LINC01559 promotes lung adenocarcinoma metastasis by disrupting the ubiquitination of vimentin.

BACKGROUND: Distant metastasis is the major cause of lung adenocarcinoma (LUAD)-associated mortality. However, molecular mechanisms involved in LUAD metastasis remain to be fully understood. While the role of long non-coding RNAs (lncRNAs) in cancer development, progression, and treatment resistance is being increasingly appreciated, the list of dysregulated lncRNAs that contribute to LUAD pathogenesis is also rapidly expanding. METHODS: Bioinformatics analysis was conducted to interrogate publicly available LUAD datasets. In situ hybridization and qRT-PCR assays were used to test lncRNA expression in human LUAD tissues and cell lines, respectively. Wound healing as well as transwell migration and invasion assays were employed to examine LUAD cell migration and invasion in vitro. LUAD metastasis was examined using mouse models in vivo. RNA pulldown and RNA immunoprecipitation were carried out to test RNA-protein associations. Cycloheximide-chase assays were performed to monitor protein turnover rates and Western blotting was employed to test protein expression. RESULTS: The expression of the lncRNA LINC01559 was commonly upregulated in LUADs, in particular, in those with distant metastasis. High LINC01559 expression was associated with poor outcome of LUAD patients and was potentially an independent prognostic factor. Knockdown of LINC01559 diminished the potential of LUAD cell migration and invasion in vitro and reduced the formation of LUAD metastatic lesions in vivo. Mechanistically, LINC01559 binds to vimentin and prevents its ubiquitination and proteasomal degradation, leading to promotion of LUAD cell migration, invasion, and metastasis. CONCLUSION: LINC01559 plays an important role in LUAD metastasis through stabilizing vimentin. The expression of LINC01559 is potentially an independent prognostic factor of LUAD patients, and LINC01559 targeting may represent a novel avenue for the treatment of late-stage LUAD.

Obstructive sleep apnea promotes the progression of lung cancer by modulating cancer cell invasion and cancer-associated fibroblast activation via TGFß signaling.

OBJECTIVE: Obstructive sleep apnea (OSA) is associated with severity of pneumonia; however, the mechanism by which OSA promotes lung cancer progression is unclear. METHODS: Twenty-five lung cancer patients were recruited to investigate the relationship between OSA and cancer-associated fibroblast (CAFs) activation. Lung cancer cells (A549) and WI38 fibroblast cells were used to explore the hypoxia-induced TGFß expression using qPCR, Western blot, and ELISA. Wound healing and transwell assays were performed to evaluate cancer cell migration and invasion. A549 or A549-Luc + WI38 xenograft mouse models were established to detect the intermittent hypoxia (IH) associated with lung tumor growth and epithelial-mesenchymal transition (EMT) in vivo. RESULTS: OSA promotes CAF activation and enrichment in lung cancer patients. Hypoxia (OSA-like treatment) activated TGFß signaling in both lung cancer cells and fibroblasts, which promoted cancer cell migration and invasion, and enriched CAFs. IH promoted the progression and EMT process of lung cancer xenograft tumor. Co-inoculation of lung cancer cells and fibroblast cells could further promote lung cancer progression. CONCLUSIONS: IH promotes lung cancer progression by upregulating TGFß signaling, promoting lung cancer cell migration, and increasing the CAF activation and proportion of lung tumors.

SNHG3/WISP2 Axis Promotes Hela Cell Migration and Invasion via Activating Wnt/ß-Catenin Signaling.

BACKGROUND/AIM: Cervical cancer (CC) poses a significant threat to women's health and has a relatively poor prognosis due to local invasion and metastasis. It is, therefore, crucial to elucidate the molecular mechanisms of CC metastasis. SNHG3 has been implicated in various tumor metastasis processes, but its involvement in CC has not been thoroughly studied. Our study aimed to investigate the role of SNHG3 in metastasis and elucidate its underlying mechanisms in CC. MATERIALS AND METHODS: LncRNA SNHG3 expression in CC tissues was analyzed using TCGA and GSE27469 databases. Normal cervical epithelial cells and CC cell lines were used to detect mRNA expression of SNHG3 via quantitative reverse transcription polymerase chain reaction (qRT-PCR). With RNA interference (RNAi) technology, antisense oligonucleotides (ASO) can act on HeLa cells to knockdown target gene expression. The influence of SNHG3 on cell migration and invasion were determined by wound healing and transwell assays. Transcriptome sequencing (RNA-seq) was used to seek abnormally expressed genes between SNHG3 knockdown cells and control cells. The expressions of epithelial-mesenchymal transition (EMT) and Wnt/ß-catenin signaling related proteins were detected using western blot. RESULTS: SNHG3 was obviously up-regulated in CC tissues and cell lines, and ectopic expression of SNHG3 was associated with lymph node metastasis of CC. Knockdown of SNHG3 significantly inhibited cell migration and invasion in CC. Further molecular mechanism studies showed that SNHG3 knockdown could down-regulate the expression of WNT1 Inducible Signaling Pathway Protein 2 (WISP2) so as to inhibit the activation of the Wnt/ß-catenin signaling pathway, and regulated the expression of EMT-related markers, that promoted the protein expression of E-cadherin, as well as decreased the expression of N-cadherin and vimentin. CONCLUSION: SNHG3 appears to exert a pro-metastatic effect in CC, as evidenced by inhibition of cell migration and invasion upon SNHG3 knockdown. EMT also appears to be attenuated. Of interest is the down-regulation of WISP2 following SNHG3 knockdown leads to the inactivation of the Wnt/ß-catenin signaling pathway.

Intermittent hypoxia inhibits anti-tumor immune response via regulating PD-L1 expression in lung cancer cells and tumor-associated macrophages.

Accumulating evidence has shown an increased tumor incidence and reduced survival rate in cancer patients with obstructive sleep apnea (OSA). Although intermittent hypoxia is known to play a crucial role, the molecular mechanism by which intermittent hypoxia accelerates lung cancer progression remains to be elucidated.A lung cancer xenograft mouse model was established by subcutaneously injecting LLC cells into C57BL/6 mice. The tumor-bearing mice were exposed to either normoxia or intermittent hypoxia and received either IgG2a, anti-programmed death ligand-1 (PD-L1), PX-478, or anti-PD-L1 + PX-478 treatment.A significant upregulation of tumor associated macrophages (TAMs) papulation and PD-L1 levels was observed in lung adenocarcinoma patients with OSA. We further confirmed that hypoxia-inducible factor-1 alpha (HIF-1α) regulates PD-L1 at transcriptional levels, mainly through binding to the hypoxia response element 4. Using a lung cancer xenograft mouse model, we observed that intermittent hypoxia exposed tumors grew faster and bigger with upregulated HIF-1α and PD-L1 expression, enhanced TAMs and Treg populations, and reduced cytotoxic T cells and cytokine secretion. Finally, we found a combination of PX-478 and anti-PD-L1 exerted an encouraging tumor inhibition effect compared to single treatment. Combination therapies based on HIF-1α and PD-L1 blockade might serve as a promising strategy to treat lung cancer patients with OSA.

KIF14 mediates cabazitaxel-docetaxel cross-resistance in advanced prostate cancer by promoting AKT phosphorylation.

Docetaxel is a first-line chemotherapy drug for castration-resistant prostate cancer (CRPC); yet, some CRPC patients develop docetaxel drug resistance. Cabazitaxel is approved in the post-docetaxel treatment setting. However, recent studies suggested cross-resistance between the development of drug resistance and current treatments. In this study, we used docetaxel-resistant cell lines DU145/DTX50 and PC-3/DTX30 to measure the responses to cabazitaxel. Our findings demonstrated that docetaxel resistance could lead to cross-resistance to cabazitaxel. After docetaxel-resistant cells were treated with cabazitaxel, transcriptome analysis was performed, and the results were analyzed in combination with survival analysis and correlation analysis with Gleason score to screen the cross-resistance genes. The continuously increased expression of kinesin family member 14 (KIF14) was identified as the main cause of cross-resistance to cabazitaxel in docetaxel-resistant cells. Silencing the expression of KIF14 could restore the sensitivity of resistant PCa cells to docetaxel and cabazitaxel, attenuate proliferation and promote apoptosis of the resistant PCa cells. Notably, the depressed expression of KIF14 inhibited the phosphorylation of Akt located downstream. In summary, KIF14 mediates the cross-resistance between docetaxel and cabazitaxel, and targeting KIF14 could be an effective measurement for reversing docetaxel or cabazitaxel chemotherapy failure or enhancing the anti-tumor effects of docetaxel or cabazitaxel.

The N-Myc-responsive lncRNA MILIP promotes DNA double-strand break repair through non-homologous end joining.

The protooncoprotein N-Myc, which is overexpressed in approximately 25% of neuroblastomas as the consequence of MYCN gene amplification, has long been postulated to regulate DNA double-strand break (DSB) repair in neuroblastoma cells, but experimental evidence of this function is presently scant. Here, we show that N-Myc transcriptionally activates the long noncoding RNA MILIP to promote nonhomologous end-joining (NHEJ) DNA repair through facilitating Ku70-Ku80 heterodimerization in neuroblastoma cells. High MILIP expression was associated with poor outcome and appeared as an independent prognostic factor in neuroblastoma patients. Knockdown of MILIP reduced neuroblastoma cell viability through the induction of apoptosis and inhibition of proliferation, retarded neuroblastoma xenograft growth, and sensitized neuroblastoma cells to DNA-damaging therapeutics. The effect of MILIP knockdown was associated with the accumulation of DNA DSBs in neuroblastoma cells largely due to decreased activity of the NHEJ DNA repair pathway. Mechanistical investigations revealed that binding of MILIP to Ku70 and Ku80 increased their heterodimerization, and this was required for MILIP-mediated promotion of NHEJ DNA repair. Disrupting the interaction between MILIP and Ku70 or Ku80 increased DNA DSBs and reduced cell viability with therapeutic potential revealed where targeting MILIP using Gapmers cooperated with the DNA-damaging drug cisplatin to inhibit neuroblastoma growth in vivo. Collectively, our findings identify MILIP as an N-Myc downstream effector critical for activation of the NHEJ DNA repair pathway in neuroblastoma cells, with practical implications of MILIP targeting, alone and in combination with DNA-damaging therapeutics, for neuroblastoma treatment.

CD47 blockade enhances therapeutic efficacy of cisplatin against lung carcinoma in a murine model.

Cisplatin (CDDP) is the first generation of platinum-based drug and is widely used to treat many cancers due to its potency. The present study aims to explore the effects of CDDP on lung carcinoma and its relationship with macrophage phagocytosis. In in vitro study, murine and human lung cancer cell lines were applied and treated with CDDP, CD47 antibody (aCD47), or CDDP plus aCD47. In in vivo study, a tumor xenograft animal model was treated with CDDP, aCD47, or CDDP plus aCD47. Real-time PCR was applied to determine the mRNA expressions. Enzyme-linked immunosorbent assay (ELISA), Western blotting, and Immunofluorescent staining were applied to determine the protein expressions. Flow cytometry was applied to analyze cell apoptosis, phagocytosis, and specific cell populations. CDDP enhanced the expressions of CD47 in lung cancer cells. Interestingly, the blockage of CD47 enhanced the macrophages' phagocytic activity on the CDDP-treated tumor cells. The treatment of CDDP and aCD47 exhibited anti-tumor effects and prolonged the LLC tumor-bearing mice survival time. Mechanistic studies revealed that the treatment of CDDP and aCD47 regulated the phagocytic activity of macrophage, percentage of CD8+ T cells, and cytokines (tumor growth factor (TGF)-ß, interleukin (IL)12p70, and interferon (IFN)-γ) in the tumor-bearing model. CD47 blockade enhanced therapeutic efficacy of cisplatin against lung carcinoma in vivo and in vitro.

ABCA8-mediated efflux of taurocholic acid contributes to gemcitabine insensitivity in human pancreatic cancer via the S1PR2-ERK pathway.

The development of resistance to anticancer drugs is believed to cause chemotherapy failure in pancreatic cancer (PC). The efflux of anticancer drugs mediated by ATP-binding cassette (ABC) transporters is a widely accepted mechanism for chemoresistance, but for ABCA subfamily members, which are characterized by their ability to transport lipids and cholesterol, its role in chemoresistance remains unknown. Here we found that the expression of ABCA8, a member of ABCA subfamily transporters, was significantly increased in human PC cells after gemcitabine (GEM) treatment, as well as in established GEM-resistant (Gem-R) PC cells. Importantly, ABCA8 knockdown reversed the chemoresistance phenotype of Gem-R cells, whereas ABCA8 overexpression significantly decreased the sensitivity of human PC cells to GEM, both in vitro and in vivo, demonstrating an important role of ABCA8 in regulating chemosensitivity. Moreover, our results showed that treatment with taurocholic acid (TCA), an endogenous substrate of ABCA8, also induced GEM insensitivity in PC cells. We further demonstrated that ABCA8 mediates the efflux of TCA out of PC cells, and that extracellular TCA activates extracellular signal-regulated kinase (ERK) signaling via the sphingosine 1-phosphate receptor 2 (S1PR2), which is responsible for ABCA8-induced GEM ineffectiveness. Together, these findings reveal a novel TCA-related mechanism of ABCA subfamily transporter-mediated chemoresistance that goes beyond the role of a drug pump and suggest ABCA8 or the TCA-S1RP2-ERK pathway as potential targets for improving the effectiveness of and overcoming the resistance to chemotherapy in PC.

Cadherin 13 Inhibits Pancreatic Cancer Progression and Epithelial-mesenchymal Transition by Wnt/ß-Catenin Signaling.

Cadherin 13 (CDH13) is an atypical cadherin that exerts tumor-suppressive effects on cancers derived from epithelial cells. Although the CDH13 promoter is frequently hypermethylated in pancreatic cancer (PC), the direct impact of CDH13 on PC is unknown. Accordingly, the expression of CDH13 in PC cell lines and paired PC tissues was examined by immunohistochemistry, quantitative real-time PCR and western blotting. Our findings showed that CDH13 was downregulated in PC tissues and cell lines. Moreover, cell proliferation, migration and invasion were detected by CCK-8 assay, transwell migration assay and transwell invasion assay, respectively. Xenograft tumor experiments were used to determine the biological function of CDH13 in vivo. As revealed by our data, CDH13 overexpression significantly inhibited the proliferation, migration and invasion of human PC cells in vitro. The inhibitory effect of CDH13 on PC was further confirmed in animal models. Mice subcutaneously or orthotopically transplanted with CDH13-overexpressing CFPAC-1 cells developed significantly smaller tumors with less liver metastases and mesenteric metastases than those of the control group. Next, transcriptomics and western blot analysis were used to identify the underlying mechanisms. Further molecular mechanism studies showed that CDH13 overexpression inhibited the activation of the Wnt/ß-catenin signaling pathway and regulated the expression of epithelial-mesenchymal transition (EMT)-related markers. Our results indicated that CDH13 displayed an inhibitory effect on PC and suggested that CDH13 might be a potential biomarker and a new therapeutic target for PC.

Exposure to an enriched environment promotes the terminal maturation and proliferation of natural killer cells in mice.

The maturation of natural killer (NK) cells is critical for the acquisition of robust effector functions and the immune response to tumors. However, the influence of psychological stress on NK-cell maturation remains unknown. In this study, we investigated the alteration of NK-cell maturation in response to enriched environment (EE) exposure, which induced eustress, or positive stress, in mice. Analysis of markers representing distinct mature stages revealed that EE promoted the terminal maturation of NK cells both centrally in the bone marrow and peripherally in the spleen and blood. Additionally, EE increased CD27+ immature and intermediate-mature NK cell proliferation in the bone marrow. Furthermore, EE exposure brought about a similar promoting effect on NK-cell maturation in tumor-bearing mice. In tumor-bearing mice, EE substantially enhanced the proliferative potential of splenic CD27+ NK cells compared to those in the bone marrow. EE-housed mice displayed a tumor-resistant phenotype and an increased proportion of intratumoral NK cells, especially CD11b+ CD27- mature NK cells, while splenectomy abolished the tumor-retardant effect caused by EE and EE-induced NK-cell infiltration into tumors. Given that our previous study demonstrated an important role for NK cells in EE-induced tumor inhibition, the findings of this study further indicate that the enhanced maturation and proliferation of splenic NK cells may contribute to EE-induced tumor inhibition to some extent. Taken together, the results of this study suggest a positive modulating effect of environment-induced eustress on NK-cell maturation, with potential implications for understanding how eustress boosts NK-cell antitumor immunity.

Renoprotective effects of propofol on the expression of iNOS protein in rats with ischemia reperfusion injury.

AIMS: This study aims to explore the pathogenesis of myocardial ischemia-reperfusion injury and the treatment method. METHODS: Myocardial Ischemia-reperfusion rat model was established in this study. They were divided into three groups: sham operation (SO) group, IRI control (C) group and IRI with propofol (A) group (n = 9). Myocardial infarct size was compared with HE staining method. TUNEL assay was used to detect cell apoptosis. Changes in the expression of iNOS were detected using real-time PCR and Western blotting methods. RESULTS: Myocardial infarct size of control group and propofol group was 53.03 ± 8.90% and 34.73 ± 7.20% respectively, there were significant differences between them (P < 0.01). Apoptotic index of two groups was 0.21 ± 0.02 and 0.31 ± 0.05, with statistical significance (P < 0.05). The expression levels of iNOS in propofol group reduced significantly (P < 0.05). CONCLUSION: The levels of iNOS increase in IRI rats, suggesting that the severity of myocardial failure may be correlated with iNOS; propofol can specifically inhibit iNOS and thus protect the myocardial function.

[Arsenic trioxide restores ERα expression in ERα-negative human breast cancer cells and its treatment efficacy in combination with tamoxifen in xenografts in nude mice].

OBJECTIVE: To study the demethylation effect of arsenic trioxide (As2O3) on ERα-negative human breast cancer MDA-MB-435s cells and its possible mechanisms, and to observe its treatment efficacy in combination with tamoxifen (TAM) after ERα re-expression. METHODS: MTT assay was used to examine the inhibitory effect of As2O3 treatment alone or in combination with TAM on cell proliferation. A nude mouse xenograft model was used to further examine the treatment efficacy in vivo. MSP was used to detect the methylation status of ERα gene after treated with As2O3 in MDA-MB-435s cells and the transplanted tumor tissues. RT-PCR was used to detect the mRNA expression of DNMT1 and Erα. Western bolt was used to detect the DNMT1 and ERα protein expression. The diameter of xenograft tumors was measured weekly, and the tumor growth curve was drawn. RESULTS: The level of proliferation of the MDA-MB-435s cells was significantly suppressed after treatment with different concentration of As2O3 alone or As2O3 combined with TAM, and the 4 µmol/L As2O3 + TAM treatment for 72 h showed the highest inhibition rate (62.6%). 1, 2, 4 µmol/L As2O3 had demethylation effect on MDA-MB-435s cells, and the DNMT1 mRNA and protein expression was inhibited and accompanied by ERα mRNA and protein re-expression. The unmethylation specific bands of ERα gene were enhanced after treated by As2O3 alone or As2O3 combined with TAM in the xenograft tumors. The expression of DNMT1 mRNA and protein was inhibited, and accompanied by ERα mRNA and protein re-expression. An significant decrease of volume and weight of the xenograft tumors in the As2O3 treated alone or combined with TAM groups was observed compared with those of the normal saline group or TAM alone group (P < 0.05), and the 4 mg/kg As2O3 + TAM group had the highest inhibition rate of tumor weight (79.5%) and volume (76.4%). CONCLUSIONS: ERα can be re-expressed in ERα-negative breast cancer MDA-MB-435s cells after treated with As2O3 by inhibiting the DNMT1 activity. MDA-MB-435s cells are re-sensitized to endocrine therapy after ERα re-expression. As2O3 combined with TAM may provide a new therapeutic approach for patients with ERα-negative breast cancer in the clinic.