Potential for the embryonic morphogen Nodal as a prognostic and predictive biomarker in breast cancer

Various classification schemes have been developed to categorize the heterogeneity of breast cancer in an attempt to better predict disease stage, progression potential and outcome. Traditionally, the diagnosis of breast cancer has been based on histological criteria [1]. Moreover, defined architectural features like those described in the Nottingham Grading system for invasive breast cancer, which includes tubule formation, mitoses and nuclear pleomorphism, are used to classify the differentiation status of breast cancer - with poor differentiation being the hallmark of high grade, more aggressive disease [2]. Over the years, with advances in molecular medicine, the incorporation of markers, such as oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), have proven to be especially valuable not only for stratifying certain types of breast cancers in distinct functional groups [3], but also for planning and predicting the outcome with respect to specific treatment options [4, 5]. Due to the heterogeneity within specific subgroups of breast cancer and the interobserver variability with detection frequencies, not all breast cancers can be successfully classified into specific risk groups based on the expression profile of these traditional markers alone [3]. For instance, adenoid cystic carcinoma and secretory carcinoma are generally hormone receptor negative, but have favourable prognosis and low recurrence rates [6, 7]. Further confounding, the expression profile of these markers not only varies within areas of the same lesion but also during the course of disease in the same patient [8]. Additional studies are needed, therefore, to identify novel biomarkers, based on the molecular underpinnings of disease progression that can be used to predict outcome and response to therapy in a larger population of patients, especially those in the high risk category.

Data from gene expression microarrays have led to the molecular stratification of breast cancer into subgroups, such as luminal and non-luminal tumours [9]. Even with this approach, it is difficult to obtain unequivocal consensus on breast cancer classification among observers [10]. Given the heterogeneous subpopulations comprising breast cancer tissue, a major concern is whether results from this type of broad gene expression analysis can be confidently designated as the 'genetic signature' of a specific breast cancer type and whether this approach can be applied to all breast cancer patients. With the introduction of the cancer stem cell theory, different markers have been reported to identify cancer stem cells (CSCs) with the prospect of exploiting these putative CSCs markers as therapeutic targets [11]. In breast cancer, the role of the 'CD44high/CD24low' expression profile, proposed by some to represent a unique subpopulation of breast CSCs [12], has been challenged by others who postulate that not every breast cancer cell with this particular expression profile possesses the properties of CSCs [13]. This may be due to the genetic heterogeneity within the 'CD44high/CD24low' population [14], which suggests a much broader functional variability for this population. Nevertheless, advances in the field of CSC research have enabled us to characterize the re-emergence of specific embryonic signalling pathways in cancer cells, thus contributing to our understanding of the molecular mechanisms that regulate cancer cell plasticity and aggressiveness [15].

One of the embryonic pathways recently described by our group to have profound implications in cancer progression is Nodal [16, 17]. Nodal, a member of the TGF-beta superfamily, plays a major role in the maintenance of pluripotency in embryonic stem cells and subsequent organ development [18‐20]. Typically, Nodal binds to the Cripto-1-Alk4/7-ActRIIB receptor complex resulting in Smad2/3/4-dependent gene activation [18]. The Nodal co-receptor, Cripto-1, has been shown to enhance the proliferation, migration and invasion of human breast cancer cells and non-transformed mouse mammary epithelial cells in vitro and in vivo [21]. In human breast cancers, Cripto-1 expression was also found to correlate with progressive disease [22]. There is also a growing body of evidence which indicates that Nodal expression re-emerges in a number of human cancers, such as melanoma, glioma, breast, endometrial and prostate cancers [16, 17, 23‐25]. Interestingly, in some of these reports, Nodal expression was detected in the context of very low or barely detectable Cripto-1, raising the question as to whether Cripto-1 and Nodal can exert their cancer promoting effects independently of each other [17, 26]. Nevertheless, the clinical significance of Nodal expression in cancer has yet to be thoroughly explored. In this study, we examined the expression of Nodal in tissue samples from patients with benign and malignant breast disease and compared the levels of Nodal with the available patient data to determine clinical correlations. These immunohistochemical findings were further explored in human breast cancer cell lines treated with a Nodal blocking antibody to determine biological effects for target validation. Collectively, these data suggest the potential for Nodal as a biomarker for invasive disease and a novel therapeutic target in breast cancer.

In this study, immunohistochemistry was used to detect the TGF-beta-related morphogen Nodal in breast tissue samples from over 400 patients (N = 431). The analyses revealed the strongest Nodal staining in a significantly greater percentage of patients with malignant breast disease compared to patients with benign breast disease (P < 0.0001). Immunofluorescence analysis confirmed Nodal expression in a subpopulation of cells in the two human breast cancer lines studied. Nodal is translated as a precursor form consisting of a signal peptide and pro-domain. Studies using mouse Nodal suggest that the pro-form is subsequently cleaved to a much less stable mature form by extracellular proprotein convertases [31]. Both pro-Nodal and mature Nodal are known to be functionally active [32]. It is impossible to determine which form of Nodal protein is detected in breast tissues and cancer cell lines by immunostaining, since both species are detected by the commercially available Nodal antibody utilized in this study. However, Western blot analysis of cell lysates and conditioned medium from the two breast cancer cell lines detected bands corresponding to the molecular weight of approximately 36 KD reported for pro-Nodal [25]. The smaller mature Nodal (approximately 12-13 KD) was not observed in either cell lysates or conditioned medium, most likely due to the instability of this form. Like most TGF-beta ligands, mature Nodal is capable of forming homodimers; therefore, it is tempting to speculate that the additional band of approximately 24 KD detected in conditioned medium by Western blotting (Figure 2B) could represent a more stable, homodimeric form of mature Nodal. Further studies will be necessary to address whether these secreted form(s) of cancer cell-derived Nodal may affect neighbouring non-Nodal expressing cells. In a previous study, however, we demonstrated that when exogenous recombinant Nodal is added to non-Nodal expressing cells, these cells increase Smad-2 activation [17]. Also, anchorage independent growth that was inhibited in cancer cells treated with Nodal Morpholino treatment, was rescued when these cells were treated with recombinant Nodal protein [16, 17], thus supporting the potential for Nodal paracrine effects.

In the category of patients diagnosed with breast cancer (N = 138), the degree of Nodal staining showed a significant correlation with poorly differentiated (P = 0.008), advanced stage (P = 0.0003) and lymph node positive (P = 0.009) breast cancer. These observations are in agreement with previous studies describing Nodal expression in aggressive melanoma and glioma and advanced stage endometrial and prostate cancers [16, 17, 23‐25]. Nodal and Cripto-1 (the co-receptor for Nodal) have both been observed in different cancer cell lines, including aggressive human breast cancer cells [17, 21]. There is still much debate, however, as to whether Nodal and Cripto-1 can affect cancer cells along distinct pathways or are more likely to function synergistically to propagate downstream signalling events responsible for tumour aggressiveness. Indeed, future studies are needed to specifically address these possibilities. Nevertheless, the present study is the first comprehensive report demonstrating the clinical association between Nodal expression and progression of breast cancer in patient tissues.

Particularly curious is the significant correlation found between higher Nodal expression and non-smoker status (P = 0.01). Although it is not clear how smoking can influence the expression of Nodal, it is interesting to note that tobacco smoke has been shown to have deleterious effects on human embryonic stem cells [33]. Thus, one could speculate that since Nodal expression represents the re-emergence of embryonic signalling, initiated perhaps in a subset of breast cancer cells that share certain phenotypic characteristics with stem cells, it is possible that toxicity from tobacco smoke could negatively affect Nodal expression in these stem cell-like breast cancer cells.

It is well established that hormone activity can play a role during tumorigenesis in a variety of responsive tissues [34‐37]. Although the relationship between Nodal expression and hormone activity during development, especially in the mammary gland, has not been clearly defined in rigorously controlled studies, a recent report on prostate cancer found that Nodal was capable of reducing the endogenous expression of androgen regulated genes [25]. From this study, one could speculate that a possible effect of Nodal is to regulate differentiation by promoting cell plasticity, which would eventually lead to increased aggressiveness in prostate cancer. In fact, Nodal has been shown to regulate the plastic, endothelial phenotype in melanoma during vasculogenic mimicry [16, 28]. Most noteworthy, when Nodal gene expression is down-regulated in tumour cells, the plastic phenotype is diminished, and a more differentiated and less tumorigenic cell phenotype emerges [16, 17].

Similar to prostate, hormones play an important role in the development, differentiation and tumorigenesis of breast tissue [38, 39]. The expression status of ER or PR in breast cancer represents a useful clinical tool for prognosticating patient survival and predicting the benefit from specific hormonal therapy [40]. However, not all breast cancer patients express these hormone receptors, thus highlighting the need for novel biomarkers that would facilitate universal clinical decisions. Our study did not detect any correlation between Nodal expression and ER or PR status, which was available in 102/138 of the breast cancer cases analysed. However, Nodal was detected in all 138 breast cancer cases, including the samples from patients in which ER or PR status was negative or undetermined. Our results suggest that Nodal could represent a novel biomarker detectable across various stages of breast cancer progression, with the potential to expand the classification scheme based on ER, PR or HER2 status.

Previously, we reported that interference with Nodal signalling can significantly reduce Nodal-dependent cancer cell activities, such as migration and invasion, tumorigenicity and anchorage independent growth [16, 17, 28, 41]. In particular, we showed that it is possible to significantly reduce Nodal expression in human breast cancer cells by exposing them to a human embryonic stem cell conditioned microenvironment containing a Nodal inhibitor, Lefty [17]. Furthermore, knockdown of Nodal with anti-Nodal Morpholino can significantly reduce tumour growth rate and increase apoptosis in an in vivo orthotopic human breast cancer xenograft model [41]. Here, we extend these findings by demonstrating that treatment of human metastatic breast cancer cells with a Nodal blocking antibody decreases Nodal expression levels and Smad-2 phosphorylation and reduces cell proliferation and increases apoptosis by reducing cellular levels of pHH3, PCNA and BCL2α. These treatments also led to reduced anchorage independent colony formation in soft agar, further supporting the anti-tumorigenic effect of targeting Nodal. This is in agreement with a previous study where Nodal blocking antibodies were shown to inhibit the colony forming ability of human melanoma cells in soft agar and significantly reduce the ability of these tumour cells to colonize in the lungs of Nude mice [29].

Our results indicate that the expression of Nodal is associated with advanced stage, invasive human breast cancer. We also describe the inhibitory effects, with underlying mechanistic insights, of a Nodal blocking antibody on human breast cancer cells, extending previous reports showing target validation of Nodal in human cancer. These findings suggest a potential role for Nodal as a novel prognostic biomarker and a promising target for anti-Nodal therapy in breast cancer.