Small Cell Lung Cancer Tissues Research Articles

Clinical significance of serum miR-25 in non-small-cell lung cancer

ABSTRACTBackground: MicroRNAs (miRNAs) are becoming recognized as novel diagnostic and prognostic biomarkers in several malignancies, including non-small-cell lung cancer (NSCLC). miR-25 is overexpressed in small cell lung cancer (SCLC) and NSCLC tissues, and high miR-25 expression is associated with poorer overall survival of women with lung ADC. We hypothesised links between serum miR-25 levels and clinicopathological characteristics, diagnosis and prognosis of NSCLC patients.Methods: Serum miR-25 was determined by real-time quantitative polymerase chain reaction in 128 NSCLC patients and 128 healthy controls, and links between miR-25 level and cliniopathological characteristics including diagnosis and prognosis were explored.Results: Median (IQR) serum miR-25 levels were significantly increased in NSCLC compared to healthy controls at 0.86 relative units (0.14–1.78) versus 0.23 (0.08–0.96) (P < 0.001). Using a cut-off of 0.67 units, miR-25 had a sensitivity of 76.4%, specificity of 84.6%, accuracy of 72.6%, positive predictive value of 92.8% and negative predictive value of 68.5% for the diagnosis of NSCLC. High serum miR-25 level was significantly associated with gender (P = 0.042), tumour stage (P = 0.014) and lymph node metastasis (P < 0.001). In multivariate analyses, miR-25 was an independent prognostic factor for overall survival and relapse-free survival.Conclusions: Serum levels of miR-25 could improve NSCLC screening, and be a useful diagnostic and prognostic marker of NSCLC.

SBF2-AS1: An oncogenic lncRNA in small-cell lung cancer.

The long noncoding RNAs (lncRNAs) SBF2 antisense RNA 1 (SBF2-AS1) was found to act as an oncogenic lncRNA in non-small-cell lung cancer (NSCLC), but the role of SBF2-AS1 in small-cell lung cancer (SCLC) was still unclear. The purpose of this study was to provide the clinical significance and biological function of SBF2-AS1 in SCLC. In our results, SBF2-AS1 was found to be upregulated in SCLC tissues compared with NSCLC tissues or adjacent normal lung tissues. Besides, SBF2-AS1 expression was also elevated in SCLC cell lines compared with the normal bronchial epithelial cell line or NSCLC lines. Moreover, high expression of SBF2-AS1 was associated with clinical stage, tumor size, lymph node metastasis and distant metastasis in SCLC patients. Survival analysis showed SCLC patients with high expression of SBF2-AS1 had shorter overall survival than patients with low expression of SBF2-AS1, and high expression of SBF2-AS1 acted as an independent poor prognostic factor for overall survival in SCLC patients. The study in vitro suggested inhibition of SBF2-AS1 obviously depressed cell proliferation, migration, and invasion in SCLC. In conclusion, SBF2-AS1 acts as a novel oncogenic lncRNA in SCLC.

FLI1 Exonic Circular RNAs as a Novel Oncogenic Driver to Promote Tumor Metastasis in Small Cell Lung Cancer.

The aberrantly upregulated Friend leukemia virus integration 1 (FLI1) is closely correlated with the malignant phenotype of small cell lung cancer (SCLC). It is interesting to note that the CRISPR gene knockout by Cas9 gRNAs that target the FLI1 coding region and the posttranscriptional knockdown by shRNAs that target the 3' region of FLI1 mRNA yielded distinct antimetastasis effects in SCLC cells. This study attempts to examine if FLI1 exonic circular RNAs (FECR) function as a new malignant driver that determines the metastatic phenotype in SCLC. The clinical relevance of FECRs was examined in 56 primary SCLC tissues and 50 non-small cell lung cancer (NSCLC) tissues. The prognostic value of FECRs was examined by measuring serum exosomal FECRs in a longitudinal cohort of patients with SCLC. The oncogenic activity of FECRs was investigated in both SCLC cell lines and animal xenograft studies. Finally, we explored the molecular mechanisms underlying these noncoding RNAs as a malignant driver. Therapeutic comparison of CRISPR Cas9 knockout and shRNA knockdown of FLI1 identified FECRs as a new noncanonical malignant driver in SCLC. Using RNA FISH and quantitative PCR, we found that FECR1 (exons 4-2-3) and FECR2 (exons 5-2-3-4) were aberrantly upregulated in SCLC tissues (P < 0.0001), and was positively associated with lymph node metastasis (P < 0.01). Notably, serum exosomal FECR1 was associated with poor survival (P = 0.038) and clinical response to chemotherapy. Silencing of FECRs significantly inhibited the migration in two highly aggressive SCLC cell lines and reduced tumor metastasis in vivo. Mechanistically, we uncovered that FECRs sequestered and subsequently inactivated tumor suppressor miR584-3p, leading to the activation of the Rho Associated Coiled-Coil Containing Protein Kinase 1 gene (ROCK1). This study identifies FLI1 exonic circular RNAs as a new oncogenic driver that promotes tumor metastasis through the miR584-ROCK1 pathway. Importantly, serum exosomal FECR1 may serve as a promising biomarker to track disease progression of SCLC.

MicroRNA-485-5p suppresses the proliferation, migration and invasion of small cell lung cancer cells by targeting flotillin-2

ABSTRACT This study is aimed to elucidate the mechanisms underlying the role of miR-485-5p in small cell lung cancer (SCLC). The expression of miR-485-5p were quantified with real time quantitative PCR and it was found that the level of miR-485-5p was lower in SCLC tissues than normal tissues. In cultured SCLC cell lines, overexpression of miR-485-5p reduced cell proliferation, migration, and invasion in vitro, whereas knockdown of miR-485-5p performed contrary. FLOT2 expression was obviously upregulated and negatively correlated with miR-485-5p expression level in SCLC tissues. Overexpression of miR-485-5p significantly inhibited the protein expression of flotillin-2 (FLOT2) in cultured SCLC cells. Luciferase reporter assay confirmed that FLOT2 was a direct target of miR-485-5p in SCLC cells. It is concluded that miR-485-5p, as a tumor suppressor, inhibits the growth and metastasis in SCLC by targeting FLOT2. Upregulation of miR-485-5p expression may be an attractive strategy for SCLC therapy.

CDYL promotes the chemoresistance of small cell lung cancer by regulating H3K27 trimethylation at the CDKN1C promoter.

Rationale: Chemoresistance frequently occurs in patients with small cell lung cancer (SCLC) and leads to a dismal prognosis. However, the mechanisms underlying this process remain largely unclear.Methods: The effects of chromodomain Y-like (CDYL) on chemoresistance in SCLC were determined using Western blotting, immunohistochemistry, cell counting kit-8 assays, flow cytometry, and tumorigenicity experiments, and the underlying mechanisms were investigated using mRNA sequencing, chromatin immunoprecipitation-qPCR, electrophoretic mobility shift assays, co-immunoprecipitation, GST pull down assays, bisulfite sequencing PCR, ELISA, and bioinformatics analyses.Results: CDYL is expressed at high levels in chemoresistant SCLC tissues from patients, and elevated CDYL levels correlate with an advanced clinical stage and a poor prognosis. Furthermore, CDYL expression is significantly upregulated in chemoresistant SCLC cells. Using gain- and loss-of-function methods, we show that CDYL promotes chemoresistance in SCLC in vitro and in vivo. Mechanistically, CDYL promotes SCLC chemoresistance by silencing its downstream mediator cyclin-dependent kinase inhibitor 1C (CDKN1C). Further mechanistic investigations showed that CDYL recruits the enhancer of zeste homolog 2 (EZH2) to regulate trimethylation of lysine 27 in histone 3 (H3K27me3) at the CDKN1C promoter region and promotes transcriptional silencing. Accordingly, the EZH2 inhibitor GSK126 de-represses CDKN1C and decreases CDYL-induced chemoresistance in SCLC.Principal conclusions: Based on these results, the CDYL/EZH2/CDKN1C axis promotes chemoresistance in SCLC, and these markers represent promising therapeutic targets for overcoming chemoresistance in patients with SCLC.

H3K27me3 induces multidrug resistance in small cell lung cancer by affecting HOXA1 DNA methylation via regulation of the lncRNA HOTAIR.

The long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) serves as a powerful predictor of tumor progression and overall survival in patients. Our previous studies showed that HOTAIR modulated HOXA1 DNA methylation by reducing DNMT1 and DNMT3b expression in drug-resistant small cell lung cancer (SCLC). Moreover, H3 lysine 27 trimethylation (H3K27me3) is catalyzed by enhancer of zeste homolog 2 (EZH2) and plays a critical role in SCLC chemoresistance. However, it is not completely clear whether H3K27me3 affects HOXA1 DNA methylation or whether this effect is mediated by HOTAIR. The levels of EZH2 and H3K27me3 were identified in SCLC tissues by immunohistochemical (IHC) staining and in SCLC multidrug-resistant cells by Western blotting. Cell counting kit-8 (CCK-8) and flow cytometry were used to detect and analyze the biological function of H3K27me3. Then, we assessed the role of HOTAIR in the regulation of EZH2 and H3K27me3 by using lentivirus and small interfering RNA. Further, bisulfite sequencing PCR was conducted to detect the methylation levels of HOXA1 DNA. Finally, Western blotting was performed to examine the regulatory role of H3K27me3 in controlling HOTAIR expression in SCLC. In this study, we found that EZH2 and H3K27me3 levels were markedly higher in SCLC tissues and multidrug-resistant SCLC cells. The results indicated that H3K27me3 was related to multidrug resistance. HOTAIR overexpression and knockdown showed that EZH2 and H3K27me3 were regulated by HOTAIR. Moreover, H3K27me3 affected HOXA1 DNA methylation levels. Strikingly, we found that H3K27me3 acted as a negative feedback regulator of HOTAIR. Our study showed that H3K27me3 affects HOXA1 DNA methylation via HOTAIR regulation, indicating that H3K27me3 may be a potential therapy target for SCLC chemoresistance.

BLACAT1 predicts poor prognosis and serves as oncogenic lncRNA in small-cell lung cancer.

Bladder cancer-associated transcript 1 (BLACAT1) is a novel identified long noncoding RNA (lncRNA) in bladder cancer, and has been suggested to function as an oncogenic lncRNA in several types of human cancer. However, its involvement in the progression of small-cell lung cancer (SCLC) remained unknown. The aim of our study was to investigate the clinical value and biological function in SCLC. In our results, BLACAT1 expression was increased in SCLC tissues and cell lines compared with paired adjacent normal tissues and bronchial epithelial cell lines, respectively. In addition, BLACAT1 high-expression was obviously associated with advanced clinical stage, large tumor size, more lymph node metastasis, present distant metastasis, and poor prognosis. Furthermore, multivariate analysis indicated that high-expression of BLACAT1 acted as an independent poor prognostic factor for overall survival in SCLC cases. The loss-of-function studies suggested that of BLACAT1 suppressed SCLC cell proliferation, migration, and invasion, and induced G0/G1 phase arrest. In conclusion, BLACAT1 is associated with the malignant status and prognosis in patients with SCLC, and functions as an oncogenic lncRNA in regulating cell proliferation and motility, suggesting BLACAT1 may act as a potential target for SCLC prevention and treatment.

The 180 splice variant of NCAM-containing exon 18-is specifically expressed in small cell lung cancer cells.

The Neural Cell Adhesion Molecule (NCAM) is a glycoprotein expressed as 120, 140 and/or 180 kDa isoforms, all derived through alternative splicing of a single gene. NCAM 120 contains no intracellular domain, whereas NCAM 140 and 180 have different intracellular domains determined by alternative splicing of exon 18. NCAM has been described as a biomarker to discriminate small cell lung cancer (SCLC) from non-SCLC (NSCLC). However, peripheral blood mononuclear cells (PBMC) also express NCAM. We studied the expression of NCAM splice variants in cell lines, tumor tissues and control cells. Using reverse transcriptase-PCR we evaluated the expression of NCAM exon 18 splice variants in lung cancers cell lines, control cell lines, PBMC of healthy controls and SCLC tissue. In addition we studied the expression of the NCAM exon 18 encoded protein (E18) in SCLC by immunocytochemistry and flow cytometry using an E18-specific monoclonal antibody obtained by hybridoma fusion of E18-immunized mouse spleen cells. Finally we looked at immune responses to E18 in mice. We found expression of RNA encoding the NCAM 180 variant in all SCLC cell lines. NCAM exon 18 was not expressed in 23/28 (82%) of the other tumor and leukemia cell lines tested and PBMC. Next, we also evaluated the expression of NCAM exon 18 in human SCLC tissue. Expression of NCAM exon 18 in 8 of the 10 (80%) SCLC biopsy samples was found. The newly raised E18-specific antibodies stained NCAM at the adherent junctions between adjacent cells in SCLC cell lines. The data demonstrate the intracellular location of E18 in SCLC. Furthermore, a specific cytotoxic T cell (CTL) response and significant antibody titers were found in mice upon immunization with recombinant E18 and its encoding DNA. The results of this study can be applied in the diagnosis and immunotherapy of SCLC. A larger study investigating E18 as a marker for SCLC is indicated.

Lysine‑specific demethylase 2 contributes to the proliferation of small cell lung cancer by regulating the expression of TFPI‑2.

The present study aimed to investigate the effect of lysine-specific demethylase 2 (LSD2) in small cell lung cancer (SCLC) and explore its underlying regulatory mechanism. Cell growth was tested by MTT assay and mRNA and protein expression was determined by quantitative polymerase chain reaction (q-PCR) and western blot analysis, respectively. Chromatin immunoprecipitation (ChIP) was used to investigate the degree of H3K4me2 enrichment in the promoter region of tissue factor pathway inhibitor-2 (TFPI-2). SCLC tissues and cell lines presented significantly higher expression of LSD2 and DNA methyltransferase 3B (DNMT3B) and lower expression of TFPI-2 compared with the controls. In H1417 cells LSD2 overexpression increased the mRNA and protein expression of DNMT3B, while inhibiting the mRNA and protein expression of TFPI-2. Following transfection with short interfering (si) RNA-DNMT3B, the expression of TFPI-2 increased in H1417 cells. The results of ChIP demonstrated that compared with the controls, H3K4me1 enrichment in the TFPI-2 promoter region was to a lower degree in the H1417 cells with LSD2 overexpression and a higher degree in the H1417 cells with LSD2 silencing. MTT assays revealed that LSD2 overexpression significantly promoted the growth of H69, DMS-114 and H1417 cells, which was contradictory to the effect on LSD2 silencing. Compared with the LSD2 overexpression cells, SCLC cells with simultaneous overexpression of LSD2 and TFPI-2 demonstrated a decreased proliferation. These results suggest that LSD2 achieves a promoting effect on SCLC by indirectly regulating TFPI-2 expression through the mediation of DNMT3B expression or through the regulation of the demethylation of H3K4me1 in the promoter region of the TFPI-2 gene.

Preclinical Evaluation of SCC244 (Glumetinib), a Novel, Potent, and Highly Selective Inhibitor of c-Met in MET-dependent Cancer Models.

Because the receptor tyrosine kinase c-Met plays a critical role in tumor growth, metastasis, tumor angiogenesis, and drug resistance, the c-Met axis represents an attractive therapeutic target. Herein, we report the first preclinical characterization of SCC244, a novel, potent, and highly selective inhibitor of c-Met kinase. SCC244 showed subnanomolar potency against c-Met kinase activity and high selectivity versus 312 other tested protein kinases, making it one of the most selective c-Met inhibitors described to date. Moreover, this inhibitor profoundly and specifically inhibits c-Met signal transduction and thereby suppresses the c-Met-dependent neoplastic phenotype of tumor and endothelial cells. In xenografts of human tumor cell lines or non-small cell lung cancer and hepatocellular carcinoma patient-derived tumor tissue driven by MET aberration, SCC244 administration exhibits robust antitumor activity at the well-tolerated doses. In addition, the in vivo antitumor activity of SCC244 involves the inhibition of c-Met downstream signaling via a mechanism of combined antiproliferation and antiangiogenic effects. The results of the current study provide a strong foundation for the clinical investigation of SCC244 in patients with tumors harboring c-Met pathway alterations. Mol Cancer Ther; 17(4); 751-62. ©2017 AACR.

Noncatalytic Endosialidase Enables Surface Capture of Small-Cell Lung Cancer Cells Utilizing Strong Dendrimer-Mediated Enzyme-Glycoprotein Interactions.

Enumeration of circulating tumor cells (CTCs) of small-cell lung cancer (SCLC) patients has been shown to predict the disease progress and long-term survival. Most CTC detection methods rely on epithelial surface markers, such as epithelial cell adhesion molecule (EpCAM). However, this marker in SCLC is reported to be often downregulated after a variety of phenotypic changes, which impairs the reliability of EpCAM-based CTC detections. In this regard, the development of an alternative CTC detection method involving different CTC surface markers is in demand. In this study, we evaluated, for the first time to our knowledge, the feasibility of detecting SCLC CTCs using a noncatalytic endosialidase (EndoN Trap, EndoNt). This noncatalytic enzyme was chosen due to its high affinity to polysialic acid (polySia), a cell-surface glycan, that is highly expressed by SCLC tissue. Furthermore, this enzyme-based system was integrated into our dendrimer-mediated CTC capture platform to further enhance the capture efficiency via multivalent binding. We found that the EndoNt-immobilized surfaces could specifically capture polySia-positive SCLC cells and the binding between SCLC cells and EndoNt surfaces was further stabilized by dendrimer-mediated multivalent binding. When compared to the EpCAM-based capture, EndoNt significantly improved the capture efficiency of polySia-positive SCLC cells under flow due to its higher binding affinity (lower dissociation rate constants). These findings suggest that this enzyme-based CTC capture strategy has the potential to be used as a superior alternative to the commonly used EpCAM-based methods, particularly for those types of cancer that overexpress polySia.

Transcription factor E2F1 promotes EMT by regulating ZEB2 in small cell lung cancer

BackgroundEpithelial-mesenchymal transition (EMT) is an early event in tumour invasion and metastasis, and widespread and distant metastasis at early stages is the typical biological behaviour in small cell lung cancer (SCLC). Our previous reports showed that high expression of the transcription factor E2F1 was involved in the invasion and metastasis of SCLC, but the role of E2F1 in the process of EMT in SCLC is unknown.MethodsImmunohistochemistry was performed to evaluate the expressions of EMT related markers. Immunofluorescence was used to detect the expressions of cytoskeletal proteins and EMT related markers when E2F1 was silenced in SCLC cell lines. Adenovirus containing shRNA against E2F1 was used to knock down the E2F1 expression, and the dual luciferase reporter system was employed to clarify the regulatory relationship between E2F1 and ZEB2.ResultsIn this study, we observed the remodelling of cytoskeletal proteins when E2F1 was silenced in SCLC cell lines, indicating that E2F1 was involved in the EMT in SCLC. Depletion of E2F1 promoted the expression of epithelial markers (CDH1 and CTNNB1) and inhibited the expression of mesenchymal markers (VIM and CDH2) in SCLC cell lines, verifying that E2F1 promotes EMT occurrence. Next, the mechanism by which E2F1 promoted EMT was explored. Among the CDH1 related inhibitory transcriptional regulators ZEB1, ZEB2, SNAI1 and SNAI2, the expression of ZEB2 was the highest in SCLC tissue samples and was highly consistent with E2F1 expression. ChIP-seq data and dual luciferase reporter system analysis confirmed that E2F1 could regulate ZEB2 gene expression.ConclusionOur data supports that E2F1 promotes EMT by regulating ZEB2 gene expression in SCLC.

A long non-coding RNA HOTTIP expression is associated with disease progression and predicts outcome in small cell lung cancer patients

BackgroundDespite progress in treatment of small cell lung cancer (SCLC), the biology of the tumor still remains poorly understood. Recently, we globally investigated the contributions of lncRNA in SCLC with a special focus on sponge regulatory network. Here we report lncRNA HOTTIP, which is specifically amplified in SCLC, is associated with SCLC proliferation and poor prognosis of patients.MethodsRT-qPCR was used to investigate the expression of HOTTIP in SCLC tissues and cell lines. The role of HOTTIP in SCLC cell proliferation was demonstrated by CCK8 assay, colony formation assay, flow cytometry analysis and in vivo SCLC xenograft model in nude mice through HOTTIP loss- and gain-of-function effects. Western blot assay was used to evaluate gene expression in cell lines at protein level. RNA pull-down, Mass spectrometry and RNA binding protein immunoprecipitation (RIP) were performed to confirm the molecular mechanism of HOTTIP involved in SCLC progression.ResultsWe found that HOTTIP was overexpressed in SCLC tissues, and its expression was correlated with the clinical stage and the shorter survival time of SCLC patients. Moreover, HOTTIP knockdown could impair cell proliferation, affect the cell cycle and inhibit tumor growth of mice, while HOTTIP overexpression might enhance cell proliferation and cell cycle in vitro and in vivo. Mechanistic investigations showed that HOTTIP functions as an oncogene in SCLC progression by sponging miR-574-5p and affecting the expression of polycomb group protein EZH1.ConclusionsOverall, we identified that HOTTIP was involved in SCLC tumorigenesis through the ceRNA network “HOTTIP/miR-574-5p/EZH1”. Our findings not only illuminate how HOTTIP confers an oncogenic function in SCLC pathogenesis, but also underscore a novel gene expression governing hallmarks in the disease.

CD13 as target for tissue factor induced tumor vascular infarction in small cell lung cancer

ObjectivesZinc-binding protease aminopeptidase N (CD13) is expressed on tumor vascular cells and tumor cells. It represents a potential candidate for molecular targeted therapy, e.g. employing truncated tissue factor (tTF)-NGR, which can bind CD13 and thereby induce tumor vascular infarction. We performed a comprehensive analysis of CD13 expression in a clinically well characterized cohort of patients with small cell lung cancer (SCLC) to evaluate its potential use for targeted therapies in this disease. Material and methodsCD13 expression was analyzed immunohistochemically in 27 SCLC patients and correlated with clinical course and outcome. In CD-1 nude mice bearing human HTB119 SCLC xenotransplants, the systemic effects of the CD13-targeting fusion protein tTF-NGR on tumor growth were tested. Results and conclusionIn 52% of the investigated SCLC tissue samples, CD13 was expressed in tumor stroma cells, while the tumor cells were negative for CD13. No prognostic effect was found in the investigated SCLC study collective with regard to overall survival (p>0.05). In CD-1 nude mice, xenografts of CD13 negative HTB119 SCLC cells showed CD13 expression in the intratumoral vascular and perivascular cells, and the systemic application of CD13-targeted tissue factor tTF-NGR led to a significant reduction of tumor growth. We here present first data on the expression of CD13 in SCLC tumor samples. Our results strongly recommend the further investigation of tTF-NGR and other molecules targeted by NGR-peptides in SCLC patients. Considering the differential expression of CD13 in SCLC samples pre-therapeutic CD13 analysis is proposed for testing as investigational predictive biomarker for patient selection.

MHC class II expression in lung cancer

BackgroundImmunotherapy is an exciting development in lung cancer research. In this study we described major histocompatibility complex (MHC) Class II protein expression in lung cancer cell lines and patient tissues. MethodsWe studied MHC Class II (DP, DQ, DR) (CR3/43, Abcam) protein expression in 55 non-small cell lung cancer (NSCLC) cell lines, 42 small cell lung cancer (SCLC) cell lines and 278 lung cancer patient tissues by immunohistochemistry (IHC). ResultsSeven (12.7%) NSCLC cell lines were positive for MHC Class II. No SCLC cell lines were found to be MHC Class II positive. We assessed 139 lung cancer samples available in the Hirsch Lab for MHC Class II. There was no positive MHC Class II staining on SCLC tumor cells. MHC Class II expression on TILs in SCLC was significantly lower than that on TILs in NSCLC (P＜0.001). MHC Class II was also assessed in an additional 139 NSCLC tumor tissues from Medical University of Gdansk, Poland. Patients with positive staining of MHC Class II on TILs had longer regression-free survival (RFS) and overall survival (OS) than those whose TILs were MHC Class II negative (2.980 years, 95% CI 1.628–4.332 vs. 1.050 years, 95% CI 0.556–1.554, P=0.028) (3.230 years, 95% CI 2.617–3.843 vs. 1.390 years, 95% CI 0.629–2.151, P=0.014). ConclusionsMHC Class II was expressed both in NSCLC cell lines and tissues. However, MHC Class II was not detected in SCLC cell lines or tissue tumor cells. MHC Class II expression was lower on SCLC TILs than on NSCLC TILs. Loss of expression of MHC Class II on SCLC tumor cells and reduced expression on SCLC TILs may be a means of escaping anti-cancer immunity. Higher MHC Class II expression on TILs was correlated with better prognosis in patients with NSCLC.

Visualization and quantitation of epidermal growth factor receptor homodimerization and activation with a proximity ligation assay.

ObjectivesActivation of the epidermal growth factor receptor (EGFR) results from receptor homodimerization and autophosphorylation and confers sensitivity to tyrosine kinase inhibitors in some tumors. However, the visual detection and quantitation of activated EGFR in the clinical setting has not been established.Materials and MethodsA proximity ligation assay (PLA) was applied to detect EGFR homodimers in non–small cell lung cancer (NSCLC) cell lines and tissue specimens.ResultsPLA signals corresponding to EGFR homodimers were higher in NSCLC cell lines and tissue specimens positive for activating EGFR mutations than in those wild type (WT) for EGFR. Stimulation with EGF in NSCLC cells WT for EGFR or forced overexpression of EGFR in Ba/F3 cells resulted in marked EGFR homodimerization. The extent of EGFR homodimerization appeared related to that of EGFR autophosphorylation in NSCLC cells WT for EGFR.ConclusionPLA may provide a new tool for detection and quantitation of EGFR homodimers in NSCLC and other tumors.

Downregulation of USP32 inhibits cell proliferation, migration and invasion in human small cell lung cancer.

Ubiquitin specific protease 32 (USP32) is a highly conserved but uncharacterized gene, which has been reported to be associated with growth of breast cancer cells. However, the role of USP32 in human small cell lung cancer (SCLC) has not been uncovered. The aim of this study was to investigate and evaluate the clinical significance of USP32 in patients with SCLC. Expression of USP32 was firstly investigated using public online data sets and then determined in SCLC tissues and cell lines using quantitative real-time PCR, Western blotting and immunohistochemical staining. SCLC cells were transfected with a small-interfering RNA targeting USP32 mRNA and analysed for cell viability, proliferation ability, cell cycle distribution, apoptosis and invasion. USP32 was found to be overexpressed in SCLC tissues compared with normal tissues. High USP32 expression was significantly correlated with disease stage and invasion. In vitro experiments demonstrated that silencing of USP32 caused a significant decrease in the proliferation and migration rate of cells. Furthermore, USP32 silencing arrested cell cycle progression at G0/G1 phase via decreasing CDK4/Cyclin D1 complex and elevating p21. In addition, downregulation of USP32 significantly induced cell apoptosis by activating cleaved caspase-3 and cleaved PARP, as well as inhibiting cell invasiveness via altering epithelial mesenchymal transition expression. Our results suggest for the first time that USP32 is important for SCLC progression and might be a potential target for molecular therapy of SCLC.

Small cell lung cancer: The immune microenvironment and prognostic impact of checkpoint expression.

8569 Background: To date, immunotherapy has had limited success in small cell lung cancer (SCLC), despite the tumor’s high mutation load. Little is understood of the immune tumor microenvironment in SCLC due to a paucity of resected tumor. We present a SCLC cohort and describe the prognostic impact of checkpoint expression. Methods: SCLC tissue microarrays with triplicate cores from 105 SCLC specimens underwent IHC assessment for PD-L1, PD-L2, LAG3, TIM3, FoxP3, CD4 and CD8 on tumor and/or tumor infiltrating lymphocytes (TILs). Checkpoint positivity was defined &gt; 5% tumor expression or TIL expression in &gt; 5% of the total core area. Associations with clinicopathologic characteristics and survival were assessed. A Cox model was used for univariate and multivariate survival analysis. Results: Tumor expression of PD-L1 was positive (+) in 17/95 (18%), PD-L2+ in 2/96 (2%), and TIM3+ or LAG3+ in no cases. TILs expressed PD-L1+ in 64/95 (67%), PD-L2+ in 22/96 (22%), TIM3+ in 57/96 (59%) and LAG3+ in 43/96 (45%). FoxP3+ lymphocytes were found in all samples (range 0.02 – 2.98% of total core). TIL expression of PD-L1, PD-L2, TIM3 and LAG3 were all significantly correlated (p value ≤0.001 for all comparisons), and were associated with high FoxP3+ expression. All four checkpoints were expressed on TILs in 20/105 (19%) patients. PD-L1+ and PD-L2+, but not TIM3 or LAG3, on TILs were significantly higher in limited stage compared with extensive stage SCLC (76% v 52%, p 0.045 and 28% v 7%, p 0.02 respectively). There was no association between stage and tumor expression. TIL expression of PD-L1, PD-L2, TIM3 and LAG3 were all associated with improved prognosis. PD-L1+ median OS: 17.2 v 7.9 months (HR 0.36; 95%CI 0.22 – 0.6; p &lt; 0.001). Univariate analysis showed stage and TIL expression of PD-L1, PD-L2, TIM3 and LAG3 were associated with improved survival, but only stage and PD-L1+ TILs remained significant on multivariate analysis (p &lt; 0.01). Conclusions: Immune checkpoint molecules are frequently expressed in SCLC-associated TILs, but not the tumor itself. TIL expression of checkpoint molecules is associated with improved survival. Limited tumor expression of PD-L1 and an exhausted immune cell phenotype may contribute to immunotherapy failure.

Role of TGF-β1 in multi-drug resistance in small cell lung cancer and its clinical significance

To investigate the role of transforming growth factor β1 (TGF-β1) in multi-drug resistance in small cell lung cancer and its clinical significance. Methods: The mRNA and protein expressions of TGF-β1 in H69 and H69AR cells were detected by real-time PCR and Western blot, respectively. After silence of TGF-β1, the sensitivity of H69AR to drugs was detected by CCK8 assay. The expressions of TGF-β1 in lung cancer and paracarcinoma tissues were examined by QRT-PCR and immunohistochemistry. The relationship of TGF-β1 expression with clinical pathological features and prognosis of patients was studied. Results: Compared to H69, the mRNA and protein expressions of TGF-β1 in H69AR cells were significantly increased by (5.93±0.47) and (8.49±1.92) folds, respectively (P<0.01). Transfection of TGF-β1 siRNA resulted in a decrease of TGF-β1 expression by 70.432% in H69AR cells (F=21.20, P<0.01) and an increase insensitivity to chemotherapeutic agents of H69AR cells (t=4.576, P<0.05). Compare with the paracarcinoma tissues, the expression of TGF-β1 was significantly increased in small cell lung cancer tissues (t=13.925, P<0.01), which was closely related with clinical stage, chemosensitivity and overall survival (all P<0.05), but not related with gender, age (both P>0.05). Conclusion: TGF-β1 is involved in the regulation of small cell lung cancer multidrug resistance, which may be a potential marker to evaluate the chemosensitivity and clinical prognostic for small cell lung cancer.

Increased HAGLR expression promotes non-small cell lung cancer proliferation and invasion via enhanced de novo lipogenesis.

Lung cancers are broadly classified into small cell lung cancer and non-small cell lung cancer, with non-small cell lung cancer one of the leading causes of cancer-associated deaths worldwide. Presently, the mechanisms underlying lung tumorigenesis remain incompletely understood. Accumulating evidence indicates that abnormal expression of long non-coding RNAs is associated with tumorigenesis in multiple cancers, including lung cancer. HAGLR messenger RNA of non-small cell lung cancer tissues was significantly higher. Moreover, high levels of HAGLR expression were associated with non-small cell lung cancer tumor lymph node metastasis status, stage, and poor overall survival. Inhibition of HAGLR in non-small cell lung cancer cells suppressed cell proliferation and invasion. RNA interference-mediated downregulation of HAGLR also decreased levels of fatty acid synthase, with fatty acid synthase levels positively correlated with HAGLR expression in non-small cell lung cancer specimens. In addition, the cellular free fatty acid content of cancer cells was decreased following HAGLR knockdown. HAGLR depletion significantly inhibited the growth of non-small cell lung cancer cells in vivo. Furthermore, the expression levels of p21 and matrix metallopeptidase-9 (MMP-9) were dysregulated when HAGLR expression was suppressed. Our results suggest that HAGLR is an important regulator of non-small cell lung cancer cell proliferation and invasion, perhaps by regulating fatty acid synthase. Therefore, targeting HAGLR may be a possible therapeutic strategy for non-small cell lung cancer.