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

Molecular Aspects of Cancer Targets of Mutations: Oncogenes & Tumor Suppressor Genes

  • 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.

  • Mutation Agent ==> Lesion ==> Repair or Mutation

Learning Objectives

  1. Understand the difference between an oncogene and a tumor suppressor gene.

  2. Understand the difference between normal and transformed cells.

  3. Describe the signal transduction pathway.

  4. Describe how the first oncogene was identified.

  5. Understand the difference between an acutely transforming virus and a non-transforming virus.

Mutations and Cancer

  • Only ~1% of the human genome codes for proteins (exons).

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

  • Mutations in 2 types of genes can lead to cancer:

    • Oncogenes

    • Tumor Suppressor genes

Normal vs. Transformed Cells

Normal Cells:

  • Require growth factors

  • Exhibit density-dependent inhibition of growth

  • Anchorage dependence

  • Finite proliferative life span

  • Adhesiveness

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 è morphology

  • Form tumors in vivo

Transformed Phenotype

  • The transformed phenotype exhibits one or more properties of a cancer cell

  • Lack density-dependent inhibition of growth è foci

  • RSV-induced focus on top of normal chicken fibroblasts

  • RSV = Rous Sarcoma Virus (causes tumors in chickens)

  • Form tumors in nude mice (lack a thymus è immune compromised)

  • Anchorage independence

Proto-oncogenes and Tumor Suppressor Genes

  • Proto-oncogene: A normal gene that can be changed into an oncogene.

  • Proto-oncogene è mutation è oncogene

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

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

Cell Cycle Regulation

  • Oncogenes and tumor suppressor genes regulate the cell cycle:

    • M (mitosis)

    • G2

    • G1 (cell growth)

    • S (synthesis)
      DNA repair genes

Oncogenes vs. Tumor Suppressor Genes

  • Oncogenes:

    • 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:

    • Are like brakes

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

Signal Transduction Pathway

  • Oncogenes activate the Signal Transduction Pathway and push the cell cycle forward.

  • Tumor Suppressor Genes Act as brakes on the signal transduction pathway & cell cycle
    Cell proliferation

Signal Transduction Pathway & Cell Cycle

  • 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, TF, cyclins.

Protein Kinases and Phosphatases

  • Protein Kinase: Kinase adds a phosphate (PO4)(PO_4) to Ser (S), Thr (T) and Tyr (Y).

  • Protein Phosphatase: Phosphatase removes the (PO4)(PO_4).

  • Kinases tend to be very specific, (PO4)(PO_4)’g only specific aa consensus sequences.

  • Phosphatases tend to be non-specific, acting on a wide range of phosphoproteins.

  • There are >500 kinases in the human kinome, but <200 protein phosphatase genes.

  • Many more Ser/Thr kinases than Tyr kinases.

Serine(Ser,S)Threonine(Thr,T)Tyrosine(Tyr,Y)Serine (Ser,S) Threonine (Thr,T) Tyrosine (Tyr,Y)

CH2OHCH_2 OH

CH<em>2−CH</em>3OHCH<em>2-CH</em>3 OH

CH2OHCH_2 OH

SerThrTyrSer Thr Tyr

OHOH

ProteinKinaseProteinPhosphataseProtein Kinase Protein Phosphatase

O=P−OO=P-O

O−O-

O−O-

SerThrTyrSer Thr Tyr

ATPADPPiATP ADP Pi

ProteinPhosphorylationProtein Phosphorylation

Retroviruses

  • 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.

Rous Sarcoma Virus (RSV)

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

  • Extracted the tumors and processed them and 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

  • First demonstration that a virus can cause cancer

Rous Sarcoma Virus (RSV) vs. Avian Leukosis Virus (ALV)

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

    • In vivo: tumors in 1-2 weeks

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

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

    • In vivo: minimal tumors in 2-3 months

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

Identification of v-onc

  • 1970’s: Several labs show Rous sarcoma virus (RSV) has an “extra” gene that is not found in ALV.

  • They call it v-onc.

  • Virus can infect but not transform the cell

  • Virus can infect and transform the cell

ALV “captured” c-src è RSV

  • ALV “captured” c-src è RSV

  • c stands for cellular

  • Src stands for Sarcoma

  • v-src is different from c-src because it has no introns, mutated

ALV vs. RSV Genes

  • Gag: assembles the virus

  • Pol: reverse transcriptase. Converts the single-stranded viral RNA genome into ds RNA and then DNA.

  • Env: envelope protein on the outside of the virus, has role in infecting the host.

  • v-src: oncogene in RSV

Human Oncogenes

  • Many human oncogenes were first identified as acutely transforming retroviruses in animals

  • There are no known human retroviruses that cause cancer. (HIV causes cancer indirectly)

Identification of c-Src

  • Southern blot analysis using a radiolabeled v-Src probe

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

  • They called it c-Src for cellular Src, as opposed to v-Src, viral Src.

Origin of v-src

  • Mid-1970s: Duesberg shows that RSV acquired the v-onc gene (which he called v-src) from the cells infected by the virus!

  • But, the gene in the cells is slightly different from the viral oncogene and is called c-src.

  • Retroviruses: can pick up normal cellular genes via the mRNA intermediate.

  • Viruses undergo high mutation rates and change the gene into a viral oncogene ==> V-SRC

  • Virus Host