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Epigenetic silencing of lncRNA MORT in 16 TCGA cancer types

We have previously described a hominid-specific long non-coding RNA, MORT (also known as ZNF667-AS1, Gene ID: 100128252), which is expressed in all normal cell types, but epigenetically silenced during cancer-associated immortalization of human mammary epithelial cells. Initial analysis of The Cancer Genome Atlas (TCGA) showed that 15 of 17 cancer types, which represent the 10 most common cancers in women and men, display DNA methylation associated MORT silencing in a large fraction of their tumors. In this study we analyzed MORT expression and DNA methylation state in the remaining 16 TCGA cancer types not previously reported. Seven of the 16 cancer types showed DNA methylation linked MORT silencing in a large fraction of their tumors. These are carcinomas (cervical cancer, and cancers of esophagus, stomach, and bile duct), and the non-epithelial tumors mesothelioma, sarcoma, and uterine carcinosarcoma. Together with the findings from our previous report, MORT expression is silenced by aberrant DNA methylation in 22 of 33 of TCGA cancer types. These 22 cancers include most carcinoma types, blood derived cancers and sarcomas. In conclusion, results suggest that the MORT gene is one of the most common epigenetic aberrations seen in human cancer. Coupled with the timing of MORT gene silencing during in vitro epithelial cell immortalization and its occurrence early in the temporal arc of human carcinogenesis, this provides strong circumstantial evidence for a tumor suppressor role for MORT.


Introduction
MORT was originally found as a transcript silenced during in vitro immortalization of human mammary epithelial cells 1 . Like a significant majority of lncRNAs, MORT's molecular function remains enigmatic. The MORT gene is specific to higher primates, is expressed in all normal human cell types, and MORT RNA is located predominantly in the cytoplasm 1 . Analysis of MORT expression and the DNA methylation state of its promoter in 17 cancer types from The Cancer Genome Atlas (TCGA) 2 , which represent the 10 most frequent cancers in males and females, showed MORT is epigenetically silenced in 15 of 17 these cancers 1 . Based on the data from the original in vitro study 1 , we predicted epigenetic MORT silencing occurs early in human carcinogenesis and therefore could be seen in premalignant lesions, such as ductal carcinoma in situ of the breast and colonic adenomas. We used data from clinical samples from published genomic data sets 3-8 to address this possibility, and indeed, MORT loss occurs prior to or at the stage of pre-malignancy and not thereafter 9 . Taken together these facts suggest that MORT transcript has a tumor suppressive role and is not simply an epigenetic "passenger error." Since our previous analysis of MORT in TCGA datasets was not exhaustive and only reported on 17 out of 33 TCGA cancer types, the goal of this short study was to extend our earlier work and complete the analysis of MORT DNA methylation associated gene silencing in the final 16 TCGA cancer types.

Methods
We integrated the MORT expression level and the DNA methylation state of its promoter region using TCGA data as described before 1 . The Illumina HiSeq RNA-seq and Human-Methylation450 DNA methylation data for samples of 16 TCGA cancer types listed in Table 1 were downloaded from the GDC data portal. The data were analyzed in the R programming environment, version 3.4.2 10 . The mean RNA-Seq rpkm values for the two exons constituting the MORT RNA were plotted against the mean DNA methylation beta value of the 7 CpGs from the MORT promoter region for the individual samples of each cancer type. The Spearman correlation coefficient rho between the MORT RNA level and the DNA methylation of MORT promoter was calculated using the function cor.test.

Results and discussion
Seven of sixteen analyzed cancer types (CESC, CHOL, ESCA, MESO, SARC, STAD, and UCS) show strong MORT silencing by DNA methylation (Figure 1). The negative correlation rho between MORT expression and DNA methylation in these cancers is below -0.5; the DNA methylation level in some tumor samples of these cancers exceeds 0.5 beta (> 50% DNA methylation), and a large fraction of the tumor samples in these cancer types have very low to no MORT expression level ( Figure 1). The correlation of MORT expression and promoter DNA methylation in the remaining nine cancer types is also negative; however, the maximum level of the DNA methylation  of MORT promoter in some of these cancers is either very low (UVM), or a very few tumor samples have MORT silenced (ACC, KICH, OV, and THYM), and some of the cancer types (GBM, LGG, PCPG, and TGCT) do not appear to display MORT gene silencing ( Figure 1).
The analysis presented shows DNA methylation associated MORT gene silencing in 7 of 16 TCGA cancer types. Compared to the 17 TCGA cancer types presented in our original study, 1 most the 16 cancer types presented here lack their respective normal tissues samples and some of them have lower amounts of tumor samples (Table 1). Nevertheless, the distribution of MORT expression and DNA methylation data in tumor samples clearly indicates MORT silencing in multiple cancer types ( Figure 1).
Cervical tumors (CESC) have high proportion of MORT silencing ( Figure 1); more than 75% of 304 cervical tumor samples have MORT promoter DNA hypermethylated and MORT silenced. Using TCGA data, a recent study found MORT downregulated in cervical cancer 11 , but surprisingly did not report on or hypothesize potential mechanisms for this transcriptional repression. Here we confirm and extend their initial analysis of MORT silencing in cervical cancer and show further that this silencing is strongly linked to aberrant DNA methylation of the MORT promoter.
Combined together with the findings from our previous report 1 , Table 2 shows MORT is silenced by DNA methylation in In summary, our findings show that the MORT gene is one of the most common epigenetic aberrations seen in human cancer. Coupled together with MORT silencing occurring early in the temporal arc of human carcinogenesis it strongly supports a tumor suppressive role for MORT.

Data availability
Illumina HiSeq RNA-seq and HumanMethylation450 DNA methylation data for TCGA cancer types used in the present study can be downloaded from the GDC data portal.

Competing interests
No competing interests were disclosed.

Grant information This work was supported by the Maynard Chair in Breast Cancer
Epigenomics at the University of Arizona Cancer Center and the Cancer Center Support Grant (P30 CA023074).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors utilize the TCGA database to extend their previous studies on MORT that has been primarily characterized in breast cancer, but also observed to undergo silencing in 15 out of the 17 most common cancers. The current work takes this analysis deeper into the 33 TCGA cancer types and perform a more thorough analysis of the DNA methylation associated with the 16 cancer types evaluated here.
SUGGESTIONS FOR IMPROVEMENT: -The authors make a bold statement that "the MORT gene is one of the most common epigenetic aberrations seen in human cancer". This statement is not supported by the data presented. In order to make a statement of this level, the authors would have to provide comparisons to other documented genes that undergo a high frequency of epigenetic alteration.
-This statement becomes more difficult to make when considering that several of the tumors present do not have normal counterparts for comparison. We therefore do not know if those tissue types whether the DNA methylation is abnormal or typical for the tissue type.
-Authors should consider revising some of the scales on the graphs in Figure 1 to highlight the lack of methylation (for example, in testicular germ cell tumors). Similarly, the cutoff for methylation could be shown by a dotted line or similar feature.
-The location of methylation relative to the MORT gene would be informative. Of the tumor types that are methylated, are the same regions methylated across types?
-The authors should be cautious not to overstate their conclusions throughout the text since the conclusions are speculative without experiments to support their statements. Nevertheless, these correlations present interesting speculation and avenues for further investigations.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly No competing interests were disclosed. Competing Interests: I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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