Salah Boudjadi

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.

Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells

Summary Integrin α6Aβ4 is up-regulated in colorectal cancers. Knockdown of α6A in adenocarcinoma cell lines revealed a sustained reduction of cell growth both in cellulo and in xenografts as well as a repression of a number of Wnt/β-catenin pathway end points.

Integrin α1β1 expression is controlled by c-MYC in colorectal cancer cells.

The α1β1 collagen receptor is only present in a few epithelial cell types. In the intestine, it is specifically expressed in proliferating crypt cells. This integrin has been reported to be involved in various cancers where it mediates the downstream activation of the Ras/ERK proliferative pathway. We have recently shown that integrin α1β1 is present in two-thirds of colon adenocarcinomas, but the mechanism by which ITGA1 expression is regulated is not known. DNA methylation, involved in ITGA1 repression during megakaryocyte differentiation, is not the mechanism of ITGA1 regulation in colorectal cancer cells. Our in silico analysis of the ITGA1 promoter revealed two response elements for MYC, an oncogenic factor known to regulate cancer cell proliferation, invasion and migration. In situ, the expressions of both MYC and ITGA1 are localized in the lower crypt of the normal colon and correlate in 72% of the 65 analyzed colorectal cancers. MYC pharmacological inhibition or downregulation of expression with short hairpin RNA in HT29, T84 and SW480 cells resulted in reduced ITGA1 expression at both the transcript and protein levels. Chromatin immunoprecipitation assays revealed that MYC was bound to the chromatin region of the ITGA1 proximal promoter, whereas MYC overexpression enhanced ITGA1 promoter activity that was reduced with MAD co-transfection or by the disruption of the response elements. We concluded that MYC is a key regulating factor for the control of ITGA1 expression.

Integrin α1 subunit is up-regulated in colorectal cancer

BackgroundColorectal cancer remains one of the leading causes of death from cancer in industrialized countries. Integrins are a family of heterodimeric glycoproteins involved in bidirectional cell signaling and participate in the regulation of cell shape, adhesion, migration, differentiation, gene transcription, survival and proliferation. The 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, but nothing is known about its expression in the colon mucosa, or in colorectal cancer.ResultsIn the present study, we demonstrated that in the colon mucosa, α1 is present in the basolateral domain of the proliferative cells of the crypt, and in the surrounding myofibroblasts. We found higher levels of α1 mRNA in 86% of tumours compared to their corresponding matched margin tissues. Immunohistochemical analysis showed that α1 staining was moderate to high in 65% of tumour cells and 97% of the reactive cells surrounding the tumour cells vs 23% of normal epithelial cells.ConclusionOur findings suggest an active role for the α1β1 integrin in colorectal cancer progression.

Epigenetics in Intestinal Epithelial Cell Renewal

A controlled balance between cell proliferation and differentiation is essential to maintain normal intestinal tissue renewal and physiology. Such regulation is powered by several intracellular pathways that are translated into the establishment of specific transcription programs, which influence intestinal cell fate along the crypt‐villus axis. One important check‐point in this process occurs in the transit amplifying zone of the intestinal crypts where different signaling pathways and transcription factors cooperate to manage cellular proliferation and differentiation, before secretory or absorptive cell lineage terminal differentiation. However, the importance of epigenetic modifications such as histone methylation and acetylation in the regulation of these processes is still incompletely understood. There have been recent advances in identifying the impact of histone modifications and chromatin remodelers on the proliferation and differentiation of normal intestinal crypt cells. In this review we discuss recent discoveries on the role of the cellular epigenome in intestinal cell fate, development, and tissue renewal. J. Cell. Physiol. 231: 2361–2367, 2016.

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.

MYC Regulates α6 Integrin Subunit Expression and Splicing Under Its Pro-Proliferative ITGA6A Form in Colorectal Cancer Cells

The α6 integrin subunit (ITGA6) pre-mRNA undergoes alternative splicing to form two splicing variants, named ITGA6A and ITGA6B. In primary human colorectal cancer cells, the levels of both ITGA6 and β4 integrin subunit (ITGB4) subunits of the α6β4 integrin are increased. We previously found that the upregulation of ITGA6 is a direct consequence of the increase of the pro-proliferative ITGA6A variant. However, the mechanisms that control ITGA6 expression and splicing into the ITGA6A variant over ITGA6B in colorectal cancer cells remain poorly understood. Here, we show that the promoter activity of the ITGA6 gene is regulated by MYC. Pharmacological inhibition of MYC activity with the MYC inhibitor (MYCi) 10058-F4 or knockdown of MYC expression by short hairpin RNA (shRNA) both lead to a decrease in ITGA6 and ITGA6A levels in colorectal cancer cells, while overexpression of MYC enhances ITGA6 promoter activity. We also found that MYC inhibition decreases the epithelial splicing regulatory protein 2 (ESRP2) splicing factor at both the mRNA and protein levels. Chromatin immunoprecipitation revealed that the proximal promoter sequences of ITGA6 and ESRP2 were occupied by MYC and actively transcribed in colorectal cancer cells. Furthermore, expression studies in primary colorectal tumors and corresponding resection margins confirmed that the up-regulation of the ITGA6A subunit can be correlated with the increase in MYC and ESRP2. Taken together, our results demonstrate that the proto-oncogene MYC can regulate the promoter activation and splicing of the ITGA6 integrin gene through ESRP2 to favor the production of the pro-proliferative ITGA6A variant in colorectal cancer cells.

Neoexpression of a functional primary cilium in colorectal cancer cells.

The Hedgehog (HH) signaling pathway is involved in the maintenance of numerous cell types both during development and in the adult. Often deregulated in cancers, its involvement in colorectal cancer has come into view during the last few years, although its role remains poorly defined. In most tissues, the HH pathway is highly connected to the primary cilium (PC), an organelle that recruits functional components and regulates the HH pathway. However, normal epithelial cells of the colon display an inactive HH pathway and lack a PC. In this study, we report the presence of the PC in adenocarcinoma cells of primary colorectal tumors at all stages. Using human colorectal cancer cell lines we found a clear correlation between the presence of the PC and the expression of the final HH effector, GLI1, and provide evidence of a functional link between the two by demonstrating the recruitment of the SMO receptor to the membrane of the primary cilium. We conclude that the primary cilium directly participates in the HH pathway in colorectal cancer cells.

In silico Analysis and Site-directed Mutagenesis of Promoters

[Abstract] In normal as in cancerous cells, gene expression is tightly regulated by transcription factors, which are responsible for upor down-regulation of thousands of targets involved in different cell processes. Transcription factors can directly regulate the expression of genes by binding to specific DNA sequences known as response elements. Identification of these response elements is important to characterize targets of transcription factors in order to understand their contribution to gene regulation. Here, we describe in silico analysis coupled to selected mutagenesis and promoter gene reporter assay procedures to identify and analyze response elements in the proximal promoter sequence of genes.

ITGA1 (integrin, alpha 1)

The α1 integrin subunit (SU) belongs to a large family of α and β subunits that are noncovalently linked to constitute αβ transmembrane units. To date, 18 α and 8 β subunits are known to form 24 αβ units (Takada et al., 2007; Barczyk et al., 2010) which are involved in cell-cell and cell-matrix attachment and can drive inside-out and outside-in cell signaling (Shattil et al., 2010). Integrins are known to participate in different cell processes including cell shape, differentiation, migration, survival and proliferation (Giancotti, 1997; Vachon, 2011; Beauséjour et al., 2012). The α1 SU was discovered in 1986 as the Very Late Antigen 1 (VLA1) and is highly expressed in activated lymphocytes in the joints of patients with rheumatoid arthritis (Hemler et al., 1986). In fibroblasts, α1 is known to activate the RAS/ERK proliferative pathway and has a pro-invasive function in certain cancers. In megakaryocyte differentiation α1 is silenced by DNA methylation but not histone modification (Cheli et al., 2007). Different transcription factors involved in cancer progression can bind to the ITGA1 promoter. Integrin α1 transcriptional regulation remains to be further defined.