Head and Neck Cancers - Research Paper Example

?The relationship between SC35 and E2F1 in the apoptotic response of head and neck cancer cells following cisplatin treatment Literature Background: Head and neck cancers are the sixth most common amongst all cancers globally and each year almost 100,800 new cases are diagnosed in Europe alone (Leemans, Braakhuis, & Brakenhoff, 2011). Over the last several decades, an increase in the incidence of these cancers has been observed which has several implications for the health care system, as these cancers are not only associated with significant morbidity and mortality but also pose an economic burden since they incur enormous health care related costs. The impact of head and neck cancers on the health care system can be gauged by the fact that each year almost 500,000 new cases of head and neck cancers occur globally, with almost two thirds of the cases occurring in developing nations (Marur & Forastiere, 2008; Lung, Tascau, Almasan, & Muresan, 2007). There are several risk factors for the development of head and neck cancer but amongst those, the most important are tobacco use and alcohol consumption (Forastiere, Koch, Trotti, & Sidransky, 2001). Studies have revealed that the consumption of tobacco and alcohol has the strongest association with these cancers and these factors have a synergistic effect in the causation of head and neck cancers (Leemans, Braakhuis, & Brakenhoff, 2011). It has been found that tobacco usage increases the risk of developing head and neck cancer from five to up to twenty five-fold (Marur & Forastiere, 2008). Moreover, the use of both tobacco and alcohol leads to a forty times greater risk for these cancers (Marur & Forastiere, 2008). Other important risk factors which have a role in the aetiology of these cancers include infection with certain strains of Human Papilloma Virus (HPV) (Forastiere, Koch, Trotti, & Sidransky, 2001) and inherent predisposing factors such as Fanconi’s anemia or a genetic predisposition to develop such cancers (Leemans, Braakhuis, & Brakenhoff, 2011). The most common histological type of head and neck cancers is squamous cell carcinomas (Marur & Forastiere, 2008) and the common locations include the oral cavity, nasopharynx, oropharynx, larynx and hypopharynx (reviewed by Leemans, Braakhuis, & Brakenhoff, 2011). Over the last few years, there has been an increase in the incidence of cancers occurring at the base of the tongue and the tonsils. This changing trend has been attributed to the increase in the occurrence of HPV-associated squamous cell carcinoma (discussed above) which occurs due to HPV infection following oral sex, which has become an increasingly popular practice amongst the younger generation (Marur & Forastiere, 2008). More recently, it has become common practice to classify head and neck tumors into two main subcategories based in the underlying aetiologies and risk factors, viz. HPV-positive and HPV-negative head and neck tumors. Studies have revealed that these tumors belonging to these two categories differ in not only the aetiology and causative factors but also have different underlying molecular mechanisms, which cause them to have different levels of tumor severity and prognosis (Leemans, Braakhuis, & Brakenhoff, 2011). Head and neck cancer is a heterogeneous disease, which can arise due to several different molecular mechanisms, each of which have different implications for the cancer invasiveness, severity, response to treatment, prognosis and patient survival rates (Leemans, Braakhuis, & Brakenhoff, 2011). Cancers are shown to be clonal replications of cells that have acquired certain genetic alterations which cause them to undergo unchecked cellular proliferation. These genetic alterations can occur in two main classes of genes which are important in the cell cycle, viz. proto-oncogenes and tumor suppressor genes (Forastiere, Koch, Trotti, & Sidransky, 2001) In the case of tumors of the head and neck, studies have revealed that the most commonly implicated genetic change in tumors of this region is the loss of region 9p21 of the chromosomes which leads to inactivation of the p16 gene. This inactivation leads to downstream alterations in the cell cycle since the p16 gene is responsible for the inhibition of cyclin-dependent kinases (CDKs) which are important in the regulation of cell cycle (Forastiere, Koch, Trotti, & Sidransky, 2001). Another important genetic alteration which plays an important role in the aetiology of head and tumors is the mutation of p53 tumor suppressor gene which is located at 17p13. The p53 mutation in these cells increases the invasiveness of the tumor and predisposes the tumor cells to further genetic progression (Forastiere, Koch, Trotti, & Sidransky, 2001). There are two main molecular pathways which have been implicated in the aetiology of tumors of the head and neck region, viz. (1) the MDM2 and P53 pathway; and (2) the pathway involving CDKN2A (p16), RB1, CCND1 (Cyclin D1) and E2F1 (Braakhuis, Brakenhoff, & Leemans, 2005). Both these pathways are discussed in detail below. E2F and its role in the etiology of Head and Neck Cancers: E2F is a transcription factor which plays an important role in the aetiology of head and neck squamous cell cancers. This important transcription factor plays an important role in the regulation of the cell cycle and in apoptosis, i.e., programmed cell death (T?mar, Csuk, Remenar, Repassy, & Kasler, 2005). It has been found that E2F transcription factor is involved in promoting the transcription of the genes involved in DNA synthesis. Moreover, this transcription factor also enables cells to progress from the G1 phase of the cell cycle into the S phase (Zhang, Liu, & al., 2000). Studies have revealed that tumors which display an over-expression of this transcription factor have a higher potential to relapse locally after treatment (T?mar, Csuk, Remenar, Repassy, & Kasler, 2005). The E2F1 transcription factor has been found to have two possible mechanisms whereby it can contribute towards the aetiology of head and neck cancers. It has been found that in normal cells, E2F1 is involved in regulation of the expression of a particular class of cyclins, viz. the G1 cyclins which include cyclin D1. Studies have elucidated that E2F1 is involved in the upregulation of cyclin D1 in normal cells (T?mar, Csuk, Remenar, Repassy, & Kasler, 2005). Moreover, E2F1 also induces the ARF/CDKN2A pathway, resulting in the neutralization of MDM2, which helps in the stabilization of the p-53 tumor suppressor gene. This mechanism enables to keep cellular proliferation in check via the sensitization of the cell to apoptosis (T?mar, Csuk, Remenar, Repassy, & Kasler, 2005). A defect in either of the two aforementioned functions of the E2F1 gene can cause the cell to undergo genetic transformation and convert into neoplastic cells, however, which of the two mechanisms has a more significant role in the etiology of head and neck tumor still remains to be elucidated (T?mar, Csuk, Remenar, Repassy, & Kasler, 2005). Since E2F plays such a significant role in the control of cell progression through various stages of the cell cycle and in apaoptosis, it is of prime importance that the levels and expression of E2F is higly regulated in the human body in order to keep cellular proliferation in check under normal conditions. D-type cyclins have been shown to play a role in the regulation of the activity of E2F1 via the activation of Cdk4/Cdk6 (Zhang, Liu, & al., 2000). This activation is achieved via binding of Cyclin D1 to the Cdk4/Cdk6 complex. The activated complex is then involved in the phosphorylation of pRb (which is a product of the Rb gene), which causes the dissociation of the pRb-E2F complex, resulting in the release of E2F. Any alterations in the aforementioned pathway can lead to overexpression of E2F1 in the cells resulting in uncontrolled cellular proliferation which results in the development of various neoplams, including and not limited to head and neck cancers (Zhang, Liu, & al., 2000). More recently, a novel mechanism of E2F in the regulation of apoptosis has been elucidated. It has been found that E2F is involved in the transcriptional upregulation of this splicing factor, SC35 (Korotayev & Ginsberg, 2008). This function of E2F in the regulation of splicing factor SC35 is discussed further in the folowing discussion of SC35 and its relation with E2F (Zhang, Liu, & al., 2000). SC35 and its relation with E2F1: SC35 is a protein that belongs to a family of splicing factors which have been found to be rich in serine/arginine residues (Korotayev & Ginsberg, 2008). These splicing factors have been found to serve important functions in both consitiutive and alternative splicing in normal human cells. Since splicing, in particular alternative splicing, is an important step in the regulation of apoptosis, SC35, plays an important role in the regulation of apoptosis in normal human cells. It has been discovered that E2F1 is involved in the regulation of the expression of SC35. This is achieved via the binding of E2F1 to the promoter region of th SC35 gene, which causes the activation of this gene and promotes SC35 expression (Korotayev & Ginsberg, 2008). SC35 in turn is then involved in the alternative splicing of various proteins involved in mediating apoptosis in the cells such as c-FLIP, Bcl-X and Caspases 8 and 9. Via the alternative splicing of these apoptotic genes, SC35 is involved in the increased expression of the pro-apoptotic splice variants of these genes in favor of the anti-apoptotic variants, thus promoting apoptosis in cells (Korotayev & Ginsberg, 2008). Merdzhanova et al. (2008) elucidated that the response to DNA dmanaging agents, such as the alkylating agents methylmethanesulfonate and cyclophosphamide, the E2F mediated expression of SC35 is upregulated (Merdzhanova, et al., 2008). This has important implications for the treatment of neoplams. It has been found that tumor cells develop the ability to become resistant to chemotherapeutic agents via shifting the balance of splice variants of apoptotic genes in the favor of anti-apoptotic splice variants (Merdzhanova, et al., 2008). Since SC35 expression meditaed via E2F1 is increased in the presence of DNA damaging agents, it can be postulated that this pathway is a strong determinant of the response of cancer cells to various chemotherapeutic agents. This study, thus, focuses on unravelling the relationship between SC35 and E2F1 in the apoptotic response of head and neck cancer cells following cisplatin treatment. Aims and Objectives: This study aims to elucidate whether the mechanism of resistance in head and neck cancer cells relates to a defective apoptotic response due to changes in the E2F1/SC35 axis by using human head and neck cancer cell line HN5 and a cisplatin-resistant variant – HN5CisR and comparing them. We hypothesize that there is a down-regulation of this gene in cisplatin resistant cells as revealed by an initial microarray screen. Moreover, this study aims to compare the expression levels of SC35 and E2F1 in the drug sensitive and drug resistant lines constitutively and when challenged with sub-lethal doses of cisplatin and to examine the apoptotic response in those cell lines. In addition, the study will investigate the effects of a siRNA approach to silence SC35 in head and neck cancer cells to determine whether this approach modulates the apoptotic response. Experimental plan: In order to address the above-mentioned hypotheses and objectives, the following experimental methodology will be used. The HN5 human head and neck cell lines provided from Dr Helen Coley, will inititally be cultured using the standard culture medium (RPMI with stable glutamine) which consists of vitamins, amino acids, growth factors, cytokines or serum (Zhang, Liu, Johnson, & Klein-Szanto, 2000). The cell lines then obtained will be treated with the chemotherapeutic agent (cisplatin). The cisplatin resistant cell line-HN5CisR was developed by my supervisor Dr Helen Coley. Cells will be grown at 370C. The apoptotic response in both cell lines will be compared using flow cytometry with the Annexin V assay combined with propidium iodide staining. This will enable us to evaluate and quantify the proportion of cells undergoing apoptosis. To determine the expression levels of SC35 and E2F1 in the drug sensitive and drug resistant cell lines in order to address the second objective of this study, whole cell lysates will be prepared and then will be subjected to SDS-PAGE analysis for western immuno-blotting. The expression levels of different proteins such as E2F1, SC35 and key apoptotic players such as caspase 9 will then be determined and compared in both the cell lines. Lastly, the effect of a siRNA approach directed against the SC35 gene on the apoptotic response in the HN5 cell line will be examined using flow cytometry. Figure 1: Experimental Protocol . References Braakhuis, B. J., Brakenhoff, R. H., & Leemans, C. R. (2005). Head and neck cancer: molecular carcinogenesis. Annals of Oncology . Forastiere, A., Koch, W., Trotti, A., & Sidransky, D. (2001). Head and Neck Cancer. The New England Journal of Medicine , 1890-1900. Fearon, E., & Vogelstein, B. (1990). A genetic model of colorectal tumorigenesis. Cell , 759-767. Goldenberg, D., Lee, J., & Koch, W. e. (2004). Habitual risk factors for head and neck cancer. Otolaryngol Head Neck Surgery , 986-993. Korotayev, K., & Ginsberg, D. (2008). Many pathways to apoptosis: E2F1 regulates splicing of apoptotic genes. Cell Death and Differentiation , 1813–1814. Kutler, D. I. (2003). High incidence of head and neck squamous cell carcinoma in patients with fanconi anemia. Archives Otolaryngoly Head Neck Surgery , 106-112. Leemans, C. R., Braakhuis, B. J., & Brakenhoff, R. H. (2011). The molecular biology of head and neck cancer. Nature Reviews . Lung, T., Tascau, O. C., Almasan, H. A., & Muresan, O. (2007). Head and neck cancer, epidemiology and histological aspects – Part 1: A decade’s results 1993–2002. Journal of Cranio-Maxillofacial Surgery , 120-125. Lydiatt, W., Davidson, P., Schantz, S., Carvana, S., & Chaganti, R. (1998). 9p21 deletion correlates with recurrence in head and neck cancer. Head and Neck , 113-118. Marur, S., & Forastiere, A. A. (2008). Head and Neck Cancer: Changing Epidemiology, Diagnosis, and Treatment. Mayo Clinic Proceedings , 489-501. Mercatante, D., & Kole, R. (2000). Modification of alternative splicing pathways as a potential approach to chemotherapy. Pharmacologic Therapy , 237-243 Merdzhanova, G., Edmond, V., Seranno, S. D., Broeck, A. V., Corcos, L., Brambilla, C., et al. (2008). E2F1 controls alternative splicing pattern of genes involved in apoptosis through upregulation of the splicing factor SC35. Cell Death and Differentiation , 1815–1823. Mouth Cancer Foundation. (2011, March 5). Retrieved March 4, 2011, from Mouth Cancer Foundation: http://www.rdoc.org.uk/ Riet, P. V., Nawroz, H., & Hruban, R. (1994). Frequent loss of chromosome 9p21-22 early in head and neck cancer progression. Cancer Research , 1156-1158. Shiboski, C., Schmidt, B., & Jordan, R. (2005). Tongue and tonsil carcinoma: increasing trends in the U.S. population ages 20-44 years. Cancer , 1843-1849. T?mar, J., Csuk, O., Remenar, E., Repassy, G., & Kasler, M. (2005). Progression of head and neck squamous cell cancer. Cancer and Metastasis Reviews , 107-127. Zhang, S. Y., Liu, S. C., & al., D. G. (2000). E2F-1 Gene Transfer Enhances Invasiveness of Human Head and Neck Carcinoma Cell Lines. Cancer Research , 5972-5976. Read More