Mutations in Transformation
Cancer is caused by the accumulation of mutations leading to transformation and immortality. These processes cause a vast array of phenotypic changes which will turn differentiated cells with tightly regulated growth cycles into cells with a mesenchyme-like phenotype allowing them to evade death signals and invade tissue. Coupled with anchorage-independent growth, cancer cells can quickly become a powerful weapon, invading other tissues of the body. Underlying these phenotypic changes are genetic changes – alterations of specific genes within the genome. Not every mutation in a gene will contribute to cancer progression; rather, mutations that promote growth and increase survival over normal cells are selected for. These specific genes vary in importance and prevalence in cancer progression and fall into two broad categories based on whether their expression increases or decreases cancer progression. These two categories are known as oncogenes and tumor suppressor genes. In this chapter we will focus our discussion on oncogenes, and in Chapter 3 we will investigate tumor suppressor genes.
Introduction to Oncogenes
Oncogenes are defined as mutated genes causing the transformation of normal cells into cancer cells (1). The wild-type form, known as proto-oncogenes, are present in all healthy cells to regulate controlled growth and development. A proto-oncogene becomes a cancer-causing oncogene when it acquires a dominant mutation, or several mutations. These mutations will affect the protein structure or protein expression. The perceived higher activity of oncogenes is therefore due to gain-of-function mutations or over-expression. (1). To better understand the function and significance of oncogenes in cancer, we will review the history of oncogene discovery and identification.
Retroviral oncogenes have played a central role in the understanding of cancer as a genetic disease. As early as 1958, assays were developed to facilitate the investigation of Rous Sarcoma Virus (RSV), an avian sarcoma virus (2). Further research led to the finding that RSV had the potential to transform primary fibroblasts (2), suggesting that it was carrying a tumorigenic component. In 1976, this component was identified as the gene, v-src, which originated from a cellular genome (3). This landmark study was the first to demonstrate that a cellular gene could cause cancer and was the first step towards our understanding of oncogenes today. V-src is a viral oncogene derived from the cellular proto-oncogene c-src, which RSV uses to promote proliferation of its host cell. Normally c-src, a tyrosine kinase, contains two key regulatory domains at its C-terminus. These two regulatory domains are absent in the viral version of this gene, leading to constitutive activation of v-src and its downstream targets (2). Thus, the RSV is able to induce cellular transformation because it carries a mutated gain-of-function version of a cellular gene, which results in uncontrolled growth.
So, how exactly did RSV acquire the v-src oncogene? As a retrovirus, RSV carries genetic information in the form of RNA. Upon infecting a cell, it uses its encoded reverse transcriptase enzyme to make a DNA copy of its RNA. The DNA copy is then translocated into the nucleus of the host cell and is inserted into the cellular genome (4). It is thought that for proliferation to occur, the retroviral DNA must incorporate itself directly adjacent to the c-src gene. Upon transcription of the viral genome, the RNA polymerase will continue to transcribe the c-src gene. The viral RNA transcript along with the c-src gene is then packaged into a viral particle. In other words, RSV steals a gene from its host. However, in order to become oncogenic, the c-src gene had to acquire alterations, which would confer a gain-of-function mutation and turn it into the oncogenic v-src. This likely happened quickly, as the poor proofreading ability of viral reverse transcriptase and the high proliferative ability of viruses allow for quick antigenic drift (5,6).
Another retrovirus-associated oncogene that was found to have a connection with human tumor oncogene is the Avian Myelocytomatosis Virus (AMV). This connection was discovered during the 1980s with the identification of human DNA that was homologous to the v-myc oncogene of AMV (10).
Normally, in non-cancerous cells proto-oncogene c-myc is highly regulated via different mechanisms since it is a transcriptional factor involved in processes such as cell cycle and apoptosis (11). The activation of the proto-oncogene c-myc depends on extracellular signals such as growth factors that initiate a signaling cascade, and allow the expression of c-myc by activating its transcriptional promoters. However, once present in the AMV genome the expression of v-myc is completely regulated by the virus’s transcriptional promoters that are completely unresponsive to cellular regulatory mechanisms since they lack the c-myc regulatory regions. Therefore expression of v-myc appears to be much higher and constitutive (12).
Beyond retroviral studies, many investigations into other forms of cancer have contributed to our understanding of oncogenes and their role in cancer. Initial success with src intensified interest in the field and soon researchers were racing to identify new oncogenes within a variety of cancers. In 1982, Burkitt’s lymphoma was discovered to be caused by a translocation event where the MYC gene is positioned under the control of an immunoglobulin enhancer leading to its over-expression (2,7). When MYC is complexed with MAX (myc-associated factor X) the heterodimer functions as a transcription factor and is directly involved with the regulation of several thousand cellular factors so therefore up-regulation of it is associated with uncontrolled cellular replication and apoptosis (2). A summary of these discoveries can be found in Figure 2.1.2 below.
Over-expression of a proto-oncogene due to mutations is not the only origin of oncogenes. It is also possible that a mutation or accumulation of mutations would modify the structure of a protein causing it to become overly active. An example would be the point mutation that was discovered in the ras oncogenes. RAS proteins are GTPases that are involved in signal transduction that would ultimately activate genes that part-take in cell growth, differentiation, and survival (13). The point mutation in RAS protein causes the proteins to become slightly different in structure, which results in active oncoproteins regardless of the presence of a signal (14).
Given the significance of oncogenes to our understanding of cancer, and thus future anti-cancer therapies, effective methods to identify oncogenes are of paramount importance. The greatest advancement in the identification of oncogenes came with the completion of the human genome project and subsequent development of tools enabling researchers to examine gene expression relative to this standard (8,9). Since then a variety of techniques including chromatin immunoprecipitation, genome scanning for copy number determination, and microarrays have expanded the field to build a formidable repertoire of tools for the study of oncogenes. We will cover these in more detail in the subsequent section experimental techniques.
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