Relationship Between P16and Characteristics Of Cervical Cancer Stem Cells

Background and objectivesCervical cancer is a common cancer in women, with high incidence second to breast cancer. Five hundred thousands of new cases of cervical cancer are occurred each year in word and80%of them are found in developing countries. Persistent infections with high-risk human papillomaviruses (HPV) are primary causes of cervical cancer but only few of the infected patients will develop to cancer.Genes of high-risk HPV is related to cervical cancer directly. The genes of HPV virus could integrate into host genome, which is the initial factor of cervical cancer. E6and E7proteins of HPV virus interact with protein P53and pRB. E6protein combines with P53by through cell ubiquitin ligase E6AP, which lead to degradation of P53and dysfunction of P14-MDM2-P53pathway and uncontrolled cell proliferation. E7can combine with pRb and lead to dissociation of pRb and E2F. Increased number of E2F protein can induce aberrant control of P16-cyclinD-CDK4/6-pRb-E2F pathway, initiate Gl-S progress and cell proliferation,p16gene acts as tumor suppressor gene in many kinds of carcinomas. Down regulation of p16caused by gene aberrations such as gene mutations, deletion and promoter methylation, is related to carcinogenesis. Relationship between cervical cancer and p16gene is also supported by cumulative data. However p16gene expression is upregulated in cervical cancer, which is different from many other cancers. To explore the role of p16gene in carcinogenesis of cervical cancer, expressions of p16gene and its related gene including Rb, c-myc and Terc were observed.Like other malignant tumors metastasis and recurrence are serious problem in treatment of cervical cancer, which decrease the therapy efficacy and survival of cancer patients, especially for patients in advanced stage. At the end of last century stem cell theory was founded and accepted by most of people. And the hypothesis of cancer stem cell (CSC) becomes hotpoint among oncologists. It’s thought CSC might be main mechanism of cancer genesis. Cumulated evidence in tumors shows the exist of CSC in leukemia and solid tumors. Common sense about CSC is that it’s kinds of subtypes of tumor cells owing the characteristics of self-renew, multi-direction differentiation, new tumor formation and resistance to radiation and chemical therapy. They have the abilities of anti-apoptosis and high invasion potential, which is the underlined mechanism of tumor recurrence.Advance in CSC of cervical cancer includes the finding of surface markers (ABCG2, ALDH), drug-resistance, and tumor formation in vivo. All these features are similar to the CSCs in many other tumors. Also some embryo stem or adult stem markers such as Oct4, Nanog, and Nestin, were upregulated in cervical cancer stem cells. More and more published data support that CSC is the determined factor of metastasis of cervical cancer.To understand the regulation mechanism of p16in cervical cancer stem cell, lentivirus p16-shRNA vector was constructed to establish stable p16shRNA expression cell line from parental Hela cervical cancer cell line. After silencing of p16gene expression, cancer stem cell characteristics of Hela were observed.Methods1. Specimens of193cervical infection lesions,52cervical cancers,193cases of benign lesions of cervix and normal cervical mucosa from40healthy volunteers were collected from in-/out-patients departments, by either surgery or biopsy.2. Expressions of pl6and pRb proteins were detected using immunohistochemical assay, and Terc and c-myc genes by fluorescence in situ hybridazation (FISH), in three different types of tissues.3. pLenti6/BLOCK-iT-DEST vector system was used to construct p16gene shRNA vector. Hela parental cell line was transfected using constructed vector above for stable gene silencing cell line, p16gene expersstion was identified by both RT-PCR and western-blot methods.4. Hela cell was cultured in serum free conditioned medium to induce cell spheres in which p16gene expression was observed. Cell sphere formation was also observed in p16gene silencing Hela cells.5. Reported cervical cancer stem cell markers including CD44, CD24and ABCG2(by Flow cytometry), and CK17(western-blot) were detected in Hela cells after p16gene silence.6. In vitro assays including cancer cell sphere formation and invasion assay in Transwell were observed in Hela cells with p16gene silence. Tumor model was established using different number of Hela cells with or without p16gene silence by subcutaneous injection, and tumor size and weight was observed.Results1. Positive rates of p16protein, Terc and c-myc genes were different in chronic cervicitis, cervical intraepithelial neoplasia (CIN) and cervical cancer (p=0.000). Gene expressions increased along with the severity of the histological grades. pRb protein expression down-regulated along with the severity of the histological grades(p=0.000).2. There were no significant difference between p16, pRb or Terc gene and the histological grades, clinical stages and metastasis status of cervical lesions. Expression of c-myc gene in G3grade was significantly higher than that in G2/G1(P0.05).3. Among3shRNA vectors designed for p16gene silencing, strongest inhibition effect was found by using sh289vector after p16gene expression identification. It showed no effect on p14ARF expression and used for next experiments.4. Cancer cell spheres were cultured by serum-free medium form Hela parental cell line. Cells collected from sphere showed higher p16gene expressions both in mRNA and protein levels.5. After p16gene silence, cell number of CD44+/CD24-and ABCG2decreased by FACS, but no obvious alteration for CK17protein expression by western-blot. Cell numbers passed through Matrigel decreased after p16gene silence compared to Hela parental cells.6. More cancer cell was needed for tumor model formation after p16gene silence in nude mice. Tumor size and weight in mice established with p16gene silence Hela cells were less than those with Hela parental cell model.Conclusions1. In carcinogenesis of cervical cancer, from chronic cervicitis, CIN to cervical cancer, p16gene up-regulation was an early event and aberrant alteration happened in CIN stage. There were no relationships between p16gene expression and clinical stages, biological grades and metastasis. Expressions of pRb protein decreased, and c-myc and Terc genes decreased along with the grades of cervical lesions.2. High expression of p16gene is related to pRb, c-myc and Terc genes.3. p16-sh289could inhibit p16gene expression but no effect on p14ARF.4. High level p16gene expression is found in Hela tumor sphere cells. Sphere formation, tumor cell invasion could be inhibited by p16gene silence. Other cancer stem cell markers including CD44+/CD24-and ABCG2could be inhibited by p16gene silence.5. Tumor cell invasion ability in vitro and tumor formation in vivo could be suppressed by p16gene silence.