Ret inhibition decreases growth and metastatic potential of estrogen receptor positive breast cancer cells

Abstract

We show that elevated levels of Ret receptor are found in different sub-types of human breast cancers and that high Ret correlates with decreased metastasis-free survival. The role of Ret in ER+ breast cancer models was explored combining in vitro and in vivo approaches. Our analyses revealed that ligand-induced Ret activation: (i) stimulates migration of breast cancer cells; (ii) rescues cells from anti-proliferative effects of endocrine treatment and (iii) stimulates expression of cytokines in the presence of endocrine agents. Indeed, we uncovered a positive feed-forward loop between the inflammatory cytokine IL6 and Ret that links them at the expression and the functional level. In vivo inhibition of Ret in a metastatic breast cancer model inhibits tumour outgrowth and metastatic potential. Ret inhibition blocks the feed-forward loop by down-regulating Ret levels, as well as decreasing activity of Fak, an integrator of IL6-Ret signalling. Our results suggest that Ret kinase should be considered as a novel therapeutic target in subsets of breast cancer.

Keywords: Fak; IL6; Ret; endocrine-therapy; metastasis.

Figure 1. Ret analysis in breast cancer

1. A. Representative images of negative, moderate and strong Ret immunohistochemical staining in a tissue microarray of human breast cancer are shown.

2. B,C. Kaplan–Meier analyses of the metastasis–free survival and overall survival. Patients with a high Ret score (High Ret, n = 66) have a significantly shorter metastasis-free survival and overall survival rate compared to the low Ret score (Low Ret, n = 23). Hazard ratios (HR) plus corresponding 95% confidence intervals (95%-CI) and p values, as well as the number of patients at each time point (No. at risk) are depicted.

Figure 2. GDNF induces cell migration and proliferation of breast cancer cells

1. MCF7, MCF7/Aro and J110 tumour cells were stimulated with GDNF (10 ng/ml) for 15 min, lysates were prepared and pY and total Ret levels were analysed by western blots (WB) or by Ret immonoprecipitations (IP) followed by WB.

2. Chemotactic response of MCF7 and J110 cells to GDNF was measured in transwell assays. Lower wells contained 0.5% FBS alone (Control) or supplemented with GDNF (10 ng/ml). Migrated cells were fixed and stained. Representative pictures are shown (100×). The mean migrated cell number was determined by counting 4 fields of duplicate wells in 4 independent experiments. Error bars represent s.e.m. *p < 0.05 by t-test.

3. Serum-starved confluent T47D cultures were scratched and either left in control medium or stimulated with GDNF/GFRα1 (10 ng/ml/100 ng/ml). Migration into the wound was monitored for 24 h in 6 regions of the scratch. Recovered area was quantified using ImageJ. Results of 3 experiments are shown by the mean ± s.e.m. ***p < 0.001 by t-test.

4. Lysates from T47D parental cells, Ret KD (siRet1 and siRet2) and control cells (siLacZ) stimulated (+) or not (−) for 15 min with GDNF/GFRα1 were analysed by westerns with the indicated antibodies.

5. Ret KD and control T47D cells were assessed in the wound closure assay as described in C. ***p < 0.001 by t-test.

6. Steroid deprived MCF7/Aro cultures were treated 6 days with GDNF (10 ng/ml) or/and the estrogen precursor (Δ4A, 1 nM) in the absence or presence of letrozole (100 nM), fulvestrant (100 nM) or tamoxifen (100 nM). Proliferation was assessed by counting viable cells. Results are shown as means of triplicate values ± s.d. *p < 0.05, ** p < 0.01, *** p < 0.001 by ANOVA using Tukey's test.

Figure 3. Fulvestrant, IL6 and Ret interactions

1. IL6 levels (pg/ml) were measured by ELISA in 4-day conditioned medium (CM) of MCF7/Aro cultures treated as indicated. Results represent the mean ± s.d. of triplicate determinations from 3 independent experiments. *p < 0.05 by t-test.

2. Steroid-deprived MCF7/Aro or J110 cells were treated 6 or 3 days, with 10 nM Δ4A or E2, respectively, in the presence or absence of 100 nM fulvestrant. Lysates were analysed by WB with the indicated antibodies.

3. Steroid-deprived MCF7/Aro cells were treated for 6 days with EtOH (−) or 10 nM Δ4A ± 100 nM Fulvestrant, in the presence of IgG control or IL6-blocking (Anti-IL6) antibodies at 1 µg/ml. Lysates were analysed by WB with the indicated antibodies.

4. Serum-deprived MCF7 cells were treated 24 h with IL6 (100 ng/ml). Total RNA was extracted and qRT-PCR was performed with Ret and actin specific primers. Cell lysates were analysed by WB with the indicated antibodies. *p < 0.05 by t-test.

Figure 4. Analysis of IL6- and GDNF induced migration and signalling

1. Serum-deprived MCF7 cells were pre-incubated with DMSO or NVP-BBT594 (50 nM), then seeded into the upper chamber of a transwell. Lower wells contained 0.5% FBS alone (Control) or supplemented with GDNF (10 ng/ml), IL6 (100 ng/ml) or the combination. Migrated cells were fixed, stained and counted. Data shown are the mean of 5 independent experiments; error bars represent s.e.m. *p < 0.05 ***p < 0.001 ANOVA using t-test.

2. MCF7 cells were pre-incubated with DMSO or NVP-BBT594 (50 nM), then treated 15 min with IL6 (100 ng/ml) or GDNF (10 ng/ml). Ret IPs were probed with specific antibodies for pY then reprobed for Ret in a WB analysis.

3. Serum-deprived MCF7 cells were pre-incubated with DMSO or NVP-AST487 (100 nM) then seeded into the upper chamber of a transwell. Lower wells contained 0.5% FBS alone (Control) or supplemented with EGF (10 ng/ml). Migrated cells were quantified as in panel A. Data shown are the mean of three independent experiments; error bars represent s.e.m. *p < 0.05 by t-test.

4. Serum-deprived MCF7 KD (shRet1) or control (shLacZ1) cells were seeded into the upper chamber of a transwell. Lower wells contained 0.5% FBS alone (control) or supplemented with IL6 (100 ng/ml). Migrated cells were quantified as in panel A. Data shown are the mean for three independent experiments; error bars represent s.e.m. *p < 0.05 by t-test.

5. Steroid-deprived, serum-deprived MCF7 cells were seeded into the upper chamber of a transwell. Lower wells contained 0.5% FBS medium plus E2 (10 nM) + fulvestrant (100 nM) (E2 + Ful), GDNF (10 ng/ml) or IL6 (100 ng/ml). An IL6 blocking antibody or IgG control antibody were added at 1 µg/ml. After 24 hours, migrated cells were quantified as in panel A. Data shown are the mean of three independent experiments; error bars represent s.e.m. *p < 0.05, **p < 0.01 by t-test.

Figure 5. Ret knock-down (KD) or Ret inhibition reduces in vivo tumour growth and metastasis in breast cancer models

1. Independent T47D Ret KD cell lines (shRet1.5 and shRet8.1) or control cell lines (shLacZ1 and shLacZ4) were injected in E2-pellet-bearing BALB/c nude mice (n = 6–8). Growth was monitored for 48 days and tumour size was calculated. *p < 0.05 by t-test.

2. Groups of 100 mm³ J110-tumour bearing FVB/N mice were randomized and treated 12 days daily with vehicle (N-methylpyrrolidone/PEG300) or Ret inhibitor NVP-AST487 (50 mg/Kg/day); tumour weight at the end of the experiment was determined (n = 8–9). Bars represent mean ± s.e.m. *p < 0.02 by t-test.

3. Groups of J110-tumour bearing mice were randomized and treated with vehicle, Ret inhibitor NVP-AST487 (50 mg/kg/day) or fulvestrant (3 mg/week). After 10 days (combo arrow), the fulvestrant-treatment group was randomized to continue with fulvestrant or combination treatment (Fulvestrant + AST487); tumour volume was determined every 2 days. Points represent mean ± s.e.m. A representative experiment of 2 is shown. *p < 0.05 by t-test.

4. Upper panel, lysates from J110 tumours harvested 8 h after the last treatment with vehicle or inhibitor (AST487) (panel B), were analysed by WB with the indicated antibodies. Lower panel, tumours from vehicle- or fulvestrant-treated mice (panel C) were harvested and paraffin sections were stained for ERα⋅ Representative pictures of four tumours are shown (400×). Scale bars: 12 µm.

5. Quantification of metastatic foci in lungs from animals at experiment termination (panel C). Two independent experiments with n = 9–15 mice were analysed. Number of foci was normalized by tumour gram at the experiment-end and represented as metastatic index (media ± s.d of number of lung foci/tumour gram). *p < 0.05 by Mann–Whitney test.

6. Quantification of metastatic foci in lungs from animals at experiment termination (Supporting Information Fig S5C). Two independent experiments with n = 6–10 mice were analysed. Number of foci was expressed as indicated in E. *p < 0.05 by Mann–Whitney test.

Figure 6. Protein analyses on J110 tumours

1. Tumour lysates from 3 mice per treatment group (experiment shown in Fig 5) were analysed by WB using the indicated antibodies. On the right, quantification of additional western analyses with the indicated phospho-protein/protein was performed using imageJ in 7–20 independent tumours for each group, from 2–3 independent experiments. Data shown are the mean ± s.e.m. *p < 0.05 or **p < 0.01 by Mann–Whitney test.

2. Tumours from vehicle- or NVP-AST487-treated mice were harvested at the end of the experiment (Fig 5). Paraffin blocks were prepared and sections were stained for pY705Stat3 (pStat3, 400×). The proportion of the tumour area showing pStat3 immunoreactivity was quantified using ImageJ. For each group n = 10 tumours from two independent experiments were examined and five fields per tumour were analysed. **p < 0.01 by Mann–Whitney test. Representative pictures are shown. Scale bars: 12 µm.

3. J110 cells were stimulated or not with GDNF (10 ng/ml) and IPs performed with a Ret specific antibody followed by WB analyses using Ret and Fak antibodies. WB on whole cell extracts (WCE) with the indicated antibodies is also shown.

Figure 7. Analysis of pFak, pStat3 and migration in breast tumour cells

1. A–C. Cultures of MCF7 or J110 cells were pre-incubated with DMSO or the indicated inhibitors for: Fak NVP-TAE836 (400 nM) or PF573228 (1 µM); Ret NVP-AST487 (100 nM) and NVP-BBT594 (50 nM), or Jak1/2 INCB18424 (1 µM). Cell lysates from cultures treated 15 min with IL6 (100 ng/ml) or GDNF (10 ng/ml) were analysed by WB using the indicated antibodies.

2. D. Ret KD MCF7 cells (shRet1) and control cells (shLacZ1) were stimulated 15 min with IL6 (100 ng/ml). Cell lysates were analysed by WB using the indicated antibodies. On the right, quantification of the western analyses was performed using imageJ in three independent experiments. Data shown are the mean ± s.e.m. *p < 0.05 by t-test.

3. E. Serum-deprived MCF7 cells were pre-incubated with DMSO or the Jak1/2 inhibitor INCB18424 (1 µM) then cells were seeded into the upper chamber of transwells. Lower wells contained 0.5% FBS alone (Control) or supplemented with GDNF (10 ng/ml) or IL6 (100 ng/ml). Migrated cells were fixed, stained and counted. Data shown are the mean of three experiments; error bars represent s.e.m. *p < 0.05 by t-test.

4. F. Confluent cultures of T47D cells were pre-incubated with DMSO or the Fak inhibitor NVP-TAE836 (500 nM), then scratched and exposed to GDNF/GFRα1 (10 ng/ml/100 ng/ml) or left untreated. The recovered area of the wound was quantified using ImageJ over a 20 h time-course. The results from three experiments are shown by the mean ± s.e.m. **p < 0.01 by t-test.

Figure 8. Model of the IL6-Ret interaction

1. Ret+/ER+ tumours treated with endocrine therapy might simultaneously be exposed to factors such as IL6 that promote migration. When IL6 levels are high, e.g. in fulvestrant conditions (1), Ret expression increases (2) and Ret activation also stimulates IL6 production (3), setting up a positive feed-forward loop. In Ret-inhibitor treated tumours (4) the loop is broken since Ret levels decrease.

2. Total RNA was extracted from J110 tumours treated as indicated (n = 8–12, upper panel and n = 4–6, lower panel) from 2 independent experiments. qRT-PCR was performed using specific primers for IL6 and Ret mRNA. Expression levels were normalized to cytokeratin 18 (CK18). Columns represent means of the values ± s.e.m. *p < 0.05, **p < 0.01 by Mann–Whitney test.

3. Fak is an intracellular mediator of the IL6-Ret interaction; Fak activity is essential for both IL6 and Ret to stimulate migration. A direct interaction of Ret and gp130 was not observed. However, the Ret-complexed Fak might be primed to respond and a transient complex of the receptors and their associated kinases might form in response to IL6 (dotted line) and signal to Stat3. (•) pY.