Inhibition of RET increases the efficacy of antiestrogen and is a novel treatment strategy for luminal breast cancer

Abstract

Purpose: Recent findings suggest that combination treatment with antiestrogen and anti-RET may offer a novel treatment strategy in a subset of patients with breast cancer. We investigated the role of RET in potentiating the effects of antiestrogen response and examined whether RET expression predicted the ability for tyrosine kinase inhibitor (TKI) to affect extracellular signal-regulated kinase 1/2 (ERK1/2) activation in primary breast cancer.

Experimental design: Growth response, ERK1/2 activation, Ki-67, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were assessed in breast cancer cell lines in vitro and in xenografts with vandetanib and/or tamoxifen. Thirty tumors with matched normal breast tissue were evaluated for RET expression and response to TKI treatment.

Results: Vandetanib potentiated the inhibitory effect of tamoxifen in hormone responsive (P = 0.01) and hormone insensitive (P < 0.001) estrogen receptor α (ERα)-positive breast cancer cells. Vandetanib significantly repressed tumorigenesis of MCF-7 xenografts (P < 0.001), which displayed decreased activation of ERK1/2 and AKT. Vandetanib and tamoxifen reduced the growth of established tumors with a greater effect of dual therapy compared with single agent (P = 0.003), with tamoxifen-reducing proliferative index and vandetanib-inducing apoptosis. In primary breast cancers, RET expression correlated with the ERα-positive subtype. Relative decrease in ERK1/2 phosphorylation with TKI treatment was 42% (P < 0.001) in RET-positive tumors versus 14% (P = ns) in RET-negative tumors.

Conclusions: Vandetanib potentiated the antigrowth effects of tamoxifen in breast cancer, which was mediated through RET activation. RET predicted response to TKI therapy with minimal effects on ERK1/2 activation in RET-negative tumors. The preclinical data support evaluation of antiestrogen in combination with TKI as a potential treatment strategy for RET-positive luminal breast cancer.

Conflict of interest statement

All authors have no conflict of interest with this manuscript.

©2014 AACR.

Figures

FIGURE 1. Anti-RET Reduces Luminal Breast Cancer Viability and Sensitizes to Anti-ERα

A. Treatment of MCF-7 resulted in reduced phosphorylated RET with no change in total RET. Treatment of hormone responsive MCF-7 with vandetanib or tamoxifen reduces cell viability after 48 hours (p<0.001). Treatment with vandetanib sensitizes hormone sensitive MCF-7 to tamoxifen treatment such that treatment with tamoxifen results in a larger reduction in viability in the presence of vandetanib (−67.6%) then with control treatment (−31.6%), p=0.01 using the paired t-test. Knockdown of RET causes a reduction in viability compared to non-targeting (NT) siRNA (p<0.001) and results in an increase in tamoxifen sensitivity (−10.8% to −31.3%, paired t-test p=0.01) which is not increased with both RET knockdown and vandetanib treatment (−37.3%, paired t-test p=ns), confirming that the effects are mediated by RET. B. Treatment of hormone resistant luminal breast cancer cells, BT-474 with vandetanib reduces phosphorylated RET with no effect on total RET expression. Vandetanib reduces cell viability (p<0.001) with no significant effect with tamoxifen alone (p=ns). Similar to MCF-7, treatment with vandetanib increases sensitivity to tamoxifen, −40.4% vs −4.7%, p<0.001 with paired t-test. Knockdown of RET reduces viability compared to NT siRNA (p=0.03) and sensitizes hormone resistant BT-474 to tamoxifen treatment (−23.7% compared to −5.4%, paired t-test p<0.001). Treatment with vandetanib under conditions of RET knockdown did not result in a significant reduction in viability or change in tamoxifen sensitivity (−23.7% vs −27.6%, paired t-test p=ns). All westerns were performed in triplicate with representative blots shown. All MTT experiments were performed in technical and biologic triplicate with mean and standard deviation reported. P values were calculated using unpaired (straight lines) and paired (brackets) student’s t-test where appropriate.

FIGURE 2. Vandetanib Inhibits MCF-7 Xenograft formation

A. Ten mice were treated with vandetanib, 25 mg/kg daily by oral gavage from the time of tumor injection, and had reduced tumor formation and growth compared to ten vehicle gavaged animals. Mean tumor volume is shown with error bars representing the standard deviation. B. Protein from xenograft tumors demonstrates decreased activation of the RET pathway downstream targets ERK1/2 and AKT in vandetanib treated animals compared to control treated. Three tumors from each group were analyzed by western blot and representative blots are shown. C. Immunohistochemistry (IHC) was performed on all 20 tumors with representative slides shown and quantified with graphs representing the mean and standard deviation of the spread of data for the treatment and vehicle groups. IHC of tumors demonstrates no change in proliferative index (Ki-67) with vandetanib treatment. Vandetanib caused a significant induction of apoptosis measured by TUNEL positive cells, and resulted in a reduction in microvessel density measured by CD31. Stars indicate p<0.05. D. Treatment with vandetanib reduces detectable tumor cells in the lung. The p values were calculated using the Student’s t-test for continuous variable data.

FIGURE 3. Dual Treatment with Vandetanib and Tamoxifen Reduces Xenograft Tumor Growth Greater Than Either Agent Alone

A. Daily gavage with either vandetanib (9 mice) or tamoxifen (8 mice) resulted in a significant reduction in tumor growth in established xenografts compared to vehicle gavage (10 mice). Animals treated with a combination of both vandetanib and tamoxifen (9 mice) had significantly reduced tumor growth compared to either agent alone (p=0.02). Mean relative tumor volume (normalized to the volume at the time of randomization to treatment) is reported with error bars representing the standard deviation. The p values were calculated using the Student’s t-test to compare treatment groups. B. Mice in the dual treatment group were significantly less likely to have progression of the primary tumor site than single agent or control treated animals, p=0.003. The p value was calculated using analysis of variation (ANOVA). C. Protein from xenograft tumors demonstrates that treatment with vandetanib reduces phosphorylation of ERK1/2 and AKT which are downstream signalers in the RET pathway. Treatment with tamoxifen reduced expression of the estrogen response gene GREB-1. Dual treatment (Van + Tam) resulted in decreased activation of RET and ERα pathways. Three tumors were analyzed from each treatment group and representative western blots are shown.

FIGURE 4. Vandetanib Induces Apoptosis, Tamoxifen Reduces Proliferative Index and Both Effects in Dual Treated Tumors

A. Immunohistochemistry (IHC) demonstrates that RET expression is not changed by vandetanib, tamoxifen or dual (Van + Tam) treatment. Vandetanib treatment induces TUNEL, tamoxifen treatment reduces Ki-67 positivity, and both effects are seen with dual treatment. IHC was performed on slides from all xenograft tumors with representative slides shown and graphs represent the mean and standard deviation for each group. B. Animals treated with vandetanib, tamoxifen, or dual treated had reduced tumor burden in the lung. The p values were calculated using the Student’s t-test for continuous variables.

FIGURE 5. RET Expression is Associated with ERα Positive Breast Cancers

A. RNA extracted from primary human breast cancers and patient matched normal breast tissue demonstrates significantly higher mean RET expression in ERα-positive tumors compared to ERα-negative tumors and normal breast tissue. The data represent values from 20 ERα-positive, 10 ERα-negative tumors, and the 30 samples of matched normal breast tissue, which are overlaid for some data points. The p values were calculated using the Student’s t-test. B. When a cutoff of 0.5 fold RET expression relative to MCF-7 is used, significantly more ERα-positive tumors are RET positive compared to ERα-negative and normal breast tissue, p=0.001. The p value was calculated using the analysis of variation (ANOVA).

Figure 6. TKI Treatment Reduces ERK1/2 Activation in Primary Breast Tumors and Response is Associated with RET Expression

A. Immunohistochemistry of resected tumors demonstrating a representative RET-positive and RET-negative sample. B. Representative western blots showing a greater reduction in ERK1/2 activation and no effect on AKT activation with sunitinib treatment in a RET-positive tumor compared to a RET-negative tumor. Western blots for ERK1/2, p-ERK1/2, AKT, and p-AKT were performed on all 30 tumors with representative blots shown. C. Fresh primary human breast cancer tissue treated in vitro with sunitinib had a significant reduction in mean ERK1/2 activation relative to control treated tumor tissue. Patient matched normal breast tissue did not have reduced ERK1/2 activation with sunitinib treatment. ERK1/2 activation was quantified for all 30 tumors and matched normal breast tissue. D. The reduction in ERK1/2 activation in response to sunitinib treatment was significantly greater in RET expressing tumors. The p values calculated in C and D was performed using the Student’s t-test.