Vijayababu M. Radhakrishnan

14-3-3γ Induces Oncogenic Transformation by Stimulating MAP Kinase and PI3K Signaling

The 14-3-3 proteins are a set of highly conserved scaffolding proteins that have been implicated in the regulation of a variety of important cellular processes such as the cell cycle, apoptosis and mitogenic signaling. Recent evidence indicates that the expression of some of the family members is elevated in human cancers suggesting that they may play a role in tumorigenesis. In the present study, the oncogenic potential of 14-3-3γ was shown by focus formation and tumor formation in SCID mice using 14-3-3γ transfected NIH3T3 mouse fibroblast cells. In contrast, 14-3-3σ, a putative tumor suppressor, inhibited NIH3T3 transformation by H-ras and c-myc. We also report that activation of both MAP kinase and PI3K signaling pathways are essential for transformation by 14-3-3γ. In addition, we found that 14-3-3γ interacts with phosphatidylinositol 3-kinase (PI3K) and TSC2 proteins indicating that it could stimulate PI3K signaling by acting at two points in the signaling pathway. Overall, our studies establish 14-3-3γ as an oncogene and implicate MAPK and PI3K signaling as important for 14-3-3γ induced transformation.

Curcumin inhibits interferon-γ signaling in colonic epithelial cells

Curcumin (diferulolylmethane) is an anti-inflammatory phenolic compound found effective in preclinical models of inflammatory bowel diseases (IBD) and in ulcerative colitis patients. Pharmacokinetics of curcumin and its poor systemic bioavailability suggest that it targets preferentially intestinal epithelial cells. The intestinal epithelium, an essential component of the gut innate defense mechanisms, is profoundly affected by IFN-γ, which can disrupt the epithelial barrier function, prevent epithelial cell migration and wound healing, and prime epithelial cells to express major histocompatibility complex class II (MHC-II) molecules and to serve as nonprofessional antigen-presenting cells. In this report we demonstrate that curcumin inhibits IFN-γ signaling in human and mouse colonocytes. Curcumin inhibited IFN-γ-induced gene transcription, including CII-TA, MHC-II genes (HLA-DRα, HLA-DPα1, HLA-DRβ1), and T cell chemokines (CXCL9, 10, and 11). Acutely, curcumin inhibited Stat1 binding to the GAS cis-element, prevented Stat1 nuclear translocation, and reduced Jak1 phosphorylation and phosphorylation of Stat1 at Tyr(701). Longer exposure to curcumin led to endocytic internalization of IFNγRα followed by lysosomal fusion and degradation. In summary, curcumin acts as an IFN-γ signaling inhibitor in colonocytes with biphasic mechanisms of action, a phenomenon that may partially account for the beneficial effects of curcumin in experimental colitis and in human IBD.

pTyr421 cortactin is overexpressed in colon cancer and is dephosphorylated by curcumin: involvement of non-receptor type 1 protein tyrosine phosphatase (PTPN1).

Cortactin (CTTN), first identified as a major substrate of the Src tyrosine kinase, actively participates in branching F-actin assembly and in cell motility and invasion. CTTN gene is amplified and its protein is overexpressed in several types of cancer. The phosphorylated form of cortactin (pTyr421) is required for cancer cell motility and invasion. In this study, we demonstrate that a majority of the tested primary colorectal tumor specimens show greatly enhanced expression of pTyr421-CTTN, but no change at the mRNA level as compared to healthy subjects, thus suggesting post-translational activation rather than gene amplification in these tumors. Curcumin (diferulolylmethane), a natural compound with promising chemopreventive and chemosensitizing effects, reduced the indirect association of cortactin with the plasma membrane protein fraction in colon adenocarcinoma cells as measured by surface biotinylation, mass spectrometry, and Western blotting. Curcumin significantly decreased the pTyr421-CTTN in HCT116 cells and SW480 cells, but was ineffective in HT-29 cells. Curcumin physically interacted with PTPN1 tyrosine phosphatases to increase its activity and lead to dephosphorylation of pTyr421-CTTN. PTPN1 inhibition eliminated the effects of curcumin on pTyr421-CTTN. Transduction with adenovirally-encoded CTTN increased migration of HCT116, SW480, and HT-29. Curcumin decreased migration of HCT116 and SW480 cells which highly express PTPN1, but not of HT-29 cells with significantly reduced endogenous expression of PTPN1. Curcumin significantly reduced the physical interaction of CTTN and pTyr421-CTTN with p120 catenin (CTNND1). Collectively, these data suggest that curcumin is an activator of PTPN1 and can reduce cell motility in colon cancer via dephosphorylation of pTyr421-CTTN which could be exploited for novel therapeutic approaches in colon cancer therapy based on tumor pTyr421-CTTN expression.

Hypomethylation of the 14-3-3σ promoter leads to increased expression in non-small cell lung cancer.

The 14‐3‐3 proteins are a set of seven highly conserved proteins that have recently been implicated in having a role in human tumorigenesis. However, the mechanism by which 14‐3‐3 proteins may act in this capacity is not well understood. In this study, we examined the expression of one of the 14‐3‐3 family members, 14‐3‐3σ, since it was shown previously to be aberrantly altered in human tumors. Using quantitative rtPCR and immunohistochemistry, we found that the expression levels of 14‐3‐3σ were elevated in the majority of human non‐small cell lung cancers (NSCLC) we examined. Surprisingly, we found that the 14‐3‐3σ gene was hypomethylated in lung tumors relative to normal lung tissue suggesting that decreased DNA methylation resulted in increased expression of 14‐3‐3σ in NSCLC. We also determined the gene copy number for 14‐3‐3σ in tumor samples and found no significant correlation with elevated mRNA expression. And also no mutations were found in 14‐3‐3σ gene. Overall, our data suggest that misregulated expression of 14‐3‐3σ gene may be due to altered methylation status.

P53 suppresses expression of the 14-3-3gamma oncogene

Background14-3-3 proteins are a family of highly conserved proteins that are involved in a wide range of cellular processes. Recent evidence indicates that some of these proteins have oncogenic activity and that they may promote tumorigenesis. We previously showed that one of the 14-3-3 family members, 14-3-3gamma, is over expressed in human lung cancers and that it can induce transformation of rodent cells in vitro.MethodsqRTPCR and Western blot analysis were performed to examine 14-3-3gamma expression in non-small cell lung cancers (NSCLC). Gene copy number was analyzed by qPCR. P53 mutations were detected by direct sequencing and also by western blot. CHIP and yeast one hybrid assays were used to detect p53 binding to 14-3-3gamma promoter.ResultsQuantitative rtPCR results showed that the expression level of 14-3-3gamma was elevated in the majority of NSCLC that we examined which was also consistent with protein expression. Further analysis of the expression pattern of 14-3-3gamma in lung tumors showed a correlation with p53 mutations suggesting that p53 might suppress 14-3-3 gamma expression. Analysis of the gamma promoter sequence revealed the presence of a p53 consensus binding motif and in vitro assays demonstrated that wild-type p53 bound to this motif when activated by ionizing radiation. Deletion of the p53 binding motif eliminated p53s ability to suppress 14-3-3gamma expression.ConclusionIncreased expression of 14-3-3gamma in lung cancer coincides with loss of functional p53. Hence, we propose that 14-3-3gammas oncogenic activities cooperate with loss of p53 to promote lung tumorigenesis.

Activation of Phosphatidylinositol 3-Kinase (PI3K) and Mitogen-activated Protein Kinase (MAPK) Signaling and the Consequent Induction of Transformation by Overexpressed 14-3-3γ Protein Require Specific Amino Acids within 14-3-3γ N-terminal Variable Region II

Background: 14-3-3 proteins are highly conserved phosphoamino acid-binding proteins that may play a role in tumorigenesis. Results: The oncogenic activity of 14-3-3γ requires a small variable domain in the N terminus. Conclusion: The biological activity of 14-3-3 proteins involves protein motifs outside of the phosphoamino acid binding domain. Significance: Understanding how structure determines 14-3-3 function is crucial to understanding how they function in tumorigenesis. Members of the 14-3-3 superfamily regulate numerous cellular functions by binding phosphoproteins. The seven human isoforms (and the myriad of other eukaryotic 14-3-3 proteins) are highly conserved in amino acid sequence and secondary structure, yet there is abundant evidence that the various isoforms manifest disparate as well as common functions. Several of the human 14-3-3 isoforms are dysregulated in certain cancers and thus have been implicated in oncogenesis; experimentally, 14-3-3γ behaves as an oncogene, whereas 14-3-3σ acts as a tumor suppressor. In this study, we sought to localize these opposing phenotypes to specific regions of the two isoforms and then to individual amino acids therein. Using a bioinformatics approach, six variable regions (VRI–VRVI) were identified. Using this information, two sets of constructs were created in which N-terminal portions (including either VRI–IV or only VRI and VRII) of 14-3-3γ and 14-3-3σ were swapped; NIH3T3 cells overexpressing the four chimeric proteins were tested for transformation activity (focus formation, growth in soft agar) and activation of PI3K and MAPK signaling. We found that the specific phenotypes of 14-3-3γ are associated with the N-terminal 40 amino acids (VRI and VRII); in like fashion, VRI and VRII of 14-3-3σ dictated its tumor suppressor function. Using individual amino acid substitutions within the 14-3-3γ VRII, we identified two residues required for and two contributing to the γ-specific phenotypes. Our observations suggest that isoform-specific phenotypes are dictated by a relatively few amino acids within variable regions.

Experimental colitis is associated with transcriptional inhibition of Na+/Ca2+ exchanger isoform 1 (NCX1) expression by interferon γ in the renal distal convoluted tubules.

Background: Defective renal Ca2+ reabsorption contributes to impaired systemic Ca2+ homeostasis and loss of bone density in IBD. Results: During colitis, IFNγ represses NCX1 expression and activity in a Stat1-dependent manner. Conclusion: IFNγ contributes to the reduced renal Ca2+ reabsorption during colitis by repressing basolateral NCX1. Significance: IFNγ inhibits renal NCX1 to contribute to negative systemic Ca2+ balance and increased bone resorption in IBD patients. NCX1 is a Na+/Ca2+ exchanger, which is believed to provide a key route for basolateral Ca2+ efflux in the renal epithelia, thus contributing to renal Ca2+ reabsorption. Altered mineral homeostasis, including intestinal and renal Ca2+ transport may represent a significant component of the pathophysiology of the bone mineral density loss associated with Inflammatory Bowel Diseases (IBD). The objective of our research was to investigate the effects of TNBS and DSS colitis and related inflammatory mediators on renal Ncx1 expression. Colitis was associated with decreased renal Ncx1 expression, as examined by real-time RT-PCR, Western blotting, and immunofluorescence. In mIMCD3 cells, IFNγ significantly reduced Ncx1 mRNA and protein expression. Similar effects were observed in cells transiently transfected with a reporter construct bearing the promoter region of the kidney-specific Ncx1 gene. This inhibitory effect of IFNγ is mediated by STAT1 recruitment to the proximal promoter region of Ncx1. Further in vivo study with Stat1−/− mice confirmed that STAT1 is indeed required for the IFNγ mediated Ncx1 gene regulation. These results strongly support the hypothesis that impaired renal Ca2+ handling occurs in experimental colitis. Negative regulation of NCX1- mediated renal Ca2+ absorption by IFNγ may significantly contribute to the altered Ca2+ homeostasis in IBD patients and to IBD-associated loss of bone mineral density.

Expression of Cav1.3 calcium channel in the human and mouse colon: posttranscriptional inhibition by IFNγ

It has been hypothesized that apically expressed L-type Ca2+ channel Cav1.3 (encoded by CACNA1D gene) contributes toward an alternative TRPV6-independent route of intestinal epithelial Ca2+ absorption, especially during digestion when high luminal concentration of Ca2+ and other nutrients limit TRPV6 contribution. We and others have implicated altered expression and activity of key mediators of intestinal and renal Ca2+ (re)absorption as contributors to negative systemic Ca2+ balance and bone loss in intestinal inflammation. Here, we investigated the effects of experimental colitis and related inflammatory mediators on colonic Cav1.3 expression. We confirmed Cav1.3 expression within the segments of the mouse and human gastrointestinal tract. Consistent with available microarray data (GEO database) from inflammatory bowel disease (IBD) patients, mouse colonic expression of Cav1.3 was significantly reduced in trinitrobenzene sulfonic acid (TNBS) colitis. In vitro, IFNγ most potently reduced Cav1.3 expression. We reproduced these findings in vivo with wild-type and Stat1-/- mice injected with IFNγ. The observed effect in Stat1-/- suggested a noncanonical transcriptional repression or a posttranscriptional mechanism. In support of the latter, we observed no effect on the cloned Cav1.3 gene promoter activity and accelerated Cav1.3 mRNA decay rate in IFNγ-treated HCT116 cells. While the relative contribution of Cav1.3 to intestinal Ca2+ absorption and its value as a therapeutic target remain to be established, we postulate that Cav1.3 downregulation in IBD may contribute to the negative systemic Ca2+ balance, to increased bone resorption, and to reduced bone mineral density in IBD patients.

Abstract A61: 14-3-3gamma transforming ability is determined by a motif outside the conserved phosphoamio-acid binding domain

The 14-3-3 proteins are a set of seven highly conserved scaffolding proteins that have been implicated as having a role in human tumorigenesis. However, despite the striking similarity of amino acid sequences and structures of the eukaryotic 14-3-3 family of proteins, there is increasing evidence that the seven human isoforms manifest disparate functions. For instance 14-3-3gamma functions as an oncogene and 14-3-3sigma acts as a tumor suppressor. To gain insight into how specific functions are manifested we constructed chimeric proteins by swaping various regions between gamma and sigma. Chimeric proteins were then utilized in NIH3T3 cell transformation assays. We found that transforming ability was associated with a small variable 40 amino acid region in the N-terminus of the gamma protein and that the transforming ability of gamma was dominant to the tumor suppressor activity of sigma. Importantly, the transforming motif was located outside the conserved 14-3-3 phosphoamino acid binding motif. Hence, transforming ability of 14-3-3 proteins is determined by a variable region that does not involve the phosphoamino acid binding site. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A61.