Inhibition of RET Increases the Efficacy of Antiestrogen and Is a Novel Treatment Strategy for Luminal Breast Cancer

Breast cancer has an annual incidence of 226,000 and accounts for approximately 40,000 deaths in the United States, making it the second most common cause of cancer-related deaths in women (1). Approximately 75% of breast cancers belong to the luminal subtypes, characterized by expression of the estrogen receptor α (ERα; refs. 2 and 3). Systemic treatment strategies for these patients rely on hormone therapy; however, patients with luminal breast cancers that are hormone insensitive have limited treatment options. Patients with luminal breast cancer have a favorable prognosis measured by rates of recurrence and disease specific long-term survival relative to other breast cancer subtypes (4, 5). However, roughly one third of hormone receptor positive breast cancers have little response to antiestrogen treatment or develop hormone resistance after initial response (6–8). Recently the BOLERO2 trial demonstrated improved response in women with advanced hormone receptor positive breast cancer treated with the mTOR inhibitor everolimus combined with the aromatase inhibitor exemestane, with median progression-free survival improved by 6 months compared with exemestane alone (9). In addition, luminal breast cancers have relatively poor response to neoadjuvant chemotherapy measured by conversion to breast conserving operations, axillary clearance, and pathologic complete response, indicating an underlying lack of responsiveness to cytotoxic chemotherapies (10–12). Hormone resistant and locally advanced disease are 2 common clinical scenarios in which targeted molecular therapy could improve treatment options for patients with luminal breast cancer.

One marker of aggressive tumors within the luminal subtype is expression of the RET proto-oncogene (13). The RET gene encodes a receptor tyrosine kinase (RTK), constitutively activated mutants of which cause the multiple endocrine neoplasia type 2 syndromes and familial medullary thyroid carcinoma (14–16). Wild-type RET is expressed in breast cancer with a strong association with ERα expression (17–19); the RET gene is transcriptionally regulated by TFAP2C, which is a key transcriptional regulator of the luminal phenotype (20–23). The RET receptor is activated by glial cell line–derived neurotrophic factor (GDNF), which has been shown in breast cancer models to result in activation of signal transduction pathways, including extracellular signal–regulated kinase 1/2 (ERK1/2) and AKT, leading to increased proliferation and cell survival (13, 18, 24).

Significant interaction between RET and ERα pathways has been previously described, with increased response to estrogen stimulation observed in the presence of functional RET (13, 19). RET has additionally been associated with resistance to tamoxifen and aromatase inhibitors, and increased expression has been demonstrated in hormone-resistant cell lines and primary tumors (25, 26). Previously we reported that the combination therapy with antiestrogen and anti-RET in luminal breast cancer had a greater effect on cell growth than either therapy alone (24). In addition, we found that antagonism of RET with a tyrosine kinase inhibitor (TKI) primarily acted to reduce growth through induction of apoptosis, while anti-ERα acted primarily through a reduction in cell proliferation, forming the biologic basis for dual treatment. However, a recent preclinical study using combination therapy with Fulvestrant and the RET inhibitor AST487 failed to demonstrate improved response with combination therapy (27). However, metastatic disease in mice with J110 tumors treated with tamoxifen demonstrated some improved response with the addition of AST487, for which the authors suggested a mechanism involving interleukin (IL)-6 signaling. Based on these findings, we sought to further characterize the effects of anti-RET treatment using vandetanib in sensitizing luminal breast cancers to the antiestrogen effects of tamoxifen. Furthermore, this study was designed to provide additional preclinical data by assessing the effect of TKI treatment in fresh, primary breast cancer tumors in vitro and establishing a correlation between effects of TKI with RET expression.

The MCF-7 and BT-474 cell lines were obtained from the American Type Culture Collection. Cells were grown using Dulbecco's modified Eagle medium with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO₂. The cells were not tested and authenticated by the authors and were passed for less than 6 months since obtaining the cells.

Vandetanib was obtained from AstraZeneca Pharmaceuticals. For in vitro studies, vandetanib was dissolved in dimethyl sulfoxide to a final concentration of 10 μmol/L. Z-4-hydroxytamoxifen was obtained from Sigma-Aldrich and dissolved in ethanol to a final concentration of 10 μmol/L for in vitro studies. GDNF was purchased from Sigma-Aldrich and dissolved in PBS and dissolved to a final concentration of 2.5 ng/mL.

Vandetanib and tamoxifen for xenograft studies were solubilized in 1% Tween 80 in deionized water.

Cells were plated on a 48-well plate at 5,000 cells per well in technical triplicate. Cells were allowed to adhere overnight and the appropriate hormone and/or drug containing media added. Samples were allowed to grow for 24 to 48 hours, after which they were incubated with MTT (0.5 mg/mL) for 3 hours at 37°C and crystals solubilized in 10% SDS in 0.01 M HCL for 3 hours at 37°C and read on an Infinite 200 Pro plate reader (Tecan) at an absorbance wavelength of 570 nm. Samples were averaged over 3 biologic replicates.

Fifty female Nu/J mice 6 weeks old (Jackson Laboratory) were implanted with a 1.7-mg 30-day release estrogen pellet (Innovative Research), which were left in place in all treatment groups for the duration of the experiment. The following day 1.5 × 10⁶ exponentially dividing MCF-7 cells in 50% Matrigel (BD Biosciences) were injected subcutaneously into the right flank. Ten mice were randomized to receive treatment with vandetanib, 25 mg/kg by daily oral gavage, starting on the day of tumor injection. Ten mice were randomized to the control group with daily vehicle (1% Tween 80 in deionized water) daily gavage. The remaining 30 mice were randomized to receive tamoxifen (50 mg/kg), vandetanib (25 mg/kg), or both by daily gavage when tumors reached 0.5 cm in diameter. Four mice did not develop tumors within 2 weeks of injection and were not randomized to treatment or included in the analysis. In total, 9 mice were randomized to both the vandetanib and dual treatment groups and 8 mice were randomized to tamoxifen only treatment. Tumor volumes were recorded daily and calculated using the formula a × b², where “a” represents the tumor diameter in longest dimension and “b” is the tumor length orthogonal to “a.” Mice in the vandetanib from injection group were euthanized 3 weeks following tumor injection, and all other groups were euthanized 10 days after randomization. Tumors were harvested immediately following euthanasia and placed in aliquots of formalin for immunohistochemistry (IHC) and radioimmunoprecipitation assay buffer (RIPA) for Western blot analysis.

Formalin fixed, paraffin-embedded sections were evaluated by hematoxylin and eosin stain, and IHC was performed for Ki-67 (Dako), terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL; Millipore), CD 31 (BD Biosciences), and RET (Cell Signaling Technology) with appropriate positive and negative controls. Samples were interpreted and scored by a blinded attending pathologist (R.W. Askeland). RET expression was scored according to percentage of positive cells with 500 cells counted per slide. Ki-67 was scored according to percentage of positive cells with 500 nuclei counted per slide. TUNEL was quantified using positive cells per high-powered field (HPF) with 10 fields counted per sample. Microvascular density was quantified using antimouse CD31 with 10 HPF counted per slide and vessels only included if they exhibited a typical morphology with lumen as previously described (28).

Fresh tumor samples with corresponding normal breast tissue were collected from 30 patients with invasive breast cancer from the University of Iowa Breast Molecular Epidemiologic Resource (B-MER), an institutional review board approved tumor bank. Patients who received chemotherapy before resection were excluded. Tissue was obtained in the surgical pathology suite within 15 minutes of resection and was immediately aliquoted in RNAlater (Ambion) and frozen at −80°C or minced sharply and placed fresh into minimal media containing sunitinib (Sigma-Aldrich) at 500 nmol/L or control media without drug. Samples were incubated for 30 minutes at 37°C and total protein collected as described later.

Total protein was isolated from resected xenograft and primary tumors using RIPA buffer with Halt protease inhibitor cocktail (Thermo Scientific) and PhosStop phosphatase inhibitor (Roche). The antibodies used for Western blot analysis were: RET, ERK, p-ERK, AKT, and p-AKT (Cell Signaling Technologies), pRET, recognizing human phospho-RET Y905 (catalog no. AF3269; R and D Systems, Inc.), GREB-1 (Abcam), and glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnologies).

Following euthanasia, the right lower lobe of the mouse lung was resected and total RNA was isolated from the whole lung tissue using the RNeasy Mini Kit (Qiagen). Primary tumor RNA was extracted using the TRizol method. Total RNA was converted to cDNA using the Superscript III Kit (Invitrogen) using random hexamer primers. Quantitative PCR was performed according to standard TaqMan Fast protocol (Applied Biosystems). To quantify the tumor burden in the lung RT-PCR was performed using primers specific for human and mouse GAPDH (Applied Biosystems) as previously described (29). Primary tumor sample RNA was analyzed using TaqMan primer/probes for RET (Applied Biosystems) with 18S rRNA as endogenous control. RET expression of samples was normalized to MCF-7 expression. RT-PCR was performed in technical triplicate for each sample.

Statistical analysis was performed using the 2-sided Student T test for continuous variables. Frequency association of categorical variables was performed using the Fisher exact test for comparisons between 2 groups, and using ANOVA when more than 2 groups were compared. All statistical calculations were performed using R (a standard statistical program). Statistical significance was defined as P < 0.05.

Previously we have demonstrated that inhibiting RET signaling with gene knockdown, or pharmacologic inhibition with sunitinib or vandetanib resulted in decreased proliferation in luminal breast cancer, and that effects can be combined with antiestrogen treatment (24). To further evaluate the relationship of dual treatment, we investigated the effects of tamoxifen in the presence and absence of intact RET signaling, through gene knockdown or the more RET-specific TKI, vandetanib. Treatment of hormone sensitive MCF-7 cells with vandetanib resulted in a reduction in phosphylated RET without change in total RET, and reduced viability at 48 hours (P < 0.001; Fig. 1). Cell viability was significantly reduced with tamoxifen alone (mean reduction 31.6%, P < 0.001). In the presence of vandetanib, tamoxifen response was significantly augmented (mean reduction 67.6%, P < 0.001), demonstrating a greater than 2-fold increase in the effect of tamoxifen in the presence of vandetanib, paired t test P = 0.01 (Fig. 1A). Parallel experiments were performed using siRNA knockdown of RET. Knockdown of RET resulted in a significant reduction in viability (P < 0.001 compared with siNT). Treatment with tamoxifen with intact RET reduced viability 10.8% (P < 0.001 compared with no tamoxifen), whereas treatment with tamoxifen after knockdown of RET resulted in a 31.3% reduction in viability (paired t test P < 0.001). Finally, the addition of vandetanib resulted in a small, nonstatistically significant reduction in growth and potentiation of tamoxifen under conditions of RET knockdown (paired t test P = ns), demonstrating that effects of vandetanib to sensitize to tamoxifen treatment in hormone responsive MCF-7 are mediated by inhibition of RET signaling. Although tamoxifen can alter RET expression, RET expression was confirmed by Western blot analysis in all treatment groups (Supplementary Fig. S1).

Similar to MCF-7, vandetanib treatment of the hormone-resistant luminal breast cancer cell line BT-474 resulted in a reduction of phosphorylated RET without change in total RET expression (Fig. 1B). Treatment of BT-474 cells with tamoxifen alone failed to result in a significant reduction in viability (P = ns). However, in the presence of vandetanib, tamoxifen resulted in a significant reduction in viability (mean reduction 40.4%, P < 0.001; Fig. 1B). Hence, the addition of vandetanib resulted in an 8-fold augmentation in the cell viability effects of tamoxifen (−4.7% vs. −40.4%, paired t test P < 0.001). Parallel experiments demonstrated that knockdown of RET reduced cell viability compared with nontargeting siRNA (P = 0.03). Similar to vandetanib treatment, compared with NT transfection, knockdown of RET increased the effects of tamoxifen treatment (−5.4% vs. −23.7%, paired t test P < 0.001). Vandetanib treatment with or without tamoxifen under conditions with knockdown of RET failed to demonstrate any additional effects on cell viability compared with RET knockdown alone (paired t test P = ns). Hence, the data support a model where the ability of vandetanib to augment tamoxifen effects on cell viability are mediate through RET. These results demonstrate that inhibition of RET signaling can reduce proliferation in 2 ways—through direct effects and through increasing sensitivity to antiestrogen. Of particular clinical significance, anti-RET treatment increased antiestrogen effects in both hormone sensitive and hormone-resistant luminal breast cancer.

To test the observed effects of vandetanib in vivo, we studied tumorigenesis using a xenograft model. Athymic mice injected with MCF-7 cells had a decreased rate of tumor formation when treated with daily gavage of vandetanib from the time of tumor cell injection (Fig. 2A). Mice treated with vandetanib were not noted to have obvious weight loss compared with the control treated mice, and no drug-specific toxicity, including diarrhea or visible rash, were observed. To investigate the effects of vandetanib treatment on RET downstream pathways, protein from xenograft tumors was evaluated using phosphorylation-specific Western blot analysis for ERK1/2 and AKT. Tumors from animals treated with vandetanib had a reduction in activation of ERK1/2 and AKT, which demonstrates that vandetanib acted to abrogate RET signaling in the xenograft tumors (Fig. 2B). IHC demonstrated no significant change in mean Ki-67 proliferative index in the vandetanib treated tumors (Fig. 2C). Staining for TUNEL showed a significantly higher number of apoptotic cells per high powered field in the vandetanib treated group compared with the vehicle-treated group (1.28 vs. 0.69 cells/HPF, P < 0.001). Evaluation of microvascular density using CD31 staining demonstrated a significant reduction in vessel formation in tumors from the vandetanib-treated animals compared with the control (3.7 vs. 6.45 vessels/HPF, P = 0.008). These results demonstrate that vandetanib reduced tumor growth through decreased activation of RET downstream mediators and a subsequent increase in apoptosis with effects on angiogenesis also noted.

Because of the observed effects of growth reduction in primary MCF-7 xenograft tumor sites by systemic therapy with vandetanib, we investigated the effects of systemic treatment on disease burden in the mouse lung. RNA from the lungs of mice that were not injected with human tumor cells did not demonstrate amplification of the quantitative PCR probe for human GAPDH (Fig. 2D). Treatment with vandetanib in lung tissue evaluated at 22 days postinoculation resulted in 97% reduction in tumor cells in the lung compared with vehicle-treated animals, P < 0.01. These results show decreased tumor cells in the lung of treated mice, which could represent decreased tumor in the lung parenchyma, a reduction in micrometastases or a reduction in the number of circulating tumor cells.

To test the potential synergistic effects of anti-RET and antiestrogen treatment in vivo, we investigated the effects of treatment with vandetanib, tamoxifen, or dual treatment on established MCF-7 xenografts. Treatment with single agent of either vandetanib or tamoxifen resulted in a significant reduction in the growth of established xenografts, P < 0.001 (Fig. 3A). Combination therapy with both vandetanib and tamoxifen resulted in significantly reduced mean tumor growth compared with either agent alone (P = 0.02). All of the mice in the vehicle-treated group had progression (defined as a tumor volume on day 10 greater than tumor volume on randomization). By comparison, 87.5% of mice in each of the single drug treatment groups progressed; however, only 33% of animals in the dual treatment group had tumor progression, P = 0.003 by ANOVA (Fig. 3B).

Protein was harvested from xenograft tumors and effects of single and dual treatment on downstream markers of RET and ERα activation was assessed by Western blot analysis. Tumors from mice treated with vandetanib had reduced activation of ERK1/2 and AKT (Fig. 3C). Tumors from mice treated with tamoxifen had decreased expression of GREB-1, an estrogen response gene (30), which is necessary for estrogen-dependent growth (31). These results demonstrate the efficacy of systemic treatment to reduce RET and ERα downstream activation and highlight the distinct pathways of RET and ERα signaling, informing the rationale for use of dual receptor therapy.

Prior studies have shown that RET is an estrogen response gene, so we tested the effect of vandetanib, tamoxifen, or dual treatment on RET expression. IHC for RET expression in the xenografts demonstrated no significant change in RET expression in any of the treatment groups (Fig. 4). Similar to the tumorigenesis experiment, analysis of tumor tissue from mice with established tumors treated with vandetanib showed no significant differences in Ki-67 positivity on IHC compared with tumors from control animals. However, tumors from vandetanib-treated animals demonstrated a significant induction in TUNEL-positive apoptotic cells compared with control-treated animals (0.99 cells/HPF vs. 0.69 cells/HPF, P = 0.03; Fig. 4A). Systemic treatment with tamoxifen resulted in a similar reduction in growth as vandetanib; however, IHC demonstrated that tamoxifen treatment resulted in a significant reduction in proliferative index compared with vehicle-treated animals (47% vs. 61%, P = 0.02), without changes in apoptosis determined by TUNEL. Dual treatment with tamoxifen and vandetanib resulted in a larger reduction in tumor growth rate (Fig. 3A), decreased progression compared with single treatment (Fig. 3B) and both a reduction in Ki-67 (via antiestrogen) and an induction of apoptosis (via anti-RET), P < 0.05 (Fig. 4A). These results provide further evidence that ERα and RET drive cell growth through distinct pathways that can be simultaneously targeted by a dual treatment strategy. Treatment of mice with established xenografts with tamoxifen, vandetanib, or both similarly resulted in significant reduction of lung tumor burden, P < 0.05; however, there was no increased effect seen with dual treatment (Fig. 4B).

Previously it has been reported that RET is expressed in breast cancer in association with the ERα gene expression cluster (19, 32). To further characterize RET expression in breast cancers and normal breast tissue, we obtained samples from 20 ERα-positive tumors, 10 ERα-negative tumors with 30 patient matched normal breast tissue samples and analyzed RET expression by quantitative RT-PCR. Mean RET expression in ERα-positive tumors was 8 times higher than expression in ERα-negative tumors, and 20 times higher than mean expression in corresponding normal breast tissue (Fig. 5A; P < 0.001). Using a threshold of 0.5-fold expression relative to MCF-7, 15 of 20 (75%) of ERα-positive tumors were RET positive, 1 of 10 (10%) of ERα-negative tumors were RET positive, and 0 of 30 normal breast tissue samples were positive for RET, P = 0.001 (Fig. 5B). These results demonstrate that RET is more highly expressed in ERα-positive tumors, and that it is relatively poorly expressed in normal breast, thus making RET an ideal molecule for targeted therapy.

Previously we have reported that activation of ERK1/2 is a marker of RET pathway activation and that in vitro inhibition or gene knockdown of RET resulted in decreased phosphorylated ERK1/2 (24). To examine the response to TKI treatment across a spectrum of breast cancer, primary tumors were obtained fresh from resection specimens and treated in vitro with sunitinib or vehicle, in parallel. Tumor tissue was assessed for RET expression by RT-PCR, IHC, and Western blot analysis and samples were analyzed for phosphorylated ERK1/2 and AKT by Western blot analysis (Fig. 6A and B). Primary breast tumors demonstrated a significant mean reduction in phosphorylated ERK1/2 compared with control treated (29% reduction, P < 0.001), whereas normal breast tissue treated with sunitinib did not show decreased activation of ERK1/2 (Fig. 6C). The data were stratified by RET expression and compared for RET-negative and RET-positive tumors. Where they could be compared, expression of RET protein by Western blot analysis was confirmed in samples with RET RNA expression (Supplementary Fig. S2). The RET-negative tumors demonstrated no significant effect of ERK1/2 activation with TKI treatment. By contrast, the RET-positive tumors demonstrated a significant reduction in ERK1/2 activation with sunitinib, P < 0.001. Comparing tumor samples with differential RET expression determined by quantitative RT-PCR, there was a significantly greater effect in the reduction in mean ERK1/2 activation with sunitinib treatment in RET-positive compared with RET-negative tumors (42% vs. 14%, P = 0.01; Fig. 6D). Although sunitinib has activity against multiple RTKs, response measured by activation of ERK1/2 was highly correlated with RET expression. Samples were also measured for activated AKT by phosphorylation-specific Western blot analysis. Treatment with sunitinib did not significantly reduce activated AKT in all tumors (mean reduction 9%, P = 0.43) or significantly reduce activated AKT in RET positive tumors (10% reduction) relative to RET negative tumors (8% reduction, P = 0.62). These results could indicate that the observed effects are independent of the PI3K/AKT/mTOR pathway, or that our 30-minute treatment was insufficient to induce reduced activation of this pathway in primary tumors.

There are a subset of patients with advanced luminal breast cancer that have limited treatment options because of poor response to cytotoxic chemotherapy or the development of hormone resistance. Developing successful treatment options for these patients will rely on a better understanding of molecular determinants of tumor growth, progression, and hormone resistance. Investigation of these factors will allow identification of patients with more aggressive tumors predicted to have worse disease-related outcomes, as well as inform subsequent development of targeted therapies. Recent investigation has demonstrated that expression of the RET proto-oncogene in luminal breast cancer is involved in proliferation and survival of tumor cells, and overexpression and activation has been implicated in the development of hormone resistance (25, 26). Previously we have reported there are independent pathways regulated by RET and ERα, which can be targeted independently with dual receptor–targeted therapy to reduce in vitro growth in luminal breast cancer (24). Herein, the effect of dual treatment is further clarified in hormone sensitive and hormone-resistant luminal breast cancer lines and shown in a xenograft model to increase efficacy over single-agent treatment to prevent tumor growth and progression. In addition, analysis of primary tumors demonstrated that response to the TKI, sunitinib, is associated with RET expression and that RET is both a target and a marker for patient selection for this type of treatment.

Previously TKIs have been investigated as systemic therapy in patients with breast cancer. Clinical trials in breast cancer with TKIs have focused directed therapy based on anti-VEGF or anti-EGFR activity. One TKI with activity against RET, sunitinib, has been examined in a limited number of clinical trials in breast cancer focused on unresectable metastatic disease. Some response has been demonstrated in these highly selected patients. Most investigators have suggested that combination therapy be considered as monotherapy failed to demonstrate improved response compared with more conventional agents (33–37). Vandetanib has been examined in small series of patients with breast cancer with refractory metastatic disease. In one trial of 46 patients with previously treated breast cancer, doses of 100 or 300 mg were well tolerated but demonstrated limited activity as monotherapy (38). In a recent trial, 35 patients were treated with combination therapy using vandetanib in combination with docetaxel, but vandetanib was not shown to improve the response rate (39). However, the small number of patients limited the ability to determine whether or not there was a response to treatment attributable to vandetanib. In a phase I trial using vandetanib in combination with cyclophosphamide and methotrexate, vandetanib at a daily dose of 200 mg was well tolerated and objective response was reported, although determining the efficacy of vandetanib was not possible because of the study design (40). Several factors are important when considering these limited earlier studies. First, these studies did not consider RET as the molecular target and combining tumors with and without RET expression would likely mask effects without subgroup analysis. Second, the studies were limited to highly selected patients with aggressive chemoresistant cancer. RET expression is strongly linked to ERα-positive breast cancers, which is a group that would likely be underrepresented in this cohort. In addition, the prior studies with vandetanib have been uniformly underpowered and have not utilized RET expression as a data stratification factor, which could mask a quantifiable response to treatment in RET expressing tumors. Finally, utilization of RET-specific endpoints, such as ERK1/2 or AKT activation, or tumor apoptotic rate, which we have shown to be associated with xenograft response, could be more informative biomarkers of response to vandetanib treatment in luminal breast cancer.

Demonstration of systemic TKI therapy as an efficacious treatment in breast cancer relies on the identification and selection of patients most likely to have a response. RET expression levels have been shown in one study to be predictive of induction of apoptosis with sunitinib treatment in liver cancer xenografts (41). Previously we have shown that although the available molecules with anti-RET activity have activity against multiple receptors, in vitro activity of the TKIs sunitinib and vandetanib are significantly reduced in the absence of functional RET (24). In this manuscript, we further these investigations by showing that blocking RET potentiated the effect of antiestrogen therapy in hormone responsive and hormone insensitive ERα-positive breast cancer. Furthermore, we established RET as a molecular marker for response to TKI treatment by demonstrating that only RET-positive breast tumors reduced ERK1/2 activation in response to TKI treatment. Although further characterization is needed to confirm RET as a marker of cellular TKI response, these data indicate that RET expressing breast cancers could be most likely to respond to treatment and that results of future investigation should be stratified according to RET expression. In this study, we identify 53% of tumors and 75% of ERα-positive tumors as RET positive, which is within the published range of 25% to 100% (18, 19, 25, 27). In addition, we have identified that vandetanib reduced microvessel formation in luminal breast cancer xenografts. The mechanism leading to reduced microvasculature with vandetanib treatment, which could be mediated at least in part by VEGF family receptors, as well as the relative contribution of this effect on the observed reduction in tumor growth, require further study. Similar findings have previously been shown in models of glioblastoma and hepatocellular carcinoma (42, 43). This effect is believed to be mediated by VEGF family receptors and indicates that antitumor results seen in luminal breast cancer xenografts with vandetanib treatment could be because of antagonism of multiple receptors. Vandetanib has additionally been shown to have antitumor activity in non–small cell lung cancer (NSCLC), which is thought to be mediated by EGFR, which is expressed in luminal breast cancer and normal breast epithelium (44, 45). Interestingly, the effects of vandetanib in NSCLC were increased in dual treatment with antiestrogen, indicating some interaction between ERα and RTK (RET, EGFR, or others) pathways in multiple cancer types. More recently, the effects of dual EGFR and ERα antagonism were investigated in breast cancer, and the efficacy of EGFR inhibition with lapatinib when added to letrozole has been shown in ERα-positive, HER-2–negative metastatic breast cancer, with effects most pronounced in low ERα-expressing ERα-positive tumors (46). In addition, in lung cancer cell lines, a recent study showed benefit of combining anti-EGFR and antiestrogen (47). Although multiple studies have shown interaction between EGFR and ERα pathways (48, 49), further study is needed to elucidate the role of RET, EGFR, and VEGFRs in determining growth characteristics, interaction with hormone signaling pathways, and mediating the antitumor effects of vandetanib in luminal breast cancer.

Based on these findings vandetanib should be investigated in breast cancer using RET as a marker for selection and stratification. The majority of RET-expressing breast cancers belong to the luminal subtype, and selection of patients in this subtype allows for combination with antiestrogen, which could allow for enhancement of antiproliferative effects with dual treatment. Systemic TKI therapy in breast cancer with RET as the therapeutic target has not been previously investigated in combination with antiestrogen, which could demonstrate increased efficacy compared with single-agent therapy. Establishment of a new treatment paradigm in luminal breast cancer based on anti-RET and ant-estrogen could improve outcomes for a large number of patients with limited treatment options.

No potential conflicts of interest were disclosed.

Conception and design: P.M. Spanheimer, J.-M. Park, G.W. Woodfield, R.J. Weigel

Development of methodology: P.M. Spanheimer, J.-M. Park, G.W. Woodfield, R.J. Weigel

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P.M. Spanheimer, J.-M. Park, R.W. Askeland, G.W. Woodfield, J.P. De Andrade, A.R. Cyr, A. Thomas, R.J. Weigel

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P.M. Spanheimer, J.-M. Park, M.V. Kulak, J.P. De Andrade, A. Thomas, R.J. Weigel

Writing, review, and/or revision of the manuscript: P.M. Spanheimer, J.-M. Park, R.W. Askeland, J.P. De Andrade, A.R. Cyr, S.L. Sugg, A. Thomas, R.J. Weigel

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P.M. Spanheimer, J.-M. Park, M.V. Kulak, R.J. Weigel

Study supervision: R.J. Weigel

The authors thank AstraZeneca, LC, for providing vandetanib for this work.

This work was supported by the NIH grants R01CA109294 (PI: R.J. Weigel), T32CA148062 (PI: R. J. Weigel), and by a generous gift from the Kristen Olewine Milke Breast Cancer Research Fund. P.M. Spanheimer and J.P. De Andrade were supported by the NIH grant T32CA148062.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.