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Background Annexin A1 is expressed specifically on the tumour vasculature surface. Intravenously injected IF7 targets tumour vasculature via annexin A1. We tested the hypothesis that IF7 overcomes the blood–brain barrier and that the intravenously injected IF7C(RR)-SN38 eradicates brain tumours in the mouse. Methods (1) A dual-tumour model was generated by inoculating luciferase-expressing melanoma B16 cell line, B16-Luc, into the brain and under the skin of syngeneic C57BL/6 mice. IF7C(RR)-SN38 was injected intravenously daily at 7.0 μmoles/kg and growth of tumours was assessed by chemiluminescence using an IVIS imager. A similar dual-tumour model was generated with the C6-Luc line in immunocompromised SCID mice. (2) IF7C(RR)-SN38 formulated with 10% Solutol HS15 was injected intravenously daily at 2.5 μmoles/kg into two brain tumour mouse models: B16-Luc cells in C57BL/6 mice, and C6-Luc cells in nude mice. Results (1) Daily IF7C(RR)-SN38 injection suppressed tumour growth regardless of cell lines or mouse strains. (2) Daily injection of Solutol-formulated IF7C(RR)-SN38 led into complete disappearance of B16-Luc brain tumour in C57BL/6 mice, whereas this did not occur in C6-Luc in nude mice. Conclusions IF7C(RR)-SN38 crosses the blood–brain barrier and suppresses growth of brain tumours in mouse models. Solutol HS15-formulated IF7C(RR)-SN38 may have promoted an antitumour immune response.

Zika virus (ZIKV) is a member of the Flaviviridae family and is considered a major health threat causing cases of microcephaly in newborns and Guillain-Barré syndrome in adults. Here we targeted a transient deep and hydrophobic pocket of the super-open conformation of ZIKV NS2B-NS3 protease to overcome the limitations of the orthosteric inhibitors. After virtual docking screening of approximately 7 million compounds against the novel allosteric site we selected the top seven candidates and tested them in an enzymatic assay. Six out of seven top candidates selected by the docking screen inhibited ZIKV NS2B-NS3 protease proteolytic activity at low micromolar concentrations, as well as suppressing viral replication. These six compounds, targeting the selected protease pocket conserved in ZIKV as well as several other Flaviviruses, have opened an opportunity for a new kind of drug candidate that might be useful to treat several flaviviral infections.

The effect of Bone Morphogenetic Proteins (BMPs) on glioblastoma propagation cells (GPCs) is being debated [1-5]. We observed that exposure of GPCs to BMP2/4 initiates a rapid proteasomal degradation of the pluripotency factor SOX2 followed by the differentiation of GPCs into glial cells using 5 primary human glioblastoma lines tested. Enforced expression of SOX2 in GPCs antagonized BMP2/4-induced gliogenesis and reduction of proliferation. Both outcomes are consistent with the role of SOX2 in normal neurogenesis [6-8]. Using gene expression analysis, we observed that level of SOX2 in different glioblastoma lines tracks with the levels of oncogenes H/K/N-RAS and OLIG2. Our work provides support for BMP2/4-SOX2 axis in GPCs and points to the proteasome-regulated degradation of SOX2 that initiates glycogenic differentiation similar to that observed during normal development [6, 7]. Our work supports a potential for developing novel therapeutic strategies aimed at differentiation of GPC into non-tumorigenic glia.

Zika virus (ZIKV) and dengue virus (DENV) are arthropod-borne pathogenic flaviviruses that co-circulate in many countries. To understand some of the pressures that influence ZIKV evolution, we mimic the natural transmission cycle by repeating serial passaging of ZIKV through cultured mosquito cells and either DENV-naive or DENV-immune mice. Compared with wild-type ZIKV, the strains passaged under both conditions exhibit increased pathogenesis in DENV-immune mice. Application of reverse genetics identifies an isoleucine-to-valine mutation (I39V) in the NS2B proteins of both passaged strains that confers enhanced fitness and escape from pre-existing DENV immunity. Introduction of I39V or I39T, a naturally occurring homologous mutation detected in recent ZIKV isolates, increases the replication of wild-type ZIKV in human neuronal precursor cells and laboratory-raised mosquitoes. Our data indicate that ZIKV strains with enhanced transmissibility and pathogenicity can emerge in DENV-naive or -immune settings, and that NS2B-I39 mutants may represent ZIKV variants of interest.

Zika virus (ZIKV) is a mosquito-borne pathogen classified by the World Health Organization (WHO) as a public health emergency of international concern in 2016, and it is still identified as a priority disease. Although most infected individuals are asymptomatic or show mild symptoms, a risk of neurologic complications is associated with infection in adults. Additionally, infection during pregnancy is directly linked to microcephaly and other congenital malformations. Since there are no currently available vaccines or approved therapeutics for this virus, there is a critical unmet need in developing treatments to prevent future ZIKV outbreaks. Towards this end, we performed a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral entry or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses identified single amino acid substitions in the N-terminal region of non-structural protein NS4A arguing this to be the likely drug target. These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically-approved NS5A inhibitors for hepatitis C virus (HCV). Taken together, SBI-0090799 represents a promising lead candidate for the development of an antiviral treatment against ZIKV infection for the mitigation of severe complications and potential resurgent outbreaks of the virus. IMPORTANCE This study describes the elucidation of (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as selective and potent inhibitor of Zika virus (ZIKV) replication using a high throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel non-enzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this non-structural protein mirrors the identification and development of hepatitis C virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been part of a curative strategy for HCV.

The comprehensive characterization of mechanisms safeguarding cell fate identity in differentiated cells is crucial for 1) our understanding of how differentiation is maintained in healthy tissues or misregulated in disease states and 2) to improve our ability to use direct reprogramming for regenerative purposes. To uncover novel fate-stabilizing regulators, we employed a genome-wide TF siRNA screen followed by a high-complexity combinatorial evaluation of top performing hits, in a cardiac reprogramming assay in mouse embryonic fibroblasts, and subsequently validated our findings in cardiac, neuronal and iPSCs reprogramming assays in primary human fibroblasts and adult endothelial cells. This approach identified a conserved set of 4 TFs (ATF7IP, JUNB, SP7, and ZNF207 [AJSZ]) that robustly opposes cell fate reprogramming, as demonstrated by up to 6-fold increases in efficiency upon AJSZ knockdown in both lineage- and cell type-independent manners. Mechanistically, ChIP-seq and single-cell ATAC-seq analyses, revealed that AJSZ bind to both open and closed chromatin in a genome-wide and regionalized fashion, thereby limiting reprogramming TFs access to target DNA and ability to remodel the chromatin. In parallel, integration of ChIP-seq and RNA-seq data followed by systematic functional gene testing, identified that AJSZ also promote cell fate stability by proximally down-regulating a conserved set of genes involved in the regulation of cell fate specification (MEF2C), proteome remodeling (TPP1, PPIC), ATP homeostasis (EFHD1), and inflammation signaling (IL7R), thereby limiting cells ability to undergo large-scale phenotypic changes. Finally, simultaneous knock-down of AJSZ in combination with cardiac reprogramming TFs overexpression improved heart function by 250% as compared to no treatment and 50% as compared to MGT, 1 month after myocardial infarction. In sum, this study uncovers a novel evolutionarily conserved mechanism mediating cell fate stability in differentiated cells and also identifies AJSZ as promising therapeutic targets for regenerative purposes in adult organs.Significance StatementDifferentiated cells can be converted from one cell type into another by overexpressing lineage-determining transcription factors. Direct lineage reprogramming represents a promising strategy for regenerative medicine, but current clinical applications remain limited by the low yield of the reprogramming process. Here, we present the identification and detailed mechanistic study of a novel mechanisms opposing reprogramming process and promoting cell fate stability. Using a highthroughput screening technique, we identified four transcription factors that act as blockades to cell type change. By tracking chromatin and gene expression changes, we reconstructed a conserved pathway mediating cell fate stability in differentiated cells.

Cellular heterogeneity in cancer is linked to disease progression and therapy response, although the mechanisms regulating distinct cellular states within tumours are not well understood. To address this, we identified melanin pigment content as a major source of phenotypic and functional heterogeneity in melanoma and compared RNAseq data from high (HPC) and low pigmented melanoma cells (LPC), revealing the polycomb repressor complex protein, EZH2, as a master regulator of these states. EZH2 protein, but not RNA expression, was found to be upregulated in LPCs and inversely correlated with melanin in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA strategy or DZNep, MS1943 that reduces EZH2 protein levels, significantly inhibited cell growth in LPCs by hampering ribosome biogenesis. In addition, decline in EZH2 protein level induces pigmented cell phenotype by inducing melanin biosynthesis. Proteasomal inhibitor, MG132 treatment induced EZH2 protein levels in HPCs prompted us to look for differentially regulated ubiquitin system proteins in HPC vs LPCs. UBE2L6, E2 conjugating enzyme has been shown to be downregulated significantly in LPCs by UHRF1-mediated CpG methylation. Both biochemical assays and animal studies demonstrated that UBE2L6 expression decline, in turn, promotes EZH2 protein stability due to lack of ubiquitination on K381 residue in LPCs. UBR4 cooperates with UBE2L6 to facilitate this ubiquitination process. Targeting UHRF1/UBE2L6/UBR4 axis can be a better treatment option to trigger HPC state in melanoma in which conventional EZH2 inhibitors are ineffective.

Purpose: The glycoprotein Trop-2 is frequently overexpressed in human malignancies and linked to worse outcome. A Trop2-targeting antibody-drug conjugate, sacituzumab govitecan, has been approved by the FDA for patients with unresectable locally advanced or metastatic triple-negative breast cancer. Trop-2 enhances Akt phosphorylation in numerous cancer cell lines, while the opposite findings are also reported suggesting the cell context-dependent effect of Trop-2. Blockade of PI3K/Akt pathway mediates anti-tumor mechanism of anti-HER2 agents. In this study, we aim to investigate the role of Trop-2 in HER2-positive breast cancer. Experimental design: The archival samples comprising different breast cancer subtypes were analyzed using immunohistochemical staining. The public databases, including, Metabric, KM plotter, ROC plotter, were used to verify the prognostic role and clinical significance of Trop-2 in HER2-positive breast cancer. In vitro tumor models, the transwell migration, invasion and sphere assays, were applied for evaluation of cancer progression. The apoptotic cells were determined by flow cytometry using Annexin V/propidium iodide double staining. Results: We analyzed a cohort of 986 breast cancer patients and found that HER2-positive breast cancer harbored higher Trop-2 protein expression than other breast cancer subtypes. Higher levels of Trop-2 and phospho-Akt in HER2-positive breast cancer tissues compared with normal tissues. Trop-2 expression was correlated with tumor stage and phospho-Akt level. HER2-positive breast cancer patients with high level of Trop-2 protein were linked to worse recurrence-free survival. Consistently, data from the public databases exhibited patients with higher level of Trop-2 transcript had shorter recurrence-free survival and overall survival of HER2-positive breast cancer. Overexpression of Trop-2 increased cell migration, invasion and sphere number in SK-BR-3 and BT-474 cells. In contrast, knockdown of Trop-2 suppressed cell motility and sphere formation. Trop-2-expressing cells had better cell viability compared to control cells in the presence of lapatinib or neratinib treatment. Lapatinib- and neratinib-induced cell apoptosis was rescued by Trop-2 overexpression. Notably, patients responded to anti-HER2 therapy had lower Trop-2 transcript expression than non-responders. Conclusion: These results suggested that the oncogenic role of Trop-2 in HER2-positive breast cancer and Trop-2 expression might confer resistance to anti-HER2 therapy. Citation Format: Chun-Yu Liu, Ji-Lin Chen, Pei-Yi Chu, Chun-Teng Huang, Wan-Lun Wang, Chi-Cheng Huang, Po-Han Lin, Ling-Ming Tseng. Significance of Trop-2 in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5127.

IF7 peptide, which binds to the annexin A1 (ANXA1) N-terminal domain, functions as a tumor vasculature-targeted drug delivery vehicle after intravenous injection. To enhance IF7 stability in vivo, we undertook mirror-image peptide phage display using a synthetic D-peptide representing the Anxa1 N-terminus as target. Peptide sequences were identified, synthesized as D-amino acids, and designated as dTIT7, which was shown to bind the ANXA1 N-terminus. Whole body imaging of mouse brain tumors modeled with near infrared fluorescent IRDye-conjugated dTIT7 showed fluorescent signals in brain and kidney. Furthermore, orally-administered geldanamycin (GA)-conjugated dTIT7 suppressed brain tumor growth. Ours is a proof-of-concept experiment showing that Anxa1-binding D-peptide could be developed as an orally-administrable, tumor vasculature-targeted therapeutic.Role of each author: MN designed and performed experiments, analyzed data, and wrote the manuscript; HMA and DLJ produced recombinant ANXA1 protein; KY conducted NMR analysis and data analysis; TOA designed, performed and analyzed LC-MS/MS data; MN, TS, IKT, YS, and TY analyzed peptide-binding assays and performed in silico structural analysis; CTU produced lentivirus for luciferase expression; CNH performed peptide binding assays, tissue culture and animal experiments; and MNF supervised the project and wrote the manuscript.

Context The impact of hospice care services on the utilization of life-sustaining treatments during end-of-life care in terminally ill patients has not been extensively studied. Objectives To determine the impact of hospice care services on the utilization of life-sustaining treatments during the last three months of life among patients with cancer. Methods This nationwide population-based cohort study identified adults with cancer diagnosis from the Taiwan Registry for Catastrophic Illness, 2006–2016. Life-sustaining treatments included cardiopulmonary resuscitation, intubation, mechanical ventilation support, nasogastric tube feeding, and total parenteral nutrition. Hospice care services consisted of hospice inpatient care, hospice-shared care, and hospice home care. The association of hospice care services with the utilization of life-sustaining treatments was determined using multiple logistic regression. Results Of 516,409 patients with cancer, 310,722 (60.2%) patients used life-sustaining treatments during the last three months of life. After adjusting for covariates, patients with hospice care services were less likely to receive life-sustaining treatments during the last three months of life than those without the services (adjusted odds ratio [AOR]: 0.70; 95% CI: 0.69–0.71). While type of life-sustaining treatments were considered, hospice care services were associated with a lower likelihood of receiving cardiopulmonary resuscitation (AOR: 0.125; 95% CI: 0.118–0.131), endotracheal intubation (AOR: 0.204; 95% CI: 0.199–0.210), mechanical ventilation support (AOR: 0.265; 95% CI: 0.260–0.270), nasogastric tube feeding (AOR: 0.736; 95% CI: 0.727–0.744), and total parenteral nutrition (AOR: 0.86; 95% CI: 0.84–0.88). Conclusion Hospice care services were associated with a lower likelihood of receiving life-sustaining treatments during the last three months of life in patients with cancer.

One of the major challenges of the current Zika virus (ZIKV) epidemic is to prevent congenital foetal abnormalities, including microcephaly, following ZIKV infection of pregnant women. Given the urgent need for ZIKV prophylaxis and treatment, repurposing of approved drugs appears to be a viable and immediate solution. We demonstrate that the common anti-malaria drug chloroquine (CQ) extends the lifespan of ZIKV-infected interferon signalling-deficient AG129 mice. However, the severity of ZIKV infection in these mice precludes the study of foetal (vertical) viral transmission. Here, we show that interferon signalling-competent SJL mice support chronic ZIKV infection. Infected dams and sires are both able to transmit ZIKV to the offspring, making this an ideal model for in vivo validation of compounds shown to suppress ZIKV in cell culture. Administration of CQ to ZIKV-infected pregnant SJL mice during mid-late gestation significantly attenuated vertical transmission, reducing the ZIKV load in the foetal brain more than 20-fold. Given the limited side effects of CQ, its lack of contraindications in pregnant women, and its worldwide availability and low cost, we suggest that CQ could be considered for the treatment and prophylaxis of ZIKV.

The recent re-emergence of Zika virus (ZIKV)1, a member of the Flaviviridae family, has become a global emergency. Currently, there are no effective methods of preventing or treating ZIKV infection, which causes severe neuroimmunopathology and is particularly harmful to the developing fetuses of infected pregnant women. However, the pathology induced by ZIKV is unique among flaviviruses, and knowledge of the biology of other family members cannot easily be extrapolated to ZIKV. Thus, structure-function studies of ZIKV proteins are urgently needed to facilitate the development of effective preventative and therapeutic agents. Like other flaviviruses, ZIKV expresses an NS2B-NS3 protease, which consists of the NS2B cofactor and the NS3 protease domain and is essential for cleavage of the ZIKV polyprotein precursor and generation of fully functional viral proteins. Here, we report the enzymatic characterization of ZIKV protease, and we identify structural scaffolds for allosteric small-molecule inhibitors of this protease. Molecular modeling of the protease-inhibitor complexes suggests that these compounds bind to the druggable cavity in the NS2B-NS3 protease interface and affect productive interactions of the protease domain with its cofactor. The most potent compound demonstrated efficient inhibition of ZIKV propagation in vitro in human fetal neural progenitor cells and in vivo in SJL mice. The inhibitory scaffolds could be further developed into valuable research reagents and, ultimately, provide a roadmap for the selection of efficient inhibitors of ZIKV infection.

Triple negative breast cancer (TNBC) is an aggressive cancer for which prognosis remains poor. Combination therapy is a promising strategy for enhancing treatment efficacy. Blockade of STAT3 signaling may enhance the response of cancer cells to conventional chemotherapeutic agents. Here we used a SHP-1 agonist SC-43 to dephosphorylate STAT3 thereby suppressing oncogenic STAT3 signaling and tested it in combination with docetaxel in TNBC cells. We first analyzed messenger RNA (mRNA) expression of SHP-1 gene (PTPN6) in a public TNBC dataset (TCGA) and found that higher SHP-1 mRNA expression is associated with better overall survival in TNBC patients. Sequential combination of docetaxel and SC-43 in vitro showed enhanced anti-proliferation and apoptosis associated with decreased p-STAT3 and decreased STAT3-downstream effector cyclin D1 in the TNBC cell lines MDA-MB-231, MDA-MB-468, and HCC-1937. Ectopic expression of STAT3 reduced the increased cytotoxicity induced by the combination therapy. In addition, this sequential combination showed enhanced SHP-1 activity compared to SC-43 alone. Furthermore, the combination treatment-induced apoptosis was attenuated by small interfering RNA (siRNA) against SHP-1 or by ectopic expression of SHP-1 mutants that caused SC-43 to lose its SHP-1 agonist capability. Moreover, combination of docetaxel and SC-43 showed enhanced tumor growth inhibition compared to single-agent therapy in mice bearing MDA-MB-231 tumor xenografts. Our results suggest that the novel SHP-1 agonist SC-43 enhanced docetaxel-induced cytotoxicity by SHP-1 dependent STAT3 inhibition in human triple negative breast cancer cells. TNBC patients with high SHP-1 expressions show better survival. Docetaxel combined with SC-43 enhances cell apoptosis and reduces p-STAT3. SHP-1 inhibition reduces the enhanced effect of docetaxel-SC-43 combination. Docetaxel-SC-43 combination suppresses xenograft tumor growth and reduces p-STAT3.TNBC patients with high SHP-1 expressions show better survival. Docetaxel combined with SC-43 enhances cell apoptosis and reduces p-STAT3. SHP-1 inhibition reduces the enhanced effect of docetaxel-SC-43 combination. Docetaxel-SC-43 combination suppresses xenograft tumor growth and reduces p-STAT3.

Enhancing the tumour suppressive activity of protein phosphatase 2A (PP2A) has been suggested to be an anti-leukaemic strategy. KIAA1524 (also termed CIP2A), an oncoprotein inhibiting PP2A, is associated with disease progression in chronic myeloid leukaemia and may be prognostic in cytogenetically normal acute myeloid leukaemia. Here we demonstrated that the selective proteasome inhibitor, carfilzomib, induced apoptosis in sensitive primary leukaemia cells and in sensitive leukaemia cell lines, associated with KIAA1524 protein downregulation, increased PP2A activity and decreased p-Akt, but not with the proteasome inhibition effect of carfilzomib. Ectopic expression of KIAA1524, or pretreatment with the PP2A inhibitor, okadaic acid, suppressed carfilzomib-induced apoptosis and KIAA1524 downregulation in sensitive cells, whereas co-treatment with the PP2A agonist, forskolin, enhanced carfilzomib-induced apoptosis in resistant cells. Mechanistically, carfilzomib affected KIAA1524 transcription through disturbing ELK1 (Elk-1) binding to the KIAA1524 promoter. Moreover, the drug sensitivity and mechanism of carfilzomib in xenograft mouse models correlated well with the effects of carfilzomib on KIAA1524 and p-Akt expression, as well as PP2A activity. Our data disclosed a novel drug mechanism of carfilzomib in leukaemia cells and suggests the potential therapeutic implication of KIAA1524 in leukaemia treatment.

Recurrent triple-negative breast cancer (TNBC) needs new therapeutic targets. Src homology region 2 domain-containing phosphatase-1 (SHP-1) can act as a tumor suppressor by dephosphorylating oncogenic kinases. One major target of SHP-1 is STAT3, which is highly activated in TNBC. In this study, we tested a sorafenib analogue SC-60, which lacks angiokinase inhibition activity, but acts as a SHP-1 agonist, in TNBC cells. SC-60 inhibited proliferation and induced apoptosis by dephosphorylating STAT3 in both a dose- and time-dependent manner in TNBC cells (MDA-MB-231, MDA-MB-468, and HCC1937). By contrast, ectopic expression of STAT3 rescued the anticancer effect induced by SC-60. SC-60 also increased the SHP-1 activity, but this effect was inhibited when the N-SH2 domain (DN1) was deleted or with SHP-1 point mutation (D61A), implying that SHP-1 is the major target of SC-60 in TNBC. The use of SC-60 in combination with docetaxel synergized the anticancer effect induced by SC-60 through the SHP-1/STAT3 pathway in TNBC cells. Importantly, SC-60 also displayed a significant antitumor effect in an MDA-MB-468 xenograft model by modulating the SHP-1/STAT3 axis, indicating the anticancer potential of SC-60 in TNBC treatment. Targeting SHP-1/p-STAT3 and the potential combination of SHP-1 agonist with chemotherapeutic docetaxel is a feasible therapeutic strategy for TNBC.

Objectives Cancerous inhibitor of protein phosphatase 2A (CIP2A) has emerged as a therapeutic determinant mediating the anti-cancer effects of several new agents. We investigated the efficacy and mechanism of TD52, an erlotinib derivative with minimal p-EGFR inhibition but significant CIP2A downregulation, in triple-negative breast cancer (TNBC) cells. Methods TNBC lines were used for in vitro studies. Cell apoptosis was examined by flow cytometry and Western blot. Signal transduction pathways in cells were assessed by Western blot. In vivo efficacy of TD52 was tested in xenograft nude mice. Results We explored the CIP2A mRNA expression in a publically available database and found that higher levels of CIP2A mRNA is associated with worse recurrence-free survival in patients with TNBC. TD52-enhanced apoptosis accompanied with CIP2A downregulation and CIP2A overexpression protected cells from TD52-mediated apoptosis. The activity of protein phosphatase 2A (PP2A) was also increased in TD52-treated cells. TD52-induced apoptosis and p-Akt downregulation was attenuated by PP2A antagonist okadaic acid. Furthermore, TD52 indirectly downregulated CIP2A transcription via disturbing the binding of Elk1 to the CIP2A promoter. Importantly, TD52 showed anti-tumour activity in mice bearing TNBC xenograft tumours and downregulated CIP2A and p-Akt in these xenografted tumours. Interestingly, higher Elk1 mRNA expression was also associated with worse recurrence-free survival in TNBC patients by Kaplan–Meier survival analysis. Conclusion Our findings indicated that EGFR-independent pharmacological modulation on Elk1/CIP2A signalling mediates the apoptotic effect of TD52 in TNBC cells, suggesting the potential therapeutic strategy.

Background. Late onset depression (LOD) often occurs in the context of vascular disease and may be associated with risk of dementia. Aspirin is widely used to reduce the risk of cardiovascular disease and stroke. However, its role in patients with LOD and risk of dementia remains inconclusive. Materials and Methods. A population-based study was conducted using data from National Health Insurance of Taiwan during 1996–2009. Patients fulfil diagnostic criteria for LOD with or without subsequent dementia (incident dementia) and among whom users of aspirin (75 mg daily for at least 6 months) were identified. The time-dependent Cox proportional hazards model was applied for multivariate analyses. Propensity scores with the one-to-one nearest-neighbor matching model were used to select matching patients. Cumulative incidence of incident dementia after diagnosis of LOD was calculated by Kaplan–Meier Method. Results. A total of 6028 (13.4%) and 40,411 (86.6%) patients were defined as, with and without diagnosis of LOD, among whom 2,424 (41.9%) were aspirin users. Patients with LOD had more comorbidities such as cardiovascular diseases, diabetes, and hypertension comparing to those without LOD. Among patients with LOD, aspirin users had lower incidence of subsequent incident dementia than non-users (Hazard Ratio = 0.734, 95% CI 0.641–0.841, p<0.001). After matching aspirin users with non-users by propensity scores-matching method, the cumulative incidence of incident dementia was significantly lower in aspirin users of LOD patients (p=0.022). Conclusions. Aspirin may be associated with a lower risk of incident dementia in patients with LOD. This beneficial effect of aspirin in LOD patients needs validation in prospective clinical trials and our results should be interpreted with caution.

High-content phenotypic screening has become the approach of choice for drug discovery due to its ability to extract drug-specific multi-layered data. In the field of epigenetics, such screening methods have suffered from a lack of tools sensitive to selective epigenetic perturbations. Here we describe a novel approach, Microscopic Imaging of Epigenetic Landscapes (MIEL), which captures the nuclear staining patterns of epigenetic marks and employs machine learning to accurately distinguish between such patterns. We validated the MIEL platform across multiple cells lines and using dose-response curves, to insure the fidelity and robustness of this approach for high content high throughput drug discovery. Focusing on noncytotoxic glioblastoma treatments, we demonstrated that MIEL can identify and classify epigenetically active drugs. Furthermore, we show MIEL was able to accurately rank candidate drugs by their ability to produce desired epigenetic alterations consistent with increased sensitivity to chemotherapeutic agents or with induction of glioblastoma differentiation., eLife digest Each cell contains a complete copy of a person’s genes coded in their DNA. However, for a cell to perform its specific role, it only needs a small fraction of this genetic information. The mechanisms that control which genes a cell is using fall under the umbrella of ‘epigenetics’ (meaning beyond genetics). These mechanisms involve changes in the way that DNA is organized inside the cell nucleus and changes in how accessible different parts of the genome are to various cellular components. DNA is long and fragile so, to maintain its integrity, it is wrapped around protein complexes called histones. Adding chemical modifications to histones is one of the main epigenetic mechanisms that cells use to regulate which genes are turned on and off. Several methods allow researchers to read patterns of histone modification and use this information to derive what state a cell is in and how it might behave. Improving these methods is of particular interest in drug development, where this information could reveal the effects, and side-effects, of new treatments. Unfortunately, existing techniques are costly in both time and money, and they are not well suited to analyzing epigenetic changes caused by the large numbers of compounds tested during drug development. To overcome this barrier, Farhy et al. have developed a new system called ‘Microscopic Imaging of the Epigenetic Landscape’ (MIEL for short). The system allows them to quickly analyze the epigenetic changes caused by each of a large number of different chemical compounds when they are used on cells. MIEL tags different histone modifications by staining each with a different color, and then uses automated microscopy to produce images. It then extracts information from these images using advanced image analysis tools. The changes induced by different drugs can then be compared and categorized using machine learning algorithms. To test the MIEL system, Farhy et al. grew brain cancer cells (derived from human tumors) in the lab, and treated them with compounds that target proteins involved in histone modifications. Using their newly created pipeline, Farhy et al. were able to identify the unique epigenetic changes caused by these compounds, and train the system to correctly predict which type of drug the cells had been treated with. In a different set of experiments Farhy et al. demonstrate the utility of their new pipeline in identifying drugs that induce a set of epigenetic changes associated with a reduced ability to regrow tumors. This new system could help screen thousands of compounds for their epigenetic effects, which could aid the design of new treatments for many diseases.

With the advent of automatic cell imaging and machine learning, high-content phenotypic screening has become the approach of choice for drug discovery because it can extract drug-specific multi-layered data, which could be compared to known profiles. In the field of epigenetics, such screening approaches have suffered from a lack of tools sensitive to selective epigenetic perturbations. Here we describe a novel approach, Microscopic Imaging of Epigenetic Landscapes (MIEL), which captures the nuclear staining patterns of epigenetic marks (e.g., acetylated and methylated histones) and employs machine learning to accurately distinguish between such patterns. We validated the fidelity and robustness of the MIEL platform across multiple cells lines using dose-response curves. We employed MIEL to uncover the mechanism by which bromodomain inhibitors synergize with temozolomide-mediated killing of human glioblastoma lines. To explore alternative, non-cytotoxic, glioblastoma treatment, we screen the Prestwick chemical library and documented the power of MIEL platform to identify epigenetically active drugs and accurately rank them according to their ability to produce epigenetic and transcriptional alterations consistent with the induction of glioblastoma differentiation.

Purpose: Stimulator of interferon genes (STING) is an upstream regulator of interferon and NF-κB, the key mediators of immune and inflammatory responses associated with cancer, triggered by cytosolic DNA. Downregulation of STING in breast tumor tissues has been reported. STING elicits caspase-8-dependent cell apoptosis via NF-κB in breast cancer cells. STING agonists are considered as potent anti-cancer agents. The current study aims to assess the biological functions of STING in breast cancer progression. Experimental design: Data of mRNA expression levels and recurrence-free survival in patients with breast cancer from The Cancer Genome Atlas and Kaplan Meier-plotter databases were analyzed. Transwell assays were used to evaluate cell migrated and invaded abilities. Sphere formation was performed in stem cell-selective medium and ultra-low attachment plates. Two STING agonists, ADU-S100 and diABZI STING agonist-1 trihydrochloride, were used for in vitro studies. The molecular events were examined by Western blot analysis. Results: Data from public database showed that lower transcripts levels of STING in Her2 and luminal B breast cancer tissues than normal breast tissues. Luminal-type breast cancer cell lines harbored low STING transcripts compared with other subtypes. Importantly, breast cancer patients with low expression levels of STING was associated with worse recurrence-free survival. Results of immunoblotting indicated that breast cancer cell lines have lower STING protein expressions than normal MCF10A cells. Knockdown of STING led to aggressive phenotypes of MCF10A and BT474 cells. In contrast, overexpression of STING suppressed the abilities of migration, invasion and sphere formation of MCF7 and BT474 cells. The markers of epithelial were increased while the markers of mesenchymal and stemness were decreased by STING overexpression. STING agonist slightly suppressed cell viability of breast cancer cells but not MCF10A cells. Treatment of STING agonists reduced cell migrated ability with upregulation of E-cadherin and γ-catenin. Conclusion: These findings suggest that STING serves tumor-suppressive effects in breast cancer. Citation Format: Chun-Yu Liu, Ji-Lin Chen, Chun-Teng Huang, Pei-Yi Chu, Mei-Fang Tseng, Yu-Hsuan Lee, Wan-Lun Wang, Ling-Ming Tseng. Pleiotropic anti-cancer effects of STING in estrogen receptor positive breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2486.

Triple-negative breast cancer (TNBC) is a complex disease associated with the aggressive phenotype and poor prognosis. TNBC harbors heterogeneous molecular subtypes with no approved specific targeted therapy. It has been reported that HER receptors are overexpressed in breast cancer including TNBC. In this study, we evaluated the efficacy of varlitinib, a reversible small molecule pan-HER inhibitor in TNBC. Our results showed that varlitinib reduced cell viability and induced cell apoptosis in most TNBC cell lines but not in MDA-MB-231 cells. MEK and ERK inhibition overcame resistance to varlitinib in MDA-MB-231 cells. Varlitinib inhibited HER signaling which led to inhibition of migration, invasion and mammosphere formation of TNBC cells as well as significant suppression of tumor growth of MDA-MB-468 xenograft mouse model. In summary, these results suggest that HER signaling plays an important role in TNBC progression and that pan-HER inhibition is potentially an effective treatment for TNBC patients.

The Zika virus (ZIKV) global epidemic prompted the World Health Organization to declare it a 2016 Public Health Emergency of International Concern. The overwhelming experience over the past several years teaches us that ZIKV and the associated neurological complications represent a long-term world-wide challenge to public health. Although the number of ZIKV cases in the Western Hemisphere has dropped since 2016, the need for basic research and anti-ZIKV drug development remains strong. Re-emerging viruses like ZIKV are an ever-present threat in the 21st century where fast transcontinental travel lends itself to viral epidemics. Here, we first present the origin story for ZIKV and review the rapid progress researchers have made toward understanding of the ZIKV pathology and in the design, re-purposing, and testing-particularly in vivo-drug candidates for ZIKV prophylaxis and therapy ZIKV. Quite remarkably, a short, but intensive, drug-repurposing effort has already resulted in several readily available FDA-approved drugs that are capable of effectively combating the virus in infected adult mouse models and, most importantly, in both preventing maternal-fetal transmission and severe microcephaly in newborns in pregnant mouse models.

Adjuvant tamoxifen reduces the recurrence rate of estrogen receptor (ER)-positive breast cancer. Previous in vitro studies have suggested that tamoxifen can affect the cancerous inhibitor of protein phosphatase 2A (CIP2A)/protein phosphatase 2A (PP2A)/phosphorylation Akt (pAkt) signaling in ER-negative breast cancer cells. In addition to CIP2A, SET nuclear proto-oncogene (SET) oncoprotein is another intrinsic inhibitor of PP2A, participating in cancer progression. In the current study, we explored the clinical significance of SET, CIP2A, PP2A, and Akt in patients with ER-positive breast cancer receiving adjuvant tamoxifen. A total of 218 primary breast cancer patients receiving adjuvant tamoxifen with a median follow-up of 106 months were analyzed, of which 17 (7.8%) experienced recurrence or metastasis. In an immunohistochemical (IHC) stain, SET overexpression was independently associated with worse recurrence-free survival (RFS) (hazard ratio = 3.72, 95% confidence interval 1.26–10.94, p = 0.017). In silico analysis revealed mRNA expressions of SET, PPP2CA, and AKT1 significantly correlated with worse RFS. In vitro, SET overexpression reduced tamoxifen-induced antitumor effects and drove luciferase activity in an Estrogen receptor element (ERE)-dependent manner. In conclusion, SET is a prognostic biomarker in patients with primary ER-positive breast cancer receiving adjuvant tamoxifen and may contribute to the failure of the tamoxifen treatment by modulating the ER signaling. Our study warrants further investigation into the potential role of SET in ER-positive breast cancer.

The mechanisms by which noninvasive vagal nerve stimulation (nVNS) affect central and peripheral neural circuits that subserve pain and autonomic physiology are not clear, and thus remain an area of intense investigation. Effects of nVNS vs sham stimulation on subject responses to five noxious thermal stimuli (applied to left lower extremity), were measured in 30 healthy subjects (n=15 sham and n=15 nVNS), with fMRI and physiological galvanic skin response (GSR). With repeated noxious thermal stimuli a group × time analysis showed a significantly (p < .001) decreased response with nVNS in bilateral primary and secondary somatosensory cortices (SI and SII), left dorsoposterior insular cortex, bilateral paracentral lobule, bilateral medial dorsal thalamus, right anterior cingulate cortex, and right orbitofrontal cortex. A group × time × GSR analysis showed a significantly decreased response in nVNS group (p < .0005) in bilaterally in SI, lower and mid medullary brainstem, and inferior occipital cortex. Finally, nVNS treatment showed decreased activity in pronociceptive brainstem nuclei (e.g. the reticular nucleus and rostral ventromedial medulla) and key autonomic integration nuclei (e.g. the rostroventrolateral medulla, nucleus ambiguous, and dorsal motor nucleus of the vagus nerve). In aggregate, noninvasive vagal nerve stimulation reduced the physiological response to noxious thermal stimuli and impacted neural circuits important for pain processing and autonomic output.

The resistance of Glioblastoma (GBM) to conventional cytotoxic drugs has prompted novel therapeutic strategies, including differentiating tumor propagating cells (TPCs) into less tumorigenic cells using small molecule inducers of TPC differentiation. However, high-throughput screening for such molecules is hampered by the lack of robust markers of GBM differentiation. To obtain a signature of differentiated TPCs, we developed “Microscopic Imaging of Epigenetic Landscapes” (MIEL), which captures patterns of nuclear staining for epigenetic marks to derive feature-fingerprints of individual cells. We confirmed MIEL’s ability to accurately distinguish multiple cell fates and identified a multiparametric epigenetic signature of differentiated TPCs. Critically, we validated epigenetic imaging-based signature using global gene expression thus providing the proof of principle for the MIEL’s ability to select and prioritize small molecules, which induce TPC differentiation.

The re-emergence of Zika virus (ZIKV) and Ebola virus (EBOV) poses serious and continued threats to the global public health. Effective therapeutics for these maladies is an unmet need. Here, we show that emetine, an anti-protozoal agent, potently inhibits ZIKV and EBOV infection with a low nanomolar half maximal inhibitory concentration (IC50) in vitro and potent activity in vivo. Two mechanisms of action for emetine are identified: the inhibition of ZIKV NS5 polymerase activity and disruption of lysosomal function. Emetine also inhibits EBOV entry. Cephaeline, a desmethyl analog of emetine, which may be better tolerated in patients than emetine, exhibits a similar efficacy against both ZIKV and EBOV infections. Hence, emetine and cephaeline offer pharmaceutical therapies against both ZIKV and EBOV infection.

The outbreak of the Zika virus (ZIKV) has been associated with increased incidence of congenital malformations. Although recent efforts have focused on vaccine development, treatments for infected individuals are needed urgently. Sofosbuvir (SOF), an FDA-approved nucleotide analog inhibitor of the Hepatitis C (HCV) RNA-dependent RNA polymerase (RdRp) was recently shown to be protective against ZIKV both in vitro and in vivo. Here, we show that SOF protected human neural progenitor cells (NPC) and 3D neurospheres from ZIKV infection-mediated cell death and importantly restored the antiviral immune response in NPCs. In vivo, SOF treatment post-infection (p.i.) decreased viral burden in an immunodeficient mouse model. Finally, we show for the first time that acute SOF treatment of pregnant dams p.i. was well-tolerated and prevented vertical transmission of the virus to the fetus. Taken together, our data confirmed SOF-mediated sparing of human neural cell types from ZIKV-mediated cell death in vitro and reduced viral burden in vivo in animal models of chronic infection and vertical transmission, strengthening the growing body of evidence for SOF anti-ZIKV activity.

We tested the efficacy of lapatinib, a dual tyrosine kinase inhibitor which interrupts the HER2 and epidermal growth factor receptor (EGFR) pathways, in a panel of triple-negative breast cancer (TNBC) cells, and examined the drug mechanism. Lapatinib showed an anti-proliferative effect in HCC 1937, MDA-MB-468, and MDA-MB-231 cell lines. Lapatinib induced significant apoptosis and inhibited CIP2A and p-Akt in a dose and time-dependent manner in the three TNBC cell lines. Overexpression of CIP2A reduced lapatinib-induced apoptosis in MDA-MB-468 cells. In addition, lapatinib increased PP2A activity (in relation to CIP2A inhibition). Moreover, lapatinib-induced apoptosis and p-Akt downregulation was attenuated by PP2A antagonist okadaic acid. Furthermore, lapatinib indirectly decreased CIP2A transcription by disturbing the binding of Elk1 to the CIP2A promoter. Importantly, lapatinib showed anti-tumor activity in mice bearing MDA-MB-468 xenograft tumors, and suppressed CIP2A as well as p-Akt in these xenografted tumors. In summary, inhibition of CIP2A determines the effects of lapatinib-induced apoptosis in TNBC cells. In addition to being a dual tyrosine kinase inhibitor of HER2 and EGFR, lapatinib also inhibits CIP2A/PP2A/p-Akt signaling in TNBC cells.

The outbreak of the Zika virus (ZIKV) has been associated with increased incidence of congenital malformations. Although recent efforts have focused on vaccine development, treatments for infected individuals are needed urgently. Sofosbuvir (SOF), an FDA-approved nucleotide analog inhibitor of the Hepatitis C (HCV) RNA-dependent RNA polymerase (RdRp) was recently shown to be protective against ZIKV both in vitro and in vivo. Here, we show that SOF protected human neural progenitor cells (NPC) and 3D neurospheres from ZIKV infection-mediated cell death and importantly restored the antiviral immune response in NPCs. In vivo, SOF treatment post-infection (p.i.) decreased viral burden in an immunodeficient mouse model. Finally, we show for the first time that acute SOF treatment of pregnant dams p.i. was well-tolerated and prevented vertical transmission of the virus to the fetus. Taken together, our data confirmed SOF-mediated sparing of human neural cell types from ZIKV-mediated cell death in vitro and reduced viral burden in vivo in animal models of chronic infection and vertical transmission, strengthening the growing body of evidence for SOF anti-ZIKV activity.

Background Currently, the association between human immunodeficiency virus (HIV) infection and subsequent development of autoimmune hemolytic anemia (AIHA) remains unclear. This nationwide population-based cohort study aimed to determine the association between incident AIHA and HIV infection in Taiwan. Methods During 2000-2012, we identified people aged ≧15 years living with HIV (PLWH) from the Taiwan Centers for Disease Control HIV Surveillance System. Individuals were considered to be infected with HIV on the basis of positive results of an HIV type 1 Western blot. Age- and sex-matched controls without HIV infection were selected from the Taiwan National Health Insurance Research Database for comparison. All patients were followed until 31 December 2012 and observed for occurrence of AIHA. Results Of 171468 subjects (19052 PLWH and 152416 controls), 30 (0.02%) had incident AIHA during a mean follow-up of 5.45 years, including 23 PLWH (0.12%) and 7 controls (0.01%). After adjustment for age, sex, and comorbidities, HIV infection was found to be an independent risk factor of incident AIHA (adjusted hazard ratio, 20.9; 95% confidence interval, 8.34-52.3). Moreover, PLWH who were receiving highly active antiretroviral therapy were more likely to develop AIHA than those who were not receiving these drugs (adjusted hazard ratio, 16.2; 95% confidence interval, 3.52-74.2). Conclusions Our study suggests that HIV infection is an independent risk factor for incident AIHA.

International audience; Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix–loop–helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation—Tcf3 and Foxa2—and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1–4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

Purpose: Tryptophan-kynurenine pathway involves in inflammation, immune response and tumorigenesis, in which kynurenine 3-monooxygenase (KMO), an outer mitochondrial membrane protein, mediating kynurenine metabolism. Previous studies indicated KMO showed increased activity in breast cancer. Triple-negative breast cancer (TNBC) tumors exhibited elevated levels of tryptophan metabolites compared to estrogen receptor positive breast cancers. We aimed to study the role of KMO in human TNBC. Experimental design: The gene alterations and transcripts of enzymes in kynurenine metabolism were analyzed from The Cancer Genome Atlas (TCGA) database. Immunohistochemical staining for KMO was performed and a H-score was assigned to quantify protein expression. Epithelial-mesenchymal transition (EMT) phenotypes were examined by transwell assay and EMT markers expressions. Stemness properties were assessed by mammosphere assay and pluripotent genes expressions. The molecular events were analyzed by Western blot, quantitative real-time PCR and luciferase reporter assay. Tumor growth and metastasis were conducted in nude mice and NOD-SCID mice by subcutaneous and tail vein injection respectively. Results: TCGA databases showed KMO but not KYNU and KAT2 was amplified in breast cancer. Both the data from TCGA and our in-house IHC-based tissue-microarray exhibited increased KMO expression in TNBC compared to normal tissue. In vitro, overexpression of KMO in TNBC cells resulted in increased cell growth and colony formation. The abilities migration and invasion as well as EMT markers expressions of TNBC cells were elevated by KMO overexpression. In addition, KMO increased mammosphere formation, pluripotent genes expressions and promoter activities. However, inhibition of KMO enzymatic activity by KMO inhibitors did not affect cancer progression or mitochondrial respiration of TNBC cells. KMO upregulated β-catenin, the upstream regulator of pluripotent genes, CD44 and Nanog expressions. Mechanistically, data showed KMO expressed in both cytosol and nuclear fractions and was associated with β-catenin. KMO enhanced pluripotent genes expressions through β-catenin upregulation. Importantly, KMO knockdown suppressed tumor growth and expressions of β-catenin, CD44 and Nanog in TNBC tumors. Moreover, KMO knockout significantly decreased lung metastasis in vivo. Conclusion: Our data indicated KMO can play an oncogenic role in TNBC, acting as a novel regulator of pluripotent genes via β-catenin and promoted TNBC progression. Citation Format: Chun-Yu Liu, Tzu-Ting Huang, Ji-Lin Chen, Chia-Han Lee, Wan-Lun Wang, Ka-Yi Lau, Chun-Teng Huang, Pei-Yi Chu, Hsin-Chen Lee, Ling-Ming Tseng. Kynurenine 3-monooxygenase (KMO) acts as a novel oncoprotein in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1711.

Antibiotics susceptibility of blood stream infections. (DOCX 16Â kb)

Objectives Triple negative breast cancer (TNBC) lacks specific drug targets and remains challenging. Palbociclib, a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor is approved for metastatic estrogen receptor (ER)-positive and human epithermal growth factor 2 (HER2)-negative breast cancer. The nature of cell cycle inhibition by palbociclib suggests its potential in TNBC cells. Retinoblastoma (RB, a known substrate of CDK4/6) pathway deregulation is a frequent occurrence in TNBC and studies have revealed that pharmacological CDK4/6 inhibition induces a cooperative cytostatic effect with doxorubicin in RB-proficient TNBC models. In addition, recent studies reported that anti-androgen therapy shows preclinical efficacy in androgen-receptor (AR)-positive TNBC cells. Here we examined the effect of palbociclib in combination with an anti-androgen enzalutamide in TNBC cells. Method MDA-MB-453, BT-549, MDA-MB-231 and MDA-MB-468 TNBC cell lines were used for in vitro studies. Protein expressions were assessed by Western blot analysis. Cytostatic effect was examined by MTT assay. Cell cycle and apoptosis were examined by flow cytometry. Results Palbociclib showed inhibitory effect in RB-proficient TNBC cells, and enzalutamide inhibited cell viability in AR-positive TNBC cells. Enzalutamide treatment could enhance the palbociclib-induced cytostatic effect in AR-positive/RB-proficient TNBC cells. In addition, palbociclib-mediated G1 arrest in AR-positive/RB-proficient TNBC cells was attenuated by RB knockdown. Conclusion Our study provided a preclinical rationale in selecting patients who might have therapeutic benefit from combining CDK4/6 inhibitors with AR antagonists.

Triple-negative breast cancer (TNBC) remains difficult to treat and urgently needs new therapeutic options. Nintedanib, a multikinase inhibitor, has exhibited efficacy in early clinical trials for HER2-negative breast cancer. In this study, we examined a new molecular mechanism of nintedanib in TNBC. The results demonstrated that nintedanib enhanced TNBC cell apoptosis, which was accompanied by a reduction of p-STAT3 and its downstream proteins. STAT3 overexpression suppressed nintedanib-mediated apoptosis and further increased the activity of purified SHP-1 protein. Moreover, treatment with either a specific inhibitor of SHP-1 or SHP-1-targeted siRNA reduced the apoptotic effects of nintedanib, which validates the role of SHP-1 in nintedanib-mediated apoptosis. Furthermore, nintedanib-induced apoptosis was attenuated in TNBC cells expressing SHP-1 mutants with constantly open conformations, suggesting that the autoinhibitory mechanism of SHP-1 attenuated the effects of nintedanib. Importantly, nintedanib significantly inhibited tumor growth via the SHP-1/p-STAT3 pathway. Clinically, SHP-1 levels were downregulated, whereas p-STAT3 was upregulated in tumor tissues, and SHP-1 transcripts were associated with improved disease-free survival in TNBC patients. Our findings revealed that nintedanib induces TNBC apoptosis by acting as a SHP-1 agonist, suggesting that targeting STAT3 by enhancing SHP-1 expression could be a viable therapeutic strategy against TNBC.