| BackgroundExtrahepatic cholangiocarcinoma(CCA), which is derived from the epithelium ofextrahepatic bile ducts, has a devastating prognosis because of early invasion andmetastasis. The incidence of this cancer has been increasing in recent years. Surgicalresection is the main option for curative treatment; however, lymph node or distantmetastases are present in many patients, thus limiting the effectiveness of surgery andworsening the prognosis. Furthermore, many patients who undergo surgery present withrecurrent disease within2year. Their survivals, therefore, vary to a large extent. Earlymetastasis and varying prognosis highlight the need of defining novel targets forantimetastatic therapy and survival prediction.Laminin332, a large heterotrimeric glycoprotein consisting of laminin α3, β3, and γ2chains. Over15laminin isoforms with similar gene sequence and structure are presentlyknown. The expression of laminin isoforms are present with cell and tissue specificity.Laminin332is a major component of the basement membrane in some epithelial tissues andplays important biological roles in the adhesion of epithelial cells to basement membrane,migration, growth and differentiation via receptors. Lamininγ2are able to be secreted asmonomer and unique to laminin332according to the structure of laminin. its expression iscommonly considered as an indicator of the presence of laminin332. Overexpression oflamininγ2has been reported in many human cancers, and is significantly associated withinvasion and metastases. Some studies indicated that the expression of lamininγ2might be aprognostic marker in tumor cells. Although its aberrant expression has been correlated withmalignant transformation and poor prognosis in intrahepatic CCA, the clinical significanceof lamininγ2in extrahepatic CCA, which is the more common form of CCA that accountsfor80%-90%of all CCAs and possesses distinct morphology, etiopathogenesis, andmolecular signatures, is still unclear. HypothesisLamininγ2might promote cell invasion in extrahepatic CCA, and would be a riskfactor indicating poor prognosis of patients following surgical resection.Materials and Methods1. We enrolled a total of72extrahepatic CCA patients undergoing surgical resection atSouthwest Hospital from2005to2010. A total of72cancerous samples,16matchedparacancerous samples,28lymph node metastatic samples, and7liver metastatic samplesfrom these patients were collected. In addition,8chronic inflammatory bile duct samplesfrom patients with hepatolithiasis and5biliary adenoma samples were also collected fromSouthwest Hospital. QBC939cell lines were builted in Institute of Hepatobiliary Surgery ofSouthwest Hospital from an extrahepatic CCA patient.2. Immunohistochemistry(IHC) was used to study the expression of lamininγ2inparaffin-embedded clinical samples. The expression differences of lamininγ2were analysedin cancerous samples, paracancerous samples, chronic inflammatory bile duct samples,biliary adenoma samples. Correlations between lamininγ2expression and clinicopathologicfeatures in extrahepatic CCAs were analyzed. The expression of lamininγ2was comparedbetween primary cancerous samples and metastatic samples.3. The date of death and tumor recurrence for72patients was recorded. The overallsurvival time was computed as the time from the date of first pathologic diagnosis to thedate of death or last follow-up. Recurrence-free survival time was computed from the dateof first pathologic diagnosis to the date of recurrence. Correlations between the expressionof lamininγ2and overall survival and recurrence were analyzed by using Kaplan-Meier andCox models.4. QBC939cells were transfected with siRNA targeting lamininγ2. The impact oflamininγ2inhibition on ability of invasion and migration were evaluated by cell invasionand wound-healing assay in vitro.Results1. In total,36of72cancerous samples were defined as lamininγ2positive, whichexhibited two staining patterns:28samples showed positive stromal staining and24samples showed positive cytoplasmic staining. Among the positive cancerous samples,16of which showed both stromal and cytoplasmic staining. None of the16matching paracancerous samples showed either stromal or cytoplasmic expression of lamininγ2in thebile ducts. In addition, all8inflammatory bile duct samples and5biliary adenoma samplesstained negative for lamininγ2. Clinicopathologic features of72extrahepatic CCAs weregrouped by stromal or cytoplasmic lamininγ2expression. The correlation betweenlamininγ2expression in extrahepatic CCA and clinicopathologic features of patients wereanalyzed. The results showed that stromal expression of lamininγ2was significantlyassociated with lymph node metastasis (P=0.01). In contrast, cytoplasmic expression oflamininγ2in tumor cells markedly correlated with lymph node and distant metastases(P=0.02and P=0.01). Positive cytoplasmic staining of lamininγ2was observed in17of28lymph node metastatic samples， which was significantly higher than that observed in theprimary samples (P=0.01).7liver metastatic samples were examined for lamininγ2cytoplasmic expression. The positive staining rate (4/7,57%) in these samples was higherthan that of the primary lesions. Together, the data suggested that aberrant expression oflamininγ2was associated with tumor invasion and metastasis in patients with extrahepaticCCA.2. Kaplan-Meier analysis showed that stromal lamininγ2expression was significantlyassociated with poor overall survival and early recurrence in extrahepatic CCA patientsafter surgical resection (P=0.007and P=0.004). Cytoplasmic lamininγ2expression was alsocorrelated with poor overall survival and early recurrence (P=0.008and P=0.006). Thepatients were then divided into four groups: both stromally and cytoplasmically positive(Stroma+Cyto+), only stromally positive (Stroma-Cyto+), only cytoplasmically positive(Stroma-Cyto+), and both stromally and cytoplasmically negative (Stroma-Cyto-). Furtheranalysis indicated that aberrant expression of lamininγ2, regardless of its cellularlocalization, was significantly associated with a poor prognosis. Consistent with the aboveinterpretation, log-rank test also showed that aberrant expression of lamininγ2wassignificantly associated with poor overall survival and early recurrence (p=0.001andp<0.001). Cox model analysis indicated that lamininγ2expression was an independentpredictor of poor overall survival and early recurrence (HR=2.465,95%CI=1.391-4.368,p=0.002; HR=2.679,95%CI=1.547-4.638, p<0.001). Taken together, our data revealed thataberrant expression of lamininγ2might be a risk factor and indicator of poor prognosis inextrahepatic CCA patients after surgical resection. 3. Immunofluorescence staining showed that lamininγ2was expressed in thecytoplasm of QBC939cells. Knockdown of lamininγ2via siRNA transfection led to asignificant reduction in the number of invading cells in the transwell assay. Consistently,QBC939migration was also significantly reduced by lamininγ2knockdown, as assessed bythe wound-healing assay. These data showed that the knockdown of lamininγ2inhibitedextrahepatic CCA cell invasion and migration in vitro.ConclusionIn conclusion, our findings indicate that aberrant expression of lamininγ2correlateswith poor prognosis and promotes tumor invasion in extrahepatic CCA. |
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