The incidence and mortality rate of gastric cancer are among the highest in the world, and the mainland is a major country in stomach cancer, accounting for 43.9% of new cases of gastric cancer and 48.6% of deaths worldwide [1]. The treatment of stomach cancer has come a long way over the past few decades, but the mortality rate is still high. Therefore, exploring the molecular mechanism of gastric cancer occurrence and finding effective therapeutic targets are the key to improving prognosis.
N6-adenylate methylation (m6A) is a common post-transcriptional modification of RNA that is primarily used to maintain the stability of mRNA. M6A-modified mRNAs that want to perform specific biological functions require methylated reading proteins.
The family protein 1 (YTHDF1) containing the YTH domain is an important m6A reading protein. Recent studies have shown that YTHDF1 can promote the development of gastric cancer through curl protein 7 (FZD7) and ubiquitin-specific peptidase 14 (USP14)[2]. So, what role does YTHDF1 play in the gastric tumor microenvironment?
Recently, Professor Yu Jun from the University of Chinese in Hong Kong and his team published important research results in the Journal of Cancer Immunotherapy, and they found that YTHDF1 is overexpressed in gastric cancer and promotes the progression of gastric cancer by inducing tumor cell proliferation and inhibiting dendritic cell-mediated anti-tumor immune response. In addition, YTHDF1 deletion promotes overexpression of interferon receptor 1 (IFNGR1) and JAK-STAT1 signaling pathways in tumor cells, thereby increasing the sensitivity of anti-tumor immunity. In the future, YTHDF1 is expected to become a new target for gastric cancer immunotherapy [3].
Screenshot of the first page of the paper
Let's take a look at how this research is conducted.
First, the researchers collected tissue samples from 101 patients with stomach cancer diagnosed at prince of Wales Hospital from 2015 to 2018 to form a cohort 1 to detect the expression of YTHDF1 mRNA, while collecting tissue samples of 278 patients with gastric cancer diagnosed from 1998 to 2006 to form a cohort 2 to detect YTHDF1 protein expression levels.
Subsequently, the research team also selected tumor tissue samples and paracancerous normal tissue samples from gastric cancer patients from the TCGA database (of which the unpaired group: 32 cases of normal tissue next to cancer and 375 cases of tumor tissue; paired group: 27 cases of tumor tissue and normal tissue of the same patient) were used to analyze the expression of m6A-related genes in the tumor tissue and paracancerous normal tissue of gastric cancer patients.
The results showed that upregulation of YTHDF1 expression was observed in both the paired group and the tumor tissue in the unpaired group. At the same time, the researchers also observed that both YTHDF1 mRNA levels and YTHDF1 protein levels were expressed in the tumor tissues of gastric cancer patients in gastric cancer patients in the tissue samples of cohort 1 and cohort 2 collected at the Prince of Wales Hospital.
So, is the expression of YTHDF1 related to the prognosis of patients with stomach cancer?
In Cohort 1, the K-M curve showed that YTHDF1 high mRNA expression was significantly associated with low survival in patients with gastric cancer (P=0.0164), and in multivariate Cox regression analysis, YTHDF1 high mRNA expression was an independent adverse prognostic factor in patients with gastric cancer (P=0.002). In cohort 2, it was observed that overexpression of the YTHDF1 protein correlated with low survival in patients with gastric cancer (P=0.039).
YTHDF1 is overexpressed in gastric cancer, and its expression is associated with low survival in patients with gastric cancer.
A: YTHDF1 mRNA level expression and its relationship with the survival rate of gastric cancer patients; B: expression of YTHDF1 mRNA level in the TCGA cohort; C:YTHDF1 protein expression and the survival rate of gastric cancer patients.
In order to further explore the biological function of YTHDF1 in gastric cancer, the researchers examined the expression of YTHDF1 protein in 12 human gastric cancer cell lines and found that the expression of YTHDF1 protein was relatively high in the three human gastric cancer cell lines AGS, BGC823, and MKN74. The researchers intended to select these three cell lines for knockdown of the YTHDF1 gene, and found that the knockdown of YTHDF1 significantly inhibited the proliferation of AGS, BGC823, and MKN74 cells. At the same time, the researchers knocked out the YTHDF1 gene on the mouse gastric cancer cell line YTN16, and a similar phenomenon was observed. Subsequent functional tests have shown that YTHDF1 knockdown/knockdown significantly inhibits the proliferation and colony formation capacity of human and mouse gastric cancer cells.
YTHDF1 knockdown inhibits gastric cancer cell line proliferation
A: Western blot confirmed knock-down effect of YTHDF1 gene in cell lines
B: Effect of YTHDF1 gene knockdown on gastric cancer cell viability
Effect of C: Effect of YTHDF1 gene knockdown on colony formation ability of gastric cancer cells
The above in vitro experimental results show that YTHDF1 has obvious pro-cancer effects. To explore whether YTHDF1 has the same pro-cancer effect in vivo, the researchers inoculated MKN74 cells (MKN74-shNC) transfected with a blank plasmid and two stable cell lines MKN74-shYTHDF1-1 and MKN74-shYTHDF1-2, which are knocked down YTHDF1 expression, into mouse skin, respectively.
The results showed that mice inoculated with MKN74-shYTHDF1-1 and MKN74-shYTHDF1-2 had significantly reduced the size and weight of subcutaneous tumors compared with mice inoculated with MKN74-shNC. This conclusion was further validated in mice inoculated with YTN16-sgNC cells transfected with blank plasmids and knockout (YTN16-sgYTHDF1-1 and YTN16-sgYTHDF1-2) cells.
Mouse subcutaneous tumorigenesis experiment
D: Effect of MKN74 cells and YTHDF1 low-expression cells on subcutaneous tumorigenesis in NSG mice
Effect of E: NSG mice inoculated with YTN16 cells and YTHDF1 low-expression cells on subcutaneous tumorigenesis
In vitro and in vitro experiments have shown that YTHDF1 can significantly promote the proliferation of gastric cancer cells, in order to clarify the relevant molecular mechanism of its occurrence, the researchers further sequenced RNA sequencing YTN16-sgNC cells and YTHDF1 gene knockout cells.
The analysis showed that in the YTHDF1 gene knockout cells, 575 gene expressions were upregulated and 866 gene expressions were down-regulated. Gene enrichment analysis revealed deletions of immunomodulatory pathway genes in YTHDF1 knockout cells, including the CTLA-4 checkpoint pathway and the CD209 DC pathway. At the same time, when the researchers used the KEGG database for gene enrichment analysis, they found that the JAK-STAT signaling pathway gene expression was upregulated and the T cell receptor signaling pathway gene expression was downregulated in the YTHDF1 gene knockout cells. These results suggest that YTHDF1 may exert immunomodulatory effects in vivo.
Next, to explore whether the pro-cancer effects of YTHDF1 depend on the immune system, the team used immunocomputent homologous mouse model C57BL/6 mice to inoculate YTN16-sgNC cells and YTHDF1 gene knockout cells under their skin, respectively. Throughout the 6-week experiment, the tumors of mice inoculated with YTN16-sgNC cells continued to grow, and the mice inoculated with YTHDF1 gene knockout cells had tumors growing under the skin after 1 week of inoculation, but the subcutaneous tumors of mice inoculated with YTN16-sgYTHDF1-1 began to gradually shrink after 2 weeks. This suggests that the pro-cancer effects of YTHDF1 depend at least in part on a functioning immune system.
After 2 weeks, the tumor volume changes
Since knockout of the YTHDF1 gene accelerates the elimination of subcutaneous tumors in mice with a normal immune system, the researchers wondered whether YTHDF1 in tumors can regulate anti-tumor immunity in vivo. Therefore, they inoculated YTN16-sgNC cells and YTHDF1 gene knockout cells in C57BL/6 mice, respectively, and collected tumors after 2 weeks to detect immune cell infiltration in the tumor microenvironment by flow cytometry.
The results showed that mice inoculated with YTHDF1 gene knockout cells had significant reductions in tumor size and weight compared with YTN16-sgNC cells. Flow cytometry analysis showed that the proportion of infiltrative immune cells (CD45+ cells) in tumor tissues in the YTHDF1 gene knockout group was significantly higher than that in the YTN16-sgNC group (P
In homologous mouse models, YTHDF1 deletion induces tumor elimination and increases immune cell infiltration
T cells (CD3+ lymphocytes) are known to be an important component of adaptive tumor immunity, so the researchers further analyzed the infiltration of T lymphocytes in C57BL/6 mouse tumor tissues. The results showed that the proportion of CD3+ cells and CD45+ cells in the YTHDF1 gene knockout group was significantly higher than that of the YTN16-sgNC group, and the number and proportion of CD4+ T cells and CD8+ T cells in the infiltrative immune cells were also higher than those in the YTN16-sgNC group.
Since interferon γ (IFNγ) is secreted by activated lymphocytes[4], the researchers further examined the expression level of IFNγ in tumor tissue. Compared with tumor tissues in the YTN16-sgNC group, IFNγ was significantly higher in tumor tissues in the YTHDF1 gene knockout group. Interestingly, pathway enrichment analysis showed that AFTER knockout of YTHDF1 in YTN16 cells, IFNGR1 was the most upregulated gene in the JAK-STAT signaling pathway, and YTHDF1 deletion was found in gastric cancer cell lines to upregulate IFNGR1, JAK1, JAK2, and STAT1 protein expression. The above results show that YTHDF1 gene knockout further activates the JAK-STAT1 pathway by upregulating IFNGR1, thereby enhancing tumor immune surveillance.
The deletion of YTHDF1 in tumor cells activates adaptive anti-tumor immunity
As can be seen from the above, knocking down YTHDF1 expression in tumor cells can enhance adaptive T cell anti-tumor immunity, so what is the impact of YTHDF1 expression on immunosuppressive cells in tumors?
Myeloid suppressors (MDSCs) are tumor-infiltrating immune cells with immunosuppressive capacity, and there is growing evidence that the high invasiveness of MDSCs is associated with poor prognosis and treatment resistance[5]. Therefore, the researchers examined the infiltration of MDSCs in YTN16 tumor tissue by flow cytometry and found that the proportion of MDSCs in tumor tissues and infiltrating immune cells in the YTHDF1 gene knockout group was lower than that in tumor tissues in the YTN16-sgNC group.
Adaptive anti-tumor immunity relies mainly on dendritic cells (DCs), which present antigens from tumor cells to T lymphocytes, and previous results have confirmed that tumor cells can promote infiltration of adaptive T lymphocytes after knocking down YTHDF1 expression. So, will YTHDF1 also increase DC infiltration after knocking down?
So the researchers also observed the infiltration of DC in the tumor tissue of C57BL/6 mice. The results showed that the proportion of infiltrative DC in tumor tissues in the YTHDF1 gene knockout group was significantly higher than that in the YTN16-sgNC group, and the expression of class II molecules (MHCII) of the DC surface histocompatibility complex was increased, and MHCII expression was the basic feature of DC cell maturation, and mature DC could secrete interleukin-12 (IL-12) to enhance adaptive anti-tumor immunity. Subsequently, the study found that IL-12 was significantly higher in the tumor tissue of the YTHDF1 knockout group than in the YTN16-sgNC group. In addition, in the TCGA cohort, DC tumor infiltration was inversely correlated with YTHDF1 mRNA expression.
Deletion of YTHDF1 in tumor cells recruits mature dendritic cells
These results suggest that YTHDF1 deletion in tumor cells can induce recruitment of mature DCs, thereby promoting infiltration of CD4+ T cells and CD8+ T cells, and increasing the secretion of cytotoxic cytokine IL-12.
To further evaluate whether the expression of YTHDF1 in tumor tissue affects the systemic anti-tumor immune response, the research team inoculated YTN16-sgNC cells and knocked down YTHDF1 expression of stable cells YTN16-sgYTHDF1-1 in the same mouse. Subsequently, depending on the inoculation, the researchers divided the mice into three groups, namely the YTN16-sgNC group, the YTN16-sgYTHDF1-1 group, and the mixed group. In both the YTN16-sgNC and YTN16-sgYTHDF1-1 groups, the same cells are seeded separately onto both sides of the mouse. In the mixed group, YTN16-sgNC and YTN16-sgYTHDF1-1 cells were seeded to the left and right sides of the same mouse, respectively.
The results showed that after 8 weeks of inoculation, there was tumor growth in the YTN16-sgNC group and no tumor growth in the YTN16-sgYTHDF1-1 group, while the mice in the mixed group had tumor growth on both sides, but the right tumor volume was significantly smaller than that on the left, and western blotting experiments confirmed that the sgYTHDF1-1 tumor retained the loss of YTHDF1 protein. This suggests that YTN16-sgNC tumors may lead to systemic suppression of anti-tumor immunity, which in turn allows YTN16-sgYTHDF1-1 tumors to survive.
Tumor volume changes after inoculation of 3 groups of mice
Now that YTHDF1 deletion eliminates tumors in immunocompetent hosts, the researchers next wonder if it can trigger a persistent systemic anti-tumor immune response that prevents future tumor implantation. So they divided the mice into two groups, YTN16-sgNC cells and YTN16-sgYTHDF1-1 cells were seeded to the right dorsal side of the mice, after 2 weeks of inoculation, the right dorsal tumor was excised, and then YTN16-sgNC cells were injected into the left side. The results showed that there was tumor growth on the left side of the mouse group previously inoculated with YTN16-sgNC cells, and no tumor growth was seen in the previously inoculated YTN16-sgYTHDF1-1 cell group. These results suggest that mice inoculated with YTN16-sgYTHDF1-1 cells adequately trigger the host's anti-tumor immunity, thereby preventing tumor implantation that is subsequently inoculated with normal YTHDF1 cells.
Overall, this study showed that YTHDF1 is overexpressed in gastric cancer and exerts a pro-cancer effect; YTHDF1 deletion induces DC cell recruitment and increases MHCII expression and IL-12 secretion, thereby promoting infiltration of CD4+ and CD8 T+ cells and increasing the secretion of interferon γ; YTHDF1 deletion mediates overexpression of the IFNGR1 and JAK-STAT1 signaling pathways in tumor cells, which may help restore anti-tumor immunity sensitivity. This study provides a new strategy for effective immunotherapy for gastric cancer, and YTHDF1 is expected to become a new target for gastric cancer immunotherapy.
bibliography:
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3.Bai X, Wong CC, Pan Y, et al. Loss of YTHDF1 in gastric tumors restores sensitivity to antitumor immunity by recruiting mature dendritic cells. J Immunother Cancer. 2022;10(2):e003663. doi:10.1136/jitc-2021-003663
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Responsible Editor | Zhang Jinxu