MicroRNA Expression in a Readily Accessible Common Hepatic Artery Lymph Node Predicts Time to Pancreatic Cancer Recurrence Postresection.

Lymph node involvement in pancreatic adenocarcinoma (PAC) predicts postresection survival, but early lymph node metastasis detection is not easily accomplished. We assessed a panel of microRNAs (miRNAs) in a common hepatic artery lymph node (station 8) that is readily accessible during pancreatoduodenectomy (PD) to determine if increased miRNA levels correlate with postresection recurrence. Station 8 lymph nodes overlying the common hepatic artery collected during PD were assayed for miRNA-10b, miRNA-30c, miRNA-21, and miRNA-155 and cytokeratin-19 (CK19), an epithelial cell marker, using quantitative PCR. Expression was correlated with disease recurrence, recurrence-free survival (RFS), and overall survival (OS). Station 8 lymph nodes from 37 patients (30 periampullary carcinomas (PCs), 2 chronic pancreatitis, 5 other cancers) exhibited increased miRNA-10b levels in 14/30 PCs, and in 10 of these 14 patients, cancer recurred during the study period (2012-2015). High miRNA-10b was also associated with shorter RFS (42.5 vs. 92.4 weeks, p < 0.05) but not OS, whereas miRNA-30c, miRNA-21, and miRNA-155 levels and CK19 mRNA levels in station 8 nodes were variable and did not correlate with RFS or OS. We conclude that elevated miRNA-10b levels in station 8 lymph nodes could be utilized to assess risk for early disease progression in patients with periampullary tumors.

Combined targeting of TGF-ß, EGFR and HER2 suppresses lymphangiogenesis and metastasis in a pancreatic cancer model.

Pancreatic ductal adenocarcinomas (PDACs) are aggressive with frequent lymphatic spread. By analysis of data from The Cancer Genome Atlas, we determined that ~35% of PDACs have a pro-angiogenic gene signature. We now show that the same PDACs exhibit increased expression of lymphangiogenic genes and lymphatic endothelial cell (LEC) markers, and that LEC abundance in human PDACs correlates with endothelial cell microvessel density. Lymphangiogenic genes and LECs are also elevated in murine PDACs arising in the KRC (mutated Kras; deleted RB) and KIC (mutated Kras; deleted INK4a) genetic models. Moreover, pancreatic cancer cells (PCCs) derived from KRC tumors express and secrete high levels of lymphangiogenic factors, including the EGF receptor ligand, amphiregulin. Importantly, TGF-ß1 increases lymphangiogenic genes and amphiregulin expression in KRC PCCs but not in murine PCCs that lack SMAD4, and combinatorial targeting of the TGF-ß type I receptor (TßRI) with LY2157299 and EGFR/HER2 with lapatinib suppresses tumor growth and metastasis in a syngeneic orthotopic model, and attenuates tumor lymphangiogenesis and angiogenesis while reducing lymphangiogenic genes and amphiregulin and enhancing apoptosis. Therefore, this combination could be beneficial in PDACs with lymphangiogenic or angiogenic gene signatures.

Overview of pre-clinical and clinical studies targeting angiogenesis in pancreatic ductal adenocarcinoma.

The importance of angiogenesis in pancreatic ductal adenocarcinoma (PDAC) and its therapeutic potential have been explored in both pre-clinical and clinical studies. Human PDACs overexpress a number of angiogenic factors and their cognate high-affinity receptors, and anti-angiogenic agents reduce tumor volume, metastasis, and microvessel density (MVD), and improve survival in subcutaneous and orthotopic pre-clinical models. Nonetheless, clinical trials using anti-angiogenic therapy have been overwhelmingly unsuccessful. This review will focus on these pre-clinical and clinical studies, the potential reasons for failure in the clinical setting, and ways these shortcomings could be addressed in future investigations of angiogenic mechanisms in PDAC.

Angiogenic gene signature in human pancreatic cancer correlates with TGF-beta and inflammatory transcriptomes.

Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, but overexpress pro-angiogenic factors and exhibit regions of microvasculature. Using RNA-seq data from The Cancer Genome Atlas (TCGA), we previously reported that ~12% of PDACs have an angiogenesis gene signature with increased expression of multiple pro-angiogenic genes. By analyzing the recently expanded TCGA dataset, we now report that this signature is present in ~35% of PDACs but that it is mostly distinct from an angiogenesis signature present in pancreatic neuroendocrine tumors (PNETs). These PDACs exhibit a transcriptome that reflects active TGF-ß signaling, and up-regulation of several pro-inflammatory genes, and many members of JAK signaling pathways. Moreover, expression of SMAD4 and HDAC9 correlates with endothelial cell abundance in PDAC tissues. Concomitantly targeting the TGF-ß type I receptor (TßRI) kinase with SB505124 and JAK1-2 with ruxolitinib suppresses JAK1 phosphorylation and blocks proliferative cross-talk between human pancreatic cancer cells (PCCs) and human endothelial cells (ECs), and these anti-proliferative effects were mimicked by JAK1 silencing in ECs. By contrast, either inhibitor alone does not suppress their enhanced proliferation in 3D co-cultures. These findings suggest that targeting both TGF-ß and JAK1 signaling could be explored therapeutically in the 35% of PDAC patients whose cancers exhibit an angiogenesis gene signature.

Pathophysiological role of microRNA-29 in pancreatic cancer stroma.

Dense fibrotic stroma associated with pancreatic ductal adenocarcinoma (PDAC) is a major obstacle for drug delivery to the tumor bed and plays a crucial role in pancreatic cancer progression. Current, anti-stromal therapies have failed to improve tumor response to chemotherapy and patient survival. Furthermore, recent studies show that stroma impedes tumor progression, and its complete ablation accelerates PDAC progression. In an effort to understand the molecular mechanisms associated with tumor-stromal interactions, using in vitro and in vivo models and PDAC patient biopsies, we show that the loss of miR-29 is a common phenomenon of activated pancreatic stellate cells (PSCs)/fibroblasts, the major stromal cells responsible for fibrotic stromal reaction. Loss of miR-29 is correlated with a significant increase in extracellular matrix (ECM) deposition, a major component in PDAC stroma. Our in vitro miR-29 gain/loss-of-function studies document the role of miR-29 in PSC-mediated ECM stromal protein accumulation. Overexpression of miR-29 in activated stellate cells reduced stromal deposition, cancer cell viability, and cancer growth in co-culture. Furthermore, the loss of miR-29 in TGF-ß1 activated PSCs is SMAD3 dependent. These results provide insights into the mechanistic role of miR-29 in PDAC stroma and its potential use as a therapeutic agent to target PDAC.

TCGA data and patient-derived orthotopic xenografts highlight pancreatic cancer-associated angiogenesis.

Pancreatic ductal adenocarcinomas (PDACs) overexpress pro-angiogenic factors but are not viewed as vascular. Using data from The Cancer Genome Atlas we demonstrate that a subset of PDACs exhibits a strong pro-angiogenic signature that includes 37 genes, such as HDAC9, that are overexpressed in PDAC arising in KRC mice, which express mutated Kras and lack RB. Moreover, patient-derived orthotopic xenografts can exhibit tumor angiogenesis, whereas conditioned media (CM) from KRC-derived pancreatic cancer cells (PCCs) enhance endothelial cell (EC) growth and migration, and activate canonical TGF-ß signaling and STAT3. Inhibition of the type I TGF-ß receptor with SB505124 does not alter endothelial activation in vitro, but decreases pro-angiogenic gene expression and suppresses angiogenesis in vivo. Conversely, STAT3 silencing or JAK1-2 inhibition with ruxolitinib blocks CM-enhanced EC proliferation. STAT3 disruption also suppresses endothelial HDAC9 and blocks CM-induced HDAC9 expression, whereas HDAC9 re-expression restores CM-enhanced endothelial proliferation. Moreover, ruxolitinib blocks mitogenic EC/PCC cross-talk, and suppresses endothelial p-STAT3 and HDAC9, and PDAC progression and angiogenesis in vivo, while markedly prolonging survival of KRC mice. Thus, targeting JAK1-2 with ruxolitinib blocks a final pathway that is common to multiple pro-angiogenic factors, suppresses EC-mediated PCC proliferation, and may be useful in PDACs with a strong pro-angiogenic signature.

Pancreatic cancer stroma: friend or foe?

Pancreatic cancer desmoplasia is thought to confer biological aggressiveness. In this issue of Cancer Cell, Özdemir and colleagues and Rhim and colleagues demonstrate that targeting the stroma results in undifferentiated, aggressive pancreatic cancer that responds to checkpoint blockade or antiangiogenic therapy, uncovering a protective role by stroma in this cancer.

Selective inhibition of pancreatic ductal adenocarcinoma cell growth by the mitotic MPS1 kinase inhibitor NMS-P715.

Most solid tumors, including pancreatic ductal adenocarcinoma (PDAC), exhibit structural and numerical chromosome instability (CIN). Although often implicated as a driver of tumor progression and drug resistance, CIN also reduces cell fitness and poses a vulnerability that can be exploited therapeutically. The spindle assembly checkpoint (SAC) ensures correct chromosome-microtubule attachment, thereby minimizing chromosome segregation errors. Many tumors exhibit upregulation of SAC components such as MPS1, which may help contain CIN within survivable limits. Prior studies showed that MPS1 inhibition with the small molecule NMS-P715 limits tumor growth in xenograft models. In cancer cell lines, NMS-P715 causes cell death associated with impaired SAC function and increased chromosome missegregation. Although normal cells appeared more resistant, effects on stem cells, which are the dose-limiting toxicity of most chemotherapeutics, were not examined. Elevated expression of 70 genes (CIN70), including MPS1, provides a surrogate measure of CIN and predicts poor patient survival in multiple tumor types. Our new findings show that the degree of CIN70 upregulation varies considerably among PDAC tumors, with higher CIN70 gene expression predictive of poor outcome. We identified a 25 gene subset (PDAC CIN25) whose overexpression was most strongly correlated with poor survival and included MPS1. In vitro, growth of human and murine PDAC cells is inhibited by NMS-P715 treatment, whereas adipose-derived human mesenchymal stem cells are relatively resistant and maintain chromosome stability upon exposure to NMS-P715. These studies suggest that NMS-P715 could have a favorable therapeutic index and warrant further investigation of MPS1 inhibition as a new PDAC treatment strategy.

Molecular imaging in pancreatic cancer--a roadmap for therapeutic decisions.

Pancreatic ductaladeno carcinoma (PDAC) is a deadly cancer characterized by multiple molecular alterations, the presence of an intense stroma, poor perfusion, and resistance to therapy. In addition to standard imaging techniques, experimental imaging strategies, such as those utilizing molecular probes, nanoparticle-based agents, and tagged antibodies are actively being explored experimentally. It is hoped that advances in these technologies will allow for detecting PDAC at an early stage, and could serve to validate experimental therapies, rapidly identify non-responders, and assist in the design of novel therapeutic strategies tailored to the patient's molecular profile.