molecular genetics and cancer biology

Meshari AlzahraniR1 Urology Resident | KAMC-NGHA-Jeddah

16 November 2014

& Cancer BiologyMolecular Genetics

Lecture Roadmap

Introduction

Basic Molecular Genetics

Tumor Suppressor Genes and Oncogenes

The Cell Cycle

DNA Methylation

DNA Damage and Repair

Chromosomal Abnormalities and Genetic Instability

Telomeres and Telomerase

Apoptosis

Stem Cells and Cancer

Why This Lecture !

Despite decades of intensive biomedical research, cancer remains a significant cause of morbidity and mortality worldwide.

Campbell-Walsh Urology 10th Edition

World Cancer Day – 4th February 2014

By 2025, there will be more than 20 million new cancer cases per year, compared with 14.1 million in 2012, according to the World Cancer

Report 2014, released on 3 February by the World Health

Organization’s International Agency for Research on Cancer.

IARC World Cancer Report 2014

IARC World Cancer Report 2014

cancer ribbon

• However, significant advances in the diagnosis and treatment of certain Genitourinary (GU) cancers have been made.

• For example, the cure rate for testicular cancer now approaches 100%.

• Unfortunately, this cancer is unusual in its responsiveness to therapy and is relatively uncommon

(Einhorn, 2002; Horwich et al, 2006)


• We have had less success with the more prevalent GU malignancies, such as prostate, bladder, and renal cancers—the second, eighth and tenth most common cancers, respectively.


In Saudi Arabia, prostate cancer is the 6th most common cancer among men of all ages and the most common cancer among men over the age of 75.

It accounts for 6.1% of all newly diagnosed cases among males in year 2010 with an age - standardized incidence rate of 5.5/100,000 among the male population.

Stage at the time of diagnosis is localized in 43.9% of cases with the remainder being either locally advanced, metastatic or unknown.

Saudi Cancer Registry Annual Report, 2010

Bladder cancer ranked 13 among the most common cancer diagnosis in Saudi Arabia, affecting 3.6/100,000 men and 1/100,000 women.

In 2010, there were an estimated 243 new cases of bladder cancer accounting for 2.5% of all newly diagnosed cases.

It affected 193 (78.4%) males and 50 (21.6%) females with a male : female ratio of 385:100.

The most common histological subtypes is TCC (82%) followed by SCC(4%).

Saudi Cancer Registry Annual Report, 2010

In 1953

Co-discoverer of the structure of the DNA molecule

Francis Crick

James Watson

The molecular characteristics of DNA were first described in 1953 (Watson and Crick, 1953).

This molecule serves as the blueprintfor determination of structure and

function of all living organisms.

““

Somatic cell is any biological cell forming the body of an organism

Germ cells are cells that give rise to gametes

Gametes : is a cell that fuses with another cell during fertilization (conception) in organisms that sexually reproduce, witch carry half the genetic information of an individual.

Stem cells are cells that can divide through mitosis and differentiate into diverse specialized cell types.



• somatic cells contain DNA arranged in chromosomes.

• If a somatic cell contains chromosomes arranged in pairs, it is called diploid and the organism is called a diploid organism.

• The gametes of diploid organisms contain only single unpaired chromosomes and are called haploid.

• Each pair of chromosomes comprises one chromosome inherited from the father and one inherited from the mother


• In humans, somatic cells contain 46 chromosomes organized into 23 pairs.

• By contrast, gametes of diploid organisms contain only half as many chromosomes.

• In humans, this is 23 unpaired chromosomes.• When two gametes (i.e. a spermatozoon and an

ovum) meet during conception, they fuse together, creating a zygote.

• Due to the fusion of the two gametes, a human zygote contains 46 chromosomes (i.e. 23 pairs)


• Chromosome: A distinct segment of linear DNA containing a large number of genes. In humans there are 23 such segments, each containing hundreds to thousands of genes.

• Gene: A segment of DNA that contributes to the formation of a protein, including both the introns(noncoding regions) and the exons (coding regions), as well as the regulatory regions preceding and following the coding regions.

Mitosis & Meiosis

DNA is composed of 3 basic components:

Base : Pyrimidine or purine

Sugar : (2-deoxyribose)

Phosphate


The Nucleic Acid alphabet consists of 4 bases:

1. purines adenine (A)

2. purines guanine (G)

3. pyrimidines thymine (T)

4. pyrimidines cytosine (C).

5. There is a fifth base that can be found in DNA known as 5-methylcytosine (5-mC).

Uracil (U) is substituted for thymine in the case of RNA.

The combination of a base and a sugar (deoxyribose) is referred to as a nucleoside

nucleoside

DNA : deoxyribonucleic acid

Nucleobases

Hydrogen bonding occurs specifically between the purine adenine (A) and the pyrimidinethymine (T) and between the purine guanine (G) and the pyrimidine cytosine (C)

The connection between repeating phosphates and sugars creates a Helical Chain.

In the RNA molecule, adenine base pairs with uracil (U).

The combination of a sugar phosphate group and a base constitutes a nucleotide.

The double helix is made from two polynucleotide chains, each of which consists of a series of 5′– to 3′–sugar phosphate links that form a backbone from which the bases protrude.

The double helix maintains a constant width because purines always face pyrimidines in complementary A-T and G-C base pairs, respectively.

TranscriptionTranscription is the first step in converting DNA into protein

↓

During the process of transcription, linear DNA is converted to linear messenger RNA (mRNA)

↓

The process of translation consists of the conversion of linear mRNA to a linear set of

amino acids that will eventually form a functional protein

↓

Single strand of RNA is copied from one of the strands of DNA.

↓

The sugar element in the RNA molecule is ribose and the pyrimidine uracil substitutes for thymine

↓

RNA polymerase II is the enzyme that synthesizes the first copy of RNA

This primary strand of RNA is called heterogeneous nuclear RNA (hnRNA)

↓

hnRNA contains coding sequences (exons) of DNA and noncoding sequences (introns).

RNA : ribonucleic acid

During transcription, a section of one DNA strand, or the other, is used as a template for the synthesis of mRNA. This synthesis always occurs in a 5′ to 3′ direction.

Protein Translation

Translation of mRNA into protein occurs in the cytoplasm where ribosomes are located.

Two other forms of RNA are important for protein translation: transfer RNA (tRNA) and ribosomal RNA (rRNA)

The mRNA message is translated in segments of three adjacent nucleotides called a codon

Each codon is translated into one of 20 amino acids

The information contained in DNA is transcribed into RNA and then translated into protein.Transcription of RNA is tightly regulated and is tissue specific.A single gene can encode for multiple unique proteins by including or excluding certain exonsin the mRNA transcript by alternative splicing.Post-transcriptional gene regulation can occur by a mechanism involving the expression of noncoding RNAs that have the capability of binding to and degrading mRNAs

Key Points

Summary

Body cells

↓

loss

↓

cell proliferation

↓

maintaining tissue and organ homeostasis

How can Normal cell become Cancer Cell ?

1. Genetic instability2. Autonomous growth3. Insensitivity to internal and external antiproliferative signals4. Resistance to apoptosis and other forms of induced cell

suicide5. Unlimited cell division potential6. The ability to induce new blood vessel formation , a process

termed angiogenesis.7. Locally invasive behavior, which uniquely distinguishes

malignant from benign neoplasms.8. Evasion of the immune system.

(Hanahan and Weinberg, 2000; Solimini et al, 2007; Luo et al,2009).

In addition, cancer cells need to cope with various cellular stresses that are byproducts of their abnormal physiology.

Finally, many cancers develop an additional, lethal attribute—the ability to leave the site of the primary tumor to colonize and thrive in distant organs or tissues as metastases.

(Hanahan and Weinberg, 2000; Solimini et al, 2007; Luo et al,2009).

Our knowledge of the molecular genetics of cancer is rapidly expanding, providing new insights that are just beginning to be successfully exploited for use in novel diagnostic, prognostic, and therapeutic applications.


Tumor Suppressor Genes & Oncogenes

Tumor Suppressor Genes

Tumor suppressor genes (antioncogene) regulate cellular growth and play a critical role in the normal processes of the cell cycle.

These genes are also important for DNA repair and cell signaling.

The absence of tumor suppressor gene function may lead to dysregulation of normal growth control and malignancy.

Loss of function of both alleles of a tumor suppressor gene is typically required for carcinogenesis.

This functional loss can occur by :1. homozygous deletion

2. loss of one allele and mutational inactivation of the second allele,

3. mutational events involving both alleles,

4. loss of one allele and inactivation of the second allele by DNA methylation

• The “two-hit” hypothesis was first proposed in cases of retinoblastoma, which required mutations in both alleles for disease manifestation (Knudson, 1971).

“two-hit” hypothesis

• Specific types of mutations in certain gene however, may not follow this two-hit rule and can function as dominant negative mutations that produce altered protein.

• Mutant protein products have been reported to inhibit function of normal protein from unaltered alleles (Baker et al, 1990).

• Mutation of a single allele may result in haploinsufficiency, causing increased carcinogen susceptibility as in the case of the TP27 Kip1 gene (Fero et al, 1998).

Oncogenes

• Oncogenes (proto-oncogene): are associated with cellular proliferation and are the mutated form of normal genes.

• Two oncogenes that have been found to be overexpressed in a variety of cancers include :

c-MYC and c-MET(Wong et al, 1986; Bottaro et al, 1991).

Key Point

Mutations in DNA can lead to changes in protein function or expression that increase the potential for cancer initiation, progression, or metastasis.

Tumor suppressor genes regulate and control cellular growth.

Oncogenes promote cell growth.

Loss of tumor suppressor gene function can occur primarily by :

(1) homozygous deletion

(2) loss of one allele and mutational inactivation of the second allele

(3) mutational events involving both alleles

(4) loss of one allele and inactivation of the second allele by DNA methylation.

Key Point

Key Point

Certain tumor suppressor genes do not follow the “two-hit” hypothesis and may be inactivated by dominantnegative mutations or haploinsufficiency.

Proto-oncogenes can be converted to oncogenes by :

(1) mutation of the proto-oncogene resulting in an activated form of the gene

(2) gene amplification

(3) chromosomal rearrangement.

The Cell Cycle

the cell cycle takes approximately 24 hours to completeHartwell et al,1974

checkpoint mechanisms closely monitorDNA integrity as well as certain critical cell cycle events.If problems are detected (e.g., DNA damage), the cell cycle will pause to allow repair

(Hartwell and Weinert, 1989).

If repair is not possible, normal cells often will commit cellular suicide through an active process termed apoptosis.

Sequential activation of cyclin-dependent kinasecomplex (CDKC, cyclin-CDK) is critical to the orderly

progression of cell replication.

Many oncogenes and tumor suppressors exert their effects by interfering with cell cycle checkpoints and apoptotic pathways, allowing cancer cells to divide continuously and accumulate.

Loss of the ability to respond appropriately to damaged DNA is particularly dangerous, because it fosters genetic instability, a key attribute of cancer cells.

Loss of DNA damage checkpoint controls results in an increased mutation rate, accelerating the mutation of cancer-associate genes, thus contributing to carcinogenesis and disease progression.

(Bartek et al, 1999).

Some Oncogenes associate with GU cancers

Recourse Associate with Oncogenes

(Gil et al, 2005).prostate Ca.c-MYC

(Pisters et al, 1997)RCCc-MET

(Schmidt et al, 1997).hereditary RCCMET proto-oncogene

(Schmitz-Drager et al, 1997)bladder Ca.c-MYC

(Horowitz et al, 1990)bladder Ca.pRB

• The cell cycle consists of an ordered, unidirectional series of events, the main goal of which is to replicate the cell’s genome and partition one copy into each of two resulting daughter cells.

• The cell cycle is divided up into 4 phases; G1, S, G2, and M phase.

• The transition from G1 into S is critically dependent on phosphorylation of the pRB tumor suppressor protein.

Key Point

Mutations in pRB are common in some urologic malignancies (Bladder Ca.)

Phase-specific phosphorylation of substrate proteins by cyclin-dependent kinases (CDK) orchestrate progression through the cell cycle.

The activities of CDKs are dependent upon their association with specific cyclin proteins.

Cyclins accumulate and are rapidly degraded in a phase-specific manner, thus assuring the proper sequencing and irreversibility of key events throughout the cell cycle.

Key Point

Primary points of cell cycle control are the G1S and G2M checkpoints.

Checkpoints employ cyclin-dependent kinaseinhibitor proteins (CDK1) to pause the cell cycle in response to a variety of stress signals, including DNA damage, cell– cell contact, cytokine release, and hypoxia.

Key Point

The TP53 tumor suppressor protein is a key player in cell cycle checkpoints, responding to DNA damage by signaling cell cycle arrest and repair of the damage.

If the DNA damage cannot be repaired, TP53 may trigger cell death (apoptosis).

Key Point

• TP53 is the most commonly mutated gene in cancer and plays a prominent role in genitourinary malignancies.

• Defects in cell cycle checkpoints lead to unregulated cell proliferation and genetic instability.

Key Point

DNA Methylation

• The covalent modification of the C-5 position of cytosine is mediated by DNA (cytosine-5) methyltransferase, resulting in the formation of 5-methylcytosine.

• Methylation of cytosine occurs primarily at the CpG palindrome in DNA.

• One important role for methylation is genomic imprinting, which results in monoallelic gene expression without altering the genetic sequence.

• Loss of imprinting (LOI) is a reduction in the methylation of the normally methylated allele, which can lead to activation of the normally silent copy of a growthpromoting gene.

(Feinberg and Tycko, 2004).

• Changes in global levels and regional patterns of DNA methylation are among the earliest and most frequent events known to occur in human cancer.

(Jones and Baylin, 2002)

• Three major pathways by which DNA methylationmay result in genetic dysregulation in human caner include

(1) inherent mutational effects of 5-methylcytosine

(2) epigenetic effects of promoter methylation on gene transcription,

(3) potential gene activation and induction of chromosomal instability by DNA hypomethylation

(Gonzalgo and Jones, 1997).

Recourse %Mutation Abnormal methylation gene

(Lee et al, 1994)>90% PCa*≈ 70% PIN*

CpG islandGSTP1*

(Kuzmin et al, 2002)60% to 70% RASSF1ARASSF1A*

GSTP1 glutathione-S-transferase Pi RASSF1A RAS association domain-containing protein 1Pca Prostate CancerPIN prostatic intraepithelial neoplasim

DNA Methylation and Prostate Cancer

Role of DNA Methylationin Bladder Cancer

Recourse Type %Mutation Abnormal methylation

gene

(Greenblatt et al, 1994)(Rideout et al, 1990;

Tornaletti and Pfeifer, 1995).(Spruck et al,1994b)

urothelial dysplasiacarcinomain situ (CIS)invasive bladder Ca

24C→T transition

TP53

(Chan et al, 2002; Chang et al,2003)

primary urothelialcarcinomas

27-60TP16 alleleTP16

(Graff et al, 1995; Horikawa et al, 2003)

high-grade urothelialcarcinoma,

-Hypermethylation

CDH1

(Lee et al, 2001)(Maruyama et al, 2001).

primary bladder tumorsHigh tumor grade, nonpapillarygrowth patternmuscle invasive disease

97RASSF1ARASSF1A

(Kimura et al,2003)(Jurgens et al, 1996).

Bladder Ca.Hypomethylation

DNMT1DNMT3A, DNMT3B

Methylation occurs specifically at CpG dinucleotides in the genome.

The presence of 5-methylcytosine in DNA can result in spontaneous deamination to thymine and formation of C→T transition mutations.

DNA methylation can affect gene function by mutational events or epigenetic mechanisms.

Methylation of CpG islands associated with the promoter region of genes may result in down-regulation of transcription and suppression of gene expression.

Loss of methylation of normally methylated genes can lead to the potential for gene expression.

Key Point

DNA damage & repair

DNA damage does not often lead to malignancy, because the cell possesses multiple repair mechanisms.

Defects in DNA repair facilitate the accumulation of the mutations critical for tumor formation and progression.

The cell cycle and the DNA damage response (DDR) are closely integrated. In response to DNA damage, the first step is to arrest the cell cycle so that the DNA can be repaired. TP53 plays a key role at this interface.

Key Point

Nucleotide excision repair (NER) is a major defense against

DNA damage caused by ultraviolet radiation and chemical exposure.

Base excision repair (BER) repairs damage caused by spontaneous

deamination of bases, radiation, oxidative stress alkylating agents, and replication errors.

Key Point

Mismatch repair (MMR) removes nucleotides mispaired by DNA polymerase.

Double-stranded break repair (DSBR) is a major defense against DNA damage caused by ionizing radiation, free radicals, and chemicals.

Many syndromes involving inherited defects in DNA repair exhibit marked increases in cancer susceptibility; strongly linking genomic instability and cancer.

Key Point

Chromosome Abnormalities & Genetic Instability

The chromosomal changes seen in solid tumors can be broken down into two main classes:

1. changes in the number of whole chromosomes

2. changes in chromosomal structure.

Specific Chromosomal Rearrangementsin Genitourinary Malignancies

Recurrent Gene Rearrangements

Recourse oncogeneCancer

Tomlins andcolleagues (2005),

•ETS transcriptionfactor family members

Pca

(Hemesath et al, 1994; Argani et al, 2005).

MITF/TFE family translocation carcinomas

RCC

(Atkin and Baker, 1982; Rodriguez et al,

1993; Rosenberg et al, 1998; Verdorfer et al, 2004).

shortarm of chromosome 12

Testicular Cancer

(Smith et al, 1996).HPC familiesHereditary Prostate Cancer

chromosome 8Sporadic Prostate Cancer

(Gnarra et al, 1994; Shuin et al, 1994).

germ line mutations VHL gene

von Hippel-Lindau syndrom

sporadic ccRCC

(Tsai et al, 1990; Cairns et al, 1993; Linnenbachet al, 1993)

chromosome 9

RAS familyBladder Ca. transitional cell

typ (urothelial cell carcinomas)

Risk factor Cancer

Family history is one of the strongest prostate cancerrisk factors

HPC

• first degree relatives of patients with bladder cancer are at increased risk of developing the disease• high-risk families are very rare and lack clear mendelianinheritance patterns, precluding classical linkage analysis.• Bladder cancer is therefore not considered a familialDisease

Bladder Cancer

structural rearrangements, as well as intratumoral heterogeneity in these aberrations, are hallmarks of most human solid tumors.

The extent of chromosomal abnormalities typically correlates with disease severity and aggressiveness.

Recurrent structural rearrangements occur in prostate (ETS

Key Point

family gene fusions), renal (MITF/TFE family translocation carcinomas), and testicular cancers (isochromosome 12p).

Copy number alteration in a particular gene, coupled with changes in the other allele is strong evidence for that gene functioning as a disease-relevant oncogene or tumor suppressor gene.

Key Point

Genes discovered to have germ line mutations that cause familial forms of cancer may also be involved in the sporadic form of the disease (e.g., VHL in ccRCC).

High-density single nucleotide polymorphism (SNP) microarrays have been used in genome-wide association studies (GWAS) to identify DNA sequence variants associated with cancer risk.

Key Point

Telomere & Telomerase

• Telomeres contain stretches of terminal, noncoding, repetitive DNA that cap the ends of each chromosome, thereby stabilizing them.

• Telomere DNA repeats are progressively lost as cells divide and as a result of oxidative DNA damage at the telomeres.

• Normal cells monitor their telomere lengths and permanently exit the cell cycle (cellular senescence) or commit suicide (apoptosis) in response to telomere shortening. This tumor-suppressive telomere length checkpoint involves TP53 and pRB.

• Loss of telomere length checkpoints can lead to critical telomere shortening that initiates chromosomal instability, thus contributing to carcinogenesis.

• A majority of cancers and premalignant lesions have abnormally short telomeres.

• Most cancers express the enzyme telomerase, which restabilizes the telomeres and allows unlimited cell division potential (“immortalization”), thus telomerase represents an attractive therapeutic target.

Apoptosis

• Apoptosis:programmed cell death

• Apoptosis is a rapid, orderly, programmed form of cell death that is used by multicellularorganisms to eliminate unwanted cells. Through this process, cells are preprogrammed to commit suicide in response to various internal and external signals.

• Apoptosis is believed to play an important role in tumor suppression because many of the signals that induce apoptosis arise from potentially tumorigeniccell stresses such as DNA damage.

• Cancer is characterized by interruptions in the normal process of apoptosis, resulting in inappropriate cell survival.

• Apoptosis is mediated by a conserved family of proteases known as caspases. Initiator caspases begin caspase proteolytic cascades that result in the activation of downstream executioner caspases, which, in turn, target several cellular proteins.

• Two main apoptotic pathways have been identified:

In the intrinsic pathway, BCL-2 family membersmodulate the release of cytochrome c from mitochondria, which then participates in the activation of initiator caspases.

The extrinsic pathway activates caspases in response to signals from extracellular “death receptors.”

• In addition to its functions in cell cycle arrest and DNA repair, TP53 also plays a key role in apoptosis.

• BCL-2 is a classic inhibitor of the mitochondrial pathway of apoptosis and is overexpressed in some genitourinary malignancies.

• Therapeutic response is often dependent upon the integrity of apoptotic pathways in cancer cells. Most TGCT retain intact DDR, wild-type TP53, and apoptotic responses, providing high cure rates with DNA-damaging agents.

• Novel agonists and antagonists of apoptosis, such as ceramide and clusterin, may be successfully controlled to combat cancer.

Cancer Stem Cells

Stem cells are defined by their ability to differentiate along multiple lineages and their immortality.

Cancer is believed to be a stem cell disease in which a small population of cancer stem cells maintains the larger tumor.

The hedgehog signaling pathway is required for regeneration of prostate epithelium and has been implicated in transformation of prostate progenitor cells.

Cancer may ultimately be eradicated by targeting only the cancer stem cell.

References

• Campbell-Walsh Urology – 10th Edition -Chapter 18 page 530

molecular genetics and cancer biology

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