Exam 3: Neoplasia III

Question 1

How is clonality of neoplasms established clinically (see starred citations). How do we know cancer is clonal?

Answer 1

X-linked isoenzyme markers are evidence of monoclonality of neoplasms
Established clinically by:
1) Specific translocations: veryify clonality
2) Lymphocytic neoplasms: gene rearrangements establish clonality→ immunoglobulin receptor of B cell proliferation, Tcell receptor gene for T cell proliferations

Question 2

List oncogenes from lecture/text. Know general function of each (growth factor, transduction, transcription, etc.) How many “mutated” alleles are required for self-sufficiency of growth signals?

Answer 2

Only need mutation of 1 single allele to produce self-sufficiency in growth! Cell produces growth factor to which it responds: Autocrine stimulation. Product is overexpressed secondary to signal transduction pathway gene.

Examples: 
ERB-B1 (EGFR)→ growth factor; 
ERB-B2 (Her-2 neu)→ growth factor; 
RET→ growth factor; 
RAS→ signal transduction; 
cABL→ signal transduction; 
MYC→ transcription

Question 3

List growth factor oncogenes and related tumors.

Answer 3

ERB B1: Variably expresses in some carcinomas (lung, head, neck); may be over expressed in gliomas
therapy: Monoclonal antibody targeted at mutated EGFR

ERB B2 (HER-2 nu): Amplified in 25% of breast cancers and ovarian cancer; correlates with poor prognosis (increased incidence of metastases and death)
therapy: Responds to monoclonal antibody: Trastuzamab (Herceptin)

RET: Activated by point mutations; involved with neural development; associated with familial and sporadic conditions of thyroid and endocrine cancers

cKIT: Mutated in gastrointestinal stromal tumors; encodes for receptor of stem cell factor (steel)

Question 4

Describe RAS as to actions. How is it activated? In which tumors? What other member of this pathway are closely associated with another tumor?

Answer 4

RAS (most commonly mutated oncogene). Mutated in up to 30% of all human tumors; frequent in myeloid leukemia and adenocarcinomas (most pancreatic carcinomas: colon, endometrial, thyroid etc). Arise as point mutations and are associated with chemical injury.

Activation of RAS: Inactive RAS is bound by GDP→ GTP binds and activates RAS causing transduction (MAP kinase pathway)
Inhibition of RAS: GAP (GTPase activating proteins: NF-1) bind to RAS to inactivate it. Mutated RAS evades GAP and remains bound to GTP in the active state→ persistent signal transduction.
Associated with: BRAF→ mutations in BRAF are common in melanoma

Question 5

What is the function of ABL and how is it activated? What is the associated tumor? Specific therapy?

Answer 5

ABL is a nonreceptor tyrosine kinase protooncogene that is normally dampened.
Activation: When ABL (on chromosome 9) fuses with BRC (on chromosome 22)→ cABl fusion gene is produced and dampening is lost→ leads to signal transduction and cancer development.
Associated tumor: chronic myelogenous leukemia, and acute lymphoblastic leukemias
Treatment: Imatinib mesylate tyrosine kinase inhibitor

Question 6

What type of neoplasia is associated with JAK2-STAT?

Answer 6

JAK2 tyrosine kinase is activated by a point mutation in its negative regulatory domain. Neoplasias associated with JAK2-STAT are myeloproliferative disorders such as polycythemia vera and primary myelofibrosis (proliferation of myeloid cells in bone marrow)

Question 7

List tumors associated with MYC dysregulation. Where given, know the mechanism of activation, method of detection and related tumor.

Answer 7

MYC is a protooncogene that is present in many normal cells. In normal fxn, it binds DNA and causes transcription of CDKs. MYC levels should normally decrease when the cell cycle begins.
MYC dysregulation: MYC is persistently over-expressed causing tumors
1) Burkitt lymphoma (B cell lymphoma)→ MYC arises from t(8:14)
2) Lung , breast and colon cancers→ MYC amplified
3) Neuroblastomas→ N-MYC amplified
4) Small-cell lung cancer→ L-MYC amplified

Question 8

Review cell cycle. Know cited tumor suppressor genes involved in the cell cycle and how each is activated and works.

Answer 8

Checkpoints delay cell cycle progression:
G1/S→ point of commitment to DNA synthesis phase; check for DNA damage; delay for repair or p53 dependent apoptosis
G2/M→ point of commitment to mitosis; monitors completion of repair important to radiation damaged cells; sensor of damage: RAD and ATM proteins
Defects of these systems are major causes of genetic instability in cancer cells

Regulation of the cell cycle: Cell cycle is regulated by cyclins, cyclin-dependent kinases (CDKs) and their inhibitors. CDKs are activated by phosphorylation and are synthesized during specific phases of the cell cycle. CDKs are rapidly eliminated by ubiquitin-proteosome pathway. (Cyclins D, E, A and B are sequential)

Tumor suppressor genes of the cell cycle:
1) RB gene: activated RB halts the advancement of G1→ S checkpoint
2) CIP/WAF family (p21, p27, p57): blocks actions of several cyclin/CDK complexes
P21 induced by p53
P27 responds to TGF-β
3) INK4 family (p15, p16, p18, p19): p16INK4a binds to cyclin D-CDK4; inhibitory to RB

Question 9

Review the concept of gatekeeper pathway to malignant transformation. List tumor suppressor genes. How many alleles are usually involved with a suppressor gene are implicated in a tumor?

Answer 9

Tumor suppressor genes are referred to as gate keeper genes→ regulator of cell growth and brakes to cell proliferation. They are also involved in cell differentiation.
Tumor suppressor genes include: RB and p53

Loss of fxn is a key event in almost all human cancers→ loss of both alleles is involved in transformation. Haploinsufficiency (loss of a single allele) is implicated in some cancers.

Question 10

Define LOH.

Answer 10

Loss of heterozygosity (LOH)→ Person born with 1 defective/mutated copy of a gene. Heterozygous individuals are normal. Occurs in familial cancer syndromes. LOH is associated with cancer: RB; WT1→Wilm’s tumor (nephroblastoma); VHL→ von Hippel Lindau (clear cell renal carcinoma)

Question 11

Know functions of RB (be specific). How is its function lost? What cancer is most associated with this gene?

Answer 11

RB is activated/inactivated by phosphorylation; (active= hypophosphorylated; inactive= hyperphosphorylated). Activated RB inhibits the G1→S checkpoint. Expression of cyclin E (s-phase gene) is dependent on E2F transcription factor→ RB hypophosphorylated will bind and inhibit E2F.
Many oncogenic viruses act by neutralizing growth/inhibitory activities of RB such as→ SV40, polyomavirus, adenovirus, HPV (high risk for malignancy types→ E7 protein) activity is increased.

Loss of function: RB is mutated/inactive in many cancers including breast and retinoblastoma. RB function is lost by:

1) RB mutation
2) Gene silencing by hypermutation
3) Cyclin D,CDK4 or p16INK4a mutations

Question 12

Know details of actions of p53, including chromosomal location, causes of inactivation, including familial.

Answer 12

P53 is the guardian of the genome; it is a central cellular monitor and critical gatekeeper. Gene TP53 is located on chromosome 17p13.1 (50% of all cancers demonstrate homozygous loss of p53 including breast, lung and colon cancers etc.) Inactivating point mutations occur in about 80% of cancer.
Activation of p53 pathway: triggered by anoxia, oncogene expression, damage to DNA and other stressors. Has a short t 1/2 which is maintained by regulator protein MDM2.
Cellular stress results in:
1) Modification→ released from regulator
2) Activation→ transcription of genes that: arrest cell cycle (quiescence); permanent cell arrest (senescence) or cause apoptosis.
Sensors of DNA damage: ATM and Rad4 related (ATR)
If DNA is damaged and unrepaired→ cell cycle is arrested permanently
Inactivation of p53: if p53 is mutated (tobacco, UV light etc.), it is non-fxnal
Familial p53 mutation (LiFraumeni Syndrome): is a germ line heterozygous mutation of p53 that can increase a persons risk of developing cancer by 25x. Results in many types of cancer such as breast, brain tumors, sarcomas, leukemia and adrenal cortical tumors.

Question 13

What tumors are most strongly associated with APC/beta catenin pathway changes? How do beta catenin and APC interact? Which is the oncogene?

Answer 13

Adenomatous polyposis coli genes (APC) is a class of tumor suppressors whose main fxn is to down-regulate growth-promoting signals. APC is a cytoplasmic protein that functions to regulate β-catenin by degrading it after its normal function is complete.
2 main functions of β-catenin:
1)	Binds E-cadherin→ maintains cell to cell cohesion (adhesion)
2)	It is a component of the WNT signaling pathway→ translocates to the nucleus as a transcription activator.

Mutations of APC (on 5q21 loci) result in→ the loss of both alleles (homozygous loss) and an individual who inherits this mutation develops thousands of adenomatous polyps in the colon and possible development of colon and hepatic cancers. APC mutations are the cause of 70-80% of colon cancers (some cancers are a result of β-catenin)

Question 14

What tumors are most associated with TGF-beta/SMAD alterations?

Answer 14

Transforming growth factor β (TGF-β) functions to inhibit cell proliferation mostly by regulating RB pathways. When TGF-β is bound by a ligand, it becomes activate and causes a phosphorylation and activation of receptor SMADs (RSMADs). When R-SMADs is active, it enters the nucleus to bind SMAD-4 and activates transcription genes (CDKIs, p21 and p15/INK4b). It inhibits CDKs, cyclins, and MYC.
Associated tumors: pancreatic, gastric and colon cancers.

Question 15

What is the function of NF-1. Review disease due to inherited NF-1 mutation (lecture 1).

Answer 15

Neurofibromatosis type I (NF-1) occurs with inheritance of one mutant allele for the NF-1 gene. Individuals who inherit the mutant allele of the NF-1 gene develop numerous benign neurofibromas and optic nerve gliomas as a result of inactivation of the second copy of the gene. NF-1 codes for neurofibromin which is a GTPase activating protein that inactivates RAS. Mutation of NF-1 leads to continuously active RAS protein.

Question 16

What are the clinical characteristics of NF-2 mutations?

Answer 16

Neurofibromatosis type II (NF-2) is similar to NF-1 but is less common than type I. Individuals that inherit this mutation develop benign bilateral schwannomas (benign neoplasms composed of the fibrous elements of a nerve) of the acoustic nerve. Mutations of NF-2 occurs in some sporadic CNS tumors.
Gene product of NF-2 gene: The product of the NF-2 gene is merlin which is related to a family of membrane cytoskeleton-associated proteins. Merlin deficiency leads to carcinogenesis (unknown mechanism) and cells lacking this protein are not capable of establishing stable cell-to-cell junctions and are insensitive to normal growth arrest signals.

Question 17

Know functions and cancer related to VHL.

Answer 17

Cancers related to VHL: Germline mutations of the von Hippel-Lindau (VHL) gene on chromosome 3p are associated with hereditary renal cell cancers, pheochromocytomas, hemangioblastomas of the central nervous system, retinal angiomas and renal cysts.
Functions of VHL: VHL protein is part of a ubiquitin ligase complex. A critical substrate for this activity is HIF1-α (hypoxia-inducible transcription factor 1α). HiF1-α is hydroxylated and binds to the VHL protein→ leads to ubiquitination and proteasomal degradation. Process is oxygen dependent.
Functions of HIF1-α: Once activated, it translocates to the nucleus and turns on many genes (VEGF and PDGF).
Lack of VHL activity: prevents ubiquitination and degradation of HIF1-α and produces increased levels of angiogenic growth factors.

Question 18

Know functions, familial syndrome and cancers related to PTEN

Answer 18

PTEN (phosphatase and tensin homologue) is a membrane-associated phosphatase encoded by a gene on chromosome 10q23. PTEN acts as a tumor suppressor by stopping the pro-survival/pro-growth PI3K/AKT pthw.
Loss of PTEN function: Mutation of chromosome 10q23 which encodes PTEN results in Cowden syndrome→ autosomal dominant disorder that results in frequent, benign outgrowths, tumors of the skin and appendages and increased incidence of epithelial cancers (breast, endometrium, and thyroid).

Question 19

Know the cancer related to WT-1.

Answer 19

The WT-1 gene is located on chromosome 11p13. WT1 protein is a transcriptional activator of gene involved in renal and gonadal differentiation. It regulates the mesenchymal-to-epithelial transition that occurs in kidney development.
WT-1 deficiency: results in a tumorigenic effect→ Wilm’s tumor (nephroblastoma and pediatric renal cancer). Adult tumors (leukemias and breast carcinomas etc.) may over-express WT-1. Abnormalities of this gene and tumor may be inherited or sporadic.

Question 20

21) Know the cancer related to PTCH.

Answer 20

PTCH1 and PTCH2 are tumor suppressor genes that encode a cell membrane protein (PATCHED) which functions as a receptor for a family of proteins called Hedgehog. The PATCHED/Hedgehog pthw regulate several genes including TGF-β, PDGFRA and PDGFRB.
Mutations in PTCH: result in Gorlin syndrome→ an inherited condition known as basal cell carcinoma syndrome. Sporadic mutations are related to UV exposure.

Question 21

How do cancer cells evade apoptosis? What tumor is strongly associated with evasion of apoptosis and what gene is involved?

Answer 21

Apoptosis occurs secondary to a variety of signals including DNA damage and loss of cell adhesion to basement membrane (anoikis).
2 pathways of apoptosis:
1) Extrinsic pathway: FAS-FAS ligand
2) Intrinsic pathway apoptosis: release of mitochondrial cytochrome c
Release of mitochondrial cytochrome c initiates apoptosis.
Pro-apoptotic signal: BAX, BAK
Anti-apoptotic signal: BCL2, BCL-XL
Balance between these 2 signals is regulated by BH3-only proteins (BAD, BID, PUMA)
Evasion of apoptosis: cancers that have reduced FAS on surface evade apoptosis. Cancer cells may inhibit caspase 8 via FLIP. Cells that over express BCL-2 (B-cell lymphoma, follicular type) prevent tumor cells from being apoptosed. t(14:18) activates BCL-2 transcription.
P53 is an important pro-apoptotic gene→ p53 activity is regulated by BAX. If p53 is mutated, BAX is not transcribed and p53 will not have apoptotic activity in cells expressing DNA damage→ unregulated cell growth

Question 22

Review functions of telomerase with respect to neoplasia.

Answer 22

Normal cells: Somatic cells have a fixed capacity for doubling; the telomers shorten after every cell division because these cells lack telomerase. P53 dependent checkpoints are activated by short telomers→ cell apoptosis.
Mutated cells: Cells with disabled p53 checkpoints may have many DNA breaks and or shorten telomers leading to additional genomic instability. In 90% of cancer cells, telomerase is upregulated which allows continued tumor cell replication.

Question 23

What factors are pro-angiogenic? Any inhibitors?

Answer 23

Proangiogenic factors:

1) VEGF→ transcription controlled by hypoxia-inducing factor (HIF-1a), RAS-MAP, and MYC. VEGF increases ligands that activate the Notch pthw (controls branching and density of vessels). Bevacizumab an inhibitor of VEGF is used as a cancer therapy.
2) Basic fibroblastic growth factor (bFGF)→ released by proteases from ECM

Question 24

List factors that are anti-angiogenic.

Answer 24

Antiangiogenic factors produced or induced by tumor cells:
1) Thrombospondin-1→ induced by p53

Agents produced in response to tumor by proteolytic cleavage of ECM:

1) Angiostatin
2) Endostatin
3) Vasculostatin

Question 25

26) Use figures in text/handout to understand how cancer invades. Know specific molecules involved in this process (include those not bolded).

Answer 25

The ability of a tumor to invade and metastasize into tissue occurs in a series of events:

1) Invasion of the ECM→ tumor cells detach (dissociate) from one another due to the loss of E-cadherin and reduced catenin protein (link with cytoskeleton)

2) Degradation of ECM→The basement membrane is degraded along with interstitial connective tissue by matrix metalloproteinases (type 4 collagenase- MMP9), cathepsin D, or urokinase plaminogen activator. The tumor cells may either secrete proteolytic enzymes themselves or induce stromal cells (fibroblasts and inflammatory cells) to secrete proteases.
Degradation of ECM consists of: remodeling, release of growth factors, activation of VEGF and chemotaxis
Ameboid migration: a second method of invasion in which the cell squeezes through spaces in the matrix instead of cutting its way through it. Process is much faster.

3) Attachment→ Changes in attachment of tumor cells to ECM proteins (increases in the # of laminin and fibronectin receptors as well as a loss of polarity of these receptors).

4) Migration→ Cells attach to the matrix at the leading edge, detach from the matrix at the trailing edge and contract the actin cytoskeleton to ratchet forward. Movement is directed by Autocrine motility factor and chemotactic factors from the ECM
Results in: Stromal cells are altered due to the tumor→ supports successful cancer establishment

Question 26

27) Review the process of metastasis. What factors are involved in metastatic sites?

Answer 26

Mechanisms of metastasis:

1) Intravasation→ vascular invasion
2) Formation of tumor embolus→ platelets and coagulation factors are involved
3) Adhesion to endothelium→ via adhesion molecule CD44
4) Extravasation→ grow out through wall of the vessel
5) Induction of angiogenesis→ neovascularization
6) Growth in metastatic location

Metastatic site tropism: What draws cancer cells to certain locations?
Endothelial cells of various organs express different ligands for adhesion molecules. Chemokines of target tissues may attract cancer cells (most cancer cells have specific chemokine receptors). Target organs may also release chemoattractants (IGF’s I and II).

Preferred metastatic sites for each tumor type:
Natural drainage patterns explains some but not all sites of tumor metastases
1) Prostate→ to lumbar vertebrae
2) Bronchogenic carcinoma→ to adrenals and brain
3) Neuroblastomas→ to liver and bone
4) Breast→ to bone, liver and lung

Question 27

List DNA repair genes/systems.

Answer 27

1) Mismatch repair
2) Nucleotide excision repair
3) Recombination repair

Question 28

What are the functions and neoplasm most associated with mismatch repair genes, MSH, MLH and PMS?

Answer 28

Mismatch repair genes are the “spell-checkers” of DNA. They proofread and correct defects. Mutations lead to accumulation or errors in oncogenes and tumor suppressor genes.

Hereditary nonpolyposis colon cancer (HNPCC) is a result of errors due to or caused by mismatch repair genes. Inheriting this disease requires 2 mutated alleles for disease. Germline mutations in MSH2 (2p16) and MLH1 (3p21) account for up to 30% of cases of HNPCC. Mutations in the PMS gene is related to HNPCC type 3 syndrome.

Question 29

What condition is associated with nucleotide excision repair defects?

Answer 29

Xeroderma Pigmentosum→ results when an individual inherits defective nucleotide excision repair genes. These individuals are at increased risk of skin cancer when they are exposed to UV light which causes cross liking of pyrimidines preventing normal DNA replication.

Question 30

What genes are involved in homologous recombination repair? What are their associations?

Answer 30

1) BRCA1 & BRCA2→ These mutations occur in familial breast-ovarian cancer syndromes. Both are involved in homologous recombination of DNA repair. Women with mutations in BRCA1 have a substantially higher risk of epithelial ovarian cancers and men have a slightly higher risk or prostate cancer. Mutations of BRCA2 result in increased risk of breast cancer in both men and women as well as cancer of the ovary, prostate, pancreas, bile ducts, stomach, and melanocytes.
2) ATM→ aka “Bloom syndrome” or “Fanconi anemia” is an autosomal recessive disorder that is characterized by hypersensitivity to other DNA-damaging agents (ionizing radiation and DNA cross-linking agents.) Results in a predisposition to cancer and developmental defects.

Question 31

Describe the Warburg effect and clinical significance/use.

Answer 31

Warburg effect→ occurs when cancer cells shift their glucose metabolism away from mitochondrial directed (utilizing oxygen) to that of aerobic gylcolysis. Occurs even in the presence of abundant oxygen. This may serve as an advantage to cancer cells that are in hypoxic microenvironments. Mutations in many genes have shown to shift metabolism (PTEN, RAS, p53, and MYC)

Clinical Significance: The glucose-hunger of tumors is used to visualize them using PET scanning. Patients are injected with 18F-fluorodeoxyglucose which is preferentially taken up by cancer cells and dividing cells (bone marrow cells).