Abstract 1392: Development of a novel mouse model for intrahepatic cholangiocarcinoma

Cholangiocarcinoma (CCA) is a poorly understood cancer of the biliary epithelium and the second most common type of liver cancer. Surgery currently offers the only potential for cure; however, most patients present with advanced disease and are therefore unresectable. The Cancer Genome Atlas (TCGA) analysis and other recent genomic studies have revealed discrete epigenetic perturbations amongst CCAs originating from different anatomic sites. Intrahepatic cholangiocarcinoma (ICC) arises from the intrahepatic bile ducts, and a subset of ICCs is characterized by loss-of-function mutations in the gene encoding for the chromatin regulatory factor BRCA associated protein 1 (BAP1). Loss of this protein may be associated with global epigenomic and transcriptomic alterations that ultimately contribute to tumor progression and metastasis dissemination. While elucidating the molecular pathogenesis of ICC may identify potential targeted therapies and improve early detection, inquiry into this disease has been hampered by a lack of genetically faithful animal models. We developed a genetically engineered mouse model (GEMM) of ICC that incorporates an inactivating mutation in BAP1 combined with Kras activation. An Albumin-Cre promoter was used to induce hepatoblast-specific mutations. Mutant Kras cooperates with loss of BAP1 and results in lethal hepatic transformation and dose-dependent survival. Kras activation alone results in extended disease latency and survival > 50 weeks. Loss of BAP1 alone or heterozygous loss of BAP1 combined with mutant Kras shortens disease latency, with mice surviving 39 weeks on average. A significant reduction in survival is seen with homozygous loss of BAP1 and Kras activation (Kras BAP1L/L). These mice survive on average 23 weeks (p ≤ 0.0045). Histopathologic evaluation of Kras BAP1L/L mice demonstrates focal biliary precursor lesions, frank ICC, and hepatocellular carcinoma (HCC). Mice with heterozygous deletion of BAP1 and Kras activation, loss of BAP1 alone, or Kras activation alone develop HCC only. Given the bipotential nature of hepatoblasts, the ICC phenotype of our GEMM may be enhanced by inducing biliary tree-specific mutations. Adenoviral Cre enzyme (Ad-Cre) is used to achieve such combinatorial specificity, and a novel surgery was developed whereby retrograde biliary tree administration of this enzyme is performed. Surgery utilizing GFP-tagged adeno-associated virus confirms administration targeted to the biliary tree. Retrograde biliary tree injection of Ad-Cre into ROSAmT/mG mice demonstrates Cre recombinase expression within cholangiocytes, thereby establishing proof-of-principle. Ad-Cre injection in Kras BAP1L/L mice to induce cholangiocyte-specific BAP1 deletion and Kras activation is ongoing. Citation Format: Rebecca Marcus, Wai Chin Foo, Anirban Maitra, Sonal Gupta. Development of a novel mouse model for intrahepatic cholangiocarcinoma [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 1392. doi:10.1158/1538-7445.AM2017-1392