In recent decades, substantial improvements have been made in therapeutic interventions for BC, which have increased the survival and quality of life of patients(25). Chemotherapy, as the broadest application of tumour treatment, remains the cornerstone of adjuvant therapy for BC and is widely used in BC patients with high metastatic burden and local advanced disease(26). Nevertheless, recent studies have indicated that the response rate of BC patients with the HR+/HER2- subtype to chemotherapy is low(27). Hence, the demand for elucidating the mechanism of HR+/HER2- BC insensitivity to chemotherapy is crucial. In this study, we first discovered that m6A modifications and METTL3 expression were inhibited by chemotherapy; thus, we evaluated the function of METTL3 in regulating HR+/HER2- BC progression, metastasis, and drug resistance. Based on our findings, we defined the METTL3/CDKN1A/EMT and METTL3/BAX/caspase3/8/9 axes as novel pathways involved in a potential mechanism of HR+HER2- BC chemoresistance (Figure 6F).
m6A modification, one type of RNA epigenetic modification, has been identified on almost all types of RNAs and has been implicated in a variety of cellular processes, including mRNA stability, splicing, location, and translation, RNA-protein interactions, and pri-miRNA processes(28–33). An increasing number of studies have addressed the pathological significance of m6A dysregulation in human diseases, especially in cancers(28–30). The results of our current study showed that the overall level of m6A modification was significantly downregulated after chemotherapy in HR+/HER2− BC patients, and treatment of MCF-7 and T47D cells with DOX, PTX and DDP also resulted in a decrease in m6A modifications. However, the levels of m6A modification were not affected by drug intervention in MDA-MB-231 cells. Therefore, our results suggested that chemotherapy-induced changes in m6A levels are a biological difference between HR+HER2- BC and TNBC, especially in terms of responsiveness to chemotherapy. Various studies indicate that the m6A modification affects drug sensitivity by regulating ABC transporters either directly at the transcript level or via upstream signalling pathways(31). Recent studies also indicated that the m6A modification is involved in the maintenance of CSCs in tumours, leading to drug resistance and recurrence(32). It has also been shown that m6A modifications can affect the response of BC to endocrine therapy(22). However, there are few studies on the relationship between m6A and chemotherapy response. Therefore, considering the potential role of the m6A RNA modification in the development of chemoresistance, it is necessary to illustrate the relationship between these two phenomena.
METTL3, a key component of the N6-methyltransferase complex, has been reported to play an important role in many tumour types(33–38). Previous studies reported that METTL3 plays an oncogenic role in acute myeloid leukaemia through diverse downstream targets(38), whereas other studies suggested that either increased or decreased METTL3 expression could promote the self-renewal and tumorigenicity of glioma stem-like cells, respectively(37, 39). Regarding METTL3 in BC, data from the literature have suggested that METTL3 can promote BC progression by targeting Bcl-2, HBXIP or SOX2(36, 40, 41) and that METTL3 could promote adriamycin resistance by accelerating pri-miRNA-221-3p maturation(42). However, our results illustrated that chemotherapy-mediated depletion of METTL3 plays a significant protective role in tumour progression and drug tolerance. Reasonable explanations for these contradictory phenomena could be attributed to recognition by different m6A readers(43). We speculated that the m6A modification and METTL3 expression protect some critical genes from degradation or restrain the role of oncogenes by enhancing their recognition by “readers”. However, this hypothesis needs more study. In summary, the decreased METTL3 expression is secondary to chemotherapy, which is consistent with the clinical medication pattern, and HR+HER2- BC is the only BC subtype to exhibit this expression pattern. Therefore, METTL3 can be used as a biomarker to predict the sensitivity of HR+HER2- BC to chemotherapy and as a novel target for combination therapy to reverse chemotherapy resistance.
Our results further showed that METTL3 regulates the proliferation, apoptosis, migration and drug resistance of HR+/HER2− BC through multiple signalling pathways. On the one hand, METTL3 can affect the m6A modification of BAX mRNA, thereby promoting activation of the pro-apoptotic caspase cascade and (consequently) apoptosis. Apoptosis is an important mechanism to mitigate the uncontrolled growth of tumour cells and is mainly regulated by the Bcl2 protein family(44). The Bcl2 protein family can be divided into two categories according to their functions: one plays a pro-apoptotic role and includes BAX and Bak, whereas the other plays an anti-apoptotic role and includes Bcl2. Both pathways promote caspase cascades that eventually lead to cell death(44). Our experiment found that METTL3 can promote the expression of BAX and the subsequent activation of caspase3, 8, and 9, leading to apoptosis.
On the other hand, downregulation of METTL3 can regulate CDKN1A expression to affect the EMT process and promote cell proliferation. CDKN1A is one of the key molecules involved in cell cycle progression and was first identified as a tumour suppressor(45). Later, it was found to be involved in pathways related to tumorigenesis and development, such as cell death, DNA replication/repair, gene transcription and cell motility(46). It is believed that the dual role of CDKN1A depends on its cellular localization(47). When in the nucleus, CDKN1A functions a tumour suppressor. However, when CDKN1A is concentrated in the cytoplasm, p53-impaired or p53-deficient cells may acquire carcinogenic properties, which may inhibit apoptosis and promote cell migration and proliferation(48). Studies have shown that miR-33b-3p can promote the survival and cisplatin resistance of A549 human lung cancer cells by targeting CDKN1A after DNA damage(49). It was also found that miR-520g mediated the resistance of colorectal cancer cells to 5-fluorouracil (5-FU) or oxaliplatin by downregulating of CDKN1A expression(50). These studies suggest that the presence of CDKN1A protects cancer cells from apoptosis after anti-cancer therapy. Therefore, in this study, the changes in CDKN1A expression were caused by chemical drugs, which may stimulate the translocation of CDKN1A protein from the nucleus to the cytoplasm, thereby activating downstream related pathways to reduce the sensitivity of cells to chemotherapy drugs; however, the specific mechanism is still unknown. In short, the mechanisms of interaction between cell signalling pathways and epigenetic elements are diverse and complex and merit further exploration and verification.
This study still has some shortcomings, such as the small sample size and lack of follow-up data. The direct intermolecular regulatory mechanism by which METTL3 affects tumour progression and survival was not clarified in detail and we will be further studied in the future.