Gérald Bernatchez

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 β 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 β (ERβ) 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 ERβ deficiency on intestinal tumorigenesis in the ApcMin/+ 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. ERβ−/− mice, treated with estradiol (E2) or vehicle. Specifically in female, ERβ deficiency was found to be associated with higher adenoma multiplicity in the small intestine, but not in the colon. Furthermore, tumors from ERβ−/−ApcMin/+ female mice were on average significantly larger than those from control ApcMin/+ mice. Higher steady‐state proliferation in epithelial cells of the jejunum and colon from ERβ−/−ApcMin/+ vs. ApcMin/+ female mice was confirmed by BrdU incorporation assay. Interestingly, functional categorization of microarray results revealed the TGFβ signaling pathway to be modulated in colonocytes, especially for the WT + E2 vs. WT + Vehicle and the ERβ−/− + E2 vs. WT + E2 comparisons. Using quantitative PCR analysis, we observed transcripts from ligands of the TGFβ pathway to be upregulated in colonocytes from E2‐treated WT and ERβ−/− mice and downregulated in ERβ‐deficient mice, mostly in an E2‐independent manner. Therefore, our results demonstrate that ERβ deficiency enhances small intestinal tumorigenesis and suggest that modulation of the TGFβ signaling pathway could contribute to the protective role of estrogens on intestinal tumorigenesis.

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 p27Kip1 were observed in cathepsin B‐deficient tumors as well as an increase in cyclin B1. Finally, cathepsin B colocalized with p27Kip1 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.

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.

Chemopreventive effect of ERβ-Selective agonist on intestinal tumorigenesis in ApcMin/+ Mice†

Epidemiological and experimental evidence suggests that estrogen replacement therapy reduces the risk of colon cancer in postmenopausal women. Estrogen receptor beta (ERβ) 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)β pathway by estrogen in mice colonocytes. We therefore wanted to investigate the effects of ERβ agonist treatment on intestinal tumorigenesis in ApcMin/+ mice. Weaned ApcMin/+ mice were injected subcutaneously three times a week for 12 wk with vehicle or ERβ‐selective agonist, diarylpropionitrile (DPN, 5 mg/kg). DPN administration resulted in a significant reduction in small intestinal polyp multiplicity in both ApcMin/+ 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 TGFβ pathway was also studied in colonocytes. DPN treatment increased expression of TGFβ1 and TGFβ3 transcripts, levels of nuclear and phosphorylated Smad2 as well as p27 cell‐cycle inhibitor, a TGFβ pathway target gene. Our results demonstrate that DPN treatment reduces intestinal tumorigenesis in ApcMin/+ mice. Furthermore, we suggest that positive regulation of the TGFβ pathway by ERβ activation could contribute to the protective role of estrogen in intestinal tumor development.

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.

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.

Involvement of the Integrin α1β1 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 α1β1 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 α1β1 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 α1β1 is involved in tumor cell proliferation, survival and migration. This finding suggests that α1β1 contributes to CRC progression.

Abstract 5090: α1β1 is pro-proliferative and promigratory integrin and its expression is upregulated by the MYC oncogenic factor in colorectal cancer

Background Colorectal cancer (CRC) is a multi-step process that involves successive mutation, epigenetic alteration and gene dysregulation. Integrins are a family of heterodimeric glycoproteins involved in bidirectional cell signaling and participate in the regulation of cell shape, adhesion, migration, survival and proliferation. The integrin α1 subunit is known to be involved in RAS/ERK proliferative pathway activation and plays an important role in mammary carcinoma cell proliferation and migration. In the small intestine, α1 is present in the crypt proliferative compartment and absent in the villus. In mouse models, the α1β1 integrin, together with the Kras oncogenic factor, potentiates tumor growth. Very little is known about α1β1 function in CRC. Aims: As we have recently shown that α1 is present in 65% of CRC (Boudjadi et al, 2013), that its expression is controlled by the MYC oncogenic factor and that the expressions of α1 and MYC correlate in 72.3% of colon adenocarcinomas (Boudjadi et al, Oncogene 2015) we postulated that integrin α1β1 has a pro-tumoral contribution to CRC related to α1 function. METHODS: α1β1 function was studied in HT29, T84 and SW480 CRC cell lines using shRNA silencing targeting α1 (shα1) compared to an shRNA control (shCtrl). Cell proliferation was assessed by cell count and BrdU incorporation. Migration was tested by the scratch test assay. For the anoikis test, cells were kept in suspension without serum for 24 hours on poly-2-hydroxyethyl methacrylate (polyHEMA)-coated dishes and were then lysed and subjected to caspase3 activity measurement and cleaved PARP expression. To test tumorigenic capacity, shα1 and shCtrl HT29 cells were injected into the dorsal subcutaneous tissue of female CD1 nu/nu mice. The tumor volume was assessed by external measurement. After resection, α1 knockdown was confirmed at the mRNA and protein levels. RESULTS: In HT29, T84 and SW480 cells, α1 mRNA silencing resulted in reduced cell growth and proliferation compared to the control. Caspase3 activity measurement and cleaved PARP expression in HT29 and T84 cells showed that resistance to anoikis was altered in shα1 cells compared to shCtrl. Wound healing was delayed in shα1 HT29 and T84 cells compared to shCtrl. Moreover, tumor development in xenografts was reduced in HT29 shα1 cells. Histopathological analysis showed extensive necrosis areas and low mitotic index in shα1 tumors compared to the shCtrl tumors. CONCLUSION: Our results show that α1β1 is involved in tumor cell proliferation, survival and migration. This finding suggests that α1β1 is involved in colorectal cancer progression. (Supported by the CIHR) Citation Format: Salah Boudjadi, Gerald Bernatchez, Blanche Senicourt, Marco Beausejour, Pierre-Henri Vachon, Julie Carrier, Jean-Francois Beaulieu. α1β1 is pro-proliferative and promigratory integrin and its expression is upregulated by the MYC oncogenic factor in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5090.

Abstract 2045: Intriguing role of estrogen-related receptor alpha (ERRα) in colorectal cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Besides its well-known function in energetic metabolism, the nuclear receptor Estrogen-Related Receptor Alpha (ERRα) has also been shown to be overexpressed in breast, ovarian, uterine, prostate and colon cancer and to be associated to a poor prognosis in some of these cancer types. Interestingly, an ERRα splice variant mainly expressed in non cancerous tissues has been reported. This variant missing the fifth exon (ERRα Δ5) is still expected to interact with DNA and form dimers but cannot recruit coactivators, therefore being devoid of any transcriptional activity. AIM: Determine the role of ERRα as well as ERRα Δ5 in intestinal tumorigenesis. METHODS: shRNA-mediated silencing of ERRα and overexpression of ERRα Δ5 were performed by lentivirus infection in DLD1 and HCT116 human colon cancer cells. The interaction between both proteins was investigated as well as their respective roles in various tumorigenic processes. RESULTS: Silencing of ERRα by shRNA reduced proliferation of DLD1 and HCT116 cells as measured by growth kinetics and anchorage-independent growth assays in soft agar. We also observed that ERRα knockdown cells grew into smaller tumors when subcutaneously implanted in NUDE mice. Furthermore, FACS-scan analysis revealed that ERRα silencing delays G1 to S phase transition in colon cancer cells. Differential expression of a splice variant of ERRα (ERRα Δ5) in cancer vs normal tissues would support the importance of ERRα role in colorectal carcinogenesis. This is the case at least for colon tumors as cancerous samples display lower amount of ERRα Δ5 and higher amount of ERRα compared to adjacent paired normal tissues, as measured by Western Blot analysis. Interaction between both variants has been characterized by immunoprecipitation assays, confirming the conserved dimerization potential of ERRα Δ5 variant with the full length ERRα. Furthermore, luciferase assays revealed that ERRα Δ5 is transcriptionaly inactive and its expression inhibits the transcriptional activity of ERRα on its target genes, suggesting that this variant acts as a dominant negative for ERRα. Interestingly, the reintroduction of ERRα Δ5 in human colon cancer cells leads to the reduction of cell growth and soft agar colony formation. CONCLUSION: Strong ERRα expression is associated with cancerous tissues and is required for the intense proliferation of colon cancer cells. Interestingly, the poorly active splice variant ERRα Δ5 is lost in cancerous colon tissues and has antiproliferative properties when reintroduced in colon cancer cells. Since this splice variant could interact with ERRα and inhibit its activity, ERRα Δ5 reduction of expression in cancerous tissue could allow ERRα to fully promote colon cancer cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2045. doi:10.1158/1538-7445.AM2011-2045