Neoplasia part ii

Neoplasia II

Dr Bahoran Singh

Molecular Basis of CancerSome fundamental principles • Nonlethal genetic damage lies at the heart of

carcinogenesis• Tumor is formed by the clonal expansion of a single

precursor cell that has incurred genetic damage (i.e., tumors are monoclonal)

• Four classes of normal regulatory genes are principal targets of genetic damage

1. The growth-promoting proto-oncogenes, 2. The growth-inhibiting tumor suppressor genes,3. Genes that regulate programmed cell death (apoptosis)4. Genes involved in DNA repair

• Carcinogenesis is a multistep process at both the phenotypic and the genetic levels, resulting from the accumulation of multiple mutations

• Carcinogenesis results from the accumulation of complementary mutations in a stepwise fashion over time.

• Malignant neoplasms have several phenotypic attributes referred to as cancer hallmarks.• Mutations that contribute to the development of the malignant phenotype are referred to as driver mutations.• Loss of function mutations in genes that maintain genomic integrity appear to be a common early step on the road to malignancy, particularly in solid tumors.

  Development of a cancer through stepwise acquisition of complementary mutations.

• Once tumor evolved, its progression under the pressure of Darwinian selection (survival of the fittest).

• Selection of fittest explain the natural history of cancer and changes in tumor behaviour following therapy.

• In addition to genetic mutations, epigenetic aberrations also contribute to the malignant properties of cancer cells ( like DNA methylation and histone modification.)

Cellular and Molecular Hallmarks of Cancer

The eight key changes in cell physiology that together determine malignant phenotype

1. Self-sufficiency in growth signals2. Insensitivity to growth-inhibitory signals3. Altered cellular metabolism (Warburg effect)4. Evasion of apoptosis5. Limitless replicative potential ( immortality)6. Sustained angiogenesis7. Ability to invade and metastasize8. Ability to evade the host immune response.

Hallmarks of cancer

Normal Cell Cycle and Its Regulation

SELF-SUFFICIENCY IN GROWTH SIGNALS: ONCOGENES

• Genes that promote autonomous cell growth in cancer cells are called oncogenes

• Oncogenes are created by mutations in proto-oncogenes• Ability to promote cell growth in the absence of normal

growth-promoting signals• Their products, called oncoproteins, resemble the normal

products of proto-oncogenes except 1. Oncoproteins are often devoid of important internal

regulatory elements2. Their production in the transformed cells does not

depend on growth factors or other external signals

Under physiologic conditions cell proliferation can be readily resolved into the following steps• Binding of a growth factor to its specific receptor • Transient and limited activation of the growth factor

receptor• Activation of several signal-transducing proteins • Transmission of the transduced signal across the

cytosol to nucleus via second messengers or by cascade of signal transduction molecules

• Induction and activation of nuclear regulatory factors that initiate DNA transcription

• Entry and progression of the cell into the cell cycle, ultimately resulting in cell division

Proto-oncogenes, Oncogenes, and Oncoproteins

• Proto-oncogenes have multiple roles, participating in cellular functions related to growth and proliferation

• Oncoproteins encoded by oncogenes generally serve functions similar to their normal counterparts

• Mutations convert proto-oncogenes into constitutively active cellular oncogenes involved in tumorogenesis because oncoproteins endow cell with self-sufficiency in growth

Growth Factors• Normal cells require stimulation by growth factors to

undergo proliferation• Many cancer cells acquire the ability to synthesize

same growth factors to which they are responsive, generating an autocrine loop

• In most instances the growth factor gene itself is not altered or mutated

• Products of other oncogenes that lie along many signal transduction pathways, cause overexpression of growth factor genes

• Increased growth factor production is not sufficient for neoplastic transformation

• Growth factor driven proliferation contributes to malignant phenotype by increasing the risk of spontaneous or induced mutations in the proliferating cell population

Growth FactorsCATEGORY Proto-

OncogeneMode of Activation in Tumor

Associated Human Tumor

Growth Factors

PDGF- β chain PDGFB Overexpression Astrocytoma

Fibroblast growth factors

HST1FGF3

OverexpressionAmplification

osteosarcomaStomach cancerBladder cancerBreast cancerMelanoma

TGF- α TGFA Overexpression Astrocytoma

HGF HGF Overexpression Hepatocellular carcinomasThyroid cancer

Growth Factor Receptors

• Oncogenic versions of these receptors are associated with constitutive dimerization and activation without binding to the growth factor

• Hence, the mutant receptors deliver continuous mitogenic signals to the cell, even in the absence of growth factor in the environment

• Growth factor receptors can be constitutively activated in tumors by multiple different mechanisms, including mutations, gene rearrangements, and overexpression

• Far more common than mutations of these proto-oncogenes is overexpression of normal forms of growth factor receptors.

• Point mutations of RAS family genes constitute the most common type of abnormality involving proto oncogenes in human tumors.

Growth Factor ReceptorsCATEGORY Proto-Oncogene Mode of Activation in

TumorAssociated Human Tumor

EGF-receptor family

ERBB1 (EGFR)ERBB2 (HER)

MutationAmplification

Adenocarcinoma of lungBreast carcinoma

FMS-like tyrosine kinase 3

FLT3 Point mutation Leukemia

Receptor for neurotrophicfactors

RET Point mutation Multiple endocrine neoplasia 2A and B, familial medullary thyroid carcinomas

PDGF receptor PDGFRB Overexpression, translocation

Gliomas, leukemias

Receptor for KIT ligand

KIT Point mutation Gastrointestinal stromal tumors, seminomas, leukemias

ALK receptor ALK Translocation, fusion gene formation Point mutation

Adenocarcinoma of lung, certain lymphomasNeuroblastoma

Signal-Transducing Proteins• Oncoproteins that mimic the function of normal

cytoplasmic signal-transducing proteins have been found

• Most such proteins are strategically located on the inner leaflet of the plasma membrane

• They receive signals from outside the cell ( by GFR activation) and transmit them to the cell's nucleus

• Biochemically, the signal-transducing proteins are heterogeneous

Proteins Involved in Signal TransductionCATEGORY Proto-Oncogene Mode of Activation in

TumorAssociated Human Tumor

GTP-binding (G) proteins

KRAS

HRASNRAS

GNAQGNAS

Point mutation

Point mutationPoint mutation

Point mutationPoint mutation

Colon, lung, and pancreatic tumorsBladder and kidney tumorsMelanomas, hematologic malignanciesUveal melanomaPituitary adenoma, other endocrine tumors

Nonreceptor tyrosinekinase

ABL Translocation

Point mutation

Chronic myelogenous leukemiaAcute lymphoblastic leukemia

RAS signal transduction

BRAF Point mutation, Translocation

Melanomas, leukemias, colon carcinoma, others

Notch signal transduction

NOTCH1 Point mutation, TranslocationGene rearrangement

Leukemias, lymphomas, breast carcinoma

JAK/STAT signaltransduction

JAK2 Translocation Myeloproliferative disordersALL

Receptor-mediated signaling

• Most well-studied example of a signal-transducing oncoprotein is the RAS genes

• Normal RAS proteins are tethered to the cytoplasmic aspect of the plasma membrane, as well as the endoplasmic reticulum and Golgi membranes

• Normally RAS proteins flip back and forth between an excited signal-transmitting and a quiescent state

• Activated RAS stimulates downstream regulators of proliferation, as mitogen-activated protein (MAP)

• The removal of GDP and its replacement by GTP during RAS activation are catalyzed by a family of guanine nucleotide–releasing proteins

• Conversely, the GTPase activity intrinsic to normal RAS proteins is dramatically accelerated by GTPase-activating proteins (GAPs)

• These widely distributed proteins bind to the active RAS and augment its GTPase activity leading to termination of signal transduction

• GAPs function as “brakes” that prevent uncontrolled RAS activity

Growth factor signaling pathways in cancer

Alterations in Nonreceptor Tyrosine Kinases• Mutations occur in several non-receptor-associated

tyrosine kinases, which normally function in signal transduction pathways that regulate cell growth.

• As with receptor tyrosine kinases, in some instances the mutations take form of chromosomal translocations or rearrangements .

• They create fusion genes encoding constitutively active tyrosine kinases.

The chromosomal translocation and associated oncogenes inBurkitt lymphoma and chronic myelogenous leukemia

Transcription Factors• All signal transduction pathways converge to the

nucleus.• In nucleus large bank of responder genes that

orchestrate cell's orderly advance through the mitotic cycle, get activated.

• Ultimate consequence of signaling through oncogenes like RAS or ABL is inappropriate and continuous stimulation of nuclear transcription factors that drive growth-promoting genes

• Transcription factors contain specific amino acid sequences or motifs that allow them to bind DNA or to dimerize for DNA binding.

• Binding of these proteins to specific sequences in the genomic DNA activates transcription of genes.

• Growth autonomy may thus occur as a consequence of mutations affecting genes that regulate transcription.

Nuclear Regulatory ProteinsCATEGORY Proto-Oncogene Mode of Activation

in TumorAssociated Human Tumor

Transcriptional activators

MYCNMYC

TranslocationAmplifiation

Burkitt lymphomaNeuroblastoma

The MYC Oncogene• MYC proto-oncogene expressed in all eukaryotic cells.• The MYC proto-oncogene belongs to the immediate early

response genes, which are rapidly induced by RAS/MAPK siganaling when quiescent cells receive a signal to divide.

• After a transient increase of MYC messenger RNA, the expression declines to a basal level.

• Range of activities modulated by MYC includes histone acetylation, reduced cell adhesion, increased cell motility, increased telomerase activity, increased protein synthesis, decreased proteinase activity, & changes in cellular metabolism that enable a high rate of cell division.

• MYC interacts with components of the DNA-replication machinery, and plays a role in the selection of origins of replication

• Thus, overexpression drive activation of more origins than needed for normal cell division, or bypass checkpoints involved in replication

• Leading to genomic damage and accumulation of mutations

• MYC activates the expression of many genes that are involved in cell growth.

• Some MYC target genes, like D cyclins, are directly Involved in cell cycle progression.

• It up regulates the expression of rRNA genesand rRNA processing, so enhancing assembly of ribosomes needed for protein synthesis.

• It upregulates a program of gene expression that leads to metabolic reprogramming and the Warburg effect.

• MYC upregulates expression of telomerase.• It can reprogram somatic cells into pluripotent stem

cells.

• MYC may also enhance self-renewal, block differentiation, or both

• Persistent expression, overexpression, dysregulation of MYC expression resulting from translocation of the gene (Burkitt lymphoma) & MYC amplification (neuroblastomas, breast, colon,& lung ) commonly found in tumors.

• Constitutive RAS/MAPK signaling (many cancers), Notch signaling (several hematologic cancers), Wnt signaling (colon carcinoma), and Hedgehog signaling (medulloblastoma) all transform cells in part through upregulation of MYC.

Amplification of the NMYC gene in human neuroblastomas

Normally present on chromosome 2p, becomes amplified and is seen either as extra chromosomal double minutes or as a chromosomally integrated, homogeneous staining region (HSR). The integration involvesother autosomes, such as 4, 9, or 13.

Cyclins and Cyclin-Dependent Kinases

• The CDK-cyclin complexes phosphorylate crucial target proteins that drive the cell through the cell cycle

• Mishaps affecting the expression of cyclin D or CDK4 seem to be a common event in neoplastic transformation

• Cyclin D genes overexpressed in Ca of breast, esophagus, liver, and a subset of lymphomas

• Amplification of the CDK4 gene occurs in melanomas, sarcomas, and glioblastomas

Role of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors in regulating the cell cycle.

• While cyclins arouse the CDKs, inhibitors (CDKIs), silence the CDKs & exert negative control .

• CIP/WAF family of CDKIs, composed of three proteins, called p21 , p27 , and p57 inhibits the CDKs broadly.

• INK4 family of CDIs, made up of p15, p16, p18 , and p19, selectively effects on cyclin D/CDK4 and cyclin D/CDK6.

• Expression of these inhibitors is down-regulated by mitogenic signaling pathways, thus promoting the progression of the cell division.

• Internal controls of the cell cycle called checkpoints• Two main cell cycle checkpoints, one at the G1/S

transition and the other at G2/M.• S phase is the point of no return in the cell cycle.G1/S checkpoint • Checks for DNA damage before point of no return in

cell cycle.• If damage is present DNA-repair machinery and

mechanisms that arrest the cell cycle activated.

• The delay in cell cycle progression provides the time needed for DNA repair.

• If the damage is not repairable, apoptotic pathways activated to kill the cell.

• Thus, the G1/S checkpoint prevents the replication of cells that have defects in DNA, which would be perpetuated as mutations or chromosomal breaks in the progeny of the cell.

• Gain of function mutations in D cyclin genes andCDK4, oncogenes that promote G1/S progression.

G2/M checkpoint

• Monitors the completion of DNA replication and checks whether cell can safely initiate mitosis and separate sister chromatids.

• This checkpoint is particularly important in cells exposed to ionizing radiation.

• Cells damaged by ionizing radiation activate the G2/M checkpoint and arrest in G2.

• Defects in this checkpoint give rise to chromosomal abnormalities

• To function properly, cell cycle checkpoints require sensors of DNA damage, signal transducers, and effector molecules

• Sensors and transducers of DNA damage seem to be similar for G1/S and G2/M checkpoints.

• They include, as sensors, proteins of the RAD family and ataxia telangiectasia mutated (ATM) and as transducers, the CHK kinase families.

• In G1/S checkpoint, cell cycle arrest mediated through p53, which induces cell cycle inhibitor p21.

• Arrest of cell cycle by G2/M checkpoint involves both p53-dependent and p53-independent mechanisms.

• Defects in cell cycle checkpoint components are a major cause of genetic instability in cancer cells.

Cell Cycle Components and Inhibitors

TUMOR SUPPRESSOR GENESINSENSITIVITY TO GROWTH INHIBITION AND ESCAPE

FROM SENESCENCE• Failure of growth inhibition is one of the fundamental

alterations in the process of carcinogenesis.• Whereas oncogenes drive the proliferation of cells, the

products of tumor suppressor genes apply brakes to cell proliferation.

• Tumor suppressor proteins form a network of checkpoints that prevent uncontrolled growth.

• Many tumor suppressors, such as RB and p53, are part of a regulatory network that recognizes genotoxic stress from any source, and responds by shutting down proliferation.

• Expression of an oncogene in an otherwise completely normal cell leads to quiescence, or to permanent cell cycle arrest (oncogene-induced senescence), rather than uncontrolled proliferation.

• Ultimately, the growth-inhibitory pathways may lead the cells into apoptosis.

• Another set of tumor suppressors seem to be involved in cell differentiation, causing cells to enter a post-mitotic, differentiated pool without replicative potential.

• Similar to mitogenic signals, growth-inhibitory, pro-differentiation signals originate outside the cell and use receptors, signal transducers, and nuclear transcription regulators .

Tumor Suppressor Genes and Associated Familial Syndromes and Cancers

Gene Protein function Familial syndrome Sporadic Cancer

Retinoblastoma gene: Governor of Proliferation.

• RB locus on chromosome 13q14 .• RB protein,product of RB gene, is ubiquitously

expressed nuclear phosphoprotein that plays a key role in regulating the cell cycle.

• Active hypophosphorylated state in quiescent cells and inactive hyperphosphorylated state in the G1/S cell cycle transition .

• It is a key negative regulator of G1 /S cell cycle transition.

• In G1, however, cells can exit the cell cycle, either temporarily quiescence or permanently senescence.

• In G1, therefore, diverse signals integrated to determine whether the cell should enter the cell cycle, exit the cell cycle and differentiate, or die.

• RB is a key node in this decision process as once cells cross the G1 checkpoint can be paused cell cycle for a time, but they are obligated to complete mitosis.

• Mechanism of Retinoblastoma in initiating DNA replication by activating cyclin E gene transcription.

The role of RB in regulating the G1-S checkpoint of the cell cycle.

• If RB is absent (gene mutations) or its ability to regulate E2F transcription factors is derailed, the molecular brakes on the cell cycle are released, and the cells move through the cell cycle.

• The mutations of RB genes found in tumors are localized to a region of the RB protein, called the “RB pocket,” that is involved in binding to E2F.

• However, the versatile RB protein has also been shown to bind to a variety of other transcription factors that regulate cell differentiation .

Pathogenesis of retinoblastoma. Two mutations of the RB locus on chromosome 13q14 lead to neoplastic proliferation of the retinal cells. In the sporadic form, both RB

mutations in the tumor-founding retinal cell are acquired. In the familial form, all somatic cells inherit one mutated copy of RB gene from a carrier parent, and as a result only one

additional RB mutation in a retinal cell is required for complete loss of RB function.

TWO HIT HYPOTHESIS

• RB stimulates myocyte-, adipocyte-, melanocyte-, and macrophage-specific transcription factors.

• Thus, the RB pathway couples control of cell cycle progression at G1 with differentiation, which may explain how differentiation is associated with exit from the cell cycle.

• In addition to these dual activities, RB can also induce senescence.

p53: Guardian of the Genome

• The p53 gene is located on chromosome 17p13.1, and most common target for genetic alteration in human tumors.

• A tumor suppressor gene that regulates cell cycle progression, DNA repair, cellular senescence, and apoptosis.

• Homozygous loss of p53 occurs in virtually every type of cancer, including carcinomas of the lung, colon, and breast—the three leading causes of cancer death.

• In most cases, the inactivating mutations affect both p53 alleles and are acquired in somatic cells.

• Less commonly, some individuals inherit one mutant p53 allele.

• Inheritance of one mutant allele predisposes individuals to develop malignant tumors because only one additional “hit” is needed to inactivate the second, normal allele.

• Such individuals, said to have the Li-Fraumeni syndrome, have a 25-fold greater chance of developing a malignant tumor by age 50 than the general population.

• p53 mutations are common in a variety of human tumors suggests that the p53 protein functions as a critical gatekeeper against the formation of cancer.

• Indeed, p53 acts as a “molecular policeman” that prevents the propagation of genetically damaged cells.

• P53 protein is a transcription factor that is at the center of a large network of signals that sense cellular stress, such as DNA damage, shortened telomeres, and hypoxia.

• Many activities of the p53 protein are related to its function as a transcription factor.

• Several hundred genes have been shown to be regulated by p53 in numerous different contexts.

• 80% of the p53 point mutations present in human cancers are located in the DNA-binding domain of the p53 protein.

• In non-stressed, healthy cells, p53 has a short half-life (20 minutes), because of its association with MDM2, a protein that targets it for destruction.

• When the cell is stressed, by an assault on its DNA, p53 undergoes post-transcriptional modifications that release it from MDM2 and increase its half-life.

• Unshackled from MDM2, p53 also becomes activated as a transcription factor.

• Hundreds of genes whose transcription is triggered by p53 grouped into two broad categories-

• 1. Causes cell cycle arrest • 2. Causes apoptosis• If DNA damage can be repaired during cell cycle

arrest, the cell reverts to a normal state.• If the repair fails, p53 induces apoptosis or

senescence.

The role of p53 in maintaining the integrity of the genome

• p53-mediated cell cycle arrest may be considered the primordial response to DNA damage.

• It occurs late in the G1 phase and is caused mainly by p53-dependent transcription of the CDK inhibitor CDKN1A (p21).

• p21 inhibits cyclin-CDK complexes and phosphorylation of RB, thereby preventing cells from entering G1 phase.

• p53 also inducing certain proteins, such as GADD45 (growth arrest and DNA damage), that help in DNA repair.

• p53 can stimulate DNA-repair pathways by transcription-independent mechanisms as well.

• If DNA damage is repaired successfully, p53 up-regulates transcription of MDM2, leading to its own destruction and thus releasing the cell cycle block.

• If the damage cannot be repaired, the cell may enter p53-induced senescence or undergo p53-directed apoptosis.

• p53-induced senescence • It is a permanent cell cycle arrest.• Characterized by specific changes in morphology and

gene expression that differentiate it from quiescence or reversible cell cycle arrest.

• Senescence requires activation of p53 and/or RB and expression of their mediators, such as the CDK inhibitors, and is generally irreversible.

• Mechanisms of senescence involve epigenetic changes that result in the formation of heterochromatin at different loci throughout the genome.

• These senescence-associated heterochromatin foci include pro-proliferative genes regulated by E2F.

• This drastically and permanently alters expression of these E2F targets.

• Like all p53 responses, senescence may be stimulated in response to a variety of stresses, such as unopposed oncogene signaling, hypoxia, and shortened telomeres

• p53-induced apoptosis of cells with irreversible DNA damage is the ultimate protective mechanism against neoplastic transformation.

• p53 directs the transcription of several pro-apoptotic genes such as BAX and PUMA.

• It appears that affinity of p53 for the promoters and enhancers of DNA-repair genes is stronger than its affinity for pro-apoptotic genes.

• Thus, the DNA-repair pathway is stimulated first, while p53 continues to accumulate.

• If the DNA damage is not repaired, enough p53 accumulates to stimulate transcription of the pro-apoptotic genes and the cell dies.

• p53 activates transcription of the mir34 family of miRNAs.

• miRNAs, bind to cognate sequences in the 3 ′untranslated region of mRNAs, preventing translation.

• Blocking targets of mir34s include pro-proliferative genes such as cyclins, and anti-apoptotic genes such as BCL2 to induce growth arrest and apoptosis.

• With loss of p53 function, DNA damage goes unrepaired, driver mutations accumulate in oncogenes and other cancer genes, and the cell leads to malignant transformation.

• It also has therapeutic implication like wild type TP53 are more likely killed by radiation and chemotherapy.

• Eg. Testicular teratocarcinomas and childhood acute lymphoblastic leukemias have wild typeTP53 alleles.

• Lung cancers and colorectal cancers, which frequently carry TP53 mutations, are relatively resistant to chemotherapy and irradiation.

APC: Gatekeeper of Colonic Neoplasia.

• A tumor suppressors.• Main function is to down-regulate growth-promoting

signals.• Germ-line mutations at the APC (5q21) loci are

associated with familial adenomatous polyposis.• All individuals born with one mutant allele develop

thousands of adenomatous polyps in the colon during their teens or 20s (familial adenomatous polyposis).

• One or more of these polyps undergoes malignant transformation, giving rise to colon cancer.

• As with other tumor suppressor genes, both copies of the APC gene must be lost for a tumor to arise.

• 70% to 80% of nonfamilial colorectal carcinomas and sporadic adenomas also show homozygous loss of the APC gene.

• Implicating APC loss in the pathogenesis of colonic tumors.

• APC is a component of the WNT signaling pathway.• Major role in controlling cell fate, adhesion, and cell

polarity during embryonic development.• WNT signaling also required for self-renewal of

hematopoietic stem cells.• WNT signals through a family of cell surface

receptors called frizzled (FRZ), and stimulates several pathways.

• The central one involving β-catenin and APC.

The role of APC in regulating the stability and function of β-catenin

• Loss of cell-cell contact, such as in a wound or injury to the epithelium, disrupts the interaction between E-cadherin and β-catenin.

• This allows β-catenin to travel to the nucleus and stimulate proliferation.

• Re-establishment of these E-cadherin leads to β-catenin again being sequestered at the membrane and reduction in the proliferative signal.

• This mechanism known as contact-inhibition mechanism.

• Loss of contact inhibition, by mutation of the E-cadherin/β-catenin axis, or by other methods, is characteristic of carcinomas.

• Loss of cadherins can favor the malignant phenotype by allowing easy disaggregation of cells, which can then invade locally or metastasize.

• Reduced cell surface expression of E-cadherin has been seen in Ca esophagus, colon, breast, ovary, and prostate.

CDKN2A INK4a/ARF

• Also called the CDKN2A gene locus.• INK4a/ARF locus encodes two protein products.1. p16/INK4a CDKI, which blocks cyclin D/CDK2-mediated

phosphorylation of RB, keeping the RB checkpoint in place.

2. p14/ARF, activates the p53 pathway by inhibiting MDM2 and preventing destruction of p53.

• Both protein products function as tumor suppressors.• Mutation or silencing by hypermethylation of the gene

of this locus impacts both the RB and p53 pathways.

The TGF-β Pathway

• Occur in most normal epithelial, endothelial, and hematopoietic cells.

• TGF-β is a potent inhibitor of proliferation.• It regulates cellular processes by binding to a serine-

threonine kinase complex composed of TGF-β receptors I and II.

• Dimerization of the receptor upon ligand binding leads to activation of the kinase and phosphorylation of receptor SMADs (R-SMADs).

• Upon phosphorylation, R-SMADs can enter the nucleus, bind to SMAD-4, and activate transcription of genes, including the CDKIs p21 and p15/INK4b.

• In addition, TGF-β signaling leads to repression of c-MYC, CDK2, CDK4, and cyclins A and E.

• These changes result in decreased phosphorylation of RB and cell cycle arrest.

• In many forms of cancer the growth-inhibiting effects of TGF-β pathways are impaired by mutations in the TGF-β signaling pathway.

• These mutations may affect the type II TGF-β receptor or interfere with SMAD molecules.

• Mutations affecting the type II receptor are seen in cancers of the colon, stomach, and endometrium.

• Mutational inactivation of SMAD4 is common in pancreatic cancers. In 100% of pancreatic cancers and 83% of colon cancers, at least one component of the TGF-β pathway is abnormal.

NF1 gene

• Individuals who inherit one mutant allele of the NF1 gene develop numerous benign neurofibromas.

• Inactivation of the second copy of the gene results in optic nerve gliomas.

• This condition is called neurofibromatosis type 1.• Some of the neurofibromas later develop into

malignant peripheral nerve sheath tumors.

• Neurofibromin, a GTPase-activating domain, regulating signal transduction through RAS proteins.

• Neurofibromin facilitates conversion of RAS from an active to an inactive state.

• With loss of neurofibromin function, RAS is trapped in an active, signal-emitting state.

NF2 gene

• Germline mutations in the NF2 gene predispose to the development of neurofibromatosis type 2.

• Individuals develop benign bilateral schwannomas of the acoustic nerve.

• Somatic mutations affecting both alleles of NF2 have also been found in sporadic meningiomas and ependymomas.

• Neurofibromin 2 or merlin, homologous with the red cell membrane cytoskeletal protein 4.1.

• Also related to the ERM (ezrin, radixin, and moesin) family of membrane cytoskeleton-associated proteins.

• Cells lacking merlin incapable of establishing stable cell-to-cell junctions and insensitive to normal growth arrest signals by contact inhibtion.

VHL gene

• Von Hippel-Lindau (VHL) gene located on chromosome 3p.

• Germline mutations associated with hereditary renal cell cancers, pheochromocytomas, hemangioblastomas of CNS, retinal angiomas, and renal cysts.

• VHL protein is part of a ubiquitin ligase complex. A critical substrate for this activity is HIF1α (hypoxia-inducible transcription factor 1α).

• In presence of 02, HIF1α is hydroxylated and binds to VHL protein, l/t ubiquitination and proteasomal degradation.

• In hypoxic environments the reaction cannot occur, and HIF1α escapes recognition by VHL and subsequent degradation.

• HIF1α then translocate to the nucleus and turn on many genes, such as the growth/angiogenic factors vascular endothelial growth factor (VEGF) and PDGF.

• Lack of VHL activity prevents ubiquitination and degradation of HIF1α and is associated with increased levels of angiogenic growth factors.

WT1 gene

• WT1 gene, located on chromosome 11p13.• Associated with development of Wilms' tumor, a

pediatric kidney cancer.• Both inherited and sporadic forms of Wilms' tumor

occur & mutational inactivation of WT1 locus seen in both forms.

• The WT1 protein is a transcriptional activator of genes involved in renal and gonadal differentiation.

• It regulates the mesenchymal-to-epithelial transition that occurs in kidney development.

• Tumorigenic effect of WT1 deficiency intimately connected with role of gene in the differentiation of genitourinary tissues.

• WT2, located on 11p15, is associated with the Beckwith-Wiedemann syndrome .

PTEN gene: a tumor suppressor gene

• PTEN (phosphatase and tensin homologue) is a membrane-associated phosphatase.

• Encoded by gene on chromosome 10q23.• It is mutated in Cowden syndrome : an autosomal

dominant marked by frequent benign growth like skin appendages.

• Has increased incidence of epithelial cancer particularly of breast, endometrium, and thyroid.

• It act by serving a break on the PI3K/AKT arm of receptor tyrosine kinase pathway.

Growth-Promoting Metabolic Alterations:The Warburg Effect

• Even in presence of ample oxygen Cancer cells have a distinct form of cellular metabolism characterized by high level of glucose uptake and increased conversion of glucose to lactose (fermentation) via the glycolytic pathway.

• This is called Warburg effect or aerobic glycolysis.

• Glucose hunger of the cancer cell is visualized by PET scan.

• “Warburg metabolism” is not cancer specific, but instead is a general property of growing cells that becomes “fixed” in cancer cells.

• This metabolism occur In cancer cell because aerobic glycolysis provides rapidly dividing tumor cells with metabolic intermediates that are needed for the synthesis of cellular components, whereas mitochondrial oxidative phosphorylation does not.

• Metabolic reprogramming is produced by signaling cascades downstream of growth factor receptors.

Relation between pro- growth signaling factors and metabolism

• PI3K/AKT signaling- it up regulate the activity of glucose transporters and multiple glycolytic enzymes leading to- – Increase glycolysis.– Shunting of mitochondrial intermediates to

pathways leading to lipid biosynthesis.– stimulates factors that are required for protein

synthesis.

• Receptor tyrosine kinase activity –• It transmit growth signal to nucleus.• This causing the alteration of M2 isoform of

pyruvate kinase ( catalyze the last step in glycolytic pathway, PEP pyruvate.) causing phosphorylation of M2 isoform, attenuating its activity.

• This create a damming effect leading to build-up of upstream glycolytic intermediates.

• Post-mitotic tissues with high demand for ATP such as the brain express M1 isoform which is insensitive to growth factor signaling pathways.

• MYC – it causes changes in gene expression that support anabolic metabolism and cell growth.

• It upregulates the multiple glycolytic enzymes and glutaminase , required for mitochondrial utilization of glutamine.

• Tumor suppressors often inhibit metabolic pathways that support growth like breaking effect of PTEN on PI3K/AKT pathway.

• p53 target gene that inhibit glucose uptake, glycolysis, lipogenesis, and generation of NADPH

Metabolism and cell growth Quiescent cells rely mainly on the Krebs cycle for ATP production; if starved, autophagy. When stimulated by growth factors, normal cells markedly upregulate glucose and glutamine uptake, which provide carbon sourcesfor synthesis of nucleotides, proteins, and lipids. In cancers, oncogenic mutations

involving growth factor signaling pathways and other key factors such asMYC deregulate these metabolic pathways, an alteration known as the Warburg effect.

EVASION OF APOPTOSIS

• Apoptosis represents a barrier that must be surmounted for cancer to occur.

• Signals, ranging from DNA damage to loss of adhesion to the basement membrane (anoikis), can trigger apoptosis.

• Two distinct programs that activate apoptosis, the extrinsic and intrinsic pathways.

• CD95 receptor–induced and DNA damage–triggered pathways of apoptosis .

Intrinsic and extrinsic pathways of apoptosis

• (1) Loss of p53, leading to reduced function of pro-apoptotic factors such as BAX.

• (2) Reduced egress of cytochrome c from mitochondria as a result of upregulation of anti-apoptotic factors such as BCL2, BCL-XL, and MCL-1.

• (3) Loss of apoptotic peptidase activating factor 1 (APAF1).

• (4) Upregulation of inhibitors of apoptosis (IAP).

• (5) Reduced CD95 level.• (6) Inactivation of death-induced

signaling complex. FADD, Fas-associated via death domain.

Limitless Replicative Potential: The StemCell–Like Properties of Cancer Cells

• All cancers contain cells that are immortal and have limitless replicative potential.

• Three interrelated factors appear critical to the immortality of cancer cells:

• (1) evasion of senescence;• (2) evasion of mitotic crisis; • (3) the capacity for self-renewal.

Evasion of senescence

• Normal human cells have the capacity to divide 60 to 70 times.

• The senescent state is associated with upregulation of tumor suppressors such as p53 and INK4a/p16 (perhaps in response to the accumulation of DNA damage over time).

• Maintaining RB in a hypophosphorylated state.

Evasion of mitotic crisis

• Mitotic crisis- This phenomenon has been ascribed to progressive shortening of telomeres at the ends of chromosomes.

• When the telomeric DNA is eroded, the exposed chromosome ends are “sensed” as double-stranded DNA breaks. By p53 and cell arrest occur.

• if p53 is dysfunctional, the nonhomologous endjoining pathway is activated and may join the “naked” ends of two chromosomes.

Escape of cells from senescence and mitotic catastrophe caused by telomere shortening In the absence of checkpoints, DNA repair pathways, such as the nonhomologous end-joining (NHEJ) pathway are inappropriately activated, leading to the formation ofdicentric chromosomes.

• Inappropriately activated repair system results in dicentric chromosomes that are pulled apart at anaphase.

• Resulting in new double-stranded DNA breaks.• The resulting genomic instability from the repeated

bridge-fusion-breakage cycles eventually produces mitotic catastrophe, characterized by massive cell death.

Self-renewal

• Self-renewal means that each time a stem cell divides at least one of the two daughter cells remains a stem cell.

• Symmetric division - both daughter cells remain stem cells. Occur during embryogenesis.

• Asymmetric division - only one daughter cell remains a stem cell. the non– stem cell daughter proceeds along some differentiation pathway, losing “stemness” but gaining one or more functions in the process.

• Cancers cells are immortal and have limitless proliferative capacity, they too must contain cells that self-renew, so-called cancer stem cells.

• Cancer stem cells arises from transformation of tissue stem cells like in CML and conversion of conventional somatic cells to transformed cells with acquired property of stemness. eg Acute myeloid leukemia cancer stem cells in this disease arise from more differentiated hematopoietic progenitors that acquire an abnormal capacity for self-renewal.

Origins of cells with self-renewing capacity in cancer.

ANGIOGENESIS

• New blood vessel formation in adults k/a angiogenesis or neovascularization

• Angiogenesis is controlled by a balance between angiogenesis promoters and inhibitors; in angiogenic tumors this balance is skewed in favor of promoters.

Two machenisms• New vessels sprout from previously existing

capillaries( capillary growth)• Vasculogenesis, in which endothelial cells are

recruited from the bone marrow (EPC)

Angiogenesis from pre existing vessels

• The local balance of angiogenic and antiangiogenic factors is influenced by several factors-

• Relative lack of oxygen due to hypoxia stabilizes HIF1α, an oxygen-sensitive transcription factor which then activates the transcription of the proangiogenic cytokines VEGF and bFGF.

• Transcription of VEGF is also influenced by signals from the RAS-MAP kinase pathway, and gain-of-function mutations in RAS or MYC upregulate the production of VEGF.

• NO---> vasodilation• VEGF---> ↑permeability• MMP---> proteolytic degredation of basement memb• Plasminogen Activator---> disruption of cell to cell

contact between endothelial cells• EC migration towards angiogenic stimuli.• Proliferation of endothelial cells just behind leading

front of migrating cells.

• Maturation of endothelial cells by growth inhibition & remodeling into capillary tubes.

• Recruitment of periendothelial cells ( pericytes & smooth muscles from mature vessels)

(B) Angiogenesis from Endothelial Precursor cells

1. ECP---> angioblasts2. Hematopoetic/ ECP---> hemangioblasts

• EPC----> moblise from BM --> site of tumor/injury --> EPC diffrentiates into endothelial cells---> forms a mature network by linking to existing vessels

• EPC expresses markers of 1. Hematopetic stem cells 2. VEGFR 23. V E cadherin• Leads to re- endothelization of vascular implant and

neovascularization.

• Like normal tissues, tumors require delivery of oxygen and nutrients and removal of waste products.

• Cancer cells can stimulate neo-angiogenesis.• Tumor vasculature is abnormal, however with leaky

& dilated vessels & haphazard connection pattern.• Neovascularization has dual effect on tumor growth1. Perfusion supplies nutrients and oxygen.2. Newly formed endothelial cells stimulate the

growth of adjacent tumor cells by secreting growth factors, such as IGFs, PDGF, and GM-CSF.

• Angiogenic switch involves increased production of angiogenic factors and/or loss of angiogenic inhibitors.

• These factors may be produced directly by the tumor cells themselves or by inflammatory cells macrophages or other stromal cells associated with the tumors.

• Proteases, elaborated from tumor cells directly / from stromal cells involved in regulating the balance between angiogenic and anti-angiogenic factors.

• Many proteases can release the proangiogenic basic fibroblast growth factors (bFGF) stored in the ECM.

• Three potent angiogenesis inhibitors—angiostatin, endostatin, and vasculostatin—are produced by proteolytic cleavage of plasminogen, collagen, and transthyretin, respectively.

• The angiogenic switch is controlled by several physiologic stimuli, such as hypoxia..

• VEGF also increases expression of ligands activating Notch signaling pathway, which plays crucial role in regulating branching and density of the new vessels

INVASION AND METASTASIS• Invasion and metastasis are biologic hallmarks of

malignant tumors.• Major cause of cancer-related morbidity and

mortality..• Millions of cells released into circulation each day

from 10 tumor, only few produces metastases .• Each step in the process is subject to a multitude of

controls.

• Metastatic cascade divided into two phases(1) Invasion of the extracellular matrix (ECM)(2) Vascular dissemination, homing of tumor cells, and

colonizationInvasion of Extracellular Matrix• Initiates the metastatic cascade and is an active

process that can be resolved into several steps• “Loosening up” of tumor cell–tumor cell interactions• Degradation of ECM• Attachment to novel ECM components• Migration and invasion of tumor cells

• Dissociation of cancer cells from one another is often the result of alterations in intercellular adhesion molecules and is the first step in the process of invasion.

• E-cadherins mediate the homotypic adhesion of epithelial cells, serving to both hold the cells together and to relay signals between the cells.

• Degradation of the basement membrane and interstitial connective tissue is the second step in invasion.

• By proteolytic enzymes themselves or by inducing stromal cells (fibroblasts and inflammatory cells) to elaborate proteases.

• Matrix metalloproteinases (MMPs), cathepsin D, and urokinase plasminogen activator.

• The third step in invasion involves changes in attachment of tumor cells to ECM proteins.

• Normal epithelial cells have receptors help to maintain the cells in a resting, differentiated state.

• Loss of adhesion in normal cells leads to induction of apoptosis.

• Locomotion is the final step of invasion, propelling tumor cells through the degraded basement membranes and zones of matrix proteolysis.

The metastatic cascade. Sequential steps involved in the hematogenous spread of a tumor

Sequence of events in the invasion of epithelial basement membranes by tumor cells.

Vascular Dissemination and Homing of Tumor Cells

• Within the circulation, tumor cells tend to aggregate in clumps.

• Homotypic adhesions among tumor cells & heterotypic adhesion between tumor cells and platelets l/t formation of platelet-tumor aggregates enhancing tumor cell survival and implantability.

• Tumor cells may also bind and activate coagulation factors, resulting in the formation of emboli.

• Arrest and extravasation of tumor emboli at distant sites involves adhesion to the endothelium, followed by egress through the basement membrane.

• Involved adhesion molecules (integrins, laminin receptors) and proteolytic enzymes.

• Particularly CD44 adhesion molecule, which is expressed on normal T lymphocytes and is used by these cells to migrate to selective sites in the lymphoid tissue.

• Such migration is accomplished by the binding of CD44 to hyaluronate on high endothelial venules, and overexpression of CD44 may favor metastatic spread.

• At the new site, tumor cells must proliferate, develop a vascular supply, and evade the host defenses.

Molecular Genetics of Metastasis Development

• Four models are presented1. Metastasis is caused by rare variant clones that

develop in the primary tumor.2. Metastasis is caused by the gene expression pattern

of most cells of the primary tumor, referred to as a metastatic signature.

3. Combination of A and B, in which metastatic variants appear in a tumor with a metastatic gene signature.

4. Metastasis development is greatly influenced by the tumor stroma, which may regulate angiogenesis, local invasiveness, and resistance to immune elimination, allowing cells of the primary tumor, as in C, to become metastatic.

Mechanisms of metastasis development within a primary tumor

• A. Metastasis is caused by rare variant clones that develop in the primary tumor.

• B. Metastasis is caused by the gene expression pattern of most cells of the primary tumor, referred to as a metastatic signature.

• C. A combination of A & B.• D. Metastasis influenced by

stroma which may regulate angiogenesis, local invasiveness, and resistance to immune elimination.

Evasion of Host Defense• Tumor cells can be recognized as “foreign” and

eliminated by the immune system.• Immune surveillance- it is the normal function

of the immune system is to constantly “scan” the body for emerging malignant cells and destroy them.

• Cancer immunoediting– it is the ability of the immune system to shape and mold the immunogenic properties of tumor cells in a fashion that ultimately leads to the darwinian selection of subclones that are best able to avoid immune elimination.

• Tumor Antigens`- Antigens found in tumors that elicit an immune response.

• Classes of tumor antigens-• 1. Products of mutated genes• 2. Overexpressed or aberrantly expressed cellular

proteins• 3. Tumor antigens produced by oncogenic viruses• 4. Oncofetal antigens• 5. Altered cell surface glycolipids and glycoproteins• 6. Cell type–specific differentiation antigens

• Products of mutated genes- due to genetic alteration in proto- oncogene and tumor suppressor gene, the new mutated protein is produced that is non self.

• These may enter the class I MHC antigen-processing pathway and be recognized by CD8+ T cells. Or may enter class II antigen-processing pathway in antigen presenting cells that have phagocytosed dead tumor cells, and thus be recognized by CD4+ T cells

Tumor antigens recognized by CD8+ T cells

• 2. Overexpressed or aberrantly expressed cellular proteins- Tumor antigens may also be normal cellular proteins that are abnormally expressed in tumor cell.

• Eg. Tyrosinase- when produced in excess amount

• Cancer testis antigen- genes that are silent in all tissues except germ cells in the adult testis

• Melanoma antigen gene (MAGE) family expressed by variety of tumor like melanoma, lung , liver, stomach..

• Oncofetal antigens- proteins that are expressed at high levels on cancer cells and in normal developing (fetal) tissues.

• These are specific and can serve as markers that aid in tumor diagnosis and clinical management.

• Eg. Carcinoembryonic antigen (CEA) and α-fetoprotein (AFP).

• Altered cell surface glycolipids and glycoproteins- these altered molecules include gangliosides , blood group antigens and mucin.

• Tumors often have dysregulated expression of the enzymes that synthesize these carbohydrate side chains, which leads to the appearance of tumor-specific epitopes on the carbohydrate side chains

• Eg. CA-125 and CA-19-9- in ovarian carcinoma

• Cell type–specific differentiation antigens- antigen that are normally present on the cell of origin.

• They are called differentiation antigens because they are specific for particular lineages or differentiation stages of various cell types.

• Their importance is as potential targets for immunotherapy and for identifying the tissue of origin of tumors

• Eg . Monoclonal antibody like anti CD20 have broad cytocidal activity against mature B-cell lymphomas and leukemias

• Anti CD30 in CD30positive lymphoma• Toxin-conjugated antibodies specific for HER2

Antitumor Effector Mechanisms

• Cell-mediated immunity is the dominant antitumor mechanism in vivo.

• Cytotoxic T lymphocytes- CD8+ CTLs have protective role against virus-associated neoplasms (e.g., EBV- and HPV-induced tumors).

• Natural killer cells- lymphocytes that are capable of destroying tumor cells without prior sensitization.

• Provide first line of defense against tumor cells.

• Macrophages- Activated macrophages exhibit cytotoxicity against tumor cells in vitro.

• Interferon-γ, a cytokine secreted by T cells and NK cells, is a potent activator of macrophages.

Immune Surveillance and Escape

• Persons with congenital immunodeficiencies develop cancers at about 200 times the rate in immunocompetent individuals.

• Mechanism of tumor cell to escape or evade the immune system-

• 1. Selective outgrowth of antigen-negative variants

• 2. Loss or reduced expression of MHC molecules- by this escape by attack of cytotoxic T cells

• 3. Activation of immunoregulatory pathways• Tumor cells actively inhibit tumor immunity by

engaging normal pathways of immune regulation that serve as “checkpoints” in immune responses.

• Mechanism-1. Downregulate the expression of costimulatory factors on antigen-presenting cells eg in dendritic cells.

• 2. Upregulate the expression of PD-L1 and PD-L2, cell surface proteins that activate the programmed death-1 (PD-1) receptor on effector T cells. PD-1, like CTLA-4, may inhibit T cell activation.

• Secretion of immunosuppressive factors by cancer cells-

• TGF-β- a potent immunosuppressant• Galectins- sugar-rich lectin-like factors that

skew T-cell responses so as to favor immunosuppression.

• Interleukin-10, prostaglandin E2, VEGF inhibit the diapedesis of T cells from the vasculature into the tumor bed.

• Induction of regulatory T cells (Tregs)• factors that favor the development of

immunosuppressive regulatory T cells,

Mechanisms by which tumors evade the immune system

Genomic Instability

• These are genetic alteration that increase the mutation rate and expedite the acquisition of driver mutation.

• Mutations in DNA-repair genes themselves are not oncogenic, but their abnormalities greatly enhance the occurrence of mutations in other genes during the process of normal cell division.

• Genomic instability occurs when both copies of the DNA repair gene are lost;

• Types of DNA repair system– Mismatch repair– Nucleotide excision repair– Recombination repair

• Defect in any these of mechanism contributes to different types of cancer

Hereditary Nonpolyposis Colon Cancer Syndrome.

• It is an autosomal dominant disorder characterized by familial carcinomas of the colon affecting predominantly the cecum and proximal colon.

• It occur due to defect in DNA mismatch repair.• These proteins acts as a spell checker.• Eg. if there is an erroneous pairing of G with T

rather than the normal A with T, the mismatch-repair factors correct the defect.

• HNPCC syndrome inherit one abnormal copy of a mismatch repair gene.

• when cells acquire loss of- function mutations, in their normal alleles with loss of “proofreading” function .

• These errors gradually accumulate and may activate proto- oncogene or deactivate tumor suppressor gene.

• So DNA-repair genes behave like tumor suppressor genes in their mode of inheritance.

• Mismatch-repair defects has microsatellite instability.

• Microsatellites are tandem repeats of one to six nucleotides found throughout the genome.

• These are constant in normal people but may be increase or decrease in length in HNPCC.

• DNA mismatch-repair genes involved in HNPCC are MSH2 and MLH1, genes encoding TGF-β receptor II, TCF component of the β-catenin pathway, & BAX.

Xeroderma Pigmentosum

• It is a disorder of DNA repair.• Increased risk for the development of cancers

of the skin particularly following exposure to the UV light.

• UV radiation causes cross-linking of pyrimidine residues, preventing normal DNA replication.

Diseases with Defects in DNA Repair by Homologous Recombination

• Hypersensitivity to DNA-damaging agents, such as ionizing radiation( Bloom syndrome and ataxia-telangiectasia) or DNA cross-linking agents, such as many chemotherapeutic drugs (Fanconi anemia).

• Gene mutated in ataxia telangiectasia ATM recognize and respond to DNA damage by ionizing radiation.

• Mutation of BRCA1 and BRCA2 in breast cancer.

Cancers Resulting from Mutations Induced by Regulated Genomic Instability: Lymphoid Neoplasms

• B cells and T cells express a pair of gene product ,RAG1 and RAG2, it carry out V(D)J segment recombination, permitting the assembly of functional antigen receptor genes.

• Eg. lymphoid cells mutation.

Cancer-Enabling Inflammation

• Infiltrating cancers provoke a chronic inflammatory reaction leading to “wounds that do not heal.”

• The cancer-enabling effects of inflammatory cells are following:

• Release of factors that promote proliferation- secreted by infiltrating lymphocytes

• Eg. EGF and proteases.

• Removal of growth suppressors- growth is suppressed by cell- cell and cell-ECM interaction.

• Proteases degrade adhesion molecule, and remove this barrier.

• Enhanced resistance to cell death – • Detachment of epithelial cells from basement

membranes and from cell-cell interactions can lead to death called anoikis.

• Tumor-associated macrophages may prevent anoikis by expressing adhesion molecules like integrins.

• Inducing angiogenesis- due to release of angiogenic factors like VEGF.

• Activating invasion and metastasis- Proteases foster tissue invasion by remodeling the ECM, while TNF and EGF may directly stimulate tumor cell motility.

• TGF-β, may promote epithelial-mesenchymal transitions, process of invasion and metastasis.

• Evading immune destruction- immunosuppressive factors like TGF- β, suppress the function of CD8+ cytotoxic T cells.

Dysregulation of Cancer-Associated Genes

• Chromosomal Changes-• 1. Chromosomal Translocations – defined as

a chromosome abnormality caused by rearrangement of parts between nonhomologous chromosomes.

• Translocations can activate proto-oncogenes in two ways:

• 1. By promoter or enhancer substitution-• Translocation results in overexpression of a

protooncogene by swapping its regulatory elements with those of another gene.

• 2. By formation of a fusion gene- in which coding sequences of two genes are fused in part or in whole, leading to the expression of a novel chimeric protein with oncogenic properties.

• Overexpression of a proto-oncogene caused by translocation is exemplified by Burkitt lymphoma

• Overexpression of MYC located on chromosome 8q24.

Oncogenes Created by TranslocationsMalignancy Translocation Affected Genes

Chronic myelogenous leukemia (CML)

(9;22)(q34;q11) ABL 9q34BCR 22q11

Acute myeloid leukemia (AML)

(8;21)(q22;q22)(15;17)(q22;q21)

AML 8q22ETO 21q22PML 15q22RARA 17q21

Burkitt lymphoma (8;14)(q24;q32) MYC 8q24IGH 14q32

Mantle cell lymphoma (11;14)(q13;q32) CCND1 11q13IGH 14q32

Follicular lymphoma (14;18)(q32;q21) IGH 14q32BCL2 18q21

Ewing sarcoma (11;22)(q24;q12) FLI1 11q24EWSR1 22q12

Prostatic adenocarcinoma (7:21)(p22;q22)(17:21)(p21;q22)

TMPRSS2 (21q22.3)ETV1 (7p21.2)ETV4 (17q21)

• The Philadelphia chromosome- characteristic of CML and a subset of B-cell acute lymphoblastic leukemias is an example of chromosomal rearrangement that creates a fusion gene encoding a chimeric oncoprotein.

• The ABL gene on chromosome 9 and BCR on chromosome 22.

• There is Non-homologous end-joining then leads to a reciprocal translocation that creates an oncogenic BCR-ABL fusion gene.

• The BCR-ABL fusion gene encodes a chimeric BCR-ABL protein with constitutive tyrosine kinase activity.

• Fusion gene encode nuclear factor that regulate transcription or chromatin structure.

• Eg. Acute promyelocytic leukemia (APML) associated with reciprocal translocation between chromosomes 15 and 17 that produces a PML-RARA fusion gene.

• Mechanism of action of fusion gene- • The fusion gene encodes a chimeric protein

consisting of part of a protein called PML and part of the retinoic acid receptor-α (RARα).

Molecular pathogenesis of acute promyelocytic leukemia and basis for response to all-trans retinoic acid. ATRA

• Deletions -Deletion of specific regions of chromosomes is associated with the loss of particular tumor suppressor genes.

• eg. RB gene deletion on 13q14- retinoblastoma• VHL gene deletion on 3p in RCC.• Few deletion causing activation of proto- oncogene

like in T- cell acute lymphoblastic leukemias have small deletions of chromosome 1, leading to overexpression of the TAL1 transcription factor.

• Gene Amplification- A selective increase in the number of copies of a gene coding for a specific protein without a proportional increase in other genes.

• Overexpression of oncogenes may also result from reduplication and amplification of their DNA sequences.

• Two mutually exclusive patterns are seen:• (1) double minutes.• 2. homogeneous staining regions

• The affected chromosomal regions lack a normal pattern of light and dark-staining band appearing homogeneous in karyotypes.

• Eg. NMYC in neuroblastoma and ERBB2 in breast cancers.

• Chromothrypsis- phenomenon by which up to thousands of clustered chromosomal rearrangements occur in a single event in localised and confined genomic regions in one or a few chromosomes.

Epigenetic Changes

• epigenetics” refers to factors other than the sequence of DNA that regulate gene expression.

• It include histones modifications catalyzed by enzymes associated with chromatin regulatory complexes;

• DNA methylation, a modification created by DNA methyltransferases;

• epigenetic alterations in cancers can be divided into the following categories:

• 1. Silencing of tumor suppressor genes by local hypermethylation of DNA - hypermethylation of the promoters of tumor suppressor genes that results in their transcriptional silencing.

• Eg. Hypermethylation of CDKN2A,which encodes p14/ARF and p16/INK4a( tumor suppressor gene)

• Global changes in DNA methylation- this is abnormal patterns of DNA methylation throughout the genomes. in the form of hypermethylation or hypomethylation.

• Eg. Acute myeloid leukemia• The consequence is altered expression of multiple

genes may be overexpressed or underexpressed compared to normal.

• Hypomethylated genomes also exhibit chromosomal instability

• Changes in histones these changes in histones near genes that influence cellular behavior.

• mutations that affect the activities of protein complexes that “write”, “read” and “erase” histone marks, or that position nucleosomes on DNA.

Epigenomic Regulatory Genes that Mutated in Cancer

Gene(s) Function Tumor (Approximate Frequency of Mutation)

DNMT3A DNA methylation Acute myeloid leukemia (20%)

MLL1 Histone methylation Acute leukemia in infants (90%)

MLL2 Histone methylation Follicular lymphoma (90%)

CREBBP/ EP300 Histone acetylation Diffuse large B cell lymphoma (40%)

ARID1A Nucleosomepositioning/chromatin remodeling

Ovarian clear cell carcinoma (60%),endometrial carcinoma (30%-40%)

SNF5 Nucleosomepositioning/chromatinremodeling

Malignant rhabdoid tumor (100%)

PBRM1 Nucleosomepositioning/chromatinremodeling

Renal carcinoma (30%)

Noncoding RNAs and Cancer

• OncomiRs- miR-200 promote epithelial-mesenchymal transitions which is important in invasiveness and metastasis.

• miR-155- overexpressed in many human B cell lymphomas and indirectly upregulate genes that promote proliferation, including MYC.

• Tumor suppressive miRs- miR-15 and miR-16 in chronic lymphocytic leukemia,

• their loss leads to upregulation of the anti-apoptotic protein BCL-2.

• Tumor suppressive properties of mIR processing factors families that are prone to the development of an unusual collection of neoplasms have heterozygous germline defects in DICER.

• DICER encodes endonuclease, required for the processing and production of functional mIRs.

Molecular Basis of Multistep Carcinogenesis

• cancers result from the stepwise accumulation of multiple mutations that act in complementary ways to produce a fully malignant tumor.

• Eg Colon cancer- colon epithelial hyperplasia formation of adenomas progressively enlarge malignant transformation.

• inactivation of the APC gene activation of RAS loss of a tumor suppressor gene on 18q and loss of TP53.

Molecular model for the evolution of colorectal cancers through the adenoma-carcinoma sequence

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