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HOTAIR is a negative prognostic factor and exhibits pro-oncogenic activity in pancreatic cancer.

ABSTRACT: HOTAIR is a long intervening non-coding RNA (lincRNA) that associates with the Polycomb Repressive Complex 2 (PRC2) and overexpression is correlated with poor survival for breast, colon and liver cancer patients. In this study, we show that HOTAIR expression is increased in pancreatic tumors compared with non-tumor tissue and is associated with more aggressive tumors. Knockdown of HOTAIR (siHOTAIR) by RNA interference shows that HOTAIR has an important role in pancreatic cancer cell invasion, as reported in other cancer cell lines. In contrast, HOTAIR knockdown in Panc1 and L3.6pL pancreatic cancer cells that overexpress this lincRNA decreased cell proliferation, altered cell cycle progression and induced apoptosis, demonstrating an expanded function of HOTAIR in pancreatic cancer cells compared with other cancer cell lines. Results of gene array studies showed that there was minimal overlap between HOTAIR-regulated genes in pancreatic cells and breast cancer cells, and HOTAIR uniquely suppressed several interferon-related genes and gene sets related to cell cycle progression in pancreatic cancer cells and tumors. Analysis of selected genes suppressed by HOTAIR in Panc1 and L3.6pL cells showed by knockdown of EZH2 and chromatin immunoprecipitation assays that HOTAIR-mediated gene repression was both PRC2-dependent and -independent. HOTAIR knockdown in L3.6pL cells inhibited tumor growth in mouse xenograft model, further demonstrating the pro-oncogenic function of HOTAIR in pancreatic cancer.

SUBMITTER: Kim K

PROVIDER: S-EPMC3484248 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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HOTAIR is a negative prognostic factor and exhibits pro-oncogenic activity in pancreatic cancer.

Kim K K Jutooru I I Chadalapaka G G Johnson G G Frank J J Burghardt R R Kim S S Safe S S

Oncogene 20120521 13

HOTAIR is a long intervening non-coding RNA (lincRNA) that associates with the Polycomb Repressive Complex 2 (PRC2) and overexpression is correlated with poor survival for breast, colon and liver cancer patients. In this study, we show that HOTAIR expression is increased in pancreatic tumors compared with non-tumor tissue and is associated with more aggressive tumors. Knockdown of HOTAIR (siHOTAIR) by RNA interference shows that HOTAIR has an important role in pancreatic cancer cell invasion, as ...[more]

PMID: 22614017

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Project description:BackgroundHOX transcript antisense RNA (HOTAIR), which is expressed from the homebox C gene (HOXC) locus, is capable of reprogramming chromatin organisation and promoting cancer cell metastasis and can simultaneously bind the polycomb repressive complex 2, which enhances H3K27 trimethylation, and the LSD1-CoREST-REST complex, which is critical for H3K4 demethylation. Clinically, the overexpression of HOTAIR is a powerful predictor of the tumour progression and overall survival in patients with diverse cancers. The relationship between HOTAIR and oesophageal squamous cell carcinoma (ESCC), however, remains unclear. We investigated the role of HOTAIR in the pathogenesis of ESCC.MethodsWe used quantitative real-time PCR to determine the level of HOTAIR in ESCC cell lines and 100 ESCC samples from patients; 56 adjacent non-neoplastic tissues were used as controls. We measured the effect of HOTAIR knockdown and overexpression in ESCC cell lines using colony formation assays, anchorage-independent growth assays, the CCK-8 assay, transwell migration and invasion assays, and Annexin V-binding assays. We analysed the growth of ESCC xenograft tumours in nude mice. Changes in the gene expression and methylation levels in ESCC cell lines were analysed using gene expression microarrays and the Infinium HumanMethylation450K BeadChip assay, respectively. results: The levels of HOTAIR were increased in ESCC cell lines and patient samples compared with the controls; the expression levels correlated with the disease stage and survival time. The knockdown of HOTAIR in the KYSE510 and KYSE180 ESCC cell lines using small hairpin RNAs (shRNAs) reduced the ability of the cells to form foci, migrate, and invade the extracellular matrix in culture, altered cell cycle progression, and increased the sensitivity of the cells to apoptosis. The HOTAIR knockdown reduced cancer cell metastasis in vivo, and the tumours formed by HOTAIR-silenced ESCC cells were smaller, both in size and weight, than the tumours and metastases formed by the shRNA vector control cells in a mouse xenograft model. The results of the gene microarray study showed that HOTAIR reprogrammed the gene expression profile of ESCC cells, and the gene ontology analysis revealed an enrichment in genes that are important for tumorigenesis, such as genes involved in cell migration and the regulation of the cell cycle. Comparing the gene expression profiles and DNA methylation analysis between the KYSE180 and KYSE180_HOTAIR cells revealed that only a small proportion of the methylation changes were correlated with gene expression changes.ConclusionHOX transcript antisense RNA is upregulated in ESCC cell lines and patient samples, and promotes ESCC cell proliferation and tumour metastasis in mice. The knockdown of HOTAIR resulted in significant changes in gene expression, and data analysis suggested that HOTAIR-mediated gene regulation has a critical role in ESCC progression and is a novel epigenetic molecular target for treating ESCC patients.

Project description:Rationale: Alternative therapeutic strategies based on tumor-specific molecular targets are urgently needed for triple-negative breast cancer (TNBC). The protease cathepsin D (cath-D) is a marker of poor prognosis in TNBC and a tumor-specific extracellular target for antibody-based therapy. The identification of cath-D substrates is crucial for the mechanistic understanding of its role in the TNBC microenvironment and future therapeutic developments. Methods: The cath-D substrate repertoire was investigated by N-Terminal Amine Isotopic Labeling of Substrates (TAILS)-based degradome analysis in a co-culture assay of TNBC cells and breast fibroblasts. Substrates were validated by amino-terminal oriented mass spectrometry of substrates (ATOMS). Cath-D and SPARC expression in TNBC was examined using an online transcriptomic survival analysis, tissue micro-arrays, TNBC cell lines, patient-derived xenografts (PDX), human TNBC samples, and mammary tumors from MMTV-PyMT Ctsd-/- knock-out mice. The biological role of SPARC and its fragments in TNBC were studied using immunohistochemistry and immunofluorescence analysis, gene expression knockdown, co-culture assays, western blot analysis, RT-quantitative PCR, adhesion assays, Transwell motility, trans-endothelial migration and invasion assays. Results: TAILS analysis showed that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro, cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion. Conclusions: Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited SPARC proteolysis, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments. Our study will pave the way for the development of strategies for targeting bioactive fragments from matricellular proteins in TNBC.

Dataset Information

HOTAIR is a negative prognostic factor and exhibits pro-oncogenic activity in pancreatic cancer.

Publications

HOTAIR is a negative prognostic factor and exhibits pro-oncogenic activity in pancreatic cancer.

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