The Cell Cycle

I wanted to give a simplified overview of the cell signaling that drives a cell through the growth cycle before we get into oncology and look at all the things that can go wrong. Most cells will spend the bulk of their time in the G0 phase of the cell cycle. This means they are mature and carrying out their daily functions. It is only when they are given growth signals do they enter the cell cycle and replicate. The cell cycle is highly controlled and regulated. There is a massive amount of regulation on controlling cell division. This is to prevent uncontrolled growth which leads to cancer.

There are genes that promote growth and we call them proto oncogenes. They are capable of driving cancer, but they won't when they are normal and healthy. A proto oncogene is one that normally helps drive the cell through the cell cycle. When they mutate, they can break free from regulation and drive uncontrolled growth. Then they are called oncogenes. There are other genes that block cell growth without the proper signals to advance the cell through the cell cycle. We call these tumor suppressor genes. These can be lost by mutations that render them inactive. These act like the breaks for the cell cycle and prevent the cell from going through the cell cycle unless they have the proper signals. The loss of a tumor suppressor gene can allow uncontrolled cell growth.

The signal for the cell to enter the cell cycle comes in the form of growth factors. There are a ton of them like Epidermal Growth Factor, Vascular Endothelial Growth Factor and many more. These growth factors all have the same role but for different tissues. It all starts when the growth factor binds to its receptor on the cell surface. It will initiate a signaling cascade of the growth pathways inside the cell. They are the MAPK and mTOR pathways that mainly drive cell growth. These 2 pathways will activate key transcription factors that will enter the nucleus and activate key genes. Those genes will produce proteins that will help push the cell into the cell cycle. At the same time, they will release the tumor suppressor genes like GSK3-Beta that prevent the cell going through the cell cycle. The combination of growth gene activation and tumor suppressor protein suppression will allow the cell cycle to start.

The deactivation of GSK3-Beta will allow the first Cyclin with Cyclin D to build within the cell which pushes the cell into the G1 phase of the cycle. At the End of the G1 phase the cell will go through a checkpoint to ensure all the DNA is ready to be copied and no problems exist with the DNA. The DNA Repair pathways act as another set of breaks for the cell cycle. If anything goes wrong with the DNA the DDR pathways will stop the cell cycle. If the DNA is good, the cell proceeds to the S phase of the cycle.

In the S phase of the cycle, the DNA will go through the long process of copying itself. Every chromosome has to be copied and checked for quality. There are several cyclins and proteins that regulate the initiation and termination of the DNA synthesis. These are things like Cyclin E and Cyclin A along with Cyclin Dependent Kinases like CDK1 and CDK2. These pathways are complex and mutations in the many proteins that drive them can lead to errors in the DNA or over activation of the cell cycle.

That last stage is the G2 phase of the cell cycle. This is where the DNA is checked before the cell is allowed to enter the process of Mitosis. There is a checkpoint at the end of the G2 phase called the G2/M checkpoint. Here the DDR pathways will make sure the DNA is good before allowing the cell to proceed to Mitosis.

Mitosis is the complex process by which each and every chromosome will line up and be separated into 2 groups. Each new cell has to get a copy of each and every chromosome. Errors anywhere in the cell cycle can cause cancer. There are dozens of proteins, transcription factors, enzymes and checkpoints that could mutate to create either gain of function mutations in the proto-oncogenes that drive the cell cycle, or they can be a loss of function mutation in a tumor suppressor gene. Either of these types of mutation can push the cell into the cell cycle and drive cancer growth.

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What is Cancer?

The definition of cancer is uncontrolled growth. It is a genetically driven disease. It begins with the accumulation of genetic mutations in a cell over time. One mutation alone will not lead to cancer. Some people are born with enough mutated genes to give them cancer at birth. Some people are born with genes that give them a predisposition toward cancer, but that does not mean they will certainly get it. They are at higher risk. Genetic mutations in key proto oncogenes or tumor suppressor genes can give a person a higher risk for cancer. It takes a series of mutations over time to accumulate all the traits of cancer in one cell. There are two major factors that contribute to the formation of cancer with genetics and environment.

Genetics is what we are born with, and we could have a higher risk for cancer in our genes. Environmental exposure and lifestyle choices contribute to the accumulated mutations to our genes over the course of our lifetimes. We can inherit specific genes that give us a predisposition toward a specific type of cancer. That does not mean we will get that cancer, but it can significantly increase the odds. The environment plays a major role in causing cell mutations. The biggest factor in cancer development are acquired mutations over time caused by chemicals or pollutants. We call these factors carcinogens. Some types of carcinogens in cancer are UV light from the sun or chemicals from cigarette smoking. Even the Oxidative stress caused by our own immune systems can cause mutations. Chronic Inflammation is a large cause of cancer.

Surveillance toward cancer is the job of the immune system. The immune system is designed to find and kill any mutated cells, and it does so all the time without us ever knowing it. The formation of a tumor begins when all the genetic variables come together for a mutated cell to escape the immune defenses and grow uncontrolled.

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Types and Incidence

A solid tumor gets named based on the tissue in which it begins. A Carcinoma is found in the epithelial tissue that makes up the skin that lines both the outside and inside of the body. Sarcoma is found in the connective tissues and bones. About 85% to 90% of solid tumors are Carcinomas made up of organ tissue epithelial cells. The other 10% to 15% are Sarcomas made up of bone and connective tissue cancers. Carcinoma is further divided into two subcategories of Adenocarcinoma and Squamous Cell Carcinoma. The Adenocarcinoma forms inside the tissue of the organs while the Squamous Cell Carcinoma forms in the skin that covers the organs. Most solid tumors will fall into the Carcinoma.

Blood Cancers are made up of Leukemia and Lymphoma. Leukemia is the cancer of the blood and starts in the bone marrow. It can spread into the circulation and even evolve to become a Lymphoma. Leukemia is a cancer of White Blood Cells. Leukemia is broken down in myelocytic and lymphocytic forms. The myelocytic form is made up of the cell lines that come from granulocytes, mainly neutrophils, while the lymphocytic form is made up of cells from the T cell or B cell lines. Acute Leukemia develops rapidly while chronic develops over the long term. You will never see a child with chronic leukemia. All childhood leukemia is acute. Lymphoma is a cancer that originates in the lymph nodes of the lymph system. It's a solid tumor and gets named based on which cell lines it originates in and which parts of the lymph nodes where it begins. Lymphoma is broken down into two kinds of Hodgkin's Lymphoma accounting for 5% of lymphoma and Non Hodgkin Lymphoma (NHL) accounting for 95% of lymphoma. NHL is further broken down into many subtypes.

About 14 million new cancers are diagnosed around the world each year and about 8 million people die from cancer each year. Lung cancer makes up about 12% of all cancers and affects about 235,000 people in the US each year. About 85% of lung cancer falls into Non Small Cell Lung cancer (NSCLC). It makes up the largest subset with small cell lung cancer making about 15%. Many lung cancers are diagnosed when they are advanced as they are hard to detect early. Up to 80% of all lung cancers stem from smoking. There are a lot of environmental contributing factors for lung cancers, especially occupational for firefighters.

Breast cancer makes up about 12% of all cancers and affects about 285,000 people in the US each year. Breast cancer is the second leading cause of cancer deaths in women. Annual screening has led to a significant reduction in deaths related to breast cancer. About 70% to 80% of breast cancers are ductal cancers. This cancer has a huge genetic component with the BRCA gene which dramatically drives up the risk of breast cancer. Age and genetics are the largest contributing factors to breast cancer.

Prostate cancer makes up about 8% of all cancers and affects about 248,000 men in the US each year. It leads to about 27,000 deaths each year. It is the second leading cause of cancer death for men. It is another cancer where screening can catch it early and significantly improve outcomes. The highest risk factor is age with 60% of cases in men over 65. There is a PSA test that can easily be used yearly as a screen.

Colon cancer makes up 10% of all cancers and affects about 105,000 people in the US each year. This is one of those cancers that can be caught and treated early with testing. The survival rate is high when caught early. Still, around 50,000 people die every year from Colon Cancer. The top risks for Colon cancer are age, diet, lifestyle such as smoking, and other illnesses like Inflammatory Bowel Disease.

Liver cancer makes up about 700,000 cases worldwide every year and 42,000 in the US. It is far more common outside the US. The death rate is extremely high in Liver cancer as it goes undetected often until it's too late. It is twice as common for men as women. Chronic viruses of the liver like Hepatitis are the leading cause of liver cancer. Drinking and cirrhosis is another major factor that contributes to liver cancer.

Skin cancer is one of the largest cancers in America with Melanoma making up about 1% of skin cancers. Melanoma affects about 77,000 people in the US each year. This one is of particular interest as it has a high genetic component with the BRAF V600E mutations. The number one risk factor is sun exposure.

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DNA Damage Repair Pathways

On average, when the DNA polymerase copies the DNA during the S phase of the cell cycle, it makes a mistake once in every 1 billion base pairs. That means each cell makes about 3 mistakes each time it replicates. The DNA polymerase has proofreading ability and catches the vast majority of these errors and fixes them, but some errors to get through. Most of the DNA damage will come from environmental events like UV light, Radical Oxygen Species, and chemicals.

There is a set of proteins and enzymes that are designed to find and repair different kinds of DNA damage. If it is just a single base error, then the DNA Damage Repair (DDR) proteins will undergo Base Excision Repair (BER). This is where the specific base is removed and fixed. It can be a deamination of a cytosine, a depurination or even an alkylation of a base. The BER enzymes will flip the base out from the DNA and cut it loose from the carbon bonds. One of these DNA repair enzymes is Uracil DNA Glycosylase (UNG). This targets and repairs deamination events which occur thousands of times a day from environmental factors. This happens when the A or T loses its amino group. The UNG repairs these deamination events.

If damage involves two bases, like those two bases becoming bound to each other called a pyrimidine dimer, then the Nucleotide Excision Repair (NER) kicks in. The binding together of two bases is a common type of damage from things like chemicals and UV light. When that happens the NER repair will remove and replace a section of the DNA strand. It binds to a single strand of the DNA about 2 twists apart or 24 bases and removes a section of the stand. NER will fix what is called bulky groups in the DNA. These are large multi ringed groups from carcinogens and chemicals. They get into the DNA and need to be removed by NER. This is a process which chemotherapy drugs exploit.

When both strands of DNA get broken in what is called a Double Stranded Break (DSB), other sets of repair proteins will go to work. The process of Non Homologous End Joining is the most inaccurate way for DNA repair. This process basically takes two ends of DNA and glues them back together. It doesn't even care if they belong together. A second method can be used for DSB with Homology Directed Repair. This will take the matching chromosome and use it as a template to repair the break. Homology Directed Repair (HDR) is only available when the cell is going through mitosis and a sister chromatid is available to use as a template for directing repair. The vast majority of the time, the cell will be using Non Homologous End Joining (NHEJ). The Double Stranded Break (DSB) is the worst possible damage to the DNA as the process of NHEJ will typically do the repair by adding or removing bases. These are called insertions and deletions (Indels) and they can throw off the entire gene.

When it is only 1 strand of DNA that gets broken, the DNA will undergo Single Strand Break Repair. This is the pathway where the famous BRCA gene exists. It takes the good strand of DNA and uses it as a template to repair the broken strand.

The DDR machinery is designed to find and fix the damaged DNA. When something gets too bad for these mechanisms, then the cell will undergo programmed cell death. Mutations in the DDR repair mechanisms will lead to genetic instability which will allow the cell to accumulate more mutations at a faster rate. Loss of DNA repair will lead to chromosomal rearrangements.

There is a new approach to targeting cancer called Synthetic Lethality. This uses the defects in the DDR repair pathways that allow cancer to gain faster mutations against the cancer to push it into such instability that it initiates programmed cell death called Apoptosis. This is like the bar stool approach. If you have a four legged bar stool and a leg breaks, it might have only three legs, but it will most likely still stand up. If you take out another leg, then it becomes unstable and collapses. The process of Synthetic Lethality looks for mutated DDR pathways that promote cancer mutations. Then it looks for another point in the DDR pathways that can be knocked out to cause the cell to become so unstable it undergoes cell death.

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Genetic Abnormalities

Point Mutations are one of the most common mutations in cancer. This is where a single base gets changed in the DNA. This can lead to an entirely new amino acid being encoded into the protein changing its behavior. Point Mutations come in 3 kinds. The first is the silent mutation. This where a base gets changed, but it still encodes the same amino acid. This can happen as there is some redundancy in the coding of amino acids. Some amino acids have multiple codons that create them. The second type of point mutation is the Nonsense mutation. This is where a stop codon is now placed early in the gene. You end up with only a truncated version of the protein which can be non functional or only partially functional. The final point mutation is the one we will deal with most of the time in cancer. This is where a single base changes and causes a different amino acid to be encoded. This is called a Missense mutation. These will often result in a protein with an altered function.

When this point mutation occurs in a proto oncogene, then it becomes an oncogene which will drive cancer. These mutations are labeled in a specific format. First is the letter for the amino acid that was supposed to be encoded. Then the number for the location of that amino acid in the protein. Last will be the letter for the new amino acid that was encoded. An example would be G12C. So, G12C means that a Glycine at the 12th amino acid in the sequence was replaced with a Cysteine. In proteins, the structure of a protein determines function. A single change like this can completely change its function. When this occurs in a proto oncogene, you can get increased growth signals that lead to rapid cell proliferation and cancer. The alternative is a point mutation that can occur in a tumor suppressor gene like p53 and render it non functional. This causes the loss of that tumor suppressor gene. This removes the protection and brakes on the system which can lead to uncontrolled growth.

The last possible mutation of a single base is called the frameshift mutation. Since every 3 bases makes up a single amino acid, if one base is removed or inserted, then the whole sequence of amino acids shifts. That makes it encode an all new set of amino acids from that point forward.

Now that we looked at DNA level mutations, we are now going to look at mutations that can occur at the chromosomal level. These can cause a whole gene to be inserted, deleted, inverted, duplicated or even translocated. The first set of mutations are insertions or deletions. This is where a single gene gets deleted or duplicated. A duplication of a gene can happen many times. This is sometimes the case in cancer. When a gene for a growth factor gets copied 20 times. In the same way, the deletion of a tumor suppressor gene will lead to a loss of function for that gene. The other alternative is a gene inversion. That means the gene gets flipped around so it is now read backwards.

The last chromosomal level change can be what is called a translocation. This is where one section of DNA gets taken from one chromosome and placed on another. When a gene for a growth signal ends up being translocated to a region of the chromosome where it is placed behind a promoter which is highly active, you now have a highly active growth signal. This is what occurs in the famous Philadelphia Chromosome with the 9/22 translocation. This is where part of chromosome 9 and part of chromosome 22 are exchanged. The gene BCR gets translocated so that it is fused with the ABL gene which results in hyper active growth signaling. This is the most common translocation in Chronic Lymphocytic Leukemia (CLL). There are many other kinds of translocations that can occur. Not all translocations are reciprocal. That is where 2 segments of a chromosome are exchanged. Sometimes they are one sided. That is where a section of one chromosome just gets broken off and placed on another.

The last issue with chromosomes is called Aneuploidy. This occurs when the cells go through mitosis. We know all chromosomes have 2 copies with one set coming from each parent. We have 23 pairs of chromosomes. During mitosis the DNA gets copied so that each new cell gets a full set. That means after the DNA is copied, that cell has 4 copies of each of the chromosomes. When an error occurs during mitosis, you can end up with too many or too few chromosomes ending up in a single daughter cell. This can lead to dosing issues with having too many growth signals in that new cell. You might end up with 1 daughter cell with 1 copy of a chromosome and another with 3 copies.

All these possible mutations that can happen will often happen in cancer at some point. This brings us to the concept of chromothripsis. This is where a single mutation leads to genomic instability. That allows more mutations to happen which leads to more mutations to happen. Then you end up with cancer cells who have chromosomes and DNA that are all scrambled up. This creates an environment of rapid evolution. The tumor becomes a battlefield of survival of the fittest. Every new cell has new mutations which battles with every other cancer cell for survival. The tumor will have a ton of cells dying as they fail, but some will survive and thrive. This battle for resources inside the tumor is what eventually leads to invasion and metastasis. This wide possibility of mutations demonstrates the vast genetic variation within a single tumor let alone within different patients. There can be hundreds of mutations inside a single tumor and not every cell will have them all.

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Cancer Genes

A Proto-oncogene is one of those genes that drives cell growth through the cell cycle which can mutate and become an oncogene that drives cancer growth. These are the many genes that make up the growth receptors and signal transduction pathways inside a cell. There are a lot of these growth factor receptors on the surface of cells. They fall into families like Epidermal Growth Factor, Vascular Endothelial Growth Factor, Platelet Derived Growth Factor, Fibroblast Growth Factor, and many more. When mutations occur in these receptors, they can stay in the on state even when there are no growth signals around to activate them. This drives the cell into the cell cycle and promotes cell proliferation.

The next set of proto oncogenes occupy the transduction pathways that translate the activation of the growth receptor into the nucleus. These genes are the cascades of proteins and enzymes inside the cell that translate receptor activation into gene activation inside the nucleus. These are broken down into specific pathways that control specific functions of the cell. They fall into the Mitogen Activated Protein Kinase (MAPK) pathway and the mammalian Target of Rapamycin (mTOR) pathway. There are others like the Wnt and Hedgehog pathways. These pathways are made up of many key proto-oncogenes that can mutate like RAS, RAF, MEK, ERK, PI3K, AKT, and so many more. When these proteins mutate the growth of the cell can be locked into an always on state. There are groups of tumor suppressor genes in these same pathways like NF-1 deactivates RAS and PTEN deactivates PI3K. These tumor suppressor genes ensure when things get turned on that they also get turned back off. The loss of them can leave the pathway turned on all the time.

The last set of proto oncogenes are in the control of the cell cycle. The cell cycle is governed by the Cyclins and the Cyclin Dependent Kinases (CDK). There are many other proteins and enzymes in the cell cycle checkpoints. Mutations in any of them can allow the cell to advance through the cycle without proper growth signals. Any of these Cyclins or CDK proteins can become oncogenes when they mutate. The tumor suppressor genes are those that block the cell from growing when it's not required. Many of the growth pathways are regulated by factors that prevent them from being activated without the proper growth signals. In the cell cycle these are genes like p53, p21 and Retinoblastoma.

The most famous of tumor suppressor genes is the p53 gene. It is known as the guardian of the genome. It is responsible for ensuring the integrity of the DNA and will stop cell growth when there is damage to DNA. It can even initiate cell death if damage is too bad. p53 is activated by all of the DDR pathways to arrest the cell cycle for DNA damage repair. There are other internal checkpoints the cell must clear during the growth cycle to ensure everything is good before allowing it to move to the next stage of growth.

The loss of a key tumor suppressor gene is called a loss of function mutation. If you have 1 good copy of a tumor suppressor gene, it will still function. It takes a loss of both alleles of these genes to lose function. Many times in cancer a tumor suppressor gene like p53 is lost, but not by mutation. They become silenced by epigenetic forces such as methylation from carcinogens. The cell growth cycle is held in a delicate balance between these growth forces and tumor suppressor forces. When there is a mutation that tips the scales in either direction, you get increased cellular growth.

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Targeted Therapies

Current Chemotherapies take a very broad approach to trying to stop cancer. They tend to have good efficacy but carry a lot of side effects. They attempt to block cancer by inhibiting cell replication and growth. Some of them interfere with the process of mitosis by blocking the process through inhibiting the centrosomes and spindles. Other chemotherapy agents will damage DNA to a point of cell death. Some do this by cross linking the DNA strands. These approaches are very broad and have side effects on healthy cells. They cause a lot of toxicity related to hair loss, GI problems and low cell counts in the blood. They inhibit all rapidly replicating cells good and bad. This includes some of the most rapidly developing cells in the body which are all the blood cells like red blood cells, platelets and white blood cells. This leads to anemia, clotting issues and risk of infections. This led to the concept of targeted therapies for fighting cancer. Targeted therapies have been a growing and evolving area of cancer treatment. Currently, it only affects a minor amount of patients as Chemotherapy, Surgery and Radiation still work for about 50% of patients.

Targeted therapies have grown in use over the past decade. For all they do, they still only apply to about 20% of cancer patients. They are very powerful drugs for those patients who do benefit from them. Many of these drugs are a simple pill or a few pills each day. They are really easy to use for both doctors and patients. They do have side effects based on their gene of target, but not nearly as much as chemotherapy. There are a lot of different targeted therapy approaches so I will start with one that should now be very familiar to us. The growth pathways that drive cancer become the first group of targeted therapies. Targeted therapies started by targeting the growth receptors using antibodies. They bind to the receptor and block the binding of the growth factor. This works for cancers where they produce too many receptors or growth factors. The downside with this approach is it can't reach cancers that are driven by the pathways inside the cell. The proteins inside the cell mutate into an "always on" state. They no longer need a growth factor to stay active. That is where targeting growth pathways started.

The first growth pathway to target was the receptor, but instead of using an antibody, they blocked the kinase that was responsible for activating the receptor. This prevented its activation. These drugs quickly took over as targeted therapies against growth factors. Antibodies required IV administration, but a small molecule inhibitor could be a pill just a few times a day. Next came the pathways that transduce the signal from the receptor to the nucleus of the cell. These are the MAPK and mTOR pathways with PI3K, RAS, RAF, and MEK. These are the most common mutated oncogenes that lead to the proteins that drive cancer growth. The first generations of these inhibitors were more toxic as they blocked the entire protein. They inhibited the Normal form (Wildtype) as well as the mutated forms of these proteins. Some of the proteins they inhibited were extremely toxic like PI3Ka.

The newer generations are focused on targeting just the mutated forms of the proteins. This has been very effective, but cancer always finds a way to mutate and gain resistance to these targeted inhibitors. This constant resistance by cancer leads to 2nd, 3rd and even 4th generation targeted therapies for the same target as the cancer mutates the sites where these drugs engage the target proteins. The newest generation of inhibitors in the growth pathways have been using AI to guide smarter development to increase efficacy and reduce toxicity. The latest data from Relay in FGFR2 is very promising for these AI guided drug developers.

The last major frontier in pathways has been those inside the cell like the cell cycle and DDR pathways. There are cancers where the oncogenes are driving things like Cyclin E which pushes the cell into the cell cycle. This has brought with it a new phase of targeted drugs that focus on the cell cycle like CDK2 or Wee1. There are a lot of new targets going on in the cell cycle now. I am sure they will not all be successful, but we will learn a lot from these attempts. The latest target has been the DNA Damage Repair pathways. Failure in this process leads to genomic instability which drives rapid mutation through possible chromosomal rearrangements. Losses of genes like p53 and BRCA1 are powerful cancer drivers. There is a lot of renewed interest in DDR genes. The major challenge to them is they are tumor suppressors. You just can't develop a drug that inhibits something. They need to be expressed, but they are being suppressed already in cancer.

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Cancer Epigenetics

The term epigenetics means on top of genetics. This is the study of how DNA is packaged, regulated and expressed. The basic unit of DNA packaging is the nucleosome. The nucleosome includes all the histone proteins and DNA wrapped around them. It's about 150 base pairs of DNA wrapped twice around eight histone proteins. It also includes the small segment of DNA that links to the next nucleosome. There are two H2a, two H2b, two H3 and two H4 histone proteins made into an octamer. The tails on the histone proteins can be modified with different chemical groups like Acetyl groups, methyl groups, phosphates, and ubiquitination.

The patterns of modifications on these histone tails can increase or decrease the expression of that gene. If you add a methyl group to the promoter of the gene on the DNA, it can silence the gene. This happens to tumor suppressor genes that lead to cancer. If you add that methyl group to the right place on the histone tail, it will increase expression of that gene. The addition and removal of acetyl groups on the histone tails will modify the charge on the histones and its ability to bind the negatively charged DNA. This addition or removal of acetyl groups is done by enzymes and will allow or deny access to that section of DNA for gene transcription. This is done by Histone Deacetylases (HDAC) and Histone Acetyltransferase (HAT) enzymes.

The way the DNA is packaged plays a big role in how genes are expressed, but it is not the only way the DNA gets regulated without actual DNA modification. The process for epigenetic regulation of genes is methylation of the promoter region. This is different from histone methylation which increases the expression of a gene. The DNA methylation occurs in the promoter region of the gene. The promoter region of the gene has regions called CpG rich islands. These are regions where more than 50% of the bases are cytosine and guanine rich. These CpG regions can become methylated by the addition of the methyl groups to the bases. This blocks the binding of the RNA polymerase and prevents expression of that gene. This can happen from environmental factors. There is an enzyme called DNA methyltransferase (DNMT) that adds these methyl groups. There are 2 versions. The first will add new methyl groups to the DNA adding to current methylation patterns on the DNA. The second only replicates current DNA methylation patterns as the DNA gets copied. So why in the world do cells need to methylate DNA anyway? This is how genes get silenced as the body develops and genes need to be turned off.

The process of Histone modification will alter how DNA is packaged and how genes within that Histone are expressed. The process of DNA methylation controls the promoter of that gene and if that gene gets transcribed or not. Epigenetics is all about understanding how our environment can alter the way our DNA expresses certain genes. It's about how the choices we make can alter the way our DNA expresses certain genes.

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Tumor Heterogeneity

A tumor begins with just one mutation, but as the cells continue to replicate, it will begin to develop more and more mutations. Each generation will have new mutations the generation before it did not have. This is the concept of the progression of cancer. A typical tumor will have at least 6 mutations before it even becomes cancer. There are several attributes we studied in the Hallmarks of Cancer each tumor must acquire before it becomes cancerous and can metastasize. This brings up the concept of Tumor Heterogeneity. The number of different mutations across a tumor can be dozens or even hundreds. Not every cell will have every mutation as each new generation of cells will gain mutations the generation before it did not have. The data even shows that cells that travel to distant parts of the body to create metastasis can evolve differently from the tumor they originated from.

Tumor heterogeneity can be very different across tumors within a single patient, and patients can have very wide tumor heterogeneity from one another. Cancer is a mutation in cells and those mutations can take any form and be completely random. The amount of tumor heterogeneity depends on the tumor type. Blood cancers seem to have less mutations and different antigens than solid tumors. They stem from immune cells which typically have clear antigens to target that are not on other healthy tissues. In B cell cancers, we can target BCMA, CD19 and CD22. These therapies will kill all B cells and have no effect on other healthy tissues. A human can live without B cells with supportive care. That makes it a very easy to treat cancers compared to solid tumors. When it comes to solid tumors, the amount of tumor heterogeneity often depends on the type of tumor; some will have only a handful of different mutations while others will have hundreds of mutations.

There are 3 types of Tumor Heterogeneity we should understand. The first type of tumor heterogeneity which is the amount of different mutations within a single tumor or intratumor heterogeneity. Then there is inter-tumor heterogeneity that can be different mutations between different tumors within the same patient such as a tumor in the Lung and one in the Brain. Then there is inter patient tumor heterogeneity. That is the concept that one patient's tumor will mutate differently from another patient's tumor.

Chemotherapies do so well in cancer because they target rapidly dividing cells regardless of their mutations. That is one of their major benefits despite their many side effects. Targeted therapies will often go after a specific mutation in a tumor, but not all cells in the tumor may have that mutation. The concept of tumor heterogeneity is very important in cancer as it makes it one of the most difficult diseases to treat.

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Immune Surveillance

The immune system has the main role of removing pathogens, but it also has the role of removing harmful or unwanted cells that have mutated. This is because the immune system is the primary system for finding and removing mutated cells that are missed by DNA repair. The concept is called Immune Surveillance. There are cells within the immune system whose job it is to recognize a mutated cell and kill it.

The first is Antigen Presenting Cells. They clean up the dead cells inside a developing tumor. They can process these pieces of mutated proteins into antigens and present them to helper T cells. This provokes an immune response toward that antigen and clears tumor cells. The same T cell response which is designed to kill virally infected cells can be directed toward killing mutated cancer cells. This concept has brought about a whole new focus in biotechnology around redirecting these killer T cells for cancer therapies.

The other cell that is part of immune surveillance is the Natural Killer (NK) cell. These cells come with a group of receptors that are designed to find cells that are under stress. This can be things like proteins that are only supposed to be inside cells suddenly appearing on the surface of a cell. It could be just a lower level of MHC I expression on the cell surface. The NK cell is known as the primary immune surveillance cell. The NK cell has a host of receptors for detecting mutated cells. It is capable of triggering cell death just like cytotoxic T cells.

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Tumor Microenvironment

There is a complex set of interactions that must go on between a tumor and its surroundings. It has to interact with the tissue cells, it needs nutrients, it needs oxygen, it needs to survive and proliferate in a hostile environment. One of the many things it has to achieve to exist and thrive is to overcome the immune system and its natural ability to find, target and destroy tumors. There are 3 key cells in the immune system that are designed to find and kill cells that are infected or defective. They are the Natural Killer (NK) cell, the Cytotoxic T cell, and the Macrophage. .

The NK cells deploy a set of damage receptors called Damage Associated Molecular Patterns (DAMPs) that recognize stressed cells. This can be things like Mic-a or Mic-b and the lack of MHC I. Normal cells will express MHC I on their surface that tells the immune system they are healthy. These MHC I receptors are made up of an alpha chain and a beta chain. Defective cells will often display just 1 chain called MIC-a for the MHC I related alpha chain or MIC-b for the MHC I related beta chain. This is just one of the many ways NK cells can recognize an unhealthy cancer cell.

The Cytotoxic T cells can detect Tumor Associated Antigens (TAA) which are just mutated versions of the normal self proteins. This can cause T cell activation and a cell mediated response. The Cytotoxic T cells will bind to and kill tumor cells that display their antigen. The last cells that are key are the Antigen Presenting Cells (APCs) like the Dendritic cells and Macrophage. The tumor not only competes with the healthy tissue for survival, but the cancer cells will battle each other in a survival of the fittest. There is a lot of cell death going on in the tumor. This naturally allows APCs like the Macrophage to clean up these dead cells and process TAA antigens. They can present these antigens to the T cells to activate a response.

One of the hallmarks of tumor progression is the tumor evolves mechanisms to evade the immune response. There are multiple ways the tumor can evade or redirect the immune system. The most common is the tumor cells will release signals that will induce T regulatory cells and Myeloid Derived Suppressor Cells (MDSC) to protect the tumor by promoting tolerance. These cells will release signals that inactivate the immune cells like T cells, NK cells and APCs. This is one of these hardest effects to overcome in the tumor microenvironment. One effective way to clear all the miss directed regulatory cells is the use of a lymphodepletion regiment like Flu/Cy. This works in cell therapies to clear the battlefield.

The next mechanism the tumor cells will deploy is the expression of suppressive receptors like PD-1, CTLA-4 and CD-47. These will engage with T cells, NK cells and Macrophages and inhibit them from tumor killing. We call them checkpoints as they stop the immune response. This inhibitory response by a cell is designed to protect healthy cells from accidental killing by the immune system. The cancer cells will over express these inhibitory receptors to protect them from the immune system. There is a huge amount of development going on to understand all the interactions between the immune cells and the tumor. There has been some great success with PD-1 and CTLA-4 and a lot of disappointments along the way trying to develop checkpoint inhibitors. Some of the early developing checkpoints are LAG-3, TIM-3, TIGIT and CD47. All of these are natural receptors used by healthy cells to block the immune system from killing them.

The critical role of the immune system in protecting us from cancer has been well established by following patients who are immunosuppressed from cancer treatments or organ transplants. Immunocompromised patients have a significantly higher risk for cancer. Even HIV patients will have higher risk of cancer related to the immune suppression by the virus. Some cancer patients get secondary cancers while their immune systems are suppressed from chemotherapy. The role of the immune system in oncology is now well established. The many interactions that play a role in cancer surviving in a healthy immune system is still developing, but establishing therapies to return the balance back to the immune system can help fight cancer.

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