Hypoxia Suppresses High Fat Diet-Induced Steatosis And Development Of Hepatic Adenomas.

PURPOSE: Nonalcoholic fatty liver disease (NAFLD) is considered the most common form of silent liver disease in the United States and obesity is associated with increased risk of NAFLD. Obstructive sleep apnea (OSA) which is common in obese individuals is associated with a greater incidence of NAFLD, which in turn, increases the risk for hepatocellular carcinoma (HCC). It is unclear how obesity, OSA and NAFLD interrelate nor how they collectively contribute to an increased risk for developing HCC. PATIENTS AND METHODS: Male BALB/c mice were exposed to diethylnitrosamine and phenobarbital followed by 48 weeks of either standard chow diet (chow), chow with hypoxia, high-fat diet, or a combination of hypoxia and high-fat diet. We noninvasively monitored tumor development using micro-CT imaging. We tracked the total weight gained throughout the study. We evaluated liver histology, fat accumulation, carbonic anhydrase 9 (CA9) and hypoxia-inducible factor 1-alpha (HIF-1α) expression, as well as, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). RESULTS: A high-fat diet without hypoxia led to the development of obesity that induced hepatic steatosis and promoted tumorigenesis. Animals on a high-fat diet and that were also exposed to hypoxia had lower total weight gain, lower steatosis, lower serum AST and ALT levels, and fewer number of hepatic adenomas than a high-fat diet without hypoxia. CONCLUSION: These findings suggest that hypoxia abrogates obesity, hepatic steatosis, and hepatic tumorigenesis related to a high-fat diet.

Intraperitoneal injections as an alternative method for micro-CT contrast enhanced detection of murine liver tumors.

Micro-computed tomography (micro-CT) coupled with tissue, or vascular, specific contrast agent has emerged as a powerful tool for detecting and monitoring tumor growth in the liver of murine animals. Intravenous injections of contrast agents can be technically challenging and lead to errors that can considerably influence the outcome of a preclinical study, prompting an alternative method. Here we assessed the effectiveness of intraperitoneal injections of polyiodinated triglycerides emulsions (Fenestra LC) in micro-CT imaging of young SCID (8 weeks) and old BALB/c (48 weeks) mice with xenograft or carcinogen-induced liver tumors, respectively, and determined an optimal acquisition time. Utilizing an intraperitoneal injection is a viable alternative administration route for using Fenestra in detection and quantification of murine liver tumor burden.

Measuring DNA content in live cells by fluorescence microscopy.

BACKGROUND: Live-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell's integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique. RESULTS: Presented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm's accurate assessment of DNA content was validated by parallel flow cytometric studies. CONCLUSIONS: This method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.

Double-negative (CD27-IgD-) B cells are expanded in NSCLC and inversely correlate with affinity-matured B cell populations.

BACKGROUND: The presence of B cells in early stage non-small cell lung cancer (NSCLC) is associated with longer survival, however, the role these cells play in the generation and maintenance of anti-tumor immunity is unclear. B cells differentiate into a variety of subsets with differing characteristics and functions. To date, there is limited information on the specific B cell subsets found within NSCLC. To better understand the composition of the B cell populations found in NSCLC we have begun characterizing B cells in lung tumors and have detected a population of B cells that are CD79A+CD27-IgD-. These CD27-IgD- (double-negative) B cells have previously been characterized as unconventional memory B cells and have been detected in some autoimmune diseases and in the elderly population but have not been detected previously in tumor tissue. METHODS: A total of 15 fresh untreated NSCLC tumors and 15 matched adjacent lung control tissues were dissociated and analyzed by intracellular flow cytometry to detect the B cell-related markers CD79A, CD27 and IgD. All CD79A+ B cells subsets were classified as either naïve (CD27-IgD+), affinity-matured (CD27+IgD-), early memory/germinal center cells (CD27+IgD+) or double-negative B cells (CD27-IgD-). Association of double-negative B cells with clinical data including gender, age, smoking status, tumor diagnosis and pathologic differentiation status were also examined using the logistic regression analysis for age and student's t-test for all other variables. Associations with other B cell subpopulations were examined using Spearman's rank correlation. RESULTS: We observed that double-negative B cells were frequently abundant in lung tumors compared to normal adjacent controls (13 out of 15 cases), and in some cases made up a substantial proportion of the total B cell compartment. The presence of double-negative cells was also found to be inversely related to the presence of affinity-matured B cells within the tumor, Spearman's coefficient of - 0.76. CONCLUSIONS: This study is the first to observe the presence of CD27-IgD- double-negative B cells in human NSCLC and that this population is inversely correlated with traditional affinity-matured B cell populations.

Key Strategies for Building Research Capacity of University Faculty Members.

Universities are under pressure to increase external research funding, and some federal agencies offer programs to expand research capacity in certain kinds of institutions. However, conflicts within faculty roles and other aspects of university operations influence the effectiveness of particular strategies for increasing research activity. We review conventional approaches to increasing research, focusing on outcomes for individual faculty members and use one federally-funded effort to build cancer-related research capacity at a public university as an example to explore the impact of various strategies on research outcomes. We close with hypotheses that should be tested in future formal studies.

The receptor tyrosine kinase AXL promotes migration and invasion in colorectal cancer.

The receptor tyrosine kinases (RTKs) TYRO3, AXL and MERTK (TAM) have well-described oncogenic functions in a number of cancers. Notwithstanding, TAM RTKs are also potent and indispensable inhibitors of inflammation. The combined deletion of Axl and Mertk in mice enhances chronic inflammation and autoimmunity, including increased inflammation in the gut and colitis-associated cancer. On the other hand, deletion of Tyro3 increases the risk of allergic responses. Therefore, the indiscriminate inhibition of these TAM RTKs could result in undesirable immunological diseases. Here we show that AXL, but not MERTK or TYRO3 expression is enhanced in late stage colorectal cancer (CRC) and AXL expression associates with a cell migration gene signature. Silencing AXL or the inhibition of AXL kinase activity significantly inhibits tumor cell migration and invasion. These results indicate that the selective inhibition of AXL alone might confer sufficient therapeutic benefit in CRC, while preserving at least some of the beneficial, anti-inflammatory effects of MERTK and TYRO3 RTKs.

The induction of endoreduplication and polyploidy by elevated expression of 14-3-3γ.

Several studies have demonstrated that specific 14-3-3 isoforms are frequently elevated in cancer and that these proteins play a role in human tumorigenesis. 14-3-3γ, an isoform recently demonstrated to function as an oncoprotein, is overexpressed in a variety of human cancers; however, its role in promoting tumorigenesis remains unclear. We previously reported that overexpression of 14-3-3γ caused the appearance of polyploid cells, a phenotype demonstrated to have profound tumor promoting properties. Here we examined the mechanism driving 14-3-3γ-induced polyploidization and the effect this has on genomic stability. Using FUCCI probes we showed that these polyploid cells appeared when diploid cells failed to enter mitosis and subsequently underwent endoreduplication. We then demonstrated that 14-3-3γ-induced polyploid cells experience significant chromosomal segregation errors during mitosis and observed that some of these cells stably propagate as tetraploids when isolated cells were expanded into stable cultures. These data lead us to conclude that overexpression of the 14-3-3γ promotes endoreduplication. We further investigated the role of 14-3-3γ in human NSCLC samples and found that its expression is significantly elevated in polyploid tumors. Collectively, these results suggests that 14-3-3γ may promote tumorigenesis through the production of a genetically unstable polyploid intermediate.

Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells.

Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects.

Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells.

BACKGROUND: The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention. OBJECTIVE: We investigated how APC inactivation influences the regulation of FXR expression in colonic mucosal cells. We hypothesized that APC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation. METHODS: Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J and Apc-deficient (Apc(Min) (/+)) male mice, respectively. The expression of Fxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC and HT-29 cells carrying inactivated APC. RESULTS: In Apc(Min) (/+) mice, constitutive CpG methylation of the Fxrα3/4 promoter was linked to reduced (60-90%) baseline Fxr, Ibabp, and Shp and increased Cox-2 expression in apparently normal adjacent mucosa and colon tumors. Apc knockdown in HCT-116 cells increased cellular myelocytomatosis (c-MYC) and lowered (∼50%) FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA induced FXR expression and lowered CpG methylation of FXR. CONCLUSIONS: We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells, leading to reduced expression of downstream targets (SHP, IBABP) involved in BA homeostasis while increasing the expression of factors (COX-2, c-MYC) that contribute to inflammation and colon cancer.

Comparative analysis of 14-3-3 isoform expression and epigenetic alterations in colorectal cancer.

BACKGROUND: The 14-3-3 family is a group of intracellular proteins found in all eukaryotic organisms. Humans have seven isoforms that serve as scaffolds to promote interactions of regulatory phospho-proteins involved in many vital cellular processes and previous studies have shown that disturbances in native 14-3-3 levels can contribute significantly to the development of various cancers. METHODS: DNA and RNA was extracted from frozen tissue samples collected by the Human Cooperative Tissue Network. RNA samples were reverse transcribed and subjected to qRT-PCR analysis using fluorescently labelled probes. Genomic DNA was treated with bisulfite and cloned into bacterial vectors for subsequent high-resolution sequencing. Mammalian NIH3T3 cells were transformed with 14-3-3 eta and Ras expression vectors synthesized from cDNA. Colonies were counted and transforming capability assessed after 21 days of growth. Cell lysates were analyzed by western blot to verify protein expression. RESULTS: Here we examined normal and cancerous 14-3-3 expression levels of all seven isoforms in a cohort of sporadic colorectal adenocarcinomas and in a group of tumors and their matched normals using qRT-PCR analysis. We found a statistically significant decrease in the levels of 14-3-3 sigma, eta, and zeta observed among adenocarcinomas compared to normal tissue. A parallel analysis of microarray data from the TCGA dataset confirmed that expression of sigma and eta were down-regulated in colon tumors. To explore the mechanisms behind 14-3-3 expression changes, we examined the methylation status of the sigma, eta, and zeta gene promoters in selected samples. Our data identified novel CpG methylation sites in the eta promoter consistent with epigenetic silencing of both 14-3-3 sigma and eta isoforms during colon tumorigenesis. Because epigenetic silencing is the hallmark of a tumor suppressor we tested eta in focus formation assays and found that it is capable of suppressing ras-induced transformation of NIH3T3 cells. CONCLUSION: To our knowledge, this is the first study to identify the 14-3-3 eta gene as a tumor suppressor and that its expression is suppressed in colon tumors by DNA hypermethylation. These data suggest a link between 14-3-3 expression levels and the development of colon cancers.

Differential regulation of EGFR-MAPK signaling by deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) in colon cancer.

A high-fat diet coincides with increased levels of bile acids. This increase in bile acids, particularly deoxycholic acid (DCA), has been strongly associated with the development of colon cancer. Conversely, ursodeoxycholic acid (UDCA) may have chemopreventive properties. Although structurally similar, DCA and UDCA present different biological and pathological effects in colon cancer progression. The differential regulation of cancer by these two bile acids is not yet fully understood. However, one possible explanation for their diverging effects is their ability to differentially regulate signaling pathways involved in the multistep progression of colon cancer, such as the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway. This review will examine the biological effects of DCA and UDCA on colon cancer development, as well as the diverging effects of these bile acids on the oncogenic signaling pathways that play a role in colon cancer development, with a particular emphasis on bile acid regulation of the EGFR-MAPK pathway.

Using logistic regression to improve the prognostic value of microarray gene expression data sets: application to early-stage squamous cell carcinoma of the lung and triple negative breast carcinoma.

BACKGROUND: Numerous microarray-based prognostic gene expression signatures of primary neoplasms have been published but often with little concurrence between studies, thus limiting their clinical utility. We describe a methodology using logistic regression, which circumvents limitations of conventional Kaplan Meier analysis. We applied this approach to a thrice-analyzed and published squamous cell carcinoma (SQCC) of the lung data set, with the objective of identifying gene expressions predictive of early death versus long survival in early-stage disease. A similar analysis was applied to a data set of triple negative breast carcinoma cases, which present similar clinical challenges. METHODS: Important to our approach is the selection of homogenous patient groups for comparison. In the lung study, we selected two groups (including only stages I and II), equal in size, of earliest deaths and longest survivors. Genes varying at least four-fold were tested by logistic regression for accuracy of prediction (area under a ROC plot). The gene list was refined by applying two sliding-window analyses and by validations using a leave-one-out approach and model building with validation subsets. In the breast study, a similar logistic regression analysis was used after selecting appropriate cases for comparison. RESULTS: A total of 8594 variable genes were tested for accuracy in predicting earliest deaths versus longest survivors in SQCC. After applying the two sliding window and the leave-one-out analyses, 24 prognostic genes were identified; most of them were B-cell related. When the same data set of stage I and II cases was analyzed using a conventional Kaplan Meier (KM) approach, we identified fewer immune-related genes among the most statistically significant hits; when stage III cases were included, most of the prognostic genes were missed. Interestingly, logistic regression analysis of the breast cancer data set identified many immune-related genes predictive of clinical outcome. CONCLUSIONS: Stratification of cases based on clinical data, careful selection of two groups for comparison, and the application of logistic regression analysis substantially improved predictive accuracy in comparison to conventional KM approaches. B cell-related genes dominated the list of prognostic genes in early stage SQCC of the lung and triple negative breast cancer.

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 (pTyr(421)) 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 pTyr(421)-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 pTyr(421)-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 pTyr(421)-CTTN. PTPN1 inhibition eliminated the effects of curcumin on pTyr(421)-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 pTyr(421)-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 pTyr(421)-CTTN which could be exploited for novel therapeutic approaches in colon cancer therapy based on tumor pTyr(421)-CTTN expression.

Development and evaluation of a biomedical search engine using a predicate-based vector space model.

Although biomedical information available in articles and patents is increasing exponentially, we continue to rely on the same information retrieval methods and use very few keywords to search millions of documents. We are developing a fundamentally different approach for finding much more precise and complete information with a single query using predicates instead of keywords for both query and document representation. Predicates are triples that are more complex datastructures than keywords and contain more structured information. To make optimal use of them, we developed a new predicate-based vector space model and query-document similarity function with adjusted tf-idf and boost function. Using a test bed of 107,367 PubMed abstracts, we evaluated the first essential function: retrieving information. Cancer researchers provided 20 realistic queries, for which the top 15 abstracts were retrieved using a predicate-based (new) and keyword-based (baseline) approach. Each abstract was evaluated, double-blind, by cancer researchers on a 0-5 point scale to calculate precision (0 versus higher) and relevance (0-5 score). Precision was significantly higher (p<.001) for the predicate-based (80%) than for the keyword-based (71%) approach. Relevance was almost doubled with the predicate-based approach-2.1 versus 1.6 without rank order adjustment (p<.001) and 1.34 versus 0.98 with rank order adjustment (p<.001) for predicate--versus keyword-based approach respectively. Predicates can support more precise searching than keywords, laying the foundation for rich and sophisticated information search.

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.

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.

The significance of p53 isoform expression in serous ovarian cancer.

Epithelial ovarian cancer still carries a high mortality rate and remains the leading cause of gynecologic cancer death in the USA, despite decades of research. Hence, there is considerable interest in developing biomarkers that could be used to stratify patients for subsequent treatment. Mutation of the p53 tumor suppressor gene occurs very frequently (∼96%) in high-grade serous ovarian cancer. However, loss of p53 has not proven to be a reliable prognostic marker. Recent evidence indicates that the truncated p53 protein isoforms can regulate activated p53 and thus may play a role in tumorigenesis. In the article by Hofstetter et al., the authors examined the relationship between the expression of two p53 isoforms (Δ133p53 and Δ40p53) and prognosis in patients with serous ovarian cancer. Their findings indicate that Δ133p53 constitutes an independent prognostic marker for improved recurrence-free and overall survival. Intriguingly, this relationship was observed in patients whose tumors expressed a mutant p53, suggesting that Δ133p53 might suppress the actions of mutant p53. The mutational status of p53 alone did not have prognostic significance. These studies suggest that mutant p53 activity may be counteracted by Δ133p53, which leads to a more favorable prognosis in advanced serous ovarian carcinomas. Novel therapeutic approaches could be built upon these findings.

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

BACKGROUND: 14-3-3 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, 14-3-3 gamma, is over expressed in human lung cancers and that it can induce transformation of rodent cells in vitro. METHODS: qRTPCR and Western blot analysis were performed to examine 14-3-3 gamma 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 14-3-3 gamma promoter. RESULTS: Quantitative rtPCR results showed that the expression level of 14-3-3 gamma was elevated in the majority of NSCLC that we examined which was also consistent with protein expression. Further analysis of the expression pattern of 14-3-3 gamma 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 p53's ability to suppress 14-3-3 gamma expression. CONCLUSION: Increased expression of 14-3-3 gamma in lung cancer coincides with loss of functional p53. Hence, we propose that 14-3-3 gamma's oncogenic activities cooperate with loss of p53 to promote lung tumorigenesis.

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. © 2011 Wiley-Liss, Inc.

Loss of HSF1 results in defective radiation-induced G(2) arrest and DNA repair.

HSF1 is a transcription factor that plays a key role in the response to heat stress and was previously shown by us to also be essential for importation of p53 into the nucleus. Here we extend these studies and show that loss of HSF1 renders cells more sensitive to killing by ionizing radiation. Cells that lack a functional HSF1 were unable to arrest in G(2) after exposure to ionizing radiation, suggesting that HSF1 activity was essential for activation of this cell cycle checkpoint. In addition, cells with no HSF1 showed a reduced capacity to repair radiation-induced double-stranded DNA breaks. We found that in these cells 53BP1 did not accumulate at sites of DNA damage, suggesting that HSF1 was also essential for the functioning of this DNA damage mediator. Taken together our results indicate that HSF1 plays an important role in checkpoint activation and DNA repair and suggest that there is overlap between the heat stress response pathway and the pathway that responds to ionizing radiation.

P53 is transported into the nucleus via an Hsf1-dependent nuclear localization mechanism.

Loss of p53 function can occur through disruption of its ability to localize to the nucleus. Previously we showed through characterization a set of mutant cell lines that lacked the ability to import p53 into the nucleus that nuclear translocation of p53 appeared to be mechanistically different from that of the SV40 T-antigen (SV40TAg). Here we extend that work by examining nuclear importation of p53 and SV40TAg using both in vivo and in vitro assays for nuclear localization. We show that disruption of microtubule polymerization using colchicine suppresses nuclear localization of p53 but not of SV40TAg. We also show, for the first time, that the heat shock transcription factor (Hsf1), is required for establishment of the microtubule network in cells and for nuclear localization of p53. In contrast, SV40TAg does not interact with polymerized microtubules suggesting that it is transported into the nucleus through an alternative mechanism. Interestingly, lacking of Hsf1 expression and suppressing Hsf1 by siRNA also made cells more resistant to the cytotoxic effects of paclitaxel. Hence, loss of Hsf1 activity not only suppressed p53 function, but also led to reduced sensitivity to killing by drugs that target microtubules.