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// Bangrong Cao 1,2 , Lin Feng 1 , Dan Lu 1 , Yan Liu 1 , Yu Liu 1 , Suping Guo 1 , Naijun Han 1 , Xiangyang Liu 3 , Yousheng Mao 3 , Jie He 3 , Shujun Cheng 1 , Yanning Gao 1 and Kaitai Zhang 1 1 State Key Laboratory of Molecular Oncology, Department of Etiology and Carcinogenesis, Cancer Institute & Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China 2 Department of Basic Research, Sichuan Cancer Hospital & Institute, Chengdu, Sichuan Province, China 3 Department of Thoracic Surgical Oncology, Cancer Institute & Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China Correspondence to: Kaitai Zhang, email: // Yanning Gao, email: // Shujun Cheng, email: // Keywords : negative surgical margin, epithelial-to-mesenchymal transition, prognosis, non-small-cell lung cancer, gene-expression signature Received : June 13, 2016 Accepted : July 19, 2016 Published : July 29, 2016 Abstract It is hypothesized that the molecular status in negative surgical margin (NSM) is associated with prognosis of cancer patients. In this study, the prognostic relevance of Epithelial-to-Mesenchymal Transition (EMT) molecular events in NSMs in patients with NSCLC was investigated. EMT model was developed, in which the mesenchymal transition of human immortalized bronchial epithelial cell line was induced by TGF-beta1. Gene expression of EMT-induced cells and NSMs from 60 lung squamous cell carcinoma (SCC) patients was profiled by microarray and validated by quantitative RT-PCR. Two independent cohorts (lung SCC, n = 50; NSCLC, n = 54) were employed to validate the prognostic value of candidate genes. A set of 1490 genes were identified in EMT model in vitro . An EMT-like gene-expression pattern by 33 essential genes was optimized in NSMs, and was significantly associated with tumor progression. The 33 genes also exhibited a site-dependent field cancerization effect in the normal-appearing airways adjacent to NSCLCs. In the independent lung SCC cohort, the EMT-like active pattern indicated poor outcome of patients ( n = 50, log-rank p = 0.009). Furthermore, in the NSCLC cohort, patients with EMT-like active pattern had shorter predictive survival time ( n = 54, log-rank p = 0.02). In conclusion, the existence of EMT-like gene expression in NSMs, may play critical role in tumor progression and be a potential biomarker for prognosis in patients with NSCLC.

Multiple synchronous lung cancers (MSLCs) present a clinical dilemma as to whether individual tumours represent intrapulmonary metastases or independent tumours. In this study we analyse genomic profiles of 15 lung adenocarcinomas and one regional lymph node metastasis from 6 patients with MSLC. All 15 lung tumours demonstrate distinct genomic profiles, suggesting all are independent primary tumours, which are consistent with comprehensive histopathological assessment in 5 of the 6 patients. Lung tumours of the same individuals are no more similar to each other than are lung adenocarcinomas of different patients from TCGA cohort matched for tumour size and smoking status. Several known cancer-associated genes have different mutations in different tumours from the same patients. These findings suggest that in the context of identical constitutional genetic background and environmental exposure, different lung cancers in the same individual may have distinct genomic profiles and can be driven by distinct molecular events., Some patients present with multiple lung tumours but it is unclear whether these are metastases or individual lesions. Here, the authors use genomics techniques to demonstrate in six patients that multiple tumours have individual genetic profiles and represent separate tumours.

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.

Background Exposure to cigarette smoke is a major risk factor for lung cancer, but how it induces cancer is unclear. The overexpressed in lung cancer 1 ( OLC1 ) gene is one of 50 candidate lung cancer genes identified by suppression subtractive hybridization as having higher expression in squamous cell carcinoma (SCC) than normal lung epithelia. Methods We used immunohistochemistry (IHC) to measure OLC1 protein levels in primary lung cancer samples from 559 patients and used fluorescence in situ hybridization to measure OLC1 copy number in primary SCC samples from 23 patients. We compared OLC1 protein expression in SCC samples of 371 patients with and without a smoking history using the Pearson � 2 test. We assayed OLC1 protein levels by immunoblotting in H1299 human lung cancer cells, immortalized human bronchial epithelial cells, and primary cultured normal human bronchial epithelial cells that were treated with cigarette smoke condensate. We assayed tumor formation in athymic mice using NIH3T3 mouse fibroblast cells transfected with OLC1 (eight mice) and analyzed apoptosis and colony formation of H1299 and H520 lung cancer cells transfected with scrambled (negative) or OLC1 small interfering RNAs (siRNAs) (s1). Results OLC1 protein was overexpressed in 387 of 464 (83.4%) of primary lung cancers, as detected by IHC, and OLC1 was amplified in 14 of 23 (60%) of SCC samples. OLC1 protein overexpression was more common in SCC patients with a smoking history than those without (77.1% vs 45.8%, P < .001). In addition, cigarette smoke condensate increased OLC1 protein levels in H1299 cells, immortalized human bronchial epithelial cells, and primary cultured normal human bronchial epithelial cells. Overexpression of OLC1 induced tumor formation in athymic mice (control vs OLC1, 0% vs 100%). Knockdown of OLC1 increased apoptosis (mean percentage of apoptotic H1299 cells, s1 vs negative: 30.3% vs 6.4%, difference = 23.9%, 95% confidence interval [CI] = 19.1% to 28.5%, P = .002; mean percentage of apoptotic H520 cells, s1 vs negative: 21.6% vs 4.9%, difference = 16.7%, 95% CI = 10.6% to 22.8%, P = .007) and decreased colony formation (mean no. of colonies of H1299 cells transfected with siRNAs, negative vs s1: 84 vs 4, difference = 80, 95% CI = 71 to 88, P < .001; mean no. of colonies of H520 cells transfected with siRNAs, negative vs s1: 103 vs 24, difference = 79, 95% CI = 40 to 116, P = .005). Conclusions OLC1 is a candidate oncogene in lung cancer whose expression may be regulated by exposure to cigarette smoke. J Natl Cancer Inst 2008;100: 1592 – 1605

Background Estimation of the survival of patients with lung squamous cell carcinoma (SCC) on the basis of histopathology is inadequate. The aim of this study was to identify genomic regions with potential value for estimating the prognosis of these patients. Patients and Methods Depending on their survival time, 100 patients with primary lung SCC were separated into high- or low-risk prognostic groups, and their copy number aberrations (CNAs) were analyzed using array-comparative genomic hybridization (array-CGH). Results We identified 123 CNA regions that were significantly associated with survival. Among these regions, some have been reported previously (eg, amplifications of 8p12, 3q27.1, and loss of 9p21.3 and 13q34) but others have never been reported. For example, gains of 3q27.1, 5p13.2, and 5p13.3 were found to be associated with a favorable prognosis, but patients harboring gains of 11q23.3, 11q13.1, and 14q32.3, and deletions of 3p21.3 and 9p21.3 tended to have poor survival. Among the 123 CNA regions, 41 were further selected to construct a survival estimation model that could effectively separate SCC patients into high- or low-risk groups with an accuracy of 92%, sensitivity of 90%, and specificity of 94%. The results of the array-CGH were further validated in an independent cohort of 45 formalin-fixed, paraffin-embedded specimens using real-time polymerase chain reaction. Conclusion A number of CNA regions were found to be associated with the survival of SCC patients, and we were able to construct a model to estimate prognosis on the basis of these regions. Assessment of these CNAs could potentially assist in clinical decision-making regarding adjuvant therapy after surgery.

Effective biomarkers for early detection of ovarian cancer are needed. Our study previously showed that basement membrane protein, nidogen-1 plasma level was significantly increased in ovarian cancer patients. This study aimed to examine the plasma levels of nidogen-1 in a large patient population to evaluate its effectiveness in ovarian serous carcinoma and expression in tumor tissues.The concentration of nidogen-1 in circulating plasma specimens of 265 ovarian serous cancer patients and 98 healthy individuals were assayed by enzyme linked immunosorbent assay. The medical records of 265 ovarian serous cancer cases were reviewed retrospectively. The expression status of nidogen-1 in tumor tissues of 44 ovarian serous carcinoma patients was examined by immunohistochemical analysis. For statistical analysis, we used the Mann-Whitney U test, Fisher's exact test and receiver operating characteristic.Protein levels of nidogen-1 were considerably raised in the plasma from ovarian serous cancer patients compared with those in healthy controls (P0.001), especially elevated in patients with advanced stage and those received neoadjuvant chemotherapy followed by interval debulking surgery. However, it was irrelevant to the grade, chemotherapy sensitivity or residual tumor of the ovarian serous carcinoma cases investigated (P0.05). Receiver operating characteristic curve analysis for nidogen-1 showed that it could discriminate patients with ovarian serous carcinomas from healthy controls [areas under the curve (AUC): 0. 65, 95%CI, 0.59-0.71], but CA125 was superior (AUC: 0. 98, 95%CI, 0.96-0.99). The immunohistochemical staining result showed that nidogen-1 protein was localized both in the cancer cell cytoplasm and intercellular substance, mainly expressed in extracellular matrix of ovarian serous carcinoma tissues (the positive rate was 77.3%).Our study suggests that plasma nidogen-1 may be used as a diagnostic biomarker for ovarian serous carcinoma and can reflect the tumor burden.

The transmembrane and secreted protein delta-like 1 homolog (DLK1) belongs to the EGF-like family. It is widely accepted that DLK1 plays important roles in regulating cell differentiation, such as adipogenesis and osteogenesis. Aberrant expression of DLK1 has been found in various types of human cancers, including lung cancer. A previous study in this lab has revealed that DLK1 is associated with tumor invasion, although the mechanism is still unknown. To explore the potential effects that DLK1 might have on invasion, DLK1 was overexpressed or knocked down in the human lung cancer cell lines. The protein's influences on cell invasion were subsequently evaluated. A transwell assay showed that DLK1 overexpression significantly promoted cancer cell invasion. Western blotting and gelatin zymography analysis indicated that DLK1 could affect both matrix metalloproteinase-9 (MMP9) expression and its extracellular activity. An analysis of NOTCH1 and HES1 gene expression and Notch intracellular domain (NICD) nuclear translocation during DLK1 stimulation or depletion demonstrated that DLK1 could activate Notch signaling in lung cancer cells. Additionally, the elevated expression of MMP9 induced by DLK1 stimulation could be significantly decreased by inhibiting Notch signaling using γ-secretase inhibitor (GSI). The data presented in this study suggest that DLK1 can promote the invasion of lung cancer cells by upregulating MMP9 expression, which depends on Notch signaling.

Objective: Considerable evidence has showed that apoptosis is involved in both cancer development and inhibition. A new assay (terminal deoxynucleotidyl tansferase, TdT) was recently reported to have advantages in the detection of apoptosis. In this study, this assay was used to investigate antitumor drug-induced apoptosis in human cancer cells. Methods: TdT assay, DNA gel electrophoresis, electron and light microscopy were used to observe apoptosis. Results: Our results showed that cisplatin-induced apoptosis in both HL-60 and SV40T-transformed human bronchial epithelial cells was detected with a good dosage and time response. The occurrence of the apoptosis was preceded by the decrease of bcl-2 mRNA expression. With the TdT assay, apoptotic cells were observed in ovarian tumor of patients treated with carboplatin. Conclusion: TdT assay may be applicable to monitor apoptosis in human cancers induced by chemotherapy, and to evaluate tumor cell response during treatment.

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.

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.

To identify differentially expressed genes in lung squamous cell carcinomas (SCCs), the suppression subtractive hybridization method (SSH) was performed comparing six lung tumour tissues and 10 morphologically normal bronchial epithelial tissues. A cDNA library consisting of 220 upregulated genes in tumour tissue was established and named as LSCC (lung squamous cell carcinoma). Of them, six were tested using semi-quantitative reverse transcription-PCR on 27 pairs of tumour tissue and normal lung tissue. Differential expression was confirmed in five of these six genes, including IGFBP5, SQLE, RAP2B, CLDN1, and TBL1XR1. The elevated mRNA expression of RAP2B, CLDN1 and TBL1XR1, three genes located on chromosome 3q, were further validated in 64.3% (18/28), 82.1% (23/28), and 75.0% (21/28) of lung SCC tumour tissues, respectively, by quantitative real-time reverse transcription-PCR analysis. Moreover, western blot analysis showed that the protein expression of TBL1XR1 was also upregulated in 53.3% (8/15) of lung SCC tumour samples, as well as in five lung cancer cell lines and in one human immortalized bronchial epithelial cell line. All the initial characteristics of these genes were first reported in the lung SCCs. The differentially expressed genes reported in this study will provide a valuable resource for understanding the pathogenesis of lung SCCs and for discovery of novel diagnostic or therapeutic targets.

PURPOSE: Expression of targeting protein for Xklp2 (TPX2), a microtubule-associated protein, is tightly cell cycle regulated. Abnormally expressed TPX2 has been reported in various malignancies, but less is known in lung cancer. The present study appraised the significance of TPX2 aberrant expression for tumorigenesis and progression of human squamous cell carcinoma (SCC) in lung. EXPERIMENTAL DESIGN AND RESULTS: The expressive status of TPX2 was firstly examined with lung cancer (L, PAa, and PG) and immortalized bronchial epithelial (C45, M-BE, Tr, and Y-BE) cell lines, and TPX2 expression was detected at both RNA and protein levels by reverse transcription-PCR and Western blotting, respectively. Immunofluorescence staining on M-BE cells showed that the subcellular localization of TPX2 protein is in nucleus at interphase and mitotic spindle at metaphase. Immunohistochemical analyses were subsequently done on the precancerous lesions derived from 114 patients and the tumor tissues of 432 patients with SCC in lung. Extremely low levels of TPX2 protein were found in the normal bronchial epithelia and alveoli, whereas gradually increased TPX2 protein levels were observed in the squamous metaplasia, dysplasia, carcinoma in situ, and invasive tumor tissues. Statistical analysis showed that the TPX2 immunohistochemistry labeling index was correlated with the differentiation grade, stage, and lymphous metastasis of SCC in lung and that TPX2 overexpression is significantly associated with decreased 5-year survival rate of the patients. CONCLUSIONS: Aberrant expression of TPX2 may play important role(s) in both malignant transformation of respiratory epithelium and progression of squamous cell lung cancer and could serve as a prognostic predictor for the disease.

Purpose: The extracellular matrix (ECM) molecule osteopontin is implicated in many pathologic processes, including inflammation, cell proliferation, ECM invasion, tumor progression, and metastasis. The present study evaluated the clinical and biological importance of osteopontin in human lung cancer. Experimental Design and Results: Tissue microarrays derived from non–small cell lung cancer (NSCLC) patients were analyzed immunohistochemically. Osteopontin protein expression was observed in 64.5% (205 of 318) of primary tumors and 75.5% (108 of 143) of lymph node metastases, but in only 27.9% (12 of 43) of normal-appearing bronchial epithelial and pulmonary tissues. Osteopontin expression was associated with tumor growth, tumor staging, and lymph node invasion. In vitro osteopontin enhanced ECM invasion of NSCLC cells, and an osteopontin antibody abolished this effect. We further analyzed osteopontin levels in circulating plasma derived from 158 patients with NSCLC, 54 patients of benign pulmonary disease, and 25 healthy donors, and found that the median osteopontin levels for the three groups were 319.1, 161.6, and 17.9 ng/mL, respectively. Conclusions: Overexpression of osteopontin is common in primary NSCLC and may be important in the development and progression of the cancer. Osteopontin levels in the plasma may serve as a biomarker for diagnosing or monitoring patients with NSCLC.