Tumor pathogenesis Oncogenes Tumor suppressor genes Invasion and Metastasis

*IntroductionCarcinogensis is multistep process involving the multiple genetic changes including the activation of cooperating oncogenes and the inactivation of tumor suppressors in somatic cells

Usually, a single oncogene is not enough to turn a normal cell into a cancer cell, and many mutations in a number of different genes may be required to make a cell cancerous.

Figure 2. Intracellular Signaling Networks Regulate the Operations of the Cancer Cell. An elaborate integrated circuit operates within normal cells and is reprogrammed to regulate hallmark capabilities within cancer cells. Separate subcircuits, depicted here in differently colored fields, are specialized to orchestrate the various capabilities. At one level, this depiction is simplistic, as there is considerable crosstalk between such subcircuits. In addition, because each cancer cell is exposed to a complex mixture of signals from its microenvironment, each of these subcircuits is connected with signals originating from other cells in the tumor microenvironment, as outlined in Figure 5. (Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell 2011, 144:646)

Michael R. Stratton. Exploring the Genomes of Cancer Cells: Progress and Promise. Science 331, 1553 (2011).

Michael R. Stratton. Exploring the Genomes of Cancer Cells: Progress and Promise. Science 331, 1553 (2011).

Oncogene

Concept: An oncogene is a gene that when mutated or expressed at abnormally-high levels contributes to converting a normal cell into a cancer cell.

Cellular oncogene (c-onc)--- proto-oncogene proto-oncin normal physiologic version--- Oncogenealtered in cancer Viral oncogene v-onc

Proto-oncogenes have been identified at all levels of the various signal transduction cascades that control cell growth, proliferation and differentiation:extracellular proteins function as growth factors,membrane proteins as cell surface receptors cellular proteins that relay signals proteins in nucleus, which activate the transcription and promote the cell cycle

This signaling process involves a series of steps that:begin from the extracellular environment to cell membrane;involve a host of intermediaries in the cytoplasm; end in the nucleus with the activation of transcription factors that help to move the cell through its growth cycle.Fuctions of proto-oncogenes

Growth factors, e.g. V-sis, PDGF-b, int-2Receptor Tyrosine Kinases, e.g. Her-2/neu/ erbb2,Membrane Associated Non-Receptor Tyrosine Kinases, e.g. src, LckG-Protein Coupled Receptors e.g. MasMembrane Associated G-Proteins , e.g. RasSerine/Threonine Kinases e.g. RafNuclear DNA-Binding/Transcription Factors, e.g. myc, fosOthersApoptosis regulators, e.g. Bcl-2,Regulators of cell cycle, e.g. Cyclin D1, CDK4Classification of proto-oncogenes

Mechanisms of Oncogene ActivationGene amplification, e.g. myc, CCND1Point mutation, e.g. ras,Chromosomal rearrangement or translocation the transcriptional activation of proto-onc.the creation of fusion genes, e.g. abl-bcrViral insertion activation, e.g. c-Myc

AmplificationTranslocation

*CHROMOSOMAL REARRANGEMENTS OR TRANSLOCATIONS

NeoplasmTranslocationProto-oncogene

Burkitt lymphoma t(8;14)80% of casesc-myc1 t(8;22) 15% of cases t(2;8) 5% of cases

Chronic myelogenoust(9;22)90-95% of cases bcr-abl2leukemia

Acute lymphocytict(9;22)10-15% of cases bcr-abl2Leukemia

1c-myc is translocated to the IgG locus, which results in its activated expression

2bcr-abl fusion protein is produced, which results in a constitutively active abl kinase

* GENE AMPLIFICATION

OncogeneAmplification Source of tumor

c-myc ~20-foldleukemia and lung carcinoma

N-myc 5-1,000-foldneuroblastomaretinoblastoma

L-myc 10-20-foldsmall-cell lung cancer

c-abl ~5-foldchronic myoloid leukemia

c-myb 5-10-foldacute myeloid leukemiacolon carcinoma

c-erbB ~30-foldepidermoid carcinoma

K-ras 4-20-foldcolon carcinoma 30-60-foldadrenocortical carcinoma

Mechanisms of action of oncogenes

RasLocates on chromosome 11, codes for a protein with GTPase activityrelays signals by acting as a switch: When receptors on the cell surface are stimulated, Ras is switched on and transduces signals that tell the cell to grow. If the cell-surface receptor is not stimulated, Ras is not activated and so the pathway that results in cell growth is not initiated.mutated in about 30% of human cancers so that it is permanently switched on, telling the cell to grow regardless of whether receptors on the cell surface are activated or not.

Her2/neu/erbB-2This gene was discovered by three different groups. That is why it has three different names.It is a member of EGFR superfamily, also be a receptor tyrosine kinasesDr. Slamon (UCLA) described the role of Her2/neu in breast cancer and ovarian cancer.Overexpression, amplification, rare translocationsNo ligand is known

Ras relays signals from the cell surface receptors to the nucleusRas relays signals by acting as a switch

ProspectA breakthrough for our understanding of the molecular and genetic basis of cancerProvided important knowledge concerning the regulation of normal cell proliferation, differentiation, and programed cell death. The identification of oncogene abnormalities has provided tools for the molecular diagnosis and monitoring of cancer. Oncogenes represent potential targets for new types of cancer therapies.

Tumor suppressor genesConcept: genes that sustain loss-of function mutations in the development of cancer

TSGsTranscriptional factor: p53, WT1,Direct transcription regulator: Rb, APCInhibitor of cell cylcle kinase: p16INK4A, p19ARF, Cell structural components: NF2Phosphatase: PTENPotential mediator of mRNA processing: VHLComponents involved in DNA repair: MSH2, MLH1, BRCA1, p53

TUMOR SUPPRESSOR GENES

Disorders in which gene is affected

Gene (locus) Function Familial Sporadic

DCC (18q) cell surface unknowncolorectal interactions cancer

WT1 (11p) transcription Wilms tumorlung cancer

Rb1 (13q) transcription retinoblastomasmall-cell lung carcinoma

p53 (17p) transcription Li-Fraumeni breast, colon, syndrome & lung cancer

BRCA1(17q) transcriptionalbreast cancerbreast/ovarian tumorsBRCA2 (13q) regulator/DNA repair

Mechanism for the inactivation of TSGsMutation: point mutation or frameshift mutation, p53Deletion: LOH (loss of heterozygosity) or homozygous deletion, RbViral oncoprotein inactivation: p53, RbPromoter hypermethylation, histone modification changes: p16

Rb function

Rb regulates G1/S transitionRb inactivation by viral oncoprotein

*Normal CellsTumor cellsKNUDSON TWO HIT HYPOTHESIS IN SPORADIC CASESRBRBInactivation of a tumor suppressor gene requires two somatic mutations.

P53Function as gatekeeperInactivation of p53 in cancer LOH on 17p13 in a number of tumorsPoint mutation on exon 5-8 hot-spot (Dominant negative mutation)MDM2 negative regulation viral-oncogene products inactivation

Invasion and Metastasis

Stepwise Malignant Progression of Cancer

The process of metastasis consists of sequential linked steps Growth at primary site and angiogenesis Tumor cell invasion Lymphatic and hematogenous metastasis Growth at secondary site and angiogenesis

Mechanisms involved in tumor cell invasion1.Loss of cell-to cell cohesive forces2. Secretion of ECM-degrading enzymes3. Active Locomotion4. Tumor angiogenesis5. Metastasis-related genes

5. Metastasis-enhancing Genes: Oncogenes,CD44, Integrin1, CEA, MMP2, u-PA, etc

1. Loss of cell-to cell cohesive forces: Cell adhesion molecules (CAMs:ECM

E-cadherin: Expression Loss of cell-cell adhesionIncreased cell motility Integrins Expression Immunoglobin superfamilyNCAM, VCAM-1,CEA, DCC, etc Selectins CD44 variants

2. Secretion of ECM-degrading enzymes Matrix Metalloproteinases (MMPs)20 Tissue inhibitors of metalloproteinases (TIMPs) 4 Plasminogen Activators (PAs) :urokinase-type, tissue-type PA PA inhibitors (PAIs): 3Metastasis-associated proteinases

Cell invasion of the extracellular matrix

The MMP family (at least 23 members)Classification

Interstitial Collagenase MMP-1MMP-5MMP-8 MMP-13 (Gelatinase )MMP-2 MMP- 9Stromelysin,MMP-3 MMP-7 MMP-10 MMP-11FN(LN) Membrane-type MMPs, MT-MMPs ,MT1-MMPMT2-MMPMT3-MMPMT4-MMPMT-MMPsMMPMMPFN

MMPs share the following common characteristics:

TIMP: TIMPs play a key role in maintaining the balance between ECM deposition and its degradation by binding tightly to and regulating MMP actions Four isoforms: TIMP 1-4

uPAuPAR-initiated signal transduction and consequencesPlasminogen/Plasmin System

3. Active Locomotion E- cadherin Growth factors and receptors, Autocrine motility factor (AMF), Autotaxin (ATX), Cytoskeletal proteins ECM components (laminin, LN, etc

4. Tumor angiogenesis factors (TAFs)angiogenin, etc Inhibitorsangiostatin, etc

Models of Tumour Angiogenesis

Endogenous angiogenesis inhibitors

5. Metastasis-enhancing Genes:Oncogenes, CD44, Integrin1, CEA, MMP2, u-PA, etc

Metastasis Promoting Genes - I

GeneTissue SiteFunctionARM-1LymphomaPromotes adhesion of tumor cells to the endotheliumATXBreast, Liver, Lung, Melanoma, Teratocarcinomacytoskeletal reorganization and motility; G-protein coupled receptor activationCD44Multiple sitescell-cell interactions; activates HGF/c-Met pathwayCox2Breast, Colorectal, GastricProstaglandin synthase; induces VEGFCyr61BreastMediates adhesion; Erb-B2/3/4 pathwayEzrinLiver, Ovary, Pancreas, Prostate, UterusMembrane-cytoskeletal linker; RHO and RAC interactionsHMG-I(Y)Breast, Cervical, Colorectal, Prostate, Skin, Thyroid, UterusRegulated by EGF and MMP-9Laminin-5Multiple sitesEGF and TGF-a induce expression of laminin subunits; cell adhesion, motilityc-MetMultiple sitesActivated by HGF; Modulates Ras and PI3 kinase

Metastasis Promoting Genes - II

GeneTissue SiteFunctionMTA1Breast, Cervix, Melanoma, OvaryNeucleosome remodeling; histone deacetylase complexOncostatin MLungActivates PKA-dependent pathwayPP2ANot determinedActivated by p38/MAPK; inhibits MEK1, MEK2, and MMP-1RAGEGastric, Lung, Pancreatic, Renaltransmembrane receptor; activates p21, MAPKs, NF-6B, cdc42/racS100A4Breast, Colorectal, ProstateCalcium-binding protein; activates c-erbB-2S100A9Colon, Gastric, SkinCalcium-binding protein; Modulates Mac-1 integrin receptor through G-proteinSemaphorinsGastric, Leukemia, Lung, Skincell-cell interactions; Receptor crosstalk with c-Met binding semaphorin receptor, plexin Thymosin-b15Prostateactin binding; motilityWnt-5aBreast, Colon, Lung, Melanoma, Pancreas, ProstatePKC activation with associated changes in cytoskeleton, cell adhesion, and motility

6. Metastasis-Suppressor Genes Identifiednm23, KAI 1, TIMPs, E-cadherin, Kiss, etcModified from JNCI 2000; 92:1717

The nm23 gene family The first metastasis suppressor gene identified was nm23 Eight members of the human nm23 family have been reported and are found in multiple subcellular compartments.

Biochemical functions nm23 proteins posses multiple biochemical functions1. Interaction with numerous proteins Tiam1, Ras, cytoskeletal protein2. A NDPKinase activity3. DNA nuclease4. Serine or histidine protein kinase inhibition of the Map kinase pathway and correlated with motility suppression.

Nucleoside diphosphate kinase (NDPKinase) activityThe nm23-H1 gene product has been identified as the NDPKA isoformThe nm23-H2 gene product has been identified as the NDPKB isoformNDPKs: catalyze the phosphorylation of nucleoside diphosphates to the corresponding nucleoside triphosphates, mainly at the expense of the ATP synthesized through oxidative phosphorylation

KAI1 / CD 82Names : KAI1 / CD82, (C33, R2, IA4)Gender : Transmembrane GlycoproteinMember of the tetraspanin or transmembrane 4 superfamily (TM4SF)Contains an internalization sequence at its C-terminus (YSKV)Current Address :Cell membrane (lymphocytes, epithelial cells)LysosomesVesiclesLigands ?Biological Function :motilityinvasivenesscell-cell interactionsParticularitySignal Pathways : ?

High level of KAI1/CD82 is a good prognosis factor or associated with low grade histology :prostatelungpancreascarcinomacolonprostatelung carcinomacolonhepatomabreastlung (non-small-cell carcinoma)bladder cancerovarymelanomaKAI1/CD82 expression is inversely related to the metastatic potential :Transfection of tetraspanin reduces metastatic potentialmelanoma

prostatebreastB16MDA-MB-435 *AT6.1, AT6.3MDA-MB-231KAI1 / CD 82 and CancerCorrelationsExperimental Data(from Boucheix & Rubinstein , 2001

Loss of KAI-1 Expression in Prostate Cancer

KAI-1 Functions

+ (Promotes)- (Inhibits)Cell AggregabilityCell AdhesionInvasionMotilityMetastasis

RTKCDH1E G F RRASCRSP3RAFMEKERKGrowthDifferentiationApoptosisInflammationDifferentiationSurvivalb-CateninKAI1RhoGDI2FASKISS1NFkBASK1MKK7JNKMKK4MKK6p38MKK3ApoptosisCytokinesSurvivalAngiogenesisTXNIPTXNHIFVAVKSRNME1MotilityInvasionGPR54IGFR1METPI3KPLCAKTBADPTENRhoRacMetastasis Suppressor Pathways

Metastasis Facts Up to 70% of patients with invasive cancer have overt or occult metastases at diagnosis. Acquisition of the invasive and metastatic phenotype is an early event in cancer progression. Millions of tumor cells are shed daily into the circulation. Less than 0.01% of circulating tumor cells successfully initiate a metastatic focus. Angiogenesis is a ubiquitous and early event that is necessary for and promotes metastatic dissemination. Invasion and angiogenesis use the same signal transduction programs and gene expression cassettes. Circulating tumor cells can be detected in patients who do not develop overt metastatic disease. Metastases may be as susceptible to anti- cancer therapy as their primary tumors?

Metastasis Therapeutic Targets and Agents A. Targeted Therapeutics

Target Example Agents Effects Growth factors C225 (anti-EGFR) Block growth factor signaling Tyrphostins (anti-RTK)Cell adhesion Anti-avb3 (Vitaxin) Blocks endothelial cell avb3 peptidomimetics interaction with matrix may regulate MP activationProteolysis MMPIs uPAR-I Blocks degradation of matrix, blocks activation of proteases, growth factorsMotility Taxanes Blockade of microtubule cycling

B. Signal Inhibitors: Blockade of signals necessary for angiogenesis , invasion, and metastasis

Agent Target ActivityCAI Calcium influx Inhibits adhesion, motility, angiogenesisSqualamine Inhibits NHE-3 Anti-angiogenicPI3K inhibitors Inhibit motility, proliferation, promote MAPK inhibitors Inhibit invasion, proliferation

Figure 4. The Cells of the Tumor Microenvironment. (Upper) An assemblage of distinct cell types constitutes most solid tumors. Both the parenchyma and stroma of tumors contain distinct cell types and subtypes that collectively enable tumor growth and progression. Notably, the immune inflammatory cells present in tumors can include both tumor-promoting as well as tumor-killing subclasses.(Lower) The distinctive microenvironments of tumors. The multiple stromal cell types create a succession of tumormicroenvironments that change as tumors invade normal tissue and thereafter seed and colonize distant tissues.The abundance, histologic organization, and phenotypic characteristics of the stromal cell types, as well as of theextracellular matrix (hatched background), evolve during progression, thereby enabling primary, invasive, and then metastatic growth. The surrounding normal cells of the primary and metastatic sites, shown only schematically, likely also affect the character of the various neoplastic microenvironments. (Not shown are the premalignant stages in tumorigenesis, which also have distinctive microenvironments that are created by the abundance and characteristics of the assembled cells.) (Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell 2011, 144:646)Tumor Microenvironment

Figure 5. Signaling Interactions in the Tumor Microenvironment during Malignant Progression(Upper) The assembly and collective contributions of the assorted cell types constituting the tumor microenvironment are orchestrated and maintained byreciprocal heterotypic signaling interactions, of which only a few are illustrated.(Lower) The intracellular signaling depicted in the upper panel within the tumor microenvironment is not static but instead changes during tumor progression asa result of reciprocal signaling interactions between cancer cells of the parenchyma and stromal cells that convey the increasingly aggressive phenotypes thatunderlie growth, invasion, and metastatic dissemination. Importantly, the predisposition to spawn metastatic lesions can begin early, being influenced by thedifferentiation program of the normal cell-of-origin or by initiating oncogenic lesions. Certain organ sites (sometimes referred to as fertile soil or metastaticniches) can be especially permissive for metastatic seeding and colonization by certain types of cancer cells, as a consequence of local properties that are eitherintrinsic to the normal tissue or induced at a distance by systemic actions of primary tumors. Cancer stem cells may be variably involved in some or all of thedifferent stages of primary tumorigenesis and metastasis.(Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell 2011, 144:646)

The diagnosis and therapy of cancer

Figure 6. Therapeutic Targeting of the Hallmarks of Cancer.Drugs that interfere with each of the acquired capabilities necessary for tumor growth and progression have been developed and are in clinical trials or in some cases approved for clinical use in treating certain forms of human cancer. Additionally, the investigational drugs are being developed to target each of theenabling characteristics and emerging hallmarks depicted in Figure 3, which also hold promise as cancer therapeutics. The drugs listed are but illustrative examples; there is a deep pipeline of candidate drugs with different molecular targets and modes of action in development for most of these hallmarks. (Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell 2011, 144:646).

Three linesof accomplishments are converging toenable clinical researchers to investigatetherapies that target the molecular andgenetic abnormalities detected in an individualpatients cancer: (i) We have identifiedmost of the few hundred genes thatare mutated or abnormally expressed inhuman cancers. (ii) Genome sequencingtechnology will soon enable us to screenthe mutations in a human cancer biopsyin less than a week, at a cost of a few thousanddollars. Abnormalities in cellularproteins and RNA can already be detectedin that time interval and at a reasonablecost. (iii) Pharmaceutical and biotechnologycompanies have more than 800 newexperimental drugs and biological agentsin the pipeline that are designed to targetthe products of abnormal genes found inhuman cancers

**#16*#15DPINC33 KAI1 mAb*16d*