Proteasome Inhibitor PS-341 Research Articles

P97 inhibits integrated stress response-induced neuronal apoptosis after subarachnoid hemorrhage in mice by enhancing proteasome function

Neuronal apoptosis is a common pathological change in early brain injury after subarachnoid hemorrhage (SAH), and it is closely associated with neurological deficits. According to previous research, p97 exhibits a remarkable anti-cardiomyocyte apoptosis effect. p97 is a critical molecule in the growth and development of the nervous system. However, it remains unknown whether p97 can exert an anti-neuronal apoptosis effect in SAH. In the present study, we examined the role of p97 in neuronal apoptosis induced after SAH and investigated the underlying mechanism. We established an in vivo SAH mice model and overexpressed the p97 protein through transfection of the mouse cerebral cortex. We analyzed the protective effect of p97 on neurons and evaluated short-term and long-term neurobehavior in mice after SAH. p97 was found to be significantly downregulated in the cerebral cortex of the affected side in mice after SAH. The site showing reduced p97 expression also exhibited a high level of neuronal apoptosis. Adeno-associated virus-mediated overexpression of p97 significantly reduced the extent of neuronal apoptosis, improved early and long-term neurological function, and repaired the neuronal damage in the long term. These neuroprotective effects were accompanied by enhanced proteasome function and inhibition of the integrated stress response (ISR) apoptotic pathway involving eIF2α/CHOP. The administration of the p97 inhibitor NMS-873 induced a contradictory effect. Subsequently, we observed that inhibiting the function of the proteasome with the proteasome inhibitor PS-341 blocked the anti-neuronal apoptosis effect of p97 and enhanced the activation of the ISR apoptotic pathway. However, the detrimental effects of NMS-873 and PS-341 in mice with SAH were mitigated by the administration of the ISR inhibitor ISRIB. These results suggest that p97 can promote neuronal survival and improve neurological function in mice after SAH. The anti-neuronal apoptosis effect of p97 is achieved by enhancing proteasome function and inhibiting the overactivation of the ISR apoptotic pathway.

A Landmark Paper That Introduced Proteasome Inhibition in Myeloma.

The ongoing therapeutic revolution in multiple myeloma care can be traced to the turn of the millennium with the unanticipated discovery in 1999 that the cereblon binding small molecule thalidomide had profound clinical effectiveness and, simultaneously, the emergence of a new class of targeted therapies inhibiting the proteasome, both of which ultimately target ubiquitinated protein degradation. These contemporaneous discoveries forever changed the landscape of multiple myeloma care, substantially extending survival. Foreshadowing this seismic change, Nobel Prize winning work on the proteasome ubiquitin pathway had stimulated the development of highly specific proteasome inhibitor small molecules, particularly PS-341 (later named bortezomib). An abundance of the proteasome in hematologic malignancies had been recognized and thus PS-341 was logically being explored in relevant preclinical models. Concurrent with phase I trials, which were soon to prove the significant clinical relevance of preclinical models, the laboratory of Dr. Kenneth Anderson and colleagues at Dana-Farber, in partnership with Dr. Julian Adams and scientists at ProScript (later Millennium Pharmaceuticals) first demonstrated that the proteasome inhibitor PS-341 inhibited growth, induced apoptosis, and overcame drug resistance in human multiple myeloma cells. This landmark paper in Cancer Research set the stage for a paradigm shift in how multiple myeloma was managed across all stages of the disease, which changed the lives of patients worldwide. See related article by Hideshima and colleagues, Cancer Res 2001;61:3071-6.

Abstract 2781: Mechanism study of surufatinib on the optimized strategy of AG plus PD-1 antibody treatment in pancreatic cancer

Abstract Background: Gemcitabine combined with albumin-bound paclitaxel (AG) is the standard treatment for pancreatic cancer. However, it provides no benefit after combined AG with PD-1 antibody, which needs to be optimized urgently. Surufatinib (SUR) is a novel small-molecule inhibitor that observably inhibits VEGFR 1, 2, 3, fibroblast growth factor receptor 1 (FGFR 1), and colony stimulating factor-1 receptor (CSF-1R), which simultaneously shows anti-angiogenic activity and immune microenvironment activation. Herein, we aimed to explore the synergistic effect and related mechanism of SUR plus AG and PD-1 antibody (SAP) regimen. Methods: Surgical tumor specimens were collected and analyzed using single-cell sequencing and immunohistochemistry technology. Orthotopic transplantation mice model of pancreatic cancer and co-culture system of pancreatic cancer cells and tumor-associated macrophages (TAMs) were successively established to reveal the potential molecular mechanism of SUR in optimizing the effects of AG plus PD-1 antibody, by western blot, immunofluorescence and ubiquitination experiments. Results: Tumor CSF-1 levels were markedly promoted in pancreatic cancer patients after the treatment of gemcitabine, while an increased infiltration of M2 polarized TAMs (M2-TAMs) was induced. A time-dependent upregulation of M2-TAMs infiltration and PD-L1 expression were also observed after the treatment of AG regimen, which might interfere the therapeutic benefits of AG plus PD-1. In co-culture system, tumor PD-L1 expression levels were distinctly down-regulated after the treatment of SAP regimen, while the decreased infiltration of M2-TAMs was observed. In pancreatic cancer cell, PD-L1 protein synthesis were obviously inhibited by SUR, instead of PD-L1 mRNA expression. After the pancreatic cancer cells pretreated with cycloheximide to inhibit protein synthesis, the half-life period of PD-L1 was ulteriorly reduced by SUR. Inversely, the inhibitory action of SUR on PD-L1 expression was reversed by proteasome inhibitor PS-341, rather than lysosomal inhibitor chloroquine. As a result, it indicated that the protein stability of PD-L1 might interfered by SUR via ubiquitin-proteasome pathway. Particularly, when compared with AG plus PD-1 antibody treatment in tumor-bearing model, SAP regimen significantly down-regulated the expression levels of CSF1R, CD163 and PD-L1, and inhibited tumor growth by promoting tumor cell apoptosis and improving the tumor immune microenvironment. Conclusions: Forceful evidence supported that PD-L1 expression could be interfered by surufatinib via ubiquitin-proteasome pathway and M2-TAMs regulation. Herein, additional administration of surufatinib might improve the immunosuppression and drug resistance caused by AG, and increase the curative effect of AG plus PD-1 antibody treatment in clinical practice. Citation Format: Song Gao, Wenbo Zhu, Xiaofan Guo, Yuexiang Liang, Hongwei Wang, Peijun Xu, Yuning Song, Jihui Hao, Shaoshi Wen, Xing Lv, Jiancai Zhou. Mechanism study of surufatinib on the optimized strategy of AG plus PD-1 antibody treatment in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2781.

OAB-032: Targeting GCK in RAS-mutant multiple myeloma offer a promising therapeutic approach

<h3>Background</h3> Next generation sequencing revealed frequent mutations of NRAS, KRAS or BRAF in up to 50% of newly diagnosed multiple myeloma (MM) patients. <h3>Methods</h3> Specific K- or N-RAS knockdowns led to strong decrease in MM cell viability if harbored the respective oncogenic isoform, but minimal effects were observed if only the wild-type isoform was present, presenting RAS signaling as the key driving oncogenes in the mutation bearing patients. Unfortunately, no inhibitor for RAS mutation is available for clinical use in MM. Therefore, key component in the RAS/MAPK pathway may represent an alternative therapeutic target for MM. Germinal center kinase (GCK) is an upstream activator in the MAPK pathway. <h3>Results</h3> Our data showed that GCK (MAP4K2) knockdown in MM cells induced MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins. Moreover, GCK silencing only led to significantly decreased GCK mRNA, however, did not affect IKZF1 expressions at mRNA level. We next tested the effects of GCK inhibitor TL4-12 on MM. Given the fact that RASMut MM cells have higher GCK expression level and are more sensitive to GCK knockdown, we compared mutated RAS MM cells with wild-type RAS MM cells. TL4-12 significantly inhibited the growth of mutated RAS MM cells, with IC50 5-10 fold lower compared to wild-type RAS MM cell lines. IKZF1/3 are the key targets of the immunomodulatory drugs (IMiDs), which are the backbone of MM therapy. IMiDs bind to cereblon (CRBN) and induce IKZF1/3 protein degradation, which subsequently lead to MM cell growth inhibition. Our data showed that GCK knockdown also downregulates IKZF1 protein level, indicating that IKZF1 is under the regulation of GCK. Concomitant treatment with proteasome inhibitor PS-341 blocked TL4-12 induced IKZF1 downregulation, suggesting that inhibition of GCK induces IKZF1 protein degradation. However, since IMiDs-resistant RPMI-8226 and JJN3 MM cells are sensitive to GCK inhibition, we hypothesized that the IKZF1 degradation mechanism induced by GCK inhibition is different from IMiDs and independent of CRBN. To confirm this hypothesis, we silenced CRBN in N-Rasmut H929 MM cells and examined the response to IMiDs and GCK inhibitor. CRBN silencing in H929 cells resulted in lenalidomide resistance. In contrast, CRBN silencing failed to rescue H929 MM from TL4-12 induced proliferation inhibition and IKZF1 downregulation, confirming that GCK regulated IKZF1 and cell growth is independent of CRBN. <h3>Conclusions</h3> Taken together, our data demonstrated that GCK inhibition induces cell growth inhibition and triggers apoptosis especially in RASmut MM cells. Importantly, GCK inhibition downregulates IKZF1 via a CRBN-independent mechanism. Our findings thus provide a rationale for the clinical evaluation of targeting GCK in RASmut MM patients and further mechanistic insight into the role of GCK in MM tumorigenesis as well as drug resistance.

Arsenic Trioxide and Interferon-Alpha Eradicate Adult T Cell Leukemia Initiating Cells through PML/p53 Senescence and Activation of Innate Immunity

Adult T cell leukemia (ATL) is an aggressive chemo-resistant T cell malignancy secondary to chronic HTLV-I infection. The viral oncoprotein Tax initiates ATL in transgenic mice. We previously showed that arsenic trioxide (As) and interferon-α (IFN) combination, known to trigger proteasome mediated Tax degradation and apoptosis in ATL cells, cures murine ATL through clearance of leukemia initiating cells (LIC). We also showed that the triple combination of As, IFN and zidovudine is highly effective in the treatment of chronic ATL patients, inducing a shift from an immunosuppressive Treg/Th2 profile at diagnosis (high IL-10; low IL2; low IFN-γ) towards a Th-1 immunocompetent profile (high IFN-γ; high IL2; low IL-10).To dissect the molecular basis of As/IFN-induced ATL LIC eradication, primary ATL SCID mice were treated with As/IFN for 3 days. Spleen derived ATL cells were injected into secondary recipient SCID mice left untreated and sacrificed on a weekly basis. Whereas, secondary mice derived from untreated primaries succumbed from ATL between 3 and 4 weeks after transplantation, we observed a biphasic growth intersected by a spontaneous regression phase in untreated secondary mice transplanted from As/IFN treated primary mice. This process was associated with telomere shortening, DNA damage, activation of the PML/P53 axis, and senescence in tumor cells. Interestingly, injection of spleen derived ATL cells from secondary SCID sacrificed before ATL senescence, into untreated tertiary and quaternary SCID, resulted in normal ATL development, indicating that senescence of ATL cells in secondary SCID mice resulted in a change of the microenvironment that culminated in ATL cell apoptosis and loss of ATL LIC activity. Furthermore, injection of spleen derived ATL cells from untreated secondary SCID sacrificed before or after ATL senescence, into untreated tertiary NSG mice, resulted in normal ATL development. These NSG mice, contrary to SCID mice, lack natural killer (NK) cells and have an impaired macrophage activity. In agreement with these results, injection of spleen derived ATL cells from treated primary SCID into untreated secondary NSG mice, resulted in normal ATL development. RT-PCR and ELISA analysis of sorted CD25 positive (ATL) or negative (microenvironment) cells revealed treatment-induced upregulation of MIP1, RANTES, and IFN-γ in the CD25 negative fraction in untreated secondary SCID, following ATL senescence, and concomitant with apoptosis and loss of LIC activity. Conversely, and as previously seen in treated ATL patients, IL-10 expression was rapidly and dramatically downregulated in the CD25 positive fraction in untreated secondary SCID mice. Addition of the proteasome inhibitor PS-341 to AS/IFN treatment of primary mice reverted all above results, presumably indicating the role of As/IFN-induced Tax degradation in the observed phenotypes.Collectively, these results demonstrate that As/IFN-induced, PML/p53 dependent, senescence of ATL cells resulted in activation of the innate immunity that culminated in apoptosis and loss of ATL LIC activity. The striking similarity of these results with patients' data suggests a critical role for innate immunity in ATL cure. DisclosuresHermine:Novartis: Research Funding; Hybrigenics: Research Funding; Celgene: Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; INatherys: Equity Ownership, Research Funding.

Proteasome Inhibitor PS-341 Effectively Blocks Infection by the Severe Fever with Thrombocytopenia Syndrome Virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever disease caused by SFTSV, a newly discovered phlebovirus that is named after the disease. Currently, no effective vaccines or drugs are available for use against SFTSV infection, as our understanding of the viral pathogenesis is limited. Bortezomib (PS-341), a dipeptide-boronic acid analog, is the first clinically approved proteasome inhibitor for use in humans. In this study, the antiviral efficacy of PS-341 against SFTSV infection was tested in human embryonic kidney HEK293T (293T) cells. We employed four different assays to analyze the antiviral ability of PS-341 and determined that PS-341 inhibited the proliferation of SFTSV in 293T cells under various treatment conditions. Although PS-341 did not affect the virus absorption, PS-341 treatment within a non-toxic concentration range resulted in a significant reduction of progeny viral titers in infected cells. Dual-luciferase reporter assays and Western blot analysis revealed that PS-341 could reverse the SFTSV-encoded non-structural protein (NS) mediated degradation of retinoic acid-inducible gene-1 (RIG-I), thereby antagonizing the inhibitory effect of NSs on interferons and blocking virus replication. In addition, we observed that inhibition of apoptosis promotes virus replication. These results indicate that targeting of cellular interferon pathways and apoptosis during acute infection might serve as the bases of future therapeutics for the treatment of SFTSV infections.

Post-transcriptional regulation of cancer/testis antigen MAGEC2 expression by TRIM28 in tumor cells

BackgroundCancer/testis antigen MAGEC2 (also known as HCA587) is highly expressed in a wide variety of tumors and plays an active role in promoting growth and metastasis of tumor cells. However, little is known for the regulation of MAGEC2 expression in cancer cells.MethodsWestern blotting and quantitative RT-PCR were performed to analyze MAGEC2 expression. Co-immunoprecipitation assay was applied for detecting the endogenous interaction of MAGEC2 and TRIM28 in tumor cells. Overexpression and knockdown assays were used to examine the effects of TRIM28 on the expression of MAGEC2 protein. Immunohistochemistry (IHC) staining was performed in hepatocellular carcinoma patients to evaluate the association between the expression of MAGEC2 and TRIM28. Proteasome inhibitors MG132 or PS-341 and lysosome inhibitor Chloroquine (CQ) were used to inhibit proteasomal or lysosomal-mediated protein degradation respectively.ResultsWe demonstrate that MAGEC2 interacts with TRIM28 in melanoma cells and MAGEC2 expression in tumor cells depends on the expression of TRIM28. The expression level of MAGEC2 protein was significantly reduced when TRIM28 was depleted in tumor cells, and no changes were observed in MAGEC2 mRNA level. Furthermore, expression levels of MAGEC2 and TRIM28 are positively correlated in MAGEC2-positive human hepatocellular carcinoma tissues (p = 0.0011). Mechanistic studies indicate that the regulatory role of TRIM28 on MAGEC2 protein expression in tumor cells depends on proteasome-mediated pathway.ConclusionsOur findings show that TRIM28 is necessary for MAGEC2 expression in cancer cells, and TRIM28 may serve as a new potential target for immunotherapy of cancer.

Synergistic activity of the histone deacetylase inhibitor trichostatin A and the proteasome inhibitor PS-341 against taxane-resistant ovarian cancer cell lines.

Although a combination of platinum- and taxane-based chemotherapy is recommended for at least 70% patients with ovarian cancer as treatment subsequent to surgery, the initial response to the chemotherapy is not durable and tumors become resistant. Histone deacetylase and proteasome inhibitors are novel therapeutic agents. However, the moderate antitumoral effect of the inhibitors has restricted their clinical use when used as single agents. The aim of the present study was to investigate the synergistic activity of trichostatin A (TSA) and PS-341 in ovarian cancer cells, along with the investigation of the molecular mechanisms of taxane resistance. The taxane-sensitive ovarian cancer A2780 cell line and its resistant variant, A2780T, were treated with taxane, TSA and PS-341 at various concentrations. An Annexin V assay was performed to determine the levels of cell viability and apoptosis, while flow cytometry and immunofluorescence staining for the mitotic phase-specific protein phosphorylated-histone H3 (Ser10) were used for cell cycle detection. The effects of combined TSA and PS-341 on cell cycle-associated proteins were tested by western blot analysis. Furthermore, the present study examined the apoptosis and cell cycle arrest induced by the 3 agents subsequent to overexpression or downregulation of cyclin B1 in A2780 and A2780T cells, respectively. It was found that TSA interacted synergistically with PS-341, resulting in a marked increase in apoptosis and the rate of G2/M arrest in A2780T cells. A lower basal level of cyclin B1 expression and the incompetence of the upregulation of the cyclin may explain the taxane resistance found in A2780T cells. Collectively, the combination of TSA and PS-341 increased cyclin B1 expression level regardless of the basal expression level, resulting in the proliferation inhibition and apoptosis in A2780 and A2780T cells, which raised the possibility that a combination of the two drugs may represent a novel strategy for the treatment of ovarian cancer, particularly in taxane-resistant ovarian cancer.

Abstract 4545: The E3 ubiquitin ligase COP1 controls STAT3 turnover and its loss leads to increased STAT3 stabilization and activation in prostate cancer

Abstract The E3 ubiquitin ligase COP1 acts as a tumor suppressor and is deleted in a small percentage of prostate cancers. We reported that COP1 was repressed in prostate tumors through miRNA-mediated silencing, which represents an alternative and more frequent event than genetic deletion. COP1 controls ubiquitination and turnover of c-Jun and ETV1 and its loss has been associated with over-expression of these transcription factors. In this study, we identify STAT3 as a novel substrate of COP1 and report that concomitant deregulation of COP1 and STAT3 leads to prostate cancer progression. In a panel of prostate cancer cell lines, low expression of COP1 was associated with increased level of STAT3 protein and a more aggressive phenotype. Knockdown of COP1 in normal prostate epithelial RWPE-1 cells increased total (tSTAT3) and phosphorylated STAT3 (pSTAT3) and promoted tumorigenic properties. Conversely, over-expression of COP1 in DU145 cells, expressing low level of COP1, reversed the transformed phenotype and reduced tSTAT3 and pSTAT3 level and activation. COP1/STAT3 anti-correlation suggested that STAT3 was a substrate of COP1 for ubiquitination and degradation by the ubiquitin-proteasome system. Consistently, the proteasome inhibitor PS-341 prevented down-regulation of STAT3 in response to COP1 in DU145 cells. Furthermore, COP1 knockdown delayed significantly STAT3 protein turnover in RWPE-1 cells, indicating that COP1 regulated STAT3 degradation in these cells. Co-IP showed that COP1 and STAT3 directly interacted in RWPE-1 and DU145 cells. Interestingly, co-IP with the wild type and phosphorylation defective Y705F mutant STAT3 showed that the interaction with COP1 did not depend on STAT3 phosphorylation. Furthermore, the level of ubiquitinated STAT3 in RWPE1 cells was reduced after COP1 knockdown, whereas increased in DU145 cells after COP1 over-expression. These data provided evidence of COP1-dependent ubiquitination and degradation of STAT3 in normal prostate epithelial cells and, conversely, STAT3 accumulation and activation in prostate cancer cells with loss of COP1. To assess the clinical relevance of these findings, we examined the level of COP1, tSTAT3 and pSTAT3 by IHC in primary prostate tumors (n = 136) from patients with long-term clinical follow up. Low COP1 levels were significantly associated with high tSTAT3 expression (Fisher test p&amp;lt;0.001). Furthermore, pSTAT3 was prevalently observed in the group of COP1 negative/STAT3 high tumors indicative of STAT3 activation. The combination of low COP1 and high STAT3 expression was associated with significantly higher risk of disease recurrence after prostatectomy (p≤0.01) marking patients at risk of poor clinical outcome. Collectively, this study identifies STAT3 as a substrate of COP1 and provides evidence of a novel pathway leading to STAT3 activation in human tumors. Citation Format: Cecilia Dallavalle, George Thalmann, Carlo V. Catapano, Giuseppina M. R. Carbone. The E3 ubiquitin ligase COP1 controls STAT3 turnover and its loss leads to increased STAT3 stabilization and activation in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4545.

Proteasome inhibitor PS-341 limits macrophage necroptosis by promoting cIAPs-mediated inhibition of RIP1 and RIP3 activation

Apoptotic and necrotic macrophages have long been known for their existence in atherosclerotic lesions. However, the mechanisms underlying the choice of their death pattern have not been fully elucidated. Here, we report the effects of PS-341, a potent and specific proteasome inhibitor, on the cell death of primary bone marrow-derived macrophages (BMDMs) in vitro. The results showed that PS-341 could not induce macrophage apoptosis or promote TNF-induced macrophage apoptosis, on the other hand, PS-341 could significantly inhibit macrophage necroptosis induced by TNF and pan-caspase inhibitor z-VAD treatment. Remarkably, high-dose of PS-341 showed similar inhibitory effects on macrophage necroptosis comparable to that of kinase inhibition of RIP1 through specific inhibitor Nec-1 or inhibition of RIP3 via specific genetical ablation. Furthermore, the degradation of cellular inhibitor of apoptosis proteins (cIAPs) was suppressed by PS-341, which could antagonize the activation of RIP1 kinase via post-translational mechanism. Further evidences demonstrated reduced levels of both RIP1 and RIP 3 upon PS-341 treatment, concomitantly, a more strong association of RIP1 with cIAPs and less with RIP3 was found following PS-341 treatment, these findings suggested that PS-341 may disrupt the formation of RIP1-RIP3 complex (necrosome) through stabilizing cIAPs. Collectively, our results indicated that the proteasome-mediated degradation of cIAPs could be inhibited by PS-341 and followed by limited RIP1 and RIP3 kinase activities, which were indispensable for necroptosis, thus eliciting a significant necroptosis rescue in BMDMs in vitro. Overall, our study has identified a new role of PS-341 in the cell death of BMDMs and provided a novel insight into the atherosclerotic inflammation caused by proteasome-mediated macrophage necroptosis.

Next-generation proteasome inhibitor MLN9708 sensitizes breast cancer cells to doxorubicin-induced apoptosis.

Doxorubicin (Dox), one of the most effective chemotherapy drug for cancer treatment, is limited by its severe side effects and chemoresistance. Dox induces DNA damage and leads to significant proteomic changes in the cancer cells, which makes the ubiquitin-proteasome system a potential target to enhance the efficacy of Dox therapy. The unsuccessful clinical trials of proteasome inhibitor PS-341 (bortezomib) in solid tumors led to the invention of MLN9708 (ixazomib), an orally bioavailable next-generation proteasome inhibitor with improved pharmacokinetic and pharmacodynamic features. In this preclinical study, we used eight human breast cancer cell lines, which represent the major molecular subtypes of breast cancer, to validate the cytotoxic effects of MLN9708, alone and in combination with Dox. We found that MLN9708 had cytotoxic effects, induced autophagy and MKP-1 expression, and enhanced Dox-induced apoptosis in these cell lines. MLN9708 also enhanced Dox-induced JNK and p38 phosphorylation and inhibited Dox-induced IκBα degradation. Our in vitro results suggest that MLN9708 has antitumor effects in breast cancer and can sensitize breast cancer cells to Dox treatment. This promising combination may be an effective and feasible therapeutic option for treating breast cancer and warrants clinical validation.

A microRNA-mediated decrease in eukaryotic initiation factor 2α promotes cell survival during PS-341 treatment.

MicroRNAs (miRs) play pivotal roles in carcinogenesis and endoplasmic reticulum (ER) that performs the folding, modification and trafficking of proteins targeted to the secretory pathway. Cancer cells often endure ER stress during tumor progression but use the adaptive ER stress response to gain survival advantage. Here we report: (i) A group of miRs, including miR-30b-5p and miR-30c-5p, are upregulated by proteasome inhibitor PS-341 treatment, in HepG2 and MDA-MB-453 cells. (ii) Two representative PS-341-induced miRs: miR-30b-5p and miR-30c-5p are found to promote cell proliferation and anti-apoptosis in both tumor cells. (iii) eIF2α is confirmed as the congenerous target of miR-30b-5p and miR-30c-5p, essential to the anti-apoptotic function of these miRs. (iv) Upregulation of miR-30b-5p or miR-30c-5p, which occurs latter than the increase of phosphorylated eIF2α (p-eIF2α) in the cell under ER stress, suppresses the p-eIF2α/ATF4/CHOP pro-apoptotic pathway. (v) Inhibition of the miR-30b-5p or miR-30c-5p sensitizes the cancer cells to the cytotoxicity of proteasome inhibition. In conclusion, we unravels a new miRs-based mechanism that helps maintain intracellular proteostasis and promote cell survival during ER stress through upregulation of miR-30b-5p and miR-30c-5p which target eIF2α and thereby inhibit the p-eIF2α/ATF4/CHOP pro-apoptotic pathway, identifying miR-30b-5p and miR-30c-5p as potentially new targets for anti-cancer therapies.

Proteasomal inhibition potentiates drugs targeting DNA topoisomerase II

The reaction mechanism of DNA topoisomerase II (TOP2) involves a covalent double-strand break intermediate in which the enzyme is coupled to DNA via a 5′-phosphotyrosyl bond. This normally transient enzyme-bridged break is stabilised by drugs such as mitoxantrone, mAMSA, etoposide, doxorubicin, epirubicin and idarubicin, which are referred to as TOP2 poisons. Removal of topoisomerase II by the proteasome is involved in the repair of these lesions. In K562 cells, inhibiting the proteasome with MG132 significantly potentiated the growth inhibition by these six drugs that target topoisomerase II, and the highest level of potentiation was observed with mitoxantrone. Mitoxantrone also showed the greatest potentiation by MG132 in three Nalm 6 cell lines with differing levels of TOP2A or TOP2B. Mitoxantrone was also potentiated by the clinically used proteasome inhibitor PS341 (Velcade). We have also shown that proteasome inhibition with MG132 in K562 cells reduces the rate of removal of mitoxantrone or etoposide stabilised topoisomerase complexes from DNA, suggesting a possible mechanism for the potentiation of topoisomerase II drugs by proteasomal inhibition.

Disulfiram sensitizes pituitary adenoma cells to temozolomide by regulating O6-methylguanine-DNA methyltransferase expression.

O6-methylguanine-DNA methyltransferase (MGMT) activity is responsible for temozolomide (TMZ) resistance in patients harboring aggressive pituitary adenomas. Recently, disulfiram (DSF) has been shown to induce the loss of MGMT protein and increase TMZ efficacy in glioblastoma cells, while CD133+ nestin+ cells isolated from the cell population have been implicated as pituitary adenoma stem-like cells. However, whether DSF is able to potentiate the cytotoxic effects of TMZ on human pituitary adenoma cells has not been investigated to date. In the present study, CD133+ nestin+ phenotype cells were isolated from primary cultured human pituitary adenoma cells using microbeads. It was found that DSF reduced MGMT protein expression and sensitized human pituitary adenoma cells and stem-like cells to TMZ in vitro, while the proteasome inhibitor PS-341 abrogated the inhibitory effect of DSF on MGMT in vitro. The sensitizing effect of DSF was also verified in primary cultured human pituitary adenoma cells in vivo. The results of the present study suggested that DSF can increase the efficacy of the anti-tumor effect of TMZ on human pituitary adenoma cells and CD133+ nestin+ stem like cells via the ubiquitin-proteasomal MGMT protein elimination route. DSF combined with TMZ may be an effective therapeutic strategy against aggressive pituitary adenomas.

IMiD® Compounds Affect the Hematopoiesis Via CRBN Dependent Degradation of IKZF1 Protein in CD34+ Cells

Introduction: Lenalidomide (LEN) and pomalidomide (POM) are derivatives of thalidomide (IMiD® compounds) and currently used to treat multiple myeloma (MM) and B-cell malignancies. We have shown before that IMiD® compounds shift lineage commitment of CD34+ cells towards myeloid development by affecting critical transcription factors such as GATA1 and PU.1 with concomitant inhibition of cell maturation resulting in anemia and neutropenia. Nonetheless the underlying pathomechanism is still unknown. Recently, IMiD® compounds were shown to bind to cereblon (CRBN) in MM cells, which is the substrate recognition component of cullin-dependent ubiquitin ligase and LEN leads to ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IZKF3 by the CRBN-CRL4 ubiquitin ligase. We investigated here the role of CRBN, IKZF1 and IKZF3 in IMiD®-induced effects on lineage commitment and maturation of CD34+ cells.Methods and Results: By western blot analysis we found that CRBN and IKZF1, but not IKZF3 are expressed in CD34+ cells. Treatment of CD34+ cells with LEN and POM for only 1h almost completely decreased the expression of IKZF1 without affecting CRBN protein expression. By using a thalidomide analog bead assay, we found that IMiD® compounds directly bind CRBN in CD34+ cells. In contrast to our protein studies, IKZF1-mRNA level was not altered in real-time PCR, suggesting that IMiD® compounds regulate IKZF1 at post-transcriptional level. Treatment with proteasome inhibitors MG132, PS341 or MLN4924 which function as cullin-dependent ubiquitin ligase inhibitors blocked LEN and POM induced IKZF1 degradation, confirming that IMiD® compounds induce ubiquitination and subsequent protein degradation of endogenous IKZF1. Next we generated lentiviral constructs to knockdown the expression of CRBN in CD34+ cells. Knockdown of CRBN in CD34+ cells induced resistance to POM-induced IKZF1 downregulation and subsequently reversed the POM-induced lineage shift in colony-formation assays, suggesting that POM-induced degradation of IKZF1 in HSC requires CRBN.Knowing that PU.1 and GATA-1 are critically involved in the IMID-induced lineage shift in CD34+ cells we demonstrated in chromatin immunoprecipitation assays that IKZF1 binds to promoter regions of PU.1 and GATA-1, suggesting that PU.1 and GATA-1 are direct downstream targets of IKZF1 in CD34+ cells .Conclusion: Our findings show that CRBN and IKZF1 mediate the effects of IMiD® compounds on hematopoietic progenitors. IMiD® compounds promote CRBN dependent degradation of IKZF1 protein in CD34+ cells that subsequently decreases transcription factors such as GATA1 and PU.1 which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Our findings that IMiD® compounds mediate their effects via CRBN and IKZF1 provide for the first time the pathomechanism how LEN and POM affect hematopoiesis and induce neutropenia, thrombocytopenia as well as anemia. DisclosuresLentzsch:Bristol Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Research Funding.

Correction: Induction of Cell Cycle Arrest and Apoptosis by the Proteasome Inhibitor PS-341 in Hodgkin Disease Cell Lines Is Independent of Inhibitor of Nuclear Factor-κB Mutations or Activation of the CD30, CD40, and RANK Receptors

Correction: Induction of Cell Cycle Arrest and Apoptosis by the Proteasome Inhibitor PS-341 in Hodgkin Disease Cell Lines Is Independent of Inhibitor of Nuclear Factor-κB Mutations or Activation of the CD30, CD40, and RANK Receptors

Proteasome inhibitor PS-341 attenuates flow-induced pulmonary arterial hypertension

PS-341, a proteasome inhibitor, is suggested to prevent the vascular remodeling induced by high-flow pulmonary artery hypertension (PAH), but the mechanism remains unclear. The aim of the current study was to investigate the effects and possible mechanism of PS-341 on hypertension-induced vascular remodeling. Male Sprague-Dawley rats were subjected to surgical methods to produce a shunt model of PAH. Three days after the surgical procedure, the animals randomly assigned to four groups (n = 10 in each group): I: sham group; II: shunt group; III: vehicle; IV: treated group. Eight weeks postoperative, the hemodynamics data were measured through Swan-Ganz catheter; the protein expression level of proliferating cell nuclear antigen, nuclear factor-κB (NF-κB), inhibitor of nuclear factor-κB (I-κBα), transforming growth factor beta-β (TGF-β), drosophila mothers against decapentaplegic protein (Smad) and vascular endothelia growth factor (VEGF) were investigated by immunohistochemical and Western blotting; the mRNA expression level of Ubiquitin (Ub), Smad3, TGF-β1and Smad2 in lung were performed to detect by real-time reverse transcription-polymerase chain reaction analysis. The results showed that hemodynamic data and right ventricular hypertrophy were significantly improved (P < 0.05), the expression level of Ub, NF-κB, TGF-β1, Smad2 and VEGF were decreased (P < 0.05), but the level of I-κBα was increased in PS-341 treated group as compared with the shunt and vehicle groups (P < 0.05). In conclusion, the present study indicated that PS-341 could significantly improve the lung damage, attenuate pulmonary vascular remodeling induced by high blood PAH model. The mechanism may be mediated by inhibition of NF-κB and TGF-β/Smad signaling pathway and modulation the effect of VEGF.

Degradation of NF-κB, p53 and other regulatory redox-sensitive proteins by thiol-conjugating and -nitrosylating drugs in human tumor cells

The ionized cysteines present on the surfaces of many redox-sensitive proteins play functionally essential roles and are readily targeted by the reactive oxygen and reactive nitrogen species. Using disulfiram (DSF) and nitroaspirin (NCX4016) as the model compounds that mediate thiol-conjugating and nitrosylating reactions, respectively, we investigated the fate of p53, nuclear factor-kappaB (NF-κB) and other redox-responsive proteins following the exposure of human cancer cell lines to the drugs. Both drugs induced glutathionylation of bulk proteins in tumor cells and cell-free extracts. A prominent finding of this study was a time- and dose-dependent degradation of the redox-regulated proteins after brief treatments of tumor cells with DSF or NCX4016. DSF and copper-chelated DSF at concentrations of 50-200 µM induced the disappearance of wild-type p53, mutant p53, NF-κB subunit p50 and the ubiquitin-activating enzyme E1 (UBE1) in tumor cell lines. DSF also induced the glutathionylation of p53. The recombinant p53 protein modified by DSF was preferentially degraded by rabbit reticulocyte lysates. The proteasome inhibitor PS341 curtailed the DSF-induced degradation of p53 in HCT116 cells. Further, the NCX4016 induced a dose-dependent disappearance of the UBE1 and NF-κB p50 proteins in cell lines, besides a time-dependent degradation of aldehyde dehydrogenase in mouse liver after a single injection of 150 mg/kg. The loss of p53 and NF-kB proteins correlated with decreases in their specific binding to DNA. Our results demonstrate the hitherto unrecognized ability of the non-toxic thiolating and nitrosylating agents to degrade regulatory proteins and highlight the exploitable therapeutic benefits.

Menin Missense Mutants Encoded by the MEN1 Gene that Are Targeted to the Proteasome: Restoration of Expression and Activity by CHIP siRNA

In multiple endocrine neoplasia type 1 (MEN1) characterized by tumors of parathyroid, enteropancreas, and anterior pituitary, missense mutations in the MEN1 gene product, menin, occur in a subset of cases. The mutant proteins are degraded by the proteasome. However, whether their expression and activity can be restored is not known. Our objective was to functionally characterize a panel of 16 menin missense mutants, including W423R and S443Y identified in new MEN1 families, with respect to protein stability, targeting to the proteasome and restoration of expression by proteasome inhibitors and expression and function by small interfering RNA technology. Flag-tagged wild-type (WT) and missense menin mutant expression vectors were transiently transfected in human embryonic kidney (HEK293) and/or rat insulinoma (Rin-5F) cells. The majority of mutants were short-lived, whereas WT menin was stable. Proteasome inhibitors MG132 and PS-341 and inhibition of the chaperone, heat-shock protein 70 (Hsp70), or the ubiquitin ligase, COOH terminus of Hsp70-interacting protein (CHIP), by specific small interfering RNA, restored the levels of the mutants, whereas that of WT menin was largely unaffected. Inhibition of CHIP restored the ability of mutants to mediate normal functions of menin: TGF-β up-regulation of the promoters of its target genes, the cyclin-dependent kinase inhibitors p15 and p21 as well as TGF-β inhibition of cell numbers. When the levels of missense menin mutants that are targeted to the proteasome are normalized they may function similarly to WT menin. Potentially, targeting specific components of the proteasome chaperone pathway could be beneficial in treating a subset of MEN1 cases.

Synergistic Efficacy in Human Ovarian Cancer Cells by Histone Deacetylase Inhibitor TSA and Proteasome Inhibitor PS-341

Histone deacetylase inhibitors and proteasome inhibitor are all emerging as new classes of anticancer agents. We chose TSA and PS-341 to identify whether they have a synergistic efficacy on human ovarian cancer cells. After incubated with 500 nM TSA or/and 40 nM PS-341, we found that combined groups resulted in a striking increase of apoptosis and G2/M blocking rates, no matter in A2780, cisplatin-sensitive ovarian cancer cell line OV2008 or its resistant variant C13*. This demonstrated that TSA interacted synergistically with PS-341, which raised the possibility that combined the two drugs may represent a novel strategy in ovarian cancer.