Activation of c-Jun NH2-terminal kinase is required for gemcitabine's cytotoxic effect in human lung cancer H1299 cells

Although gemcitabine is a potent therapeutic agent in the treatment of human non-small cell lung cancer (NSCLC), resistance to gemcitabine is common. In this study, we investigated the molecular mechanisms involved in acquired gemcitabine resistance against NSCLC cells. Gemcitabine-resistant NSCLC H1299 cells (H1299/GR) were selected by long-term exposure of parental H1299 cells to gemcitabine. The median inhibitory concentrations of gemcitabine in H1299 and H1299/GR cells were 19.4 and 233.1 nM, respectively. Gemcitabine induced activation of c-Jun NH2-terminal kinase (JNK) in parental H1299 cells but not in H1299/GR cells after 48 h. Blocking JNK activation by pretreatment with SP600125, a specific JNK inhibitor, or by transfection with dominant-negative JNK vectors abrogated gemcitabine-induced apoptosis in parental H1299 cells as evidenced by interruption of caspase activation. Transient transfection with a JNKK2-JNK1 plasmid expressing constitutive JNK1 partially restored the effect of gemcitabine in H1299/GR cells. Our results indicate that gemcitabine-induced apoptosis in human NSCLC H1299 cells requires activation of the JNK signaling pathway. Attenuated JNK activation may contribute to development of acquired gemcitabine resistance in cancer cells.     

STAMBPL1 promotes the progression of lung adenocarcinoma by inhibiting DHRS2 expression

BackgroundLung cancer is responsible for the majority of cancer deaths in the world. We found a significant increase of STAMBPL1 expression in lung adenocarcinoma (LUAD) tissues and cells. However, its mechanism has not been clarified.MethodsLUAD tissues and adjacent normal tissues were collected from 62 patients treated in the First Affiliated Hospital of Wenzhou Medical University from August 2018 to August 2021. In vivo, the clinical data and STAMBPL1 expression of 62 patients with LUAD were analyzed by qPCR. In vitro, cell experiments were carried out after STAMBPL1 knockdown in A549 and H1299 cells to determine cell growth, migration rate, evasiveness, colony-forming ability, and apoptosis. Gene sequencing was used to explore the expression of various genes in A549 and H1299 cells to verify that DHRS2 was up-regulated after STAMBPL1 knockdown; cell experiments further detected the role of the DHRS2 gene after DHRS2 overexpression in A549 and H1299 cells. A rescue experiment was conducted to certify that STAMBPL1 promotes NSCLC progression by regulating DHRS2 expression.ResultsAfter STAMBPL1 knockdown by siRNA. Migration, invasion, colony formation, and proliferation of siRNA groups were suppressed than those of NC groups in A549 and H1299 cells, while the cell apoptosis rate of siRNA groups increased significantly. By using gene-sequence analysis, we found that the expression level of the DHRS2 gene was up-regulated in STAMBPL1 siRNA groups, compared with STAMBPL1 NC (negative control) groups in A549 and H1299, which was verified by qPCR and WB. Further experiments showed that the DHRS2 OE group was suppressed in cell proliferation, migration, and invasion in the A549 and H1299 cell lines compared to the DHRS2 NC group, while DHRS2 OE group was significantly enhanced in the cell apoptosis in the A549 and H1299 cell lines. According to the rescue experiment, cell proliferation, migration, and invasion of the STAMBPL1 SI+DHRS2 SI group were enhanced compared with the STAMBPL1 SI+DHRS2 NC group in A549 and H1299 cells, while the STAMBPL1 SI+DHRS2 OE group were further decreased.ConclusionsThe expression of STAMBPL1 mRNA is significantly up-regulated in LUAD, promoting the progression of LUAD by down-regulating the expression of DHRS2 and acting as a potential biomarker of LUAD.     

Knockdown of LncRNAZFAS1 suppresses cell proliferation and metastasis in non-small cell lung cancer

ABSTRACT

To evaluate the effects of LncRNAZFAS1 on cell proliferation and tumor metastasis in non-small cell lung cancer (NSCLC), we detected the expression level of LncRNAZFAS1 in NSCLC-related tissues and cells. qRT-PCR results revealed that LncRNAZFAS1 in tumor tissues was significantly higher than that in normal lung tissue, especially significantly up-regulated in stage III / IV and in metastatic NSCLC tissues. LncRNAZFAS1 expression was dramatically up-regulated in 4 NSCLC-related cells (A549, SPC-A1, SK-MES-1, and NCI-H1299), with having the highest expression level in A549 cells. Furthermore, we implemented a knockdown of LncRNAZFAS1 in A549 cells, and the results of CCK8 and Transwell assays suggested that knockdown of LncRNAZFAS1 significantly inhibited NSCLC cell proliferation and metastasis. Next, we constructed a tumor xenograft model to evaluate the effect of LncRNAZFAS1 on the NSCLC cell proliferation in vivo. The results indicated that knockdown of LncRNAZFAS1 dramatically inhibited A549 cells proliferation and repressed tumor growth. Additionally, knockdown of LncRNAZFAS1 drastically weakened the expressions of MMP2, MMP9 and Bcl-2 proteins, whereas noticeably strengthened the expression of BAX protein. Our results altogether suggest that knockdown of LncRNAZFAS1 has a negative effect on the proliferation and metastasis of NSCLC cell, which implying LncRNAZFAS1 is a potential unfavorable biomarker in patients with NSCLC.     

Loss of BCAA catabolism enhances Rab1A-mTORC1 signaling activity and promotes tumor proliferation in NSCLC

BackgroundNon-small cell lung cancer (NSCLC) is a leading cause of cancer death. Branched-chain amino acid (BCAA) homeostasis is important for normal physiological metabolism. Branched-chain keto acid dehydrogenase kinase (BCKDK) is a rate-limiting enzyme involved in BCAA degradation. BCAA metabolism has been highlighted in human cancers. The aberrant activation of mTORC1 has been implicated in tumor progression. Rab1A is a small GTPase, an activator of mTORC1, and an oncogene. This study aimed to reveal the specific role of BCKDK-BCAA-Rab1A-mTORC1 signaling in NSCLC.MethodsWe analyzed a cohort of 79 patients with NSCLC and 79 healthy controls. Plasma BCAA assays, immunohistochemistry, and network and pathway analyses were performed. The stable cell lines BCKDK-KD, BCKDK-OV A549, and H1299 were constructed. BCKDK, Rab1A, p-S6 and S6 were detected using western blotting to explore their molecular mechanisms of action in NSCLC. The effects of BCAA and BCKDK on the apoptosis and proliferation of H1299 cells were detected by cell function assays.ResultsWe demonstrated that NSCLC was primarily involved in BCAA degradation. Therefore, combining BCAA, CEA, and Cyfra21-1 is clinically useful for treating NSCLC. We observed a significant increase in BCAA levels, downregulation of BCKDHA expression, and upregulation of BCKDK expression in NSCLC cells. BCKDK promotes proliferation and inhibits apoptosis in NSCLC cells, and we observed that BCKDK affected Rab1A and p-S6 in A549 and H1299 cells via BCAA modulation. Leucine affected Rab1A and p-S6 in A549 and H1299 cells and affected the apoptosis rate of H1299 cells.In conclusion, BCKDK enhances Rab1A-mTORC1 signaling and promotes tumor proliferation by suppressing BCAA catabolism in NSCLC, suggesting a new biomarker for the early diagnosis and identification of metabolism-based targeted approaches for patients with NSCLC.     

Down‐regulation of MTHFD2 inhibits NSCLC progression by suppressing cycle‐related genes

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a bifunctional enzyme located in the mitochondria. It has been reported to be overexpressed in several malignancies. However, the relationship between the expression of MTHFD2 and non‐small cell lung cancer (NSCLC) remains largely unknown. In this study, we found that MTHFD2 was significantly overexpressed in NSCLC tissues and cell lines. Knockdown of MTHFD2 resulted in reduced cell growth and tumorigenicity in vitro and in vivo. Besides, the mRNA and protein expression level of cell cycle genes, such as CCNA2, MCM7 and SKP2, was decreased in MTHFD2 knockdown H1299 cells. Our results indicate that the inhibitory effect of MTHFD2 knockdown on NSCLC may be mediated via suppressing cell cycle‐related genes. These findings delineate the role of MTHFD2 in the development of NSCLC and may have potential applications in the treatment of NSCLC.     

E2F Inhibition Synergizes with Paclitaxel in Lung Cancer Cell Lines

The CDK/Rb/E2F pathway is commonly disrupted in lung cancer, and thus, it is predicted that blocking the E2F pathway would have therapeutic potential. To test this hypothesis, we have examined the activity of HLM006474 (a small molecule pan-E2F inhibitor) in lung cancer cell lines as a single agent and in combination with other compounds. HLM006474 reduces the viability of both SCLC and NSCLC lines with a biological IC₅₀ that varies between 15 and 75 µM, but with no significant difference between the groups. Combination of HLM006474 with cisplatin and gemcitabine demonstrate little synergy; however, HLM006474 synergizes with paclitaxel. Surprisingly, we discovered that brief treatment of cells with HLM006474 led to an increase of E2F3 protein levels (due to de-repression of these promoter sites). Since paclitaxel sensitivity has been shown to correlate with E2F3 levels, we hypothesized that HLM006474 synergy with paclitaxel may be mediated by transient induction of E2F3. To test this, H1299 cells were depleted of E2F3a and E2F3b with siRNA and treated with paclitaxel. Assays of proliferation showed that both siRNAs significantly reduced paclitaxel sensitivity, as expected. Taken together, these results suggest that HLM006474 may have efficacy in lung cancer and may be useful in combination with taxanes.     

The Tumor Suppressor Gene TUSC2 (FUS1) Sensitizes NSCLC to the AKT Inhibitor MK2206 in LKB1-dependent Manner

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.     

Inhibition of RAC1-GEF DOCK3 by miR-512-3p contributes to suppression of metastasis in non-small cell lung cancer

MicroRNAs are a class of small non-coding RNAs regulating gene expression. In this study, we demonstrated that retinoic acid (RA) treatment increases the expression of miR-512-3p. Overexpression of miR-512-3p inhibited cell adhesion, migration, and invasion in non-small cell lung cancer (NSCLC) cell lines A549 and H1299. miR-512-3p inhibitor partially reversed these effects in H1299 cells stably expressing miR-512. We identified DOCK3, a RAC1-GEF (guanine nucleotide exchange factor), as a target gene of miR-512-3p. Overexpression of miR-512-3p led to the decrease of DOCK3 protein but not its mRNA. Knockdown of DOCK3 resulted in similar effects on adhesion, migration, and invasion as observed of miR-512-3p overexpression. Active RAC1 pull-down assay indicated that overexpression of miR-512-3p could decrease the activity of RAC1 with a higher efficiency than that of DOCK3 knockdown. Furthermore, expression of miR-512-3p was suppressed in most NSCLC patient tumor samples compared to its paired normal controls, suggesting that miR-512-3p might play a crucial role in lung cancer development. In conclusion, our results supported that miR-512-3p could inhibit tumor cell adhesion, migration, and invasion by regulating the RAC1 activity via DOCK3 in NSCLC A549 and H1299 cell lines.     

Rsf-1 is overexpressed in non-small cell lung cancers and regulates cyclinD1 expression and ERK activity

Rsf-1 (HBXAP) was recently reported to be overexpressed in various cancers and associated with the malignant behavior of cancer cells. However, the expression of Rsf-1 in primary lung cancer and its biological roles in non-small cell lung cancer (NSCLC) have not been reported. The molecular mechanism of Rsf-1 in cancer aggressiveness remains ambiguous. In the present study, we analyzed the expression pattern of Rsf-1 in NSCLC tissues and found that Rsf-1 was overexpressed at both the mRNA and protein levels. There was a significant association between Rsf-1 overexpression and TNM stage (p=0.0220) and poor differentiation (p=0.0013). Furthermore, knockdown of Rsf-1 expression in H1299 and H460 cells with high endogenous Rsf-1 expression resulted in a decrease of colony formation ability and inhibition of cell cycle progression. Rsf-1 knockdown also induced apoptosis in these cell lines. Further analysis showed that Rsf-1 knockdown decreased cyclin D1 expression and phospho-ERK levels. In conclusion, Rsf-1 is overexpressed in NSCLC and contributes to malignant cell growth by cyclin D1 and ERK modulation, which makes Rsf-1 a candidate therapeutic target in lung cancer.     

Dichloroacetate alters Warburg metabolism,inhibits cell growth,and increases the X-ray sensitivity of human A549 and H1299 NSC lung cancer cells

We investigated whether altering Warburg metabolism (aerobic glycolysis) by treatment with the metabolic agent dichloroacetate (DCA) could increase the X-ray-induced cell killing of the radiation-resistant human non-small-cell lung cancer (NSCLC) cell lines A549 and H1299. Treatment with 50 mM DCA decreased lactate production and glucose consumption in both A549 and H1299, clear indications of attenuated aerobic glycolysis. In addition, we found that DCA treatment also slowed cell growth, increased population-doubling time, and altered cell cycle distribution. Furthermore, we report that treatment with 50 mM DCA significantly increased single and fractionated X-ray-induced cell killing of A549 and H1299 cells. Assay of DNA double-strand break repair by neutral comet assays demonstrated that DCA inhibited both the fast and the slow kinetics of X-ray-induced DSB repair in both A549 and H1299 NSCL cancer cells. Taken together the data suggest a correlation between an attenuated aerobic glycolysis and enhanced cytotoxicity and radiation-induced cell killing in radiation-resistant NSCLC cells.     

NUSAP1 knockdown inhibits cell growth and metastasis of non-small-cell lung cancer through regulating BTG2/PI3K/Akt signaling

Non-small-cell lung cancer (NSCLC) is the most common malignancy along with high mortality rate worldwide. Recently, nucleolar and spindle-associated protein 1 (NUSAP1) has been reported to be involved in the malignant progression of several cancers. However, in NSCLC, the biological function of NUSAP1 and its molecular mechanism have not been reported. Here, our findings indicated that the NUSAP1 messenger RNA expression level was remarkably upregulated in NSCLC tissues compared with that of adjacent normal tissues. We also found that NUSAP1 gene expression was notably upregulated in NSCLC cell lines (A549, 95-D, H358, and H1299) compared with that of normal human bronchial epithelial cell line (16HBE). Subsequently, the biological function of NUSAP1 was investigated in A549 and H358 cells transfected with NUSAP1 small interfering RNA (siRNA), respectively. Results showed that NUSAP1 knockdown inhibited NSCLC cell proliferation, and promoted cell apoptosis. Furthermore, the number of cell migration and invasion was significantly suppressed by NUSAP1 knockdown. In addition, our results indicated that NUSAP1 knockdown increased the gene expression of B-cell translocation gene 2 (BTG2), but decreased the expression levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT). BTG2 siRNA partly abrogates the effect of NUSAP1 knockdown on BTG2 gene expression. Fumonisin B1 (FB1), a AKT activator, reversed the effect of NUSAP1 knockdown on the biological function in NSCLC. Taken together, NUSAP1 knockdown promotes NSCLC cell apoptosis, and inhibits cell proliferation, cell migration, and invasion, which is associated with regulating BTG2/PI3K/Akt signal pathway. Our findings suggest that NUSAP1 is a promising molecular target for NSCLC treatment.     

Prediction of nonsmall cell lung cancer sensitivity to cisplastin and paclitaxel based on marker gene expression

The goal of the present study was to define gene expression signatures that predict a chemosensitivity of nonsmall cell lung cancer (NSCLC) to cisplatin and paclitaxel. To generate a set of candidate genes likely to be predictive, current knowledge of the pathways involved in resistance and sensitivity to individual drugs was used. Forty-four genes coding proteins belonging to the following categories—ATP-dependent transport proteins, detoxification system proteins, reparation system proteins, tubulin and proteins responsible for its synthesis, cell cycle, and apoptosis proteins—were considered. Eight NSCLC cell lines (A549, Calu1, H1299, H322, H358, H460, H292, and H23) were used in our study. For each NSCLC cell line, a cisplatin and paclitaxel chemosensitivity, as well as an expression level of 44 candidate genes, were evaluated. To develop a chemosensitivity prediction model based on selected genes’ expression level, a multiple regression analysis was performed. The model based on the expression level of 11 genes (TUBB3, TXR1, MRP5, MSH2, ERCC1, STMN, SMAC, FOLR1, PTPN14, HSPA2, GSTP1) allowed us to predict the paclitaxel cytotoxic concentration with a high level of correlation (r = 0.91, p < 0.01). However, no model developed was able to reliably predict sensitivity of the NSCLC cells to cisplatin.     

Epidermal growth factor receptor mutation in combination with expression of MIG6 alters gefitinib sensitivity

Background  

Epidermal growth factor receptor (EGFR) signaling plays an important role in the regulation of cell proliferation, survival, metastasis, and invasion in various tumors. Earlier studies showed that the EGFR is frequently overexpressed in non-small-cell lung cancer (NSCLC) and EGFR mutations at specific amino acid residues in the kinase domain induce altered responsiveness to gefitinib, a small molecule EGFR tyrosine kinase inhibitor. However, the mechanism underlying the drug response modulated by EGFR mutation is still largely unknown. To elucidate drug response in EGFR signal transduction pathway in which complex dynamics of multiple molecules involved, a systematic approach is necessary. In this paper, we performed experimental and computational analyses to clarify the underlying mechanism of EGFR signaling and cell-specific gefitinib responsiveness in three H1299-derived NSCLC cell lines; H1299 wild type (H1299WT), H1299 with an overexpressed wild type EGFR (H1299EGFR-WT), and H1299 with an overexpressed mutant EGFR L858R (H1299L858R; gefitinib sensitive mutant).     

PPDPF Promotes the Progression and acts as an Antiapoptotic Protein in Non-Small Cell Lung Cancer

Resistance to radiotherapy is frequently observed in the clinic and leads to poor prognosis of non-small cell lung cancer (NSCLC). How to overcome resistance to radiotherapy is a challenge in the treatment of NSCLC. In this study, PPDPF was found to be upregulated in NSCLC tissues and cell lines, and its expression negatively correlated with the overall survival of patients with NSCLC. PPDPF promoted the growth, colony formation and invasion of lung cancer cells. Moreover, knockout of PPDPF inhibited tumorigenesis in the KL (Kras^G12D; LKB1^f/f) mouse model of lung cancer. Additionally, overexpression of PPDPF led to radioresistance in lung cancer cells, and knockdown of PPDPF sensitized lung cancer cells to radiotherapy. Mechanistically, PPDPF interacted with BABAM2 (an antiapoptotic protein) and blocked its ubiquitination by MDM2, thus stabilizing BABAM2 and promoting the radioresistance of lung cancer cells. Our present study suggested PPDPF as a therapeutic target in NSCLC.     

Influence of p53 expression on sensitivity of cancer cells to bleomycin

In this study, we determined whether p53 expression affected the sensitivity of non–small cell lung cancer (NSCLC) and colon cancer cells to bleomycin (BLM). Two human NSCLC cell lines (A549 expressing wild‐type p53 and p53‐null H1299) and two colon cancer cell lines (HCT116 p53+/+ and its p53 deficient subline HCT116 p53?/?) were subjected to treatment with BLM. Cells were treated with various concentrations of BLM, and cellular viability was assessed by formazan assay. To investigate the role of p53 in BLM sensitivity, we transduced cells with adenovirus with wild‐type p53, dominant‐negative p53, and GFP control, and analyzed the effect on cellular viability. Cells expressing wild‐type p53 were more sensitive to BLM than p53‐null cells in both NSCLC and colon cancer cells. Sensitivity to BLM of the cells with wild‐type p53 was reduced by overexpression of dominant‐negative p53, while BLM sensitivity of p53‐null cells was increased by wild‐type p53 in both NSCLC cells and colon cancer cells. In conclusion, our data show that p53 sensitizes all four cancer cells lines tested to BLM toxicity and overexpression of p53 confers sensitivity to the cytotoxic activity of the anticancer agent in p53‐null cells. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:260–269, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20334     

mTOR inhibitors radiosensitize PTEN‐deficient non‐small‐cell lung cancer cells harboring an EGFR activating mutation by inducing autophagy

Clinical resistance to gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), in patients with lung cancer has been linked to acquisition of the T790M resistance mutation in activated EGFR or amplification of MET. Phosphatase and tensin homolog (PTEN) loss has been recently reported as a gefitinib resistance mechanism in lung cancer. The aim of this study was to evaluate the efficacy of radiotherapy in non‐small‐cell lung cancer (NSCLC) with acquired gefitinib resistance caused by PTEN deficiency to suggest radiotherapy as an alternative to EGFR TKIs. PTEN deficient‐mediated gefitinib resistance was generated in HCC827 cells, an EGFR TKI sensitive NSCLC cell line, by PTEN knockdown with a lentiviral vector expressing short hairpin RNA‐targeting PTEN. The impact of PTEN knockdown on sensitivity to radiation in the presence or absence of PTEN downstream signaling inhibitors was investigated. PTEN knockdown conferred acquired resistance not only to gefitinib but also to radiation on HCC827 cells. mTOR inhibitors alone failed to reduce HCC827 cell viability, regardless of PTEN expression, but ameliorated PTEN knockdown‐induced radioresistance. PTEN knockdown‐mediated radioresistance was accompanied by repression of radiation‐induced cytotoxic autophagy, and treatment with mTOR inhibitors released the repression of cytotoxic autophagy to overcome PTEN knockdown‐induced radioresistance in HCC827 cells. These results suggest that inhibiting mTOR signaling could be an effective strategy to radiosensitize NSCLC harboring the EGFR activating mutation that acquires resistance to both TKIs and radiotherapy due to PTEN loss or inactivation mutations. J. Cell. Biochem. 114: 1248–1256, 2013. © 2012 Wiley Periodicals, Inc.