Biochemistry of Cancer

A research assignment for my Biochemistry of Cancer class, answering some questions on biochemical principles in regards to cancer.​ A rather long read for anyone interested...

1. Discuss the importance of protein kinase inhibitors in cancer therapy.
Protein kinases are a class of enzymes capable of phosphorylating other molecules, enzymes, and proteins, providing regulatory function through phosphorylation. Many regulatory and metabolic pathways in the cell rely on phosphorylation to activate or inhibit (depending on the circumstances) an enzyme and thus phosphorylation can be said to control the progress of metabolites through a given pathway. Their important regulatory function means that mutations in a kinase that change its phosphorylating activity can wreak havoc on the cell’s natural regulation patterns: that increased kinase function will result in overexpression of proteins activated by the kinase and underexpression of proteins inactivated by the kinase. The opposite effects are true if kinase function is decreased, resulting in underexpression of proteins the kinase activates and overexpression of proteins the kinase inhibits.
These unregulated cellular pathways can lead to a wide range of phenotypes, depending on the mutations present in a particular cell, but commonly result in cancer due to abnormal cell expression and proliferation. A single mutation in a cycle-dependent kinase responsible for keeping the cell in the G0 phase, for example, can push the cell past DNA damage checkpoints and into replication prematurely, allowing naturally occurring mutations to be replicated before they can be repaired. This eventually compounds into cellular genomes housing a collection of mutations, which can easily lead to cancerous growth if these mutations are pro-oncogenic, such as preventing apoptosis, encouraging angiogenesis, or helping the cell avoid immune intervention, to name a few possibilities.
Over 120 protein kinases have been connected to oncogenesis, which can result in tumor cell proliferation, genomic instability, tumor angiogenesis, and increased survival of cancerous cells, providing selective growth advantages to them. The sheer amount of different kinases and their applications as well as the number of possible mutations and their cellular effects mean that abnormal protein kinases can make cancers with a wide variety of behaviors. This would make kinase inhibition practically impossible to achieve, however, kinases maintain similar structural characteristics, especially in their ATP binding pocket (the active site of the enzyme) and the surrounding hydrophobic areas on the surface of the enzyme. Because of this, a successful kinase inhibitor would not need to have perfect specificity, and can theoretically be used against many cancer types, as well as interfere with compensatory signaling pathways which could lead to drug resistance and eventually render inhibitors non-functional. The hydrophobic areas surrounding the active site of a kinase means that any inhibitor would not need to function as a competitive inhibitor for ATP (which would be very difficult due to the high concentration of ATP in the cell) as long as it has an affinity for the surrounding hydrophobic areas in addition to binding to the active site. Protein kinase inhibitors provide a large number of potential targets in the sense that they allow for the avoidance of a lot of variety and specificity found in pathway levels downstream of kinase intervention, which can yield a strong clinical response with much lower levels of toxicity than traditionally used chemotherapy drugs and similar therapeutics because they would not affect healthy cells. Functional protein kinase inhibitors would allow for the reduction of overactive protein kinases and therefore reduce the amount of cancer-like behavior exhibited by affected cells, without adversely affecting other tissues in the body that exhibit normal protein kinase expression, which is ideal for a therapeutic.

2. Explain how genomic profiling can help improve cancer therapy.
Genomic profiling utilizes next-generation sequencing techniques to identify all mutations present in a cell’s genome, which can be used to identify all the mutations within a cancerous tumor to determine its behavior (Loberg, 2022). Once a cancer has been profiled, that information provides an in-depth understanding of a person's cancer’s specific mutations. It therefore helps in the selection of the most appropriate and effective therapeutics available to the patient and their medical team. Being able to understand the underlying mutations and behaviors of cancer immediately (as opposed to gaining this understanding through a trial-and-error-based approach) can reduce the time between cancer detection and successful treatment, as well as allow for very directed treatments that are effective on the cancerous cells while dealing the least amount of damage to the patient’s healthy cells and tissues.
An example close to my heart of the potential benefits of genomic profiling is that of my grandfather, who was diagnosed with leukemia in the late 1990s and went through years of (various) treatments available at the time, with variable success. He was by no means ‘getting better’ but the progression of his cancer slowed dramatically, and he was able to live another six years past doctors’ initial prognoses because of these treatments. What eventually killed him was not the leukemia, it was septic shock in response to an aggressive new treatment that his body was simply too weak to handle, despite it showing promise in fighting his cancer.
Today, a man in the same position my grandfather was in back then not only has countless more therapeutic options at the disposal of his doctors but also the ability for them to quickly and efficiently determine which of these therapeutics is best for him by utilizing genomic profiling. Finding the right treatment quicker gives cancer less time to progress and mutate, making it easier to fight as well as ensuring the patient is in a healthier position to fight it, and therefore more able to withstand the treatment, resulting in better outcomes and shorter treatment times. It also helps to minimize the number of detrimental side effects–ranging from more short-term effects such as nausea to long-term heart problems or increased risk of secondary cancers (Mayo Clinic Staff, 2022)–patients must worry about in addition to their cancer.

3. Describe the adaptive immune response in cancer.
Adaptive immune response is the term used to describe the process by which a cancer changes its phenotype in response to the body’s immune response against it (Ribas, 2015). In other words, the cancer adapts and expresses immunity against the natural defenses meant to destroy it. These natural defenses come in the form of T-cells, which initiate, maintain, and regulate immune responses throughout the body. The different types of T-cells (cytotoxic/CD8+, helper, and regulatory) control how immune responses are delivered. Helper T-cells are responsible for extracellular signaling that coordinates other immune cells, cytotoxic T-cells are those that are capable of destroying tumor cells, and regulatory T-cells can reduce the activity of other T-cells (Cleveland Clinic, n.d.).
In adaptive immune response examples, the cancer cells are recognized by T-cells, but the cancer can inhibit the effect of the T-cells and protect themselves by taking over the mechanisms employed and limiting the inflammatory and immune response (Ribas, 2015). Adaptive immune resistance can emerge as the result of cancer cells continuing to accumulate mutations and developing a variety of phenotypes within a single tumor (Kim, 2022), which makes it harder for T-cells to target all cancerous cells present in a tissue. Cancers can develop a variety of resistance mechanisms, so being able to identify the specific means of resistance present can allow for targeted immunotherapy to be designed and employed to inhibit the inhibition of immune response, theoretically restoring normal immune function to the T-cells in regards to the cancer cells.

4. What is meant by “adoptive” cancer therapy? What are the positive and negative aspects of this type of therapy?
There are three types of adoptive cancer therapy–In vitro-activated and expanded cancer antigen-specific host T-cell populations, chimeric antigen receptor-modified T-cells (CAR T-cells), and allogeneic bone marrow transfer. The specifics of each method are different, but they involve the same basic concept: utilizing T-cells to attack cancer. 
Allogeneic bone marrow transfer is the most colloquially known of the three methods and involves the replacement of the recipient's (the cancer patient’s) hematopoietic system with that of a donor. The donor is specifically selected to have a different type of hematopoietic system than the patient, which has a robust effect on hematologic cancers and is usually very successful in this regard. However, there is a very high risk of Graft-Versus Leukemia Disease (GVLD) which results when the patient (the recipient)’s body recognizes the donor tissue as foreign and mounts a potentially life-threatening immune response against it. GVLD makes the theoretically simple procedure very challenging because of the need for immunosuppressive therapies to minimize the chance of GVLD, which must be balanced to still allow for the anti-cancer activity provided by the donor tissue.
In vitro-activated and expanded cancer antigen-specific host T-cell populations and chimeric antigen receptor-modified T-cells (CAR T-cells) both involve the introduction of T-cells into the patient’s body to fight cancer. In the first method, a patient’s T-cells are collected and selected or modified to isolate those that are reactive to the patient’s cancer. These chosen T-cells are then allowed to grow to a large population before being reintroduced into the patient’s tumor. This treatment has no adverse immune response where the body may attack the re-introduced cells because they are identified as originating in the patient.
CAR T-cells, on the other hand, are products of genetic engineering, where endogenous T-cells are reinfused into the patient after being modified for antigen specificity and anti-cancer activity by selecting an antigen present on the surface of the cancer cells and then creating a monoclonal antibody for that antigen, or using recombinant DNA techniques to clone genes that specify for antigen binding domains. This allows them to immediately recognize the target and attack the cancer, as opposed to natural T-cells which require multiple types of receptor-ligand interactions to function against the cancer. The greatest downside of both these techniques is the need for chemotherapy treatment between the harvesting of the T-cells and the infusion of the selected T-cells to ensure that those T-cells that are present can work against cancer. Another negative is that these techniques require the tumor to be located in an easily accessible location that physically allows for the harvesting and reintroduction of T-cells. This is even though the type of tissue theoretically has no limiting effect on the technique, it simply must be accessible to enable the physical interaction between the T-cells and the cancer.

5. What are the two types of vaccinations that are used in cancer therapy? What immune responses do they elicit and how are they effective in cancer treatment?
Prophylactic vaccines can prevent cancers resulting from infection with microbes that have been identified as either directly or indirectly oncogenic. There are two very effective prophylactic vaccines: for human papillomavirus (HPV) which is a major cause of cervical cancer and hepatitis B virus (HBV) which is an indirect (but major contributing) factor in liver cancer development. Many other microbes and viruses have been identified as having some sort of connection to a variety of cancers, which is the first step in developing vaccines to protect against them. These are ‘traditional’ vaccines similar to those for other non-cancer-causing viruses, meaning that despite the challenge of developing a vaccine, the fact that it is a cancer-linked virus should not make it more difficult than other viruses for which vaccines have been developed.
Therapeutic cancer vaccines are slightly different. They are much more difficult to develop because they involve taking blood mononuclear cells from the patient and altering them to initiate an immune response in the patient. Effectively, these must be made on a case-by-case basis, although they follow the same procedural formula. This is achieved by culturing the mononuclear cells with chimeric protein that codes for a tumor-associated antigen which will target the cancerous cells as well as cells in the surrounding tissue that exhibit the associated antigen. A particularly successful therapeutic vaccine has been developed for metastatic castration-resistant prostate cancer called sipuleucel-t. The main challenge of this type of vaccine is that it is difficult to induce an immune attack that is both strong and durable enough to result in sustained remission or elimination of the cancer it is targetting.

6. Compare and contrast the therapies presented for metastatic melanoma in the selected readings. Why might one therapy be selected over the other?
A handful of promising therapies for metastatic melanoma were presented in the selected readings, namely cytokine treatments, immune checkpoint inhibitors (in the form of antibodies), and immunotherapy using a patient’s T-cells. The consensus seems to be that a combined approach is the most effective in treating metastatic melanoma, starting with the excision of any initial cancerous growths present. This allows for the removal of a majority of the cancerous cells (in terms of sheer number) enabling additional therapies to target metastatic cells and new tumor foci.
Spontaneous remission in metastatic melanoma has been described, likely involving anti-tumor immune responses, which may be why immune-based treatments have been deemed successful in targeting metastatic melanoma. Treatment with cytokines, such as IL-2, is capable of inducing the proliferation of T-cells and NK cells (natural killer cells, another type of lymphocyte), as well as lysing autologous tumor cells in vitro. IL-2 specifically showed a low effectiveness rate, and the high doses that were required resulted in dramatic side effects–system toxicity, hemodynamic instability, and high risk of infection–but the drug was approved by the FDA because of the lack of more promising alternatives at the time. Now, administration of IL-2 is highly limited because of the advent of more effective and safer therapies.
Immune checkpoint inhibitors are antibodies capable of enhancing the body’s anti-tumor immune response by blocking inhibitory immune checkpoints. Some, such as ipilimumab, improved overall survival rates but were capable of causing significant autoimmune toxicity, while newer antibodies, such as PD-1 and PD-L1, maintain the improved survival rates of ipilimumab treatment while showing decreased toxicity, likely because their target interactions occur in peripheral tissues which allow them to disperse throughout the body.
    Immunotherapy involving the transfer of selected or otherwise altered T-cells from the patient has shown efficacy against melanoma, but not metastatic melanoma, likely because this treatment requires the reintroduction of T-cells into the tumor directly, which is not feasible in metastatic cancer. On the whole, newer treatments show higher efficacy in treating metastatic melanoma than their predecessors while avoiding many of the extreme side effects.

​7. Describe the process of apoptosis.
Apoptosis can also be called ‘programmed cell death’ and refers to a set of pathways naturally occurring in the cell which, when certain criteria are met, are activated and result in cell death. Apoptosis is a completely natural and commonplace process, occurring all over the body millions of times each day (D’Arcy, 2019). It can be triggered either by the cell itself (intrinsic) or by intracellular signaling from nearby cells in the tissue or immune cells (extrinsic). There is a large variability in the stimuli and conditions that trigger and can result in apoptosis, meaning it is important to note that not one single stimulus can work to induce apoptosis in all applications (Elmore, 2007).
The general pathway apoptosis initiation follows relies on the activation of cysteine-aspartic proteases called caspases (D’Arcy, 2019). Upon the detection of cell damage, initiator caspases are activated, which then go on to activate executioner caspases which can amplify the signal by initiating several metabolic pathways to fragment DNA, destroy intercellular structural proteins, stop ligand expression, and eventually form apoptotic bodies which can be taken up by other cells to reuse the materials encased within (D’Arcy, 2019). The type and origin of the initial stimulus signaling apoptosis determines which of the many initiator caspases is activated (to name two examples, caspase 8 is activated by an extrinsic pathway of cell signaling, and caspase 9 is activated by a cytotoxic stimulus), but all initiator caspases activate executioner caspases which converge into one standard execution pathway (D’Arcy, 2019).
Cells have systems of intracellular sensors that allow them to detect positive and negative signaling pathways to trigger intrinsic apoptosis: positive signaling directly triggers the process, and negative signaling–the absence of a ‘pro-survival’ signal–allows pro-apoptotic molecules to activate and initiate apoptosis.
Necrosis (sometimes called oncosis) is another process of cell death that can coexist with apoptosis and sometimes be initiated by the same stimuli, though it is most commonly associated with a shock of stimuli such as high heat or radiation exposure (Elmore, 2007). If apoptosis is ‘programmed’ death, resulting in the organized repackaging and reallocation of cell materials and resources without collateral tissue damage or inflammation, necrosis is ‘uncontrolled’ death, resulting in the disorganized spillage of cell materials into the intracellular space, which can potentially cause a domino effect of damage spread through tissues (D’Arcy, 2019). While the two processes result in cell death, apoptosis is a more preferred, controlled method.

8. How can targeting apoptosis be used as a therapy for cancer?
Degenerative diseases can be characterized as having too much caspase activation–and subsequently too much apoptosis of healthy cells–which should be avoided, however too little apoptosis results in uncontrolled cell growth and proliferation resulting in cancer (D’Arcy, 2019). Death receptors, membrane proteins to which death ligands from immune cells bind–triggering apoptosis extrinsically–are in the tumor necrosis factor (TNF) superfamily (D’Arcy, 2019), which, as their name suggests, play an important role in tumor destruction (National Institute of Health, 2018). When immune cells identify cellular abnormalities or antigens suggesting the presence of cancer, they are meant to release death ligands that bind to the TNF death receptors and induce apoptosis, and failure to do so results in further proliferation and potential mutation of the cancer.
Many oncogenes–such as BCL-2, BCL-XL, and BCL-W (D’Arcy, 2019)–function as apoptosis inhibitors, preventing extrinsic apoptosis signaling from immune cells at some stage of the pathway (recognition by the immune cells, production of death ligands, blocking of the signal from death receptors, etc) so external influence in the form of therapeutics targeting apoptosis would allow for the reduction of cancerous growth by returning apoptosis levels to normal. Targeting apoptosis can take two forms: inhibiting apoptosis inhibitor genes such as those listed above, or activating apoptosis directly, both of which would theoretically result in increased apoptosis levels.

9. A researcher tells you that they are testing a compound for being anti-apoptotic. Outline 3 experiments that you think they should do to prove the compound is anti-apoptotic.
Anti-apoptotic compounds should be capable of preventing caspase cascade initiation, as caspases are the compounds responsible for triggering the apoptosis pathway. There are three types of caspase initiation mechanisms–the extrinsic death receptor pathway, and the intrinsic mitochondrial and endoplasmic reticular pathways–so an anti-apoptotic compound should be able to inhibit at least one of these mechanisms, or at the very least one of the accessory methods of triggering apoptosis which has been identified (O’Brien, 2008).
A common means by which anti-apoptotic compounds function is by expressing BCL-2, BCL-XL, or BCL-W enzymes, which inhibit apoptosis (D’Arcy, 2019) by preventing extrinsic signaling from reaching target cells. One experiment to prove the compound is anti-apoptotic would involve testing for the expression of these enzymes before and after exposure to the compound. An increase in expression would indicate the compound is anti-apoptotic.
Alternatively, an anti-apoptotic compound may inhibit or dismantle caspases, preventing their activation which triggers the processes by which the cell is deconstructed. Caspases, specifically caspase-3, are the last step in the signaling pathway that leads to the triggering of apoptosis in most cells (Brenner, 2009). This type of activity can be explored by isolating caspases and determining the compound's effects on them, or harvesting caspases from treated and untreated cells (which should be otherwise identical) and analyzing any changes.
BH3 proteins have been identified as isolating BCL-2 and similar anti-apoptotic enzymes and releasing pro-apoptotic BAX and BAK proteins (Brenner, 2009). Consequently, BH3 proteins can be said to have pro-apoptotic effects. An anti-apoptotic compound, therefore, may also exhibit behavior involving the inhibition of BH3 protein expression, either inactivating BH3 proteins or preventing their transcription. This can be tested by monitoring cell expression of BH3 proteins/protein products before and after exposure to the compound.

10. Discuss the mutations/genetic changes that need to occur for a cell to become metastatic.
For a cell to become metastatic, it must first become cancerous. A series of gene mutations must arise which lead to a loss of normal growth control. This can include mutations allowing for increased cellular proliferation, decreased DNA proofreading, an immunity to intrinsic and extrinsic apoptosis triggering pathways, decreased apoptosis, increased angiogenesis, and the ability to dedifferentiate and redifferentiate. When normal growth control is lost, a contained mass of cancerous cells forms, referred to as initiation.
The next step for an ‘initiated’ cancerous growth on the path to metastasis is called promotion, referring to the presence of alterations in cell signaling pathways changing the cell-environment interactions. While initiation is focused on what is happening inside the cell, promotion refers to changes that allow the cancerous cell to change its attachments to neighboring cells and the extracellular matrix and break off the mass, losing adhesion with the initial tumor. Additionally, cancerous cells can begin to produce–or recruit neighboring cells to produce–enzymes that can allow them to break through the membranes that delineate cancerous cells from the surrounding tissue, allowing them to migrate out of the initial tumor.
Further genetic damage (karyotypic instability increases dramatically at this stage) makes the stage of progression as the newly liberated cancerous cells become capable of intravasation and can enter the bloodstream or lymphatic systems, providing relatively easy access to the entire body. The number of cells that can enter the bloodstream and the places they settle and perform extravasation (exiting the bloodstream or lymphatic system) determines where and how many new tumor foci may form. At this point, the cancer has reached metastasis and can continue to spread from the initial tumor site or any of the new foci.

11. Why is angiogenesis important for metastasis? How can angiogenesis inhibitors help in cancer therapy?
All metabolically active tissues in the body require close proximity to blood capillaries in order to participate in the nutrient and metabolite exchange that is necessary for function. This means that throughout one’s lifetime, existing blood vessel vasculature must be able to grow and change, which is achieved by a process called angiogenesis. Changes in metabolic activity naturally trigger angiogenesis, and subsequently cause changes in the capillary of a tissue (Adair, 2010). This involves both cell-to-cell and cell-extracellular matrix interactions, which are controlled by the presence of growth factors and other types of signaling molecules released by cells and identified by blood vasculature cells.
Cancerous growths accumulate a large sum of mutations that disrupt normal cellular behavior and function, but in order to metastasize these abnormal cells must be able to migrate into other tissues and perform intravasation by infiltrating blood vessel vasculature (or alternatively the lymphatic system). This allows cells to be transported throughout the body and form new tumor foci after extravasation into novel tissues. The increased proliferation of cancerous cells and the growth signals they emit can trigger angiogenesis, leading to blood vessel vasculature forming new offshoots that grow more and more closely to the tumor. This makes nutrient transport to the cancerous cells more effective and makes it even easier for the cells to continue their uncontrolled growth, creating a positive feedback loop in which the cancer grows and the vasculature gets closer to accommodate this growth, making it increasingly easier for the cancer to perform intravasation.
This is why tumor growth is considered angiogenesis-dependent (Adair, 2010). Therapies that control angiogenesis (specifically those that inhibit it, in the interest of cancer therapeutics) would need to target endothelial or pericyte cells, which line the capillaries, lymphatic cells that make up the lymphatic system, fibroblasts, which synthesize the extracellular matrix, or other cells which are directly involved in vasculature structure or growth signaling. Reducing angiogenesis can greatly reduce (if not entirely prevent) metastasis.

12. How could you target metastasis for cancer therapy?
Metastasis into new tumor foci accounts for 90% of lethal cancer outcomes, meaning it is an important research target. Metastasis-promoting genes help cells from initial tumors disperse into other tissues by assisting with intravasation and extravasation and improving cells’ resistance to the body’s immune response and other metabolic stressors, or otherwise assist new tumor foci cells in adapting to their new host tissue environments (Ganesh, 2021). Research has also identified concordant mutational patterns present in primary and metastatic cancers, and associated some acquired from epigenetic and transcriptional changes as metastasis-associated mutations (Ganesh, 2021). Therapies designed to inhibit these genes and mutational patterns could show promise in preventing further metastasis or stopping the development of metastasis in cancer.
Metastasis-initiating cells (MICs) are those cells capable of initiating new tumor foci, and show high levels of plasticity, allowing them to traverse vascular membranes and adapt to new tissue environments in part because of performing EMT/MET. Epithelial-mesenchymal transition (EMT) is when an epithelial cell initially loses its extracellular adhesions and is enabled to move through tissues until mesenchymal-epithelial transition (MET), the reverse process, occurs and metastatic growth can be initiated. EMT is caused by the repression of epithelial genes at the transcriptional level (Ganesh, 2021), so therapeutics that either inhibit these repressors or otherwise maintain activation of epithelial genes would be able to prevent EMT and therefore MET.
Most therapeutic efforts thus far have been focused on preventing the colonization of new tissues and the establishment of new tumor foci, because it is difficult to create an effective therapeutic that can stop cancerous cells from entering and traveling through the circulatory system (Ganesh, 2021). It is also incredibly difficult to trial new drugs meant to directly target metastasis unless it is in patients with no other viable options available to them, due to ethical implications as well as financial limitations and the unpredictable nature of metastasis formation (Ganesh, 2021). These reasons are why chemotherapy remains the main treatment for metastasis treatment, although targeted therapy approaches are becoming more prevalent.

Works Cited
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