Natasha G. Deane

Claudin-1 regulates cellular transformation and metastatic behavior in colon cancer

Disruption of the cell-cell junction with concomitant changes in the expression of junctional proteins is a hallmark of cancer cell invasion and metastasis. The role of adherent junction proteins has been studied extensively in cancer, but the roles of tight junction (TJ) proteins are less well understood. Claudins are recently identified members of the tetraspanin family of proteins, which are integral to the structure and function of TJs. Recent studies show changes in expression/cellular localization of claudins during tumorigenesis; however, a causal relationship between claudin expression/localization and cancer has not been established. Here, we report an increased expression of claudin-1 in human primary colon carcinoma and metastasis and in cell lines derived from primary and metastatic tumors. We also report frequent nuclear localization of claudin-1 in these samples. Genetic manipulations of claudin-1 expression in colon cancer cell lines induced changes in cellular phenotype, with structural and functional changes in markers of epithelial-mesenchymal transition. Furthermore, we demonstrate that changes in claudin-1 expression have significant effects on growth of xenografted tumors and metastasis in athymic mice. We further provide data suggesting that the regulation of E-cadherin expression and beta-catenin/Tcf signaling is a possible mechanism underlying claudin-1-dependent changes.

Experimentally Derived Metastasis Gene Expression Profile Predicts Recurrence and Death in Patients With Colon Cancer

BACKGROUND & AIMS Staging inadequately predicts metastatic risk in patients with colon cancer. We used a gene expression profile derived from invasive, murine colon cancer cells that were highly metastatic in an immunocompetent mouse model to identify patients with colon cancer at risk of recurrence. METHODS This phase 1, exploratory biomarker study used 55 patients with colorectal cancer from Vanderbilt Medical Center (VMC) as the training dataset and 177 patients from the Moffitt Cancer Center as the independent dataset. The metastasis-associated gene expression profile developed from the mouse model was refined with comparative functional genomics in the VMC gene expression profiles to identify a 34-gene classifier associated with high risk of metastasis and death from colon cancer. A metastasis score derived from the biologically based classifier was tested in the Moffitt dataset. RESULTS A high score was significantly associated with increased risk of metastasis and death from colon cancer across all pathologic stages and specifically in stage II and stage III patients. The metastasis score was shown to independently predict risk of cancer recurrence and death in univariate and multivariate models. For example, among stage III patients, a high score translated to increased relative risk of cancer recurrence (hazard ratio, 4.7; 95% confidence interval, 1.566-14.05). Furthermore, the metastasis score identified patients with stage III disease whose 5-year recurrence-free survival was >88% and for whom adjuvant chemotherapy did not increase survival time. CONCLUSION A gene expression profile identified from an experimental model of colon cancer metastasis predicted cancer recurrence and death, independently of conventional measures, in patients with colon cancer.

Smad4-mediated signaling inhibits intestinal neoplasia by inhibiting expression of β-catenin.

BACKGROUND & AIMS Mutational inactivation of adenomatous polyposis coli (APC) is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of β-catenin, a mediator of Wnt signaling. Progression of CRC also involves inactivation of signaling via transforming growth factor β and bone morphogenetic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss of the transcription factor Smad4 on levels of β-catenin messenger RNA (mRNA) and Wnt signaling. METHODS We used microarray analysis to associate levels of Smad4 and β-catenin mRNA in colorectal tumor samples from 250 patients. We performed oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T) and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480 colon cancer cells. We analyzed adenomas from (APC(Δ1638/+)) and (APC(Δ1638/+)) × (K19Cre(ERT2)Smad4(lox/lox)) mice by using laser capture microdissection. RESULTS In human CRC samples, reduced levels of Smad4 correlated with increased levels of β-catenin mRNA. In Smad4-depleted cell lines, levels of β-catenin mRNA and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T cells increased binding of RNA polymerase II to the β-catenin gene. Expression of Smad4 in SW480 cells reduced Wnt signaling and levels of β-catenin mRNA. In mice with heterozygous disruption of Apc(APC(Δ1638/+)), Smad4-deficient intestinal adenomas had increased levels of β-catenin mRNA and expression of Wnt target genes compared with adenomas from APC(Δ1638/+) mice that expressed Smad4. CONCLUSIONS Transcription of β-catenin is inhibited by BMP signaling to Smad4. These findings provide important information about the interaction among transforming growth factor β, BMP, and Wnt signaling pathways in progression of CRC.

Oncogenic Ras and Transforming Growth Factor-β Synergistically Regulate AU-Rich Element–Containing mRNAs during Epithelial to Mesenchymal Transition

Colon cancer progression is characterized by activating mutations in Ras and by the emergence of the tumor-promoting effects of transforming growth factor-β (TGF-β) signaling. Ras-inducible rat intestinal epithelial cells (RIE:iRas) undergo a well-described epithelial to mesenchymal transition and invasive phenotype in response to H-RasV12 expression and TGF-β treatment, modeling tumor progression. We characterized global gene expression profiles accompanying Ras-induced and TGF-β–induced epithelial to mesenchymal transition in RIE:iRas cells by microarray analysis and found that the regulation of gene expression by the combined activation of Ras and TGF-β signaling was associated with enrichment of a class of mRNAs containing 3′ AU-rich element (ARE) motifs known to regulate mRNA stability. Regulation of ARE-containing mRNA transcripts was validated at the mRNA level, including genes important for tumor progression. Ras and TGF-β synergistically increased the expression and mRNA stability of vascular endothelial growth factor (VEGF), a key regulator of tumor angiogenesis, in both RIE:iRas cells and an independent cell culture model (young adult mouse colonocyte). Expression profiling of human colorectal cancers (CRC) further revealed that many of these genes, including VEGF and PAI-1, were differentially expressed in stage IV human colon adenocarcinomas compared with adenomas. Furthermore, genes differentially expressed in CRC are also significantly enriched with ARE-containing transcripts. These studies show that oncogenic Ras and TGF-β synergistically regulate genes containing AREs in cultured rodent intestinal epithelial cells and suggest that posttranscriptional regulation of gene expression is an important mechanism involved in cellular transformation and CRC tumor progression. (Mol Cancer Res 2008;6(7):1124–36)

Targeted Imaging of Colonic Tumors in Smad3 / Mice Discriminates Cancer and Inflammation

The peripheral benzodiazepine receptor (PBR) is a trans-mitochondrial membrane protein that modulates steroid biosynthesis. Recently, up-regulation and nuclear localization of PBR has been shown to be associated with colon, prostate, and breast cancer. PBR has been targeted by the exogenous synthetic ligand, PK11195, for various purposes including imaging. To capitalize on these observations, we developed a high-throughput, noninvasive, in vivo imaging approach to detect spontaneously arising colonic tumors in mice using a novel PBR-targeted molecular imaging agent (NIR-conPK11195). NIR-conPK11195 localized and was retained in colonic adenomas and carcinomas in Smad3−/− mice but not in non-neoplastic hamartomas or chronically inflamed colonic tissue. Using a fluorescence signal-to-noise ratio of ≥4-fold 13 h after injection of the agent, we detected colonic tumors with a sensitivity of 67% and a specificity of 86% in a cohort of 37 Smad3−/− mice and control littermates. Furthermore, using oral administration of dextran sulfate to induce colonic inflammation, we showed that the clearance profile of NIR-conPK11195 distinguished transient uptake in inflammatory tissue from longer term retention in tumors. Taken together, these results indicate that NIR-conPK11195 is a promising optical molecular imaging tool to rapidly screen for colonic tumors in mice and to discriminate inflammation from cancer. (Mol Cancer Res 2007;5(4):341–9)

Enhanced Tumor Formation in Cyclin D1 × Transforming Growth Factor β1 Double Transgenic Mice with Characterization by Magnetic Resonance Imaging

Transgenic mice that overexpress cyclin D1 protein in the liver develop liver carcinomas with high penetrance. Transforming growth factor β (TGF-β) serves as either an epithelial cell growth inhibitor or a tumor promoter, depending on the cellular context. We interbred LFABP-cyclin D1 and Alb-TGF-β1 transgenic mice to produce cyclin D1/TGF-β1 double transgenic mice and followed the development of liver tumors over time, characterizing cellular and molecular changes, tumor incidence, tumor burden, and tumor physiology noninvasively by magnetic resonance imaging. Compared with age-matched LFABP-cyclin D1 single transgenic littermates, cyclin D1/TGF-β1 mice exhibited a significant increase in tumor incidence. Tumor multiplicity, tumor burden, and tumor heterogeneity were higher in cyclin D1/TGF-β1 mice compared with single transgenic littermates. Characteristics of cyclin D1/TGF-β1 livers correlated with a marked induction of the peripheral periductal oval cell/stem cell compartment of the liver. A number of cancerous lesions from cyclin D1/TGF-β1 mice exhibited unique features such as ductal plate malformations and hemorrhagic nodules. Some lesions were contiguous with the severely diseased background liver and, in some cases, replaced the normal architecture of the entire organ. Cyclin D1/TGF-β1 lesions, in particular, were associated with malignant features such as areas of vascular invasion by hepatocytes and heterogeneous hyperintensity of signal on T2-weighted magnetic resonance imaging. These findings demonstrate that TGF-β1 promotes stem cell activation and tumor progression in the context of cyclin D1 overexpression in the liver.

Transforming Growth Factor β Regulates P-Body Formation through Induction of the mRNA Decay Factor Tristetraprolin

ABSTRACT Transforming growth factor β (TGF-β) is a potent growth regulator and tumor suppressor in normal intestinal epithelium. Likewise, epithelial cell growth is controlled by rapid decay of growth-related mRNAs mediated through 3′ untranslated region (UTR) AU-rich element (ARE) motifs. We demonstrate that treatment of nontransformed intestinal epithelial cells with TGF-β inhibited ARE-mRNA expression. This effect of TGF-β was promoted through increased assembly of cytoplasmic RNA processing (P) bodies where ARE-mRNA localization was observed. P-body formation was dependent on TGF-β/Smad signaling, as Smad3 deletion abrogated P-body formation. In concert with increased P-body formation, TGF-β induced expression of the ARE-binding protein tristetraprolin (TTP), which colocalized to P bodies. TTP expression was necessary for TGF-β-dependent P-body formation and promoted growth inhibition by TGF-β. The significance of this was observed in vivo, where colonic epithelium deficient in TGF-β/Smad signaling or TTP expression showed attenuated P-body levels. These results provide new insight into TGF-βs antiproliferative properties and identify TGF-β as a novel mRNA stability regulator in intestinal epithelium through its ability to promote TTP expression and subsequent P-body formation.

Regulation of metastasis in colorectal adenocarcinoma : A collision between development and tumor biology

Metastases account for the vast majority of fatalities due to colon cancer. The metastatic phenotype, or the ability of normally polarized epithelial cells to separate from their tissue of origin, invade basement membrane, survive transit through the bloodstream and re-colonize at distal sites recapitulates a developmental process known as EMT. Regulators of EMT in adult epithelial cells, including the TGF-β and Wnt signal transduction systems, are important mediators of embryonic tissue development, tissue homeostasis, wound healing and they have important roles in tumor cell behaviors that are still being defined. Research into the potential epigenetic and reversible nature of EMT, and the downstream effector signaling pathways leading to EMT may enable the identification of novel therapeutic interventions to disrupt the metastatic process. Through a better understanding of the biological mechanisms that contribute to metastasis lies the hope of translating these discoveries into new treatments that will improve the survival of cancer patients.

Nuclear Factor of Activated T-cell Activity Is Associated with Metastatic Capacity in Colon Cancer

Metastatic recurrence is the leading cause of cancer-related deaths in patients with colorectal carcinoma. To capture the molecular underpinnings for metastasis and tumor progression, we performed integrative network analysis on 11 independent human colorectal cancer gene expression datasets and applied expression data from an immunocompetent mouse model of metastasis as an additional filter for this biologic process. In silico analysis of one metastasis-related coexpression module predicted nuclear factor of activated T-cell (NFAT) transcription factors as potential regulators for the module. Cells selected for invasiveness and metastatic capability expressed higher levels of NFATc1 as compared with poorly metastatic and less invasive parental cells. We found that inhibition of NFATc1 in human and mouse colon cancer cells resulted in decreased invasiveness in culture and downregulation of metastasis-related network genes. Overexpression of NFATc1 significantly increased the metastatic potential of colon cancer cells, whereas inhibition of NFATc1 reduced metastasis growth in an immunocompetent mouse model. Finally, we found that an 8-gene signature comprising genes upregulated by NFATc1 significantly correlated with worse clinical outcomes in stage II and III colorectal cancer patients. Thus, NFATc1 regulates colon cancer cell behavior and its transcriptional targets constitute a novel, biologically anchored gene expression signature for the identification of colon cancers with high risk of metastatic recurrence.

Four jointed box 1 promotes angiogenesis and is associated with poor patient survival in colorectal carcinoma.

Angiogenesis, the recruitment and re-configuration of pre-existing vasculature, is essential for tumor growth and metastasis. Increased tumor vascularization often correlates with poor patient outcomes in a broad spectrum of carcinomas. We identified four jointed box 1 (FJX1) as a candidate regulator of tumor angiogenesis in colorectal cancer. FJX1 mRNA and protein are upregulated in human colorectal tumor epithelium as compared with normal epithelium and colorectal adenomas, and high expression of FJX1 is associated with poor patient prognosis. FJX1 mRNA expression in colorectal cancer tissues is significantly correlated with changes in known angiogenesis genes. Augmented expression of FJX1 in colon cancer cells promotes growth of xenografts in athymic mice and is associated with increased tumor cell proliferation and vascularization. Furthermore, FJX1 null mice develop significantly fewer colonic polyps than wild-type littermates after combined dextran sodium sulfate (DSS) and azoxymethane (AOM) treatment. In vitro, conditioned media from FJX1 expressing cells promoted endothelial cell capillary tube formation in a HIF1-α dependent manner. Taken together our results support the conclusion that FJX1 is a novel regulator of tumor progression, due in part, to its effect on tumor vascularization.