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HER3 as biomarker and therapeutic target in pancreatic cancer: new insights in pertuzumab therapy in preclinical models.

Thomas G, Chardès T, Gaborit N, Mollevi C, Leconet W, Robert B, Radosevic-Robin N, Penault-Llorca F, Gongora C, Colombo PE, Lazrek Y, Bras-Goncalves R, Savina A, Azria D, Bazin H, Pèlegrin A, Larbouret C - Oncotarget (2014)

Bottom Line: We correlated in vitro and in vivo HER3 expression and neuregulin dependency with the inhibitory effect of pertuzumab on cell viability and tumor progression.HER3 knockdown in BxPC-3 cells led to resistance to pertuzumab therapy.Further studies in clinical samples are required to confirm these findings and the interest of combining anti-HER2 and anti-HER3 therapeutic antibodies.

View Article: PubMed Central - PubMed

Affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, F-34298, France; INSERM, Unit 896, Montpellier, F-34298, France; Université Montpellier1, Montpellier, F-34298, France; ICM, Montpellier, France. Institut Roche de Recherche et Médecine Translationnelle, Boulogne Bilancourt, France.

ABSTRACT
The anti-HER2 antibody pertuzumab inhibits HER2 dimerization and affects HER2/HER3 dimer formation and signaling. As HER3 and its ligand neuregulin are implicated in pancreatic tumorigenesis, we investigated whether HER3 expression could be a predictive biomarker of pertuzumab efficacy in HER2low-expressing pancreatic cancer. We correlated in vitro and in vivo HER3 expression and neuregulin dependency with the inhibitory effect of pertuzumab on cell viability and tumor progression. HER3 knockdown in BxPC-3 cells led to resistance to pertuzumab therapy. Pertuzumab treatment of HER3-expressing pancreatic cancer cells increased HER3 at the cell membrane, whereas the anti-HER3 monoclonal antibody 9F7-F11 down-regulated it. Both antibodies blocked HER3 and AKT phosphorylation and inhibited HER2/HER3 heterodimerization but affected differently HER2 and HER3 homodimers. The pertuzumab/9F7-F11 combination enhanced tumor inhibition and the median survival time in mice xenografted with HER3-expressing pancreatic cancer cells. Finally, HER2 and HER3 were co-expressed in 11% and HER3 alone in 27% of the 45 pancreatic ductal adenocarcinomas analyzed by immunohistochemistry. HER3 is essential for pertuzumab efficacy in HER2low-expressing pancreatic cancer and HER3 expression might be a predictive biomarker of pertuzumab efficacy in such cancers. Further studies in clinical samples are required to confirm these findings and the interest of combining anti-HER2 and anti-HER3 therapeutic antibodies.

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Effect of pertuzumab and of the anti-HER3 antibody 9F7-F11 on HER2, HER3 and downstream signaling pathways in BxPC-3 cellsCells were pre-incubated with pertuzumab [A], 9F7-F11 (B) or both antibodies (C) (50 μg/ml/each antibody for the indicated time) and then with 100 ng/ml NRG1ß1 for 10 minutes. The expression level of phosphorylated and total HER2, HER3, AKT and ERK was then analyzed by western blotting. Effect of pertuzumab and 9F7-F11 on (D) HER2/HER3, (E) HER2/HER2 and (F) HER3/HER3 dimer formation in NIH/3T3 HER2/HER3 cells. 105 cells/well were incubated with increasing concentrations (1 to 100 μg/ml) of antibodies in serum-free medium for 30 minutes. The TR-FRET signal was expressed as Delta F665 (%) and then as dimer percentage (see “Materials and Methods”). Data are the mean ± SEM of 3 experiments performed in triplicate. P, ***<0.001, ** p < 0.01, n.s., not significant.
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Figure 5: Effect of pertuzumab and of the anti-HER3 antibody 9F7-F11 on HER2, HER3 and downstream signaling pathways in BxPC-3 cellsCells were pre-incubated with pertuzumab [A], 9F7-F11 (B) or both antibodies (C) (50 μg/ml/each antibody for the indicated time) and then with 100 ng/ml NRG1ß1 for 10 minutes. The expression level of phosphorylated and total HER2, HER3, AKT and ERK was then analyzed by western blotting. Effect of pertuzumab and 9F7-F11 on (D) HER2/HER3, (E) HER2/HER2 and (F) HER3/HER3 dimer formation in NIH/3T3 HER2/HER3 cells. 105 cells/well were incubated with increasing concentrations (1 to 100 μg/ml) of antibodies in serum-free medium for 30 minutes. The TR-FRET signal was expressed as Delta F665 (%) and then as dimer percentage (see “Materials and Methods”). Data are the mean ± SEM of 3 experiments performed in triplicate. P, ***<0.001, ** p < 0.01, n.s., not significant.

Mentions: To assess the mechanisms underlying the role of HER3 in pancreatic cancer response to pertuzumab, HER2/HER3 expression, phosphorylation and downstream signaling were studied in NRG1β1-stimulated BxPC-3 cells after pertuzumab addition (Figure 5A). As expected, NRG1β1 stimulation induced phosphorylation of HER2 and HER3 and of the downstream signaling molecules ERK1/2 and AKT. Pertuzumab did not affect HER2 and HER3 expression during the first hour of treatment and only slightly after 24h of treatment in comparison to untreated cells. Conversely, it increased HER2 phosphorylation even after 24h of treatment and completely abrogated HER3, AKT and ERK1/2 phosphorylation. These results were confirmed in vivo in BxPC-3 tumor xenografts isolated from antibody-treated and control mice (Supplementary Figure. 1).


HER3 as biomarker and therapeutic target in pancreatic cancer: new insights in pertuzumab therapy in preclinical models.

Thomas G, Chardès T, Gaborit N, Mollevi C, Leconet W, Robert B, Radosevic-Robin N, Penault-Llorca F, Gongora C, Colombo PE, Lazrek Y, Bras-Goncalves R, Savina A, Azria D, Bazin H, Pèlegrin A, Larbouret C - Oncotarget (2014)

Effect of pertuzumab and of the anti-HER3 antibody 9F7-F11 on HER2, HER3 and downstream signaling pathways in BxPC-3 cellsCells were pre-incubated with pertuzumab [A], 9F7-F11 (B) or both antibodies (C) (50 μg/ml/each antibody for the indicated time) and then with 100 ng/ml NRG1ß1 for 10 minutes. The expression level of phosphorylated and total HER2, HER3, AKT and ERK was then analyzed by western blotting. Effect of pertuzumab and 9F7-F11 on (D) HER2/HER3, (E) HER2/HER2 and (F) HER3/HER3 dimer formation in NIH/3T3 HER2/HER3 cells. 105 cells/well were incubated with increasing concentrations (1 to 100 μg/ml) of antibodies in serum-free medium for 30 minutes. The TR-FRET signal was expressed as Delta F665 (%) and then as dimer percentage (see “Materials and Methods”). Data are the mean ± SEM of 3 experiments performed in triplicate. P, ***<0.001, ** p < 0.01, n.s., not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196190&req=5

Figure 5: Effect of pertuzumab and of the anti-HER3 antibody 9F7-F11 on HER2, HER3 and downstream signaling pathways in BxPC-3 cellsCells were pre-incubated with pertuzumab [A], 9F7-F11 (B) or both antibodies (C) (50 μg/ml/each antibody for the indicated time) and then with 100 ng/ml NRG1ß1 for 10 minutes. The expression level of phosphorylated and total HER2, HER3, AKT and ERK was then analyzed by western blotting. Effect of pertuzumab and 9F7-F11 on (D) HER2/HER3, (E) HER2/HER2 and (F) HER3/HER3 dimer formation in NIH/3T3 HER2/HER3 cells. 105 cells/well were incubated with increasing concentrations (1 to 100 μg/ml) of antibodies in serum-free medium for 30 minutes. The TR-FRET signal was expressed as Delta F665 (%) and then as dimer percentage (see “Materials and Methods”). Data are the mean ± SEM of 3 experiments performed in triplicate. P, ***<0.001, ** p < 0.01, n.s., not significant.
Mentions: To assess the mechanisms underlying the role of HER3 in pancreatic cancer response to pertuzumab, HER2/HER3 expression, phosphorylation and downstream signaling were studied in NRG1β1-stimulated BxPC-3 cells after pertuzumab addition (Figure 5A). As expected, NRG1β1 stimulation induced phosphorylation of HER2 and HER3 and of the downstream signaling molecules ERK1/2 and AKT. Pertuzumab did not affect HER2 and HER3 expression during the first hour of treatment and only slightly after 24h of treatment in comparison to untreated cells. Conversely, it increased HER2 phosphorylation even after 24h of treatment and completely abrogated HER3, AKT and ERK1/2 phosphorylation. These results were confirmed in vivo in BxPC-3 tumor xenografts isolated from antibody-treated and control mice (Supplementary Figure. 1).

Bottom Line: We correlated in vitro and in vivo HER3 expression and neuregulin dependency with the inhibitory effect of pertuzumab on cell viability and tumor progression.HER3 knockdown in BxPC-3 cells led to resistance to pertuzumab therapy.Further studies in clinical samples are required to confirm these findings and the interest of combining anti-HER2 and anti-HER3 therapeutic antibodies.

View Article: PubMed Central - PubMed

Affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, F-34298, France; INSERM, Unit 896, Montpellier, F-34298, France; Université Montpellier1, Montpellier, F-34298, France; ICM, Montpellier, France. Institut Roche de Recherche et Médecine Translationnelle, Boulogne Bilancourt, France.

ABSTRACT
The anti-HER2 antibody pertuzumab inhibits HER2 dimerization and affects HER2/HER3 dimer formation and signaling. As HER3 and its ligand neuregulin are implicated in pancreatic tumorigenesis, we investigated whether HER3 expression could be a predictive biomarker of pertuzumab efficacy in HER2low-expressing pancreatic cancer. We correlated in vitro and in vivo HER3 expression and neuregulin dependency with the inhibitory effect of pertuzumab on cell viability and tumor progression. HER3 knockdown in BxPC-3 cells led to resistance to pertuzumab therapy. Pertuzumab treatment of HER3-expressing pancreatic cancer cells increased HER3 at the cell membrane, whereas the anti-HER3 monoclonal antibody 9F7-F11 down-regulated it. Both antibodies blocked HER3 and AKT phosphorylation and inhibited HER2/HER3 heterodimerization but affected differently HER2 and HER3 homodimers. The pertuzumab/9F7-F11 combination enhanced tumor inhibition and the median survival time in mice xenografted with HER3-expressing pancreatic cancer cells. Finally, HER2 and HER3 were co-expressed in 11% and HER3 alone in 27% of the 45 pancreatic ductal adenocarcinomas analyzed by immunohistochemistry. HER3 is essential for pertuzumab efficacy in HER2low-expressing pancreatic cancer and HER3 expression might be a predictive biomarker of pertuzumab efficacy in such cancers. Further studies in clinical samples are required to confirm these findings and the interest of combining anti-HER2 and anti-HER3 therapeutic antibodies.

Show MeSH
Related in: MedlinePlus