Hechun Lin

Integrative Analyses Identify Osteopontin, LAMB3 and ITGB1 as Critical Pro-Metastatic Genes for Lung Cancer

Objective To explore the key regulatory genes associated with lung cancer in order to reduce its occurrence and progress through silencing these key genes. Methods To identify the key regulatory genes involved in lung cancer, we performed a combination of gene array and bioinformatics analyses to compare gene transcription profiles in 3 monoclonal cell strains with high, medium or low metastatic abilities, which were separated from the SPC-A-1sci and SPC-A-1 cell lines by limiting dilution monoclone assay. We then analyzed those genes’ biological activities by knocking down their expression in SPC-A-1sci cells using siRNA and lenti-viral shRNA vectors, followed by determinations of the invasion and migration capabilities of the resulting cell lines in vitro as well as their potential for inducing occurrence and metastasis of lung cancer in vivo. To examine the clinical relevance of these findings, we analyzed the expression levels of the identified genes in human lung cancer tissues (n = 135) and matched adjacent normal tissues by immunohistochemical (IHC) staining. Results Three monoclonal cell strains characterized with high, medium or low metastatic abilities were successfully selected. Gene array and bioinformatics analyses implied that osteopontin, LAMB3 and ITGB1 were key genes involved in lung cancer. Knockdown of these genes suppressed human lung cancer cell invasion and metastasis in vitro and in vivo. Clinical sample analyses indicated that osteopontin, LAMB3 and ITGB1 protein expression levels were higher in lung cancer patients, compared to non-cancerous adjacent tissues, and correlated with lymphatic metastasis. Conclusions We confirmed that osteopontin, LAMB3 and ITGB1 played important roles in the occurrence and metastasis of lung cancer, thus provided important clues to understanding the molecular mechanism of metastasis and contributing to the therapeutic treatment of lung cancer.

MiRNA-10a is upregulated in NSCLC and may promote cancer by targeting PTEN

MicroRNAs (miRNAs) are involved in human cancer including non-small cell lung cancer (NSCLC). In this study, we compared miRNA expression microarray of SPC-A-1sci (high metastatic) and SPC-A-1 (weakly metastatic) cells. We found that miRNA-10a was up-regulated in NSCLC compared with corresponding normal tissues. High expression of miR-10a was associated with tumor node metastasis and lymph node metastasis. Furthermore, overexpression of miR-10a promoted NSCLC cell proliferation, migration and invasion in vitro. We found that PTEN was a direct target of miR-10a in NSCLC. Also miR-10a activated the PTEN/AKT/ERK pathway. We suggest that miR-10a contributes to NSCLC by targeting PTEN.

miRNA-200c inhibits invasion and metastasis of human non-small cell lung cancer by directly targeting ubiquitin specific peptidase 25

BackgroundGrowing evidence indicates that miR-200c is involved in carcinogenesis and tumor progression in non-small-cell lung cancer (NSCLC). However, its precise biological role remains largely elusive.MethodsThe functions of miR-200c and USP25 in migration/invasion and lung metastasis formation were determined by transwell and tail vein injection assays, respectively. The potential regulatory targets of miR-200c were determined by prediction tools, correlation with target protein expression, and luciferase reporter assay. The mRNA expression levels of miR-200c and USP25 were examined in NSCLC cell lines and patient specimens using quantitative reverse transcription-PCR. The protein expression levels of USP25 were examined in NSCLC cell lines and patient specimens using western blot and immunohistochemical staining.ResultsWe demonstrated that over-expression of miR-200c inhibited NSCLC cells migration, invasion, epithelial-mesenchymal transition (EMT) in vitro and lung metastasis formation in vivo. Further studies revealed that USP25 was a downstream target of miR-200c in NSCLC cells as miR-200c bound directly to the 3’-untranslated region of USP25, thus reducing both the messenger RNA and protein levels of USP25. Silencing of the USP25 gene recapitulated the effects of miR-200c over-expression. Clinical analysis indicated that miR-200c was negatively correlated with clinical stage, lymph node metastasis in NSCLC patients. Moreover, USP25 protein and mRNA level expressions were higher in NSCLC patients, compared to healthy control, and correlated with clinical stage and lymphatic node metastasis.ConclusionsThese findings indicate that miR-200c exerts tumor-suppressive effects for NSCLC through the suppression of USP25 expression and suggests a new therapeutic application of miR-200c in the treatment of NSCLC.

SWATH™- and iTRAQ-based quantitative proteomic analyses reveal an overexpression and biological relevance of CD109 in advanced NSCLC☆

UNLABELLED To identify cancer-related proteins, we used isobaric tags in a relative and absolute quantitation (iTRAQ) proteomic approach and SWATH™ quantification approach to analyze the secretome of an isogenic pair of highly metastatic and low metastatic non-small-cell lung cancer (NSCLC) cell lines. In addition, we compared two groups of pooled serum samples (12 early-stage and 12 late-stage patients) to mine data for candidates screened by iTRAQ-labeled proteomic analysis. A total of 110 proteins and 71 proteins were observed to be significantly differentially expressed in the cell line secretome and NSCLC sera, respectively. Among these proteins, CD109 was found to be highly expressed in both the highly metastatic cell line secretome and the group of late-stage patients. A sandwich ELISA assay also demonstrated an elevation of serum CD109 levels in individual NSCLC patients (n=30) compared with healthy subjects (n=19). Furthermore, CD109 displayed higher expression in lung cancer tissues compared with their matched noncancerous lung tissues (n=72). In addition, the knockdown of CD109 influenced several NSCLC cell bio-functions, for instance, depressing cell growth, affecting cell cycle phases. These phenomena suggest that CD109 plays a critical role in NSCLC progression. BIOLOGICAL SIGNIFICANCE We simultaneously applied two quantitative proteomic approaches-iTRAQ-labeling and SWATH™-to analyze the secretome of metastatic cell lines, in order to explore the cancer-associated proteins in conditioned media. In this study, our results indicate that CD109 plays a critical role in non-small-cell lung cancer (NSCLC) progression, and is overexpressed in advanced NSCLC.

Quantitative proteomic analysis of the metastasis-inhibitory mechanism of miR-193a-3p in non-small cell lung cancer.

Background: microRNAs can repress the expression of target genes by destabilizing their mRNAs or by inhibiting their translation. Our previous findings suggested that miR-193a-3p inhibited the progression of NSCLC both in vitro and in vivo. However, the biological processes and molecular pathways through which this miRNA exerts its positive effects are unknown. Methods: To explore the molecular mechanisms by which miR-193a-3p inhibited NSCLC metastasis, we investigated the changes in the protein profile of SPC-A-1sci (highly metastatic) cells in response to the up-regulation of miR-193a-3p expression using a proteomics approach (iTRAQ combined with NanoLC-MS/MS). Changes in the profiles of the expressed proteins were verified using western blotting and were analyzed using the DAVID and STRING programs. Results: In the two replicated experiments, 4962/4946 proteins were identified, and the levels of expression of 4923/4902 proteins were quantified. In total, 112 of these proteins were differentially expressed. Among them, the up-regulated levels of expression of two of the 62 proteins with up-regulated expression (PPP2R2A and GSN) and the down-regulated levels of expression four of the 50 proteins with down-regulated expression (LMNB2, UHRF1, G3BP1, and HNRNPU) were verified using western blotting. The bioinformatics analysis revealed the interactions and signaling networks of these differentially expressed proteins. Conclusion: miR-193a-3p inhibited the metastasis of lung cancer cells by deregulating the expression of tumor-related proteins. These findings may improve the understanding of the molecular mechanisms underlying the metastatic-inhibitory effect of miR-193a-3p on lung cancer cells.

Quantitative proteomic analysis identifies CPNE3 as a novel metastasis-promoting gene in NSCLC.

To discover metastasis-associated proteins within cancer cells, we used the isobaric tags for relative and absolute quantitation (iTRAQ) approach combined with nano liquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) analysis to identify proteins that were differentially expressed between lung adenocarcinoma cancer cell lines SPC-A-1sci cells with high metastatic potential and parent SPC-A-1 cells with low metastatic potential. By employing biological and technical replicates, we identified 5818 nonredundant proteins and quantified 5443 proteins, 256 of which were differentially expressed in the two cell lines. Through si-RNA-mediated functional screens, Myosin heavy chain 9 (MYH9) and Copine III (CPNE3) were indicated as positively correlating with the migration and invasion properties of SPC-A1sci cells, and the same function of CPNE3 was confirmed in another lung cancer cell line, H1299. Furthermore, overexpressing CPNE3 promoted nonsmall-cell lung cancer (NSCLC) cell line (SPC-A-1 and XL-2) migration and invasion in vitro. Moreover, the targeted knock-down of CPNE3 inhibited the in vivo metastatic abilities of H1299 cells in mouse models. Lastly, immunohistochemistry revealed that the CPNE3 expression level was positively correlated with the clinical stage and TNM classification in NSCLC patients. Taken together, our results indicate that CPNE3 could play a critical role in NSCLC metastasis.

Hepatic stellate cells activated by acidic tumor microenvironment promote the metastasis of hepatocellular carcinoma via osteopontin.

Extracellular pH of solid tumor is generally acidic due to excessive glycolysis and poor perfusion. But whether acidic tumor microenvironment influenced the stromal cells infiltrating in tumor remains unknown. As the predominant progenitor of stromal cells in liver, the number of activated hepatic stellate cells (HSCs) was found positively correlated to the acidification level in the tumor tissues of HCC patients in our study. Whereas, in vitro acidic culture condition and in vivo co-implanting xenograft model were adopted to study the response of HSCs and its influence on HCC progression. HSCs were activated under acidic culture condition depending on the phosphorylation of cellular signal-regulated kinase (ERK). Acidity-activated HSCs promoted HCC metastasis in vitro and in vivo. Osteopontin (OPN) excretion from HSCs was increased under acidic condition and proved to promote the migration of HCC cells. Furthermore, the expression level of OPN was significantly associated with myofibroblasts and the combination of α-SMA with OPN was a powerful predictor for poor prognosis of HCC patients. Activation of HSCs in acidic tumor microenvironment represents a novel mechanism for HCC metastasis and provides a potential therapeutic strategy for HCC.

MicroRNA-148a Suppresses Invasion and Metastasis of Human Non-Small-Cell Lung Cancer.

Background/Aims: microRNAs (miRNAs) are noncoding RNAs that regulate multiple targets through either the degradation of mRNAs or the inhibition of protein translation, thereby altering several functions simultaneously. Growing evidence indicates that miRNAs are involved in carcinogenesis and tumor progression in non-small-cell lung cancer (NSCLC). Methods: In this study, the mRNA expression levels of miR-148a were examined in NSCLC cell lines and patient specimens using quantitative reverse transcription-PCR. The functions of miR-148a in migration/invasion and lung metastasis formation were determined by using transwell and tail vein injection assays, respectively. Results: We demonstrated that miR-148a was down-regulated in NSCLC metastatic samples, and its expression was suppressed in NSCLC compared with the corresponding nonmalignant lung tissues. Clinical analysis indicated that miR-148a expression was lower in NSCLC patients compared with nonmalignant lung tissues . Decreased miR-148a was significantly associated with tumor node metastasis stage and lymph node metastasis. Furthermore, functional assays showed that miR-148a expression suppressed NSCLC cell invasive and migratory abilities in vitro and suppressed cancer metastasis in vivo, while inhibition of miR-148a enhanced NSCLC cell invasion and lung metastasis formation in a mouse model. Conclusions: Evidence from this study demonstrated that miR-148a exerts tumor-suppressive effects in NSCLC and suggests a new therapeutic option for NSCLC.

Enriched Environment Inhibits Mouse Pancreatic Cancer Growth and Down-regulates the Expression of Mitochondria-related Genes in Cancer Cells

Psycho-social stress has been suggested to influence the development of cancer, but it remains poorly defined with regard to pancreatic cancer, a lethal malignancy with few effective treatment modalities. In this study, we sought to investigate the impacts of enriched environment (EE) housing, a rodent model of “eustress”, on the growth of mouse pancreatic cancer, and to explore the potential underlying mechanisms through gene expression profiling. The EE mice showed significantly reduced tumor weights in both subcutaneous (53%) and orthotopic (41%) models, while each single component of EE (inanimate stimulation, social stimulation or physical exercise) was not profound enough to achieve comparative anti-tumor effects as EE. The integrative transcriptomic and proteomic analysis revealed that in response to EE, a total of 129 genes in the tumors showed differential expression at both the mRNA and protein levels. The differentially expressed genes were mostly localized to the mitochondria and enriched in the citrate cycle and oxidative phosphorylation pathways. Interestingly, nearly all of the mitochondria-related genes were down-regulated by EE. Our data have provided experimental evidence in favor of the application of positive stress or of benign environmental stimulation in pancreatic cancer therapy.

MetaLnc9 Facilitates Lung Cancer Metastasis via a PGK1-Activated AKT/mTOR Pathway

Long noncoding RNAs (lncRNA) participate in carcinogenesis and tumor progression in lung cancer. Here, we report the identification of a lncRNA signature associated with metastasis of non-small cell lung cancer (NSCLC). In particular, elevated expression of LINC00963 (MetaLnc9) in human NSCLC specimens correlated with poor prognosis, promoted migration and invasion of NSCLC cells in vitro, and enhanced lung metastasis formation in vivo Mechanistic investigations showed that MetaLnc9 interacted with the glycolytic kinase PGK1 and prevented its ubiquitination in NSCLC cells, leading to activation of the oncogenic AKT/mTOR signaling pathway. MetaLnc9 also interacted with P54nrb/NonO (NONO) to help mediate the activity of CRTC, a coactivator for the transcription factor CREB, reinforcing a positive feedback loop for metastasis. Taken together, our results establish MetaLnc9 as a driver of metastasis and a candidate therapeutic target for treating advanced NSCLC. Cancer Res; 77(21); 5782-94. ©2017 AACR.