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Purpose: Numerous biomarkers for pancreatic cancer have been reported. We determined the extent to which such biomarkers are expressed throughout metastatic progression, including those that effectively predict biologic behavior. Experimental Design: Biomarker profiling was performed for 35 oncoproteins in matched primary and metastatic pancreatic cancer tissues from 36 rapid autopsy patients. Proteins of significance were validated by immunolabeling in an independent sample set, and functional studies were performed in vitro and in vivo. Results: Most biomarkers were similarly expressed or lost in expression in most samples analyzed, and the matched primary and metastases from a specific patient were most similar to each other than to other patients. However, a subset of proteins showed extensive interpatient heterogeneity, one of which was p38 MAPK. Strong positive pp38 MAPK immunolabeling was significantly correlated with improved postresection survival by multivariate analysis (median overall survival 27.9 months, P = 0.041). In pancreatic cancer cells, inhibition of functional p38 by SB202190 increased cell proliferation in vitro in both low-serum and low-oxygen conditions. High functional p38 activity in vitro corresponded to lower levels of pJNK protein expression, and p38 inhibition resulted in increased pJNK and pMKK7 by Western blot analysis. Moreover, JNK inhibition by SP600125 or MKK7 siRNA knockdown antagonized the effects of p38 inhibition by SB202190. In vivo, SP600125 significantly decreased growth rates of xenografts with high p38 activity compared with those without p38 expression. Conclusions: Functional p38 MAPK activity contributes to overall survival through JNK signaling, thus providing a rationale for JNK inhibition in pancreatic cancer management. Clin Cancer Res; 20(23); 6200–11. ©2014 AACR.

Thioredoxin reductase 1 (TR1) controls the redox state of protein thiols in mammalian cells and has been shown to have roles in both preventing and promoting cancer. To define the role of this selenoenzyme in hepatocellular carcinoma development, we examined tumor incidence in the liver of mice with tissue-specific knockout of mouse TR1 subjected to the liver carcinogen, diethylnitrosamine (DEN). TR1-deficient livers manifested ~90% tumor incidence compared with ~16% in control livers. The TR1-dependent effect was observed independent of sex, and, in control mice, tumorigenesis did not affect the expression of TR1. On the other hand, we observed upregulation of another selenoenzyme, glutathione peroxidase 2 (GPx2), and components of the glutathione (GSH) system, including those that generate reduced GSH. Overall, this study shows that TR1 protects against chemically induced hepatocarcinogenesis via the control of the cellular redox state, whereas its role in promoting this type of cancer is minimal.

Selenium is an essential micronutrient for humans and animals, and is thought to provide protection against some forms of cancer. These protective effects appear to be mediated, at least in part, through selenium-containing proteins (selenoproteins). Recent studies in a mouse colon cancer cell line have shown that the 15 kDa selenoprotein (Sep15) may also play a role in promoting colon cancer. The current study investigated whether the effects of reversing the cancer phenotype observed when Sep15 was removed in mouse colon cancer cells, were recapitulated in HCT116 and HT29 human colorectal carcinoma cells. Targeted down-regulation of Sep15 using RNAi technology in these human colon cancer cell lines resulted in similarly decreased growth under anchorage-dependent and anchorage-independent conditions. However, the magnitude of reduction in cell growth was much less than in the mouse colon cancer cell line investigated previously. Furthermore, changes in cell cycle distribution were observed, indicating a delayed release of Sep15 deficient cells from the G(0)/G(1) phase after synchronization. The potential mechanism by which human colon cancer cells lacking Sep15 revert their cancer phenotype will need to be explored further.

Selenoproteins mediate much of the cancer-preventive properties of the essential nutrient selenium, but some of these proteins have been shown to also have cancer-promoting effects. We examined the contributions of the 15kDa selenoprotein (Sep15) and thioredoxin reductase 1 (TR1) to cancer development. Targeted down-regulation of either gene inhibited anchorage-dependent and anchorage-independent growth and formation of experimental metastases of mouse colon carcinoma CT26 cells. Surprisingly, combined deficiency of Sep15 and TR1 reversed the anti-cancer effects observed with down-regulation of each single gene. We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells. Interestingly, components of the Wnt/β-catenin signaling pathway were up-regulated in cells lacking both TR1 and Sep15. These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells. Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer.

Selenium has long been known to have a role in preventing cancer, but only in recent years has a deficiency in this element also been shown to function in preventing cancer. Selenoproteins have also been shown to serve as selenium-containing components in cancer prevention, but we are learning that specific members of this protein class can also function in cancer promotion. The involvement of two of these selenoproteins, thioredoxin reductase 1 and the 15 kDa selenoprotein, in promoting cancer is examined in this chapter.

9 páginas, 8 figuras -- PAGS nros. 24982-24990, The von Hippel-Lindau (VHL) tumor suppressor gene regulates extracellular matrix deposition. In VHL negative renal cancer cells, VHL(-), the lack of fibronectin matrix assembly is thought to promote and maintain tumor angiogenesis allowing vessels to infiltrate tumors. Therefore, and considering the importance of this process in tumor growth, we aimed to study why VHL(-) renal cancer cells fail to form a proper extracellular matrix. Our results showed that VHL(-) cells were not defective in fibronectin production and that the fibronectin produced by these cells was equally functional in promoting cell adhesion and matrix assembly as that produced by VHL(+) cells. We have previously reported that VHL(-) cells fail to form β1 integrin fibrillar adhesions and have a diminished organization of actin stress fibers; therefore, we aimed to study if the small GTPase family is involved in this process. We found that activation of the RhoA GTPase was defective in VHL(-) cells, and this was possibly mediated by an increased activation of its inhibitor, p190RhoGAP. Additionally, the expression of constitutively active RhoA in VHL(-) cells resulted in formation of a fibronectin matrix. These results strongly suggest an important role for RhoA in some of the defects observed in renal cancer cells, This work was supported in part by SAF2006-04013 from the Ministerio de Educación y Ciencia (MEC) (to M. J. C.), SAF2007-60592 and RECAVA C03/01 (to M. O. L.), and PI060400 from the Ministerio de Sanidad y Consumo and the Fundacion de Investigacion Medica Mutua Madrileña (to A. G. -P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact

Background: Dysregulation of DNA repair mechanisms are known to play a major role in tumor progression. Moreover, the tumor microenviroment itself has been shown to enhance this phenomenon. For example, studies using reporter genes and endogenous loci have demonstrated increased mutation rates in cells grown in tumors compared to the identical cells grown in culture, or a higher frequency of point mutations in cancer cells cultured under hypoxic conditions, a common feature during tumor growth. An accepted mechanism for these observations is hypoxia-induced down-regulation of MLH1, MSH2 or MSH6 leading to an increased mutation rate, or to hypoxia-induced repression of DNA double strand break repair. Methods: Expression microarrays were performed using RNA isolated from nine pancreatic cancer cell lines cultured under normoxic (Nx, 20% O2) or hypoxic conditions (Hx, 1% O2) for 24 hours to identify differentially expressed genes. Retroviruses encoding oligos for HIF-1 or HIF-2 knockdown were used to generate stable pancreatic cancer cell lines in which HIF-1 or HIF-2, respectively, expression was blocked. Alternatively, Hx was induced by culture of cells inside an air-tight chamber with inflow and outflow valves that was infused with a mixture of 1% O2, 5% CO2 and 94% N2. Protein levels of HIF-1, HIF-2, MLH1, MSH2, MSH6 and CHK1 were determined by western blotting. Cell proliferation was measured by BrdU incorporation and cell cycle analysis by flow cytometry of propidium iodide stained cells. G2/M check point was studied analyzing Histone H3 phosphorylation by flow cytometry and DNA damage was determined by immunofluorescence of cells stained with anti phospho γ-H2AX antibody (γ-H2AX foci formation) Results: We first compared the gene expression profiles of 9 different pancreatic cancer cell lines each grown under normoxic versus hypoxic conditions. As previously described, hypoxia led to down-regulation of the mRNA levels of the mismatch repair proteins MLH1, MSH2 and MSH6 as well as down-regulation of several genes involved in the DNA damage response to double strand breaks such as Brca1, Rad51, Ku70 and Ku80. Of interest, a novel downregulated gene identified was CHK1, a master coordinator of DNA repair following genotoxic stress. Growth of pancreatic cancer cells under hypoxic conditions was confirmed to decrease CHK1 mRNA and protein levels and stable knockdown of HIF-1a but not HIF-2 abolished these effects. In contrast to what have been previously reported in other cancer cell types, hypoxia had no effects on cell proliferation in pancreatic cancer cells as assessed by cell cycle analysis or by BrdU incorporation.To determine if hypoxic down-regulation of CHK1 protein levels has functional effects, we determined the ability of pancreatic cancer cells to arrest in the G2/M checkpoint after ionizing radiation. We found that cells cultured under hypoxia were significantly less arrested on G2/M check point after radiation compared to the same irradiated cells grown under normoxic conditions. This hypoxic effect on G2/M checkpoint was abolished on HIF-1 knockdown cells. Finally, pancreatic cancer cells grown under hypoxic conditions accumulated more DNA damage 24 hours after radiation compared to the same irradiated cells grown under normoxic conditions. Conclusion: Hypoxic conditions decrease CHK1 mRNA and protein levels in pancreatic cancer cells in a HIF1-dependent manner. Impairment on CHK1 function may promote the mutator phenotype of pancreatic cancer cells during tumor progression by allowing unchecked cell divisions despite ongoing DNA damage. This finding also has significance for the use of CHK1 inhibitors for this tumor type. Citation Format: Salvador Naranjo-Suarez, Elsa Callen, Yi Zhong, Alvin Makohon-Moore, Andre Nussenzweig, Christine Iacobuzio-Donahue. Hypoxia-induced CHK1 repression may enhance the mutator phenotype of pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A68.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2α did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF. (Cancer Res 2006; 66(3): 1553-60)

The essential nutrient selenium appears to have cancer preventive properties that are partly mediated through selenoproteins. This study investigated the role of the 15 kDa selenoprotein (Sep15) in cancer by examining Sep15 knockout in mice. Weanling homozygous Sep15 knockout, heterozygote and wild type littermate controls (N=12/genotype) were given four weekly subcutaneous injections of the known colon carcinogen azoxymethane (10 mg/kg). Sep15 knockout mice developed significantly (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1868. doi:10.1158/1538-7445.AM2011-1868