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Precise methods for risk stratification to guide adjuvant chemotherapy for stage III colon cancers are needed. Here, we combined circulating tumor DNA (ctDNA) with consensus molecular subtype (CMS) to improve risk stratification in stage III colon cancers.We conducted a prospective, observational cohort study of 165 patients with stage III colon cancers. Somatic variants in tumor tissues and plasmas collected pre- and post-chemo were detected via a targeted sequencing panel of 197 cancer-related genes. CMSs classification was characterized using a targeted RNA sequencing panel of 788 genes.We analyzed 151 pre-chemo and 124 post-chemo plasmas, while 130 patients were CMSs classified. ctDNA was detectable in 15.9% pre-chemo and 8.9% post-chemo samples. Significantly worse recurrence-free survival (RFS) was seen if ctDNA was detectable in pre-chemo samples (hazard ratio [HR], 3.585; P 0.001) or in post-chemo samples (HR, 3.337; P = 0.005). Pre-chemo ctDNA (HR, 5.538; P 0.001) and post-chemo ctDNA status (HR, 3.272; P = 0.037) remained independently associated with RFS in multivariate analysis. According to the redefined recurrence risk stratification, mid-risk patients (ctDNA-negative with CMS4/T4 or N2 tumors) were 5.3 times (HR, 5.269; P = 0.025) more likely to relapse than low-risk patients (ctDNA-negative with CMS1-3/T3N1 tumors), while high-risk patients (ctDNA-positive) were 14.6 times (HR, 14.590; P 0.001) more likely to relapse.Postoperative ctDNA indicating residual disease, combined with CMSs classification and clinical risk reflecting the intrinsic characteristics of tumors, can redefine risk stratification of stage III colon cancers and better predict relapse.

396 Background: PD-1 inhibitor combined with chemotherapy is one of the standard regimens for first line gastric. The addition of anti-vascularization agents has been reported to help improve the overall survival (OS) in GC/GEJC patients (pts) through vascular-normalization in the microenvironment. In this phase I/IIa study, we aim to explore the safety and efficacy of tislelizumab combined with anlotinib and XELOX (TAXE regimen) as first line treatment for advanced GC/GEJC. Methods: This prospective, multicenter, phase I/IIa study planned to enroll 9 patients in phase I and 57 patients in Phase IIa. The aim of this Phase 1 study was to determine dose-limiting toxicities (DLTs), maximum tolerated dose (MTD) and safety of 8 mg, 10 mg or 12 mg anlotinib in combination with XELOX and tislelizumab. The maximum tolerable dosage (MTD) would be administered to all pts in phase II. The primary and objective response rate(ORR). Secondary endpoints included disease control rate (DCR), duration of response (DoR), progression-free survival (PFS), OS, and safety. The exploring endpoint was to evaluate the association between the efficacy and the expression of biomarkers including PD-L1, microsatellite instability profile, and genome stability (GS). Results: From march 2021 to August 2022, 38 pts were enrolled (9 pts in phase 1). Phase 1 was completed without dosage limiting toxicities (DLTs). 12mg anlotinib was recommended for phase 2. Thirty two (84.21%) pts had at least one treatment-related adverse events (TRAEs), and 7 patients (18.42%) had at least one grade 3/4 TRAEs. Anemia (15 pts, 39.47%), leukopenia (12 pts, 31.58%) and fatigue (10 pts, 26.32%) were the most common TRAEs. Two pts achieved complete response (CR), 20 pts achieved partial response (PR), and 7 pts achieved stable disease (SD). The ORR was 75.86% (95% CI: 57.89%-87.78%), and the DCR was 100%. Conclusions: The preliminary results of this Phase I/IIa trial by using tislelizumab combined with anlotinib and XELOX had shown great potential in improving the response rate in advanced GC/GEJC patients with manageable toxicity. Clinical trial information: NCT04963088 .

The discovery of long noncoding RNAs (lncRNAs) has increased our understanding of the development and progression of many cancers, but their contributions to non-small cell lung cancer (NSCLC) remain poorly understood. Here, we profiled lncRNA expression in NSCLC and investigated in detail the molecular function of one upregulated lncRNA, LINC01234. LINC01234 was overexpressed in NSCLC compared with normal lung tissue and correlated positively with poor prognosis. Downregulation of LINC01234 impaired cell proliferation in vitro and tumor growth in vivo. RNA pull-down/mass spectrometry experiments showed that LINC01234 interacted with the RNA-binding protein heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1), which, in turn, led to the recruitment of DiGeorge syndrome critical region gene 8 (DGCR8), a subunit of the microRNA (miRNA) microprocessor complex. Accordingly, depletion of either LINC01234 or HNRNPA2B1 reduced the processing of several miRNA precursors, including primary microRNA (pri-miR)-106b. miR-106b-5p enhanced NSCLC cell growth by downregulating cryptochrome 2 (CRY2), thereby increasing c-Myc expression. Finally, we found that activated c-Myc binds to the LINC01234 promoter to increase its transcription, creating a c-Myc-LINC01234-HNRNPA2B1-miR-106b-5p-CRY2-c-Myc positive-feedback loop. We identified numerous lncRNAs with dysregulated expression in NSCLC and demonstrated a novel oncogenic axis involving LINC01234, HNRNPA2B1, miR-106b-5p, CRY2, and c-Myc. Components of this axis may be potential novel targets for NSCLC.

Lung cancer is the leading cause of cancer-related death worldwide. Owing to the difficulty in early diagnosis and the lack of effective treatment strategies, the 5-year survival rates for lung cancer remain very low. With the development of whole genome and transcriptome sequencing technology, long non-coding RNA (lncRNA) has attracted increasing attention. LncRNAs regulate gene expression at the epigenetic, transcriptional and post-transcriptional levels and are widely involved in a variety of diseases, including tumorigenesis. In lung cancer studies, multiple differentially expressed lncRNAs have been identified; several lncRNAs were identified as oncogenic lncRNAs with tumor-driving effects, while other lncRNAs play a role in tumor inhibition and are called tumor-suppressive lncRNAs. These tumor-suppressive lncRNAs are involved in multiple physiological processes such as cell proliferation, apoptosis, and metastasis and thus participate in tumor progression. In this review, we discussed the oncogenic and tumor-suppressive lncRNAs in lung cancer, as well as their biological functions and regulatory mechanisms. Furthermore, we found the potential significance of lncRNAs in clinical diagnosis and treatment.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib, have been established as first-line treatments for non-small cell lung cancer (NSCLC) patients and have exhibited notable clinical efficacy. However, resistance to TKIs has become one of the major obstacles in improving the therapeutic efficacy of patients with NSCLC. This study aims to investigate the role of the long non-coding RNA (lncRNA) LINC01116 in gefitinib resistance of NSCLC and explore its underlying mechanism. In this study, we found that LINC01116 is upregulated in the gefitinib-resistant NSCLC cells and tissues. Loss- and gain-of-function assays uncovered that LINC01116 downregulation sensitized gefitinib resistance, whereas the overexpression of LINC01116 conferred PC9/R cells to gefitinib resistance. Moreover, LINC01116 silencing increased IFI44 expression. Overexpression of IFI44 reversed the resistance to gefitinib in PC9/R cells, and rescue experiments confirmed that LINC01116 affects the gefitinib resistance of PC9/R cells partly dependent on regulating IFI44 expression. Moreover, downregulation of LINC01116 increased the sensitivity of PC9/R cells to gefitinib in vivo. Our study demonstrates that LINC01116 plays a critical role in gefitinib resistance of NSCLC cells by affecting IFI44 expression, providing a novel therapeutic target to overcome TKI resistance in NSCLC.

Undifferentiated large-cell lung cancer is a rare type of non-small cell lung cancer (NSCLC) with a poor prognosis. It is insensitive to chemotherapy and easily develops drug resistance. Analysis of the Surveillance, Epidemiology, and End Results (SEER) database showed that patients with stage IV undifferentiated large-cell lung cancer had a median overall survival (OS) of only 4 months and that those who received chemotherapy had a median OS of only 5 months longer than those who did not. For the first time, we report a case of advanced large-cell undifferentiated lung cancer with rare tonsil metastasis. The patient developed resistance after 3 months of platinum-based systemic chemotherapy and local treatment. Antiangiogenic therapy has been continuously progressing and has shown certain efficacy in treating many malignant tumors, such as lung cancer. However, there are no relevant studies or case reports on antiangiogenic therapy in the treatment of undifferentiated large-cell lung cancer. Anlotinib, an orally delivered small-molecule antiangiogenic tyrosine kinase inhibitor (TKI), was administered to this patient after chemotherapy resistance occurred, and the outcome was assessed as continued stable disease (SD). As of the last follow-up evaluation, the progression-free survival (PFS) of the patient was 21.5 months, and the OS was 27.5 months. Retrospective immunohistochemical analysis showed that the patient was positive for one of the targets of anlotinib (PDGFR). In general, the findings in this case suggest that anlotinib may be an option with good efficacy for patients with large-cell undifferentiated lung cancer after chemotherapy resistance that may have good efficacy and also suggest that PDGFR may be the target underlying this effect.

Lung cancer is the most common cancer all around the world, with high morbidity and mortality. Long noncoding RNA (lncRNA) has been reported to have a critical role in non-small-cell lung cancer (NSCLC) proliferation and migration. In the present study, we analyzed The Cancer Genome Atlas (TCGA) data, and we found that lncRNA Small Nucleolar RNA Host Gene 17 (SNHG17) was upregulated in NSCLC driven by the amplification of copy number, indicating the special role of SNHG17 in NSCLC. The full exact length of SNHG17 was determined by rapid amplification of cDNA ends (RACE). We modulated SNHG17 expression by RNAi and a series of functional assays were performed. Flow cytometry was used to explore the involvement of SNHG17 in NSCLC cell apoptosis. Results showed that the knockdown of SNHG17 inhibited the proliferation and migration and promoted the apoptosis of NSCLC cells. We acquired the global gene expression profile regulated by SNHG17 in A549 through RNA sequencing (RNA-seq) assays. We found 637 genes were upregulated while 581 genes were downregulated. We selected three genes (FOXA1, XAF1, and BIK) that were closely related to proliferation and apoptosis, and we confirmed their altered expression in A549 and PC-9 cells treated with small interfering RNA si-SNHG17. Our findings indicated gene amplification-driven lncRNA SNHG17 promotes cell proliferation and migration in NSCLC, suggesting its potential value as a biomarker in NSCLC. Keywords: gene amplification, lncRNA, proliferation, migration, NSCLC

Lung cancer is the most common cancer globally and is associated with high morbidity and mortality. Gefitinib has been widely used for treating advanced non-small-cell lung cancer (NSCLC). However, acquired resistance usually develops, although we still know little about the mechanism underlying this. In the present study, we found that the lncRNA UCA1 was upregulated in NSCLC tissues and cells with acquired gefitinib resistance, indicating the special role of UCA1 in gefitinib resistance. Knockdown of UCA1 promoted the sensitivity to gefitinib both in vitro and in vivo by suppressing cell proliferation and inducing apoptosis. Moreover, UCA1 could interact with EZH2 (enhancer of zeste homolog 2) to epigenetically reduce the expression of CDKN1A. Taking the obtained findings together, our study suggests that UCA1 is important for NSCLC to develop gefitinib resistance, and is a potential biomarker for gefitinib resistance and a therapeutic target for advanced NSCLC.

Background Long noncoding RNAs (lncRNAs) are known to regulate tumorigenesis and cancer progression, but their contributions to non-small-cell lung cancer (NSCLC) metastasis remain poorly understood. Our previous and other studies have revealed the involvement of upregulated LINC01234 in regulating gastric cancer and colon cancer cells proliferation, and we aimed to investigate whether LINC01234 overexpression also contribute to cancer cells metastasis in this study. Methods We collect the NSCLC tissues and adjacent non-tumor tissues and analyzed expression levels of LINC01234 by quantitative reverse-transcription PCR. LINC01234 were knocked down by using siRNAs or shRNAs, and overexpressed by transfection with overexpression vector; RNA levels of miRNA were downregulated or upregulated with inhibitors or mimics. Transwell assays were used to evaluate cell migration and invasive ability; in vivo metastasis experiments were performed to investigate the effect of LINC01234 on NSCLC cells metastasis. Luciferase reporter, RIP, and ChIP assays were used to determine the regulation of LINC01234 on its targets. Results LINC01234 expression is increased in NSCLC tissues, and its upregulation is associated with metastasis and shorter survival in NSCLC. Downregulation of LINC01234 impairs cell migration and invasion in vitro, and inhibits cells metastasis in vivo by acting as a competing endogenous RNA for the miR-340-5p and miR-27b-3p. LINC01234 also interacts with the RNA-binding proteins LSD1 and EZH2, leading to histone modification and transcriptional repression of the anti-proliferative genes BTG2. Conclusions Taken together, our findings identify two oncogenic regulatory axes in NSCLC centering on LINC01234: one involving miR-340-5p/miR-27b-3p in the cytoplasm and the second involving EZH2, LSD1, and BTG2 in the nucleus. Our study indicates that these genes may be targeted to reduce or prevent NSCLC metastasis.

// Chen-Chen Wei 1, * , Feng-Qi Nie 1, * , Li-Li Jiang 1, 2, * , Qin-Nan Chen 1 , Zhen-Yao Chen 1 , Xin Chen 1 , Xuan Pan 3 , Zhi-Li Liu 4 , Bin-Bin Lu 1 , Zhao-Xia Wang 1 1 Department of Oncology, Second Affiliated Hospital, Nanjing Medical University, Nanjing, People’s Republic of China 2 Department of Oncology, Haimen People’s Hospital, Haimen, People’s Republic of China 3 Department of Medical Oncology, Nanjing Medical University Affiliated Cancer Hospital of Jiangsu Province, Cancer Institution of Jiangsu Province, Nanjing, People’s Republic of China 4 Department of Oncology, The Affiliated Jiangyin Hospital, School of Medicine, Southeast University, Jiangyin, People’s Republic of China * These authors contributed equally to this work and should be regarded as joint first authors Correspondence to: Zhao-Xia Wang, email: zhaoxiawang88@126.com Keywords: pseudogene, DUXAP10, LSD1, proliferation, migration and invasion Received: June 06, 2016 Accepted: November 21, 2016 Published: December 23, 2016 ABSTRACT Pseudogenes have been considered as non-functional transcriptional relics of human genomic for long time. However, recent studies revealed that they play a plethora of roles in diverse physiological and pathological processes, especially in cancer, and many pseudogenes are transcribed into long noncoding RNAs and emerging as a novel class of lncRNAs. However, the biological roles and underlying mechanism of pseudogenes in the pathogenesis of non small cell lung cancer are still incompletely elucidated. This study identifies a putative oncogenic pseudogene DUXAP10 in NSCLC, which is located in 14q11.2 and 2398 nt in length. Firstly, we found that DUXAP10 was significantly up-regulated in 93 human NSCLC tissues and cell lines, and increased DUXAP10 was associated with patients poorer prognosis and short survival time. Furthermore, the loss and gain of functional studies including growth curves, migration, invasion assays and in vivo studies verify the oncogenic roles of DUXAP10 in NSCLC. Finally, the mechanistic experiments indicate that DUXAP10 could interact with Histone demethylase Lysine specific demethylase1 (LSD1) and repress tumor suppressors Large tumor suppressor 2 (LATS2) and Ras-related associated with diabetes (RRAD) transcription in NSCLC cells. Taken together, these findings demonstrate DUXAP10 exerts the oncogenic roles through binding with LSD1 and epigenetic silencing LATS2 and RRAD expression. Our investigation reveals the novel roles of pseudogene in NSCLC, which may serve as new target for NSCLC diagnosis and therapy.

Background: Long non-coding RNA with a length of more than 200 nucleotides, called long non-coding RNA (LncRNAs), lncRNAs can regulate its activity or change its location by interfering with the activity of mRNA and directly binding to proteins. It can also affect the expression of downstream genes by inhibiting RNA polymerase, And as a competitive endogenous RNAs to regulate gene expression, thus playing an important role in the biological process. Colorectal cancer is a malignant lesion of colorectal mucosa caused by environmental, genetic and epigenetic factors. The occurrence of colorectal cancer is a multi-stage, multi-gene process, but its pathogenesis is not fully understood. With the discovery of new molecular and epigenetic mechanisms in CRC, lncRNA has become a biological target for diagnostic, prognostic and therapeutic applications. Methods: Therefore, by screening the GEO database, we found that the expression of Linc01003 in cancer tissues was significantly different from that in paracancerous tissues, and then the expression was detected in 60 pairs of colorectal cancer tissues, five colorectal cancer cell lines and normal intestinal epithelial cell lines. In order to verify the phenotype of linc01003 by knockdown and overexpression experiments, and to explore the mechanism of linc01003 regulating mechanism of proliferation and apoptosis, the upstream transcription factors were predicted by database and verified by related experiments. The related downstream targets were screened by high throughput sequencing and verified by qRT-PCR, Western blot, RIP, ChIP, RNA-FISH and other experiments. Results: Highly expressed linc01003 is closely related to more advanced pathological stages and shorter life cycles.The results showed that the expression of LncRNA Linc01003 induced by transcription factor FOXP1 was significantly increased in colorectal cancer tissues and cells. Knockdown of Linc01003 in colorectal cells could significantly inhibit cell proliferation and induce apoptosis, while overexpression could promote cell proliferation. This can also be proved in animal experiments. After high throughput sequencing, it was found that the expression of crude oncogene HKDC1 was up-regulated and the expression of tumor suppressor gene DIC was down-regulated. Then through bioinformatics prediction and RIP experiments, it was proved that linc01003 binds to EZH2 and AGO2 proteins. CHIP experiments showed that Linc01003 could bind EZH2 to participate in the transcriptional regulation of HKDC1. Luciferase reporter gene confirmed that linc01003 competitive adsorption of miR-106b-5p promoted SLC25A10( DIC) expression. Therefore, we conclude that long non-coding RNA Linc01003 with high expression is associated with later stage and worse prognosis, and transcription factor FOXP1 can induce high expression of long non-coding RNA Linc01003. The high expression of linc01003 promotes the proliferation and inhibits the apoptosis of colorectal cancer cells. Lin01003 promotes the proliferation of colorectal cancer cells through EZH2/HKDC1 and miR-106b-5p/DIC regulatory axes. Conclusions: Therefore, we conclude that the transcription factor FOXP1 induced linc01003 promotes proliferation and inhibits apoptosis of colorectal cancer cells through EZH2/HKDC1 and miR-106b-5p/DIC regulatory axis . It is expected to be a diagnostic marker for CRC and a potential biotherapeutic target. Funding Statement: This study was supported by grants from the National Natural Sciences Foundation of China (grant no. 81772603), Key talents of young medicine in Jiangsu Province(grant no.QNRC2016662), Nanjing Health Science and Technology Development Medical Key Technology Development Project (grant no.YKK18190). Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: All patients provided written informed consent, and all the experiments were approved by the Research Ethics Committee of Nanjing Medical University. This study was conducted in strict accordance with the guidelines of the National Institutes of Health (NIH) on the use of laboratory animals. Our plan has been approved by the Animal Experimental Ethics Committee of Nanjing Medical University.

In recent years, polymer micelles have been investigated as targeted drug delivery systems for treating tumors. However, passive targeted drug delivery systems failed in accumulation at dispersive tumor metastasis sites, which led to a poor prognosis of tumor metastasis. The chemokine receptor CXCR4, which is often highly expressed in metastatic tumor cells, regulates tumor cell trafficking during the metastasis process and is related to the invasive response of tumor cells, is a potential targeting site. In this study, the BKT-140 peptide, a specific CXCR4 inhibitor, was conjugated on the surface of chitosan based polymer micelles (BKT-CSOSA). After peptide modification, in CXCR4 high expressing cells BKT-CSOSA showed a considerable increase of cell uptake by receptor-mediated cell internalization. However, in CXCR4 low expressing cells there was no obvious change in uptake efficiency but the main uptake pathway was altered from clathrin-mediated endocytosis into macropinocytosis. Doxorubicin loaded BKT-CSOSA micelles (BKT-CSOSA/DOX) showed an enhanced anti-metastasis effect on cell migration and cell invasion in vitro. It was further confirmed that BKT-CSOSA/DOX micelles decreased tumor metastasis sites’ formation in the lung. Both CSOSA and BKT-CSOSA micelles were mainly located in the liver, spleen and lung in vivo, but BKT-CSOSA showed an enhanced accumulation in the lung. This study demonstrates that BKT-140 modification notably increased the cell internalization of polymer micelles in metastatic tumor cells and enhanced the inhibition effect on lung metastasis sites’ formation.

9531 Background: Inevitable progression on 3rd-generation EGFR-tyrosine kinase inhibitor (TKI) osimertinib of EGFR-mutated lung cancer patients represents a great challenge in clinic. Previous studies have revealed that one-third of the resistant mechanisms are due to acquired EGFR secondary mutations, mainly on C797, L718 and L792 residues. Our study aims to gain insights into novel mechanisms of acquired resistance to osimertinib. Methods: We performed genomic profiling on a total of 1,058 EGFR-mutated lung cancer patients with progressed disease on osimertinib, and a cohort of 1,803 patients who received only 1st-generation EGFR TKIs upon progression. Recurrent EGFR mutations with a significant enrichment in the osimertinib group were identified. We further established and applied molecular dynamic simulation-based computational model of the mutant EGFR protein to predict its sensitivity to osimertinib. Results: As expected, compared with 1st-TKIs alone group, EGFR mutations, including C797S/G (22.1% vs. 0.5%), L718Q/V (6.2% vs. 0.3%), L792F/H (4.4% vs. 0.3%), were significantly more enriched in the osimertinib cohort. Our computational model has also successfully predicted their sensitivities to osimertinib: WT (-35.19 kcal/mol) > L792F (-34.10 kcal/mol) > L718Q (-30.33kcal/mol) > C797S (-28.02 kcal/mol), which are consistent with our previous in vitro validations. Importantly, a total of 14 low-frequency EGFR mutations were exclusively observed in the osimertinib group, seven of which, including EGFR G796S(n = 6), V802F(n = 3), T725M(n = 2), Q791L/H(n = 2), P794S/R(n = 2), were predicted to dramatically reduce the binding affinity of osimertinib to EGFR. Of note, analysis of the pretreatment samples of two patients supported that EGFR V802F and Q791L/H were acquired during osimertinib treatment. Interestingly, EGFR G796S was predicted to be sensitive to gefitinib, suggesting the possibility of administration of gefitinib in patients with acquired EGFR G796S to first-line osimertinib treatment. Further in vitro functional validations are currently ongoing. Conclusions: Our study represents the largest EGFR-mutated lung cancer cohort so far to investigate osimertinib resistance in a real-world setting, and has uncovered a list of recurrent low-frequency EGFR mutations that may confer acquired resistance to osimertinib. Our in silico structural model was proved to be powerful and robust in the prediction of osimertinib sensitivity of EGFR mutants.

Background: Lung cancer is the most common cancer all around the world with high morbidity and mortality. Long non-coding RNA(LncRNA) has been reported to be a critical role in non-small cell lung cancer (NSCLC) proliferation and migration. Methods: In the present study, we analyzed the TCGA data to figure out the expression of SNHG17 in NSCLC. RACE was used to acquired the exact sequence of SNHG17. We modulated SNHG17 expression by RNA interference and a series of functional assays such as MTT, transwell and EdUstaining assays were performed. Flow cytometry was used to explore the involvement of SNHG17 in NSCLC cell apoptosis The global gene expression profile regulated by SNHG17 in NSCLC cells was acquired through RNA-Seq assays and several potential downstream genes were confirmed by qRT-PCR. Results: We found that lncRNA SNHG17 was up-regulated in NSCLC driven by the amplification ofcopy number,indicating the special role of SNHG17 in NSCLC.Full exact length of SNHG17 was determined. Knockdown of SNHG17 inhibited proliferation, migration and promoted apoptosis of NSCLC cells. We acquired the global gene expression profile regulated by SNHG17 in A549. We found 637 genes were up-regulated while 581 genes were down-regulated. We selected three genes(FOXA1, XAF1, BIK) which were closely releated to proliferation and apoptosis and confirmed their altered expression in A549 and PC-9 treated by si-SNHG17. Conclusion: Our findings indicated gene amplification driven-long noncoding RNA SNHG17 promotes cell proliferation and migration in NSCLC,suggesting its potential value as biomarker in NSCLC. Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 81672307, 81703056, 81702266;), the Medical Innovation Team Foundation of the Jiangsu Provincial Enhancement Health Project (No. CXTDA2017021), the “333 high class Talented Man Project” (No.BRA2016509), China Postdoctoral Science Foundation (2017M610339) and Jiangsu Planned Projects for Postdoctoral ResearchFunds (1701041A). Declaration of Interest: None.

Wilms’ tumor is associated with a high treatment success rate, but there is still a risk of recurrence. Cisplatin, which is one of the chemotherapeutic agents used for its treatment, is associated with a very high rate of resistance. Par-4 (prostate apoptosis response 4) is a tumor suppressor, which is capable of sensitizing tumor cells to chemotherapy. Therefore, the aim of this study was to determine whether combined treatment with Par-4 and cisplatin is effective for inhibiting growth of Wilms’ tumor. Wilms’ tumor and control cell samples were collected and analyzed by immunofluorescence assay and immunohistochemistry. Total proteins extracted from cultured cells were analyzed using western blotting and flow cytometry. In addition, a mouse xenograft model was established. We discovered significantly low expression of Par-4 in the tumor tissue, which was positively correlated with high expression of GRP78 (glucose-regulated protein 78). In addition, we found that ectopic Par-4 co-localized with cell surface GRP78 and induced high expression of the endoplasmic reticulum proteins ATF4 and BAX, which activated the endoplasmic reticulum apoptosis pathway. Moreover, treatment with ectopic Par-4 and cisplatin suppressed xenograft growth in nude mice. In conclusion, our results showed that Par-4 overexpression and cisplatin had a synergistic effect on SK-NEP-1 cells, as a result of which cell growth was inhibited and cellular apoptosis was induced. Thus, in vitro and in vivo upregulation of Par-4 expression is indispensable for the trafficking of GRP78 to the cell membrane and subsequent apoptosis of cancer cells.

Mounting evidence demonstrates that long non-coding RNAs (lncRNAs) are novel transcripts governing multiple biological processes, and their dysregulation is involved in the development and progression of multiple types of cancers. Small Nucleolar RNA Host Gene 20 (SNHG20) is a 2183 bp lncRNA, and its overexpression predicts poor prognosis in colorectal cancer and hepatocellular carcinoma. However, the clinical relevance of SNHG20 and its molecular mechanisms affecting cancer cell phenotype have not been documented. Here, we found that SNHG20 was upregulated in non-small cell lung cancer (NSCLC) tissues compared with normal samples. Higher SNHG20 expression was significantly associated with advanced tumor, lymph node and metastases (TNM) stage and tumor size, as well as poorer overall survival. Moreover, knockdown of SNHG20 repressed NSCLC cell proliferation, migration and induced cell apoptosis. Mechanistic investigations revealed that SNHG20 could interact with EZH2 (enhancer of zeste homolog 2), thereby repressing P21 expression. Furthermore, rescue experiments indicated that SNHG20 functioned as an oncogene partly via repressing p21 in NSCLC cells. Taken together, our findings demonstrate that SNHG20 is a new candidate for use in NSCLC diagnosis, prognosis and therapy.

Background/Aims: Dysregulation of microRNAs is correlated with tumor development. The aim of this study is to investigate the clinicopathologic and prognostic significance of microRNA (miR)-409-3p and its tumor suppressor roles in lung adenocarcinoma (LAD). Methods: Quantitative real-time PCR (qRT-PCR) was performed to detect miR-409-3p expression in LAD tissues and corresponding noncancerous tissues. Additionally, the correlations of miR-409-3p expression with clinicopathologic factors and prognosis of patients were statistically analyzed. Next, we investigated whether miR-409-3p could function as a tumor suppressor in LAD cells via regulation of Akt signaling by targeting receptor tyrosine kinase (c-Met). Results: MiR-409-3p was significantly downregulated in LAD tissues compared with corresponding noncancerous tissues. Low miR-409-3p expression was observed to be significantly correlated with poorer tumor differentiation, advanced pTNM stage and higher incidence of lymph node metastasis. Multivariate Cox regression analyses showed that miR-409-3p expression was an independent prognostic factor for LAD patients. Functional analyses indicated that miR-409-3p could inhibit growth, induce apoptosis, reduce migration and invasion in LAD cells via inactivation of Akt signaling by targeting c-Met. Conclusions: MiR-409-3p was an independent prognostic factor and functioned as a tumor suppressor in LAD via regulation of Akt signaling by targeting c-Met.

MicroRNAs act as important gene regulators in human genomes, and their aberrant expression is linked to many malignancies. Aberrant expression of miR-206 has been frequently reported in cancer studies; however, the role and mechanism of its function in breast cancer remains unclear. Quantitative real-time PCR was performed to detect the relative expression levels of miR-206 in breast cancer and normal breast tissues. Lower expression of miR-206 in breast cancer tissues was associated with larger tumour size and a more advanced clinical stage. Further in vitro observations showed that the enforced expression of miR-206 in MCF-7 breast cancer cells inhibited cell growth by blocking the G1/S transition and suppressed cell proliferation and colony formation, implying that miR-206 functions as a tumour suppressor in the progression of breast cancer. Interestingly, Luciferase assays first revealed that miR-206 inhibited cyclinD2 expression by targeting two binding sites in the 3'-untranslated region of cyclinD2 mRNA. qRT-PCR and Western blot assays verified that miR-206 reduced cyclinD2 expression at both the mRNA and protein levels. A reverse correlation between miR-206 and cyclinD2 expression was noted in breast cancer tissues. Altogether, our results identify a crucial tumour suppressive role of miR-206 in the progression of breast cancer, at least partly via up-regulation of the expression of cyclinD2, and suggest that miR-206 might be a candidate prognostic predictor or an anticancer therapeutic target for breast cancer patients.

Neogambogic acid (NGA), an active ingredient in garcinia, can inhibit the growth of some solid tumors and result in an anticancer effect. We hypothesize that NGA may be responsible for the inhibition of proliferation of human breast cancer cell line MCF-7 cells. To investigate its anticancer mechanism in vitro, MCF-7 cells were treated with various concentrations of NGA. Results of MTT (methyl thiazolyl tetrazolum) assay showed that treatment with NGA significantly reduced the proliferation of MCF-7 cells in a dose-dependent manner. NGA could increase the expression of the apoptosis-related proteins FasL, caspase-3, caspase-8, caspase-9, and Bax and decrease the expression of anti-apoptotic protein Bcl-2 accompanied by the mitochondrial transmembrane damage. The antiproliferative effect of NGA on MCF-7 cells is due to the G(0)/G(1) arrest, increased apoptosis and activation of Fas/FasL and cytochrome C pathway. These results provide an important insight into the cellular and molecular mechanisms through which NGA impairs the proliferation of breast cancer cells.

Background and Aims Ovexpression of microRNA-21 (miR-21) is found in various human cancers. Our aim is to investigate the association of miR-21 expression with the sensitivity of breast cancer cells to doxorubicin (ADR). Methods The half maximal inhibitory concentration (IC 50 ) value of ADR in resistant MCF-7/ADR or parental MCF-7 cells was determined by MTT assay. TaqMan RT-PCR or Western blot assay was performed to detect the expression of mature miR-21 and tumor suppressor gene (PTEN) protein. MCF-7 or MCF-7/ADR cell line was transfected with miR-21mimic or inhibitor. The IC 50 value of ADR was determined. Flow cytometry and TUNEL assays were performed to analyze apoptosis. The activity of caspase-3 was analyzed. Results The IC 50 of ADR in MCF-7 and MCF-7/ADR cells was 0.21 ± 0.05 and 16.5 ± 0.08 μmol/L, respectively. We showed that upregulation of miR-21 in MCF-7/ADR cells was concurrent with downregulation of PTEN protein. MiR-21 mimic or inhibitor could obviously affect the sensitivity of breast cancer cells to ADR. Moreover, miR-21 inhibitor could enhance caspase-3-dependent apoptosis in MCF-7/ADR cells. Overexpression of PTEN could mimic the same effects of miR-21 inhibitor in MCF-7/ADR cells and PTEN-siRNA could increase the resistance of MCF-7 cells to ADR. MiR-21 inhibitor could increase PTEN protein expression and the luciferase activity of a PTEN 3′ untranslated region-based reporter construct in MCF-7/ADR cells. PTEN-siRNA could partially reverse the increased chemosensitivity of MCF-7/ADR cells induced by miR-21 inhibitor. Conclusions Dysregulation of miR-21 plays critical roles in the ADR resistance of breast cancer, at least in part via targeting PTEN.

(−)-Gossypol, a natural BH3-mimetic and small-molecule Bcl-2 inhibitor, shows promise in ongoing phase II clinical trials for human cancers. However, whether (−)-gossypol plays functional roles in tumor angiogenesis has not been directly elucidated yet. In this study, we showed that (−)-gossypol dose dependently inhibited the expression of VEGF, Bcl-2, and Bcl-xL in human prostate cancer cells (PC-3 and DU 145) and primary cultured human umbilical vascular endothelial cells (HUVEC) in vitro. Notably, the growth of human prostate tumor PC-3 xenografts in mice was significantly suppressed by (−)-gossypol at a dosage of 15 mg/kg/d. This inhibitory action of (−)-gossypol in vivo was largely dependent on suppression of angiogenesis in the solid tumors, where VEGF expression and microvessel density were remarkably decreased. Furthermore, (−)-gossypol inhibited VEGF-induced chemotactic motility and tubulogenesis in HUVECs and human microvascular endothelial cells and suppressed microvessel sprouting from rat aortic rings ex vivo. When examined for the mechanism, we found that (−)-gossypol blocked the activation of VEGF receptor 2 kinase with the half maximal inhibitory concentration of 2.38 μmol/L in endothelial cells. Consequently, the phosphorylation of key intracellular proangiogenic kinases induced by VEGF was all suppressed by the treatment, such as Src family kinase, focal adhesion kinase, extracellular signal-related kinase, and AKT kinase. Taken together, the present study shows that (−)-gossypol potently inhibits human prostate tumor growth through modulating VEGF signaling pathway, which further validates its great potential in clinical practice. Mol Cancer Ther; 10(5); 795–805. ©2011 AACR.

Tumor angiogenesis is one of the hallmarks of the development in malignant neoplasias and metastasis. Many angiogenesis inhibitors are small molecules from natural products. Indirubin, the active component of a traditional Chinese herbal medicine, Banlangen, has been shown to exhibit antitumor and anti-inflammation effects. But its roles in tumor angiogenesis, the key step involved in tumor growth and metastasis, and the involved molecular mechanism is unknown. Here, we identified that indirubin inhibited prostate tumor growth through inhibiting tumor angiogenesis. Using chick chorioallantoic membrane (CAM) assay and mouse corneal model, we found that indirubin inhibited angiogenesis in vivo. We also showed the inhibition activity of indirubin in endothelial cell migration, tube formation and cell survival in vitro. Furthermore, indirubin suppressed vascular endothelial growth factor receptor 2-mediated Janus kinase (JAK)/STAT3 signaling pathway but had little effects on the activity of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase in endothelial cell. Our study provided the first evidence for antitumor angiogenesis activity of indirubin and the related molecular mechanism. Our investigations suggested that indirubin was a potential drug candidate for angiogenesis related diseases.

Understanding the molecular basis and target of traditional medicine is critical for drug development. Celastrol, derived from Trypterygium wilfordii Hook F. (“Thunder of God Vine”), a traditional Chinese medicine plant, has been assigned anticancer activities, but its mechanism is not well understood. Here, we investigated whether Celastrol could inhibit angiogenesis-mediated tumor growth and, if so, through what mechanism. When given s.c. to mice bearing human prostate cancer (PC-3 cell) xenografts, Celastrol (2 mg/kg/d) significantly reduced the volume and the weight of solid tumors and decreased tumor angiogenesis. We found that this agent inhibited vascular endothelial growth factor (VEGF)–induced proliferation, migration, invasion, and capillary-like structure formation by primary cultured human umbilical vascular endothelial cells (HUVEC) in a dose-dependent manner. Furthermore, Celastrol abrogated VEGF-induced sprouting of the vessels from aortic rings and inhibited vascular formation in the Matrigel plug assay in vivo. To understand the molecular mechanism of these activities, we next examined the signaling pathways in treated HUVECs and PC-3 tumor cells. Celastrol suppressed the VEGF-induced activation of AKT, mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase (P70S6K). Additionally, we found that Celastrol inhibited the proliferation of prostate cancer cells and induced apoptosis, and these effects correlated with the extent of inhibition of AKT/mTOR/P70S6K signaling. Taken together, our results suggest that Celastrol targets the AKT/mTOR/P70S6K pathway, which leads to suppression of tumor growth and angiogenesis. Cancer Res; 70(5); 1951–9

Polo-like kinase 1 is a serine/threonine kinase which plays an essential role in mitosis and malignant transformation. The aim of this study was to investigate the prognostic significance of polo-like kinase 1 expression and determine its possibility as a therapeutic target in non-small cell lung cancer. Semi-quantitative RT-PCR assay was performed to detect polo-like kinase 1 mRNA expression in non-small cell lung cancer cells or tissues. Immunohistochemistry was performed to detect polo-like kinase 1 protein expression in 100 non-small cell lung cancer tissue samples, and the associations of polo-like kinase 1 expression with clinicopathological factors or prognosis of non-small cell lung cancer patients were evaluated. RNA interference was employed to inhibit endogenous polo-like kinase 1 expression and analyzed the effects of polo-like kinase 1 inhibition on the malignant phenotypes of non-small cell lung cancer cells including growth, apoptosis, radio- or chemoresistance. Also, the possible molecular mechanisms were also investigated. The levels of polo-like kinase 1 mRNA expression in non-small cell lung cancer cell lines or tissues were significantly higher than those in normal human bronchial epithelial cell line or corresponding non-tumor tissues. High polo-like kinase 1 expression was significantly correlated with advanced clinical stage, higher tumor classification and lymph node metastasis of non-small cell lung cancer patients (P=0.001, 0.004 and 0.001, respectively). Meanwhile, high polo-like kinase 1 protein expression was also an independent prognostic molecular marker for non-small cell lung cancer patients (hazard ratio: 2.113; 95% confidence interval: 1.326-3.557; P=0.017). Polo-like kinase 1 inhibition could significantly inhibit in vitro and in vivo proliferation, induce cell arrest of G(2)/M phase and apoptosis enhancement in non-small cell lung cancer cells, which might be activation of the p53 pathway and the Cdc25C/cdc2/cyclin B1 feedback loop. Further, inhibition of polo-like kinase 1 could enhance the sensitivity of non-small cell lung cancer cells to taxanes or irradiation. Thus, polo-like kinase 1 might be a prognostic marker and a chemo- or radiotherapeutic target for non-small cell lung cancer.

Tumor growth depends on angiogenesis and inducing angiogenesis is one of the most important hallmarks in the cancer development. Treatment with small molecules that inhibit angiogenesis has been an effective strategy for anti-cancer therapy. Some anti-angiogenic factors are derived from traditional Chinese herbs. Usnic acid (UA), an active compound mainly found in lichens, has shown some biological and physiological activities. However, the role and mechanism of UA in tumor angiogenesis are still unknown. The aim of this study was to assess the effects of UA on tumor angiogenesis. In this study, we demonstrated that UA strongly inhibited in vivo angiogenesis in a chick embryo chorioallantoic membrane assay and vascular endothelial growth factor-induced mouse corneal angiogenesis model. In a mouse xenograft tumor model, UA suppressed Bcap-37 breast tumor growth and angiogenesis without affecting mice body weight. In an in vitro assay, UA not only significantly inhibited endothelial cell proliferation, migration and tube formation, but also induced morphological changes and apoptosis in endothelial cells. In addition, UA inhibited Bcap-37 tumor cell proliferation. Moreover, western blot analysis of cell signaling molecules indicated that UA blocked vascular endothelial growth factor receptor (VEGFR) 2 mediated Extracellular signal-regulated protein kinases 1 and 2(ERK1/2) and AKT/P70S6K signaling pathways in endothelial cells. These results provided the first evidence of the biological function and molecular mechanism of UA in tumor angiogenesis.

Cucurbitacin E (CuE, α-elaterin), a tetracyclic triterpenes compound from folk traditional Chinese medicine plants, has been shown to inhibit cancer cell growth, inflammatory response and bilirubin-albumin binding. However, the effects of CuE on tumor angiogenesis and its potential molecular mechanism are still unknown. Here, we demonstrated that CuE significantly inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, migration and tubulogenesis in vitro and blocked angiogenesis in chick embryo chorioallantoic membrane assay and mouse corneal angiogenesis model in vivo. Furthermore, we found that CuE remarkably induced HUVEC apoptosis, inhibited tumor angiogenesis and suppressed human prostate tumor growth in xenograft tumor model. Finally, we showed that CuE blocked vascular endothelial growth factor receptor (VEGFR) 2-mediated Janus kinase (Jak) 2-signal transducer and activator of transcription (STAT) 3 signaling pathway in endothelial cells and suppressed the downstream protein kinases, such as extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Therefore, our studies provided the first evidence that CuE inhibited tumor angiogenesis by inhibiting VEGFR2-mediated Jak-STAT3 and mitogen-activated protein kinases signaling pathways and CuE is a potential candidate in angiogenesis-related disease therapy.

Recent evidence indicates that long noncoding RNAs (lncRNAs) have a critical role in the regulation of cellular processes such as differentiation, proliferation, and metastasis. These lncRNAs are dysregulated in a variety of cancers and many function as tumor suppressors; however, the regulatory factors involved in silencing lncRNA transcription are poorly understood. In this study, we showed that epigenetic silencing of lncRNA SPRY4 intronic transcript 1 (SPRY4-IT1) occurs in non-small-cell lung cancer (NSCLC) cells through direct transcriptional repression mediated by the Polycomb group protein enhancer of zeste homolog 2 (EZH2). SPRY4-IT1 is derived from an intron within SPRY4, and is upregulated in melanoma cells; knockdown of its expression leads to cell growth arrest, invasion inhibition, and elevated rates of apoptosis. Upon depletion of EZH2 by RNA interference, SPRY4-IT1 expression was restored, and transfection of SPRY4-IT1 into NSCLC cells resulted in a significant antitumoral effect, both in culture and in xenografted nude mice. Moreover, overexpression of SPRY4-IT1 was found to have a key role in the epithelial–mesenchymal transition through the regulation of E-cadherin and vimentin expression. In EZH2-knockdown cells, which characteristically showed impaired cell proliferation and metastasis, the induction of SPRY4-IT1 depletion partially rescued the oncogenic phenotype, suggesting that SPRY4-IT1 repression has an important role in EZH2 oncogenesis. Of most relevance, translation of these findings into human NSCLC tissue samples demonstrated that patients with low levels of SPRY4-IT1 expression had a shorter overall survival time, suggesting that SPRY4-IT1 could be a biomarker for poor prognosis of NSCLC.

Background Aberrant expression of long noncoding RNAs (lncRNAs) has frequently been reported in cancer studies, including those of colorectal cancer (CRC). Increasing evidence suggests that lncRNAs are significantly correlated with the pathogenesis, development and metastasis of cancer. Loc554202 is a 2166-bp transcript on human chromosome 9p21.3, the expression of which is dysregulated in breast and lung cancer cells. However, its role in CRC remains under investigation. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to assess the relative expression of Loc554202 in CRC cell lines and tissues. Gain and/or loss of function approaches were used to investigate the potential functional roles in cell proliferation and apoptosis in vitro and in vivo. qRT-PCR, western-blotting and immunohistochemistry were used to evaluate the mRNA and protein expression of apoptosis-related factors. Results Loc554202 was significantly downregulated in cancerous tissues and CRC cell lines compared with adjacent normal tissue and a normal human intestinal epithelial cell line. Low Loc554202 expression was closely associated with advanced pathologic stage and a larger tumor size. The overexpression of Loc554202 decreased the cell proliferation and induced apoptosis in vitro and hindered tumorigenesis in vivo. Loc554202 regulated cell apoptosis partly through the activation of specific caspase cleavage cascades. Conclusion Our results suggest that Loc554202 may play an important role in the progression of CRC and could be a candidate prognostic biomarker or a target for new cancer therapies. Electronic supplementary material The online version of this article (doi:10.1186/s13046-015-0217-7) contains supplementary material, which is available to authorized users.