Receptor-interacting protein (RIP) plays a critical role in tumor necrosis factor-␣ (TNF-␣)-induced IB kinase (IKK) activation and subsequent activation of transcription factor NF-B. However, the molecular mechanism by which RIP mediates TNF-␣-induced NF-B activation is not completely defined. In this study, we have found that TAK1 is recruited to the TNF-␣ receptor complex in a RIP-dependent manner following the stimulation of TNF-␣ receptor 1 (TNF-R1). Moreover, a forced recruitment of TAK1 to TNF-R1 in the absence of RIP is sufficient to mediate TNF-␣-induced NF-B activation, indicating that the major function of RIP is to recruit its downstream kinases to the TNF-R1 complex. Interestingly, we also find that TAK1 and MEKK3 form a functional complex, in which TAK1 regulates autophosphorylation of MEKK3. The TAK1-mediated regulation of MEKK3 phosphorylation is dependent on the kinase activity of TAK1. Although TAK1-MEKK3 interaction is not affected by overexpressed TAB1, TAB1 is required for TAK1 activation and subsequent MEKK3 phosphorylation. Together, we conclude that TAK1 is recruited to the TNF-R1 complex via RIP and likely cooperates with MEKK3 to activate NF-B in TNF-␣ signaling.The mitogen-activated protein kinases (MAPKs) 2 are a family of serine/threonine kinases that have been shown to function in a wide variety of biological processes (1). Transforming growth factor-␤-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase kinase 3 (MEKK3) are known to act as a MAP3K in the c-Jun N-terminal kinase and the p38 MAPK cascades (2, 3). In addition, it has been shown that both kinases are involved in the nuclear factor B (NF-B) pathway (4 -8). However, the molecular mechanism of this function is still unclear, mainly due to the poor characterization of MAP3K activation under the physiological conditions. NF-B is a family of transcription factors involved in inflammation and innate immunity (9). In unstimulated cells NF-B is sequestered in the cytoplasm through an interaction with a family of inhibitory proteins, IB. In response to extracellular stimuli, the IB proteins are phosphorylated by the IB kinase (IKK) complex, then ubiquitinated and rapidly degraded, which leads to the nuclear localization and activation of NF-B (10, 11).One of the most potent NF-B activators is tumor necrosis factor-␣ (TNF-␣), a major proinflammatory cytokine. TNF-␣ functions through two distinct surface receptors, a 55-kDa receptor 1 (TNF-R1) and a 75-kDa receptor 2 (TNF-R2). TNF-R1 plays the predominant role in induction of cellular responses by soluble TNF-␣ (12). The binding of TNF-␣ to TNF-R1 leads to the recruitment of TNF-R1-associated death domain (TRADD), and TRADD further recruits TNF-receptor-associated factor 2 (TRAF2) (13) and Receptor-interacting protein (RIP) (14,15). RIP interacts directly with TRADD via its death domain (14). It has been demonstrated that TRAF2 plays an essential role in IKK recruitment to the TNF-R1 complex (16), but IKK activation requires the presence of RIP in the same comple...

Recent advances demonstrate a critical yet poorly understood role for the pancreatic stellate cell (PSC) in the pathogenesis of chronic pancreatitis (CP) and pancreatic cancer (PC). Progress in this area has been hampered by the availability, fidelity, and/or reliability of in vitro models of PSCs. We examined whether outgrowth cultures from human surgical specimens exhibited reproducible phenotypic and functional characteristics of PSCs. PSCs were cultured from surgical specimens of healthy pancreas, CP and PC. Growth dynamics, phenotypic characteristics, soluble mediator secretion profiles and co-culture with PC cells both in vitro and in vivo were assessed. Forty-seven primary cultures were established from 52 attempts, demonstrating universal α-smooth muscle actin and glial fibrillary acidic protein but negligible epithelial surface antigen expression. Modification of culture conditions consistently led to cytoplasmic lipid accumulation, suggesting induction of a quiescent phenotype. Secretion of growth factors, chemokines and cytokines did not significantly differ between donor pathologies, but did evolve over time in culture. Co-culture of PSCs with established PC cell lines resulted in significant changes in levels of multiple secreted mediators. Primary PSCs co-inoculated with PC cells in a xenograft model led to augmented tumor growth and metastasis. Therefore, regardless of donor pathology, outgrowth cultures produce PSCs that demonstrate consistent growth and protein secretion properties. Primary cultures from pancreatic surgical specimens, including malignancies, may represent a reliable source of human PSCs. Evidence continues to accumulate implicating the intense desmoplastic reaction common to both chronic pancreatitis (CP) and pancreatic cancer (PC) is not simply a bystander process from a dynamic inflammatory state, but rather represents a complex interplay within the microenvironment responsible for the initiation and maintenance of key pathologic feedback systems. 1 Pancreatic stellate cells (PSCs) appear to represent a critical determinant in the development and maintenance of this desmoplastic microenvironment in pancreatic disease states. 2-4 However, recent investigations have questioned the role of PSCs in cancer. Specifically, depletion of activated PSCs in genetically engineered mouse models of PC has paradoxically demonstrated pro-tumor effects. 5,6 While disparate from prior observations showing the tumor promoting abilities of activated PSCs, 3,7-9 these investigations still implicate an interplay between PSCs, other cells within the tumor microenvironment, and the cancer. 5,6 Given these disparate findings, further investigation into the biology of PSCs in various disease states is essential and necessitates a high degree of experimental standardization. To this effect, an expert panel recently published guidelines regarding PSC isolation, identification, and activation. 10 Human PSCs are most typically isolated via the outgrowth method, whereby direct culture of minced pancreatic s...

The pancreatic adenocarcinoma (PDAC) microenvironment is largely comprised of fibrotic tumor associated stroma (TAS) that contributes to the lethal biology of PDAC. microRNA (miRNA) are small non-coding RNAs that regulate gene expression. We hypothesized that interactions between PDAC cells and TAS cells within the microenvironment modulate miRNA expression and thus, tumor biology. We observed that miR-205 and members of the miR-200 family (miR-200a, -200b, -200c, -141 and miR-429) were exclusively expressed in PDAC cells, consistent with an epithelial miRNA signature, while miR-145 and miR-199 family members (miR-199a and -199b) were solely expressed in TAS cells, consistent with a stromal miRNA signature. This finding was confirmed by qRT-PCR of RNA obtained by laser-capture microdissection of surgical specimens. Using an in vitro co-culture model, we further demonstrated regulation of miRNA expression by cell-cell contact. Forced expression in TAS cells of miR-200b/-200c and miR-205 to mimic these observed changes in miRNA concentrations induced secretion of GM-CSF and IP10, and notably inhibited migration. These data suggest interactions within the tumor microenvironment alter miRNA expression, which in turn have a functional impact on TAS.

No abstract

We previously reported that within the pancreatic ductal adenocarcinoma (PDAC) microenvironment, miR-145 and miR-199a are exclusively expressed in tumor-associated stroma (TAS) cells, but these miRNAs are present in PDAC cells following co-culture with TAS cells. We hypothesized that miRNAs function as paracrine signals via exosomal exchange between TAS cells and adjacent PDAC cells. Primary cultures of human TAS and PDAC cells were employed. Membrane-bound microparticles were isolated from TAS conditioned, serum-free culture media by sequential ultracentrifugation followed by ultrafiltration. Exosomes and microvesicles were then assayed for particle size distribution using nanoparticle tracking analysis and electronic microscopy. miRNA expression levels were determined using quantitative PCR. miRNA transfection was performed with RNAiMax reagents. Cell viability was measured by Alamar Blue. Statistics were performed using Prism 6 software. Following transfection of human TAS cells with cel-miR-39, a nonhuman miRNA, we demonstrated that miRNA exchanges occurred between TAS cells and neighboring PDAC cells via a process that is not dependent upon cell-cell contact. We next confirmed the presence and enrichment of miR-145-5p in TAS-cell-derived exosomes (8-fold higher concentrations in exosomes than parental cells, p<0.05). Feeding of TAS-derived exosomes or transfection of miR-145-5p mimics into PDAC cells led to dose-dependent decreases in PDAC cell viability (p<0.05). Taken together, our data suggest that stroma derived exosomes deliver miRNAs to adjacent PDAC cells and may function as tumor-suppressing paracrine signals in the case of miR-145. This finding provides a potential explanation for the observation that stroma depletion paradoxically accelerates PDAC progression in murine models. Citation Format: Song Han, Sayali Belsare, DongYu Zhang, Mark Beveridge, Carlos Rinaldi, Jose G. Trevino, Thomas D. Schmittgen, Steven J. Hughes. Exosomal delivery of stroma-derived miR-145 inhibits pancreatic cancer cell proliferation [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 4322. doi:10.1158/1538-7445.AM2017-4322