TRIM44 promotes BRCA1 functions in HR repair to induce Cisplatin Chemoresistance in Lung Adenocarcinoma by Deubiquitinating FLNA.

Tripartite motif-containing 44 (TRIM44) has recently been implicated in various pathological processes in numerous cancers, including lung adenocarcinoma (LUAD); however, its functional roles in chemoresistance are poorly understood. Herein, TRIM44 knockdown sensitized LUAD cells to cisplatin and enhanced cisplatin-induced apoptosis. Microarray analysis indicated that the "Role of BRCA1 in DNA damage" and the BRCA1 gene expression were positively regulated by TRIM44, which was further verified by immunofluorescence, qRT-PCR, and Western blotting. BRCA1 depletion effectively abolished TRIM44-modulated cisplatin resistance and regulation of homologous recombination (HR) repair. Interestingly, TRIM44 interacted with FLNA, an upstream regulator of BRCA1 as specified by STRING V 11.5, and facilitated its stability and deubiquitination. FLNA was also found to be required for the functions of TRIM44 in drug resistance. Using animal models, overexpression of TRIM44 was shown to confer resistance to cisplatin in a BRCA1- and FLNA-dependent manner. TRIM44 expression levels in tissues from cisplatin-resistant LUAD patients were significantly higher than those in tissues from cisplatin-sensitive LUAD patients. Collectively, our study results demonstrate that the TRIM44/FLNA/BRCA1 axis is involved in cisplatin chemoresistance, providing potential therapeutic targets for LUAD patients with cisplatin resistance.

A novel EHD1/CD44/Hippo/SP1 positive feedback loop potentiates stemness and metastasis in lung adenocarcinoma.

BACKGROUND: There is growing evidence that endocytosis plays a pivotal role in cancer metastasis. In this study, we first identified endocytic and metastasis-associated genes (EMGs) and then investigated the biological functions and mechanisms of EMGs. METHODS: Cancer stem cells (CSCs)-like characteristics were evaluated by tumour limiting dilution assays, three-dimensional (3D) spheroid cancer models. Microarray analysis was used to identify the pathways significantly regulated by mammalian Eps15 homology domain protein 1 (EHD1) knockdown. Mass spectrometry (MS) was performed to identify EHD1-interacting proteins. The function of EHD1 as a regulator of cluster of differentiation 44 (CD44) endocytic recycling and lysosomal degradation was determined by CD44 biotinylation and recycling assays. RESULTS: EHD1 was identified as a significant EMG. Knockdown of EHD1 suppressed CSCs-like characteristics, epithelial-mesenchymal transition (EMT), migration and invasion of lung adenocarcinoma (LUAD) cells by increasing Hippo kinase cascade activation. Conversely, EHD1 overexpression inhibited the Hippo pathway to promote cancer stemness and metastasis. Notably, utilising MS analysis, the CD44 protein was identified as a potential binding partner of EHD1. Furthermore, EHD1 enhanced CD44 recycling and stability. Indeed, silencing of CD44 or disruption of the EHD1/CD44 interaction enhanced Hippo pathway activity and reduced CSCs-like traits, EMT and metastasis. Interestingly, specificity protein 1 (SP1), a known downstream target gene of the Hippo-TEA-domain family members 1 (TEAD1) pathway, was found to directly bind to the EHD1 promoter region and induce its expression. Among clinical specimens, the EHD1 expression level in LUAD tissues of metastatic patients was higher than that of non-metastatic patients. CONCLUSIONS: Our findings emphasise that EHD1 might be a potent anti-metastatic target and present a novel regulatory mechanism by which the EHD1/CD44/Hippo/SP1 positive feedback circuit plays pivotal roles in coupling modules of CSCs-like properties and EMT in LUAD. Targeting this loop may serve as a remedy for patients with advanced metastatic LUAD.

Identification of Differentially Expressed Circular RNAs as miRNA Sponges in Lung Adenocarcinoma.

BACKGROUND: Circular RNAs (circRNAs) may function as the decoys for microRNAs (miRNAs) or proteins, the templates for translation, and the sources of pseudogene generation. The purpose of this study is to determine the diagnostic circRNAs, which are related to lung adenocarcinoma (LUAD), that adsorb miRNAs on the basis of the competing endogenous RNA (ceRNA) hypothesis. METHODS: The differentially expressed circRNAs (DEcircRNAs) in LUAD were revealed by the microarray data (GSE101586 and GSE101684) that were obtained from the Gene Expression Omnibus (GEO) database. The miRNAs that were targeted by the DEcircRNAs were predicted with the CircInteractome, and the target mRNAs of the miRNAs were found by the miRDB and the TargetScan. The ceRNA network was built by the Cytoscape. The potential biological roles and the regulatory mechanisms of the circRNAs were investigated by the Gene Ontology (GO) enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The expression of the host genes of circRNAs was examined by the Ualcan. The survival analysis was performed by the Kaplan-Meier plotter. RESULTS: In comparison with normal lung tissues, LUAD tissues contained 7 overlapping cancer-specific DEcircRNAs with 294 miRNA response elements (MREs). Among the 7 DEcircRNAs, 3 circRNAs (hsa_circ_0072088, hsa_circ_0003528, and hsa_circ_0008274) were upregulated and 4 circRNAs (hsa_circ_0003162, hsa_circ_0029426, hsa_circ_0049271, and hsa_circ_0043256) were downregulated. A circRNA-miRNA-mRNA regulatory network, which included 33 differentially expressed miRNAs (DEmiRNAs) and 2007 differentially expressed mRNAs (DEmRNAs), was constructed. These mRNAs were enriched in the biological function of cell-cell adhesion, response to hypoxia, and stem cell differentiation and were involved in the PI3K-Akt signaling, HIF-1 signaling, and cAMP signaling pathways. CONCLUSION: Our results indicated that 7 DEcircRNAs could have diagnostic value for LUAD. Additionally, the circRNAs-mediated ceRNA network might provide a novel perspective into unraveling the pathogenesis and progression of LUAD.

LINC02678 as a Novel Prognostic Marker Promotes Aggressive Non-small-cell Lung Cancer.

Non-small-cell lung carcinoma (NSCLC) is considered to be a fatal disease and characterized by a poor prognosis. Long non-coding RNAs (lncRNAs) have been reported to act as biomarkers and therapeutic targets in solid tumors. However, the expression of lncRNAs and their clinical relevance in NSCLC remain undetermined. The gene expression data profiled in The Cancer Genome Atlas and Gene Expression Omnibus (GSE81089) were employed to screen differentially expressed lncRNAs in NSCLC. LINC02678 was found to be upregulated in NSCLC and exhibited hypomethylation of the promoter region in NSCLC tissues. LINC02678 (also called RP11-336A10.5) was associated with poorer overall survival and relapse-free survival in NSCLC patients. In vitro models of gain- and loss-of-function demonstrated that LINC02678 promotes NSCLC progression by promoting NSCLC cell proliferation and cell cycle progression, as well as inducing NSCLC cell migration, invasion and epithelial-mesenchymal transition. LINC02678 was primarily located in the nucleus and could bind with the enhancer of zeste homolog 2 (EZH2). Moreover, we found that LINC02678 knockdown impaired the occupancy capacity of EZH2 and trimethylation of lysine 27 on histone 3 (H3K27me3) at the promoter region of cyclin dependent kinase inhibitor 1B (CDKN1B) and E-cadherin, as confirmed by ChIP-qPCR. A mouse transplantation model further demonstrated that LINC02678 could promote the tumorigenic and metastatic capacities of NSCLC cells. We identified LINC02678 as a tumor promoter in NSCLC, which enhanced the growth and metastasis of NSCLC cells by binding with EZH2, indicating that LINC02678 may serve as a potential biomarker for cancer diagnosis and treatment.

Identification of Differentially Expressed and Prognostic lncRNAs for the Construction of ceRNA Networks in Lung Adenocarcinoma.

BACKGROUND: Long noncoding RNAs (lncRNAs) could function as competitive endogenous RNAs (ceRNAs) to competitively adsorb microRNAs (miRNAs), thereby regulating the expression of their target protein-coding mRNAs. In this study, we aim to identify more effective diagnostic and prognostic markers for lung adenocarcinoma (LUAD). METHODS: We obtained differentially expressed lncRNAs (DElncRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) for LUAD by using The Cancer Genomes Atlas (TCGA) portal. Weighted gene coexpression network analysis (WGCNA) was performed to unveil core gene modules associated with LUAD. The Cox proportional hazards model was performed to determine the prognostic significance of DElncRNAs. The diagnostic and prognostic significance of DElncRNAs was further verified based on the receiver operating characteristic curve (ROC). Cytoscape was used to construct the ceRNA networks comprising the lncRNAs-miRNAs-mRNAs axis based on the correlation obtained from the miRcode, miRDB, and TargetScan. RESULTS: Compared with normal lung tissues, 2355 DElncRNAs, 820 DEmiRNAs, and 17289 DEmRNAs were identified in LUAD tissues. We generated 8 WGCNA core modules in the lncRNAs coexpression network, 5 modules in the miRNAs, and 12 modules in the mRNAs coexpression network, respectively. One lncRNA module (blue) consisting of 441 lncRNAs, two miRNA modules (blue and turquoise) containing 563 miRNAs, and one mRNA module (turquoise), which consisted of 15162 mRNAs, were mostly significantly related to LUAD status. Furthermore, 67 DEmRNAs were found to be tumor-associated as well as the target genes of the DElncRNAs-DEmiRNAs axis. Survival analyses showed that 6 lncRNAs (LINC01447, WWC2-AS2, OGFRP1, LINC00942, LINC01168, and AC005863.1) were significantly correlated with the prognosis of LUAD patients. Ultimately, the potential ceRNA networks including 6 DElncRNAs, 4 DEmiRNAs, and 22 DEmRNAs were constructed. CONCLUSION: Our study indicated that 6 DElncRNAs had the possibilities as diagnostic and prognostic biomarkers for LUAD. The lncRNA-mediated ceRNA networks might provide novel insights into the molecular mechanisms of LUAD progression.

CUEDC1 inhibits epithelial-mesenchymal transition via the TßRI/Smad signaling pathway and suppresses tumor progression in non-small cell lung cancer.

Lung cancer remains the most lethal cancer worldwide because of its high metastasis potential. Epithelial-mesenchymal transition (EMT) is known as the first step of the metastasis cascade, but the potential regulatory mechanisms of EMT have not been clearly established. In this study, we first found that low CUEDC1 expression correlated with lymph node metastasis in non-small cell lung cancer (NSCLC) patients using immunohistochemistry (IHC). CUEDC1 knockdown promoted the metastasis of NSCLC cells and EMT process and activated TßRI/Smad signaling pathway. Overexpression of CUEDC1 decreased the metastatic potential of lung cancer cells and inhibited the EMT process and inactivated TßRI/Smad signaling pathway. Immunoprecipitation (IP) assays showed that Smurf2 is a novel CUEDC1-interacting protein. Furthermore, CUEDC1 could regulate Smurf2 expression through the degradation of Smurf2. Overexpression of Smurf2 abolished CUEDC1 knockdown induced-EMT and the activation of TßRI/Smad signaling pathway, while siRNA Smurf2 reversed CUEDC1 overexpression-mediated regulation of EMT and TßRI/Smad signaling pathway. Additionally, CUEDC1 inhibited proliferation and promoted apoptosis of NSCLC cells. In vivo, CUEDC1-knockdown cells promoted metastasis and tumor growth compared with control cells. In conclusion, our findings indicate that the crucial role of CUEDC1 in NSCLC progression and provide support for its clinical investigation for therapeutic approaches.

Chinese Medicines for Preventing and Treating Radiation-Induced Pulmonary Injury: Still a Long Way to Go.

Thoracic radiotherapy is a mainstay of the treatment for lung, esophageal, and breast cancers. Radiation-induced pulmonary injury (RIPI) is a common side effect of thoracic radiotherapy, which may limit the radiotherapy dose and compromise the treatment results. However, the current strategies for RIPI are not satisfactory and may induce other side effects. Chinese medicines (CMs) have been used for more than a thousand years to treat a wide range of diseases, including lung disorders. In this review, we screened the literature from 2007 to 2017 in different online databases, including China National Knowledge Infrastructure (CNKI), Chongqing VIP, Wanfang, and PubMed; summarized the effectiveness of CMs in preventing and treating RIPI; explored the most frequently used drugs; and aimed to provide insights into potential CMs for RIPI. Altogether, CMs attenuated the risk of RIPI with an occurrence rate of 11.37% vs. 27.78% (P < 0.001) compared with the control groups. We also found that CMs (alone and combined with Western medical treatment) for treating RIPI exerted a higher efficacy rate than that of the control groups (78.33% vs. 28.09%, P < 0.001). In the screened literature, 38 CMs were used for the prevention and treatment of RIPI. The top five most frequently used CMs were Astragali Radix (with a frequency of 8.47%), Ophiopogonis Radix (with a frequency of 6.78%), Glycyrrhizae Radix et Rhizome (with a frequency of 5.08%), Paeoniae Radix Rubra (with a frequency of 5.08%), and Prunellae Spica (with a frequency of 5.08%). However, further high-quality investigations in CM source, pharmacological effects and underlying mechanisms, toxicological aspects, and ethical issues are warranted. Taken together, CMs might have a potential role in RIPI prevention and treatment and still have a long way to investigate.

PHLPP2 as a novel metastatic and prognostic biomarker in non-small cell lung cancer patients.

BACKGROUND: PH domain and leucine-rich repeat protein phosphatase 2 (PHLPP2) has been reported to be a potent tumor suppressor in many human cancers. However, PHLPP2 has not been fully researched as a putative clinical prognostic biomarker of lung cancer. METHODS: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases including data on 1383 non-small cell lung cancer (NSCLC) patients were used to determine PHLPP2 expression. PHLPP2 expression was then examined by immunohistochemistry, and its clinical significance analyzed in 134 NSCLC patients, including 73 patients with adenocarcinoma and 81 with squamous cell carcinoma. RESULTS: We found PHLPP2 expression to be less pronounced in NSCLC tissue samples than that in nontumoral lung tissues according to data taken from TCGA and GEO datasets; this outcome was further validated by immunohistochemistry assay. The low PHLPP2 expression level was found to be associated with the presence of lymph node metastasis (P = 0.003). Importantly, PHLPP2 was found to be an independent indicator of prognosis for overall (hazard ratio [HR] = 0.520, 95% confidence interval [Cl] = 0.327-0.827; P = 0.006) and disease-free survival (HR = 0.489, 95% Cl = 0.308-0.775; P = 0.002) in patients with surgically-resected NSCLC by multivariate analysis. CONCLUSION: Taken together, our findings show that PHLPP2 is a robust clinical marker for NSCLC survival and could serve as a potential therapeutic target.

LRH1 as a promising prognostic biomarker and predictor of metastasis in patients with non-small cell lung cancer.

BACKGROUND: LRH1, which promotes the malignant transformation of carcinoma, has recently been documented in several types of malignancies. However, LRH1 has not been assessed as a potential clinical biomarker in any cancer. METHODS: LRH1 expression was tested in fresh-frozen tissue samples with quantitative real-time PCR and Western blot analysis. Surgically resected tumor tissues were collected from 156 non-small cell lung cancer (NSCLC) patients: 75 with adenocarcinoma and 81 with squamous cell carcinoma. Subsequently, the immunohistochemical expression of LRH1 was examined, and its clinical significance was evaluated. RESULTS: LRH1 overexpression was observed in NSCLC carcinoma tissues compared to adjacent normal lung tissues. LRH1 expression was correlated with poorer differentiation (P = 0.023), pathological tumor classification (P < 0.001), advanced pathological tumor node metastasis stage (P = 0.017), adenocarcinoma subtype (P = 0.031), and positive lymph node metastasis (P < 0.001). Multivariate analysis demonstrated that LRH1 expression status was an independent prognostic factor for overall (hazard ratio 1.372, 95% confidence interval 1.225-1.617; P = 0.003) and disease-free survival (hazard ratio 1.497, 95% confidence interval 1.059-2.115; P = 0.011) in patients who suffered from resectable NSCLC. CONCLUSION: The results of our study indicate that LRH1 predicts NSCLC progression, metastasis, and a dismal prognosis, emphasizing its promising role as a novel target in NSCLC therapies.

TNFAIP8 promotes the proliferation and cisplatin chemoresistance of non-small cell lung cancer through MDM2/p53 pathway.

BACKGROUND: The highly refractory nature of non-small cell lung cancer (NSCLC) to chemotherapeutic drugs is an important factor resulting in its poor prognosis. Recent studies have revealed that tumour necrosis factor alpha-induced protein 8 (TNFAIP8) is involved in various biological and pathological processes of cells, but their underlying mechanisms in processes ranging from cancer development to drug resistance have not been fully elucidated. METHODS: TNFAIP8 expression in clinical NSCLC samples was examined through immunohistochemistry (IHC). After adjusting for patients' characteristics with propensity score matching, Kaplan-Meier analysis and Cox regression analysis were performed for comparison of patients' survival according to the TNFAIP8 level. Lentiviral transfection with TNFAIP8-specific shRNAs was used to establish stable TNFAIP8 knockdown (TNFAIP8 KD) NCI-H460, A549 and cis-diamminedichloroplatinum II resistant A549 (A549/cDDP) cell lines. Cell proliferation and viability were assessed by CCK-8 assay. Cell cycle was examined by flow cytometry. Multiple pathways regulated by TNFAIP8 KD were revealed by microarray analysis. RESULTS: We found that high TNFAIP8 expression was associated with advanced pT stage, advanced pTNM stage, lymph node metastasis and unfavourable survival in NSCLC patients. TNFAIP8 shRNAs reduced in vitro cancer cell proliferation and in vivo tumor growth. Additionally, TNFAIP8 KD increased the sensitivity of NSCLC cells to cisplatin in vitro and in vivo. Conversely, up-regulation of TNFAIP8 promoted the proliferation and drug resistance to cisplatin of NSCLC cells. TNFAIP8 influences cancer progression pathways involving the MDM2/p53 pathway. Indeed, we observed that TNFAIP8 KD mediated the MDM2 downregulation and the p53 ubiquitination, thereby decreasing the degradation of p53 protein. shRNA p53 reversed TNFAIP8 shRNA-mediated regulation of cell proliferation, cell cycle, cisplatin sensitivity, and expression levels of RAD51, a DNA repair gene. CONCLUSION: Our work uncovers a hitherto unappreciated role of TNFAIP8 in NSCLC proliferation and cisplatin chemoresistance that is mediated through the MDM2/p53 pathway. These findings might offer potential therapeutic targets for reversing cisplatin resistance in NSCLC patients with high TNFAIP8 expression.

[Role of Hippo Signaling Pathway in Lung Cancer].

Lung cancer is the leading cause of cancer related mortality in the world, with more than 1 million deaths per year, accounting for about one fifth of all cancer deaths worldwide. Over the past years, lung cancer treatment has been based on surgery, radiation therapy, chemotherapy, targeted therapies, and immunotherapy, but the improvement is not very perfect. Therefore, it has become clear that additional therapeutic strategies are urgently required to provide an improved survival benefit for patients. In recent years, Hippo signaling pathway has become a popular direction in the field of cancer research. When the Hippo pathway is active, the core Hippo kinase, such as MST/MOB and LATS1/2, inhibit the two transcriptional co-activators, YAP/TAZ. And YAP/TAZ are phosphorylated and sequestered in the cytoplasm. Dysregulation of the Hippo pathway drives multiple aspects of lung tumor initiation and progression. Moreover, the potential value of this pathway is getting more and more prevalent in clinical application. In this review, we summarize the molecular mechanism and the core components, upstream or downstream targets of Hippo signaling pathway which contribute the formation of lung cancer and discuss the therapeutic potential of targeted strategies in lung cancer. Additionally, we highlight the prospect of research on Hippo signaling pathway in the future.