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The vascular endothelial growth factor VEGF-D promotes metastasis by inducing lymphangiogenesis and dilatation of the lymphatic vasculature, facilitating tumor cell extravasion. Here we report a novel level of control for VEGF-D expression at the level of protein translation. In human tumor cells, VEGF-D colocalized with eIF4GI and 4E-BP1, which can program increased initiation at IRES motifs on mRNA by the translational initiation complex. In murine tumors, the steady-state level of VEGF-D protein was increased despite the overexpression and dephosphorylation of 4E-BP1, which downregulates protein synthesis, suggesting the presence of an internal ribosome entry site (IRES) in the 5′ UTR of VEGF-D mRNA. We found that nucleolin, a nucleolar protein involved in ribosomal maturation, bound directly to the 5′UTR of VEGF-D mRNA, thereby improving its translation following heat shock stress via IRES activation. Nucleolin blockade by RNAi-mediated silencing or pharmacologic inhibition reduced VEGF-D translation along with a subsequent constriction of lymphatic vessels in tumors. Our results identify nucleolin as a key regulator of VEGF-D expression, deepening understanding of lymphangiogenesis control during tumor formation. Cancer Res; 76(15); 4394–405. ©2016 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is extremely rich in stroma. By secreting tremendous amounts of soluble and un-soluble proteins, stromal cancer-associated fibroblasts (CAFs) can induce tumor chemoresistance by activating survival signals in cancer cells and preventing drug delivery through increased fibrosis. Our results demonstrate that chemoresistance triggered by the CAF secretome is strongly reduced upon inhibition of CAF protein synthesis. This is achieved by impinging upon the PI3K/mTOR/4E-BP1 pathway that we found highly activated in primary cultures of alpha-SMA-positive CAFs isolated from human PDAC. CAFs, but not normal pancreatic fibroblasts, express primarily the sstr1 somatostatin receptor subtype whose activation by the somatostatin analogue SOM230 (Pasireotide) strongly inhibits the PI3K/mTOR/4E-BP1 pathway and the resulting synthesis of secreted proteins including IL-6 and collagen-I. As a consequence, the growth and chemoresistance of pancreatic tumors provided by co-injection of CAFs in nude mice are overcome when chemotherapy (such as gemcitabine) is combined to pasireotide treatment. While gemcitabine or pasireotide alone have marginal effects, the combined treatment markedly decreases fibrosis and strongly enhances cancer cell apoptosis due to increased cell sensitivity to gemcitabine. We propose that selective inhibition of CAF protein synthesis and secretion with sstr1-directed pharmacological compounds represents an anti-stromal targeted therapy with a promising potential in chemosensitization. Citation Format: Camille Duluc, Siham Moatassim, Mounira Chalabi, Aurélie Perraud, Yvan Martineau, Florence Breibach, Marie-Bernadette Delisle, Muriel Mathonnet, Herbert A. Schmid, Stéphane Pyronnet, Corinne Bousquet. Pasireotide reduces chemoresistance in pancreatic tumor cells by inhibiting the synthesis and secretion of growth factors from tumor associated fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 402. doi:10.1158/1538-7445.AM2015-402

Hemidesmosomes (HDs) play a critical role in epithelia integrity by anchoring epithelial cells to the basement membrane (BM). These structures, present at the basal cell surface, link the intracellular intermediate filament network with the extracellular laminins of the BM. Interestingly, HDs are frequently lost in cancer cells, thereby facilitating epithelial tumor cell detachment from the BM. Surprisingly, we here provide evidence that a strong immunoreactivity for the integrin α6β4 and BP180, two specific hemidesmosomal proteins, is observed in human pancreatic ductal adenocarcinoma cells, where their function served paradoxically to stimulate migration and invasion. Using human pancreatic cancer cells in culture, we described molecular events explaining why these proteins are hijacked from their primary function and, importantly, how to force back the reassembly of HDs, thereby decreasing cancer cell migration and invasion. This is achieved by introducing in these cells the somatostatin receptor sst2, whose expression is lost in 90% of pancreatic cancers, and which we have shown to have oncosuppressive functions including inhibition of tumor progression and metastasis. Cell treatment with somatostatin forced HDs reassembly by stimulating two separate critical events, as demonstrated in pancreatic cancer cells and in migrating keratinocytes: 1- the dephosphorylation of the α6β4 integrin which drives its relocalization from lamellipodia, where it is delocalised upon its phosphorylation by growth factors in migrating cells, to HDs; 2- the inhibition of the cleavage of BP180 into its pro-migratory BP120 form, which is here shown to account for the strong BP180 immunoreactivity observed in human pancreatic tumor samples as compared to normal pancreas where the full-length BP180 form is mostly expressed. BP180 cleavage into BP120 is here shown to rely at least on the activity of the MMP-9 metalloprotease whose activity is inhibited by sst2. Strikingly, knocking-down BP180 expression (siRNA) impairs somatostatin-induced HDs assembly in sst2-expressing cells. Interestingly, BP180 siRNA partially revert sst2 inhibitory role on in vitro and in vivo cell migration and invasion, as demonstrated using the chick chorioallatoic membrane model whereby tumor progression of pancreatic cancer cell xenografts is monitored. We have identified novel and original mechanisms for somatostatin-activated sst2 to revert cancer cell pro-migratory phenotype by facilitating HD assembly and cancer cell anchorage to the BM. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5181.

Inactivation of the transcription factor Smad4/DPC4 gene is a frequent event that correlates with aggressiveness of pancreatic adenocarcinomas. We have previously shown that the stress-inducible translational inhibitor 4E-BP1 is a target of Smad4 essential for TGFbeta-mediated inhibition of cell proliferation. Here, we show that 4E-BP1 expression parallels that of Smad4 through the graded stages of pancreatic cancer progression. 4E-BP1 amount transiently increases in human PanIN-1 and PanIN-2 lesions, but is dramatically diminished in PanIN-3 and invasive carcinomas. A similar pattern of 4E-BP1 expression is seen in PanINs from pancreas of mice that carry a pancreas-specific mutated Kras allele (Kras-Pdx1/cre mice). Furthermore, we show in vitro that 4E-BP1 expression is efficiently induced under hypoxia in Smad4+/+, but not in Smad4−/− pancreatic duct cells. We further reveal that, at the molecular level, Smad4 cooperates with HIF1-alpha to induce 4E-BP1 gene transcription in hypoxic cells. Thus, it appears that pancreatic duct cells which carry Smad4 loss are no longer capable of inducing 4E-BP1 under stress conditions. This may have important consequences in pancreatic carcinogenesis as 4E-BP1 is viewed as a stress-inducible factor whose function is to block cellular protein synthesis to concentrate resources on an appropriate stress response aimed at restraining carcinogenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3149.