The Relationship Between HDPR1 With Catenin Family Members Each Other In Non-Small Cell Lung Cancer

IntroductionThere is emerging evidence that the abnormal activation of WNT signaling pathway is linked with the development of many human tumors. Dishevelled (DVL) is a critical positive regulator in WNT signaling pathway. A recent study characterized Dapper (Dpr) which interacted with DVL and functioned as an antagonist of DVL in Xenopus. The roles of Dpr in WNT signaling pathway are still controversial. Some reports indicated that Dpr inhibited the classical WNT/β-catenin and non-classical WNT/JNK pathways, but others demonstrated that Dpr activated the WNT signaling. The Dpr gene was expressed in lower vertebrates (such as zebra-fish) as well as in human. Dpr gene is highly conserved through evolution, indicating that Dpr may have important physiological functions.HDPR1, which was first identified in 2003, is the human homologue of Dpr and is composed of 799 amino acids. HDPR1 gene was deleted in human astrocytoma. When 72 cases of primary hepatocellular carcinoma (HCC) were examined by RT-PCR, HDPR1 mRNA expression was down-regulated in 31 specimens (43%) as compared to the corresponding non-tumorous livers. Down-regulation of HDPR1 mRNAin HCC was associated with cytoplasmic accumulation ofβ-catenin. These results suggested that HDPR1 may function as a tumor suppressor. We recently reported that DVL expression was associated with cytoplasmic accumulation ofβ-catenin in NSCLC. These data indicated that cytoplasmic accumulation ofβ-catenin may be also associated with HDPR1 expression in lung cancer. p120ctn is another member of catenin subfamily and plays an important role in the regulation of cell-cell adhesion. We recently reported that p120ctn expression was down-regulated in NSCLC and knockdown of p120ctn promoted the invasion and metastasis of lung cancer cells through down-regulation of E-cadherin expression. It was reported that the protein stability of p120ctn was dependent on Frodo (the Xenopus homologue of Dapper) in Xenopus embryogenesis. More studies will be required to investigate the relationship between HDPR1 and p120ctn in human tumors.In this study, we examined HDPR1 expression in NSCLC and analyzed the relationship between HDPR1 expression and clinicopathological factors of patients with NSCLC. We also explored the effects of HDPR1 gene on p120ctn andβ-catenin expression, and on the invasive ability of lung cancer cells.Materials and MothodsParaffin specimens (n=120) were obtained from patients with lung cancer who underwent surgery or biopsy at the First Affiliated Hospital of China Medical University between 1998 and 2006. Follow-up data was obtained from review of the patients' medical record. According to the WHO (2004) histological classification criteria, there were 55 cases of squamous cell carcinoma and 65 cases of adenocarcinoma. The TNM staging system of the International Union Against Cancer (UICC) in 2002 was used to classify specimens as stagesⅠ(n=39),Ⅱ(n=25),Ⅲ(n=44) andⅣ(n=12). None of the patients had received radiotherapy or chemotherapy before surgical resection or biopsy.42 cases (included in the 120 cases) of tumor and paired non-tumorous tissues (distant from the primary tumor) were quickly frozen in-70℃until protein and RNA extraction. The 42 paired cases were chosen according to the date of undergoing surgery in sequence. Additionally,30 normal lung tissues from the 42 cases were used for immunohistochemical staining.In this study, immunohistochemical analysis in 120 NSCLC tissues was performed to examine the expression of HDPR1. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis were used to examine mRNA and protein expression of HDPR1 between tumor tissues and corresponding non-tumorous tissues. The Pearson Chi-Square test was used to examine the correlation between the expression of HDPR1 and clinicopathological factors. The Kaplan-Meier method was used to estimate the probability of patient survival. The Cox's proportional hazard regression model was used to estimate the possible prognostic significance of cliniciopathological variables. Furthermore, RNAi technique was performed in LH7 lung cancer cell line to explore the regulation mechanism among HDPR1, p120ctn andβ-catenin, as well as the invasive ability of lung cancer cells.ResultsIn 30 cases of normal lung tissues, HDPR1 was expressed in the cytoplasm of bronchial epithelial cells (≥3 score, defined as normal expression of HDPR1). However, in the 120 NSCLC specimens, HDPR1 expression was significantly reduced (<3 score) in 82 samples (68.3%). In the 58 poorly differentiated samples, HDPR1 expression was reduced in 46 samples (79.3%). In the 56 cases with pTNM stages III and IV, HDPR1 expression was reduced in 44 samples (78.6%). In the 51 cases with lymph node metastasis, reduced expression of HDPR1 was observed in 41 samples (80.4%). In the 120 NSCLC cases, patients with reduced HDPR1 expression had a significantly lower survival time than patients with normal expression of HDPR1 (31.538±3.637, 52.057±6.687, respectively; P=0.008). Western blotting and RT-PCR analysis in tissues showed that HDPR1 protein and mRNA expression in lung cancer tissues were significantly lower as compared to those in the corresponding non-tumorous lung tissues (P=0.019, n=42; P=0.001, n=42).β-catenin was expressed in the membrane of normal bronchial epithelial cells. In 120 cases of lung cancer,81 cases had abnormalβ-catenin expression (67.5%). Statistical analysis showed that reduced HDPR1 expression was closely correlated with abnormal expression ofβ-catenin (Correlation coefficient=0.211, P=0.018). Increasing HDPR1 siRNA concentrations resulted in a significant up-regulation ofβ-catenin protein expression. In 120 lung cancer samples,91 samples were observed with abnormal pl20ctn expression (75.8%). Statistical analysis showed that the reduced expression of HDPR1 was closely correlated with abnormal p120ctn expression (Correlation coefficient= 0.198, P=0.027). Knockdown of HDPR1 gene led to down-regulation of p120ctn protein expression, but had no effect on pl20ctn mRNA. Matrigel Invasion Assay showed that the group co-transfected with HDPR1 siRNA and p120ctn cDNAhad fewer invasive cells than that transfected with HDPR1 siRNA (P<0.05).Conclusions1,HDPR1 expression was significantly reduced in NSCLC. Reduced HDPR1 expression was associated with poor differentiation, high pTNM stage, lymph node metastasis in NSCLC.2,Reduced expression of HDPR1 was an independent hazard factors for the prognosis of patients with lung cancer.3,Reduced HDPR1 expression was associated with abnormal expression ofβ-catenin. Knockdown of HDPR1 induced cytoplasmic accumulation ofβ-catenin in lung cancer cell line.4,Reduced HDPR1 expression was associated with abnormal p120ctn expression. Knockdown of HDPR1 increased the invasive ability of lung cancer cells through down-regulating p120ctn.