BackgroundHuman pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies in the world and despite great efforts in research types of treatment remain limited. A frequently detected alteration in PDACs is a truncated O-linked N-acetylgalactosamine (GalNAc) glycosylation with expression of the Tn antigen. Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations. Tn antigen is a tumor associated glycan detected in 75-90 % of PDACs and up to 67 % in its precursor lesions. Since the role of Tn antigen expression in PDAC is insufficiently understood we analyzed the impact of COSMC mediated Tn antigen expression in two human PDAC cell lines on cellular oncogenic properties.MethodsForced expression of Tn antigen on O-glycosylated proteins in pancreatic cancer cells was induced by lentiviral-mediated knockdown of the COSMC chaperone, which prevented O-glycan elongation beyond the initial GalNAcα1- residue on O-linked glycoproteins. Altered O-GalNAc glycosylation was analyzed in human pancreatic cancer cell lines Panc-1 and L3.6pl using Western and Far-Western blot as well as immunocytochemical techniques. To assess the biological implications of COSMC function on oncogenic properties, cell viability assays, scratch assays combined with live cell imaging, migration and apoptosis assays were performed. Lectin based glycoprotein enrichment with subsequent mass spectrometric analysis identified new cancer O-GalNAc modified proteins. Expression of Tn antigen bearing Nucleolin in patient derived PDAC tumor specimens was evaluated and correlated with clinicopathological data.ResultsTn antigen expression was induced on various O-GalNAc glycoproteins in COSMC deficient cell lines compared to the control. Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells. Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines. Interestingly, immunohistochemical staining and co-localization studies of patient derived PDACs revealed poor survival for patients with strong co-localization of Tn antigen and Nucleolin (p = 0.037).ConclusionThis study substantiates the influence of altered O-glycan (Tn/STn) expression on oncogenic properties in pancreatic cancer and identifies O-GalNAc modified Nucleolin as novel prognostic marker.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0386-1) contains supplementary material, which is available to authorized users.

The incidence of pancreatic ductal adenocarcinoma (PDAC) nearly equals its mortality rate, partly because most PDACs are intrinsically chemoresistant and thus largely untreatable. It was found recently that chemoresistant PDAC cells overexpress the Notch-2 receptor and have undergone epithelial-mesenchymal transition (EMT). In this study, we show that these two phenotypes are interrelated by expression of Midkine (MK), a heparin-binding growth factor that is widely overexpressed in chemoresistant PDAC. Gemcitabine, the front-line chemotherapy used in PDAC treatment, induced MK expression in a dose-dependent manner, and its RNAi-mediated depletion was associated with sensitization to gemcitabine treatment. We identified an interaction between the Notch-2 receptor and MK in PDAC cells. MK-Notch-2 interaction activated Notch signaling, induced EMT, upregulated NF-kB, and increased chemoresistance. Taken together, our findings define an important pathway of chemoresistance in PDAC and suggest novel strategies for its clinical attack. Cancer Res; 71(14);

Mammary oncogenesis is profoundly influenced by signaling pathways controlled by estrogen receptor A (ERA). Although it is known that ERA exerts its oncogenic effect by stimulating the proliferation of many human breast cancers through the activation of target genes, our knowledge of the underlying transcriptional mechanisms remains limited. Our published work has shown that the in vivo activity of LIM homeodomain transcription factors (LIM-HD) is critically regulated by cofactors of LIM-HD proteins (CLIM) and the ubiquitin ligase RING finger LIM domain-interacting protein (RLIM). Here, we identify CLIM and RLIM as novel ERA cofactors that colocalize and interact with ERA in primary human breast tumors. We show that both cofactors associate with estrogen-responsive promoters and regulate the expression of endogenous ERA target genes in breast cancer cells. Surprisingly, our results indicate opposing functions of LIM cofactors for ERA and LIM-HDs: whereas CLIM enhances transcriptional activity of LIM-HDs, it inhibits transcriptional activation mediated by ERA on most target genes in vivo. In turn, the ubiquitin ligase RLIM inhibits transcriptional activity of LIM-HDs but enhances transcriptional activation of endogenous ERA target genes. Results from a human breast cancer tissue microarray of 1,335 patients revealed a highly significant correlation of elevated CLIM levels to ER/progesterone receptor positivity and poor differentiation of tumors. Combined, these results indicate that LIM cofactors CLIM and RLIM regulate the biological activity of ERA during the development of human breast cancer.

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Previous studies reported that Follistatin-like 3 (FSTL3) is abundantly expressed in several solid tumors and participate in the regulation of cell metabolism. However, the clinico-pathological significance, biological role and molecular mechanism of FSTL3 in colorectal cancer (CRC) is still unclear. Here we report that the expression level of FSTL3 in colon cancer specimens was significantly higher, compared to normal tissue and interestingly, the expression of FSTL3 was related to lymph node metastasis, tumor stage, tumor size, and intravascular emboli (IVE). As an upstream molecular event, we found that transcriptional regulation of FSTL3 was highly dependent on YAP1 de-phosphorylation events and that increased FSTL3 expression readily activated the β-Catenin pathway, which is a well-known signaling hub that promotes EMT processes and aerobic glycolysis in cancer cells. We found that elevated FSTL3 expression strongly promotes migration, invasion and metastatic formation of CRC cells by directly activating β-Catenin -mediated EMT and aerobic glycolysis. In the xenograft mouse model, FSTL3 expression was linked to increased metastatic formation of CRC cells. Together, the activation of YAP1 induces FSTL3 expression. FSTL3-mediated β-Catenin pathway activation promotes EMT and aerobic glycolysis and therefore affecting the invasive and metastatic capacity of CRC cells. The abundant FSTL3 expression is a poor prognostic factor and pharmacological targeting of YAP1 can counteract FSTL3 expression, suggesting a promising therapeutic target for anti-metastatic strategies in patients suffering from CRC.

BackgroundPancreatic cancer is still associated with devastating prognosis. Real progress in treatment options has still not been achieved. Therefore new models are urgently needed to investigate this deadly disease. As a part of this process we have established and characterized a new human pancreatic cancer cell line.MethodsThe newly established pancreatic cancer cell line PaCa 5061 was characterized for its morphology, growth rate, chromosomal analysis and mutational analysis of the K-ras, EGFR and p53 genes. Gene-amplification and RNA expression profiles were obtained using an Affymetrix microarray, and overexpression was validated by IHC analysis. Tumorigenicity and spontaneous metastasis formation of PaCa 5061 cells were analyzed in pfp-/-/rag2-/- mice. Sensitivity towards chemotherapy was analysed by MTT assay.ResultsPaCa 5061 cells grew as an adhering monolayer with a doubling time ranging from 30 to 48 hours. M-FISH analyses showed a hypertriploid complex karyotype with multiple numerical and unbalanced structural aberrations. Numerous genes were overexpressed, some of which have previously been implicated in pancreatic adenocarcinoma (GATA6, IGFBP3, IGFBP6), while others were detected for the first time (MEMO1, RIOK3). Specifically highly overexpressed genes (fold change > 10) were identified as EGFR, MUC4, CEACAM1, CEACAM5 and CEACAM6. Subcutaneous transplantation of PaCa 5061 into pfp-/-/rag2-/- mice resulted in formation of primary tumors and spontaneous lung metastasis.ConclusionThe established PaCa 5061 cell line and its injection into pfp-/-/rag2-/- mice can be used as a new model for studying various aspects of the biology of human pancreatic cancer and potential treatment approaches for the disease.

In mice, the ubiquitin ligase RLIM/Rnf12 is a critical survival factor for mammary milk-producing alveolar cells, but little is known about how its activity is regulated. It is shown here that RLIM shuttles between the nucleus and cytoplasm in a phosphorylation-dependent manner, and shuttling is important for its alveolar survival function.

This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4.