CDKN2A somatic copy number amplification in normal tissues surrounding gastric carcinoma reduces cancer metastasis risk in droplet digital PCR analysis.

BACKGROUND: The CDKN2A gene is frequently affected by somatic copy number variations (SCNVs, including deletions and amplifications [SCNdel and SCNamp]) in the cancer genome. Using surgical gastric margin tissue samples (SMs) as the diploid reference in SCNV analysis via CDKN2A/P16-specific real-time PCR (P16-Light), we previously reported that the CDKN2A SCNdel was associated with a high risk of metastasis of gastric carcinoma (GC). However, the status of CDKN2A SCNVs in SMs and their clinical significance have not been reported. METHODS: Peripheral white blood cell (WBC) and frozen GC and SM tissue samples were collected from patients (n = 80). Droplet digital PCR (ddPCR) was used to determine the copy number (CN) of the CDKN2A gene in tissue samples using paired WBCs as the diploid reference. RESULTS: A novel P16-ddPCR system was initially established with a minimal proportion (or limit, 10%) of the detection of CDKN2A CN alterations. While CDKN2A SCNamp events were detected in both SMs and GCs, fewer CDKN2A SCNdel events were detected in SMs than in GCs (15.0% vs. 41.3%, P = 4.77E-04). Notably, significantly more SCNamp and fewer SCNdel of the CDKN2A gene were detected in SMs from GC patients without metastasis than in those from patients with lymph node metastasis by P16-ddPCR (P = 0.023). The status of CDKN2A SCNVs in SM samples was significantly associated with overall survival (P = 0.032). No cancer deaths were observed among the 11 patients with CDKN2A SCNamp. CONCLUSION: CDKN2A SCNVs in SMs identified by P16-ddPCR are prevalent and significantly associated with GC metastasis and overall survival.

Somatic CDKN2A copy number variations are associated with the prognosis of esophageal squamous cell dysplasia.

BACKGROUND: Somatic copy number variations (SCNVs) in the CDKN2A gene are among the most frequent events in the dysplasia-carcinoma sequence of esophageal squamous cell carcinoma. However, whether CDKN2A SCNVs are useful biomarkers for the risk stratification and management of patients with esophageal squamous cell dysplasia (ESCdys) is unknown. This study aimed to investigate the characteristics and prognostic value of CDKN2A SCNVs in patients with mild or moderate (m/M) ESCdys. METHODS: This study conducted a prospective multicenter study of 205 patients with a baseline diagnosis of m/M ESCdys in five high-risk regions of China (Ci County, Hebei Province; Yanting, Sichuan Province; Linzhou, Henan Province; Yangzhong, Jiangsu Province; and Feicheng, Shandong Province) from 2005 to 2019. Genomic DNA was extracted from paraffin biopsy samples and paired peripheral white blood cells from patients, and a quantitative polymerase chain reaction assay, P16-Light, was used to detect CDKN2A copy number. The cumulative regression and progression rates of ESCdys were evaluated using competing risk models. RESULTS: A total of 205 patients with baseline m/M ESCdys were enrolled. The proportion of ESCdys regression was significantly lower in the CDKN2A deletion cohort than in the diploid and amplification cohorts (18.8% [13/69] vs. 35.0% [28/80] vs. 51.8% [29/56], P  <0.001). In the univariable competing risk analysis, the cumulative regression rate was statistically significantly lower ( P = 0.008), while the cumulative progression rate was higher ( P = 0.017) in ESCdys patients with CDKN2A deletion than in those without CDKN2A deletion. CDKN2A deletion was also an independent predictor of prognosis in ESCdys ( P = 0.004) in the multivariable analysis. CONCLUSION: The results indicated that CDKN2A SCNVs are associated with the prognosis of ESCdys and may serve as potential biomarkers for risk stratification.

Driving effect of P16 methylation on telomerase reverse transcriptase-mediated immortalization and transformation of normal human fibroblasts.

BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase (TERT) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT. However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16-specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION: P16 methylation drives TERT-mediated immortalization and transformation of normal human cells that may contribute to cancer development.

LncRNA miR663AHG represses the development of colon cancer in a miR663a-dependent manner.

The MIR663AHG gene encodes both miR663AHG and miR663a. While miR663a contributes to the defense of host cells against inflammation and inhibits colon cancer development, the biological function of lncRNA miR663AHG has not been previously reported. In this study, the subcellular localization of lncRNA miR663AHG was determined by RNA-FISH. miR663AHG and miR663a were measured by qRT-PCR. The effects of miR663AHG on the growth and metastasis of colon cancer cells were investigated in vitro and in vivo. CRISPR/Cas9, RNA pulldown, and other biological assays were used to explore the underlying mechanism of miR663AHG. We found that miR663AHG was mainly distributed in the nucleus of Caco2 and HCT116 cells and the cytoplasm of SW480 cells. The expression level of miR663AHG was positively correlated with the level of miR663a (r = 0.179, P = 0.015) and significantly downregulated in colon cancer tissues relative to paired normal tissues from 119 patients (P < 0.008). Colon cancers with low miR663AHG expression were associated with advanced pTNM stage (P = 0.021), lymph metastasis (P = 0.041), and shorter overall survival (hazard ratio = 2.026; P = 0.021). Experimentally, miR663AHG inhibited colon cancer cell proliferation, migration, and invasion. The growth of xenografts from RKO cells overexpressing miR663AHG was slower than that of xenografts from vector control cells in BALB/c nude mice (P = 0.007). Interestingly, either RNA-interfering or resveratrol-inducing expression changes of miR663AHG or miR663a can trigger negative feedback regulation of transcription of the MIR663AHG gene. Mechanistically, miR663AHG could bind to miR663a and its precursor pre-miR663a, and prevent the degradation of miR663a target mRNAs. Disruption of the negative feedback by knockout of the MIR663AHG promoter, exon-1, and pri-miR663A-coding sequence entirely blocked these effects of miR663AHG, which was restored in cells transfected with miR663a expression vector in rescue experiment. In conclusion, miR663AHG functions as a tumor suppressor that inhibits the development of colon cancer through its cis-binding to miR663a/pre-miR663a. The cross talk between miR663AHG and miR663a expression may play dominant roles in maintaining the functions of miR663AHG in colon cancer development.

Detection of somatic copy number deletion of the CDKN2A gene by quantitative multiplex PCR for clinical practice.

Background: A feasible method to detect somatic copy number deletion (SCND) of genes is still absent to date. Methods: Interstitial base-resolution deletion/fusion coordinates for CDKN2A were extracted from published articles and our whole genome sequencing (WGS) datasets. The copy number of the CDKN2A gene was measured with a quantitative multiplex PCR assay P16-Light and confirmed with whole genome sequencing (WGS). Results: Estimated common deletion regions (CDRs) were observed in many tumor suppressor genes, such as ATM, CDKN2A, FAT1, miR31HG, PTEN, and RB1, in the SNP array-based COSMIC datasets. A 5.1 kb base-resolution CDR could be identified in >90% of cancer samples with CDKN2A deletion by sequencing. The CDKN2A CDR covers exon-2, which is essential for P16INK4A and P14ARF synthesis. Using the true CDKN2A CDR as a PCR target, a quantitative multiplex PCR assay P16-Light was programmed to detect CDKN2A gene copy number. P16-Light was further confirmed with WGS as the gold standard among cancer tissue samples from 139 patients. Conclusion: The 5.1 kb CDKN2A CDR was found in >90% of cancers containing CDKN2A deletion. The CDKN2A CDR was used as a potential target for developing the P16-Light assay to detect CDKN2A SCND and amplification for routine clinical practices.

Corrigendum: P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7.

[This corrects the article DOI: 10.3389/fcell.2022.993525.].

P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7.

Background: It is well known that P16 INK4A , P14 ARF , P15 INK4B mRNAs, and ANRIL lncRNA are transcribed from the CDKN2A/2B locus. LncRNA P14AS is a lncRNA transcribed from antisense strand of P14 ARF promoter to intron-1. Our previous study showed that P14AS could upregulate the expression level of ANRIL and P16 INK4A and promote the proliferation of cancer cells. Because polycomb group protein CBX7 could repress P16 INK4A expression and bind ANRIL, we wonder whether the P14AS-upregulated ANRIL and P16 INK4A expression is mediated with CBX7. Results: In this study, we found that the upregulation of P16 INK4A , P14 ARF , P15 INK4B and ANRIL expression was induced by P14AS overexpression only in HEK293T and HCT116 cells with active endogenous CBX7 expression, but not in MGC803 and HepG2 cells with weak CBX7 expression. Further studies showed that the stable shRNA-knockdown of CBX7 expression abolished the P14AS-induced upregulation of these P14AS target genes in HEK293T and HCT116 cells whereas enforced CBX7 overexpression enabled P14AS to upregulate expression of these target genes in MGC803 and HepG2 cells. Moreover, a significant association between the expression levels of P14AS and its target genes were observed only in human colon cancer tissue samples with high level of CBX7 expression (n = 38, p < 0.05), but not in samples (n = 37) with low level of CBX7 expression, nor in paired surgical margin tissues. In addition, the results of RNA immunoprecipitation (RIP)- and chromatin immunoprecipitation (ChIP)-PCR analyses revealed that lncRNA P14AS could competitively bind to CBX7 protein which prevented the bindings of CBX7 to both lncRNA ANRIL and the promoters of P16 INK4A , P14 ARF and P15 INK4B genes. The amounts of repressive histone modification H3K9m3 was also significantly decreased at the promoters of these genes by P14AS in CBX7 actively expressing cells. Conclusions: CBX7 expression is essential for P14AS to upregulate the expression of P16 INK4A , P14 ARF , P15 INK4B and ANRIL genes in the CDKN2A/2Blocus. P14AS may upregulate these genes' expression through competitively blocking CBX7-binding to ANRIL lncRNA and target gene promoters.

TTC22 promotes m6A-mediated WTAP expression and colon cancer metastasis in an RPL4 binding-dependent pattern.

WTAP, an essential component of the RNA N-6-methyladenosine (m6A) modification complex, guides METLL3-METLL14 heteroduplexes to target RNAs in the nuclear speckles of mammalian cells. Here, we show that TTC22 is widely coexpressed with WTAP and FTO in many human tissues by mining Genotype-Tissue Expression (GTEx) datasets. Our results indicate that the direct interaction of TTC22 with 60S ribosomal protein L4 (RPL4) promotes the binding of WTAP mRNA to RPL4, enhances the stability and translation efficiency of WTAP mRNA, and consequently increases the level of WTAP protein. Also, WTAP mRNA itself is an m6A target and YTHDF1 is characterized as an essential m6A binding protein interacting with m6A-modified WTAP mRNA. TTC22 triggers a positive feedback loop between WTAP expression and WTAP mRNA m6A modification, leading to an increased m6A level in total RNA. The knockdown of RPL4, WTAP, or YTHDF1 expression diminishes the TTC22-induced increase in the m6A level of total RNA. Thus, TTC22 caused dramatic expression changes in genes related to metabolic pathways, ribosomal biogenesis, the RNA spliceosome, and microorganism infections. Importantly, TTC22 upregulates the expression of SNAI1 by increasing m6A level and thus promotes lung metastases of colon cancer cells in mice. In conclusion, our study showed that TTC22 upregulates WTAP and SNAI1 expression, which contributes to TTC22-induced colon cancer metastasis.

Kaiso phosphorylation at threonine 606 leads to its accumulation in the cytoplasm, reducing its transcriptional repression of the tumour suppressor CDH1.

It is well known that the Kaiso protein (encoded by the ZBTB33 gene) is a transcription factor, and Kaiso-P120ctn [P120 catenin (CTNND1)] interaction increases the translocation of Kaiso from the nucleus into the cytoplasm. However, the regulatory mechanisms of Kaiso compartmentalisation are far from clear. Here, we reported that RAC-alpha serine/threonine-protein kinase (AKT1) could phosphorylate threonine residue 606 (T606) within the RSSTIP motif of Kaiso in the cytoplasm. The T606-phosphorylated Kaiso (pT606-Kaiso) could directly bind to 14-3-3 family proteins, and depletion of T606 phosphorylation by T606A mutation abolished most of the Kaiso-14-3-3 binding. In addition, the Kaiso-P120ctn interaction was essential for pT606-Kaiso accumulation in the cytoplasm. Notably, enforced stratifin (14-3-3σ; SFN) overexpression could increase pT606-Kaiso accumulation in the cytoplasm and de-repress the transcription of Kaiso target gene cadherin 1 (CDH1), which is a tumour suppressor. Decreased amounts of both pT606-Kaiso and CDH1 proteins were frequently observed in human gastric cancer tissues compared to paired normal controls. The mRNA levels of 14-3-3σ and Kaiso target gene CDH1 showed highly significant positive correlations in both human normal tissues and cancer cell lines by bioinformatics analyses. Furthermore, Kaiso T606A mutant (unable to be phosphorylated) significantly increased the migration and invasion of cancer cells in vitro and promoted the growth of these cells in vivo. In conclusion, Kaiso could be phosphorylated at T606 by AKT1 and pT606-Kaiso accumulates in the cytoplasm through binding to 14-3-3/P120ctn, which de-represses the Kaiso target gene CDH1 in normal tissues. Decreased Kaiso phosphorylation might contribute to the development of gastrointestinal cancer. The status of Kaiso phosphorylation is a determinant factor for the role of Kaiso in the development of cancer.

Feasibility of using P16 methylation as a cytologic marker for esophageal squamous cell carcinoma screening: A pilot study.

BACKGROUND: Early diagnosis and treatment of esophageal squamous cell dysplasia (ESCdys) and esophageal squamous cell carcinoma (ESCC) could significantly reduce the incidence and mortality of ESCC. This pilot study aimed to investigate whether P16/CDKN2A methylation could serve as a cytologic biomarker for early detection of ESCdys and ESCC. METHODS: Paired esophageal biopsy and cytology specimens (exfoliated cells) were obtained from subjects at different stages of ESCC development. The methylation status of P16 gene in these two specimen types was determined using a 115-bp MethyLight assay. Categorical data were compared by the Chi-square test. Logistic regression was performed to assess adjusted odds ratios of P16 methylation associated with ESCC and ESCdys. Prediction models for identifying individuals at risk of ESCC and high-grade ESCdys (high-grade intraepithelial neoplasia, HGIN) were developed by multivariable logistic regression. Diagnostic performance was evaluated using receiver operating characteristic (ROC) analysis. Internal validation of the prediction models was performed using the 1000-bootstrap resample. RESULTS: A total of 105 subjects with diagnoses ranging from normal mucosa through ESCC were included in this study. An increase in P16 methylation frequency was observed with increasing severity of esophageal lesions (p for trend <0.001). In the adjusted logistic regression models, P16 methylation in cytology specimens was positively associated with ESCC and ESCdys risk, whereas P16 methylation in biopsy specimens was only associated with a higher risk of developing ESCC. The predictive capacity of base model I (AUC, 0.816) for ESCC and HGIN was significantly increased by adding P16 methylation in cytology specimens (model III; AUC, 0.882; p = 0.043), but not P16 methylation in biopsy specimens (model II; AUC, 0.850; p = 0.225). Bootstrap validation showed optimism-corrected AUC of 0.789 for model I, 0.822 for model II, and 0.854 for model III. CONCLUSION: P16 methylation as a cytologic marker was associated with the ESCC development and has the potential for application in minimally invasive ESCC screening.

Evaluation of the Impact of Intratumoral Heterogeneity of Esophageal Cancer on Pathological Diagnosis and P16 Methylation and the Representativity of Endoscopic Biopsy.

BACKGROUND: P16 methylation is expected to be potential diagnostic and therapeutic targets for esophageal cancer (EC). The intratumoral heterogeneity (ITH) of EC has been mentioned but has not been quantitatively measured yet. We aimed to clarify the impact of ITH on pathological diagnosis and P16 methylation, and the concordance between endoscopic biopsy and the corresponding surgically resected tissue. METHODS: We designed a systematic sampling method (SSM) compared with a general sampling method (GSM) to obtain EC tumor tissue, tumor biopsy, and normal squamous epithelium biopsy. MethyLight assay was utilized to test P16 methylation. All specimens obtained by the SSM were pathologically diagnosed. RESULTS: A total of 81 cases were collected by the GSM, and 91.4% and 8.6% of them were esophageal squamous cell carcinomas (ESCCs) and esophageal adenocarcinomas (EADs), respectively. Nine SSM cases were 100.0% ESCCs. The positive rates of P16 methylation of the GSM tumor and normal tissues were 63.0% (51/81) and 32.1% (26/81), respectively. For SSM samples, tumor tissues were 100.0% (40/40) EC and 85.0% (34/40) P16 methylated; tumor biopsy was 64.4% (29/45) diagnosed of EC and 68.9% P16 methylated; the corresponding normal biopsies were 15.7% (8/51) dysplasia and 54.9% (28/51) P16 methylated. The concordance of pathological diagnosis and P16 methylation between tumor biopsy and the corresponding tumor tissue was 75.0% and 62.5%, respectively. CONCLUSION: The SSM we designed was efficient in measuring the ITH of EC. We found inadequate concordance between tumor biopsy and tissue in pathological diagnosis and P16 methylation.

CDKN2A Deletion Leading to Hematogenous Metastasis of Human Gastric Carcinoma.

INTRODUCTION: Somatic copy number deletion (SCND) of CDKN2A gene is the most frequent event in cancer genomes. Whether CDKN2A SCND drives human cancer metastasis is far from clear. Hematogenous metastasis is the main reason of human gastric carcinoma (GC) death. Thus, prediction GC metastasis is eagerly awaited. METHOD: GC patients (n=408) enrolled in both a cross-sectional and a prospective cohorts were analysed. CDKN2A SCND was detected with a quantitative PCR assay (P16-Light). Association of CDKN2A SCND and GC metastasis was evaluated. Effect of CDKN2A SCND by CRISPR/Cas9 on biological behaviors of cancer cells was also studied. RESULTS: CDKN2A SCND was detected in 38.9% of GCs from patients (n=234) enrolled in the cross-sectional cohort. Association analysis showed that more CDKN2A SCND was recognized in GCs with hematogenous metastasis than those without (66.7% vs. 35.7%, p=0.014). CDKN2A SCND was detected in 36.8% of baseline pN0M0 GCs from patients (n=174) enrolled in the prospective study, the relationship between CDKN2A SCND and hematogenous metastasis throughout the follow-up period (62.7 months in median) was also significant (66.7% vs. 34.6%, p=0.016). Using CDKN2A SCND as a biomarker for predicting hematogenous metastasis of GCs, the prediction sensitivity and specificity were 66.7% and 65.4%. The results of functional experiments indicated that CDKN2A SCND could obviously downregulate P53 expression that consequently inhibited the apoptosis of MGC803 GC and HEK293T cells. This may account for hematogenous metastasis of GCs by CDKN2A SCND. CONCLUSION: CDKN2A SCND may drive GC metastasis and could be used as a predictor for hematogenous metastasis of GCs.

Chidamide increases the sensitivity of Non-small Cell Lung Cancer to Crizotinib by decreasing c-MET mRNA methylation.

Introduction: Crizotinib is a kinase inhibitor targeting c-MET/ALK/ROS1 used as the first-line chemical for the treatment of non-small cell lung cancer (NSCLC) with ALK mutations. Although c-MET is frequently overexpressed in 35-72% of NSCLC, most NSCLCs are primarily resistant to crizotinib treatment. Method: A set of NSCLC cell lines were used to test the effect of chidamide on the primary crizotinib resistance in vitro and in vivo. Relationships between the synergistic effect of chidamide and c-MET expression and RNA methylation were systemically studied with a battery of molecular biological assays. Results: We found for the first time that chidamide could sensitize the effect of crizotinib in a set of ALK mutation-free NSCLC cell lines, especially those with high levels of c-MET expression. Notably, chidamide could not increase the sensitivity of NSCLC cells to crizotinib cultured in serum-free medium without hepatocyte growth factor (HGF; a c-MET ligand). In contrast, the addition of HGF into the serum-/HGF-free medium could restore the synergistic effect of chidamide. Moreover, the synergistic effect of chidamide could also be abolished either by treatment with c-MET antibody or siRNA-knockdown of c-MET expression. While cells with low or no c-MET expression were primarily resistant to chidamide-crizotinib cotreatment, enforced c-MET overexpression could increase the sensitivity of these cells to chidamide-crizotinib cotreatment. Furthermore, chidamide could decrease c-MET expression by inhibiting mRNA N6-methyladenosine (m6A) modification through the downregulation of METTL3 and WTAP expression. Chidamide-crizotinib cotreatment significantly suppressed the activity of c-MET downstream molecules. Conclusion: Chidamide downregulated c-MET expression by decreasing its mRNA m6A methylation, subsequently increasing the crizotinib sensitivity of NSCLC cells in a c-MET-/HGF-dependent manner.

RNA N-6-methyladenosine enzymes and resistance of cancer cells to chemotherapy and radiotherapy.

Aim: As one of the early adaptive mechanisms by which cells respond to environmental changes, RNA modification appears to be a very promising target for cancer treatment. Results: RNA modifications are currently a hot topic in epigenetic research. Emerging experimental studies show that expression alterations of multiple m6A enzymes, including demethylase FTO, methyltransferase METTL3 and WTAP, mediate the development of resistance of cancer cells to various treatments. A set of small molecular chemical drugs targeted to these m6A enzymes are under development. Intervention of RNA m6A methylation is a possible therapeutic strategy to overcome drug resistance. Conclusions: RNA m6A methylation may play a crucial role in drug resistance development and intervention in cancer cells.

Cross talk between oxidative stress and hypoxia via thioredoxin and HIF-2α drives metastasis of hepatocellular carcinoma.

Oxidative stress and hypoxia are two opposite microenvironments involved in HCC metastasis. Thioredoxin (TXN) and hypoxia-inducible factor 2α (HIF-2α) are typical proteins involved in these two different microenvironments, respectively. How these two factors interact to influence the fate on tumor cells remains unknown. Hypoxia facilitated HCC cells withstood oxidative stress and eventually promoted HCC cells metastasis, in which TXN and HIF-2α were mostly involved. Upregulation of TXN/HIF-2α correlated with poor HCC prognosis and promoted HCC metastasis both in vitro and in vivo. Epithelial-mesenchymal transition (EMT) process was involved in TXN/HIF-2α-enhanced invasiveness of HCC cells. Additionally, the stability and activity of HIF-2α were precisely regulated by TXN via SUMOylation and acetylation, which contributed to HCC metastasis. Our data revealed that the redox protein TXN and HIF-2α are both associated with HCC metastasis, and the fine regulation of TXN on HIF-2α contributes essentially during the process of metastasis. Our study provides new insight into the interaction mechanism between hypoxia and oxidative stress and implies potential therapeutic benefits by targeting both TXN and HIF-2α in the treatment of HCC metastasis.

Characterization of novel LncRNA P14AS as a protector of ANRIL through AUF1 binding in human cells.

BACKGROUND: The CDKN2A/B locus contains crucial tumor suppressors and a lncRNA gene ANRIL. However, the mechanisms that coordinately regulate their expression levels are not clear. METHODS: Novel RNAs transcribed from the CDKN2A gene were screened by CDKN2A-specific RNA capture deep-sequencing and confirmed by Northern blotting and clone-sequencing. Long non-coding RNA (lncRNA) binding proteins were characterized by RNA pull-down combined with mass spectrometry and RNA immunoprecipitation. LncRNA functions in human cells were studied using a set of biological assays in vitro and in vivo. RESULTS: We characterized a novel lncRNA, P14AS with its promoter in the antisense strand of the fragment near CDKN2A exon 1b in human cells. The mature P14AS is a three-exon linear cytoplasmic lncRNA (1043-nt), including an AU-rich element (ARE) in exon 1. P14AS decreases AUF1-ANRIL/P16 RNA interaction and then increases ANRIL/P16 expression by competitively binding to AUF1 P37 and P40 isoforms. Interestingly, P14AS significantly promoted the proliferation of cancer cells and tumor formation in NOD-SCID mice in a P16-independent pattern. Moreover, in human colon cancer tissues, the expression levels of P14AS and ANRIL lncRNAs were significantly upregulated compared with the paired normal tissues. CONCLUSION: A novel lncRNA, P14AS, transcribed from the antisense strand of the CDKN2A/P14 gene, promotes colon cancer development by cis upregulating the expression of oncogenic ANRIL.

Impact of GFRA1 gene reactivation by DNA demethylation on prognosis of patients with metastatic colon cancer.

BACKGROUND: The expression of the membrane receptor protein GFRA1 is frequently upregulated in many cancers, which can promote cancer development by activating the classic RET-RAS-ERK and RET-RAS-PI3K-AKT pathways. Several therapeutic anti-GFRA1 antibody-drug conjugates are under development. Demethylation (or hypomethylation) of GFRA1 CpG islands (dmGFRA1) is associated with increased gene expression and metastasis risk of gastric cancer. However, it is unknown whether dmGFRA1 affects the metastasis of other cancers, including colon cancer (CC). AIM: To study whether dmGFRA1 is a driver for CC metastasis and GFRA1 is a potential therapeutic target. METHODS: CC and paired surgical margin tissue samples from 144 inpatients and normal colon mucosal biopsies from 21 noncancer patients were included in this study. The methylation status of GFRA1 islands was determined by MethyLight and denaturing high-performance liquid chromatography and bisulfite-sequencing. Kaplan-Meier analysis was used to explore the effect of dmGFRA1 on the survival of CC patients. Impacts of GFRA1 on CC cell proliferation and migration were evaluated by a battery of biological assays in vitro and in vivo. The phosphorylation of AKT and ERK proteins was examined by Western blot analysis. RESULTS: The proportion of dmGFRA1 in CC, surgical margin, and normal colon tissues by MethyLight was 68.4%, 73.4%, and 35.9% (median; nonparametric test, P = 0.001 and < 0.001), respectively. Using the median value of dmGFRA1 peak area proportion as the cutoff, the proportion of dmGFRA1-high samples was much higher in poorly differentiated CC samples than in moderately or well-differentiated samples (92.3%% vs 55.8%, Chi-square test, P = 0.002) and significantly higher in CC samples with distant metastasis than in samples without (77.8% vs 46.0%, P = 0.021). The overall survival of patients with dmGFRA1-low CC was significantly longer than that of patients with dmGFRA1-high CC (adjusted hazard ratio = 0.49, 95% confidence interval: 0.24-0.98), especially for 89 CC patients with metastatic CC (adjusted hazard ratio = 0.41, 95% confidence interval: 0.18-0.91). These data were confirmed by the mining results from TCGA datasets. Furthermore, GFRA1 overexpression significantly promoted the proliferation/invasion of RKO and HCT116 cells and the growth of RKO cells in nude mice but did not affect their migration. GFRA1 overexpression markedly increased the phosphorylation levels of AKT and ERK proteins, two key molecules in two classic GFRA1 downstream pathways. CONCLUSION: GFRA1 expression is frequently reactivated by DNA demethylation in CC tissues and is significantly associated with a poor prognosis in patients with CC, especially those with metastatic CC. GFRA1 can promote the proliferation/growth of CC cells, probably by the activation of AKT and ERK pathways. GFRA1 might be a therapeutic target for CC patients, especially those with metastatic potential.

DNA hydroxymethylation increases the susceptibility of reactivation of methylated P16 alleles in cancer cells.

It is well established that 5-methylcytosine (5mC) in genomic DNA of mammalian cells can be oxidized into 5-hydroxymethylcytosine (5hmC) and other derivates by DNA dioxygenase TETs. While conversion of 5mC to 5hmC plays an important role in active DNA demethylation through further oxidation steps, a certain proportion of 5hmCs remain in the genome. Although 5hmCs contribute to the flexibility of chromatin and protect bivalent promoters from hypermethylation, the direct effect of 5hmCs on gene transcription is unknown. In this present study, we have engineered a zinc-finger protein-based P16-specific DNA dioxygenase (P16-TET) to induce P16 hydroxymethylation and demethylation in cancer cells. Our results demonstrate, for the first time, that although the hydroxymethylated P16 alleles retain transcriptionally inactive, hydroxymethylation could increase the susceptibility of reactivation of methylated P16 alleles.

P16 methylation increases the sensitivity of cancer cells to the CDK4/6 inhibitor palbociclib.

The P16 (CDKN2Aink4a) gene is an endogenous CDK4/6 inhibitor. Palbociclib (PD0332991) is an anti-CDK4/6 chemical for cancer treatment. P16 is most frequently inactivated by copy number deletion and DNA methylation in cancers. It is well known that cancer cells with P16 deletion are more sensitive to palbociclib than those without. However, whether P16 methylation is related to palbociclib sensitivity is not known. By analyzing public pharmacogenomic datasets, we found that the IC50 of palbociclib in cancer cell lines (n = 522) was positively correlated with both the P16 expression level and P16 gene copy number. Our experimental results further showed that cancer cell lines with P16 methylation were more sensitive to palbociclib than those without. To determine whether P16 methylation directly increased the sensitivity of cancer cells to palbociclib, we induced P16 methylation in the lung cancer cell lines H661 and HCC827 and the gastric cancer cell line BGC823 via an engineered P16-specific DNA methyltransferase (P16-Dnmt) and found that the sensitivity of these cells to palbociclib was significantly increased. The survival rate of P16-Dnmt cells was significantly lower than that of vector control cells 48 hrs post treatment with palbociclib (10 µM). Notably, palbociclib treatment also selectively inhibited the proliferation of the P16-methylated subpopulation of P16-Dnmt cells, further indicating that P16 methylation can increase the sensitivity of cells to this CDK4/6 inhibitor. These results were confirmed in an animal experiment. In conclusion, inactivation of the P16 gene by DNA methylation can increase the sensitivity of cancer cells to palbociclib.

A panel of DNA methylated markers predicts metastasis of pN0M0 gastric carcinoma: a prospective cohort study.

BACKGROUND: The aim of this prospective study was to evaluate the feasibility of predicting GC metastasis using CDH1, GFRA1, P16 and ZNF382 DNA methylation as biomarkers. METHODS: 198 GC patients without metastasis at the time of surgery resection were recruited into the double-blind cohort (NCT02159339). Gene methylation was analysed using MethyLight assays. GC metastasis and survival data were obtained from 178 patients with 94.7% compliance during follow-up. RESULTS: Twenty six cases of metastasis and 5 cases of recurrence were observed in 178 cases (17.4%) during the follow-up (median, 62.7 months). The GC metastasis rate for GFRA1 methylation-positive patients was significantly reduced compared with GFRA1 methylation-negative patients (odds ratio [OR]: 0.23, 95% confidence interval [CI] 0.08-0.66). Similar results were also observed using ZNF382 methylation as a predictor (OR: 0.17, 95% CI 0.06-0.47). A risk score including methylation of GFRA1 and ZNF382 was generated. The metastasis rate was significantly increased in high-risk GC patients (OR: 4.71, 95% CI: 1.85-12.00). GC patients with high risk had a shorter overall survival, especially for patients with stage I GC (P = 0.024). CONCLUSIONS: The combination of GFRA1 and ZNF382 methylation is a biomarker panel for the prediction of GC metastasis.