The Relationship Of Growth Factor Pgrn And Cervical Cancer And Its Signal Transduction Mechanisms In Cervical Cancer And Radiation-inducible Rbap48Contributes To Radiosensitivity Of Cervical Cancer Cells

Part One:The relationship of growth factor PGRN and cervical cancer and its signal transduction mechanisms in cervical cancer cellsCervical cancer is one of the most common neoplastic diseases among women, with a combined worldwide incidence of approximately one-half million new cases annually and rates of morbidity and fatality second only to breast cancer.85%of the cervical cancer cases occurred in developing world and there are150thousand new cases and20thousand death cases of cervical cancer annually in China. In addition, in recent years the average cervical cancer patient has become progressively younger. Approximately90%of cervical cancer cases as associated with human papillomavirus (HPV) infection, particularly with high risk HPV types such as HPV16and HPV18, indicating that HPV infection is the most important cause for cervical carcinomas. HPV is a small DNA virus with a genome of approximately8000base pairs. The tumorigenesis of cervical cancer is associated with upregulated expression of the viral oncogenes E6and E7. The E6and E7proteins inactivate two tumor suppressor proteins, p53and pRb respectively.Progranulin (PGRN), a pluripotent secreted growth factor, was first discovered in trophoblasts of the preimplantation embryo. PGRN is abundantly expressed in rapidly cycling epithelial cells, in cells of the immune system, in neurons, and in chondrocytes. High levels of PGRN expression are also found in varieties of human cancers and contribute to tumorigenesis in breast cancer, ovarian carcinoma, and multiple myeloma. PGRN plays a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, inflammation, and host defense.PGRN supports tumor growth by increased cell proliferation, decreased cell apoptosis, and greater invasiveness through the extracellular matrix (ECM). Each of these actions requires the activity of the Erk and PI3K signal transduction pathways. PGRN may activate phosphorylation of focal adhesion kinase (FAK) in integrin signaling pathways. In bladder cancer cells, PGRN promoted the association of FAK with paxillin and Erk, indicating, PGRN may have a link with ECM signaling machinery.To better understand the function of PGRN, we need to probe the mechanisms that control PGRN expression, how actions of PGRN are mediated, and what the consequences are. Unveiling the mystery of these proteins may represent an alternative for the development of therapeutic strategies.The relationship of PGRN and cervical cancer has not been reported. Here, we first investigated the expression and regulatory role of PGRN in cervical cancer. 1. Expression of PGRN in cervical cancerImmunohistochemisty was carried out to determine the expression of PGRN in cervical cancer tissues, which showed that PGRN is significantly higher expressed in cervical cancer tissue compared with normal cervix tissue. Western blot and Elisa assays were used to detect the level of intracellular and secreted PGRN in HeLa (HPV18+), SiHa (HPV16+) and human cervical mucosa epithelial H8cell lines. PGRN were powerfully detectable in both cell lysate and medium supernatant of three cell lines. Intracellular and secreted PGRN in H8cells is lower than those in Siha and HeLa cells. The different expression of PGRN among cervical mucosa epithelial cells and cervical cancer cells suggested PGRN may play an important role in malignancy on cervical cancer. To explore the cause of high expression of PGRN in cervical cancer, the effect of HPV oncogenes on the expression of PGRN were investigated. Western blot assay indicated that HPV16E7, but not E6, increased the expression of PGRN in RPE1cells. Other potential inducing factors were also detected. IL-6and estrogen can effectively stimulate PGRN expression in cervical epithelial cell lines.2. PGRN contributes to cervical cancer cells proliferation in vitro and in vivoTo determine the effect of PGRN on the proliferation of cervical caner cells, H8cells were administrated with rhPGRN or transfected with pc-DNA3.1-PGRN and HeLa cells were transfected with PGRN siRNA. The proliferation assay, colony formation assay and scratch assay showed that PGRN can regulate cell proliferation, colony formation, and migration of cervical mucosa epithelial cells. In human tumor xenografts mice model, intraperitoneal injection or tumor injection of recombinant PGRN promoted the tumor formation in BALB/c nude mice transplanted with H8cells, and HeLa cells transfected with PGRN siRNA inhibited tumor growth in mice. In the allografts mice model, a mouse lung cancer cell line TC-1, which was obtained by cotransformation of HPV-16E6/E7and activated ras oncogene to primary mouse lung epithelial cells of C57BL/6, were injected into WT and PGRN KO mice respectively. Deficiency of endogenous PGRN in host mice effectively inhibited tumor growth. To elucidate the molecular mechanism of PGRN in regulation of tumor growth, cell cycle and apoptosis of cervical cancer cells, the expression profiles of p53, and Rb respond to PGRN treatment were analyzed by western blot assay. Stimulation with rhPGRN markedly enhanced p53and Rb expression expression of H8cells.3. The regulatory role of PGRN on the PI3K/Akt/mTOR pathwayGrowth factors, mitogens and hormones activate the PI3K signaling pathway and consequently mTOR. AKT, p70S6K and FOXO1are the members of mTOR signalling pathway. As a growth factor, whether PGRN can stimulate mTOR signalling pathway is still unknown. In this study, the regulatory role of PGRN in phosphorylation of Akt, mTOR and activation of mTORC1and mTORC2were investigated. PGRN strongly induced phosphorylation of mTOR in a PI3K/Akt dependent manner, and subsequently enhanced activity of mTORC1and mTORC2, including phosphorylation of4E-BP1, p70S6K, Akt (Ser473) and FoxO1.In conclusion, PGRN is highly expressed in cervical cancer tissue, cell lysate and cell culture supernatant, which might be regulate by HPV16E7, IL-6and estrogen. PGRN regulates cell proliferation and malignant transformation of human cervical derived epithelial cells in vitro and in vivo. PGRN stimulation enhanced the activation of mTOR and determined the activity of its downstream signaling molecules. This study provides the first evidence linking PGRN and cervical cancer, gives an idea that PGRN plays critical role in cervical carcinogenesis, and both in vitro and in vivo studies will contribute to the PGRN targeting therapeutic strategy to cancer, including cervical cancer. Part Two:Radiation-inducible protein RbAp48contributes to radiosensitivity of cervical cancer cellsCervical cancer is one of the most common neoplastic diseases among women, with a combined worldwide incidence of approximately one-half million new cases annually and rates of morbidity and fatality second only to breast cancer. In addition, in recent years the average cervical cancer patient has become progressively younger. Over90%of cervical carcinomas are positive for human papillomavirus (HPV) DNA, indicating that HPV infection is the most important cause for cervical cancer. The human papillomavirus (HPV) is a non-enveloped double-stranded DNA virus that belongs to the Papillomaviridae family. The two primary oncoproteins of high risk HPV types are E6and E7. Viral genome integration into host DNA genome increases E6and E7expression to promote cellular proliferation and the chance of malignancy. RbAp48, a48-kDa protein, initially identified as a retinoblastoma-binding protein, has been shown to play an important role in nucleosome assembly and histone modification. E2F-1and RbAp48are physically associated in the presence of Rb and HDAC, indicating that RbAp48is involved in the transcriptional regulation of E2F-responsive genes. Our previously studies have demonstrated that RbAp48is a critical mediator that controls HPV16transforming activity in HPV-induced cervical carcmogenesis. Radical hysterectomy is the principal treatment for cervical cancer, but the therapeutic efficacy of radiation is limited by the occurrence of radiation resistance. RbAp48has also been predicted as a radiosensitive gene, overexpression of RbAp48induces radiosensitization in breast cancer and melanoma cell lines. However, the role of RbAp48in radiation sensitivity of cervical cancer cells is still unknown.1. RbAp48is an irradiation inducible protein in cervical cancer To verify whether RbAp48is also sensitive to radiation in cervical cancer, the expression of RbAp48was investigated in cervical cancer cells exposed to irradiation. Real-time PCR and western blotting were performed to evaluate the mRNA and protein level of RbAp48in SiHa, Caski and SiHa with different dose of irradiation. The data showed radiation treatment significantly facilitated the expression of RbAp48both in mRNA and protein levels in three cervical cancer cell lines compared with untreated control cells. It’s indicated RbAp48is an irradiation inducible protein in cervical cancer cells.2. RbAp48contributes to radiosensitivity of cervical cancer cells in vitro To detect whether RbAp48is related with radiation in cervical cancer, we changed RbAp48gene expression in SiHa, Caski and HeLa cells by RbAp48expression plasmid and siRNA targeting RbAp48. CCK-8assay, cell number counting analysis, survival assay and soft agar assay indicated that combination of RbAp48overexpression and radiation treatment had a much more remarkable inhibitory effect on cell proliferation and viability of cervical cancer cells, and reduced RbAp48expression significantly increased proliferation and viability of cervical cancer cells in response to various doses of radiation treatment. These results indicated that alteration of RbAp48expression affected the survival and viability of cervical cancer cells after radiation treatment.3. The mechanism of RbAp48regulated radiosensitivity of cervical cancer cells To elucidate the molecular mechanism of RbAp48-mediated radiosensitivity of cervical cancer cells, flow cytometry analysed cell cycle distribution and apoptosis in response to radiation. Meanwhile, real time RT-PCR analysis was employed to assess the mRNA levels of p53, Rb and caspase-8in cervical cancer cell lines with alterative expression of RbAp48treated with radiation. RbAp48-overexpressed cells treated with radiation exhibited more remarkable G2/M phase arrest and apoptosis compared with radiation treated control. RbAp48-silenced cervical cancer cells displayed more dramatic reduction in radiation-induced G2/M phase arrest and apoptosis compared with control siRNA-transfected cell lines. RbAp48-overexpression markedly enhanced radiation-induced mRNA levels of p53, Rb and caspase-8in cervical cancer cells. Radiation combined with RbAp48-silencing sharply inhibited the mRNA levels of p53, Rb and caspase-8in cervical cancer cells compared with that in cells treated with radiation alone. The data of this part indicated that RbAp48contributes to the radiosensitivity of cervical cancer cells by inducing G2/M phase arrest, cell apoptosis and mRNA levels of p53, Rb and caspase-8.4. Adenovirus-mediated overexpression of RbAp48enhances radiosensitivity of cervical cancer cells and tumor growth in vivo To evaluate the potential role of RbAp48as a therapeutic target in radiation therapy of cervical cancer, the RbAp48expression adenovirus, Ad5-RbAp48, was constructed. Infection of Ad5-RbAp48markedly enhanced the expression of RbAp48in cervical cancer cells and promoted the radiosensitivity of cervical cancer cells in vitro. A combination of Ad5-RbAp48infection and irradiation more decreased tumor size and weight than mice treated with Ad5-EGFP infection and irradiation, regardless of the cell lines used to transplant. These data suggested that adenovirus-mediated RbAp48delivery might be a potential adjuvant approach to the radiation therapy of cervical cancer.In conclusion, RbAp48is a radiation inducible protein, and contributes to radiosensitivity of cervical cancer cells. Identification of RbAp48as a novel critical mediator in the radiosensitivity of cervical cancer as well as the elucidation of molecular events involved provide insights into the processes regulating cellular response of cervical cancer to radiation therapy. Considering its potent ability in promotion of radiosensitivity, RbAp48also has great potential to be employed as a therapeutic target for irradiation treatment of cancer, including cervical carcinoma.