SEMA6A/RhoA/YAP axis mediates tumor-stroma interactions and prevents response to dual BRAF/MEK inhibition in BRAF-mutant melanoma.

BACKGROUND: Despite the promise of dual BRAF/MEK inhibition as a therapy for BRAF-mutant (BRAF-mut) melanoma, heterogeneous responses have been observed in patients, thus predictors of benefit from therapy are needed. We have previously identified semaphorin 6A (SEMA6A) as a BRAF-mut-associated protein involved in actin cytoskeleton remodeling. The purpose of the present study is to dissect the role of SEMA6A in the biology of BRAF-mut melanoma, and to explore its predictive potential towards dual BRAF/MEK inhibition. METHODS: SEMA6A expression was assessed by immunohistochemistry in melanoma cohort RECI1 (N = 112) and its prognostic potential was investigated in BRAF-mut melanoma patients from DFCI and TCGA datasets (N = 258). The molecular mechanisms regulated by SEMA6A to sustain tumor aggressiveness and targeted therapy resistance were investigated in vitro by using BRAF-mut and BRAF-wt melanoma cell lines, an inducible SEMA6A silencing cell model and a microenvironment-mimicking fibroblasts-coculturing model. Finally, SEMA6A prediction of benefit from dual BRAF/MEK inhibition was investigated in melanoma cohort RECI2 (N = 14). RESULTS: Our results indicate higher protein expression of SEMA6A in BRAF-mut compared with BRAF-wt melanoma patients and show that SEMA6A is a prognostic indicator in BRAF-mut melanoma from TCGA and DFCI patients cohorts. In BRAF-mut melanoma cells, SEMA6A coordinates actin cytoskeleton remodeling by the RhoA-dependent activation of YAP and dual BRAF/MEK inhibition by dabrafenib+trametinib induces SEMA6A/RhoA/YAP axis. In microenvironment-mimicking co-culture condition, fibroblasts confer to melanoma cells a proliferative stimulus and protect them from targeted therapies, whereas SEMA6A depletion rescues the efficacy of dual BRAF/MEK inhibition. Finally, in BRAF-mut melanoma patients treated with dabrafenib+trametinib, high SEMA6A predicts shorter recurrence-free interval. CONCLUSIONS: Overall, our results indicate that SEMA6A contributes to microenvironment-coordinated evasion of melanoma cells from dual BRAF/MEK inhibition and it might be a good candidate predictor of short-term benefit from dual BRAF/MEK inhibition.

Up-regulation of activating and inhibitory NKG2 receptors in allogeneic and autologous hematopoietic stem cell grafts.

BACKGROUND: Hematopoietic Stem Cell Transplantation (HSCT) is known to induce the inhibitory immune receptor NKG2A on NK cells of donor origin. This occurs in allogeneic recipients, in both the haploidentical and HLA-matched settings. METHODS: To gain further insight, not only NKG2A, but also the activating receptors NKG2C and NKG2D were assessed by flow cytometry. Immunophenotyping was carried out not only on CD56(+) but also on CD8(+) lymphocytes from leukemia and lymphoma patients, receiving both HLA-matched (n = 7) and autologous (n = 5) HSCT grafts. Moreover, cognate NKG2 ligands (HLA-E, MICA, ULBP-1, ULBP-2 and ULBP-3) were assessed by immunohistochemistry in diagnostic biopsies from three autotransplanted patients, and at relapse in one case. RESULTS: All the NKG2 receptors were simultaneously up-regulated in all the allotransplanted patients on CD8(+) and/or CD56(+) cells between 30 and 90 days post-transplant, coinciding with, or following, allogeneic engraftment. Up-regulation was of lesser entity and restricted to CD8(+) cells in the autotransplantation setting. The phenotypic expression ratio between activating and inhibitory NKG2 receptors was remarkably similar in all the patients, except two outliers (a long survivor and a short survivor) who surprisingly displayed a similar NKG2 activation immunophenotype. Tumor expression of 2 to 3 out of the 5 tested NKG2 ligands was observed in 3/3 diagnostic biopsies, and 3 ligands were up-regulated post-transplant in a patient. CONCLUSIONS: Altogether, these results are consistent with a dual (activation-inhibition) NK cell re-education mode, an innate-like T cell re-tuning, and a ligand:receptor interplay between the tumor and the immune system following HSCT including, most interestingly, the up-regulation of several activating NKG2 ligands. Turning the immune receptor balance toward activation on both T and NK cells of donor origin may complement ex vivo NK cell expansion/activation strategies in unmanipulated patients.

Monoclonal antibodies to HLA-E bind epitopes carried by unfolded ß2 m-free heavy chains.

Since HLA-E heavy chains accumulate free of their light ß2 -microglobulin (ß2 m) subunit, raising mAbs to folded HLA-E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA-E-restricted mAbs (MEM-E/02, MEM-E/07, MEM-E/08, DT9, and 3D12) were tested on denatured, acid-treated, and natively folded (both ß2 m-associated and ß2 m-free) HLA-E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet ß2 m-free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM-E/02, MEM-E/07, and MEM-E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4-6 residues and are "hidden" in folded HLA-E heterodimers. They contain at least one HLA-E-specific residue that cannot be replaced by single substitutions with polymorphic HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G residues. Finally, also the MEM-E/02 and 3D12 epitopes are spatially distinct. In summary, HLA-E-specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA-E heterodimers, and HLA-E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies.

Antibody-drug conjugates: targeting melanoma with cisplatin encapsulated in protein-cage nanoparticles based on human ferritin.

A novel antibody-drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt-Pt-Ep1). The HFt-Pt-Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4(+) melanoma cell line, but not to a CSPG4(-) breast carcinoma cell line. As compared to the cisplatin-containing ferritin nanoparticle alone (HFt-Pt), which inhibited thymidine incorporation more efficiently in breast carcinoma than melanoma cells, the mAb-derivatized HFt-Pt-Ep1 nanoparticle had a 25-fold preference for the latter. A similar preference for melanoma was observed upon systemic intravenous administration of HFt-Pt-Ep1 to nude mice xenotransplanted with pre-established, palpable melanoma and breast carcinoma tumors. Thus, we have been able to determine precise combinations and stoichiometric relationships between mAbs and nanoparticle protein cages, whereby the latter lose their tropism for ubiquitously distributed cellular receptors, and acquire instead remarkably lineage-selective binding. HFt-Pt-Ep1 is therefore an interesting model to improve the therapeutic index of antiblastic therapy in a tumor such as melanoma, which at its advanced stages is totally refractory to mono- and combination-chemotherapy.

Artichoke polyphenols induce apoptosis and decrease the invasive potential of the human breast cancer cell line MDA-MB231.

The human breast cancer cell line, estrogen receptor negative, MDA-MB231, was used to evaluate the antitumor effect of polyphenolic extracts from the edible part of artichokes (AEs). Treatment of cancer cells reduced cell viability and inhibited cell growth in a dose-dependent manner. Importantly, AEs did not have any effect on normal breast epithelial cell line, MCF10A. Chlorogenic acid (ChA), the most representative component of the polyphenolic fraction of artichoke, had no prominent effects on the cell death rate of MDA-MB231 cells. The addition of AEs to the cells, rather than ChA, triggered apoptosis via a mitochondrial and a death-receptor pathway, as shown by the activation of caspase-9 and caspase-8, respectively. Furthermore, an increase of the Bax:Bcl2 ratio and up-regulation of cyclin-dependent kinase inhibitor, p21(WAF1), crucial apoptotic players, were documented. According to our data on activation of caspase-9, a loss of mitochondrial transmembrane potential (Ψ(m)) was shown. Cell motility and invasion capabilities were remarkably inhibited by AEs-treatment in highly invasive MDA-MB231 cells. In addition, a significant decrease of proteolytic activity of metalloproteinase-2 protein (MMP-2), involved in degrading components of the extracellular matrix, was detected. Our findings indicate that AEs reduced cell viability, inhibited cell growth, triggered apoptotic mechanisms, and showed inhibitory properties against the invasive behavior of MDA-MB231 cancer cell line. Altogether, these data indicate the potential chemopreventive activity of artichoke polyphenolic extracts.

Altered expression of endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 in transformed non-lymphoid human tissues.

The endoplasmic reticulum (ER) aminopeptidases ERAP1 and ERAP2 contribute to generate HLA class I binding peptides. Recently, we have shown that the expression of these enzymes is high and coordinated (with each other and with HLA class I molecules) in immortalized B cells, but variable and imbalanced in human tumour cell lines of various non-lymphoid lineages. Herein, this issue was investigated in vivo by testing ERAP1 and ERAP2 expression in normal non-lymphoid tissues and their malignant counterparts. ERAP1 and ERAP2 were detected exclusively in the epithelial cells of over half of the tested normal tissues. Four ERAP1/ERAP2 phenotypes (+/+, -/-, +/- and -/+) were detected, and the presence of either or both enzymes was not necessarily associated with HLA class I expression. In more than 160 neoplastic lesions, the expression of either or both aminopeptidases was retained, lost (most frequently, particularly ERAP1) or acquired as compared to the normal counterparts, depending on the tumour histotype. The double-negative (-/-) phenotype was the most frequent, and significantly (P = 0.013) associated with a lack of detectable HLA class I antigens. In selected neoplastic lesions, ERAP1 and ERAP2 were also tested for their enzymatic (peptide-trimming) activities. Expression and function were found to correlate, indicating that immunohistochemistry detects active enzymes in vivo. Thus, dissociation in the expression of ERAP1, ERAP2 and HLA class I may already be present in some normal tissues, but malignant transformation causes additional losses, gains and imbalances in specific tumour histotypes, and these alter the peptide-trimming ability of tumour cells in vivo.

Antioxidative and apoptotic properties of polyphenolic extracts from edible part of artichoke (Cynara scolymus L.) on cultured rat hepatocytes and on human hepatoma cells.

Cultured rat hepatocytes and human hepatoma HepG2 cells were used to evaluate the hepatoprotective properties of polyphenolic extracts from the edible part of artichoke (AE). The hepatocytes were exposed to H2O2generated in situ by glucose oxidase and were treated with either AE, or pure chlorogenic acid (ChA) or with the well known antioxidant, N, N'-diphenyl-p-phenilenediamine (DPPD). Addition of glucose oxidase to the culture medium caused depletion of intracellular glutathione (GSH) content, accumulation of malondialdehyde (MDA) in the cultures, as a lipid peroxidation indicator, and cell death. These results demonstrated that AE protected cells from the oxidative stress caused by glucose oxidase, comparable to DPPD. Furthermore, AE, as well as ChA, prevented the loss of total GSH and the accumulation of MDA. Treatment of HepG2 cells for 24 h with AE reduced cell viability in a dose-dependent manner, however, ChA had no prominent effects on the cell death rate. Similarly, AE rather than ChA induced apoptosis, measured by flow cytometric analysis of annexin and by activation of caspase-3, in HepG2 cells. Our findings indicate that AE had a marked antioxidative potential that protects hepatocytes from an oxidative stress. Furthermore, AE reduced cell viability and had an apoptotic activity on a human liver cancer cell line.

Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems.

BACKGROUND: Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. METHODS: Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs) to ErbB-2 that involves their functional expression in (a) bacteria, (b) transient as well as stable transgenic tobacco plants, and (c) a newly developed cell-free transcription-translation system. RESULTS: An ScFv (ScFv800E6) was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T), displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 x 10(8) M(-1)) only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining. CONCLUSION: ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology, and a candidate for future in vivo studies.

Expression of endoplasmic reticulum aminopeptidases in EBV-B cell lines from healthy donors and in leukemia/lymphoma, carcinoma, and melanoma cell lines.

Peptide trimming in the endoplasmic reticulum (ER), the final step required for the generation of most HLA class I-binding peptides, implicates the concerted action of two aminopeptidases, ERAP1 and ERAP2. Because defects in the expression of these peptidases could lead to aberrant surface HLA class I expression in tumor cells, we quantitatively assayed 14 EBV-B cell lines and 35 human tumor cell lines of various lineages for: 1) expression and enzymatic activities of ERAP1 and ERAP2; 2) ER peptide-trimming activity in microsomes; 3) expression of HLA class I H chains and TAP1; and 4) surface HLA class I expression. ERAP1 and ERAP2 expression was detectable in all of the EBV-B and tumor cell lines, but in the latter it was extremely variable, sometimes barely detectable, and not coordinated. The expression of the two aminopeptidases corresponded well to the respective enzymatic activities in most cell lines. A peptide-trimming assay in microsomes revealed additional enzymatic activities, presumably contributed by other unidentified aminopeptidases sharing substrate specificity with ERAP2. Interestingly, surface HLA class I expression showed significant correlation with ERAP1 activity, but not with the activity of either ERAP2 or other unidentified aminopeptidases. Transfection with ERAP1 or ERAP2 of two tumor cell lines selected for simultaneous low expression of the two aminopeptidases resulted in the expected, moderate increases of class I surface expression. Thus, low and/or imbalanced expression of ERAP1 and probably ERAP2 may cause improper Ag processing and favor tumor escape from the immune surveillance.

Impaired assembly results in the accumulation of multiple HLA-C heavy chain folding intermediates.

Class I MHC H chains assemble with beta2-microglobulin (beta2m) and are loaded with peptide Ags through multiple folding steps. When free of beta2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in this study, detected a preferential association of L31-reactive, beta2m-free H chains with calnexin in beta2m-defective cells, and with calreticulin and TAP in beta2m-expressing cells. In beta2m-defective cells, the accumulation of calnexin-bound H chains stoichiometrically exceeded their overall accumulation, a finding that supports both chaperoning preferences and distinct sorting abilities for different class I folds. No peptide species, in a mass range compatible with that of the classical class I ligands, could be detected by mass spectrometry of acidic eluates from L31-reactive HLA-Cw1 H chains. In vitro assembly experiments in TAP-defective T2 cells, and in cells expressing an intact Ag-processing machinery, demonstrated that L31 H chains are not only free of, but also unreceptive to, peptides. L31 and HC10, which bind nearly adjacent linear epitopes of the alpha1 domain alpha helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the alpha1 domain alpha helix and peptide-anchoring residues of the class I H chain. Thus, unlike certain murine free H chains, L31-reactive H chains are not the immediate precursors of conformed class I molecules. A model inferring their precursor-product relationships with other known class I intermediates is presented.

Ligand-regulated association of ErbB-4 to the transcriptional co-activator YAP65 controls transcription at the nuclear level.

It has been proposed that ligand-dependent Regulated Intramembrane Proteolysis (RIP) of ErbB-4 receptors generates 80 kDa Intra-Cellular Domains (E4.ICDs) that relocate to the nuclear compartments where they implement the signaling abilities of the ErbB-4 receptors. The E4.ICD may directly regulate gene transcription or, in an alternative scenario, the tyrosine kinase activity of E4.ICDs may target proteins involved in transcriptional regulation upon its relocation into the nucleus. We have identified the transcriptional coactivator YAP65, here referred as YAP (Yes Associated Protein), as binding partner of ErbB-4 in a two hybrid screening in yeast. Interaction between YAP and ErbB-4 occurs via the WW domain of YAP and the PPPPY at positions 1297-1301 and the PPPAY at positions 1052-1056 of the amino acid sequence of the Cyt-1 isoform of ErbB-4. Stechiometry of binding is regulated by the ligand-dependent phosphorylation of Tyr 1056 in the PPPAYTPM module that function as "biochemical switch" to decrease the association of YAP to ErbB-4. In principle, this novel interaction highlights new mechanisms of signaling propagation from the ErbB-4 receptors, offering supporting evidences that the E4.ICDs forms released following ligand-receptor engagement may recruit YAP and relocate to the nucleus to implement or regulate transcription.

Feedback inhibition by RALT controls signal output by the ErbB network.

The ErbB-2 interacting protein receptor-associated late transducer (RALT) was previously identified as a feedback inhibitor of ErbB-2 mitogenic signals. We now report that RALT binds to ligand-activated epidermal growth factor receptor (EGFR), ErbB-4 and ErbB-2.ErbB-3 dimers. When ectopically expressed in 32D cells reconstituted with the above ErbB receptor tyrosine kinases (RTKs) RALT behaved as a pan-ErbB inhibitor. Importantly, when tested in either cell proliferation assays or biochemical experiments measuring activation of ERK and AKT, RALT affected the signalling activity of distinct ErbB dimers with different relative potencies. RALT deltaEBR, a mutant unable to bind to ErbB RTKs, did not inhibit ErbB-dependent activation of ERK and AKT, consistent with RALT exerting its suppressive activity towards these pathways at a receptor-proximal level. Remarkably, RALT deltaEBR retained the ability to suppress largely the proliferative activity of ErbB-2.ErbB-3 dimers over a wide range of ligand concentrations, indicating that RALT can intercept ErbB-2.ErbB-3 mitogenic signals also at a receptor-distal level. A suppressive function of RALT deltaEBR towards the mitogenic activity of EGFR and ErbB-4 was detected at low levels of receptor occupancy, but was completely overcome by saturating concentrations of ligand. We propose that quantitative and qualitative aspects of RALT signalling concur in defining identity, strength and duration of signals generated by the ErbB network.