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Selective inhibition of RET mediated cell proliferation in vitro by the kinase inhibitor SPP86.

Alao JP, Michlikova S, Dinér P, Grøtli M, Sunnerhagen P - BMC Cancer (2014)

Bottom Line: Herein, we have further investigated the effect of the lead compound SPP86 on RET mediated signaling and proliferation.We compared the effects of SPP86 on RET-induced signaling and proliferation in thyroid cancer cell lines expressing RET-PTC1 (TPC1), or the activating mutations BRAFV600E (8505C) and RASG13R (C643).Additionally, RET- FAK crosstalk may play a key role in facilitating PTC1/RET and GDNF- RET induced activation of Akt and MAPK signaling in TPC1 and MCF7 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, SE-405 30 Göteborg, Sweden. John.P.Alao@cmb.gu.se.

ABSTRACT

Background: The RET tyrosine kinase receptor has emerged as a target in thyroid and endocrine resistant breast cancer. We previously reported the synthesis of kinase inhibitors with potent activity against RET. Herein, we have further investigated the effect of the lead compound SPP86 on RET mediated signaling and proliferation. Based on these observations, we hypothesized that SPP86 may be useful for studying the cellular activity of RET.

Methods: We compared the effects of SPP86 on RET-induced signaling and proliferation in thyroid cancer cell lines expressing RET-PTC1 (TPC1), or the activating mutations BRAFV600E (8505C) and RASG13R (C643). The effect of SPP86 on RET- induced phosphatidylinositide 3-kinases (PI3K)/Akt and MAPK pathway signaling and cell proliferation in MCF7 breast cancer cells was also investigated.

Results: SPP86 inhibited MAPK signaling and proliferation in RET/PTC1 expressing TPC1 but not 8505C or C643 cells. In TPC1 cells, the inhibition of RET phosphorylation required co-exposure to SPP86 and the focal adhesion kinase (FAK) inhibitor PF573228. In MCF7 cells, SPP86 inhibited RET- induced phosphatidylinositide 3-kinases (PI3K)/Akt and MAPK signaling and estrogen receptorα (ERα) phosphorylation, and inhibited proliferation to a similar degree as tamoxifen. Interestingly, SPP86 and PF573228 inhibited RET/PTC1 and GDNF- RET induced activation of Akt and MAPK signaling to a similar degree.

Conclusion: SPP86 selectively inhibits RET downstream signaling in RET/PTC1 but not BRAFV600E or RASG13R expressing cells, indicating that downstream kinases were not affected. SPP86 also inhibited RET signaling in MCF7 breast cancer cells. Additionally, RET- FAK crosstalk may play a key role in facilitating PTC1/RET and GDNF- RET induced activation of Akt and MAPK signaling in TPC1 and MCF7 cells.

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SPP86 inhibits RET- mediated proliferation. (A) Estrogen- deprived and serum starved MCF7 cells were left untreated or exposed to 1 ng/ml E2 and/or 10 ng/ml GDNF alone or in combination with 1 μg/ml SPP86 in phenol red- free media for 7 days. Proliferation was expressed as fold increase in growth relative the untreated control population. The data represent the mean of 3 experiments ± S. E; *p <0.05 treated vs. untreated for each series. (B) Estrogen- deprived and serum starved MCF7 cells were cultured in the presence of 1 nM E2 (estrogen) together with 10 ng/ml of GDNF or 10 ng/ml insulin in the presence or absence of 1 μM SPP86 for 72 h. Proliferation was expressed as fold increase in growth relative the untreated control population. The data represent the mean of 3 experiments ± S. E; *p <0.05. (C) Estrogen- deprived and serum starved MCF7 cells exposed to increasing concentrations of SPP86 or 4-OHT in phenol red- free media containing 1 ng/ml E2 and 10 ng/ml GDNF for 7 days. Viability was expressed as a percentage of the untreated control population. The data in each panel represent the mean of 3 experiments ± S. E.; *p <0.05, **p <0.0001.
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Fig5: SPP86 inhibits RET- mediated proliferation. (A) Estrogen- deprived and serum starved MCF7 cells were left untreated or exposed to 1 ng/ml E2 and/or 10 ng/ml GDNF alone or in combination with 1 μg/ml SPP86 in phenol red- free media for 7 days. Proliferation was expressed as fold increase in growth relative the untreated control population. The data represent the mean of 3 experiments ± S. E; *p <0.05 treated vs. untreated for each series. (B) Estrogen- deprived and serum starved MCF7 cells were cultured in the presence of 1 nM E2 (estrogen) together with 10 ng/ml of GDNF or 10 ng/ml insulin in the presence or absence of 1 μM SPP86 for 72 h. Proliferation was expressed as fold increase in growth relative the untreated control population. The data represent the mean of 3 experiments ± S. E; *p <0.05. (C) Estrogen- deprived and serum starved MCF7 cells exposed to increasing concentrations of SPP86 or 4-OHT in phenol red- free media containing 1 ng/ml E2 and 10 ng/ml GDNF for 7 days. Viability was expressed as a percentage of the untreated control population. The data in each panel represent the mean of 3 experiments ± S. E.; *p <0.05, **p <0.0001.

Mentions: Since these observations suggested that SPP86 disrupts ERα- RET crosstalk, we investigated the effect of SPP86 on the proliferation of MCF7 cells. Estrogen deprived and serum starved cells were cultured in the presence of 1 ng/ml β- estradiol (E2) or 10 ng/ml GDNF alone and in combination in the presence of 1 μM SPP86 for 7 days. SPP86 effectively inhibited E2 and/or GDNF- induced proliferation (p <0.05) (Figure 5A). In contrast, SPP86 did not inhibit proliferation when MCF7 cells were co-exposed to 1 ng/ml E2 and 5 ng/insulin under similar conditions (Figure 5B). We next compared the effect of SPP86 and tamoxifen on the proliferation of MCF7 cells. Estrogen deprived and serum starved cells were cultured in the presence of 1 ng/ml β- estradiol (E2) and 10 ng/ml GDNF with increasing doses of either SPP86 or tamoxifen, in medium containing 1 ng/ml β- estradiol (E2) and 10 ng/ml GDNF and incubated for 7 days. In these experiments, SPP86 and tamoxifen inhibited proliferation to a similar degree with IC50 values of 1.0 and 1.4 μM respectively (Figure 5C).Figure 5


Selective inhibition of RET mediated cell proliferation in vitro by the kinase inhibitor SPP86.

Alao JP, Michlikova S, Dinér P, Grøtli M, Sunnerhagen P - BMC Cancer (2014)

SPP86 inhibits RET- mediated proliferation. (A) Estrogen- deprived and serum starved MCF7 cells were left untreated or exposed to 1 ng/ml E2 and/or 10 ng/ml GDNF alone or in combination with 1 μg/ml SPP86 in phenol red- free media for 7 days. Proliferation was expressed as fold increase in growth relative the untreated control population. The data represent the mean of 3 experiments ± S. E; *p <0.05 treated vs. untreated for each series. (B) Estrogen- deprived and serum starved MCF7 cells were cultured in the presence of 1 nM E2 (estrogen) together with 10 ng/ml of GDNF or 10 ng/ml insulin in the presence or absence of 1 μM SPP86 for 72 h. Proliferation was expressed as fold increase in growth relative the untreated control population. The data represent the mean of 3 experiments ± S. E; *p <0.05. (C) Estrogen- deprived and serum starved MCF7 cells exposed to increasing concentrations of SPP86 or 4-OHT in phenol red- free media containing 1 ng/ml E2 and 10 ng/ml GDNF for 7 days. Viability was expressed as a percentage of the untreated control population. The data in each panel represent the mean of 3 experiments ± S. E.; *p <0.05, **p <0.0001.
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Fig5: SPP86 inhibits RET- mediated proliferation. (A) Estrogen- deprived and serum starved MCF7 cells were left untreated or exposed to 1 ng/ml E2 and/or 10 ng/ml GDNF alone or in combination with 1 μg/ml SPP86 in phenol red- free media for 7 days. Proliferation was expressed as fold increase in growth relative the untreated control population. The data represent the mean of 3 experiments ± S. E; *p <0.05 treated vs. untreated for each series. (B) Estrogen- deprived and serum starved MCF7 cells were cultured in the presence of 1 nM E2 (estrogen) together with 10 ng/ml of GDNF or 10 ng/ml insulin in the presence or absence of 1 μM SPP86 for 72 h. Proliferation was expressed as fold increase in growth relative the untreated control population. The data represent the mean of 3 experiments ± S. E; *p <0.05. (C) Estrogen- deprived and serum starved MCF7 cells exposed to increasing concentrations of SPP86 or 4-OHT in phenol red- free media containing 1 ng/ml E2 and 10 ng/ml GDNF for 7 days. Viability was expressed as a percentage of the untreated control population. The data in each panel represent the mean of 3 experiments ± S. E.; *p <0.05, **p <0.0001.
Mentions: Since these observations suggested that SPP86 disrupts ERα- RET crosstalk, we investigated the effect of SPP86 on the proliferation of MCF7 cells. Estrogen deprived and serum starved cells were cultured in the presence of 1 ng/ml β- estradiol (E2) or 10 ng/ml GDNF alone and in combination in the presence of 1 μM SPP86 for 7 days. SPP86 effectively inhibited E2 and/or GDNF- induced proliferation (p <0.05) (Figure 5A). In contrast, SPP86 did not inhibit proliferation when MCF7 cells were co-exposed to 1 ng/ml E2 and 5 ng/insulin under similar conditions (Figure 5B). We next compared the effect of SPP86 and tamoxifen on the proliferation of MCF7 cells. Estrogen deprived and serum starved cells were cultured in the presence of 1 ng/ml β- estradiol (E2) and 10 ng/ml GDNF with increasing doses of either SPP86 or tamoxifen, in medium containing 1 ng/ml β- estradiol (E2) and 10 ng/ml GDNF and incubated for 7 days. In these experiments, SPP86 and tamoxifen inhibited proliferation to a similar degree with IC50 values of 1.0 and 1.4 μM respectively (Figure 5C).Figure 5

Bottom Line: Herein, we have further investigated the effect of the lead compound SPP86 on RET mediated signaling and proliferation.We compared the effects of SPP86 on RET-induced signaling and proliferation in thyroid cancer cell lines expressing RET-PTC1 (TPC1), or the activating mutations BRAFV600E (8505C) and RASG13R (C643).Additionally, RET- FAK crosstalk may play a key role in facilitating PTC1/RET and GDNF- RET induced activation of Akt and MAPK signaling in TPC1 and MCF7 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, SE-405 30 Göteborg, Sweden. John.P.Alao@cmb.gu.se.

ABSTRACT

Background: The RET tyrosine kinase receptor has emerged as a target in thyroid and endocrine resistant breast cancer. We previously reported the synthesis of kinase inhibitors with potent activity against RET. Herein, we have further investigated the effect of the lead compound SPP86 on RET mediated signaling and proliferation. Based on these observations, we hypothesized that SPP86 may be useful for studying the cellular activity of RET.

Methods: We compared the effects of SPP86 on RET-induced signaling and proliferation in thyroid cancer cell lines expressing RET-PTC1 (TPC1), or the activating mutations BRAFV600E (8505C) and RASG13R (C643). The effect of SPP86 on RET- induced phosphatidylinositide 3-kinases (PI3K)/Akt and MAPK pathway signaling and cell proliferation in MCF7 breast cancer cells was also investigated.

Results: SPP86 inhibited MAPK signaling and proliferation in RET/PTC1 expressing TPC1 but not 8505C or C643 cells. In TPC1 cells, the inhibition of RET phosphorylation required co-exposure to SPP86 and the focal adhesion kinase (FAK) inhibitor PF573228. In MCF7 cells, SPP86 inhibited RET- induced phosphatidylinositide 3-kinases (PI3K)/Akt and MAPK signaling and estrogen receptorα (ERα) phosphorylation, and inhibited proliferation to a similar degree as tamoxifen. Interestingly, SPP86 and PF573228 inhibited RET/PTC1 and GDNF- RET induced activation of Akt and MAPK signaling to a similar degree.

Conclusion: SPP86 selectively inhibits RET downstream signaling in RET/PTC1 but not BRAFV600E or RASG13R expressing cells, indicating that downstream kinases were not affected. SPP86 also inhibited RET signaling in MCF7 breast cancer cells. Additionally, RET- FAK crosstalk may play a key role in facilitating PTC1/RET and GDNF- RET induced activation of Akt and MAPK signaling in TPC1 and MCF7 cells.

Show MeSH
Related in: MedlinePlus