Tumor pathogenesis Oncogenes

Tumor suppressor genes

Invasion and Metastasis

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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

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 ）

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 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

Classification of proto-oncogenes

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

Amplification

Translocation

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CHROMOSOMAL REARRANGEMENTS OR TRANSLOCATIONS

Neoplasm Translocation Proto-oncogene

Burkitt lymphoma t(8;14) 80% of cases c-myc1

t(8;22) 15% of cases t(2;8) 5% of cases

Chronic myelogenous t(9;22) 90-95% of cases bcr-abl2

leukemia

Acute lymphocytic t(9;22) 10-15% of cases bcr-abl2

Leukemia

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

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GENE AMPLIFICATION

Oncogene Amplification Source of tumor

c-myc ~20-fold leukemia and lung carcinoma

N-myc 5-1,000-fold neuroblastomaretinoblastoma

L-myc 10-20-fold small-cell lung cancer

c-abl ~5-fold chronic myoloid leukemia

c-myb 5-10-fold acute myeloid leukemiacolon carcinoma

c-erbB ~30-fold epidermoid carcinoma

K-ras 4-20-fold colon carcinoma 30-60-fold adrenocortical carcinoma

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-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 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

TSGsTranscriptional 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 unknown colorectal interactions cancer

WT1 (11p) transcription Wilm’s tumor lung cancer

Rb1 (13q) transcription retinoblastoma small-cell lung carcinoma

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

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RB RB

RBLOH

RBMutation

Normal Cells

Tumor cells

KNUDSON TWO HIT HYPOTHESIS IN SPORADIC CASES

RB RB

Inactivation of a tumor suppressor gene requires two somatic mutations.

P53Function as gatekeeper

Bax

Inactivation of p53 in cancer •LOH on 17p13 in a number of tumors

•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. Growth at primary site and angiogenesis2. Tumor cell invasion3. Lymphatic and hematogenous metastasis4. Growth at secondary site and angiogenesis

Mechanisms involved in tumor cell invasion

1.Loss of cell-to cell cohesive forces2. 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; G-protein 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, Uterus

Membrane-cytoskeletal linker; RHO and RAC interactions

HMG-I(Y) Breast, Cervical, Colorectal, Prostate, Skin, Thyroid, Uterus

Regulated by EGF and MMP-9

Laminin-5 Multiple sites EGF and TGF-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-15 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 Identifiednm23, 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) activityThe 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

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

Ligands ?

Biological Function :motilityinvasivenesscell-cell interactionsParticularity

Signal Pathways : ?

High level of KAI1/CD82 is a good prognosis factor or associated with low grade histology :

prostate lungpancreas carcinomacolon

prostatelung carcinomacolonhepatomabreastlung (non-small-cell carcinoma)bladder cancerovarymelanoma

KAI1/CD82 expression is inversely related to the metastatic potential :

Transfection of tetraspanin reduces metastatic potential

melanoma

prostatebreast

B16MDA-MB-435 *AT6.1, AT6.3MDA-MB-231

KAI1 / CD 82 and Cancer

Correlations Experimental Data

(from Boucheix & Rubinstein , 2001

Loss of KAI-1 Expression in Prostate CancerLoss of KAI-1 Expression in Prostate Cancer

KAI-1 Functions

+ (Promotes) - (Inhibits)

Cell AggregabilityCell Adhesion

InvasionMotilityMetastasis

RTK

CDH1

EGFR

RASCRSP3

RAF

MEK

ERK

GrowthDifferentiation

ApoptosisInflammationDifferentiation

Survival

-Catenin

KAI1

RhoGDI2

FAS

KISS1

NFB

ASK1

MKK7

JNK

MKK4

MKK6

p38

MKK3

ApoptosisCytokines

SurvivalAngiogenesis

TXNIP

TXN

HIF

VAV

KSR

NME1

MotilityInvasion

GPR54

IGFR1

MET

PI3KPLC

AKT

BAD

PTENRho

Rac

Metastasis Suppressor PathwaysMetastasis 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-v3 (Vitaxin) Blocks endothelial cell v3 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 individualpatient’s 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