Candidate Tumor Suppressor Gene Research Articles

ING3 is required for ATM signaling and DNA repair in response to DNA double strand breaks.

Inhibitor of Growth 3 (ING3) is a candidate tumor suppressor gene whose expression is lost in tumors such as hepatocellular carcinoma, head and neck squamous cell carcinoma and melanoma. In the present study, we show that ING3-depleted human cells and yeast cells deleted for its ortholog YNG2 are sensitive to DNA damage suggesting a conserved role in response to such stress. In human cells, ING3 is recruited to DNA double strand breaks and is required for ATM activation. Remarkably, in response to doxorubicin, ATM activation is dependent on ING3 but not on TIP60, whose recruitment to DNA breaks also depends on ING3. These events lead to ATM-mediated phosphorylation of NBS1 and the subsequent recruitment of RNF8, RNF168, 53BP1, and BRCA1, which are major mediators of the DNA damage response. Accordingly, upon genotoxic stress, DNA repair by non-homologous end joining (NHEJ) or homologous recombination (HR) were impaired in absence of ING3. Finally, immunoglobulin class switch recombination (CSR), a physiological mechanism requiring NHEJ repair, was impaired in the absence of ING3. Since deregulation of DNA double strand break repair is associated with genomic instability, we propose a novel function of ING3 as a caretaker tumor suppressor involved in the DNA damage signaling and repair.

Frameshift mutation of candidate tumor suppressor genes QK1 and TMEFF2 in gastric and colorectal cancers.

Both QKI and TMEFF2 genes are considered putative tumor suppressor genes (TSGs). In gastric (GC) and colorectal (CRC) cancers, downregulation of their expressions is known to be frequent. However, QKI and TMEFF2 mutations that could potentially inactivate their functions are not reported in cancers. In a genome database, we observed that both QKI and TMEFF2 harbor mononucleotide repeats, which could be mutated in cancers with high microsatellite instability (MSI-H). For this, we studied 79 GCs and 124 CRCs for the mutations and their intratumoral heterogeneity (ITH). Six of 34 GCs (17.6%) and 10 of 79 CRCs (12.7%) with MSI-H exhibited QKI frameshift mutations while five of 79 CRCs (6.3%) with high MSI (MSI-H) exhibited TMEFF2 frameshift mutations. However, we found no such mutation in microsatellite stable/low MSI (MSS/MSI-L) cancers within the mononucleotide repeats. We also studied ITH for the detected frameshift mutations in 16cases of CRCs and detected that QKI and TMEFF2 frameshift mutations showed regional ITH in 2 (12.5%) and 1 (6.3%) cases, respectively. Our data show that candidate TSG genes QKI and TMEFF2 harbor mutational ITH as well as the frameshift mutations in GC and CRC with MSI-H. From this observation, frameshift mutations of QKI and TMEFF2 may play a role in tumorigenesis through their TSG inactivation in GC and CRC.

TES functions as a Mena-dependent tumor suppressor in gastric cancer carcinogenesis and metastasis

BackgroundIn our previous study, we identified a candidate tumor suppressor gene, testin LIM domain protein (TES), in primary gastric cancer (GC). TES contains three LIM domains, which are specific interacting regions for the cell adhesion and cytoskeleton regulatory proteins. Mena is a known cytoskeleton regulator that regulates the assembly of actin filaments and modulates cell adhesion and motility by interacting with Lamellipodin (Lpd). Therefore, we hypothesized that TES plays a role as tumor suppressor in GC through interacting with Mena. This study aimed to investigate the tumor suppressive functions of TES in GC.MethodsWe explored the tumor suppressive effect of TES in GC by in vitro cell proliferation assay, colony formation assay, cell cycle analysis, Transwell assays, and in vivo tumorigenicity and metastasis assays. The interaction of TES and Mena was investigated through immunoprecipitation-based mass spectrometry. We also analyzed the expression of TES and Mena in 172 GC specimens using immunohistochemistry and investigated the clinicopathological and prognostic significance of TES and Mena in GC.ResultsTES suppressed GC cell proliferation and colony formation, induced cell cycle arrest, and inhibited tumorigenicity in vitro. Additionally, it inhibited GC cell migration and invasion in vitro and suppressed metastasis in vivo. TES interacted with Mena, and inhibited the interaction of Mena with Lpd. Transwell assays suggested that TES suppressed migration and invasion of GC cells in a Mena-dependent fashion. In GC patients with high Mena expression, the expression of TES was associated with tumor infiltration (P = 0.005), lymph node metastasis (P = 0.003), TNM stage (P = 0.003), and prognosis (P = 0.010). However, no significant association was observed in GC patients with low Mena expression.ConclusionsWe believe that TES functions as a Mena-dependent tumor suppressor. TES represents a valuable prognostic marker and potential target for GC treatment.

Protocadherin 17 is a tumor suppressor and is frequently methylated in nasopharyngeal carcinoma.

PurposeSeveral PCDH genes were shown to be downregulated or silenced in carcinomas and act as candidate tumor suppressor genes. However, the functions of PCDH17 in nasopharyngeal carcinoma (NPC) remain unclear. Here, we investigated the PCDH17 promoter methylation status and its impact on the expression and functions of PCDH17 in NPC.Patients and methodsTo determine the mRNA levels and promoter methylation status of PCDH17 in NPC cell lines as well as 42 NPC patient specimens, we performed reverse transcription PCR, methylation-specific PCR, and bisulfite genome sequencing. The effects of ectopic PCDH17 expression in NPC cell lines were determined by colony formation, cell proliferation, wound healing, in vitro human umbilical vein endothelial cells tube formation, migration, invasion, cell cycle, and apoptosis assays and an in vivo subcutaneous tumor model.ResultsPCDH17 expression was almost absent or significantly reduced in 100% of the NPC cell lines (5/5). However, 5-aza-2′-deoxycytidine and trichostatin A treatment restored PCDH17 expression. Promoter methylation was involved in PCDH17 silencing. Ectopic expression of PCDH17 in silenced NPC cells reduced colony formation, cell migration, angiogenesis, VEGF secretion, and tumorigenicity.ConclusionPCDH17 plays a tumor suppressor role in NPC. PCDH17 methylation may be a tumor-specific event and can be used as an epigenetic biomarker for NPC.

Elucidating the pathogenesis of esophageal adenocarcinoma through meta-analysis of public data.

71 Background: Esophageal adenocarcinoma (EAC) is the sixth most common cause of cancer-related deaths worldwide. It commonly arises in the setting of reflux disease and Barett’s esophagus. It remains incurable and holds a poor prognosis. Dissecting the genetic signature of EAC can pave new therapeutic avenues. Methods: We employed our Search Tag Analyze Resource (STARGEO) platform to conduct meta-analysis using the National Center for Biotechnology’s (NCBI) Gene Expression Omnibus (GEO). We tagged 151 tumor samples from EAC patients and 62 normal esophageal samples as a control. We then analyzed the signature in Ingenuity Pathway Analysis, restricting genes that showed statistical significance (p < 0.05) and an absolute experimental log ratio greater than 0.15 between tumor and control. Results: Our analysis revealed granulocyte adhesion and diapedesis and FXR/RXR signaling as top canonical pathways. TGFB1, TNF, and beta-estradiol were top upstream regulators with predicted activation. TGFB1 and TNF expression have been correlated with poor prognosis in EAC. Also, beta-estradiol has tumorigenic activity in several cancers but has not been investigated in EAC. Among the top upregulated genes were oncogenic genes such as tetraspanin 8, the antiapoptotic factor OLFM4, and the protease cathepsin E (CTSE). SPINK1, a trypsin inhibitor with recently suggested role in cancer, and the choline transporter SLC44A4, a drug target for pancreatic cancer, were also upregulated. The most downregulated genes included alcohol dehydrogenase 7, associated with EAC in alcohol-drinkers, and the pro-apoptotic gene CRCT1. We also found downregulation of the serine peptidase inhibitor SPINK7. SPINK7 is involved in maintaining epithelial-barrier integrity and is implicated in eosinophilic esophagitis pathogenesis. Lastly, there was downregulation of the candidate tumor suppressor gene transglutaminase 3. Conclusions: Despite screening efforts, EAC incidence and mortality continues to increase as does the need for better treatment. This meta-analysis defines the significant gene expression changes within causal disease processes to provide markers for detection, prognostic insight, and therapeutic value for EAC.

Epigenomic profiling of myelofibrosis reveals widespread DNA methylation changes in enhancer elements and ZFP36L1 as a potential tumor suppressor gene that is epigenetically regulated.

In this study we interrogated the DNA methylome of myelofibrosis patients using high-density DNA methylation arrays. We detected 35,215 differentially methylated CpG, corresponding to 10,253 genes, between myelofibrosis patients and healthy controls. These changes were present both in primary and secondary myelofibrosis, which showed no differences between them. Remarkably, most differentially methylated CpG were located outside gene promoter regions and showed significant association with enhancer regions. This aberrant enhancer hypermethylation was negatively correlated with the expression of 27 genes in the myelofibrosis cohort. Of these, we focused on the ZFP36L1 gene and validated its decreased expression and enhancer DNA hypermethylation in an independent cohort of patients and myeloid cell-lines. In vitro reporter assay and 5’-azacitidine treatment confirmed the functional relevance of hyper-methylation of ZFP36L1 enhancer. Furthermore, in vitro rescue of ZFP36L1 expression had an impact on cell proliferation and induced apoptosis in SET-2 cell line indicating a possible role of ZFP36L1 as a tumor suppressor gene in myelofibrosis. Collectively, we describe the DNA methylation profile of myelofibrosis, identifying extensive changes in enhancer elements and revealing ZFP36L1 as a novel candidate tumor suppressor gene.

JAK2 regulates mismatch repair protein-mediated epigenetic alterations in response to oxidative damage.

At sites of chronic inflammation epithelial cells undergo aberrant DNA methylation that contributes to tumorigenesis. Inflammation is associated with an increase in reactive oxygen species (ROS) that cause oxidative DNA damage, which has also been linked to epigenetic alterations. We previously demonstrated that in response to ROS, mismatch repair proteins MSH2 and MSH6 recruit epigenetic silencing proteins DNA methyltransferase 1 (DNMT1) and polycomb repressive complex 2 (PRC2) members to sites of DNA damage, resulting in transcriptional repression of tumor suppressor genes (TSGs). However, it was unclear what signal is unique to ROS that results in the chromatin binding of MSH2 and MSH6. Herein, we demonstrate that in response to hydrogen peroxide (H2 O2 ), JAK2 localizes to the nucleus and interacts with MSH2 and MSH6. Inhibition or knockdown of JAK2 reduces the H2 O2 -induced chromatin interaction of MSH2, MSH6, DNMT1, and PRC2 members, reduces H2 O2 -induced global increase in trimethylation of lysine 27 of histone H3 (H3K27me3), and abrogates oxidative damage-induced transcriptional repression of candidate TSGs. Moreover, JAK2 mRNA expression is associated with CpG island methylator phenotype (CIMP) status in human colorectal cancer. Our findings provide novel insight into the connection between kinase activation and epigenetic alterations during oxidative damage and inflammation. Environ. Mol. Mutagen. 60:308-319, 2019. © 2018 Wiley Periodicals, Inc.

Epigenetically Silenced Candidate Tumor Suppressor Genes in Prostate Cancer: Identified by Modeling Methylation Stratification and Applied to Progression Prediction.

Recent studies have shown that epigenetic alterations, especially the hypermethylated promoters of tumor suppressor genes (TSGs), contribute to prostate cancer progression and metastasis. This article proposes a novel algorithm to identify epigenetically silenced TSGs (epi-TSGs) for prostate cancer. Our method is based on the perception that the promoter CpG island(s) of a typical epi-TSG has a stratified methylation profile over tumor samples. In other words, we assume that the methylation profile resembles the combination of a binary distribution of a driver mutation and a continuous distribution representing measurement noise and intratumor heterogeneity. Applying the proposed algorithm and an existing method to The Cancer Genome Atlas prostate cancer data, we identify 57 candidate epi-TSGs. Over one third of these epi-TSGs have been reported to carry potential tumor suppression functions. The negative correlations between the expression levels and methylation levels of these genes are validated on external independent datasets. We further find that the expression profiling of these genes is a robust predictive signature for Gleason scores, with the AUC statistic ranging from 0.75 to 0.79. The identified signature also shows prediction strength for tumor progression stages, biochemical recurrences, and metastasis events. We propose a novel method for pinpointing candidate epi-TSGs in prostate cancer. The expression profiling of the identified epi-TSGs demonstrates significant prediction strength for tumor progression. The proposed epi-TSGs identification method can be adapted to other cancer types beyond prostate cancer. The identified clinically significant epi-TSGs would shed light on the carcinogenesis of prostate adenocarcinomas.

Methylation of Epstein-Barr Virus-Associated Gastric Cancer Suppressor Genes

Objective: To identify the methylation silenced tumor suppressor genes in Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC). Methods: EBV-positive (GT38, PT and SNU719) and negative (SGC7901) gastric cancer cell lines were selected and treated with 5-Aza-CdR. Then real-time fluorescence quantitative PCR was used to validate the results of microarray, and methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) were adopted to detect the CpG island methylation levels of gene promoters. Results: The expression levels of 6 differentially expressed genes (H19, LOXL1, ARMCX2, LXN, CDH3 and MMP7) before and after 5-Aza-CdR treatment were confirmed by real-time qPCR, which are consistent with the results of microarray analysis. There were different degrees of methylation in LOXL1 gene promoter in EBVaGC. GT38 and PT were fully methylated, and SGC7901 and HGC-27 was unmethylated, suggesting that this gene is a candidate methylation silenced tumor suppressor gene. The methylation rate of LOXL1 in EBVaGC was significantly higher than that in EBV-negative gastric cancer (EBVnGC). Conclusion: The promoter region of candidate tumor suppressor gene LOXL1 shows high methylation status, indicating that EBV critically accounts for the methylation of LOXL1 gene regulatory region. EBV is involved in the pathogensis of EBVaGC that aberrant methylation occurred in promoter CpG island, which inactivates tumor suppressor genes.

Targeting NPRL2 to enhance the efficacy of Olaparib in castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) lacks effective treatment, and studies have shown that PARPi inhibitors, such as Olaparib, are somewhat effective; however, the efficacy of Olaparib in CRPC still needs to be further improved. Nitrogen permease regulator-like 2 (NPRL2) is reported to be a tumor suppressor candidate gene and is closely related to the DNA repair pathway, which can affect the sensitivity of many chemotherapeutic drugs. However, there is no research on whether NPRL2 is associated with sensitivity to Olaparib. Hence, in the present study, we examined the NPRL2 expression levels in several PCa cell lines (LNCaP, PC3, and enzalutamide-resistant LNCaP, named LNPER) by Western blot. In addition, we investigated the role of NPRL2 expression and silencing in cell proliferation and in the regulation of ataxia telangiectasia mutated (ATM), which can mediate DNA repair and sensitivity to Olaparib. Furthermore, we performed in vitro and in vivo experiments to determine the mechanism of action of NPRL2 in adjusting Olaparib sensitivity. Our findings demonstrated that the NPRL2 expression level was upregulated in PCa cells, especially CRPC cells. NPRL2 overexpression promoted growth and resistance to Olaparib, and NPRL2 silencing inhibited proliferation, enhanced sensitivity to Olaparib, and increased CRRL2 expression in PCa cells. In addition, the Olaparib-induced growth delay in NPRL2-silenced PC3 tumors in mice correlated with ATM signaling downregulation, an apoptosis increase and migration/invasion suppression. Our results indicate that NPRL2 silencing enhances sensitivity to Olaparib treatment in CRPC and that NPRL2 may serve as a potential therapeutic target and predict resistance to Olaparib in CRPC.

A Genome-Wide CRISPR Screen Implicates MYC Dysregulation in TCF3-PBX1 B-ALL

Introduction: Acute lymphoblastic leukaemia (ALL) is the most common paediatric cancer, of which, precursor B-cell ALL (B-ALL) accounts for approximately 80% of diagnoses. B-ALL is a heterogeneous disease, with patients characterised and risk stratified according to their cytogenetic profile. TCF3-PBX1 B-ALL was associated with a poor prognosis, but on current therapies outcome has greatly improved. However, approximately 10% of these patients relapse with their disease and at this stage have a dismal prognosis (Moorman et. al. Lancet Oncology 2010). Thus, this subset of patients pose a clinical challenge, and further understanding of disease mechanisms in relapsed TCF3-PBX1 is required to aid the discovery of novel targets for therapy.Methods: Clustered regularly-interspaced short palindromic repeats (CRISPR) technology was utilised as a whole genome CRISPR knockout (GeCKO) screen (Sanjana et. al. Nature Methods 2014) for genome-wide identification of candidate oncogene and tumour suppressor genes (TSGs) in B-ALL cell lines, including 697 (relapsed TCF3-PBX1), REH (ETV6-RUNX1), NALM16 (hypodiploid) and HAL-01 (TCF3-HLF). The GeCKO screens were performed at 300-fold library coverage, with transduced and selected cells harvested at day 0, weeks 2, 4, 6 and 8. DNA was sequenced across the integrated sgRNA region. The abundance of sgRNA constructs was analysed over time using the model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK) program (Li et. al. Genome Biology 2014) to identify candidate oncogenes and TSGs. Pathway analysis was performed for the identification of significantly dysregulated pathways in TCF3-PBX1 using the 697 data set and the MaGeCK Gene Set Enrichment Analysis pathway program.Results: Whole genome CRISPR screening in the 697 cell line successfully identified 2213 candidate oncogenes (false discovery rate (FDR) <0.05, p value <0.012) and 5 candidate TSGs (FDR <0.3, p value < 6.7x10-5). Amongst the significant candidate oncogenes was MYC (FDR = 3.0x10-6, p value = 2.3x10-8, rank 16) and MaGeCK Gene Set Enrichment pathway analysis using the Molecular Signatures Database (MSigDB) Hallmarks data set, identified HALLMARK_MYC_TARGETS_V1 and HALLMARK_MYC_TARGETS_V2 as the two most significant negatively regulated pathways within the knockout screen (FDR of 1.79x10-55 and 9.876x10-21, respectively). These data indicate a role for MYC dysregulation in TCF3-PBX1 B-ALL. Additionally, the identification of the intriguing TSG candidates CREBBP (FDR = 0.059, p value = 5.5x10-6, rank 2), MLXIP (FDR = 0.24, p value = 3.4x10-5, rank 3), HIF1A (FDR = 0.29, p value = 6.7x10-5, rank 4) and ARNT (FDR = 0.02, p value = 1x10-6, rank 1) further highlighting the importance of MYC signalling in TCF3-PBX1 B-ALL. HIF1A inhibits MYC by direct interaction, induction of the repressor MXI1 and by coordinating the degradation of MYC by the proteasome (Zhang et. al. Cancer Cell 2007, Corn et. al. Cancer Biol. Ther. 2005, Gordan et. al. Cancer Cell 2007). ARNT dimerises with HIF1A and is responsible for the recruitment of transcriptional coactivators for the transcriptional output of HIF1A (Partch et. al. Proc Natl Acad Sci USA 2011), while CREBBP has been reported to have a role in MYC inhibition in G1, preventing the progression from G1 to S phase (Rajabi et. al. J. Biol. Chem. 2005), in addition to interaction with the candidate TSG, HIF1A (Ema et. al. EMBO J. 1999, Bhattacharya et. al. Genes Dev. 1999, Park et. al. Mol. Pharmacol. 2008). Despite the role of MLXIP in glycolysis in B-ALL (Wernicke et. al. Leuk Res. 2012), and MLXIP knockout being synthetic lethal in MYC-overexpressing neuroblastoma cell lines (Carroll et. al. Cancer Cell 2015), CRISPR mediated knockout of MLXIP in the 697 TCF3-PBX1 cell line promoted growth. This may be because knockout of MLXIP reduces the number of MLXIP-MLX heterodimers to compete with MYC-MAX heterodimers at E-box sequences, permitting transcriptional activation of MYC-target genes (Carroll et. al. Cancer Cell 2015). This study has identified a number of potential mechanisms by which MYC deregulation can occur in TCF3-PBX1 B-ALL, highlighting the essential role of MYC in disease maintenance. MYC inhibition offers a potential avenue for targeted therapy in relapsed TCF3-PBX1 B-ALL, which warrants further investigation. DisclosuresNo relevant conflicts of interest to declare.

Action and clinical significance of CCAAT/enhancer-binding protein delta in hepatocellular carcinoma.

CCAAT/enhancer-binding protein delta (CEBPD) is associated with the regulation of apoptosis and cell proliferation and is a candidate tumor suppressor gene. Here, we investigated its role in hepatocellular carcinoma (HCC). We observe that CEBPD mRNA expression is significantly downregulated in HCC tumors as compared with adjacent tissues. Protein levels of CEBPD are also lower in tumors relative to adjacent tissues. Reduced expression of CEBPD in the tumor correlates with worse clinical outcome. In both Huh7 and HepG2 cells, shRNA-mediated CEBPD knockdown significantly reduces cell proliferation, single cell colony formation and arrests cells in the G0/G1 phase. Subcutaneous xenografting of Huh7 in nude mice show that CEBPD knockdown results in smaller tumors. Gene expression analysis shows that CEBPD modulates interleukin-1 signaling. We conclude that CEBPD expression uncouples cancer compartment expansion and clinical outcome in HCC, potentially by modulating interleukin-1 signaling. Thus, although our results support the notion that CEBPD acts as a tumor suppressor in HCC, its action does not involve impairing compartment expansion per se but more likely acts through improving anticancer immunity.

A genome-wide Drosophila epithelial tumorigenesis screen identifies Tetraspanin 29Fb as an evolutionarily conserved suppressor of Ras-driven cancer.

Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, Ras mutations alone are insufficient for tumorigenesis, therefore it is paramount to identify cooperating cancer-relevant signaling pathways. We devised an in vivo near genome-wide, functional screen in Drosophila and discovered multiple novel, evolutionarily-conserved pathways controlling Ras-driven epithelial tumorigenesis. Human gene orthologs of the fly hits were significantly downregulated in thousands of primary tumors, revealing novel prognostic markers for human epithelial tumors. Of the top 100 candidate tumor suppressor genes, 80 were validated in secondary Drosophila assays, identifying many known cancer genes and multiple novel candidate genes that cooperate with Ras-driven tumorigenesis. Low expression of the confirmed hits significantly correlated with the KRASG12 mutation status and poor prognosis in pancreatic cancer. Among the novel top 80 candidate cancer genes, we mechanistically characterized the function of the top hit, the Tetraspanin family member Tsp29Fb, revealing that Tsp29Fb regulates EGFR signaling, epithelial architecture and restrains tumor growth and invasion. Our functional Drosophila screen uncovers multiple novel and evolutionarily conserved epithelial cancer genes, and experimentally confirmed Tsp29Fb as a key regulator of EGFR/Ras induced epithelial tumor growth and invasion.

Significance of hydroxymethylglutaryl-CoA synthase 2 on prognosis of esophageal squamous cell carcinoma

Objective: To explore the roll and function of hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) in the development and progression of human esophageal squamous cell carcimoma(ESCC). Method: Using immunohistochemistry, the expression of HMGCS2 was determined in 150 primary ESCC patients from July 2002 to December 2005 in the People's Hospital of Linzhou City, Henan Province. And HMGCS2 over-expression ESCC cell lines were established to verify HMGCS2 gene function. Result: In 150 cases of ESCCs, the expression rate of HMGCS2 was 58% (87/150), which was lower than 72% (108/150) in paired normal tissues, the difference was statistically significant (P=0.013). HMGCS2 down-regulated expression was associated with tumor cell differentiation (P=0.022), pT status (P=0.036), pN status (P=0.017) and TNM stage(P=0.012). The 5-years disease-specific survival (DSS) in down HMGCS2 expression group (14 months) was poorer than those in normal expression group (20 months; P=0.002). In addition, multivariate Cox regression analysis showed that HMGCS2 expression (Wald=7.136, P=0.008) was an independent risk factor for DSS. Furthermore, functional studies demonstrated that HMGCS2 gene could suppress the tumorigenic ability of ESCC cells (OD: 0.79±0.04 vs 1.25±0.68; P=0.01), the formation of colone (number of colones: 30±10 vs 189±15, P=0.002), and cell motility (number of cells: 27±14 vs 222±40, P=0.009). Conclusion: HMGCS2 can inhibit the proliferation and migration of ESCC cells, and could be an important candidate tumor suppressor gene for ESCC.

Overexpression of MYCT1 Inhibits Proliferation and Induces Apoptosis in Human Acute Myeloid Leukemia HL-60 and KG-1a Cells in vitro and in vivo.

MYC target 1 (MYCT1), a direct target gene of c-Myc, is a novel candidate tumor suppressor gene first cloned from laryngeal squamous cell carcinoma. The downregulation of MYCT1 has been reported to be associated with carcinogenesis. However, the role of MYCT1 in the development and progress of acute myeloid leukemia (AML) remains unknown and requires further investigation. In this study, we first found that the expression level of MYCT1 was significantly lower in the bone marrow (BM) derived from AML patients than that from healthy individuals. The low expression of MYCT1 in AML BM may be due to the hypermethylation in its promoter. MYCT1 expression was strongly associated with French–American–British classifications of AML. The low expression level of MYCT1 was more often observed in patients of M1, M5 and M6 types. In vitro, lentiviral particles carrying the complete CDS of MYCT1 gene were used to mediate the forced overexpression of MYCT1 in two AML cell lines, HL-60 and KG-1a. MYCT1 overexpression significantly inhibited cell proliferation, arrested cell cycle at G0/G1 phase, and downregulated the expression of cyclins D and E. Moreover, MYCT1 overexpression triggered apoptosis in AML cells, which was accompanied by enhanced cleavage of caspase-3 and -9, upregulated expression of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), and downregulated Bcl-2. Finally, in BALB/c nude mice bearing xenograft tumors generated by HL-60 and KG-1a cells, we noted that the intratumoral injection of MYCT1 lentivirus repressed tumor growth and led to massive apoptosis. In summary, our results reveal that MYCT1’s promoter is hypermethylated and its expression is downregulated in the BM of AML patients. MYCT1 plays a tumor-suppressive role, and it may serve as a promising target for the genetic therapeutic strategy in treating AML.

Identification of Hyper-Methylated Tumor Suppressor Genes-Based Diagnostic Panel for Esophageal Squamous Cell Carcinoma (ESCC) in a Chinese Han Population.

DNA methylation-based biomarkers were suggested to be promising for early cancer diagnosis. However, DNA methylation-based biomarkers for esophageal squamous cell carcinoma (ESCC), especially in Chinese Han populations have not been identified and evaluated quantitatively. Candidate tumor suppressor genes (N = 65) were selected through literature searching and four public high-throughput DNA methylation microarray datasets including 136 samples totally were collected for initial confirmation. Targeted bisulfite sequencing was applied in an independent cohort of 94 pairs of ESCC and normal tissues from a Chinese Han population for eventual validation. We applied nine different classification algorithms for the prediction to evaluate to the prediction performance. ADHFE1, EOMES, SALL1 and TFPI2 were identified and validated in the ESCC samples from a Chinese Han population. All four candidate regions were validated to be significantly hyper-methylated in ESCC samples through Wilcoxon rank-sum test (ADHFE1, P = 1.7 × 10-3; EOMES, P = 2.9 × 10-9; SALL1, P = 3.9 × 10-7; TFPI2, p = 3.4 × 10-6). Logistic regression based prediction model shown a moderately ESCC classification performance (Sensitivity = 66%, Specificity = 87%, AUC = 0.81). Moreover, advanced classification method had better performances (random forest and naive Bayes). Interestingly, the diagnostic performance could be improved in non-alcohol use subgroup (AUC = 0.84). In conclusion, our data demonstrate the methylation panel of ADHFE1, EOMES, SALL1 and TFPI2 could be an effective methylation-based diagnostic assay for ESCC.

NPRL2 enhances autophagy and the resistance to Everolimus in castration-resistant prostate cancer.

Nitrogen permease regulator-like 2 (NPRL2) is reported to be a tumor suppressor candidate gene and involved in the mTOR signaling and drug resistance in several cancers. However, the role of NPRL2 in regulating the resistance to Everolimus (EVS), an inhibitor of the mTOR, in castration-resistant prostate cancer (CRPC) is still unclear. Therefore, in present study, we evaluated the role of NPRL2 and its potential resistance to EVS in CRPC. NPRL2 expression levels in prostate tissues, including benign prostate hyperplasia (BPH) tissues, primary prostate cancer (PCa) tissues, CRPC tissues, and several PCa cell lines (LNCaP, PC3, and enzalutamide-resistant LNCaP, named LNPER) were be evaluated by immunohistochemistry, RT-PCR, and Western blot. Furthermore, we employed the loss or gain function of NPRL2 to determine the role of NPRL2 in regulating the proliferation, sensitivity to EVS, the mTOR signaling, autophagy in CRPC. Lastly, relationship between NPRL2 expression level and the efficacy of EVS were evaluated in mice tumor xenograft models. NPRL2 expression level is upregulated in PCa, particularly in the CRPC. NPRL2 over-expression promoted the proliferation, resistance to EVS, and NPRL2 silencing inhibited proliferation, enhanced sensitivity to EVS in PC3 and LNPER cells. Moreover, NPRL2-silencing increased the activity of mTOR signaling, and the autophagy attenuation induced by NPRL2-silencing in EVS-treated CRPC cells was associated with the increase of apoptosis. In addition, the growth prevention of NPRL2-silencing LNPER tumors in mice induced by EVS-treatment was associated with the autophagy attenuation and apoptosis increase. NPRL2 may act as a pro-growth factor in PCa. The high levels of NPRL2 expression in CRPC promote resistance to EVS by enhancing autophagy. NPRL2 may be a new therapeutic target for intervention of CRPC and a biomarker for predicting resistance to EVS in CRPC.

Widespread intronic polyadenylation inactivates tumour suppressor genes in leukaemia.

DNA mutations are known cancer drivers. Here, we investigated if mRNA events that are upregulated in cancer can functionally mimic the outcome of genetic alterations. 3′-seq or RNA-seq were applied to normal and malignant B cells from chronic lymphocytic leukemia (CLL; N = 59)1–3. We discovered widespread upregulation of truncated mRNAs and proteins in primary CLL cells that were not generated by genetic alterations but occurred through intronic polyadenylation (IPA). IPA-generated truncated mRNAs were recurrent (N = 330) and predominantly affected genes with tumor-suppressive functions. The IPA-generated truncated proteins often lack the tumor-suppressive functions of the corresponding full-length proteins (DICER, FOXN3), and several even acted in an oncogenic manner (CARD11, MGA, CHST11). In CLL, inactivation of tumor-suppressor genes (TSGs) through aberrant mRNA processing is substantially more prevalent than loss of TSGs through genetic events. We further identified novel TSG candidates that are inactivated by IPA in leukemia and by truncating DNA mutations in solid tumors4,5. These genes are understudied in cancer as their overall mutation rates are lower than those of well-known TSGs. Our findings show the need to go beyond genomic analyses in cancer diagnostics, as mRNA events that are silent at the DNA level are widespread contributors to cancer pathogenesis through inactivation of TSGs.

Transformed Follicular Lymphoma (TFL) Predicts Outcome in Advanced Endometrial Cancer.

Background: Transformed follicular lymphoma (TFL, ZC3H12D) was identified as a candidate tumor suppressor gene that contributes to cell-cycle arrest through regulation of Rb phosphorylation, but the clinical impact of TFL is unknown. The goal of this study was to evaluate the prognostic significance of TFL expression in advanced endometrial cancer.Methods: Tissue samples were obtained from 103 patients with Federation Internationale des Gynaecologistes et Obstetristes stage III-IV endometrial cancer. Associations between TFL expression and outcomes were evaluated using the Kaplan-Meier method and multivariate Cox proportional hazards regression models.Results: There were 24 TFL-low cases (23.3%) and the 10-year progression-free survival (PFS) and overall survival (OS) in these cases were lower than those for patients with normal TFL expression in univariate analysis (PFS, P = 0.003; OS, P = 0.106). In multivariate analysis, TFL status was a significant predictor for PFS [HR = 2.76; 95% confidence interval (CI), 1.45-5.28; P = 0.002] and OS (HR = 1.94; 95% CI, 0.91-4.11; P = 0.085), adjusted for covariates. The TFL gene maps to human chromosome 6q25.1, where estrogen receptor alpha (ERα) gene ESR1 is also located. Lack of ERα expression is a poor prognostic factor in early endometrial cancer. Among 41 ERα-low patients, 10-year PFS was significantly lower in 15 TFL-low cases (univariate analysis, P = 0.055; multivariate analysis, HR = 4.70; 95% CI, 1.68-13.20; P = 0.003).Conclusions: We identified TFL as a strong independent prognostic factor, regardless of ERα status.Impact: An investigation of the mechanism underlying tumor suppression by TFL may lead to new therapies for patients with advanced endometrial cancer. Cancer Epidemiol Biomarkers Prev; 27(8); 963-9. ©2018 AACR.

ECRG4: a new potential target in precision medicine.

Given the rapid development in precision medicine, tremendous efforts have been devoted to discovering new biomarkers for disease diagnosis and treatment. Esophageal cancer-related gene-4 (ECRG4), which is initially known as a new candidate tumor suppressor gene, is emerging as a sentinel molecule for gauging tissue homeostasis. ECRG4 is unique in its cytokine-like functional pattern and epigenetically-regulated gene expression pattern. The gene can be released from the cell membrane upon activation and detected in liquid biopsy, thus offering considerable potential in precision medicine. This review provides an updated summary on the biology of ECRG4, with emphasis on its important roles in cancer diagnosis and therapy. The future perspectives of ECRG4 as a potential molecular marker in precision medicine are also discussed in detail.