HOXA5 counteracts stem cell traits by inhibiting Wnt signaling
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ABSTRACT: Here we identify HOXA5 as an important repressor of intestinal stem cell fate in vivo and identify a reciprocal feedback between HOXA5 and Wnt signaling. HOXA5 is suppressed by the Wnt pathway to maintain stemness and becomes active only outside the intestinal crypt where it inhibits Wnt signaling to enforce differentiation. In colon cancer, HOXA5 is down-regulated and its re-expression induces loss of the cancer stem cell phenotype preventing tumor progression and metastasis. Tumor regression by HOXA5 induction can be triggered by retinoids, which represents a tangible means to treat colon cancer by eliminating cancer stem cells. We use microarrays to identify HOXA5 signature in SW480 human colon carcinoma cell line
Project description:Homeobox genes comprise a family of regulatory genes that contain a common homeobox domain and act as transcription factors. Changes in the expression of homeobox genes are observed in many cancers, including leukaemia, colon, skin, prostate, breast, and ovarian cancers. Recent studies indicate that homeobox A5 (HOXA5) may serve as a tumour suppressor gene in breast cells. However, the precise role of HOXA5 in lung cancer remains unclear. Using microarrays and an invasion/metastasis lung adenocarcinoma cell line model, we showed that the expression of HOXA5 is negatively correlated with cancer cell invasion ability. The ectopic expression of HOXA5 in highly invasive cancer cells suppressed cell migration, invasion, and filopodia formation in vitro and inhibited metastatic potential in vivo. The knockdown of HOXA5 expression via a HOXA5-specific siRNA was able to promote the invasiveness of lung cancer cells. Furthermore, HOXA5 expression was associated with improved overall survival and disease-free survival in non-small cell lung cancer patients with wild-type EGFR (P = 0.0199 and 0.0345, respectively). Genome-wide transcriptome and pathway analyses indicated that these effects of HOXA5 may be associated with the regulation of cytoskeletal remodelling. In summary, our studies provide new insights into how HOXA5 may contribute to the suppression of metastasis in lung cancer. We used microarrays to profile the global gene expression of HOXA5-overexpressing cells compared with mock control cells and identified the pathways involved in HOXA5-induced biofunctional alterations.
Project description:Homeobox A5 (HOXA5) is a transcription factor in mammalian and can regulate cell differentiation, proliferation and apoptosis as well as tumorigenesis. However, little is known on whether and how HOXA5 can regulate the malignant behaviors of cholangiocarcinoma. The methylation levels of HOXA5 were evaluated by methylation microarray and bisulfite sequencing PCR. We found that hypermethylation in the HOXA5 promoter down-regulated HOXA5 expression in extrahepatic cholangiocarcinoma (ECCA) tissues, which was correlated with worse overall survival. HOXA5 over-expression significantly inhibited the proliferation and tumor growth.
Project description:Homeobox A5 (HOXA5) is a transcription factor in mammals and can regulate cell differentiation, proliferation and apoptosis as well as tumorigenesis. However, little is known about whether and how HOXA5 can regulate the malignant behaviors of cholangiocarcinoma. The expression profiles were analyzed by RNA microarray. We found MXD1 was upregulated upon HOXA5 overexpression. Besides, p53 pathway was activated by HOXA5 overexpression.
Project description:HOXA5 inducible cell line Hs578T was established and cultured as described previously (MCB 2004. 24:924-935). 2×106 cells were seeded onto a 10-cm cell culture dish at 24h before induction. Two batches of RNA from HOXA5-inducible cells (0 h, 6 h and 9 h) were purified for repeating the microarray hybridization experiments once using Affymatrix Chips.Total RNA was extracted using TRIzol reagent (Gibco BRL, Life Technologies, Grand Island, NY) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA). Keywords: time-course
Project description:Hoxa5 plays numerous roles in development, but its downstream molecular effects are mostly unknown. We applied bulk RNA_seq assays to characterize the transcriptional impact of the loss of Hoxa5 gene function in seven different biological contexts, including developing respiratory and musculoskeletal tissues that present phenotypes in Hoxa5 mouse mutants. This global analysis revealed few shared transcriptional changes, suggesting that HOXA5 acts mainly via the regulation of context_specific effectors. However, Hox genes themselves appeared as potentially conserved targets of HOXA5 across tissues. Notably, a trend toward reduced expression of HoxA genes was observed in Hoxa5 null mutants in several tissue contexts. Comparative analysis of epigenetic marks along the HoxA cluster in lung tissue from two different Hoxa5 mutant mouse lines revealed limited effect of either mutation indicating that Hoxa5 gene targeting did not significantly perturb the chromatin landscape of the surrounding HoxA cluster. Combined with the shared impact of the two Hoxa5 mutant alleles on phenotypes and Hox expression, these data argue against the contribution of local cis effects to Hoxa5 mutant phenotypes and support the notion that the HOXA5 protein acts in trans in the control of Hox gene expression.
Project description:Wnt/b-catenin pathway is a key modulator of intestinal homeostasis by regulating stem cell biology during gut organogenesis and in adult life. DICKKOPF (DKK)-1 is a secreted inhibitor of Wnt/b-catenin signaling from plasma membrane receptors. We found that in human intestine, DKK-1 locates within the nucleus of differentiated enterocytes and enteroendocrine cells but not of proliferating and stem cells at the bottom of the crypts. DKK-1 is also nuclear in colon cancer cells locating at sites of active gene transcription. ChIP-seq and transcriptomic analysis both confirmed that DKK-1 binds chromatin and regulates gene expression. Thus, we demonstrate that DKK-1 is a multifunctional protein that inhibits Wnt/b-catenin signaling at plasma membrane and controls specific genes in the nucleus, suggesting that these functions are relevant for intestinal homeostasis. Analysis of DKK-1 location and associated roles in human colon cancer cells and crypts of small and large bowel.
Project description:HOXA5 is a transcription factor in mammalian and can regulate cell differentiation, proliferation and apoptosis as well as tumorigenesis. However, little is known on whether and how HOXA5 can regulate the malignant behaviors of cholangiocarcinoma. The binding pattern of HOXA5 was evaluated by CUT & Tag assay. The differential peaks were analysed to assess the role of HOXA5 in the regulation of the key genes involved in cell proliferation and DNA replication.
Project description:HOXA5 inducible cell line Hs578T was established and cultured as described previously (MCB 2004. 24:924-935). 2Ã106 cells were seeded onto a 10-cm cell culture dish at 24h before induction. Two batches of RNA from HOXA5-inducible cells (0 h, 6 h and 9 h) were purified for repeating the microarray hybridization experiments once using Affymatrix Chips.Total RNA was extracted using TRIzol reagent (Gibco BRL, Life Technologies, Grand Island, NY) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA).
Project description:The effect of ER-stress and subsequent activation of the unfolded protein response was investigated in colon cancer stem cells and more differentiated colon cancer cells Human colon cancer derived spheroid cultures with a TCF/LEF driven GFP reporter for Wnt signaling activity were treated for 24 hours with SubAB or protease-dead SubABco (1µg/ml) Treated spheroid cultures were sorted into colon-cancer stem cell fraction (highest 10% Wnt-GFP) and differentiated colon cancer fraction (10% Lowest(Wnt Low) for micro array