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. 2011 Sep 6;105(6):796-806.
doi: 10.1038/bjc.2011.321. Epub 2011 Aug 16.

Targeting both Notch and ErbB-2 signalling pathways is required for prevention of ErbB-2-positive breast tumour recurrence

K Pandya  1 , K Meeke, A G Clementz, A Rogowski, J Roberts, L Miele, K S Albain, C Osipo
Affiliations

Targeting both Notch and ErbB-2 signalling pathways is required for prevention of ErbB-2-positive breast tumour recurrence

K Pandya et al. Br J Cancer. .

Abstract

Background: We reported that Notch-1, a potent breast oncogene, is activated in response to trastuzumab and contributes to trastuzumab resistance in vitro. We sought to determine the preclinical benefit of combining a Notch inhibitor (γ-secretase inhibitor (GSI)) and trastuzumab in both trastuzumab-sensitive and trastuzumab-resistant, ErbB-2-positive, BT474 breast tumours in vivo. We also studied if the combination therapy of lapatinib plus GSI can induce tumour regression of ErbB-2-positive breast cancer.

Methods: We generated orthotopic breast tumour xenografts from trastuzumab- or lapatinib-sensitive and trastuzumab-resistant BT474 cells. We investigated the antitumour activities of two distinct GSIs, LY 411 575 and MRK-003, in vivo.

Results: Our findings showed that combining trastuzumab plus a GSI completely prevented (MRK-003 GSI) or significantly reduced (LY 411 575 GSI) breast tumour recurrence post-trastuzumab treatment in sensitive tumours. Moreover, combining lapatinib plus MRK-003 GSI showed significant reduction of tumour growth. Furthermore, a GSI partially reversed trastuzumab resistance in resistant tumours.

Conclusion: Our data suggest that a combined inhibition of Notch and ErbB-2 signalling pathways could decrease recurrence rates for ErbB-2-positive breast tumours and may be beneficial in the treatment of recurrent trastuzumab-resistant disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Trastuzumab (Trast) plus a γ-secretase inhibitor (GSI) prevents or reduces tumour recurrence. ErbB-2-overexpressing BT474 xenografts were generated in 56 ovariectomised, athymic nude mice by injecting 5 × 106 cells into both mammary fat pads. Once tumours reached a mean tumour cross-sectional area of 0.20 cm2, mice were randomised and treated with vehicle (100 μl sterile PBS injected i.p. 1 day per week and 200 μl 2% carboxymethylcellulose), 10 mg kg−1 trastuzumab in 100 μl PBS injected i.p. once weekly, 5 mg kg−1 LY 411 575 GSI (A) or 100 mg kg−1 MRK-003 GSI (B) in 200 μl 2% carboxymethylcellulose, fed by oral gavage, three days on, 4 days off, or trastuzumab plus LY 411 575 GSI or MRK-003 GSI. Tumour area (length × width) was measured weekly using Vernier calipers. The measurements were performed up to 12 or 19 weeks. The treatments were stopped and tumour recurrence was measured up to an additional 105 days or 98 days in mice that specifically showed complete tumour regression (A and B). Results from (A and B) show mean tumour cross-sectional area ((area × Π)/4) on the y axis and time in weeks on the x axis. Error bars are s.d. of the mean for 12 mice bearing tumours in the response phase of the study and 8 mice for the recurrent phase of the study. The results from (A and B) also demonstrate mice bearing recurrent tumours on the y axis and treatments on the x axis. *Statistically significant differences between mean slopes of the curve for trastuzumab plus GSI vs GSI alone. **Statistically significant differences between mean slopes of the curve for trastuzumab vs trastuzumab plus GSI in recurrent tumours. Linear regression analyses were performed for tumour growth curves in (A and B).
Figure 2
Figure 2
Kaplan–Meier curve of the percentage of tumour-free mice among Trast1, Trast1+MRK-003 GSI, Trast2, and Trast2+LY 411 575 GSI treatment groups using a log rank (Mantel–Cox) test. *Statistically significant differences between Trast1 and Trast1 + MRK-003 GSI. **Statistically significant differences between Trast2 and Trast2 + LY 411 575 GSI.
Figure 3
Figure 3
Activity of the Notch-1 signalling pathway in BT474 tumours. (A and B) In a separate experiment, 1 mg of snap-frozen tumours were homogenised and lysed in Trizol reagent and total RNA extracted using the Trizol protocol as described previously (Osipo et al, 2008). Total RNA was reverse transcribed to total cDNA using the Applied Biosystems kit. Real-time PCR was performed using human-specific primers to detect transcripts from Notch target genes: human HEY1 and human Deltex1. Human-specific 18S rRNA was detected for normalisation. Results are mean relative transcripts levels compared with vehicle (Control) after normalisation to 18S rRNA. Error bars are s.d. of the mean for five independent tumour samples. *Statistically significant differences between MRK-003 or LY 411 575 GSI and vehicle (Control). **Statistically significant differences between trastuzumab (Trast) and Control. ***Statistically significant differences between GSI + Trast and Trast alone. #Statistically significant differences between Trast-treated tumours and recurrent tumours previously treated with Trast. ##Statistically significant differences between GSI + Trast and Trast alone in recurrent tumours.
Figure 4
Figure 4
BT474 xenograft histology and signalling pathways. Five mice were euthanised and tumours excised at week 12 as shown in Figure 1B. One half of the tumours were immediately fixed in formalin and the remaining half snap-frozen in liquid nitrogen for future study. Fixed tumours were paraffin embedded and sectioned for H/E staining (upper panel), Ki67 (middle panel), and TUNEL (lower panel) assays. All sections were photographed at × 40 magnification using a light microscope. Four panels are shown: BT474 tumours treated with vehicle, trastuzumab (Trast), MRK-003 GSI, or Trast plus GSI. Shown are representative photographs based on at least three tumours. (B) Quantification of Ki67- and TUNEL-positive cells of three tumours using 60 high-powered fields (HPFs) × at 40 magnification. The y axis represents the number of Ki67- or TUNEL-positive cells for 60 HPFs. The bar graphs are mean±s.d.. *Statistical significance between trastuzumab and trastuzumab plus MRK-003 GSI (Trast + MRK-003). **Statistical significance between MRK-003 GSI and Trast + MRK-003 GSI. (C) Expression of ErbB-2 and downstream signalling pathways in BT474 tumours. Bits of tumours (1 mg) that were snap-frozen in liquid nitrogen were homogenised and lysed in RIPA buffer containing protease and phosphatase inhibitors. Cellular debris was removed by centrifugation at 1000 g for 5 min at 4 °C. Supernatants were collected and 25 μg of total protein loaded onto a 7% SDS–PAGE gel followed by western blotting to detect tyrosine phosphorylated ErbB-2 or HER2 (PY1248-HER2), total HER2, P-ERK1/2, total ERK1/2, P-AKT1, total AKT1, PTEN, and actin proteins. The western blot shown is a representative of three independent tumour samples with similar results. Density of bands corresponding to PY-HER2 and total HER2 for each treatment groups was quantified using ImageJ program (NIH, Bethesda, MD, USA) and expressed as a PY-HER2/HER2 ratio of area of the peak.
Figure 5
Figure 5
Lapatinib plus MRK-003 GSI reduces tumour growth. (A) ErbB-2-overexpressing BT474 xenografts were generated in 40 ovariectomised, athymic nude mice by injecting 5 × 106 cells into both mammary fat pads. Once tumours reached a mean tumour cross-sectional area of 0.25 cm2, mice were randomised and treated with vehicle, lapatinib (LAP), MRK-003 GSI, or LAP plus GSI. Tumour area (length × width) was measured weekly using Vernier calipers. The measurements were performed up to 13 weeks. Results show mean tumour cross-sectional area ((area × Π)/4) on the y axis and time in weeks on the x axis. Error bars are s.d. of the mean for 10 mice bearing tumours. (B) Fixed tumours were paraffin embedded and sectioned for H/E staining (upper panel), Ki67 (middle panel), and TUNEL (lower panel) assays. All sections were photographed at × 40 using a light microscope. Four panels are shown: BT474 tumours treated with vehicle, lapatinib (LAP), MRK-003 GSI, and LAP + GSI. The photographs of a single tumour sample are representative of three tumours with similar results. (C) Quantification of Ki67- and TUNEL-positive cells of three tumours using 60 high-powered fields (HPFs) at × 40 magnification. The y axis represents the number of Ki67- or TUNEL-positive cells per 60 HPFs. The bar graphs are mean±s.d. *Statistical differences compared with vehicle control. **Statistical differences between LAP and LAP + GSI. ***Statistical difference between MRK-003 GSI and LAP + GSI. Statistical analysis was performed using a two-sided, nonpaired Student's t-test. (D) Bits of tumours (1 mg) that were snap-frozen in liquid nitrogen were homogenised and lysed in RIPA buffer containing protease and phosphatase inhibitors. Cellular debris was removed by centrifugation at 1000 g for 5 min at 4 °C. Supernatants were collected and 25 μg of total protein loaded onto a 7% SDS–PAGE gel followed by western blotting to detect tyrosine phosphorylated ErbB-2 or HER2 (PY1248-HER2), total HER2, P-ERK1/2, total ERK1/2, P-AKT1, total AKT1, and actin proteins. Densitometry was performed on PY-HER2 and total HER2 bands for each treatment groups using ImageJ program and expressed as a PY-HER2/HER2 ratio of area of the peak. Western blotting was performed on at least three independent tumour samples. Representative western blots are shown with similar results. *Statistically significant differences between mean slopes of the curve for LAP plus GSI and GSI alone. **Statistically significant differences between mean slopes of the curve for LAP and vehicle control. ***Statistically significant differences between mean slopes of the curve for LAP plus GSI and LAP alone. Linear regression analyses were performed for tumour growth curve.
Figure 6
Figure 6
GSI partially restores trastuzumab sensitivity in resistant tumours. (A and B) The exact same protocol as described in Figure 1 was used to generate trastuzumab-resistant tumours in athymic, nude mice using BT474 trastuzumab-resistant cells. (A) LY 411 575 GSI was used. (B) MRK-003 GSI was used. Tumour area (length × width) was measured weekly using Vernier calipers. The measurements were performed up to 15 or 10 weeks, respectively. Results show mean tumour cross-sectional area ((area × Π)/4) on the y axis and time in weeks on the x axis. Error bars are s.d. of the mean for 10 mice bearing tumours. *Statistically significant differences between GSI and Trast + GSI. **Statistically significant differences between Trast and Trast + GSI.
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