Suping Guo

Prognostic significance of matrix metalloproteinase-1 levels in peripheral plasma and tumour tissues of lung cancer patients

Matrix metalloproteinase-1 (MMP-1) participates in a variety of physiological and pathological processes. We previously found that MMP-1 was one of the lung cancer-related proteins detectable in peripheral blood. To validate our preliminary observations and explore the clinical significance of MMP-1 for lung cancer further, we carried out the present study. The concentrations of MMP-1 in circulating plasma specimens of 170 lung cancer patients and 70 healthy individuals were measured by an enzyme-linked immunosorbance assay. The expression status of the MMP-1 in archival tissue samples from 122 lung cancer patients was examined by immunohistochemical analysis. The correlation between the MMP-1 levels and prognosis of the lung cancer patients was then assessed statistically. Protein levels of MMP-1 were considerably raised in the plasma from lung cancer patients relative to those in healthy controls. The high plasma MMP-1 levels were associated with advanced-stage of the disease and significantly lower overall survival rate of the patients. Coincidently, MMP-1 protein extraordinarily overexpressed in the tumour tissues of lung cancer; and the up-regulated MMP-1 was associated with the progression (including tumour size, staging and lymphatic invasion), especially with decreased survival rate of the patients. Statistic analysis revealed that MMP-1 protein levels had an independent influence on survival. MMP-1 levels were elevated in both tumour tissue and blood; the latter may serve as an independent predictor for survival of lung cancer patients. MMP-1 protein levels in plasma/serum thus represent a potential and clinically relevant biomarker for the prognosis of patients with lung cancers.

The ovarian cancer‐derived secretory/releasing proteome: A repertoire of tumor markers

Ovarian cancer is the most lethal gynecological malignancy worldwide, and early detection of this disease using serum or plasma biomarkers may improve its clinical outcome. In the present study, a large scale protein database derived from ovarian cancer was created to enable tumor marker discovery. First, primary organ cultures were established with the tumor tissues and corresponding normal tissues obtained from six ovarian cancer patients, and the serum‐free conditioned medium (CM) samples were collected for proteomic analysis. The total proteins from the CM sample were separated by SDS‐PAGE, digested with trypsin and then analyzed by LC‐MS/MS. Combining data from the tumor tissues and the normal tissues, 1129 proteins were identified in total, of which those categorized as “extracellular proteins” and “plasma membrane proteins” accounted for 21.4% and 16.9%, respectively. For validation, three secretory proteins (NID1, TIMP2, and VCAN) involved in “organ development”‐associated subnetwork, showed significant differences between their levels in the circulating plasma samples from ovarian cancer patients and healthy women. In conclusion, this ovarian cancer‐derived protein database provides a credible repertoire of potential biomarkers in blood for this malignant disease, and deserves mining further.

Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma

For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ. Moreover, 19% of the proteins were characterized as extracellular or membrane-bound. For validation, secretory proteins involved in transforming growth factor-β signaling pathways were validated in plasma samples. Alphafetoprotein (AFP), metalloproteinase (MMP)1, osteopontin (OPN), and pregnancy-specific beta-1-glycoprotein (PSG)9 were significantly increased in HCC patients. The overall performance of MMP1 and OPN in the diagnosis of HCC remained greater than that of AFP. In addition, this study represents the first report of MMP1 as a biomarker with a higher sensitivity and specificity than AFP. Thus, this study provides a valuable resource of the HCC secretome with the potential to investigate serological biomarkers. MMP1 and OPN could be used as novel biomarkers for the early detection of HCC and to improve the sensitivity of biomarkers compared with AFP.

Metastatic potential of lung squamous cell carcinoma associated with HSPC300 through its interaction with WAVE2

The small protein, HSPC300 (haematopoietic stem/progenitor cell protein 300), is associated with reorganization of actin filaments and cell movement, but its activity has not been reported in human cancer cells. Here, we investigated the association of HSPC300 expression with clinical features of lung squamous cell carcinoma. High levels of HSPC300 protein were detected in 84.1% of tumour samples, and in 30.8% of adjacent morphologically normal tissues. The number of primary tumours with elevated HSPC300 levels was significantly higher in primary tumours with lymph node metastases as opposed to those without, and also in tumours from patients with more advanced disease. HSPC300 modulates the morphology and motility of cells, as siRNA knockdown caused the reorganization of actin filaments, decreased the formation of pseudopodia, and inhibited the migration of a lung cancer cell line. We further showed that HSPC300 interacted with the WAVE2 protein, and HSPC300 silencing resulted in the degradation of WAVE2 in vitro. HSPC300 and WAVE2 were co-expressed in approximately 85.7% of primary tumours with lymph node metastases. We hypothesize that HSPC300 is associated with metastatic potential of lung squamous cell carcinoma through its interaction with WAVE2.

Suppression of non-small cell lung cancer proliferation and tumorigenicity by DENND2D

DENND2D was identified as being down-regulated in lung cancer using a lung cancer low-expression suppression subtractive hybridization (SSH) library. In this study, DENND2D down-regulation has been observed not only in non-small cell lung cancer (NSCLC) cell lines and lung squamous cell carcinoma (SCC) tissues, but also in immortalized human bronchial epithelial (IHBE) cell lines and precancerous lesions, indicating that the down-regulation of DENND2D may be an early event in lung cancer. The relative DNA copy number and mRNA and protein expression levels of DENND2D were determined in vitro, and they revealed a complicated regulatory network at the genomic, transcriptional and translational levels. Over-expression of DENND2D significantly suppressed the proliferation of NSCLC cells in vitro and in vivo by inducing apoptosis. These results indicate that DENND2D might function as a tumor suppressor-like gene to prevent the survival and expansion of cells with genetic damage through apoptosis mechanism, and absence of DENND2D might play a permissive role, as an early event, in tumorigenesis.

Molecular cytogenetic alterations in the early stage at human bronchial epithelial cell carcinogenesis.

Lung carcinogenesis is a multi‐step process involving activation of oncogenes and inactivation of tumor suppress genes. Many molecular and cytogenetic alterations occur in the early stages of carcinogenesis. We have developed an effective culture system for human bronchial epithelial cells and lung cancer cells. Four immortalized human bronchial epithelial cell lines were established by transfecting the epithelial cells with plasmid DNA containing the early region of SV40. Some molecular and cytogenetic alterations, such as 3p‐, 2q‐, 9p‐, c‐myc translocation t(8;14) (q23; q32), were found in one immortalized bronchial epithelial cell line M when approaching malignant transformation. An increase in cell proliferation and decrease of apoptosis were noted in the late passages of the immortalized cell line M. Some molecular cytogenetic alterations were also observed in human primary non‐small cell lung cancers. Molecular cytogenetic alterations during the early stage of carcinogenesis of human bronchial epithelial cells may be useful as biomarkers for both diagnosis and intermediate endpoint of chemoprevention of lung cancer. J. Cell. Biochem. Suppls. 28/29:74–80.

Sp1 transcriptionally regulates BRK1 expression in non-small cell lung cancer cells

Following a previous study reporting that BRK1 is upregulated in non-small cell lung cancer (NSCLC), the present study sought to clarify the role of specificity protein 1 (Sp1) in the transcriptional regulation of the BRK1 gene. Therefore, a construct, named F8, consisting of the -1341 to -1 nt sequence upstream of the start codon of the BRK1 gene inserted into pGL4.26 was made. A series of truncated fragments was then constructed based on F8. Segment S831, which contained the -84 to -1 nt region, displayed the highest transcriptional activity in the A549, H1299 and H520 NSCLC cell lines. Bioinformatic analysis showed a potential Sp1-binding element at -73 to -64 nt, and a mutation in this region suppressed the transcriptional activity of S831. Then the RNAi assays of Sp1 and its coworkers Sp3 and Sp4 were performed, and suppression of Sp1 by siRNA inhibited the mRNA expression of BRK1. Both an electrophoretic mobility shift assay (EMSA) and a chromatin immunoprecipitation (ChIP) assay demonstrated that Sp1 bound to the promoter area of the BRK1 gene. Our data identified a functional and positive Sp1 regulatory element from -73 to -64 nt in the BRK1 promoter, which may likely explain the overexpression of BRK1 in NSCLC.