Atypical protein kinase C iota is an oncogene in human non-small cell lung cancer

Protein kinase C (PKC) isozymes have long been implicated in carcinogenesis. However, little is known about the functional significance of these enzymes in human cancer. We recently showed that the atypical PKC (aPKC) isozyme PKCiota is overexpressed in human non-small cell lung cancer (NSCLC) cells and that PKCiota plays a critical role in the transformed growth of the human lung adenocarcinoma A549 cell line in vitro and tumorigenicity in vivo. Here we provide compelling evidence that PKCiota is an oncogene in NSCLC based on the following criteria: (a) aPKCiota is overexpressed in the vast majority of primary NSCLC tumors; (b) tumor PKCiota expression levels predict poor survival in patients with NSCLC; (c) the PKCiota gene is frequently amplified in established NSCLC cell lines and primary NSCLC tumors; (d) gene amplification drives PKCiota expression in NSCLC cell lines and primary NSCLC tumors; and (e) disruption of PKCiota signaling with a dominant negative PKCiota allele blocks the transformed growth of human NSCLC cells harboring PKCiota gene amplification. Taken together, our data provide conclusive evidence that PKCiota is required for the transformed growth of NSCLC cells and that the PKCiota gene is a target for tumor-specific genetic alteration by amplification. Interestingly, PKCiota expression predicts poor survival in NSCLC patients independent of tumor stage. Therefore, PKCiota expression profiling may be useful in identifying early-stage NSCLC patients at elevated risk of relapse. Our functional data indicate that PKCiota is an attractive target for development of novel, mechanism-based therapeutics to treat NSCLC.     

Overexpression of osteopontin is associated with more aggressive phenotypes in human non-small cell lung cancer.

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.     

Aberrant methylation of CXCL12 in non-small cell lung cancer is associated with an unfavorable prognosis

Chemokines play an important role in the pathogenesis of non-small cell lung cancer (NSCLC). However, aberrant methylation of CXCL12 has not been examined in NSCLC. CXCL12 mRNA expression and methylation were examined in 17 NSCLC cell lines by RT-PCR and methylation-specific PCR (MSP). MSP was performed on 236 tumor specimens from NSCLC patients who received curative intent surgery. CXCL12 and CXCR4 protein expression was examined in 90 of the 236 NSCLC specimens by immunohistochemistry. Down-regulation of CXCL12 expression was found in 10 of 17 (59%) NSCLC cell lines compared with normal bronchial cells. Treatment of 8 expression-negative cell lines with a demethylating agent restored expression in all cases. Twelve cell lines (71%) showed aberrant methylation, and good concordance between methylation and expression was present. Aberrant methylation occurred in 85 out of 236 (36%) primary NSCLCs in a tumor-specific manner. In multivariate analysis, CXCL12 methylation correlated strongly and independently with prognosis both in all patients with NSCLCs and in those with stage I NSCLCs (hazard ratio=1.68, P=0.015 and hazard ratio=3.58, P=0.017). Secreted protein CXCL12 and its receptor CXCR4 were abundant in NSCLC cells (72 out of 90, 80%; 57 out of 90, 63%) and correlated with the progression of NSCLCs. In conclusion, epigenetic silencing of CXCL12 is a frequent event in NSCLCs, and could be an independent and powerful prognostic marker in patients with NSCLCs and those with stage I disease. Analysis for CXCL12 may provide novel opportunities for prognosis and therapy of resected NSCLCs.     

An in vitro chemoresponse assay defines a subset of colorectal and lung carcinomas responsive to cetuximab

Cetuximab is a chimeric monoclonal antibody for the epidermal growth factor receptor (EGFR) that may provide benefit to select cancer patients; however, identification of the characteristics of those patients who may benefit from its use is not complete. The ChemoFx? drug response marker (DRM) is an in vitro assay that can provide drug response data on tumor specimens before any patient treatment is initiated. We determined the feasibility of using the ChemoFx DRM to test tumor samples for sensitivity to cetuximab. We exposed four non-small cell lung carcinoma (NSCLC) cell lines (H358, H520, HCC827, and H1666) to cetuximab and determined their sensitivity using the ChemoFx DRM and, in parallel, EGFR status using immunocytochemistry, Western blotting, and In-Cell Western (TM) analysis. We used the ChemoFx DRM to determine cetuximab sensitivity of primary NSCLC and colorectal tumor samples. The ChemoFx DRM distinguished between cetuximab-sensitive and -resistant cell lines. Cetuximab sensitivity was not dependent on EGFR mutational status; H358 cells were non-responsive to cetuximab yet contain wild-type EGFR, whereas H1666 cells were intermediately responsive to cetuximab and contain wild-type EGFR. HCC827 (EGFR-mutant) cells were intermediately responsive and, as expected, H520 cells (EGFR-null) were non-responsive to cetuximab. ChemoFx-determined cetuximab sensitivity of primary NSCLC and colorectal tumor samples was 9.0% and 7.5%, respectively. Use of the ChemoFx DRM is feasible for determining cetuximab sensitivity. The ChemoFx-determined cetuximab responses of primary NSCLC and colorectal tumor specimens were similar to published response rates of patients to treatment with cetuximab monotherapy.     

非小细胞肺癌组织PD-L1的表达及其与临床因素的关系

目的:探索程序性死亡配体1 (programmed death-ligand 1,PD-L1)在中国非小细胞肺癌(non-small cell lung carcinoma,NSCLC)患者肿瘤组织中的表达水平及影响因素.方法:免疫组织化学法检测2008年4月至2014年8月天津医科大学肿瘤医院122例NSCLC初治患者肿瘤组织中PD-L1、PD-1和CD3+T细胞表达情况,采用x2和kruskal-wallis检验分析PD-L1表达在临床因素中分布差异性,用Person检验和Spearman检验分析PD-L1表达与EGFR基因型、CD3+T细胞数量及淋巴细胞PD-1表达的相关性,以及原发灶与淋巴结PD-L1表达相关性.结果:所有患者原发灶肿瘤细胞PD-L1表达百分比中位值1.5％(0～93.2％),PD-L1表达在TNM分期分布上有统计学差异(P =0.003),与TNM分期呈显著正相关(r=0.273,P=0.002),与性别、年龄、有无吸烟史、肿瘤最大径、病理类型、CEA水平分布无显著相关(P ＞0.05);PD-L1表达水平与CD3+T细胞数量、淋巴细胞PD-1表达水平无相关性,PD-L1表达阴性、低表达和高表达与表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变亦无显著相关(P ＞0.05);48例有淋巴结转移的NSCLC患者原发灶与相应转移淋巴结肿瘤细胞PD-L1表达水平无统计学相关性(P＞0.05).结论:NSCLC患者原发灶肿瘤细胞PD-L1表达在TNM分期分布上有差异,与CD3+T细胞数量、淋巴细胞PD-1表达水平、EGFR基因突变情况无相关性;原发灶与相应转移淋巴结之间肿瘤细胞PD-L1的表达亦没有相关性.     

Soluble epidermal growth factor receptor isoforms in non-small cell lung cancer tissue and in blood

Epidermal growth factor receptor (EGFR) is implicated in tumor development and is highly expressed in many human tumors. EGFR overexpression has been observed in both premalignant lesions and in malignant lung tumors, as well as in 40-80% of patients with non-small cell lung cancer (NSCLC). EGFR is a 170-kDa transmembrane glycoprotein with an extracellular ligand-binding domain and a cytoplasmic domain with intrinsic tyrosine kinase activity. Soluble forms of EGFR (sEGFR) containing the extracellular domain have been described both in conditioned media from EGFR overexpressing cells as well as in peripheral blood. However, very little is known regarding the molecular function and the biochemical properties of these circulating EGFR isoforms. This study investigates the expression of sEGFR in lung cancer cultured cells and NSCLC patients with the aim of identifying clinically relevant isoforms specifically produced by tumor cells. Proteomic approaches including OFFGEL electrophoresis and Western blotting analysis were used to assess the sEGFR expression pattern in primary lung tumor samples, normal counterparts and matched plasma. We discover that the isoelectric points of sEGFR isoforms in NSCLC biopsy tissue differ from those of the isoforms present in healthy tissue and detected in the plasma of all subjects. These results demonstrate, for the first time, the existence of sEGFR isoforms specifically produced by NSCLC tumor cells which could represent a new potential biomarker for diagnosis and therapy of lung tumors. However, our observations indicate that more highly sensitive and specific quantitative assays are needed in order to reliably detect the tumor-associated sEGFR isoforms in plasma samples.     

CD1c+ and CD303+ dendritic cells in peripheral blood, lymph nodes and tumor tissue of patients with non-small cell lung cancer

Dendritic cells (DCs) are the most potent antigen presenting cells, which can stimulate a cellular immune response against malignant tumor cells. Many authors have described the phenomenon of tumor infiltration by dendritic cells and emphasized an immunosuppressive tumor influence on DC function. In the present study, we examined the presence of myeloid CD1c+ (BDCA-1+) dendritic cells and lymphoid/plasmacytoid CD303+ (BDCA-2+) dendritic cells in peripheral blood, lymph nodes and cancer tissue of patients with non-small cell lung cancer (NSCLC). Fifty male patients treated surgically for NSCLC stages I-IIIa without neoadjuvant chemotherapy were included. Employing a multiparameter flow cytometry for CD1c, CD19, CD123 and CD303, we observed an accumulation of immature DCs in the tissues involved in the neoplasmatic process with the predominance of lymphoid/plasmacytoid over myeloid DCs. Moreover, in peripheral blood NSCLC patients had a significantly lower percentage of CD1c+ DCs than healthy donors. Our results suggest that NSCLC cells might hamper the maturation of DCs, thus escaping an efficient immune response.     

Carbonic anhydrase IX in early-stage non-small cell lung cancer.

PURPOSE: Tumor hypoxia is associated with poor prognosis and increased tumor aggressiveness. Carbonic anhydrase (CA) IX, an endogenous marker for tumor hypoxia, catalyzes the hydration of carbon dioxide into carbonic acid and contributes to the pH regulation of tumor cells. Therefore, CA IX might allow tumors to acclimate to a hypoxic microenvironment, promoting tumor cell proliferation. We hypothesized that CA IX expression is related to tumor cell proliferation and poor disease-free survival in patients with early-stage non-small-cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: CA IX expression was measured in 75 resected NSCLC tumors to assess prognostic implications for disease-free survival. The relationship of CA IX expression with microvessel density (MVD) and proliferation (Ki-67) index was assessed via colocalization analysis. RESULTS: All patients had operable NSCLC (stage I, 58; stage II, 17). CA IX expression was present in 54 (72%) of 75 patients and was associated with tumor necrosis (P < 0.05). CA IX-positive tumor areas showed greater cell proliferation as measured by Ki-67 index (P < 0.05) and less MVD (P < 0.05) than did CA IX-negative areas in colocalization analysis. The percentage of CA IX-positive tumor cells was significantly related to postoperative recurrence and poor disease-free survival (P < 0.05). Ki-67 index and pathologic stage were also independent prognostic factors for worse disease-free survival (P < 0.05). CONCLUSIONS: CA IX expression of tumor cells may be an indicator for poor disease-free survival in early-stage NSCLC.     

Elevated expression of carcinoembryonic antigen-related cell adhesion molecule 1 promotes progression of non-small cell lung cancer.

PURPOSE: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) has recently been implicated in cancer development and progression. This study was performed to assess whether CEACAM-1 expression in primary tumors is correlated to long-term survival in patients with operable non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Primary tumors of 145 consecutive patients with completely resected NSCLC (pT(1-4) pN(0-2) M(0) R(0)) were stained immunohistochemically using the monoclonal anti-CEACAM-1 antibody 4D1/C2. The prognostic relevance of CEACAM-1 expression was evaluated by univariate Kaplan-Meier and multivariate Cox regression analysis. The median follow-up period was 72 months (range, 10-130 months). RESULTS: Normal bronchiolar epithelium present in all sections exhibited no immunostaining. In contrast, 73 tumors (50.4%) showed between 1 and 66% CEACAM-1 positive tumor cells, and 72 tumors (49.6%) exhibited even a higher percentage of positive tumor cells. A high CEACAM-1 expression rate (i.e., >/=66% positive tumor cells) was more frequent in adenocarcinomas than in squamous cell carcinomas (61.9 versus 35.7%, respectively). Multivariate Cox regression analysis demonstrated that CEACAM-1 represents an independent prognosticator for cancer-related survival (P = 0.018; relative risk, 1.8; 95% confidence interval, 1.1-2.8). Subgroup analysis revealed that a high CEACAM-1 expression rate was of significant prognostic impact in pN(1)-pN(2) patients (n = 60; P = 0.024), pT(3)-pT(4) patients (n = 22; P = 0.009), and stage IIa-IIIa patients (n = 69; P = 0.012). CONCLUSIONS: The absence of CEACAM-1 in normal lung tissue and its expression in tumor cells argues against a tumor-suppressive role of CEACAM-1 in NSCLC. The correlation between elevated CEACAM-1 expression and an unfavorable prognosis indicates rather that CEACAM-1 might promote lung cancer progression.     

Prognostic impact of the primary tumor location based on the hilar structures in non-small cell lung cancer with mediastinal lymph node metastasis

The status of mediastinal lymph node metastasis is one of the main factors determining the treatment strategy for non-small cell lung cancer (NSCLC), but the primary tumor location is not considered crucial in the tumor-node-metastasis (TMN) classification at present. The aim of this study was to estimate the prognostic value of the primary tumor location on the basis of the hilar structures in NSCLC with mediastinal lymph node metastasis. We retrospectively reviewed the cases of 337 consecutive patients who underwent surgical resection for NSCLC between 1995 and 2004, divided the pN2 NSCLC cases (n=40) into central- and peripheral-type tumors according to the distance of the primary tumor from the first branch of the extrapulmonary bronchus, and compared the surgical outcomes between these tumor groups. Eighteen and twenty-two cases were classified as central- and peripheral-type tumors, respectively. The 5-year survival rate was significantly better for patients with central-type tumors than peripheral-type tumors (51.5% vs. 21.2%, P=0.034). The location-specific prognostic tendency was noted irrespective of the presence (n=13) or absence of skip metastasis. In a multivariate Cox analysis of the N2 NSCLC cases, the primary tumor location was a significant (P=0.026) prognostic factor for overall survival. In conclusion, evaluation of the primary tumor location based on the hilar structures is useful to predict the prognosis in N2 NSCLC.     

Profound coordinated alterations of intratumoral NK cell phenotype and function in lung carcinoma

Both the innate and adaptive immune systems contribute to tumor immunosurveillance in mice and humans; however, there is a paucity of direct evidence of a role for natural killer (NK) cells in this important process. In this study, we investigated the intratumoral phenotypic profile and functions of NK cells in primary human tumor specimens of non-small cell lung carcinoma (NSCLC). We used in situ methods to quantify and localize NK cells using the NKp46 marker and we characterized their phenotype in blood, tumoral, and nontumoral samples of NSCLC patients. Intratumoral NK cells displayed a profound and coordinated alteration of their phenotype, with a drastic reduction of NK cell receptor expression specifically detected in the tumoral region. According to their altered phenotype, intratumoral NK cells exhibited profound defects in the ability to activate degranulation and IFN-γ production. We found that the presence of NK cells did not impact the clinical outcome of patients with NSCLC. Finally, we showed that tumor cells heterogeneously express ligands for both activating and inhibitory NK receptors. Taken together, our results suggest that the NSCLC tumor microenvironment locally impairs NK cells, rendering them less tumorcidal and thereby supportive to cancer progression.     

Tanshinone IIA inhibits cell growth by suppressing SIX1-induced aerobic glycolysis in non-small cell lung cancer cells

Aerobic glycolysis plays a key role in cancer cell metabolism and contributes to tumorigenesis, including that of non-small cell lung cancer (NSCLC). Tanshinone IIA (Tan IIA), an active compound of Salvia miltiorrhiza, exhibits antitumor properties. Multiple mechanisms are involved in the antitumor action of Tan IIA in lung cancer, such as inhibiting cell growth, promoting cell apoptosis and influencing cellular metabolism. However, the effects of Tan IIA on NSCLC cells and its mechanisms of action remain unclear. The present study shows Tan IIA dose-dependently attenuated the growth of NSCLC cells and in vitro in a dose-dependent manner. Moreover, Tan IIA markedly decreased the ATP level, glucose uptake and lactate production in the NSCLC cells in vitro. Tan IIA also inhibited tumor growth in a xenograft model in vivo. Mechanically, Tan IIA treatment decreased sine oculis homeobox homolog 1 (SIX1) mRNA and protein levels, thus leading to the downregulation of pyruvate kinase isozyme M2, hexokinase 2 and lactate dehydrogenase A (LDHA) expression in A549 cells. SIX1 knockdown with small interfering-RNA inhibited glycolysis in NSCLC cells, suggesting that SIX1 plays a role in the antitumor effect of Tan IIA on NSCLC cells. More importantly, it was demonstrated that SIX1 expression was stimulated in patients with NSCLC and was positively correlated with the LDH serum level. Finally, SIX1 low expression levels predicted the poor prognosis of patients with NSCLC. In conclusion, the present study showed that Tan IIA functioned as an anti-glycolysis agent in NSCLC cells by downregulating SIX1 expression and inhibiting cell proliferation.     

全基因组芯片筛查非小细胞肺癌组织多药耐药相关基因

背景与目的筛查与非小细胞肺癌多药耐药相关的基因,为非小细胞肺癌的个体化治疗提供理论依据。方法将手术切除的肺癌组织细胞进行原代培养。首先,采用MTT法检测诺维本、吉西他滨、多西他赛、紫杉醇及顺铂对非小细胞肺癌组织细胞的抑制率和敏感性。再利用全基因组芯片筛选人高度敏感组和耐药组间的差异表达基因。结果共筛选出差异表达基因212个,与耐药组相比,高度敏感组中上调基因168个,下调基因44个。结论利用全基因组芯片筛查出212个可能与非小细胞肺癌多药耐药相关的基因,用于指导临床个体化治疗。     

The Induction of Cytotoxic T Lymphocytes against HLA-A Locus-matched Lung Adenocarcinoma in Patients with Non-small Cell Lung Cancer

To induce cytotoxic T lymphocytes (CTL) against non-small cell lung cancer (NSCLC) efficiently, the induction of CTL was attempted using HLA-A locus-shared allogeneic NSCLC cells. T cells derived from either tumor tissue specimens or the regional lymph nodes of patients with NSCLC were stimulated twice or three times with an HLA-A2/A24-positive NSCLC cell line (PC-9), and thereafter the cytotoxic activity was examined by ⁵¹Cr-release assay. In patients with HLA-A24/ adenocarcinoma, anti-PC-9 cytotoxicity was induced in all 6 patients tested. Anti-PC-9 cytotoxicity was induced in 2 out of 5 patients with HLA-A2 (A24⁻)/adenocarcinoma, in 2 out of 4 patients with HLA-A24/squamous cell carcinoma, and 1 of 2 patients with HLA-A2/squamous cell carcinoma. The cytotoxic activity was observed to kill PC-9 selectively, not other NSCLC lines, and the activity was substantially blocked by anti-MHC class I antibody, but not by anti-MHC class II antibody. The PC-9-specific CTL produced γ-interferon in response to autologous tumor cells. These results indicated that the anti-PC-9 cytotoxicity was mediated by cytotoxic T lymphocytes that may recognize the T cell epitope(s) shared and presented by HLA-A2 and/or HLA-A24-positive NSCLC.