Narayanan 7/1.qxd

Cervical cancer constitutes the second most common cancer in women. It is evident from earlier studies that epidermal growth factor (EGF) is a mitogen, in that it mimics the function of estrogen by mediating cross-talk with other oncoproteins. Although epidermal growth factor receptor (EGFR) is highly expressed in breast and ovarian tumor tissues, its regulation by the exogenous source of its ligand EGF in human papillomavirus (HPV)-associated cervical cancer remains unclear. In this study, we addressed the question of whether EGF is required for the proliferation of HPV-positive cervical cancer cells and what mechanisms are involved. To determine this, we conducted a series of studies using HPV-positive human cervical cancer cells, CaSki and HeLa, and stimulated the cells with EGF. Our findings suggest that 6 h of stimulation with 10 ng/ml of EGF is sufficient to induce cell cycle progression associated with a significant increase in DNA synthesis, EGFR, COX-2 and cyclin D1 levels. Consistently, cellular localization and Western blot analysis for p-EGFR (Try-1045) protein showed an increase after EGF stimulation. Using siRNA gene knockdown assays we have shown that cyclin D1 siRNA has a significant negative effect on EGFR and inhibit cell growth independent of COX-2 levels. In summary, our findings reveal that an exogenous EGF stimulation may enhance HPV-related cervical cancer cell proliferation by activating EGFR and cyclin D1 that is independent of COX-2 levels, suggesting that the inhibitors of EGFR and cyclin D1 may be effective against cervical cancer cell proliferation. Introduction Human cervical cancer is the second most common malignancy among women worldwide. About 500,000 new cases of cervical cancer are diagnosed each year, resulting in 250,000 deaths. It is widely accepted that cervical cancer is primarily associated with the human papillomavirus (HPV) (1), which is a site-specific DNA virus that infects the basal cell layer of the squamous mucosa and replicates during epithelial cell differentiation. More than 90% of high grade cervical intraepithelial neoplasias (CIN) contain HPV type 16 and 18 DNA (2-4). In the United States, Gardasil is currently recommended for use in girls and young women 9 through 26 years of age for the prevention of cervical cancer caused by HPV 16, 18 and other types. Although HPV infection is partly an initiating event in cervical tumorigenesis, this alone is not sufficient for the progression of invasive cancer. Estrogen is believed to be an important cofactor for cervical cancer; however its critical role as a precursor or its requirement in human cervical cancer development still remains unclear. Epidermal growth factor (EGF) is a mitogen for estrogen receptor (ER). It has been proven that EGF occasionally mimics estrogen action and cross-talk with ER-· to exert its activity. Earlier studies have indicated that the malignant transformation of the squamous cell carcinoma (SCC) of the uterine cervix is associated with several molecular events, including cell cycle aberration (5). Recent studies also suggest that the pro-inflammatory protein cyclooxygenase-2 (COX-2) is overexpressed in HPV-associated cervical cancer (6,7). Unfortunately, the complex molecular interaction between EGF, COX-2 and the cell cycle regulatory proteins, including cyclin D1 in cervical cancer has not been completely investigated. Interestingly, a few studies have indicated that the treatment of murine osteoblast cells treated with EGF increased the production of prostaglandin E2 (PGE2) resulting in the activation of epidermal growth factor receptor (EGFR) (8) and cell growth. Consistently, cutaneous squamous cell carcinomas (SCC) that overexpress EGFR have been shown to be associated with cell cycle progression and metastasis (9,10). Further studies have shown that the upregulation of EGFR is correlated with an elevated level of COX-2 and PGE2 in several of the human cancers, including the carcinomas of the cervix (11). Although EGFR seems to co-localize and INTERNATIONAL JOURNAL OF ONCOLOGY 40: 13-20, 2012 13 Epidermal growth factor-stimulated human cervical cancer cell growth is associated with EGFR and cyclin D1 activation, independent of COX-2 expression levels RAJKISHEN NARAYANAN, HYE NA KIM, NARAYANAN K. NARAYANAN, DOMINICK NARGI and BHAGAVATHI NARAYANAN New York University School of Medicine, Department of Environmental Medicine, Tuxedo, NY, USA Received January 7, 2010; Accepted April 12, 2010 DOI: 10.3892/ijo.2011.1211 _________________________________________ Correspondence to: Dr Bhagavathi A. Narayanan, New York University School of Medicine, Department of Environmental Medicine, Tuxedo, NY 10987, USA E-mail: [email protected] Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; COX-2, cyclooxygenase-2; CCND1, cyclin D1; siRNA, small interfering RNA; HPV, human papillomavirus