Shu-Li Liu

Clinicopathologic correlations of liver kinase B1, E-cadherin, and N-cadherin expression in non-small cell lung cancer.

The role of liver kinase B1 (LKB1) as a tumor suppressor has emerged from the observation of increased risk of malignancy in gastrointestinal tract in Peutz-Jeghers syndrome patients harboring LKB1 gene mutations. LKB1 gene inactivation has recently been demonstrated in a subset of lung carcinoma and has been proven to trigger epithelial-mesenchymal transition in lung adenocarcinoma cells. However, the clinicopathologic significance, particularly prognosis, of LKB1 protein expression remains largely unclear. Using immunohistochemistry, we investigated the correlations between LKB1, E-cadherin, and N-cadherin expression and clinicopathologic parameters of lung cancer patients. Immunohistochemistry on specimens of the normal bronchial epithelium revealed that LKB1 was strongly or moderately expressed in the cytoplasm, and E-cadherin was expressed clearly on the cell membrane, whereas N-cadherin was absent or only weakly expressed at the membrane and/or in the cytoplasm. In contrast, in lung cancer samples, LKB1 expression was absent or decreased in 25.7% (29/113) cases accompanied with loss of membranous E-cadherin expression (25/29, P=0.009) and increased membranous and/or cytoplasmic N-cadherin expression (18/29, P=0.007). Loss of LKB1 expression positively correlated with histologic type (P=0.001), poor differentiation (P=0.004), and adverse prognosis (P<0.001). Moreover, loss of LKB1 expression correlated with lymph node metastasis (P=0.022) in lung adenocarcinoma samples and was an independent factor that impacted lung adenocarcinoma patients’ prognosis (P=0.003). Therefore, loss of LKB1 expression correlates with epithelial-mesenchymal transition markers and may be a useful marker of poor survival for the patient with lung adenocarcinoma.

Transcription Expression and Clinical Significance of Dishevelled-3 mRNA and δ-Catenin mRNA in Pleural Effusions from Patients with Lung Cancer

Objective. To evaluate diagnostic utility of Dishevelled-3 (DVL-3) mRNA and δ-catenin mRNA expression in pleural effusions of patients with lung cancer. Methods. DVL-3 mRNA and δ-catenin mRNA levels were assessed by performing RT-PCR on pleural effusion specimens from patients with lung cancer (n = 75) and with lung benign disease (n = 51). Results. The expressions of DVL-3 mRNA and δ-catenin mRNA were significantly higher in malignant than in benign lung disease (P < 0.01) and were obviously higher than cytology in adenocarcinoma (P < 0.01). In single use, DVL-3 mRNA had the highest specificity (94.1%) and PPV (95.7%), whereas δ-catenin mRNA had the highest sensitivity (92.0%) and NPV (88.5%). When combinations of markers were evaluated together, DVL-3 mRNA and δ-catenin mRNA gave a high-diagnostic performance: sensitivity of 100.0%, NPV of 100.0%, and accuracy of 96.0%, respectively. Conclusion. As molecular markers of detecting pleural micrometastasis, DVL-3 mRNA and δ-catenin mRNA are helpful to diagnose the cancer cells in pleural effusions of patients with lung cancer.

Immunocytochemical panel for distinguishing carcinoma cells from reactive mesothelial cells in pleural effusions

Objective:  The aim of this study was to evaluate the individual and combined diagnostic utility of carcinoembryonic antigen (CEA), cytokeratin 19 fragments (CK19) and HBME‐1 in pleural effusions of patients with lung cancer.

Overexpression of HPV16 E6/E7 mediated HIF-1α upregulation of GLUT1 expression in lung cancer cells

High-risk human papillomavirus (HPV) infection may play an important role in non-small cell lung carcinoma (NSCLC) development. However, some recent studies have proved that it was not directly associated with lung cancer. The aim of this study was to evaluate the underlying molecular mechanism that HPV16 regulate the expression of GLUT1 and may promote the development of lung cancer. HPV16, HIF-1α, and GLUT1 were detected in pleural effusions of patients with lung cancer (n = 95) and with benign lung disease (n = 55) by immunocytochemistry. Western blotting and qRT-PCR were used to detect the expression chances of HPV16 E6/E7, HIF-1α, and GLUT1 in lung cancer cells. HPV16, HIF-1α, and GLUT1 were significantly more likely to be expressed in the malignant group than in the benign group as detected by immunocytochemistry (ICC), and HIF-1α was significantly correlated with HPV16 or GLUT1 in the malignant group (P < 0.01). Expression changes of E6 and E7 significantly promoted the protein expression of HIF-1α, the expression of both protein and mRNA of GLUT1, but had no effect on the expression of HIF-1α mRNA in lung cancer cells. After inhibition of HIF-1α, it obviously downregulated the expression of both protein and mRNA of GLUT1 in lung cancer cells. E6 and E7 regulated the expression of GLUT1 may be due to the mediation of HIF-1α in lung cancer cells. These results suggest that both E6 and E7 play the important role in the regulation of Warburg effect and may be a valuable therapeutic target for HPV-related cancer.

Atonal homolog 1 expression in lung cancer correlates with inhibitors of the Wnt pathway as well as the differentiation and primary tumor stage.

Atonal homolog 1 (Atoh1) is crucial to the differentiation of many cell types and participates in tumorigenesis and progression. This study investigated the role of Atoh1 in lung cancer development and its correlation with key members of the Wnt pathway. We used immunohistochemistry to examine the expressions of Atoh1, β‐catenin, Axin, chibby, and Disabled‐2 (Dab2) in 118 samples of lung cancer. We also detected the cytoplasmic and nuclear expression of Atoh1 in lung cancer tissues using western blot. Atoh1 nuclear expression was negatively correlated with differentiation level (p = 0.004) and primary tumor stage (p = 0.044) of lung cancer. Nuclear Atoh1 expression was positively correlated with nuclear expression of chibby (p < 0.001) and Dab2 (p < 0.001). Cytoplasmic Atoh1 expression was positively correlated with the cytoplasmic expression of Axin (p = 0.028), chibby (p < 0.001), and Dab2 (p < 0.001). We conclude that the nuclear expression of Atoh1 was inversely correlated with the differentiation and primary tumor stage of lung cancers. The expression and localization of Atoh1 correlated with Axin, chibby, or Dab2. Atoh1 may be a potential therapeutic target for the inhibition of growth and progression of lung cancers.

Abnormal Expression of p120-catenin and E-cadherin Is Significantly Correlated with Malignant Phenotype of Human Lung Cancer

BACKGROUND To explore the correlation between p120-catenin (p120ctn) and small GTPases in human lung cancer, and their effect on the cell-cell adhesion, we examined the expression patterns of p120ctn and Rac1, which is the core member of small GTPases, and their correlation with clinicopathological factors. METHODS S-P immunohistochemistry, Western Blot, and RT-PCR were used to detect the expression patterns of p120ctn and Rac1 in 138 patients with non-small cell lung cancer (NSCLC) and two kinds of homologous lung cancer cell lines. We also used an in vitro model to evaluate their expression, and to determine whether protein expression correlated with the invasive capacity of lung cancer cell lines. RESULTS In lung cancer, the levels of protein and mRNA expression of p120ctn were significantly lower than normal lung tissue, and Rac1 was also found to be higher in tumor tissue than in normal lung tissue. A correlation between abnormal p120ctn and overexpression of Rac1 (Correlation coefficient=0.720, P <0.001) was also associated with malignancy of lung cancer, such as poor differentiation (P =0.022), high TNM stage (P =0.010), and lymph node metastasis (P =0.009) in NSCLC patients. Abnormal expression of p120ctn and overexpression of Rac1 was significantly associated with the high metastatic capacity of BE1 cells. CONCLUSIONS Abnormal p120ctn expression correlates with Rac1 overexpression, which contributes to the malignancy-related of NSCLC.

Expression and Significance of Stem Cell Markers CK19, Notch3, CD133, P75NTR, STRO-1 and ABCG2 in Pulmonary Squamous Carcinomas

BACKGROUND Increasing reports showed that some tumor stem cells were selfrenewal and multi-lineage differentiated in tumors, similar to the normal stem cells in human body. The aim of this study is to observe the expression of stem cell markers in lung squamous carcinoma tissues. METHODS Fifty-four lung cancer specimens from surgery were analyzed for CK19, Notch3, CD133, P75NTR, STRO-1 and ABCG2 expression by using S-P immunohistochemistry. In addition, ten normal lung tissue samples were included as control. RESULTS CK19, Notch3, CD133 and ABCG2 were expressed in 54 Lung cancer tissues, without expression of P75NTR and STRO-1. The expression rate of CK19, Notch3, CD133 and ABCG2 was 66.67% (36/54), 87.04% (47/54), 50% (27/54), and 61.11% (33/54) respectively. The levels of expression of Notch3, CD133 and ABCG2 were significantly lower in high differentiation group than those in moderate and low differentiation group (P <0.05). The levels of expression of CK19, CD133 and ABCG2 were significantly higher in lymph node metastasis group than those in non-metastasis group (P <0.05). The percentage of total positive cells of four stem cell markers in serial tissue sections was lower than 2%. CONCLUSIONS There was expression of some stem cell markers in pulmonary squamous carcinomas, and there was relationship between expression degree with differentiation degree and lymph node metastasis.

[Transfection of Axin Gene Down-regulates Expressions of beta-catenin and TCF-4 and Inhibits the Proliferation and Invasive Ability of Lung Cancer Cells.].

BACKGROUND Axin is an important negative regulator of Wnt signaling pathway. It can induce the phosphorylation and degradation of beta-catenin. The reduced expression of axin or high expression of beta-catenin and TCF-4 were associated with malignant proliferation in many tumors. The aim of this study is to examine the relationships among the expressions and locations of axin, beta-catenin and other relevant molecules, and the roles of axin on proliferation, invasive ability and apoptosis of lung cancer cells. METHODS The axin cDNA was transfected into lung cancer BE1 cell line which has very low axin expression. The levels of expression and location of axin, beta-catenin and TCF-4 before and after transfection were detected using immunofluorescence. The mRNA levels of expression of axin, beta-catenin and TCF-4 were examined using reverse transcription-polymerase chain reaction (RT-PCR). The apoptosis, proliferation and invasive ability of lung cancer cells before and after transfection were examined with flow cytometry, MTT and transwell methods. RESULTS After transfection of axin gene into BE1 cells (BE1-axin cells), axin mRNA and protein were overexpressed significantly. Meanwhile, the protein expression of beta-catenin and mRNA expression of TCF-4 were decreased significantly in BE1-axin cells than that in BE1 or vector control cells. The flow cytometry revealed that the apoptosis rate of BE1-axin cells was enhanced, but MTT and Transwell assay indicated that the proliferation and invasive ability were decreased significantly in BE1-axin cells than those in BE1 or vector control cells. CONCLUSIONS The overexpression of axin could down-regulate the protein expression of beta-catenin and the transcription of TCF-4, and inhibit the proliferation and invasive ability of lung cancer cells.

Clinical value of capture of cancer cells in pleural fluid of patients with lung cancer by cytochip

BACKGROUND Immunocytochemistry is valuabale in differentiating malignant fluids from benign ones. However, the diagnostic value of a single tumor marker is limited. The aim of this study is to evaluate the clinical value of capture of cancer cells in pleural fluids of patients with lung cancer by cytochip. METHODS A new pattern cytochip was developed to immunize hybridization of cells in pleural fluids of patients with 42 lung cancers and with 20 lung benign lesions. Ten antibodies were fixed on the cytochip, they were epithelial specific antigen (ESA), CD44V6, ND-1, T cell (CD3), CD45RO, B cell (CD20), CD79a, Hodgkins cell (CD15), CD30 and macrophage (CD68). RESULTS The point of positive hybridization showed round distribution with clear border, and the shape of cell displayed well. The positive numbers of ESA, CD44V6, ND-1 were 35, 30, 38 respectively in pleural fluids of 42 patients with luog cancers; lymphocytes and neutrophils were found on the 1 ESA and 1 ND-1 respectively, and only lymphocytes were found on the 3 CD44V6 in 20 ones with lung benign lesions; the other 7 antibodies did not capture cancer cells except for lymphocytes, neutrophils and macrophages from two pleural fluids. CONCLUSIONS The cytochip could be an important practical foreground in clinic for diagnosing cancer cells in pleural fluids of patients with lung cancer.