Neoplasia (2, 3, 4)

Question 1

Tumor Progression

Answer 1

**Tumor Progression
= *Over time, Tumors Become *Progressively More Malignant.

Although Originally Monoclonal, with time, the growing Tumor is Enriched in subclones of More Malignant cells and becomes more prone to invade and metastasize.

**The Degree of Genetic Instability Sets the Speed of Tumor Progression.

____________________

4 Phases of Tumor Progression:

1) **Transformation
= Malignant Changes occur @ the Initial target Cell
(Non-lethal Genetic damage)

2) **MonoClonal Expansion
= Growth of the Transformed Cell

3) **Local Invasion
4) **Distant Spread / Metastasis

Question 2

Malignant Transformation

Answer 2

Non-Lethal *Genetic Damage
= *Lies at the Heart of *Carcinogenesis

4 Types of Targets of Genetic Damage:

1) **Growth-Promoting ProtoOncogenes:
=> Mutant Alleles are **DOMINANT
=> One Mutated Allele Can Transform the Cell, Despite the Presence of the other Normal Allele

2) **Growth-Inhibiting Suppressor Genes:
=> Mutant Alleles are **RECESSIVE
=> Both Alleles Must be Mutated to Transform the Cell
(Recessive Oncogenes)

3) **Genes that Regulate Apoptosis:
=> **RECESSIVE or Dominant
(Tumor Suppressor Genes)

4) **Genes that Regulate DNA Repair:
=> **RECESSIVE

**Mutator Phenotype:
=> The Predisposition for Mutations.
=> **Genetic Instability = **Impaired Ability to Repair DNA.
_Predisposes to mutations.

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
Hallmarks of Cancer:
*Cancer genes affect 7 Fundamental Cell Processes, which Determine the *Malignant Phenotype:
1) Defects in DNA Repair
2) Self-Sufficiency in Growth Signals
3) Insensitivity to Anti-Growth Signals
4) Evading Apoptosis
5) Unlimited Replicative Potential
6) Sustained Angiogenesis
7) Tissue Invasion and Metastasis

• *Genetic Mutations or **Epigenetic Changes in these genes are
• *Seen in Every Cancer.

**Mutations in these cancer-causing genes is conditioned by **DNA Repair.

Question 3

Tumor Monoclonality

Answer 3

Tumors contain a Progeny of cells derived from a Single Transformed Ancestral Cell:

1) **X-Linked G-6-PD Isoenzyme or 
Androgen Receptor (AR) Alleles

2) Monoclonality in B-cell Tumors by Kappa / Lambda Ratio

Question 4

Tumor Growth Kinetics

Answer 4

Time Required for a Single Transformed Cell to produce a detectable Tumor Depends on 3 Variables:

1) **Cell Cycle Division Time:
= Time to Complete Cell Cycle
(24 hrs)

2) **Cell Growth Fraction:
= Proportion of Cells in Active Proliferation

3) **Cell Loss:
= Progressive Growth Depends on the Ratio of Cell Production to Cell Loss.

____________________

During Early Tumor Growth, the Vast Majority of Cells are Proliferative.

Then, Increasing Number of Tumor Cells Stop Proliferating Due to Lack of Nutrients. So, they Differentiate and Revert to G0 Phase.
_Growth @ this time is Faster due to the Exponential Kinetics.

____________________

Doubling Time
= Time Required by Tumor to Duplicate Size or Cell Number.

Question 5

Oncogenes

Answer 5

= Genes that Induce Transformation when Expressed in cells.

**Many human cancers contain Unique DNA Sequences that can Transform Mouse Fibroblasts in Vitro.
= *Cellular Oncogenes (c-oncs)

Some Retroviruses cause rapid induction of cancer in animals.
=> Due to Unique Viral Genome Sequences
= *Viral Oncogenes (v-oncs)

Protooncogenes are Probably transduced from mammalian infected cells to retroviruses during infection, resulting in v-oncs.

Question 6

Oncogene Activation

Answer 6

Protooncogenes:
= Normal Genes that Regulate Cell Growth and Development.

**Oncogene Activation:
= Conversion of Protooncogene to C-onc

_Point Mutations cannot be seen on karyotype. Translocations, Deletions, and Amplifications can.

1) **Point Mutation:
=> *Ras encodes *GTP-Binding Protein: *Colon, *Bladder
_Ras is Inactive when bound to GDP and Active when bound to GTP.
*(RAS/RAF/MAP Pathway)

2) **Translocation:
= Rearrangement of genetic material may result in Fusion of Unrelated Genes.

=> Burkitt’s Lymphoma:
_C-myc Translocation
_Encodes Nuclear Transcription Factor

=> Chronic Myelogenous Leukemia (CML):
**Abl Translocates to next to Bcr,
Results in **Abl-Bcr Fusion Gene that encodes a Tyrosine Kinase that Signals Cell Growth.

3) **Amplification:
= **Manifold Reduplication of Normal Protooncogenes; several hundred copies.

=> **Neuroblastomas: **N-myc

=> **Breast Carcinomas:
**ERB2 (HER2-neu)

=> L-myc and N-myc:
Small Cell Lung Carcinoma

4) **Suppressor Gene Inactivation:
Via Dual Allele Mutations or
Gene Deletions.

Question 7

Cyclins, CDKs, CDKIs

Answer 7

*Cyclins:
_Transiently peak at critical times in cell cycle, then are quickly degraded (short life).

_They Form Complexes with
**Cyclin Dependent Kinases (CDKs).
=> These Complexes **Orchestrate Progression throughout Cell Growth Cycle, Regulating DNA Synthesis and Mitosis.

CDKs are Only Active when Bound to Cyclins.

**Cyclin Dependent Kinase Inhibitors (CDKIs) Inactivate CDKs.

Question 8

Growth Factors

Answer 8

= **Ubiquitous Molecules Essential for Normal Cell Growth.

Act locally in paracrine or autocrine manner.

**Normal Cells Require *Growth Factors and *10% Calf Serum for in vitro growth.

**Tumor Cells Grow Well in 1% Serum WITHOUT Growth Factors!

____________________

**4 Critical Steps of Cell Growth Cascade:

1) Growth Factor Receptor **Binding
2) Receptor **Activation

3) **Signal Transduction by
*Second Messengers
(from cell membrane to throughout cytoplasm)

4) Triggering of Nuclear **Transcription Factors

Question 9

Cell Surface Receptors

Answer 9

Intrinsic Tyrosine Kinase Receptors: (EGF, FGF, PDGF)
Linked to **IP3, PI-3, and MAP Kinase Pathways

Cytosolic Tyrosine Kinase Receptors: (Cytokines)
Linked to **JAK/STAT Pathway

G Protein Receptors:
(Chemokines, Epinephrine, Glucagon)
=> Activate G Protein Complex Linked to **cAMP and Ca2+ Pathways

Question 10

Suppressor Genes

Answer 10

Suppressor Genes Encode Proteins that **Stop Cell Proliferation:

**Phosphatases, such as **PTEN, Dephosphorylate Cell Growth Mediators.

_Phosphorylation and Dephosphorylation Events Regulate Cell Growth.

Usually, Pro-Growth Genes are Activated when Phosphorylated and Inactivated by Dephosphorylation.

_____________________

Tumor Suppressor Genes are
**RECESSIVE.

=> Cancer Develops Only when the cell becomes **HOMOZYGOUS for the Mutant Allele
(Both Alleles are Mutated)

**2 Hits are Required to Transform Cell

Inherited germline mutations affecting one allele leave the cell perfectly normal but predisposed to cancer.

*Second Hit Results in
**Loss of Heterozygosity (LOH)
and **Loss of Gene Function.

_____________________

Suppressor Genes:

**PTEN (Phosphatase)
(@ cytosol; endometrial and prostate cancers)

@ Nucleus; Inherited Mutations:

*RB: *Retinoblastomas, *Osteocarcomas

• p53: *Li-Fraumeni Syndrome;
• Multiple Carcinomas and Sarcomas
• WT-1 p16 (INK4a): *Wilms Tumor
• BRCA 1 and 2: *Breast and *Ovary Carcinomas

Question 11

p53

Answer 11

Guardian of the Genome

Located on Chromosome **17p13.1

Encodes a Protein with a DNA Binding Domain that Regulates:
_DNA Replication
_Cell Growth
_Cell Death

It **Detects DNA Damage.

If there is DNA Damage, p53 will
**Arrest Cell Cycle @ G1 by Increasing **CDKI p21

It is the **Most Commonly Mutated Gene in Human Cancers

Mutations affect the DNA Binding Domain.
___________________

**ATM is also Activated by DNA Damage.

1) ATM **Phosphorylates p53,
2) p53 then moves to the Nucleus, Binds DNA, and **Becomes an Active Transcription Factor.
3) Following Successful DNA Repair, p53 **induces MDM2
4) **MDM2 Degrades p53 and Cell Cycle Proceeds.

_**In Some Tumors, p53 is Inactivated by MDM2 Overexpression.

_p53 Has a Short Life, 20 Minutes.

5) If DNA Damage Cannot be Repaired, p53 **Stimulates Cell Death by Increasing **Bax, Triggering Apoptosis.

**Cell Lifespan is Genetically Determined. Cells commit suicide after 100 divisions.

_________________________

**Bcl-2 Protein @ Inner Mitochondrial Membrane Blocks Apoptosis, Prolonging Cell Survival.
=> Follicular Lymphomas:
Overexpression of Bcl-2

Question 12

DNA Damage and Repair

Answer 12

1) Base Modifications
(Oxidation, Alkylation)
=> **Base Excision Repair (BER)

2) Nucleotide Cross-Links and
Pyrimidine Dimers (e.g. by UV)
=> **Nucleotide Excision Repair (NER)

3) Single or Double Strand Breaks
=> **Recombination Repair

4) DNA Mismatch
=> **Mismatch Repair

Question 13

Nucleotide Excision Repair

Answer 13

Corrects Bulky DNA Cross-Links (Adducts), such as those Induced by UV Light;
*Pyrimidine Dimers.

**Xeroderma Pigmentosum:
=> Increased Risk of Skin Cancers when Exposed to UV Light.

Several Genes/Proteins are involved in NER.
_Inherited Loss of any of them leads to Xeroderma Pigmentosum.

Question 14

Recombination Repair

Answer 14

1) **Ataxia-Telangiectasia
2) **Bloom Syndrome
3) **Fanconi Anemia

**Defective DNA Recombination Repair, **Predisposition to Cancer;

_And other alterations, such as

1) Neurologic symptoms (AT),
2) Developmental Defects (Bloom),
3) and Anemia (Fanconi).

Bloom Syndrome: Predisposition to a wide variety of cancers.

Ataxia-Telangiectasia: Mutations of ATM, which Detects Double-Stranded DNA Damage and Responds by Phosphorylating/Activating p53.

Question 15

Mismatch Repair

Answer 15

Hereditary Non-Polyposis Colon Cancer Syndrome:

=> **Early Onset Familial Colon Cancer

_Due to Defective Mismatch Repair.

_Affects Proximal Colon / Cecum.

_**May Associate with *Endometrial or *Ovarian Cancer.

Mismatch Repair Mechanism:

1) MSH2-MSH6 (MutS) Recognizes Mismatch.
2) MLH1-PMS2 (MutL) Binds to MutS.
3) MutS-MutL Complex Recruits other enzymes.

Enzymes involved:
*MLH1
MLH3
*MSH2
MSH6
*PMS1
*PMS2
GTBP

Question 16

BRCA1 and BRCA2

Answer 16

**BRCA1:
=> Increases Risk of Cancers:
1) **Fallopian Tube / **Ovarian
2) and **Prostate

**BRCA2:
=> Increases Risk of Cancers:
1) **Fallopian Tube / **Ovarian
2) and **Pancreatic

Function not well defined. Both are nuclear proteins that bind to RAD51, a gene involved in Double Stranded DNA Break Repair.

Question 17

Epigenetics and Cancer

Answer 17

Epigenetic Changes such as
*Histone Modifications and
*DNA Methylation may Silence paternal or maternal Gene Alleles
= **Imprinting

Cancer is Associated with
**Selective Hypermethylation of
Promoter CpG Islands, thus Silencing Suppressor Genes.

**Demethylation of Silenced Suppressor Genes may revert its Normal Expression.

Question 18

Micro RNAs (miRNAs)

Answer 18

= Small (22 nucleotides)
Single-Stranded RNAs that Regulate **RNA-induced Silencing Complex,
=> which is a **Post-Transcriptional Regulator of Gene Expression of
cell growth, differentiation, and survival.

May Increase Oncogene Expression or Decrease Suppressor Gene Expression.

**miRNAs Profiling Shows Specific Changes in Cancer

(e.g. Bcl-2 Overexpression in some leukemias and lymphomas)

Question 19

Telomerase

Answer 19

**Telomeres = Repeated DNA Sequences @ the End of Chromosomes.

It shortens with Each Cell Division.

After ~100 Cycles, Cells Become Arrested in a Non-Dividing State
= **Replicative Cell Senescence

==> *Leading to Activation of p53
and *Apoptosis.

**Telomerase can re-synthesize Telomeres to Maintain Chromosomal Integrity.
_____________________

Telomerase is @ Germ cells,
@ Stem cells, and @ Tumor cells.

***Telomerase Activity is Absent in Most **Somatic Cells,
_But is Expressed in 90% of **Tumors.

Reactivation of Telomerase Activity and Maintenance of Telomere Length Inhibits Cell Senescence.
=> Tumor Cells Become Immortalized.

=>**Unlimited Proliferative Capacity, which increases Probability of acquiring new genetic alterations,
**Enhancing Tumor Progression.

Question 20

Sustained Angiogenesis

Answer 20

**Tumors cannot enlarge Beyond
1 - 2 mm in Diameter unless Vascularized.

Early Tumors Don’t Engage in Angiogenesis.
_An **Angiogenic Switch occurs beyond a critical size.

=> **Production of Angiogenic Factors by *Tumor Cells and by

• Tumor-Infiltrating Leukocytes:
  1) **VEGF:
• -mostly produced by Tumor cells, but also by Tumor Stroma.

2) **Basic Fibroblast Growth Factor (bFGF)
3) Also, Decreased Production of Inhibitors of Angiogenesis

Also!

4) **p53 Inhibits Angiogenesis by Inducing **Thrombospondin-1,
which **Downregulates VEGF.

=> **Mutated p53 Loses this Function, and Promotes Angiogenesis.

**Endostatin: Angiogenesis Inhibitor in Cancer Therapy.

Also, Trials with mAbs inhibiting VEGF and VEGF-R2 are promising.

Question 21

The Incredible Journey of Metastasis

Answer 21

Metastasis Cascade:

1) Local Invasion
2) Intravasation into Blood and Lymph Vessels
3) Transit through Vasculature
4) Extravasation from the Vessels
5) Formation of micrometastases
6) Growth of micrometastases into macroscopic tumors.

Can be Subdivided into 2 Phases:

1) Invasion of ECM
2) Vascular Dissemination and Homing of Tumor Cells

Question 22

Invasion of Extracellular Matrix (ECM)

Answer 22

1) Loss of Intercellular Junctions and Loss of Adhesion; Detachment.
2) Local Degradation of Basement Membrane and Interstitial Connective Tissue:
3) Attachment to ECM Proteins:

_Normal Epithelial cells express High-affinity Integrins for Basement Membrane Laminin and collagens that are polarized @ their basal surface.

_Carcinoma Cells Overexpress these Laminin Receptors all over their Cell Surface.

_Tumor Cells express Integrins that adhere to Laminin, Fibronectin, and Collagens in the ECM.

4) Locomotion:

_**Tumor cells

(1) Secrete Proteases or
(2) Induce Stromal Fibroblasts / Macrophages to Secrete Proteases that DEGRADE ECM.

_This protease activity is tightly controlled by protease inhibitors.

_@ the Front Edge of Invasive Tumors, Protease Activity Prevails over Protease Inhibition.

**Matrix Metalloproteinases (MMPs)
(such as MMP9 and MMP2)
= Are Collagenases **Overproduced by Invasive Carcinomas.
=> They Degrade Type IV Collagen.
=> They are NOT Produced by Cells of Carcinoma In Situ.

**MMP Inhibitors: potential strategy for treatment.

5) MMPs Generate **Cleavage Products of ECM **Collagens and Proteoglycans, **Promoting Angiogenesis, Tumor Cell Growth, and Tumor Cell Motility.

_Some of these products are Chemotactic to Tumor cells and Promote Tumor Cell Migration.

Question 23

Vascular Dissemination and Homing

Answer 23

1) @ in Circulation, Tumor Cells are Vulnerable to Destruction by **Immune System.

=> Tumor Cells **Aggregate in Platelet Clumps, **Hiding from Immune System.

2) Arrest and Extravasation Requires
**Adhesion to the Vascular Endothelium
=> Mediated by **CD44
(Overexpressed in Metastatic Cells)

3) *Site of Metastasis Depends on Location of Primary Tumor:

_*Certain Tumors Have Specific Tropism:
=> **Prostate Cancer for Bone;
=> Lung Cancer for Adrenals and Brain;
=> etc.

_Due to the type of
**Adhesion Molecules Produced by **Tumor, whose Ligands may be Preferentially Expressed on Certain Organ Endothelium.

_**Chemokines:
=> Breast cancer cells express CXCR4 and CCR7 Receptors for Chemokines highly expressed in those tissue to which breast cancers commonly metastasize.

Question 24

Genes of Metastasis

Answer 24

**miRNAs in **Breast Cancer:
=> **mir335 and **mir126 Suppress Metastasis in Breast Cancer,

=> **mir10b Promotes Metastasis in Breast Cancer.

**SNAIL and **TWIST are Metastasis Oncogenes:

=> **Promote Epithelial-to-Mesenchymal Transition (EMT)

_During EMT, **Carcinoma Cells **Downregulate Epithelial Markers
(E-cadherin) Gene Expression,
while **Upregulating Mesenchymal Markers
(vimentin, smooth muscle actin).

=> This Switch Leads to a
**Pro-Migratory Phenotype, which Promotes Metastasis.

Question 25

Stromal Microenvironment and Carcinogenesis

Answer 25

**Cross Talk Between ECM and Tumor cells is of vital importance.

ECM Stores Inactive Growth Factors, such as PDGF, TGF-beta, b-FGF, or VEGF, which are Released by MMPs and Exposed to Tumor Cells During Tumor Invasion.

Stromal Cells from ECM Transmit Oncogenic Signals to Tumor Cells:

=> Prostate Cancer: Smooth Muscle cells Adjacent to
Benign Epithelium Transform into
Carcinoma-Associated Fibroblasts when Exposed in Contact with Tumor Cells
= (Tumor Conspirators).

_These carcinomas have new genetic abnormalities absent in the parental tumor cell line;
=> Thus, **Tumor Stroma Can Promote Carcinogenesis.

Question 26

Carcinogenesis

Answer 26

1) **Chemical:
=> *Initiator: *Produces Permanent DNA Damage,
_Rendering the cell susceptible to Transformation.
_Initiator alone is insufficient to transform the cell.

=> *Promoter: *Induces Proliferation of *Initiated Cells
_But it is Non-tumorigenic;
_It Does not produce DNA Damage.

2) *Radiation
3) *Viruses and other Microbes

Question 27

Chemical Carcinogenesis

Answer 27

Initiation:

Permanent **DNA Damage (Mutation) Induced by Carcinogen.

Two Classes of Agents:
=> **Direct-Acting: Modifies DNA
=> **Indirect-Acting (Procarcinogen):
_Must be Modified (Metabolic Processing) to Become Carcinogenic.

Most Procarcinogens are Activated by CYP450 Dependent Mono-oxygenases.

________________________

Promotion:

When Alone, it’s Non-Muatgenic.
_Their Effects are Reversible Over Time.

Stimulate Proliferation of Target Cells.
=> **Cell Proliferation “Fixes” DNA Damage as a Permanent Mutation in a Progeny of Initiated and Clonal Cells.

=> Initiated Mutated Cells are Susceptible to Growth Promoter Effect Due to Mutations of Cell Growth Regulatory Genes.

________________________

Initiators:

1) **Alkylating Agents:
_Used in Cancer Chemotherapy:
_Chlorambucil, Cyclophosphamide, Melphalan.

2) **Polycyclic Aromatic Hydrocarbons:
_Tobacco, Animal Fats
_Benzo(a)pyrene

3) **Aromatic Amines (Dyes):
_Potent Carcinogens
@ Bladder Urothelium

4) **Natural Plant and Microbial Products:
_Aflatoxin: Product of Aspergillus Flavus that is a Potent Carcinogen
@ Liver

Promoters:

1) **Phorbol Esters:
_Derivatives of **Croton Oil

2) **Okadaic Acid:
_Inhibitor of Protein Phosphatases

3) **Wounding:
_Tissue Damage and Reactive Hyperplasia

4) **Hormones (Sex Steroids)
5) Other postulated promoters: fat in colon cancer; alcohol in cancer of mouth, pharynx, and larynx.

Question 28

Radiation Carcinogenesis

Answer 28

1) **Ultraviolet Light:
=> Induces *Pyrimidine Dimers in DNA:
_Repaired by Nucleotide Excision Repair; Defective in Xeroderma Pigmentosum.

=> Effects on Skin Result in *Increased Risk of Skin Cancers.

2) **Ionizing Radiation:
=> Increases Risk for *Leukemia,
Particularly of Myeloid Types.

=> **Iodine Exposure => Risk for **Thyroid Carcinoma.

Question 29

Oncogenic DNA Viruses

Answer 29

1) **Human Papilloma Virus (HPV):

_Causes Warts, Condyloma, and Squamous Cell Carcinoma @ Cervix

=> **E7 Binds and *Inactivates Rb
=> **E6 Binds and Inactivates p53/Bax, Blocking Apoptosis and Activating Telomerase.

2) **Epstein-Barr Virus (EBV):

**EBV Latent Membrane Protein (LMP-1):
= Constitutively Active CD40-like Receptor that Signals B cell Growth;
=> Blocks Bcl2 and Apoptosis,
=> Stimulates NFkB and JAK/STAT Pathways to Immortalize B cells.

EBV => Associated with 
_Burkitt Lymphoma,
_Some Hodgkin's Lymphomas,
_Nasopharyngeal Carcinoma,
_Post-Transplant B Cell Lymphomas

3) **Hepatitis B Virus (HBV):
=> Hepatocellular Cancer

4) *Kaposi’s Sarcoma Herpes Virus:
(**KSHV, **HHV-8)
=> Kaposi’s Sarcoma in AIDS
=> Body-Cavity based Lymphomas

Question 30

Oncogenic RNA Viruses

Answer 30

**Human T Cell Leukemia Virus-1
(HTLV-1):
=> Adult T Cell Leukemia/Lymphoma

Question 31

Oncogenic Bacteria

Answer 31

**Heliobacter Pylori:
=> Gastric Carcinoma and Lymphoma
(MALToma)