KPNA1 regulates nuclear import of NCOR2 splice variant BQ323636.1 to confer tamoxifen resistance in breast cancer

Dear Editor, Tamoxifen is a first-line treatment option for estrogen-receptor-α positive (ER+) breast cancer. Drug resistance significantly compromises its clinical efficacy. Nuclear receptor corepressor-2 (NCOR2) is a transcriptional coregulatory protein. We previously identified a novel splice variant of NCOR2, that is, BQ323636.1 (BQ), which retains only the N-terminus repression domain-1 of the NCOR2 wild-type protein (Figure S1).1 BQ nuclear overexpression is found significantly associated with tamoxifen resistance in ER+ primary breast cancer, nuclear localization being essential in modulating tamoxifen response.2 This study reports a possible molecular mechanism behind BQ nuclear localization mediated by KPNA1 (importin-α5). We generated two expression constructs in which the BQ expression vector was fused with either a nuclear-localization signal (BQ-NLS) or with a nuclear-export signal (BQ-NES), and confirmed that BQ-NLS was predominantly localized in the nucleus, further promoted cell proliferation and enhanced tamoxifen resistance (Figure S2A–D). Using cNLS Mapper,3 we identified a putative NLS (PQRRRPSLLS) in BQ (NLSBQ; Figure S3A). Through RaptorX,4 we found that the NLS in BQ had greater relative surface accessibility than for that in NCOR2 (Figure S3B), suggesting it might be more functional. By coimmunoprecipitation, only KPNA1 interacted with BQ and importin-β1 (Figure 1A). An expression construct that expressed GFP fused with NLSBQ was cloned and coimmunoprecipitation confirmed that GFP-NLSBQ could interact with KPNA1 (Figure S3C). Knockdown of KPNA1 resulted in reduced nuclear-BQ (Figures 1B, C and S4A–C) in BQ-overexpressed cells. LCC2, a tamoxifen resistant cell-line derived from MCF-7, has a high endogenous BQ-expression (Figure S5A). Knockdown of KPNA1 in LCC2 reduced BQ levels in the nucleus (Figures 1D and S5B–D). These results suggested that NLSBQ was functional and KPNA1 may mediate the nuclear import of BQ in breast cancer cells. Using GPS tool,5 it was predicted that serine in NLSBQ could be phosphorylated by AKT (Figure S6A). Constructs were created expressing GFP fused with wild-type NLSBQ (wtNLSBQ) and with mutant NLSBQ (mtNLSBQ; Figure S6B). Compared with wtNLSBQ, coimmunoprecipitation showed that the interaction between KPNA1 and mtNLSBQ was significantly compromised (Figure S6C). Furthermore, AKT inhibitor treatment reduced the interaction between KPNA1 and wtNLSBQ (Figure S6D) as well as the interaction between KPNA1 and BQ (Figure 1E). AKT inhibition compromised the nuclear import of BQ (Figure 1F). To validate that nuclear import of BQ can be modulated by AKT, we employed IGF-1 to activate AKT activity (Figure S6E) in endogenously BQ-overexpression cells LCC2 and found IGF-1 could enrich BQ levels in the nucleus (Figure 1G). These results suggest that AKT is involved in governing the subcellular localization of BQ in breast cancer via KPNA1. Knockdown of KPNA1 could recover tamoxifen sensitivity in vitro (Figure S7A–H). While KPNA5 and KPNA6 showed high similarity to KPNA1 (Figure S8A), knockdown of either did not alter tamoxifen resistance in LCC2 (Figure S8B–D), suggesting KPNA1 to be specific for mediating tamoxifen resistance. In vivo studies showed KPNA1 knockdown xenografts could recover tamoxifen response (Figure 1H–J). Therefore, knockdown of KPNA1 compromises the effect of high BQ-expression in conferring tamoxifen resistance. From our previous informatics study, we observed HIF-1α signaling pathway enrichment in BQ-overexpressing cells6 which may contribute to tamoxifen resistance.7 We confirmed that BQ-overexpression could enhance both mRNA (Figure 2A) and protein expression (Figure 2B) of HIF-1α under normal and hypoxic conditions. As expected, BQ-overexpression enhanced HIF-1α transcriptional activity as indicated by luciferase reporter assay (Figure 2C) and qPCR (Figure 2D) as revealed by the expression of HIF-1α-regulated genes, hexokinase (HK), phosphofructokinase-1 (PFK), enolase-1 (ENO1), and lactate dehydrogenase-A (LDHA).8 Moreover, knockdown of KPNA1 (Figure S9A–C) compromised the effect of BQ on HIF-1α expression (Figure 2E) and the activity of HIF-1α (Figure 2F). Similar results were obtained from LCC2 (Figure S10A, B). These results suggest that nuclear import of BQ should be important for the activity of HIF-1α. Heat shock factors HSF2 and HSF4 govern the transcription of HIF-1α.9 Through coimmunoprecipitation, we found NCOR2 could interact with HSF4 (Figure 2G). There are two HSF4 binding sites in the promoter of HIF-1α (Figure S11), namely HSE1 (–901 to –864) and HSE2 (–1457 to –1423). ChIP assay showed that HSF4 could bind to HSE1 but not to HSE2 (Figure 2H). BQ-overexpression interfered with the interaction between NCOR2 and HSF4 (Figure 2I). As expected, BQ-overexpression could reduce the amount of NCOR2 associated with HSE1 (Figure 2J) and favored the binding of HSF4 to HSE1 (Figure 2K). These results suggest a novel mechanism regarding the role of BQ on the transcriptional regulation of HIF-1α as illustrated in Figure 2L. The expression of KPNA1 and BQ in primary breast cancer samples was examined through immunohistochemistry (Figure 3A; Table S1). A positive correlation was observed between nuclear KPNA1 and nuclear BQ expression (Figure 3B). Patients with high KPNA1 had a higher nuclear BQ score (p < .05; Figure 3C). High nuclear KPNA1 expression was associated with poorer overall (p = .002; Figure 3D) and disease-specific survival (p = .029; Figure 3E). Combined analysis for both KPNA1 and BQ nuclear expression showed even greater discrimination for poor overall survival (p = .0003; Figure 3F) and disease-free survival (p = .007; Figure 3G). We also found that high nuclear expression of KPNA1 was associated with tamoxifen resistance (Figure 3H) and metastasis (Figure 3I). Cox-regression analysis (Table 1) showed cases with high nuclear-KPNA1 and high nuclear-BQ was statistically significantly associated with poorer overall survival (RR = 3.832, 95% CI 1.758, 8.353; p = .001) and disease-free survival (RR = 3.402, 95% CI 1.332, 8.693; p = .011). In conclusion, our investigation shows that nuclear import of BQ mediated by KPNA1 plays a critical role in modulating tamoxifen resistance. Nuclear-BQ in competing with NCOR2 leads to the formation of defective corepressor complex, giving rise to upregulation of HIF-1α. Thus, disruption of BQ nuclear import may be relevant to the development of therapeutic interventions in breast cancer. A recent finding that ERα repressor Neurofibromin (NF1) modulates tamoxifen resistance,10 further lends support to the importance of nuclear receptor corepressor in tamoxifen resistance. The possibility of other nuclear receptor corepressors involved remains to be investigated. These studies might help identify alternative therapeutic approaches for reducing tamoxifen resistance. We would like to thank the Faculty Core Facility of LKS Faculty of Medicine, HKU, for providing technical support in confocal microscopy. We would like to thank Dr. Carmen Wong (Department of Pathology, HKU) for sharing her expertise in experiments related to hypoxia. U.S. Khoo is Ada Chan Endowed Professor in Oncological Pathology. This project was supported by the Innovative Technology Fund, HKSAR (ITF Ref. ITS/015/13), Health and Medical Research Fund (07182046, 06171696) and the Committee on Research and Conference Grants from the University of Hong Kong Project number 201811160005, 201811159118 and 201711159147. The authors have no conflict of interest to declare. Ui-Soon Khoo holds the patent of anti-BQ323636.1 antibody (US Patent no: US 10823735; China Patent no: ZL201680051133.9). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.