The PDGF pathway in breast cancer is linked to tumour aggressiveness, triple-negative subtype and early recurrence

The platelet-derived growth factor receptor (PDGFR) pathway is a signalling network of importance for the normal development of cells of mesenchymal origin. The PDGF signalling network consists of two tyrosine kinase receptors, PDGFR alpha (PDGFRα) and PDGFR beta (PDGFRβ), and five ligands PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and PDGF-DD, which are formed from four gene products (PDGF A, B, C and D) [1]. Autocrine stimulation of the pathway is frequently observed in various neoplasms, such as gliomas [2], gastro-intestinal stromal tumours (GISTs) [3] and chronic myelomonocytic leukaemia [4]. In addition, dysregulation of paracrine PDGFR signalling can cause extracellular matrix remodelling in a tumour-promoting way to facilitate migration, invasion, angiogenesis and possibly also lymph angiogenesis [5, 6].

In breast cancer, most studies of the PDGF signalling pathway examine the expression of PDGFRα and PDGFRβ. PDGFRα expression has been described both in stroma and in tumour cells. High tumour cell PDGFRα expression has been linked to lymph node metastasis, human epidermal growth factor receptor 2 (HER2) positivity [7], high histologic grade, oestrogen receptor (ER) negativity, progesterone receptor (PR) negativity and the triple-negative breast cancer subtype (TNBC) [8, 9]. High stromal PDGFRα has been linked to HER2 positivity and high Ki67 [9]. PDGFRβ expression in breast cancer has only been reported in stroma and high expression has been associated to HER2 positivity, high Ki67 [9], high histopathologic grade, ER negativity and shorter survival [10].

The PDGF-CC ligand was discovered towards the end of the 1990s and it has been shown to be involved in tumour growth by paracrine signalling through PDGFRα in malignant melanoma and cervical carcinoma [11, 12]. However, to date little is known about the role of PDGF-CC in breast cancer. We have recently shown that tumour cell-derived PDGF-CC acts on neighbouring tumour stromal cells in mouse models, and we proposed that the PDGF signalling pathway is a regulator of breast tumour subtype with high PDGF-CC driving breast tumours towards a more basal-like phenotype [13]. Basal-like breast cancer (BLBC) is a subtype of breast cancer defined by gene expression profiling and it is characterised by the expression of genes related to basal epithelial cells such as keratin 5, keratin 17, integrin-β4 and laminin [14]. BLBC largely overlaps with the molecular surrogate breast cancer subtype TNBC, which is defined by immunohistochemistry as being negative for the ER, PR and HER2 receptors [15]. Patients with TNBC are generally younger, have larger tumours with high grade at diagnosis and are more frequently BRCA-mutation carriers [16]. They also have a poor prognosis and no targeted therapies are hitherto available. It is thus particularly desirable to find targetable tumour-driving pathways within TNBC [17]. The PDGF signalling network holds promise of such a pathway, in TNBC in particular and in breast cancer in general.

The aim of the present study was to explore the expression of PDGFRα, PDGFRβ and ligand PDGF-CC in breast cancer to elucidate if these proteins are associated with molecular surrogate subtypes, type of metastatic location and prognosis in breast cancer. A secondary aim was to explore the relation to tumour progression by investigating changes in protein expression between primary tumour, synchronous lymph node metastases and asynchronous recurrences.

We present novel data on expression of PDGFRα, PDGFRβ and ligand PDGF-CC in primary breast tumours, synchronous lymph node metastasis and asynchronous recurrences in relation to St Gallen Molecular subtypes and long-term follow-up data in a large cohort of primary breast cancer patients. Data are accumulating on the importance of the PDGF receptors α and β in breast cancer, but the presented results have been conflicting and not related to modern pathology. Furthermore, little is known concerning the role of ligand PDGF-CC. We found that high expression of the investigated members of the PDGF family correlated to several prognostic patient and tumour characteristics that indicate tumours’ inherent biological aggressiveness (e.g. younger age, increasing NHG, high Ki67 and negative ER and PR). This is in line with what has been reported in some previous studies [9, 10] but it contrasts with Weigel et al. who found no significant correlations between PDGFR and the expression of established prognostic biomarkers. Reasons for diverging data could be the use of different antibodies, different inclusion criteria and the location of marker expression.

Interestingly, we report for the first time a significant up-regulation of tumour cell PDGFRα expression in lymph node metastasis and asynchronous recurrences, as a sign of its role in tumour progression. Moreover, we found a significantly increased risk of early breast cancer recurrence amongst patients with tumours expressing increasing levels of PDGF-CC. This effect was visualised by the Kaplan–Meier survival curves which indicate that the prognosis is related to the level of PDGF-CC expression (Fig. 4c). Up-regulation of members of the PDGF signalling pathway has previously been shown to occur during epithelial-to-mesenchymal transition in in vitro/in vivo cellular models [22], and subsequent experiments in mouse tumour models present support that an autocrine PDGF/PDGFR loop contribute to tumour progression and metastasis in vivo [23]. These studies conclude that PDGFR signalling plays an essential role during cancer progression, as has also been observed in our findings.

Approximately 30% of women with primary breast cancer eventually experience distant breast cancer recurrence. We found that high expression of PDGFRα in tumour cells of the primary tumour was correlated to subsequent first distant recurrence occurring within CNS. Even if prognosis in metastatic breast cancer has improved over the past decades [24], metastasis within CNS is a major limitation of life quality and survival [25]. Deciphering the biology behind metastasis and target organs for metastatic spread is important in order to develop targeted therapies. In glioblastoma, autocrine signalling by PDGF-CC/PDGFRα has been proposed to have a role in tumour development and Lokker et al. detected concomitant expression of PDGF-CC and PDGFRα in 6/6 tested glioblastoma cell lines, and 5/5 investigated primary glioblastoma tissue samples [26]. Furthermore, PDGF-CC has been shown to increase the permeability of the blood–brain barrier (BBB) [27], and the role of the PDGFRα/PDGF-CC pathway relative to BBB dysfunction in neurological disorders was recently reviewed [28]. In our cohort, patients who developed CNS metastases had primary tumours with high expression of tumour cell PDGFRα in 67%, PDGF-CC in 50% and concomitant high ligand/receptor expression in 33% of cases, indicating an active role of the PDGF pathway in these tumours. Thus it is possible that the PDGF pathway is involved when a tumour cell crosses the BBB. The number of patients with CNS recurrence is small within this cohort but the finding is intriguing. Unfortunately, we had no tumour tissue available from CNS metastases to explore the expression of the PDGF members. However, Kim et al. recently investigated the expression of tumour cell PDGFRα in breast cancer CNS metastases and found high expression in 12/38 (32%) metastases [29]. Unfortunately, comparisons between these different studies are limited due to different antibodies and scoring systems for PDGFRα staining.

In this study we also explored concomitant expression of PDGFRα and PDGFRβ, and ligand PDGF-CC. Experiments in vitro have shown that PDGF-CC binds to and stimulates homodimers of PDGFRα, and heterodimers of PDGFRα and PDGFRβ, but not homodimers of PDGFRβ [30]. However, the binding capacity of PDGF-CC in vivo is not completely known and we thus explored all the three receptor/ligand combinations. Almost all tumours with high stromal cell PDGFRβ and high tumour cell PDGF-CC also had high PDGFRα, either in stromal or tumour cells. In contrast, more than half of the tumours with high PDGF-CC and high PDGFRα expression in tumour or stromal cells displayed low PDGFRβ. These findings support that PDGF-CC binds to and signals though receptor combinations including the PDGFRα in vivo. Concomitant PDGFRα and PDGF-CC expression varied markedly over the molecular subtypes where the TNBC displayed co-expression in 59% of the tumours, whereas the luminal subtypes only displayed co-expression in 5% (Luminal A) to 19% (Luminal B HER2+). This adds further evidence to the involvement of the PDGF signalling pathway in TNBC as indicated in previous studies [8, 9, 13] and this pathway might thus be a target for therapy in this difficult-to-treat subgroup of breast cancer.

The strength of the present study is that it is performed in a large cohort of breast cancer patients with long follow-up time (13.7 years). The study enables analysis of the PDGF signalling pathway in tumour tissue biopsies from several locations where data on clinically important biomarkers and St Gallen molecular subtypes were available, which allows to evaluate the study results in relation to modern pathology. A limitation is that there was limited tissue material available from lymph node metastasis and recurrences. Also, we recorded distant recurrence and/or breast cancer-related death in only 20% of patients during the follow-up time diminishing the power of the study.

In conclusion, we show that high expression of PDGFRα, PDGFRβ and ligand PDGF-CC is associated to several important prognostic patient and tumour characteristics in breast cancer, indicating a link to tumours with inherent biological aggressiveness. Tumour cell expression of PDGFRα is commonly up-regulated in lymph node metastases and asynchronous recurrences, whereas high expression of PDGF-CC is related to early breast cancer recurrence supporting an active role of the PDGF signalling pathway in tumour progression. Furthermore, our results indicate an intriguing connection between the PDGF pathway and metastatic spread to the CNS, which merits further exploration. In summary, our data encourage further evaluation of the PDGF receptors and ligands in breast cancer, as well as of strategies to target this pathway since evidence is compiling for its involvement in breast cancer progression.