Syrbactin Structural Analog TIR-199 Blocks Proteasome Activity and Induces Tumor Cell Death.

Multiple myeloma is an aggressive hematopoietic cancer of plasma cells. The recent emergence of three effective FDA-approved proteasome-inhibiting drugs, bortezomib (Velcade®), carfilzomib (Kyprolis®), and ixazomib (Ninlaro®), confirms that proteasome inhibitors are therapeutically useful against neoplastic disease, in particular refractory multiple myeloma and mantle cell lymphoma. This study describes the synthesis, computational affinity assessment, and preclinical evaluation of TIR-199, a natural product-derived syrbactin structural analog. Molecular modeling and simulation suggested that TIR-199 covalently binds each of the three catalytic subunits (ß1, ß2, and ß5) and revealed key interaction sites. In vitro and cell culture-based proteasome activity measurements confirmed that TIR-199 inhibits the proteasome in a dose-dependent manner and induces tumor cell death in multiple myeloma and neuroblastoma cells as well as other cancer types in the NCI-60 cell panel. It is particularly effective against kidney tumor cell lines, with >250-fold higher anti-tumor activities than observed with the natural product syringolin A. In vivo studies in mice revealed a maximum tolerated dose of TIR-199 at 25 mg/kg. The anti-tumor activity of TIR-199 was confirmed in hollow fiber assays in mice. Adverse drug reaction screens in a kidney panel revealed no off-targets of concern. This is the first study to examine the efficacy of a syrbactin in animals. Taken together, the results suggest that TIR-199 is a potent new proteasome inhibitor with promise for further development into a clinical drug for the treatment of multiple myeloma and other forms of cancer.

PEA15 impairs cell migration and correlates with clinical features predicting good prognosis in neuroblastoma.

ERK and RSK2 drive proliferation and invasion of many cancers. Phosphoprotein enriched in astrocytes 15 (PEA15) binds ERK and RSK2 and high PEA15 levels can impair ERK- and RSK2-dependent transcription. PEA15 expression also inversely correlates with cell motility and invasiveness. We therefore tested PEA15 effects on neuroblastoma cells in vitro. We further analyzed PEA15 expression in the context of clinical and genetic features of neuroblastoma in tumor samples to determine its correlation with disease progression. Affymetrix microarray analysis was performed using 24 different neuroblastoma cell lines. Cell lines expressing low to intermediate levels of PEA15 were chosen for in vitro functional studies. The cell line results were verified by Affymetrix analysis of three different neuroblastic tumor types (total of 110 samples) PEA15 overexpression inhibited neuroblastoma migration in vitro. We verified that inhibition of motility required PEA15 interaction with its binding partners ERK and RSK2. Additionally, synthetic inhibitors of RSK2 suppressed integrin-dependent migration. PEA15 expression correlates with clinical parameters and a 25% increase in patient survival rate. The highest PEA15 levels were found in low stage, more differentiated and less metastatic neuroblastic tumors, and correlated with lack of MYCN amplification. PEA15 blocks neuroblastoma migration through inhibition of ERK/RSK2 signaling. PEA15 expression levels correlate with favorable clinical features suggesting that PEA15 limits metastatic progression of neuroblastoma. Thus, PEA15 and its partners ERK and RSK2 are potential targets for the development of new therapeutics to impede progression of minimal residual disease in patients with high-risk neuroblastoma.

Metastatic progression of prostate cancer and e-cadherin regulation by zeb1 and SRC family kinases.

Expression of E-cadherin is used to monitor the epithelial phenotype, and its loss is suggestive of epithelial-mesenchymal transition (EMT). EMT triggers tumor metastasis. Exit from EMT is marked by increased E-cadherin expression and is considered necessary for tumor growth at sites of metastasis; however, the mechanisms associated with exit from EMT are poorly understood. Herein are analyzed 185 prostate cancer metastases, with significantly higher E-cadherin expression in bone than in lymph node and soft tissue metastases. To determine the molecular mechanisms of regulation of E-cadherin expression, three stable isogenic cell lines from DU145 were derived that differ in structure, migration, and colony formation on soft agar and Matrigel. When injected into mouse tibia, the epithelial subline grows most aggressively, whereas the mesenchymal subline does not grow. In cultured cells, ZEB1 and Src family kinases decrease E-cadherin expression. In contrast, in tibial xenografts, E-cadherin RNA levels increase eight- to 10-fold despite persistent ZEB1 expression, and in all ZEB1-positive metastases (10 of 120), ZEB1 and E-cadherin proteins were co-expressed. These data suggest that transcriptional regulation of E-cadherin differs in cultured cells versus xenografts, which more faithfully reflect E-cadherin regulation in cancers in human beings. Furthermore, the aggressive nature of xenografts positive for E-cadherin and the frequency of metastases positive for E-cadherin suggest that high E-cadherin expression in metastatic prostate cancer is associated with aggressive tumor growth.

Syringolin B-inspired proteasome inhibitor analogue TIR-203 exhibits enhanced biological activity in multiple myeloma and neuroblastoma.

CONTEXT: The bacterium Pseudomonas syringae pv. syringae (Pss) is a pathogen of many plant species and causes, for example, brown spot disease in bean plants (Phaseolus vulgaris). Pss excretes the syringolins, natural product molecules that act as a virulence factors and inhibit the proteasome of the host plants. OBJECTIVE: Proteasome inhibitors belong to an important class of anticancer agents and bortezomib (Velcade(®)) has been Food and Drug Administration-approved for the treatment of multiple myeloma (MM) and mantle cell lymphoma. Syringolins represent a new class of proteasome inhibitors and the present work was undertaken to design a potent syringolin-inspired analogue (TIR-203) for anticancer drug development. MATERIALS AND METHODS: TIR-203 was tested against human MM and neuroblastoma (NB) cells. Cancer cells were treated with TIR-203 at various concentrations (0-10 µM) and the cell viability was measured using the MTS assay. To determine the effects on proteasomal activities, the cell culture-based proteasome inhibition assay was used. Syringolin A (SylA) and bortezomib were included as controls. RESULTS: TIR-203 inhibited the cell proliferation of MM and NB cells in a dose-dependent manner at significantly lower concentrations than SylA. In MM cells, TIR-203 effectively inhibited the chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) activities of the proteasome. In NB cells, TIR-203 inhibited the CT-L and C-L activities, but not the T-L activity. DISCUSSION AND CONCLUSIONS: The newly designed proteasome inhibitor TIR-203 is more potent than the natural product SylA and strongly inhibits the cell viability and proteasomal activity of MM and NB cells.

The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling.

PEA-15 is a death effector domain-containing phosphoprotein that binds ERK and restricts it to the cytoplasm. PEA-15 also binds to FADD and thereby blocks apoptosis induced by death receptors. Abnormal expression of PEA-15 is associated with type II diabetes and some cancers; however, its physiological function remains unclear. To determine the function of PEA-15 in vivo, we used C57BL/6 mice in which the PEA-15 coding region was deleted. We thereby found that PEA-15 regulates T-cell proliferation. PEA-15-null mice did not have altered thymic or splenic lymphocyte cellularity or differentiation. However, PEA-15 deficient T cells had increased CD3/CD28-induced nuclear translocation of ERK and increased activation of IL-2 transcription and secretion in comparison to control wild-type littermates. Indeed, activation of the T-cell receptor in wild-type mice caused PEA-15 release of ERK. In contrast, overexpression of PEA-15 in Jurkat T cells blocked nuclear translocation of ERK and IL-2 transcription. Finally, PEA-15-null T cells showed increased IL-2 dependent proliferation on stimulation. No differences in T cell susceptibility to apoptosis were found. Thus, PEA-15 is a novel player in T-cell homeostasis. As such this work may have far reaching implications in understanding how the immune response is controlled.

ERK MAP kinase is targeted to RSK2 by the phosphoprotein PEA-15.

The ERK pathway responds to extracellular stimuli and oncogenes by modulating cellular processes, including transcription, adhesion, survival, and proliferation. ERK has diverse substrates that carry out these functions. The processes that are modulated are determined in part by the substrates that ERK phosphorylates. We demonstrate that PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) targets ERK to RSK2 and thereby enhances RSK2 activation. PEA-15 independently bound ERK and RSK2 and increased ERK association with RSK2 in a concentration-dependent manner. PEA-15 increased RSK2 activity and CREB-mediated transcription, and this process was regulated by phosphorylation of PEA-15. Finally, phorbol ester stimulation of PEA-15-null lymphocytes resulted in impaired RSK2 activation that was rescued by exogenous PEA-15 expression. Therefore, PEA-15 functions as a scaffold to enhance ERK activation of RSK2, and this activity is regulated by phosphorylation. Thus, PEA-15 can integrate signal transduction to provide a specific physiological outcome from activation of the multipotent ERK MAP kinase pathway.

Withaferin A Inhibits STAT3 and Induces Tumor Cell Death in Neuroblastoma and Multiple Myeloma.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that has been implicated in many human cancers and has emerged as an ideal target for cancer therapy. Withaferin A (WFA) is a natural product with promising antiproliferative properties through its association with a number of molecular targets including STAT3. However, the effect of WFA in pediatric neuroblastoma (NB) and its interaction with STAT3 have not been reported. In this study, we found that WFA effectively induces dose-dependent cell death in high-risk and drug-resistant NB as well as multiple myeloma (MM) tumor cells, prevented interleukin-6 (IL-6)-mediated and persistently activated STAT3 phosphorylation at Y705, and blocked the transcriptional activity of STAT3. We further provide computational models that show that WFA binds STAT3 near the Y705 phospho-tyrosine residue of the STAT3 Src homology 2 (SH2) domain, suggesting that WFA prevents STAT3 dimer formation similar to BP-1-102, a well-established STAT3 inhibitor. Our findings propose that the antitumor activity of WFA is mediated at least in part through inhibition of STAT3 and provide a rationale for further drug development and clinical use in NB and MM.

Phosphorylation is the switch that turns PEA-15 from tumor suppressor to tumor promoter.

Abnormal ERK signaling is implicated in many human diseases including cancer. This signaling cascade is a good target for the therapy of certain malignancies because of its important role in regulating cell proliferation and survival. The small phosphoprotein PEA-15 is a potent regulator of the ERK signaling cascade, and, by acting on this pathway, has been described to have both tumor-suppressor and tumor-promoter functions. However, the exact mechanism by which PEA-15 drives the outcome one way or the other remains unclear. We propose that the cellular environment is crucial in determining PEA-15 protein function by affecting the protein's phosphorylation state. We hypothesize that only unphosphorylated PEA-15 can act as a tumor-suppressor and that phosphorylation alters the interaction with binding partners to promote tumor development. In order to use PEA-15 as a prognostic marker or therapeutic target it is therefore important to evaluate its phosphorylation status.