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Tumor pathogenesis Oncogenes Tumor suppressor genes Invasion and Metastasis Introduction Carcinogensis is multistep process involving the multiple genetic changes including the activation of cooperating oncogenes and the inactivation of tumor suppressors in somatic cells 2 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. 肿瘤的信号转导通路调控异常 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-onc):in normal physiologic version --- Oncogene:altered in cancer Viral oncogene (v-onc) Fuctions of proto-oncogenes 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. Classification of proto-oncogenes Growth factors, e.g. V-sis, PDGF-b, int-2 Receptor Tyrosine Kinases, e.g. Her-2/neu/ erbb2, Membrane Associated Non-Receptor Tyrosine Kinases, e.g. src, Lck G-Protein Coupled Receptors e.g. Mas Membrane Associated G-Proteins , e.g. Ras Serine/Threonine Kinases e.g. Raf Nuclear DNA-Binding/Transcription Factors, e.g. myc, fos Others Apoptosis regulators, e.g. Bcl-2, Regulators of cell cycle, e.g. Cyclin D1, CDK4 Mechanisms of Oncogene Activation 1. Gene amplification, e.g. myc, CCND1 2. Point mutation, e.g. ras, 3. Chromosomal rearrangement or translocation the transcriptional activation of proto-onc. the creation of fusion genes, e.g. abl-bcr 4. Viral insertion activation, e.g. c-Myc Translocation Amplification CHROMOSOMAL REARRANGEMENTS OR TRANSLOCATIONS Neoplasm Translocation Proto-oncogene Burkitt lymphoma t(8;14) 80% of cases t(8;22) 15% of cases t(2;8) 5% of cases c-myc1 Chronic myelogenous leukemia t(9;22) 90-95% of cases bcr-abl2 Acute lymphocytic Leukemia t(9;22) 10-15% of cases bcr-abl2 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 15 GENE AMPLIFICATION Oncogene Amplification Source of tumor c-myc ~20-fold leukemia and lung carcinoma N-myc 5-1,000-fold neuroblastoma retinoblastoma L-myc 10-20-fold small-cell lung cancer c-abl ~5-fold c-myb 5-10-fold acute myeloid leukemia colon carcinoma c-erbB ~30-fold epidermoid carcinoma K-ras 4-20-fold 30-60-fold colon carcinoma adrenocortical carcinoma chronic myoloid leukemia 16 Mechanisms of action of oncogenes Ras Locates on chromosome 11, codes for a protein with GTPase activity relays 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-2 This 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 kinases Dr. Slamon (UCLA) described the role of Her2/neu in breast cancer and ovarian cancer. Overexpression, amplification, rare translocations No ligand is known Ras relays signals from the cell surface receptors to the nucleus Ras relays signals by acting as a switch Prospect A breakthrough for our understanding of the molecular and genetic basis of cancer Provided 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 genes Concept: genes that sustain loss-of function mutations in the development of cancer TSGs Transcriptional factor: p53, WT1, Direct transcription regulator: Rb, APC Inhibitor of cell cylcle kinase: p16INK4A, p19ARF, Cell structural components: NF2 Phosphatase: PTEN Potential mediator of mRNA processing: VHL Components 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 interactions unknown colorectal cancer WT1 (11p) transcription Wilm’s tumor lung cancer Rb1 (13q) transcription retinoblastoma small-cell lung carcinoma p53 (17p) transcription Li-Fraumeni syndrome breast, colon, & lung cancer BRCA1(17q) transcriptional breast cancer breast/ovarian tumors BRCA2 (13q) regulator/DNA repair Mechanism for the inactivation of TSGs 1. Mutation: point mutation or frameshift mutation, p53 2. Deletion: LOH (loss of heterozygosity) or homozygous deletion, Rb 3. Viral oncoprotein inactivation: p53, Rb 4. Promoter hypermethylation, histone modification changes: p16 Rb function Rb regulates G1/S transition Rb inactivation by viral oncoprotein KNUDSON TWO HIT HYPOTHESIS IN SPORADIC CASES Normal Cells RB RB RB RB RB Mutation RB LOH Tumor cells Inactivation of a tumor suppressor gene requires two somatic mutations. 29 P53 Function as gatekeeper Inactivation of p53 in cancer •LOH on 17p13 in a number of tumors Bax •Point 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 1. 2. 3. 4. Growth at primary site and angiogenesis Tumor cell invasion Lymphatic and hematogenous metastasis Growth at secondary site and angiogenesis Mechanisms involved in tumor cell invasion 1.Loss of cell-to cell cohesive forces 2. Secretion of ECM-degrading enzymes 3. Active Locomotion 4. Tumor angiogenesis 5. Metastasis-related genes 5. Metastasis-enhancing Genes: Oncogenes,CD44, Integrinβ1, CEA, MMP2, u-PA, etc 1. Loss of cell-to cell cohesive forces: Cell adhesion molecules (CAMs): 细胞粘附分子:介导细胞之间或细胞与ECM之间的选择性粘附。 • E-cadherin: Expression↓ Loss of cell-cell adhesion,Increased cell motility • Integrins: Expression↓→↑ • Immunoglobin superfamily:NCAM, 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): ~3 Metastasis-associated proteinases Cell invasion of the extracellular matrix The MMP family (at least 23 members) (1) Classification 基质金属蛋白酶 间质胶原酶(Interstitial Collagenase ),如MMP-1、MMP-5、 MMP-8、 MMP-13等,作用底物主要为间质胶原 Ⅰ、Ⅱ、Ⅲ、Ⅶ、Ⅹ 型胶原,但不能降解明胶和Ⅳ型胶原。 明胶酶(Gelatinase )又称Ⅳ型胶原酶,如MMP-2、 MMP- 9,作用底 物主要是Ⅳ型胶原和明胶,还可以降解Ⅵ、Ⅶ、Ⅷ和Ⅹ型胶原,但 不能降解间质胶原。 基质溶解素(Stromelysin),如MMP-3、 MMP-7、 MMP-10 和MMP-11 等,作用底物主要是基质中的蛋白多糖和糖蛋白,如纤维连接蛋白 (FN)、层黏连蛋白(LN)等。此外,基质溶解素对胶原的作用不同 于间质胶原酶间质和胶原酶,他们能降解Ⅳ、Ⅴ、Ⅷ、Ⅹ型胶原的 非螺旋区及Ⅰ型胶原的氨基末端。 膜型金属蛋白酶(Membrane-type MMPs, MT-MMPs ),目前已发现四 种,包括MT1-MMP、MT2-MMP、MT3-MMP和MT4-MMP。MT-MMPs主要定位 于肿瘤细胞及其基质成纤维细胞的细胞膜上,是MMP的受体,也是 MMP的激活剂,还可以降解Ⅰ、Ⅱ、Ⅳ型胶原和FN,其表达受刀豆蛋 白、癌基因等因素的调解。 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 uPA¯uPAR-initiated signal transduction and consequences Plasminogen/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 Inhibitors:angiostatin, etc Models of Tumour Angiogenesis Endogenous angiogenesis inhibitors 5. Metastasis-enhancing Genes: Oncogenes, CD44, Integrinβ1, CEA, MMP2, u-PA, etc Metastasis Promoting Genes - I Gene Tissue Site Function ARM-1 Lymphoma Promotes adhesion of tumor cells to the endothelium ATX Breast, Liver, Lung, Melanoma, Teratocarcinoma cytoskeletal reorganization and motility; Gprotein coupled receptor activation CD44 Multiple sites cell-cell interactions; activates HGF/c-Met pathway Cox2 Breast, Colorectal, Gastric Prostaglandin synthase; induces VEGF Cyr61 Breast Mediates adhesion; Erb-B2/3/4 pathway Ezrin Liver, Ovary, Pancreas, Prostate, Membrane-cytoskeletal linker; RHO and RAC Uterus interactions HMG-I(Y) Breast, Cervical, Colorectal, Prostate, Skin, Thyroid, Uterus Regulated by EGF and MMP-9 Laminin-5 Multiple sites EGF and TGF-a induce expression of laminin subunits; cell adhesion, motility c-Met Multiple sites Activated by HGF; Modulates Ras and PI3 kinase Metastasis Promoting Genes - II Gene Tissue Site Function MTA1 Breast, Cervix, Melanoma, Ovary Neucleosome remodeling; histone deacetylase complex Oncostatin M Lung Activates PKA-dependent pathway PP2A Not determined Activated by p38/MAPK; inhibits MEK1, MEK2, and MMP-1 RAGE Gastric, Lung, Pancreatic, Renal transmembrane receptor; activates p21, MAPKs, NF-6B, cdc42/rac S100A4 Breast, Colorectal, Prostate Calcium-binding protein; activates c-erbB-2 S100A9 Colon, Gastric, Skin Calcium-binding protein; Modulates Mac-1 integrin receptor through G-protein Semaphorins Gastric, Leukemia, Lung, Skin cell-cell interactions; Receptor crosstalk with c-Met binding semaphorin receptor, plexin Thymosin-b15 Prostate actin binding; motility Wnt-5a Breast, Colon, Lung, Melanoma, Pancreas, Prostate PKC activation with associated changes in cytoskeleton, cell adhesion, and motility 6. Metastasis-Suppressor Genes Identified nm23, KAI 1, TIMPs, E-cadherin, Kiss, etc Modified 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 functions 1. Interaction with numerous proteins Tiam1, Ras, cytoskeletal protein 2. A NDPKinase activity 3. DNA nuclease 4. Serine or histidine protein kinase inhibition of the Map kinase pathway and correlated with motility suppression. Nucleoside diphosphate kinase (NDPKinase) activity The nm23-H1 gene product has been identified as the NDPKA isoform The nm23-H2 gene product has been identified as the NDPKB isoform NDPKs: catalyze the phosphorylation of nucleoside diphosphates to the corresponding nucleoside triphosphates, mainly at the expense of the ATP synthesized through oxidative phosphorylation KAI1 / CD 82 Names : KAI1 / CD82, (C33, R2, IA4) Gender : Transmembrane Glycoprotein Ligands ? Signal Pathways : ? Particularity Biological Function : motility invasiveness cell-cell interactions Member of the tetraspanin or transmembrane 4 superfamily (TM4SF) Contains an internalization sequence at its C-terminus (YSKV) Current Address : Cell membrane (lymphocytes, epithelial cells) Lysosomes Vesicles KAI1 / CD 82 and Cancer Correlations High level of KAI1/CD82 is a good prognosis factor or associated with low grade histology : prostate pancreas colon lung carcinoma KAI1/CD82 expression is inversely related to the metastatic potential : Experimental Data Transfection of tetraspanin reduces metastatic potential melanoma prostate breast B16 MDA-MB-435 * AT6.1, AT6.3 MDA-MB-231 (from Boucheix & Rubinstein , 2001 prostate lung carcinoma colon hepatoma breast lung (non-small-cell carcinoma) bladder cancer ovary melanoma Loss of KAI-1 Expression in Prostate Cancer KAI-1 Functions + (Promotes) - (Inhibits) Cell Aggregability Cell Adhesion Invasion Motility Metastasis Metastasis Suppressor Pathways C D H 1 K A I 1 b-Catenin E G F R I G F R 1 F A S M E T G P R 5 4 R T K CRSP3 RAS VAV RhoGDI2 PI3K Rho Rac RAF KSR PTEN ASK1 NME1 MKK4 MKK7 KISS1 PLC TXN MEK AKT TXNIP MKK6 ERK MKK3 JNK Apoptosis Inflammation Differentiation Survival NFkB HIF BAD p38 Apoptosis Cytokines Survival Angiogenesis Motility Invasion Growth Differentiation 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 activation Proteolysis MMPIs uPAR-I Blocks degradation of matrix, blocks activation of proteases, growth factors Motility Taxanes Blockade of microtubule cycling B. Signal Inhibitors: Blockade of signals necessary for angiogenesis , invasion, and metastasis Agent Target CAI Calcium influx Squalamine Inhibits NHE-3 PI3K inhibitors MAPK inhibitors Activity Inhibits adhesion, motility, angiogenesis Anti-angiogenic Inhibit motility, proliferation, promote Inhibit invasion, proliferation Tumor Microenvironment 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 tumor microenvironments 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 the extracellular matrix (hatched background), evolve during progression, thereby enabling primary, invasive, and then 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 by reciprocal heterotypic signaling interactions, of which only a few are illustrated. (Lower) The intracellular signalin depicted in the upper panel withi the tumor microenvironment is no static but instead changes during tumor progression as a result of reciprocal signaling interactions between cancer cells of the parenchyma and stromal cells that convey the increasingly aggressive phenotypes that underlie growth, invasion, and metastatic dissemination. Importantly, the predisposition to spawn metastatic lesions can begin early, being influenced by the differentiation program of the normal cell-of-origin or by initiating oncogenic lesions. 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 the enabling 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 lines of accomplishments are converging to enable clinical researchers to investigate therapies that target the molecular and genetic abnormalities detected in an individual patient’s cancer: (i) We have identified most of the few hundred genes that are mutated or abnormally expressed in human cancers. (ii) Genome sequencing technology will soon enable us to screen the mutations in a human cancer biopsy in less than a week, at a cost of a few thousand dollars. Abnormalities in cellular proteins and RNA can already be detected in that time interval and at a reasonable cost. (iii) Pharmaceutical and biotechnology companies have more than 800 new experimental drugs and biological agents in the pipeline that are designed to target the products of abnormal genes found in human cancers