Molecular Genetics summary year 3 BMW track patient-oriented
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Course
Moleculaire Genetica
Institution
Universiteit Van Amsterdam (UvA)
Summary of all lectures of the molecular genetics course Summary of all lectures of the molecular genetics course I have completed this course with a 9 :)
Molecular Genetics
Cancer and colorectal cancer: Vogelgram 30/10/17
Introduction
Colon cancer: nr 1 cancer in the Netherlands, increasing due to screening. The survival depends on
stage the patient is in.
Multi-hit model: Cancer is a multistep process; different mutations are needed to make a tumor. At
least four hits are needed, but many more hits are possible.
TNM stage: tumor, (lymph) nodes stages, metastases. Stage 4 means bad prognosis. Early stage (1)
cancer means good prognosis. The stage also determines the treatment.
Hallmarks of cancer: resisting cell death, self-sufficient in growth signals, telomerase activity,
invasive capacities.
Research
LT: inhibits P53 and RB, two hits. Not enough to mimic cancer, cells go senescent (can’t divide)
because telomere length becomes too short, cells stop dividing. Cancer cells circumvent this stop, by
using telomerase to elongate the telomeres again.
Ras-transformation: makes it possible for cancer cells to grow on agar, independent of surroundings.
Familial Adenomatous Polyposis (FAP): Mutation in APC, because of this cancer is acquired more
easily. Patients have multiple adenomas in the colon at early age. Filled with polyps.
Mormons: inbreeding between families. Easy for locus identification. FAP was linked to APC gene.
Genetics behind colorectal cancer
Vogelgram: multistep process (millions of hits) that leads to cancer formation. Because of the
number of steps/hits, cancer takes years to develop. Four mutations are needed in the same cell to
form cancer. Samples of colorectal cancer were looked at, at different stages to find sequence of
mutations. CRISPR was used to mimic the Vogelgram process in vitro, only quadrupole mutant shows
cancerous growth, attracting stroma from the mouse. Triple mutant has limited growth and no
invasion.
1. Loss of APC: tumor suppressor, inhibits beta-catenin mediated transcription.
2. Activation of KRAS: does not have to be a mutation, but activation.
3. Loss of BMP (Smad 4): anti-growth signal, shown to block cancer growth. Mutation in BMP
receptor or Smad 4 TF is found in cancer. This way the cells don’t get the anti-growth signal.
4. Loss of p53: tumor suppressor gene is lost
MSI Microsatellite instability: see 2/11/2017
Serrated pathway: see 2/11/2017
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,Wnt signaling 30/10/2017
MMTv: breast cancer retrovirus. Virus integration induces proto-oncogenes transcription. Integrates
into INT1, hyperactivation of the proto-oncogene next to the virus. It can lead to cancer when proto-
oncogenes are next to where the virus binds. Host cell is made into production machinery for viruses.
Wnt signaling
1. Wnt: Wg gene + INT -> Wnt. Is essential for morphological development, segmentation in flies.
Binds to Frizzled and Lrp, the signal transduction gives beta-catenin/TCF activation (by blocking
inactivation by APC/Axin. Endless possibilities because of a wide variety of binding partners.
Proliferates often and is heavily regulated, therefore it is sensitive to mutations.
Wg gene: next to the INT1 that causes cancerous expression.
INT1: integration site, where the MMTv virus binds
2. Frizzled/LRP (Wnt receptors): When Wnt binds to the receptors, APC is inactivated, therefore
beta-catenin is activated. The receptors are regulated by ubiquitination.
RNF43: ubiquitinates Frizzled. Regulation of Wnt signaling.
RSPO: Wnt signaling causes low signals, but high signal when RSPO binds to RNF43 (RING).
This blocks ubiquitination by RNF43, giving a high signal when Wnt binds!
3. APC: APC binds and inactivates beta-catenin, blocking transcription.
Mutation in colorectal cancer: The APC binding region is mutated in colorectal cancers. It
cannot inhibit b-catenin and transcription. 80% of colorectal cancer patients have a mutation
in beta-catenin binding region of the APC gene. The ones that don’t have an APC mutation
have a mutation in beta-catenin. Beta-catenin and APC are mutually exclusive, which means
that they are in the same pathway.
4. Axin: complex formation of APC and beta catenin. Wnt signaling pulls Axin away from the
complex, so beta catenin is no longer suppressed and phosphorylated.
5. Beta-catenin: activate transcription for cell division, hyperproliferation. APC blocks Beta catenin
from activating transcription. APC and Beta-catenin are part of the same complex. When Wnt
signaling is active, Beta-catenin becomes dephosphorylated and free and activates TCF.
Mutation in colorectal cancer: Beta-catenin mutation in N-terminal serine or threonine
(phosphorylation sites) causes colorectal cancer. Phosphorylation on serine or threonine is
overactive, Beta catenin can now keep binding to TCF in the nucleus by itself.
GSK and CK: kinases that phosphorylate beta-catenin. Phosphorylation makes a docking
station for the E3 ubiquitin ligase complex, results in the continuous degradation of Beta-
catenin by proteasome without Wnt signaling. When Wnt is present,
6. TCF: Beta-catenin binding to TCF in the nucleus causes transcription.
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, Intestinal crypts and Wnt signaling 30/11/2017
Intestinal Crypts: receive canonical Wnt signals. Stem cells in the
intestine of the colon are in the bottom of the crypt. Stem cells
have nuclear beta-catenin activity. Wnt signaling is active there.
APC deletion: causes adenoma formation, not cancer.
TCF deletion: no proliferation. The epithelium stops dividing,
can’t absorb food.
Wnt: oncogene, ligand and morphogen. Wnt signaling is more
prominent in the colon.
Finding the target of Wnt signaling: with gene sequencing, up or
downregulated genes are looked at. They use Doxycyclin induced
activation of TCF to show the targets. Crucial targets should be expressed at the stem cells in the
crypts, where Wnt activity is also found. They found LGR5, Ascl2 and Myc.
LGR5 promoter: is induced by beta-catenin-TCF
Ascl2: Wnt signaling causes transcription of Ascl/Myc. Transgenic expression results in crypt
expansion, hyperproliferation, cancer formation. Does the same thing as APC mutation,
therefore it is a downstream transcription factor of the pathway.
Myc (oncogene): affected in all cancers. Cancer transcription. Myc deletion prevents APC-
driven tumorigenesis.
Color Tracing
CreDNA recombinase: recognizes LoxP sites in constructs, puts them together and starts cutting
genes in between. LoxP sites are opposed for inversion and head the same way to make a deletion,
the genes in between is cut out.
CreER: only active when induced by Tamoxifen. Replace LGR5 with CreER, so CreER is expressed
instead of LGR5 when beta-catenin is active. Combined with LacZ reporter gene, for coloring. Now
you can see where in the crypts the color turns up. It becomes colored when beta-catenin is active
after tamoxifen activation.
Lineage tracing: with Lgr5-GFP-ires-CreERT2 Knock-in Mouse x RosaLacZ, the progeny of these cells
shows blue coloring. Therefore, you can trace the lineage. When tamoxifen is added Cre goes to the
nucleus excises the STOP codon and Rosa26 becomes a driver for the LacZ gene, activating the gene
and giving a blue color. When Lgr5 is off the color still stays on, therefore the offspring will show the
blue color. The progenitor cell is GFP.
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