Biochemical Foundations of Pharmaceutical Biotechnology: Normal vs. Cancer Cells PDF

Biochemical foundations of pharmaceutical biotechnology Cancer: Normal cells vs. Cancer cells Audrey Minden, Ph.D. Department of Chemical Biology Ernest Mario School of Pharmacy Susan Lehman Cullman Laboratory for Cancer Research, Room 205 (848) 445-5766 [email protected] Normal vs can...

Biochemical foundations of pharmaceutical biotechnology Cancer: Normal cells vs. Cancer cells Audrey Minden, Ph.D. Department of Chemical Biology Ernest Mario School of Pharmacy Susan Lehman Cullman Laboratory for Cancer Research, Room 205 (848) 445-5766 [email protected] Normal vs cancer cells: Outline of topics I. Some basic differences between cancer cells and normal cells A. Overview of some of the characteristics that differentiate cancer cells from normal cells B. Some of the drugs used to target cancer cells based on these characteristics II. Classification of cancers III. Cancer development IV. Tumor progression V. Stem cells and Cancer stem cells A. Overview of stem cells in normal tissue B. Evidence for cancer stem cells C. The problems cancer stem cells create for treating cancer 2 Some basic differences between normal and cancer cells 1. Altered homeostasis: cells grow and divide at a faster rate than they die a. Increased cell proliferation b. Decreased cell apoptosis c. Loss of contact inhibition d. Anchorage independent growth 2. Changes in cell differentiation 3. Altered cellular metabolism 4. DNA repair abnormalities 5. Angiogenesis 6. Alteration of tumor microenvironment to one that encourages cell survival 7. Immune response changes 8. Metastasis: Cells migrate and invade surrounding tissues 9. Changes in cell shape and motility 10. Altered responses to growth factors Cells that have characteristics of cancer cells are often referred to as transformed cells 3 I. Some basic differences between normal and cancer cells 4 1. Altered homeostasis Altered homeostasis In cancer, cells may grow and divide at a faster rate than they die. This can involve increased cell proliferation, or decreased apoptosis (Figure from Alberts) 5 1a. Increased or unregulated cell proliferation Growth Control: In normal cells, cell growth and division are under strict control Cells only divide when the body needs more cells. In contrast, cancer cells can grow and divide uncontrollably, even when the body does not need more cells. (National Human Genome Research Institute, 2023) 6 1a. Increased or unregulated cell proliferation One way increased proliferation can occur is by evading the process of replicative senescence Replicative senescence: Many normal cells have a limit to how many times they can divide. Human fibroblasts, for ex, stop dividing after 25-50 divisions Replicative senescence is related to the shortening of telomeres. Telomerase is an enzyme that maintains telomeres. Many cells are deficient in telomerase. Thus, in most cells telomeres constantly shorten at every S phase and cells eventually stop replication. Cancer cells can avoid replicative senescence by 1 of 2 ways: a. They re-activate telomerase gene so that telomeres don’t shorten (this is the most common mechanism), or b. Alternative Lengthening of Telomeres (ALT): a DNA repair mechanism that elongates telomeres The result is that the cancer cells may continue to proliferate while normal cells would have stopped 7 1b. Decreased apoptosis Apoptosis: Normal cells undergo apoptosis when they become old or damaged. Too much or too little apoptosis can lead to disease. Cancer cells often evade apoptosis and continue to grow even in the presence of apoptotic signals. (National Human Genome Research Institute, 2023) 8 1b. Decreased apoptosis Even in the absence of apoptosis, cancer cell still experience death by necrosis. This can happen in the interior of a tumor, where cells are not exposed to sufficient oxygen and nutrients (Figure from Alberts) 9 1c. Loss of contact Inhibition Normal cells have contact inhibition: They stop dividing when they come into contact with other cells. Cancer cells often lack this inhibition and can pile up on top of each other to form tumors. 10 1d. Anchorage independent growth: Loss of the requirement for adhesion to a surface Most normal cells need to adhere to a surface in order to survive. For example, normal cells of the gut epithelium constantly turn over but once they are detached from the matrix, they die by apoptosis This is an important protection against cancer. Some cancer cells lose this inhibition, and can continue to grow even when they are not associated with a surface They no longer require all of the positive signals from their surroundings, that normal cells require. 11 1d. Anchorage independent growth: Loss of the requirement for adhesion to a surface Anchorage independent growth can also be observed in vitro. In vitro, normal cells must be attached to a surface, such as a culture dish (A), but will not grow in a solution where there is no substrate for them to bind to (B). This can include growth on soft agar, which is often used as a test for anchorage independent growth. A B (Figures reated with BioRender.com) 12 2. Changes in cell differentiation Cell Differentiation: Normal cells are differentiated (carry out specific functions). In contrast, cancer cells are often undifferentiated or poorly differentiated. 13 3. Altered cellular metabolism Tumors grow rapidly and have a high requirement for nutrients Their nutritional requirements are more like those of a growing embryo rather than an adult cell They consume a large amount of glucose They import glucose from the blood at rates up to 100 fold higher than normal cells The glucose is also not metabolized the same way as normal cells metabolize it Instead of using oxidative phosphorylation, which is more efficient at ATP generation, most of the glucose is metabolized by glycolysis. This leads to rapid production of new building blocks of the cells such as proteins and amino acids This Is called the Warburg effect 14 4. DNA repair abnormalities Normal cells have mechanisms to repair DNA damage. Cancer cells often have mutations that disrupt these mechanisms, leading to genetic instability and further mutations. 15 5. Angiogenesis Angiogenesis: The formation of blood vessels. Normal cells do not generally promote the growth of new blood vessels. In contrast, cancer cells can stimulate the growth of new blood vessels, which helps them obtain nutrients and oxygen from the blood supply. 16 6. Tumor microenvironment Tumor microenvironment (TME): This refers to the complex ecosystem of cells and molecules that surround the tumor, including the cancer cells themselves, the stroma, and immune cells. The TME plays an important role in tumor growth and responses to treatment The stroma can include cells such as fibroblasts (in the connective tissue supporting the tumor), immune cells, and vascular cells 17 6. Tumor microenvironment The tumor depends on communication and cooperation with the stroma As with some normal tissue, the stroma initially has fibroblasts and inflammatory white blood cells and endothelial cells that make up vessels, and smooth muscle cells As a tumor progresses, the cancer cells change this environment. They do so by secreting signaling proteins that change the characteristics of the stroma cells, and sometimes recruit or avoid certain cells. They also secrete proteolytic enzymes that modify the Extracellular Matrix (ECM) in the stroma In response, the stromal cells secrete signaling proteins that stimulate the cancer cells to divide, and proteases that further remodel the ECM Thus, tumor and stroma evolve together 18 7. Immune response changes Normal cells are recognized by the immune system as self and are not attacked. Sometimes cancer cells are recognized as foreign by the immune system and destroyed, but other times cancer cells can evade the immune system's detection, allowing them to continue to grow and divide. 19 7. Immune response changes Tumor cells can manipulate the immune system cells found in the stroma: Some tumor cells can block the activation of immune cells (WBCs) that could kill them. Cancer treatments are being developed to block this inhibition. 20 7. Immune response changes Immune Response and cancer treatment: PD-1 and other related receptors can sometimes be referred to as checkpoint molecules. Their normal role may be to help maintain a balance between the ability of the immune system to attack invaders, and the avoidance of attacking self cells. Cancer cells may exploit this system, by expressing proteins such as PD-L1, which shut down the immune response from an immune cell. (Figure from Caldwell, C. et al. 2017) 21 Drugs that block PD-1 or PD-L1 The immunotherapy drugs below are immune checkpoint inhibitors Keytruda (pembrolizumab): A PD-1 inhibitor. It binds to PD-1. It is approved for the treatment of melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, classical Hodgkin lymphoma, and others. Other drugs may block PD-L1. Drugs that block either PD-1 or PD-L1 are considered immune checkpoint inhibitors 22 8. metastasis Metastasis is a multistep process: - Cancer cells first invade local tissues and vessels - Cells then circulate through vessels - Cells eventually leave the vessels - Cells stablish new colonies at distant sites 23 8. metastasis Invasiveness is an important first step. The cell develops ragged borders and grows in a disorganized way. The cell disrupts the of adhesive mechanisms that normally keeps it tethered to a surface. The cell can then penetrate blood or lymphatic vessels and travel to distant sites. Establishment of distant colonies. This can occur when the cells penetrate blood or lymphatic vessels again, leaving the vessels and establishing new colonies. Even after these events occur, only a small percentage of the cancer cells succeed in establishing new colonies. Circulating Tumor Cells: CTC. Some techniques can detect CTCs in blood samples for diagnostic purposes. “CellSearch Circulating Tumor test” is the first and only (as of 2023) FDA approved test for use in the clinic. It is used to identify small amounts of circulating tumor cells in patients who have had certain types of cancer, to predict whether there is cancer remaining in the body. 24 8. metastasis Scientists have learned more about metastasis from mouse studies : These studies show that some cells die immediately once entering vessel or foreign tissue Others survive entry into foreign tissue but fail to proliferate Others divide a few times then stop: These are micrometastases Only very few establish full blown metastases: fewer than one in thousands or a million. The final step in colonization seems the hardest. Cells may fail to survive, or survive for only a short time. Thus only a very small portion of cancer cells are able to survive the process of full metastasis 25 9. Changes in cell shape and motility Cancer cells often change their shape They may become more rounded They may change into a shape that allows them to be more mobile, which can result in transit through the bloodstream and metastasis 10. Altered responses to growth factors Cancer cells may change their responses to growth factors They may overexpress growth factor receptors They may have mutated growth factor receptors that no longer require a stimulus to be activated In the lab they can often grow in low growth factor conditions, where normal cells would not be able to survive 26 II. Classification of cancers 27 Classification of cancers: One way to classify cancers is according to the tissues they came from: Cancers that derive from epithelial cells: Carcinomas: These are the most common type of cancer, about 85% of cancers This is likely because the cells in these tissues are highly proliferative. Most normal cell proliferation happens in the epithelia, More proliferation means more of a chance for developing mutations. Cancers that derive from white blood cells: Myeloma, Leukemia, Lymphoma Cancers that develop from connective tissue or muscle cells: Sarcomas Some cancers develop from the cells of the nervous system: Examples are Medulloblastoma and Gliomas, 28 Classification of cancers: Some types of tumors are often benign: One example is Adenoma: Adenomas derive from glandular tissue and have a glandular structure. They are most often benign, but can potentially become malignant. 29 Cancer cells often have characteristics that reflect their origins Basal cell carcinomas come from a keratinocyte cells in the skin. They usually continue to synthesize the types of proteins normally made in these cells Melanomas are derived from pigment cells of the skin, and will often continue to make pigment granules Cancer cells with different origins may have different characteristics and outcomes: For example, Basal cell carcinomas of the skin rarely metastasize, while melanomas, also a skin cancer, are more likely to metastasize. . 30 Benign vs malignant tumor cells: Cancer cells can do the following: 1. They can reproduce without the normal restraints 2. They invade and colonize other tissues A tumor made up of cells that are incapable of invading and colonizing is usually considered benign. 31 III. Cancer Development 32 Development of cancer, starting with a single cell 33 Cancer is a microevolutionary process Cancer can start from a single cell, as a result of DNA mutations The human body has trillions of cells DNA mutations occur normally, in billions of these cells each day. Some mutations may cause a selective advantage, which can be dangerous A single cell with such a mutation can become a founder of a clonal colony of mutant cells This gives rise to the primary tumor. 34 Cancer is a microevolutionary process Cancer can start from a single cell, as a result of DNA mutations As the cells derived from this single cell grow, repeated rounds of mutations can take place in the the cells within the clone If the cells continue to have a growth advantage, this clone of cells can propagate at the expense of its neighbors By the time it is detected a tumor often has a billion or more cells, In addition to primary cells, other cells are associated with the tumor: These are the cells of the tumor stroma 35 The clonal origin of cancer cells Genetic studies have been done to validate the clonal origin hypothesis Cells within a tumor usually have certain identical genetic abnormalities This supports the clonal origin for the cells in the tumor. 36 Genetic mutations and carcinogens Somatic mutations are a primary cause of cancer Thus, agents that can cause genetic mutations can be carcinogens Carcinogenesis is thus linked to mutagenesis. Types of carcinogens: Chemical carcinogens can cause simple changes to DNA Radiation such as x-rays, can cause chromosomal breaks and translocation, UV light can also cause mutations in DNA Germ line mutations (inherited mutations) also cause some types of cancer, approximately 5-10% of cancers. . 37 IV. Tumor progression 38 Tumor progression: Over time tumor cells accumulate genetic mutations and become more invasive. (Figure by Alberts) 39 Multiple mutations may be required for full progression of cancer cells Mutations occur naturally during cell replication, even without mutagens Given this high rate of mutation, why is cancer not even more common than it is? The risk of cancer increases with age, most likely due to an accumulation of genetic mutations. Even after exposure to carcinogenic agents, cancer can take years to develop, also suggesting that an accumulation of mutations is required for development of cancer. 40 Tumor progression Tumor progression occurs over time, during which new mutations have a chance to develop: A cell develops a selective advantage and grows more rapidly than its neighboring cells. As these cells progress, some cells acquire additional mutations that give them an even better advantage The larger the number of replicating cells, the better chance for the development of mutations with even better selective advantages Thus as the tumor grows, progression accelerates Over time, the original cancer cell and its offspring can diversify even more, to give rise to many genetically different subclones of cells 41 Sometimes cancer cells can evolve quickly Changes in the DNA in tumor cells compared to normal cells can be large (as seen in a karyotype), or can be small changes that can only be seen by sequencing analysis Changes can be large changes which can be seen in a karyotype, or they can be smaller genetic changes 42 Genetic changes that can be seen at the level of the karyotype Chromosomal changes in colon cancer cells A) The karyotype of a typical cancer shows many gross abnormalities in chromosome number and structure. B) The karyotype of a tumor that has few chromosomal anomalies (Figure by Alberts) 43 V. Stem cells and cancer stem cells • • • • Normal functions of stem cells Stem cells in cancer Cancer stem cells The problem with cancer stem cells 44 Normal functions of stem cells (brief summary) 45 Stem Cells When a normal stem cell divides, each daughter cell has a choice, it can remain a stem cell or it can commit to a pathway leading to differentiation. Stem cell Stem cell Differentiated cell 46 Stem Cells specialized cells 47 The role for stem cells in normal adult tissue: Adult stem cells For almost all proliferating tissues, such as the skin, renewal depends on adult stem cells. These stem cells give rise to populations of cells that are at various states of differentiation: Different layers in the skin: Adult stem cells are found at the basal layer 48 The role for stem cells in normal adult tissue: Adult stem cells Differentiated cells Stem cells are fewer in number than differentiated cells. However, the stem cells have the advantage because they maintain the ability to proliferate, while the differentiated cells have little or no capacity to differentiate. The balance between these two different types of cells is tightly controlled. The stem cells remain dormant until they are needed for renewal, while the differentiated cells do not regain the ability to proliferate. Stem cells (clinicares.com) 49 Adult stem cells and TACs In the case of some adult stem cells, a committed daughter undergoes some rounds of replication before differentiation. The resulting cells are called transit amplifying cells, or transient amplifying cells (TACs). The TACs can differentiate when called upon, and thus replace dead or damaged cells. (Figure by Alberts) (TACs) 50 Stem cells in cancer 51 Evidence for the role for stem cells in cancer: Cancers are most common in self renewing tissues The majority of cancers are in self renewing tissues such as the epidermis, epithelial lining of digestive system, bone marrow, or reproductive tracts. These are the types of tissues most likely to contain adult stem cells. This suggests that adult stem cells may contribute to tumorigenesis 52 Disruption of the normal balance between stem cells and differentiated cells in cancer Differentiated cells In cancer tissue: During cancer, mutations can subvert the normal balance between proliferative cells and differentiated cells. Mutations can lead to over-proliferation of the proliferative cells, or a decrease in the differentiated cells. Stem cells (clinicares.com) Mutations may cause the stem cells to proliferate even in the absence of a signal, or may inhibit differentiation of their daughter cells. 53 Cancer stem cells Evidence for the existence of cancer stem cells 54 Stem cells and cancer stem cells During cancer, mutations may alter proliferation of adult stem cell and Transit Amplifying Cells (TACs) process in various ways. For example, mutations can lead to over-proliferation of the stem cells or the TACs Or, they can lead to inhibition of terminal differentiation of the daughter cells, including the TACs Some mutations can even lead to the generation of new stem cells These cells – cancer stem cells - are capable of high levels of self renewal. 55 Stem cells in a normal population and in cancer Evidence for cancer stem cells Experiments were done where individual cells from a tumor were tested for their ability to give rise to new tumors when implanted into a mouse. There was less than a 1% chance that a tumor cell would give rise to new tumor in the recipient mouse. There was a subpopulation of cancer cells that were much more likely to form tumors when implanted. The cells in this subpopulation express markers that are typically found on the surface of stem cells. (Figure by Alberts) 56 The problem of cancer stem cells The existence of cancer stem cells is concerning because treatments that eliminate most of the tumor cells may still allow survival of the cancer stem cells, as they are often the more slowly dividing cells. This can lead to treatments that seem to be successful at first, only to have new tumors appear after some time 57 Resources: Useful reading material: Alberts Molecular Biology of the Cell. References for some of the figures: Helena, J.M. et al. (2018). Deoxyribonucleic Acid Damage and Repair: Capitalizing on Our understanding of the Mechanisms of Maintaining Genomic Integrity for Therapeutic Purposes. Molecular Sciences. 19, 1148; doi:10.3390/ijms19041148 Caldwell, C. et al. (2017). Identification and Validation of a PD-L1 Binding Peptide for Determination of PDL1 Expression in Tumors. Scientific Reports. 7: 13682 58