BCLAF1-induced HIF-1α accumulation under normoxia enhances PD-L1 treatment resistances via BCLAF1-CUL3 complex.

Bcl-2-associated transcription factor-1 (BCLAF1), an apoptosis-regulating protein of paramount significance, orchestrates the progression of various malignancies. This study reveals increased BCLAF1 expression in hepatocellular carcinoma (HCC) patients, in whom elevated BCLAF1 levels are linked to escalated tumor grades and diminished survival rates. Moreover, novel BCLAF1 expression is particularly increased in HCC patients who were not sensitive to the combined treatment of atezolizumab and bevacizumab, but not in patients who had tumors that responded to the combined regimen. Notably, overexpression of BCLAF1 increases HCC cell proliferation in vitro and in vivo, while the conditioned medium derived from cells overexpressing BCLAF1 strikingly enhances the tube-formation capacity of human umbilical vein endothelial cells. Furthermore, compelling evidence demonstrates that BCLAF1 attenuates the expression of prolyl hydroxylase domain protein 2 (PHD2) and governs the stability of hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions without exerting any influence on transcription, as determined by Western blot and RT‒qPCR analyses. Subsequently, employing coimmunoprecipitation and immunofluorescence, we validated the reciprocal interaction between BCLAF1 and Cullin 3 (CUL3), through which BCLAF1 actively upregulates the ubiquitination and degradation of PHD2. The Western blot and RT‒qPCR results suggests that programmed death ligand-1 (PD-L1) is one of the downstream responders to HIF-1α in HCC. Thus, we reveal the pivotal role of BCLAF1 in promoting PD-L1 transcription and, through binding to CUL3, in promoting the accumulation of HIF-1α under normoxic conditions, thereby facilitating the ubiquitination and degradation of PHD2.

Aberrant methylation and microRNA-target regulation are associated with downregulated NEURL1B: a diagnostic and prognostic target in colon cancer.

BACKGROUND: Aberrant methylation and miRNA-target-gene regulation function as important mechanisms for gene inactivation in colon carcinogenesis. Although a serious of molecular events (such as aberrant alterations of genomics and epigenetics) have been identified to be related to prognostic in colon cancer (CC) patients, beneficial biomarkers for early diagnosis and prognostic evaluation remain largely unknown. METHODS: In our study, the role of NEURL1B, including gene expression analysis, methylation characteristic, miRNA-target regulation, diagnostic and prognostic significance, were evaculated using multiple bioinformatic tools based on TCGA database and clinical samples. RESULTS: Our data showed that NEURL1B was aberrantly downregulated in CC, regardless of the mRNA level or protein level. Moreover, ROC curve and multivariate Cox regression analysis demonstrated that NEURL1B was a diagnostic and independent prognostic facter for CC patients. Of interest, methylation of NEURL1B was also high and closely associated with poor survival in CC. In addition, multiple NEURL1B-target miRNAs were found to be overexpressed in CC tissues. Thus, our findings suggested that NEURL1B participated in the pathological processes of CC as a tumor suppressor gene. Double management, including DNA methylation modification and miRNA-target regulation, were considered to be related to the downregulation of NEURL1B. Importantly, there existing be an significant intersection between miRNAs-target pathways and NEURL1B-target pathways, suggesting that miR-17 and miR-27a might promote tumor cell malignant property by targeting NEURL1B degradation via the activation of PI3K/AKT signaling pathway. CONCLUSIONS: Taking together, the first investigation of NEURL1B in CC provide us a strong evidences that it might be served as a potential biomarkers for early diagnosis and prognostic evaluation in CC.

Hypoxia-induced lncRNA EIF3J-AS1 accelerates hepatocellular carcinoma progression via targeting miR-122-5p/CTNND2 axis.

Long noncoding RNAs (lncRNAs) exert crucial roles in hepatocellular carcinoma (HCC) progression. LncRNA EIF3J-AS1 is recently reported to be highly expressed in HCC and correlates with recurrence-free survival. However, the biological function of EIF3J-AS1 and its underlying molecular mechanism are unexplored yet. In the current study, we demonstrated that EIF3J-AS1 expression was obviously upregulated in HCC tissues compared to adjacent noncancerous tissues. Moreover, the elevated levels of EIF3J-AS1 was detected in four HCC cell lines (HepG2, SMMC-7721, MHCC97H, HCCLM3) compared with the normal hepatic cell line LO2. Notably, the expression of EIF3J-AS1 was correlated with prognostic features including tumor size, vascular invasion and tumor stage. TCGA-LIHC data indicated that the upregulated expression of EIF3J-AS1 predicted poor prognosis of HCC. EIF3J-AS1 knockdown remarkably suppressed the proliferation, migration and invasion of HCC cells. Mechanistically, EIF3J-AS1 inversely regulated miR-122-5p expression via acting as a competing endogenous RNA (ceRNA) in HCC cells. Furthermore, catenin delta 2 (CTNND2) was recognized as a novel target of miR-122-5p. CTNND2 restoration partially reversed EIF3J-AS1 knockdown-induced inhibitory effects on HCC cell proliferation, migration and invasion. Importantly, we found that hypoxia induced EIF3J-AS1 and CTNND2 expression, and led to miR-122-5p downregulation in HCC cells. EIF3J-AS1 knockdown partially abolished hypoxia-induced HCC cell proliferation and mobility. In conclusion, our results provide a new insight into the molecular pathogenesis of HCC, and EIF3J-AS1 may be a potential therapeutic target for HCC.

Correction to: Bioinformatic exploration of OLFML2B overexpression in gastric cancer base on multiple analyzing tools.

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Bioinformatic exploration of OLFML2B overexpression in gastric cancer base on multiple analyzing tools.

BACKGROUND: Gastric cancer (GC) is one of the most commonly occuring gastrointestinal tumor types, and early diagnosis and operation have a notable effect on the prognosis of patients. Although certain markers, including HER2, VEGER-2, ERCC1 and Bcl-2, have been utilized in clinical practise to screen out new patients with GC, the results of using these markers remains poor. The role of olfactomedin-like 2B (OLFML2B) in GC, as a member of the olfactomedin domain-containing proteins family, is remain unclear. METHODS: In the present study, we assessed the expression of OLFML2B, including mRNA and protein level, by using The Cancer Genome Atlas (TCGA) database and 13 pairs of clinical samples between GC and NG tissues. The correlation between expression of OLFML2B and prognosis of GC was evaculated by the Kaplan-Meier plotter and OncoLnc online tools. In addition, mechanism analysis of OLFML2B in GC was explored thought bioinformatic tools, including cBioPortal and FunRich software. RESULTS: In our study, the mRNA expression of OLFML2B in GC both TCGA database and clinical samples was consistently revealed to be significantly higher compared with that in NG tissues (P < 0.0001 for TCGA database and P = 0.0034 for clinical samples), and high OLFML2B expression was found in 9 (69.23%) of 13 clinical GC by immunohistochemistry and was positively correlated with the depth of tumor invasion and clinical stage (TNM). In addition, the AUC for a ROC of 0.867 indicated a moderate diagnostic ability of OLFML2B for GC. Survival analysis from the Kaplan-Meier plotter (P = 2.6 × 10- 6) and OncoLnc (P = 0.00276) revealed that the high expression of OLFML2B was associated with a short overall survival. Futhermore, 5% (24/478) alterations of OLFML2B were identified from cBioPortal, and among them, missense mutation (14/478) was the primary type. The results from FunRich revealed that OLFML2B participated in mediating multiple biological processes including cell growth and maintenance, regulation of the cell cycle, apoptosis and cell communication through multiple signaling pathways including the M/G1 transition pathway, post-translational protein modification and DNA replication pre-initiation. CONCLUSIONS: Taken together, it could be deduced that OLFML2B may act as an oncogene in the development of GC and the overexpression of OLFML2B in GC may be used as a novel diagnostic and prognostic target for GC.

Highly frequent PIK3CA amplification is associated with poor prognosis in gastric cancer.

BACKGROUND: The phosphoinositide 3-kinase (PI3K)/Akt pathway plays a fundamental role in cell proliferation and survival in human tumorigenesis, including gastric cancer. PIK3CA mutations and amplification are two major causes of overactivation of this pathway in human cancers. However, until this work, there was no sound investigation on the association of PIK3CA mutations and amplification with clinical outcome in gastric cancer, particularly the latter. METHODS: Using direct sequencing and real-time quantitative PCR, we examined PIK3CA mutations and amplification, and their association with clinicopathological characteristics and clinical outcome of gastric cancer patients. RESULTS: PIK3CA mutations and amplification were found in 8/113 (7.1%) and 88/131 (67%) gastric cancer patients, respectively. PIK3CA amplification was closely associated with increased phosphorylated Akt (p-Akt) level. No relationship was found between PIK3CA mutations and clinicopathological characteristics and clinical outcome in gastric cancer. PIK3CA amplification was significantly positively associated with cancer-related death. Importantly, Kaplan-Meier survival curves revealed that the patients with PIK3CA amplification had significantly shorter survival times than the patients without PIK3CA amplification. CONCLUSIONS: Our data showed that PIK3CA mutations were not common, but its amplification was very common in gastric cancer and may be a major mechanism in activating the PI3K/Akt pathway in gastric cancer. Importantly, Kaplan-Meier survival curves revealed that PIK3CA amplification was significantly positively associated with poor survival of gastric cancer patients. Collectively, the PI3K/Akt signaling pathway may be an effective therapeutic target in gastric cancer.

Frequent gene amplification predicts poor prognosis in gastric cancer.

Gastric cancer is one of the most common malignancies worldwide. However, genetic alterations leading to this disease are largely unknown. Gene amplification is one of the most frequent genetic alterations, which is believed to play a major role in the development and progression of gastric cancer. In the present study, we identified three frequently amplified genes from 30 candidate genes using real-time quantitative PCR method, including ERBB4, C-MET and CD44, and further explored their association with clinicopathological characteristics and poor survival in a cohort of gastric cancers. Our data showed amplification of these genes was significantly associated with certain clinicopathological characteristics, particularly tumor differentiation and cancer-related death. More importantly, amplification of these genes was significantly related to worse survival, suggesting that these amplified genes may be significant predictors of poor prognosis and potential therapeutic targets in gastric cancer. Targeting these genes may thus provide new possibilities in the treatment of gastric cancer.

Lenvatinib Promotes the Antitumor Effect of Doxorubicin in Anaplastic Thyroid Cancer.

PURPOSE: Anaplastic thyroid cancer (ATC) is a kind of rare thyroid cancer with very poor prognosis. Doxorubicin has been approved in ATC treatment as a single agent, but monotherapy still shows no improvement of the total survival in advanced ATC. Lenvatinib was investigated with encouraging results in treating patients with radioiodine-refractory differentiated thyroid cancer (DTC). However, antitumor efficacy of combination therapy with lenvatinib and doxorubicin remains largely unclear. MATERIALS AND METHODS: The antitumor efficacy of combination therapy with lenvatinib and doxorubicin on ATC cell proliferation was assessed by the MTT assay and colony formation. Flow cytometry was employed to assess ATC cells' apoptosis and cell cycle arrest in response to combination therapy. Transwell assay was used to test the migration and invasion in response to combination therapy. Xenograft models were used to test its in vivo antitumor activity. RESULTS: Lenvatinib monotherapy was less effective than doxorubicin in treating ATC cell lines and xenograft model. The combination therapy of lenvatinib and doxorubicin significantly inhibited ATC cell proliferation and tumor growth in nude mice, and induced cell apoptosis and cell cycle arrest as compared to lenvatinib or doxorubicin monotherapy. CONCLUSION: Lenvatinib promotes the antitumor effect of doxorubicin in ATC cell and xenograft model. The lenvatinib/doxorubicin combination may be a potential candidate therapeutic approach for anaplastic thyroid cancer.

Vitamin C kills thyroid cancer cells through ROS-dependent inhibition of MAPK/ERK and PI3K/AKT pathways via distinct mechanisms.

Background: Vitamin C has been demonstrated to kill BRAF mutant colorectal cancer cells selectively. BRAF mutation is the most common genetic alteration in thyroid tumor development and progression; however, the antitumor efficacy of vitamin C in thyroid cancer remains to be explored. Methods: The effect of vitamin C on thyroid cancer cell proliferation and apoptosis was assessed by the MTT assay and flow cytometry. Xenograft and transgenic mouse models were used to determine its in vivo antitumor activity of vitamin C. Molecular and biochemical methods were used to elucidate the underlying mechanisms of anticancer activity of vitamin C in thyroid cancer. Results: Pharmaceutical concentration of vitamin C significantly inhibited thyroid cancer cell proliferation and induced cell apoptosis regardless of BRAF mutation status. We demonstrated that the elevated level of Vitamin C in the plasma following a high dose of intraperitoneal injection dramatically inhibited the growth of xenograft tumors. Similar results were obtained in the transgenic mouse model. Mechanistically, vitamin C eradicated BRAF wild-type thyroid cancer cells through ROS-mediated decrease in the activity of EGF/EGFR-MAPK/ERK signaling and an increase in AKT ubiquitination and degradation. On the other hand, vitamin C exerted its antitumor activity in BRAF mutant thyroid cancer cells by inhibiting the activity of ATP-dependent MAPK/ERK signaling and inducing proteasome degradation of AKT via the ROS-dependent pathway. Conclusions: Our data demonstrate that vitamin C kills thyroid cancer cells by inhibiting MAPK/ERK and PI3K/AKT pathways via a ROS-dependent mechanism and suggest that pharmaceutical concentration of vitamin C has potential clinical use in thyroid cancer therapy.

Examination of the expression and prognostic significance of DLGAPs in gastric cancer using the TCGA database and bioinformatic analysis.

The discs large­associated protein (DLGAP) family has been implicated in psychological and neurological diseases. However, few studies have explored the association between the expression of DLGAPs and different types of cancer. Therefore, the present study analyzed the status of DLGAPs in gastric cancer (GC) using bioinformatic tools. Analyses of data obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases revealed that there was selective upregulation of DLGAP4 and DLGAP5 expression in GC tissues when compared with normal gastric tissues. In addition, survival analysis using OncoLnc indicated that high expression of DLGAP4 was significantly correlated with shorter overall survival for all GC patients. However, Kaplan­Meier plots demonstrated that the expression of all DLGAPs, except for DLGAP3, was correlated with patient prognosis; DLGAP4 was consistently associated with GC. DLGAP4 mRNA and protein distributions were examined by reverse transcription­quantitative polymerase chain reaction and immunohistochemsitry. Furthermore, its mutation rate and associated biological processes and signaling pathways were assessed in GC with cBioPortal and FunRich analyses. Taken together, these results indicated that DLGAP4 may serve an oncogenic role in GC development and may be a monitoring target for GC prognosis.

Site-specific Hypermethylation of RUNX3 Predicts Poor Prognosis in Gastric Cancer.

BACKGROUND AND AIMS: Methylation status of RUNX3 remains largely unknown in gastric cancer (GC). The aim of this study was to prognostically evaluate the methylation level of CpG sites within RUNX3 promoter region in GC. METHODS: Using pyrosequencing, we quantitatively explored the methylation status of 8 CpG sites within RUNX3 promoter region for 76 gastric cancer and 24 normal gastric tissues. We then analyzed the association between methylation level of each CpG site and clinicopathological characteristics and outcomes in the cohort. RESULTS: Methylation of RUNX3 promoter was significantly higher in GC than normal subjects. Overall methylation level was closely associated with tumor invasion and TNM stage. Positive associations were found between hypermethylation of the following concerned sites and variables: site -1392, -1397, -1403, -1415 and tumor invasion, as well as TNM stage; site -1392 and lymph node metastasis along with number of lymph node metastases; site -1415 and cancer recurrence; site -1403, -1415 and cancer-related deaths. In multivariate analysis, tumor invasion was correlated with sites -1392 and -1397. Lymph node metastasis was associated with site -1392. Most importantly, methylation of site -1415 was associated with poor survival by using Cox survival regression. CONCLUSION: Analysis of RUNX3 gene promoter by quantitative pyrosequencing suggested methylation status of RUNX3 is different in normal and tumor tissues. RUNX3 methylation level is associated with GC, especially the methylation at site -1415 contributes to the poor prognosis in GC. Thus, RUNX3 methylation may serve as a valuable diagnostic and prognostic biomarker in GC.

Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer.

The biological function of E26 transformation-specific (ETS) transcription factor EHF/ESE-3 in human cancers remains largely unknown, particularly gastric cancer. The aim of this study was to explore the role of EHF in tumorigenesis and its potential as a therapeutic target in gastric cancer. By using quantitative RT-PCR (qRT-PCR), immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays, we investigated the expression and copy number of EHF in a cohort of gastric cancers and control subjects. Specific EHF siRNAs was used to determine the biologic impacts and mechanisms of altered EHF expression in vitro and in vivo. Dual-luciferase reporter, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) assays were performed to identify its downstream targets. Our results demonstrated that EHF was significantly upregulated and frequently amplified in gastric cancer tissues as compared with control subjects. Moreover, EHF amplification was positively correlated with its overexpression and significantly associated with poor clinical outcomes of gastric cancer patients. We also found that EHF knockdown notably inhibited gastric cancer cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice and induced cell cycle arrest and apoptosis. Importantly, we identified EHF as a new HER2 transcription factor and the modulator of HER3 and HER4 in gastric cancer. Collectively, our findings suggest that EHF is a novel functional oncogene in gastric cancer by regulating the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and may represent a potential prognostic marker and therapeutic target for this cancer.

Quantitative assessment of gene methylation and their impact on clinical outcome in gastric cancer.

BACKGROUND: Promoter methylation is an alternative mechanism of gene silencing in human tumorigenesis. Although a number of methylated genes have been found in gastric cancer, useful methylation markers for early diagnosis and prognostic evaluation of this cancer remain largely unknown. METHODS: Using quantitative methylation-specific PCR (Q-MSP), we examined promoter methylation of 6 genes, including CALCA, DAPK1, RARbeta, RASSF1A, TIMP3, and PAX6, and explored their association with clinical outcome in gastric cancer. RESULTS: We found that most of the genes investigated in the present study had significantly higher methylation level in tumor tissues than normal gastric tissues, including CALCA, RARbeta, RASSF1A, TIMP3, and PAX6. With more focus on specificity compared to sensitivity, all genes were hypermethylated in gastric cancer, ranging from 12.8% to 36.9%. Methylation of TIMP3 and PAX6 was strongly associated with differentiation and lymph node metastasis, respectively. Importantly, most of gene methylation, except for DAPK1, was closely associated with poor survival in gastric cancer. CONCLUSION: We found that a panel of genes was specifically methylated in gastric cancer, and demonstrated the effect of promoter methylation of some genes on clinical outcome in gastric cancer, indicating these methylated genes may be useful biomarkers for prognostic evaluation in this cancer.

Prognostic significance of aberrant gene methylation in gastric cancer.

Promoter methylation acts as an important alternative to genetic alterations for gene inactivation in gastric carcinogenesis. Although a number of gastric cancer-associated genes have been found to be methylated in gastric cancer, valuable methylation markers for early diagnosis and prognostic evaluation of this cancer remain largely unknown. In the present study, we used methylation-specific PCR (MSP) to analyze promoter methylation of 9 gastric cancer-associated genes, including MLF1, MGMT, p16, RASSF2, hMLH1, HAND1, HRASLS, TM, and FLNc, and their association with clinicopathological characteristics and clinical outcome in a large cohort of gastric cancers. Our data showed that all of these genes were aberrantly methylated in gastric cancer, ranging from 8% to 51%. Moreover, gene methylation was strongly associated with certain clinicopathological characteristics, such as tumor differentiation, lymph node metastasis, and cancer-related death. Of interest, methylation of MGMT, p16, RASSF2, hMLH1, HAND1, and FLNc was closely associated with poor survival in gastric cancer, particularly MGMT, p16, RASSF2 and FLNc. Thus, our findings suggested these epigenetic events may contribute to the initiation and progression of gastric cancer. Importantly, methylation of some genes were closely relevant to poor prognosis in gastric cancer, providing the strong evidences that these hypermethylated genes may be served as valuable biomarkers for prognostic evaluation in this cancer.