Identification of PSMB5 as a genetic modifier of fragile X-associated tremor/ataxia syndrome.

Fragile X­associated tremor/ataxia syndrome (FXTAS) is a debilitating late-onset neurodegenerative disease in premutation carriers of the expanded CGG repeat in FMR1 that presents with a spectrum of neurological manifestations, such as gait ataxia, intention tremor, and parkinsonism [P. J. Hagerman, R. J. Hagerman, Ann. N. Y. Acad. Sci. 1338, 58­70 (2015); S. Jacquemont et al., JAMA 291, 460­469 (2004)]. Here, we performed whole-genome sequencing (WGS) on male premutation carriers (CGG55­200) and prioritized candidate variants to screen for candidate genetic modifiers using a Drosophila model of FXTAS. We found 18 genes that genetically modulate CGG-associated neurotoxicity in Drosophila, such as Prosbeta5 (PSMB5), pAbp (PABPC1L), e(y)1 (TAF9), and CG14231 (OSGEPL1). Among them, knockdown of Prosbeta5 (PSMB5) suppressed CGG-associated neurodegeneration in the fly as well as in N2A cells. Interestingly, an expression quantitative trait locus variant in PSMB5, PSMB5rs11543947-A, was found to be associated with decreased expression of PSMB5 and delayed onset of FXTAS in human FMR1 premutation carriers. Finally, we demonstrate evidence that PSMB5 knockdown results in suppression of CGG neurotoxicity via both the RAN translation and RNA-mediated toxicity mechanisms, thereby presenting a therapeutic strategy for FXTAS.

MiR-383 sensitizes osteosarcoma cells to bortezomib treatment via down-regulating PSMB5.

BACKGROUND: Proteasome inhibition is a promising strategy for cancer therapy. Bortezomib, which primarily targets the chymotrypsin-like activity of PSMB5, has demonstrated efficacy in various tumors. However, there is variable sensitivity to bortezomib, which could be attributed, in part, to variations in the expression of proteasome subunits. METHODS AND RESULTS: In this study, we investigated whether miR-383 affects the expression of proteasome subunits in osteosarcoma (OS) cells, and if so, whether OS cells display differential sensitivity to bortezomib concerning miR-383 expression. We detected a decreased miR-383 expression in OS cells and tissues. Then we found a negative correlation between the cytotoxicity of bortezomib and the expression level of the proteasome 20S core particle subunit ß5 (PSMB5). Intriguingly, we identified PSMB5 as a direct target of miR-383. Increased expression of miR-383 resulted in decreased PSMB5 expression and increased sensitivity to bortezomib in OS cells. CONCLUSIONS: In summary, our findings present the initial comprehensive analysis of the function of miR-383 in OS. The outcomes indicate that miR-383 may augment the anticancer effect of bortezomib through PSMB5 repression, offering a novel therapeutic approach in OS and a fresh pathway for proteasome regulation.

Dihydrocelastrol induces antitumor activity and enhances the sensitivity of bortezomib in resistant multiple myeloma by inhibiting STAT3-dependent PSMB5 regulation.

Multiple myeloma (MM) is characterized by excessive aggregation of B-cell-derived malignant plasma cells in the hematopoietic system of bone marrow. Previously, we synthesized an innovative molecule named dihydrocelastrol (DHCE) from celastrol, a triterpene purified from medicinal plant Tripterygium wilfordii. Herein, we explore the therapeutic properties and latent signal transduction mechanism of DHCE action in bortezomib (BTZ)-resistant (BTZ-R) MM cells. In this study, we first report that DHCE shows antitumor activities in vitro and in vivo and exerts stronger inhibitory effects than celastrol on BTZ-R cells. We find that DHCE inhibits BTZ-R cell viability by promoting apoptosis via extrinsic and intrinsic pathways and suppresses BTZ-R MM cell proliferation by inducing G0/G1 phase cell cycle arrest. In addition, inactivation of JAK2/STAT3 and PI3K/Akt pathways are involved in the DHCE-mediated antitumor effect. Simultaneously, DHCE acts synergistically with BTZ on BTZ-R cells. PSMB5, a molecular target of BTZ, is overexpressed in BTZ-R MM cells compared with BTZ-S MM cells and is demonstrated to be a target of STAT3. Moreover, DHCE downregulates PSMB5 overexpression in BTZ-R MM cells, which illustrates that DHCE overcomes BTZ resistance through increasing the sensitivity of BTZ in resistant MM via inhibiting STAT3-dependent PSMB5 regulation. Overall, our findings imply that DHCE may become a potential therapeutic option that warrants clinical evaluation for BTZ-R MM.

20-HETE regulated PSMB5 expression via TGF-ß/Smad signaling pathway.

We previously found that 20-hydroxyeicosatetraeonic acid (20-HETE) showed an effect on proteasome activity in cytochrome P450 F2 (CYP4F2) transgenic mice. Proteasome subunit ß5 (PSMB5) is a primary subunit of the proteasome. In the current study, we examine whether 20-HETE has any affect on PSMB5. We found that PSMB5 was upregulated in the liver, but downregulated in the kidney of transgenic mice, when compared with wild-type mice. Luciferase reporter gene experiments and electrophoretic mobility shift assays (EMSA) suggested that Smad3 directly associated with the putative Smad binding element (SBE) of the Psmb5 promoter. Furthermore, the binding affinity was different between the liver and kidney, and can be regulated by 20-HETE. Compared to wild mice, both TGF-ß1 and Smad3 phosphorylation were increased in the liver but decreased in the kidney of transgenic mice. SB431542, an inhibitor of TGF-ß receptor I kinase activity, can reverse the changes induced in PSMB5 by 20-HETE in vitro. Taken together, our data demonstrated that 20-HETE upregulated the expression of PSMB5 by activating the TGF-ß/Smad signaling pathway in the liver, but downregulated the expression of PSMB5 by inhibiting the TGF-ß/Smad signaling pathway in the kidney of transgenic mice.

Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression.

Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic ß-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the ß5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.

Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits.

The proteasome is a multi-subunit proteolytic complex that plays a central role in protein degradation in all eukaryotic cells. It regulates many vital cellular processes therefore its dysfunction can lead to various pathologies including cancer and neurodegeneration. Isolation of enzymatically active proteasomes is a key step to the successful study of the proteasome regulation and functions. Here we describe a simple and efficient protocol for immunoaffinity purification of the functional 20S proteasomes from human HEK 293T cells after transient overexpression of specific proteasome subunits tagged with 3xFLAG. To construct 3xFLAG-fusion proteins, DNA sequences encoding the 20S proteasome subunits PSMB5, PSMA5, and PSMA3 were cloned into mammalian expression vector pIRES-hrGFP-1a. The corresponding recombinant proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA3-3xFLAG were transiently overexpressed in human HEK 293T cells and were shown to be partially incorporated into the intact proteasome complexes. 20S proteasomes were immunoprecipitated from HEK 293T cell extracts under mild conditions using antibodies against FLAG peptide. Isolation of highly purified 20S proteasomes were confirmed by SDS-PAGE and Western blotting using antibodies against different proteasome subunits. Affinity purified 20S proteasomes were shown to possess chymotrypsin- and trypsin-like peptidase activities confirming their functionality. This simple single-step affinity method of the 20S proteasome purification can be instrumental to subsequent functional studies of proteasomes in human cells.

The Genetic Diversity of Proteasome Genes in the T1DM Polish Population.

Autoimmune metabolic diseases generate numerous healthy and social problems. The possible association of SNPs in the ubiquitin-proteasome system (UPS) with human pathology is under intensive study. OBJECTIVE: In the present study, the genetic variations in PSMB5 (rs11543947), PSMA6 (rs2277460, rs1048990), PSMC6 (rs2295826, rs2295827) and PSMA3 (rs2348071) UPS gene cluster was investigated in type 1 diabetes and healthy donors in the Polish population. METHODS: The study comprised 105 patients with type 1 diabetes mellitus (T1DM) and 214 controls. All were genotyped by PCR and restriction digestion analysis or Sanger sequencing. RESULTS: Rs1048990 and rs2348071 were found to be neutral to T1DM (p-value: 0.499 and 0.656, respectively). According to the multiple loci genotype (MLG) analysis, the major homozygote of the tested polymorphisms had a protective effect. The most common MLG in the T1DM group was characterised by simultaneous risk factors at rs11543947, rs2277460, rs2295826 and rs2295827 (pvalue: <0.0001 vs. MGL1). Multiple locus haplotype analysis revealed a similar dependence, with common alleles at all tested loci demonstrating a protective effect, and the rare alleles increasing T1DM risk (p-value: <0.0001 vs. MLH1). CONCLUSION: Our study suggests that the proteasome gene polymorphisms rs11543947, rs2277460, rs2295826, and rs2295827 could be potential markers for T1DM susceptibility in the Polish population.

Excessive proteostasis contributes to pathology in fragile X syndrome.

In fragile X syndrome (FX), the leading monogenic cause of autism, excessive neuronal protein synthesis is a core pathophysiology; however, an overall increase in protein expression is not observed. Here, we tested whether excessive protein synthesis drives a compensatory rise in protein degradation that is protective for FX mouse model (Fmr1-/y) neurons. Surprisingly, although we find a significant increase in protein degradation through ubiquitin proteasome system (UPS), this contributes to pathological changes. Normalizing proteasome activity with bortezomib corrects excessive hippocampal protein synthesis and hyperactivation of neurons in the inferior colliculus (IC) in response to auditory stimulation. Moreover, systemic administration of bortezomib significantly reduces the incidence and severity of audiogenic seizures (AGS) in the Fmr1-/y mouse, as does genetic reduction of proteasome, specifically in the IC. Together, these results identify excessive activation of the UPS pathway in Fmr1-/y neurons as a contributor to multiple phenotypes that can be targeted for therapeutic intervention.

Ancistrocladinium A Induces Apoptosis in Proteasome Inhibitor-Resistant Multiple Myeloma Cells: A Promising Therapeutic Agent Candidate.

The N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A belongs to a novel class of natural products with potent antiprotozoal activity. Its effects on tumor cells, however, have not yet been explored. We demonstrate the antitumor activity of ancistrocladinium A in multiple myeloma (MM), a yet incurable blood cancer that represents a model disease for adaptation to proteotoxic stress. Viability assays showed a potent apoptosis-inducing effect of ancistrocladinium A in MM cell lines, including those with proteasome inhibitor (PI) resistance, and in primary MM cells, but not in non-malignant blood cells. Concomitant treatment with the PI carfilzomib or the histone deacetylase inhibitor panobinostat strongly enhanced the ancistrocladinium A-induced apoptosis. Mass spectrometry with biotinylated ancistrocladinium A revealed significant enrichment of RNA-splicing-associated proteins. Affected RNA-splicing-associated pathways included genes involved in proteotoxic stress response, such as PSMB5-associated genes and the heat shock proteins HSP90 and HSP70. Furthermore, we found strong induction of ATF4 and the ATM/H2AX pathway, both of which are critically involved in the integrated cellular response following proteotoxic and oxidative stress. Taken together, our data indicate that ancistrocladinium A targets cellular stress regulation in MM and improves the therapeutic response to PIs or overcomes PI resistance, and thus may represent a promising potential therapeutic agent.

PSMB5 overexpression is correlated with tumor proliferation and poor prognosis in hepatocellular carcinoma.

Aberrant expression of members of the proteasome subunit beta (PSMB) family (including PSMB2, PSMB4, PSMB7 and PSMB8) has been reported in hepatocellular carcinoma (HCC). However the role of PSMB5 in HCC is unclear. To address this issue, we examined the expression of PSMB5 in HCC tissues using the The Cancer Genome Atlas, International Cancer Genome Consortium and Gene Expression Omnibus databases. A quantitative real-time PCR and immunohistochemistry were performed to validate the expression of PSMB5 in HCC. The survival mutation status and immune cell infiltration of PSMB5 were also evaluated in HCC. We then examined the effect of knocking down PSMB5 expression through RNA interference in the HCC cell line Huh7. High expression of PSMB5 was observed in HCC tissues and was associated with poor prognosis. PSMB5 expression and clinical characteristics were then incorporated to build a prognostic nomogram. We observed that PSMB5 expression was closely related to the abundance of B cells, CD4+ T cells, CD8+ T cells, dendritic cell macrophages and neutrophils. Moreover silencing of PSMB5 in Huh7 significantly suppressed cell proliferation and migration at the same time as increasing apoptosis. Inhibition of the phosphatidylinositol-3-kinase/Akt/mechanistic target of rapamycin pathway was observed after PSMB5 downregulation in Huh7 cells. Our findings suggest that PSMB5 may promote the proliferation of HCC cells by inactivating the phosphatidylinositol-3-kinase/Akt/mechanistic target of rapamycin signaling pathway and thus PSMB5 may have potential as a biomarker for diagnosis and prognosis of HCC.

First Sprayable Double-Stranded RNA-Based Biopesticide Product Targets Proteasome Subunit Beta Type-5 in Colorado Potato Beetle (Leptinotarsa decemlineata).

Colorado potato beetle (CPB, Leptinotarsa decemlineata) is a major pest of potato and other solanaceous vegetables in the Northern Hemisphere. The insect feeds on leaves and can completely defoliate crops. Because of the repeated use of single insecticide classes without rotating active ingredients, many chemicals are no longer effective in controlling CPB. Ledprona is a sprayable double-stranded RNA biopesticide with a new mode of action that triggers the RNA interference pathway. Laboratory assays with second instar larvae fed Ledprona showed a dose-response where 25×10-6g/L of dsPSMB5 caused 90% mortality after 6days of initial exposure. We also showed that exposure to Ledprona for 6h caused larval mortality and decreased target messenger RNA (mRNA) expression. Decrease in PSMB5 protein levels was observed after 48h of larval exposure to Ledprona. Both PSMB5 mRNA and protein levels did not recover over time. Ledprona efficacy was demonstrated in a whole plant greenhouse trial and performed similarly to spinosad. Ledprona, currently pending registration at EPA, represents a new biopesticide class integrated pest management and insecticide resistance management programs directed against CPB.

EGCG inhibits pressure overload-induced cardiac hypertrophy via the PSMB5/Nmnat2/SIRT6-dependent signalling pathways.

AIM: Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea, exerts multiple protective effects against cardiovascular diseases, including cardiac hypertrophy. However, the molecular mechanism underlying its anti-hypertrophic effect has not been clarified. This study revealed that EGCG could inhibit pressure overload-induced cardiac hypertrophy by regulating the PSMB5/Nmnat2/SIRT6-dependent signalling pathway. METHODS: Quantitative real-time polymerase chain reaction and western blotting were used to determine the expression of mRNA and protein respectively. A fluorometric assay kit was used to determine the activity of SIRT6, a histone deacetylase. Luciferase reporter gene assay and electrophoretic mobility shift assay were employed to measure transcriptional activity and DNA binding activity respectively. RESULTS: EGCG could significantly increase Nmnat2 protein expression and enzyme activity in cultured neonatal rat cardiomyocytes stimulated with angiotensin II (Ang II) and heart tissues from rats subjected to abdominal aortic constriction. Nmnat2 knockdown by RNA interference attenuated the inhibitory effect of EGCG on cardiac hypertrophy. EGCG blocked NF-κB DNA binding activity induced by Ang II, which was dependent on Nmnat2 and the subsequent SIRT6 activation. Moreover the activation of PSMB5 (20S proteasome subunit ß-5, chymotrypsin-like) was required for EGCG-induced Nmnat2 protein expression. Additionally, we demonstrated that EGCG might interact with PSMB5 and inhibit the activation of the proteasome. CONCLUSIONS: These findings serve as the first evidence that the effect of EGCG against cardiac hypertrophy may be, at least partially, attributed to the modulation of the PSMB5/Nmnat2-dependent signalling pathway, suggesting the therapeutic potential of EGCG in the prevention and treatment of cardiac hypertrophy.

The Prognostic Value of Whole-Blood PSMB5, CXCR4, POMP, and RPL5 mRNA Expression in Patients with Multiple Myeloma Treated with Bortezomib.

Proteasome inhibitors, like bortezomib, play a key role in the treatment of multiple myeloma (MM); however, most patients eventually relapse and eventually show multiple drug resistance, and the molecular mechanisms of this resistance remain unclear. The aim of our study is to assess the expression of previously described genes that may influence the resistance to bortezomib treatment at the mRNA level (ABCB1, CXCR4, MAF, MARCKS, POMP, PSMB5, RPL5, TXN, and XBP1) and prognosis of MM patients. mRNA expression was determined in 73 MM patients treated with bortezomib-based regimens (30 bortzomib-sensitive and 43 bortezomib-refractory patients) and 11 healthy controls. RPL5 was significantly down-regulated in multiple myeloma patients as compared with healthy controls. Moreover, POMP was significantly up-regulated in MM patients refractory to bortezomib-based treatment. In multivariate analysis, high expression of PSMB5 and CXCR and autologous stem cell transplantation were independent predictors of progression-free survival, and high expression of POMP and RPL5 was associated with shorter overall survival.

The role of PSMB5 in sodium arsenite-induced oxidative stress in L-02 cells.

Endemic arsenism is widely distributed in the world, which can damage multiple organs, especially in skin and liver. The etiology is clear, but the mechanisms involved remain unknown. Ubiquitin-proteasome pathway (UPP) is the main pathway regulating protein degradation of which proteasome subunit beta type-5(PSMB5) plays a dominant role. This paper aims to study the role and mechanism of PSMB5 in sodium arsenite (NaAsO2)-induced oxidative stress liver injury in L-02 cells. Firstly, L-02 cells were exposed to different concentrations of NaAsO2 to establish a liver injury model of oxidative stress, and then mechanisms of oxidative stress were studied with carbobenzoxyl-leucyl-leucl-leucll-line (MG132) and knockdown PSMB5 (PSMB5-siRNA). The oxidative stress indicators, levels of 20S proteasome, the transcription and protein expression levels of PSMB5, Cu-Zn superoxide dismutase (SOD1), and glutathione peroxidase 1 (GPx1) were detected. The results demonstrated that NaAsO2 could induce oxidative stress-induced liver injury and the activity of 20S proteasome and the protein expression of PSMB5, SOD1, and GPx1 decreased. After MG132 or PSMB5-siRNA pretreatment, the gene expression of PSMB decreased. After MG132 or PSMB5-siRNA pretreatment, and then L-02 cells were treated with NaAsO2, the gene expression of PSMB remarkably decreased; however, the protein expression of SOD1 and GPx1 increased. Overall, NaAsO2 exposure could induce oxidative stress liver injury and low expression of PSMB5 in L-02 cells, and PSMB5 might play an important role in the regulation of oxidative stress by regulating the expression of SOD1 and Gpx1.

Transcriptional downregulation of miR-127-3p by CTCF promotes prostate cancer bone metastasis by targeting PSMB5.

Prostate cancer (PCa) is one of the most common cancers in males and particularly tends to metastasize to bone. Currently, metastatic bone disease is incurable, and new therapies need to be developed. Our study aims to determine the role of miR-127-3p in PCa metastasis to bone. The results demonstrate that miR-127-3p is markedly reduced in bone metastasis-positive PCa tissues relative to that in bone metastasis-negative PCa tissues. Furthermore, overexpressing miR-127-3p inhibits PCa cell invasion and migration in vitro by targeting the proteasome ß-subunit PSMB5. Moreover, CCCTC-binding factor (CTCF) transcriptionally inhibits miR-127-3p by interacting with the miR-127-3p promoter. Collectively, this study uncovers a novel mechanism of the CTCF/miR-127-3p/PSMB5 axis in promoting PCa bone metastasis, indicating that miR-127-3p could function as a promising therapeutic target against bone metastasis.

Curcumin inhibits proteasome activity in triple-negative breast cancer cells through regulating p300/miR-142-3p/PSMB5 axis.

BACKGROUND: Curcumin functions as a proteasome inhibitor. However, the molecular mechanisms behind this action need more detailed explanations. PURPOSE: This study aimed to investigate the inhibitory effect of curcumin on 20S proteasome activity and to elucidate its exact mechanism in triple-negative breast cancer (TNBC) MDA-MB-231 cells. METHODS: Proteasomal peptidase activities were assayed using synthetic fluorogenic peptide substrates. Knockdown or overexpression of microRNA (miRNA or miR) or protein was used to investigate its functional effect on downstream cellular processes. BrdU (5­bromo­2'-deoxyuridine) assay was performed to identify cell proliferation. Western blot and quantitative real-time PCR(qRT-PCR) were carried out to determine protein abundance and miRNA expression, respectively. Correlations between protein expressions, miRNA levels, and proteasome activities were analyzed in TNBC tissues. Xenograft tumor model was performed to observe the in vivo effect of curcumin on 20S proteasome activity. RESULTS: Curcumin significantly reduced PSMB5 protein levels, accompanied with a reduction in the chymotrypsin-like (CT-l) activity of proteasome 20S core. Loss of PSMB5 markedly inhibited the CT-l activity of 20S proteasome. Furthermore, curcumin treatment significantly elevated miR-142-3p expression. PSMB5 was a direct target of miR-142-3p and its protein levels were negatively regulated by miR-142-3p. Moreover, histone acetyltransferase p300 suppressed miR-142-3p expression. Overexpression of p300 mitigated the promotive effect of curcumin on miR-142-3p expression. The correlations among p300 abundances, miR-142-3p levels, PSMB5 expressions, and the CT-l activities of 20S proteasome were evidenced in TNBC tissues. In addition, loss of p300 and PSMB5 reduced cell proliferation. Inhibition of miR-142-3p significantly attenuated the inhibitory impact of curcumin on cell proliferation. These curcumin-induced changes on p300, miR-142-3p, PSMB5, and 20S proteasome activity were further confirmed in in vivo solid tumor model. CONCLUSION: These findings demonstrated that curcumin suppressed p300/miR-142-3p/PSMB5 axis leading to the inhibition of the CT-l activity of 20S proteasome. These results provide a novel and alternative explanation for the inhibitory effect of curcumin on proteasome activity and also raised potential therapeutic targets for TNBC treatment.

Novel cell line models to study mechanisms and overcoming strategies of proteasome inhibitor resistance in multiple myeloma.

Experimental data on resistance mechanisms of multiple myeloma (MM) to ixazomib (IXA), a second-generation proteasome inhibitor (PI), are currently lacking. We generated MM cell lines with a 10-fold higher resistance to IXA as their sensitive counterparts, and observed cross-resistance towards the PIs carfilzomib (CFZ) and bortezomib (BTZ). Analyses of the IXA-binding proteasome subunits PSMB5 and PSMB1 show increased PSMB5 expression and activity in all IXA-resistant MM cells, and upregulated PSMB1 expression in IXA-resistant AMO1 cells. In addition, sequence analysis of PSMB5 revealed a p.Thr21Ala mutation in IXA-resistant MM1.S cells, and a p.Ala50Val mutation in IXA-resistant L363 cells, whereas IXA-resistant AMO1 cells lack PSMB5 mutations. IXA-resistant cells retain their sensitivity to therapeutic agents that mediate cytotoxic effects via induction of proteotoxic stress. Induction of ER stress and apoptosis by the p97 inhibitor CB-5083 was strongly enhanced in combination with the PI3Kα inhibitor BYL-719 or the HDAC inhibitor panobinostat suggesting potential therapeutic strategies to circumvent IXA resistance in MM. Taken together, our newly established IXA-resistant cell lines provide first insights into resistance mechanisms and overcoming treatment strategies, and represent suitable models to further study IXA resistance in MM.

PSMB5 is associated with proliferation and drug resistance in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by advanced disease stage and poor prognosis. Moreover, due to the lack of therapeutic markers, TNBC patients can't benefit fully from currently available targeted therapies. METHODS: To fully understand the molecular basis of TNBC, we used gene set enrichment analysis (GSEA) to screen out the most altered functional module in TNBC, from publicly available microarray data and studied the association of the candidate gene with TNBC development. RESULTS: We found that the proteasome was significantly activated in TNBC. As compared with other breast cancer subtypes and normal tissue, proteasome subunit beta 5 (PSMB5), the key regulator of proteasome function, was overexpressed in TNBC tissue and predictive of poor prognosis. Moreover, we also found that PSMB5 knockdown induced TNBC apoptosis and significantly enhanced cancer cell sensitivity to the chemotherapeutic agents bortezomib and paclitaxel. CONCLUSIONS: Our results suggest a potential role for PSMB5 as a biomarker and therapeutic target for TNBC.

PSMB5 plays a dual role in cancer development and immunosuppression.

Tumor progression and metastasis are dependent on the intrinsic properties of tumor cells and the influence of microenvironment including the immune system. It would be important to identify target drug that can inhibit cancer cell and activate immune cells. Proteasome ß subunits (PSMB) family, one component of the ubiquitin-proteasome system, has been demonstrated to play an important role in tumor cells and immune cells. Therefore, we used a bioinformatics approach to examine the potential role of PSMB family. Analysis of breast TCGA and METABRIC database revealed that high expression of PSMB5 was observed in breast cancer tissue and that high expression of PSMB5 predicted worse survival. In addition, high expression of PSMB5 was observed in M2 macrophages. Based on our bioinformatics analysis, we hypothesized that PSMB5 contained immunosuppressive and oncogenic characteristics. To study the effects of PSMB5 on the cancer cell and macrophage in vitro, we silenced PSMB5 expression with shRNA in THP-1 monocytes and MDA-MB-231 cells respectively. Knockdown of PSMB5 promoted human THP-1 monocyte differentiation into M1 macrophage. On the other hand, knockdown PSMB5 gene expression inhibited MDA-MB-231 cell growth and migration by colony formation assay and boyden chamber. Collectively, our data demonstrated that delivery of PSMB5 shRNA suppressed cell growth and activated defensive M1 macrophages in vitro. Furthermore, lentiviral delivery of PSMB5 shRNA significantly decreased tumor growth in a subcutaneous mouse model. In conclusion, our bioinformatics study and functional experiments revealed that PSMB5 served as novel cancer therapeutic targets. These results also demonstrated a novel translational approach to improve cancer immunotherapy.

Inhibition of store-operated calcium entry by sub-lethal levels of proteasome inhibition is associated with STIM1/STIM2 degradation.

Dysfunction of the ubiquitin-proteasome system (UPS) and calcium homeostasis has been implicated in the neurodegeneration of Alzheimer's and Parkinson's diseases. The cytosolic calcium concentration is maintained by store-operated calcium entry (SOCE), which is repressed by Alzheimer's disease-associated mutants, such as mutant presenilins. We hypothesized that inhibition of UPS impacts SOCE. This study showed that pretreatment with sub-lethal levels of proteasome inhibitors, including MG-132 and clasto-lactacystin-ß-lactone (LA), reduced SOCE after depletion of endoplasmic reticulum calcium in rat neurons. With the same treatment, MG-132 and LA reduced the protein levels of stromal interaction molecule 1and 2 (STIM1/2), but not the levels of Orai1 and canonical transient receptor potential channel 1 (TRPC1). STIM1 or STIM2 protein was mobilized to lysosome by MG-132/LA treatment as observed under an immunofluorescence confocal laser microscope. In the neurons, MG-132 and LA degraded p62/SQSTM1, promoted autophagy, converted LC3I to LC3II, and promoted co-localization of LC3 and lysosomes. Rapamycin, which enhances autophagy, reduced STIM1/2 protein levels, whereas bafilomycin, which inhibits autophagy, increased their protein levels. The protein levels of STIM1/2 and the amplitude of SOCE were decreased in SH-SY5Y with decreased protein level of proteasome subunit beta type-5 induced by shRNA. We conclude that sub-lethal levels of proteasome inhibition reduce SOCE and promote autophagy-mediated degradation of STIM1/2. UPS inhibition, a common finding in neurodegenerative diseases, interferes with calcium homeostasis via repression of SOCE.