| Background:Currently,breast cancer is the leading commonly diagnosed cancer globally surpassing lung cancer.Although the advances in surgeries,chemotherapy and targeted treatment approaches for breast cancer have improved overall survival rates in recent years,the therapeutic modalities are still limited for aggressive breast cancers such as the triple-negative form.The rapidly growing field of cancer immunotherapy is expanding new horizons for antitumor therapy.Programmed death ligand-1(also known as B7-H1,CD274or PD-L1;hereafter referred to as PD-L1)is a 33?k Da type I transmembrane glycoprotein which downregulates T-cell function and cell survival by binding to programmed death-1(PD-1)receptor.PD-L1 inhibits immune-mediated rejection and assists tumor cells to evade the host immune surveillance in the tumor microenvironment.Currently,it is reported that the clinical benifit from PD-L1 inhibition therapy is limited in breast cancer.PD-L1expression may be dynamic during the treatment course and may at least partly explain why some cancer patients with tumors lacking PD-L1 expression can respond favorably to checkpoint inhibitors therapy.Therefore,investigation of upstream regulatory mechanisms of the PD-L1 expression in breast cancer is important for an in-depth understanding of the functions of this immunosuppressive molecule.This could provide us with new strategies to reduce the cellular abundance of PD-L1.Furthermore,epigenetic regulations are also crucial in controlling gene expression.RNA methylation,a type of post-transcriptional modification,has gained widespread attention.As the common principal m RNA methylation type in mammals,N~6-methyadenosine(m~6A)modifications are reversible and dynamic;these are regulated by m6A“writers”,“erasers”,and“readers”(WERs).They are relevant to RNA fate as modifications manipulate the stability,pre-m RNA splicing and translation.It is previously reported that m6A modification is also important in immunoregulation.METTL3 depletion promotes STAT1 and IRF1 m RNA expression in an m6A-YTHDF2-dependent manner,which in turn improves immunotherapeutic response by modulation of tumor-infiltrating cells in the intratumor microenvironment of colorectal cancer.Inhibition of METTL3weakens PD-1 blockade treatment by altering reprogramming of the bone marrow-derived macrophages.Moreover,FTO-mediated m6A modification is implicated in the regulation of melanoma tumorigenesis and resistance to anti-PD-1 therapy.However,the impact of m6A regulator(s)on the expression of immune-checkpoint molecules in breast cancer remains unclear and requires further investigation.However,the impact of m~6A regulator(s)on the expression of immune-checkpoint molecules in breast cancer remains unclear and requires further investigation.Objective:The purpose of this study was to determine whether m~6A regulator(s)play a role in the regulation of PD-L1 expression and reveal the specific molecular mechanism.The details are as follows:(1)Screen out the regulatory role of METTL3-mediated N~6-methyadenosine modification in PD-L1 expression;(2)Verify the regulatory role of IGF2BP3 related to METTL3-mediated m~6A modification in PD-L1 expression;(3)Confirm the effect of tumor immune surveillance of METTL3-IGF2BP3 axis via PD-L1 expression in vitro;(4)Reveal the antitumor effect of METTL3 in different animal model;(5)Clarify the expression correlation between PD-L1 with METTL3 or IGF2BP3 in tissue microarray.Methods:1.The analysis of m~6A epitranscriptomic microarray,Me RIP-seq,and expression profile data showed that the downstream targets of METTL3-mediated m6A modification;RIP-q PCR experiments revealed that the interaction between METTL3 and PD-L1 m RNA and Me RIP-q PCR assays verified the METTL3-mediated m~6A modification in four breast cancer cell lines;Through construction of m~6A site-specific mutant plasmid,luciferase reporter assay analyzed the specific methylated site of PD-L1 m RNA;Absolute quantitative assay further validated the specific m~6A methylation level of each site upon METTL3-knockdown;m RNA decay assay revealed the stability of METTL3-regulated PD-L1 m RNA;q RT-PCR,western blot,flow-through assay identified the METTL3-regulated PD-L1 expression.2.RIP-q PCR assay was used to confirm the binding relationship between IGF2BP3and PD-L1 m RNA;Futher RIP-q PCR assay revealed the binding relationship between IGF2BP3 and PD-L1 m RNA whether it was influenced by METTL3;m RNA decay assay revealed that the METTL3-IGF2BP3 axis is involved in regulating the stability of PD-L1m RNA;q RT-PCR,western blot and flow assay elucidated the regulatory role of METTL3-IGF2BP3 axis on PD-L1 expression.3.In the co-culture system of breast cancer cells with knockdown METTL3 or IGF2BP3 and immune cells(cytokine-induced killer cells),the T cell killing effect was detected by LDH release assay;The expression of IL-2 and IFN-γfor T cell activation markers in co-culture system were detected by ELISA;The expression of T cell exhausion markers,such as PD-1,TIM3 and NR4A1,was detected by q RT-PCR to reveal the of regulatory role of METTL3 or IGF2BP3 in the T cell activation process.4.Xenograft tumor model in B-NDG mice was used to reveal the antitumor effect of METTL3 and IGF2BP3;The peritoneal tumors xenografts was constructed to verify the antitumor effect of METTL3 in BALB/c mice using bioluminescence assay,and the effect of METTL3 on T-cell infiltration in the microenvironment was confirmed by immunohistochemical assay;and the effect growth and survival of The subcutaneous tumors in BALB/c mice were used to discover the regulatory role and the regulation of of METTL3 on the survival and T-cell infiltration of CD3+,CD8+and CD4+cells density in the microenvironment.5.The tissue microarray,including 140 patients with breast cancer,and clinical information was used to analyze the correlation between PD-L1 and METTL3 or IGF2BP3expression;The online database TISIDB was used to reveal the expression of METTL3 and IGF2BP3 in tumors responding to anti-PD-1/PD-L1 therapy.Results:1.The analysis of m~6A epitranscriptomic microarray,Me RIP-seq,and expression profile data showed that PD-L1 may be a direct downstream target of METTL3-mediated m~6A modification;RIP-q PCR experiments revealed that METTL3 directly interacted with PD-L1 m RNA in MDA-MB-231,HCC38,SK-BR-3,and 4T1 cells,and Me RIP-q PCR assay results showed that knockdown of METTL3 reduced the m~6A level of PD-L1 m RNA;Through prediction of methylated sites,three high confidence m~6A sites were revealed located on the CDS region of PD-L1 m RNA and plasmids with mutation vectors for a single site were constructed,and luciferase reporter assay verified that the relative luciferase activity of wide type remarkably reduced upon METTL3 knockdown,but those for mutant type groups were resistant to the effect of METTL3 silencing;Absolute quantitative PCR assay identified that METTL3 had modified on three sites;m RNA decay assay revealed that METTL3 could regulate the stability of PD-L1 m RNA;q RT-PCR,western blot,and flow assay verified that METTL3 could promote the expression level of PD-L1.2.RIP-q PCR assay was used to confirm that binding relationship between IGF2BP3directly interacted with PD-L1 m RNA;Also RIP-q PCR assay revealed the reduced binding relationship between IGF2BP3 and PD-L1 m RNA upon METTL3 knockdown;m RNA decay assay revealed that the METTL3-IGF2BP3 axis increased the stability of PD-L1m RNA;q RT-PCR,western blot and flow assay elucidated the positive regulatory role of METTL3-IGF2BP3 axis on PD-L1 expression.3.In the co-culture system of breast cancer cells with immune cells(cytokine-induced killer cells),LDH release assay revealed that knockdown METTL3 or IGF2BP3 could increase the T cell killing;The results of ELISA testing expression of markers of T cell activation including IL-2 and IFN-γshowed that T cells were activated with the increase in IL-2 and IFN-γsecretion due to METTL3 or IGF2BP3 knockdown;The expression of T cell exhausion markers,such as PD-1,TIM3 and NR4A1,were reduced upon METTL3 or IGF2BP3 knockdown detected by q RT-PCR to reveal the of regulatory role of METTL3 or IGF2BP3 in the T cell exhausion.4.Xenograft tumor model in B-NDG mice was used to reveal the antitumor effect of METTL3 and IGF2BP3,the silencing of METTL3 or IGF2BP3 could suppress tumor growth;The intraperitoneally xenografted model in BALB/c mice was constructed to verify that tumors in METTL3-silenced groups grew more slowly and prolonged the overall survival as compared to those in the control group antitumor effect of METTL3 using bioluminescence assay,and higher densities of CD3~+,CD8~+,and CD4~+T-cell infiltrations were also found in METTL3-knockdown tumors as compared to control tumors which were validated by IHC staining;In the subcutaneous xenograft models,tumoral METTL3inhibition could markedly limit tumor growth of the mice as compared to the control groups,and consistent with the results of the intraperitoneal xenografted tumors,METTL3disruption in transplanted tumors increased CD3~+,CD4~+,and CD8~+T-cell infiltrations.5.To determine the clinical correlation between PD-L1 and METTL3 or IGF2BP3,IHC staining was performed to verify the protein expression of METTL3,IGF2BP3,and PD-L1 using a tissue microarray consisting of 140 breast cancer tissues,and results showed that the PD-L1 expression was positively associated with the expression of METTL3 or IGF2BP3;Additionally,the data indicated that the correlation of METTL3 or IGF2BP3with PD-L1 was higher in HER2+(HER2 positive)and TNBC as compared to other subtypes;Besides,PD-L1-positive tissues expressed higher levels of METTL3 and PD-L1-negative tissues showed a concomitant decrease in IGF2BP3 expression;TISIDB database was used to evaluate whether METTL3 and IGF2BP3 showed differential expression in clinical data between responders and non-responders undergoing anti-PD-1/PD-L1 treatment,and results revealed that tumors of the responders expressed higher levels of METTL3 and IGF2BP3.Conclusion:1.PD-L1 was a downstream target of METTL3-mediated m~6A modification in breast cancer cells.METTL3 knockdown significantly abolished m~6A modification on the three methlated sites of CDS sequence and reduced stabilization of PD-L1 m RNA.2.IGF2BP3 mediates PD-L1 m RNA stabilization and expression in an METTL3-m~6A-dependent manner.3.Knockdown of METTL3/IGF2BP3 could enhance T cell-mediated antitumor immunity to alleviate immune surveillance of breast cancer by downregulating PD-L1expression.4.METTL3 was involved in the tumor immune escape by upregulation of PD-L1expression which inhibited intertumoral T-cell infiltration.5.The PD-L1 expression was positively correlated with METTL3 or IGF2BP3 and indicated that the interaction between PD-L1 and METTL3/IGF2BP3 in breast cancer. |
