Cholesterol inhibition enhances antitumor response of gilteritinib in lung cancer cells.

Repositioning approved antitumor drugs for different cancers is a cost-effective approach. Gilteritinib was FDA-approved for the treatment of FLT3-mutated acute myeloid leukemia in 2018. However, the therapeutic effects and mechanism of Gilteritinib on other malignancies remain to be defined. In this study, we identified that gilteritinib has an inhibitory effect on lung cancer cells (LCCs) without FLT3 mutation in vitro and in vivo. Unexpectedly, we found that gilteritinib induces cholesterol accumulation in LCCs via upregulating cholesterol biosynthetic genes and inhibiting cholesterol efflux. This gilteritinib-induced cholesterol accumulation not only attenuates the antitumor effect of gilteritinib but also induces gilteritinib-resistance in LCCs. However, when cholesterol synthesis was prevented by squalene epoxidase (SQLE) inhibitor NB-598, both LCCs and gilteritinib-resistant LCCs became sensitive to gilteritinib. More importantly, the natural cholesterol inhibitor 25-hydroxycholesterol (25HC) can suppress cholesterol biosynthesis and increase cholesterol efflux in LCCs. Consequently, 25HC treatment significantly increases the cytotoxicity of gilteritinib on LCCs, which can be rescued by the addition of exogenous cholesterol. In a xenograft model, the combination of gilteritinib and 25HC showed significantly better efficacy than either monotherapy in suppressing lung cancer growth, without obvious general toxicity. Thus, our findings identify an increase in cholesterol induced by gilteritinib as a mechanism for LCC survival, and highlight the potential of combining gilteritinib with cholesterol-lowering drugs to treat lung cancer.

CircATF6 inhibits hepatocellular carcinoma progression by suppressing calreticulin-mediated Wnt/ß-catenin signaling pathway.

Circular RNAs (circRNAs) are covalently closed, single-stranded RNAs that play critical roles in various biological processes and diseases, including cancers. However, the functions and mechanisms of circRNAs in hepatocellular carcinoma (HCC) need further clarification. Here, we identified and confirmed that circATF6 is downregulated in HCC tissues and negatively associated with the overall survival of HCC patients. Ectopic overexpression of circATF6 inhibits malignant phenotypes of HCC cells in vitro and in vivo, while knockdown of circATF6 had opposite effects. Mechanistically, we found that circATF6 bound to calreticulin (CALR) protein and acted as a scaffold to enhance the interaction of CALR with calpain2 (CAPN2), which promoted the degradation of CALR by its enzymatic activity. Moreover, we found that circATF6 inhibited HCC cells by suppressing CALR-mediated wnt/ß-catenin signaling pathway. Taken together, our findings suggest that circATF6 is a potential prognostic biomarker and therapeutic target for HCC.

LDHA contributes to nicotine induced cardiac fibrosis through autophagy flux impairment.

Cardiac fibrosis is a typical feature of cardiac pathological remodeling, which is associated with adverse clinical outcomes and has no effective therapy. Nicotine is an important risk factor for cardiac fibrosis, yet its underlying molecular mechanism remains poorly understood. This study aimed to identify its potential molecular mechanism in nicotine-induced cardiac fibrosis. Our results showed nicotine exposure led to the proliferation and transformation of cardiac fibroblasts (CFs) into myofibroblasts (MFs) by impairing autophagy flux. Through the use of drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) technology, it was discovered that nicotine directly increased the stability and protein levels of lactate dehydrogenase A (LDHA) by binding to it. Nicotine treatment impaired autophagy flux by regulating the AMPK/mTOR signaling pathway, impeding the nuclear translocation of transcription factor EB (TFEB), and reducing the activity of cathepsin B (CTSB). In vivo, nicotine treatment exacerbated cardiac fibrosis induced in spontaneously hypertensive rats (SHR) and worsened cardiac function. Interestingly, the absence of LDHA reversed these effects both in vitro and in vivo. Our study identified LDHA as a novel nicotine-binding protein that plays a crucial role in mediating cardiac fibrosis by blocking autophagy flux. The findings suggest that LDHA could potentially serve as a promising target for the treatment of cardiac fibrosis.

TRIM29 modulates proteins involved in PTEN/AKT/mTOR and JAK2/STAT3 signaling pathway and suppresses the progression of hepatocellular carcinoma.

Tripartite motif-containing 29 (TRIM29), also known as the ataxia telangiectasia group D-complementing (ATDC) gene, has been reported to play an oncogenic or tumor suppressive role in developing different tumors. So far, its expression and biological functions in hepatocellular carcinoma (HCC) remain unclear. We investigated TRIM29 expression pattern in human HCC samples using quantitative RT-PCR and immunohistochemistry. Relationships between TRIM29 expression level, clinical prognostic indicators, overall survival (OS), and disease-free survival (DFS) were evaluated by Kaplan-Meier analysis and Cox proportional hazards model. A series of in vitro experiments and a xenograft tumor model were conducted to detect the functions of TRIM29 in HCC cells. RNA sequencing, western blotting, and immunochemical staining were performed to assess the molecular regulation of TRIM29 in HCC. We found that the mRNA and protein levels of TRIM29 were significantly reduced in HCC samples, compared with adjacent noncancerous tissues, and were negatively correlated with poor differentiation of HCC tissues. Survival analysis confirmed that lower TRIM29 expression significantly correlated with shorter OS and DFS of HCC patients. TRIM29 overexpression remarkably inhibited cell proliferation, migration, and EMT in HCC cells, whereas knockdown of TRIM29 reversed these effects. Moreover, deactivation of the PTEN/AKT/mTOR and JAK2/STAT3 pathways might be involved in the tumor suppressive role of TRIM29 in HCC. Our findings indicate that TRIM29 in HCC exerts its tumor suppressive effects through inhibition of the PTEN/AKT/mTOR and JAK2/STAT3 signaling pathways and may be used as a potential biomarker for survival in patients with HCC.

Two somatic mutations in the androgen receptor N-terminal domain are oncogenic drivers in hepatocellular carcinoma.

The androgen receptor (AR) plays an important role in male-dominant hepatocellular carcinoma, and specific acquired somatic mutations of AR have been observed in HCC patients. Our previous research have established the role of AR wild type as one of the key oncogenes in hepatocarcinogenesis. However, the role of hepatic acquired somatic mutations of AR remains unknown. In this study, we identify two crucial acquired somatic mutations, Q62L and E81Q, situated close to the N-terminal activation function domain-1 of AR. These mutations lead to constitutive activation of AR, both independently and synergistically with androgens, making them potent driver oncogene mutations. Mechanistically, these N-terminal AR somatic mutations enhance de novo lipogenesis by activating sterol regulatory element-binding protein-1 and promote glycogen accumulation through glycogen phosphorylase, brain form, thereby disrupting the AMPK pathway and contributing to tumorigenesis. Moreover, the AR mutations show sensitivity to the AMPK activator A769662. Overall, this study establishes the role of these N- terminal hepatic mutations of AR as highly malignant oncogenic drivers in hepatocarcinogenesis and highlights their potential as therapeutic targets for patients harboring these somatic mutations.

Diagnostic and Prognostic Value of Protein Post-translational Modifications in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumor with high incidence and cancer mortality worldwide. Post-translational modifications (PTMs) of proteins have a great impact on protein function. Almost all proteins can undergo PTMs, including phosphorylation, acetylation, methylation, glycosylation, ubiquitination, and so on. Many studies have shown that PTMs are related to the occurrence and development of cancers. The findings provide novel therapeutic targets for cancers, such as glypican-3 and mucin-1. Other clinical implications are also found in the studies of PTMs. Diagnostic or prognostic value, and response to therapy have been identified. In HCC, it has been shown that glycosylated alpha-fetoprotein (AFP) has a higher detection rate for early liver cancer than conventional AFP. In this review, we mainly focused on the diagnostic and prognostic value of PTM, in order to provide new insights into the clinical implication of PTM in HCC.

Ezetimibe Induces Paraptosis through Niemann-Pick C1-like 1 Inhibition of Mammalian-Target-of-Rapamycin Signaling in Hepatocellular Carcinoma Cells.

Currently, hepatocellular carcinoma (HCC) is characterized by its unfavorable prognosis and resistance to conventional chemotherapy and radiotherapy. Drug repositioning, an approach aimed at identifying novel therapeutic applications for existing drugs, presents a cost-effective strategy for developing new anticancer agents. We explored the anticancer properties of Ezetimibe, a widely used oral lipid-lowering drug, in the context of HCC. Our findings demonstrate that Ezetimibe effectively suppresses HCC cell proliferation through paraptosis, an apoptotic-independent cell death pathway. The examination of HCC cells lines treated with Ezetimibe using light microscopy and transmission electron microscopy (TEM) showed cytoplasmic vacuolation in the perinuclear region. Notably, the nuclear membrane remained intact in both Ezetimibe-treated and untreated HCC cell lines. Probe staining assays confirmed that the cytoplasmic vacuoles originated from dilated endoplasmic reticulum (ER) compartments rather than mitochondria. Furthermore, a dose-dependent accumulation of reactive oxygen species (ROS) was observed in Ezetimibe-treated HCC cell lines. Co-treatment with the general antioxidant NAC attenuated vacuolation and improved cell viability in Ezetimibe-treated HCC cells. Moreover, Ezetimibe induced paraptosis through proteasome activity inhibition and initiation of the unfolded protein response (UPR) in HCC cell lines. In our in vivo experiment, Ezetimibe significantly impeded the growth of HCC tumors. Furthermore, when combined with Sorafenib, Ezetimibe exhibited a synergistic antitumor effect on HCC cell lines. Mechanistically, Ezetimibe induced paraptosis by targeting NPC1L1 to inhibit the PI3K/AKT/mTOR signaling pathway. In conclusion, our study highlights the potential of Ezetimibe as an anticancer agent by triggering paraptosis in HCC cells.

CircSCAP interacts with SF3A3 to inhibit the malignance of non-small cell lung cancer by activating p53 signaling.

BACKGROUND: Circular RNA (circRNA) has been recently identified as a critical regulator during carcinogenesis. However, the biological function and potential underlying mechanisms of circRNAs in lung cancer remain to be further elucidated. METHODS: Here, we first evaluated the differentially expressed circRNAs between tumor and the matched adjacent nontumor tissues (3 pairs) of lung cancer patients via circRNA microarray. The expression of top five dysregulated circRNAs were tested in lung cancer cell lines and the circSCAP with concordant alteration in microarray data and cell lines was selected for further investigation. Then we validated the expression level of circSCAP in tumor and corresponding adjacent tissues (161 pairs) from a lung cancer cohort by RT-PCR analysis followed by correlation and prognosis analysis between circSCAP and clinical characteristics. Non-small cell lung cancer (NSCLC) accounts for the majority of lung cancer diagnosis (about 80% in the cohort used in this study). Therefore, we focused the role of circSCAP in NSCLC in the present study. In vitro and in vivo assays were performed to study the biological function of circSCAP in NSCLC. Biotin-labeled RNA pulldown and RNA immunoprecipitation (RIP) assays were carried out to identify the proteins directly interacting with circSCAP. The molecular mechanism of circSCAP-driven tumor suppression was demonstrated by immunoblotting, immunoprecipitation and luciferase reporter assays. In vitro and in vivo rescue experiments were conducted to verify the role of the circSCAP/SF3A3/p53 signaling axis in NSCLC. RESULTS: We screened the expression profiles of human circRNAs in lung cancer tissues and found that hsa_circ_0065214 (termed as circSCAP) was significantly decreased. Kaplan-Meier analysis showed that patients with low level of circSCAP had a significantly poor prognosis. Gain- and loss-of-function experiments suggested that circSCAP played an important role in NSCLC cell proliferation, cell migration and apoptosis. Mechanistically, circSCAP directly binds to the SF3A3 protein, facilitating the reduction of SF3A3 by promoting its ubiquitin-proteasome-mediated degradation, which enhances the expression of MDM4-S to finally activate its downstream p53 signaling. CONCLUSION: These findings illustrate a novel circSCAP/SF3A3/p53 signaling axis involved in suppressing the malignance of NSCLC and provide a promising target for NSCLC prognosis prediction and treatment.

Phosphorylation of androgen receptor by mTORC1 promotes liver steatosis and tumorigenesis.

BACKGROUND AND AIMS: Androgen receptor (AR) has been reported to play an important role in the development and progression of man's prostate cancer. Hepatocellular carcinoma (HCC) is also male-dominant, but the role of AR in HCC remains poorly understood. Mechanistic target of rapamycin complex 1 (mTORC1) also has been reported to be highly activated in HCC. In this study, we aimed to explore the role of AR phosphorylation and its relationship with mTORC1 in hepatocarcinogenesis. APPROACH AND RESULTS: In vitro experiment, we observed that mTORC1 interacts with hepatic AR and phosphorylates it at S96 in response to nutrient and mitogenic stimuli in HCC cells. S96 phosphorylation promotes the stability, nuclear localization, and transcriptional activity of AR, which enhances de novo lipogenesis and proliferation in hepatocytes and induces liver steatosis and hepatocarcinogenesis in mice independently and cooperatively with androgen. Furthermore, high ARS96 phosphorylation is observed in human liver steatotic and HCC tissues and is associated with overall survival and disease-free survival, which has been proven as an independent survival predictor for patients with HCC. CONCLUSIONS: AR S96 phosphorylation by mTORC1 drives liver steatosis and HCC development and progression independently and cooperatively with androgen, which not only explains why HCC is man-biased but also provides a target molecule for prevention and treatment of HCC and a potential survival predictor in patients with HCC.

Human neutrophil peptide 1 promotes immune sterilization in vivo by reducing the virulence of multidrug-resistant Klebsiella pneumoniae and increasing the ability of macrophages.

By studying the expression in patients and cell modeling in vitro, antimicrobial peptides for Klebsiella were screened. Killing curve and membrane permeability experiments are used to study the antibacterial effect of antimicrobial peptides in vitro. Cytotoxicity-related indicators including lipopolysaccharide (LPS), capsule polysaccharide (CPS), and outer membrane protein expression were measured. Intranasal inoculation of pneumoconiosis was used to construct a mouse infection model, and the survival rate and cytokine expression level were tested. Human neutrophil peptide 1 (HNP-1) showed a significant antibacterial effect, which improved the permeability of the outer membrane of K. pneumoniae. Moreover, HNP-1 decreased LPS, CPS content, and outer membrane proteins. K. pneumoniae infection decreased antimicrobial peptide, oxidative stress, and autophagy-related genes, while HNP-1 increased these genes. After coculture with macrophages, the endocytosis of macrophages is enhanced and the bacterial load is greater in the K. pneumoniae + peptide group. Besides, higher levels of pp38 and pp65 in the K. pneumoniae + peptide group. HNP-1 rescued the cytotoxicity induced by K. pneumoniae. The survival rate is significantly improved after K. pneumoniae is treated by HNP-1. All cytokines in the peptide group were significantly higher. HNP-1 promotes immune sterilization by reducing the virulence of multidrug-resistant K. pneumoniae and increasing the ability of macrophages.

Rapamycin and trametinib: a rational combination for treatment of NSCLC.

Mammalian target of rapamycin (mTOR) is one of the most commonly activated pathways in human cancers, including lung cancer. Targeting mTOR with molecule inhibitors is considered as a useful therapeutic strategy. However, the results obtained from the clinical trials with the inhibitors so far have not met the original expectations, largely because of the drug resistance. Thus, combined or multiple drug therapy can bring about more favorable clinical outcomes. Here, we found that activation of ERK pathway was responsible for rapamycin drug resistance in non-small-cell lung cancer (NSCLC) cells. Accordingly, rapamycin-resistant NSCLC cells were more sensitive to ERK inhibitor (ERKi), trametinib, and in turn, trametinib-resistant NSCLC cells were also susceptible to rapamycin. Combining rapamycin with trametinib led to a potent synergistic antitumor efficacy, which induced G1-phase cycle arrest and apoptosis. In addition, rapamycin synergized with another ERKi, MEK162, and in turn, trametinib synergized with other mTORi, Torin1 and OSI-027. Mechanistically, rapamycin in combination with trametinib resulted in a greater decrease of phosphorylation of AKT, ERK, mTOR and 4EBP1. In xenograft mouse model, co-administration of rapamycin and trametinib caused a substantial suppression in tumor growth without obvious drug toxicity. Overall, our study identifies a reasonable combined strategy for treatment of NSCLC.

mTORC1 Promotes ARID1A Degradation and Oncogenic Chromatin Remodeling in Hepatocellular Carcinoma.

The SWI/SNF chromatin remodeling complexes control accessibility of chromatin to transcriptional and coregulatory machineries. Chromatin remodeling plays important roles in normal physiology and diseases, particularly cancer. The ARID1A-containing SWI/SNF complex is commonly mutated and thought to be a key tumor suppressor in hepatocellular carcinoma (HCC), but its regulation in response to oncogenic signals remains poorly understood. mTOR is a conserved central controller of cell growth and an oncogenic driver of HCC. Remarkably, cancer mutations in mTOR and SWI/SNF complex are mutually exclusive in human HCC tumors, suggesting that they share a common oncogenic function. Here, we report that mTOR complex 1 (mTORC1) interact with ARID1A and regulates ubiquitination and proteasomal degradation of ARID1A protein. The mTORC1-ARID1A axis promoted oncogenic chromatin remodeling and YAP-dependent transcription, thereby enhancing liver cancer cell growth in vitro and tumor development in vivo. Conversely, excessive ARID1A expression counteracted AKT-driven liver tumorigenesis in vivo. Moreover, dysregulation of this axis conferred resistance to mTOR-targeted therapies. These findings demonstrate that the ARID1A-SWI/SNF complex is a regulatory target for oncogenic mTOR signaling, which is important for mTORC1-driven hepatocarcinogenesis, with implications for therapeutic interventions in HCC. SIGNIFICANCE: mTOR promotes oncogenic chromatin remodeling by controlling ARID1A degradation, which is important for liver tumorigenesis and response to mTOR- and YAP-targeted therapies in hepatocellular carcinoma.See related commentary by Pease and Fernandez-Zapico, p. 5608.

Exploring the sulfate patterns of chondroitin sulfate/dermatan sulfate and keratan sulfate in human pancreatic cancer.

This study was designed to explore the sulfation patterns of chondroitin sulfate (CS)/dermatan sulfate (DS), and keratan sulfate (KS) and the expression of carbohydrate sulfotransferases (CHSTs) in 26 pancreatic tumor and normal tissues. CS/DS and KS profiles were simultaneously determined. Pancreatic tumor tissues exhibited increased ΔDi-0S, ΔDi-4S, and ΔDi-6S levels, with absolute ΔDi-4S content being highest, followed by ΔDi-6S. However, as for the contents of KS-6S and KS-6S,6'S, there were no significant regular change. The expression levels of CHST1 and CHST4 were 37 and 15 times higher than those in normal tissues. PCA and OPLS-DA revealed that ΔDi-4S and ΔDi-6S levels could be reliably used to differentiate between healthy and cancerous tissues. The up-regulation of CHST3, CHST12, CHST13, and CHST15 was directly correlated with C-4 and C-6 sulfation. These data provide a foundation for future studies of the role of ΔDi-4S and ΔDi-6S in the progression of pancreatic cancer.

Combination Treatment With Inhibitors of ERK and Autophagy Enhances Antitumor Activity of Betulinic Acid in Non-small-Cell Lung Cancer In Vivo and In Vitro.

Aberrant activation of the Ras-ERK signaling pathway drives many important cancer phenotypes, and several inhibitors targeting such pathways are under investigation and/or approved by the FDA as single- or multi-agent therapy for patients with melanoma and non-small-cell lung cancer (NSCLC). Here, we show that betulinic acid (BA), a natural pentacyclic triterpenoid, inhibits cell proliferation, and induces apoptosis and protective autophagy in NSCLC cells. Thus, the cancer cell killing activity of BA is enhanced by autophagy inhibition. Mitogen-activated protein kinases, and especially ERK that facilitates cancer cell survival, are also activated by BA treatment. As such, in the presence of ERK inhibitors (ERKi), lung cancer cells are much more sensitive to BA. However, the dual treatment of BA and ERKi results in increased protective autophagy and AKT phosphorylation. Accordingly, inhibition of AKT has a highly synergistic anticancer effect with co-treatment of BA and ERKi. Notably, autophagy inhibition by hydroxychloroquine (HCQ) increases the response of lung cancer cells to BA in combination with ERKi. In vivo, the three-drug combination (BA, ERKi, and HCQ), resulted in superior therapeutic efficacy than single or dual treatments in the xenograft mouse model. Thus, our study provides a combined therapy strategy that is a highly effective treatment for patients with NSCLC.

Lnc-GAN1 expression is associated with good survival and suppresses tumor progression by sponging mir-26a-5p to activate PTEN signaling in non-small cell lung cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) play vital roles in the development and progression of non-small-cell lung cancer (NSCLC); however, the role of most lncRNAs in NSCLC remains unknown. This study explored the clinical significance, biological function and underlying mechanism of lnc-GAN1 in NSCLC. METHODS: With a custom lncRNA microarray we found that lnc-GAN1 is markedly downregulated in NSCLC tissues. Then lnc-GAN1 expression level was measured using qRT-PCR in NSCLC tissues and cell lines. Survival was assessed using the Kaplan-Meier method. The biological functions of lnc-GAN1 in lung cancer cells were evaluated in vitro and in vivo. RNA fluorescence in situ hybridization and subcellular localization assays revealed the subcellular distribution of lnc-GAN1 in cells. Bioinformatic analysis was adopted to predict miRNAs and signaling pathways regulated by lnc-GAN1. RNA immunoprecipitation and Dual-luciferase reporter assays were used to assess the interaction between lnc-GAN1 and miR-26a-5p in lung cancer cells. RESULTS: lnc-GAN1 is downregulated in HCC tissues and associated with larger tumor size and poor overall survival and disease-free survival; its ectopic expression suppresses cell proliferation, colony formation, and cell cycle progression and induces apoptosis in NSCLC cells; it also inhibits tumor growth in the NSCLC xenograft model. We further proved that lnc-GAN1 is localized in cytoplasm and transcribed independently from its parental gene GAN. Mechanistically, lnc-GAN1 acts as a sponge for miR-26a-5p by two seed sequences, and the two non-coding RNAs have a negative relationship in NSCLC tissues; we further prove that PTEN is a direct target of miR-26a-5p and lnc-GAN1 inhibits cell cycle signaling pathway by activating PTEN, whose expression level correlated negatively with miR-26a-5p level but positively with lnc-GAN1 level in NSCLC samples. CONCLUSIONS: Lnc-GAN1 is downregulated and associated with poor survival of NSCLC patients, and mechanistically acts as a tumor suppressor via sponging and inhibiting miR-26a-5p to upregulate PTEN. This study provides a potential prognostic biomarker and treatment target for NSCLC.

Rab1A promotes cancer metastasis and radioresistance through activating GSK-3ß/Wnt/ß-catenin signaling in nasopharyngeal carcinoma.

Many articles have reported that Rab1A was overexpressed in a variety of human cancers and involved in tumor progression and metastasis. However, the biological function and molecular mechanism of Rab1A in nasopharyngeal carcinoma (NPC) remained unknown until now. Here we found that Rab1A overexpression is a common event and was positively associated with distant metastasis and poor prognosis of NPC patients. Functionally, Rab1A depletion inhibited the migration and EMT phenotype of NPC cells, whereas Rab1A overexpression led to the opposite effect. Furthermore, we reveal an important role for Rab1A protein in the induction of radioresistance via regulating homologous recombination (HR) signaling pathway. Mechanistically, Rab1A activated Wnt/ß-catenin signaling by inhibiting the activity of GSK-3ß via phosphorylation at Ser9. Then Wnt/ß-catenin signaling induced NPC cells radioresistance and metastasis through nuclear translocation of ß-catenin and transcription upregulation of HR pathway-related and EMT-related genes expression. In general, this study shows that Rab1A may serve as a potential biomarker for predicting prognosis in NPC patients. Targeting Rab1A and Wnt/ß-catenin signaling may hold promise to overcome NPC radioresistance.

Identification of a 4-lncRNA signature predicting prognosis of patients with non-small cell lung cancer: a multicenter study in China.

BACKGROUND: Previous findings have indicated that the tumor, nodes, and metastases (TNM) staging system is not sufficient to accurately predict survival outcomes in patients with non-small lung carcinoma (NSCLC). Thus, this study aims to identify a long non-coding RNA (lncRNA) signature for predicting survival in patients with NSCLC and to provide additional prognostic information to TNM staging system. METHODS: Patients with NSCLC were recruited from a hospital and divided into a discovery cohort (n = 194) and validation cohort (n = 172), and detected using a custom lncRNA microarray. Another 73 NSCLC cases obtained from a different hospital (an independent validation cohort) were examined with qRT-PCR. Differentially expressed lncRNAs were determined with the Significance Analysis of Microarrays program, from which lncRNAs associated with survival were identified using Cox regression in the discovery cohort. These prognostic lncRNAs were employed to construct a prognostic signature with a risk-score method. Then, the utility of the prognostic signature was confirmed using the validation cohort and the independent cohort. RESULTS: In the discovery cohort, we identified 305 lncRNAs that were differentially expressed between the NSCLC tissues and matched, adjacent normal lung tissues, of which 15 are associated with survival; a 4-lncRNA prognostic signature was identified from the 15 survival lncRNAs, which was significantly correlated with survivals of NSCLC patients. This signature was further validated in the validation cohort and independent validation cohort. Moreover, multivariate Cox analysis demonstrates that the 4-lncRNA signature is an independent survival predictor. Then we established a new risk-score model by combining 4-lncRNA signature and TNM staging stage. The receiver operating characteristics (ROC) curve indicates that the prognostic value of the combined model is significantly higher than that of the TNM stage alone, in all the cohorts. CONCLUSIONS: In this study, we identified a 4-lncRNA signature that may be a powerful prognosis biomarker and can provide additional survival information to the TNM staging system.

LncRNA CSMD1-1 promotes the progression of Hepatocellular Carcinoma by activating MYC signaling.

Emerging evidence suggests that long non-coding RNAs (lncRNA) play critical roles in the development and progression of diverse cancers including hepatocellular carcinoma (HCC), but the underlying molecular mechanisms of lncRNAs that are involved in hepatocarcinogenesis have not been fully explored. Methods: In this study, we profiled lncRNA expression in 127 pairs of HCC and nontumor liver tissues (a Discovery Cohort) using a custom microarray. The expression and clinical significance of lncCSMD1-1 were then validated with qRT-PCR and COX regression analysis in a Validation Cohort (n=260) and two External Validation Cohorts (n=92 and n=124, respectively). In vitro and in vivo assays were performed to explore the biological effects of lncCSMD1-1 on HCC cells. The interaction of lncCSMD1-1 with MYC was identified by RNA pull-down and RNA immunoprecipitation. The role of LncCSMD1-1 in the degradation of MYC protein was also investigated. Results: With microarray, we identified a highly upregulated lncRNA, lncCSMD1-1, which was associated with tumor progression and poor prognosis in the Discovery Cohort, and validated in another 3 HCC cohorts. Consistently, ectopic expression of lncCSMD1-1 notably promotes cell proliferation, migration, invasion, tumor growth and metastasis of HCC cells in in vitro and in vivo experiments. Gene expression profiling on HCC cells and gene sets enrichment analysis indicated that the MYC target gene set was significantly enriched in HCC cells overexpressing lncCSMD1-1, and lncCSMD1-1 was found to directly bind to MYC protein in the nucleus of HCC cells, which resulted in the elevation of MYC protein. Mechanistically, lncCSMD1-1 interacted with MYC protein to block its ubiquitin-proteasome degradation pathway, leading to activation of its downstream target genes. Conclusion: lncCSMD1-1 is upregulated in HCC and promotes progression of HCC by activating the MYC signaling pathway. These results provide the evidence that lncCSMD1-1 may serve as a novel prognostic marker and potential therapeutic target for HCC.

Identification of immunological subtypes of hepatocellular carcinoma with expression profiling of immune-modulating genes.

Recent studies demonstrate that immune checkpoint inhibitor (ICI) therapy has achieved success in many types of advanced cancers including advanced hepatocellular carcinoma (HCC). However, ICI therapy is beneficial in only some HCC patients, suggesting that immune-responses are highly variable in HCCs. Therefore, understanding the immune status in HCC microenvironment will facilitate ICI immunotherapy and guide patient selection for the therapy. In this study, we first analyzed the expression profile of immune-modulating genes and their relationship with survival of HCC patients using the data downloaded from The Cancer Genome Atlas - Liver Hepatocellular Carcinoma (TCGA-LIHC) database, and found that the higher expressions of CD276 (B7-H3) and CD47 were significantly associated with poor survival. Then we identified 4 immune subtypes of HCCs with different survivals by using the combination expression of B7-H3 (or CD47) and CD8. Patients with B7-H3low/CD8high or CD47low/CD8high have the best survival while ones with B7-H3high/CD8low or CD47high/CD8low have the worst survival. The 4 immune subtypes were validated in another 72 HCC patients obtained from South China. In conclusion, our findings suggest that HCC patient prognosis is associated with immunophenotypes by T cell infiltration (CD8 expression) and the expression of the adaptive immune resistance gene (B7-H3 or CD47), and this immune classification system will facilitate HCC patient selection for ICI immunotherapy.

The prognostic value of a seven-lncRNA signature in patients with esophageal squamous cell carcinoma: a lncRNA expression analysis.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been reported to be prognostic biomarkers in many types of cancer. We aimed to identify a lncRNA signature that can predict the prognosis in patients with esophageal squamous cell carcinoma (ESCC). METHODS: Using a custom microarray, we retrospectively analyzed lncRNA expression profiles in 141 samples of ESCC and 81 paired non-cancer specimens from Sun Yat-Sen University Cancer Center (Guangzhou, China), which were used as a training cohort to identify a signature associated with clinical outcomes. Then we conducted quantitative RT-PCR in another 103 samples of ESCC from the same cancer center as an independent cohort to verify the signature. RESULTS: Microarray analysis showed that there were 338 lncRNAs significantly differentially expressed between ESCC and non-cancer esophagus tissues in the training cohort. From these differentially expressed lncRNAs, we found 16 lncRNAs associated with overall survival (OS) of ESCC patients using Cox regression analysis. Then a 7-lncRNA signature for predicting survival was identified from the 16 lncRNAs, which classified ESCC patients into high-risk and low-risk groups. Patients with high-risk have shorter OS (HR: 3.555, 95% CI 2.195-5.757, p < 0.001) and disease-free survival (DFS) (HR: 2.537, 95% CI 1.646-3.909, p < 0.001) when compared with patients with low-risk in the training cohort. In the independent cohort, the 7 lncRNAs were detected by qRT-PCR and used to compute risk score for the patients. The result indicates that patients with high risk also have significantly worse OS (HR = 2.662, 95% CI 1.588-4.464, p < 0.001) and DFS (HR 2.389, 95% CI 1.447-3.946, p < 0.001). The univariate and multivariate Cox regression analyses indicate that the signature is an independent factor for predicting survival of patients with ESCC. Combination of the signature and TNM staging was more powerful in predicting OS than TNM staging alone in both the training (AUC: 0.772 vs 0.681, p = 0.002) and independent cohorts (AUC: 0.772 vs 0.660, p = 0.003). CONCLUSIONS: The 7-lncRNA signature is a potential prognostic biomarker in patients with ESCC and may help in treatment decision when combined with the TNM staging system.