Role of telomeric RAP1 in radiation sensitivity modulation and its interaction with CSC marker KLF4 in colorectal cancer.

Aims: Radiotherapy is predominantly used as one of the treatment modalities to treat local tumor in colorectal cancer (CRC). Hindrance in disease treatment can be attributed to radio-tolerance of cancer stem cells (CSCs) subsistence in the tumor. Understanding the radio-resistant property of CSCs might help in the accomplishment of targeted radiotherapy treatment and increased disease-free survival. Telomeric RAP1 contributes in modulation of various transcription factors leading to aberrant cell proliferation and tumor cell migration. Therefore, we investigated the role of RAP1 in maintaining resistance phenotype and acquired stemness in radio-resistant cells.Main methods: Characterization of HCT116 derived radio-resistant cell (HCT116RR) was performed by cell survival and DNA damage profiling. RAP1 silenced cells were investigated for DNA damage and expression of CSC markers through western blotting and Real-time PCR post-irradiation. Molecular docking and co-immunoprecipitation study were performed to investigate RAP1 and KLF4 interaction followed by RAP1 protein status profiling in CRC patient.Key findings: We established radio-resistant cells, which showed tolerance to radiotherapy and elevated expression of CSC markers along with RAP1. RAP1 silencing showed enhanced DNA damage and reduced expression of CSC markers post-irradiation. We observed strong physical interaction between RAP1 and KLF4 protein. Furthermore, higher RAP1 expression was observed in the tumor of CRC patients. Dataset analysis also revealed that high expression of RAP1 expression is associated with poor prognosis.Significance: We conclude that higher expression of RAP1 implicates its possible role in promoting radio-resistance in CRC cells by modulating DNA damage and CSC phenotype.

Radiation-induced DNA damage response and resistance in colorectal cancer stem-like cells.

Purpose: Radiation therapy is an integral part of current treatment modality for colorectal cancer. Recent studies have revealed the presence of cancer stem-like cells (CSCs) population, in different tumors are responsible for therapeutic resistance and disease relapse, including colorectal cancer with poorer survival rate. Hence, characterization of the effect of Ionizing Radiation (IR) in colorectal cancer may serve to explain possible mechanisms. Material and methods: Parental HCT116 and HCT-15 cells and derived colonospheres were irradiated and dose was optimized based on cell survival assay and cell cycle analysis. DNA damage response (DDR) was elucidated by γH2AX foci formation, COMET assay, and ATM, p-ATM, ERCC1 expression post-treatment. The expression level of developmental marker (ß-catenin), CSC markers (CD44, KLF4) and telomeric components (TRF2, RAP1, hTERT) were evaluated. Results: We observed cell survival was more in colonospheres post-irradiation and also exhibited decreased γH2AX foci, olive tail moment, increased ERCC1, and p-ATM expression than its parental counterpart which corresponds to efficient DDR. Differential expression of developmental marker, CSC markers, and telomeric components were observed after irradiation. Conclusion: This study highlighted the presence of CSC phenotype in colonospheres having increased DNA repair capacity. Differential expression of developmental marker, CSC markers and telomeric components between parental and colonospheres may contribute in radio-resistance property of CSCs.

Estrogen-related receptors alpha, beta and gamma expression and function is associated with transcriptional repressor EZH2 in breast carcinoma.

BACKGROUND: Orphan nuclear receptors ERRα, ERRß and ERRγ that belong to NR3B or type IV nuclear receptor family are well studied for their role in breast cancer pathophysiology. Their homology with the canonical estrogen receptor dictates their possible contributing role in mammary gland development and disease. Although function and regulation of ERRα, ERRγ and less about ERRß is reported, role of histone methylation in their altered expression in cancer cells is not studied. Transcriptional activity of nuclear receptors depends on co-regulatory proteins. The present study for the first time gives an insight into regulation of estrogen-related receptors by histone methylation specifically through methyltransferase EZH2 in breast cancer. METHODS: Expression of ERRα, ERRß, ERRγ and EZH2 was assessed by immunohistochemistry in four identical tissue array slides that were prepared as per the protocol. The array slides were stained with ERRα, ERRß, ERRγ and EZH2 simultaneously. Array data was correlated with expression in MERAV expression dataset. Pearson correlation coeficient r was calculated from the partial matrix expression values available at MERAV database to study the strength of association between EZH2 and three orphan nuclear receptors under study. By western blot and real time PCR, their correlated expression was studied in breast cancer cell lines MCF-7, MDA-MB-231, T47D and MDA-MB-453 including normal breast epithelial MCF-10A cells at both protein and RNA level. Regulation of ERRα, ERRß, ERRγ by EZH2 was further investigated upon overexpression and silencing of EZH2. The interaction between ERRs and EZH2 was validated in vivo by CHIP-qPCR. RESULTS: We found a negative correlation between estrogen-related receptors and Enhancer of Zeste Homolog 2, a global repressor gene. Immunohistochemistry in primary breast tumors of different grades showed a correlated expression of estrogen-related receptors and EZH2. Their correlated expression was further validated using online MERAV expression dataset where a negative correlation of variable strengths was observed in breast cancer. Ectopic expression of EZH2 in low EZH2-expressing normal breast epithelial cells abrogated their expression and at the same time, its silencing enhanced the expression of estrogen-related receptors in cancerous cells. Global occupancy of EZH2 on ERRα and ERRß was observed in-vivo. CONCLUSION: Our findings identify EZH2 as a relevant coregulator for estrogen-related receptors in breast carcinoma.