Stat3-targeted therapies overcome the acquired resistance to vemurafenib in melanomas

Abstract

Vemurafenib (PLX4032), a selective inhibitor of Braf, has been approved by the US Food and Drug Administration for the treatment of unresectable or metastatic melanoma in patients with Braf(V600E) mutations. Many patients treated with vemurafenib initially display dramatic improvement, with decreases in both risk of death and tumor progression. Acquired resistance, however, rapidly arises in previously sensitive cells. We attempted to overcome this resistance by targeting the signal transducer and activator of transcription 3 (STAT3)-paired box homeotic gene 3 (PAX3)-signaling pathway, which is upregulated, owing to fibroblast growth factor 2 (FGF2) secretion or increased kinase activity, with the Braf(V600E) mutation. We found that activation of Stat3 or overexpression of PAX3 induced resistance to vemurafenib in melanoma cells. In addition, PAX3 or Stat3 silencing inhibited the growth of melanoma cells with acquired resistance to vemurafenib. Furthermore, treatment with the Stat3 inhibitor, WP1066, resulted in growth inhibition in both vemurafenib-sensitive and -resistant melanoma cells. Significantly, vemurafenib stimulation induced FGF2 secretion from keratinocytes and fibroblasts, which might uncover, at least in part, the mechanisms underlying targeting Stat3-PAX3 signaling to overcome the acquired resistance to vemurafenib. Our results suggest that Stat3-targeted therapy is a new therapeutic strategy to overcome the acquired resistance to vemurafenib in the treatment of melanoma.

Figure 1. Introduction of Braf^V600E or N-RAS^Q61K activates Stat3-PAX3 signaling in melanocytes

Genetically engineered human melanocytes (hTERT/p53DD/CDK4(R24C)) were infected with Ad-N-RAS^Q61K, Ad-Braf^V600E or Ad-GFP. RNA and protein were collected at 24 hr after infections. (A) The mRNA expression of PAX3 was measured by quantitative RT-PCR and normalized to GAPDH. Results are expressed as the mean of the experiment done in triplicate ± SEM. Induction is calculated relative to PAX3 levels in vehicle-treated cells. (B) Protein expressions of Stat3, phospho-Stat3 and PAX3 were analyzed by western blot along with tubulin, which served as a loading control.

Figure 2. Stat3 is activated in melanoma cells with acquired resistance to vemurafenib

Sensitive and resistant A375 and UACC62 melanoma cells were stimulated by different doses of vemurafenib as indicated. Total protein was collected 6 hr after stimulation. Protein expressions of phospho--ERK1/2, Stat3, phospho-Stat3 and PAX3 were analyzed by western blot along with tubulin, which served as a loading control.

Figure 3. Overexpression of PAX3 or Stat3 inhibits the response of melanoma cells to vemurafenib

A375 and UACC62 parental cells with pcDNA-3.1-Stat3-CA plasmids or PECE-PAX3 plasmids introduction were stimulated with different doses of vemurafenib as indicated. Total protein was collected 6 hr after stimulation. Protein expressions of Stat3 and PAX3 were analyzed by western blot along with tubulin, which served as a loading control. (A) A375 cells and (B) UACC62 cells. (C) Melanoma cell growth was assessed by MTT assays. Relative growth (RG) was calculated as the ratio of treated to untreated cells at each dose for each replicate.

Figure 4. Resistant melanoma cellular growth was inhibited after silencing PAX3 or Stat3 and vemurafenib treatment

A375 and UACC62 resistant cells stably expressing control shRNA (shScr), shStat3 or shPAX3 were stimulated with different doses of vemurafenib as indicated. Total protein was collected 6 hr after stimulation. Protein expressions of Stat3 and PAX3 were analyzed by western blot along with tubulin, which served as a loading control. (A) A375R cells and (B) UACC62R cells. (C) Melanoma cells were infected with shPAX3, shStat3 or control virus and after 48 hrs, cells were subjected to MTT assays to evaluate the relative cell numbers. Relative growth (RG) was calculated as the ratio of treated to untreated cells at each dose for each replicate.

Figure 5. Vemurafenib induces FGF2 secretion

(A) Levels of FGF2 were measured in resistant melanoma cells. FGF2 levels in growth media of cultured parental and resistant melanoma cells, growing at 80% confluence in serum-free medium were examined by ELISA. FGF2 is induced by vemurafenib treatment in vitro. (B) Human primary keratinocytes and human primary fibroblast were stimulated by different doses of vemurafenib as indicated. FGF2 levels were measured by ELISA in culture media 24 hr after stimulation. Results are expressed as the mean of the experiment done in triplicate ± SEM. Induction is calculated relative to FGF2 levels in vehicle-treated cell media. (C) Schematic diagram of the FGF2-Stat3-PAX3 signaling pathway.

Figure 6. Inhibition of Stat3 overcomes the acquired resistance to vemurafenib in resistant melanoma cells

(A) M263R and M249R melanoma cells stably expressing control shRNA (shScr), shStat3 or shPAX3 were stimulated different doses of vemurafenib as indicated. (C) Sensitive and resistant A375 and UACC62 melanoma cells were stimulated with 5.0μm WP1066. Total protein was collected at 6 hr after stimulation. Protein expression of Stat3 and phospho-Stat3 were analyzed by western blot along with tubulin, which served as a loading control. A375R and UACC62R melanoma cells were stimulated with different doses of vemurafenib (B), WP1066 (D) or both (E) as indicated. Melanoma cell growth was assessed by MTT assays. Relative growth (RG) was calculated as the ratio of treated to untreated cells at each dose for each replicate (*p< 0.01; **p<0.05). A375 and UACC62 resistant cells with PECE-PAX3 plasmids were stimulated with vemurafenib and/or WP1066 as indicated. (F) Total protein was collected 6 hr after stimulation. Protein expression of phospho-Stat3 and PAX3 were analyzed by western blot along with tubulin, which served as a loading control. (G) Cells were subjected to MTT assays to evaluate the relative cell numbers. Relative growth (RG) was calculated as the ratio of treated to untreated cells at each dose for each replicate (*p< 0.01).