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Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), used for treating patients with advanced non-small-cell lung cancer (NSCLC) harboring EGFR-activating mutations or the resistant T790M mutation. However, acquired resistance to osimertinib is inevitable in EGFR-mutant NSCLC. By employing a global mass spectrometry-based phosphoproteomics approach, we identified that the activated p21-activated kinase 2 (PAK2)/β-catenin axis acts as a driver of osimertinib resistance. We found that PAK2 directly phosphorylates β-catenin and increases the nuclear localization of β-catenin, leading to the increased expression and transcriptional activity of β-catenin, which in turn enhances cancer stem-like properties and osimertinib resistance. Moreover, we revealed that HER3 as an upstream regulator of PAK2, drives the activation of PAK2/β-catenin pathways in osimertinib-resistant cells. The clinical relevance of these findings was further confirmed by examining tissue specimens from patients with EGFR-mutant NSCLC. The results demonstrated that the levels of HER3, phospho-PAK2 (p-PAK2) and β-catenin in the tissues from patients with EGFR-mutant NSCLC, that had relapsed after treatment with osimertinib, were elevated compared to those of the corresponding untreated tissues. Additionally, the high levels of HER3, p-PAK2 and β-catenin correlated with shorter progression-free survival (PFS) in patients with EGFR-TKI-treated NSCLC. We additionally observed that the suppression of PAK2 via knockdown or pharmacological targeting with PAK inhibitors markedly restored the response of osimertinib-resistant NSCLC cells to osimertinib both in vitro and in vivo. In conclusion, these results indicated that the PAK2-mediated activation of β-catenin is important for osimertinib resistance and targeting the HER3/PAK2/β-catenin pathway has potential therapeutic value in NSCLCs with acquired resistance to osimertinib.

This stud intends to assess whether exosome miR-155-5p derived from human non-small cell lung cancer cells (Hcc827) activates macrophages in lung cancer. Lung cancer Hcc827 cells were assigned into control group and expeirmental group (cultured with macrophages, THP-1 activated by exosome miR-155-5P derived from Hcc827) followed by analysis of macrophage markers inducible nitric oxide synthase (INOS), recombinant human CD163 (CD163), matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 2 (MMP2), and E-cadherin by real-time fluorescent quantitative PCR (RFQ-PCR), IL-10, IL-6 and IL-8 levels by chemiluminescence, cell invasion by Transwell assay and related protein expression by Western blot. miR-155-5p treatment significantly reduced INOS and TNF-β expressions and increased CD163, TNF-α, IL-8, IL-6 and IL-10 expressions along with enhanced cell invasion. In addition, MMP9 and MMP2 expressions in experimental group were significantly increased and E-cdherin was reduced. In conclusion, exosome miR-155-5p derived from lung cancer Hcc827 cells activates macrophages and enhanced lung cancer cell invasion.

BackgroundOne of the most diverse tumors is pancreatic cancer (PC), which makes predicting the prognosis challenging. PC development is directly related to hypoxia, angiogenesis, and immunotherapy. It is still unclear how the three features are related.MethodsThe Genotype-Tissue Expression (GTEx) and the Cancer Genome Atlas (TCGA) database were employed to obtain sequencing data for healthy pancreatic tissues and PC tissues, respectively. According to the constructed hypoxic prognostic model (HPM) and angiogenic prognostic model (APM), 4 subtypes of PC were identified. Hypoxia and angiogenesis prognostic model (HAPM) was established based on differentially expressed genes (DEGs) between high-angiogenesis/high-hypoxia (HH) and low-angiogenesis/low-hypoxia (LL) subgroups. Base on the median risk score, PC patients were separated into high-risk and low-risk groups, and clinical traits, prognosis, percentage of immune cell infiltration, PD-1 expression, and the fraction of T-cell depletion were compared between the groups. Finally, the predictive accuracy of the tumor immune dysfunction and rejection (TIDE) and tumor inflammatory signature (TIS) models, as well as HAPM, was compared.ResultWe analyzed the mRNA sequencing data from 178 PC tissues and 171 normal pancreatic tissues to obtain 9527 DEGs. We discovered 200 genes linked with hypoxia and 36 genes involved with angiogenesis through the literature. We found the core genes related with hypoxia and angiogenesis in PC by intersecting the DEGs of the HH and LL subgroups with those of PC via WGCNA. IL-17 signaling pathway, ECM-receptor interactions, cytokine receptor interactions, etc. were all enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) results of core genes. HAPM has good predictive efficiency, according to an evaluation of KM survival curves and ROC curves. The external dataset also validated the model’s ability to anticipate outcomes. Patients in the high- and low-risk groups were compared for PD1 expression and T-cell exclusion scores, which suggested that the model might be used to forecast which PC patients might benefit from immunotherapy.ConclusionsThe probable molecular processes connecting hypoxia and angiogenesis are described in this work, and a model is developed that may be utilized to forecast the prognosis for PC patients and the benefits of immunotherapy.

BackgroundThe development of HCC is often associated with extensive metabolic disturbances. Single cell RNA sequencing (scRNA-seq) provides a better understanding of cellular behavior in the context of complex tumor microenvironments by analyzing individual cell populations. MethodsThe Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data was employed to investigate the metabolic pathways in HCC. Principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) analysis were applied to identify six cell subpopulations, namely, T/NK cells, hepatocytes, macrophages, endothelial cells, fibroblasts, and B cells. The gene set enrichment analysis (GSEA) was performed to explore the existence of pathway heterogeneity across different cell subpopulations. Univariate Cox analysis was used to screen genes differentially related to The Overall Survival in TCGA-LIHC patients based on scRNA-seq and bulk RNA-seq datasets, and LASSO analysis was used to select significant predictors for incorporation into multivariate Cox regression. Connectivity Map (CMap) was applied to analysis drug sensitivity of risk models and targeting of potential compounds in high risk groups.ResultsAnalysis of TCGA-LIHC survival data revealed the molecular markers associated with HCC prognosis, including MARCKSL1, SPP1, BSG, CCT3, LAGE3, KPNA2, SF3B4, GTPBP4, PON1, CFHR3, and CYP2C9. The RNA expression of 11 prognosis-related differentially expressed genes (DEGs) in normal human hepatocyte cell line MIHA and HCC cell lines HCC-LM3 and HepG2 were compared by qPCR. Higher KPNA2, LAGE3, SF3B4, CCT3 and GTPBP4 protein expression and lower CYP2C9 and PON1 protein expression in HCC tissues from Gene Expression Profiling Interactive Analysis (GEPIA) and Human Protein Atlas (HPA) databases. The results of target compound screening of risk model showed that mercaptopurine is a potential anti-HCC drug.ConclusionThe prognostic genes associated with glucose and lipid metabolic changes in a hepatocyte subpopulation and comparison of liver malignancy cells to normal liver cells may provide insight into the metabolic characteristics of HCC and the potential prognostic biomarkers of tumor-related genes and contribute to developing new treatment strategies for individuals.

Pancreatic cancer (PC) is one of the most malignant types of cancer, and is characterized by early metastasis, limited response to chemotherapeutics, and poor prognosis. Therefore, there is an urgent need to explore new therapeutic strategies for PC treatment. Human rhomboid-like 2 (RHBDL2) is differentially expressed in cervical and breast cancer. However, the correlation between RHBDL2 and PC remains unclear. We found that RHBDL2 is highly expressed in human PC cells and tissues and is significantly associated with distant metastasis and poor survival of patients with PC. Gain- and loss-of-function assays indicated that RHBDL2 could accelerate PC cell proliferation and mobility in vitro and in vivo. The RNA-Seq results suggest that RHBDL2 may be involved in the activation of Notch signaling pathway. IMR-1 could restore the proliferation and metastatic capacity of PC cells mediated by RHBDL2. RHBDL2 interacted with and cleaved Notch1, resulting in the release of N1ICD. RHBDL2 decreased the ubiquitination level of N1ICD and collaborated with Ovarian tumor domain-containing 7B (OTUD7B) to stabilize N1ICD via the ubiquitin-proteasome pathway. RHBDL2 facilitated PC cell proliferation and mobility by stabilizing the N1ICD via the OTUD7B and activating the Notch signaling pathway. Thus, targeting this novel pathway may be a potential therapeutic strategy for PC.

Although FOXD1 has been found to be involved in the malignant processes of several types of cancers, its role in pancreatic cancer (PC) is not well understood. This study aimed to investigate the expression and function of FOXD1 in PC. We found that FOXD1 mRNA and protein expression were upregulated in PC tissues compared with non-tumor tissues, and high expression level of FOXD1 was associated with an adverse prognostic index of PC. The results of in vitro and in vivo assays indicate that overexpression of FOXD1 promotes aerobic glycolysis and the capacity of PC cells to proliferate, invade, and metastasize, whereas FOXD1 knockdown inhibits these functions. The results of mechanistic experiments suggest that FOXD1 can not only directly promote SLC2A1 transcription but also inhibit the degradation of SLC2A1 through the RNA-induced silencing complex. As a result, FOXD1 enhances GLUT1 expression and ultimately facilitates PC cell proliferation, invasion, and metastasis by regulating aerobic glycolysis. Taken together, FOXD1 is suggested to be a potential therapeutic target for PC.

Morphogenesis and organogenesis in the low organisms have been found to be modulated by a number of proteins, and one of such factor, deformed epidermal auto-regulatory factor-1 (DEAF-1) has been initially identified in Drosophila. The mammalian homologue of DEAF-1 and structurally related proteins have been identified, and they formed a family with over 20 members. The factors regulate gene expression through association with co-repressors, recognition of genomic marker, to exert histone modification by catalyze addition of some chemical groups to certain amino acid residues on histone and non-histone proteins, and degradation host proteins, so as to regulate cell cycle progression and execution of cell death. The formation of fused genes during chromosomal translocation, exemplified with myeloid transforming gene on chromosome 8 (MTG8)/eight-to-twenty one translocation (ETO) /ZMYND2, MTG receptor 1 (MTGR1)/ZMYND3, MTG on chromosome 16/MTGR2/ZMYND4 and BS69/ZMYND11 contributes to malignant transformation. Other anomaly like copy number variation (CNV) of BS69/ZMYND11 and promoter hyper methylation of BLU/ZMYND10 has been noted in malignancies. It has been reported that when fusing with Runt-related transcription factor 1 (RUNX1), the binding of MTG8/ZMYND2 with co-repressors is disturbed, and silencing of BLU/ZMYND10 abrogates its ability to inhibition of cell cycle and promotion of apoptotic death. Further characterization of the implication of ZMYND proteins in carcinogenesis would enhance understanding of the mechanisms of occurrence and early diagnosis of tumors, and effective antitumor efficacy.

Osteosarcoma (OS) is the most common bone malignant tumor. However, the genetic basis of OS pathogenesis is still not understood, and occurrence of chemo-resistance is a major reason for the high morbidity of OS patients. Recently, chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) has been identified as a gene related to malignant tumor progression. Unfortunately, its effects on OS development and drug resistance are still not understood. In the study, we attempted to investigate the effects of CHD1L on tumorigenesis and chemoresistance in OS. We found that CHD1L expression was markedly up-regulated in OS samples, especially in cisplatin (cDDP)-resistant patients. We also showed that OS cells with CHD1L knockdown were more sensitive to cDDP treatment with lower IC50 values. In addition, we found that CHD1L deletion markedly reduced cell proliferation and induced apoptosis in OS cells with cDDP resistance. Moreover, the properties of cancer stem cells were highly suppressed in cDDP-resistant OS cells following CHD1L knockdown. Furthermore, multidrug resistance protein 1 (MDR-1) expression levels were dramatically decreased in OS cells with cDDP resistance when CHD1L was suppressed. Functional analysis indicated that CHD1L knockdown clearly restrained the activation of ERK1/2, protein kinase B (AKT) and NF-κB signaling pathways in cDDP-resistant OS cells. Consistently, animal experiments suggested that CHD1L suppression mitigated cDDP resistance in the generated in vivo xenografts. Collectively, CHD1L could modulate chemoresistance of OS cells to cDDP, and thus may be inspiring findings for overcoming drug resistance in OS.

Pancreatic cancer (PC) is prone to distant metastasis in the early stage, which is attributed to the strong migration ability of tumor cells. Focal adhesion turnover is essential for cancer cell metastasis, and the integrin recycling process is a key activation pathway for focal adhesion depolymerization. To identify the key motor protein involving in the integrin β1 recycling, we screened kinesin proteins involved in integrin β1 recycling using a kinesin family siRNA library and identified kinesin family 15 (KIF15) as a key regulator. KIF15 was upregulated in metastasis PC tissues and promoted PC cell migration and invasion. We identified KIF15 as a key component mediating integrin β1/FAK signaling that accelerated FA disassembly in a FAK-Y397-dependent manner. KIF15 recruited PI3K-C2α to promote integrin β1/FAK signaling and FA disassembly in a RAB11A-dependent manner. The C-terminal tail of KIF15 is required for the PI3K-C2α interaction and RAB11A activation. In addition, we also found that SIRT1-mediated acetylation of KIF15 is essential for KIF15 phosphorylation, which is the key activation event in motor protein function. Together, these findings indicate that KIF15 interacts with PI3K-C2α to promote FA turnover in PC cells by controlling the endosome recycling of integrin β1 in a SIRT1 acetylation modification-dependent manner, eventually promoting focal adhesions turnover and distant metastasis in PC.

Aberrant activation of nuclear factor κB (NF-κB)/RELA is often found in lung adenocarcinoma (LUAD). In this study, we determined that microRNA-3613-5p (miR-3613-5p) plays a crucial role in RELA-mediated post-transcriptional regulation of LUAD cell proliferation. Expression of miR-3613-5p in clinical LUAD specimens is associated with poor prognosis in LUAD. Upregulation of miR-3613-5p promotes LUAD cell proliferation in vitro and in vivo. Our results suggested a mechanism whereby miR-3613-5p expression is induced by RELA through its direct interaction with JUN, thereby stimulating the AKT/mitogen-activated protein kinase (MAPK) pathway by directly targeting NR5A2. In addition, we also found that phosphorylation of AKT1 and MAPK3/1 co-transactivates RELA, thus constituting a RELA/JUN/miR-3613-5p/NR5A2/AKT1/MAPK3/1 positive feedback loop, leading to persistent NF-κB activation. Our findings also revealed that miR-3613-5p plays an oncogenic role in LUAD by promoting cell proliferation and acting as a key regulator of the positive feedback loop underlying the link between the NF-κB/RELA and AKT/MAPK pathways., Graphical Abstract, Aberrant activation of nuclear factor κB (NF-κB)/RELA is often found in lung adenocarcinoma (LUAD). However, RELA as a direct therapeutic target for lung cancer remains a challenge. This study investigated that microRNA-3613-5p (miR-3613-5p) plays a crucial role in RELA-mediated post-transcriptional regulation of LUAD cell proliferation.

This study was aimed at analyzing the effects of liposomal nanoparticle-based miR-27b on PPARγ and osteogenic differentiation of bone marrow mesenchymal stem cells under microgravity. The rat bone marrow mesenchymal stem cells were set as the research object, and the gyroscope was employed for simulation of microgravity. The cells were randomized into four groups, including the experimental group A (simulated microgravity+liposomal nanoparticle-mediated miR-27b transfection group), as well as the control groups: group B (simulated microgravity+negative control group), group C (simulated microgravity+transfection reagent group) and group D (normal gravity+liposomal nanoparticle-mediatedmiR-27b transfection group). After a two-week osteogenic induction in vitro, staining was performed to assess the lipogenesis rate of the samples. In addition, ALP activity and PPARγ mRNA level was detected. The number of alizarin staining-positive osteogenic nodules and ALP activity (0.21±0.44 King unit) in group A was significantly diminished compared to those in group B, C, and D. Moreover, its lipogenesis rate (9.31±1.02%) and PPARγ mRNA level (1.86±0.39) were significantly higher than those in group B, C, and D (P < 0.05). The number of alizarin staining-positive osteogenic nodules and ALP activity (0.96±0.18 King unit) in group D were significantly reduced in comparison with those in groups B and C, while the lipogenesis rate (4.86±0.77%) and PPARγ mRNA level (0.93±0.34) were significantly higher than those in group B and C (P < 0.05) without difference between group B and group C (P > 0.05). Under a microgravity condition, liposomal nanoparticle-mediated miR-27b can impede the differentiation of BMSCs into osteoblasts via regulating PPARγ signal transduction.

Dysregulation of autophagy and circular RNAs (circRNAs) are involved in the pancreatic cancer (PC) progression. However, the regulatory network between circRNAs, autophagy, and PC progression remains unknown. Herein, we demonstrated that autophagy-associated circRNA circ-autophagy related 7 (circATG7) was elevated in PC tissues compared to adjacent tissues, and in PC cells treated with EBSS and hypoxia. circATG7 expression was positively associated with tumor diameter and lymph node invasion in patients with PC. circATG7 overexpression promoted PC cell proliferation, mobility, and autophagy in vitro, while circATG7 knockdown induced the opposite effects. ATG7 inhibition attenuated the effects of circATG7 on the biological functions of PC cells. CircATG7 is located in the cell cytoplasm and nucleus. Cytoplasmic circATG7 sponged miR-766-5p and decreased its expression, and increased the expression of ATG7, a target gene of miR-766-5p. Nuclear circATG7 acted as a scaffold to increase the interaction between the human antigen R protein and ATG7 mRNA and enhanced ATG mRNA stability. Furthermore, we demonstrated that circATG7 regulates PC cell proliferation and metastasis in vivo via ATG7-dependent autophagy. In conclusion, our results demonstrated that circATG7 accelerates PC progression via miR-766-5p/ATG7 and that HUR/ATG7 depends on autophagic flux. Thus, circATG7 may be a potential therapeutic target for PC.

Inflammatory molecules and exosomes are crucial for signal transduction between tumor-associated macrophages and tumor cells. IL-6, a key inflammatory molecule secreted by M2 macrophages after polarization, can mediate malignant progression of pancreatic cancer (PC). However, the functions and mechanisms of IL-6 and tumor-derived exosomes in tumor-associated macrophages and PC remain unclear. Transcriptome chip and quantitative reverse transcription PCR experiments indicated that FGD5-AS1 induced IL-6 and high FGD5-AS1 expression correlated with the poor prognosis in PC patients. RNA pulldown, mass spectrometry, and dual luciferase reporter assays were used to identify the mechanism of exosomal FGD5-AS1 in promoting PC progression and M2 macrophage polarization. FGD5-AS1 exerted cancer-promoting functions when co-cultured with M2 macrophages. PC-derived exosomal FGD5-AS1 stimulated M2 macrophage polarization by activating STAT3/NF-κB pathway. FGD5-AS1 interacts with p300, resulting in STAT3 acetylation, thus promoting nuclear localization and transcriptional activity of STAT3/NF-κB. These data indicated that PC cells generate FGD5-AS1-rich exosomes, which cause M2 macrophage polarization to promote the malignant behaviors of PC cells. Targeting exosomal FGD5-AS1 may provide a potential diagnosis and treatment strategy for PC.

p53/p21 signaling plays a vital role in pancreatic cancer (PC) progression. ZWINT was shown to function as an oncoprotein in the progression of multiple cancers. However, the involvement of ZWINT and p53 activation in the progression of PC remains poorly understood. Bioinformatics and tissue array chip analyses were performed to evaluate ZWINT expression in pancreatic cancer. ZWINT mRNA and protein expression were evaluated in normoxia and hypoxia. CHIP was used to evaluate HIF1α interaction with the ZWINT promoter. CCK8, colony formation, EDU, and cell cycle analysis were used to examine PC cell proliferation. Immunoprecipitation and immunofluorescence were used to examine the interaction of ZWINT, MDM2, and p53. p53 activity was evaluated by q-PCR and luciferase assay. Protein degradation and ubiquitination assays were used to analyze the role of ZWINT in p53 ubiquitination. ZWINT was overexpressed in pancreatic cancer and induced in hypoxia. ZWINT promoted pancreatic cancer growth and cell cycle progression. Bioinformatic analysis revealed that ZWINT may regulate the p53 signal pathway. ZWINT interacts with p53 and promotes its ubiquitination and degradation. ZWINT promoted proliferation via p53/p21. Immunohistochemistry of clinical specimens revealed that that ZWINT expression was significantly negatively correlated with p53/p21. Our data showed that hypoxia regulates the expression of ZWINT, which activated p53/p21 signaling pathway to promote PC growth.

Background The underlying mechanism behind the tumorigenesis and progression of pancreatic cancer is not clear, and treatment failure is generally caused by early metastasis, recurrence, drug resistance and vascular invasion. Exploring novel therapeutic regimens is necessary to overcome drug resistance and improve patients outcomes. Methods Functional assays were performed to investigate the role of [6]-Paradol (6-P) in proliferation and metastasis of pancreatic cancer in vitro and in vivo. The interaction between EGFR and 6-P was tested by KEGG enrichment analysis and molecular docking analysis. qRT-PCR was performed to detect the mRNA expression of EGFR in 6-P treated groups. Involvement of the PI3K/AKT pathway was measured by western blotting. Results 6-P significantly suppressed pancreatic cancer cell proliferation and metastasis. KEGG enrichment analysis and molecular docking analysis suggested that there existed certain interaction between EGFR and 6-P. In addition, 6-P obviously decreased EGFR protein expression level but did not change the mRNA expression level of EGFR. 6-P could induce degradation of EGFR through decreasing the protein stability of EGFR and enhancing the ubiquitin-mediated proteasome-dependent degradation, 6-P-mediated EGFR degradation led to inactivation of PI3K/AKT signaling pathway. However, ectopic expression of EGFR protein resulted in resistance to 6-P-mediated inactivity of PI3K/AKT signaling and inhibition of malignant phenotype of pancreatic cancer. Inversely, erlotinib could enhance the 6-P-mediated anticancer activity. Conclusion Our data indicated that 6-P/EGFR/PI3K/AKT signaling axis might become one of the potential therapies for the treatment of pancreatic cancer.

Accumulating evidence has demonstrated the essential role of long noncoding RNAs (lncRNAs) in various types of human cancer, including pancreatic cancer (PC). However, the functions and regulatory mechanisms of nuclear receptor subfamily 2 group F member 1 antisense RNA 1 (NR2F1-AS1) that are responsible for its role in the malignant progression of PC cells remains to be investigated. In this study, the biological effects of NR2F1-AS1 and NR2F1 in PC were investigated by in vitro and in vivo experiments. The mechanisms of NR2F1-AS1 were monitored by bioinformatic predictive analysis and confirmatory experiments. Our results indicated that NR2F1-AS1 was overexpressed and positively correlated with poor survival in PC. Depletion of NR2F1-AS1 restrained PC cell proliferation, migration, invasion, and suppressed xenograft tumor growth and metastasis in vitro and in vivo. Mechanistic experiments suggested that NR2F1-AS1 positively regulated the neighboring NR2F1 gene, which subsequently activated AKT/mTOR signaling, resulting in the upregulation of hypoxia-inducible factor-1α (HIF-1α). Further investigations elucidated that NR2F1-AS1 expression was transcriptionally regulated by HIF-1α under hypoxia. These findings demonstrated that hypoxia-induced NR2F1-AS1 expression directly increased NR2F1 levels to promote PC cell proliferation, migration, and invasion by activating AKT/mTOR signaling. Together, these findings suggest that NR2F1-AS1 could be a prospective therapeutic target for PC.

Our previous work reported activating transcription factor 1 (ATF1) is a promotive factor of nasopharyngeal carcinoma (NPC) tumorigenesis. This study is to further explore the association between the human ATF1 rs11169571 polymorphism and the risk of NPC occurrence. The association between ATF1 rs11169571 and risk of NPC occurrence was investigated in clinical samples of 560 patients and 661 controls obtained from southern China with high incidence of NPC. The genotypes were detected by PCR‐RFLP. The differential expression activity of alleles ‐T and ‐C was analyzed with CNE‐2 and C666‐1 cells by luciferase reporter assay. Our data suggested that the allelic frequency and genotypes were significantly different between patients and controls. Compared to the TT homozygote, the TC and CC genotypes have been shown to be significantly decreased in NPC patients (OR = 0.494, 95% CI = 0.387‐0.629, P

Background Epithelial-mesenchymal transition (EMT) may be one of the reasons for the failure in some clinical trials regarding histone deacetylase inhibitors (HDACIs)-treated solid tumors. We investigated the effects of a pan-HDACI trichostatin A (TSA) on the proliferation and EMT of nasopharyngeal carcinoma (NPC) cells. Methods Poorly-differentiated NPC cell line CNE2 and undifferentiated C666–1 were treated with various concentrations of TSA, the cell viability was assessed by CCK-8 assay, the morphology was photographed, and the mRNA level of HDACs was assessed by semiquantitative PCR. After determination the cell cycle distributions, cells were subjected to western blotting analysis of cell cycle and EMT-associated genes expression. And the changes in migration ability were assessed by transwell migration assay and scratch wound healing assay. Finally, histone deacetylases activator ITSA-1 was used to assess the reverse of TSA-induced changes in NPC cells. Results TSA inhibited the proliferation of CNE2 and C666–1 cells in a concentration-dependent manner and arrested the cell cycle at G1 phases. TSA reduced PCNA, cyclin D1, cyclin E1, CDK2, p16 and p21 expressions and stimulated CDK6 levels. TSA stimulation for 48 h could effectively induce the EMT in CNE2 and C666–1 cells, which showed an increase of spindle-like cells and promoted expression of Vimentin and Snail1 expression in a concentration-dependent manner. Surprisingly, this short period of TSA treatment that induced EMT also impeded the migration ability of CNE2 and C666–1 cells. Interestingly, ITSA-1 rescued TSA-impeded CNE2 and C666–1 cells’ proliferation, migration and HDACs expression, also re-induced the cells to turn into epithelial cell phenotypes. Conclusions These results indicate that short-term stimulation of TSA effectively inhibits cell proliferation and induce EMT-like changes in NPC cells but not increase its invasion ability.

miR-139 has a tumor suppressor effect in many tumors. Here, we examined the suppressive role of this miRNA and its target, ROCK1, in osteosarcoma (OS), a highly malignant bone tumor that mainly affects children and adolescents. The expression of miR-139 was down-regulated in OS. Overexpression of miR-139 significantly inhibited OS cell proliferation, migration, and invasion. Ectopic expression of miR-139 down-regulated ROCK1, a target of miR-139, by direct binding to its 3' untranslated region (3'UTR). Direct siRNA-mediated silencing of ROCK1 exerted an inhibitory effect on OS cell proliferation and invasion similar to the effect of miR-139. ROCK1 transfection reversed the suppressive effect of miR-139 on OS cell proliferation and invasion. Both miR-139 and siRNA knockdown of ROCK1 significantly down-regulated β-CATENIN and p-AKT and up-regulated E-CADHERIN and p53. The data provided here show that miR-139 exerts suppressive effects on proliferation and invasion of OS cells by targeting ROCK1.

LncRNAs play essential regulatory roles in pancreatic cancer (PC) tumorigenesis and progression. We aimed to investigate the role of lncRNA CERS6-AS1 in PC. CERS6-AS1 expression was determined in PC tissues and cell lines by PCR analysis. The roles of CERS6-AS1 on proliferation, migration, invasion, and epithelial to mesenchymal transition (EMT) were confirmed via CCK-8 assay, EDU assay, transwell assay, wound healing assay, and western blot assay. Besides, the interaction between CERS6-AS1 and their target genes was verified by luciferase report assays and RIP assays. Animal assays and clinical data analysis were performed to validate the functions in vivo. We found that lncRNA CERS6-AS1 was highly expressed in PC tissues and cells. Additionally, high expression of CERS6-AS1 was obviously associated with poor prognosis. Functional assays demonstrated that CERS6-AS1 downregulation significantly inhibited PC cell growth and migration. Moreover, CERS6-AS1 exerted as a molecular sponge for miR-217-5p (miR-217), and miR-217 was confirmed as a potential target of CERS6-AS1. Subsequently, miR-217 suppressed PC cell proliferation and metastasis by directly targeting YWHAG, which interacted with RAF1 and promoted its phosphorylation, leading to RAF1-mediated ERK signaling activation and translocation of phosphorylated ERK from the cytoplasm to the nucleus. Mechanically, CERS6-AS1 silencing significantly inhibited PC cell proliferation and metastasis via a miR-217/YWHAG/RAF1 signaling axis. CERS6-AS1 exerts as a carcinogen in PC to promote malignant features and behaves as a competitive endogenous RNA for miR-217. We identified CERS6-AS1 as a potential biomarker or therapeutic target to improve PC diagnosis and treatment outcomes.

Purpose: The tripartite motif-containing family member TRIM37 is involved in a number of important biological and pathological processes, and it has recently been shown to be an essential regulator of protein ubiquitination and a contributor to tumorigenesis. We previously showed that TRIM37 is overexpressed in and promotes the proliferation and invasion of pancreatic cancer (PC). Methods: Sphere formation, flow cytometric, qRT-PCR, western blot, colony formation, EdU incorporation, mouse xenograft model, TUNEL and IHC assays were performed to detect the role of TRIM37 in stemness and chemoresistance of PC in vitro and in vivo. Bioinformatics analysis and dual-luciferase reporter assays were used to determine which intracellular pathways might mediate the effects of TRIM37 in PC cells. Immunofluorescent(IF) staining, co-immunoprecipitation(CO-IP), protein stability and ubiquitination assays were performed to investigate the relationship between TRIM37 and PTEN. Results: TRIM37 modulates the ubiquitination and degradation of the tumor suppressor phosphatase and tensin homolog (PTEN), which negatively regulates the AKT–GSK-3β–β-catenin signaling pathway, thereby sustaining aberrant activation of PC cells. High expression of TRIM37 combined with low expression of PTEN correlates with poor survival of PC patients. Conclusions: Collectively, our results suggest that inhibition of the TRIM37–AKT–GSK-3β–β-catenin axis may be a promising strategy for treatment of PC.

Background: CircRNA is a new type of non-coding RNA that has attracted much attention for involvement in the development and progression of various human diseases, especially cancer. The most reported role of circRNA in many tumors is ‘MiRNA sponge’. We aimed to investigate the role of circBVES in the proliferation and glycolysis of gastric cancer cells and its molecular mechanisms.Methods: In this study, higher CircBVES expression in gastric cancer tissues was detected by RNA sequencing. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of CircBVES in gastric cancer tissues, and the relationship between the expression of CircBVES and prognosis was further analyzed. Then, the effects of CircBVES on the growth and glycolysis of gastric cancer cells were investigated through in vitro and in vivo functional experiments. The interaction between CircBVES and miR-145-5p was detected by bioinformatics analysis, luciferase activity assay and RNA immunoprecipitation.Results: We found that the expression of CircBVES in gastric cancer tissues was evidently up-regulated, and its level was closely correlated with the prognosis of patients with gastric cancer. Inhibition of CircBVES decreased cell proliferation and glycolysis in vitro. Low expression of CircBVES inhibited tumor growth in vivo. Mechanism analysis showed that CircBVES may serve as a competitive endogenous RNA of miR-145-5p to reduce the expression of miR-145-5p in gastric cancer cells, and relieve the repressive effect of miR-145-5p on target genes HMGB3 and cycle-related proteins CCNE1 and CDK2.Conclusions: Our results suggest that CircAGFG1 may promote the progress of gastric cancer through the CircBVES / miR-145-5p / HMGB3 axis, providing a new target for the treatment of gastric cancer cells.

Drug resistance leads to tumor relapse and further progression during chemotherapy in lung cancer. Close homolog of L1 (CHL1) has been identified as a tumor suppressor in most malignancies. However, to the best of our knowledge, whether CHL1 mediates chemoresistance remains unknown. The present study observed that CHL1 was significantly downregulated in cisplatin (DDP)-resistant cells (A549/DDP) and paclitaxel (PTX)-resistant cells (A549/PTX) compared with A549 cells. When treated with or without DDP and PTX, silencing of CHL1 in A549 cells promoted the cell survival rate and clone formation, and decreased apoptosis. Whereas overexpression of CHL1 in A549/DDP and A549/PTX cells impeded the cell survival and clone formation and promoted apoptosis. Additionally, CHL1 overexpression enhanced the chemosensitivity of A549/DDP cells to DDP in vivo. Notably, the chemoresistance induced by CHL1 depletion was reversed by the Akt inhibitor SC66 in A549 cells. The results of the present study demonstrated that CHL1 enhanced sensitivity of lung cancer cells by suppressing the Akt pathway, which suggested that CHL1 may be a potential target for overcoming chemoresistance in lung cancer.

Lung adenocarcinoma (LADC), the most prevalent type of human lung cancer, is characterized by many molecular abnormalities. SH2B1, a member of the SH2-domain containing family, have recently been shown to act as tumor activators in multiple cancers, including LADC. However, the mechanisms underlying SH2B1 overexpression are not completely understood. Here, we reported that SH2B1 expression levels were significantly upregulated and positively associated with EMT markers and poor patient survival in LADC specimens. Modulation of SH2B1 levels had distinct effects on cell proliferation, cell cycle, migration, invasion, and morphology in A549 and H1299 cells in vitro and in vivo. At the molecular level, overexpression of SH2B1 resulted in the upregulation of the EMT markers, especially induced β-catenin accumulation and activated β-catenin signaling to promote LADC cell proliferation and metastasis, while silencing SH2B1 had the opposite effect. Furthermore, ectopic expression of SH2B1 in H1299 cells increased IRS1 expression level. Reduced expression of IRS1 considerably inhibited H1299 cell proliferation, migration, and invasion which were driven by SH2B1 overexpression. Collectively, these results provide unequivocal evidence to establish that SH2B1-IRS1-β-catenin axis is required for promoting EMT, and might prove to be a promising strategy for restraining tumor progression in LADC patients.

Background and purpose Asiatic acid is one of the active compounds isolated from Centella asiatica and has been used to treat many diseases, including hypertension, pulmonary fibrosis, and cancer. It exhibits anticancer effects in many cancers, such as ovarian, lung and colon cancer; however, its anticancer effects in breast cancer and the underlying mechanism are not fully understood. Chemoresistance is often induced after the use of chemotherapy, and it is a challenging problem in cancer therapy. The effects of asiatic acid on chemoresistance in breast cancer have never been studied. Therefore, the aim of the present study was to examine the anticancer effects of asiatic acid in doxorubicin-resistant breast cancer MCF-7 cells. Methods The cells were incubated with asiatic acid at 0-160 μM for 2-24 h. Cell viability and cytotoxicity were evaluated by 3-[4, 5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Florescent images were taken using a confocal microscope. P-gp function and apoptosis assays were performed using flow cytometry. Caspase activity was measured with the Caspase-Glo™ Assay System. The phosphorylation and expression of relevant proteins were assessed by western blots. Molecular docking was performed and scored by AutoDock. Cellular thermal shift assay (CETSA) was applied for experimental valuation. Results Our data demonstrated that asiatic acid induced cell death in multiple ways, including reactive oxygen species production, adenosine triphosphate (ATP) content reduction, and adaptive immunity balance via intrinsic apoptosis, AMP-activated protein kinase (AMPK), programmed death-ligand 1 (PD-L1), and indirect nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcriptional pathways, using experimental validation and in silico analysis. Moreover, asiatic acid also enhanced the sensitivity of doxorubicin-resistant MCF-7 cells to doxorubicin by improving P-glycoprotein (P-gp) function. Conclusions This study provides evidence that asiatic acid has strong anticancer effects to reverse multidrug resistance and could be developed as a promising adjuvant drug for the treatment of chemoresistant cancer.

Background: Epigallocatechin-3-gallate (EGCG) has exhibited antitumor properties in several types of cancers, including nasopharyngeal carcinoma (NPC), but the molecular mechanisms underlying this function remain incompletely understood. The aim of the present study was to characterize the global impact of EGCG on the expression of microRNAs (miRNAs) in NPC cells. Methods: Using microarray analysis, the alterations of miRNA expression profiles were investigated in EGCG-treated CNE2 cells. Furthermore, the target genes and signaling pathways regulated by EGCG-specific miRNAs were identified using target prediction program and gene ontology analysis. Results: A total of 14 miRNAs exhibited >2-fold expression changes in a dose-dependent manner after treatment with 20 μmol/L and 40 μmol/L EGCG. Totally 43, 49, and 52 target genes from these differentially expressed miRNAs were associated with the apoptosis, cell cycle regulation, and cell proliferation, respectively. A total of 66 signaling pathways, primarily involved in cancer development and lipid and glucose metabolism, were shown to be regulated by EGCG-specific miRNAs. Conclusion: EGCG induces considerable alterations of miRNA expression profiles in CNE2 cells, which provides mechanistic insights into cellular responses and antitumor activity mediated by EGCG.

Drug resistance is a serious promble during chemotherapy in lung cancer, which may lead to tumor relapse and further progression. CHL1 was a tumor suppressor in most malignancies, and it was found downregulated in NSCLC cisplatin-resistant cells H460. Thus, in this study, we investigated the role and mechanism of chemoresistance by CHL1 in lung cancer. Human lung adenocarcinoma cell lines A549 and its cisplatin resistant cells (A549/DDP) and paclitaxel resistant cells (A549/PTX) were applied in this research. CHL1 was found obvious downregulation in A549/DDP and A549/PTX cells versus A549 cells. Suppression of CHL1 in A549 cells, promoted cell survival rate and clone formation, decreased cell apoptosis when treated with or without DDP and PTX, respectively. While excessive CHL1 expression in A549/DDP and A549/PTX cells, the results were opposite. Moreover, CHL1 knockdown mediating chemoresistance was reversed by Akt inhibitor SC66 in A549 cells. In summary, overexpression of CHL1 reversed chemoresistance to cisplatin and PTX via suppressing Akt pathway in lung cancer, it was suggested that CHL1 maybe as a potential target for overcome chemoresistance in lung cancer.

// Jiahui Zhou 1 , Zunnan Huang 1 , Ziyou Wang 1 , Shumin Liu 1 , Alf Grandien 3 , Ingemar Ernberg 2 , Zhiwei He 1 and Xiangning Zhang 1 1 Department of Pathophysiology and China-America Cancer Research Institute, Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan, Guangdong, China 2 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden 3 Center for Haematology and Regenerative Medicine, Karolinska Institutet, Stockholm, Sweden Correspondence to: Zunnan Huang, email: zn_huang@yahoo.com Zhiwei He, email: Zhiweihe688@yahoo.com Xiangning Zhang, email: zhangxn_2006@126.com Keywords: nasopharyngeal carcinoma, tumor suppressor gene, BLU/ZMYND10, TRAIL, NF-κB Received: September 14, 2016 Accepted: November 19, 2016 Published: December 23, 2016 ABSTRACT A putative tumor suppressor BLU mapped on the chromosomal 3p21 region, is frequently lost in human tumors including nasopharyngeal carcinoma (NPC). To explore the underlying mechanism of tumor suppression by BLU , its potential to promote apoptosis induced by TRAIL, an effector molecule elaborated by natural killer-T (NKT) cells was investigated. BLU was re-expressed in NPC-derived HNE1 cells by recombinant adenoviral infection and the cells were challenged with recombinant TRAIL. The growth inhibition of BLU was assayed and apoptosis was examined by flow cytometry-based tetramethylrhodamine ethyl ester (TMRE) and annexin V staining, cleavage of pro-caspase-8 and poly ADP ribose polymerase (PARP). The modulation of NF-κB pathway by BLU was evaluated by the reporter activity and estimation of the level of the molecules involved such as IKKalpha, p65 NF-κB, as well as NF-κB induced anti-apoptotic factors cFLIP L and cIAP2. The expression of BLU exerted in vitro and in vivo growth inhibitory effect and promoted TRAIL-induced apoptosis. This phenomenon was validated by FACS-based assays of mitochondrial membrane potential (BLU vs. Vector 87.8% ± 7.7% and 72.1%±6.7% at 6h exposure to TRAIL) and phosphatidylserine turnover ( BLU vs. vector: 28.7%±2.9% and 22.6%±2.5%), as well as, enhanced caspapse-8 cleavage. Similar with the findings that BLU promotes chemotherapeutic agent-induced apoptosis, it also augmented death receptor-induced pathway through NF-κB pathway inhibition. In conclusion, BLU suppressed tumor formation by strengthening the antitumor immunity.

Aberrant activation of the extracellular signal‑regulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathway is frequently determined in various human tumor types, including liver cancer, and has been considered as a promising target for cancer chemoprevention and therapy. In the present study, using computer‑aided virtual screening and molecular docking, isobavachalcone (IBC), a natural chalcone compound, was identified to be an ATP‑competitive inhibitor targeting ERK1/2 and RSK2. Cell Counting Kit‑8, EdU incorporation and colony formation assays were used to detect the effects of IBC on cell viability and proliferation, and the results demonstrated that IBC effectively inhibited the proliferation of liver cancer HepG2 and Hep3B cells, whereas it had no notable cytotoxic effect on immortal liver L02 cells. Flow cytometric analysis and western blotting further revealed that IBC caused significant levels of apoptosis on liver cancer cells via the caspase‑dependent mitochondria pathway. The computer prediction was confirmed with pull‑down and in vitro kinase assays, in which IBC directly bound with ERK1/2 and RSK2, and dose‑dependently blocked RSK2 kinase activity in liver cancer cells. Treatment of HepG2 or Hep3B cells with IBC significantly attenuated epidermal growth factor‑induced phosphorylation of RSK2 and resulted in the reduced activation of its downstream substrates including cAMP response element‑binding protein, activating transcription factor 1, histone H3 and activating protein‑1. Enforced RSK2 expression in L02 cells could increase the effect of IBC on suppressing cell growth. Conversely, knockdown of RSK2 reduced the inhibitory effect of IBC on HepG2 cell proliferation. Overall, the present data indicated that ERKs/RSK2 signaling serves a pivotal role in IBC‑induced suppression of liver cancer cells and that IBC may be a potential therapeutic candidate for human cancer with elevated ERKs/RSK2 activity.

Background Non-small cell lung cancer (NSCLC), the most prevalent type of human lung cancer, is characterized by many molecular abnormalities. SH2B1, a member of the SH2-domain containing family, have recently been shown to act as tumor activators in multiple cancers. The objective of this study was to investigate the role SH2B1 and the underlying molecular mechanism in NSCLC. Methods Cell functional analysis and cell line-derived xenograft model were performed to determine SH2B1 potential roles on NSCLC cell proliferation in vitro and in vivo. In vitro assays were performed to identify signal molecular mechanisms. Subsequently, 104 patients with NSCLC undergoing primary surgical resection were recruited to evaluated expression of SH2B1 and Akt/mTOR signaling markers by immunohistochemical staining to determine their clinicopathologic significance. Results Modulation of SH2B1 expression levels had distinct effects on cell proliferation, cell cycle and apoptosis in the NSCLC cell lines A549 and H1299. At the molecular level, overexpression of SH2B1 resulted in the upregulation of the Akt/mTOR markers, p-Akt and p-mTOR, and downregulation of PTEN to promote NSCLC cell proliferation, while silencing SH2B1 had the opposite effect. In human NSCLC specimens, SH2B1 expression levels were positively associated with Akt/mTOR signaling pathway markers. Conclusions The SH2B1/Akt/mTOR/PTEN axis is required for regulating NSCLC cell proliferation and might prove to be a promising strategy for restraining tumor progression in NSCLC patients. Electronic supplementary material The online version of this article (10.1186/s12935-018-0632-x) contains supplementary material, which is available to authorized users.

Background Kruppel family member zinc binding protein 89 (ZBP-89), also known as ZNF148, regulates Bak expression via binding to GC-rich promoter domain. It is not clear if other GC-rich binding factors, such as Sp family members, can interact with ZBPp-89 on Bak expression. This study aims to elucidate the mechanism of Bak expression regulation by ZBP-89 and Sp proteins, based on in vitro experiment and The Cancer Genome Atlas (TCGA) hepatocellular carcinoma (HCC) data cohort. Methods We downloaded TCGA hepatocellular carcinoma (HCC) cohort data to analysis the association of Bak transcription level with ZBP-89 and Sp proteins transcription level. HCC cell lines and liver immortal non-tumour cell lines were used for mechanism study, including western blotting analysis, expression vector mediated gene expression and siRNA interference. Results Results showed that cancer tissues have higher Bak transcription level compared with adjacent non-cancer tissues. Bak transcription level was correlated with Sp1 and Sp3 expression level, while no correlation was found in ZBP-89 and Bak, neither Sp2 nor Sp4. Mithramycin A (MMA) induced Bak expression in a dose-dependent manner. Western blotting results showed Sp1 overexpression increased Bak expression both in liver immortal non-tumour cells and HCC cells. Interference Sp1 expression could inhibit Bak expression alone. ZBP-89 siRNA suppressed Bak expression even in the presence of MMA treatment and S1 overexpression. Additionally, Bak and Sp1 level were associated with HCC patient survival. Conclusions Bak expression required ZBP-89 and Sp1 cooperative regulation simultaneously. Electronic supplementary material The online version of this article (10.1186/s12885-018-4349-y) contains supplementary material, which is available to authorized users.

In this study, we explored the role and mechanisms of Cyclocarya paliurus polysaccharide on cell apoptosis in thyroid cancer (TC) cells. The apoptosis of thyroid cancer cells in vitro and tumor tissues in vivo induced by Cyclocarya paliurus polysaccharide was determined by MTT assay and flow cytometric assay. The downstream molecules including phosphop-protein kinase B (p-Akt), Akt, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) in tumor tissue were evaluated by western blotting. MTT and flow cytometry assay in vitro revealed Cyclocarya paliurus polysaccharide-induced apoptosis of thyroid cancer cell line in a manner of time-dependent and dose-dependent. In vivo assay showed 50 mg/kg and 100 mg/kg Cyclocarya paliurus polysaccharide significantly suppressed the proliferation of thyroid cancer in mice. Western blotting showed downregulation of p-Akt, Akt, and Bcl-2 and upregulation of Bax. These results suggest that Cyclocarya paliurus polysaccharide may enhance thyroid cancer cell apoptosis by suppressing the activation of p-Akt, Akt, and Bcl-2 and activating Bax, which provide a novel use of CPP as a thyroid cancer treatment.

Glabridin, an isoflavone isolated from licorice, owns a variety of pharmacological effects. Several reports have demonstrated that glabridin could regulate multiple cellular signaling pathways to inhibit the progression of cancer. However, the target proteins have not been elucidated yet. We used shape screening and induced fit docking to screen the protein data bank against glabridin. Braf and MEK1/2, important intermediate molecules of the braf/MEK cascade, were identified as the potential targets of glabridin. The experimental data showed that glabridin could inhibit the phosphorylation of MEK1/2 and the phosphorylation levels of downstream molecules including ERK1/2 and transcription factors ATF1 and CREB, but had no effect on the phosphorylation of braf. In particular, the in vitro pull-down assay indicated that glabridin selectively bound to braf and MEK1/2. What is more, exposure to glabridin significantly suppressed the proliferation of hepatocellular carcinoma HepG2 cell line. In addition, glabridin might arrest cell cycle in G1 through downregulation of cyclinD3, CDK2, and CDK4. In conclusion, glabridin is a potential multi-molecule-targeting inhibitor in the field of clinical prevention or treatment of cancer.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-associated mortality worldwide; however, only limited therapeutic treatments are currently available. The present study aimed to investigate the effects of cannabinoids as novel therapeutic targets in HCC. In addition, the mechanism underlying the effects of a synthetic cannabinoid, WIN55, 212-2, on the BEL7402 HCC cell line was investigated. The results demonstrated that WIN55, 212-2 induced cell cycle arrest of the BEL7402 cells at the G0/G1 phase via can nabinoid receptor 2 (CB2)-mediated down regulation of phosphorylated-extracellular signal-regulated kinases (ERK)1/2, upregulation of p27, and downregulation of cyclin D1 and cyclin-dependent kinase 4. Furthermore, inhibition of CB2 with the CB2 antagonist AM630 abrogated WIN55, 212-2-induced cell cycle arrest. Inhibition of ERK1/2 also resulted in cell cycle dysregulation and cell cycle arrest at the G0/G1 phase, which subsequently resulted in cell growth inhibition. In addition, the present study detected a significant reduction in matrix metalloproteinase-9, retinoblastoma protein and E2F1 expression, and migration inhibition by WIN treatment. These results suggested that cannabinoid receptor agonists, including WIN, may be considered as novel therapeutics for the treatment of HCC.

A large proportion of the NSCLC patients were insensitive to radiotherapy, but the exact mechanism is still unclear. This study explored the role of miR-25 in regulating sensitivity of NSCLC cells to ionizing radiation (IR) and its downstream targets. Based on measurement in tumor samples from NSCLC patients, this study found that miR-25 expression is upregulated in both NSCLC and radio-resistant NSCLC patients compared the healthy and radio-sensitive controls. In addition, BTG expression was found negatively correlated with miR-25a expression in the both tissues and cells. By applying luciferase reporter assay, we verified two putative binding sites between miR-25 and BTG2. Therefore, BTG2 is a directly target of miR-25 in NSCLC cancer. By applying loss-and-gain function analysis in NSCLC cell lines, we demonstrated that miR-25-BTG2 axis could directly regulated BTG2 expression and affect radiotherapy sensitivity of NSCLC cells.

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer-associated mortalities worldwide. MicroRNAs (miRNAs/miRs) serve important roles in tumor development, progression and metastasis. miR-221 has been reported to modulate proliferation, apoptosis, cell cycle distribution and cell migration in a variety of cancers. However, the function of miR-221 in the autophagy of cancer is unclear. In the present study, the role of miR-221 in the autophagy of CRC cells was investigated and its associated target was identified. Survival analysis using The Cancer Genome Atlas data suggested that a higher expression of miR-221 was associated with poor survival in patients with CRC. A Cell Counting kit-8 assay revealed that miR-221 promoted CRC cell proliferation. Autophagy flux analyzed by microtubule-associated protein 1 light chain 3 (LC3) turnover indicated that miR-221 reduced autophagy in CRC cells using different protease inhibitors (E64d and pepstatin A; Bafilomycin A1) in nutrient-rich medium or under starvation conditions. Tumor protein 53-induced nuclear protein 1 (TP53INP1) was identified as a potential novel target of miR-221 by bioinformative prediction. The protein expression of TP53INP1 was inversely regulated by miR-221 in CRC cells. Furthermore, luciferase activity assays were performed and indicated that miR-221 may regulate the luciferase activity of wild-type TP53INP1 without interfering with the activity of mutant TP53INP1. These data suggested that miR-221 may promote the cell proliferation of CRC via the inhibition of autophagy and targeted TP53INP1.

Osteosarcoma (OS) is a bone malignancy with high incidence. The underlying molecular mechanisms that are associated with the development of OS need further investigation. In this study, we showed that SLC34A2, a member of the solute carrier gene family, was significantly downregulated in OS patients and cell lines. Overexpression of SLC34A2 inhibited the proliferation, migration, and invasion of OS cells. Mechanistically, we found that SLC34A2 interacted with PTEN, and inactivated the PI3K/AKT signaling pathway. Collectively, our results demonstrated that SLC34A2 plays important roles in regulating the cancer cell growth of OS. The downregulation of SLC34A2 in OS patients suggested that it might be a promising target in the diagnosis and therapy of OS.

Our previous work reported the association between two single nucleotide polymorphisms (SNPs) in PIN1 promoter and nasopharyngeal carcinoma (NPC) risk with a small sample size in a low incidence area. This study investigated the association between the two SNPs and NPC risk in 733 patients and 895 controls from a high incidence area. The results indicated the genotype and allele frequencies of -842G > C and -667C > T were both significantly different between patients and controls even using the resampling statistics. The -842GC and -667TT genotypes showed a significantly increased risk of NPC (OR = 1.977, 95% CI = 1.339–2.919, P = 0.001 and OR = 1.438, 95% CI = 1.061–1.922, P = 0.019, respectively). Compared to the most common -842G-667C haplotype, -842G-667T haplotype and -842C-667C haplotype showed a significantly increased risk of NPC (OR = 1.215, 95% CI = 1.053–1.402, P = 0.008 and OR = 2.268, 95% CI = 1.530–3.362, P = 0.001, respectively). Further reporter gene expression suggested that variant -842C-667C and -842G-667T were associated with an enhanced transcriptional activity. In conclusion, our findings suggest that -842G > C and -667C > T in PIN1 promoter are associated with NPC risk; as well as the promoter activity is mediated by functional PIN1 variants.

The epidermal growth factor receptor (EGFR) family is reportedly overexpressed in bladder cancer, and tyrosine kinase inhibitors (TKIs) have been suggested as treatment. Gefitinib (Iressa®) is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and HER2 (human EGFR type 2 receptor). Both compounds compete with the binding of ATP to the tyrosine kinase domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a major clinical issue. The purpose of the present study was to use protein array analysis to compare the signaling pathway(s) induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive) and UM-UC-14 (drug-resistant) bladder cancer cells and to identify molecular markers that may be useful predictors of their efficacy. The results revealed that phosphorylation of EGFR, HER3, Met and ERK1/2 was markedly overexpressed in the sensitive cell line (UM-UC-5) and was strongly inhibited by the TKIs. Other notable differences included decreased phosphorylation of RSK, GSK3, AMPK, Akt and c-Jun by TKIs in the sensitive cells. In contrast, phosphorylated p53 was highly expressed in the resistant cell line (UM-UC-14) and TKIs had no effect in the resistant cells. Overall results suggest that phosphorylated HER3, ERK1/2 and p53 may be used as biomarkers to determine the sensitivity of bladder cancers to TKIs. In particular, a combination of these markers may be more likely to predict the sensitivity to TKIs.

Context: The epidermal growth factor receptor (EGFR) family is reportedly overexpressed in bladder cancer, and tyrosine kinaseinhibitors (TKIs) have been suggested as treatment. Gefitinib is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and HER2 (human EGFR type 2 receptor). Both compounds compete with the binding of adenosine triphosphate (ATP) to the tyrosine kinase domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a major clinical problem. Aims: To compare the apoptosis signaling pathway(s) induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive) and UM-UC-14 (drug-resistant) bladder cancer cells and to identify molecular differences that might be useful predictors of their efficacy. Materials and Methods: Cell proliferation, cell cycle and apoptosis assay were used to detect the effect of TKIs on UM-UC-5 and UM-UC-14 cells. Molecular differences for response to TKIs were examined by protein array. Results: TKIs strongly inhibited cell proliferation and induced cell cycle G1 arrest and apoptosis in UM-UC-5 cells. Most notable apoptosis molecular differences included decreased claspin, trail, and survivin by TKIs in the sensitive cells. In contrast, TKIs had no effect on resistant cells. Conclusions: Claspin, trail, and survivin might be used to determine the sensitivity of bladder cancers to TKIs.

Protein Kinase cAMP-Dependent Regulatory Type I Alpha (PRKAR1A) is a tissue-specific extinguisher that transduces a signal through phosphorylation of different target proteins. Loss of PRKAR1A was frequently observed in endocrine neoplasia and stromal cell tumors. However, a few cases were seen in epithelial tumors. Previously, we first found that PRKAR1A was downregulated in lung adenocarcinoma patients. Thus, the present study aimed to clarify its clinical implication and biological function as a tumor suppressor in lung adenocarcinoma. The low levels of PRKAR1A transcript were correlated with tumor progression and poor overall survival. The re-expression of PRKAR1A in H1299 cells suppressed the tumor cell proliferation and migration; stable knockdown (KD) of PRKAR1A in A549 cells enhanced this function both in vitro and in vivo. Moreover, KD of PRKAR1A in A549 cells promoted the statistical colonization of circulating tumor cells to the lungs in nude mice. These effects by PRKAR1A were attributed to inhibiting E-cadherin expression. Elevated E-cadherin significantly suppressed the PRKAR1A-KD induced cell proliferation and migration. Most notably, deletion of PRKAR1A inhibited E-cadherin by activating ERK/Snail signaling. In conclusion, PRKAR1A was a potent suppressor, and through the inhibition of PRKAR1A-ERK-Snail-E-cadherin axis could serve as a potential therapeutic target.

Peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1) plays an important role in cell transformation and oncogenesis. Association between PIN1 promoter polymorphisms and cancer risk was reported in several cancers. This study aimed to evaluate the association between two single nucleotide polymorphisms (SNPs, −667T>C, rs2233679 and −842G>C, rs2233678) on PIN1 promoter and risk of nasopharyngeal carcinoma (NPC). The two SNPs were genotyped using polymerase chain reaction-restriction fragment length polymorphism in a total of 334 native Chinese subjects consisting of 178 cases and 156 controls. The results indicated that the −667CT heterozygote and −667CC homozygote exhibited a significantly decreased risk of nasopharyngeal carcinoma when compared with −667TT homozygote (OR = 0.639, 95 % CI = 0.452–0.903, p = 0.011 for −667CT; and OR = 0.441, 95 % CI = 0.213–0.915, p = 0.038 for −667CC, respectively). In the −842G>C polymorphism, compared with −842GG homozygote, only −842CG heterozygote but not −842CC homozygote had a significantly decreased risk of nasopharyngeal carcinoma (OR = 0.465, 95 % CI = 0.249–0.871, p = 0.010). Genotype in the two SNPs in patients showed no significant associations with the clinicopathologic features examined. Our study showed that the minor genotypes of PIN1 promoter (−667CT, −667CC and −842CG) were associated with decreased risk of NPC in a Chinese population, suggested that PIN1 promoter polymorphisms might play an important role in NPC carcinogenesis.

Aim: The human hepatocellular carcinoma (HCC) cell line HepG2 can easily acquire resistance to doxorubicin. However, the mechanism of action is unclear. Methods: In the present study, we used confocal microscopy, flow cytometry and other methods to reveal the mechanisms by which HepG2 cells acquire doxorubicin resistance. Results: Our results showed that R-HepG2 cells, a doxorubicin-resistant sub-line of HepG2, exhibited decreased intracellular accumulation of doxorubicin and increased expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 when compared with HepG2 cells. R-HepG2 cells also harbored higher levels of glutathione and increased expression of glutathione peroxidase. Furthermore, we demonstrated that the phosphorylation of mitogen-activated protein kinases (p38 and c-jun-N-terminal kinases), IkBα and CREB were increased in R-HepG2 cells. Specific p38 inhibitor SB203580 decreased P-gp expression. The multi-kinase inhibitor sorafenib tosylate also significantly suppressed the phosphorylation of these proteins and inhibited the expression of P-gp. Conclusion: These findings reveal that the drug resistance could be acquired through mitogen-activated protein kinase-dependent upregulation of P-gp. This mechanism protects R-HepG2 cells from the anticancer action of doxorubicin.

Insulin-like growth factor-I receptor (IGF-IR) has been implicated in cancer pathophysiology. Furthermore, impairment of IGF-IR signaling in various cancer cell lines caused inhibition of the transformed phenotype as determined by the inhibition of colony formation in soft agar and the inhibition of tumor formation in athymic nude mice. Thus, the IGF-IR might be an attractive target for cancer prevention. We showed that the tea polyphenol, (−)−epigallocatechin-3-gallate (EGCG), is a small-molecule inhibitor of IGF-IR activity (IC50 of 14 μmol/L). EGCG abrogated anchorage-independent growth induced by IGF-IR overexpression and also prevented human breast and cervical cancer cell phenotype expression through inhibition of IGF-IR downstream signaling. Our findings are the first to show that the IGF-IR is a novel binding protein of EGCG and thus may help explain the chemopreventive effect of EGCG on cancer development. (Cancer Epidemiol Biomarkers Prev 2007;16(3):598–605)

Background Nurr1, a member of the orphan receptor family, plays an important role in several types of cancer. Our previous work demonstrated that increased expression of Nurr1 plays a significant role in the initiation and progression of prostate cancer (PCa), though the mechanisms for regulation of Nurr1 expression remain unknown. In this study, we investigated the hypothesis that Nemo-like kinase (NLK) is a key regulator of Nurr1 expression in PCa. Methods Immunohistochemistry and Western blot analysis were used to evaluate levels of NLK and Nurr1 in prostatic tissues and cell lines. The effects of overexpression or knockdown of Nurr1 were evaluated in PCa cells through use of PCR, Western blots and promoter reporter assays. The role of Nurr1 promoter cis element was studied by creation of two mutant Nurr1 promoter luciferase constructs, one with a mutated NF-κB binding site and one with a mutated CREB binding site. In addition, three specific inhibitors were used to investigate the roles of these proteins in transcriptional activation of Nurr1, including BAY 11–7082 (NF-κB inhibitor), KG-501 (CREB inhibitor) and ICG-001 (CREB binding protein, CBP, inhibitor). The function of CBP in NLK-mediated regulation of Nurr1 expression was investigated using immunofluorescence, co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation assays (ChIPs). Results NLK expression was inversely correlated with Nurr1 expression in prostate cancer tissues and cell lines. Overexpression of NLK suppressed Nurr1 promoter activity, leading to downregulation of Nurr1 expression. In contrast, knockdown of NLK demonstrated opposite results, leading to upregulation of Nurr1. When compared with the wild-type Nurr1 promoter, mutation of NF-κB- and CREB-binding sites of the Nurr1 promoter region significantly reduced the upregulation of Nurr1 induced by knockdown of NLK in LNCaP cells; treatment with inhibitors of CREB, CBP and NF-κB led to similar results. We also found that NLK directly interacts with CBP, that knockdown of NLK significantly increases the recruitment of CBP to both NF-κB- and CREB-binding sites, and that regulation of NLK on Nurr1 expression is abrogated by knockdown of CBP. Conclusions Our results suggest that NLK inhibits transcriptional activation of Nurr1 gene by impeding CBP’s role as a co-activator of NF-κB and CREB in prostate cancer.

Paxillin is a substrate of the Src tyrosine onco-kinase and is involved in cell transformation, cell spreading, migration, and cancer development mediated through the mitogen-activated protein kinase signaling cascades. Here, we showed that paxillin plays a key role in skin cell transformation induced by epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). To investigate the mechanism of paxillin's role in cell transformation, we established a paxillin knockdown stably transfected cell line by introducing small interfering RNA-paxillin (si-paxillin). The si-paxillin cells displayed a dramatic suppression of cell proliferation and anchorage-independent cell transformation induced by EGF or TPA compared with si-mock control cells. In si-paxillin cells, decreased activator protein-1 (AP-1)–dependent luciferase activity corresponded with suppressed AP-1 DNA binding activity. Importantly, knockdown of paxillin inhibited EGF- or TPA-induced c-Jun phosphorylation at Ser63 and Ser73. Furthermore, total c-Jun protein level was dramatically decreased in si-paxillin cells and was dependent on serum deprivation time. The down-regulation of c-Jun was restored in si-paxillin cells by treatment with the proteasome inhibitor lactacystin but not by the lysosome inhibitor leupeptin. These results clearly provided evidence that paxillin regulates c-Jun protein level and plays a key role in cell transformation most likely through the regulation of c-Jun stability. (Cancer Res 2006; 66(11): 5968-74)