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Pancreatic cancer is characterized by abundant desmoplasia, a dense stroma composed of extra-cellular and cellular components, with cancer associated fibroblasts (CAFs) being the major cellular component. However, the tissue(s) of origin for CAFs remains controversial. Here we determine the tissue origin of pancreatic CAFs through comprehensive lineage tracing studies in mice. We find that the splanchnic mesenchyme, the fetal cell layer surrounding the endoderm from which the pancreatic epithelium originates, gives rise to the majority of resident fibroblasts in the normal pancreas. In a genetic mouse model of pancreatic cancer, resident fibroblasts expand and constitute the bulk of CAFs. Single cell RNA profiling identifies gene expression signatures that are shared among the fetal splanchnic mesenchyme, adult fibroblasts and CAFs, suggesting a persistent transcriptional program underlies splanchnic lineage differentiation. Together, this study defines the phylogeny of the mesenchymal component of the pancreas and provides insights into pancreatic morphogenesis and tumorigenesis. Cancer-associated fibroblasts (CAFs) are the main component of the stroma in pancreatic cancer, but their tissue of origin remains to be defined. Here the authors perform lineage tracing and single cell RNA sequencing in mice and suggest the splanchnic mesenchyme as the tissue of origin for CAFs.

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function. Arginine methylation has been reported to regulate AKT-associated signalling. Here, the authors show that PRMT5-mediated arginine methylation promotes AKT activity and induces tumourigenesis.

Angiogenesis is a complex process orchestrated by both growth factors and cell adhesion and is initiated by focal degradation of the vascular basement membrane with subsequent migration and proliferation of endothelial cells. The Ras/Raf/MEK/ERK pathway is required for EC function during angiogenesis. Although in vitro studies implicate ERK1 and ERK2 in endothelial cell survival, their precise role in angiogenesis in vivo remains poorly defined. Cre/loxP technology was used to inactivate Erk1 and Erk2 in endothelial cells during murine development, resulting in embryonic lethality due to severely reduced angiogenesis. Deletion of Erk1 and Erk2 in primary endothelial cells resulted in decreased cell proliferation and migration, but not in increased apoptosis. Expression of key cell cycle regulators was diminished in the double knockout cells, and decreased DNA synthesis could be observed in endothelial cells during embryogenesis. Interestingly, both Paxillin and Focal Adhesion Kinase were expressed at lower levels in endothelial cells lacking Erk1 and Erk2 both in vivo and in vitro, leading to defects in the organization of the cytoskeleton and in cell motility. The regulation of Paxillin and Focal Adhesion Kinase expression occurred post-transcriptionally. These results demonstrate that ERK1 and ERK2 coordinate endothelial cell proliferation and migration during angiogenesis.

Engineered oncolytic viruses are used clinically to destroy cancer cells and have the ability to boost anticancer immunity. Phosphatase and tensin homolog deleted on chromosome 10 loss is common across a broad range of malignancies, and is implicated in immune escape. The N-terminally extended isoform, phosphatase and tensin homolog deleted on chromosome 10 alpha (PTENα), regulates cellular functions including protein kinase B signaling and mitochondrial adenosine triphosphate production. Here we constructed HSV-P10, a replicating, PTENα expressing oncolytic herpesvirus, and demonstrate that it inhibits PI3K/AKT signaling, increases cellular adenosine triphosphate secretion, and reduces programmed death-ligand 1 expression in infected tumor cells, thus priming an adaptive immune response and overcoming tumor immune escape. A single dose of HSV-P10 resulted in long term survivors in mice bearing intracranial tumors, priming anticancer T-cell immunity leading to tumor rejection. This implicates HSV-P10 as an oncolytic and immune stimulating therapeutic for anticancer therapy. Oncolytic viruses are a promising therapeutic approach for cancer treatment. The authors demonstrate the efficacy of an engineered HSV-1 expressing PTENα as an oncolytic and immune stimulating therapy against brain cancer metastases.

Background Pancreatic ductal adenocarcinoma (PDAC) presents with a high mortality rate. Two important features of PDAC contribute to this poor outcome. The first is metastasis which occurs in ~ 80% of PDAC patients. The second is cachexia, which compromises treatment tolerance for patients and reduces their quality of life. Although various mouse models of PDAC exist, recapitulating both metastatic and cachectic features have been challenging. Methods Here, we optimize an orthotopic mouse model of PDAC by altering several conditions, including the subcloning of parental murine PDAC cells, implantation site, number of transplanted cells, and age of recipient mice. We perform spatial profiling to compare primary and metastatic immune microenvironments and RNA sequencing to gain insight into the mechanisms of muscle wasting in PDAC-induced cachexia, comparing non-metastatic to metastatic conditions. Results These modifications extend the time course of the disease and concurrently increase the rate of metastasis to approximately 70%. Furthermore, reliable cachexia endpoints are achieved in both PDAC mice with and without metastases, which is reminiscent of patients. We also find that cachectic muscles from PDAC mice with metastasis exhibit a similar transcriptional profile to muscles derived from mice and patients without metastasis. Conclusion Together, this model is likely to be advantageous in both advancing our understanding of the mechanism of PDAC cachexia, as well as in the evaluation of novel therapeutics.

The development of cachexia in the setting of cancer or other chronic diseases is a significant detriment for patients. Cachexia is associated with a decreased ability to tolerate therapies, reduction in ambulation, reduced quality of life, and increased mortality. Cachexia appears intricately linked to the activation of the acute phase response and is a drain on metabolic resources. Work has begun to focus on the important inflammatory factors associated with the acute phase response and their role in the immune activation of cachexia. Furthermore, data supporting the liver, lung, skeletal muscle, and tumor as all playing a role in activation of the acute phase are emerging. Although the acute phase is increasingly being recognized as being involved in cachexia, work in understanding underlying mechanisms of cachexia associated with the acute phase response remains an active area of investigation and still lack a holistic understanding and a clear causal link. Studies to date are largely correlative in nature, nonetheless suggesting the possibility for a role for various acute phase reactants. Herein, we examine the current literature regarding the acute phase response proteins, the evidence these proteins play in the promotion and exacerbation of cachexia, and current evidence of a therapeutic potential for patients.

Osteoporosis results from an imbalance in skeletal remodeling that favors bone resorption over bone formation. Bone matrix is degraded by osteoclasts, which differentiate from myeloid precursors in response to the cytokine RANKL. To gain insight into the transcriptional regulation of bone resorption during growth and disease, we generated a conditional knockout of the transcription factor nuclear factor of activated T cells c1 (Nfatc1). Deletion of Nfatc1 in young mice resulted in osteopetrosis and inhibition of osteoclastogenesis in vivo and in vitro. Transcriptional profiling revealed NFATc1 as a master regulator of the osteoclast transcriptome, promoting the expression of numerous genes needed for bone resorption. In addition, NFATc1 directly repressed osteoclast progenitor expression of osteoprotegerin, a decoy receptor for RANKL previously thought to be an osteoblast-derived inhibitor of bone resorption. \"Cherubism mice\", which carry a gain-of-function mutation in SH3-domain binding protein 2 (Sh3bp2), develop osteoporosis and widespread inflammation dependent on the proinflammatory cytokine, TNF-alpha. Interestingly, deletion of Nfatc1 protected cherubism mice from systemic bone loss but did not inhibit inflammation. Taken together, our study demonstrates that NFATc1 is required for remodeling of the growing and adult skeleton and suggests that NFATc1 may be an effective therapeutic target for osteoporosis associated with inflammatory states.

Classical NF-kappaB signaling functions as a negative regulator of skeletal myogenesis through potentially multiple mechanisms. The inhibitory actions of TNFalpha on skeletal muscle differentiation are mediated in part through sustained NF-kappaB activity. In dystrophic muscles, NF-kappaB activity is compartmentalized to myofibers to inhibit regeneration by limiting the number of myogenic progenitor cells. This regulation coincides with elevated levels of muscle derived TNFalpha that is also under IKKbeta and NF-kappaB control. Based on these findings we speculated that in DMD, TNFalpha secreted from myotubes inhibits regeneration by directly acting on satellite cells. Analysis of several satellite cell regulators revealed that TNFalpha is capable of inhibiting Notch-1 in satellite cells and C2C12 myoblasts, which was also found to be dependent on NF-kappaB. Notch-1 inhibition occurred at the mRNA level suggesting a transcriptional repression mechanism. Unlike its classical mode of action, TNFalpha stimulated the recruitment of Ezh2 and Dnmt-3b to coordinate histone and DNA methylation, respectively. Dnmt-3b recruitment was dependent on Ezh2. We propose that in dystrophic muscles, elevated levels of TNFalpha and NF-kappaB inhibit the regenerative potential of satellite cells via epigenetic silencing of the Notch-1 gene.

Chronic pancreatitis (CP) is a fibro-inflammatory disease leading to pain, maldigestion, and pancreatic insufficiency. No therapeutic options exist due to a limited understanding of the biology of CP pathology. Recent findings implicate pancreatic stellate cells (PSC) as prominent mediators of inflammatory and fibrotic processes during CP. Here, we utilized primary and immortalized PSC obtained from mice and patients with CP or pancreatic cancer to examine the effect of Jak/STAT and MAPK pathway inhibition in vitro . The well-characterized caerulein model of CP was used to assess the therapeutic efficacy of Jak1/2 inhibition in vivo . Treatment of cultured PSC with the Jak1/2 inhibitor ruxolitinib reduced STAT3 phosphorylation, cell proliferation, and expression of alpha-smooth muscle actin (α-SMA), a marker of PSC activation. Treatment with the MAPK inhibitor, MEK162, had less consistent effects on PSC proliferation and no impact on activation. In the caerulein-induced murine model of CP, administration of ruxolitinib for one week significantly reduced biomarkers of inflammation and fibrosis. These data suggest that the Jak/STAT pathway plays a prominent role in PSC proliferation and activation. In vivo treatment with the Jak1/2 inhibitor ruxolitinib reduced the severity of experimental CP, suggesting that targeting Jak/STAT signaling may represent a promising therapeutic strategy for CP.

Collagen deposition contributes to both high mammographic density and breast cancer progression. Low stromal PTEN expression has been observed in as many as half of breast tumors and is associated with increases in collagen deposition, however the mechanism connecting PTEN loss to increased collagen deposition remains unclear. Here, we demonstrate that Pten knockout in fibroblasts using an Fsp-Cre;Pten loxP/loxP mouse model increases collagen fiber number and fiber size within the mammary gland. Pten knockout additionally upregulated Sparc transcription in fibroblasts and promoted collagen shuttling out of the cell. Interestingly, SPARC mRNA expression was observed to be significantly elevated in the tumor stroma as compared to the normal breast in several patient cohorts. While SPARC knockdown via shRNA did not affect collagen shuttling, it notably decreased assembly of exogenous collagen. In addition, SPARC knockdown decreased fibronectin assembly and alignment of the extracellular matrix in an in vitro fibroblast-derived matrix model. Overall, these data indicate upregulation of SPARC is a mechanism by which PTEN regulates collagen deposition in the mammary gland stroma.