Jesse J. Smith

Claudin-2 expression increases tumorigenicity of colon cancer cells: role of epidermal growth factor receptor activation

Claudin-2 is a unique member of the claudin family of transmembrane proteins, as its expression is restricted to the leaky epithelium in vivo and correlates with epithelial leakiness in vitro. However, recent evidence suggests potential functions of claudin-2 that are relevant to neoplastic transformation and growth. In accordance, here we report, on the basis of analysis of mRNA and protein expression using a total of 309 patient samples that claudin-2 expression is significantly increased in colorectal cancer and correlates with cancer progression. We also report similar increases in claudin-2 expression in inflammatory bowel disease-associated colorectal cancer. Most importantly, we demonstrate that the increased claudin-2 expression in colorectal cancer is causally associated with tumor growth as forced claudin-2 expression in colon cancer cells that do not express claudin-2 resulted in significant increases in cell proliferation, anchorage-independent growth and tumor growth in vivo. We further show that the colonic microenvironment regulates claudin-2 expression in a manner dependent on signaling through the EGF receptor (EGFR), a key regulator of colon tumorigenesis. In addition, claudin-2 expression is specifically decreased in the colon of waved-2 mice, naturally deficient in EGFR activation. Furthermore, genetic silencing of claudin-2 expression in Caco-2, a colon cancer cell line, prevents the EGF-induced increase in cell proliferation. Taken together, these results uncover a novel role for claudin-2 in promoting colon cancer, potentially via EGFR transactivation.

Tumor Suppressor Function of the Plasma Glutathione Peroxidase Gpx3 in Colitis-Associated Carcinoma

The glutathione peroxidases, a family of selenocysteine-containing redox enzymes, play pivotal roles in balancing the signaling, immunomodulatory, and deleterious effects of reactive oxygen species (ROS). The glutathione peroxidase GPX3 is the only extracellular member of this family, suggesting it may defend cells against ROS in the extracellular environment. Notably, GPX3 hypermethylation and underexpression occur commonly in prostate, gastric, cervical, thyroid, and colon cancers. We took a reverse genetics approach to investigate whether GPX3 would augment inflammatory colonic tumorigenesis, a process characterized by oxidative stress and inflammation, comparing Gpx3(-/-) mice in an established two-stage model of inflammatory colon carcinogenesis. Gpx3-deficient mice exhibited an increased tumor number, though not size, along with a higher degree of dysplasia. In addition, they exhibited increased inflammation with redistribution toward protumorigenic M2 macrophage subsets, increased proliferation, hyperactive WNT signaling, and increased DNA damage. To determine the impact of acute gene loss in an established colon cancer line, we silenced GPX3 in human Caco2 cells, resulting in increased ROS production, DNA damage and apoptosis in response to oxidative stress, combined with decreased contact-independent growth. Taken together, our results suggested an immunomodulatory role for GPX3 that limits the development of colitis-associated carcinoma.

HDAC inhibitors regulate claudin-1 expression in colon cancer cells through modulation of mRNA stability

Expression and cellular distribution of claudin-1, a tight junction protein, is dysregulated in colon cancer and its overexpression in colon cancer cells induced dedifferentiation and increased invasion. However, the molecular mechanism(s) underlying dysregulated claudin-1 expression in colon cancer remains poorly understood. Histone deacetylase (HDAC)-dependent histone acetylation is an important mechanism of the regulation of cancer-related genes and inhibition of HDACs induces epithelial differentiation and decreased invasion. Therefore, in this study, we examined the role of HDAC-dependent epigenetic regulation of claudin-1 in colon cancer. In this study, we show that sodium butyrate and Trichostatin A (TSA), two structurally different and widely used HDAC inhibitors, inhibited claudin-1 expression in multiple colon cancer cell lines. Further studies revealed modulation of claudin-1 mRNA stability by its 3′-UTR as the major mechanism underlying HDAC-dependent claudin-1 expression. In addition, overexpression of claudin-1 abrogated the TSA-induced inhibition of invasion in colon cancer cells suggesting functional crosstalk. Analysis of mRNA expression in colon cancer patients, showed a similar pattern of increase in claudin-1 and HDAC-2 mRNA expression throughout all stages of colon cancer. Inhibition of claudin-1 expression by HDAC-2-specific small interfering RNA further supported the role of HDAC-2 in this regulation. Taken together, we report a novel post-transcriptional regulation of claudin-1 expression in colon cancer cells and further show a functional correlation between claudin-1 expression and TSA-mediated regulation of invasion. As HDAC inhibitors are considered to be promising anticancer drugs, these new findings will have implications in both laboratory and clinical settings.

Claudin-7 expression induces mesenchymal to epithelial transformation (MET) to inhibit colon tumorigenesis.

In normal colon, claudin-7 is one of the highly expressed claudin proteins and its knockdown in mice results in altered epithelial cell homeostasis and neonatal death. Notably, dysregulation of the epithelial homeostasis potentiates oncogenic transformation and growth. However, the role of claudin-7 in the regulation of colon tumorigenesis remains poorly understood. Using a large colorectal cancer (CRC) patient database and mouse models of colon cancer, we found claudin-7 expression to be significantly downregulated in cancer samples. Most notably, forced claudin-7 expression in poorly differentiated and highly metastatic SW620 colon cancer cells induced epithelial characteristics and inhibited their growth in soft agar and tumor growth in vivo. By contrast, knockdown of claudin-7 in HT-29 or DLD-1 cells induced epithelial-to-mesenchymal transition (EMT), colony formation, xenograft-tumor growth in athymic mice and invasion. Importantly, a claudin-7 signature gene profile generated by overlapping the DEGs (differentially expressed genes in a high-throughput transcriptome analysis using claudin-7-manipulated cells) with human claudin-7 signature genes identified high-risk CRC patients. Furthermore, Rab25, a colon cancer suppressor and regulator of the polarized cell trafficking constituted one of the highly upregulated DEGs in claudin-7 overexpressing cells. Notably, silencing of Rab25 expression counteracted the effects of claudin-7 expression and not only increased proliferation and cell invasion but also increased the expression of p-Src and mitogen-activated protein kinase–extracellular signal–regulated kinase 1/2 that were suppressed upon claudin-7 overexpression. Of interest, CRC cell lines, which exhibited decreased claudin-7 expression, also exhibited promoter DNA hypermethylation, a modification associated with transcriptional silencing. Taken together, our data demonstrate a previously undescribed role of claudin-7 as a colon cancer suppressor and suggest that loss of claudin-7 potentiates EMT to promote colon cancer, in a manner dependent on Rab25.

ERBB4 is over-expressed in human colon cancer and enhances cellular transformation.

The ERBB4 receptor tyrosine kinase promotes colonocyte survival. Herein, we tested whether ERBB4s antiapoptotic signaling promotes transformation and colorectal tumorigenesis. ERBB4 alterations in a The Cancer Genome Atlas colorectal cancer (CRC) data set stratified survival, and in a combined Moffitt Cancer Center and Vanderbilt Medical Center CRC expression data set, ERBB4 message levels were increased at all tumor stages. Similarly, western blot and immunohistochemistry on additional CRC tissue banks showed elevated ERBB4 protein in tumors. ERBB4 was highly expressed in aggressive, dedifferentiated CRC cell lines, and its knockdown in LIM2405 cells reduced anchorage-independent colony formation. In nude mouse xenograft studies, ERBB4 alone was insufficient to induce tumor establishment of non-transformed mouse colonocytes, but its over-expression in cells harboring Apc(min) and v-Ha-Ras caused a doubling of tumor size. ERBB4-expressing xenografts displayed increased activation of survival pathways, including epidermal growth factor receptor and Akt phosphorylation and COX-2 expression, and decreased apoptotic signals. Finally, ERBB4 deletion from mouse intestinal epithelium impaired stem cell replication and in vitro enteroid establishment. In summary, we report that ERBB4 is over-expressed in human CRC, and in experimental systems enhances the survival and growth of cells driven by Ras and/or WNT signaling. Chronic ERBB4 over-expression in the context of, for example, inflammation may contribute to colorectal carcinogenesis. Tumors with high receptor levels are likely to have enhanced cell survival signaling through epidermal growth factor receptor, PI3K and COX-2. These results suggest ERBB4 as a novel therapeutic target in a subset of CRC.

Does intentional support of degree programs in general surgery residency affect research productivity or pursuit of academic surgery

OBJECTIVE Many residents supplement general surgery training with years of dedicated research, and an increasing number at our institution pursue additional degrees. We sought to determine whether it was worth the financial cost for residency programs to support degrees. DESIGN We reviewed graduating chief residents (n = 69) in general surgery at Vanderbilt University from 2001 to 2010 and collected the data including research time and additional degrees obtained. We then compared this information with the following parameters: (1) total papers, (2) first-author papers, (3) Journal Citation Reports impact factors of journals in which papers were published, and (4) first job after residency or fellowship training. SETTING The general surgery resident training program at Vanderbilt University is an academic program, approved to finish training 7 chief residents yearly during the time period studied. PARTICIPANTS Chief residents in general surgery at Vanderbilt who finished their training 2001 through 2010. RESULTS We found that completion of a degree during residency was significantly associated with more total and first-author publications as compared with those by residents with only dedicated research time (p = 0.001 and p = 0.017). Residents completing a degree also produced publications of a higher caliber and level of authorship as determined by an adjusted resident impact factor score as compared with those by residents with laboratory research time only (p = 0.005). Degree completion also was significantly correlated with a first job in academia if compared to those with dedicated research time only (p = 0.046). CONCLUSIONS Our data support the utility of degree completion when economically feasible and use of dedicated research time as an effective way to significantly increase research productivity and retain graduates in academic surgery. Aggregating data from other academic surgery programs would allow us to further determine association of funding of additional degrees as a means to encourage academic productivity and retention.

Myeloid translocation genes differentially regulate colorectal cancer programs

Myeloid translocation genes (MTGs), originally identified as chromosomal translocations in acute myelogenous leukemia, are transcriptional corepressors that regulate hematopoietic stem cell programs. Analysis of The Cancer Genome Atlas (TCGA) database revealed that MTGs were mutated in epithelial malignancy and suggested that loss of function might promote tumorigenesis. Genetic deletion of MTGR1 and MTG16 in the mouse has revealed unexpected and unique roles within the intestinal epithelium. Mtgr1−/− mice have progressive depletion of all intestinal secretory cells, and Mtg16−/− mice have a decrease in goblet cells. Furthermore, both Mtgr1−/− and Mtg16−/− mice have increased intestinal epithelial cell proliferation. We thus hypothesized that loss of MTGR1 or MTG16 would modify Apc1638/+-dependent intestinal tumorigenesis. Mtgr1−/− mice, but not Mtg16−/− mice, had a 10-fold increase in tumor multiplicity. This was associated with more advanced dysplasia, including progression to invasive adenocarcinoma, and augmented intratumoral proliferation. Analysis of chromatin immunoprecipitation sequencing data sets for MTGR1 and MTG16 targets indicated that MTGR1 can regulate Wnt and Notch signaling. In support of this, immunohistochemistry and gene expression analysis revealed that both Wnt and Notch signaling pathways were hyperactive in Mtgr1−/− tumors. Furthermore, in human colorectal cancer (CRC) samples MTGR1 was downregulated at both the transcript and protein level. Overall our data indicates that MTGR1 has a context-dependent effect on intestinal tumorigenesis.

MTG16 is a tumor suppressor in colitis-associated carcinoma

MTG16 is a member of the myeloid translocation gene (MTG) family of transcriptional corepressors. While MTGs were originally identified in chromosomal translocations in acute myeloid leukemia, recent studies have uncovered a role in intestinal biology. For example, Mtg16-/- mice have increased intestinal proliferation and are more sensitive to intestinal injury in colitis models. MTG16 is also underexpressed in patients with moderate/severe ulcerative colitis. Based on these findings, we postulated that MTG16 might protect against colitis-associated carcinogenesis. MTG16 was downregulated at the protein and RNA levels in patients with inflammatory bowel disease and in those with colitis-associated carcinoma. Mtg16-/- mice subjected to inflammatory carcinogenesis modeling exhibited worse colitis and increased tumor multiplicity and size. Loss of MTG16 also increased severity of dysplasia, apoptosis, proliferation, DNA damage, and WNT signaling. Moreover, transplantation of WT marrow into Mtg16-/- mice failed to rescue the Mtg16-/- protumorigenic phenotypes, indicating an epithelium-specific role for MTG16. While MTG dysfunction is widely appreciated in hematopoietic malignancies, the role of this gene family in epithelial homeostasis, and in colon cancer, was unrealized. This report identifies MTG16 as an important modulator of colitis and tumor development in inflammatory carcinogenesis.

Abstract 3076: BVES, a novel adhesion molecule, acts as tumor modifier through modulation of tight-junction-associated signaling

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BVES is novel adhesion protein that functions as a modulator of tight junction (TJ) formation with TJs level directly correlating with BVES expression. TJs, in addition to regulating paracellular passage of molecules, can also directly regulate Rho activity and transcriptional activation of cell cycle regulatory genes (ERBB2, PCNA, CDK4, and Cyclin-D1). Hence, alteration in BVES expression also modulates TJ associated signaling. Interestingly, overexpressing a dominant negative BVES induced human corneal epithelial cells to exhibit increased mesenchymal characteristics. Together, these observations led us to hypothesize that increasing BVES expression in carcinoma cells would induce an epithelial-like phenotype. To evaluate the function of BVES in carcinoma, a semi-adherent LIM2405 human colorectal cancer cell line was stably transfected with wild type BVES (WT-LIM2405) or empty vector (V-LIM2405) as control. Multiple stably transfected clonal isolates of LIM2405 overexpressing BVES exhibited increased epithelial features as evidenced by: i) uniform monolayer formation, ii) decreased vimentin and increased cytokeratin, Zo-1, and occludin expression, iii) decreased cellular motility (66% reduction, p<0.001), and iv) decreased colony number in soft-agar assays (53.5%, p<0.01). In addition, WT-LIM2405 cells progressively displayed reduced cellular proliferation, prolonged cell-doubling time (35%), and decreased S-phase and increased G1 fractions. This was more apparent with increasing confluence, suggesting that contact inhibition is restored with BVES expression. To evaluate the affect of BVES on tumor growth in vivo, V-LIM2405 and WT-LIM2405 cells were grown as orthotopic xenografts in athymic mice. WT-LIM2405 tumor growth was significantly attenuated (86%, p<0.001). Intra-tumoral proliferation rates were equivalent, however apoptosis was increased in WT-LIM2405 tumors (TUNEL positive cells/HPF, p=0.04). We next surveyed BVES expression in colorectal carcinoma. 80% of human CRCs samples exhibited at least a 40% reduction in BVES protein by immunoblotting. In addition, immunolocalization studies showed disorganized or absent BVES and ZO-1 staining only in carcinomatous regions. BVES expression is decrease not only in colorectal cancer (p=0.004), but also in breast (p=0.018) and thyroid carcinoma (p=0.20). Global expression analysis of 250 staged CRC samples compared with normal and adenoma colon specimens showed a decrease in BVES expression as early as carcinoma in situ (p<0.05). These findings indicate endogenous BVES expression is decrease in carcinoma and restoration of its’ expression induces an epithelial-like phenotype in colorectal cancer cells, thus implicating BVES as a tumor suppressor. Further investigation to determine the role of BVES in epithelial cancer biology is warranted and may lead to novel insight into targeted therapies. 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 3076.