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1.
Mol Carcinog ; 53 Suppl 1: E130-9, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23996472

ABSTRACT

Recently, a reciprocal relationship between calcitriol and epithelial-to-mesenchymal transition has been described. Therefore, we hypothesized that calcitriol (1α,25-dihydroxyvitamin D3) would enhance radiation sensitivity in colorectal cancer regulated by epithelial mesenchymal transition. Vitamin-D receptor, E-cadherin and vimentin protein as well as E-cadherin, Snail and Slug mRNA levels were assessed in a panel of human colorectal cancer cell lines at baseline and in response calcitriol. We defined cell lines as calcitriol sensitive based on demonstrating an enhanced epithelial phenotype with increased E-cadherin, reduced vimentin and decreased expression of Snail and Slug as well as decreased cellular migration in response to calcitriol. In calcitriol sensitive cells, including DLD-1 and HCT116, 24 h calcitriol pre-treatment enhanced the radiation sensitivity by 2.3- and 2.6-fold, respectively, at 4 Gy (P < 0.05). In contrast, SW620 cells with high baseline mesenchymal features including high Slug and vimentin expression with low E-cadherin expression demonstrated no significant radiation sensitizing response to calcitriol treatment. Similarly, transfection of Slug in the calcitriol sensitive colon cancer cell lines, DLD-1 and HCT 116, completely inhibited the radiation sensitizing effect of calcitriol. Collectively, we demonstrate that calcitriol can enhance the therapeutic effects of radiation in colon cancer cells and Slug expression mitigates this observed effect potentially representing an effective biomarker for calcitriol therapy.


Subject(s)
Calcitriol/pharmacology , Colorectal Neoplasms/pathology , Epithelial-Mesenchymal Transition/drug effects , Radiation-Sensitizing Agents/pharmacology , Transcription Factors/genetics , Blotting, Western , Cadherins/genetics , Cadherins/metabolism , Calcium Channel Agonists/pharmacology , Cell Adhesion/drug effects , Cell Adhesion/radiation effects , Cell Movement/drug effects , Cell Movement/radiation effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/radiotherapy , Epithelial-Mesenchymal Transition/radiation effects , Fluorescent Antibody Technique , Gamma Rays , Humans , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Calcitriol/genetics , Receptors, Calcitriol/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Snail Family Transcription Factors , Transcription Factors/metabolism , Tumor Cells, Cultured , Vimentin/genetics , Vimentin/metabolism
2.
Breast Cancer Res ; 15(4): R70, 2013.
Article in English | MEDLINE | ID: mdl-23971998

ABSTRACT

INTRODUCTION: MicroRNAs are small non-coding RNAs that are involved in the post-transcriptional negative regulation of mRNAs. MicroRNA 510 (miR-510) was initially shown to have a potential oncogenic role in breast cancer by the observation of its elevated levels in human breast tumor samples when compared to matched non-tumor samples. Few targets have been identified for miR-510. However, as microRNAs function through the negative regulation of their direct targets, the identification of those targets is critical for the understanding of their functional role in breast cancer. METHODS: Breast cancer cell lines were transfected with pre-miR-510 or antisense miR-510 and western blotting and quantitative real time PCR were performed. Functional assays performed included cell growth, migration, invasion, colony formation, cytotoxicity and in vivo tumor growth. We performed a PCR assay to identify novel direct targets of miR-510. The study focused on peroxiredoxin 1 (PRDX1) as it was identified through our screen and was bioinformatically predicted to contain a miR-510 seed site in its 3' untranslated region (3'UTR). Luciferase reporter assays and site-directed mutagenesis were performed to confirm PRDX1 as a direct target. The Student's two-sided, paired t-test was used and a P-value less than 0.05 was considered significant. RESULTS: We show that miR-510 overexpression in non-transformed and breast cancer cells can increase their cell growth, migration, invasion and colony formation in vitro. We also observed increased tumor growth when miR-510 was overexpressed in vivo. We identified PRDX1 through a novel PCR screen and confirmed it as a direct target using luciferase reporter assays. The reintroduction of PRDX1 into breast cancer cell lines without its regulatory 3'UTR confirmed that miR-510 was mediating its migratory phenotype at least in part through the negative regulation of PRDX1. Furthermore, the PI3K/Akt pathway was identified as a positive regulator of miR-510 both in vitro and in vivo. CONCLUSIONS: In this study, we provide evidence to support a role for miR-510 as a novel oncomir. We show that miR-510 directly binds to the 3'UTR of PRDX1 and blocks its protein expression, thereby suppressing migration of human breast cancer cells. Taken together, these data support a pivotal role for miR-510 in breast cancer progression and suggest it as a potential therapeutic target in breast cancer patients.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Peroxiredoxins/genetics , 3' Untranslated Regions , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation , Disease Models, Animal , Female , Humans , Oxidation-Reduction , Peroxiredoxins/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , Signal Transduction , Tumor Burden , Tumor Stem Cell Assay , Xenograft Model Antitumor Assays
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