L8: Cellular Oncogenes/TSG

Question 1

Using the example of colon cancer, show that cancer is a multistep process

Answer 1

Normal colon cell -> Chromosome 5q gene loss/mutation -> Increased cell growth -> Ras gene mutation (Adenoma 1) -> Chromosome 18 loss/mutation in DCC TSG (Adenoma 2) -> Chromosome 17 loss/Mutation in p53 TSG (Adenoma 3) -> Carcinoma -> Other chromosomal losses -> Metastasis

Question 2

What are protooncogenes and oncogenes (+ eg)

Answer 2

• POG: A gene that normally functions to control cell division and may become a cancer gene (oncogene) by mutation
• Oncogene: A gene that induces or continues uncontrolled cell proliferation
• Acts in a autosomal dominant fashion (1 mutation is enough for cancer development)
• Eg. Ras oncogene: single nucleotide mutation from Gly (normal) to Val (mutated)

Question 3

How was oncogene discovered?

Answer 3

• Rous sarcoma virus first discovered in 1911 by Peyton Rous
• Observed that cancer can be transmitted
• Induced tumours in healthy chickens by injecting a preparation of cell free filtered extract from chicken tumours.
• Later discovered that the causative agent is the rous sarcoma virus (RSV): a retrovirus which reverse transcribes its RNA genome into cDNA before integrating into the host DNA.
• The first confirmed oncogene was discovered in 1970 and was termed src (pronounced sarc as in sarcoma)
• Src was first discovered as an oncogene in a chicken retrovirus.
• Src encoded for RTK, causing sarcoma in chicken
• Src contributes to embryonic development and cell growth

Question 4

What are tumour suppressor genes?

Answer 4

• Tumor suppressor gene encodes for products/proteins that inhibit cell proliferation.
• Mutant versions in cancer cells have lost their function.
• Both alleles of a tumor suppressor gene must be inactivated to change the behavior of the cell.

Question 5

**What are some examples of TSG?

Answer 5

• Retinoblastoma; pRB (nuclear phosphoprotein)
• Wilm’s tumor; WT-1
• Li-Fraumeni syndrome; p53 (transcription factor): germline mutation
• Colon carcinoma; DCC
• Breast cancer; BRCA1

Question 6

What is the normal functioning of Rb protein/Using the example of Rb gene, how does it lead to uncontrolled cell growth and eventually retinoblastoma?

Answer 6

Normal functioning during growth suppression:
- E2F is a transcription factor that mediates growth-dependent activation of genes required to make the transition into and through S phase
- Rb binds and inactivates E2F under conditions of growth suppression
- But there are several ways to alleviate growth suppression resulting in controlled or uncontrolled cell growth

Growth relief:
1) Binding of GF during G1 phase causes phosphorylation of Rb protein (2 additional phosphate groups) -> E2F cannot bind to Rb -> Rb cannot inactivate E2F and cells proliferate
2) Adenovirus E1A oncoprotein binds to Rb protein -> replacing E2F -> E2F not inactivated -> cell proliferation
3) Gene mutation on Rb gene and affects binding pocket on Rb protein -> releases E2F and E2F cannot be inactivated -> cell proliferation

Question 7

What are the features of retinoblastoma?

Answer 7

• 1 in 20,000 children
• Most common eye tumor in children
• Occurs in heritable (2 eyes) and non-heritable (1 eye) forms
• Identifying at-risk infants substantially reduces morbidity and mortality

Question 8

What is p53 TSG?

Answer 8

• Frequently found mutated in human tumors
• p53 protein functions as a transcription factor that regulates cell-cycle and DNA repair genes
• UV irradiation causes cell-cycle arrest in G1 that is dependent on p53
• And cells that contain a mutated p53 cannot be arrested and go into S phase and replicate damaged DNA
• LOF mutations of p53 result in the replication of cells with damaged DNA -> Further accumulation of other mutations affecting oncogenes and tumor suppressor genes -> increased likelihood of cancer

Question 9

What are the downstream pathways of p53?

Answer 9

• p53 is also a transcription factor so it binds to DNA and trigger downstream transcriptional activation of genes
  1) Apoptosis -> cell death through synthesis of Bax, Apaf1 etc
  2) Angiogenesis and metastasis pathways -> Inhibition of angiogenesis and metastasis through synthesis of maspin
  3) Arrest/repair pathway through p21/GADD45
• if p53 is mutated, these pathways cannot be triggered

Question 10

What are the genes responsible for tumourigenic cell growth?

Answer 10

• For normal growth: Balance between POG and TSG
• In cancer: Overexpression of oncogenes + LOF of TSG -> cannot suppress uncontrolled cell proliferation -> malignant transformation
• For oncogene (usually dominant, GOF)
• cellular POG that have been mutated (and “activated”)
• cellular POG captured by retroviruses and have been mutated in the process (and “activated”)
• virus-specific genes that behave like cellular POG that have been mutated to oncogenes (i.e., “activated”)
• For TSG (usually recessive, LOF)
• Deletion -> LOF of a cellular gene or chromosome region
• Inactivating point mutation -> LOF of gene function

Question 11

What are the 4 ways of oncogene activation?

Answer 11

• Altered gene function: Base substitutions
• Amplification
• Altered regulation
• Viral insertion

Question 12

What is an example of altered gene function of oncogene?

Answer 12

[Refer to slide]
- Amino acid substitutions in Ras family proteins at positions 12, 59 and 61 (can result in activation)
- Normal cells: c-ras - Gly (12), Ala (59), Glutamine (61)
- Made up of 3 different types of Ras: H-ras, K-ras, N-ras
- Mutations at any of the 3 amino acids affect GTPase active site -> inactivating GTPase -> Ras constitutively active
- Mutation in H-ras (lung carcinoma) - Change in aa 61 from Gln to Leu
- Mutation in H-ras (bladder carcinoma) - Change in aa 12 from Gly to Val
- Mutation in K-ras (lung carcinoma) - Change in aa 12 from Gly to Cys or Arg
- Mutation in K-ras (colon carcinoma) - Change in aa 12 from Gly to Val
- Mutation in N-ras (neuroblastoma) - Change in aa 61 from Gln to Lys
- Mutation in N-ras (Lung) - Change in aa 61 from Gln to Arg
- For murine sarcoma virus, require 2 mutations to convert to tumourigenesis strain
H-ras (Harvey strain): Change in aa 12 from Gly to Arg and 59 from Ala to Thr
K-ras (Kirsten strain): Change in aa 12 from Gly to Ser and 59 from Ala to Thr

Question 13

What is an example of oncogene amplification?

Answer 13

• Burkitt lymphoma
• Reciprocal translocation involving c-myc (on chromosome 8) and 1 of the immunoglobulin heavy chain on chromosome 14/22/2
• Translocation (8;14) (80% of cases), t(8;22) (15% of cases), t(2;8) (5% of cases)
• Normal function in lymphocytes: Immunoglobulin enhancer is often active in lymphocytes to produce antibodies-> this activates the immunoglobulin heavy chain gene promoter
• Due to translocation: c-myc gene is brought close to the immunoglobulin enhancer -> causing c-myc promoter to be activated in these cells when enhancer is activated -> one of the expression of c-myc results in abnormal proliferation causing lymphoma due to increased expression of c-myc

Question 13

What is an example of altered regulation of oncogene?

Answer 13

• Chronic myelogenous leukemia
• Translocation of chromosome 9 (Abl) and chromosome 22 (Bcr) results in “Philadephia chromosome”
• Fusion of BCR (multi-domain signalling protein) and Abl (non-receptor tyrosine kinase), producing a chimeric gene/protein made up of both Bcr gene at 5’ end and Abl gene at 3’ end
• Constitutive kinase activity causing dysregulation of cell proliferation and apoptosis

Question 14

What is an example of viral insertion oncogene?

Answer 14

1) Adenovirus, dsDNA, encode for E1A and E1B oncogenes
2) Papovavirus: SV40 (monkey), polyoma (human), dsDNA, encode for T antigens
-> virus carry their own genes (dsDNA) -> designed to stimulate own growth and proliferation -> stimulating uncontrolled cell proliferation
3) Retrovirus, ssRNA (transcribed to cDNA), insert itself into host genome -> mutate cellular proto-oncogenes

Question 15

What are the examples of retrovirus oncogenes?

Answer 15

[Refer to slide]
- Rous sarcoma virus (v-src) -> c-src (src), (from viral oncogene v-src to cellular POG c-src)
- Simian sarcoma (v-sis) -> c-sis (sis)
- Harvey murine sarcoma (v-H-ras) -> c-H-ras (H-ras)
- Kirsten murine sarcoma (v-K-ras) -> c-K-ras (K-ras)
- FBJ murine osteosarcoma (v-fos) -> c-fos (fos)
- Avian myelocytomatosis (v-myc) -> c-myc (myc)
- Abelson leukemia virus (v-abl) -> c-abl (abl)
- Avian erythroblastosis (v-erbB) -> c-erbB (erbB)
-> viral oncogenes are 80-90% homologous to cellular POG

Question 16

How does retroviruses convert a regulatory gene to a viral oncogene?

Answer 16

1) A normal cell is infected with a retrovirus.
2) The retrovirus integrates its genome into the host cell genome, near the proto-oncogene, via long terminal repeats.
3) Using the host cell replication machinery, forming viruses encapsulates POG and viral genome.
4) Mutation occurs in the newly formed retroviruses (since viruses are prone to mutations), creating oncogene from POG.
5) Retroviruses with oncogene invades normal cells.
6) Normal cells now produce defective regulatory protein, transforming cells to abnormal cells, thus increasing mutations and the risk of cancer.

Question 17

What are the different classifications of oncogenes?

Answer 17

1) Growth factors - sis: For wound healing (normal functioning); causes connective tissue cancer when abnormal
2) Growth factor receptors: erbB, fms, trk
3) Intracellular transducers: src, abl, raf, gsp, ras
4) Nuclear transcription factors: jun, fos, myc, erbA

Question 18

How does the different types of oncogenes play a role in the pathway?

Answer 18

• Secreted growth factors binds to GF receptors
• Activates cytoplasmic signal transduction proteins and downstream signalling
• Downstream signalling molecules such as nuclear protein transcription factors enter nucleus and upregulate/active cell growth genes
• Causing cell proliferation

Question 19

Give an example of how GF receptors contribute to cancer.

Answer 19

• Using the example of erbB/EGFR, which is a tyrosine receptor kinase
• Under normal circumstances, erbB is inactive. GF binds to EGF-binding domain on EGFR , activating the tyrosine kinase and downstream reactions.
• When EGFR is mutated, tyrosine kinase is constitutively active without any GF binding to the binding domain.
• Leads to increased cell proliferation
• Mutated in breast, stomach and ovarian cancers

Question 20

Give an example of how cytoplasmic signal transducer protein contribute to cancer.

Answer 20

[Refer to slides]
Under normal functioning
- the c-ras family contains three genes: H-ras, K-ras, and N-ras
- these genes encode for Ras proteins which are small G-proteins
- When RTK is activated by ligand, it activates G proteins in the intracellular domain
- G proteins transmit growth signals from cell surface receptors to GEF (Guanylate exchange factors (GEFs) such as Sos)
- This catalyzes the exchange of GDP bound to Ras proteins with GTP, leading to the activation of Ras (Ras-GTP)
- This signals downstream molecules.
- The Ras-GTP complex is inactivated by GAP which hydrolyzes GTP back to GDP

Under abnormal conditions:
- mutations in the c-ras genes inactivate the Ras GTPase
- mutated Ras proteins are constitutively active
- constitutively active Ras proteins result in uncontrolled cell growth, leading to cancer

Question 21

What phenomenon is needed to maintain carcinogenesis?

Answer 21

• Oncogenic addiction
• Continued activity of specific over expressed oncogene is necessary for the maintenance of malignant phenotype, throughout the whole process of carcinogenesis

Question 22

What are some examples of oncogenic addiction?

Answer 22

1) Myc: Transgenic mouse is overexpressed with myc oncogene, which induces formation of malignant osteogenic sarcoma. Loss of overexpression of myc oncogene leads to differentiation and formation of normal osteocytes. (Myc needs to be overexpressed for a long time for malignancy and maintain malignancy)
2) Bcr-Abl gene: Conditional activation of Bcr-Abl gene in transgenic mouse resulted in development of leukemia. Subsequent deactivation leads to apoptosis of cancer cells.
-> Removal of oncogene can lead to either apoptosis of cancer cells or reversal back to normal phenotype.

Question 23

What are some examples that opposes oncogenic addiction?

Answer 23

Myc and ras:
- Not always necessary that continued overexpression is required for maintenance of malignant phenotype
- Overexpressed oncogenes shows their effects by causing genomic instability (high frequency of mutations such as chromosome translocation, point mutation and change in nucleotides)
- Previous subset of c-myc dependent tumour cells can escape myc dependence by activating endogenous ras oncogenes (dependent on another oncogene by inducing genomic instability)
-> Loss of overexpression of myc oncogene may not reduce the tumour bulk/cause apoptosis/reversal of cancer cells back to normal phenotypes

Question 24

How does non coding RNA play a role in cancer (+ eg of CLL/AML)?

Answer 24

[Refer to slides - microRNA genes, class of ncRNA,play an important role in regulating gene expression - Using the example of molecular alterations in chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML) - Deletion or downregulation of microRNA (miR)-15a/miR-16-1 cluster, located at chromosome 13q14.3 and directly involved in the regulation of BCL2 and MCL1 expression, represent an early event in the pathogenesis of CLL - During the evolution of malignant clones, other microRNAs (miRs) can be deleted (such as miR-29) or overexpressed (such as miR-155), contributing to the aggressiveness of B-cell CLL - Such abnormalities can influence the expression of other protein-coding genes (PCGs), as TCL1 oncogene, directly regulated by miR-29 and miR-181, or affect other noncoding RNAs (ncRNAs) - The consequences of this steady accumulation of abnormalities are represented by the reduction of apoptosis and the induction of survival and proliferation of malignant B cells, leading to the evolution of more aggressive clones - Members of the miR-29 family, lost in AML and in other tumor types as lung cancer, have also been shown to directly target MCL1 and DNMT3A and B.

Question 25

What is gene therapy?

Answer 25

- Insertion of genes into an individual's cells and tissues to treat a disease, such as a hereditary disease in which a deleterious mutant allele is replaced with a functional one

Question 26

How is gene therapy done?

Answer 26

1) A 'corrected' (functional one) gene is inserted into the genome to replace an 'abnormal' disease-causing gene. 2) A carrier called a vector must be used to deliver the therapeutic gene to the patient's target cells. 3) The vector then carries the therapeutic gene into the patient's target cell. 4) The target cells become infected with the viral vector. 5) The vector's genetic material is inserted into the target cell. 6) Functional proteins are created from the therapeutic gene, leading to the cell returning to a normal state.

Question 27

What are the uses of gene therapy?/How can gene therapy be used as treatment?

Answer 27

- Replace missing or defective genes - Deliver genes that speed the destruction of cancer cells (apoptosis) - Supply genes that causes reversal of cancer cells back to normal cells (eg. insert copy of TSG to have functional TSG) - Deliver bacterial or viral genes as a form of vaccination - Provide genes that impede growth of new tissue (angiogenesis) - Deliver genes that stimulate the healing of damaged tissue

Question 28

What are the types of gene therapy and how is it usually done (+eg)

Answer 28

Germline GT - sperm cells, ova, stem cell precursors of sperm/ova - Newly introduced gene is passed on to the offspring - Remains controversial - Introduced gene must be incorporated into the chromosomes by genetic recombination Somatic GT - Recipient's genome is changed but not passed on to next generation - Most gene therapy has been directed at somatic cells - Ex vivo: cells are modified outside the body and transplanted back into pt again (esp for blood cancers as they are the easiest to remove and return eg. leukemia, sickle cell anemia) - In vivo: genes are changed while cells are in the body - Recombination based approaches in vivo are uncommon as most DNA constructs have a very low probability.

Question 29

What are some examples of in vivo gene therapy?

Answer 29

- Infusion of adenoviral vectors into the trachea and bronchi of cystic fibrosis patients. - Injection of a tumor mass with a vector carrying the gene for a cytokine or toxin. - Injection of a dystrophin gene directly into the muscle of muscular dystrophy patients.

Question 30

What vector is usually used for in vivo gene therapy and why?

Answer 30

- Virus = carrier of desired gene (vector). - Virus genome is manipulated to remove disease-causing genes and introduce therapeutic genes. - Viral methods have proved to be the most efficient to date. - Many viral vectors can stable integrate the desired gene into the target cell’s genome.

Question 31

What are the characteristics of an ideal vector?

Answer 31

- Insert size: one or more genes. - Targeted: limited to a cell type. - No immune response. - Stable: not mutated. - Production: easy to produce high concentrations - Can be Regulated: produce enough protein to cause an effect.

Question 32

What are the different types of vectors?

Answer 32

RNA viruses (Retroviruses - Most common) - Murine leukemia virus (MLV) - Lentivirus eg. Human immunodeficiency virus (HIV) - Human T-cell lymphotropic viruses (HTLV) DNA viruses - Adenoviruses (2nd most common) - Adeno-associated viruses - Herpes simplex virus (HSV) - Pox viruses Non-viral vectors - Liposomes - Naked DNA - Liposome-polycation complexes - Peptide delivery systems

Question 33

What are the advantages and disadvantages of retroviruses?

Answer 33

Adv: - Wide host range - Able to stably integrate into target host genome - Long term expression of transgene Disadv: - Small capacity in carry therapeutic genes - Infectivity limited to dividing cells (Cannot affect non-dividing which makes up 1-5% of the tumour bulk) - Inactivated by complement cascade - Random integration into the host genome, may disrupt endogenous genome of the host cell and lead to oncogene activation -> increase tumour bulk

Question 34

What are the adv and disadv of using adenoviruses?

Answer 34

Adv: - Efficiency of transduction is high - High level gene expression - Slightly increased capacity for exogenous DNA than retroviruses Disadv: - Expression may be transient (Does not integrate into the genome and thus not replicated during cell division) - Cell-specific targeting difficult to achieve - Virus uptake is ubiquitous - Safety - Limited approach as it can only be used with certain tissues and require large amounts of DNA (since it is not replicated so it only targets that tumour bulk at that time)

Question 35

What are the non-viral approaches for gene therapy?

Answer 35

Liposome: - Creation of artificial lipid sphere with an aqueous core - Carries therapeutic DNA, capable of passing the DNA through the target cell's membrane Nano-engineered substances - Eg. Ormosil (stands for organically modified silica/silicate) - Used as DNA vector and can deliver DNA loads to specifically targeted cells in living animals

Question 36

What are the 4 main strategies of cancer gene therapies?

Answer 36

1) Artificial killing of cancer cells using suicide gene - Insert a gene encoding a toxin (eg. diphtheria A chain) or gene conferring sensitivity to drug (eg. herpes simplex thymidine kinase; to use this eg.) into tumour cells. - Virus-originated HSV-TK (suicide gene, without inducing much immune response) is different from mammals - Its product, thymidine kinase, can metabolize the non-toxic prodrug ganciclovir (GCV) into monophosphate derivative, then phosphorylate further into GCV triphosphate (active form) -> similar to purine analog/gemcitabine - Metabolite triphosphate is incorporated into replicating DNA and acts as DNA synthesis inhibitor 2) Stimulate natural killing of cancer cells - Enhance the immunogenicity of the tumor by (for e.g. inserting genes encoding foreign antigens). - Increase anti-tumor activity of immune system cells by (for e.g. inserting genes that encode cytokines). - Induce normal tissues to produce anti-tumor substances (e.g. interleukins, interferon). - Protect surrounding normal tissue from side effects of chemotherapy. One example is the multidrug-resistant protein-1, which is encoded by the human ABCBI gene named as MDR1 gene. It stimulates the cellular pump to remove cytotoxic drugs from normal cell cytoplasm to the outside, thus protecting normal cells from chemotherapy’s side effects. The MDR1 gene is minimally expressed in malignant cells; thus, chemotherapeutic medications entering the cytoplasm will remain at a higher concentration, leading to apoptosis. [Decrease side effects of chemotherapy; BUT in some cases when MDR1 is overexpressed, it causes chemoresistance] 3) Tumours resulting from oncogene activation - Gene silencing, selectively inhibit expression of oncogene - Deliver gene specific antisense oligonucleotide or ribozyme to inactivate/cleave oncogene mRNA - Not a good example, can inactivate 1-2 genes but not sure if it is enough to kill cancer (many mutations) 4) Tumours arising from inactivation of TSG - Gene editing/gene replacement - Insert WT TSG -> Should choose well characterized treatments eg. CAR T-cell amd GCV**

Question 37

What are some problems with gene therapy?

Answer 37

1. Short Lived - Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy from being long lived - Would require multiple rounds of therapy. 2. Immune Response - New things introduced leads to immune response, eliminating virus - Increased immune response when a repeat offender enters, making gene therapy inefficient/no therapeutic effect 3. Viral Vectors - May have toxic, immune, inflammatory response - May induce disease once viral vector is injected 4. Multigene Disorders - Difficult to treat diseases such as cancer, Heart disease, and diabetes as there is more than one gene mutated; would have to introduce more than one gene. - May induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis.

Question 38

What are the other difficulties associated with gene therapy (technical difficulties)

Answer 38

- Even for single-gene disorders, there are other difficulties associated with GT, technical difficulties - Eg. Cystic fibrosis, even though the genetic basis is well characterised, presence of mucus in lungs makes it difficult to deliver the genes to the target lung cells - Delivery of genes may also be difficult if the disease is present in many sites

Question 38

Case study/successful example of gene therapy

Answer 38

- X-linked severe combined immunodeficiency (X-SCID) - Case study: On September 14, 1990, at the U.S. National Institutes of Health, W. French Anderson M.D. and his colleagues R. Michael Blaese, M.D., C. Bouzaid, M.D., and Kenneth Culver, M.D., performed the first approved gene therapy procedure on four-year old Ashanthi DeSilva. - Ashanthi DeSilva was born with a rare genetic disease called severe combined immunodeficiency (SCID). - Removed WBC from her body -> let the cells grow in the lab -> insert the missing gene into the cells -> infused the genetically modified blood cells back into her bloodstream - Is still in good health as of 2007 (attending college) - Important study as it showed that gene therapy can be successful without adverse consequences

Question 39

What are the different ways gene therapy be done and achieved/succeed?

Answer 39

1) Replace a mutated gene that causes disease with a healthy copy of the gene 2) Inactivating/KO a mutated gene that is functioning abnormally 3) introduce a new gene into the body to fight the disease

Question 40

Future of gene therapy

Answer 40

- Current: Used for treating or curing existing conditions - Future: Shifting to prevention once we know which genes contribute/causes cancer - However, even though there have been 175 clinical trials with 2000 patients treated, we still have no conclusive evidence for GT

Question 41

What are the ethical issues associated with GT?

Answer 41

1. How can “good” and “bad” uses of gene therapy be distinguished? 2. Who decides which traits are normal and which constitute a disability or disorder? 3. Will the high costs of gene therapy make it available only to the wealthy? 4. Could the widespread use of gene therapy make society less accepting of people who are different? 5. Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?

Question 42

With an example, describe the first FDA approved GT treatment.

Answer 42

- [To include in exam] **Chimeric antigen receptor T cell (CAR-T) therapy for B-cell acute lymphoblastic leukemia, the most common blood cancer in children [Link to L6] - Disease is cured, not just treated - Using immune system to fight, which is currently the most promising way of fighting tumours. - Cancer cells arise from normal cells, so the immune system doesn't always recognize that anything is wrong. - A pioneering group of drugs already approved by the FDA, called checkpoint inhibitors. - Checkpoint inhibitors remove the brakes on the immune system and allow it to attack tumor cells that it normally wouldn’t. - CAR-T therapy works to co-opt the immune system in a different way. - It involves removing a patient's blood and essentially replacing it with a population of blood cells stacked with cancer-fighting immune cells known as T cells. - Using GT to change patients’ bone marrow cells -> making blood and immune cells, to recognize cancer cells.