Anti-tumour efficacy of capecitabine in a genetically engineered mouse model of pancreatic cancer.

Capecitabine (CAP) is a 5-FU pro-drug approved for the treatment of several cancers and it is used in combination with gemcitabine (GEM) in the treatment of patients with pancreatic adenocarcinoma (PDAC). However, limited pre-clinical data of the effects of CAP in PDAC are available to support the use of the GEMCAP combination in clinic. Therefore, we investigated the pharmacokinetics and the efficacy of CAP as a single agent first and then in combination with GEM to assess the utility of the GEMCAP therapy in clinic. Using a model of spontaneous PDAC occurring in Kras(G12D); p53(R172H); Pdx1-Cre (KPC) mice and subcutaneous allografts of a KPC PDAC-derived cell line (K8484), we showed that CAP achieved tumour concentrations (∼25 µM) of 5-FU in both models, as a single agent, and induced survival similar to GEM in KPC mice, suggesting similar efficacy. In vitro studies performed in K8484 cells as well as in human pancreatic cell lines showed an additive effect of the GEMCAP combination however, it increased toxicity in vivo and no benefit of a tolerable GEMCAP combination was identified in the allograft model when compared to GEM alone. Our work provides pre-clinical evidence of 5-FU delivery to tumours and anti-tumour efficacy following oral CAP administration that was similar to effects of GEM. Nevertheless, the GEMCAP combination does not improve the therapeutic index compared to GEM alone. These data suggest that CAP could be considered as an alternative to GEM in future, rationally designed, combination treatment strategies for advanced pancreatic cancer

SCRIB expression is deregulated in human prostate cancer, and its deficiency in mice promotes prostate neoplasia

Loss of cellular polarity is a hallmark of epithelial cancers, raising the possibility that regulators of polarity have a role in suppressing tumorigenesis. The Scribble complex is one of at least three interacting protein complexes that have a critical role in establishing and maintaining epithelial polarity. In human colorectal, breast, and endometrial cancers, expression of the Scribble complex member SCRIB is often mislocalized and deregulated. Here, we report that Scrib is indispensable for prostate homeostasis in mice. Scrib heterozygosity initiated prostate hyperplasia, while targeted biallelic Scrib loss predisposed mice to prostate intraepithelial neoplasia. Mechanistically, Scrib was shown to negatively regulate the MAPK cascade to suppress tumorigenesis. Further analysis revealed that prostate-specific loss of Scrib in mice combined with expression of an oncogenic Kras mutation promoted the progression of prostate cancer that recapitulated the human disease. The clinical significance of the work in mice was highlighted by our observation that SCRIB deregulation strongly correlated with poor survival in human prostate cancer. These data suggest that the polarity network could provide a new avenue for therapeutic intervention

[Review] Cian Duffy (2014) My purpose was humbler, but also higher: Thomas De Quincey’s 'System of the heavens’, popular science and the sublime

Review of article: Duffy Cian, Romanticism, Volume 20 Issue 1, Page 1-14, ISSN 1354-991x Available Online Mar 201

Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer

An autochthonous model of pancreatic ductal adenocarcinoma (PDA) permitted the analysis of why immunotherapy is ineffective in this human disease. Despite finding that PDA-bearing mice had cancer cell-specific CD8+ T cells, the mice, like human patients with PDA, did not respond to two immunological checkpoint antagonists that promote the function of T cells: anti-cytotoxic T-lymphocyte-associated protein 4 (α-CTLA-4) and α-programmed cell death 1 ligand 1 (α-PD-L1). Immune control of PDA growth was achieved, however, by depleting carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). The depletion of the FAP+ stromal cell also uncovered the antitumor effects of α-CTLA-4 and α-PD-L1, indicating that its immune suppressive activity accounts for the failure of these T-cell checkpoint antagonists. Three findings suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) explained the overriding immunosuppression by the FAP+ cell: T cells were absent from regions of the tumor containing cancer cells, cancer cells were coated with the chemokine, CXCL12, and the FAP+ CAF was the principal source of CXCL12 in the tumor. Administering AMD3100, a CXCL12 receptor chemokine (C-X-C motif) receptor 4 inhibitor, induced rapid T-cell accumulation among cancer cells and acted synergistically with α-PD-L1 to greatly diminish cancer cells, which were identified by their loss of heterozygosity of Trp53 gene. The residual tumor was composed only of premalignant epithelial cells and in flammatory cells. Thus, a single protein, CXCL12, from a single stromal cell type, the FAP+ CAF, may direct tumor immune evasion in a model of human PDA

TLR9 ligation in pancreatic stellate cells promotes tumorigenesis

Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis

Loss of the Tumor Suppressor Pten Promotes Proliferation of Drosophila melanogaster Cells In Vitro and Gives Rise to Continuous Cell Lines

In vivo analysis of Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and development. While in vivo analysis of mutants has been a strength of the field, analyzing fly cells in culture is valuable for cell biological, biochemical and whole genome approaches in which large numbers of homogeneous cells are required. An efficient genetic method to derive Drosophila cell lines using expression of an oncogenic form of Ras (RasV12) has been developed. Mutations in tumor suppressors, which are known to cause cell hyperproliferation in vivo, could provide another method for generating Drosophila cell lines. Here we screened Drosophila tumor suppressor mutations to test if they promoted cell proliferation in vitro. We generated primary cultures and determined when patches of proliferating cells first emerged. These cells emerged on average at 37 days in wild-type cultures. Using this assay we found that a Pten mutation had a strong effect. Patches of proliferating cells appeared on average at 11 days and the cultures became confluent in about 3 weeks, which is similar to the timeframe for cultures expressing RasV12. Three Pten mutant cell lines were generated and these have now been cultured for between 250 and 630 cell doublings suggesting the life of the mutant cells is likely to be indefinite. We conclude that the use of Pten mutants is a powerful means to derive new Drosophila cell lines

Gamma secretase inhibition promotes hypoxic necrosis in mouse pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease that is refractory to medical intervention. Notch pathway antagonism has been shown to prevent pancreatic preneoplasia progression in mouse models, but potential benefits in the setting of an established PDA tumor have not been established. We demonstrate that the gamma secretase inhibitor MRK003 effectively inhibits intratumoral Notch signaling in the KPC mouse model of advanced PDA. Although MRK003 monotherapy fails to extend the lifespan of KPC mice, the combination of MRK003 with the chemotherapeutic gemcitabine prolongs survival. Combination treatment kills tumor endothelial cells and synergistically promotes widespread hypoxic necrosis. These results indicate that the paucivascular nature of PDA can be exploited as a therapeutic vulnerability, and the dual targeting of the tumor endothelium and neoplastic cells by gamma secretase inhibition constitutes a rationale for clinical translation

Somatic p16INK4a loss accelerates melanomagenesis

Loss of p16INK4a–RB and ARF–p53 tumor suppressor pathways, as well as activation of RAS–RAF signaling, is seen in a majority of human melanomas. Although heterozygous germline mutations of p16INK4a are associated with familial melanoma, most melanomas result from somatic genetic events: often p16INK4a loss and N-RAS or B-RAF mutational activation, with a minority possessing alternative genetic alterations such as activating mutations in K-RAS and/or p53 inactivation. To generate a murine model of melanoma featuring some of these somatic genetic events, we engineered a novel conditional p16INK4a-null allele and combined this allele with a melanocyte-specific, inducible CRE recombinase strain, a conditional p53-null allele and a loxP-stop-loxP activatable oncogenic K-Ras allele. We found potent synergy between melanocyte-specific activation of K-Ras and loss of p16INK4a and/or p53 in melanomagenesis. Mice harboring melanocyte-specific activated K-Ras and loss of p16INK4a and/or p53 developed invasive, unpigmented and nonmetastatic melanomas with short latency and high penetrance. In addition, the capacity of these somatic genetic events to rapidly induce melanomas in adult mice suggests that melanocytes remain susceptible to transformation throughout adulthood

Inactivation of TIF1γ Cooperates with KrasG12D to Induce Cystic Tumors of the Pancreas

Inactivation of the Transforming Growth Factor Beta (TGFβ) tumor suppressor pathway contributes to the progression of Pancreatic Ductal AdenoCarcinoma (PDAC) since it is inactivated in virtually all cases of this malignancy. Genetic lesions inactivating this pathway contribute to pancreatic tumor progression in mouse models. Transcriptional Intermediary Factor 1 gamma (TIF1γ) has recently been proposed to be involved in TGFβ signaling, functioning as either a positive or negative regulator of the pathway. Here, we addressed the role of TIF1γ in pancreatic carcinogenesis. Using conditional Tif1γ knockout mice (Tif1γlox/lox), we selectively abrogated Tif1γ expression in the pancreas of Pdx1-Cre;Tif1γlox/lox mice. We also generated Pdx1-Cre;LSL-KrasG12D;Tif1γlox/lox mice to address the effect of Tif1γ loss-of-function in precancerous lesions induced by oncogenic KrasG12D. Finally, we analyzed TIF1γ expression in human pancreatic tumors. In our mouse model, we showed that Tif1γ was dispensable for normal pancreatic development but cooperated with Kras activation to induce pancreatic tumors reminiscent of human Intraductal Papillary Mucinous Neoplasms (IPMNs). Interestingly, these cystic lesions resemble those observed in Pdx1-Cre;LSL-KrasG12D;Smad4lox/lox mice described by others. However, distinctive characteristics, such as the systematic presence of endocrine pseudo-islets within the papillary projections, suggest that SMAD4 and TIF1γ don't have strictly redundant functions. Finally, we report that TIF1γ expression is markedly down-regulated in human pancreatic tumors by quantitative RT–PCR and immunohistochemistry supporting the relevance of these findings to human malignancy. This study suggests that TIF1γ is critical for tumor suppression in the pancreas, brings new insight into the genetics of pancreatic cancer, and constitutes a promising model to decipher the respective roles of SMAD4 and TIF1γ in the multifaceted functions of TGFβ in carcinogenesis and development

Activated K-ras and INK4a/Arf Deficiency Cooperate During the Development of Pancreatic Cancer by Activation of Notch and NF-κB Signaling Pathways

BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, suggesting that novel strategies for the prevention and treatment of PDAC are urgently needed. K-ras mutations are observed in >90% of pancreatic cancer, suggesting its role in the initiation and early developmental stages of PDAC. In order to gain mechanistic insight as to the role of mutated K-ras, several mouse models have been developed by targeting a conditionally mutated K-ras(G12D) for recapitulating PDAC. A significant co-operativity has been shown in tumor development and metastasis in a compound mouse model with activated K-ras and Ink4a/Arf deficiency. However, the molecular mechanism(s) by which K-ras and Ink4a/Arf deficiency contribute to PDAC has not been fully elucidated. METHODOLOGY/PRINCIPAL FINDINGS:To assess the molecular mechanism(s) that are involved in the development of PDAC in the compound transgenic mice with activated K-ras and Ink4a/Arf deficiency, we used multiple methods, such as Real-time RT-PCR, western blotting assay, immunohistochemistry, MTT assay, invasion, EMSA and ELISA. We found that the deletion of Ink4a/Arf in K-ras(G12D) expressing mice leads to PDAC, which is in part mediated through the activation of Notch and NF-κB signaling pathways. Moreover, we found down-regulation of miR-200 family, which could also play important roles in tumor development and progression of PDAC in the compound transgenic mice. CONCLUSIONS/SIGNIFICANCE:Our results suggest that the activation of Notch and NF-κB together with the loss of miR-200 family is mechanistically linked with the development and progression of PDAC in the compound K-ras(G12D) and Ink4a/Arf deficient transgenic mice