Lung cancer continues to increase in incidence every year and remains the most commonly diagnosed and leading cause of cancer-related deaths worldwide. Despite the understanding of the genetic alteration and advanced medical treatments such as the development of target therapy or immunotherapy, non-small cell lung cancer (NSCLC), which accounts for about 80% of lung cancers, has a poor long-term prognosis and a high mortality rate. Therefore, it is necessary to study the mechanisms of malignant transformation of NSCLC including non-genomic mechanisms. Similar to the epigenome, in which DNA and histones undergo modification, the epitranscriptome, in which RNA undergoes modification, has attracted much attention as a recent biological discovery. Of these, N6-methyladenosine (m⁶A), the methylation of RNA, has been the most studied ¹. At least 24 proteins are known to be involved in its modification ². Although these proteins have been reported to play important roles across various steps of human carcinogenesis, the involvement of m⁶A-related proteins in the malignant behavior of NSCLC pathogenesis has not been fully elucidated. In 2015, our laboratory started a study about the prognostic impact of m⁶A-related proteins and their molecular biological mechanisms in NSCLC. We have previously reported that m⁶A-reader proteins, YTHDF1 and YTHDF2, are associated with the tumor microenvironment of NSCLC and are favorable prognostic factors ³. Since our research members are respiratory physicians, we have often had to suspend research after mid-2020 because of the need to be on the front lines against the COVID-19 pandemic. We overcame these difficulties and were finally able to share our research results with the public. In the current article, we focused on the m⁶A demethylase ALKBH5 and FTO to analyze protein expression and prognosis, and to elucidate the mechanism of m⁶A-mediated malignant behavior of cancer.

First, we generated tissue microarrays of resected NSCLC specimens and quantified protein expression by immunostaining using ALKBH5 and FTO antibodies to analyze the relationship with prognosis (n = 607). The results showed that ALKBH5 was highly expressed in tumor sites, and high expression of ALKBH5 was associated with shorter survival. In addition, there was no significant association between FTO expression and prognosis. After adjusting for confounding factors, high expression of ALKBH5 was an independent prognostic factor associated with poor overall survival in NSCLC. Next, we analyzed cell proliferation, migration, cell cycle, and apoptosis in lung cancer cell lines with knockdown of ALKBH5 and FTO cells. Knockdown of ALKBH5 decreased the cell proliferation of PC9 and A549 cells, while knockdown of FTO did not decrease the proliferative ability. In addition, knockdown of ALKBH5 promoted G1 cell cycle arrest and increased the number of apoptotic cells. Furthermore, we analyzed the proliferative and migratory ability of immortalized airway epithelial cells overexpressing ALKBH5 and found that overexpression of ALKBH5 increased the cell proliferation of immortalized cell lines. The quantitative changes of m⁶A depending on the expression of ALKBH5 were also evaluated by liquid chromatography-mass spectrometry. Consequently, the amount of m⁶A changed in an ALKBH5 expression dependent. From these results, it was derived that ALKBH5, which is one of the two m⁶A demethylases and decreases m⁶A modification, is associated with shorter survival, and promotes cell proliferation. In other words, at this point, ALKBH5 was suggested to be associated with the malignant transformation of cancer.

Next, we performed comprehensive differential gene expression analysis using microarrays in ALKBH5 knockdown -PC9 cells. Two types of siRNAs were used for the knockdown, and 129 differentially expressed genes common to both types of siRNAs were identified. Target genes were narrowed down to 10 genes in the literature, and then further narrowed down to 6 genes by quantitative polymerase chain reaction (qPCR). Among these six genes, CDKN1A, TIMP3, E2F1, and CCNG2 were identified as potential target genes of ALKBH5 based on the results of qPCR of MeRIP products. Among these genes, increased protein expression of CDKN1A (p21) and TIMP3 was observed upon knockdown of ALKBH5 in lung cancer cells. We also knocked down these target genes such as CDKN1A (p21) and TIMP3. Consequently, the decrease in cell proliferation observed with knockdown of ALKBH5 was partially rescued by knockdown of CDKN1A and TIMP3. These results identify CDKN1A or TIMP3 as important downstream of ALKBH5 that are involved in the cell proliferation via m⁶A.

Since the expression of CDKN1A and TIMP3, which we identified as target genes of ALKBH5, is reduced by knockdown of ALKBH5, we hypothesized that the reader protein targeting these target transcripts should have a function to enhance RNA stability. To test this hypothesis, we examined whether knockdown of ALKBH5 increases the stability of CDKN1A and TIMP3 and whether knockdown of IGF2BP1, IGF2BP2, and IGF2BP3 (IGF2BPs), which have been reported to increase RNA stability, decreases the stability of CDKN1A and TIMP3. At least one of the IGF2BPs reduced the stability of mRNA in ALKBH5 knockdown cells. In addition, the increased expression of CDKN1A and TIMP3 and decreased cell proliferation ability seen with knockdown of ALKBH5 was offset by knockdown of both ALKBH5 and one of the IGF2BPs. We conclude that high expression of ALKBH5 in NSCLC reduces m⁶A modification of CDKN1A and TIMP3, and the reduction of m⁶A decreases the opportunity for recognition of m⁶A by IGF2BPs, destabilizing target transcripts such as CDKN1A (p21) and TIMP3.

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2. Zhou Z, Lv J, Yu H, et al. Mechanism of RNA modification N6-methyladenosine in human cancer. Mol Cancer. 2020;19(1):104.
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