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Targeting c-Met in melanoma: mechanism of resistance and efficacy of novel combinatorial inhibitor therapy.

Etnyre D, Stone AL, Fong JT, Jacobs RJ, Uppada SB, Botting GM, Rajanna S, Moravec DN, Shambannagari MR, Crees Z, Girard J, Bertram C, Puri N - Cancer Biol. Ther. (2014)

Bottom Line: Treatment with everolimus, resulted in 56% growth inhibition, and a triple combination of SU11274, everolimus and XAV939, resulted in 95% growth inhibition in RU cells.The V600E BRAF mutation was found to be positive only in MU cells.Combination treatment with a c-Met TKI and a BRAF inhibitor displayed a synergistic effect in reducing MU cell viability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences; University of Illinois College of Medicine; Rockford, IL USA.

ABSTRACT
Numerous tyrosine kinase inhibitors (TKIs) targeting c-Met are currently in clinical trials for several cancers. Their efficacy is limited due to the development of resistance. The present study aims to elucidate this mechanism of c-Met TKI resistance by investigating key mTOR and Wnt signaling proteins in melanoma cell lines resistant to SU11274, a c-Met TKI. Xenografts from RU melanoma cells treated with c-Met TKIs SU11274 and JNJ38877605 showed a 7- and 6-fold reduction in tumor size, respectively. Resistant cells displayed upregulation of phosphorylated c-Met, mTOR, p70S6Kinase, 4E-BP1, ERK, LRP6, and active β-catenin. In addition, GATA-6, a Wnt signaling regulator, was upregulated, and Axin, a negative regulator of the Wnt pathway, was downregulated in resistant cells. Modulation of these mTOR and Wnt pathway proteins was also prevented by combination treatment with SU11274, everolimus, an mTOR inhibitor, and XAV939, a Wnt inhibitor. Treatment with everolimus, resulted in 56% growth inhibition, and a triple combination of SU11274, everolimus and XAV939, resulted in 95% growth inhibition in RU cells. The V600E BRAF mutation was found to be positive only in MU cells. Combination treatment with a c-Met TKI and a BRAF inhibitor displayed a synergistic effect in reducing MU cell viability. These studies indicate activation of mTOR and Wnt signaling pathways in c-Met TKI resistant melanoma cells and suggest that concurrent targeting of c-Met, mTOR, and Wnt pathways and BRAF may improve efficacy over traditional TKI monotherapy in melanoma patients.

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Figure 2. Oral TKI treatment reduces tumor size in vivo. Five million RU-P melanoma cells were injected subcutaneously into the hind flanks of nu/nu mice. Tumors were allowed to develop for a week after which daily oral doses of JNJ38877605 or vehicle were given for 3 wk. (A)Treatment with JNJ38877605 reduced tumor size by 6-fold when compared with control mice. (B) Immunostaining of control and JNJ38877605-treated RU-P tumor xenografts with p-c-Met antibody showed decrease in p-c-Met after treatment with JNJ38877605. (C) Immunostaining of control and JNJ38877605 treated RU-P tumor xenografts with CD31 antibody indicate treatment with JNJ38877605 decreased the number of blood vessels in melanoma. There was an 80% (± 2%) decrease in the number of blood vessels when counted in 10 microscopic fields after treatment with JNJ38877605. (D) Immunostaining of control and JNJ38877605-treated RU-P tumor xenografts with VEGF and TSP1 antibody showed a decrease in VEGF and an increase of TSP1 with JNJ38877605 treatment suggesting decreased angiogenesis.
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Figure 2: Figure 2. Oral TKI treatment reduces tumor size in vivo. Five million RU-P melanoma cells were injected subcutaneously into the hind flanks of nu/nu mice. Tumors were allowed to develop for a week after which daily oral doses of JNJ38877605 or vehicle were given for 3 wk. (A)Treatment with JNJ38877605 reduced tumor size by 6-fold when compared with control mice. (B) Immunostaining of control and JNJ38877605-treated RU-P tumor xenografts with p-c-Met antibody showed decrease in p-c-Met after treatment with JNJ38877605. (C) Immunostaining of control and JNJ38877605 treated RU-P tumor xenografts with CD31 antibody indicate treatment with JNJ38877605 decreased the number of blood vessels in melanoma. There was an 80% (± 2%) decrease in the number of blood vessels when counted in 10 microscopic fields after treatment with JNJ38877605. (D) Immunostaining of control and JNJ38877605-treated RU-P tumor xenografts with VEGF and TSP1 antibody showed a decrease in VEGF and an increase of TSP1 with JNJ38877605 treatment suggesting decreased angiogenesis.

Mentions: To study the therapeutic efficacy of JNJ38877605, an orally bioavailable c-Met TKI, in vivo studies were performed. Mice bearing RU-P melanoma cell tumor xenografts were treated orally with 20 mg/kg JNJ38877605 or vehicle for three weeks. Similar to SU11274, it was determined that JNJ38877605 significantly reduced tumor size by 6-fold (124 ± 57 mm2 and 17 ± 11 mm2, P < 0.03), as compared with control (vehicle) (Fig. 2A). Tumors treated with JNJ38877605 showed a significant reduction in expression of p-c-Met (Y1234/1235), as seen by IHC in small residual tumor nodules (Fig. 2B). These results indicate that the reduction in p-c-Met after administration of JNJ38877605 has a significant effect on tumor proliferation. Treatment with JNJ38877605 also resulted in 80% ± 2% (P < 0.001) reduction in blood vessels, as seen by CD31 staining, suggesting that inhibition of vessel formation may be one of the mechanisms by which JNJ38877605 inhibits tumor growth (Fig. 2C). Similar to SU11274 treatment, JNJ38877605 decreased VEGF expression and increased TSP-1 expression, as seen by IHC (Fig. 2D). These data indicate that JNJ38877605 could be a promising orally administered therapeutic option for treating HGF-producing melanoma.


Targeting c-Met in melanoma: mechanism of resistance and efficacy of novel combinatorial inhibitor therapy.

Etnyre D, Stone AL, Fong JT, Jacobs RJ, Uppada SB, Botting GM, Rajanna S, Moravec DN, Shambannagari MR, Crees Z, Girard J, Bertram C, Puri N - Cancer Biol. Ther. (2014)

Figure 2. Oral TKI treatment reduces tumor size in vivo. Five million RU-P melanoma cells were injected subcutaneously into the hind flanks of nu/nu mice. Tumors were allowed to develop for a week after which daily oral doses of JNJ38877605 or vehicle were given for 3 wk. (A)Treatment with JNJ38877605 reduced tumor size by 6-fold when compared with control mice. (B) Immunostaining of control and JNJ38877605-treated RU-P tumor xenografts with p-c-Met antibody showed decrease in p-c-Met after treatment with JNJ38877605. (C) Immunostaining of control and JNJ38877605 treated RU-P tumor xenografts with CD31 antibody indicate treatment with JNJ38877605 decreased the number of blood vessels in melanoma. There was an 80% (± 2%) decrease in the number of blood vessels when counted in 10 microscopic fields after treatment with JNJ38877605. (D) Immunostaining of control and JNJ38877605-treated RU-P tumor xenografts with VEGF and TSP1 antibody showed a decrease in VEGF and an increase of TSP1 with JNJ38877605 treatment suggesting decreased angiogenesis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128856&req=5

Figure 2: Figure 2. Oral TKI treatment reduces tumor size in vivo. Five million RU-P melanoma cells were injected subcutaneously into the hind flanks of nu/nu mice. Tumors were allowed to develop for a week after which daily oral doses of JNJ38877605 or vehicle were given for 3 wk. (A)Treatment with JNJ38877605 reduced tumor size by 6-fold when compared with control mice. (B) Immunostaining of control and JNJ38877605-treated RU-P tumor xenografts with p-c-Met antibody showed decrease in p-c-Met after treatment with JNJ38877605. (C) Immunostaining of control and JNJ38877605 treated RU-P tumor xenografts with CD31 antibody indicate treatment with JNJ38877605 decreased the number of blood vessels in melanoma. There was an 80% (± 2%) decrease in the number of blood vessels when counted in 10 microscopic fields after treatment with JNJ38877605. (D) Immunostaining of control and JNJ38877605-treated RU-P tumor xenografts with VEGF and TSP1 antibody showed a decrease in VEGF and an increase of TSP1 with JNJ38877605 treatment suggesting decreased angiogenesis.
Mentions: To study the therapeutic efficacy of JNJ38877605, an orally bioavailable c-Met TKI, in vivo studies were performed. Mice bearing RU-P melanoma cell tumor xenografts were treated orally with 20 mg/kg JNJ38877605 or vehicle for three weeks. Similar to SU11274, it was determined that JNJ38877605 significantly reduced tumor size by 6-fold (124 ± 57 mm2 and 17 ± 11 mm2, P < 0.03), as compared with control (vehicle) (Fig. 2A). Tumors treated with JNJ38877605 showed a significant reduction in expression of p-c-Met (Y1234/1235), as seen by IHC in small residual tumor nodules (Fig. 2B). These results indicate that the reduction in p-c-Met after administration of JNJ38877605 has a significant effect on tumor proliferation. Treatment with JNJ38877605 also resulted in 80% ± 2% (P < 0.001) reduction in blood vessels, as seen by CD31 staining, suggesting that inhibition of vessel formation may be one of the mechanisms by which JNJ38877605 inhibits tumor growth (Fig. 2C). Similar to SU11274 treatment, JNJ38877605 decreased VEGF expression and increased TSP-1 expression, as seen by IHC (Fig. 2D). These data indicate that JNJ38877605 could be a promising orally administered therapeutic option for treating HGF-producing melanoma.

Bottom Line: Treatment with everolimus, resulted in 56% growth inhibition, and a triple combination of SU11274, everolimus and XAV939, resulted in 95% growth inhibition in RU cells.The V600E BRAF mutation was found to be positive only in MU cells.Combination treatment with a c-Met TKI and a BRAF inhibitor displayed a synergistic effect in reducing MU cell viability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences; University of Illinois College of Medicine; Rockford, IL USA.

ABSTRACT
Numerous tyrosine kinase inhibitors (TKIs) targeting c-Met are currently in clinical trials for several cancers. Their efficacy is limited due to the development of resistance. The present study aims to elucidate this mechanism of c-Met TKI resistance by investigating key mTOR and Wnt signaling proteins in melanoma cell lines resistant to SU11274, a c-Met TKI. Xenografts from RU melanoma cells treated with c-Met TKIs SU11274 and JNJ38877605 showed a 7- and 6-fold reduction in tumor size, respectively. Resistant cells displayed upregulation of phosphorylated c-Met, mTOR, p70S6Kinase, 4E-BP1, ERK, LRP6, and active β-catenin. In addition, GATA-6, a Wnt signaling regulator, was upregulated, and Axin, a negative regulator of the Wnt pathway, was downregulated in resistant cells. Modulation of these mTOR and Wnt pathway proteins was also prevented by combination treatment with SU11274, everolimus, an mTOR inhibitor, and XAV939, a Wnt inhibitor. Treatment with everolimus, resulted in 56% growth inhibition, and a triple combination of SU11274, everolimus and XAV939, resulted in 95% growth inhibition in RU cells. The V600E BRAF mutation was found to be positive only in MU cells. Combination treatment with a c-Met TKI and a BRAF inhibitor displayed a synergistic effect in reducing MU cell viability. These studies indicate activation of mTOR and Wnt signaling pathways in c-Met TKI resistant melanoma cells and suggest that concurrent targeting of c-Met, mTOR, and Wnt pathways and BRAF may improve efficacy over traditional TKI monotherapy in melanoma patients.

Show MeSH
Related in: MedlinePlus