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I) What is cancer metastasis?

• Cancer defines as a population of cells that have lost their normal controls of growth and differentiation and are proliferating without check.

• Metastasis is the process by which a tumor

cell leaves the primary tumor, travels to a distant site via the circulatory system, and establishes a secondary tumor.

Invasione e metastasi

• L’approccio riduzionistico alla biologia del tumore si è concentrato sui meccanismi molecolari alterati della cellula tumorale

• Questa scelta ha portato ad un ritardo nell’affrontare lo studio delle metastasi, malgrado rappresentino la più grave complicazione delle malattie tumorali

Relazioni cliniche e patologiche

• Il rischio di metastatizzazione aumenta con: – dimensioni del tumore primario – l’invasione regionale dei linfonodi – (inversamente) grado di differenziamento del

tumore – profondità dell’invasione tumorale oltre il

normale confine tessutale (es. melanoma)

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Preferential metastatic sites

Primary tumour Common distant site (s) Breast’ adenocarcinoma Bone, brain, adrenal

Prostate adenocarcinoma Bone

Lung small cell carcinoma Bone, brain, liver

Skin cutaneous melanoma Brain, liver, Bowel

Thyroid adenocarcinoma Bone

Kidney clear cell carcinoma Bone, liver, thyroid

Testis carcinoma Liver

Bladder carcinoma Brain

Neuroblastoma Liver, adrenal

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Reason for organ selectivity

Mechanistic theory: determined by the pattern of blood flow.

“Seed and soil” theory: the provision of a

fertile environment in which compatible tumor cells could grow

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Determining factors

• Appropriate growth factors or extracellular matrix environment

• Compatible adhesion sites on the endothelial lumenal surface

• Selective chemotaxis at which the organ producing some soluble attraction factors to the tumor cells

Implicazioni del modello di progressione tumorale

• Importanza principale data alla diagnosi ed eradicazione precoce del tumore

• Dissezione linfonodale (segno di estensione precoce), radiazione locale

• Chemioterapia adiuvante per eliminare le cellule residue rimaste dopo l’eradicazione chirurgica

La geografia della progressione tumorale

• Le cellule che danno inizio a metastasi hanno già accumulato le mutazioni “progressor” nel tumore primario

• Diverse cellule (con diverso spettro di mutazioni progressor) danno origine a diverse metastasi

Modelli alternativi (anni ’80)

• Fin dall’inizio del processo tumorale, c’è una predeterminazione rispetto alla capacità o meno di dare metastasi

• I tumori sono una malattia sistemica fin dall’inizio (nei casi che danno origine a metastasi)

Un puzzle molecolare

• Se le metastasi hanno origine da una piccola frazione di cellule nella massa tumorale primaria, come è possibile che si possa predire il destino clinico (metastasi o meno) analizzando il tumore primario in toto?

• The genes that specify the metastasis would not seem to confer increased proliferative benefit at the primary site. That is, there is no reason to think that a metastatic phenotype enables cells to proliferate more effectively within the primary tumour mass, thereby increasing their representation in the overall tumour-cell population. Hence, rare cells in the primary tumour mass that happen to acquire metastatic capability will remain rare.

Alternative model: Weinberg and Bernards, 2002

• Reasoning like this drives us to consider a quite different mechanistic model: namely, that the tendency to metastasize is largely determined by the identities of mutant alleles that are acquired relatively early during multistep tumorigenesis.

• It is already apparent that there are several alternative genetic paths that cells can take en route to forming a primary tumour. Thus, a particular phenotype required early in tumorigenesis by an evolving tumour cell can be acquired through the mutation of any one of several alternative growth-controlling genes.

• We suggest that a subset of the mutant alleles acquired by incipient tumour cells early in tumorigenesis confer not only the selected replicative advantage, but also, later in tumorigenesis, the proclivity to metastasize. This proclivity will become manifest only much later in tumour progression, in the context of yet other mutations that have struck the genomes of descendant cells.

Evidenze in supporto • In some small, well-localized primary human breast cancers,

individual carcinoma cells are clearly detectable in the bone marrow.

• Furthermore, DNA-microarray analysis reveals that the gene-expression pattern of metastatic tumour cells is often strikingly similar to that of the cells confined to the primary tumour mass from which they were derived, implying that the dominant cell population in the primary tumour mass is phenotypically and possibly genotypically (almost) identical to the cells in the metastases.

• (già discusso) Equally relevant are other studies in which the gene-expression profiles of the dominant populations of breast-cancer cells within a primary tumour mass have been used to predict, with 90% accuracy, whether the tumour will remain localized or whether the patient will experience metastases and disease relapse. Here, once again, the metastatic behaviour of these cancer cells seems to be determined relatively early in tumorigenesis.

• Finally, several well-studied oncogenes, including ras and myc, the proliferative powers of which are well documented, can function in certain mouse models of tumorigenesis to enable

Implicazioni • First, the tendency of a tumour eventually to metastasize is already pre-

ordained by the spectrum of mutations that progenitor cells acquire relatively early in tumorigenesis; that is, some cancers start out 'on the wrong foot'.

• Second, genes and genetic changes specifically and exclusively involved in orchestrating the process of metastasis do not exist. Instead, the genes for metastasis are largely those that cancer biologists have been studying intensively for a generation: the oncogenes and tumour-suppressor genes.

• Third, because important components of the genotype of metastasis are already implanted in cells relatively early in tumorigenesis, even relatively small primary tumour cell populations may already have the ability to dispatch metastatic pioneers to distant sites in the body.

• The implications for the usefulness of early clinical detection of breast cancer are unsettling

Eppure... • Apparentemente lo screening e la diagnosi

precoce hanno diminuito la mortalità tumorale

• Il fenomeno delle metastasi dormienti (micrometastasi possono rimanere non clinicamente rilevabili per decenni dopo la rimozione del tumore primario) è più facilmente spiegabile con un modello in cui occorrono ulteriori mutazioni genetiche al sito metastatico

Cascata metastatica

•Diminuita aderenza intercellulare

•Aumentata motilità ed invasione dello stroma

•Ingresso nei vasi e sopravvivenza in circolo

•Crescita in organi distanti

Cancer cells need to change their epithelial properties, to lose their adhesion and to

penetrate through potent physical barriers

basal lamina connective tissue

Figure 14.13a The Biology of Cancer (© Garland Science 2007)

EMT Epithelial to Mesenchymal Transition

sea urchin embryo

EMT in Tumor Progression

Major changes during EMT

- Loss of E-cadherin

- Cell shape changes driven by Rho GTPases

- MMPs

Figure 13.12d The Biology of Cancer (© Garland Science 2007)

cadherin actin

Figure 14.15b The Biology of Cancer (© Garland Science 2007)

Adopting changes typical to EMT

Figure 14.19c The Biology of Cancer (© Garland Science 2007)

Epithelial marker

Mesenchymal marker

Rho family proteins promote actin remodeling

Svitkina and Borisy JCB 99

TGF beta and EMT

MMPs (matrix metalloproteinases) help the cancer cells to invade the ECM

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Components of invasion

a) Matrix degrading enzymes

b) Cell adhesion

c) Cell motility

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a) Matrix degrading enzymes

• Required for a controlled degradation of components of the extracellular matrix (ECM)

• The proteases involved in this process are classified into serine-, cysteine-, aspartyl-, and metalloproteinase.

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MMP family

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Matrix metalloproteinases (MMP)

• 16 members, subdivided into 4 groups, based on their structural characteristics and substrate specificities

• Soluble and secreted groups; collagenase, gelatinase and stromelysins

• Membrane type (MT-MMP) group are anchored in the plasma membrane

• A zinc ion in the active centre of the protease is required for their catalytic activities.

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Regulation of MMP

• MMP is controlled by an increased expression on a transcriptional level.

• MMPs are calcium-dependent proteases, which are synthesized as a inactive proenzymes and are activated by the cleavage of a propeptide.

• MMP activity is regulated by specific inhibitors, the tissue inhibitors of MMP (TIMPs). Binding TIMP to MMP is in a 1:1 stoichiometry.

• MMP2 and MMP9, which cleave type IV collagen the major constituent of basement membrane, are believed to be of special importance

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Serine proteases

• Serine protease involved in ECM degradation are plasmin, plasminogen activators and cathepsin G.

• Plasmin is believed to be the most important serine protease, firstly because its ability to degrade several matrix components like gelatin, fibronectin or laminin, and secondly by the possible activation of numerous proforms of MMPs by propeptide cleavage.

• Plasmin is synthesized in its inactive proform, plasminogen, which can be converted to plasmin by plasminogen activator.

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Plasminogen activator

• Two main types : urokinase (uPA) and tissue (tPA).

• uPA is bound to the surface of tumor cells by means of a specific receptor (uPAR)

• There are specific inhibitors (PAI-1 and PAI-2) for the PA.

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Interaction between tumour cells and the surrounding connective tissue

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Cell adhesion and metastasis

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HGF induce cell scattering and invasion

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Signalling pathways responsible for MET-dependent invasive growth

Angiogenesi tumorale

• Le cellule di mammifero devono essere localizzate ad un massimo di 200µM di distanza dalla microvascolatura

• Questa distanza corrisponde al limite massimo di diffusione dell’ossigeno

• Per una crescita ulteriore, c’é bisogno di formare nuovi vasi sanguigni

• Simili restrizioni sono in atto anche nella massa tumorale

Tumor Angiogenesis and Neovasculature

A, Tumors less than 1 mm3 receive oxygen and nutrients by diffusion from host vasculature. B, Larger tumors require new vessel network. Tumor secretes angiogenic factors that stimulate migration, proliferation, and neovessel formation by endothelial cells in adjacent established vessels. C, Newly vascularized tumor no longer relies solely on diffusion from host vasculature, facilitating progressive growth.

Ca in situ e nuovi vasi sanguigni

Ca invasivo

Lo “switch” angiogenico

• I tumori producono fattori pro-angiogenici ed anti-angiogenici

• Nella prima fase della crescita tumorale, il bilancio é tale da non favorire l’angiogenesi

• Lo “switch” avviene quando I fattori pro-angiogenici prendono il sopravvento

• L’ulteriore crescita tumorale e la metastatizzazione sono angiogenesi-dipendenti

Segnali di attivazione dello “switch”

• Stress metabolico (ipossia, basso pH, ipoglicemia): tutte condizioni che si creano nell’habitat tumorale quando avviene un accrescimento di massa senza angiogenesi

• Infiammazione • Stress in generale

Struttura e funzione dei vasi tumorali

• I meccanismi di regolazione dell’angiogenesi sono molto meno stringenti

• I vasi tumorali hanno caratteristiche strutturali diverse da quelli normali: in alcuni casi, il lume é rivestito da un mosaico di cellule endoteliali e tumorali (con ovvie implicazioni per il processo di metastatizzazione)

• I vasi tumorali sono tortuosi, dilatati, molto ramificati: questo provoca ipossia locale nel tumore, indirettamente provoca problemi di distribuzione dei farmaci anti-tumorali, ed inoltre porta ad ulteriore stimolazione della produzione di fattori pro-angiogenici

Quiescenza tumorale

• Non necessariamente la crescita dei tumori segue un’evoluzione spontanea fulminante

• Alcuni tumori possono restare in uno stato di quiescenza per anni, probabilmente per l’espressione di un’elevata quantitá di fattori antiangiogenici (endostatina, angiostatina, ecc.)

Terapia anti-angiogenica • Si basa sul presupposto (dimostrato sperimentalmente in

diversi modelli animali) che l’inibizione dell’angiogenesi impedisca l’espansione e metastatizzazione tumorali

• Risultati nei modelli animali: la terapia anti-angiogenica non é in grado di “uccidere” le cellule tumorali esistenti (stasi tumorale)

• Gli inibitori dell’angiogenesi sembrano “normalizzare” I vasi tumorali. Questo potrebbe aumentare l’efficacia di farmaci chemioterapici, facendo sí che raggiungano l’intera massa tumorale