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Combinatorial targeting of FGF and ErbB receptors blocks growth and metastatic spread of breast cancer models.

Issa A, Gill JW, Heideman MR, Sahin O, Wiemann S, Dey JH, Hynes NE - Breast Cancer Res. (2013)

Bottom Line: The results show that in vivo these breast cancer models become dependent upon co-activation of FGFR and ErbB receptors for PI3K pathway activity.The work presented here shows that in the breast cancer models examined, the combination of dovitinib + NVP-BEZ235 or dovitinib + AEE788 results in strong inhibition of tumor growth and a block in metastatic spread.The resultant decrease in mitosis and increase in apoptosis was consistently stronger in the dovitinib + AEE788 treatment-group, suggesting that targeting ErbB receptors has broader downstream effects compared to targeting only PI3K/mTOR.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Targeting receptor tyrosine kinases (RTKs) with kinase inhibitors is a clinically validated anti-cancer approach. However, blocking one signaling pathway is often not sufficient to cause tumor regression and the effectiveness of individual inhibitors is often short-lived. As alterations in fibroblast growth factor receptor (FGFR) activity have been implicated in breast cancer, we examined in breast cancer models with autocrine FGFR activity the impact of targeting FGFRs in vivo with a selective kinase inhibitor in combination with an inhibitor of PI3K/mTOR or with a pan-ErbB inhibitor.

Methods: Using 4T1 or 67NR models of basal-like breast cancer, tumor growth was measured in mice treated with an FGFR inhibitor (dovitinib/TKI258), a PI3K/mTOR inhibitor (NVP-BEZ235) or a pan-ErbB inhibitor (AEE788) individually or in combination. To uncover mechanisms underlying inhibitor action, signaling pathway activity was examined in tumor lysates and transcriptome analysis carried out to identify pathways upregulated by FGFR inhibition. Anti-phosphotyrosine receptor antibody arrays (P-Tyr RTK) were also used to screen 4T1 tumors.

Results: The combination of dovitinib + NVP-BEZ235 causes tumor stasis and strong down-regulation of the FRS2/Erk and PI3K/Akt/mTOR signaling pathways. P-Tyr RTK arrays identified high levels of P-EGFR and P-ErbB2 in 4T1 tumors. Testing AEE788 in the tumor models revealed that the combination of dovitinib + AEE788 resulted in blockade of the PI3K/Akt/mTOR pathway, prolonged tumor stasis and in the 4T1 model, a significant decrease in lung metastasis. The results show that in vivo these breast cancer models become dependent upon co-activation of FGFR and ErbB receptors for PI3K pathway activity.

Conclusions: The work presented here shows that in the breast cancer models examined, the combination of dovitinib + NVP-BEZ235 or dovitinib + AEE788 results in strong inhibition of tumor growth and a block in metastatic spread. Only these combinations strongly down-regulate the FGFR/FRS2/Erk and PI3K/Akt/mTOR signaling pathways. The resultant decrease in mitosis and increase in apoptosis was consistently stronger in the dovitinib + AEE788 treatment-group, suggesting that targeting ErbB receptors has broader downstream effects compared to targeting only PI3K/mTOR. Considering that sub-classes of human breast tumors co-express ErbB receptors and FGFRs, these results have implications for targeted therapy.

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Anti-phosphotyrosine receptor antibody (P-Tyr RTK) array analysis of 4T1 cells and tumors. (A-B) P-RTK analysis on lysates of 4T1 cultures either growing in low serum (Vehicle) or stimulated for 15 minutes with 20 ng/ml EGF, 12 nM HRG or 20 ng/ml platelet-derived growth factor (PDGF) (Ligands). Lysates from individual ligand-treated cells were pooled prior to analysis. (B) Quantification of the signal intensity of the indicated p-RTKs is shown in comparison to positive control, set as 1. (C-D) Three control (Vehicle) and 50 mg/kg dovitinib-treated (TKI258) 4T1 tumors were harvested after 3 days treatment, and lysates were pooled and analyzed on P-RTK filters. (D) Quantification of the intensity of signal of indicated p-RTKs is shown, in comparison to positive control set as 1.
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Figure 4: Anti-phosphotyrosine receptor antibody (P-Tyr RTK) array analysis of 4T1 cells and tumors. (A-B) P-RTK analysis on lysates of 4T1 cultures either growing in low serum (Vehicle) or stimulated for 15 minutes with 20 ng/ml EGF, 12 nM HRG or 20 ng/ml platelet-derived growth factor (PDGF) (Ligands). Lysates from individual ligand-treated cells were pooled prior to analysis. (B) Quantification of the signal intensity of the indicated p-RTKs is shown in comparison to positive control, set as 1. (C-D) Three control (Vehicle) and 50 mg/kg dovitinib-treated (TKI258) 4T1 tumors were harvested after 3 days treatment, and lysates were pooled and analyzed on P-RTK filters. (D) Quantification of the intensity of signal of indicated p-RTKs is shown, in comparison to positive control set as 1.

Mentions: Blocking FGFR activity in combination with PI3K/mTOR inhibition was very effective in decreasing tumor growth. Our next goal was to uncover a tyrosine kinase receptor that when inhibited would block PI3K pathway activity. To approach this, we used anti-phosphotyrosine receptor antibody arrays (P-Tyr RTK) to screen for activity across a panel of RTKs in 4T1 cultured cells and tumors. In lysates from cell cultures, high basal levels of P-ErbB2 and P-platelet-derived growth factor receptor alpha (PDGFRa) were detected in vehicle control cells and their P-Tyr content increased in response to their activating ligands Heregulin (HRG), PDGF and EGF (Figure 4A, quantified in 4B); although P-EGFR (B1 to 2) was only visible on a longer exposure. The other RTKs, including FGFR2 (B9 to 10) and FGFR3 (B11 to 12), showed little or no P-Tyr. Using mass spectrometry and a phospho-proteomic screen, we have previously shown that FGFR-1, -2 and -3, which are expressed in the cells, each contain Tyr-P [9]; apparently their level is too low to detect with this RTK array. High levels of P-ErbB2 and P-PDGFRa were also detected in 4T1 tumor lysates (Figure 4C, vehicle). Interestingly, novel P-RTKs that were not detected in lysates from cell cultures, including P-EGFR, P-macrophage-stimulating protein receptor (P-MSPR), and to a lesser extent P-VEGFR3 (D7 to 8) and P-musk receptor (P-MuSK) (D9 to 10), were also found in the tumors (Figure 4C, vehicle, quantified in 4D). Tumors were analyzed 10 days after 4T1 injection so there would be sufficient time for ligands from the tumor environment [18] to influence their activity.


Combinatorial targeting of FGF and ErbB receptors blocks growth and metastatic spread of breast cancer models.

Issa A, Gill JW, Heideman MR, Sahin O, Wiemann S, Dey JH, Hynes NE - Breast Cancer Res. (2013)

Anti-phosphotyrosine receptor antibody (P-Tyr RTK) array analysis of 4T1 cells and tumors. (A-B) P-RTK analysis on lysates of 4T1 cultures either growing in low serum (Vehicle) or stimulated for 15 minutes with 20 ng/ml EGF, 12 nM HRG or 20 ng/ml platelet-derived growth factor (PDGF) (Ligands). Lysates from individual ligand-treated cells were pooled prior to analysis. (B) Quantification of the signal intensity of the indicated p-RTKs is shown in comparison to positive control, set as 1. (C-D) Three control (Vehicle) and 50 mg/kg dovitinib-treated (TKI258) 4T1 tumors were harvested after 3 days treatment, and lysates were pooled and analyzed on P-RTK filters. (D) Quantification of the intensity of signal of indicated p-RTKs is shown, in comparison to positive control set as 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Anti-phosphotyrosine receptor antibody (P-Tyr RTK) array analysis of 4T1 cells and tumors. (A-B) P-RTK analysis on lysates of 4T1 cultures either growing in low serum (Vehicle) or stimulated for 15 minutes with 20 ng/ml EGF, 12 nM HRG or 20 ng/ml platelet-derived growth factor (PDGF) (Ligands). Lysates from individual ligand-treated cells were pooled prior to analysis. (B) Quantification of the signal intensity of the indicated p-RTKs is shown in comparison to positive control, set as 1. (C-D) Three control (Vehicle) and 50 mg/kg dovitinib-treated (TKI258) 4T1 tumors were harvested after 3 days treatment, and lysates were pooled and analyzed on P-RTK filters. (D) Quantification of the intensity of signal of indicated p-RTKs is shown, in comparison to positive control set as 1.
Mentions: Blocking FGFR activity in combination with PI3K/mTOR inhibition was very effective in decreasing tumor growth. Our next goal was to uncover a tyrosine kinase receptor that when inhibited would block PI3K pathway activity. To approach this, we used anti-phosphotyrosine receptor antibody arrays (P-Tyr RTK) to screen for activity across a panel of RTKs in 4T1 cultured cells and tumors. In lysates from cell cultures, high basal levels of P-ErbB2 and P-platelet-derived growth factor receptor alpha (PDGFRa) were detected in vehicle control cells and their P-Tyr content increased in response to their activating ligands Heregulin (HRG), PDGF and EGF (Figure 4A, quantified in 4B); although P-EGFR (B1 to 2) was only visible on a longer exposure. The other RTKs, including FGFR2 (B9 to 10) and FGFR3 (B11 to 12), showed little or no P-Tyr. Using mass spectrometry and a phospho-proteomic screen, we have previously shown that FGFR-1, -2 and -3, which are expressed in the cells, each contain Tyr-P [9]; apparently their level is too low to detect with this RTK array. High levels of P-ErbB2 and P-PDGFRa were also detected in 4T1 tumor lysates (Figure 4C, vehicle). Interestingly, novel P-RTKs that were not detected in lysates from cell cultures, including P-EGFR, P-macrophage-stimulating protein receptor (P-MSPR), and to a lesser extent P-VEGFR3 (D7 to 8) and P-musk receptor (P-MuSK) (D9 to 10), were also found in the tumors (Figure 4C, vehicle, quantified in 4D). Tumors were analyzed 10 days after 4T1 injection so there would be sufficient time for ligands from the tumor environment [18] to influence their activity.

Bottom Line: The results show that in vivo these breast cancer models become dependent upon co-activation of FGFR and ErbB receptors for PI3K pathway activity.The work presented here shows that in the breast cancer models examined, the combination of dovitinib + NVP-BEZ235 or dovitinib + AEE788 results in strong inhibition of tumor growth and a block in metastatic spread.The resultant decrease in mitosis and increase in apoptosis was consistently stronger in the dovitinib + AEE788 treatment-group, suggesting that targeting ErbB receptors has broader downstream effects compared to targeting only PI3K/mTOR.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Targeting receptor tyrosine kinases (RTKs) with kinase inhibitors is a clinically validated anti-cancer approach. However, blocking one signaling pathway is often not sufficient to cause tumor regression and the effectiveness of individual inhibitors is often short-lived. As alterations in fibroblast growth factor receptor (FGFR) activity have been implicated in breast cancer, we examined in breast cancer models with autocrine FGFR activity the impact of targeting FGFRs in vivo with a selective kinase inhibitor in combination with an inhibitor of PI3K/mTOR or with a pan-ErbB inhibitor.

Methods: Using 4T1 or 67NR models of basal-like breast cancer, tumor growth was measured in mice treated with an FGFR inhibitor (dovitinib/TKI258), a PI3K/mTOR inhibitor (NVP-BEZ235) or a pan-ErbB inhibitor (AEE788) individually or in combination. To uncover mechanisms underlying inhibitor action, signaling pathway activity was examined in tumor lysates and transcriptome analysis carried out to identify pathways upregulated by FGFR inhibition. Anti-phosphotyrosine receptor antibody arrays (P-Tyr RTK) were also used to screen 4T1 tumors.

Results: The combination of dovitinib + NVP-BEZ235 causes tumor stasis and strong down-regulation of the FRS2/Erk and PI3K/Akt/mTOR signaling pathways. P-Tyr RTK arrays identified high levels of P-EGFR and P-ErbB2 in 4T1 tumors. Testing AEE788 in the tumor models revealed that the combination of dovitinib + AEE788 resulted in blockade of the PI3K/Akt/mTOR pathway, prolonged tumor stasis and in the 4T1 model, a significant decrease in lung metastasis. The results show that in vivo these breast cancer models become dependent upon co-activation of FGFR and ErbB receptors for PI3K pathway activity.

Conclusions: The work presented here shows that in the breast cancer models examined, the combination of dovitinib + NVP-BEZ235 or dovitinib + AEE788 results in strong inhibition of tumor growth and a block in metastatic spread. Only these combinations strongly down-regulate the FGFR/FRS2/Erk and PI3K/Akt/mTOR signaling pathways. The resultant decrease in mitosis and increase in apoptosis was consistently stronger in the dovitinib + AEE788 treatment-group, suggesting that targeting ErbB receptors has broader downstream effects compared to targeting only PI3K/mTOR. Considering that sub-classes of human breast tumors co-express ErbB receptors and FGFRs, these results have implications for targeted therapy.

Show MeSH
Related in: MedlinePlus