The intestinal epithelial cell differentiation marker intestinal alkaline phosphatase (ALPi) is selectively induced by histone deacetylase inhibitors (HDACi) in colon cancer cells in a Kruppel-like factor 5 (KLF5)-dependent manner.

The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect.

Global protein profiling reveals anti-EGFR monoclonal antibody 806-modulated proteins in A431 tumor xenografts.

An important mediator of tumorigenesis, the epidermal growth factor receptor (EGFR) is expressed in almost all non-transformed cell types, associated with tumor progression, angiogenesis and metastasis. The significance of the EGFR as a cancer therapeutic target is underscored by the clinical development of several different classes of EGFR antagonists, including monoclonal antibodies (mAb) and tyrosine kinase inhibitors. Extensive preclinical studies have demonstrated the anti-tumor effects of mAb806 against tumor xenografts overexpressing EGFR. EGF stimulation of A431 cells induces rapid tyrosine phosphorylation of intracellular signalling proteins which regulate cell proliferation and apoptosis. Detailed understanding of the intracellular signalling pathways and components modulated by mAbs (such as mAb806) to EGFR, and other growth factor receptors, remain limited. The use of fluorescence 2D difference gel electrophoresis (2D DIGE), coupled with sensitive MS-based protein profiling in A431 tumor (epidermoid carcinoma) xenografts, in combination with mAb806, revealed proteins modulating endocytosis, cell architecture, apoptosis, cell signalling pathways and cell cycle regulation, including Dynamin-1-like protein, cofilin-1 protein, and 14-3-3 protein zeta/delta. Further, we report various proteins, including Interferon-induced protein 53 (IFI53), and Oncogene EMS1 (EMS1) which have roles in the tumor microenvironment, regulating cancer cell invasiveness, angiogenesis and formation of metastases. These findings contribute to understanding the underlying biological processes associated with mAb806 therapy of EGFR-positive tumors, and identifying further potential protein markers that may contribute in assessment of the treatment response.

Villin expression is frequently lost in poorly differentiated colon cancer.

Colorectal cancers (CRCs) are classified as having microsatellite instability (MSI) or chromosomal instability (CIN); herein termed microsatellite stable (MSS). MSI colon cancers frequently display a poorly differentiated histology for which the molecular basis is not well understood. Gene expression and immunohistochemical profiling of MSS and MSI CRC cell lines and tumors revealed significant down-regulation of the intestinal-specific cytoskeletal protein villin in MSI colon cancer, with complete absence in 62% and 17% of MSI cell lines and tumors, respectively. Investigation of 577 CRCs linked loss of villin expression to poorly differentiated histology in MSI and MSS tumors. Furthermore, mislocalization of villin from the membrane was prognostic for poorer outcome in MSS patients. Loss of villin expression was not due to coding sequence mutations, epigenetic inactivation, or promoter mutation. Conversely, in transient transfection assays villin promoter activity reflected endogenous villin expression, suggesting transcriptional control. A screen of gut-specific transcription factors revealed a significant correlation between expression of villin and the homeobox transcription factor Cdx-1. Cdx-1 overexpression induced villin promoter activity, Cdx-1 knockdown down-regulated endogenous villin expression, and deletion of a key Cdx-binding site within the villin promoter attenuated promoter activity. Loss of Cdx-1 expression in CRC lines was associated with Cdx-1 promoter methylation. These findings demonstrate that loss of villin expression due to Cdx-1 loss is a feature of poorly differentiated CRCs.

Intestinal epithelial-specific PTEN inactivation results in tumor formation.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of phosphatidylinositol 3-kinase (PI3K) signaling that is frequently inactivated in colorectal cancer through mutation, loss of heterozygosity, or epigenetic mechanisms. The aim of this study was to determine the effect of intestinal-specific PTEN inactivation on intestinal epithelial homeostasis and tumorigenesis. PTEN was deleted specifically in the intestinal epithelium, by crossing PTEN(Lox/Lox) mice with villin(Cre) mice. PTEN was robustly expressed in the intestinal epithelium and maximally in the differentiated cell compartment. Targeted inactivation of PTEN in the intestinal epithelium of PTEN(Lox/Lox)/villin(Cre) mice was confirmed by genotyping, immunohistochemistry, and qPCR. While intestinal-specific PTEN deletion did not have a major effect on cell fate determination or proliferation in the small intestine, it did increase phosphorylated (p) protein kinase B (AKT) expression in the intestinal epithelium, and 19% of animals developed small intestinal adenomas and adenocarcinomas at 12 mo of age. These tumors demonstrated pAKT and nuclear ß-catenin staining, indicating simultaneous activation of the PI3K/AKT and Wnt signaling pathways. These findings demonstrate that, while PTEN inactivation alone has a minimal effect on intestinal homeostasis, it can facilitate tumor promotion upon deregulation of ß-catenin/TCF signaling, further establishing PTEN as a bona fide tumor suppressor gene in intestinal cancer.

MUC2 mucin deficiency alters inflammatory and metabolic pathways in the mouse intestinal mucosa.

The mucus layer in the intestine affects several aspects of intestinal biology, encompassing physical, chemical protection, immunomodulation and growth, thus contributing to homeostasis. Mice with genetic inactivation of the Muc2 gene, encoding the MUC2 mucin, the major protein component of mucus, exhibit altered intestinal homeostasis, which is strictly dependent on the habitat, likely due to differing complements of intestinal microbes. Our previous work established that Muc2 deficiency was linked to low chronic inflammation resulting in tumor development in the small, large intestine including the rectum. Here, we report that inactivation of Muc2 alters metabolic pathways in the normal appearing mucosa of Muc2-/- mice. Comparative analysis of gene expression profiling of isolated intestinal epithelial cells (IECs) and the entire intestinal mucosa, encompassing IECs, immune and stromal cells underscored that more than 50% of the changes were common to both sets of data, suggesting that most alterations were IEC-specific. IEC-specific expression data highlighted perturbation of lipid absorption, processing and catabolism linked to altered Pparα signaling in IECs. Concomitantly, alterations of glucose metabolism induced expression of genes linked to de novo lipogenesis, a characteristic of tumor cells. Importantly, gene expression alterations characterizing Muc2-/- IECs are similar to those observed when analyzing the gene expression signature of IECs along the crypt-villus axis in WT B6 mice, suggesting that Muc2-/- IECs display a crypt-like gene expression signature. Thus, our data strongly suggest that decreased lipid metabolism, and alterations in glucose utilization characterize the crypt proliferative compartment, and may represent a molecular signature of pre-neoplastic lesions.

A gene expression profile that defines colon cell maturation in vitro.

Colonic epithelial cells undergo cell cycle arrest, lineage specific differentiation, and apoptosis, as they migrate along the crypt axis toward the lumenal surface. The Caco-2 colon carcinoma cell line models many of these phenotypic changes, in vitro. We used this model system and cDNA microarray analysis to characterize the genetic reprogramming that accompanies colon cell differentiation. The analyses revealed extensive yet functionally coordinated alterations in gene expression during the differentiation program. Consistent with cell differentiation reflecting a more specialized phenotype, the majority of changes (70%) were down-regulations of gene expression. Specifically, Caco-2 cell differentiation was accompanied by the coordinate down-regulation of genes involved in cell cycle progression and DNA synthesis, which reflected the concomitant reduction in cell proliferation. Simultaneously, genes involved in RNA splicing and transport, protein translation, folding, and degradation, were coordinately down-regulated, paralleled by a reduction in protein synthesis. Conversely, genes involved in xenobiotic and drug metabolism were up-regulated, which was linked to increased resistance of differentiated cells to chemotherapeutic agents. Increased expression of genes involved in extracellular matrix deposition, lipid transport, and lipid metabolism were also evident. Underlying these altered profiles of expression, components of signal transduction pathways, and several transcription factors were altered in expression.

Gene expression profiling-based prediction of response of colon carcinoma cells to 5-fluorouracil and camptothecin.

5-Fluorouracil (5-FU) is the most common chemotherapeutic agent used in the treatment of colorectal cancer, yet objective response rates are low. Recently, camptothecin (CPT) has emerged as an effective alternative therapy. Decisive means to determine treatment, based on the likelihood of response to each of these agents, could greatly enhance the management of this disease. Here, the ability of cDNA microarray-generated basal gene expression profiles to predict apoptotic response to 5-FU and CPT was determined in a panel of 30 colon carcinoma cell lines. Genes whose basal level of expression correlated significantly with 5-FU- and CPT-induced apoptosis were selected, and their predictive power was assessed using a "leave one out" jackknife cross-validation strategy. Selection of the 50 genes best correlated with 5-FU-induced apoptosis, but not 50 randomly selected genes, significantly predicted response to this agent. Importantly, this gene expression profiling approach predicted response more effectively than four previously established determinants of 5-FU response: thymidylate synthase and thymidine phosphorylase activity; and p53 and mismatch repair status. Furthermore, reanalysis of the database demonstrated that selection of the 149 genes best correlated with CPT-induced apoptosis maximally and significantly predicted response to this agent. These studies demonstrate that the basal gene expression profile of colon cancer cells can be used to predict and distinguish response to multiple chemotherapeutic agents and establish the potential of this methodology as a means by which rational decisions regarding choice of therapy can be approached.

Altered dynamics of intestinal cell maturation in Apc1638N/+ mice.

Novel imaging of active transcription sites in interphase nuclei of intestinal epithelial cells in situ showed that key genes associated with Wnt and Notch signaling were dynamically regulated as the cells underwent normal maturation during their migration along the mouse crypt-villus axis (CVA). However, oscillating patterns of activation of these genes were displaced along this axis in the histologically normal intestinal mucosa of Apc(1638N/+) mice before tumor development. Gene expression profiling then showed that the normal reprogramming of cells along the CVA was dampened in the Apc(1638N/+) mice, with an overrepresentation of c-myc target genes among those loci affected in the mutant mice. Moreover, in the Apc(1638N/+) mice, there was a perturbed pattern of expression of lineage-specific markers along the CVA consistent with transcription site repression of the Math1 gene, and genes encoding enzymes of every step of the tricarboxylic acid cycle were downregulated in the crypt of Apc(1638N/+) mice compared with WT, but not in the villus. These changes may alter energy metabolism and generate a pseudohypoxic state, suggested by elevated expression of Hif1alpha and its target genes. Thus, although intestinal tumors develop in Apc(1638N/+) mice on focal loss or inactivation of the WT allele, our results show that in the Apc(1638N/+) mouse, inheritance of only a single WT Apc allele perturbs the dynamic and complex reprogramming underlying normal cell maturation, which links epithelial function and homeostasis with architectural organization of the intestine.

Gene expression profiling of primary and metastatic colon cancers identifies a reduced proliferative rate in metastatic tumors.

The objective of this study was to gain insights into the biological basis of the metastatic process by characterizing the gene expression differences between primary and metastatic colon cancers. Recent studies have demonstrated that few new mutational changes are acquired during the metastatic progression of colon tumors [Jones et al., Proc Natl Acad Sci USA 105 (11): 4283-4288, 2008]. However, the extent to which epigenetic and transcriptional changes occur between primary and metastatic colon cancer remains unknown. We approached these issues using Affymetrix microarrays to assess the similarities and differences in gene expression profiles between macro-dissected primary and metastatic colon tumors. Unexpectedly, we found that expression of a number of cell proliferation markers were reduced in the liver metastases of colon tumors when compared to primary tumors. This finding was validated by immunohistochemical staining of Ki67 and Cyclin D1 in Formalin-Fixed Paraffin-Embedded (FFPE) section of the same samples, and in an independent cohort of FFPE matched tumor and metastatic tissue samples. These results indicate that significant transcriptional differences exist between primary and metastatic colon tumors, and demonstrate that metastatic lesions have a lower proliferative rate compared to primary tumors. These findings may have implications for interpreting differences in response rates between primary and metastatic lesions and suggest that measurement of expression-based biomarkers in metastatic tissue will be most informative for understanding the basis of response of metastatic tumors to therapeutic intervention.

Apoptotic sensitivity of colon cancer cells to histone deacetylase inhibitors is mediated by an Sp1/Sp3-activated transcriptional program involving immediate-early gene induction.

Histone deacetylase inhibitors (HDACi) induce growth arrest and apoptosis in colon cancer cells and are being considered for colon cancer therapy. The underlying mechanism of action of these effects is poorly defined with both transcription-dependent and -independent mechanisms implicated. We screened a panel of 30 colon cancer cell lines for sensitivity to HDACi-induced apoptosis and correlated the differences with gene expression patterns induced by HDACi in the five most sensitive and resistant lines. A robust and reproducible transcriptional response involving coordinate induction of multiple immediate-early (fos, jun, egr1, egr3, atf3, arc, nr4a1) and stress response genes (Ndrg4, Mt1B, Mt1E, Mt1F, Mt1H) was selectively induced in HDACi sensitive cells. Notably, a significant percentage of these genes were basally repressed in colon tumors. Bioinformatics analysis revealed that the promoter regions of the HDACi-induced genes were enriched for KLF4/Sp1/Sp3 transcription factor binding sites. Altering KLF4 levels failed to modulate apoptosis or transcriptional responses to HDACi treatment. In contrast, HDACi preferentially stimulated the activity of Spl/Sp3 and blocking their action attenuated both the transcriptional and apoptotic responses to HDACi treatment. Our findings link HDACi-induced apoptosis to activation of a Spl/Sp3-mediated response that involves derepression of a transcriptional network basally repressed in colon cancer.

DR5 receptor mediates anoikis in human colorectal carcinoma cell lines.

As human colorectal cancer (CRC) cells metastasize to distant sites, they are susceptible to detachment-induced cell death or anoikis - a form of apoptosis that occurs when anchorage-dependent CRC cells go into suspension. Our goal was to identify whether tumor necrosis factor receptor apoptosis-inducing ligand (TRAIL) receptors mediate anoikis in human CRC cells. First, we assessed whether caspases of the extrinsic (caspase-8) or intrinsic (caspase-9) death pathways were involved. Caspase-8 was cleaved during exposure to suspension culture in four CRC lines, and cell death was inhibited by caspase-3 and caspase-8 inhibitors but not by a caspase-9 inhibitor. Gene transcripts in macrophage inflammatory protein-101 (MIP-110), a weakly metastatic human CRC, were increased at least 2-fold for TRAIL-R2 (DR5) and TRAIL after 24 h of suspension culture compared with cells in monolayer culture. The increased expression of DR5 was confirmed at the protein level at 24 h, and exposure of MIP-101 cells to an antagonistic antibody to DR5 decreased caspase-8 activation. The antagonistic antibody to DR5 inhibited anoikis in four human CRC lines. Treatment with an antagonistic DR4 antibody or a neutralizing antibody to TRAIL ligand did not reduce anoikis consistently. Knockdown of DR5 or TRAIL also inhibited anoikis, whereas exogenous TRAIL or FasL did not consistently increase anoikis. In summary, DR5 receptor mediates death signals for anoikis in human CRC cells through the extrinsic apoptotic pathway.

Dietary induction of colonic tumors in a mouse model of sporadic colon cancer.

A defined rodent "new Western diet" (NWD), which recapitulates intake levels of nutrients that are major dietary risk factors for human colon cancer, induced colonic tumors when fed to wild-type C57Bl/6 mice for 1.5 to 2 years from age 6 weeks (two-thirds of their life span). Colonic tumors were prevented by elevating dietary calcium and vitamin D(3) to levels comparable with upper levels consumed by humans, but tumorigenesis was not altered by similarly increasing folate, choline, methionine, or fiber, each of which was also at the lower levels in the NWD that are associated with risk for colon cancer. The NWD significantly altered profiles of gene expression in the flat colonic mucosa that exhibited heterogeneity among the mice, but unsupervised clustering of the data and novel statistical analyses showed reprogramming of colonic epithelial cells in the flat mucosa by the NWD was similar to that initiated by inheritance of a mutant Apc allele. The NWD also caused general down-regulation of genes encoding enzymes involved in lipid metabolism and the tricarboxylic acid cycle in colonic epithelial cells before tumor formation, which was prevented by the supplementation of the NWD with calcium and vitamin D(3) that prevented colon tumor development, demonstrating profound interaction among nutrients. This mouse model of dietary induction of colon cancer recapitulates levels and length of exposure to nutrients linked to relative risk for human sporadic colon cancer, which represents the etiology of >90% of colon cancer in the United States and other Western countries.

Proteomic changes during intestinal cell maturation in vivo.

Intestinal epithelial cells undergo progressive cell maturation as they migrate along the crypt-villus axis. To determine molecular signatures that define this process, proteins differentially expressed between the crypt and villus were identified by 2D-DIGE and MALDI-MS. Forty-six differentially expressed proteins were identified, several of which were validated by immunohistochemistry. Proteins upregulated in the villus were enriched for those involved in brush border assembly and lipid uptake, established features of differentiated intestinal epithelial cells. Multiple proteins involved in glycolysis were also upregulated in the villus, suggesting increased glycolysis is a feature of intestinal cell differentiation. Conversely, proteins involved in nucleotide metabolism, and protein processing and folding were increased in the crypt, consistent with functions associated with cell proliferation. Three novel paneth cell markers, AGR2, HSPA5 and RRBP1 were also identified. Notably, significant correlation was observed between overall proteomic changes and corresponding gene expression changes along the crypt-villus axis, indicating intestinal cell maturation is primarily regulated at the transcriptional level. This proteomic profiling analysis identified several novel proteins and functional processes differentially induced during intestinal cell maturation in vivo. Integration of proteomic, immunohistochemical, and parallel gene expression datasets demonstrate the coordinated manner in which intestinal cell maturation is regulated.

Quantitative rather than qualitative differences in gene expression predominate in intestinal cell maturation along distinct cell lineages.

Several cell types are present in the intestinal epithelium that likely arise from a common precursor, the stem cell, and each mature cell type expresses a unique set of genes that characterizes its functional phenotype. Although the process of differentiation is intimately linked to the cessation of proliferation, the mechanisms that dictate intestinal cell fate determination are not well characterized. To investigate the reprogramming of gene expression during the cell lineage allocation/differentiation process, we took advantage of a unique system of two clonal derivatives of HT29 cells, Cl16E and Cl19A cells, which spontaneously differentiate as mucus producing goblet and chloride-secreting cells, respectively, as a function of time. By profiling gene expression, we found that these two cell lines show remarkably similar kinetics of change in gene expression and common clusters of coordinately regulated genes. This demonstrates that lineage-specific differentiation of intestinal epithelial cells is characterized overall by the sequential recruitment of functionally similar gene sets independent of the final phenotype of the mature cells.

Gene expression profiling of intestinal epithelial cell maturation along the crypt-villus axis.

BACKGROUND & AIMS: To define the genetic reprogramming that drives intestinal epithelial cell maturation along the crypt-villus axis, enterocytes were sequentially isolated from the villus tip to the crypts of mouse small intestine. METHODS: Changes in gene expression were assessed using 27,405-element complementary DNA microarrays (14,685 unique genes) and specific changes validated by Western blotting. RESULTS: A total of 1113 genes differentially expressed between the crypt and villus were identified. Among these, established markers of absorptive and goblet cell differentiation were up-regulated in villus cells, whereas Paneth cell markers were maximally expressed in crypt cells. The 1113 differentially expressed genes were significantly enriched for genes involved in cell cycle progression, RNA processing, and translation (all predominantly down-regulated during maturation) and genes involved in cytoskeleton assembly and lipid uptake (predominantly up-regulated during maturation). No enrichment for apoptosis-regulating genes was observed. We confirmed that Wnt signaling was maximal in the proliferative compartment and observed a decrease in MYC and an increase in MAD and MAX expression during the maturation program. Consistent with these changes, the 1113 genes were enriched for MYC targets, establishing the importance of this network in intestinal cell maturation. CONCLUSIONS: This database serves as a resource for understanding the molecular mechanisms of intestinal cell maturation and for dissection of how perturbations in the maturation process can lead to changes in gastrointestinal physiology and pathology, particularly intestinal tumorigenesis.

Oncogenic Ki-ras inhibits the expression of interferon-responsive genes through inhibition of STAT1 and STAT2 expression.

Endogenous interferon gamma (IFNgamma) promotes the host response to primary tumors, and IFNgamma-insensitive tumors display increased tumorigenicity and can evade tumor surveillance mechanisms. Here we demonstrate that activating mutations of Ki-ras are sufficient to inhibit the expression of STAT1 and STAT2, transcription factors required for signaling by IFNs, providing a potential mechanism for the insensitivity of tumors to IFNs. We demonstrated that colon cancer cell lines with Ki-ras mutations display reduced expression of IFN-responsive genes compared with the cell lines that have retained wild type Ras and that inactivation of the mutant Ki-ras allele in the HCT116 colon cancer cell line is sufficient to restore the expression of STAT1, STAT2, and IRF-9. Accordingly, the expression of 27 interferon-inducible genes was reduced in HCT116 cells compared with the isogenic clones with targeted deletion of the mutant Ki-ras allele, Hkh2 and Hke-3. The expression of IFNgamma receptors did not differ among the isogenic cell lines. IFNgamma stimulated transcription of a STAT1-dependent reporter gene was impaired by RasV12, demonstrating a transmodulation of IFN/STAT signaling by activated Ras. Finally, we demonstrated that the expression of RasV12 in 293T cells is sufficient to inhibit the endogenous expression of STAT1 and STAT2, confirming the negative regulation of IFN signaling by oncogenic Ras. Our data demonstrate that the signaling initiated by activated Ki-ras interferes with the IFN/STAT signaling pathway and modulates the responsiveness of cancer cells to interferons. Furthermore, the data suggest that tumors harboring activating Ki-ras mutations may escape tumor surveillance mechanisms due to reduced responsiveness to IFNgamma.

Application of gene expression profiling to colon cell maturation, transformation and chemoprevention.

Methods for high-throughput analysis of profiles of gene expression that assay thousands of genes simultaneously are powerful approaches for understanding and classifying cell and tissue phenotype. This includes analysis of normal pathways of cell maturation and their perturbation in transformation, the sensitivity and mechanism of response of normal and tumor cells to physiological and pharmacological agents, and modulation of tumor risk and progression by nutritional factors. However, the complex data generated by such approaches raise difficulties in analysis. We will describe some of the methods we have used in analyzing databases generated in a number of projects in our laboratories. These include: the role of k-ras mutations in colon cell transformation; the role of p21(WAF1/cip1) in intestinal tumor formation and response to sulindac; the development of the absorptive and goblet cell lineages; sensitivity of colonic cells to chemotherapeutic agents; mechanisms that regulate c-myc expression utilizing novel methods of transcriptional imaging; and interaction of nutritional and genetic factors in modulation of intestinal tumor formation.