Adjacent Normal Lung Tissues Research Articles

Decreased expression of long non-coding RNA LINC00261 is a prognostic marker for patients with non-small cell lung cancer: a preliminary study.

The previous study found that long non-coding RNA LINC00261 (LINC00261) was significantly down-regulated in non-small cell lung cancer (NSCLC). However, the function of LINC00261 in the progression of NSCLC has not been reported. The present work aimed to explore the prognostic value of LINC00261 in patients with NSCLC. The expression of LINC00261 was determined in NSCLC tissues and matched normal lung tissues by quantitative Real-time PCR (qRT-PCR). Furthermore, we evaluated the relationship of LINC00261 and clinicopathological features with survival of patients with NSCLC. Finally, univariate and multivariate Cox regression analyses were used to explore whether LINC00261 was an independent predictor of survival. We found that the LINC00261 expression level in NSCLC tissues was suppressed compared with that in adjacent normal lung tissues (p < 0.01). Low expression of LINC00261 was found to significantly correlate with TNM stage (p = 0.005), lymph node status (p = 0.020), and distant metastasis (p = 0.004). Then, Kaplan-Meier analysis demonstrated that low LINC00261 expression level was associated with poorer overall survival (p = 0.0013). Furthermore, multivariate analysis showed that low expression of LINC00261 was an independent adverse prognostic factor of NSCLC (p = 0.004). We firstly provided evidence that LINC00261 expression was associated with poor prognosis of NSCLC patients and may serve as an independent prognostic indicator.

High expression of CIP2A protein is associated with tumor aggressiveness in stage I-III NSCLC and correlates with poor prognosis.

The aim of this work was to examine the expression of cancerous inhibitor of protein phosphatase 2A (CIP2A) in non-small cell lung cancer (NSCLC) and analyze its correlation with clinical outcomes. CIP2A protein levels were detected by immunohistochemistry (IHC). One hundred and eighty-four of 209 (88.3%) primary stage I–III NSCLC specimens and 4 of 38 (10.5%) adjacent normal lung tissue specimens expressed CIP2A protein. High expression of CIP2A was detected in 38.8% (81/209) of the NSCLC specimens. Patients diagnosed histologically with late-stage NSCLC (p<0.001) and malignant nodes (p=0.001) exhibited high CIP2A expression. Univariate analysis using the log-rank test identified CIP2A expression as a prognostic predictor for overall survival (p=0.005). In multivariate analyses using the Cox regression test, CIP2A expression, T stage, N stage, histological type, and chemotherapy were identified as independent prognostic factors (p=0.007, 0.001, 0.003, <0.001, and <0.001, respectively). Furthermore, Kaplan–Meier survival curves demonstrated that high CIP2A expression indicated poor prognosis in the subgroup of patients with squamous cell carcinoma (p=0.008). Similar results were noted in the subgroup of patients with adenocarcinoma, but the results did not reach statistical significance (p=0.084). We also used univariate analysis and multivariate analysis to assess the prognostic factors for overall survival in the subgroup of patients who received postoperative chemotherapy. CIP2A expression was also an independent prognostic factor in NSCLC patients who received postoperative chemotherapy (p=0.009), along with histological type (p=0.001) and N stage (p=0.034). In conclusion, adding to the accumulating evidence, our research suggested that the CIP2A expression is associated with aggressiveness and correlates with poor prognosis in NSCLC. Our findings also indicated that CIP2A might be a potential therapeutic target against NSCLC.

MiR-9-5p promotes cell growth and metastasis in non-small cell lung cancer through the repression of TGFBR2

BackgroundIncreasing evidence indicates that the dysregulation of microRNAs (miRNAs) play critical roles tumor progression and metastasis, but very few papers had reported the function of miR-9-5p in lung cancer, especially in NSCLCs. MethodsIn this study, we investigated the role of miR-9-5p in non-small cell lung cancers (NSCLCs). MiR-9-5p level were analyzed in 62 clinical NSCLC lung tissue samples and adjacent normal lung tissues by RT-PCR. The target of miR-9-5p was predicted by TargetScan and luciferase reporter assay was used to verify the binding site of miR-9-5p on TGFBR2 mRNA. MTT assay, wound healing assay and invasion assay were performed in both miR-5p inhibitor transfected A549 and miR-5p mimic transfected SK-MES-1 cells. To further investigate whether TGFBR2 is the major target of miR-9-5p, we used TGFBR2 siRNA to transfect A549 and SK-MES-1 cells with miR-9-5p inhibitor or miR-9-5p mimic transfection. Western blot were then used to analyze TGFBR2, p-smad2 and p-smad3 protein expressions after transfection. ResultsResults indicated that NSCLC patients’ tissues had a significantly higher expression of miR-9-5p compared to adjacent normal lung tissues. MiR-9-5p mimic transfection promoted proliferation, metastasis and invasion abilities in both A549 and SK-MES-1 cells. Conversely, miR-9-5p inhibitor transfection showed the decreased abilities of these cells. Luciferase reporter assay indicated that TGFBR2 is a direct target of miR-9-5p and the up-regulation of TGFBR2 suppressed cell proliferation, metastasis and invasion. The knock down of TGFBR2 abrogated the effect of miR-9-5p in down-regulating p-smad2 and p-smad3 expressions, which indicated that TGFBR2 is the major target of miR-9-5p in NSCLC cells. ConclusionsOur finding indicated that miR-9-5p promotes the proliferation, metastasis and invasion of NSCLC cells by down-regulating TGFBR2 expression.

MARCH8 is associated with poor prognosis in non-small cell lung cancers patients.

MARCH8 belongs to a family of membrane-associated RING-CH (MARCH) ubiquitin ligases. The functions of MARCH8 have been thoroughly investigated but its mechanism of action remains unknown. In this study, we detected the expression of MARCH8 protein in NSCLC samples and identified MARCH8 mRNA expression through a TCGA database. In addition, we analyzed the correlation between MARCH8 and the clinical characteristics of NSCLC patients and their prognosis.(www.kmplot.com). The roles of MARCH8 in proliferation, migration, and metastasis were further explored through ectopic expression analysis and western blot analysis; its mechanism of expressionwas also explored. We discovered that MARCH8 was downregulated in NSCLC tissues compared to adjacent normal lung tissues. Overexpression of MARCH8 inhibited NSCLC cell proliferation and metastasis via the PI3K and mTOR signaling pathways; this also increased apoptosis of A549 and H1299 cells. Our results indicated that MARCH8 plays crucial roles in NSCLC against carcinogenesis and progression; therefore, MARCH8 might be a predictive factor and an attractive therapeutic target for NSCLC patients.

Expression of CXC chemokine ligand 12 and CXC chemokine receptor 4 in non-small cell lung cancer tissues and clinical implication

Objective By detecting the expression of CXC chemokine ligand 12 (CXCL12) and CXC chemokine receptor 4 (CXCR4) in non-small cell lung cancer (NSCLC) tissues and adjacent normal lung tissues, we try to investigate the correlation between the expression of CXCL12/CXCR4 and clinical parameters. Methods Immunohistochemical staining method was used to detect the expression of CXCL12 and CXCR4 in cancer tissues and matched normal lung tissues adjacent to carcinoma of 62 cases of NSCLC. Under the microscope at high magnification, positive staining (+ ) was recorded when the number of positive cells accounted for more than or equal to 10%, and negative staining (-) was recorded when the number of positive cells accounted for less than 10%. The positive expression rate and the relationship between the various clinical parameters were analyzed. Results The positive expression rate of CXCL12 in NSCLC tissues and normal lung tissue adjacent to carcinoma was 73.8% and 15.0% respectively (P=0.040). The positive expression of CXCR4 was detected in 33 cases and 2 cases of NSCLC tissues and normal lung tissue adjacent to carcinoma respectively (P=0.030). The positive expression rate of CXCL12 in stage Ⅲa-Ⅲb was 90.9%, and 55.5% in stage Ⅱa-Ⅱb. The positive expression rate of CXCR4 in Ⅲa-Ⅲb was 90.9%, and 55.5% in stage Ⅱa-Ⅱb (P=0.030). The positive expression rate of CXCL12 in mediastinal lymph node with metastasis was 94.7%, and that in the mediastinal lymph node without metastasis was 56.5%. The expression of CXCL12 and CXCR4 in NSCLC tissues was consistent. Conclusion CXCL12 and CXCR4 expression in NSCLC tissues has the effect to promote cancer. The positive expression of CXCL12 and CXCR4 is associated with TNM staging and mediastinal lymph node metastasis. The specific binding of CXCL12 and CXCR4 may promote lymph node metastasis. Key words: CXC chemokine ligand 12; CXC chemokine receptor 4; Non-small-cell lung carcinoma; Immunohistochemisty

KLF15 promotes the proliferation and metastasis of lung adenocarcinoma cells and has potential as a cancer prognostic marker.

Lung adenocarcinoma (LADC)is a general form of non-small cell lung cancer that represents a significant threat to public health worldwide. The 5-year-survival rate for LADC is currently below 15%. The transcription factor KLF15, also called kidney-enriched KLF (KKLF), has been proven to play a role in inhibiting proliferation and diversification of carcinoma cells, including those of the endometrium, pancreas and breast, but the involvement of KLF15 in LADC has not previously been studied. In this study, we compared the in vitro expression of KLF15 in human LADC tissues and adjacent normal lung tissues. Expression of KLF15 was found to be abnormally high in LADC tissues and cells compared with adjacent non-tumorous tissues, and was correlated with tumor TNM stage and tumor differentiation (P = 0.003, P = 0.001, respectively). The effect of KLF15 on cell growth and migration were explored in vitro by Western Blotting, CCK8 and colony formation assays, flow cytometry analysis and transwell migration assays, and in vivo by analysis of tumorigenesis in 5-week old BALB/c nude mice. Knockdown of KLF15 significantly upregulated the protein levels of cleaved caspase-3, caspase-7, caspase-8 and PARP, thereby inducing apoptosis. Downregulation of KLF15 in A549 and NCI-H1650 cell lines resulted in these cell lines exhibiting markedly slower growth rates when injected subcutaneously into the flank of nude mice, compared with the comparator control groups (P < 0.05). Collectively, our findings suggest that KLF15 may have a significant effect on LADC cell survival, and that it represents a potential therapeutic and preventive biomarker for LADC prognosis and treatment.

Genetic variation at the microRNA binding site of CAV1 gene is associated with lung cancer susceptibility.

Single nucleotide polymorphism (SNP) may influence the genesis and development of cancer in a variety of ways depending on their location. Here we conducted a study in Chinese female non-smokers to investigate the relationship between rs1049337, rs926198 and the risk or survival of lung cancer. Further, we explored whether rs1049337 could alter the binding affinity between the mRNA of CAV1 and the corresponding microRNAs. Finally, we evaluated the relationship between expression level of CAV1 and prognosis of lung cancer. The results showed that the rs1049337-C allele and rs926198-C allele were the protective alleles of lung cancer risk. Haplotype analysis indicated that the C-C haplotype (constructed by rs1049337 and rs926198) was a protective haplotype for lung cancer risk. The result of luciferase reporter assay showed that rs1049337 can affect the binding affinity of CAV1 mRNA to the corresponding microRNAs both in A549 cell line and H1299 cell line. Compared with C allele, T allele had a relatively decreased luciferase activity. Compared with paired normal adjacent tissue or normal lung tissue, lung cancer tissue showed a relatively low level of CAV1. Refer to those patients at early stage of lung cancer, the expression level of CAV1 in patients at late stage of lung cancer was relatively low. In conclusion, the results indicated that rs1049337, it's a SNP located at 3′UTR region of CAV1 may affect lung cancer risk by altering the binding affinity between the mRNA of CAV1 and the corresponding microRNAs.

PKCζ, MMP‑2 and MMP‑9 expression in lung adenocarcinoma and association with a metastatic phenotype.

The aim of the present study was to investigate protein kinase C ζ type (PKCζ), matrix metalloproteinase (MMP)‑2 and MMP‑9 expression in lung adenocarcinoma and to define their association with invitro invasion and metastatic capacity. PKCζ, MMP‑2 and MMP‑9 expression was assessed by immunohistochemistry in 110cases of lung adenocarcinoma. PKCζ small interfering (si)RNA was transfected into A549 cells, and western blotting was used to confirm PKCζ‑knockdown in transfected cells and to measure MMP‑2 and MMP‑9 levels. A Transwell invasion assay was used to detect invitro invasive capacity. The rates of positive PKCζ, MMP‑2 and MMP‑9 staining in lung adenocarcinoma tissues were 52.73, 55.45 and 61.82%, respectively. PKCζ expression was increased in malignant tissues compared with adjacent normal lung tissues and was associated with lymph node metastasis (P<0.05), although it was not associated with any other clinicopathological parameters, including sex, age, tumor size, smoking status or distant metastases (all P>0.05). PKCζ, MMP‑2 and MMP‑9 expression was markedly decreased in siPKCζ‑treated A549 cells, which exhibited a significantly decreased invasive capacity in the Transwell invasion assay (P<0.05). In conclusion, PKCζ promoted lung adenocarcinoma invasion and metastasis, and its expression was associated with MMP‑2 and MMP‑9 expression. PKCζ may be a potential target for gene therapy in lung adenocarcinoma.

The mechanisms for lung cancer risk of PM2.5 : Induction of epithelial-mesenchymal transition and cancer stem cell properties in human non-small cell lung cancer cells.

Fine particulate matter (PM2.5 ) is a major component of air pollutions that are closely associated with increased risk of lung cancer. However, the role of PM2.5 in the etiology of lung cancer is largely unknown. In this study, we performed acute (24 hours) and chronic (five passages) exposure models to investigate the carcinogenetic mechanisms of PM2.5 by targeting the induction of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) properties in human non-small cell lung cancer cell line A549. We found that both acute and chronic PM2.5 exposure enhanced cell migration and invasion, decreased mRNA expression of epithelial markers and increased mRNA expression of mesenchymal markers. Chronic PM2.5 exposure further induced notable EMT morphology and CSC properties, indicating the developing process of cell malignant behaviors from acute to chronic PM2.5 exposure. CSC properties induced by chronic PM2.5 exposure characterized with increased cell-surface markers (CD44, ABCG2), self-renewal genes (SOX2 and OCT4), side population cells and neoplastic capacity. Furthermore, the levels of three stemness-associated microRNAs, Let-7a, miR-16 and miR-34a, were found to be significantly downregulated by chronic PM2.5 exposure, with microarray data analysis from TCGA database showing their lower expression in human lung adenocarcinoma tissues than that in the adjacent normal lung tissues. These data revealed that the induction of EMT and CSC properties were involved in the lung cancer risk of PM2.5 , and implicated CSC properties and related microRNAs as possible biomarkers for carcinogenicity prediction of PM2.5 .

MicroRNA-125a-3p downregulation correlates with tumorigenesis and poor prognosis in patients with non-small cell lung cancer

MicroRNA (miR)-125a-3p is derived from the 3′-end of pre-miR-125a, which is associated with several types of cancer, such as gastric and prostate cancer, and glioma. The aim of the present study was to identify the prognostic significance of miR-125a-3p expression levels in patients with NSCLC. The gene expression omnibus database was used to analyze miR-125a-3p expression in NSCLC in silico, and 148 NSCLC samples and 30 adjacent normal lung tissue specimens were analyzed for the expression of miR-125a-3p by qPCR. The results showed that the expression levels of miR-125a-3p in the adjacent normal tissues was higher than the expression level in the NSCLC tissues. There were several clinical parameters demonstrated to be associated with miR-125a-3p expression, such as lymph node metastasis, tumor node metastasis classification of malignant tumor stage and tumor diameter. Furthermore, high expression levels of miR-125a-3p with chemotherapy prolonged the overall survival rate and disease free survival rate compared with untreated patients with low expression of miR-125a-3p. Thus, miR-125a-3p is a significant prognostic biomarker for patients with NSCLC, from which a novel therapeutic strategy to combat NSCLC may be derived.

Lamin B2 binding to minichromosome maintenance complex component 7 promotes non-small cell lung carcinogenesis.

We investigated the role of lamin B2 in non-small cell lung cancer (NSCLC). We detected higher lamin B2 expression in 20 NSCLC tumor tissues obtained from The Cancer Genome Atlas than in adjacent normal lung tissues. LMNB2-RNAi knockdown in A549 and H1299 NSCLC cells inhibited colony formation, cell proliferation and G1-S cell cycle progression while increasing apoptosis. LMNB2 overexpression had the opposite effects. Tumor xenograft experiments showed diminished tumor growth with LMNB2 knockdown H1299 cells than with controls. Yeast two-hybrid studies revealed minichromosome maintenance complex component 7 (MCM7) to be a binding partner of lamin B2, which was confirmed by co-immunoprecipitation and co-localization studies. Lamin B2 binding enhanced DNA binding and helicase activities of MCM7. Deletion analysis with MCM7-N, MCM7-M or MCM7-C mutant proteins showed that lamin B2 binds to the C-terminus of MCM7, and competes with the binding of the tumor suppressor retinoblastoma (RB) protein. Immunohistochemical analysis of 150 NSCLC patient samples revealed that both lamin B2 and MCM7 levels positively correlated with histological grade and tumor TNM stage. Moreover, high lamin B2 and MCM7 levels correlated with shorter overall survival of NSCLC patients. In sum, these results show that lamin B2 interaction with MCM7 promotes NSCLC progression.

The clinical and prognostic value of polo-like kinase 1 in lung squamous cell carcinoma patients: immunohistochemical analysis.

Polo-like kinase 1 (PLK1) has been suggested to serve as an oncogene in most human cancers. The aim of our study is to present more evidence about the clinical and prognostic value of PLK1 in lung squamous cell carcinoma patients. The status of PLK1 was observed in lung adenocarcinoma, lung squamous cell carcinoma, and normal lung tissues through analyzing microarray dataset (GEO accession numbers: GSE1213 and GSE 3627). PLK1 mRNA and protein expressions were detected in lung squamous cell carcinoma and normal lung tissues by using quantitative real-time PCR (qRT-PCR) and immunohistochemistry. In our results, the levels of PLK1 in lung squamous cell carcinoma tissues were higher than that in lung adenocarcinoma tissues. Compared with paired adjacent normal lung tissues, the PLK1 expression was increased in lung squamous cell carcinoma tissues. Furthermore, high expression of PLK1 protein was correlated with differentiated degree, clinical stage, tumor size, lymph node metastasis, and distant metastasis. The univariate and multivariate analyses showed PLK1 protein high expression was an unfavorable prognostic biomarker for lung squamous cell carcinoma patients. In conclusion, high expression of PLK1 is associated with the aggressive progression and poor prognosis in lung squamous cell carcinoma patients.

TMEM17 depresses invasion and metastasis in lung cancer cells via ERK signaling pathway.

Transmembrane protein 17(TMEM17) is a newly identified protein, its expression pattern and clinicopathological relevance is still unclear. In this study, western blot assay was performed in 20 paired lung cancer samples and found that TMEM17 protein levels were lower in lung cancer tissues than that in the corresponding normal lung tissues (p=0.010). Immunohistochemistry staining in 143 cases lung cancer specimens also showed that TMEM17 expression in lung cancer tissues were significantly lower than adjacent normal lung tissues (35.7% vs 63.2%, p<0.001). And negative TMEM17 expression was significantly associated with poor histological differentiation (p=0.027), advanced TNM stages (p=0.006), positive lymph node metastasis (p=0.002) and poor prognosis (p=0.002). After overexpressing TMEM17, levels of p-ERK and its downstream molecules, p-P90RSK and Snail, were down-regulated, while levels of Occludin and Zo-1 were up-regulated, which result in the inhibition of invasion and migration ability of lung cancer cells. The effects were reversed by the incorporation of specific ERK inhibitor PD98059. In conclusion, loss of TMEM17 correlates with the development of non-small cell lung cancer (NSCLC) and predicts adverse clinical outcome of NSCLC patients. The effect of TMEM17 on inhibiting invasion and migration may attribute to restoring Occludin and Zo-1 expression through inactivating ERK-P90RSK-Snail pathway.

MicroRNA-383-5p acts as a prognostic marker and inhibitor of cell proliferation in lung adenocarcinoma by cancerous inhibitor of protein phosphatase 2A.

Lung cancer is the leading cause of cancer-associated mortality worldwide. MicroRNAs (miRNAs/miRs) serve a role in the occurrence and development of lung cancer. The aim of the present study was to analyze the expression and function of the proliferation-associated miR-383-5p in lung adenocarcinoma (LAC). Samples of human LAC and matched adjacent normal lung tissues were surgically removed, and miR-383-5p expression and the pathological characteristics of lung adenocarcinoma were investigated. The present study revealed that miR-383-5p expression level was significantly decreased in LAC tissues and its expression levels were markedly associated with tumor size and differentiation. Overexpression of miR-383-5p in A549 and H1299 LAC cell lines inhibited cell proliferation by G1 cell cycle phase arrest and induction of apoptosis. Cancerous inhibitor of protein phosphatase 2A (CIP2A), a potential target gene of miR-383-5p, was inversely associated with miR-383-5p expression level in LAC tissues and cell lines. Furthermore, the results of the present study demonstrated that CIP2A was directly regulated by miR-383-5p and the restoration of CIP2A expression reversed the inhibitory effects of miR-383-5p on LAC cell proliferation. In conclusion, the results of the present study demonstrated that miR-383-5p was downregulated in LAC tissues. By targeting CIP2A, miR-383-5p exerts its anti-proliferative function in LAC, suggesting its use a potential novel potential prognostic biomarker and therapeutic target for LAC.

Abstract 3494: Dissecting the cellular expression and subcellular localization of lncRNAs implicated as prognostic biomarkers in non-small cell lung carcinomas by RNA ISH

Abstract Background: Long non-coding RNAs (lncRNAs) are involved in many biological processes, including epigenetic modification, cell differentiation, and apoptosis. More recently, lncRNAs have emerged as unique biomarkers that may be associated with a physiological or diseased state, and several lncRNAs have been identified as prognostic markers in a wide variety of human cancers. Because lncRNAs do not translate into protein, their discovery in the tumor is entirely dependent upon RNA detection. The majority of lncRNAs are also expressed at very low levels compared to mRNAs and may be more heterogeneously expressed than mRNAs. Therefore, detection of lncRNAs in tumor biopsies requires a highly sensitive and specific detection method that can discern single-cell and subcellular localization of lncRNA expression. Methods: Single-molecule RNA in situ hybridization (ISH) is a well-suited method for the detection of lncRNAs in tumor biopsies because it allows for the visualization of single RNA molecules with morphological context. To interrogate the expression pattern of lncRNAs within the tumor and its microenvironment, we performed RNA ISH using the RNAscope assay on FFPE tissue microarrays consisting of archived tissue samples of 53 primary non-small cell lung carcinoma (NSCLC) tumors and 4 adjacent normal lung tissues. We examined the expression of 4 lncRNAs which have been implicated as potential prognostic markers of lung cancer: AFAP-AS1, ANRIL, HOTAIR, and UCA1. Results: These lncRNAs were detected in approximately 40-60% of the 53 NSCLC tumor cores and in none of the 4 adjacent normal lung tissues. Expression of these 4 lncRNAs was observed predominantly in tumor cells, with little to no detection in the stroma. Some lncRNAs displayed a heterogeneous expression pattern, with some tumor cell foci displaying strong lncRNA signal while other foci in the same tumor did not display signal. Furthermore, some lncRNAs displayed heterogeneous cell expression in the same foci, with some cells expressing very high levels of lncRNA while other cells in the same foci had little to no lncRNA expression. An evaluation of each lncRNA in serial sections revealed that 6 of the 53 NSCLC tumors expressed all 4 lncRNAs in the same tumor foci. Lastly, little to no signal was observed for the prostate cancer-specific lncRNA PCA3 in the NSCLC tumors and no PCA3 expression was detected in adjacent normal lung tissues. Conclusions: These results demonstrate the ability of the RNAscope ISH assay to detect potential lncRNA biomarkers in lung cancer samples in situ, allowing for the identification of expression heterogeneity within the tissue environment and precise tumor expression patterns of lncRNAs. Identification of the subcellular localization and cell-to-cell expression patterns of lncRNAs can facilitate greater understanding about their specific biological roles in cancer and other diseases. Citation Format: Courtney M. Anderson, Na Li, Xiao-Jun Ma, Emily Park. Dissecting the cellular expression and subcellular localization of lncRNAs implicated as prognostic biomarkers in non-small cell lung carcinomas by RNA ISH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3494. doi:10.1158/1538-7445.AM2017-3494

Protein regulator of cytokinesis-1 expression: prognostic value in lung squamous cell carcinoma patients.

Protein regulator of cytokinesis-1 (PRC1) has been shown to participate in the completion of cytokinesis, and it is dysregulated in cancer processes. However, its relevance in lung squamous cell carcinoma (SCC) remained largely unknown. We aimed to study the expression pattern of PRC1 and assess its clinical significance in lung SCC. PRC1 protein expression in human lung SCC and adjacent normal lung tissues was detected by immunohistochemistry. PRC1 expression was assessed in association with clinicopathological features and clinical outcomes of lung SCC patients. In lung SCC tissues, PRC1 protein expression was significantly higher than those in paired normal lung tissues. The lung SCC patients with PRC1 overexpression had an advanced pathological stage (TNM stage), positive lymph node metastasis, and a shorter overall survival (OS) time more frequently than patients with low PRC1 expression. Additional, PRC1 expression was also shown to be poor as a prognostic factor for OS in patients with lung SCC. Our study indicated that aberrant expression of PRC1 may point to biochemical recurrence in lung SCC. This highlights its potential as a valuable prognostic marker for lung SCC.

Abstract 4451: Elevated USP22 is a potential therapeutic target for human non-small cell lung cancer

Abstract Elevated ubiquitin-specific protease 22 (USP22) is associated with enhanced malignancy, therapeutic resistance, and poor prognosis of various human cancers. In this study, the clinicopathological significance of USP22, therapeutic implication of targeting USP22, and underlying molecules of USP22 knockdown were investigated in human non-small cell lung cancer (NSCLC). Immunohistochemistry analysis revealed that USP22 protein was dramatically elevated in ~ 67% of 197 NSCLC tissues compared with adjacent normal lung tissues. Importantly, statistical analysis demonstrated that elevated USP22 was positively associated with advanced cancer stage and a trend to poor prognosis of NSCLC patients. Moreover, siRNA-mediated knockdown of USP22 induced a cell cycle arrest in G1/S phase and significantly suppressed cell proliferation and soft agar colony formations in NSCLC cell lines. In addition, knockdown of USP22 significantly enhanced cisplatin-induced genotoxic effects in NSCLC cell lines. Furthermore, USP22 silencing substantially down-regulated SIRT1 and upregulated p53 tumor suppressor in NSCLC cells, indicating downregulation of USP22 may boost p53-mediated tumor suppression and DNA damage response in NSCLC cells. Knockdown of USP22 also increased global histone H2B monoubiquitylation, H3K4 and H3K79 trimethylation leading to global gene expression profile changes in these NSCLC cells. These results suggest that targeting USP22 will have broad anti-cancer effects via altering multiple cancer signaling pathways. Taken together, data of this study further indicate that the elevated USP22 may potentially represent a prognostic factor and therapeutic target for NSCLC patients. Note: This abstract was not presented at the meeting. Citation Format: Keqiang Zhang, Tommy R Tong, Xinwei Yun, Rebecca Nelson, Nardi Isaac, Ravi Salgia, Dan Raz. Elevated USP22 is a potential therapeutic target for human non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4451. doi:10.1158/1538-7445.AM2017-4451

Coal Mine Dust Desquamative Chronic Interstitial Pneumonia: A Precursor of Dust-Related Diffuse Fibrosis and of Emphysema.

Background:Diseases associated with coal mine dust continue to affect coal miners. Elucidation of initial pathological changes as a precursor of coal dust-related diffuse fibrosis and emphysema, may have a role in treatment and prevention. Objective:To identify the precursor of dust-related diffuse fibrosis and emphysema. Methods:Birefringent silica/silicate particles were counted by standard microscope under polarized light in the alveolar macrophages and fibrous tissue in 25 consecutive autopsy cases of complicated coal worker's pneumoconiosis and in 21 patients with tobacco-related respiratory bronchiolitis. Results:Coal miners had 331 birefringent particles/high power field while smokers had 4 (p<0.001). Every coal miner had intra-alveolar macrophages with silica/silicate particles and interstitial fibrosis ranging from minimal to extreme. All coal miners, including those who never smoked, had emphysema. Fibrotic septa of centrilobular emphysema contained numerous silica/silicate particles while only a few were present in adjacent normal lung tissue. In coal miners who smoked, tobacco-associated interstitial fibrosis was replaced by fibrosis caused by silica/silicate particles. Conclusion:The presence of silica/silicate particles and anthracotic pigment-laden macrophages inside the alveoli with various degrees of interstitial fibrosis indicated a new disease: coal mine dust desquamative chronic interstitial pneumonia, a precursor of both dust-related diffuse fibrosis and emphysema. In studied coal miners, fibrosis caused by smoking is insignificant in comparison with fibrosis caused by silica/silicate particles. Counting birefringent particles in the macrophages from bronchioalveolar lavage may help detect coal mine dust desquamative chronic interstitial pneumonia, and may initiate early therapy and preventive measures.

Alex3 suppresses non-small cell lung cancer invasion via AKT/Slug/E-cadherin pathway.

Alex3, is a newly identified mitochondrial protein, regulates mitochondrial dynamics and is involved in neural development. However, its expression pattern and clinicopathological relevance in human tumors are still unclear. In this study, Immunohistochemistry assay was performed in 109 cases of lung cancer samples and found that Alex 3 expression in lung cancer tissues was significantly lower than adjacent normal lung tissues (28.4% vs 52.6%, p < 0.001). Sequent statistical analysis indicated that negative Alex3 expression was significantly associated with advanced tumor-node-metastasis stages (p = 0.001), positive lymph node metastasis (p = 0.005), and poor prognosis (p = 0.008). After overexpression of Alex3, levels of p-AKT and Slug were downregulated, while level of E-cadherin was upregulated, which results in the inhibition of invasion and migration ability of lung cancer cells. In conclusion, reduction of Alex3 correlates with the development of non-small cell lung cancer and predicts adverse clinical outcome of non-small cell lung cancer patients. The effect of Alex3 on inhibiting invasion and migration may attribute to upregulation of E-cadherin expression through AKT-Slug pathway inactivation.

Abstract 4424: Cancer stem cells-related microRNAs have prognostic implications in non-small cell lung cancer

Abstract Background: Lung cancer stem cells (CSCs) are a small population of stem-like cells that remains largely unknown. MicroRNAs (miRNAs) regulation of CSCs gene expression has been reported, constituting a promising therapeutic target. The aim of this study was to isolate and analyze differential expression of a set of miRNAs in tumorspheres from lung cancer cell lines and tumor tissue from resectable non-small cell lung cancer (NSCLC) patients and to determine the prognostic implications of these miRNAs in a cohort of resected-NSCLC patients. Methods: Lung CSC-related miRNAs (miR-145-5p, miR-188-5p, miR-218-5p, miR-34a-5p, miR-21-5p and miR-125a-5p) were analyzed in cells from seven NSCLC tumor samples and six cell lines (H1650, H1993, H480, H358, A549 and PC9) grown in monolayer and as spheroids by RTqPCR using TaqMan® microRNA assays. The expression levels of these six miRNAs were also analyzed in paired fresh-frozen tumor and normal adjacent lung tissue samples (N=178) from resected-NSCLC patients. Statistical analyses were considered significant at p&amp;lt;0.05. Results: miRNA expression analysis of cultured cells revealed an increased expression of miR-125a (p=0.04) and miR-188 (p=0.05) in lung tumorspheres from tumor samples and cell lines compared to their paired-adherent cells. Moreover, lungspheres from patients showed elevated expression levels of miR-21 when compared with their paired monolayer-cell cultures (p= 0.028). Interestingly, miR-125a, miR-188 and miR-21 had prognostic value when were analyzed in patients’ tissue samples. We found that those patients with higher levels (above the median) of miR-188 and miR-21 had a significant reduction in relapse-free survival (RFS, 23.67 vs 66.97 months; p= 0.009 and 24.03 vs 56.83 months, p= 0.042, respectively) and overall survival (OS, 42.9 vs NR months; p= 0.002 and 42.6 vs 82.60 months; p= 0.043, respectively), whereas the group of patients with higher levels of miR-125a had a worse outcome (OS 51.90 vs NR; p= 0.014). A signature combining the expression of miR-188 and miR-21 was able to give more significant prognostic information (RFS, 16.97 vs. 56.83 months; p= 0.006 and OS, 29.90 vs. NR; p&amp;lt;0.0001). The multivariate analysis including clinico-pathological and analytical variables revealed this miRNAs signature as an independent prognostic biomarker for RFS (HR 2.170 [1.372-3.431]; p= 0.001) and OS (HR 3.256 [1.907-5.561]; p&amp;lt;0.0001). Conclusions: Lung tumorspheres had increased levels of the CSC-related miRNAs miR-125a, miR-188 and miR-21, highlighting their role in the CSC biology. The analyses performed in a large cohort of resectable NSCLC patients show that a two miRNAs-signature (miR-188 and miR-21) was an independent prognostic marker for RFS and OS. Supported by grants RD06/0020/1024 and RD12/0036/0025 from RTICC-FEDER, PI12-02838 and PI15-00753 from ISCIII, TRACE (TRA09-0132) and Beca Roche Oncohematología. Note: This abstract was not presented at the meeting. Citation Format: Sandra Gallach Garcia, Alejandro Herreros-Pomares, Silvia Calabuig-Fariñas, Eva Escorihuela, Atilio Navarro, Aminta Isabel Martínez, Alberto Jacobo Cunquero, Eloisa Jantus-Lewintre, Carlos Camps. Cancer stem cells-related microRNAs have prognostic implications in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4424. doi:10.1158/1538-7445.AM2017-4424