R KPNA1 regulates nuclear import of NCOR2 splice variant BQ323636.1 to confer tamoxifen resistance in breast cancerDear Editor, Tamoxifen is a first-line treatment option for estrogenreceptor-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 nuclearlocalization 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 (NLS BQ ; 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 NLS BQ was cloned and coimmunoprecipitation confirmed that GFP-NLS BQ 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 BQexpression (Figure S5A). Knockdown of KPNA1 in LCC2 reduced BQ levels in the nucleus . These results suggested that NLS BQ was functional and KPNA1 may mediate the nuclear import of BQ in breast cancer cells.

Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels are members of the voltage‐gated cation channel family known to be expressed in the heart and central nervous system. Ivabradine, a small molecule HCN channel‐blocker, is FDA‐approved for clinical use as a heart rate‐reducing agent. We found that HCN2 and HCN3 are overexpressed in breast cancer cells compared with normal breast epithelia, and the high expression of HCN2 and HCN3 is associated with poorer survival in breast cancer patients. Inhibition of HCN by Ivabradine or by RNAi, aborted breast cancer cell proliferation in vitro and suppressed tumour growth in patient‐derived tumour xenograft models established from triple‐negative breast cancer (TNBC) tissues, with no evident side‐effects on the mice. Transcriptome‐wide analysis showed enrichment for cholesterol metabolism and biosynthesis as well as lipid metabolism pathways associated with ER‐stress following Ivabradine treatment. Mechanistic studies confirmed that HCN inhibition leads to ER‐stress, in part due to disturbed Ca2+ homeostasis, which subsequently triggered the apoptosis cascade. More importantly, we investigated the synergistic effect of Ivabradine and paclitaxel on TNBC and confirmed that both drugs acted synergistically in vitro through ER‐stress to amplify signals for caspase activation. Combination therapy could suppress tumour growth of xenografts at much lower doses for both drugs. In summary, our study identified a new molecular target with potential for being developed into targeted therapy, providing scientific grounds for initiating clinical trials for a new treatment regimen of combining HCN inhibition with chemotherapy.

BackgroundWhile the efficacy of immune checkpoint inhibitors (ICIs) is increasingly recognized in advanced gastric cancer (aGC), overall survival (OS) has not been consistently improved across the different randomized controlled trials (RCTs). This meta-analysis aimed to quantify the efficacy and safety of ICI and explore potential predictive tumor tissue biomarkers in aGC.MethodsA random-effect pairwise meta-analysis was used to evaluate the primary outcome of OS. Sensitivity analysis was performed to investigate the effects of ICIs on PD-L1 status, TMB, MSI-H, and the Asian patient population. We extracted the OS Kaplan–Meier curves from the included trials to compare the effect of PD-L1 status on response to ICIs using DigitizeIt 2.5 and Guyot’s algorithm.ResultsA pairwise meta-analysis of seven RCTs included in this study showed that ICIs were more effective than the comparator in improving OS (pooled HR: 0.84). We demonstrated that PD-1 ICIs were additive when combined with the comparator arm (pooled HR: 0.79). A sensitivity analysis showed that PD-1 ICIs were associated with better OS outcomes in the Asian patient population as monotherapy (pooled HR: 0.66) or in combination with chemotherapy (pooled HR: 0.83). We demonstrated that tumors with PD-L1 ≥1 (P = 0.02) and PD-L1 ≥10 (P = 0.006) derived OS benefit from ICI monotherapy. Equally, MSI-H (P <0.00001) and TMB-high (P <0.0001) tumors derived favorable survival benefits from ICIs.Conclusions and relevanceThe results of this meta-analysis suggest that ICIs result in improved OS outcomes in aGC. The benefits varied with different ethnicities, class of ICI, PD-L1 expression, MSI status, and TMBSystematic Review Registrationhttps://www.crd.york.ac.uk/prospero, identifier (CRD42019137829).