Reduced expression of luminal breast cancer biomarkers, including ER, FOXA1, and GATA3, has consistently correlated with tamoxifen resistance 21. Our study elucidates a mechanism involving N6AMT1-mediated alteration of p110α expression in tamoxifen resistance. We show N6AMT1 is downregulated, in tamoxifen-resistant cells, in a manner transcriptionally regulated by FOXA1 (forkhead box A1). As a member of the forkhead box transcription factor family, FOXA1 acts as a pioneer factor, binding to silent chromatin target sites and dominantly initiating regulatory cascades 24. Its significance is further underscored as a recognized biomarker for luminal breast cancer, where it modulates ER 25 and is pivotal in hormone responses, both during breast development and in ER-positive breast cancer scenarios 26. Around 50% of ER binding sites overlap with FOXA1 binding sites 27. Clinically, it has been observed that elevated FOXA1 expression in luminal A-type breast cancer patients is associated with enhanced survival rate and a more favorable response to endocrine therapies 18. Conversely, low FOXA1 expression is linked with the maintenance of tumor cell stemness, an upsurge in IL-6 expression, and the mediation of tamoxifen resistance 28. The FOXA1 promoter region contains estrogen response elements (EREs), suggesting that FOXA1 is a ER-regulated gene 29. In this context, our results suggest that prolonged tamoxifen exposure could reduce FOXA1 expression by disrupting the intricate regulatory network of ER-associated genes, providing an explanation for the reduced FOXA1 levels in TamR cells. Subsequently, the observed downregulation of N6AMT1, that occurs as a result of decreased FOXA1 levels, leads to increased expression of p110α and concomitant tamoxifen resistance.
Previous research has primarily focused on PIK3CA mutation status, particularly in two hotspots: exon 9 (E542K and E545K) encoding the helix domain, and exon 20 (H1047R) encoding the kinase domain 30–32. These mutations, as evidenced by both in vitro and in vivo models, possess pronounced oncogenic potential 33–36. While a meta-analysis has correlated them with unfavorable survival outcomes 37, a comprehensive analysis encompassing 10,319 breast cancer patients paints a different picture, associating PIK3CA mutations with enhanced survival rates 38. The activation of the PI3K/AKT pathway by PIK3CA mutations is a well-documented mechanism contributing to tamoxifen resistance 14. Yet, this fails to elucidate the resistance observed in patients devoid of PIK3CA mutations. Our data posits a novel perspective: the downregulation of N6AMT1 potentially escalates the levels of p110α and p-AKT (T308), thereby attenuating tamoxifen sensitivity. Notably, our experiments indicate that N6AMT1 overexpression or stable knockdown, both in vitro and in vivo, inversely affects p110α and p-AKT (T308) levels to regulate tamoxifen sensitivity. In transgenic mouse models of breast cancer (MMTV and HER2/Neu), PIK3CA knockout inhibits tumor growth 39. Additionally, p110α overexpression might play a role in the resistance of Snu-5 gastric xenografts to tyrosine kinase MET inhibitors 40. Collectively, these insights suggest that the surge in p110α levels not only amplifies PI3K activity but also activates the PI3K/AKT pathway. This could explain the observed correlation between altered p110α expression and the dynamics of the PI3K/AKT pathway in tamoxifen resistance in our study.
N6AMT1 primarily functions as a glutamine/lysine methyltransferase, modifying Gln185 of eRF1 to regulate translation in eukaryotes, and modifying Lys12 of histone H4 to regulate gene transcription 5,6. Our 6mA-IP-qPCR assays showed no discernible shifts in 6mA levels within the exon-coding region of PIK3CA, irrespective of the presence or absence of tamoxifen resistance and the reduced N6AMT1 expression. Additionally, RT-qPCR showed no significant changes in PIK3CA mRNA levels, suggesting that alterations in p110α expression is not associated with 6mA modification or transcriptional regulation, but rather with protein stability and post-translational modifications.
Previous research has shown that NEDD4L, an E3 ligase, enhances p110α degradation through poly-ubiquitination and proteasomal degradation 23. NEDD4L belongs to the HECT E3 ubiquitin ligase family and interacts with substrates via its four WW domains 41. Dysregulation of NEDD4L is observed in various cancers, and reduced levels are associated with tumor development 42–44. In breast cancer cells, miR-106b-25 can downregulate NEDD4L, leading to increased NOTCH1 and activation of tumor-initiating cells 45. Our study shows that N6AMT1 overexpression upregulates NEDD4L mRNA and protein levels, sensitizing breast cancer cells to tamoxifen. Conversely, N6AMT1 knockdown has the opposite effect. These findings suggest that N6AMT1 may regulate p110α via NEDD4L, highlighting the role of NEDD4L as a tumor suppressor in tamoxifen sensitivity. Although the role of N6AMT1 in the transcriptional regulation of NEDD4L remains to be fully elucidated, our 6mA-IP-qPCR assays showed no significant changes in the 6mA levels within the exon-coding regions of NEDD4L after N6AMT1 overexpression or stable knockdown, while ChIP-qPCR assays revealed that N6AMT1 may bind to the promoter region of NEDD4L and co-IP assays showed that knockdown of N6AMT1 led to a decrease in monomethylation levels of histone H4. Based on these, we propose that N6AMT1, as a KMT9, might regulate NEDD4L transcription through H4K12 monomethylation modification 5. Unfortunately, the unavailability of the H4K12me1 antibody from the Schüle Laboratory has prevented further verification. Nevertheless, this limitation does not impact our conclusion that the alteration in p110α expression related to N6AMT1 might be dependent on NEDD4L.
Furthermore, we validated the role of the N6AMT1-p110α pathway in clinical samples and patient-derived organoid (PDO) models. The PDO model accurately reproduces the clinical response by maintaining the tumor phenotype and genotype, making it suitable for preclinical drug discovery and validation 46. A66, a potent and specific inhibitor of wild-type p110α, reduces phosphorylation of Akt on T308 47. A synergistic effect of the p110α inhibitor A66 with tamoxifen was only observed in the PDO model of case 1, which exhibited a resistance mechanism similar to our study (downregulation of N6AMT1 and upregulation of p110α). These findings suggest that targeting p110α could be a promising approach to overcoming tamoxifen resistance associated with an N6AMT1-p110α pathway. Clinical trials have explored the use of alpelisib, a PI3Kα-selective inhibitor, in hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer with PIK3CA mutations. The SOLAR-1 trial (NCT02437318) demonstrates a significant progression-free survival (PFS) benefit when alpelisib is added to fulvestrant treatment 48. However, clinical trials specifically focusing on patients with p110α expression alterations have not been reported. Our study provides a theoretical basis for understanding tamoxifen resistance mechanisms in patients without PIK3CA mutations. Further investigations should evaluate the therapeutic efficacy of p110α inhibition in preclinical models with varying p110α expression levels before advancing to clinical trials.
This study has certain limitations. The mechanisms underlying the downregulation of FOXA1 and the regulation of NEDD4L by N6AMT1 remain unknown. Additionally, the use of cell line models (MCF-7, T47D) and a limited number of clinical samples (153) and PDOs (2) cannot fully capture the complexity and heterogeneity of luminal breast cancers. Therefore, future studies with a larger representation of tumors are needed to validate the role of the N6AMT1-p110α pathway in luminal breast cancers, especially those with p110α expression alterations associated with tamoxifen resistance.
In conclusion, our study illustrates that N6AMT1 confers tamoxifen resistance by altering p110α expression in luminal breast cancer. These results suggest that the N6AMT1-p110α pathway might not only predict tamoxifen sensitivity, but also serve as a viable target for overcoming tamoxifen resistance in luminal breast cancer.