throughout the course we've talked about the potential effects of mutations on protein structure and function in this video i want to spend a bit more time talking about the clinical relevance of genetic mutation to cancer and how the disruption of normal gene expression can lead to this disease when i say gene expression here i mean that in the broadest sense making the gene product usually a protein with the correct structure and function in the right amounts at the right times as we've discussed before this could be controlled at multiple levels from gene transcription rna processing rna export rna degradation translation protein degradation disruption in any of these processes could disrupt normal expression of the product of the gene i'll provide an overview of proto-oncogenes and tumor suppressor genes which are both associated with cancer we'll look at some of the most important members of each class and talk about dominant acting versus recessive mutations previously we've discussed how changes in the coding sequence of a gene can cause changes in the amino acid sequence of a protein and depending on where in the protein that occurs and what the particular amino acid change is that can have a dramatic effect on the protein structure and properties and these altered characteristics cause a physiological change in cells that leads to disease example we saw earlier in the course is sickle cell disease caused by mutation in the beta globin gene a single base pair change that caused replacement of the normal charged glutamic acid residue with a nonpolar valine amino acid causes aggregation of the hemoglobin molecules and formation of long crystals of hemoglobin within the red blood cells these distort the biconcave disk structure of normal red blood cells and cause them to take on this elongated crescent shape which causes blockages in blood vessels failure to perfuse tissues and organs and all the assorted health problems we've talked about before but mutations that occur outside of coding regions can also cause disease if we think about that beta-globin gene we could have a mutation in the coding sequence say an exon that changes that gag codon to gtg and causes the glutamate to valene substitution in the protein but there are all kinds of ways that mutations in non-coding regions of genes can disrupt appropriate expression of a gene product as well we've talked about the regulatory elements that transcription factors bind to to activate or repress gene transcription mutations in these can lead to disruption of the level of protein expression too much or too little and depending on the protein that can be very problematic likewise what if there's a mutation in the cap site or the poly a signal sequence these could disrupt five prime cap and three prime poly a tail addition and processing of the mrna from this gene which will lead to more rapid degradation and therefore less gene product finally we know that splicing has to occur in a very precise fashion to avoid altering the reading frame of the mrna otherwise synthesis of the protein is massively disrupted usually causing premature truncation but also sometimes frame shifts that lead to extensive missense and proteins with the wrong amino acid sequence so mutations in both non-coding regions and coding regions can disrupt the ability of the cell to produce a functional protein product and in the right amounts and in the right circumstances any of these kinds of genetic mutations can lead to disease so with that understanding let's talk first about proto-oncogenes genes that encode proteins that promote cell survival or cell division are referred to as proto-oncogenes very often these are involved with signaling pathways including those that transduce signals from growth factor receptors like the receptor tyrosine kinases we talked about earlier in the course under normal circumstances these signaling pathways promotes cell division and proliferation only when growth factor stimulates the pathway the prefix proto means first or earliest form so the original unmutated version of this type of gene we call a proto-oncogene the prefix onco means tumor or more broadly cancer so an oncogene is a gene that promotes cancer but it only does so if a mutation causes it to misbehave converting the normal proto-oncogene into a cancer-causing oncogene one example of an oncogene is the ras family of signaling proteins which are gtpa's g-proteins the structure of one of the human versions hras is shown here in the upper right these signaling proteins are small but mighty they function as enzymatic on off switches in many important cellular processes including regulating the cytoskeleton and therefore cell shape and adhesion promoting cell survival and triggering mitotic cell division and cellular proliferation along with many other functions just like the heterotrimeric g proteins we discussed earlier in the course in our signaling topic the activated status of ras is determined by whether it's in its gdp or gtp bound state normally these are very tightly regulated and are only switched on in response to specific signals for example activation of a growth factor receptor like we see here in the diagram but mutations in ras that cause it to be hyperactive for example remaining in the gtp-bound state too long cause over-stimulation of these cell survival and cell division pathways leading to tumor formation ras mutations are some of the most common in human cancers overall they are found in about 25 percent of all cancers and in some types of cancer like pancreatic cancer they are the most prevalent mutation found occurring in over 90 percent of cases typically mutations that convert proto-oncogenes to oncogenes are gain of function rather than loss-of-function mutations causing the protein to acquire a new function like remaining in the on state even when no growth factor is around as a result these mutations are typically dominant you only need one hit causing an activating mutation in one of your ras alleles to see the effects we're focusing on ras here because it's one of the most common cancer-causing mutations but we should recognize that mutations in any of these other elements of the signaling pathway upstream of ras or downstream could also lead to over proliferation of cells or failure to undergo apoptosis when they should for example imagine a mutation that changes the structure of the growth factor receptor so that it is always in its ligand-bound structural state this would cause hyperactivation of the receptor and the signaling molecules downstream including ras because of its prevalence in human cancer brass protein signaling has been a prime target of research into cancer therapeutics for over 30 years without success largely because of the complexity of the signaling pathways ras is involved in drugs that seem to work well to block grass in the lab end up having little clinical impact for this reason ras has been referred to as being undruggable however that could potentially be changing new therapies that target ras in different ways for example disrupting the enzymatic modifications required for it to remain associated with the cell membrane could prove more promising so stay tuned on the other side of the cell proliferation equation we've got tumor suppressor genes in contrast to proto-oncogenes the normal function of tumor suppressor genes is to limit cell division and proliferation to promote cellular differentiation and promote apoptosis so these are acting in opposition to the effects of proto-oncogenes to balance their effects and maintain homeostasis and get rid of damaged cells through apoptosis under normal circumstances the most famous of these tumor suppressor genes and the one that is most central to our discussion of cancer genetics is a protein called p53 p53 is a transcription factor it regulates expression of a whole host of genes involved with among other things apoptosis a rest of the cell cycle in the case of dna or other significant cellular damage dna repair pathways and promoting differentiation p53 itself is activated by lots of different types of cellular stress including dna damage hyperactive or overexpressed oncogenes oxidative stress and shortening of the telomeres which as we've talked about before is associated with cellular senescence or apoptosis so when things are going wrong in the cell p53 activates a set of cellular responses to either fix the problem or get rid of the cell if the problem can't be solved because of its central role in making sure that dna damage is kept to a minimum and that cells don't divide if dna damage can't be fixed p53 is referred to as the guardian of the genome and loss of function mutations are found in nearly all cancers it's by far the most commonly mutated gene in human cancers here we're looking at histograms showing the mutation frequency for the p53 amino acid sequence in 16 different cancer types the color coding here at the bottom highlights different regions of the protein different functional domains i won't go into all of this but what we can see here is that a majority of mutations in all of these cancers happen in this pink region right in the middle which makes up the dna binding domain of the p53 protein this is the part of the protein that actually interacts with dna to control the expression of all those genes we just talked about most of these are loss of function mutations that destroy the ability of p53 to sit down on dna and regulate transcription of its target genes as a result p53 mutations are typically recessive as long as the other copy of p53 the other allele is normal the cell will still have functional p53 protein which will still do its job if you're unlucky and inherit both copies of chromosome 17 which is where p53 is found from a parent with a mutation something called uniparental disomi then you have no functional p53 protecting the cell if you have just one mutant allele then in order to completely lose the guardian function of p53 a second hit that eliminates that other allele is required this could occur through a non-disjunction that causes loss of the entire chromosome or recombination errors that cause deletions of part of the chromosome that contains the p53 locus or breaks in the dna or a sporadic mutation or some other mechanism and this is generally true for tumor suppressors in contrast to oncogenes where typically we're seeing activation and gain of function dominant mutations mutations in tumor suppressors in general are loss of function and therefore act in a recessive fashion since most p53 mutations result in a loss of function they also are referred to as undruggable from a chemotherapy perspective it's hard to find a drug that will restore function to an inactive or abnormal protein however targeting regulators of p53 has been a more successful approach in those types of cancer where wild-type p53 protein is still present in these strategies the goal is to activate the p53 protein to stimulate it to do its normal job thereby overcoming some mutation and some other growth regulating protein in the cell one strategy is to inhibit inhibitors of p53 thereby activating the p53 pathway to understand how this works we have to take a step back and talk a bit more about how p53 expression is regulated in cells remember that p53 is a transcription factor to do its job it has to be in the nucleus so that it can bind to dna and activate its target genes which protect the cell and suppress tumor formation in general a regulatory protein called mdm2 causes p53 protein to be exported out of the nucleus into the cytoplasm mdm2 also promotes ubiquitination of p53 which as we've discussed previously in the course is the kiss of death for proteins it targets the ubiquitinated protein to the proteasome for degradation so mdm2 causes p53 to leave the nucleus come out into the cytoplasm where mdm2 ubiquitinates it causing it to be degraded this is yet another example of negative feedback regulation because it turns out that the mdm2 gene is one of p53's target genes p53 activates expression of the mdm2 gene which encodes the mdm2 protein which targets p53 for degradation the logic of this system is clear this prevents p53 from being overprotective in a sense you wouldn't want cell division to always be suppressed sometimes it's necessary you don't want p53 always promoting apoptosis that would be bad so this negative feedback loop is p53 self-limiting its own effects in a concentration dependent manner when there's lots of p53 the mdm2 gene gets activated which leads to more mdm2 protein which decreases the concentration of p53 in the cell to treat cancers that have wild-type p53 but some other mutation that's promoting growth inhibitors of mdm2 can be used to block this effect thereby ramping up p53 concentrations and promoting its growth limiting effects this is just one therapeutic target on the p53 pathway there are others but this gives you a sense of how this strategy might work incidentally this same therapeutic approach of inhibiting mdm2 is being tested as a way to decrease the side effects of other chemotherapeutics treating cancers that don't have wild-type p53 as you may already know many chemotherapy agents work by targeting actively dividing cells which obviously targets the cancer cells for destruction but a side effect is that normal actively dividing cells in the body are also affected so we get off-target effects of chemotherapy like nausea and vomiting because the dividing cells in the stomach lining that normally replace that cell population every day or so are now also being killed as well as hair loss because dividing cells in the hair follicles are killed so to reduce this effect in addition to the chemotherapy being used to treat the cancer an ndm2 inhibitor can be added to increase p53 in all cells of the body this causes normal body cells to go into cell cycle arrest so they are no longer sensitive to the effects of the chemotherapy agent while the cancer cells still get killed off because they lack wild-type p53 very clever in this video we've reviewed how mutations in coding and non-coding regions of the genome can potentially cause disease and looked specifically at oncogenes and tumor suppressor genes as cancer-causing agents in this one topic we've revisited so many concepts covered in the course everything from protein structure to genetics to enzyme inhibition dna mutation and repair negative feedback loops and apoptosis and mitosis i hope you've enjoyed seeing this example of how it all ties together