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dependent ceU-cell adhesion due, not to the lack of E-cadherin expression,

b u t rather the absence of a-catenin. Re-introduction of a-catenin restores

adhesion (Breen et al., 1993). The lack of a-catenin expression also

in in vitro assays, and is linked to unfavourable prognosis for patients w ith colon cancer (Vermeulen et al., 1995; Raftopoulos et al., 1998; Vermeulen

et al., 2000). In vivo assays, however, fail to show a correlation betw een invasiveness and lack of a-catenin, suggesting that environm ental factors

are also involved (Hoorde et al., 2000). Expression of a-catenin is linked to

that of E-cadherin in L cells, which do not norm ally express cadherins.

Forced expression of cadherin in these cells, prom otes stabilisation of a-

catenin, restoring cell-cell adhesion (Nagafuchi et al., 1987). Moreover,

w hen overexpressed in this cadherin-deficient cell Une, a-catenin inhibits

W nt 3a-induced transcription (Takahashi et al., 2000). So far, no tum ours

have been found in which a-catenin is present and E-cadherin is not,

suggesting that alterations in the a-catenin gene affect only the expression

of this protein w ithout interfering w ith E-cadherin levels. These findings

indicate that the absence of a-catenin, and not that of E-cadherin,

correlates w ith the pathology of these tum ours (Gofuku et al., 1999).

O varian cells lacking functional a-catenin also have im paired ceU-

cell adhesion, which is restored after artificial expression of this protein.

M ore im portantly, expression of a-catenin slows the grow th rate of these

cells and also decreases their ability to form tum ours w hen injected into

nu d e mice (Bullions et al., 1997). Taken together, these results indicate that

d o w n regulation of a-catenin m ay be im portant, not only for cell-cell

adhesion, b u t also for cell grow th and invasion.

1.6- O ther players in tumorigenesis

1.6.1- PI3K and PTEN

The activity of lipid kinases, such as phosphatidylinositol 3 kinase

(PI3K), is essential for m any cellular processes. M utations in the pllOy

colorectal adenocarcinomas and in colon cancer cell lines. The

overexpression of this subunit restores norm al grow th in cells expressing

m utated APC, p53, p-catenin or K-ras (Sasaki et al. 2000).

Protection from apoptosis requires the activation of PK B/A kt and

negative regulation of the tum our suppressor PTEN (phosphatase and

tensin homologue deleted from chromosome 10). PTEN, by

dephosphorylating phosphatidylinositol 3,4,5 triphosphate (PIP-3),

antagonises the activity of PI3K, inhibiting ceU grow th and thus

functioning as a tum our suppressor. The activation of PK B/Akt by PI3K

inhibits apoptosis by preventing the release of cytochrome c from

m itochondria. Activated Akt also phosphorylates BAD and caspase-9,

inactivating these tw o pro-apoptotic factors (reviewed by Di Cristofano et

PandoHli, 2000). M oreover, activated Akt inactivates GSK3, resulting in

cychnDl expression (Sun et al., 1999). Cells that have lost PTEN expression

have higher levels of cyclin D l, a p-catenin/Tcf target gene. Expression of

PTEN in these cells results in inhibition of p-catenin/Tcf-dependent

transcription, possibly by prom oting degradation of p-catenin (Paramio et

al., 1999; Persad et al., 2001).

1.6.2- ILK

Another player in the pathw ay described above is the Integrin-

Linked Kinase (ILK). ILK is a serine/threonine kinase that transmits

signals activated by cell-extracellular matrix interactions. Overexpression

of ILK in epithelial cells results in nuclear localisation of p-catenin and

activation of p-catenin/Tcf-dependent transcription (Novak et al., 1998).

The activation of W nt-signalling by ILK is a consequence of its ability to

1.7- Cross ta lk between different signalling pathw ays 1.7.1- W n t and TGF-p signalling

M utations in APC are not sufficient to trigger malignancy and

further genetic alterations are needed for the developm ent of

invasiveness. Such alterations are represented for example, by loss of a

response to TGF-p (transforming grow th factor-p) in colon cancers cells

(Thiagalingam et al., 1996; Grady et al., 1999). TGF-p inhibits cell grow th

and m ay be involved in m aintaining the norm al cycle of cell renew al in

the intestinal epithelium. Activation of TGF-p induces the form ation of a

complex containing Smad 2 /3 and Smad 4, which associates w ith

transcription factors and activates specific gene targets. The Smad 4 and

Smad 2 genes are lost in some colorectal tum ours (reviewed by Bienz and

Clevers, 2000). Further evidence for the involvem ent of this signalling

pathw ay in tumorigenesis comes from mice doubly-heterozygous for

m utations in APC and Smad 4, which develop large intestinal tum ours

that are both highly proliferative and invasive (Takatu et al., 1998).

Cooperation betw een W nt and TGF-p signalling also occurs in the

frog embryo, w here both pathw ays are necessary for the establishment

of the dorsal signalling centre (Spemann's organizer). Beta-catenin/Tcf

complexes associate w ith Smad 4, leading to expression of homeobox

XTwin gene (Nishita et al., 2000). The collaboration betw een W nt and

TGF-p signaUmg w ould result in differential expression of specific genes

depending on w hether or not Smad 4 is present in the p-catenin/Tcf

complex. For instance, the prom oter regions of Twin and Ubx contain

both W nt/W g and TGF-p recognition elements (Hecht and Kemler,

1.7.2- Involvem ent o f Ras

Point m utations that activate the K-ras proto-oncogne are found in at least 50% of hum an colorectal cancers an d they play an essential role in

tumorigenesis. D isruption of activated K-ras in colon cancer cell lines changes their m orphology, abrogates their ability to evade contact

inhibition, causes loss of anchorage-independent grow th, slows the

grow th rate, inhibits the form ation of tum ours in n u d e mice and reduces

expression of c-myc (Shirasawa et al., 1993). The ras pathw ay is also involved in the lack of response of colon cancer cells to TGF- signals

(Calonge et Massague, 1999).

1.7.3- p53

P53 is a tum our suppressor gene m utated in m ost cancers

(reviewed by Damalas et al., 1999). Functional p53 helps to m aintain

cellular genomic stability by eliminating cells w ith DNA dam age or by

helping repair this damage. The cellular level of p53 is normally low, since

p53 is degraded in the ubiquitin-proteasom e pathw ay (Maki et al., 1996).

In response to stress, p53 levels rise and it moves to the nucleus, causing

cell cycle arrest or induction of apoptosis. Overexpression of p-catenin

results in high levels of p53 protein (but not mRNA), due to competition

betw een these tw o proteins for the com ponents of the degradation

pathw ay (Damalas et al., 1999). p-catenin also competes w ith p53 for the

binding to the co-activator CBP/p300, which is necessary for activation of

apoptosis by p53 (Miyagishi et al., 2000).

More recently p53 was show n to be involved in the activation of an

alternative degradation pathw ay for p-catenin. A p53-inducible ubiquitin-

conjugating enzyme, Siah, binds to the carboxyl term inus of APC and induces

p-catenin. Unlike p-TrCP, Ebi interacts w ith p-catenin, independently of GSK3

phosphorylation (M atsuzawa and Reed, 2001). Thus, Siah dow nregulates p-

catenin signaUmg in cells that express p-catenin lacking GSK3 phosphorylation

sites, but does not dow nregulate this signalling pathw ay in ceUs that express

truncated APC (Liu et al., 2001). These results indicate the existence of a

degradation pathw ay that is APC-dependent, b u t GSK3-independent.

1.7.4- W n t and Retinoic acid signalling

Retinoids (vitamin A derivatives) have a strong effect on ceU

proliferation, differentiation and carcinogenesis. Treatm ent of ceUs w ith

retinoic acid (RA) decreases p-catenin-dependent transcription through a

direct association betw een the retinoic acid receptor (RAR) and p-catenin.

Moreover, this association induces transcription of RAR-responsive

promoters, suggesting that p-catenin can w ork as a co-factor in the RA

signalling pathw ay (Easwaran et al., 1999). More recently, it has been

show n that treatm ent of colon cancer ceUs that express vitam in D receptor

(VDR) w ith vitam in D3 results in dow n regulation of p-catenin-dependent

transcription, prom oting ceU differentiation. This inhibition is due to the

competition betw een ligand-activated VDR and Tcf for binding to p-

catenin (Palmer et al., 2001).

The intim ate connection betw een ceU-cell adhesion and Wnt-

signalling pathw ay has highlighted the im portance of the proteins

involved in regulating the levels of p-catenin, both during developm ent

and in tum our progression. A lpha-catenin is ideaUy placed to regulate

these processes, since it is found in ceU adhesion complexes and associates

CHAPTER II- Materials and Methods