Studies On Functions And Radiosensitization Of Tumor Suppressor Gene Tob1 In Breast Cancer

Breast cancer is one of the most common malignancies of women in China and the world,which results from genetic or epigenetic changes of both malignant cells and the host cells interacting with the tumor, involving numerous cross-talking pathways. The complex underlying mechanisms lead to poor understanding of breast carcinogenesis, so as to difficulties in finding efficient, safe and selective therapeutics of breast cancer.Tumor suppress genes can reduce and inhibit the alteration of normal cells'turning into tumor cells, and the functions of their protein products fall into several categories including repression of genes essential for the cell cycle, coupling the cell cycle to DNA damage, inducing apoptosis, blocking loss of contact inhibition, and inhibiting metastasis etc. Many studies suggest that any mutation of one or more tumor suppressor genes will increase the probability of carcinogenesis. Over the last decade, the growing knowledge of tumor suppressor genes and proteins associated with the development and progression of breast cancer has provided us the foundation on mechanisms contributing to breast carcinogenesis, as well as the better opportunities for therapy of this disease.Tob1 (transducer of ERBB2, 1) is a member of the TOB/BTG antiproliferative protein family characterized by its anti-proliferative activity with the abilities to regulate cell cycle negatively in the periphery. Some previous studies suggest that Tob1 plays a role in the control of G1-S progression by suppressing cyclin D1 expression, and therefore may act as a tumor suppressor gene. However, it remains unknown whether and how Tob1functions in breast carcinogenesis ,and it is the purpose of this study to elucidate these issues.By using Western Blot, Northern Blot and immunohistochemistry staining assays, it first demonstrated a lower or un-detectable level of Tob1 expression in human breast cancer cell lines and human breast cancer tissues compared to normal controls in this study. Enhanced expression of Tob1 notably inhibited in vitro growth of breast cancer cells. By using animal models with nude mice, it was found that Tob1 over-expression significantly suppressed tumor growth and angiogenesis. Taken together with in vitro, in vivo experiments and clinic data, all these results suggest that Tob1 may function as a tumor suppressor in breast cancer development and progression.In order to study the specific pathways and underlying mechanism(s) contributing to Tob1 anti-tumor activity in breast cancer, the effects of Tob1 on cell cycle regulation and apoptosis was examined by flow-cytometry assay, and the result showed an increased expression of Tob1 which caused a cell cycle G0/G1 and G2/M arrest ,and programmed cell death (apoptosis) induction associated with a down-regulation of cyclin D1, a key regulator of G1-to-S phase progression of the cell cycle, and a up-regulation of Bax, a pro-apoptotic Bcl-2 family protein.Abnormal activation of Estrogen/ER singling plays an important role in breast cancer development, progression and therapy. By using Co-immunoprecipitation, Western blot and GST pull-down assays, a series of recombinant vectors expressing wild type, truncation and"LXXLL"mutation Tob1 were constructed to analyze in vitro and in vivo association of Tob1 with ER. It was found that Tob1's interacting with ER required the N-terminus of Tob1 but not the LXXLL motif within the N-terminus. Luciferase promoter assays revealed that Tob1 significantly inhibited the estrogen-driven ER transcription activation, proposing Tob1 as an important mediator in the estrogen/ER signaling pathway in breast carcinogenesis.Tob1 was also found to be associated with cyclin D1, one of the important downstream regulators of estrogen/ER signaling pathway. Demonstrated by in vitro and in vivo binding assays, Tob1 also suppressed the activity of cyclin D1 promoter. Together, these results provided a potential pathway for Tob1's mediating cell cycle progression and cell growth.Finally, the results revealed for the first time that Tob1 increased sensitivity of breast cancer cells to ionizing radiation, by ways of reduction or inhibition of expression of critical proteins in DNA repair process such as DNA-dependent protein kinase (DNA-PK), Ku70, Ku80 and X-ray-sensitive complementation group 4 (XRCC4) as well as enhancement of radiation-mediated Bax expression.In summary, it has been demonstrated that Tob1 either functions as a tumor suppressor gene in breast cancer progression by mediating cell cycle progression and apoptosis induction or acts as a radiosensitizor in breast cancer therapy by regulating DNA damage repair. Therefore, it will put forward our understanding in promotion of diagnosis and radiotherapy for breast cancer.