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.].

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.

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.

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.

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.

Coordinated transcription of ANRIL and P16 genes is silenced by P16 DNA methylation.

OBJECTIVE: To investigate the relationship between the transcription of ANRIL, P15, P14 and P16 at the same locus and the regulation mechanism of ANRIL. METHODS: Publicly available database of Cancer Cell Line Encyclopedia (CCLE) was used in bioinformatic analyses. Methylation of CpG islands was detected by denaturing high performance liquid chromatography (DHPLC). Gene transcript levels were determined using quantitative real-time polymerase chain reaction (qRT-PCR) assays. An engineered P16-specific transcription factor and DNA methyltransferase were used to induce P16-specific DNA demethylation and methylation. RESULTS: The expression level of ANRIL was positively and significantly correlated with that of P16 but not with that of P15 in the CCLE database. This was confirmed in human cell lines and patient colon tissue samples. In addition, ANRIL was significantly upregulated in colon cancer tissues. Transcription of ANRIL and P16 was observed only in cell lines in which the P16 alleles were unmethylated and not in cell lines with fully methylated P16 alleles. Notably, P16-specific methylation significantly decreased transcription of P16 and ANRIL in BGC823 and GES1 cells. In contrast, P16-specific demethylation re-activated transcription of ANRIL and P16 in H1299 cells (P<0.001). Alteration ofANRIL expression was not induced by P16 expression changes. CONCLUSIONS: ANRIL and P16 are coordinately transcribed in human cells and regulated by the methylation status of the P16 CpG islands around the transcription start site.

SRF promotes gastric cancer metastasis through stromal fibroblasts in an SDF1-CXCR4-dependent manner.

It has been suggested that the overexpression of serum response factor (SRF) in cancer cells may promote cancer metastasis. However, the exact pathway by which SRF promotes metastasis has not been clarified. Here we showed that SRF promotes gastric cancer (GC) metastasis through stromal fibroblasts in an SDF1-CXCR4-dependent manner. SRF expression was significantly increased in metastatic GCs compared with the non-metastatic GCs (n=50, p=0.013). Immuno-staining indicated that SRF was primarily expressed in a-smooth muscle actin (αSMA)-expressing periglandular fibroblasts in GCs. The conditioned medium (CM) from CCD18Co fibroblasts stably transfected with the SRF vector (CCD18Co-SRF) significantly enhanced migration of MKN45 gastric cancer cells. In contrast, the CM from CCD18Co fibroblasts, in which SRF was downregulated, inhibited mobility of MKN45 cells. Similar results were observed in cultured BGC823 cells even when they were treated with the NIH3T3-SRF CM. When MKN45 cells and SRF-upregulated or downregulated CCD18Co cells were simultaneously co-injected into the tail veins of NOD-SCID mice, a significant increase or decrease was, respectively, observed in the experimental pulmonary metastasis of cancer cells. Furthermore, SRF overexpression significantly upregulated `SMA and stromal cell derived factor1 (SDF1) expression in these fibroblasts, and an anti-SDF1 antibody or the SDF1 receptor CXCR4-specific inhibitor AMD3100 treatment completely reversed the SRF-enhanced migration of cancer cells. Quantitative RT-PCR demonstrated that the expression level of SRF was positively correlated with that of SDF1 in 92 GC samples (r=0.63, p<0.001). In conclusion, SRF promote GC metastasis by facilitating myofibroblast-cancer cell crosstalk in an SDF1-CXCR4 dependent manner, and maybe a therapeutic target.