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«Dipak Sapkota Dissertation for the degree of Philosophiae Doctor (PhD) University of Bergen, Norway 2011 UNIVERSITETET I BERGEN S100 gene family ...»

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S100 Gene Family Members in

Oral Squamous Cell Carcinomas (OSCCs):

Functional Characterization of S100A14 in

Proliferation and Invasion of OSCC Derived Cells

Dipak Sapkota

Dissertation for the degree of Philosophiae Doctor (PhD)

University of Bergen, Norway



S100 gene family members in oral squamous cell carcinomas (OSCCs):

Functional characterization of S100A14 in proliferation and invasion of OSCC derived cells Dipak Sapkota Dissertation for the degree of Philosophiae Doctor (PhD) at the University of Bergen 2011 2 ‘This work is dedicated to my parents, brother and sisters’ 3 4 Table of contents ACKNOWLEDGEMENTS





1.1. Oral squamous cell carcinoma (OSCC)

1.2. Potentially malignant oral disorders

1.3. Molecular biology of OSCC

1.4. S100 gene family members

1.4.1. S100A14

1.4.2. Expression profile and sub-cellular localization of S100A14

1.4.3. Biological functions of S100A14

1.5. Cell cycle regulation

1.5.1. p53, p21 and p27 proteins

1.5.2. S100 proteins, cell cycle regulation and tumor growth

1.6. Tumor invasion and regulatory molecules: Role of MMPs

1.6.1. Role of S100 proteins in tumor invasion and metastasis



3.1. Patients, tissue specimens and cells

3.2. mRNA and protein analyses

3.3. Selection of the endogenous control

3.4. Modulation of S100A14 expression in vitro

3.5. Functional assays






5 6 Acknowledgements I would like to express my sincere gratitude to my supervisors, Associate Professor Salah O.

Ibrahim and Professor Endre N. Vasstrand for giving me the opportunity to take this PhD. I am especially thankful to Salah O. Ibrahim for the initiation of this project, and his constant guidance and support during my study.

I am deeply grateful to Dr. Ove Bruland and Associate Professor Daniela E. Costea for introducing me to the field of molecular biology and cell culture, respectively. I will never forget their friendly support, guidance and encouragement.

I wish to thank Professor Anne C. Johannessen for allowing me to use the facilities in Oral Pathology lab and for providing me the opportunity to be a part of the wonderful environment at Oral Pathology. I would also like to thank Professor Anders Molven and Professor Bjørn Tore Gjersten for providing me with their laboratory facilities.

I am grateful to Mrs. Gunnvor Øijordsbakken and Mrs. Inger Ottesen for their excellent technical assistance. I am also thankful to Dr.Oleg Tsinkalovsky for his assistance during cell sorting and for the humorous moments we shared in the cell culture lab. I am thankful to Dr. Therese Bredholt, Dr. Janice Nigro and Sjur Huseby for their valuable help in different laboratory techniques.

I thank my colleagues and staffs at BBB 5th Floor, Oral Pathology, Dept. of Clinical Dentistry and Centre for International Health. I am grateful to Dr. Øyvind Halskau for his help and useful technical advices. I thank Associate Professor Niels Aarsaether for fruitful discussions. I thank Professor Ian F Pryme for his help in linguistic improvement of the thesis.

I am extremely grateful to my Nepali friends, Narottam, Kamal, Keshav, Rajib, Himalaya, Ridaya, Chirag and members of the Nepali Bergen Society for making my stay in Bergen easier and for the great social moments we shared.

Finally, I would like to express my love and gratitude to my parents, TR Sapkota and Ambika Sapkota for their love, care, and tireless efforts for my education. Special thanks to my younger brother, Rajendra, my sisters, Sita, Santi, Parbati and Laxmi and my special one, Sunita Sharma, for their love, care and support.

This study was supported by the Norwegian State Educational Loan Fund (Quota Programme), Meltzer’s fond, Norsk Dental Depots fond and the Norwegian Research Council.

Bergen, September, 2010 Dipak Sapkota

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It is generally accepted that carcinogenic substances in the form of tobacco, alcohol or oncogenic viruses, etc cause genetic changes resulting in conversion of normal oral epithelium to a potentially malignant (dysplastic) lesion, and subsequently into the invasive oral squamous cell carcinoma (OSCC). However, the precise molecular mechanism underlying OSCC carcinogenesis remains unclear. Several members of the multifunctional Ca2+ binding S100 proteins have been described in connection with a range of human cancers, including OSCCs. Studies from our group, using high throughput genomic and proteomic methods, have previously identified differential expression of members of the S100 proteins in OSCCs from different populations. S100A14 is a recently identified member of the S100 protein family. Although differential expression of S100A14 has been described in different human cancers, its biological roles in carcinogenesis have not been well characterized. This study aimed (i) to examine the mRNA expression profile of 16 of the S100 gene family members in OSCCs and (ii) to characterize the possible role(s) of S100A14 in proliferation and invasion of OSCC derived cells.

We identified significant down-regulation of S100A4, S100A6, S100A8 and S100A14 mRNAs in OSCCs compared to their pair-wised controls. Down-regulation of S100A14 was further validated at the protein level in OSCC archival tissues using immunohistochemistry, and in an in vitro oral cancer progression model both at the mRNA and protein levels. To investigate the functional roles of S100A14, we employed retroviral vector mediated overexpression and siRNA mediated knock-down of the endogenous S100A14 in two OSCC derived cell-lines (CaLH3 and H357). S100A14 over-expression resulted in a significant reduction in CaLH3 cell proliferation due to G1-phase cell cycle arrest, but not apoptosis.

This G1- arrest was found to be associated with nuclear accumulation of the tumor suppressor protein p53 and p53-dependent up-regulation of p21. These findings suggest a functional link

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OSCC derived cells and support the idea that S100A14 might function as a tumor suppressor protein working in the p53 pathway.

Characterization of the role of S100A14 in tumor invasion showed that overexpression of S100A14 resulted in a significant decrease in the invasive potential of the OSCC derived CaLH3 and H357 cell-lines, whereas siRNA mediated knock-down resulted in a significant increase in the invasive potential of the CaLH3 cell-line in vitro. PCR array and validation using qRT-PCR and gelatin zymography revealed that S100A14 over-expression was associated with down-regulation of MMP1 and MMP9 mRNAs in both CaLH3 and H357 cell-lines and suppression of MMP9 activity in the CaLH3 cell-line. Additionally, an inverse correlation between mRNA expression levels of MMP1 and MMP9 with S100A14 was found in OSCC tissue samples. These findings suggest that S100A14 negatively regulates expression and activity of MMP1 and MMP9 and that might be responsible for the S100A14 mediated regulation of tumor cell invasion. In conclusion, findings of this work suggest that differential expression of several of the S100 gene family members is a common genetic alteration in OSCCs. S100A14, similar to other members of the S100 family, is involved in key cellular functions such as cell cycle regulation and tumor cell invasion indicating a tumor suppressor role for S100A14. Uncontrolled cell proliferation and invasion, characteristics of OSCCs, might therefore be related to altered expression of S100A14 frequently observed in these cancers.

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This thesis is based on the following original publications referred to in the text by their roman numerals Paper I Sapkota D, Bruland O, Bøe OE, Bakeer H, Elgindi OAA, Vasstrand EN, Ibrahim SO (2008).

Expression profile of the S100 gene family members in oral squamous cell carcinomas. J Oral Pathol Med 37: 607-615.

Paper II Sapkota D, Costea DE, Blø M, Bruland O, Lorens JB, Vasstrand EN, Ibrahim SO. S100A14 induces G1-arrest through nuclear accumulation of p53 and p53 –dependent up-regulation of p21 in oral carcinoma derived cells (manuscript).

Paper III Sapkota D, Bruland O, Costea DE, Haugen H, Vasstrand EN, Ibrahim SO. S100A14 regulates the invasive potential of oral squamous cell carcinoma derived cell-lines in vitro by modulating expression of matrix metalloproteinases, MMP1 and MMP9 (Eur J Cancer, In Press).

Papers I and III are reprinted with permission from John Wiley and Sons and Elsevier publishers respectively. All rights reserved.

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1.1. Oral squamous cell carcinoma (OSCC) Head and neck cancers encompass malignancies that arise in the oral cavity, nasal cavity, para nasal sinuses, pharynx and larynx. Oral cancers represent 40% of all head and neck cancers and consist of the malignancies arising in the lip, tongue, floor of the mouth, gingiva, palate and buccal mucosa [1, 2]. The most frequent neoplasms arising from the oral epithelium are oral squamous cell carcinomas (OSCCs), representing more than 90% of all oral cancers.

Combined with pharyngeal cancers, oral cancers rank as the sixth most common type of cancer world-wide, being the third most common in the developing countries (for example,

South and Southeast Asian countries: Pakistan, India, Sri Lanka, Taiwan; African countries:

Sudan, etc) [3]. This wide geographical variation in the incidence of OSCCs has been linked with the country specific risk factors, for example: betel quid and smokeless tobacco in South and Southeast Asian countries [4] and toombak in the Sudan [5, 6]. Several etiological factors namely use of tobacco (smoked and smokeless) and alcohol, infection with human papilloma virus (HPV) or herpes simplex virus (HSV), dietary deficiencies or imbalances (micronutrient deficiency), genetic predisposition, etc have been linked with the development of OSCCs.

Based on the available global evidence, Warnakulasurya S. has categorized risk factors for OSCCs into established, strongly suggestive, possible and speculative factors (Table 1) [7].

Table 1: Suggested risk factors for OSCCs (adapted from Warnakulasurya S [7])

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Oral carcinogenesis is believed to evolve as a multi-step process where the majority of OSCCs are thought to be preceded by or associated with potentially malignant (dysplastic) oral epithelial lesions. Several terms ‘pre-cancer’, ‘precursor lesions’, ‘intra-epithelial neoplasia’ and ‘potentially malignant’ have been used broadly and interchangeably to describe the clinical presentation of oral lesions that may have the potential to transform into cancer. Recently, the term ‘potentially malignant disorders’ has been recommended when referring to these lesions [8]. Leukoplakia (white patches) and erythoplakia (red patches), the most common form of potentially malignant oral disorders, carry high risk of malignant transformation. Malignant transformation rates up to 36% and 50% have been reported respectively for leukoplakia and erythoplakia [reviewed in 9]. Malignant transformation rates of these lesions are often correlated with the severity of the histological (dysplastic) changes.

In addition, several molecular alterations have been identified and correlated with the malignant potential of these lesions. Despite the progress in the field of molecular biology, no single or a set of molecular markers can reliably predict the malignant transformation rates of oral premalignant lesions [10].

1.3. Molecular biology of OSCC Genetic damage lies central to the carcinogenesis process. It is generally accepted that alterations (usually mutations) in three classes of genes namely, oncogenes, tumor-suppressor genes and stability genes are responsible for the development of human cancers [reviewed in 11]. Oncogenes are the genes whose protein products are either produced in higher amounts or have higher activity, hence acting in a dominant manner (gain of function). Oncogenes can be activated by chromosomal translocations or insertional mutagenesis, gene amplifications or by activating point mutations/deletions. On the other hand, tumor suppressor genes are the genes whose protein products have loss of function due to mutations and are recessive in

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or insertional mutations or epigenetic silencing result in loss of function of tumor suppressor genes. The stability genes or caretakers are involved in the repair of genetic damage induced during normal cell division or due to exposure with carcinogenic environment. Stability genes are also inactivated in a manner similar to that of the tumor suppressor genes [11, 12].

Mutations in these classes of genes ultimately result in the development of an invasive cancer characterized by self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, invasion and metastasis to local and distant organs, limitless replicative potential and sustained angiogenesis [13].

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