Chapter 18: Genetics of Cancer

and Cell-Cycle Regulation

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

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

Cancer Rates in US

Age and Cancer

• Note log scale for incidence rate

Abnormal Cell Growth

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

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.

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

Oncogenes are identified through their dominant transforming effects

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

Normal and Cancer Karyotypes

• Chromosome painting

(a) is a normal cell

(b) is a “very messed up” cancer cell

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)

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

Multistep Process…Continued

• Cancers often develop in progressive steps– From mildly aberrant cells to malignant– See figure 18-3– Process called tumorigenesis

Tumorigenesis of Cervical

Cancer

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

Genome Instability

• Double minutes (DMs)– Miniature chromosome

s giving many copies of rgion

• Homogeneous staining regions (HSRs)– Tandem gene duplicati

ons

Chromosomal Translocation in CML

Xeroderma Pigmentosum

• Failure to remove pyrimidine dimers from DNA– Excision repair defect

• Patients often develop skin cancer and must stay out of sunlight

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

HNPCC Pedigree

• Colon, Stomach endometiral, pancreatic, bladder• Orange also other cancers, multiple slashes unknown cause

of death

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.

Role of cell division in tumor progression

Proto-oncogenes can be converted into oncogenes

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

Defects in Cell Cycle Regulation

• Cell cycle

• 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

Cell Cycle

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?

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.)

Cell cycle regulatory genes can be oncogene

s or tumor suppressors

Cyclins as oncogenes

2. Oncogenes that encode growth factors or their receptors

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

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

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

Cyclin Levels

Activation of CDKs

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)

A. CDK inhibitors activated by:• P53 (p21 = cip1)• Vitamin D (p21)• Adhesion (p27)• TGF (p15, p27)

B. Rb protein family:pRb, p107, p130

Cell cycle checkpoint proteins regulate G1/S transition

Induction of p15 results in inhibition of Cdk4,6/cyclin D complexes and cell cycle arrest at G1.

P15 and Smads are tumor suppressors

p15 gene

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

Oncogenes encoding signaling molecules: ras

ERb-B is an oncogene in glioblastomas, renal cell, salivary gland, squamous cell, breast, gastric, ovarian, and esophageal carcinomasRas is an oncogene in bladder, breast, lung, and head and neck carcinomas

Protooncogene

First human activated protooncogene

Homolog of Harvey-ras and Kirsten-ras from rat sarcoma

Ras is activated by a point mutation in the GTP binding domain

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 gro

wth factor receptor to nucleus– G protein

– Mutant form lacks GTPase activity and remains active

• See figure 18-11

Ras Pathway• Growth factor binds receptor• Receptor exchanges GTP for

GDP on Ras– Ras activated

• RasRafMekMap Kinasetranscription factors genes turned on

Mutant Ras Protein

• Single amino acid changes create N-ras and K-ras variants

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

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 D

NA/cellular damage is great

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

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, i

nactivating them– Free E2F and the other regulators turn on >30 genes req

uired for transition to S phase

Familial Retinoblastoma

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

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

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)

Retroviruses• ssRNA chromosome• Chromosome copied to DNA by reverse transcriptase upo

n 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 e

xpression

– Bring v-onc as part of viral chromosome

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

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

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 associ

ation• Papillomaviruses HPV 16 and 18, hepatitis B virus, Epstei

n-Barr virus, Human T-cell leukemia virus are examples of cancer-associated viruses

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

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…

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???)

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

Ras activates Raf kinase.

Raf activates MAPKs.

MAPKs phosphorylate and activate transcription factors Elk-1, SRF, AP-1 that regulate Cyclin expression

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

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

Chronic myelogenous leukemia (CML) is a clonalhematopoetic stem cell disorder

Oncogenes that code for cytoplasmic protein kinases involved in cell proliferation: 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.

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.

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.

Genetic mechanisms underlying Retinoblastoma

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

Rb mechanism of action in cell cycle regulation

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

• 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.

DNA damage induces p53 expression

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.

P53 mechanism of action in cell cycle regulation

Bax, Noxa, PUMA

Fas

DR4,5

Cell cycle regulation by tumor suppressors

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

APC = adaptor, brings GSK-3 close to β-catenin so it can be Phosphorylated and degradedNo APC = lots free β-catenin = increase in gene transcription = cell proliferation

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

Suppression of cell survival by PTEN

Genetic Alterations in Colon Carcinoma

1. Loss of APC leads to early adenoma

2. Constitutive activation of ras leads to intermediate adenoma

3. Loss of smads and TGFR lead to late adenoma

4. Loss of p53 leads to carcinoma

5. Other alterations can lead to metastasis

Apoptosis• Programmed cell death, cell suicide• Pathway should be activated if “something goes wr

ong”– 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

Control of Apoptosis

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

Two Major Pathways of Apoptosis: 1. Receptor-regulated

2. Mitochondrial

From:“The biochemistryof apoptosis”Hengartner, 2000 Nature407:770-776

Differentiation, development and programmed cell death.1999, The molecular basis of cell cycle and growth control. Ed. Stein, Baserga and Denhardt, 305-307, Wiley-Liss, Inc.

Structure of Bcl-XL with a BH3 peptide bound.

Adams and Cory, 1998. Science 281:1322-1326.

APaf-1= apoptosis protease activating factor-1

CARD=caspase recruitment domain

Apoptosome: contains cytochrome C, ATP, caspase9, APaf-1

Oncogneic activity of Bcl-2 and Bcl-xL

Oncogenes encoding cell survival signals: akt

Akt activity in blocking apoptosis