Hearing dysfunction in heterozygous M itf Mi‐wh /+ mice, a model for W aardenburg syndrome type 2 and T ietz syndrome

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/95364/1/pcmr12030.pd

Analysis of SOX10 Function in Neural Crest-Derived Melanocyte Development: SOX10-Dependent Transcriptional Control of Dopachrome Tautomerase

AbstractSOX10 is a high-mobility-group transcription factor that plays a critical role in the development of neural crest-derived melanocytes. At E11.5, mouse embryos homozygous for the Sox10Dom mutation entirely lack neural crest-derived cells expressing the lineage marker KIT, MITF, or DCT. Moreover, neural crest cell cultures derived from homozygous embryos do not give rise to pigmented cells. In contrast, in Sox10Dom heterozygous embryos, melanoblasts expressing KIT and MITF do occur, albeit in reduced numbers, and pigmented cells eventually develop in nearly normal numbers both in culture and in vivo. Intriguingly, however, Sox10Dom/+ melanoblasts transiently lack Dct expression both in culture and in vivo, suggesting that during a critical developmental period SOX10 may serve as a transcriptional activator of Dct. Indeed, we found that SOX10 and DCT colocalized in early melanoblasts and that SOX10 is capable of transactivating the Dct promoter in vitro. Our data suggest that during early melanoblast development SOX10 acts as a critical transactivator of Dct, that MITF, on its own, is insufficient to stimulate Dct expression, and that delayed onset of Dct expression is not deleterious to the melanocyte lineage

17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model

<div><p>Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in <i>BRAF</i> (<i>BRAF</i>^<i>V600E</i>) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAF^V600E and BRAF wildtype (BRAF^WT) melanomas, although there were conflicting reports about the dependence of BRAF^V600E and BRAF^WT upon HSP90 activity for stability. Here, we demonstrate that BRAF^WT and CRAF are bound by HSP90 in BRAF^WT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAF^WT in the majority of NRAS mutant melanoma cells. The highly-selective BRAF^V600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAF^WT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAF^WT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAF^V600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAF^WT cell lines studied as well as in B16F10 mouse melanoma cells. <i>In vivo</i>, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of tumor growth compared to either therapy alone. Our studies support a role for 17-AAG and HSP90 inhibition in enhancing cellular immunotherapy for melanoma.</p></div

17AAG promotes the inhibition of Tyrp1 specific CD4+ T cell treated melanoma tumor growth.

<p>(A) Cultured B16 mouse melanoma cell lines were incubated with 17-AAG (1.0 μM) for 72 h, and cell lysates collected and studied for protein and phosphoprotein expression following SDS-PAGE and Western transfer. (B) The C57BL6 mice were subcutaneously injected with 2 x 10⁵ B16 mouse melanoma cells. Five days after B16 tumor cell injection, the mice received intraperitoneal injection of either 17AAG or vehicle at a dose of 75 mg/Kg body weight x 5 consecutive days. On seventh day after tumor challenge, all mice were irradiated one time with 550 rads (5.5Gy). 50% of 17AAG and 50% of Vehicle treated tumor bearing mice received 2 x 10⁵ numbers of TYRP1-specific CD4+ T cells (T4 cells) were injected intravenously. The 17-AAG dose of 75 mg/kg x 5 consecutive days was repeated every 2 weeks following initial administration of 17-AAG until the end of experiment. (C) The time dependent effect on the B16 melanoma tumor growth in C57BL6 mice treated either with Vehicle, 17AAG, Vehicle+T4 cells or 17AAG+T4 cells were determined by measuring their growth at regular intervals till the end of the experiment (n = 6). Data is a representative of two independent experiments. (D) The survival curve analysis shows that mice receiving no T4 cell transfer and only T4 cell transfer had no surviving mice at day 40. (*P<0.05 is a comparison between Vehicle+T4 cells and 17AAG+T4 cells as indicated by dotted lines).</p

Molecular interactions between BRAF, CRAF, and HSP90 in human melanoma cells.

<p>(A) Mass spectroscopic identification of HSP90α and HSP90β as a binding partner of BRAF in human melanoma cells. An immunoprecipitate of BRAF from Mel-Juso melanoma cell lysate was incubated with an anti-BRAF monoclonal antibody, electrophoresed, and subjected to Western blot analysis (left) and Coomassie Blue staining (right). Excision of an ~85 kDa Coomassie-stained band (box) followed by mass spectroscopic analysis revealed peptides corresponding of α- and β-isoforms of HSP90. (B) Co-immunoprecipitation of HSP90 from human melanoma cell lysate with mouse monoclonal anti-BRAF. Following electrophoresis of an anti-BRAF immunoprecipitate, Western blotting (left panel) with monoclonal anti-HSP90 demonstrates HSP90 in the immunoprecipitation complex (BRAF) compared to a control immunoprecipitate with murine IgG (mIgG). (Right panel) Reprobing the membrane in (left panel) with anti-BRAF (right panel) confirms the presence of BRAF in the immunoprecipitate. (C) Immunoprecipitation conditions on stability of HSP90-BRAF interaction. Monoclonal anti-BRAF was incubated with Mel-Juso cell lysate under the following conditions: Condition A, PBS, pH 7.4, 0.1% SDS, 0.5% sodium deoxycholate; Condition B, 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1% NP-40, 0.25% sodium deoxycholate; Condition C, 50 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 1% NP-40 (TENSV) (5); Condition D, 10 mM HEPES, pH 7.35, 20 mM sodium molybdate [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191264#pone.0191264.ref031" target="_blank">31</a>]. (D) (Left panel) Co-immunoprecipitation of HSP90 with either BRAF or CRAF in human melanoma cells. Mel-Juso cell lysate was incubated with either monoclonal anti-BRAF or anti-CRAF. Following electrophoresis and Western transfer, the blot was reprobed with anti-HSP90. (Right panel) Co-immunoprecipitation of CRAF with BRAF. The blot after stripping was reprobed with anti-CRAF. The faint band above the CRAF band is residual signal from previous probing with anti-HSP90.</p

Effect of 17-AAG on cell proliferation in human melanoma cells.

<p>(A) Inhibition of melanoma cell viability with increasing concentrations of 17-AAG. Human melanoma cells (A375, SK-Mel-28, Mel-Juso, SK-Mel-30, and SK-Mel-2) were incubated with increasing concentrations (0, 0.1, 0.3, 1.0 μM) of 17-AAG for 48 h. Relative cell number was assessed by differential absorbance at 550 and 690 nm using the MTT assay. (B) Time-dependent growth inhibition of human melanoma cells with 0.3 μM 17-AAG. Human melanoma cells lines were incubated with 0.3 μM 17-AAG for 12, 24, 36, 60, 70, and 84 h before determination of relative cell number using the MTT assay. (**P<0.01; *P<0.05).</p