Cancer Genetics Comprehensive Notes

Cancer Genetics

Slides by Rebecca de Kraa, Cytogeneticist, Haematology Department, PathWest Laboratories, Fiona Stanley Hospital. Presented by Paul Ellery (P.Ellery@curtin.edu.au)

Learning Objectives

• Understand cancer as a disease of abnormal gene function and expression:
  • Understand carcinogenesis as a multi-step process involving genetic & epigenetic changes in cellular division and regulation.
• Understand oncogenes and tumour suppressor genes:
  • Mechanisms involved in oncogenic activation & their cancer-causing potential.
  • How oncogenes act in a dominant transforming way.
  • Functions of tumour suppressor genes.
  • Knudson’s Two-Hit Hypothesis using tumour suppressor genes.
• Understand the underlying principles of the hallmarks of cancer and be able to give examples of these.

Definition of Cancer

• Disease of multicellular organisms.
• Abnormal proliferation of cells.
• Invasion of local tissue; can metastasize.
• Can cause significant morbidity/death.
• Abnormal gene function.
• Dysregulation of proliferation, differentiation & apoptosis.
• Clonality of tumour cell population is favored.

History of Cancer

• ~1600BC (Karkinos)
• ~300BC

Cancer – Timeline of advances in cancer research

• 1960: Philadelphia chromosome
• 1970: First oncogene, src
• 1971: Knudson’s “2-Hit” Hypothesis
• 1979: Tp53 gene
• 1980’s: The role of cyclins & cyclin-dependent kinases
• 1983: Polymerase chain reaction (PCR)
• 1984: Epstein Barr virus (EBV)
• 1990: FDA-approved gene therapy
• 1994 - 2003: Human Genome Project
• 2000s: Next-generation sequencing
• 2001: 1st Targeted Therapy – Imatinib mesylate for CML
• 2017: CAR T-cell Therapies

Cancer Classification

1. Primary site in the body where the cancer 1st developed:
   • Lungs ….. 8664 deaths in 2022 in Australia
   • Skin
   • Colon and Rectum
   • Female Breasts
   • Cervix and Uterus
   • Simply indicated where the cancer is located but doesn’t specify the type of tissue involved
2. Histological type – tissue type in which the cancer originates:
   • Carcinomas
   • Sarcomas
   • Leukaemia
   • Lymphoma
   • Myeloma
   • Mixed Types

Aetiology

• Environmental factors that can lead to genetic changes & cause cancer to develop:
  • Chemicals (e.g., pesticides, chemotherapy)
  • Radiation
  • Diet and Exercise
  • Infection – Retroviruses
  • Physical Agents
  • Hormones

Terminology

• “hereditary” cancers: very rare, germline origin
• “familial” clusters in families: combination germline & acquired
• “acquired” (sporadic): not germline origin, all acquired mutations

Carcinogenesis

• Process by which normal cells are transformed into cancer cells.
• Characterized by changes at the:
  • Cellular level
  • Genetic level
  • Epigenetic level
  • Through abnormal cell division

Theories of Carcinogenesis

1. Knudson’s Two-Hit Hypothesis
   • Especially true for the recessive nature of tumour suppressor genes
2. Multi-step process that involves > 1 mutation
   • Chemical Carcinogen leads to Irreversible change but not yet cancer. Selection/growth advantage, Multi-step tumour progression (Form benign or precancerous lesions), Epigenetic changes. Local invasion/metastases to distant parts, DNA instability increases with each step. Promoters contribute by mechanisms cells to acquire more mutations metabolism&repair processes altered.
3. SMT vs. TOFT
   • Somatic mutation Theory (SMT)
     • Carcinogenic agents new mutations or already mutated genes affect cell growth, differentiation or function.
   • Tissue Organization Field Theory (TOFT)
     • Agrees with SMT, but further proposes that carcinogenic agents disrupt interactions between cells that maintain the tissue architecture; it’s organization, repair and regulation.
4. Variant updates of SMT and TOFT
   • Epigenetic modifications – genetic changes other than mutations that involve:
     • epigenetic factors:
       • proteins/molecules e.g. growth factors
       • adjacent stromal cells e.g. endothelial cells
       • extracellular matrix (ECM) framework surrounding tumour cells
     • epigenetic mechanisms
       • hypermethylation of genes e.g. TAF, as seen in some breast Ca
   • Stem cell model

Cancer Stem cell theories

• Normal stem cell to Mutated stem cell, or self-renewal genes turned on to Loss of regulated cell division to Cancer stem cell
• Normal progenitor cell to Mutated progenitor, or self-renewal genes turned on to De-differentiated cell to Cancer stem cell
  *National Institute of Health resource for stem cell research

Cell cycle regulation

• Cyclins
• Growth factors & growth factor receptors
• Signalling transduction pathways
• Transcriptional regulation
• Apoptosis
• Checkpoints
• Telomeres and telomerase
• Extracellular matrix
• DNA Mismatch repair

Cancer-associated genes – 1. Proto-oncogenes and oncogenes

• Proto-oncogene – regulates cell growth & differentiation potential to become cellular oncogene
  • Involved in signal transduction & execution of mitogenic signals
  • e.g. myc involved in cell regulation - encodes for transcription factor, eg’s ras, wnt
• Oncogene ( or cellular oncogene c-onc) – potential to increase the malignancy of a cell, once it becomes activated, constitutively expressed
  • eg’s c-myc, k-ras

Cancer-associated genes - Classification of Oncogenes

Mechanisms of oncogene activation – a. Mutations

• alter structure of proto-oncogene oncogene
• dominant gain-of-function
• involve protein regulatory regions uncontrolled continuous activity of the mutated protein
• types of mutations:
  • Point mutations
  • Deletions
  • Insertions
  • Integration of proviral DNA from a retrovirus

Example of a point mutation

• DNA sequence analysis of K-ras gene at codon 12 wild type of K-ras G to A mutation

Example of a deletion

• If 1 copy is deleted, it leads to expression/change of function (due to dominant gain-of function)
• Eg. “Oncogenic activation of the Notch1 gene by deletion of its promoter in Ikaros-deficient T-ALL”
• Note: gene dosage effect associated with deletions where obvious deletions in chromosomes represent an increased number of genes deleted eg 5q- syndrome in MDS. This region contains multiple oncogenes and tumour suppressor genes.

Example of a mutation involving integration of proviral DNA from a retrovirus

• Retrovirus = RNA virus (can reverse transcribe RNA into proviral DNA) e.g. HTLV-1 activate TAX gene adult T-cell leukaemia/lymphoma (severe)
• TAX involved in proliferation

Mechanisms of oncogene activation – b. Gene Amplification = oncogene onc present

• Mechanism: repeated copying in DNA replication process expansion in copy number increase in gene expression deregulated cell growth

Example of gene amplification resulting in dmins

• Amplification can result in:
  • double minutes (d-mins; mi-nutes)
  • Intracellular, but not a chromosome
  • homogenously staining regions (hsrs)
• Amplified regions can contain > 100’s copies e.g. c-myc is amplified in small-cell lung ca, breast/ovarian ca and leukaemias.

Case study example of double minutes (dmins)

• Case 1: Acute Myeloid Leukaemia patient with double minutes

Mechanisms of oncogene activation – c. Chromosomal rearrangements

• Recurrent chromosomal rearrangements are often detected in haematological malignancies & some solid tumours
• Types:
  • Numerical Gains/losses (egs trisomies, monosomies) eg trisomy 8 (clone = at least 2 cells), monosomy 7 (clone = at least 3 cells)
  • Structural – any structural aberration (clone = at least 2 cells)
    • Intrachromosomal Inversions – segments within a chromosome, reversed end to end
    • Duplications/deletions/insertions
    • Others eg rings, markers
    • Interchromosomal Translocations – reciprocal exchange of chromosomal material
    • Insertions
• When these rearrangements happen, oncogenes can be activated by:
  1. De regulated expression of oncogenes via regulatory control of an immunoglobulin gene IGH@ Proto-onco/onc is moved close to an immunoglobulin gene and falls under its control deregulated expression neoplastic transformation
  2. Formation of novel hybrid fusion genes with transforming activity Juxtaposition of 2 different genes to form a novel fusion gene codes for chimeric protein transforming activity

Case study 1

• Patient with Mantle Cell Lymphoma t(11;14)(q13;q32) G-band and directed metaphase FISH using IGH-CCND1 dual colour dual fusion probe (CCND1 R, IGH G)
  CCND1(aka BCL1) involved in G1 to S transition

Case study 2

*Patient with Acute Lymphoblastic Leukaemia t(11;14)(q23;q32) - karyotype. CCND1(aka BCL1) involved in G1 to S transition

Case study

• CML patient with t(9;22)(q34;q11.2) by G-band and directed metaphase FISH using dual colour dual fusion BCR-ABL1 probe (ABL1 R, BCR G)
• ABL1 gene encodes tyrosine kinase activity Involved in cellular differentiation
• BCR located at 22q11 – unknown function
• BCR::ABL1 fusion gene encodes chimeric protein BCR-ABL1

Cancer-associated genes - Tumour Suppressor genes

• Suppress cellular growth/survival when needed to prevent tumours forming.
• Outcomes triggered by tumour suppressor activation:
  • Arrest cell cycle to inhibit cell division
  • Induce cell cycle to DNA damage repair mechanisms
  • Promote apoptosis if damage cannot be repaired
  • Induce senescence
• Two most important tumour suppressor genes; TP53 and RB
• Other eg’s: APC, BRCA1, BRCA2
• Follow Knudson’s 2-Hit Hypothesis e.g. RB
• Usually recessive nature – both copies mutated before function is affected
• The exception is TP53 it can be; recessive or dominant negative

Recessive and Dominant Negative TP53 mutations

• Two normal TP53 genes.
• Homozygous recessive mutation
• Dominant negative mutation. Heterozygous recessive mutation.
• No TP53 produced
• Non-functional TP53
• Altered TP53

Difference between oncogenes & tumour suppressors

Cancer-associated genes - DNA Mismatch-Repair genes (same family as Tumour suppressor genes)

• The DNA mismatch-repair system normally recognizes and repairs errors that arise during replication and recombination e.g. insertions of nucleotides.
• But what happens if the repair is faulty?
  • Results in ineffective repair unstable genome
• What can go wrong with DNA Mismatch-repair genes?
  • Mutations
  • Hypermethylation of promoter regions of some of the genes
  • e.g. mutation of hMSH2 gene causes faulty repair microsatellite instability a lot of complex steps colorectal ca

Hallmarks of Cancer

• 2000 Hanahan and Weinberg
• 6 biological capabilities acquired by cancer cells:
  • (1) Sustaining growth signalling
  • (2) Evading growth suppressors
  • (3) Resisting apoptosis
  • (4) Enabling replicative immortality
  • (5) Inducing angiogenesis
  • (6) Activating invasion and metastasis
• 2011 sequel of 4 more
  • 1) abnormal metabolic pathways
  • 2) evading the immune system
  • 3) chromosome abnormalities & unstable DNA
  • (4) inflammation

Sustaining Growth Signalling

• Cancer cells can sustain growth by:
  1. producing their own growth factor molecules e.g. glioblastomas - PDGF
  2. send their own growth signals
     • send their own signals to normal cells in ECM around tumour react and supply tumour with GF e.g. E-cadherin/catenin complex
     • receptor proteins at the cancer cell surface hyper responsive to usually limited supply growth signalling e.g. increased HER2/neu receptors in some breast cancers
• Outcome: circumvent limited pathway & keep growth signalling switched on

Evading Growth Suppressors

• Function of a growth suppressor is to control growth (regulatory pathways/factors)
• Many of these are dependent on tumour suppressor genes eg RB and TP53
• Defects in pathways/genes cancer cells able to resist inhibitory signals that would usually stop their growth
  Mutated RB gene RB inactivated growth suppressor evaded, proliferates

Resisting Apoptosis

• BCL2/TP53 are involved in cellular apoptosis
• BCL2 has both anti- and pro- apoptotic functions
• TP53 can promote apoptosis and DNA repair
• Apoptosis can be overcome if:
  1. Over expression of BCL2 (translocation, controlled by IGH)
  2. Mutation/loss of TP53

Enabling Replicative Immortality

• Multiply forever!
• Normally: cells have limited # growth/division cycles before senescence is reached or a crisis phase leads to cell death
• So what causes some cells to bypass this?
• Telomeres involved immortalization
  • Telomeres protect ends of chromosomes
  • As cells reach end of lifespan, telomeres shorten genome instability/apoptosis
  • Telomerase, maintains telomere length is almost absent in normal cells but in 90% immortalized cells, including cancer cells
• Genetic mechanisms are unclear, but a combination of changes occur: loss of TP53 and RB pathway function & activation of RAS or myc telomerase genomic stability multiply forever

Inducing Angiogenesis

• Formation of new blood vessels.
• Balanced by inducers and inhibitors
  • e.g. Inducers VEGF-A which bind to receptors on endothelial cells
  • Inhibitor TSP-1 regulated by TP53
• For cancer cells to grow they need a blood supply. During carcinogenesis an “angiogenic switch” is tripped and remains on. Inducers & inhibitors control this switch.
• e.g. TP53 loss or mutation can dysregulate TSP-1 and induce angiogenesis. as seen in growth of breast and melanoma cancers

Activating invasion and metastasis

• Tissue invasion: localized
• Metastasis: distant areas attach to ECM/ conscript normal cells for support
• Activated by changes in molecules needed for cell adhesion: - cadherins & integrins
  • e.g. E-cadherin – assemble epith cells sheets & maintain integrity mutation or of E-cadherin by some cancer cells cells to detach activates invasion and metastasis
  • e.g. integrins - mediate cell attachment/integrity & send signals to regulate this, - involved in the motility of cells. expression of integrins have been correlated with metastatic progression in breast, prostate and lung ca.
• Genetic alteration in cadherins/integrins or factors that regulate/effect their pathways activation of invasion/metastasis