SMAD- signaloinnista metastasointien yhteydessä

http://www.nature.com/bjc/journal/v99/n6/full/6604562a.html

British Journal of Cancer (2008) 99, 957–965. doi:10.1038/sj.bjc.6604562 www.bjcancer.com
Published online 19 August 2008

Smad7 induces hepatic metastasis in colorectal cancer

S K Halder^1,2, G Rachakonda^1,2, N G Deane¹ and P K Datta¹

¹Departments of Surgery and Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN, USA

Received 28 April 2008; Revised 22 July 2008; Accepted 23 July 2008
Advance online publication 19 August 2008

• Vaikka SMAD-signaloinnilla  tiedetään olevan tuumorikasvua vaimentava rooli,   järjestelmällä on kuitenkin  osoitettu olevan myös osuutta metastaasin edistäjänä  rintasyövässä ja  melanoman luumetastaaseissa. 
• Sitävastoin SMAD4 -mutaatio tai sen  alentuneet pitoisuudet   kolorektaalisyövässä  korreloivat  suoraan  huonoon elossapysymiseen ja lisääntyneeseen metastasoitumiseen.  Kuitenkaan aiemmin ei ole selvitetty, mikä on SMAD- signaloinnin rooli kolorektaalikarsinooman metastasoitumisessa.

Although Smad signalling is known to play a tumour suppressor role, it has been shown to play a prometastatic function also in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer is directly correlated to poor survival and increased metastasis. However, the functional role of Smad signalling in metastasis of colorectal cancer has not been elucidated.

•  Aiemmin tämä tutkijaryhmä on osoittanut, että inhibitorisen SMAD  ( I-SMAD)  tekijän SMAD7 liiallinen ilmeneminen colonin  adenokarsinomasoluissa  vaikuttaa tumorigeenisesti  blokeeraamalla TGFbeta-indusoiman kasvunestymän ja apoptoosin. 
• Nyt tässä  työssään  tutkijat ovat havainneet, että  SMAD-signaloinnin   kumoaminen SMAD7-  tekijällä aiheuttaa maksametastaaseja  perna-injektiomallissa. PCR maksametastaasien genomisesta DNA:sta viittaa siihen, että SMAD7-proteiinia ilmentäviä  soluja migroituu  maksaan. 
•  TGFbeeta reseptorityyppiiä II  ilmenee lisääntyneenä maksametastaaseissa ja se korreloi  SMAD2 fosforylaatioon ja tumakertymään.

 We previously reported that overexpression of Smad7 in colon adenocarcinoma (FET) cells induces tumorigenicity by blocking TGF-β-induced growth inhibition and apoptosis.

 Here, we have observed that abrogation of Smad signalling by Smad7 induces liver metastasis in a splenic injection model. Polymerase chain reaction with genomic DNA from liver metastases indicates that cells expressing Smad7 migrated to the liver.

Increased expression of TGF-β type II receptor in liver metastases is associated with phosphorylation and nuclear accumulation of Smad2. Immunohistochemical analyses have suggested poorly differentiated spindle cell morphology and higher cell proliferation in Smad7-induced liver metastases.

Interestingly, we have observed increased expression and junctional staining of Claudin-1, Claudin-4 and E-cadherin in liver metastases.

Therefore, this report demonstrates, for the first time, that blockade of TGF-β/Smad pathway in colon cancer cells induces metastasis, thus supporting an important role of Smad signalling in inhibiting colon cancer metastasis.

Keywords:

TGF-β1; Smad7; metastasis; liver mets; immunohistochemistry

Metastasis is the major cause of cancer morbidity and mortality, and accounts for 90% of cancer deaths. Despite the fact that metastasis ultimately kills the host, the mechanisms leading to tumour invasion and metastasis have been less characterized than those resulting in tumour initiation. Cancer development and metastasis is a multistep process that involves local tumour growth and invasion followed by dissemination to, and re-establishment at, the distant sites (Liotta et al, 1991; Oft et al, 2002).

 There is compelling evidence indicating that TGF-β has complex roles in tumour suppression and progression that are context- and stage-dependent. Therefore, elucidating the molecular pathways essential for tumour metastasis is a higher priority in the pathobiology of cancer to design small molecule drugs.

Family members of TGF-β initiate signalling from the cell surface by binding to a heteromeric complex of two distinct but related serine/threonine kinase receptors. Binding of the ligand to the type II receptor (TβRII) results in recruitment and phosphorylation of the type I receptor (TβRI).
After being activated, TβRI propagates the signal to a family of intracellular signal mediators known as Smads. Smad proteins are classified according to their structure and function in signalling by TGF-β family members.

Receptor-regulated Smads, Smad2 and Smad3, are phosphorylated and activated by TβRI. Then they form complexes with common Smad (Smad4) and translocate to the nucleus for regulating the expression of target genes. Given the involvement of TGF-β in regulation of cellular homoeostasis, it is expected that there are also a number of feedback mechanisms regulating this process. 

The activity of the inhibitory Smad, Smad7, is regulated at many levels, suggesting that they serve as key regulators for fine-tuning the responses to TGF-β signalling.
Smad7 normally resides in the nucleus and moves to the cytoplasm in response to TGF-β. 

TGF-β not only enhances the expression of Smad7 transcription but also mobilises a pre-existing nuclear pool of Smad7 to inhibit TGF-β receptors (Itoh et al, 1998). Smad7 also interfere with TGF-β/Smad signalling through the recruitment of E3 ubiquitin ligases leading to the degradation of receptors and through the interaction with GADD34 that inactivate TβRI (Shi et al, 2004). Smad7 expression can also be induced by other signalling inputs, which interfere with TGF-β signalling.

Genes involved in oncogenic pathways are generally classified as either tumour suppressors or promoters, depending on their function in regulating cell growth, differentiation and death. TGF-β and its downstream signal transducers are well documented for such paradoxical characters. Genetic manipulation of the TGF-β pathway in tumour cells and experimental animal models validated the metastasis-promoting function of TGF-β in the late stage of cancer progression (Letterio, 2005). 

Carcinogen-induced tumours that develop in TGF-β haploid mice often maintain the wild-type TGF-β allele and these tumours in fact produce higher level of TGF-β than tumours from the wild-type mice (Tang et al, 1998). However, after development, the transgenic tumours rapidly acquire a spindle cell phenotype, overexpress TGF-β, and metastasise (Cui et al, 1996). These observations have led to the speculations that during cancer progression, TGF-β may reverse its role from an inhibitor of tumour growth to a tumour promoter (Reiss and Barcellos-Hoff, 1997; Massague et al, 2000; Derynck et al, 2001). 

Although complete or partial loss of TGF-β signals is permissive for early stages of tumour development, active TGF-β signalling with selective loss of growth inhibitory response of TGF-β may be advantageous for the progression and metastasis of cancer.

 SMAD pathway
The tumour suppressor Smad pathway has been shown to mediate the prometastatic function of TGF-β in the development of breast cancer bone metastasis (Kang et al, 2005). In addition, blockade of Smad pathway by overexpression of the inhibitory Smad, Smad7 impairs bone and lung metastases (Azuma et al, 2005; Javelaud et al, 2007). In contrast, Smad4 mutation and its reduced level in colorectal cancer are directly correlated to poor prognosis and increased metastasis (Miyaki et al, 1999). Furthermore, upregulation of Smad7 in colorectal cancer has been correlated with poor survival (Korchynskyi et al, 1999). However, nothing is known about the functional role of Smad signalling in colorectal cancer metastasis to the liver. The purpose of this study is to understand the stage-specific duality of TGF-β and Smad function, and the molecular mechanism underlying the role of Smad7 in the metastasis of colon cancer. In an experimental model of colon cancer liver metastasis, we have shown that the stable expression of Smad7 enhances liver metastasis. We have observed a diffuse positive Ki67 staining and poorly differentiated spindle cell morphology in the liver metastases. The expression of junctional proteins is increased in the liver metastases, the expression of some of which has been shown to be elevated in the metastases of human cancers. Our data provide the first evidence that Smad signalling plays a protective role in TGF-β-induced colorectal cancer metastasis.