Functional And Mechanistic Study Of Macrophages Decreasing Doxorubicin Sensitivity In Breast Cancer Via METTL14-CCL3-CCR5 Pathway

[Background]Breast cancer,the cancer with the highest incidence among women,ranks second only to lung cancer in terms of malignant tumors’ mortality rates.It exhibits high heterogeneity,with significant differences in prognosis among different pathological types and molecular subtypes.Triple-negative breast cancer(TNBC),characterized by low differentiation,rapid progression,and high likelihood of recurrence,represents the subtype with the worst prognosis.Chemotherapy,primarily utilizing DNA-damaging agents such as anthracyclines and platinum drugs due to the lack of surface receptors on tumor cells,remains the mainstay of TNBC treatment,resulting in stable efficacy and significant improvement in patient outcomes.However,due to tumor heterogeneity,a considerable number of TNBC patients develop primary and acquired resistance to DNA-damaging agents,posing a significant challenge to their clinical use.Thus,identifying crucial drug targets through basic research is essential to address this issue.Tumor-associated macrophages(TAMs)are among the most abundant immune cells infiltrating the breast cancer microenvironment.During chemotherapy,most drugs accumulate in TAMs.Clarifying the effects and mechanisms of chemotherapy drugs on TAMs holds promise for overcoming resistance to breast cancer chemotherapy.N6-methyladenosine(m6A)modification,as the most abundant RNA modification,plays a crucial role in regulating RNA transcription,transport,translation,and other biological functions,thereby maintaining cell viability.However,m6 A also plays a significant role in the occurrence and development of tumors.Regulation of m6 A methylation levels,m6 A methyltransferases,recognition proteins,and demethylases can modulate tumor cell functions,thus offering immense prospects in cancer therapy.Moreover,m6 A modification directly participates in regulating immune cells in the tumor microenvironment,reshaping tumor immune responses.However,its mechanisms in regulating macrophages and its impact on breast cancer chemotherapy resistance remain incompletely understood,necessitating further in-depth research and exploration to reverse TNBC chemotherapy resistance.[Objective]Investigating the impact and mechanisms of tumor-associated macrophages on the sensitivity of breast cancer to doxorubicin treatment;elucidating the crucial role of METTL14 in assisting breast cancer cells to resist doxorubicin damage in tumor-associated macrophages,and exploring the feasibility of targeted inhibition of macrophage METTL14 expression to sensitize breast cancer to doxorubicin.[Methods And Result]1.Doxorubicin Induces Upregulation of m6 A Levels in Tumor-Associated Macrophages and Promotes Doxorubicin Resistance in Breast Cancer1)By constructing a mouse model of mammary cancer with macrophage clearance,we confirmed that tumor-associated macrophages(TAMs)promote breast cancer progression.Macrophages induced by doxorubicin facilitated tumor growth in mice;2)Dot blot experiments revealed that doxorubicin,cisplatin,and carboplatin all led to increased mRNA m6 A methylation levels in THP-1,iBMDM,and RAW264.7macrophage cell lines.In vivo,TAMs exhibited elevated m6 A levels following doxorubicin chemotherapy;3)Through Western blot,MTT assays,and flow cytometry apoptosis analysis,we demonstrated that utilizing the methyltransferase METTL3-METTL14 inhibitor STM2457 to suppress m6 A levels in doxorubicin-stimulated macrophages significantly enhanced breast cancer sensitivity to doxorubicin chemotherapy.2.Doxorubicin Increases TNBC Drug Sensitivity by Upregulating Macrophage Methyltransferase METTL14 Expression1)Western blot analysis revealed upregulation of METTL14 expression in RAW264.7,iBMDM,and mouse bone marrow-derived macrophages(BMDM)upon doxorubicin treatment;2)Using lentiviral infection,we established iBMDM shMETTL3,shMETTL14,and shMETTL3-METTL14 cell lines.MTT proliferation assays and flow cytometry apoptosis analysis demonstrated that inhibiting METTL14 expression in macrophages significantly enhanced breast cancer cell sensitivity to doxorubicin;3)Dot blot assays were employed to assess changes in m6 A levels in macrophages following combined treatment with doxorubicin and the ATM inhibitor KU-55933,indicating that KU-55933 could inhibit m6 A levels in macrophages;4)Protein immunoprecipitation experiments confirmed that doxorubicin-induced DNA damage phosphorylated METTL14 via ATM,and exhibited by the ATM inhibitor KU-55933;5)Immunofluorescence and nuclear-cytoplasmic fractionation Western blot assays validated that the upregulation of METTL14 expression induced by doxorubicin primarily occurred in the nucleus of macrophages.3.Doxorubicin Regulates Macrophage CCL3 Secretion through METTL141)Cytokine array analysis showed that doxorubicin significantly upregulated macrophage CCL3 expression levels,which could be significantly inhibited by STM2457;2)RT-qPCR and flow cytometry confirmed the upregulation of macrophage CCL3 expression by doxorubicin at mRNA and protein levels;3)Actinomycin D mRNA stability experiments demonstrated that METTL14 positively regulates CCL3 mRNA expression;4)RIP-qPCR results indicated a significant enhancement of the binding between METTL14 and CCL3 mRNA by doxorubicin;5)MeRIP-qPCR results suggested that METTL14 bound to CCL3 mRNA through m6 A modification and doxorubicin enhanced their binding;6)Using SRAMP prediction,m6 A modification sites on CCL3 mRNA were identified.Dual luciferase reporter gene assays were conducted with p GL-CCL3 wild-type and p GL-CCL3 mutant plasmids at m6 A modification sites,revealing that the binding of METTL14 and CCL3 mainly occurs on adenine at position 7 of its mRNA;7)Lentiviral infection was used to restore CCL3 in iBMDM shMETTL14 cells.Experimental methods including Western blot and flow cytometry apoptosis detection confirmed that replenishing CCL3 in METTL14-knockdown cell lines mitigates the sensitization effect induced by METTL14 inhibition.4.Doxorubicin Reduces TNBC Sensitivity by Regulating the Macrophage CCL3-CCR5 Axis through METTL141)Western blot analysis confirmed high expression of the CCR5 receptor in breast cancer cell lines;2)MTT cell proliferation assays revealed that CCL3 had no significant effect on breast cancer cell proliferation in vitro but could reduce the sensitivity of breast cancer cells to doxorubicin;3)Western blot analysis demonstrated that CCL3 reduced the expression of DNA damage and apoptosis markers,while adding a CCR5 inhibitor increased the expression of these markers;4)RNA-seq analysis explored differential gene expression and pathways in 4T1 cells treated with doxorubicin alone versus in combination with CCL3 or the CCR5 inhibitor Maraviroc;5)Western blot analysis confirmed that administering CCL3 to breast cancer cells or coculturing breast cancer cells with macrophage supernatants overexpressing CCL3 activated the MAPK,AMPK,PI3K-Akt pathways;6)MTT assays revealed that inhibiting the AMPK pathway increased the sensitivity of breast cancer cells to doxorubicin chemotherapy;7)Flow cytometry apoptosis assays and Western blot analysis confirmed that inhibiting the AMPK pathway increased apoptosis levels in breast cancer cells,upregulated the expression of apoptosis markers,and upregulated the expression of DNA damage markers.5.Inhibiting Macrophage METTL14 Levels Increases Doxorubicin Sensitivity in TNBC1)qRT-PCR experiments were conducted to assess changes in macrophage M1 and M2 markers under different tumor-associated macrophage induction conditions.The results indicated a significant upregulation of the iBMDM M2 marker levels under coculture or IL-4 induction conditions;2)Using a direct contact coculture method,morphological characteristics of macrophages were observed.It was found that after coculture with breast cancer cells,macrophages exhibited no significant proliferation but showed pseudopodia and clear differentiation;3)A mixed tumor model of tumor-associated macrophages and breast cancer cells was established.Recording tumor growth changes and plotting growth curves revealed that inhibiting METTL14 expression in macrophages weakened the doxorubicin-induced pro-tumor effect of macrophages;4)Immunofluorescence staining of mouse tumor tissues revealed that doxorubicin increased the proportion of macrophages positive for METTL14 and CCL3;5)Immunofluorescence staining of clinical samples from triple-negative breast cancer patients before and after neoadjuvant chemotherapy also showed an increased proportion of macrophages positive for METTL14 and CCL3,further corroborating the above experiments.[Conclusion]1.TAMs promote the progression of TNBC and decrease TNBC sensitivity to doxorubicin through m6 A modification;2.Mechanistically,doxorubicin regulates TAM-mediated reduction of TNBC drug sensitivity through the ATM-METTL14-CCL3-CCR5 axis;3.METTL14 can serve as a crucial target to enhance doxorubicin therapy,as inhibiting macrophage METTL14 expression can increase drug sensitivity in breast cancer.