Dr.Bharathi Sengodan M.D., Lecture 3 :Molecular Basis of Cancer (2) Learning objectives: This lecture provides an understanding of The classification, action and the effects of the mutated oncogenes (common) and tumour suppressor genes The classification, action and the effects of the mutated oncogenes (common) and tumour suppressor genes Learning outcomes At the end of the lecture student will be able to Classify oncogenes and tumour suppressor gene Classify oncogenes and tumour suppressor gene Describe the action and oncogenic effects of RAS & MYC genes Describe the action and oncogenic effects of RAS & MYC genes Describe the action and effects of mutated tumour suppressor genes [P53 &APC] Describe the action and effects of mutated tumour suppressor genes [P53 &APC]

Dr.Bharathi Sengodan M.D., Oncogenes – Go genes Stop gene Tumor suppressor genes-Stop gene Genes that promote NORMAL cells growth are called proto-oncogenes. Genes that promote NORMAL cells growth are called proto-oncogenes. Mutations convert proto- oncogenes into constitutively active cellular oncogenes that are involved in tumor development Mutations convert proto- oncogenes into constitutively active cellular oncogenes that are involved in tumor development Inactivation of one normal allele of a gene locus produces tumours (Heterozygous) Tumor suppressor genes apply brakes to cell proliferation & form checkpoints to prevent uncontrolled growth Failure of growth inhibition is one of the fundamental alterations in the process of carcinogenesis. Both normal alleles of a gene locus should be inactivated (2 hits), to produce tumours (Homozygous) [Exception: Loss of gene function caused by damage to a single allele is called haploinsufficiency ]

Dr.Bharathi Sengodan M.D., ONCOGENES The primary function of oncogenes is to disrupt the cell's growth control systems and cause continuous division The primary function of oncogenes is to disrupt the cell's growth control systems and cause continuous division They act at the level of Growth factors Growth factorsrowth factorsrowth factors Growth factor receptors Growth factor receptors Growth factor receptors Growth factor receptors Intracellular signal transducers Intracellular signal transducersntracellular signal transducersntracellular signal transducers Nuclear transcription factors Nuclear transcription factorsuclear transcription factorsuclear transcription factors Cell cycle regulators Cell cycle regulators [classification] [classification]

Dr.Bharathi Sengodan M.D., The RAS Oncogene Point mutation of RAS family genes is the single most common abnormality of dominant oncogenes in human tumors. Point mutation of RAS family genes is the single most common abnormality of dominant oncogenes in human tumors. Approximately 15% to 20% of all human tumors contain mutated versions of RAS proteins. Approximately 15% to 20% of all human tumors contain mutated versions of RAS proteins. Normal RAS proteins are tethered to the cytoplasmic aspect of the plasma membrane, and they flip back and forth between an activated, signal-transmitting form and an inactive, quiescent state. Normal RAS proteins are tethered to the cytoplasmic aspect of the plasma membrane, and they flip back and forth between an activated, signal-transmitting form and an inactive, quiescent state. Several distinct mutations of RAS have been identified in cancer cells colon and pancreas - mutations of KRAS, colon and pancreas - mutations of KRAS, bladder tumors have HRAS mutations, bladder tumors have HRAS mutations, hematopoietic tumors bear NRAS mutations. hematopoietic tumors bear NRAS mutations.

Dr.Bharathi Sengodan M.D., In the inactive state, RAS proteins bind guanosine diphosphate (GDP) In the inactive state, RAS proteins bind guanosine diphosphate (GDP) When cells are stimulated -RAS becomes activated by exchanging GDP for GTP When cells are stimulated -RAS becomes activated by exchanging GDP for GTP Activated RAS acts on the MAP kinase pathway by recruiting the cytosolic protein RAF-1 Activated RAS acts on the MAP kinase pathway by recruiting the cytosolic protein RAF-1 The activated MAP kinases target nuclear transcription factors and promote mitogenesis The activated MAP kinases target nuclear transcription factors and promote mitogenesis In normal cells, the activated signal-transmitting stage of the RAS protein is transient because of GTPase activity In normal cells, the activated signal-transmitting stage of the RAS protein is transient because of GTPase activity GTPase hydrolyzing GTP to GDP, thereby returning RAS to its quiescent ground state GTPase hydrolyzing GTP to GDP, thereby returning RAS to its quiescent ground state GTPase activity is accelerated by GTPase-activating proteins (GAPs). GAPs function as "brakes" GTPase activity is accelerated by GTPase-activating proteins (GAPs). GAPs function as "brakes" Mutations of RAS gene reduces the braking action of GAPs and Mutant RAS proteins are "trapped" in their excited GTP-bound form, causing a pathologic activation of the mitogenic signaling pathway and excessive proliferation of cells. Mutations of RAS gene reduces the braking action of GAPs and Mutant RAS proteins are "trapped" in their excited GTP-bound form, causing a pathologic activation of the mitogenic signaling pathway and excessive proliferation of cells. by GTPase

Dr.Bharathi Sengodan M.D., Proteins involved in signal transduction - Nonreceptor Tyrosine Kinases Proteins involved in signal transduction - Nonreceptor Tyrosine Kinases ABL gene ABL gene is translocated from its normal abode on chromosome 9 to 22 where it fuses with the BCR gene (t 9:22) ABL gene is translocated from its normal abode on chromosome 9 to 22 where it fuses with the BCR gene (t 9:22) BCR-ABL fusion gene, has potent and constitutive tyrosine kinase activity BCR-ABL fusion gene, has potent and constitutive tyrosine kinase activity BCR-ABL translocation is seen in Chronic myeloid leukemia BCR-ABL translocation is seen in Chronic myeloid leukemia Treatment of CML is revolutionized by the development of imatinib mesylate, a "designer" drug that targets the BCR-ABL tyrosine kinase. Treatment of CML is revolutionized by the development of imatinib mesylate, a "designer" drug that targets the BCR-ABL tyrosine kinase.imatinib mesylate imatinib mesylate Nuclear Transcription Factor Nuclear Transcription Factor MYC Gene MYC Gene Eg: C-MYC,N-MYC,L-MYC The molecular basis of MYCThe molecular basis of MYC MYC interacts with components of the DNA- replication, and are involved in proliferation Overexpression of MYC may drive activation of more origins than needed for normal cell division or act in concert to reprogram somatic cells into pluripotent stem cellsOverexpression of MYC may drive activation of more origins than needed for normal cell division or act in concert to reprogram somatic cells into pluripotent stem cells Burkitt lymphoma, a B-cell tumor (t 8:14) and Burkitt lymphoma, a B-cell tumor (t 8:14) and Breast, colon, lung, and many other carcinomas are related to MYC dysregulationBreast, colon, lung, and many other carcinomas are related to MYC dysregulation

Dr.Bharathi Sengodan M.D., Tumor suppressor genes Failure of growth inhibition is one of the fundamental alterations in the process of carcinogenesis. Failure of growth inhibition is one of the fundamental alterations in the process of carcinogenesis. Tumor suppressor genes apply brakes to cell proliferation Tumor suppressor genes apply brakes to cell proliferation Part of a regulatory network that recognizes genotoxic stress from any source, and responds by shutting down proliferation or prod the cells into apoptosis. Part of a regulatory network that recognizes genotoxic stress from any source, and responds by shutting down proliferation or prod the cells into apoptosis. Another set of tumor suppressors seem to be involved in cell differentiation, causing cells to enter a postmitotic, differentiated pool without replicative potential. Another set of tumor suppressors seem to be involved in cell differentiation, causing cells to enter a postmitotic, differentiated pool without replicative potential. Similar to mitogenic signals, growth-inhibitory, pro- differentiation signals originate outside the cell and use receptors, signal transducers, and nuclear transcription regulators to accomplish their effects [Classification] Similar to mitogenic signals, growth-inhibitory, pro- differentiation signals originate outside the cell and use receptors, signal transducers, and nuclear transcription regulators to accomplish their effects [Classification][Classification]

Dr.Bharathi Sengodan M.D., RB gene Approximately 60% of retinoblastomas are sporadic, and the remaining are familial. Approximately 60% of retinoblastomas are sporadic, and the remaining are familial. To explain the inherited and sporadic occurrence of retinoblastomas, Knudson proposed his “two-hit” hypothesis of oncogenesis To explain the inherited and sporadic occurrence of retinoblastomas, Knudson proposed his “two-hit” hypothesis of oncogenesis Knudson's hypothesis can be stated as follows Two mutations (hits), involving both alleles of RB at chromosome locus 13q14, are required to produce retinoblastoma. Two mutations (hits), involving both alleles of RB at chromosome locus 13q14, are required to produce retinoblastoma. In the sporadic form both mutations at the RB locus are acquired by the retinal cells after birth. In the familial form, all somatic cells inherit one mutant RB gene from a carrier parent. The second mutation affects the RB locus in one of the retinal cells after birth.

Dr.Bharathi Sengodan M.D., The role of RB in regulating the G1-S checkpoint of the cell cycle. Hypophosphorylated RB in complex with the E2F transcription factors binds to DNA, recruits chromatin-remodeling factors (histone deacetylases and histone methyltransferases), and inhibits transcription of genes whose products are required for the S phase of the cell cycle. When RB is phosphorylated by the cyclin D–CDK4, cyclin D–CDK6, and cyclin E–CDK2 complexes, it releases E2F. The latter then activates transcription of S-phase genes. The phosphorylation of RB is inhibited by CDKIs, because they inactivate cyclin-CDK complexes.

Dr.Bharathi Sengodan M.D., p53: Guardian of the Genome. The official name of the gene is TP53 and the protein is p53; for the sake of simplicity, we refer to both as “p53” The official name of the gene is TP53 and the protein is p53; for the sake of simplicity, we refer to both as “p53” p53 acts as a “molecular policeman” that prevents the propagation of genetically damaged cells. p53 acts as a “molecular policeman” that prevents the propagation of genetically damaged cells. Over 50% of human tumors contain mutations in this gene. Over 50% of human tumors contain mutations in this gene. Homozygous loss of p53 occurs in virtually every type of cancer, including carcinomas of the lung, colon, and breast Homozygous loss of p53 occurs in virtually every type of cancer, including carcinomas of the lung, colon, and breast In most cases, the inactivating mutations affect both p53 alleles and are acquired in somatic cells (not inherited). Less commonly, some individuals inherit one mutant p53 allele. Such individuals, said to have the Li- Fraumeni syndrome, who have a 25- fold greater chance of developing a malignant tumor by age 50

Dr.Bharathi Sengodan M.D., In addition to somatic and inherited mutations, p53 functions can be inactivated by other mechanisms. In addition to somatic and inherited mutations, p53 functions can be inactivated by other mechanisms. 1. The transforming proteins of several DNA viruses, including the E6 protein of HPV, can bind to and promote the degradation of p53 2. Function of the p53 pathway is blocked by mutation in another gene that regulates p53 function - MDM2 and MDMX stimulate the degradation of p53.These proteins are frequently overexpressed in 33% of human sarcomas, thereby causing functional loss of p53 in these tumors.

Dr.Bharathi Sengodan M.D.,

APC/β-Catenin Pathway. Adenomatous polyposis coli genes (APC) represents a class of tumor suppressors whose main function is to down- regulate growth-promoting signals. Adenomatous polyposis coli genes (APC) represents a class of tumor suppressors whose main function is to down- regulate growth-promoting signals. Germ-line mutations at the APC (5q21) loci are associated with familial adenomatous polyposis, in which all individuals born with one mutant allele develop thousands of adenomatous polyps in the colon during their teens or 20s AND one or more of these polyps undergoes malignant transformation, giving rise to colon cancer. Germ-line mutations at the APC (5q21) loci are associated with familial adenomatous polyposis, in which all individuals born with one mutant allele develop thousands of adenomatous polyps in the colon during their teens or 20s AND one or more of these polyps undergoes malignant transformation, giving rise to colon cancer. As with other tumor suppressor genes, both copies of the APC gene must be lost for a tumor to arise. As with other tumor suppressor genes, both copies of the APC gene must be lost for a tumor to arise.

Dr.Bharathi Sengodan M.D., The role of APC in regulating the stability and function of β-catenin. APC and β-catenin are components of the WNT signaling pathway. In resting cells, β-catenin forms a macromolecular complex containing the APC protein & leads to the destruction of β-catenin, and levels of β-catenin are low. B, When cells are stimulated by WNT molecules, the destruction complex is deactivated, β-catenin degradation does not occur, and cytoplasmic levels increase. β-catenin translocates to the nucleus, where it binds to TCF, a transcription factor that activates genes involved in cell cycle progression. C, When APC is mutated or absent, the destruction of β-catenin cannot occur. β-catenin translocates to the nucleus and coactivates genes that promote entry into the cell cycle

Dr.Bharathi Sengodan M.D., Targeted therapy Molecular alteration in ERB B2 is specific for the cancer cells, new therapeutic agents consisting of monoclonal antibodies against ERB B2 have been developed and are currently in use clinically. Molecular alteration in ERB B2 is specific for the cancer cells, new therapeutic agents consisting of monoclonal antibodies against ERB B2 have been developed and are currently in use clinically. Another example is the blockage of receptor tyrosine kinase activity of c-KIT in stromal tumors of the gastrointestinal tract. Another example is the blockage of receptor tyrosine kinase activity of c-KIT in stromal tumors of the gastrointestinal tract. This type of therapy, directed to a specific alteration in the cancer cell, is called targeted therapy This type of therapy, directed to a specific alteration in the cancer cell, is called targeted therapy Various therapeutic modalities aimed at increasing normal p53 activity in tumor cells that retain this type of activity or selectively killing cells with defective p53 function are being investigated. Various therapeutic modalities aimed at increasing normal p53 activity in tumor cells that retain this type of activity or selectively killing cells with defective p53 function are being investigated.

Dr.Bharathi Sengodan M.D., Dysregulation of cancer associated genes Genetic damage which activates oncogenes or inactivates tumor suppressor genes - subtle or involve segments of chromosomes Genetic damage which activates oncogenes or inactivates tumor suppressor genes - subtle or involve segments of chromosomes Abnormalities in chromosomes which are associated with neoplasms include Abnormalities in chromosomes which are associated with neoplasms include  Chromosomal changes (Translocation & Deletions)  Gene amplification  Epigenetic changes

Dr.Bharathi Sengodan M.D., Chromosomal changes Translocation Burkitt’s lymphoma -MYC gene translocated to IgH gene.(t8:14)Burkitt’s lymphoma -MYC gene translocated to IgH gene.(t8:14) Mantle cell lymphoma- (t11;14) (Cyclin D1 to IgH gene)Mantle cell lymphoma- (t11;14) (Cyclin D1 to IgH gene) Chronic myeloid leukemia CML – (t 9:22) -BCR ABL translocationChronic myeloid leukemia CML – (t 9:22) -BCR ABL translocation Acute leukemias : AML – (t8;21) /ALL-(t15;17 )Acute leukemias : AML – (t8;21) /ALL-(t15;17 ) Ewing sarcoma – (t11;22)Ewing sarcoma – (t11;22) Prostatic adenocarcinoma - (t21;21)Prostatic adenocarcinoma - (t21;21) Certain neoplasms have non random & karyotypic abnormalities - e.g.-leukemias, lymphomas.non random & karyotypic abnormalities - e.g.-leukemias, lymphomas. Aneuploidy & chromosomal instability –initiating events in tumor growth.Aneuploidy & chromosomal instability –initiating events in tumor growth. Deletions. Chromosomal deletions are the second most prevalent structural abnormality in tumor cells. Compared with translocations, deletions are more common in nonhematopoietic solid tumors. Deletions involving chromosome 13q14, the site of the RB gene, are associated with retinoblastoma. Deletions of 17p, 5q, and 18q have all been noted in colorectal cancers; Deletion of 3p, noted in several tumors, is extremely common in small-cell lung carcinomas

Dr.Bharathi Sengodan M.D., Gene Amplification Over expression of protoonogenes may result from reduplication & amplification of DNA sequences. Over expression of protoonogenes may result from reduplication & amplification of DNA sequences. Readily detected microscopically as- Readily detected microscopically as- Double minutes (dms)- small chromosome like structures &Homogenous staining regions (HSRs). E.g.- ERB-B2 in breast cancers, N-MYC in neuroblastoma. E.g.- ERB-B2 in breast cancers, N-MYC in neuroblastoma. Epigenetic changes Epigenetics refers to reversible, heritable changes in gene expression that occur without mutation.Epigenetics refers to reversible, heritable changes in gene expression that occur without mutation. Such changes involve post- translational modifications of histones and DNA methylation, both of which affect gene expressionSuch changes involve post- translational modifications of histones and DNA methylation, both of which affect gene expression Leads to silencing of genesLeads to silencing of genes E.g- BRCA1 in breast cancer, VHL in renal cell carcinomas, and the MLH1 mismatch-repair gene in colorectal cancerE.g- BRCA1 in breast cancer, VHL in renal cell carcinomas, and the MLH1 mismatch-repair gene in colorectal cancer