Limits...
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.

Show MeSH

Related in: MedlinePlus

Figure 4. Upregulation of p-c-Met and active β-catenin in MU-R cells. (A) Cells were starved overnight in media supplemented with 0.5% BSA, and then treated with or without 10 µM SU11274 for 24 h. Cells were stimulated with 40 ng/mL of HGF for 7.5 min, after which immunoblotting analysis was performed. Upregulation of p-c-Met Y1003 (2.4-fold) and p-c-Met Y1234/1235 (1.5-fold) in MU-R cells in absence of HGF was observed, and a 3–5-fold increase in p-c-Met Y1234/1235 was seen after HGF treatment. (B) In MU-R cells, HGF induced p-c-Met and active β-catenin signaling was prolonged by 30 min compared with MU-P cells. Cells were starved for 24 h and then stimulated with 40 ng/ml HGF. Immunoblotting indicated that in MU-R cells, HGF activated p-c-Met (Y1234/1235) and basal levels of active β-catenin were also 3-fold higher in the absence of HGF and remained high (2.5-fold) for 7.5 min after HGF treatment in MU-R cells compared with those in MU-P cells at 0 min incubation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4128856&req=5

Figure 4: Figure 4. Upregulation of p-c-Met and active β-catenin in MU-R cells. (A) Cells were starved overnight in media supplemented with 0.5% BSA, and then treated with or without 10 µM SU11274 for 24 h. Cells were stimulated with 40 ng/mL of HGF for 7.5 min, after which immunoblotting analysis was performed. Upregulation of p-c-Met Y1003 (2.4-fold) and p-c-Met Y1234/1235 (1.5-fold) in MU-R cells in absence of HGF was observed, and a 3–5-fold increase in p-c-Met Y1234/1235 was seen after HGF treatment. (B) In MU-R cells, HGF induced p-c-Met and active β-catenin signaling was prolonged by 30 min compared with MU-P cells. Cells were starved for 24 h and then stimulated with 40 ng/ml HGF. Immunoblotting indicated that in MU-R cells, HGF activated p-c-Met (Y1234/1235) and basal levels of active β-catenin were also 3-fold higher in the absence of HGF and remained high (2.5-fold) for 7.5 min after HGF treatment in MU-R cells compared with those in MU-P cells at 0 min incubation.

Mentions: To further investigate the mechanism of c-Met TKI resistance in MU-R cells, key signaling proteins that have significant roles in melanoma and other cancers were studied. Earlier studies indicate that Wnt/EGFR and EGFR/c-Met crosstalk promotes tumorigenesis.37,40 These studies led us to investigate the role of the Wnt pathway in mediating c-Met inhibitor resistance. MU-R cells exhibited 1.5- and 2.4-fold upregulation of p-c-Met (Y1234/1235) and p-c-Met (Y1003), respectively, in the absence of HGF compared with MU-P cells (Fig. 4A). Additionally, time course experiments for p-c-Met and active β-catenin, a transcription factor in the Wnt pathway, were conducted. A 3- to 5-fold increase in p-c-Met (Y1234/1235) expression in MU-R cells was observed following HGF stimulation relative to control. Basal levels of active β-catenin were found to be 3-fold higher in the absence of HGF and remained elevated (2.5-fold) for 7.5 min after HGF treatment in MU-R cells, compared with those in MU-P cells. Following HGF treatment, levels of p-c-Met and active β-catenin both remained elevated for at least 60 min in MU-R cells, compared with 30 min in MU-P cells (Fig. 4B). These results indicate increased stabilization of p-c-Met suggesting crosstalk with the Wnt pathway, and that upregulation of the Wnt pathway may play a role in mediating SU11274 resistance in MU-R cells.


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 4. Upregulation of p-c-Met and active β-catenin in MU-R cells. (A) Cells were starved overnight in media supplemented with 0.5% BSA, and then treated with or without 10 µM SU11274 for 24 h. Cells were stimulated with 40 ng/mL of HGF for 7.5 min, after which immunoblotting analysis was performed. Upregulation of p-c-Met Y1003 (2.4-fold) and p-c-Met Y1234/1235 (1.5-fold) in MU-R cells in absence of HGF was observed, and a 3–5-fold increase in p-c-Met Y1234/1235 was seen after HGF treatment. (B) In MU-R cells, HGF induced p-c-Met and active β-catenin signaling was prolonged by 30 min compared with MU-P cells. Cells were starved for 24 h and then stimulated with 40 ng/ml HGF. Immunoblotting indicated that in MU-R cells, HGF activated p-c-Met (Y1234/1235) and basal levels of active β-catenin were also 3-fold higher in the absence of HGF and remained high (2.5-fold) for 7.5 min after HGF treatment in MU-R cells compared with those in MU-P cells at 0 min incubation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Figure 4. Upregulation of p-c-Met and active β-catenin in MU-R cells. (A) Cells were starved overnight in media supplemented with 0.5% BSA, and then treated with or without 10 µM SU11274 for 24 h. Cells were stimulated with 40 ng/mL of HGF for 7.5 min, after which immunoblotting analysis was performed. Upregulation of p-c-Met Y1003 (2.4-fold) and p-c-Met Y1234/1235 (1.5-fold) in MU-R cells in absence of HGF was observed, and a 3–5-fold increase in p-c-Met Y1234/1235 was seen after HGF treatment. (B) In MU-R cells, HGF induced p-c-Met and active β-catenin signaling was prolonged by 30 min compared with MU-P cells. Cells were starved for 24 h and then stimulated with 40 ng/ml HGF. Immunoblotting indicated that in MU-R cells, HGF activated p-c-Met (Y1234/1235) and basal levels of active β-catenin were also 3-fold higher in the absence of HGF and remained high (2.5-fold) for 7.5 min after HGF treatment in MU-R cells compared with those in MU-P cells at 0 min incubation.
Mentions: To further investigate the mechanism of c-Met TKI resistance in MU-R cells, key signaling proteins that have significant roles in melanoma and other cancers were studied. Earlier studies indicate that Wnt/EGFR and EGFR/c-Met crosstalk promotes tumorigenesis.37,40 These studies led us to investigate the role of the Wnt pathway in mediating c-Met inhibitor resistance. MU-R cells exhibited 1.5- and 2.4-fold upregulation of p-c-Met (Y1234/1235) and p-c-Met (Y1003), respectively, in the absence of HGF compared with MU-P cells (Fig. 4A). Additionally, time course experiments for p-c-Met and active β-catenin, a transcription factor in the Wnt pathway, were conducted. A 3- to 5-fold increase in p-c-Met (Y1234/1235) expression in MU-R cells was observed following HGF stimulation relative to control. Basal levels of active β-catenin were found to be 3-fold higher in the absence of HGF and remained elevated (2.5-fold) for 7.5 min after HGF treatment in MU-R cells, compared with those in MU-P cells. Following HGF treatment, levels of p-c-Met and active β-catenin both remained elevated for at least 60 min in MU-R cells, compared with 30 min in MU-P cells (Fig. 4B). These results indicate increased stabilization of p-c-Met suggesting crosstalk with the Wnt pathway, and that upregulation of the Wnt pathway may play a role in mediating SU11274 resistance in MU-R cells.

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