Tuesday, May 17, 2011

Book Notes: Cancer Part 2

  • cancer critical genes: small subset of genes whose mutation usually causes cancer
    • proto-oncogenes: genes who, when mutant, are overactive and drive cells to become cancerous
    • tumor suppressor genes: genes who prevent cancerous cells (when mutant, they are inactive and allow cells to become cancerous)
    • DNA maintenance genes: genes who, when mutant, are inactive and allow cells to become genomically unstable, which may or may not lead to cancer
  • to test possible proto-oncogenes, you insert a copy of the oncogene (mutated version)
    • if cells become cancerous, then the inserted gene really was an oncogene
  • to test possible tumor suppressor genes, you have to knockout both copies of the gene in the cell
    • as long as cells have one copy of a tumor suppressor gene, they can withhold the cell from abnormal growth
  • tumor viruses: viruses who don't intentionally cause cancer, but by inserting their own genome into the DNA, causes a mutation that leads to cancer
    • they are usually retroviruses, who carry RNA that is reverse transcribed into DNA and then inserted
  • DNA decoding of transformed cells helped determine oncogenes
    • tumor DNA is fragmented and put with fibroblasts that already divide a lot (but are still normal)
    • if one of the fragment contains an oncogene, some of the cells will start forming masses: transformed!
    • sequencing the genome of these transformed cells compared to the non transformed cells showed the oncogene
    • Ras was the first, and is mutated in 1 of every 5 cancers
      • a point mutation prevents it from hydrolyzing GTP, so it is hyperactive and never shuts off
      • dominant effect: only one copy of the Ras gene needs to be mutated this way in order to produce an uncontrolled effect
  • studying retinoblastoma, eye cancer, helped decode tumor suppressor genes
    • there were 2 types of eye cancer, one hereditary, one just random.  The hereditary version impacted both eyes (so it must be a genomic problem in all the eye cells)
    • they found a deletion on chromosome 13, where one copy of the Rb protein was missing
    • it's hereditary because families inherit the one good copy and one bad copy, so as soon as the one good copy is mutated, they get retinoblastoma
    • the non hereditary is random and usually in one eye because there must be 2 separate events that mutate both copies of Rb in one cell
    • Rb regulates cell cycle, so mutated Rb allows inappropriate and uncontrolled cell proliferation
  • usually tumor suppressor genes are identified by screening many cancers and finding out what gene is repeatedly missing or defective
  • tumor suppressor genes can be inactivated in many ways
    • chromosome deletion, point mutation, segregation/recombination errors, etc, etc
    • loss of heterozygosity: where one copy of a cancer-critical gene is lost or deactivated, so now the cell has a predisposition to cancer (being cancer-prone)
    • epigenetics: methylation of a tumor suppressor gene causes it to be stored in heterochromatin and silenced for all the progeny
  • proto-oncogenes can be overactivated in many ways
    • point mutation, deletion, gene amplification, chromosomal rearrangement that ends up altering a gene or the regulatory sequence of the gene
  • 3 methods of cancer-critical gene identification
    1. CGH (comparative genomic hybridization): DNA fragments from normal cells and from cancers cells are fluorescently labelled
      • the labelled fragments hybridize to complementary sequences on a DNA microarray (where each spot corresponds to a known sequence of the genome)
      • where the tumor complement binds, it means that many copies of the gene exist (gene amplification)
      • where the normal complement binds, it means there was a deletion
      • where there is no light, it means that the two copies of the genome exist and are paired up (blocking any probes from hybridizing)
    2. DNA microarray can also be used with mRNA probes
    3. RNAi: siRNA can be engineered to knockout a specific gene by destroying the mRNA of that gene or inhibiting the translation
      • then you observe whether the knockout cell becomes cancerous or not
    • best test involves doing knockouts or causing overexpression of a gene in actual mice
      • mice that develop tumors soon after prove the possibility of that gene being a cancer-critical gene
  • so what are the pathways in which these cancer-critical genes actually cause cancer?
    1. cell proliferation pathways: just about any and every signaling protein can be affected, usually with proteins that activate or relay signals becoming hyperactive (proto-oncogenes) -- i.e. Ras, EGF receptor
      • the inhibitors involved in pathways tend to be the tumor suppressor genes -- i.e. Rb
        • cancer cells might eliminate Rb, or by inhibiting the inhibitor of the inhibitor of Rb (in times of stress, p16 blocks cyclinD/Cdk4 from inhibiting Rb, who inhibits E2F from continuing the cell cycle, SO, if a mutation inhibits p16, cyclinD/Cdk4 is free to inhibit Rb so that E2F happily progresses the cycle)
    2. cell growth pathways: cancer needs to be able to grow uncontrollably, otherwise uncontrolled division will just lead to smaller and smaller cells
      • PI3/Akt pathway important for growth, cancers activate this pathway in the absence of growth signals so they just grow all the time
        • overactive Akt helps with protein synthesis, glucose uptake, and lipid synthesis
        • so it's usually PTEN phosphatase who is messed up, because he's the inhibitor of Akt
    3. apoptosis pathways: apoptosis usually activates in response to DNA damage and mutations, so because cancers need mutations, they must shut off apoptosis so they can keep proliferating
      • Bcl2 is a protein that normally inhibits apoptosis
        • in a B-lymphocyte cancer, the Bcl2 gene is translocated to an active promoter, so Bcl2 is overexpressed
        • this keeps apoptosis from activating
    4. DNA damage pathways: but really the star player is p53
      • known as the guardian angel gene, p53 is super critical because it happens to be involved in 2 of the above 3 pathways, and in DNA damage systems
        • to start, p53 is normally kept at very low levels: continuously made and continuously degraded
        • when a cell is stressed or the genome is damaged, p53 levels shoot up
        • active p53 (1) stops any cell dividing to buy some time (2) activates DNA repair systems (3) or activates apoptosis when nothing can be done
        • this is the protection that prevents cells with just a few mutations from blowing over into cancer
        • p53 is a gene regulatory protein, it activates expression of other proteins that carry out the above-listed functions
        • mutation of p53 usually happens in its DNA-binding domains, so it can't bind to genes and activate transcription, and the cell can (1) divide uncontrollably (2) avoid apoptosis (3) have genetic instability w/o DNA repair and (4) ignore stresses like radiation and drug treatment
  • DNA tumor viruses are the lysogenic viruses, who integrate their genome into the host genome and sit quietly
    • virus genome contains stuff that take control of reproduction machinery to generate more viruses
    • accidents can occur that activate some components of the virus genome that activate DNA replication and division, leading to tumor formation
    • e.g. papillomavirus produces a protein E7 that blocks Rb, and an E6 protein that inhibits p53
  • Metastasis has 2 steps: invasion and re-establishment
    • EMT (epithelial to mesenchymal transition): normal organism development process where epithelial cells change from adhesive and stable to less adhesive and migratory
      • this happens when E-cadherin is lost and cells can't form junctions, so they can go out exploring and getting into blood vessels
    • colonization: cancer cells must acquire the ability to proliferate in a new environment in order to do this
      • that's why most cancer cells that escape the primary tumor don't actually form new metastases
  • CASE STUDY: COLORECTAL CANCER
  • colon epithelium has rapid turnover rate, so it is constantly growing and dividing (at a controlled rate) to replace old cells
    • renewal depends on special crypts (pockets) of stem cells in the epithelium
    • benign tumors look like small protruding masses called polyps, the first sign of abnormal growth
    • the most common mutations happen to 3 proteins: K-Ras, p53, Apc
      • Apc is an inhibitor of the Wnt pathway but causing degradation of B-catenin (recall B-catenin can go in to the nucleus to activate transcription)
      • but tumors tend to form faster with a hyperactive B-catenin rather than an inactive APC
      • the high frequency of APC mutations comes from APC being involved in other pathways as well and getting mutated affects the mitotic spindle formation, so chromosome segregation is messed up
  • one form of colon cancer is hereditary, due to a mutation in DNA mismatch repair
    • the inherited predisposition involves having one messed up copy of the repair gene and one good copy
    • once a mutation inactivates the one good copy, cells will start building mutations, increasing chances for cancer
  • tumors often have specific order of mutations
    • in most cases, Apc is one of the first to go
      • mutations w/o Apc's guardianship will build up
    • then Ras falls apart
      • cells now proliferate w/o substrate or neighbor inhibition
    • then p53 is messed up
      • now cells can mutate faster and avoid apoptosis
    • each tumor will mutate its own variation of similar genes (like messing up the Ras regulator as opposed to Ras itself), but the control mechanisms need to be disabled in a proper order
    • even different patients with the same type of cancer will have different mutations, which makes cancer treatment very difficult
  • cancer prevention or early diagnosis of a primary tumor is the best method of avoiding cancer
    • treatment is difficult because you must remove ALL the bad cells, or else even just one cancer stem cell will regenerate a tumor
    • surgery makes it difficult to get every tiny metastasis, and drug/chemical treatments also hurt good cells
  • traditional cancer treatment exploits genetic instability
    • radiation and drugs kill cancer cells because they can't repair the DNA damage, while normal cells will pause cycling (so you lose hair) but will fix the damage
    • unfortunately, cancer cells can evolve resistance to treatment (like bacteria!)
  • new treatments exploits CAUSE of genetic instability
    • first, consider that there are multiple ways to repair DNA damage
      • a single strand break is fixed by one system
      • if that system is down, the stalled replication fork that results when DNA Pol encounters the ssbreak can be fixed by homologous recombination
    • second, consider that cancer cells tend to lose some of the repair systems in order to become cancer
      • for example, let's say a cancer cell can't repair a stalled fork, but it can fix ssbreaks
    • third, consider that we can devise drugs to impair repair systems
      • in the example with the cancer cell that can't repair stalled forks, what if we give it a drug that inhibits the repair of ssbreaks?
      • now the tumor can't repair ssbreaks, and it already couldn't repair stalled forks, so it will die....
      • BUT the normal cells live, because unlike the cancer cell, they CAN repair stalled forks, even if the drug also affects them and inhibits the repair of ssbreaks
    • these treatments take advantage of the fact that normal cells have multiple options, while cancer cells have very few repair/survival options because of their mutations
  • cancer cells have still another advantage: genomic heterogeneity
    • even though they are all descended from the same ancestor, they have such a high rate of mutation that much of the population have different variations in their genome
    • this means you can't just use one type of treatment to eradicate a whole tumor
    • multidrug resistance: phenomenon where a cell, after surviving just one drug treatment, somehow is resistant to a bunch of other drugs that it's never met before
      • this happens when a gene called Mdr1 is amplified (has multiple copies now)
      • Mdr1 makes a transporter that pumps out drugs, overexpression of the protein makes sure no drugs hang around and damage the cell: they just get pumped out
  • new therapies are being developed due to our increasing knowledge
    • targeting the one hyperactive protein that a cancer may be dependent upon (oncogene addiction: the cell is addicted to/dependent on the oncogene)
    • send in antibodies that either carry toxins or enzymes that cleave pro-drugs into toxins at the cancer site (because antibodies bind so specifically, you use anti-cancer antibodies)
  • we can also engineer small molecules that target the one hyperactive protein of oncogene addiction
    • e.g. CML (chronic myelogenous leukemia) is associated with Philadelphia chromosome
    • this chromosome snaps the genes Abl and Bcr and rejoins them so they fuse into Bcr-Abl
    • the fusion protein combines the Tyr kinase of Abl and the active expression of Bcr to make a hyperactive kinase
    • this prevents apoptosis of unnecessary lymphocytes, so they build up in the blood
    • a special molecule called Gleevec blocks Bcr-Abl, and effectively eliminates CML in early diagnosis patients
    • less effective with later patients b/c cancers have usually evolved resistance to Gleevec binding
      • probably going to have to do a cocktail to fully eradicate cancerous cells
  • another approach is to target the tumor blood vessels
    • there are drugs that inhibit the VEGF receptor
    • also, endothelial cells that are about to make new blood vessels have distinguishable characteristics that we can engineer molecules to attack them specifically
  • the final method is to enhance the existing immune system to target tumors
    • we can tag cancer cells with antibodies to attract macrophages to gobble them up
  • the future of treatment is probably going to involve a simultaneous combinations of drugs and molecules, because no cell will have resistance to all of them without some time to evolve (i.e. if we used the drugs one by one, every time, there would always be some cells resistant the single drug used)


pg. 1230 - 1240

No comments:

Post a Comment