*Tumor pathogenesis Emailchenwei566@zju.edu.cn http://mypage.zju.edu.cn/566 8888

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

Molecular alterations during human colon tumor progressionThe precise contribution of hypomethylation to tumor progression remains unclear; some evidence suggests that it creates chromosomal instability.(~ 90 %)(~ 40-50 %)(~ 50-70 %)(~ 60 %)**

Molecular Basis of Multistep Carcinogenesis

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)

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), int-2 (FGF)Receptor Tyrosine Kinases, e.g. Her-2/ neu/ erbb2 (EGFR)Membrane Associated Non-Receptor Tyrosine Kinases, e.g. src, LckMembrane 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-fold chronic myeloid leukemia

c-myb 5-10-foldacute myeloid leukemiacolon carcinoma

c-erbB ~30-foldepidermoid carcinoma

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

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.

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

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

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

RBRetinoblastoma is an uncommon childhood tumorRetinoblastoma is either sporadic (60%) or familial ( 40% )Two mutations required to produce retinoblastomaBoth normal copies of the gene should be lost to produce retinoblastoma

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

P53:Common in human cancer 70%Tumor suppressor gene

DNA damage P53 activation by release from MDM2P53 results in arresting the cell cycle by increasing P21.P53 enhance repair of the DNA damage by GADD45P53 induces apoptosis by increasing level of Bax

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

P53 is called the guardian of the genome70% of human cancers have a defect in P53It has been reported with almost all types of cancers : e.g. lung, colon, breastIn most cases, mutations are acquired, but can be inhereted, e.g : Li-Fraumeni syndrome

PTEN (Phosphatase and Tensin Homolog)Pten gene located on Chrom 10 (10q23)PTEN was discovered in 1997 as the first tumor suppressor phosphatasePTEN and PI-3 Kinase act as antagonists in lipid signaling

PTEN is a lipid 3-phosphatase, which signals down the PI3 kinase/AKT proapoptotic pathway.Backman et al.

Outcomes of the Akt/PKB pathwayRole in proliferation complicatedPTEN does not merely block proliferation because studies showed that normal bacteria cells expressing PTEN can still undergo rapid proliferation (Liliental et al., 2000)Role in apoptosis more clearRe-expression of PTEN in several carcinoma cell lines induces apoptosis (Li et al, 1998)Especially important is Anoikis, a form of apoptosis that occurs when cells lose contact with the e.c.m. -mediates the cells anchorage dependence (Davies et al., 1998)

How is Pten involved in cancer?Most frequently mutated gene identified yet in endometrial cancers (33-55% of tumors examined)Of 647 Malignant Glial Tumors examined, 24% showed mutations in PTENOvarian tumors (of endometriod origin) showed mutations in 26% of tumors Prostate Carcinoma-mutations in 18% of tumors

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

Attachments of tumor cells to matrix componentsDetachment of tumor cells from each otherDegradation of ECM by collagenase enzymeMigration of tumor cells

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, Selectins CD44 variants

Cadherin ()Mediate Ca2+ - dependent homophilic cell-cell adhesionUsed in organ formation during development Primarily link epithelial and muscle cells to their neighbors

E-cadherins hold epithelial cells together expression in tumorN-cadherins expressed in nervous system are responsible for the specificity of neuronal connections

Mode of cadherin interaction

Integrin family is so named because the molecules of this family primarily mediate the cellular adherence to extracellular matrix, enabling cells and extracellular matrix to form the integration. Integrin ()

1. Structure: Heterodimeric proteins consisting of two non-covalently bound polypeptides ( and chains).2. Components of integrin family 1: VLA (very late appearing antigen) 2: LFA-1 (lymphocyte function associated antigen-1); ligand: ICAM-1 3: gpIIb3. ligands: the component of ECM including fibronectinFNvitronectinVNlamininLM

Integrin ()

IGSF (immunoglobulin superfamily)

1. Structure Transmembrane glycoproteins, with a number of extracellular domains homologous to (CCP). The extracellular region also contains a domain related to the EGF receptor and a distal C type lectin-like domain.2. Members of selectin family L-selectin: leukocytes P-selectin: platelets and megakaryocytes E-selectin: activated endothelia3. Ligand: carbohydrates, such as CD15s (sialyl-Lewisx)Selectin

CD44CD44 is expressed in a large number of mammalian cell types. The standard isoform, designated CD44s, is expressed in most cell types. CD44 splice variants containing variable exons are designated CD44v, which plays a role in tumor metastasis.CD44 is a receptor for hyaluronic acid and can also interact with other ligands, such as osteopontin, collagens, and MMPs.

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

MMP (matrix metalloproteinases) 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

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 (LN, etc

4. tumor angiogenesisNeovascularization has two main effects: Perfusion supplies oxygen and nutrientsNewly formed endothelial cells stimulate the growth of adjacent tumor cells by secreting growth factors, e.g : PDGF, IL-1Angiogenesis is required for metastasis

How do tumors develop a blood supply?Tumor-associated angiogenic factors These factors may be produced by tumor cells or by inflammatory cells infiltrating the tumor e.g. macrophagesImportant factors : Vascular endothelial growth factor( VEGF ) Fibroblast growth factor (FGF)

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

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

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?

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. Tumor Microenvironment

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).

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