The receptor tyrosine kinase Ret, a key gain-of-function mutated oncoprotein in thyroid carcinomas, has recently been implicated in other cancer types. While Ret copy number gains and mutations have been reported at low frequencies in breast tumors, we and others have reported that Ret is overexpressed in about 40% of human tumors and this correlates with poor patient prognosis. Ret activation regulates numerous intracellular pathways related to proliferation and inflammation, but it is not known whether abnormal Ret expression is sufficient to induce mammary carcinomas. Using a novel doxycycline-inducible transgenic mouse model with the MMTV promoter controlling Ret expression, we show that overexpression of wild-type Ret in the mammary epithelium produces mammary tumors, displaying a morphology that recapitulates characteristics of human luminal breast tumors. Ret-evoked tumors are estrogen receptor positive and negative for progesterone receptor. Moreover, tumors rapidly regress after doxycycline withdrawal, indicating that Ret is the driving oncoprotein. Using next-generation sequencing, we examined the levels of transcripts in these tumors, confirming a luminal signature. Ret-evoked tumors have been passaged in mice and used to test novel therapeutic approaches. Importantly, we have determined that tumors are resistant to endocrine therapy, but respond successfully to treatment with a Ret kinase inhibitor. Our data provide the first compelling evidence for an oncogenic role of non-mutated Ret in the mammary gland and are an incentive for clinical development of Ret as a cancer biomarker and therapeutic target.
In breast cancer, the oncogenic potential of several receptor tyrosine kinases (RTK), of e.g., the ErbB and FGFR families, has been studied for many years . The Rearranged during transfection (Ret) RTK is a recent player in this disease. The first oncogenic Ret mutations were rearrangements identified in thyroid cancer . Although Ret fusion proteins [3, 4] and point mutations  appear to be infrequent in breast cancer, we and others have reported that Ret RNA and protein levels are high in around 40% of primary tumors [6,7,8] and we have shown that high Ret correlates with decreased overall survival . Elevated Ret has been found in different breast cancer subtypes, including estrogen receptor positive (ER+) and Her2/ErbB2 overexpressing tumors . A higher percentage of Ret-positive tumors was found in breast tumors of patients who failed endocrine therapy , suggesting a role for Ret in resistance. Using an ER+ orthotropic Ret-dependent model, we found that Ret inhibition reduced tumor outgrowth and lung metastatic potential . These studies provided the incentive to further explore the role of Ret in breast cancer and test its potential as a therapeutic target .
Here we present our studies on Ret using a bitransgenic mouse strain with a mammary gland-specific promoter controlling Ret expression in a doxycycline-inducible system [11, 12]. We show that chronic expression of wild-type (WT) Ret leads to the development of ER+ mammary tumors, with features of the human luminal subtype.
Results and discussion
To test whether WT Ret is tumorigenic, we created a novel transgenic mouse (Fig. 1a, top) using the doxycycline regulatory system . Tissue-specific transgene expression was determined by measuring luciferase activity and only animals on doxycycline were positive (Fig. 1a, middle). Western blot (WB) analysis for Ret was performed on mammary gland lysates from Ret/MTB, MTB/− and Ret/− adult female mice following 1 week of doxycycline treatment. Ret was induced in Ret/MTB, but not in uninduced Ret/MTB-transgenics, or doxycycline treated Ret/− or MTB/− controls (Fig. 1a, bottom). No abnormalities were found in 1 week-induced Ret/MTB mammary glands (Supplementary Figure S1a).
Next, we analyzed mammary glands from Ret/MTB adult females chronically treated 8 weeks with doxycycline. Morphological examination of whole mounts (WM) revealed hyperplastic changes (Fig. 1b, top) and WB confirmed Ret expression and activation (Fig. 1b, lower). Ret-positive glands were larger than controls, with excess side branching and secondary branch extensions, resulting in an increase in areas covered by ducts and surrounding fat (Fig. 1b, top right). Control mammary glands from doxycycline-treated MTB/− and Ret/− mice, and Ret/MTB animals maintained in its absence for > 1 year, were normal (not shown). IHC with anti-phospho (p)-S10-histone 3 (pS10H3) revealed that ducts of induced glands have an increase in pS10H3-positive cells, compared with controls (Fig. 1b, middle), indicating higher Ret-driven proliferation. Prior to hyperplasia, no significant changes were seen (Supplementary Figure S1b). An analysis of markers for specific cell compartments showed that Ret-induced hyperplasia is caused by the expansion of luminal ER+ and CK8/18+ cells, and not basal CK14+ cells (Fig. 1c).
Chronic expression of Ret in mammary epithelium induces solid tumors with characteristics of human luminal breast cancer
Chronic Ret induction resulted in tumor development in Ret/MTB females. Tumors expressed the transgene, shown by bioluminescence and Ret protein; phosphorylated Ret and Erk, both indicators of activity were also detected (Fig. 1d). Since the Ret ligands neurturin (Nrtn) and artemin (Artn), and the Ret co-receptor GFRα1 are expressed in transgenic glands and tumors, these likely contribute to Ret activation (Supplementary Table S1–2 and Supplementary Figure S1c).
Transgene levels were quantified by qRT-PCR for human Ret. Ret mRNA was present in doxycycline-induced hyperplasia and tumors, but not in uninduced tissue (Fig. 1e). Ret/MTB-tumors express higher levels of Ret compared to hyperplastic glands, suggesting that not all cells in hyperplasia express the transgene. To examine this, we performed Ret IHC. Ret/MTB tumors show homogeneously high Ret levels, while Ret expression in the surrounding hyperplastic tissue is heterogeneous (Supplementary Figure S1d), as was previously shown using this system . These results suggest that chronic Ret expression is sufficient to form tumors.
Focal tumors arose stochastically in Ret/MTB females, averaging 1-2 per mouse. In virgin females, tumor incidence was 37.0% (n = 27) following 6.5 ± 1.6 months of induction. Glands of chronically doxycycline-treated MTB/−, Ret/− (n = 18), and untreated Ret/MTB-mice (n = 6), developed no tumors for > 1 year (Fig. 2a). We also compared tumor onset in nulliparous (V) and multiparous (M) Ret/MTB mice. In parous mice, tumors occurred with similar latency (6.7 ± 3.2 months), but with a higher incidence reaching 76.8% (n = 13), compared to virgins (Fig. 2a). Since the MMTV promotor contains binding sites for steroid hormone receptors that are activated during pregnancy, we consider it likely that increased transcriptional activity of MMTV-rTTA leads to increased Ret expression, which contributes to higher tumor incidence in multiparous females. In addition, the hormonal environment could impact on mammary epithelial cell fate and ensuing tumor development in the transgenics. In human breast tumors, higher levels of Ret are seen compared to normal human tissue .. The mechanism underlying this observation is not fully understood. Interestingly, it has been reported that the transcription factor TFAP2C drives Ret expression and controls ER expression as well as other luminal signature target genes . Estrogen stimulation increases Ret expression in breast tumor cell lines [6, 15]; however, cells persistently treated with anti-estrogenic drugs also show increased Ret levels . Thus, it is tempting to speculate that higher Ret levels in tumors might be related to a balance between the action of specific transcription factor and the lifetime hormonal milieu.
Histological analysis of Ret-induced mammary tumors revealed solid nodular adenocarcinomas (similar to Neu-initiated tumors ), with high vascularity and areas of hemorrhage. H&E-staining showed that tumors had similar morphologies, and recapitulated features of human luminal breast cancer (Fig. 2b, left) . IHC analyses using anti-Ret, -ErbB2, -CK8/18, -CK14 and -αSMA antibodies showed that all tumors express Ret and ErbB2, and are of luminal epithelial origin (CK8/18+; CK14/SMA-). ErbB2 levels were similar to those in normal ducts and lower than MMTV-Neu tumor levels (ErbB2-Control), indicating no ErbB2 overexpression. Ret-induced tumors express low ER levels at the periphery of the tumor, and have no detectable progesterone receptor (PR) (Fig. 2b, right). Thus, we suggest that the Ret model resembles the specific sub-group of ER+/PR-/ErbB2- tumors. Importantly, while expression of the Ret/PTC fusion  generated mammary adenocarcinomas , our work is the first to demonstrate that WT Ret plays a causal role in mammary cancer.
We used next-generation RNA sequencing (NGS) to analyze the transcriptomes of 6 independent ER+Ret-induced tumors (T1, T11, T12, T15, T16 and T18) (Fig. 2c and Supplementary Figure S2), and control mammary glands from 3 uninduced Ret/MTB- and 3 chronically induced Ret/− females. A global gene expression analysis comparing Ret/MTB-tumors with breast cancer subgroups  showed that 4/6 tumors clustered with the luminal A subtype (Fig. 2c and Supplementary Table S3). We also performed correlations between signatures in Gene Set Enrichment Analysis (GSEA) using gene sets up- or downregulated by Ret overexpression. Proliferative genes were upregulated in tumors (Fig. 2d, top), and we identified enrichment of Stat1-driven genes (Fig. 2d, bottom and Supplementary Table S4); nuclear Stat1 and Stat3 phosphorylated forms were present in some tumor areas; active Stat5 was not detected (Fig. 2e). The transcription factors Stat1 and Stat3 appear to play opposite roles in tumorigenesis. While Stat3, described as oncogenic, promotes cell survival/proliferation, motility and immune tolerance , Stat1 is considered to be a tumor suppressor , which triggers anti-proliferative and pro-apoptotic responses while enhancing anti-tumor immunity . Despite being activated downstream of common cytokine and growth factor receptors, perturbations in their balanced expression or phosphorylation levels may re-direct signals from proliferative to apoptotic, or from inflammatory to anti-inflammatory.
The intra-tumor heterogeneity in Stat1/3 activation suggests that local factors act as Stat activators. In the future, identification of these factors will be important to determine.
By examining TCGA data, we found that 81% (1556/1904) of human Ret+breast tumors express at least one of the therapeutic receptors (ER, PR, or ErbB2) (Supplementary Figure S3a), which is in line with our previous TMA analysis . In patients with ER+tumors, PR negativity is an independent predictor of recurrence and shorter survival, hence of poorer response to hormonal therapy . Thus, our model might be useful for studying therapeutic strategies targeting Ret.
Mammary carcinomas require Ret for maintenance
To examine whether established tumors require continued Ret expression for maintenance, we withdrew doxycycline from 8 tumor-bearing mice, each of which harbored one 150-400 mm3 tumor. All tumors rapidly regressed (Fig. 3a) and were undetectable after 72 h; bioluminescence and Ret-staining confirmed Ret suppression after 48 h (Fig. 3b). To gain mechanistic insight into Ret-driven tumorigenesis, we examined signaling pathway activity after 48 h of doxycycline withdrawal. In regressing tumors, IHC showed lower abundance of pT202/Y204Erk1/2 and pS240/244S6, an mTor effector that is a key component of Ret signaling . Slightly lower levels of pY705Stat3 were also observed. Furthermore, we detected a significant reduction in pY701Stat1-positive nuclei and fewer pS10H3 positive proliferating cells in regressed tumors, compared to controls (Fig. 3b). Conversely, there was an increase in cleaved caspase 3 (CC3)-positive tumor cells after doxycycline removal (Fig. 3b). Thus, reduced proliferation and increased cell death, combined with signaling pathway downregulation, appear to be responsible for the initial phases of tumor regression following Ret downregulation.
We performed NGS on RNA from 48 h-regressing tumors and compared differential gene expression with tumors growing on doxycycline. Supporting the previous results, Ret downregulation was accompanied by downregulation of Stat1- and mTor-regulated genes and a decrease in DNA replication signature (Fig. 3c and Supplementary Figure S3b).
To analyze the impact of Ret inhibition on tumor growth, in a reproducible manner with larger cohorts, we engrafted pieces of Ret‐induced tumors onto flanks of Balb/c nude females. The grafts show less vascularity than primary tumors, importantly, however, they retain all other characteristics of the primary (Supplementary Figure S4). When tumors reached approximately 100–400 mm3, mice were subjected to different treatments. First, we determined that doxycycline-withdrawal produced tumor regression in 48 h (Fig. 3d), verifying their Ret-dependency. Second, we tested the in vivo response to an ErbB2 inhibitor and as anticipated from the low ErbB2 levels, no effects were found, confirming ErbB2 independence (Supplementary Figure S5a). Third, growth of tumor grafts was insensitive to hormone deprivation, mimicked by ovariectomy (Supplementary Figure S5b), and intact tumor-bearing mice did not respond to fulvestrant (Supplementary Figure S5c), confirming their estrogen-independence. Taken together, these data show that Ret/MTB tumors are functionally similar to human ER+/PR-/ErbB2- breast cancers.
Systemic inhibition of Ret signaling causes tumor reduction
We tested the role of Ret in signaling and tumor growth by treating mice bearing Ret-induced tumor transplants with NVP-AST487, a tool compound targeting Ret and additional kinases that we and others have successfully used [7, 22]. Of note, several commercially available multi-kinase inhibitors, such as vandetanib or cabozantinib, also have activity against the Ret kinase, however, selective Ret inhibitors have not yet been developed for clinical use. Several phase II clinical trials have been initiated to investigate the therapeutic effects of these multi-kinase inhibitors in patients with advanced Ret fusion-positive NSCLC . We show here that treatment with NVP-AST487 significantly reduced tumor growth (Fig. 4a) and WB analysis showed that tumor grafts from these mice had decreased levels of phosphorylated-Ret, accompanied by modest lower abundance of active-Erk and -mTor (Fig. 4b), as well as a reduction in phospho-S6, –Stat3 and -Stat1 (Fig. 4c).
We show here that WT Ret expression in the mammary epithelium potently evokes mammary tumors and, we end by summarizing and discussing the important conclusions. Our finding that Ret causes ER+tumors suggests that Ret activity expands ER+mammary cells, which is consistent with Ret expression in human ER+tumors [7,8,9]. Moreover, in our model, we show that these Ret/MTB-evoked tumors are PR- and do not respond to endocrine treatment. This is consistent with the observation that Ret expression in human tumors is associated with tamoxifen resistance [9, 24], which has been proposed to be due to Ret-driven mTor pathway activity . Interestingly, we show that proliferation of Ret/MTB-evoked tumors is blocked by Ret-targeted therapy, which is accompanied by a modulation in mTor signaling, suggesting that our model could be useful for studying hormone resistance. In the clinic, treatment with mTor inhibitors reverts many but not all cases of established resistance  and new biomarkers to guide therapeutic decisions are being sought. Based on our results, Ret expression in ER+/PR-/ErbB2- breast tumors might be a relevant biomarker predicting hormone-resistance. In the future blocking Ret alone or in combination with an mTor inhibitor might be an alternative therapy.
We thank Dr. Giorgio Caravatti (Novartis Institutes for Biomedical Research, Basel, Switzerland) and all members of Hynes and Gattelli labs for helpful discussions.
AG was supported by grant KG101234 from Susan G. Komen for the Cure® and grant GF N° 02 from Fundación para el Progreso de la Medicina Córdoba. NEH was supported by grants from Susan G. Komen for the Cure® SAC110041, from Krebsliga beider Basel 10-2030 and by the Novartis Research Foundation.
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Nature Communications (2018)