Lecture #10 | Targets of Mutations: Oncogenes & Tumor Suppressor Genes Identification of the first Oncogene (Src)

Oncogene

A gene whose presence in certain forms and/or overactivity can stimulate the development of cancer

Tumor Suppressor Gene

A gene whose absence can lead to cancer

Do most mutations lead to cancer?

No, only 1% of the human genome codes for proteins (exons)

• most mutations do not impact protein function and do not lead to cancer

What gene mutations can lead to cancer?

Mutations in oncogenes and tumor suppressor genes

Transformed phenotype

exhibits one or more properties of a cancer cell

Characteristics of normal cells

• Require growth factors

• Exhibit density-dependent inhibition of growth

• Anchorage dependence

  • Want to have a surface to attach to

• Finite proliferative life span

  • Do not endlessly grow

• Adhesiveness

Characteristics of transformed cells

• Require fewer, if any, growth factors

• Do not exhibit density-dependent inhibition of growth

• Anchorage independence

• Infinite proliferative life span

• Lack of adhesiveness → morphology

• Form tumors in vivo

Details on the density-dependent inhibition of growth

• In normal cells, they form in a monolayer on a Petri dish

• In transformed cells that lack this density-dependent growth, they form a foci

• Does not care that there are a lot of cells around

• Can expand and impact normal cells

  • RSV causes the transformation of the cell

    

Growing on top

Which is a normal cell and transformed?

On the left → normal; aware of the space

On the right → transformed; growing on top of each other

Anchorage independence experimentation for transformed cell

Uses a nude mouse model

• lack a thymus → immune compromise

  • Lacks the immune response to fight foreign cells

  • develops tumors fast

• Lack hair

  • Allows for easy visualization of tumors

Ways that a transformed phenotype can arise

1. Activation of a proto-oncogene in to an oncogene

2. Mutation or deletion of a tumor suppressor gene

Proto-oncogene

A normal gene that can be changed into an oncogene

• Proto-oncogene → mutation → oncogene

• An activating mutation in one copy of a proto-oncogene can lead to the transformed phenotype.

When does a mutation of a tumor suppressor gene result in cancer?

Both copies of a tumor suppressor gene must be mutated for cells to be transformed

Normal function of oncogenes and tumor suppressor genes

They regular the cell cycle

• proto-oncogenes result in proliferation (division, growth) signals

• Tumor suppressors stop growth

Cell cycle

• G1 (cell growth)

  • Oncogenes

  • Tumor suppressor genes

• S (synthesis)

  • Tumor suppressor genes

  • DNA repair genes

• G2

• Mitosis

  

Oncogenes in cell cycle

• Act like the gas pedal

• Activating mutations in oncogenes tend to be dominant

  • You need to mutate only one copy to get transformation.

• Proto-oncogene to oncogene 1st mutation accelerated cell division

• Activating mutations in oncogenes are like a stuck accelerator - the gas pedal is always on!

Tumor suppressor genes in cell cycle

• Are like brakes

• Inactivating mutations in tumor suppressor genes tend to be recessive -- both copies must be altered.

  • 1st mutations: cell in susceptible to cancer

  • 2nd mutation or loss: leads to cancer

    • No brakes at all

Normal function of proto-oncogenes

Activate the signal transduction pathway and push the cell cycle forward

• stimulates cell proliferation

What do tumor suppressors normally do?

Act as brakes on the signal transduction pathway and cell cycle

• mutations cause lose of ability to inhibit cell proliferation if needed

Signal transduction pathway

• Normal cells need a signal from a growth factor (a peptide) to enter the cell cycle.

• Growth factors bind receptors on the surface of the cell.

• The receptor becomes active, often via dimerization, & sends a signal -- phosphorylation.

• This starts a cascade of short-lived phosphorylation events.

• The ultimate targets are transcription factors (TF) in the nucleus and other proteins that regulate the cell cycle.

• Proto-oncogenes can be anywhere in the ST pathway: Growth factor, receptor, kinase (enzyme that can add a phosphate), TF, cyclins.

  

Role of protein kinase

Kinase adds a phosphate (PO_4) to Ser (S), Thr (T) and Tyr (Y).

• very specific, PO4’g only specific aa consensus

  sequences

• >500 kinases in the human kinome

• Many more Ser/Thr kinases than Tyr kinases

  • STKs are more ancient and widespread, playing crucial roles in fundamental cellular processes, while TKs are a more recent innovation, often involved in more specialized signaling pathway

Role of protein phosphatase

Removes the (PO_4)

• non-specific, acting on a wide range of phosphoproteins.

• <200 protein phosphatase genes in human kinome

Retroviruses

RNA Tumor Viruses

• Retroviruses can productively infect only proliferating cells.

• HIV (aka AIDS) is a classical retrovirus

The viral RNA genome is reverse transcribed into proviral DNA which integrates randomly into the host genome.

How is RNA reverse transcribed into proviral DNA

RNA has reverse transcriptase and integrate

• after infection, these help in synthesis of negative DNA and then then RNA is removed

• + strand of viral DNA is synthesized by reverse transcriptase

• this integrates into viral dsDNA into cell chromosome to form provirus

Can infect only proliferating cells

What was the first demonstration that a virus can cause cancer?

• In 1911 Sir Francis Peyton Rous experimented with chickens with sarcomas.

  • Cancer of the breast muscle

• Extracted the tumors, grinder it up, processed in a filtrate

  • Why filtered? Make sure to get rid of other infectious agents like bacteria

• injected into another chicken and saw sarcomas in 1-2 weeks!

• Much later, the tumor extracts were found to produce virus that transformed cells.

• 50 yrs later, Rous Sarcoma Virus (RSV) was isolated -- Rous won Nobel prize 1966

Rous Sarcoma Virus

Has spike proteins

Attached on cell surface of the host

• Core made up of lipids and proteins

• SsDNA has a reverse transcriptase and integrase

  • Reverse synthesis

  • Helps with integration

• Not all retroviruses are infections

Basic structure of viral RNA genome

• gag which codes for core proteins

• pol which codes for reverse transcriptase and integrate

• and env which codes for protein

  • Not always infections

Acutely transforming virus (e.g., RSV):

• In vivo: tumors in 1-2 weeks

• In vitro: infect (& replicate) and transform cells

  • Able to produce tumors and transform cells in Petri dish

Virus can infect and transform the cell

Non acutely transforming virus (e.g. ALV):

• In vitro: infect (& replicate) but not transform cells

  • Infect but not transform cells

• In vivo: minimal tumors in 2-3 months

Can infect but not transform the cell

What is the difference between transforming and non transforming virus?

V-onc

V-onc

Viral oncogene

How does ALV → RSV

Capture c-src (cellular carcoma)

• Found in the host cell chromosomal DNA

• ALV infected a cell and accidentally integrated next to c-src

• So c-src + provirus → fused ALV-src RNA transcript

• Packaged into capsid carrying src sequences

So ability to transforms cells comes from host cell chromosome

How are v-src and c-src different?

V-src has no introns, mutated → always activated

How did they identify the cellular version of v-src

Southern blot analysis using a radio labeled v-Src probe

• They found a Src gene not just in chickens but in every other higher eukaryote they examined, including humans.