Cancer genetics 2

What’re proto-oncogenes?

• NON cancer causing genes

• It’s a normal gene that can become an oncogene

• They encode proteins involved in cell proliferation such as:

  • Signal transduction, Mitogenic signaling (growth factors in mitosis

What needs to occur to proto-oncogenes to become oncogenes?

• They need to acquire gain of function via mutations or increased expression

What’re oncogenes?

• Genes with potential to cause cancer

• They function in cell proliferation, signal transduction, mitogenic signals

• They upregulate gene expression (eg: transcription factors)

What’re the genetic alterations that cause proto-oncogenes and what do they result in?

1. Mutation: results in hyperactive protein (ex: BRAF V6OOE)

2. Gene amplification: results in overexpressed amounts of normal protein (ex: HER2 amplification)

3. Chromosome rearrangement: results in overexpressed protein, normal vs fusion (ex: BCR: ABL fusion)

What’re oncogenes in cancer?

• Hypermorphic (increased) function and/or expression

• UPREGULATED, mis -expressed (ectopic expression), enhanced activity/ function, etc

How do mutations act in oncogenes in cancer?

• They act dominantly and are usually somatic mutations

Explain amplified (double minutes) mutations in cancer

• Double minutes = not normal chromosomes, no telomeres or centromeres, highly replicated pieces of DNA

• Ex: HER2/ Neu, K-Rays, c-Myc, etc

What’s the classical breast cancer oncogene?

• Human epidermal growth factors receptor 2

  • HER2/ Neu, ERBB2

  • Proto-oncogene (17q21-22)

How does the classical breast cancer oncogene work?

• It’s a surface bound tyrosine kinase receptor which binds to growth factors inducing dimerization and signaling (phosphorylation)

• Signal transduction causes cell growth, differentiation, etc

How much do breast cancers have ERBB2 amplification?

• ~30% of them

• Causes overactive signaling and enhanced proliferation potential

What does breast cancer causes?

• Increased expression and higher disease recurrence

• Causes other cancers such as ovarian, stomach, uterine, etc

What does more HER2 look like in immunohistochemistry?

• More brown in later stages

What do breast cancer therapies (herceptin) aim to do?

• They aim to inhibit dimerization in domain with pertuzumab and trastuzumab cuz dimerization has to occur for HER2/neu to amplify/ proliferate

Whats the normal function of tumor suppressor genes?

• To inhibit/ regulate cell proliferation and work as brakes

What happens to tumor suppressor genes in cancer?

• They’re down regulated/ inactivated in cancers cuz you have to lose both alleles

• They have a recessive effect at cellular level because you have to lose both copies

  • Require loss of both functional alleles generally

How are tumor suppressor genes dominantly inherited?

• Via familial cancers through loss of heterozygosity (LOH), becuase in familial cancers through, every cell in body already has 1 hit, so you js need one more hit

What’re some tumor suppressor genes?

• TP53, RB1, APC

Whats TP53 (p53)?

• The classical TSG (guardian of the genome)

• Mutated in >50% of all cancers (has key importance in a lot of functions)

Whats retinoblastoma (RB1)?

• It’s a TSG

• Involved in cell cycle regulation, chromatin remodeling, apoptosis

Whats retinoblastoma (RB)

• A tumor suppressor genes

• Cancer of the retina

• Caused by loss of heterozygosity which would make it a familial cancer

Whats knudsons 2-hit hypothesis?

• States that 1 mutation is constitutional (inherited), 2nd allele is mutated somatically (2nd hit) starting the process of tumorigenesis for famililial cancers

• Cancer is the result of accumulated mutations

What is latency (time which cancer arises) for familiar and sporadic cancers and why?

• Early for familial, later for sporadic

• This is becuase of probabilities, its less likely to get the 2 hits via sporadic

What is occurrence of cancer for bi-lateral and uni-lateral for RB and why?

• Bi-lateral = hereditary , Uni-lateral = hereditary or sporadic

• This is becuase of probabilities, its less likely to get the 2 mutations via sporadic

How does double stranded break repair (DSB) occur?

• Naturally occurring due to:

  • V(D)J recombination - B cells - Antibody diversity

  • Collapsed/ stalled replication forks

  • Reactive oxygen species (ROS)

• Can also be caused by exogenous sources such as ionizing radiation

What’re the 2 types of DSB repair?

1. Non-homologous end joining (NHEJ)

2. Homologous recombination repair (HRR)

Explain Non homologous end joining (NHEJ)

• Error prone repair

• Glues ends back together which results in micro deletions and can promote translocations

• Can occur through cell cycle

• Predominant DSB repair pathway in mammals

Explain homologous recombination repair (HRR)

• Error free repair

• Copies a template to repair the damage, utilizes sister chromatids as template

• Restricted to specific cell-cycle stages of G2 and S because it needs completed sister chromatids to use as template

  • More prominent than NHEJ during these stages

• Secondary DSB repair pathway in mammals

Whats genome instability seen by?

• Chromosome Instability - CIN

Whats CIN (chromosome instability) defined as?

• Increased rate of chromosomes or large parts are gained/ lost

• Very large genetic changes (whole chromosome)

  • Multiple oncogenes, tumor suppressor and DNA repair genes

Why do we care about CIN?

• It’s highly prevalent in numerous cancer types (>80% of all cancers have shown CIN)

• It’s an early event that drives tumorigenesis/ oncogenesis

  • Not all cells in tumor are same which is why theres drug resistance

• Associated with highly aggressive tumors, multi drug resistance, poor patient prognosis

How is CIN assessed or measured?

• DNA content analysis

• Cytogenetic and karyotypic analyses

• Chromosome ideogram

Explain what DNA content analysis does

• Uses flow cytometry which analyzes DNA content NOT chromosome number

• It fluorescently labels DNA, it only fluoresces when associated with DNA

• Increase in fluorescence = increase in DNA content

What happens to peaks in flow cytometry?

• Abnormal amounts of chromosomes causes broadening of peaks

• Looks at DNA content NOT # of chromosomes

Whats cytogenetic and karyotypic analyses used for?

• To evaluate numerical vs structural anomalies

  • Numerical - enumerate

  • Structural - label specific chromosomes and visually identify gross chromosomal rearrangements

• Specifically assess chromosome numbers ad types

  • Requires mitotic preparation to be generated

  • Cannot be used for interphase cells

  • Counterstain and/or fluorescently labels DNA

Explain what chromosome ideograms do

• Groups chromosomes in size from large to small

• Shows centromeres position

• Depicts the binding patterns which are unique and specific for each chromosome

Whats the DNA counterstain used for in cytogenetics?

• To count chromosomes

• Quick and cheap

• Very easy to use for liquid cancer but impossible for solid tumors cuz of cellular mass predominantly in interphase

Whats chromosome specific labeling used for in cytogenetics?

• Too enumerate AND evaluate structural defects but defect must be large enough (> 1Mb of DNA)

  • Can’t detect inversions

• More expensive and time consuming

• But can use whole chromosome paints (WCP) which is chromosome specific and Special Karyotyping (SKY) which simultaneously and differently labels all chromosomes

Whats FISH used for?

• To fluorescently label probes to genomic DNA

Whats the limitation/ drawbacks of FISH?

• Very limited/ gene specific evaluation (BCR: ABL)

• Can be used to assess mitotic and interphase cells

In terms of targeting the aberrant genetic origins of cancer whats the approach of targeting oncogenes

• Targeting oncogenes is the classical approach cuz oncogenes are associated with amplification, enhanced promoter function, ectopic expression, fusion protein, etc

• Aberrant Function (hypermorphic function)

  • BCR:ABL

• Aberrant expression (hypermorphic expression)

  • B-RAF

What did company Novartis do?

• Used a tyrosine kinase inhibitor STI-571 which popped into active site and prevented phosphorylation from happening

What does phase 1 mean?

• Designed to be small clinical trials of late stage disease people basically donating their life to see if theres any adverse affects, not cure themselves but for next gen

Explain targeting B-RAF

• BRAF is an oncogene, a serine/theronine kinase involved in signal transduction

• In the MAPK pathway

  • Mitogens Activated Protein Kinase

  • Pro-proliferation

  • Anti-apoptotic

Whats the MAPK pathway and cancer?

• Constitutive pathway activation (proliferation/anti-apoptotic program)

Explain B-RAF and Melanoma

• Metastatic Melanoma (skin cancer)

• 5-year survival = 5-22% (stage 4)

• B-RAF V600E - constitutive (ectopic) activity

• Caused by extracellular stimulus (Mitogens) initiates signaling cascade to induce alteration in gene expression (i.e response)

What does MEK1 mutant do in MAPK pathway?

• It bypassed and started being resistant to MEK1 inhibitor

How is targeting defective tumor suppressor and DNA repair genes done?

• Via synthetic lethality

Explain synthetic lethality

• A RARE lethal combination of 2 independently viable mutations

• Extensively studied in model organisms (yeast)

• Now being expanded into cancer contexts

  • BRCA1 and PARP (breast and ovarian cancer) 2005

Whats the mech of synthetic lethality?

• Exploit the molecular defect in tumor suppressor or DNA repair gene by downregulating/ inhibiting a 2nd gene (synthetic lethal interactor = candidate drug target)

What’re some genetic interactions of synthetic lethality?

Whats the main summary of what cancer is?