Cancer cells: excessive birth rate

What are the 4 types of genetic changes to a gene? (pathogenic variants) which do they all lead to?

• Amplification/Deletion (large scale aneuploidy - chromosomal)

• Single gene mutation (point/ small insertion -deletion )

• Chromosomal translocation

• Epigenetic methylation

All lead to aberrant activation or inactivation of specific genes so they function incorrectly (at the wrong time)

Amplification / Deletion

can be caused by

increased number of chromosomes (>2) from errors in cell division/ mitosis

or

Formation of “double minute“ chromosomes - extra fragment of DNA carrying specific (onco)genes outside of the nuclear genome. Can be reincorporated into cell chromosomes

Leads to production of MORE proteins by having extra copies of genes

Deletion can also occur during mitosis/ aberrant DNA repair leading to loss of specific (tumor supressor genes) = less protein

Partial gene deletion - case of oncogene Epidermal Growth Factor receptor

due to deletion tyrosine kinase always dimerised which leads to phosphorylation which leads to constant proliferation

Translocation - description and case of CCND1 oncogene

Rearrangements of genetic material to juxtapose gene promoters/regulatory elements which drives inappropriate gene expression (activating mutation)

Translocation of CCND1 gene from Chr 11 to CHr 14 leading to IGH enhancer element having an effect by switching on CCND1 gene leading to excess Cyclin D1 protein which results in deregulation of cell cycle at R point

Seen frequently in Mantle cell lymphoma

Evidence of effects of excess oncogenes

• Transfection of immortilise mouse fibroblast with cancer mutation (eg DNA that carry oncogen RAS) leading to fully transformed cells that are:
  - growth factor independent

• have 3D growth (disorganised)

• lose Anoikis (an apoptotic death triggered by the detachment from the ECM/BM) → allow anchorage independent growth

• Form tumours in vivo (invasive/ metastatic)

Inactivating point mutations and epigenetic regulation - two hit hypothesis - case of retinoblastoma

retinoblastoma often caused by inheritance of one mutation in RB1 gene and acquisition of a second mutation which switches off its function (regulating cell cycle and proliferation)

Alfred Knudson’s TWO-HIT hypothesis for the inactivation of tumour suppressor gene

1. First hit- acquired inheritance or somatic mutation of one allel

2. Second hit - somatic mution/ deletion or epigenetic inactivation of second organelle

Causes total loss of RB1 encoded protein (pRb). pRb function is lost in 25% of all cancers through inactivating mutation or epigenetic changes (promoter methylation)

Oncogenes - overactive proteins

EGFR, RAS, BRAF, CCND1

Tumour supressor genes - inactivated proteins

RB1, TP53, BRCA1/2, APC

Cell cycle

Normal growth factor signalling

• Promotes entry into cell cycle

• Relieves blocks on cell cycle progression (promote replication)

• Prevent apoptosis

• Enhances synthesis of components (nucleic acids, proteins, lipids, carbs) required for cell division

EG, EGF - epidermal growth factor - mitogenic, stimulates epithelial cell migration, stimulate formation of granulation tissue

Oncogenic growth factor signalling

• Increased levels of growth factors (sometimes different ones in metastases)

• Permanent activation of growth factor receptor

• Increased levels of growth factor receptor

• Abnormal signal transduction (eg active RAS)

Abnormal behavior = advances cells through the cell cycle

Growth factor signalling pathway

growth factor receptor binds to the receptor (EGF, TGFa ) to EGFR which induces receptor dimerization, tyrosine kinase activation, phosphyrylation of the receptor

some oncogenic muatation (partial gene deletion, point mutation) lead to constitutive activation of the tyrosine kinase receptor signalling, even in the absence of growth factor

Ras pathway

Ras is an intracellular protein that is central to integrating GFR signals

GRF activation → Ras binds GTP and recruits and activates signalling protein (eg Raf, PI3K)

Signal stops when Ras convert GTP to GDP+Pi via its GTPase activity

Cancers with activating mutant Ras have reduced GTPase activity → signalling is ON

Ras activates transcription of CCND1, cyclin D which activates CDK4 to stimulate cell cycle progression. Excess oncogenic cyclin D or CDK4 (by activating mutation, amplification) occur in some cancers)

Retinoblastoma protein (pRB)

The RB exists as two forms during the cell cycle

• Weakly phosphorylated at G0 and G) - ACTIVE - halt cell cycle by suppressing transcription factor signalling

• Strongly phosphorylated (ppRb) during rest of the cell cycle - INACTIVE protein, therefore cell cycle progresses

Inactivating phosphorylation of pRB is by (oncogene) CyclinD- CKD4)

Loss of pRB (eg by gene deletion/ inactivating mutation/ methylation) leads to uncontrolled cell cycle

• By skipping the cell cycle checkpoints, mutation can be passed onto daughter cells