Histone deacetylase inhibition impairs normal intestinal cell proliferation and promotes specific gene expression.

Mechanisms that maintain proliferation and delay cell differentiation in the intestinal crypt are not yet fully understood. We have previously shown the implication of histone methylation in the regulation of enterocytic differentiation. In this study, we investigated the role of histone deacetylation as an important epigenetic mechanism that controls proliferation and differentiation of intestinal cells using the histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) on the proliferation and differentiation of human and mouse intestinal cells. Treatment of newly confluent Caco-2/15 cells with SAHA resulted in growth arrest, increased histone acetylation and up-regulation of the expression of intestine-specific genes such as those encoding sucrase-isomaltase, villin and the ion exchanger SLC26A3. Although SAHA has been recently used in clinical trials for cancer treatment, its effect on normal intestinal cells has not been documented. Analyses of small and large intestines of mice treated with SAHA revealed a repression of crypt cell proliferation and a higher expression of sucrase-isomaltase in both segments compared to control mice. Expression of SLC26A3 was also significantly up-regulated in the colons of mice after SAHA administration. Finally, SAHA was also found to strongly inhibit normal human intestinal crypt cell proliferation in vitro. These results demonstrate the important implication of epigenetic mechanisms such as histone acetylation/deacetylation in the regulation of normal intestinal cell fate and proliferation.

Modulation of stemness in a human normal intestinal epithelial crypt cell line by activation of the WNT signaling pathway.

The small intestine consists of two histological compartments composed of the crypts and the villi. The function of the adult small intestinal epithelium is mediated by four different types of mature cells: enterocytes, goblet, enteroendocrine and Paneth. Undifferentiated cells reside in the crypts and produce these four types of mature cells. The niche-related Wnt and Bmp signaling pathways have been suggested to be involved in the regulation and maintenance of the stem cell microenvironment. In our laboratory, we isolated the first normal human intestinal epithelial crypt (HIEC) cell model from the human fetal intestine and in this study we investigated the expression of a panel of intestinal stem cell markers in HIEC cells under normal culture parameters as well as under conditions that mimic the stem cell microenvironment. The results showed that short term stimulation of HIEC cells with R-spondin 1 and Wnt-3a±SB-216763, a glycogen synthase kinase 3ß (GSK3ß) inhibitor, induced ß-catenin/TCF activity and expression of the WNT target genes, cyclin D2 and LGR5. Treatment of HIEC cells with noggin, an antagonist of BMP signaling, abolished SMAD2/5/8 phosphorylation. Inducing a switch from inactive WNT/active BMP toward active WNT/inactive BMP pathways was sufficient to trigger a robust intestinal primordial stem-like cell signature with predominant LGR5, PHLDA1, PROM1, SMOC2 and OLFM4 expression. These findings demonstrate that even fully established cultures of intestinal cells can be prompted toward a CBC stem cell-like phenotype. This model should be useful for studying the regulation of human intestinal stem cell self-renewal and differentiation.

Pharmacological inhibition of polycomb repressive complex-2 activity induces apoptosis in human colon cancer stem cells.

Colorectal cancer is among the leading causes of cancer death in the USA. The polycomb repressive complex 2 (PRC2), including core components SUZ12 and EZH2, represents a key epigenetic regulator of digestive epithelial cell physiology and was previously shown to promote deleterious effects in a number of human cancers, including colon. Using colon cancer stem cells (CCSC) isolated from human primary colorectal tumors, we demonstrate that SUZ12 knockdown and treatment with DZNep, one of the most potent EZH2 inhibitors, increase apoptosis levels, marked by decreased Akt phosphorylation, in CCSCs, while embryonic stem (ES) cell survival is not affected. Moreover, DZNep treatments lead to increased PTEN expression in these highly tumorigenic cells. Taken together, our findings suggest that pharmacological inhibition of PRC2 histone methyltransferase activity may constitute a new, epigenetic therapeutic strategy to target highly tumorigenic and metastatic colon cancer stem cells.

Src family kinase inhibitor PP2 accelerates differentiation in human intestinal epithelial cells.

The proto-oncogene Src is an important protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and survival. Here, we investigated the involvement of Src family kinases (SFKs) in human intestinal cell differentiation. We first observed that Src activity peaked in early stages of Caco-2/15 cell differentiation. Inhibition of SFKs with PP2, a selective SFK inhibitor, accelerated the overall differentiation program. Interestingly, all polarization and terminal differentiation markers tested, including sucrase-isomaltase, lactase-phlorizin hydrolase and E and Li-cadherins were found to be significantly up-regulated after only 3 days of treatment in the newly differentiating cells. Further investigation of the effects of PP2 revealed a significant up-regulation of the two main intestinal epithelial cell-specific transcription factors Cdx2 and HNF1α and a reduction of polycomb PRC2-related epigenetic repressing activity as measured by a decrease in H3K27me3, two events closely related to the control of cell terminal differentiation in the intestine. Taken together, these data suggest that SFKs play a key role in the control of intestinal epithelial cell terminal differentiation.

A concerted HIF-1α/MT1-MMP signalling axis regulates the expression of the 3BP2 adaptor protein in hypoxic mesenchymal stromal cells.

Increased plasticity, migratory and immunosuppressive abilities characterize mesenchymal stromal cells (MSC) which enable them to be active participants in the development of hypoxic solid tumours. Our understanding of the oncogenic adaptation of MSC to hypoxia however lacks the identification and characterization of specific biomarkers. In this study, we assessed the hypoxic regulation of 3BP2/SH3BP2 (Abl SH3-binding protein 2), an immune response adaptor/scaffold protein which regulates leukocyte differentiation and motility. Gene silencing of 3BP2 abrogated MSC migration in response to hypoxic cues and generation of MSC stably expressing the transcription factor hypoxia inducible factor 1alpha (HIF-1α) resulted in increased endogenous 3BP2 expression as well as cell migration. Analysis of the 3BP2 promoter sequence revealed only one potential HIF-1α binding site within the human but none in the murine sequence. An alternate early signalling cascade that regulated 3BP2 expression was found to involve membrane type-1 matrix metalloproteinase (MT1-MMP) transcriptional regulation which gene silencing abrogated 3BP2 expression in response to hypoxia. Collectively, we provide evidence for a concerted HIF-1α/MT1-MMP signalling axis that explains the induction of adaptor protein 3BP2 and which may link protein binding partners together and stimulate oncogenic MSC migration. These mechanistic observations support the potential for malignant transformation of MSC within hypoxic tumour stroma and may contribute to evasion of the immune system by a tumour.

3BP2 adapter protein is required for receptor activator of NFκB ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells.

The adapter protein 3BP2 (also known as SH3BP2 and Abl SH3-binding protein 2) has been involved in leukocyte signaling and activation downstream immunoreceptors. Genetic studies have further associated 3BP2 mutations to the human disease cherubism and to inflammation and bone dysfunction in mouse. However, how wild type 3BP2 functions in macrophage differentiation remains poorly understood. In this study, using small interfering RNA-mediated silencing of 3BP2 in the RAW264.7 monocytic cell line, we show that 3BP2 was required for receptor activator of NFkappaB ligand (RANKL)-induced differentiation of RAW264.7 cells into multinucleated mature osteoclasts but not for granulocyte macrophage-colony stimulating factor/interleukin-4-induced differentiation into dendritic cells. 3BP2 silencing was associated with impaired activation of multiple signaling events downstream of RANK, including actin reorganization; Src, ERK, and JNK phosphorylation; and up-regulation of osteoclastogenic factors. In addition, 3BP2 knockdown cells induced to osteoclast by RANKL displayed a reduced increase of Src and nuclear factor of activated T cells (NFATc1) mRNA and protein expression. Importantly, 3BP2 interacted with Src, Syk, Vav, and Cbl in monocytic cells, and the introduction of constitutively active mutants of Src and NFATc1 in 3BP2-deficient cells restored osteoclast differentiation. Finally, the expression of a 3BP2 cherubism mutant was found to promote increased Src activity and NFAT-dependent osteoclast formation. Together, this study demonstrates that wild type 3BP2 is a key regulator of RANK-mediated macrophage differentiation into osteoclast through Src and NFATc1 activation.

Differential regulation of cell death in head and neck cell carcinoma through alteration of cholesterol levels in lipid rafts microdomains.

Lipid rafts are cholesterol-enriched microdomains in the plasma membrane. They act as molecular platforms that spatially organize membrane receptor molecules and are involved in the transduction of various signaling pathways. We recently reported that in the radiosensitive squamous cell carcinoma SCC61 line, gamma-irradiation results in a rearrangement of the plasma membrane rafts and signaling platforms leading to radiation-induced apoptosis in a ceramide-dependent pathway. By contrast, this reorganization was found to be defective in the radioresistant counterpart cell line, SQ20B. As the cholesterol content of lipid rafts is two times higher in SQ20B compared with SCC61 cells, we investigated the modulation of these microdomains using methyl-beta-cyclodextrin (MbetaCDX), a widely used cholesterol-depleting agent, in order to disrupt raft organization in both cells. Here, we report that MbetaCDX treatment resulted in the triggering of apoptosis in SCC61 cells involving mitochondrial events and associated with the clustering of Fas, the formation of Fas-FADD complexes and the cleavage of procaspase 8. The ligand-independent activation of this death receptor was totally absent in SQ20B cells, which remained resistant to MbetaCDX-triggered apoptosis. However, treatment of SQ20B with MbetaCDX resulted in a ligand-independent activation of the epidermal growth factor receptor (EGFR) survival pathway, as evidenced by an increased tyrosine phosphorylation of EGFR. Taken altogether, our results indicate that lipid raft integrity is intimately involved in the triggering of apoptotic cell death and/or survival pathways in head and neck carcinoma cells.