Prenylation inhibition-induced cell death in melanoma: reduced sensitivity in BRAF mutant/PTEN wild-type melanoma cells

Abstract

While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells.

Fig 1. Cell viability, clonogenic growth and apoptosis in melanoma cells after zoledronic acid treatment.

(A) Dose-response analysis of cell viability of human melanoma cell lines with different mutations after 72hs treatment with ZA measured by SRB assay; Most pronounced reduction in cell viability was observed in two NRAS mutant lines (B) Long-term effect of 10 days of 5 μM ZA treatment on clonogenic growth. Resistance was found in BRAF mutant and PTEN wild-type cells. Of note, NRAS and BRAF double mutant cells demonstrated intermediate sensitivity (C) Apoptosis induction after 25μM ZA treatment demonstrated by the proportion of TUNEL positive cells and (D) apoptosis induction of 72hs ZA treatment evaluated by the immunoblot of cleaved PARP. Limited apoptosis induction was found in BRAF mutant and PTEN wild-type cells and in the MDM2 over expressing WM239 cells. Colors green, red, blue, and dark-blue indicate triple wild-type, NRAS, BRAF, and BRAF mutant/PTEN-null mutational status of the cells, respectively. Data shown as average ± SEM are from at least 5 repeats. Asterisks indicate the lowest concentration of ZA treatment resulting in a significant difference with p < 0.05 from control by ANOVA and Dunnett’s post hoc test in the cell viability assay. Data shown as average ± SEM are from at least 3 measurements. Asterisks indicate significant difference of p < 0.05 between given mutational group and the BRAF mutant PTEN wild-type group by Kruskal-Wallis and Dunn’s post hoc test in the clonogenic assay, and p < 0.05 difference from the respective control by unpaired two tailed T test in the apoptosis assay. (C = control; Z = zoledronic acid).

Fig 2. Cell migration after zoledronic acid treatment in melanoma cells.

(A-C) Migrated distance as a function of time and (D) average migrated distance after zoledronic acid (ZA) treatment in melanoma cells measured by videomicroscopy. A profound and significant increase in migrated distance was found in all of the BRAF mutant cells. A modest but significant increase in migration was found in VM-47 triple wild-type and VM-15 NRAS mutant cells. Colors blue, red and green indicate BRAF, NRAS mutation and wild-type for these genes, respectively. Data shown as average ± SEM are from at least three independent measurements. Asterisks indicate significant difference of p < 0.05 from the respective control with unpaired two-tailed T test.

Fig 3. Activation of downstream elements of the RAS/RAF pathway in melanoma cells after zoledronic acid treatment.

(A) Representative blots of the effect of 48hs zoledronic acid (ZA) treatment on the activation of Erk1/2 and S6. (B) Quantification of the effect of ZA treatment on the activation of Erk1/2. Treatment with ZA resulted in robust increase in the phosphorylation of Erk1/2 in MEWO and M24met cells. (C) Quantification of the effect of ZA treatment on the activation of S6. After the treatment with ZA, decreased activation of S6 proteins was found only in NRAS mutant M24met and VM-15 cells. Colors blue, red and green indicate BRAF, NRAS mutation and wild-type for these genes, respectively. (C = control; ZA = zoledronic acid).

Fig 4. Proliferation and apoptosis in melanoma cells after combination treatments.

(A) Both cytotoxic compounds reduced proliferation significantly after 48hs of treatment in three cell lines irrespective of oncogenic mutation using SRB assay. An additive effect of ZA and cytotoxic treatment was measured in the NRAS mutant M24met cells. (B) Proportion of TUNEL positive cells after combined treatment with ZA and standard cytotoxic therapy (DTIC or cisplatin). No synergism was observed in any of the cell lines investigated. Data shown as average ± SEM are results of four independent measurements. Asterisks indicate significance of p < 0.05 with Kruskal-Wallis and Dunn’s multiple comparison tests.

Fig 5. Activation of downstream elements of the RAS/RAF pathway in melanoma cells after combination treatments.

(A) Representative blots of the effect of 12hs zoledronic acid (ZA) and/or DTIC/cisplatin treatment on the activation of Erk1/2 and S6. (B) Quantification of the effect of ZA and/or DTIC/cisplatin treatment on the activation of Erk1/2. Treatment with cytotoxic agents decreased the activation of Erk1/2 in double wild type MEWO and NRAS mutant M24met cells. DTIC or cisplatin and combined treatment resulted in the increase of Erk1/2 activation in BRAF mutant HT168-M1 cells. (C) Quantification of the effect of ZA and or DTIC/cisplatin treatment on the activation of S6. DTIC or cisplatin treatment increased S6 activation in HT168-M1 cells. S6 activation was also increased in M24met and in the combination treatment with cisplatin also in MEWO cells. Synergism between ZA and cytotoxic treatment was seen only in M24met cells if treated with DTIC and ZA and MEWO cells after the treatment with cisplatin and ZA.

Fig 6. In vivo effects of zoledronic acid treatment.

Effect of zoledronic acid (ZA) treatment using in vivo subcutaneous xenograft model of human melanoma cells in SCID mice (A, C, E). ZA treatment failed to show effects in the subcutaneous growth of melanoma cells with either mutation. (B, D, F) Effect of ZA treatment using in vivo spleen liver colonization model of human melanoma cells in SCID mice. ZA did not inhibit the primary tumor or metastatic growth of melanoma cells. Data shown as average ± SEM.