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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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Effect of dovitinib, AEE788 or the combination of both on 4T1 and 67NR tumors. (A) Groups of 4T1 tumor-bearing mice (n = 6) were treated for 12 days with PEG300 (Vehicle), AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788. AEE788 was administered 3×/week; dovitinib daily, and tumor volume was determined; representative of two experiments. (B) Groups of 67NR tumor-bearing mice (n = 5) were treated with PEG300 (Vehicle), AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788. Treatment was performed through days 7 to 13 and days 18 to 23. Dovitinib and vehicle were dosed daily; AEE788 was administered on days 7, 9, 11 and 13, then on day 18, 20 and 22, and tumor volume was determined. (C) Quantification of the number of metastatic foci covering lungs of mice from the experiment in Panel A. N = 6, representative of two separate experiments. (D) 4T1 tumor-bearing mice were treated with PEG300, a single dose of AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788, then sacrificed 2 hrs later. Tumor lysates were prepared from three mice per group and a western analysis for the indicated proteins and phospho-proteins (P) was performed. *P < 0.05 (Mann-Whitney U-test).
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Figure 5: Effect of dovitinib, AEE788 or the combination of both on 4T1 and 67NR tumors. (A) Groups of 4T1 tumor-bearing mice (n = 6) were treated for 12 days with PEG300 (Vehicle), AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788. AEE788 was administered 3×/week; dovitinib daily, and tumor volume was determined; representative of two experiments. (B) Groups of 67NR tumor-bearing mice (n = 5) were treated with PEG300 (Vehicle), AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788. Treatment was performed through days 7 to 13 and days 18 to 23. Dovitinib and vehicle were dosed daily; AEE788 was administered on days 7, 9, 11 and 13, then on day 18, 20 and 22, and tumor volume was determined. (C) Quantification of the number of metastatic foci covering lungs of mice from the experiment in Panel A. N = 6, representative of two separate experiments. (D) 4T1 tumor-bearing mice were treated with PEG300, a single dose of AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788, then sacrificed 2 hrs later. Tumor lysates were prepared from three mice per group and a western analysis for the indicated proteins and phospho-proteins (P) was performed. *P < 0.05 (Mann-Whitney U-test).

Mentions: We decided to focus on ErbB receptors, since ErbB2 signals strongly to the PI3K pathway through ErbB3 [19], and pan-ErbB inhibitors are in clinical use [20]. For our work, we used AEE788, which has been shown to block EGFR and ErbB2 activity [11,21]. Preliminary testing with AEE788 revealed good anti-tumor activity in the 4T1 model, and a decrease in P-ErbB2 levels was readily detected in tumor lysates from AEE788-treated mice (see Figure S6 upper panel in Additional file 1). Groups of 4T1 and 67NR tumor-bearing mice were treated long-term with AEE788 or with the combination of dovitinib + AEE788. In both the 4T1 and the 67NR tumor models we observed significantly impaired tumor outgrowth with single agent AEE788 as well as with the combination, the latter treatment consistently showing stronger anti-tumor activity (Figure 5A and 5B). The 4T1 tumor-bearing mice treated with AEE788 alone had fewer lung metastases, but there was a stronger, significant effect in mice treated with the combination. We performed intermittent dosing in the 67NR model and observed tumor stasis over the course of three weeks in the combination treated group (Figure 5B). An analysis of signaling proteins in 4T1 tumors was also undertaken. Interestingly, there was no consistent decrease in P-Akt levels in tumors from AEE788 treated mice (Figure 5D) (see also Figure S6 lower panel in Additional file 1), which was surprising since in vitro treatment of 4T1 cells with AEE788 does block this pathway (see Figure S7 in Additional file 1). Only in the combination treated group did we observe a strong decrease in P-Akt and P-S6 (Figure 5D). As expected there was also a decrease in P-FRS2 and P-Erk levels in the combination-treated group, due to dovitinib treatment (Figure 5D). Taken together the results show that, concomitant inhibition of ErbB receptors and FGFRs has strong anti-tumor activity in both the 4T1 and 67NR models. Moreover, blocking ErbB RTK activity is not sufficient to lower PI3K pathway activity, only when combined with dovitinib was this achieved.


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)

Effect of dovitinib, AEE788 or the combination of both on 4T1 and 67NR tumors. (A) Groups of 4T1 tumor-bearing mice (n = 6) were treated for 12 days with PEG300 (Vehicle), AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788. AEE788 was administered 3×/week; dovitinib daily, and tumor volume was determined; representative of two experiments. (B) Groups of 67NR tumor-bearing mice (n = 5) were treated with PEG300 (Vehicle), AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788. Treatment was performed through days 7 to 13 and days 18 to 23. Dovitinib and vehicle were dosed daily; AEE788 was administered on days 7, 9, 11 and 13, then on day 18, 20 and 22, and tumor volume was determined. (C) Quantification of the number of metastatic foci covering lungs of mice from the experiment in Panel A. N = 6, representative of two separate experiments. (D) 4T1 tumor-bearing mice were treated with PEG300, a single dose of AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788, then sacrificed 2 hrs later. Tumor lysates were prepared from three mice per group and a western analysis for the indicated proteins and phospho-proteins (P) was performed. *P < 0.05 (Mann-Whitney U-test).
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Figure 5: Effect of dovitinib, AEE788 or the combination of both on 4T1 and 67NR tumors. (A) Groups of 4T1 tumor-bearing mice (n = 6) were treated for 12 days with PEG300 (Vehicle), AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788. AEE788 was administered 3×/week; dovitinib daily, and tumor volume was determined; representative of two experiments. (B) Groups of 67NR tumor-bearing mice (n = 5) were treated with PEG300 (Vehicle), AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788. Treatment was performed through days 7 to 13 and days 18 to 23. Dovitinib and vehicle were dosed daily; AEE788 was administered on days 7, 9, 11 and 13, then on day 18, 20 and 22, and tumor volume was determined. (C) Quantification of the number of metastatic foci covering lungs of mice from the experiment in Panel A. N = 6, representative of two separate experiments. (D) 4T1 tumor-bearing mice were treated with PEG300, a single dose of AEE788 (50 mg/kg) or a combination of dovitinib (TKI, 40 mg/kg) + AEE788, then sacrificed 2 hrs later. Tumor lysates were prepared from three mice per group and a western analysis for the indicated proteins and phospho-proteins (P) was performed. *P < 0.05 (Mann-Whitney U-test).
Mentions: We decided to focus on ErbB receptors, since ErbB2 signals strongly to the PI3K pathway through ErbB3 [19], and pan-ErbB inhibitors are in clinical use [20]. For our work, we used AEE788, which has been shown to block EGFR and ErbB2 activity [11,21]. Preliminary testing with AEE788 revealed good anti-tumor activity in the 4T1 model, and a decrease in P-ErbB2 levels was readily detected in tumor lysates from AEE788-treated mice (see Figure S6 upper panel in Additional file 1). Groups of 4T1 and 67NR tumor-bearing mice were treated long-term with AEE788 or with the combination of dovitinib + AEE788. In both the 4T1 and the 67NR tumor models we observed significantly impaired tumor outgrowth with single agent AEE788 as well as with the combination, the latter treatment consistently showing stronger anti-tumor activity (Figure 5A and 5B). The 4T1 tumor-bearing mice treated with AEE788 alone had fewer lung metastases, but there was a stronger, significant effect in mice treated with the combination. We performed intermittent dosing in the 67NR model and observed tumor stasis over the course of three weeks in the combination treated group (Figure 5B). An analysis of signaling proteins in 4T1 tumors was also undertaken. Interestingly, there was no consistent decrease in P-Akt levels in tumors from AEE788 treated mice (Figure 5D) (see also Figure S6 lower panel in Additional file 1), which was surprising since in vitro treatment of 4T1 cells with AEE788 does block this pathway (see Figure S7 in Additional file 1). Only in the combination treated group did we observe a strong decrease in P-Akt and P-S6 (Figure 5D). As expected there was also a decrease in P-FRS2 and P-Erk levels in the combination-treated group, due to dovitinib treatment (Figure 5D). Taken together the results show that, concomitant inhibition of ErbB receptors and FGFRs has strong anti-tumor activity in both the 4T1 and 67NR models. Moreover, blocking ErbB RTK activity is not sufficient to lower PI3K pathway activity, only when combined with dovitinib was this achieved.

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