Novel pathways involved in cisplatin resistance identified by a proteomics approach in non-small-cell lung cancer cells

Although platinum-based chemotherapy remains the standard-of-care for most patients with advanced non-small-cell lung cancer (NSCLC), acquired resistance occurs frequently predicting poor prognosis. To examine the mechanisms underlying platinum resistance, we have generated and characterized by proteomic approach the resistant A549 CDDP-resistant (CPr-A549) and their parental A549 cells, identifying 15 proteins differentially expressed (13 upregulated and 2 downregulated in CPr-A549). In details, we highlighted a coherent network of proteins clustering together and involved in altered protein folding and endoplasmic reticulum stress, correlated with epithelial to mesenchymal transition process and cancer stem cell markers, where vimentin played a hierarchical role, ultimately resulting in increased aggressive features. By using publicly available databases we showed that the modulated proteins could contribute to NSCLC carcinogenesis and correlate with NSCLC patients prognosis and survival probability, suggesting that they can be used as novel potential prognostic/predictive biomarkers or therapeutic targets to overcome platinum-resistance.     

Overexpression of WDR79 in non‐small cell lung cancer is linked to tumour progression

WD‐repeat protein 79 (WDR79), a member of the WD‐repeat protein family, acts as a scaffold protein, participating in telomerase assembly, Cajal body formation and DNA double‐strand break repair. Here, we first report that WDR79 is frequently overexpressed in cell lines and tissues derived from non‐small cell lung cancer (NSCLC). Knockdown of WDR79 significantly inhibited the proliferation of NSCLC cells in vitro and in vivo by inducing cell cycle arrest and apoptosis. WD‐repeat protein 79 ‐induced cell cycle arrest at the G0/G1 phase was associated with the expression of G0/G1‐related cyclins and cyclin‐dependent kinase complexes. We also provide evidence that WDR79 knockdown induces apoptosis via a mitochondrial pathway. Collectively, these results suggest that WDR79 is involved in the tumorigenesis of NSCLC and is a potential novel diagnostic marker and therapeutic target for NSCLC.     

Grape proanthocyanidins induce apoptosis by loss of mitochondrial membrane potential of human non-small cell lung cancer cells in vitro and in vivo

Lung cancer remains the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) represents approximately 80% of total lung cancer cases. The use of non-toxic dietary phytochemicals can be considered as a chemotherapeutic strategy for the management of the NSCLC. Here, we report that grape seed proanthocyanidins (GSPs) induce apoptosis of NSCLC cells, A549 and H1299, in vitro which is mediated through increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase (PARP). Pre-treatment of A549 and H1299 cells with the caspase-3 inhibitor (z-DEVD-fmk) significantly blocked the GSPs-induced apoptosis of these cells confirmed that GSPs-induced apoptosis is mediated through activation of caspases-3. Treatments of A549 and H1299 cells with GSPs resulted in an increase in G1 arrest. G0/G1 phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk), cyclin-dependent kinase inhibitors (Cdki) and cyclins. Our western blot analyses showed that GSPs-induced G1 cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), and a simultaneous decrease in the levels of Cdk2, Cdk4, Cdk6 and cyclins. Further, administration of 50, 100 or 200 mg GSPs/kg body weight of mice by oral gavage (5 d/week) markedly inhibited the growth of s.c. A549 and H1299 lung tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death, increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3 in tumor xenograft cells. Based on the data obtained in animal study, human equivalent dose of GSPs was calculated, which seems affordable and attainable. Together, these results suggest that GSPs may represent a potential therapeutic agent for the non-small cell lung cancer.     

非小细胞肺癌的免疫治疗进展

肺癌是最致命的恶性肿瘤之一,也是男性肿瘤患者致死率最高的,5年生存率低于18%。尽管非小细胞肺癌(non-small cell lung cancer,NSCLC)在手术治疗、化疗、放疗以及靶向治疗方面均取得了一定的成果,但晚期NSCLC的预后依然很差。免疫治疗为NSCLC患者提供了一个新的治疗方向。免疫治疗目前主要研究方向在免疫检查点抑制剂(Ipilimumab、Nivolumab、MK-3475)和肿瘤疫苗(MAGE-A3,L-BLP25,TG4010,Belagenpumatucel-L)等。免疫治疗具有针对性强、副作用少、效率高的特点,并在Ⅱ、Ⅲ期临床试验中取得了较好的疗效,成为在手术、化疗、放疗以及靶向治疗后一种新的重要治疗手段。本文就当前非小细胞肺癌免疫治疗原理、临床试验及待解决问题作一综述。     

Ornithine aminotransferase promoted the proliferation and metastasis of non-small cell lung cancer via upregulation of miR-21

The incidence and mortality of lung cancer ranked the first among all types of cancer in China, and non-small cell lung cancer (NSCLC) is the most common type of lung cancer accounting for 85% of all lung cancers. Given that the survival rate of patients with advanced NSCLC is still poor nowadays, identification of novel therapeutic targets and the development of effective therapies are desired for the treatment of NSCLC in clinics. In this study, we reported the upregulation of ornithine aminotransferase (OAT) in NSCLC cells and clinical tumor samples as well as its association with the advanced TNM stage, metastasis, and poor tumor differentiation of lung cancer. Using different NSCLC cell lines, we demonstrated that OAT promoted the proliferation, invasion, and migration, inhibited the apoptosis, and altered cell cycle of NSCLC cells; besides, the involvement of OAT-miR-21-glycogen synthase kinase-3β signaling in the functional role of OAT in NSCLC was also revealed. Importantly, in the absence of OAT, the growth and metastasis of tumor lung cancer xenograft was significantly suppressed in the nude mice. Based on our findings, OAT may be a potential novel biomarker for the diagnosis and therapeutic outcome monitoring of NSCLC. Inhibition of OAT may also represent a new therapeutic strategy of NSCLC.     

Tumor vasculature as a therapeutic target in non-small cell lung cancer

Despite developments in conventional (chemo)radiotherapy and surgery, survival of non-small cell lung cancer (NSCLC) patients remains poor. Treatments with targeted molecular drugs offer novel therapeutic strategies. Bevacizumab, a recombinant anti-vascular endothelial growth factor (VEGF) antibody, is the antiangiogenic drug at the most advanced stage of development in the therapy of NSCLC. However, a number of questions and future challenges relating to the use of bevacizumab in NSCLC remain. Furthermore, novel agents targeting the pre-existing NSCLC vasculature (i.e. vascular disrupting agents, VDAs) or multiple tyrosine kinase inhibitors have emerged as unique drug classes delivering promising results in several preclinical and clinical studies. Herein, we review the most recent data using these novel targeted agents either alone or in combination with chemotherapy in NSCLC.     

Gene expression profiling and clinical outcome in breast cancer

Pathologic and clinical heterogeneity of breast cancer reflects the poorly documented, complex, and combinatory molecular basis of the disease and is in part responsible for therapeutic failures. The DNA microarray technique allows the analysis of RNA expression of several thousands of genes simultaneously in a sample. There are multiple potential applications of the technique in cancer research. A number of recent studies have shown the promising role of gene expression profiling in breast cancer by identifying new prognostic subclasses unidentifiable by conventional parameters and new prognostic and/or predictive gene signatures, whose predictive impact is superior to conventional histoclinical prognostic factors. In this review we describe current use of DNA microarrays in the prognosis of breast cancer. We also discuss issues that need to be addressed in the near future to allow the method to reach its full potential.     

Peripheral immune cell gene expression predicts survival of patients with non-small cell lung cancer

Prediction of cancer recurrence in patients with non-small cell lung cancer (NSCLC) currently relies on the assessment of clinical characteristics including age, tumor stage, and smoking history. A better prediction of early stage cancer patients with poorer survival and late stage patients with better survival is needed to design patient-tailored treatment protocols. We analyzed gene expression in RNA from peripheral blood mononuclear cells (PBMC) of NSCLC patients to identify signatures predictive of overall patient survival. We find that PBMC gene expression patterns from NSCLC patients, like patterns from tumors, have information predictive of patient outcomes. We identify and validate a 26 gene prognostic panel that is independent of clinical stage. Many additional prognostic genes are specific to myeloid cells and are more highly expressed in patients with shorter survival. We also observe that significant numbers of prognostic genes change expression levels in PBMC collected after tumor resection. These post-surgery gene expression profiles may provide a means to re-evaluate prognosis over time. These studies further suggest that patient outcomes are not solely determined by tumor gene expression profiles but can also be influenced by the immune response as reflected in peripheral immune cells.     

HELQ suppresses migration and proliferation of non-small cell lung cancer cells by repairing DNA damage and inducing necrosis

HELQ plays a key role in DNA damage response and cell-cycle checkpoint regulation. It has been implicated in ovarian and pituitary tumors and may play a role in germ cell maintenance. This study investigated the role of HELQ in lung cancer. The expression of HELQ in patients with non-small-cell lung cancer (NSCLC) was downregulated compared with normal human lungs. Clinical prognostic analysis of Kaplan–Meier plots revealed that patients with NSCLC with low HELQ levels had a reduced overall survival. Further, we found that HELQ depletion enhanced lung cancer cell malignancy. Furthermore, overexpression of HELQ in lung cancer cells reduced cell migration in vitro, while DNA damage repair was inhibited. Both in vitro and in vivo studies have shown that HELQ induces cell death. Mechanistically, we found that cells overexpressing HELQ showed a tendency to induce necrosis. After analyzing the database of HELQ interactors. we found that RIPK3 may interact with it and proved this conclusion by immunoprecipitation. Our findings identified the tumor suppressive role of HELQ in malignant human lung cancer and unraveled a potential therapeutic strategy for cancer treatment through HELQ activation. Moreover, HELQ may also be a predictive biomarker for the clinical predisposition, progression, and prognosis of lung cancer.     

PD-1 and PD-L1 Expression in NSCLC Indicate a Favorable Prognosis in Defined Subgroups

Background

Immunotherapy can become a crucial therapeutic option to improve prognosis for lung cancer patients. First clinical trials with therapies targeting the programmed cell death receptor PD-1 and its ligand PD-L1 have shown promising results in several solid tumors. However, in lung cancer the diagnostic, prognostic and predictive value of these immunologic factors remains unclear.

Method

The impact of both factors was evaluated in a study collective of 321 clinically well-annotated patients with non-small lung cancer (NSCLC) using immunohistochemistry.

Results

PD-1 expression by tumor infiltrating lymphocytes (TILs) was found in 22%, whereas tumor cell associated PD-L1 expression was observed in 24% of the NSCLC tumors. In Fisher’s exact test a positive correlation was found for PD-L1 and Bcl-xl protein expression (p = 0.013). Interestingly, PD-L1 expression on tumor cells was associated with improved overall survival in pulmonary squamous cell carcinomas (SCC, p = 0.042, log rank test), with adjuvant therapy (p = 0.017), with increased tumor size (pT2-4, p = 0.039) and with positive lymph node status (pN1-3, p = 0.010). These observations were confirmed by multivariate cox regression models.

Conclusion

One major finding of our study is the identification of a prognostic implication of PD-L1 in subsets of NSCLC patients with pulmonary SCC, with increased tumor size, with a positive lymph node status and NSCLC patients who received adjuvant therapies. This study provides first data for immune-context related risk stratification of NSCLC patients. Further studies are necessary both to confirm this observation and to evaluate the predictive value of PD-1 and PD-L1 in NSCLC in the context of PD-1 inhibition.     

Cytofluorimetric evaluation of DNA ploidy in lung cancer: a bronchoscopic study

The study of the biological characteristics of lung cancer is gaining more and more interest both because of their potential role as prognostic indicators and for therapeutic reasons. The DNA content estimated by flow cytometry in surgical samples of non-small cell lung cancer (NSCLC) has already been demonstrated to be correlated with survival in these patients. From July 1990 to February 1992 we analyzed the DNA distribution of bronchoscopic biopsies from 88 patients with lung cancer (18 small cell lung cancer, SCLC, and 68 NSCLC, two unspecified histology). Twenty-eight tumors (34.6%) had a diploid DNA distribution, while 53 were aneuploid (65.4%). A correlation was found between DNA ploidy and survival. Evaluation of the DNA content in bronchoscopic samples in a large series of patients could determine the role of this analysis prior to surgery in NSCLC and its value as a marker with respect to prognosis and response to therapy in SCLC.     

UHRF1 predicts poor prognosis by triggering cell cycle in lung adenocarcinoma

Accumulating evidence suggests that ubiquitin‐like with plant homeodomain and ring finger domains 1 (UHRF1) is overexpressed in non‐small cell lung cancer (NSCLC); however, the expression and function of UHRF1 in the subtype of NSCLC are still unclear. Here, we investigate the expression and prognosis traits of UHRF1 in large NSCLC cohorts and explore the molecular characters during UHRF1 up‐regulation. We find that UHRF1 is predominantly overexpressed in lung squamous cell carcinoma (SCC). Surprisingly, the up‐regulated UHRF1 is only associated with the overall survival of lung adenocarcinoma (ADC) and knockdown of UHRF1 dramatically attenuates ADC tumorigenesis. Mechanically, we identify a hub gene that includes a total of 55 UHRF1‐related genes, which are tightly associated with cell cycle pathway and yield to the poor clinical outcome in ADC patients. What's more, we observe knockdown of UHRF1 only affects ADC cells cycle and induces cell apoptosis. These results suggest that up‐regulated UHRF1 only contributes to lung ADC survival by triggering cell cycle pathway, and it may be a prognostic biomarker for lung ADC patients.     

非小细胞肺癌分子靶向药物治疗的研究进展

非小细胞肺癌(NSCLC)为最常见的肺癌病理类型,约占肺癌总数的 85%。大多数肺癌患者在确诊时已属晚期,失去手术机会, 保守治疗成为其重要治疗手段,但晚期肺癌患者的预后仍不理想。近年来,分子靶向治疗在肿瘤治疗领域取得重要进展,亦有研究显示 其在 NSCLC 的临床实践中发挥显著疗效。除表皮生长因子受体(EGFR)和间变淋巴瘤激酶(ALK)等主要基因突变之外,血管内皮生 长因子(VEGF)、ROS1、c-MET、RET、K-RAS、BRAF 也是目前 NSCLC 分子靶向治疗的相关靶点。综述 NSCLC 分子靶向药物治疗 的研究进展,旨在为该疾病的治疗提供参考。     

Revisiting the “Cdk-Centric” View of the Mammalian Cell Cycle

Since the early genetic studies in yeast, regulation of the cell cycle has been associated to the sequential activation of several proline-directed serine-threonine protein kinases by cyclins. From yeast to humans, the activiy of these cyclin-dependent kinases (Cdks) have been thought to be essential for cell cycle regulation. Recent gene-targeted mouse models for different cyclins and Cdks have shown that members of these families show a certain level of redundancy and that specific complexes are not required for the mitotic cell cycle. However, the complexity of the Cdk-cyclin network and the promiscuity of their members makes it difficult to understand the relative contribution of these proteins to the mammalian cell division cycle. Compensatory roles by non-Cdk activities and Cdk-independent functions of cyclins are increasing the complexity of the current simplistic models. We still do not know whether at least one cyclin-dependent kinase activity is required for cell cycle progression in mammalian cells. Indeed, a relevant question for cancer therapy.     

Exosome-mediated miR-7-5p delivery enhances the anticancer effect of Everolimus via blocking MNK/eIF4E axis in non-small cell lung cancer

Everolimus is a kind of mammalian target of rapamycin (mTOR) inhibitors. Activated mitogen-activated protein kinase interacting kinases/eukaryotic translation initiation factor 4E (MNK/eIF4E) axis plays a crucial role in resistance to Everolimus in non-small cell lung cancer (NSCLC). The eIF4E phosphorylation increased by mTOR inhibitors is mainly mediated by MNKs. However, the mechanisms are poorly understood. Recently, extensive reprogramming of miRNA profiles has also been found after long-term mTOR inhibitor exposure. Our previous studies have confirmed that tumor suppressor miR-7-5p is decreased in A549 cells after treatment with Everolimus. Exactly, MNK1 is the target of miR-7-5p. In this study, we investigated the biological functions and potential molecular mechanisms of miR-7-5p in the NSCLC undergoing treatment with Everolimus. We confirmed that Everolimus targeted mTORC1 inducing NSCLC cells to secrete miR-7-5p-loaded exosomes in Rab27A and Rab27B-dependent manners. Loss of intracellular miR-7-5p induced phosphorylation of MNK/eIF4E axis, but a supplement of extra exosomal miR-7-5p could reverse it. Of note, both low expression of miR-7-5p and elevated MNK1 protein were associated with a poor prognosis of NSCLC. Both endogenous miR-7-5p and exo-miR-7-5p enhanced the therapeutic efficacy of Everolimus by inhibiting the proliferation, migration, and metastasis of NSCLC in vitro and in vivo. The combination of miR-7-5p with Everolimus induced apoptosis to exhibit a synergistic anticancer therapeutic efficacy through dual abrogation of MNK/eIF4E and mTOR in NSCLC. In conclusion, Everolimus decreases the intracellular miR-7-5p by releasing of miR-7-5p loaded exosomes from NSCLC cells in Rab27A and Rab27B dependent manners. Either endogenous miR-7-5p or exo-miR-7-5p combined with Everolimus can enhance the anticancer efficacy by targeting MNK/eIF4E axis and mTOR. Besides, both low levels of miR-7-5p and positive expression of MNK1 act as independent poor prognostic biomarkers for NSCLC. Therefore, restoring miR-7-5p carried by exosome may be a promising novel combined therapeutic strategy with Everolimus for NSCLC.Subject terms: Drug development, Growth factor signalling, Oncogenesis     

CXCL5 regulation of proliferation and migration in human non-small cell lung cancer cells

Epithelial neutrophil-activating peptide-78 (CXCL5), a member of the subgroup of CXC-type chemokine family, is an inflammatory factor involved in the progression of lung cancer, but the underlying mechanism remains unclear. In this study, we investigated the effects of CXCL5 on proliferation and migration in non-small cell lung cancer (NSCLC) using tissue microarrays from NSCLC patients and H460 cells transfected with a CXCL5-interfered lentivirus vector or stimulated with recombinant CXCL5. We observed that the expression of CXCL5 was significantly higher in lung cancer cell lines, and high CXCL5 was associated with high chemokine (C-X-C motif) receptor 2 expression and was significantly associated with poor differentiation. The high expression of CXCL5 was associated with poor NSCLC prognosis and was an independent predictive factor. Furthermore, downregulation of CXCL5 in H460 cells significantly reduced proliferation and migration. Recombinant CXCL5 promoted H460 cell proliferation and movement by activating MAPK/ERK1/2 and PI3K/AKT signaling. Our study elucidates the important role of CXCL5 in the progression and prognosis of NSCLC. These findings suggested that CXCL5 might be a potential biomarker and novel therapeutic target for lung cancer.     

Long non‐coding RNA SNHG15 promotes CDK14 expression via miR‐486 to accelerate non‐small cell lung cancer cells progression and metastasis

Long non‐coding RNAs (lncRNAs) have been validated to play important role in multiple cancers, including non‐small cell lung cancer (NSCLC). In present study, our team investigate the biologic role of SNHG15 in the NSCLC tumorigenesis. LncRNA SNHG15 was significantly upregulated in NSCLC tissue samples and cells, and its overexpression was associated with poor prognosis of NSCLC patients. In vitro, loss‐of‐functional cellular experiments showed that SNHG15 silencing significantly inhibited the proliferation, promoted the apoptosis, and induced the cycle arrest at G0//G1 phase. In vivo, xenograft assay showed that SNHG15 silencing suppressed tumor growth of NSCLC cells. Besides, SNHG15 silencing decreased CDK14 protein expression both in vivo and vitro. Bioinformatics tools and luciferase reporter assay confirmed that miR‐486 both targeted the 3′‐UTR of SNHG15 and CDK14 and was negatively correlated with their expression levels. In summary, our study conclude that the ectopic overexpression of SNHG15 contribute to the NSCLC tumorigenesis by regulating CDK14 protein via sponging miR‐486, providing a novel insight for NSCLC pathogenesis and potential therapeutic strategy for NSCLC patients.