Understanding transcriptional control mechanisms of the ATM, TRIM29, TWIST1 pathway in cultured breast cancer cells

Breast cancer is the most common cancer in women. Breast cancer is accounting for 14% of deaths caused by cancer and 23% of total cancer cases.The formation process and mechanisms of the cancer cells are very complex. There are many studiesto understand how to control and expression of genes associated with cancer. Understanding the mechanisms in cancer formation can help to develop different methods for the treatment of cancer. The most important point the treatment of cancer is early diagnosis and multifaceted treatment.Human gene maps have been obtaining and are being tested to understand for cancer predisposition rate in future. The main purpose of this study is that to describe transcriptional relationship between TRIM29tumor suppressor gene, TWIST1 oncogene and ATM, which is a candidate to be breast cancer marker gene, in breast cancer and mammary epithelial cell lines. We aimedfirstly to understand the transcriptional relationship between ATM and TRIM29. Therefore TRIM29 abundance were testedin nine different breast epithelial cell lines in the laboratory.Seven of these cells are breast tumor cells, one of these cell is non-tumorigenic mammary epithelium-derived cell and last one is human mammary epithelium cells. We observed that TRIM29 was only detected in the tumor lines SKBr3, MDA-MB-468, and non-tumorigenic mammary epithelium-derived (MCF-10A) and human mammary epithelium cells (HMEC). SKBr3, MDA-MB-468 and HMEC were cultured for this study. Also we used BT-549 and MDA-MB-231 cell lines which do not express TRIM29 as control during study. We used shRNA mediated gene knockdown to diminish expression of ATM in SKBr3, MDA-MB-468, MDA-MB-231 and HMEC cell lines. We measured a sharp decrease expression in TRIM29 protein and mRNA, unlike increasing expression in TWIST1 protein and mRNA fallowing ATM knockdown. Then we determined transcription factors controlled by the ATM. These transcription factors are NF-κB, HIF-1α Sp1 and ATF. We obtained SKBr3, MDA-MB-468, MDA-MB-231 and HMEC cell lines which have diminished expression of NF-κB and HIF-1α. Then we tested TRIM29 and TWIST1 expression in HIF-1α knockdown cell lines. Also we analysed expression of HIF-1αand TRIM29 in NF-κB knockdown cells. We found out that expression of TRIM29 mRNA and protein decreasedfallowing HIF-1α and NF-κB knockdownunlike mRNA and protein expression of TWIST1 increased in HIF-1α knockdown. TWIST1 mRNA and protein expression was stable fallowing ATM knockdown in MDA-MB-231 cell,on the other hand TWIST mRNA expression decreased in HIF-1α knockdown for same cell line. The effect of knockdown process on target geneswere tested by RT-PCR, real timeqPCR and western-blot techniques. SKBr3, MDA-MB-468 and HMEC cell lines were incubated under hypoxia to understand how to effect increased expression of HIF-1α on TRIM29 and TWIST1.MDA-MB-231 and BT-549 cells exposed to hypoxia were analyzed TWIST1 exchange. We showed that, in SKBr3, MDA-MB-468 and HMEC cell lines, TRIM29 is upregulation in response to hypoxia and also expression of TWIST1 did not change in cell lines which express TRIM29 in response to hypoxia. We observed increases in TWIST1 mRNA and protein expression in MDA-MB-231 and BT-549 cells during hypoxia. We found out that TRIM29 is upragulating in response to hypoxia. Then we examined TRIM29 hypoxic response in ATM and HIF-1αknockdown SKBr3 and MDA-MB-468 cells. We showed that hypoxia inducibile expression of TRIM29 is ATM and HIF-1α dependent. To understand the relationship between TRIM29 and TWIST1 we constituted TRIM29 and TWIST knockdown SKBr3, MDA-MB-468 and HMEC cells and tested mRNA and protein expression for those cells. We concluded that TRIM29 and TWIST antogonize the activity of each other. We observed that TRIM29 knockdown results in upregulation of TWIST1 and knockdown of TWIST1 drives up TRIM29 expression in cultured breast cancer and mammary epithelial cells.