1. Chapter 18: Genetics of Cancer and Cell-Cycle Regulation

2. What is Cancer? Large number of complex diseases
Behave differently depending upon cell type from which originate
Age on onset, invasiveness, response to treatment
Common general properties
Abnormal cell growth/division (cell proliferation)
If only this is a benign tumor
When grow in culture without contact inhibition are referred to as transformed
Spread to other regions of body (metastasis)
Malignant tumors

3. Cancer Second leading cause of disease in Western Countries
1 million new cases per year in U.S.
500,000 per year die
War “declared” on cancer approximately 30 years ago
Slowly treatments are changing/improving based upon better genetic understanding of the varieties

4. Cancer Rates in US

6. Age and Cancer
Note log scale for incidence rate

7. Abnormal Cell Growth

8. Cancer is a Genetic Disease
Genome alterations
One nucleotide to large-scale chromosome rearrangements, amplifications and deletions
Mostly in somatic cells (unless associated with inherited risk—about 1% of total)
Alter cellular functions
DNA repair, cell division , apoptosis, cellular differentiation and cell-cell contact/communication

9. Molecular Biology of Neoplasia
Retroviral oncogenes:
Abl, akt, erbB, ets, fos, kit, myb, myc, raf, ras, rel, src, yes
Proto-oncogene: normal gene that can undergo a genetic change to become cancerous.
Oncogene: a gene that causes a normal gene to become cancerous. Mutant overactive form of a proto-oncogene.
Tumor suppressor gene: a recessive mutation in an inhibitory gene, thus inactivating the gene. Loss of function causes tumorigenesis.

10. Functions of Cancer Causing Genes/Alleles
Many disrupt control of cell cycle
Oncogenes
Proto-oncogenes
Normal genes that if mutated may act to make a cell cancerous
Recessive, cancer causing forms active and stimulates cell division
C-oncogenes and v-oncogenes
Tumor suppressors
Genes whose products act to regulate cell cycle
Loss of gene product function contributes to cancer process
Recessive, commonly involved with inherited risk
About 200 proto-oncogenes and tumor suppressor genes

11. Oncogenes are identified through their dominant transforming effects

12. Changes observed when a normal tissue culture cell is transformed by a tumor virus or an expression vector carrying the oncogene
1. Alterations in the nucleus
2. Plasmamembrane related abnormalities
3. Adherence abnormalities
4. Growth and division abnormalities
5. Defective differentiation
6. Inability to undergo apoptosis following DNA damage

13. Normal and Cancer Karyotypes
Chromosome painting
(a) is a normal cell
(b) is a “very messed up” cancer cell

14. Clonal Origin of Tumors
Tumor arises from a single cell
Burkitt’s lymphoma
Translocation involving chromosome 8 (myc) and either chromosomes 2, 14, or 22 (near an immunoglobulin gene
All cells from a patient have breakpoints at exactly the same points as shown by DNA sequence analysis
Cancer cells in tumors of females all use same X chromosome (same one in Barr body)

15. Multistep Process Cancer requires mutation of multiple genes
Age relationship with cancer consistent with this
If one mutation caused cancer then rate would be constant independent of age
It increases dramatically with age…
Delay between carcinogen exposure and onset
5-8 year delay between carcinogen exposure (Hiroshima and Nagasaki) and onset of leukemia
15 year delay between tuberculosis X-ray treatment and onset of breast cancer

16. Multistep Process…Continued
Cancers often develop in progressive steps
From mildly aberrant cells to malignant
See figure 18-3
Process called tumorigenesis

17. Tumorigenesis of Cervical Cancer

18. Properties of Cancer Cells
Genetic instability
Mutator phenotype
Duplicating, losing and translocating chromosomes or portions of them common
Chronic myelogenous leukemia (CML)
Chromosome 9/chromosome 22 translocation
BCR gene fused to ABL (protein kinase)
Mutant signal transduction protein stimulates cells constantly to proliferate

19. Genome Instability Double minutes (DMs)
Miniature chromosomes giving many copies of rgion
Homogeneous staining regions (HSRs)
Tandem gene duplications

20. Chromosomal Translocation in CML

21. Xeroderma Pigmentosum
Failure to remove pyrimidine dimers from DNA
Excision repair defect
Patients often develop skin cancer and must stay out of sunlight

22. HFNPCC Hereditary nonpolyposis colorectal cancer
Higher than normal rates of colon (first noted) but also elevated rates of ovary, uterine and kidney cancers
1/200 persons, autosomal dominant
Eight genes associated and four involve mismatch repair systems

23. HNPCC Pedigree Colon, Stomach endometiral, pancreatic, bladder
Orange also other cancers, multiple slashes unknown cause of death

24. Role of cell division in tumor progression
Tumors arise from cells with DNA damage or mutant DNA that divide uncontrollably.
Cancer cells lose normal restraints for replication of damaged DNA and G1/S progression of cells with damaged DNA.
Increased probability of tumor progression by further genetic change.

25. Proto-oncogenes can be converted into oncogenes

26. Proteins that control cell growth (proto-oncogenes and cell cycle checkpoints)
1. Growth factors and receptors: (EGF/EGFR, IGF/IGFR, PDGF/PDGFR)
2. Intracellular transducers:
GTP binding proteins: Ras
Protein kinases: Src, Raf
3. Intracellular receptors: ER, RAR
4. DNA repair proteins: BRCA1
5. Cell cycle control proteins: cyclins, cdkis, Rb, p53
6. Transcription factors: myc, jun. fos, myb
7. Anti apoptotic proteins: Akt, Bcl-2
Proto-oncogene may become converted to an oncogene by a mutation that results in increased intrinsic activity of the protein product

27. Defects in Cell Cycle Regulation
G1, S, G2, M phases
Progression through cycle is regulated and specific blocks or checkpoints exist
Nondividing cell (quiescent) is in an extended G1 phase called G0
Cancer cells never enter G0

28. Cell Cycle

30. Cell Cycle Checkpoints
G1/S
Monitors cell size and for DNA damage
G2/M
Replication complete, DNA damage? M
Spindle fibers connected, etc.? G0
Does body require more of my type of cell?

31. Regulators of Cell Cycle
Cyclins and cyclin-dependent kinases (CDKs)
Cyclins synthesized and destroyed in a precise pattern
A cyclin bind to a specific CDKs, activating it
Other proteins phosphorylated/activated
CDK4/cyclinD activate transcription factors for genes such as DNA polymerase delta and DNA ligase
CDK1/cyclinB trigger events of early mitosis (chromosome condensation, nuclear membrane breakdown, etc.)

32. Cell cycle regulatory genes can be oncogenes or tumor suppressors

33. Cyclin dependent kinase regulation

36. Cyclins as oncogenes

37. 2. Oncogenes that encode growth factors or their receptors

38. Tel/PDGFR Oncogene activation
Translocation of Tel transcription factor onto PDGF causes dimerization (activation) in the absence of correct ligand. Found in leukemias

39. mutant receptor – always active even without binding ligand
mutant Gsα – always active – always signaling to adenylate cyclase

40. Steroid hormone receptors as oncogenes: ER, AR, myl (RAR)
Steroid hormone receptors can act as nuclear transcription factors and activate transcription of other oncogenes such as cyclins, myc, myb

44. Cyclin Levels

45. Activation of CDKs

52. Tumor Suppressor Genes
Mutations in tumor suppressor genes are usually recessive in their effects on the individual cell.
1. Cell Cycle Regulators:
Cdkis (cyclin-depndent kinase inhibitors)
Rb (Retinoblastoma protein)
P53
2. Signaling proteins: APC (adenomatous polyposis coli)
3. DNA damage repair proteins: BRCA1 (Breast cancer gene)
4. Enzymes: PTEN (phosphatase and tensin homolog deleted in chromosome ten)

53. Cell cycle checkpoint proteins regulate G1/S transition
A. CDK inhibitors
activated by:
P53 (p21 = cip1)
Vitamin D (p21)
Adhesion (p27)
TGFb (p15, p27)
B. Rb protein family:
pRb, p107, p130

54. TGF beta Signaling p15 gene
Induction of p15 results in inhibition of Cdk4,6/cyclin D complexes and cell cycle arrest at G1.
P15 and Smads are tumor suppressors

56. Oncogenes/Proto-oncogenes
Cyclin D1 and Cyclin E are proto-oncogenes
Often amplified or over expressed due to other mutations (e.g. translocation) in many cancers
cyclinD1 allows for DNA replication (S phase)
Over expression seems to contribute to cell’s progression from G0 phase and begin division

57. Oncogenes encoding signaling molecules: ras

58. Tyrosine kinase receptors activate Ras
ERb-B is an oncogene in glioblastomas, renal cell, salivary gland, squamous cell, breast, gastric, ovarian, and esophageal carcinomas
Ras is an oncogene in bladder, breast, lung, and head and neck carcinomas

59. Ras is activated by a point mutation in the GTP binding domain
Protooncogene
First human activated protooncogene
Homolog of Harvey-ras and Kirsten-ras from rat sarcoma

60. Ras Regulation of Cell Cycle Progression

61. ras Proto-oncogenes Involved in signal transduction pathway
As are many proto-oncogene products
ras family genes mutated in 40% of all cancers
Involved in signal transduction pathway from growth factor receptor to nucleus
G protein
Mutant form lacks GTPase activity and remains active
See figure 18-11

62. Ras Pathway Growth factor binds receptor
Receptor exchanges GTP for GDP on Ras
Ras activated
RasRafMekMap Kinasetranscription factors genes turned on

63. Mutant Ras Protein
Single amino acid changes create N-ras and K-ras variants

64. p53 Tumor Suppressor Gene
Mutated (inactivated) in more than 50% of all cancers
p53 regulates (activates or represses) transcription of more than 50 different genes
p53 regulated by Mdm2 (prevents the phosphorylations and acetylations that activate inactive p53)
Activated p53 levels rise rapidly if DNA is damaged or repair intermediates accumulate

65. P53 Function
Activated p53 acts as transcription factor to turn on genes that
arrest the cell cycle so DNA can be repaired
Initiates synthesis of p21, which inhibits CDK4/cyuclinD1 complex, blocking entry into S phase
Genes expressed which retard rate of DNA replication
Other products block G2/M progression
Initiate apoptosis if DNA cannot be readily repaired
Turns on Bax gene, represses Bcl2 gene
Bax homodimers activate process of cell destruction
Cancer cells lacking p53 do not initiate pathway even if DNA/cellular damage is great

66. RB1 Tumor Suppressor Gene
Retinoblastoma 1 gene
Involved in breast, bone, lung, bladder and retinal cancers (among others)
Inheriting one mutated (inactivated) copy of gene increases chances of retinoblastoma formation from 1/14,000-20,000 to 85% (plus increases other cancer rates)
Loss of second copy in a cell eliminates function
Normal cells unlikely to lose both good copies

67. pRB Function
Tumor suppressor protein that controls the G1/S checkpoint
Found in nucleus and activity regulated by level of phosphorylation (by CDK4/cyclinD1 complex)
Nonphosphorylated version binds to TFs such as E2F, inactivating them
Free E2F and the other regulators turn on >30 genes required for transition to S phase

68. Familial Retinoblastoma

69. Inherited Predisposition for Cancer
About 1-2% of cancer has an inherited or familial component
50 different forms known at present
Inherited in Mendelian fashion but most all genes/alleles are recessive
Second copy must be mutated in a somatic cell
Called loss of heterozygosity (and loss of function)
Loss of second copy in germ line lethal
RB1 and APC (lost in FAP, familial adenomatous polyposis) are examples of such genes

70. Multistep Development of Colon Cancer
APC loss causes cells to partially escape cell cycle regulation, DCC seems to be involved in cell adhesion and differentiation

71. Transforming Viruses Viruses discovered to cause cancer in animals
Acute transforming viruses
Commonly but not always retroviruses
Rous sarcoma virus (RSV) discovered by Francis Peyton Rous discovered in 1910 as a causative agent of chicken sarcomas (solid tumors of muscle, bone or fat)
Many years later shown to be retrovirus
Nobel Prize in 1966 (link of viruses to cancer)

72. Retroviruses ssRNA chromosome
Chromosome copied to DNA by reverse transcriptase upon entry into cell
DNA integrated into host cell chromosome
Provirus
Provirus has strong promoter elements in U5 and U3 terminal sequences
U5 expresses gag, pol and env
Oncogenic when
Integrate near proto-oncogene and cause inappropriate or over expression
Bring v-onc as part of viral chromosome

73. Retroviruses
Many transforming retroviruses are defective in the sense that one or more of gal/pol/env have been deleted to make room for the v-onc

74. Viral Oncogenes Most v-onc genes have normal cellular counterparts
If simply mutated to the oncogenic form and not in a virus are called c-onc

75. Human Cancer-Associated Viruses
To date no acute transforming retroviruses have been discovered in humans
Viruses can contribute to but not be the sole cause of human cancer
However, up to 15% of all cancers have a viral association
Papillomaviruses HPV 16 and 18, hepatitis B virus, Epstein-Barr virus, Human T-cell leukemia virus are examples of cancer-associated viruses

76. Human Viruses Associated With Cancer
Non-retroviral varieties
Many of these v-onc genes act to stimulate the cell cycle (viruses needs host replication apparatus to multiply

77. V-onc Gene Product Action
Some v-onc gene products have their transforming effect by binding and thereby “taking out” certain tumor suppressor gene products
Cell division required to provide replication apparatus for virus
Bad, but does open some interesting treatment possibilities…

78. Environmental Agents and Cancer
Natural and man-made carcinogens
Chemicals, radiation, chronic infections
30% of cancer deaths associated with cigarettes
Seems to preferentially mutate proto-oncogene and tumor suppressor genes
Red meat consumption
How cooked?
Alcohol-based inflammation of the liver
Aflatoxin (mold on peanuts)
UV light or ionizing radiation
Radon gas (up to 50% of radiation exposure???)

94. Oncogenes that code for cytoplasmic protein kinases involved in cell proliferation: src, raf

95. ERbB2/Ras oncogenic signaling
Ras activates Raf kinase.
Raf activates MAPKs.
MAPKs phosphorylate and activate transcription factors Elk-1, SRF, AP-1 that regulate Cyclin expression

96. Cyclins Oncogenes that encode nuclear transcription factors:
AP-1: fos, jun
Cyclins

97. Translocation of c-myc in Burkitt’s lymphoma
Translocation of c-myc transcription factor gene to Ig loci
c-myc normally induced in response to growth factors
Translocation results in constitutive activation because of being under control of Ig promoters of the B-cells

98. Oncogenes that code for cytoplasmic protein kinases involved in cell proliferation: abl
Chronic myelogenous leukemia (CML) is a clonal
hematopoetic stem cell disorder

99. Translocation of Abl
The cytogenetic hallmark of all phases of CML is the Philadelphia (Ph) chromosome. The Ph chromosome is a shortened chromosome 22 that results from a reciprocal translocation between chromosomes 9 and 22.
Fusion of the c-abl oncogene from chromosome 9 with sequences from chromosome 22, the breakpoint cluster region (bcr) gives fused bcr-abl gene.

100. Conversion of abl-protooncogene into an oncogene in myelogenous leukemia
Depending on the site of the breakpoint in bcr, different fusion proteins are produced: p185 (185 kDa), p210 (210 kDa), or rarely p230.

101. How does bcr-Abl fusion protein expression cause leukemia?
c-abl, the cellular homolog of the transforming protein found in the Abelson murine leukemia virus (v-abl), encodes for a nonreceptor tyrosine kinase.
The c-abl protein has tightly regulated kinase activity and shuttles between the nucleus and cytoplasm. Abl is a Src family tyrosine kinase that has SH2, SH3 domains and can activate MAPK signaling.
bcr-abl fusion proteins are exclusively cytoplasmic and have enhanced tyrosine kinase activity that causes excess proliferation of myeloid cells.

102. Genetic mechanisms underlying Retinoblastoma

103. How to lose the remaining good copy of a tumor suppressor gene

104. Retinoblastoma

105. Rb mechanism of action in cell cycle regulation

107. Phosphorylation of Rb by Cdk results in activation of E2F family transcription factors
Cyclins
No functional Rb = no inhibition of E2F activity = unchecked cell division

108. Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome associated with soft-tissue sarcoma, breast cancer, leukemia, osteosarcoma, melanoma, and cancer of the colon, pancreas, adrenal cortex, and brain. Individuals with LFS are at increased risk for developing multiple primary cancers.
More than 50% of individuals diagnosed clinically have an identifiable disease-causing mutation in the P53 gene. Of these mutations, 95% can be detected by sequence analysis,
Genetic counseling.  LFS is inherited in an autosomal dominant manner. Offspring of an affected individual have a 50% chance of inheriting the disease-causing mutation.

109. DNA damage induces p53 expression

110. P53 mechanism of action in cell cycle regulation
DNA damage increases p53 expression of normal cells.
P53 acts as a transcription factor to induce p21 (cip1), a cdki. Blocks cell cycle progression.
If the DNA damage cannot be repaired p53 will induce transcription of Bax, PUMA, pro-apoptotic Bcl-2 family members.

111. Cell cycle regulation by tumor suppressors
Bax, Noxa, PUMA
Fas
DR4,5

112. Replication of damaged DNA can lead to chromosomal abnormalities, gene amplification, and gene loss

113. APC in Wnt signaling
APC = adaptor, brings GSK-3 close to β-catenin so it can be Phosphorylated and degraded
No APC = lots free β-catenin = increase in gene transcription = cell proliferation

114. DNA damage repair by BRCA1
This pathway is dysfunctional in some breast cancers
BRCA, the breast cancer gene, missing in only 5% of breast cancers, BRCA (-) genotype has 60% risk of breast cancer by age 70.
ATM, ataxia-telangiectasia mutated

115. Suppression of cell survival by PTEN

116. Genetic Alterations in Colon Carcinoma
Loss of APC leads to early adenoma
Constitutive activation of ras leads to intermediate adenoma
Loss of smads and TGFbR lead to late adenoma
Loss of p53 leads to carcinoma
Other alterations can lead to metastasis

117. Apoptosis Programmed cell death, cell suicide
Pathway should be activated if “something goes wrong”
Especially involving DNA/chromosome damage
Involves proteases called caspases
Regulated by Bcl2 and BAX
BAX homodimer promotes apoptosis, Bcl2 homodimer blocks apoptosis
Some cancer cells overproduce Bcl2 and are resistant to some chemotherapies and radiation treatment
Proteins involved in cell cycle checkpoints regulate pathway

118. Control of Apoptosis

119. Oncogenes encoding products that affect apoptosis
TRANSFORMATION
Initial event
Immortalization
Inhibition of apoptosis
PROMOTION
Altered DNA repair
Successive accumulation of mutations
Cell cycle promotion
Inhibition of apoptosis
PROGRESSION
Genomic instability
Other mutations
Irregular expression of apoptosis
Drug resistance

120. Two Major Pathways of Apoptosis: 1. Receptor-regulated
2. Mitochondrial
From:
“The biochemistry
of apoptosis”
Hengartner, 2000 Nature
407:

123. Differentiation, development and programmed cell death
Differentiation, development and programmed cell death.1999, The molecular basis of cell cycle and growth control. Ed. Stein, Baserga and Denhardt, , Wiley-Liss, Inc.
Bcl-2 family
Anti-apoptic signaling
Bax is prodeath
dimerize and result in death signals

124. Oncogneic activity of Bcl-2 and Bcl-xL
Structure of Bcl-XL with a BH3 peptide bound.
Adams and Cory, Science 281:
APaf-1= apoptosis protease activating factor-1
CARD=caspase recruitment domain
Apoptosome: contains cytochrome C, ATP, caspase9, APaf-1

125. Oncogenes encoding cell survival signals: akt

126. Akt activity in blocking apoptosis