Pan-RAF and MEK vertical inhibition enhances therapeutic response in non-V600 BRAF mutant cells

BACKGROUND: Currently, there are no available targeted therapy options for non-V600 BRAF mutated tumors. The aim of this study was to investigate the effects of RAF and MEK concurrent inhibition on tumor growth, migration, signaling and apoptosis induction in preclinical models of non-V600 BRAF mutant tumor cell lines. METHODS: Six BRAF mutated human tumor cell lines CRL5885 (G466 V), WM3629 (D594G), WM3670 (G469E), MDAMB231 (G464 V), CRL5922 (L597 V) and A375 (V600E as control) were investigated. Pan-RAF inhibitor (sorafenib or AZ628) and MEK inhibitor (selumetinib) or their combination were used in in vitro viability, video microscopy, immunoblot, cell cycle and TUNEL assays. The in vivo effects of the drugs were assessed in an orthotopic NSG mouse breast cancer model. RESULTS: All cell lines showed a significant growth inhibition with synergism in the sorafenib/AZ628 and selumetinib combination. Combination treatment resulted in higher Erk1/2 inhibition and in increased induction of apoptosis when compared to single agent treatments. However, single selumetinib treatment could cause adverse therapeutic effects, like increased cell migration in certain cells, selumetinib and sorafenib combination treatment lowered migratory capacity in all the cell lines. Importantly, combination resulted in significantly increased tumor growth inhibition in orthotropic xenografts of MDAMB231 cells when compared to sorafenib - but not to selumetinib - treatment. CONCLUSIONS: Our data suggests that combined blocking of RAF and MEK may achieve increased therapeutic response in non-V600 BRAF mutant tumors

Immunohistochemical staining of radixin and moesin in prostatic adenocarcinoma

<p>Abstract</p> <p>Background</p> <p>Some members of the Protein 4.1 superfamily are believed to be involved in cell proliferation and growth, or in the regulation of these processes. While the expression levels of two members of this family, radixin and moesin, have been studied in many tumor types, to our knowledge they have not been investigated in prostate cancer.</p> <p>Methods</p> <p>Tissue microarrays were immunohistochemically stained for either radixin or moesin, with the staining intensities subsequently quantified and statistically analyzed using One-Way ANOVA or nonparametric equivalent with subsequent Student-Newman-Keuls tests for multiple comparisons. There were 11 cases of normal donor prostates (NDP), 14 cases of benign prostatic hyperplasia (BPH), 23 cases of high-grade prostatic intraepithelial neoplasia (HGPIN), 88 cases of prostatic adenocarcinoma (PCa), and 25 cases of normal tissue adjacent to adenocarcinoma (NAC) analyzed in the microarrays.</p> <p>Results</p> <p>NDP, BPH, and HGPIN had higher absolute staining scores for radixin than PCa and NAC, but with a significant difference observed between only HGPIN and PCa (p = < 0.001) and HGPIN and NAC (p = 0.001). In the moesin-stained specimens, PCa, NAC, HGPIN, and BPH all received absolute higher staining scores than NDP, but the differences were not significant. Stage 4 moesin-stained PCa had a significantly reduced staining intensity compared to Stage 2 (p = 0.003).</p> <p>Conclusions</p> <p>To our knowledge, these studies represent the first reports on the expression profiles of radixin and moesin in prostatic adenocarcinoma. The current study has shown that there were statistically significant differences observed between HGPIN and PCa and HGPIN and NAC in terms of radixin expression. The differences in the moesin profiles by tissue type were not statistically significant. Additional larger studies with these markers may further elucidate their potential roles in prostatic neoplasia progression.</p

Rab-GTPase binding effector protein 2 (RABEP2) is a primed substrate for Glycogen Synthase kinase-3 (GSK3)

Glycogen synthase kinase-3 (GSK3) regulates many physiological processes through phosphorylation of a diverse array of substrates. Inhibitors of GSK3 have been generated as potential therapies in several diseases, however the vital role GSK3 plays in cell biology makes the clinical use of GSK3 inhibitors potentially problematic. A clearer understanding of true physiological and pathophysiological substrates of GSK3 should provide opportunities for more selective, disease specific, manipulation of GSK3. To identify kinetically favourable substrates we performed a GSK3 substrate screen in heart tissue. Rab-GTPase binding effector protein 2 (RABEP2) was identified as a novel GSK3 substrate and GSK3 phosphorylation of RABEP2 at Ser200 was enhanced by prior phosphorylation at Ser204, fitting the known consensus sequence for GSK3 substrates. Both residues are phosphorylated in cells while only Ser200 phosphorylation is reduced following inhibition of GSK3. RABEP2 function was originally identified as a Rab5 binding protein. We did not observe co-localisation of RABEP2 and Rab5 in cells, while ectopic expression of RABEP2 had no effect on endosomal recycling. The work presented identifies RABEP2 as a novel primed substrate of GSK3, and thus a potential biomarker for GSK3 activity, but understanding how phosphorylation regulates RABEP2 function requires more information on physiological roles of RABEP2

Glycogen Synthase Kinase 3 Beta (GSK3β) Phosphorylates the RNAase III Enzyme Drosha at S300 and S302

The canonical microRNA (miRNA) pathway commences with the enzymatic cleavage of the primary gene transcript (pri-miRNA) by the RNAase III enzyme Drosha in the nucleus into shorter pre-miRNA species that are subsequently exported to the cytoplasm for further processing into shorter, mature miRNA molecules. Using a series of reporter constructs, we have previously demonstrated that phosphorylation of Drosha at Ser 300 and 302 was required for its nuclear localization. Here, we identify GSK3β as the culprit kinase. We demonstrate that Drosha is unable to selectively localize to the nucleus in cells deficient in GSK3β. These findings expand the substrate base of GSK3β to include a central component of the miRNA biogenesis pathway

Domain Swapping and Different Oligomeric States for the Complex Between Calmodulin and the Calmodulin-Binding Domain of Calcineurin A

BACKGROUND: Calmodulin (CaM) is a ubiquitously expressed calcium sensor that engages in regulatory interactions with a large number of cellular proteins. Previously, a unique mode of CaM target recognition has been observed in the crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A. METHODOLOGY/PRINCIPAL FINDINGS: We have solved a high-resolution crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A in a novel crystal form, which shows a dimeric assembly of calmodulin, as observed before in the crystal state. We note that the conformation of CaM in this complex is very similar to that of unliganded CaM, and a detailed analysis revels that the CaM-binding motif in calcineurin A is of a novel '1-11' type. However, using small-angle X-ray scattering (SAXS), we show that the complex is fully monomeric in solution, and a structure of a canonically collapsed CaM-peptide complex can easily be fitted into the SAXS data. This result is also supported by size exclusion chromatography, where the addition of the ligand peptide decreases the apparent size of CaM. In addition, we studied the energetics of binding by isothermal titration calorimetry and found them to closely resemble those observed previously for ligand peptides from CaM-dependent kinases. CONCLUSIONS/SIGNIFICANCE: Our results implicate that CaM can also form a complex with the CaM-binding domain of calcineurin in a 1 ratio 1 stoichiometry, in addition to the previously observed 2 ratio 2 arrangement in the crystal state. At the structural level, going from 2 ratio 2 association to two 1 ratio 1 complexes will require domain swapping in CaM, accompanied by the characteristic bending of the central linker helix between the two lobes of CaM

CTLA-4 Activation of Phosphatidylinositol 3-Kinase (PI 3-K) and Protein Kinase B (PKB/AKT) Sustains T-Cell Anergy without Cell Death

The balance of T-cell proliferation, anergy and apoptosis is central to immune function. In this regard, co-receptor CTLA-4 is needed for the induction of anergy and tolerance. One central question concerns the mechanism by which CTLA-4 can induce T-cell non-responsiveness without a concurrent induction of antigen induced cell death (AICD). In this study, we show that CTLA-4 activation of the phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death. CTLA-4 ligation induced PI 3K activation as evidenced by the phosphorylation of PKB/AKT that in turn inactivated GSK-3. The level of activation was similar to that observed with CD28. CTLA-4 induced PI 3K and AKT activation also led to phosphorylation of the pro-apoptotic factor BAD as well as the up-regulation of BcL-XL. In keeping with this, CD3/CTLA-4 co-ligation prevented apoptosis under the same conditions where T-cell non-responsiveness was induced. This effect was PI 3K and PKB/AKT dependent since inhibition of these enzymes under conditions of anti-CD3/CTLA-4 co-ligation resulted in cell death. Our findings therefore define a mechanism by which CTLA-4 can induce anergy (and possibly peripheral tolerance) by preventing the induction of cell death

Trypanosoma brucei Glycogen Synthase Kinase-3, A Target for Anti-Trypanosomal Drug Development: A Public-Private Partnership to Identify Novel Leads

Over 60 million people in sub-Saharan Africa are at risk of infection with the parasite Trypanosoma brucei which causes Human African Trypanosomiasis (HAT), also known as sleeping sickness. The disease results in systemic and neurological disability to its victims. At present, only four drugs are available for treatment of HAT. However, these drugs are expensive, limited in efficacy and are severely toxic, hence the need to develop new therapies. Previously, the short TbruGSK-3 short has been validated as a potential target for developing new drugs against HAT. Because this enzyme has also been pursued as a drug target for other diseases, several inhibitors are available for screening against the parasite enzyme. Here we present the results of screening over 16,000 inhibitors of human GSK-3β (HsGSK-3) from the Pfizer compound collection against TbruGSK-3 short. The resulting active compounds were tested for selectivity versus HsGSK-3β and a panel of human kinases, as well as their ability to inhibit proliferation of the parasite in vitro. We have identified attractive compounds that now form potential starting points for drug discovery against HAT. This is an example of how a tripartite partnership involving pharmaceutical industries, academic institutions and non-government organisations such as WHO TDR, can stimulate research for neglected diseases

Synergy between inhibitors of androgen receptor and MEK has therapeutic implications in estrogen receptor-negative breast cancer

Introduction: Estrogen receptor-negative (ER-) breast cancer is a heterogeneous disease with limited therapeutic options. The molecular apocrine subtype constitutes 50% of ER-tumors and is characterized by overexpression of steroid response genes including androgen receptor (AR). We have recently identified a positive feedback loop between the AR and extracellular signal-regulated kinase (ERK) signaling pathways in the molecular apocrine subtype. In this feedback loop, AR regulates ERK phosphorylation through the mediation of ErbB2 and, in turn, ERK-CREB1 signaling regulates the transcription of AR in molecular apocrine cells. In this study, we investigated the therapeutic implications of the AR-ERK feedback loop in molecular apocrine breast cancer.Methods: We examined a synergy between the AR inhibitor flutamide and the MEK inhibitor CI-1040 in the molecular apocrine cell lines MDA-MB-453, HCC-1954 and HCC-202 using MTT cell viability and annexin V apoptosis assays. Synergy was measured using the combination index (CI) method. Furthermore, we examined in vivo synergy between flutamide and the MEK inhibitor PD0325901 in a xenograft model of the molecular apocrine subtype. The effects of in vivo therapies on tumor growth, cell proliferation and angiogenesis were assessed.Results: We demonstrate synergistic CI values for combination therapy with flutamide and CI-1040 across three molecular apocrine cell lines at four dose combinations using both cell viability and apoptosis assays. Furthermore, we show in vivo that combination therapy with flutamide and MEK inhibitor PD0325901 has a significantly higher therapeutic efficacy in reducing tumor growth, cellular proliferation and angiogenesis than monotherapy with these agents. Moreover, our data suggested that flutamide and CI-1040 have synergy in trastuzumab resistance models of the molecular apocrine subtype. Notably, the therapeutic effect of combination therapy in trastuzumab-resistant cells was associated with the abrogation of an increased level of ERK phosphorylation that was developed in the process of trastuzumab resistance.Conclusions: In this study, we demonstrate in vitro and in vivo synergies between AR and MEK inhibitors in molecular apocrine breast cancer. Furthermore, we show that combination therapy with these inhibitors can overcome trastuzumab resistance in molecular apocrine cells. Therefore, a combination therapy strategy with AR and MEK inhibitors may provide an attractive therapeutic option for the ER-/AR+ subtype of breast cancer