11-tumor-suppressors-study-guide.pdf

Biochemical foundations of pharmaceutical biotechnology Tumor Suppressor Genes Audrey Minden, Ph.D. Department of Chemical Biology Ernest Mario School of Pharmacy Susan Lehman Cullman Laboratory for Cancer Research, Room 205 (848) 445-5766 [email protected] Tumor suppressor genes: Outl...

Biochemical foundations of pharmaceutical biotechnology Tumor Suppressor Genes Audrey Minden, Ph.D. Department of Chemical Biology Ernest Mario School of Pharmacy Susan Lehman Cullman Laboratory for Cancer Research, Room 205 (848) 445-5766 [email protected] Tumor suppressor genes: Outline of topics covered: I. II. III. IV. Overview of tumor suppressors Cell fusion experiments and the identification of tumor suppressors The major categories of tumor suppressor genes The Rb tumor suppressor was the first tumor suppressors identified A. Sporadic vs familial Rb B. Types of cancers caused by Rb C. Challenges in treating cancers caused by tumor suppressors D. CDK inhibitors, Rb, and synthetic lethality V. Loss of heretozygosity (LOH) and tumor suppressors VI. Other tumor suppressor genes A. p53 1. The role for p53 2. p53 and drug treatment B. NF1 1. NF1 and cancer 2. The function of NF1 C. APC 1. Wnt, frizzled, and the APC / beta catenin pathway in normal and cancer cells 2. APC mutations in cancer D. BRCA genes 1. Functions of BRCA 2. PARP inhibitors and BRCA in cancer treatment, and synthetic lethality VII. Promoter methylation as a way to downregulate gene expression VIII. Some examples of tumor suppressors that are regulated by promoter methylation IX. The VHL tumor suppressor 2 Oncogenes compared with tumor suppressor genes (Figure from Alberts) 3 Tumor Suppressor Genes Tumor suppressor genes are genes that constrain or suppress cell proliferation They can be considered anti-growth genes. Unlike oncogenes, they are involved in cancer once the genes have mutations that disrupt their activity (inactivating mutations) or if the genes are deleted altogether. 4 Cell fusion experiments Cell fusion experiments provided some of the first clues for the presence of tumor suppressor genes: 5 Cell fusion experiments Cell fusion experiments: When cells growing adjacent to each other in culture are exposed to a fusogenic agent (such as Sendai virus or polyethylene glycol (PEG)), they initially form a heterokaryon with multiple nuclei. When this cell passes through mitosis, the two sets of parental chromosomes are pooled in a single nucleus. Figure 7.1 The Biology of Cancer (Weinberg) 6 Cell fusion experiments In this example a heterokaryon is formed by fusion of a cancer cell with a normal cell In some cases, the resulting heterokaryon is non-tumorigenic, indicating that the cancer alleles are recessive: Evidence for tumor suppressor genes Figure 7.3 The Biology of Cancer (Weinberg) 7 Types of tumor suppressors Tumor suppressor genes can be divided broadly into two types: A. Suppress normal cell cycle progression (proliferation) or induce differentiation (ex: Rb) B. Suppress the cell cycle (proliferation) in response to metabolic imbalances or genomic damage, or stress (ex: p53) 8 1. Rb is a tumor suppressor that suppresses the normal cell cycle Rb DP1 P M Rb E2F DP1 Rb G2 growth factors E2F G1 Go CyclinD Rb P CyclinD Cdk4/6 S Rb restriction point Rb DP1 E2F P P Rb Cdk4/6 interphase + E2F transcription repressed DP1 DP1 E2F transcription 9 2. p53 is a tumor suppressor that suppresses the cell cycle in response to stresses such as genomic damage DNA damage p53 levels increase p53 p53 p21 gene p21 mRNA p21 (CKI) Cyclin p21 protein p21 (CKI) Cdk Thr- P Thr+ Tyr- P P Inactive: No progression from G1 to S 10 The Rb tumor suppressor 11 1. Rb is a tumor suppressor that suppresses the normal cell cycle Rb DP1 P M Rb E2F DP1 Rb G2 growth factors E2F G1 Go CyclinD Rb P CyclinD Cdk4/6 S Cdk4/6 Rb restriction point DP1 E2F P Rb P Rb interphase + E2F transcription repressed DP1 DP1 E2F transcription 12 Retinoblastoma: Rb Rb was one of the first tumor suppressor genes discovered Its loss is associated with a retinal tumor 13 Retinoblastoma (Rb) Retinoblastoma is a tumor of the retina, arising from the photoreceptor cells, found in about 1 in 200,000 children. Two types of Retinoblastoma: Sporadic: - No family history. - Usually unilateral - Low recurrence risk Familial: - A parent also had the disease. - Usually bilateral. - Increased risk of development of other cancers later in life, especially osteosarcomas. 14 LOH The Rb gene was identified as a gene on chromosome 13 in humans Tumor suppressors: Loss of Heterozygosity at a tumor suppressor locus such as Rb leads to cancer Heterozygous Mutant allele Wild-type allele Loss of heterozygosity (LOH) Two mutant alleles 15 Rb inheritance Rb: Loss of Heterozygosity (LOH) causes cancer loss of heterozygosity (LOH) Figure 7.7 The Biology of Cancer (Weinberg) 16 Types of cancers caused by Rb mutation Loss of function of both alleles of Rb is most commonly associated with Retinoblastoma. However, Rb mutation is also, more rarely, associated with some other cancers. The most common is Osteosarcoma Treatment of cancers caused by Rb mutations In general, treatment of cancers caused by tumor suppressors can be more challenging than those caused by oncogenes. More general treatments such as chemotherapy, radiation, and surgery are often used. 17 Loss of Heterozygosity (LOH) Loss of Heterozygosity (LOH) is an important feature of Rb and all tumor suppressors Cancer can occur when there is LOH at the tumor suppressor locus 18 LOH Tumor suppressors: Loss of Heterozygosity at a tumor suppressor locus leads to cancer LOH Heterozygous Mutant allele Wild-type allele Loss of heterozygosity 19 LOH can occur by different mechanisms How does loss of heterozygosity occur? Here are several mechanisms by which it could occur: We did not discuss any details about mitotic recombination or gene conversion, you do not need to be familiar with those. Epigenetic silencing usually refers to promoter methylation 20 Some other examples of tumor suppressor genes 21 p53 22 p53 DNA damage Rb p53 DP 1 P M Rb E2 F p21 DP 1 Rb G2 Growth Factors E2 F G1 Go Rb P CyclinD CyclinD S p21 (CKI) Cdk4/6 Rb restriction point Rb P DP1 E2F P Rb Cdk4/6 interphase + E2 F transcription repressed DP 1 DP1 E2F transcription 23 p53 The p53 protein is encoded by the TP53 gene -p53 puts a break on the cell cycle in response to DNA damage -Thus it is highly protective against passing down mutated or damaged DNA to daughter cells. -Mutations in TP53 are one of the most common genetic alterations in cancer, found in about 50% of cancers. 24 p53 and drug treatment There are currently no FDA approved direct drugs targeting TP53 mutations. Research involves studying ways to restore p53 function indirectly, such as development of drugs that target pathways downstream of p53 or that modulate other proteins involved in p53 regulation. In some cases p53 may not be absent in cancer, but mutated or improperly folded. Restoring such a mutant p53 into its wild-type state is another area of investigation. Gene therapy is also an area of investigation. 25 Neurofibromatosis (NF1) 26 NF1 Neurofibromatosis (NF). NF is associated with mutation of both alleles of the NF1 gene. This familial disorder is associated with the development of benign tumors of the cell sheaths around nerves. Occasionally these can become malignant: neurofibrosarcomas. Patients are at increased risk for certain cancers 27 The NF1 gene The NF1 gene was cloned in 1990 It is transmitted similarly to Rb By sequencing the gene, its function became better understood It has sequence similarity to a yeast protein called IRA, which is a Ras GAP protein It is expressed largely in neural tissues, but is also expressed in some other tissues 28 NF1 codes for a Ras GAP Inactive Ras GDP GEF GAP (GAP stimulates hydrolysis (GTP to GDP)) Ras GTP Active In cells where NF1 is inactive by mutation, Ras stays in its active form. In this sense, mutant NF1 mimics active Ras. (NF1 is found in some cells in nervous system tissue 29 APC 30 The APC Tumor Suppressor gene (adenomatous polyposis coli) APC is a tumor suppressor gene whose mutation is frequently associated with colon cancer 31 APC APC is an important part of the Wnt signaling pathway Wnt signaling and APC Destruction complex APC is a tumor suppressor due to its role in regulating b-catenin Figure 5.24 The Biology of Cancer (Weinberg) 32 APC Destruction complex is inactive Figure 6.26b The Biology of Cancer (Weinberg) c-Myc, CyclinD1,... 33 Wnt pathway in the healthy colon Wnt levels are high near the bottoms of the crypts, after having been are released by surrounding cells. This keeps b-catenin levels high in the cells at the bottom (because Wnt prevents b-catenin’s degradation) These cells at the bottom of the crypt obtain a stem cell like state, they can proliferate and are not very differentiated As the progeny of these cells migrate upward, they move away from the high Wnt levels In cells that are farther from the crypt, b-catenin levels go down, and proliferation decreases and stops. These cells are more differentiated Figure 7.24a The Biology of Cancer (Weinberg) Wnt 34 Wnt pathway in the healthy colon The differentiated cells function briefly to form the lining of the gut, then die by apoptosis and are shed into the colonic lumen This process of out migration to cell death takes 3-4 days This system is highly protective against cancer, because cells die within days Figure 7.24a The Biology of Cancer (Weinberg) 35 Mutations in the APC gene are often present in colon cancer Destruction complex Figure 5.24 The Biology of Cancer (Weinberg) (no proliferation) Mutations in APC associated with colon cancer often consist of premature termination of the APC protein, thereby removing domains that are important for its ability to associate with b-catenin and axin. These truncated APC proteins are therefore deficient in degradation of b-catenin. 36 Mutations in the APC gene in cancer: Mutations are most common in the region of APC that binds to b-catenin Figure 7.25a The Biology of Cancer (Weinberg) 37 Wnt signaling and APC - summary Figure 5.24 The Biology of Cancer (Weinberg) (No proliferation) In summary, loss or mutation of APC causes cells in the colonic crypt to proliferate excessively (regardless of the presence or absence of Wnt. This results in too much proliferation and also gives the cells a chance to develop other mutations, such as Ras mutations. 38 APC mutation in colon cancer: Figure 7.24a The Biology of Cancer (Weinberg) 39 Resources Helpful reading material: Alberts, Molecular Biology of the Cell. Chapter 20 Weinberg, The biology of cancer, Chapters 5-7 Zheng, F. et al. (2020) . Mechanism and current progress of Poly ADP-ribose polymerase (PARP) inhibitors in the treatment of ovarian cancer. Biomedicine and Pharmacotherapy. 123: 109661 References for some of the figures: Yoshida, K., and Miki, Y. (2004). Role of BRCA1 and BRCA2 as regulators of DNA repair, transcription, and cell cycle in response to DNA damage. Cancer Sci. 95:866-71 40