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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 7.  Proposed role of mTOR and Wnt pathways in c-Met inhibitor resistance. Increased resistance to c-Met inhibitors could be due to overexpression of c-Met and consequent activation of c-Met-dependent PI3K/Akt/mTOR, RAS/RAF/MEK/ERK (MAPK) or alternative signaling pathways, such as the Wnt pathway. Upregulation of the Akt/mTOR pathway in c-Met TKI resistant cells leads to increased cell survival and proliferation. Furthermore, we postulate that in c-Met resistant cells, phosphorylation of ERK by the RAS/RAF pathway may lead to phosphorylation of GATA-6.29 Upregulated GATA-689 (Fig. S3) also stimulates the Wnt pathway,31 which may result in accumulation of β-catenin in the nucleus,78 leading to melanoma tumorigenicity.45
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Figure 7: Figure 7. Proposed role of mTOR and Wnt pathways in c-Met inhibitor resistance. Increased resistance to c-Met inhibitors could be due to overexpression of c-Met and consequent activation of c-Met-dependent PI3K/Akt/mTOR, RAS/RAF/MEK/ERK (MAPK) or alternative signaling pathways, such as the Wnt pathway. Upregulation of the Akt/mTOR pathway in c-Met TKI resistant cells leads to increased cell survival and proliferation. Furthermore, we postulate that in c-Met resistant cells, phosphorylation of ERK by the RAS/RAF pathway may lead to phosphorylation of GATA-6.29 Upregulated GATA-689 (Fig. S3) also stimulates the Wnt pathway,31 which may result in accumulation of β-catenin in the nucleus,78 leading to melanoma tumorigenicity.45

Mentions: The mTOR and Wnt signaling pathways have been shown to play a major role in conferring resistance to chemotherapy, and targeting key proteins in these pathways has become an important therapeutic approach in cancer.45,46 Therefore, the drugs everolimus and XAV939, which inhibit mTOR and Wnt pathway proteins, respectively,47,48 were selected for this combination inhibitor study. RU and MU cells were treated with everolimus or XAV939, alone and in combination with SU11274. Combination drug concentrations were determined from drug titration experiments (data not shown) and previous studies by us and other investigators.49,50 Treatment with 1 µM everolimus inhibited both RU-P and RU-R cell growth by 56% (Fig. 6A). Administration of 1 µM everolimus in combination with 5 µM SU11274 and 15 µM XAV939, a Wnt inhibitor, inhibited RU-P and RU-R cell growth by 95% (Fig. 6A). Treatment with 1 µM everolimus inhibited MU-P and MU-R cell growth 50% and 30%, respectively (Fig. 6B). Administration of 1 µM everolimus in combination with 5 µM SU11274 and 15 µM XAV939 inhibited MU-P and MU-R cell growth 78% and 65%, respectively (Fig. 6B). To verify the results of the MTT assay, cell numbers were assessed using trypan blue exclusion to examine viable cells after inhibitor treatment, and similar results were obtained (Fig. S4). Concentrations higher than the IC50 of SU11274 were used for in vitro combination experiments since the resistant cell lines were grown to develop resistance in higher concentrations of SU11274. These results suggest that mTOR and Wnt pathways may play an essential role in c-Met TKI resistance, and that simultaneous targeting of mTOR, Wnt pathways, and c-Met may prove to be an effective approach for melanoma therapy (Fig. 7). Further studies are warranted in this direction.


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 7.  Proposed role of mTOR and Wnt pathways in c-Met inhibitor resistance. Increased resistance to c-Met inhibitors could be due to overexpression of c-Met and consequent activation of c-Met-dependent PI3K/Akt/mTOR, RAS/RAF/MEK/ERK (MAPK) or alternative signaling pathways, such as the Wnt pathway. Upregulation of the Akt/mTOR pathway in c-Met TKI resistant cells leads to increased cell survival and proliferation. Furthermore, we postulate that in c-Met resistant cells, phosphorylation of ERK by the RAS/RAF pathway may lead to phosphorylation of GATA-6.29 Upregulated GATA-689 (Fig. S3) also stimulates the Wnt pathway,31 which may result in accumulation of β-catenin in the nucleus,78 leading to melanoma tumorigenicity.45
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: Figure 7. Proposed role of mTOR and Wnt pathways in c-Met inhibitor resistance. Increased resistance to c-Met inhibitors could be due to overexpression of c-Met and consequent activation of c-Met-dependent PI3K/Akt/mTOR, RAS/RAF/MEK/ERK (MAPK) or alternative signaling pathways, such as the Wnt pathway. Upregulation of the Akt/mTOR pathway in c-Met TKI resistant cells leads to increased cell survival and proliferation. Furthermore, we postulate that in c-Met resistant cells, phosphorylation of ERK by the RAS/RAF pathway may lead to phosphorylation of GATA-6.29 Upregulated GATA-689 (Fig. S3) also stimulates the Wnt pathway,31 which may result in accumulation of β-catenin in the nucleus,78 leading to melanoma tumorigenicity.45
Mentions: The mTOR and Wnt signaling pathways have been shown to play a major role in conferring resistance to chemotherapy, and targeting key proteins in these pathways has become an important therapeutic approach in cancer.45,46 Therefore, the drugs everolimus and XAV939, which inhibit mTOR and Wnt pathway proteins, respectively,47,48 were selected for this combination inhibitor study. RU and MU cells were treated with everolimus or XAV939, alone and in combination with SU11274. Combination drug concentrations were determined from drug titration experiments (data not shown) and previous studies by us and other investigators.49,50 Treatment with 1 µM everolimus inhibited both RU-P and RU-R cell growth by 56% (Fig. 6A). Administration of 1 µM everolimus in combination with 5 µM SU11274 and 15 µM XAV939, a Wnt inhibitor, inhibited RU-P and RU-R cell growth by 95% (Fig. 6A). Treatment with 1 µM everolimus inhibited MU-P and MU-R cell growth 50% and 30%, respectively (Fig. 6B). Administration of 1 µM everolimus in combination with 5 µM SU11274 and 15 µM XAV939 inhibited MU-P and MU-R cell growth 78% and 65%, respectively (Fig. 6B). To verify the results of the MTT assay, cell numbers were assessed using trypan blue exclusion to examine viable cells after inhibitor treatment, and similar results were obtained (Fig. S4). Concentrations higher than the IC50 of SU11274 were used for in vitro combination experiments since the resistant cell lines were grown to develop resistance in higher concentrations of SU11274. These results suggest that mTOR and Wnt pathways may play an essential role in c-Met TKI resistance, and that simultaneous targeting of mTOR, Wnt pathways, and c-Met may prove to be an effective approach for melanoma therapy (Fig. 7). Further studies are warranted in this direction.

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