Gut microbiome causal impacts on the prognosis of breast cancer: a Mendelian randomization study.

BACKGROUND: Growing evidence has shown that gut microbiome composition is associated with breast cancer (BC), but the causality remains unknown. We aimed to investigate the link between BC prognosis and the gut microbiome at various oestrogen receptor (ER) statuses. METHODS: We performed a genome-wide association study (GWAS) to analyse the gut microbiome of BC patients, the dataset for which was collected by the Breast Cancer Association Consortium (BCAC). The analysis was executed mainly via inverse variance weighting (IVW); the Mendelian randomization (MR) results were verified by heterogeneity tests, sensitivity analysis, and pleiotropy analysis. RESULTS: Our findings identified nine causal relationships between the gut microbiome and total BC cases, with ten and nine causal relationships between the gut microbiome and ER-negative (ER-) and ER-positive (ER+) BC, respectively. The family Ruminococcaceae and genus Parabacteroides were most apparent among the three categories. Moreover, the genus Desulfovibrio was expressed in ER- BC and total BC, whereas the genera Sellimonas, Adlercreutzia and Rikenellaceae appeared in the relationship between ER + BC and total BC. CONCLUSION: Our MR inquiry confirmed that the gut microbiota is causally related to BC. This further explains the link between specific bacteria for prognosis of BC at different ER statuses. Considering that potential weak instrument bias impacts the findings and that the results are limited to European females due to data constraints, further validation is crucial.

Assessment of the safety and efficacy of combination chemotherapy and PD-1/PD-L1 inhibitor treatment of breast cancer: A meta-analysis.

BACKGROUND: As the efficacy of programmed cell death-1/programmed death-ligand 1 (PD-1/PD-L1) inhibitors combined with chemotherapy in curing breast cancer is still controversial, this meta-analysis compares the efficacy and safety of PD-1/PD-L1 inhibitors combined with chemotherapy and chemotherapy alone in the treatment of breast cancer, which provides guidance for the clinical treatment. METHODS: Relevant studies published as of April 2022 in the various databases including EMBASE, PubMed, and Cochrane Library were selected. Randomized controlled trials (RCTs) in which control patients underwent chemotherapy alone and experimental group patients underwent combination chemotherapy and PD-1/PD-L1 inhibitor treatment were included in this investigation. Investigations without complete information, researches from which information could not be extracted, duplicate articles, animal studies, review articles, and systematic reviews were excluded. STATA 15.1 was employed for all statistical analyses. RESULTS: In total, eight eligible studies were identified, revealing that combination chemotherapy and PD-1/PD-L1 inhibitor treatment was linked to significant increases in progression-free survival (PFS) relative to chemotherapy alone (hazard ratio [HR] = 0.83, 95% confidence interval [CI]: 0.70-0.99, P = 0.032) but not overall survival (HR = 0.92, 95% CI: 0.80-1.06, P = 0.273). Pooled adverse event rates were also increased within the group of combination treatment relative to the chemotherapy group (risk ratio [RR] = 1.08, 95% CI: 1.03-1.14, P = 0.002). Specifically, nausea rates were lesser within the group of combination treatment relative to the group of chemotherapy (RR = 0.48, 95% CI: 0.25-0.92, P = 0.026). Subgroup analyses indicated that the PFS of patients who underwent combination atezolizumab or pembrolizumab and chemotherapy treatment were substantially longer than those of patients who underwent chemotherapy alone (HR = 0.79, 95% CI: 0.69-0.89, P ≤0.001; HR = 0.79, 95% CI: 0.67-0.92, P = 0.002). CONCLUSIONS: The pooled results suggest that combination chemotherapy and PD-1/PD-L1 inhibitor treatment approaches help prolong PFS in breast cancer patients, but have no statistically significant effect on overall survival (OS). Additionally, combination therapy can significantly improve complete response rate (CRR) compared with chemotherapy alone. However, combination therapy was associated with greater rates of adverse events.

OTUD1 chemosensitizes triple-negative breast cancer to doxorubicin by modulating P16 expression.

Chemotherapy remains a critical component of triple-negative breast cancer (TNBC) treatment; however, patients often develop resistance to chemotherapeutic agents. Accumulating evidence indicates that deubiquitylases (DUBs) play pivotal roles in regulating cell proliferation, differentiation, apoptosis, and tumorigenesis. Deubiquitylase OTUD1 is considered a tumor suppressor in various cancers, yet its role in doxorubicin sensitivity in breast cancer patients remains inadequately understood. In this study, we investigated the expression levels and prognostic role of OTUD1 in breast cancer. Our findings demonstrated that OTUD1 was downregulated in TNBC, and lower OTUD1 expression levels were correlated with poor prognosis. We utilized the CCK-8 cell viability assay, flow cytometric analysis, and a TNBC mouse xenograft model to examine the influence of OTUD1 on doxorubicin (DOX) chemotherapy sensitivity in vitro and in vivo. Western blot and immunohistochemistry were employed to explore the correlation between OTUD1 and P16. Our results indicated that upregulation of OTUD1 expression inhibits TNBC cell proliferation and enhances its sensitivity to doxorubicin. Additionally, rescue experiments confirmed that the chemosensitizing effect of OTUD1 overexpression could be reversed by the inhibition of P16. Therefore, our findings reveal that OTUD1 sensitizes TNBC cells to DOX by upregulating P16 expression, suggesting a potential new diagnostic biomarker and therapeutic target for the future treatment of TNBC.

Z-Guggulsterone Induces Cell Cycle Arrest and Apoptosis by Targeting the p53/CCNB1/PLK1 Pathway in Triple-Negative Breast Cancer.

Myrrh is the dried resin of Commiphora Myrrh Engl., which exerts anticancer properties. However, its effects and molecular mechanisms in triple-negative breast cancer (TNBC) remain unclear. In this study, we used network pharmacology to screen Z-Guggulsterone (Z-GS) as a characteristic active component of myrrh. Cell Counting Kit-8 proliferation assays showed that Z-GS inhibited proliferation of the TNBC cell lines MDA-MB-468 and BT-549. Transwell assays also showed that Z-GS inhibited TNBC migration and invasion phenotypes. Our network pharmacology combined with RNA-sequencing analyses showed that Z-GS affected cell cycle and apoptosis processes in TNBC cells, mainly via p53 signaling, to regulate key CCNB1 (cyclin B1), PLK1 (polo-like kinase 1), and p53 targets. Flow cytometry revealed that Z-GS arrested the cell cycle at the G2/M phase and increased apoptosis in TNBC cells. Western blotting and quantitative real-time polymerase chain reaction studies confirmed that Z-GS functioned via the p53-mediated downregulation of CCNB1 and PLK1 expression. In vivo studies showed that Z-GS effectively inhibited TNBC progression. Collectively, Z-GS exhibited potential anti-TNBC activity and may functions via the p53/CCNB1/PLK1 pathway.

Pan-cancer Analysis Identifies AIMP2 as a Potential Biomarker for Breast Cancer.

Introduction: Aminoacyl tRNA synthetase complex interacting with multifunctional protein 2 (AIMP2) is a significant regulator of cell proliferation and apoptosis. Despite its abnormal expression in various tumor types, the specific functions and effects of AIMP2 on tumor immune cell infiltration, proliferation, and migration remain unclear. Materials and Methods: To assess AIMP2's role in tumor immunity, we conducted a pan-cancer multi-database analysis using the Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Cancer Cell Lines Encyclopedia (CCLE) datasets, examining expression levels, prognosis, tumor progression, and immune microenvironment. Additionally, we investigated AIMP2's impact on breast cancer (BRCA) proliferation and migration using cell counting kit 8 (CCK-8) assay, transwell assays, and western blot analysis. Results: Our findings revealed that AIMP2 was overexpressed in 24 tumor tissue types compared to normal tissue and was associated with four tumor stages. Survival analysis indicated that AIMP2 expression was strongly correlated with overall survival (OS) in certain cancer patients, with high AIMP2 expression linked to poorer prognosis in five cancer types. Conclusion: Finally, siRNA-mediated AIMP2 knockdown inhibited BRCA cell proliferation and migration in vitro. In conclusion, our pan-cancer analysis suggests that AIMP2 may play a crucial role in tumor immunity and could serve as a potential prognostic marker, particularly in BRCA.

Real-world data on breast pathologic complete response and disease-free survival after neoadjuvant chemotherapy for hormone receptor-positive, human epidermal growth factor receptor-2-negative breast cancer: a multicenter, retrospective study in China.

BACKGROUND: The data in the real-world setting on breast pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC) for hormone receptor-positive, human epidermal growth factor receptor-2-negative (HR+, HER2-) breast cancer (BC) is limited. The present study aims to screen for some predictors and investigate the prognostic significance of breast pCR after NAC in HR+, HER2- BC in China. METHODS: This was a multicenter, retrospective study. In this study, three hundred eighty-four HR+, HER2- BC patients who received NAC were enrolled between 2010 and 2016 from Shanghai Jiaotong University Breast Cancer Database (SJTU-BCDB). These patients were dichotomized according to the presence of breast pCR after NAC. Logistic analysis was used to screen for predictors associated with breast pCR. Kaplan-Meier (K-M) curve and a propensity score matching (PSM) analysis were performed to compare the disease-free survival (DFS) between the two groups. Cox regression was used to analyze the prognostic significance of breast pCR on DFS in HR+, HER2- BC. A nomogram model was established to predict the probability of DFS at 1, 3, and 5 years after NAC. RESULTS: Fifty-seven patients (14.8%) achieved breast pCR. Univariate analysis showed that tumor size, estrogen receptor (ER), progesterone receptor (PR), and Ki67 were associated with breast pCR. Further, multivariate analysis showed that tumor size, PR, and Ki67 remained statistically significant. K-M curves showed a statistical difference between the breast pCR and non-pCR groups before PSM (p = 0.047), and a more significant difference was shown after PSM (p = 0.033). Cox regression after PSM suggested that breast pCR, adjuvant ET, clinical T stage, and Ki67 status were the significant predictive factors for DFS in HR+, HER2- BC patients. The adjusted hazards ratio (aHR) for breast pCR was 0.228 (95% CI, 0.070~0.739; p = 0.014), for adjuvant endocrine therapy was 0.217 (95% CI, 0.059~0.801; p = 0.022), for Ki67 was 1.027 (95% CI, 1.003~1.052; p = 0.027), for cT stages 2 and 3 compared with 1, the values were 1.331 (95% CI, 0.170~10.389), and 4.699 (95% CI, 0.537~41.142), respectively (p = 0.043). A nomogram was built based on these significant predictors, providing an integrated probability of DFS at 1, 3, and 5 years. The values of area under the receiver operating characteristic (ROC) curve (AUC) were 0.967, 0.991, and 0.787, at 1 year, 3 years, and 5 years, respectively, demonstrating the ability of the nomogram to predict the DFS. CONCLUSIONS: This real-world study demonstrates that tumor size, PR, and Ki67 were independent predictive factors for breast pCR in HR+, HER2- BC. Breast pCR after NAC was an independent predictor for DFS in HR+, HER2- patients, regardless of a change in nodes. Furthermore, the nomogram built in our study could predict the probability of individualized DFS in HR+, HER2- BC patients.

Evaluating white matter alterations in Parkinson's disease-related parkin S/N167 mutation carriers using tract-based spatial statistics.

Background: Genetic susceptibility plays an important role in the pathogenesis of Parkinson's disease (PD). parkin S/N167 mutations may increase the risk of PD and affect white matter fibers in the brain. This cross-sectional study explored the effects of gene polymorphisms on white matter fiber damage in PD. Methods: In all, 54 cases were enrolled in the study, including PD patients carrying parkin gene S/N167 mutations (G/A), PD patients without gene S/N167 mutations (G/G), and healthy controls (HC). The whole-brain white matter fiber skeleton was analyzed using the tract-based spatial statistics (TBSS) method. Two-way analysis of variance (ANOVA) and post hoc tests were used for data analyses. Results: Two classification methods were used; one was based on disease classification, with 26 patients in the PD group (n=12 G/G, n=14 G/A) and 28 in the HC group (n=15 G/G, n=13 G/A), and the other was based on genetic classification, with 27 patients in the G/G group and 27 in the G/A group. In the G/A group, there was a wide range of significant changes in fractional anisotropy (FA), radial diffusivity (RD), and mean diffusivity (MD) values (P<0.05). There was also a significant decrease in FA in the PD-G/A group compared with the PD-G/G and HC-G/A groups (P<0.05). Conclusions: There were more extensive brain white matter fiber damage and changes in PD patients; the G/A polymorphism may cause more extensive brain white matter damage.

Dopamine Transporter Dynamics of N-Substituted Benztropine Analogs with Atypical Behavioral Effects.

Atypical dopamine transporter (DAT) inhibitors, despite high DAT affinity, do not produce the psychomotor stimulant and abuse profile of standard DAT inhibitors such as cocaine. Proposed contributing features for those differences include off-target actions, slow onsets of action, and ligand bias regarding DAT conformation. Several 3α-(4',4''-difluoro-diphenylmethoxy)tropanes were examined, including those with the following substitutions: N-(indole-3''-ethyl)- (GA1-69), N-(R)-2''-amino-3''-methyl-n-butyl- (GA2-50), N-2''aminoethyl- (GA2-99), and N-(cyclopropylmethyl)- (JHW013). These compounds were previously reported to have rapid onset of behavioral effects and were presently evaluated pharmacologically alone or in combination with cocaine. DAT conformational mode was assessed by substituted-cysteine accessibility and molecular dynamics (MD) simulations. As determined by substituted-cysteine alkylation, all BZT analogs except GA2-99 showed bias for a cytoplasmic-facing DAT conformation, whereas cocaine stabilized the extracellular-facing conformation. MD simulations suggested that several analog-DAT complexes formed stable R85-D476 "outer gate" bonds that close the DAT to extracellular space. GA2-99 diverged from this pattern, yet had effects similar to those of other atypical DAT inhibitors. Apparent DAT association rates of the BZT analogs in vivo were slower than that for cocaine. None of the compounds was self-administered or stimulated locomotion, and each blocked those effects of cocaine. The present findings provide more detail on ligand-induced DAT conformations and indicate that aspects of DAT conformation other than "open" versus "closed" may facilitate predictions of the actions of DAT inhibitors and may promote rational design of potential treatments for psychomotor-stimulant abuse.

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats.

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ1R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ1R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di-o-tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the Bmax values of [3H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ1R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ1R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ1R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ1R antagonist CM304. Moreover, σ1R ligands had distinct effects on σ1R multimerization. CM304 increased the proportion of multimeric σ1Rs, whereas (+)-pentazocine increased monomeric σ1Rs. Together these results support the hypothesis that σ1R agonists promote dissociation of σ1R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ1R agonists in animal models.

2-Substituted 3ß-Aryltropane Cocaine Analogs Produce Atypical Effects without Inducing Inward-Facing Dopamine Transporter Conformations.

Previous structure-activity relationship studies indicate that a series of cocaine analogs, 3ß-aryltropanes with 2ß-diarylmethoxy substituents, selectively bind to the dopamine transporter (DAT) with nanomolar affinities that are 10-fold greater than the affinities of their corresponding 2α-enantiomers. The present study compared these compounds to cocaine with respect to locomotor effects in mice, and assessed their ability to substitute for cocaine (10 mg/kg, i.p.) in rats trained to discriminate cocaine from saline. Despite nanomolar DAT affinity, only the 2ß-Ph2COCH2-3ß-4-Cl-Ph analog fully substituted for cocaine-like discriminative effects. Whereas all of the 2ß compounds increased locomotion, only the 2ß-(4-ClPh)PhCOCH2-3ß-4-Cl-Ph analog had cocaine-like efficacy. None of the 2α-substituted compounds produced either of these cocaine-like effects. To explore the molecular mechanisms of these drugs, their effects on DAT conformation were probed using a cysteine-accessibility assay. Previous reports indicate that cocaine binds with substantially higher affinity to the DAT in its outward (extracellular)- compared with inward-facing conformation, whereas atypical DAT inhibitors, such as benztropine, have greater similarity in affinity to these conformations, and this is postulated to explain their divergent behavioral effects. All of the 2ß- and 2α-substituted compounds tested altered cysteine accessibility of DAT in a manner similar to cocaine. Furthermore, molecular dynamics of in silico inhibitor-DAT complexes suggested that the 2-substituted compounds reach equilibrium in the binding pocket in a cocaine-like fashion. These behavioral, biochemical, and computational results show that aryltropane analogs can bind to the DAT and stabilize outward-facing DAT conformations like cocaine, yet produce effects that differ from those of cocaine.

Membrane cholesterol modulates the outward facing conformation of the dopamine transporter and alters cocaine binding.

Clearance of synaptically released dopamine is regulated by the plasmalemmal dopamine transporter (DAT), an integral membrane protein that resides within a complex lipid milieu. Here we demonstrate that cholesterol, a major component of the lipid bilayer, can modulate the conformation of DAT and alter cocaine binding to DAT. In striatal synaptosomes and transfected cells, DAT was in cholesterol-rich membrane fractions after mild detergent extraction. After increasing the membrane cholesterol content by treatment of water-soluble cholesterol (cholesterol mixed with methyl-ß-cyclodextrin), we observed an increase in DAT binding B(max) values for cocaine analogs [(3)H]WIN35428 and [(125)I]RTI-55, but similar levels of DAT proteins on the cell surface were shown by surface biotinylation assays. Membrane cholesterol addition also markedly enhanced the accessibility of cysteine sulfhydryl moieties in DAT as probed by a membrane-impermeable maleimide-biotin conjugate. We identified cysteine 306, a juxtamembrane residue on transmembrane domain 6 (TM6) of DAT, as the intrinsic residue exhibiting enhanced reactivity. Similar effects on DAT cysteine accessibility and radioligand binding were observed with addition of zinc, a reagent known to promote the outward facing conformation of DAT. Using substituted cysteine mutants on various positions likely to be extracellular, we identified additional residues located on TM1, TM6, TM7, and TM12 of DAT that are sensitive to alterations in the membrane cholesterol content. Our findings in transfected cells and native tissues support the hypothesis that DAT adopts an outward facing conformation in a cholesterol-rich membrane environment, suggesting a novel modulatory role of the surrounding membrane lipid milieu on DAT function.

Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Modulation of bradykinin-induced calcium changes in SH-SY5Y cells by neurosteroids and sigma receptor ligands via a shared mechanism.

In this study we investigated the effects of sigma receptor ligands and neurosteroids on bradykinin-induced intracellular calcium concentration ([Ca2+]i) changes in SH-SY5Y neuroblastoma cells. [Ca2+]i levels in cells loaded with fura-2 were monitored with dual-wavelength ratiometric fluorescence measurement. Submicromolar concentrations of bradykinin elicited [Ca2+]i responses with a fast rise followed by a slow decline in these cells. Preincubation of low micromolar concentrations of the neurosteroids pregnenolone, dehydroepiandrosterone (DHEA), or the prototypic sigma (sigma) receptor agonist (+)pentazocine potentiated bradykinin-induced [Ca2+]i changes in SH-SY5Y cells. The sigma receptor antagonist haloperidol blocked the enhancing effects on [Ca2+]i by (+)pentazocine or pregnenolone. Progesterone did not significantly affect the basal [Ca2+]i level or bradykinin-induced [Ca2+]i changes in these cells. However, coincubation of progesterone with (+)pentazocine, pregnenolone, or DHEA reversed their potentiating effects. The antagonistic effects of haloperidol and progesterone on the potentiating effects of (+)pentazocine and pregnenolone suggested that these ligands might act through a common mechanism. We further showed that progesterone, pregnenolone, and DHEA competed for [3H]+pentazocine binding in SH-SY5Y cells with Ki values of 0.13 +/- 0.03 microM, 0.98 +/- 0.34 microM, and 5.2 +/- 1.4 microM, respectively. Thus, the modulation of bradykinin-induced [Ca2+]i changes by neurosteroids in these cells is likely due to their actions on sigma receptors.

Binding of sigma receptor ligands and their effects on muscarine-induced Ca(2+) changes in SH-SY5Y cells.

In human neuroblastoma SH-SY5Y cell preparations, sigma(1) receptor agonists (+)-pentazocine and 1S,2R-(-)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)cyclohexylamine (BD737) competed for [3H]haloperidol binding with K(i) values of 67+/-10 and 14+/-10 nM, respectively. (+)-Pentazocine or BD737 up to 10 microM did not affect basal levels of intracellular Ca(2+) concentration ([Ca(2+)](i)) in these cells, but they significantly reduced muscarine-induced [Ca(2+)](i) changes in a dose-related manner. However, the reduction by (+)-pentazocine was not reversed by the sigma(1) receptor antagonist haloperidol. Further studies showed (+)-pentazocine, BD737 and haloperidol could compete for [3H]quinuclidinyl benzilate binding in SH-SY5Y cells with K(i) values of 0.51+/-0.06, 0.32+/-0.07 and 4.4+/-2.3 microM, respectively. Thus, the inhibitory effects on muscarine-induced [Ca(2+)](i) changes by (+)-pentazocine and BD737 in SH-SY5Y cells were likely due to blockade of muscarinic receptors, not due to sigma(1) receptor activation by these ligands.