Is Hydrogen Sulfide a Concern During Treatment of Lung Adenocarcinoma With Ammonium Tetrathiomolybdate?

Ammonium tetrathiomolybdate (ATTM) has been used in breast cancer therapy for copper chelation, as elevated copper promotes tumor growth. ATTM is also an identified H2S donor and endogenous H2S facilitates VitB12-induced S-adenosylmethionine (SAM) generation, which have been confirmed in m6A methylation and lung cancer development. The m6A modification was recently shown to participate in lung adenocarcinoma (LUAD) progression. These conflicting analyses of ATTM's anticancer vs. H2S's carcinogenesis suggest that H2S should not be ignored during LUAD's treatment with ATTM. This study was aimed to explore ATTM's effects on LUAD cells and mechanisms associated with H2S and m6A. It was found that treatment with ATTM inhibited cell growth at high concentrations, while enhanced cell growth at low concentrations in three LUAD cell lines (A549, HCC827, and PC9). However, another copper chelator triethylenetetramine, without H2S releasing activity, was not found to induce cell growth. Low ATTM concentrations also elevated m6A content in A549 cells. Analysis of differentially expressed genes in TCGA cohort indicated that m6A writer METTL3 and reader YTHDF1 were upregulated while eraser FTO was downregulated in LUAD tissues, consistent with the findings of protein expression in patient tissues. ATTM treatment of A549 cells significantly increased METTL3/14 and YTHDF1 while decreased FTO expression. Furthermore, inhibition of m6A with shMETTL3 RNA significantly attenuated eukaryotic translation initiation factor (eIF) expressions in A549 cells. Correlation analysis indicated that small nuclear ribonucleic protein PRPF6 was positively expressed with YTHDF1 in LUAD tissues. Knockdown of YTHDF1 partially blocked both basal and ATTM-induced PRPF6 expression, as well as A549 cell growth. Lastly, ATTM treatment not only raised intracellular H2S content but also upregulated H2S-producing enzymes. Exogenous H2S application mimicked ATTM's aforementioned effects, but the effects could be weakened by zinc-induced H2S scavenging. Collectively, H2S impedes ATTM-induced anticancer effects through YTHDF1-dependent PRPF6 m6A methylation in lung adenocarcinoma cells.

Ammonium tetrathiomolybdate (ATTM) has been used in breast cancer therapy for copper chelation, as elevated copper promotes tumor growth. ATTM is also an identified H 2 S donor and endogenous H 2 S facilitates VitB 12 -induced S-adenosylmethionine (SAM) generation, which have been confirmed in m 6 A methylation and lung cancer development. The m 6 A modification was recently shown to participate in lung adenocarcinoma (LUAD) progression. These conflicting analyses of ATTM's anticancer vs. H 2 S's carcinogenesis suggest that H 2 S should not be ignored during LUAD's treatment with ATTM. This study was aimed to explore ATTM's effects on LUAD cells and mechanisms associated with H 2 S and m 6 A. It was found that treatment with ATTM inhibited cell growth at high concentrations, while enhanced cell growth at low concentrations in three LUAD cell lines (A549, HCC827, and PC9). However, another copper chelator triethylenetetramine, without H 2 S releasing activity, was not found to induce cell growth. Low ATTM concentrations also elevated m 6 A content in A549 cells. Analysis of differentially expressed genes in TCGA cohort indicated that m 6 A writer METTL3 and reader YTHDF1 were upregulated while eraser FTO was downregulated in LUAD tissues, consistent with the findings of protein expression in patient tissues. ATTM treatment of A549 cells significantly increased METTL3/14 and YTHDF1 while decreased FTO expression. Furthermore, inhibition of m 6 A with shMETTL3 RNA significantly attenuated eukaryotic translation initiation factor (eIF) expressions in A549 cells. Correlation analysis indicated that small nuclear ribonucleic protein PRPF6 was positively expressed with YTHDF1 in LUAD tissues. Knockdown of YTHDF1 partially blocked both basal and ATTM-induced PRPF6 expression, as well as A549 cell growth. Lastly, ATTM treatment not only raised intracellular H 2 S content but also upregulated H 2 S-producing enzymes. Exogenous H 2 S application mimicked INTRODUCTION Ammonium tetrathiomolybdate (ATTM), with the formula (NH 4 ) 2 MoS 4 , is a strong copper chelator. It has been clinically used in the treatment of copper toxicosis for Wilson's disease. At high concentrations, copper is also known to promote angiogenesis, metabolism and oxidative phosphorylation, thereby leading to tumor growth (1)(2)(3). Consequently, copperchelating agents, like ATTM and triethylenetetramine (TETA), have been investigated in the treatment of cancers, including breast cancer, thyroid cancer and liver cancer (4)(5)(6)(7)(8). Additionally, copper-dependent enzyme, superoxide dismutase (SOD) 1, has been reported to facilitate lung adenocarcinoma (LUAD) development (9). Therefore, ATTM therapy may theoretically be extended to LUAD.
Interestingly, our lab and others have recently discovered ATTM is a pH-dependent hydrogen sulfide (H 2 S) donor (10,11). There is no doubt that the development of ATTM as a therapeutic candidate must consider ATTM being both a copper chelator and a H 2 S releaser. It should be noted that the links between H 2 S and cancer development is still under debate (12). Some studies showed that elevated H 2 S can induce angiogenesis and tumor cell proliferation, thereby contributing to cancer development (13)(14)(15). H 2 S was also involved in VitB 12 -induced S-adenosylmethionine (SAM) generation (16), and high VitB 12 has been found to raise the risk of lung cancer (17). Notably, in lung cancer tissues, endogenous H 2 S and its producing enzymes, like cystathionine beta-synthase (CBS), cystathionine gamma lyase (CSE, also known as CTH) and 3-mercaptopyruvate sulfurtransferase (3-MST), are highly expressed thereby benefiting cancer development (18,19). N 6 -Methyladenosine (m 6 A) methylation is one major type of mRNA modification, dynamically modulated by the corresponding writers, erasers and readers (20). The m 6 A methylation can differentially influence all fundamental aspects of mRNA metabolisms, including splicing, stability, and translation efficiency, when read by different m 6 A readers (21)(22)(23). Recently, the m 6 A methylation has been implicated in cancer pathogenesis, due to its induction of cell proliferation, invasion and immune disorders (24)(25)(26)(27)(28). Analysis of the cancer genome atlas (TCGA) cohort and recent studies (29,30) indicated that m 6 A writer, methyltransferase like (METTL) 3 is upregulated in LUAD. The eraser, fat mass and obesity-associated gene (FTO), is downregulated. Both increased METTL3 and decreased FTO strongly suggest that LUAD tissues exhibit high m 6 A levels. However, no direct evidence has shown H 2 S can regulate m 6 A methylation, although H 2 S enhances SAM generation (16), a cofactor of METTL3/14 complex. We therefore hypothesize that m 6 A methylation is likely to be affected by H 2 S derived from ATTM and participate in LUAD development.
In the present study, we observed the effects of ATTM on lung adenocarcinoma cells and explored the roles of m 6 A in this process. Furthermore, we assessed the medication of H 2 S in ATTM-induced tumor cell proliferation, invasion and growth. Lastly, a purposive strategy was developed to overcome ATTM's side-effects in LUAD therapy. Cell Culture, Growth, Proliferation, and Invasion Assays Lung adenocarcinoma cell lines (A549 and HCC827) were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China), and PC9 was obtained from ATCC. The cells were maintained in RPMI-1640 medium supplemented with 10% FBS at 37 • C under an atmosphere of 5% CO 2 and 95% air. They were passaged and harvested with 0.25% trypsin every other day.

Materials
Cell number was measured with Cell Counting Kit (CCK)-8 provided by Dojindo Lab (Kyushu, Japan). A549, HCC827, and PC9 cells were plated in 96-well plates at a density of 7, 000 cells/well. When grown to ∼60-70% confluence, the cells were treated correspondingly. After the treatments, the CCK-8 solution (100 µL) at a 1:10 dilution with FBS-free medium was added to each well-followed by a 2-h incubation at 37 • C. Absorbance (A) was measured at 450 nm with a microplate reader (Molecular Devices, US).
Cell proliferation was tested with BeyoClick TM 5-Ethynyl-2 ′deoxyuridine (EdU) kit (Haimen, China). After the treatment of A549 cells with ATTM for 48 h, EdU incorporation assay was performed according to the manufacturer's instructions. TMB-derived color was measured at 630 nm with the microplate reader.
Cell invasion was observed with Transwell Migration Assay, as described (31) with modifications. RPMI-1640 medium (1% FBS) containing ATTM or Na 2 S in the absence or presence of Zn(OAc) 2 was added to the lower chambers of the 12-well format transwells (8 µm-pore, BD Biosciences). A549 cells were seeded in the upper chambers at 10 5 cells per well, following a 48 h-culture. After that, the transwells were fixed in methanol, and stained with Giemsa solution. The unmigrated cells were removed from the top of the membranes using cotton swabs. To quantify the number of migrated cells in the bottom of the membrane, four random images of each group were taken at 10× under a light microscope. Migrated cell number was counted with Image J software.

Quantification of m 6 A RNA Methylation
After treatments of A549 cells with increasing concentrations of ATTM for 24 h, total RNA was extracted using TaKaRa MiniBest kits (Kusatsu, Japan) and quantitated with NanoDrop 1000 spectrophotometer (Thermo Fisher, US). The m 6 A RNA was detected with EpiQuik m 6 A RNA Methylation Colorimetric kit (Farmingdale, US). Briefly, 200 ng of fresh extracted RNA sample was added into strip wells with RNA high binding solution, and incubated for 90 min at 37 • C. After three washes, capture antibody, detection antibody, and enhancer solution were applied in turn, and incubated for 1 h at 37 • C. After washes, color developing solution was added and incubated for 6 min in the dark. When the solution became blue in the m 6 A positive wells, stop solution was added to turn the color into yellow. Lastly, the absorbance of stable yellow was measured at 450 nm with the microplate reader.

Western Blotting for Protein Expression
After exposed to 60 µM ATTM for 24 h, A549 cells were collected and split at 4 • C. Total proteins in the lysate were quantitated with a BCA kit. Thirty micrograms of total protein sample were loaded in SDS-PAGE and electrophoresed. At the end of electrophoresis, the proteins were transferred to PVDF membranes. The membranes were blocked with 5% fat-free milk in Tris-base buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature, and then incubated with the primary antibodies against m 6 A related proteins (METTL3, METTL14, FTO, and YTHDDF1), and H 2 S-producing enzymes (CSE, CBS, and MPST), respectively, with gentle agitation overnight at 4 • C. After three washes, corresponding HRP-conjugated secondary antibodies were applied and incubated for 1.5 h at room temperature. The signal was visualized using an enhanced chemiluminescence detection system. The intensity of bands was quantified with Image J software.

Analysis of TCGA Database and Measurement of Protein Expression of LUAD Patients
Transcriptional expressions of m 6 A or H 2 S related genes in primary tumor tissues and adjacent tissues of LUAD patients, as well as correlation analysis in tumor tissues, were performed through TCGA cohort research tool (UALCAN). Furthermore, the protein expressions of METTLE3, METTL14, and FTO in LUAD patients were verified with Western blotting assay as above.

Gene Knockdown
Gene expression microarray data were downloaded at https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE76367 (29). METTL3 was knocked down through short hairpin (sh) RNA in A549 cells, and gene expression was profiled by high throughput sequencing. Selected genes of eukaryotic translation initiation factors (eIFs) were shown as the maximum vs. minimum of the ratio shMETTL3 to shGFP.
Small interfering RNA (siRNA) against human YTHDF1 (Gene ID: 54915) was synthesized by GenePharma Co., Ltd (Shanghai, China). YTHDF1 siRNA and control random nontargeting siRNA were transfected into the A549 cells using Lipofectamine 2000 (Invitrogen, USA) (32). To raise the transfection efficiency, the cells were incubated with 20 nM YTHDF1 siRNA or Control siRNA for 6 h followed by a 24-h culture. The silencing ability was evaluated by Western blotting assay.

Quantitative Polymerase Chain Reaction for PRPF6 Gene Expression
Total RNAs were extracted from A549 cells and quantitated as above. First-strand cDNA was synthesized using TaqMan SYBR R Premix Ex TaqTM II (Tli RNase H Plus) virus reverse transcriptase and 2 µg RNA template in 20 µL reaction volume. The cDNA was used for real-time PCR with Prime Script RT reagent kit with gDNA Eraser (Life Technologies, US). Actin was used to normalize the expression of PRPF6. The results were analyzed using the comparative Ct method (2-Ct with logarithmic transformation). Primer sequences were displayed as below: PRPF6 forward 5-GTCATGCGTGCCGT GATTG-3 and reverse 5-TCCAGGGCATTGTGGGCTA-3, Actin forward 5-TGGCACCCAGCACAATGAA-3 and reverse 5-CTAAGTCATAGTCCGCCTAGAAGCA-3. PCRs were carried out as follows: initial denaturation at 95 • C for 30 s, 40 cycles of 95 • C for 5 s, 60 • C for 34 s, and 95 • C for 15 s, 60 • C for 1 h, followed by a final extension at 95 • C for 15 s.

Measurement of H 2 S Levels
ATTM-mediated H 2 S generation in cells was determined with a H 2 S fluorescent probe WSP-5 (33). A549 cells were inoculated in 24-well plates and grown to 60∼70% confluence. After treated with ATTM, the cells were incubated with 10 µM WSP-5 in 1% FBS medium at 37 • C for 30 min in the dark. Cell imaging was carried out after a slight wash with PBS. The intracellular H 2 Striggered fluorescence was visualized under AMG fluorescence microscopy (Advanced Microscopy Group, US).
For H 2 S scavenging induced by Zn(OAc) 2 , CuSO 4 , FeCl 3 or VitB 12 was observed through a Unisense H 2 S micro-sensor (Tueager 1, Denmark) (34). Into 5 mL PBS buffer, fresh Na 2 S stock solution was added to produce a 100 µM Na 2 S solution. When the curve reached the peak and kept stable, the same dose of above compounds was immediately added, respectively. The curves were recorded correspondingly with the H 2 S sensor. For Zn(OAc) 2 or CuSO 4 -induced continuous H 2 S scavenging from ATTM solution was recorded for 6 h. Into 20 mL of 500 µM ATTM solution (pH 5), fresh Zn(OAc) 2 or CuSO 4 stock solutions was added, respectively, to produce a 250 µM solution. Real-time H 2 S content was monitored with the H 2 S sensor and quantified against a standard curve.

Statistical Analysis
The experiment data are presented as means ± standard deviation (SD). Significance between groups was evaluated by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test using GraphPad Prism 8 software (San Diego, US). A probability <0.05 was considered statistically significant.

Low ATTM Levels Enhanced Growth of Lung Adenocarcinoma Cells
As shown in Figure 1A, treatment with high concentrations (≥250 µM) of ATTM for 48 h remarkably reduced cell number, in three types of lung adenocarcinoma cells (A549, HCC827, and PC9). However, at low concentrations (from 60 to 125 µM), the treatment distinctively increased cell number. Notably, another copper chelator TETA, without H 2 S releasing activity, was not found to elevate cell number at the same treatment profile ( Figure 1B). Furthermore, 60-125 µM of ATTM treatment could also induce A549 cell proliferation as evaluated by EdU assay (Figure 1C). The result indicates that low ATTM concentrations are able to promote lung adenocarcinoma growth.

ATTM Induced mRNA m 6 A Methylation in Lung Adenocarcinoma A549 Cells
To understand why ATTM enhanced lung adenocarcinoma growth, A549 cells were selected as a representative in the following experiments. Since mRNA m 6 A methylation is involved in a variety of cancer growth including lung adenocarcinoma, intracellular m 6 A mRNA level was then investigated. As shown in Figure 2A, the m 6 A mRNA content was significantly elevated after the exposure of A549 cells to 60 µM ATTM for 24 h. However, the treatment duration (half of 48 h) did not alter cell number (Figure 2B) or growth status ( Figure 2C). Notably, analysis of TCGA cohort shows that LUAD condition significantly upregulates the m 6 A writer METTL3, while downregulates the m 6 A eraser FTO (Figure 2D). The result indicates that ATTM can trigger mRNA m 6 A methylation before cell growth in lung adenocarcinoma cells.

Upregulated m 6 A Methylation Was Involved in LUAD Progression and ATTM-Induced mRNA Translation in Lung Adenocarcinoma A549 Cells
To uncover the mechanisms underlying the increased m 6 A mRNA levels in ATTM-treated cells, m 6 A related proteins were detected with Western blot. As shown in Figures 3A,C, comparing with the adjacent tissues, the writer (METTL3 and METLL14) expressions were upregulated in tumor tissues of LUAD patients, while the eraser FTO expression was downregulated. Importantly, the writer (METTL3 and METLL14) expressions in A549 cells could also be upregulated, while the eraser FTO could be downregulated under ATTM treatment (Figures 3B,D), supporting the finding of the increased intracellular m 6 A content. To confirm the roles of m 6 A methylation, we investigated gene expression profile in METTL3 knockdown A549 cells. As shown in Figure 3E, shRNA-mediated METTL3 knockdown significantly inhibited the expressions of translation initiation factors, including eIFs (2B3, 3B, 3C/CL, 3D, 3IP1, 4A1, and 5/5A). This suggests that m 6 A methylation is necessary to translation process in A549 cells, and that the elevated m 6 A levels may be important for LUAD progression and ATTM-induced tumor growth.

YTHDF1 Mediated ATTM-Induced Growth in Lung Adenocarcinoma A549 Cells
Although the increased m 6 A writer METTL3/14 and the decreased eraser FTO can result in the enhancement of m 6 A content, it is the m 6 A readers that directly determine the outcome of m 6 A methylated mRNA. With TCGA cohort, we examined the four common m 6 A readers (YTHDF1, YTHDF2, YTHDF3, and YTHDC1) in LUAD tumor samples and normal samples, and found YTHDF1 highly expressed in tumor tissues ( Figure 4A). The increased YTHDF1 continuously expressed within various stages of LUAD ( Figure 4B). Importantly, it was found that treatment of A549 cells with 60 µM ATTM for 24 h remarkably upregulated YTHDF1 protein expression (Figure 4C). After knockdown of YTHDF1 with siRNA in A549 cells (Figure 4D), both basal and ATTM-triggered cell growth were attenuated ( Figure 4E).

YTHDF1-Mediated Cell Growth Was Associated With PRPF6 Induction in ATTM-Treated A549 Cells
To explore targets involved in YTHDF1-mediated A549 cell growth, we screened genes that are positively correlated with YTHDF1 in TCGA LUAD cohort. As shown in Figure 5A, PRPF6 was found to be positively expressed with YTHDF1 in LUAD tissues (r = 0.72). Similar to YTHDF1's expression profile, the increased PRPF6 expression lasted various stages of LUAD ( Figure 5B). Furthermore, qPCR analysis showed that PRPF6 mRNA level was significantly reduced after YTHDF1 knockdown in A549 cells. Additionally, the knockdown attenuated ATTMinduced PRPF6 mRNA expression ( Figure 5C). The result reveals that PRPF6 may be a potential target gene involved in YTHDF1-mediated A549 cell growth.

H 2 S Was Significant to ATTM-Induced A549 Cell Growth and m 6 A Methylation
ATTM was previously demonstrated to generate H 2 S on acid conditions in our lab. As shown in Figure 6A, the exposure of A549 cells to ATTM markedly raised intracellular H 2 S levels. With TCGA cohort, we studied endogenous H 2 S synthetase expressions and, found that CSE/CTH, CBS and MPST expressions were enhanced in tumor tissues comparing with normal samples (Figure 6B). Additionally, the exposure of A549 cells to 60 µM ATTM for 24 h obviously upregulated CBS and MPST expressions (Figures 6D,E), however, remarkable change of CSE was not found ( Figure 6C). Notably, direct H 2 S donation (Na 2 S) could also induce m 6 A methylation (Figure 6F), as well as cell growth (Figure 6G), proliferation ( Figure 6H) and invasion (Figures 6I,J), indicating H 2 S being an effector in ATTM-induced m 6 A methylation and cell growth.

H 2 S Scavenging Attenuated ATTM-Induced A549 Cell Growth and m 6 A Methylation
Since the enhanced H 2 S generation was involved in ATTMinduced A549 cell growth and m 6 A methylation, scavenging H 2 S might overcome these side-effects of ATTM. Through testing several common H 2 S scavengers, it was found that Zn(OAc) 2 and CuSO 4 had powerful ability to remove H 2 S, while the ability of VitB 12 and FeCl 3 was weak or even undetectable ( Figure 7A). Additional cell viability examination showed that both Zn(OAc) 2 and CuSO 4 were non-toxic at concentrations <25 µM (Figures 7B,C).
To make sure H 2 S is necessary to ATTM-induced biological process in A549 cells, we observed the scavenging effect of Zn(OAc) 2 or CuSO 4 on ATTM-induced H 2 S release within a period of 6 h. As shown in Figure 8A, adding 25 µM Zn(OAc) 2 or CuSO 4 time-dependently attenuated ATTM-induced H 2 S release. Notably, application of 25 µM Zn(OAc) 2 inhibited ATTM-induced cell growth (Figure 8B), proliferation ( Figure 6H) and invasion (Figures 6I,J). More importantly, Zn(OAc) 2 was also able to attenuate ATTMinduced m 6 A methylation ( Figure 8C). Collectively, the result suggests that H 2 S is necessary and sufficient to ATTM-induced biological changes, and scavenging H 2 S can potentially overcome ATTM's side-effects in lung cancer treatment.

DISCUSSION
In the present study, ATTM was found to promote cell growth, proliferation and invasion in lung adenocarcinoma cells, through YTHDF1-dependent PRPF6 m 6 A methylation. H 2 S was involved in the above effects of ATTM. Importantly, scavenging H 2 S was proved to overcome the side-effects of ATTM in lung cancer therapy.
Copper ions are known to essentially maintain organism functions by regulating activity of key enzymes, like cytochrome C oxidase and SOD. However, its aberrant increase in plasma usually leads to pathological consequences, including Wilson's disease and cancers. During cancer development, high contents of copper are believed to enhance angiogenesis and blood supply, as well as activity of mitochondrial cytochrome C oxidase, thereby promoting solid tumor growth and metastasis (1)(2)(3)35).  Consequently, chelating agents of copper ions, like ATTM and TETA, have become promising anticancer drugs. The clinical trials of ATTM in breast cancer have recently made great progress (4)(5)(6). Notably, SOD1, a copper-dependent enzyme, was reported to participate in lung cancer growth and has become a significant therapeutic target (9). Therefore, ATTM may exert anticancer effects in lung adenocarcinoma through reduction of copper ions. In this study, at high concentrations, ATTM could indeed inhibit the growth of lung adenocarcinoma cells. However, at low concentrations, ATTM distinctively promoted tumor cell growth, as well as proliferation and invasion. The result was different from the reported effects of ATTM in breast cancer (6) and BRAF-driven papillary thyroid cancer (8), which suggests that the findings in other cancers should not be simply transplanted to lung cancer.
To uncover the mechanisms underlying ATTM-induced LUAD cell growth at low concentrations, we investigated mRNA m 6 A methylation, which has been involved in different cancer growth, like liver cancer (25), endometrial cancer (26), and leukemia (36). The m 6 A writer METTL3 has also been reported to promote lung cancer survival, growth, and invasion (29). We therefore speculated that the aberrant m 6 A methylation was involved in ATTM-induced growth of LUAD cells. By measuring m 6 A content in A549 cells, we found that the treatment with ATTM for 24 h significantly enhanced intracellular m 6 A content, while it did not significantly alter cell growth, suggesting m 6 A methylation occurs earlier than cell growth. Generally, m 6 A methylated mRNA can be synthesized via methyltransferase complex m 6 A writer, mainly consisting of METTL3, METTL14, and WTAP. Meanwhile, CH 3 -can also be erased from the RNA using FTO and ALKBH5. Therefore, the process is dynamic and reversible (37). The increased METTL3 and decreased FTO in the present TCGA analysis indicate that the dysregulated writer and/or eraser may be responsible for the increased m 6 A content in lung adenocarcinoma cells. This finding was supported by protein measurement in LUAD patients. Importantly, we found that the treatment with ATTM significantly augmented METTL3, but reduced FTO protein expression. ATTM could also upregulate METTL14 expression, which was not consistent with the TCGA analysis. We hypothesize that this difference may be due to the uncertainty of gene expression between mRNA level and protein level. Notably, for the increased METTL3 and decreased FTO, the present experiment matched the TCGA analysis. Such unique expressions, we believe, contribute to ATTM-induced m 6 A increase in A549 cells.
However, the roles of m 6 A in cancer biology are complicated and conflicting, i.e., tumor growth or anti-tumor (38). In addition, the binding of target mRNA with the writers or erasers is usually instantaneous. Therefore, it may be significant to examine the roles of m 6 A readers. To date, YT521-B homology (YTH) domain family of proteins, like YTHDF1, YTHDF2, YTHDF3, and YTHDC1, have been identified as m 6 A readers. YTHDF1-mediated mRNA spicing can increase translation efficiency, whereas YTHDF2-mediated mRNA decay will inhibit gene expression (21). The TCGA analysis indicates that the expression of YTHDF1, instead of YTHDF2/3 or YTHDC1, was markedly increased in LUAD tissues. In A549 cells, the elevated m 6 A mainly induced gene translation, evidenced by shMETTL3mediated eIFs' downregulation (29). Western blot test showed that YTHDF1 expression could be upregulated by ATTM. Significantly, YTHDF1 siRNA inhibited basal and ATTMinduced cell growth, confirming the involvement of YTHDF1 in basal and environment-stimulated lung tumorigenesis.
As an m 6 A reader, it is necessary to discover YTHDF1's target genes. The TCGA analysis shows that PRPF6 (a small nuclear ribonucleic protein) is positively expressed with YTHDF1. The GEO analysis shows that PRPF6 can be attenuated through METTL3 knockdown (shGFP 1023 vs. shMETTL3 848). Additional analysis of MeT V2.0 m 6 A database indicates that m 6 A PRPF6 mRNA can be discerned and read by YTHDF1 in Hela cells via GRAC motif (R is G or A) (21). Actually, PRPF6 has been reported to promote lung cancer growth (39,40). With these bioinformatics analyses and reports, PRPF6 is probably a target gene of YTHDF1. Importantly, the present experiment showed that ATTM treatment not only induced YTHDF1 expression, but also enhanced PRPF6 mRNA abundance. Both basal and ATTMinduced PRPF6 upregulation was significantly reduced by YTHDF1 knockdown, which was supported by previous reports (39,40). As documented, the increased PRPF6 can alter the constitutive and alternative splicing of ZAK kinase, thereby activating cancer-related pathways, like AP-1, ERK, and JNK (41). In sum, it is believed that ATTM treatment upregulated METTL3/14 and downregulated FTO, raising intracellular m 6 A mRNA like PRPF6. Furthermore, these specific mRNAs were read by YTHDF1, and the spicing and translation of cancer growth-related genes were induced, thereby promoting LUAD tumor growth.
Significantly, we dissected the cause of ATTM-induced m 6 A methylation and cell growth. Copper chelating can exert anticancer effects through inhibiting angiogenesis (4,6,8). However, apart from copper chelating, ATTM can release H 2 S (10, 11). Studies have shown that the homeostasis of H 2 S is essential in organisms (42)(43)(44)(45)(46). In this study, we found that intracellular H 2 S level was markedly raised after ATTM treatment. Nevertheless, another copper chelator without H 2 S releasing ability, TETA, did not alter tumor cell growth. Direct H 2 S donation exerted similar effects to ATTM. The effects of ATTM or H 2 S could be abolished by CuSO 4 or Zn(OAc) 2 . Besides H 2 S releasing, we found ATTM induced the expressions of H 2 S-production enzymes, which have been reported to participate in survival and chemoresistance of LUAD cells (18). Therefore, H 2 S is significant and necessary for ATTM-induced m 6 A methylation and lung cancer growth.
In healthy individuals, plasma free copper ion content is <20 µM (47), but for cancer patients, the recommended daily ATTM dosage was 90 mg (48). Therefore, the initial plasma ATTM should be much higher than that of free copper ions, so the free ATTM will release H 2 S during the clinical application (11). Of course, zinc application maybe overcomes ATTM's side-effects in LUAD therapy, while copper should not be recommended because of its tumorigenesis risk.
Interestingly, during the examination of VitB 12 roles, it was found that VitB 12 enhanced ATTM-induced LUAD cell growth (Supplementary Figure 1), unlike zinc or copper's inhibitory effects. However, both our study and previous reports suggest VitB 12 is a H 2 S scavenger (34,49). In fact, it has been documented that high VitB 12 can raise the risk of lung cancer (17). H 2 S can also facilitate VitB 12 -induced SAM generation (16) that is the first substrate of METTTL3/14 complex. These findings further support H 2 S-induced m 6 A methylation promotes LUAD tumor growth.
In conclusion, the present study demonstrated that ATTM can induce m 6 A methylation through upregulation of METTL3 and downregulation of FTO in LUAD cells. YTHDF1-mediated PRPF6 expression is probably a pivotal reason. The effects of ATTM are closely associated with H 2 S generation (Figure 9). For the first time, this work reveals a potential risk and mechanism for ATTM application in LUAD treatment. Meanwhile, this is the first report on the roles of H 2 S in m 6 A methylation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Medical Ethics Committee of Guangzhou Medical University. The patients/participants provided their written informed consent to participate in this study.