Activity of the Heat Shock Protein 90 Inhibitor Ganetespib in Melanoma

Heat shock protein 90 (HSP90) is involved in the regulation of diverse biological processes such as cell signaling, proliferation and survival, and has been recently recognized as a potential target for cancer therapy. Ganetespib is a potent ATP competitive inhibitor of HSP90. Ganetespib downregulated the expression of multiple signal transducing molecules including EGFR, IGF-1R, c-Met, Akt, B-RAF and C-RAF, resulting in pronounced decrease in phosphorylation of Akt and Erk1/2 in a panel of five cutaneous melanoma cell lines including those harboring B-RAF and N-RAS mutations. Ganetespib exhibited potent antiproliferative activity on all five of these cell lines, with IC50 values between 37.5 and 84 nM. Importantly, Ganetespib is active on B-RAF mutated melanoma cells that have acquired resistance to B-RAF inhibition. Ganetespib induced apoptosis and cell cycle arrest at G1 and/or G2/M phase. Ganetespib induced cell cycle arrest was accompanied by altered expression of cyclin-dependent kinase inhibitor (CDKI) p21Cip1 and p27Kip1, cyclins B1, D1 and E, and/or cyclin-dependent kinases 1, 2 and 4. HSP90 is functionally important for melanoma cells and HSP90 inhibitors such as ganetespib could potentially be effective therapeutics for melanoma with various genetic mutations and acquired resistance to B-RAF inhibition

Concomitant Targeting of EGF Receptor, TGF-beta and Src Points to a Novel Therapeutic Approach in Pancreatic Cancer

To test the hypothesis that concomitant targeting of the epidermal growth factor receptor (EGFR) and transforming growth factor-beta (TGF-β) may offer a novel therapeutic approach in pancreatic cancer, EGFR silencing by RNA interference (shEGFR) was combined with TGF-β sequestration by soluble TGF-β receptor II (sTβRII). Effects on colony formation in 3-dimensional culture, tumor formation in nude mice, and downstream signaling were monitored. In both ASPC-1 and T3M4 cells, either shEGFR or sTβRII significantly inhibited colony formation. However, in ASPC-1 cells, combining shEGFR with sTβRII reduced colony formation more efficiently than either approach alone, whereas in T3M4 cells, shEGFR-mediated inhibition of colony formation was reversed by sTβRII. Similarly, in vivo growth of ASPC-1-derived tumors was attenuated by either shEGFR or sTβRII, and was markedly suppressed by both vectors. By contrast, T3M4-derived tumors either failed to form or were very small when EGFR alone was silenced, and these effects were reversed by sTβRII due to increased cancer cell proliferation. The combination of shEGFR and sTβRII decreased phospho-HER2, phospho-HER3, phoshpo-ERK and phospho-src (Tyr416) levels in ASPC-1 cells but increased their levels in T3M4 cells. Moreover, inhibition of both EGFR and HER2 by lapatinib or of src by SSKI-606, PP2, or dasatinib, blocked the sTβRII-mediated antagonism of colony formation in T3M4 cells. Together, these observations suggest that concomitantly targeting EGFR, TGF-β, and src may constitute a novel therapeutic approach in PDAC that prevents deleterious cross-talk between EGFR family members and TGF-β-dependent pathways

Targeting EGFR and TGF-β pathways exerts different effects on the formation and growth of tumors formed by ASPC-1 and T3M4 cells.

<p>ASPC-1 (A) and T3M4 (B) cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), sTβRII, or both EGFR-LV and sTβRII, and injected subcutaneously (one injection per mouse) into the flank region of nude mice. Tumor volumes were monitored for the indicated number of days. Values are the means ± SEM of 8 mice per group, indicated in the denominator to the right of each curve. The number of tumors that formed in each group is indicated in the numerator. *p<0.05, and **p<0.01, when compared with respective controls.</p

Effects of targeting EGFR and TGF-β pathways on phosphorylation status of src family members.

<p>T3M4 cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), and/or WPT-sTβRII (sTβRII) as indicated. Cell lysates were then analyzed with a phospho-kinase antibody array to assess the phosphorylation status of the indicated src family members. Results were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039684#s4" target="_blank">Methods</a>. Data are the means ± SEM of triplicate determinations from three independent experiments. *p<0.05, **p<0.01, and ***p<0.001 when compared with control.</p

Effects of EGFR knockdown and sTβRII expression on receptor phosphorylation and downstream signaling.

<p>(A) Effects on receptor phosphorylation. ASPC-1 and T3M4 cells were infected as indicated with shLuc-LV (shLuc), shEGFR-LV (shEGFR), WPT-sTβRII (sTβRII), or both shEGFR and sTβRII. Cell lysates were subjected to immunoblotting with antibodies directed against the indicated receptors and phospho-receptors. (B) Cells were infected as indicated in A, and cell lysates were subjected to immunoblotting with antibodies directed against the indicated proteins and phospho-proteins. Each panel shows data from a representative of at least two independent experiments. In both panels A and B, immnoblotting with an anti-ERK antibody confirmed equivalent lane loading, but not all ERK blots are shown.</p

EGFR knockdown and sTβRII expression modulate colony formation in pancreatic cancer cells.

<p>(A) ASPC-1 and T3M4 human pancreatic cancer cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), WPT-sTβRII (sTβRII), or both shEGFR and sTβRII. Cell lysates and conditioned media were then subjected to immunoblotting with anti-EGFR and anti-HA-tag antibodies, respectively, the latter serving to confirm sTβRII release by the cancer cells. An anti-ERK antibody served to assess lane loading. (B) The consequences of EGFR silencing with shEGFR and TGF-β sequestration with sTβRII were assessed by monitoring colony formation in 3-D culture (B). Data are the means ± SE of triplicate determinations from three independent experiments. *p<0.05, **p<0.01, when compared with respective controls.</p

Effect of ganetespib on B-RAF, C-RAF and N-RAS expression in melanoma cells.

<p>Cells were treated with indicated amounts of ganetespib for 48 h and subjected to Western blot analysis.</p

Antiproliferative action of ganetespib on melanoma cells.

<p>A. Ganetespib reduced viability. Cells were treated with varying amounts of ganetespib for 72 h and subjected to MTS assay. Data are expressed as mean±SD of three independent experiments. B. Mutational status and ganetespib IC50 of cell lines.</p

Downregulation of multiple signaling pathways by ganetespib in melanoma cells.

<p>A. Cells were treated with indicated amounts of ganetespib for 24 h. B-RAF and N-RAS mutational status of each cell line is indicated. B. Cells were treated with 250 nM ganetespib for 48 and 72 h. Proteins levels were determined by Western blot analysis.</p

Ganetespib induced cell cycle arrest in melanoma cells.

<p>A. Cells were treated with 250 nM ganetespib for 24 hours, stained with PI and subjected to FACS analysis. B. Bar graphs of percentage of G1, S, and G2/M populations in control and ganetespib treated cells. C. Alterations in expression of multiple cell cycle regulating proteins induced by ganetespib. Cells were treated with indicated amounts of ganetespib for 48 h and analyzed by Western blot analysis. Relative expression levels of proteins (treated vs. control cells) are indicated.</p