Introduction
Breast Cancer is still the most frequent malignant disease worldwide, and the most frequent cause of death in women (1). One out of eight women is diagnosed with breast cancer during her lifetime. Although lethality has declined in the last 40 years, still 30% of the affected patients die from the consequences of breast cancer (2).
Approximately 10–20% of all diagnosed breast cancers are characterized by a lack of expression or a only very weak expression of the hormone receptors, estrogen- and progesterone-receptor, and of the human epidermal growth factor receptor 2 (Her2) (3). These quite aggressive tumours, which are termed ‘triple-negative breast cancer’ [TNBC (4)], occur frequently in younger women, many of them show up with BRCA-1 mutations (5). Patients suffering from TNBC may develop visceral metastases, have a high risk of recurrence and a reduced overall survival (6), independent of tumour size, staging and lymph node affection (7).
Furthermore, the possibilities for treatment are sparse, as endocrine therapy with Tamoxifen or aromatase-inhibitors as well as anti-Her2 therapy with Trastuzumab are ineffective. Actually, TNBCs are treated postoperatively with a dose-dense or metronome chemotherapy using anthracyclins or taxans and radiation (8). Only in the neoadjuvant setting TNBC shows a better follow-up as non-TNBC (9). Recent therapeutic concepts introduced for example inhibitors of poly(ADP-ribose) polymerase (PARP) in addition to chemotherapy, such as Iniparib or Olaparib, which are normally responsible for the repair of single- and double-strand DNA breaks. The aim is, to accumulate severe DNA-damage within the tumour cells, that the cells stop entering mitosis, thereby stopping cell division. Thus disease-free survival can be prolonged (10). The risk of recurrence can be reduced by the additional application of VEGF inhibitors such as bevacizumab, which decelerates tumour growth by reduction of neoangiogenesis (11). However, the disadvantage of this treatment is, that bevacizumab has rather strong side effects (12,13).
From all those facts the importance of new treatment options can be explained. New therapeutic strategies could target for example inter- and intracellular signal transduction pathways, regulation of cell adhesion and proliferation. Clinical studies are carried out using cetuximab, an inhibitor of epithelial growth factor receptor (EGFR) (14,15) which seems to improve survival rates without further tumour progression (16), or the mTOR-inhibitor everolimus (17). Another treatment strategy could target the Notch1 pathway. Notch1 plays an important role in normal breast development and cell fate determination. In breast cancer tissue, especially TNBC, this pathway is activated in an aberrant manner. Additionally it has been shown, that an inhibition of Notch signalling by gamma-secretase inhibitors (GSIs) results in antitumour activity by cell cycle arrest, apoptosis and disruption of angiogenesis (18,19).
Notch-pathway members are moreover regulated by HIF1α, which thereby constitutes another possibility for TNBC treatment. Overexpression of the transcription factor HIF1α is associated with a poor prognosis for the affected patients, and in a mouse model it was shown, that it is important for promoting carcinoma onset and formation of lung metastasis. The deletion of HIF1α resulted in reduced primary tumour growth, suppression of lung metastasis and prolonged survival (20). HIF1α and XBP1 in turn form a transcriptional complex, which is known to drive tumorigenicity. XBP1 thereby regulates the expression of HIF1α targets via recruitment of polymerase II. Hence, XBP1 also plays a role in tumour progression, especially in TNBC, contributing to a poor prognosis. In a cell culture model it was shown, that an inhibition of XBP1 results in a retardation of tumour growth, giving hints to another therapeutic target (21).
Another transcription factor, which is highly expressed in tumour cells is FOXP3, a potent repressor of several oncogenes. Its involves in TNBC susceptibility and prognosis (22), but the prognosis is dependent on the cellular localisation of FOXP3: If it is located in the cytoplasm, it is a marker for poor OAS, but if it remains in the nucleus, OAS is markedly improved (23). Furthermore the Wnt/β-catenin signalling pathway could be used for therapeutical intervention, as this pathway regulates the cell cycle, cell growth and tumour progression and is responsible for poor clinical outcomes.
The Wnt/β-catenin pathway was shown to be activated in TNBC, the inhibition of Wnt-receptors by, e.g. salinomycin, is already known as therapeutic target, as it induces LRP6 degradation (24). LRP6 in turn, is frequently produced in response to obesity stimuli and induces cell proliferation by interaction with Ki67 and reduces efficiency of treatment. LRP6 is of great use in TNBC-therapy, as it has no relation with ER, PR, and Her2. Its use as drug target receptor significantly prolonged survival time in a mouse model (25). Another signalling molecule within the Wnt-signal transduction pathway, which might be of therapeutic significance, is MCL1, which modulates mitochondrial physiology, and is associated with enhanced metastasis formation and decreased DFS (26).
Discussion
The results clearly indicate that some of the analysed signal transduction molecules have a correlation to the incidence of patient and tumour traits, and are furthermore correlated to different recurrences. The novelty of this study lies in the combination of marker molecules of breast cancer tissue samples and their correlation to other tumour and patient characteristics, especially of the rather aggressive and hard to treat triple-negative breast cancer subtype.
A drawback of the presented study is of course the small number of patient samples analysed, so that the results have to be considered as preliminary. However, the data already show up a certain trend, which should be clarified by the analysis of a larger patient collective. A higher number of samples analysed would also improve the statistical significance.
Nevertheless, some of these findings are in accordance to recently published data, either in breast cancer or in other tumour entities. The correlation of HIF1α to the histological grading was already described for ovarian cancer (28), where this factor might become important for prognostic evaluation and clinical treatment. Moreover, the linkage between β-catenin and menopausal stage might be of clinical relevance, as Wang et al found, that the Wnt-pathway influenced the pathogenesis of postmenopausal osteoporosis (29). The Wnt/β-catenin pathway also contributes to radioresistence of TNBC cells. Niclosamide, a potent inhibitor of the Wnt/β-catenin pathway might be able to abolish the radioresistence of these cells, improving the TNBC-therapy (30).
Wnt/β-catenin and Notch1 are normally responsible for mammary gland morphogenesis during embryonal development and are frequently found to be upregulated during tumorigenesis, creating another treatment option (31). The role of Notch1 in lymph node affected infiltrating ductal carcinomas was already described in the literature, claiming the involvement of Notch1 in epithelial-mesenchymal transition (32) which is a rather important process in metastasis formation. Furthermore patients with high expression of Notch1 had a worse OAS and DFS (33). Recently, the microRNA, miR9, was identified to regulate Notch1 in a manner to suppress its tumorigenic capacity (34). Furthermore, the ATPase, a2V-ATPase, was identified, which is necessary for the processing of Notch1-receptor. A deficiency of this ATPase was shown to disrupt Notch signalling and mammary gland development, which might also represent a new way in breast cancer treatment (35).
However, we did not find correlations of tumour characteristics with XBP1, MCL1, LRP6 and FOXP3, and no relation could be shown between tumour size, remote metastasis formation and tumour histology and one of the investigated signalling molecules.
Additionally we investigated the coherence between the different signalling molecules and the formation of recurrence such as lymph node recurrence, local recurrence or remote metastasis formation. We found a correlation between the expression of β-catenin and the incidence of remote metastasis. It was published earlier, that β-catenin plays a role in metastasis formation by interactions with ECM (extracellular matrix)1 protein, what increased the progress on EMT and CSC (cancer stem cell) phenotype maintenance in the cancer cells (36). Furthermore, β-catenin is known to support the action of matrix-metalloproteinases (MMPs) and uPA (urokinase plasminogen activator), its inhibition thereby inhibits EMT (37). The inhibition of β-catenin seems to correlate with the inhibition of metastasis formation (38,39). However, β-catenin also plays a role in lymph node recurrence formation (40), as we also demonstrated with our experiments. An association of Notch1 and remote metastasis formation, which we found in our analysis, was also already described (41) and seems to work via angiogenesis.
Rather new are the coherence of XBP1 and lymph node recurrences and FOXP3 and local recurrences, showing ultimately, that all these signal transduction pathways are rather important in tumorigenicity and recurrence formation.
As a conclusion of the experiments, the correlation of staining of different signal transduction molecules to tumour traits, indicated that signal transduction pathways influence tumour progression and recurrence or metastasis formation. As the number of samples used in the study is rather small, the results have to be considered as preliminary and further research has to be done to verify these data. Carrying out such experiments could help to refine prognosis and find ways to inhibit tumour progression and metastasis formation and thereby might help to find new therapeutic strategies.