Dual inhibition of AKT‐mTOR and AR signaling by targeting HDAC3 in PTEN‐ or SPOP‐mutated prostate cancer

Abstract AKT‐mTOR and androgen receptor (AR) signaling pathways are aberrantly activated in prostate cancer due to frequent PTEN deletions or SPOP mutations. A clinical barrier is that targeting one of them often activates the other. Here, we demonstrate that HDAC3 augments AKT phosphorylation in prostate cancer cells and its overexpression correlates with AKT phosphorylation in patient samples. HDAC3 facilitates lysine‐63‐chain polyubiquitination and phosphorylation of AKT, and this effect is mediated by AKT deacetylation at lysine 14 and 20 residues and HDAC3 interaction with the scaffold protein APPL1. Conditional homozygous deletion of Hdac3 suppresses prostate tumorigenesis and progression by concomitant blockade of AKT and AR signaling in the Pten knockout mouse model. Pharmacological inhibition of HDAC3 using a selective HDAC3 inhibitor RGFP966 inhibits growth of both PTEN‐deficient and SPOP‐mutated prostate cancer cells in culture, patient‐derived organoids and xenografts in mice. Our study identifies HDAC3 as a common upstream activator of AKT and AR signaling and reveals that dual inhibition of AKT and AR pathways is achievable by single‐agent targeting of HDAC3 in prostate cancer

Prostate cancer-associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4

The bromodomain and extra-terminal (BET) family of proteins, comprised of four members including BRD2, BRD3, BRD4 and the testis-specific isoform BRDT, largely function as transcriptional co-activators 1–3 and play critical roles in various cellular processes, including cell cycle, apoptosis, migration and invasion 4,5. As such, BET proteins enhance the oncogenic functions of major cancer drivers by either elevating their expression such as c-Myc in leukemia 6,7 or by promoting transcriptional activities of oncogenic factors such as AR and ERG in the prostate cancer setting 8. Pathologically, BET proteins are frequently overexpressed and clinically linked to various types of human cancers 5,9,10, therefore pursued as attractive therapeutic targets for selective inhibition in patients. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed 11,12 and shown promising outcomes in early clinical trials. Despite resistance to BET inhibitor has been documented in pre-clinical models 13–15 the molecular mechanisms underlying acquired resistance are largely unknown. Here, we report that Cullin 3SPOP earmarks BET proteins including BRD2, BRD3 and BRD4 for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of BET proteins, leading to their elevated abundance in SPOP-deficient prostate cancer. As a result, prostate cancer cells and prostate cancer patient-derived organoids harboring SPOP mutations are more resistant to BET inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor suppressor role of SPOP in prostate cancer by negatively controlling BET protein stability, and also provide a molecular mechanism for BET inhibitor resistance in prostate cancer patients bearing SPOP mutations

An Optimized Tobacco Hairy Root Induction System for Functional Analysis of Nicotine Biosynthesis-Related Genes

Rhizobium rhizogenes-mediated plant hairy root induction is a convenient method for functional study of root-specific genes. To develop an optimized tobacco hairy root induction system and study gene function in nicotine biosynthesis, we investigated hairy root induction by three R. rhizogenes strains, R1601, K599, and LBA9402, on different media with leaf discs from plants of different ages, and we observed that the strain LBA9402 used for explant infection exhibited the highest hairy root induction rate with 4 and 8 week old leaf discs of the tobacco ‘Coker176’ on 2/3MS medium, and it could also be used as a cargo delivering foreign genes to hairy roots. Overexpression of MsSPL12 gene, an alfalfa (Medicago sativa) SQUAMOSA promoter binding protein-like (SPL) transcription factor, significantly improved nicotine production in transgenic hairy roots, reaching 1.38–1.85 mg/g compared to 0.5 mg/g of the controls. Expression analysis of the nicotine biosynthesis and transport-related genes responding to methyl-jasmonate (MeJA) treatment revealed a significant upregulation of NtMPO2 responsible for increased nicotine biosynthesis in MsSPL12 transgenic hairy roots. Our results establish a high-throughput approach for gene functional characterization in the hairy roots of a tobacco elite cultivar, ‘Coker176’, as well as suggest a system for efficiently manipulating tobacco nicotine biosynthesis

Overexpression of miR-506-3p Aggravates DBP-Induced Testicular Oxidative Stress in Rats by Downregulating ANXA5 via Nrf2/HO-1 Signaling Pathway

Background. Di-N-butylphthalate (DBP) is a kind of unique endocrine toxicity linked to hormonal disruptions that affects the male reproductive system and has given rise to more and more attention. However, the mechanism of DBP-induced testicular injury remains unclear. Here, the objective of this study was to investigate the potential molecular mechanism of miR-506-3p in DBP-induced rat testicular oxidative stress injury via ANXA5 (Annexin A5)/Nrf2/HO-1 signaling pathway. Methods. In vivo, a total of 40 adolescent male rats were treated from 2 weeks with 800 mg/kg/day of DBP in 1 mL/kg corn oil administered daily by oral gavage. Among them, some rats were also injected subcutaneously with 2 nmol agomir-506-3p and/or 10 nmol recombinant rat ANXA5. The pathomorphological changes of testicular tissue were assessed by histological examination, and the antioxidant factors were evaluated. Subsequently, ANXA5, Nrf2, and its dependent antioxidant enzymes, such as HO-1, NQO1, and GST, were detected by Western blotting or immunohistochemical staining. In vitro, TM3 cells (Leydig cells) were used to detect the cell activity by CCK-8 and the transfection in the DBP-treated group. Results. Differentially expressed miRNAs between the DBP-treated and normal rats were analyzed, and qRT-PCR showed miR-506-3p was highly expressed in testicular tissues of the DBP-treated rats. DBP-treated rats presented severe inflammatory infiltration, increased abnormal germ cells, and missed cell layers frequently existed in seminiferous tubules, resulted in oxidative stress and decreased testicular function. Meanwhile, upregulation of miR-506-3p aggravated the above changes. In addition, miR-506-3p directly bound to ANXA5, and overexpression of miR-506-3p could reduce the ANXA5 expression and also decrease the protein levels of Nrf2/HO-1 signaling pathway. Additionally, we found that recombinant rat ANXA5 reversed the DBP-treated testicular oxidative stress promoting injury of miR-506-3p in rats. In vivo results were reproduced in in vitro experiments. Conclusions. This study provided evidence that miR-506-3p could aggravate the DBP-treated testicular oxidative stress injury in vivo and in vitro by inhibiting ANXA5 expression and downregulating Nrf2/HO-1 signaling pathway, which might provide novel understanding in DBP-induced testicular injury therapy

Protective Effects of the Segmental Renal Artery Clamping Technique on Ischemia-Reperfusion Injury in db/db Diabetic Mice

Renal ischemia-reperfusion (I/R) injury is inevitable in partial nephrectomy and other kidney surgeries, with a higher incidence in patients with renal insufficiency. This study aimed to investigate the protective effects of precise segmental renal artery clamping (SRAC) against renal I/R injury in db/db diabetic mice, compared with conventional renal artery clamping (RAC). Grape seed extract, a powerful free radical scavenger, was administered to diabetic mice for 4 weeks before operation in subgroups (30 mg/kg/d). The unilateral renal pedicle was ligatured, and I/R injury to the contralateral kidney was induced (ischemia for 30 min followed by reperfusion for 24 h). Blood glucose value, creatinine, blood urea nitrogen, and urine microalbumin/urine creatinine ratio increased gradually and showed no preoperative statistical differences among six subgroups. These parameters were significantly lower in the SRAC than in the RAC group 24 h postoperatively. Moreover, the nonischemic area in the SRAC group expressed less KIM-1 and TNF-α mRNA and also revealed minor histopathological damage induced by I/R. These findings suggest that SRAC effectively reduces early renal injury induced by I/R and accelerates the recovery of renal function in diabetic mice. Thus, SRAC may be an ideal technique in partial nephrectomy, especially for patients with diabetic nephropathy and other renal insufficiencies

Study of the preparation of Cu-TiC composites by reaction of soluble Ti and ball-milled carbon coating TiC

In this work, Cu-TiC composites have been successfully prepared by reaction of soluble Ti and carbon coating TiC. Firstly, the ball milling of graphite and TiC mixtures is used to obtain the carbon coating TiC which has fine size and improved reaction activity. After adding the ball milled carbon coating TiC into Cu–Ti melts, the soluble Ti will easily react with the carbon coating to form TiC. This process will also improve the wettability between Cu melts and TiC core. As a result, besides the TiC prepared by reaction of soluble Ti and carbon coating, the ball milled TiC will also be brought into the melts. Some of these ball-milled TiC particles will go on being coated by the formed TiC from the reaction of Ti and the coating carbon and left behind in the composites. However, most of TiC core will be further reacted with the excessive Ti and be transformed into the newly formed TiC with different stoichiometry. The results indicate that it is a feasible method to synthesize TiC in Cu melts by reaction of soluble Ti and ball-milled carbon coating TiC. Keywords: Composites, carbon coating TiC, Cu alloys, Ball-millin