Retinoblastoma Tumor Suppressor Gene: An Overview

10.5005/jp-journals-10011-1255 Sunila Thomas, Anita Balan REVIEW ARTICLE

Retinoblastoma Tumor Suppressor Gene: An Overview Sunila Thomas, Anita Balan

ABSTRACT

GENETIC BASIS OF CANCER

A genetic basis for the development of cancer has been hypothesized for nearly a century and has been supported by familial, epidemiological and cytogenetic studies. Current view is that carcinogenesis is a multistep process involving activation of oncogenes or inactivation of tumor suppressor genes. Tumor suppressor gene is a gene whose protein product can inhibit the transformation of a normal cell to a tumor cell and therefore, whose loss of function can contribute to the malignant transformation of cell. The retinoblastoma gene (Rb) is the first tumor suppressor gene identified and plays a key role in the regulation of cell cycle. A diverse body of evidence now indicates that pRb stands in the midst of a regulatory pathway and suffers disruption during the pathogenesis of majority of human tumors, including oral cancers. However, recent studies point to a more general function of pRb. In addition to tumor suppression, Rb has a role in cellular differentiation and apoptosis. This review provides an insight into the complex functions of pRb with particular reference to its role in tumor suppression.

A genetic basis for the development of cancer has been hypothesized for nearly a century and has been supported by familial, epidemiological and cytogenetic studies. Current view is that cancers arise through a multistage evolutionary process driven by inherited and somatic mutations of cellular genes and clonal selection of variant progeny with increasingly more aggressive growth properties. Three classes of genes-proto-oncogenes, tumor suppressor genes and DNA repair genes are targeted by mutations. Proto-oncogenes are genes present in normal cells, controlling cell growth, proliferation and differentiation. Proto-oncogenes function as a positive regulator of growth and can be converted to an oncogene by mutation, deletion or overexpression. Oncogenes are genes present in human cells which have undergone mutation to produce abnormal products, or control mechanisms have altered to allow gene overexpression and thus, they have lost the normal constraints on their activity. Oncogenes may be associated with carcinogenesis, but are not sufficient for tumor formation; carcinogenesis is not a simple one-step process. Precise regulations of positive and negative signals are essential to maintain normal cell growth, and disturbance of such regulation can lead to neoplasia.

Keywords: Tumor suppressor gene, Retinoblastoma tumor suppressor gene, Rb gene, Rb protein, pRb. How to cite this article: Thomas S, Balan A. Retinoblastoma Tumor Suppressor Gene: An Overview. J Indian Aca Oral Med Radiol 2012;24(1):30-35. Source of support: Nil Conflict of interest: None declared

INTRODUCTION The etiology of development of tumor cell has been the subject of intensive scientific investigation and speculation for centuries and has spawned a fascinating litany of theories. The current paradigm of tumorigenesis has evolved into a proposition that multiple steps are necessary for tumor progression—alterations in growth promoting genes (oncogenes) and loss of growth constraining mechanisms (tumor suppressor genes). Tumor suppressor genes are negative regulators of cell growth. When their normal function is compromised, absence of their inhibitory effects can lead to unrestrained cell cycle and growth. Of recent interest has been drawn to the retinoblastoma tumor suppressor gene. The retinoblastoma gene (Rb) is a tumor suppressor and its protein product pRb is a known repressor of progression toward S-phase of the cell cycle. However, recent studies point to a more general function of pRb at both the transcriptional as well as at the cellular level. This review provides an insight into the complex functions of pRb with particular reference to its role in tumor suppression.

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TUMOR SUPPRESSOR GENES Tumor suppressor gene is defined as a gene whose product can inhibit the transformation of a normal cell to a tumor cell and therefore, whose loss of function can contribute to malignant transformation of the cell.1 To date, around 30 tumor suppressor genes and more than 100 dominant oncogenes have been identified2 (Table 1). RETINOBLASTOMA TUMOR SUPPRESSOR GENE (Rb) The existence of tumor suppressor genes was first reported by Boveri.3 Based on clinical data, the first tumor suppressor gene was hypothesized by Knudson.4 By studying the epidemiology of inherited and sporadic cases of retinoblastoma, he proposed the two hit hypothesis of tumor formation which states that at least two mutational events must occur at the same locus to result in tumorigenesis. The first tumor suppressor gene, the retinoblastoma (Rb) gene was identified by three independent investigators– JAYPEE

JIAOMR Retinoblastoma Tumor Suppressor Gene: An Overview

Friend,5 Fung6 and Lee.7 Rb gene located on chromosome 13q 148 is a large gene of about 200 kB and contains 27 exons.9 The Rb gene is one of the classes of ‘recessive cancer genes’, both copies of which must be inactivated for tumorigenesis to occur.10,4 RETINOBLASTOMA TUMOR SUPPRESSOR PROTEIN (pRb) The protein product of the Rb gene is a nuclear phosphoprotein with a molecular weight of about 105,0007 spanning 928 amino acids in length that is ubiquitously expressed in all tissues.11 The half-life of pRb is relatively long implying that functional regulation is accomplished primarily by posttranscriptional mechanisms.12 The growth suppression activity of pRb is controlled at the level of phosphorylation. The hypophosphorylated form of pRb mediates G1 arrest. Rb PATHWAY IN CELL CYCLE CONTROL pRb is a key regulator of G1 progression and possesses 16 potential sites of Cdk phosphorylation. In early G1, pRb is found in hypophosphorylated state and tightly bound to the E2F transcription factor.13 When pRb is phosphorylated by Cdk complexes, E2F is released and cell can initiate DNA synthesis. 14 The phosphorylation of pRb at the Cdk consensus sites appears to be a sequential process initiated by Cdk4 and Cdk6. This allows expression of cyclin E with the formation of active Cdk2/cyclin E complexes that then

continue the phosphorylation of pRb. This leads to disruption of the pRb-E2F interaction such that E2F is transcriptionally active, a requirement for the cell to progress from G1 into S phase. As cells progress into S phase, cyclin A is expressed and becomes the primary cyclin associated with Cdk2. Progression from G2 into mitosis requires the activity of cdc2 complexed with cyclin B, which has been shown to phosphorylate proteins regulated during mitosis. Phosphorylation of pRb is maintained throughout S and G2, and then pRb is rapidly dephosphorylated during mitosis. E2F is again sequestered by hypophosphorylated pRb during G1 (Fig. 1). Rb AND CANCER A diverse body of evidence now indicates that pRb stands in the midst of a regulatory pathway and suffers disruption during the pathogenesis of a majority of human tumors. In addition to retinoblastomas5-7 and osteosarcomas,15 mutations in the Rb gene have been detected in cancers, such as small cell lung carcinomas, prostate carcinomas, breast carcinomas, some type of leukemias and cervical carcinomas, although with lower frequency compared to the former.16-19 Children with an inherited mutant Rb1 allele, when cured from retinoblastoma, often acquire other cancers like osteosarcoma at a later age due to somatic mutations in the Rb1 locus in the affected tissues.20 The variable susceptibility to cancer in different tissues in response to loss of the retinoblastoma protein (pRb) is not known.

Table 1: Tumor suppressor genes involved in human neoplasms Gene

Chromosomal location

Cellular location

Mode of action

Neoplasm associated with somatic mutation

Rb

13 q 14

Nucleus

Transcriptional regulator

p53

17 p 13.1

Nucleus

Transcriptional factor/regulator

APC

5 q 21

Cytoplasm

Unknown

WT 1 DCC

11 p 13 18 q 21

Nucleus membrane

Transcriptional factor cell adhesion molecule

NF-1

17 q 11

Cytoplasm

NF-2

22 q 12

Inner membrane

p 21, ras. GTPase activator Cytoskeleton membrane link

Schwannomas, meningiomas

VHL

3 p 25

Cytoplasm

Inhibits transcriptional elongation

Renal cell carcinoma

Retinoblastoma, osteosarcoma, carcinomas of breast, prostate, bladder and lung Most human cancers breast, brain, sarcomas, leukemias Carcinomas of colon, stomach and pancreas Nephroblastoma Carcinomas of colon and stomach Schwannomas

Journal of Indian Academy of Oral Medicine and Radiology, January-March 2012;24(1):30-35

Neoplasm associated with inherited mutation Retinoblastoma, osteosarcoma

Li-Fraumeni syndrome, carcinomas of oral cavity

Familial adenomatosis polyposis coli, carcinoma of colon Wilms’ tumor Unknown Neurofibromatosis type 1 Neurofibromatosis type-2 schwannomas, meningiomas Von Hippel-Lindau disease, angiomas and cysts of various visceral organs

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Sunila Thomas, Anita Balan

Fig. 1: The Rb pathway

Rb ABNORMALITIES IN ORAL CANCER In order to better understand the molecular biology of head and neck cancer, Pavelic21 Li22 and Yoo23 for the first time demonstrated that Rb gene has been altered in this disease. The higher rate of altered Rb expression found in advanced oral cavity tumors strongly suggests that loss of Rb protein might be associated with tumor progression in this malignancy. Since, the tumors in which the expression of Rb protein was lost were more aggressive than tumors in which the protein was present. Pavelic24 postulated that loss of Rb protein might be an important prognostic marker for early oral cavity cancer and patients with altered Rb protein may be candidates for more aggressive therapy. Rb may be functionally inactivated by overexpression of cyclin D1 or HPV infection.25 Rb function might be abrogated as hyper phosphorylation of the Rb protein by overactivation of cyclin D1-Cdk complex or loss of Cdk4 regulator p16.26,27 Alternatively, the E7 gene of several HPV’s interacts with the Rb protein releasing the E2F transcription factor.28,29 HPV have been frequently found in verrucous carcinoma and may represent another form of functional Rb inactivation in these tumors.30,31 Abnormalities in the Rb pathway are a common mechanism of oral carcinogenesis and its expression is inversely correlated with p16/MTSI expression. 32,33 Overexpression of cyclin D1 and p53 mutations were observed with a low frequency of Rb alterations in oral cancer.34,35 Regezi36 reported an overexpression of Rb along with p53, p21 and MDM2 proteins in tongue cancers. The high incidence of lack of detectable pRb in oral and pharyngeal cancers may be explained on the basis of various mechanisms accounting for inactivation of Rb gene.37 One possible mechanism may be that overexpression of Cdk4 catalyzes phosphorylation, resulting in inactivation of pRb. Investigations of the mechanisms implicated in the deregulation of growth suppressive pathways in betel and

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tobacco-related oral tumorigenesis in the Indian population showed aberrations in the p16/pRb pathway.38 The most significant finding in this study was the lack of detectable levels of pRb protein in premalignant oral lesions. Accumulation of p16 protein was observed in nearly all the pRb negative cases. A possible explanation for this event may be the unscheduled transactivation of p16 gene and consequent accumulation of the p16 protein in the absence of pRb protein.39 The lack of pRb/p16 immunoreactivity in hyperplastic lesions indicates that inactivation of pRb/p16 is early detectable events in oral tumorigenesis. These observations together with the known association between aberrant protein expression and damage of both genes support the genetic predisposition model, which suggests that multiple genetic events are implicated in oral tumorigenesis.40 THE ROLE OF pRb IN DIFFERENTIATION Terminal differentiation is characterized by permanent cell cycle withdrawal and is, on the molecular level, accompanied by the modulation of numerous genes relevant for expression of the differentiated phenotype. Loss of pRb or inactivation of pRb by oncoviral proteins interferes with differentiation and can reactivate the cell cycle machinery even in terminally differentiated cells.41,42 The activity of pRb is also regulated at the level of transcription and increases during differentiation.43 Immunohistochemical evaluation of Rb-related tumor suppressor gene p130 in oral squamous cell carcinoma reveals that protein expression may correlate with the degree of differentiation of tumors.44 THE ROLE OF pRb IN APOPTOSIS Recent experiments point to a more complex role of pRb in that it is not only a potent growth suppressor but also can counteract apoptosis and influence the decision of a cell to differentiate. The increased sensitivity of Rb deficient cells to apoptosis can be explored in cancer therapy. The Rb-MI protein may be used to protect normal cells from apoptosis during cancer therapy. The Rb function is inactivated, either by mutation of the Rb gene or by increased Rb phosphorylation, in sporadic cancer. The inactivation of Rb should sensitize tumor cells to apoptosis, unless the tumor cells have developed compensatory mechanisms to suppress apoptosis. Rb AS A PROGNOSTIC INDICATOR The immunohistochemical evaluation of Rb protein may be of particular value in the assessment of malignant tumors and may have prognostic importance. Tumors exhibiting decreased expression of the Rb protein had more aggressive JAYPEE

JIAOMR Retinoblastoma Tumor Suppressor Gene: An Overview

biological behavior and served as an important prognostic variable in patients presenting with invasive bladder cancer and ovarian cancer.45 Allelic imbalance at the Rb locus detected in potentially malignant and malignant oral lesions suggest the prognostic value of Rb alteration in oral tumors.46,47 Pavelic48 observed that tumors in which the Rb protein was altered were more aggressive than tumors in which the protein was present and suggested that loss of Rb in oral cancer may be a prognostic variable in tumor progression. Rb AS A CONDITIONAL TUMOR SUPPRESSOR Retinoblastoma protein has a well-established function in tumor suppression, because pRb inhibits cell proliferation. The role of pRb in inhibiting both cell proliferation and cell death may seem contradictory in the context of tumor development. However, these functions of pRb may be well suited to the establishment of terminally differentiated cells that are resistant to apoptosis. In fact, Rb plays an important role in the development of muscles and neurons and these are cell types that do not proliferate and are more resistant to apoptosis. The antiapoptotic function of Rb appears to be tissue specific, indicated by the differential protection of intestinal microvilli but not spleen in LPS treated Rb-MI mice. The tumor suppressor function of Rb also exhibits tissue specificity-Rb suppresses the formation of retinoblastoma in human and pituitary tumor in mice.49 In order for the tumor cells to take advantage of the loss of Rb, they must inactivate the apoptosis mechanisms that are inhibited by Rb. Because Rb can inhibit cell proliferation and apoptosis, Wang50 consider it to be a conditional tumor suppressor gene! TUMOR SUPPRESSOR GENE THERAPY OF CANCER Human cancer is a multistep process in which key regulatory genes are damaged. Germline mutations in tumor suppressor genes p53 and Rb leave affected individuals particularly susceptible to the development of cancers by reducing the number of somatic mutations required for a cell to become malignant. The ability of a single tumor suppressor gene to revert a malignant phenotype in cancer cells presumably carrying multiple mutations demonstrates the very central and pivotal role that these genes play in determining the tumorigenic or nontumorigenic properties of mammalian cells. Loss of tumor suppressor function results in a tumorigenic cell. The reintroduction of the missing tumor suppressor function through various means will then revert the cancerous cell to a nonmalignant phenotype. In fact, Rb gene was the first tumor suppressor gene shown to suppress the malignant phenotype of cancer. Although significant

advances in understanding the role of Rb in tumor initiation and progression have occurred, as well as the demonstrated ability of delivered Rb to suppress the tumorigenic phenotype, Rb gene therapy of human cancer still lies in the future. CONCLUSION The recently discovered interactions of tumor suppressor genes with transcriptional factors, may link many of the disease carcinogenic mechanisms, indicating that damage to cell cycle control mechanism may prove to be a critical factor in carcinogenesis. It is now evident that pRb is a master regulator of cell cycle, arresting progression in mid G1-S phase. Mutations lead to functional inactivation of pRb, with loss of inhibitory control on the cell cycle. This leads to uncontrolled cell proliferation, the hallmark of malignancy. Alterations in the Rb tumor suppressor protein may serve as a useful prognostic biomarker in oral tumorigenesis. This would aid in screening and identifying patients at high risk who could be followed up and preventive measures taken to reduce the risk of progression to malignancy. In addition to tumor suppression Rb has a role in cellular differentiation and apoptosis. The increased sensitivity of Rb deficient cells to apoptosis can be explored in cancer therapy. The ability of Rb gene to revert a malignant phenotype in cancer cells demonstrates its pivotal role in gene therapy of cancer. The vista of the negative regulators of cell growth termed tumor suppressor genes is just opening. These genes will be hard to find and study since their existence is most apparent when they have undergone inactivation. Undoubtedly, the struggle to find them will be worth the effort, filling a large gap in our current picture of the growth regulatory circuitry of the cell, opening new avenues in the concept of carcinogenesis that may eventually pave the way for an ultimate cure. REFERENCES 1. Klein G. Tumor suppressor genes: Specific classes. In Encyclopedia of Cancer 1997;3:1940-49. 2. Futreal PA, Kasprzyk, Birney E, Mullikin JC, Wooster R, Stratton MR. Nature 2001;40:850-52. 3. Boveri T. Zur frage der entstehung maligner tumoren. Jena, Gustav Fischer 1914. 4. Knudson AG. Mutation and cancer: Statistical study of retinoblastoma. Proc Natl Acad Sci USA 1971;68:820-23. 5. Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 1986;323:643-46. 6. Fung YK, Murphee AL, T’Ang A, Qian J, Hinrich SH, Benedict WF. Structural evidence for the authenticity of the human retinoblastoma gene. Science 1987;236:1657-61.


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ABOUT THE AUTHORS Sunila Thomas (Corresponding Author) Reader, Department of Oral Medicine and Radiology, PMS College of Dental Science and Research, Thiruvananthapuram, Kerala, India e-mail: [email protected]

Anita Balan Professor and Head, Department of Oral Medicine and Radiology Government Dental College, Calicut, Kerala, India


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