Involvement of the Integrin α1ß1 in the Progression of Colorectal Cancer.

Integrins are a family of heterodimeric glycoproteins involved in bidirectional cell signaling that participate in the regulation of cell shape, adhesion, migration, survival and proliferation. The integrin α1ß1 is known to be involved in RAS/ERK proliferative pathway activation and plays an important role in fibroblast proliferation. In the small intestine, the integrin α1 subunit is present in the crypt proliferative compartment and absent in the villus. We have recently shown that the integrin α1 protein and transcript (ITGA1) are present in a large proportion of colorectal cancers (CRC) and that their expression is controlled by the MYC oncogenic factor. Considering that α1 subunit/ITGA1 expression is correlated with MYC in more than 70% of colon adenocarcinomas, we postulated that the integrin α1ß1 has a pro-tumoral contribution to CRC. In HT29, T84 and SW480 CRC cells, α1 subunit/ITGA1 knockdown resulted in a reduction of cell proliferation associated with an impaired resistance to anoikis and an altered cell migration in HT29 and T84 cells. Moreover, tumor development in xenografts was reduced in HT29 and T84 sh-ITGA1 cells, associated with extensive necrosis, a low mitotic index and a reduced number of blood vessels. Our results show that α1ß1 is involved in tumor cell proliferation, survival and migration. This finding suggests that α1ß1 contributes to CRC progression.

Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis.

Cathepsin B is a cysteine proteinase that primarily functions as an endopeptidase within endolysosomal compartments in normal cells. However, during tumoral expansion, the regulation of cathepsin B can be altered at multiple levels, thereby resulting in its overexpression and export outside of the cell. This may suggest a possible role of cathepsin B in alterations leading to cancer progression. The aim of this study was to determine the contribution of intracellular and extracellular cathepsin B in growth, tumorigenesis, and invasion of colorectal cancer (CRC) cells. Results show that mRNA and activated levels of cathepsin B were both increased in human adenomas and in CRCs of all stages. Treatment of CRC cells with the highly selective and non-permeant cathepsin B inhibitor Ca074 revealed that extracellular cathepsin B actively contributed to the invasiveness of human CRC cells while not essential for their growth in soft agar. Cathepsin B silencing by RNAi in human CRC cells inhibited their growth in soft agar, as well as their invasion capacity, tumoral expansion, and metastatic spread in immunodeficient mice. Higher levels of the cell cycle inhibitor p27(Kip1) were observed in cathepsin B-deficient tumors as well as an increase in cyclin B1. Finally, cathepsin B colocalized with p27(Kip1) within the lysosomes and efficiently degraded the inhibitor. In conclusion, the present data demonstrate that cathepsin B is a significant factor in colorectal tumor development, invasion, and metastatic spreading and may, therefore, represent a potential pharmacological target for colorectal tumor therapy.

Control of the human osteopontin promoter by ERRα in colorectal cancer.

Colorectal cancer is the second leading cause of death from cancer. Osteopontin (OPN) is a component of tumor extracellular matrix identified as a key marker of cancer progression. The estrogen-related receptor α (ERRα) has been implicated in endocrine-related cancer development and progression, possibly through modulation of cellular energy metabolism. Previous reports that ERRα regulates OPN expression in bone prompted us to investigate whether ERRα controls OPN expression in human colorectal cancer. Using a tissue microarray containing 83 tumor-normal tissue pairs of colorectal cancer samples, we found that tumor epithelial cells displayed higher staining for ERRα than normal mucosa, in correlation with elevated OPN expression. In addition, knocking down endogenous ERRα led to reduced OPN expression in HT29 colon cancer cells. Promoter analysis, inhibition of ERRα activity, and expression and mutation of potential ERRα response elements in the proximal promoter of human OPN showed that ERRα and its obligate co-activator, peroxisome proliferator-activated receptor γ co-activator-1 α, positively control human OPN promoter activity. Furthermore, chromatin immunoprecipitation experiments confirmed in vivo occupancy of the OPN promoter by ERRα in HT29 cells, suggesting that OPN is a direct target of ERRα in colorectal cancer. These findings suggest an additional mechanism by which ERRα participates in the development and progression of colorectal cancer, further supporting the relevance of targeting ERRα with antagonists as anticancer agents.

ERRα metabolic nuclear receptor controls growth of colon cancer cells.

The estrogen-related receptor alpha (ERRα) is a nuclear receptor that acts primarily as a regulator of metabolic processes, particularly in tissues subjected to high-energy demand. In addition to its control of energy metabolism and mitochondrial biogenesis, ERRα has recently been associated with cancer progression. Notably, increased expression of ERRα has been shown in several cancerous tissues, including breast, ovary and colon. However, additional studies are required to gain insight into the action of ERRα in cancer biology, particularly in non-endocrine-related cancers. Therefore, using a short hairpin RNA-mediated approach, we investigated whether ERRα is required for the rapid growth of colon cancer cells and to maintain their neoplastic metabolic state. Results show that silencing ERRα significantly impaired colon cancer cell proliferation and colony formation in vitro as well as their in vivo tumorigenic capacity. A pronounced delay in G1-to-S cell cycle phase transition was observed in ERRα-depleted cells in association with reduced cyclin-dependent kinase 2 activity and hyperphosphorylated state of the retinoblastoma protein along with disturbed expression of several cell cycle regulators, including p15 and p27. Interestingly, ERRα-depleted HCT116 cells also displayed significant reduction in expression of a large set of key genes to glycolysis, tricarboxylic acid cycle and lipid synthesis. Furthermore, using (14)C isotope tracer analysis, ERRα depletion in colon cancer cells resulted in reduced glucose incorporation and glucose-mediated lipogenesis in these cells. These findings suggest that ERRα coordinates colon cancer cell proliferation and tumorigenic capacity with energy metabolism. Thus, ERRα could represent a promising therapeutic target in colon cancer.

Autophagy is active in normal colon mucosa.

Recently, autophagy has been found to be strongly activated in colon cancer cells, but few studies have addressed the normal colon mucosa. The aim of this study was to characterize autophagy in normal human intestinal cells. We used the expression of LC3-II and BECN1 as well as SQSTM1 as markers of autophagy activity. Using the normal human intestinal epithelial crypt (HIEC) cell experimental model, we found that autophagy was much more active in undifferentiated cells than in differentiated cells. In the normal adult colonic mucosa, BECN1 was found in the proliferative epithelial cells of the lower part of the gland while SQSTM1 was predominantly found in the differentiated cells of the upper part of the gland and surface epithelium. Interestingly, the weak punctate pattern of SQSTM1 expression in the lower gland colocalized with BECN1-labeled autophagosomes. The usefulness of SQSTM1 as an active autophagy marker was confirmed in colon cancer specimens at the protein and transcript levels. In conclusion, our results show that autophagy is active in the colonic gland and is associated with the intestinal proliferative/undifferentiated and progenitor cell populations.

Chemopreventive effect of ERß-Selective agonist on intestinal tumorigenesis in Apc(Min/+) mice.

Epidemiological and experimental evidence suggests that estrogen replacement therapy reduces the risk of colon cancer in postmenopausal women. Estrogen receptor beta (ERß) is thought to be the principal mediator of the estrogen effect in the colon. Recent studies by our team suggested positive regulation of the transforming growth factor (TGF)ß pathway by estrogen in mice colonocytes. We therefore wanted to investigate the effects of ERß agonist treatment on intestinal tumorigenesis in Apc(Min/+) mice. Weaned Apc(Min/+) mice were injected subcutaneously three times a week for 12 wk with vehicle or ERß-selective agonist, diarylpropionitrile (DPN, 5 mg/kg). DPN administration resulted in a significant reduction in small intestinal polyp multiplicity in both Apc(Min/+) male and female mice. Furthermore, the mean diameter of small intestinal polyps was lower in DPN-treated than vehicle-treated males, along with lower BrdU incorporation indices in jejunal and colon epithelial cells of both sexes. DPN treatment also increased apoptosis in colon epithelium as measured by TUNEL assay and cleaved caspase 3 quantification. The effect of DPN on various components of the TGFß pathway was also studied in colonocytes. DPN treatment increased expression of TGFß1 and TGFß3 transcripts, levels of nuclear and phosphorylated Smad2 as well as p27 cell-cycle inhibitor, a TGFß pathway target gene. Our results demonstrate that DPN treatment reduces intestinal tumorigenesis in Apc(Min/+) mice. Furthermore, we suggest that positive regulation of the TGFß pathway by ERß activation could contribute to the protective role of estrogen in intestinal tumor development.

The PTEN phosphatase controls intestinal epithelial cell polarity and barrier function: role in colorectal cancer progression.

BACKGROUND: The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice. CONCLUSIONS/SIGNIFICANCE: Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells.

Estrogen receptor beta deficiency enhances small intestinal tumorigenesis in ApcMin/+ mice.

Clinical evidence suggests that estradiol replacement therapy reduces colon cancer risk in 'post'menopausal women. In colon epithelial cells, the estrogen receptor beta (ERbeta) is the predominant ER subtype and is thought to mediate the genomic effect of estrogens. The first aim of this study was to investigate the consequence of ERbeta deficiency on intestinal tumorigenesis in the Apc(Min/+) mouse model. Furthermore, to explore the biological mechanisms by which estrogens may influence the pathogenesis of colorectal cancer, we performed gene expression profiles in colonocytes from ovariectomized wild-type (WT) vs. ERbeta(-/-) mice, treated with estradiol (E(2)) or vehicle. Specifically in female, ERbeta deficiency was found to be associated with higher adenoma multiplicity in the small intestine, but not in the colon. Furthermore, tumors from ERbeta(-/-)Apc(Min/+) female mice were on average significantly larger than those from control Apc(Min/+) mice. Higher steady-state proliferation in epithelial cells of the jejunum and colon from ERbeta(-/-)Apc(Min/+) vs. Apc(Min/+) female mice was confirmed by BrdU incorporation assay. Interestingly, functional categorization of microarray results revealed the TGFbeta signaling pathway to be modulated in colonocytes, especially for the WT + E(2) vs. WT + Vehicle and the ERbeta(-/-) + E(2) vs. WT + E(2) comparisons. Using quantitative PCR analysis, we observed transcripts from ligands of the TGFbeta pathway to be upregulated in colonocytes from E(2)-treated WT and ERbeta(-/-) mice and downregulated in ERbeta-deficient mice, mostly in an E(2)-independent manner. Therefore, our results demonstrate that ERbeta deficiency enhances small intestinal tumorigenesis and suggest that modulation of the TGFbeta signaling pathway could contribute to the protective role of estrogens on intestinal tumorigenesis.

IL-1 beta-dependent regulation of C/EBP delta transcriptional activity.

We have previously shown that the transcription factor C/EBP delta is involved in the intestinal inflammatory response. C/EBP delta regulates several inflammatory response genes, such as haptoglobin, in the rat intestinal epithelial cell line IEC-6 in response to IL-1. However, the different C/EBP delta domains involved in IL-1 beta-mediated transcriptional activation and the kinases implicated have not been properly defined. To address this, we determined the role of the p38 MAP kinase in the regulation of C/EBP delta transcriptional activity. The IL-1-dependent induction of the acute phase protein gene haptoglobin in IEC-6 cells was decreased in response to the p38 MAP kinase inhibitor SB203580, as determined by Northern blot. Transcriptional activity of C/EBP delta was repressed by the specific inhibitor of the p38 MAP kinase, as assessed by transient transfection assays. Mutagenesis studies and transient transfection assays revealed an important domain for transcriptional activation between amino acids 70 and 108. This domain overlapped with a docking site for the p38 MAP kinase, between amino acids 75 and 85, necessary to insure C/EBP delta phosphorylation. Deletion of this domain led to a decrease in basal transcriptional activity of C/EBP delta and in p300-dependent transactivation, as assessed by transient transfection assays, and in IL-1-dependent haptoglobin induction. This unusual arrangement of a kinase docking site within a transactivation domain may functionally be important for the regulation of C/EBP delta transcriptional activity.

Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis.

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu515) to the beginning of S6 (Ala560). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro527 and Ile541 was compatible with the presence of a alpha-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions -1, 0, +1, +2 around Asp542 (high Ca2+ affinity site) were non-functional. Whole cell currents of cysteine mutants at +4 and +5 positions were however covalently inhibited by external MTSET and MTSES. Altogether, the pattern of covalent modification by MTS reagents globally supports a KcsA homology-based three-dimensional model whereby the external vestibule in ECaC-TRPV5 encompasses three structural domains consisting of a coiled structure (Glu515 to Tyr526) connected to a small helical segment of 15 amino acids (527PTALFSTFELFLT539) followed by two distinct coiled structures Ile540-Pro544 (selectivity filter) and Ala545-Ile557 before the beginning of S6.