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We have recently demonstrated that fibroblast growth factor receptor 2 (FGFR2)-mediated signalling alters progesterone receptor (PR) activity and response of oestrogen receptor α (ER)-positive (ER+) breast cancer (BCa) cell lines to anti-ER agents. Little is known about whether the crosstalk between ER and PR, shown to be modulated by the hormonal background, might also be affected by FGFR2. Here, PR-dependent behaviour of ER+ BCa cells was studied in the presence of oestrogen (E2) and progesterone (P4) and/or FGF7. In vitro analyses showed that FGF7/FGFR2 signalling: (a) abolished the effect of P4 on E2-promoted 3D cell growth and response to tamoxifen; (b) regulated ER and PR expression and activity; (c) increased formation of ER-PR complexes; and (d) reversed P4-triggered deregulation of ER-dependent genes. Analysis of clinical data demonstrated that the prognostic value of FGFR2 varied between patients with different menopausal status; that is, high expression of FGFR2 was significantly associated with longer progression-free survival (PFS) in postmenopausal patients, whereas there was no significant association in premenopausal patients. FGFR2 was found to positively correlate with the expression of JunB proto-oncogene, AP-1 transcription factor subunit (JUNB), an ER-dependent gene, only in premenopausal patients. Molecular analyses revealed that the presence of JunB was a prerequisite for FGFR2-mediated abrogation of P4-induced inhibition of cell growth. Our results demonstrate for the first time that the FGF7/FGFR2-JunB axis abolishes the modulatory effects of PR on ER-associated biological functions in premenopausal ER+ BCa. This may provide foundations for a better selection of patients for FGFR-targeting therapeutic strategies.

The role of fibroblast growth factor receptor 2 (FGFR2), an important mediator of stromal paracrine and autocrine signals, in mammary gland morphogenesis and breast cancer has been extensively studied over the last years. However, the function of FGFR2 signalling in the initiation of mammary epithelial oncogenic transformation remains elusive. Here, FGFR2-dependent behaviour of nontumorigenic model of mammary epithelial cells was studied. In vitro analyses demonstrated that FGFR2 regulates epithelial cell communication with extracellular matrix (ECM) proteins. Silencing of FGFR2 significantly changed the phenotype of cell colonies in three-dimensional cultures, decreased integrins α2, α5 and β1 protein levels and affected integrin-driven processes, such as cell adhesion and migration. More detailed analysis revealed the FGFR2 knock-down-induced proteasomal degradation of integrin β1. Analysis of RNA-seq databases showed significantly decreased FGFR2 and ITGB1 mRNA levels in breast tumour samples, when compared to non-transformed tissues. Additionally, high risk healthy individuals were found to have disrupted correlation profiles of genes associated with FGFR2 and integrin signalling, cell adhesion/migration and ECM remodelling. Taken together, our results strongly suggest that FGFR2 loss with concomitant integrin β1 degradation is responsible for deregulation of epithelial cell-ECM interactions and this process may play an important role in the initiation of mammary gland epithelial tumorigenesis.

BACKGROUND/AIM Antimicrobial peptides are part of the innate immune response, regulate inflammation and initiate acquired immunity. This study focused on theta-defensins that have been shown to have anticancer properties. MATERIALS AND METHODS RTD-2 analogs were synthesized on a peptide synthesizer. Cell viability was estimated using the MTT test. Immunoprecipitation assay was conducted to determine the molecular partner of the [Ser3,7,12,16]-RTD-2 analog. RESULTS Here, we present the biologically active [Ser3,7,12,16]-RTD-2 analog that selectively targets various types of breast cancer cells. Immunoprecipitation protein-protein interaction studies showed eleven proteins common to MDA-MB-231 and T47D cell lines. Taking into account their cellular location, it can be concluded that the synthesized peptide interacts mainly with nuclear proteins, which correlates with the obtained microscopic image. CONCLUSION Proteins that interact strongly with the [Ser3,7,12,16]-RTD-2 analog are closely related to the proteasomal protein degradation pathway. As the activity of the ubiquitin-proteasome system is markedly increased in patients with breast cancer, it is likely that selective modulation of this system may be a useful method for breast cancer treatment.

Stromal stimuli mediated by growth factor receptors, leading to ligand-independent activation of steroid hormone receptors, have long been implicated in development of breast cancer resistance to endocrine therapy. Mutations in fibroblast growth factor receptor (FGFR) genes have been associated with a higher incidence and progression of breast cancer. Increasing evidence suggests that FGFR-mediated interaction between luminal invasive ductal breast carcinoma (IDC) and its microenvironment contributes to the progression to hormone-independence. Therapeutic strategies based on FGFR inhibitors hold promise for overcoming resistance to the ER-targeting treatment. A series of excellent reviews discuss a potential role of FGFR in development of IDC. Here, we provide a concise updated summary of existing literature on FGFR-mediated signalling with an emphasis on an interaction between FGFR and estrogen/progesterone receptors (ER/PR) in IDC. Focusing on the regulatory role of tumour microenvironment in the activity of steroid hormone receptors, we compile the available functional data on FGFRs-mediated signalling, as a fundamental mechanism of luminal IDC progression and failure of anti-ER treatment. We also highlight the translational value of the presented findings and summarize ongoing oncologic clinical trials investigating FGFRs inhibition in interventional studies in breast cancer.

Interaction between fibroblast growth factor receptor 2 (FGFR2) and estrogen/progesterone receptors (ER/PR) affects resistance to anti-ER therapies, however the prognostic value of FGFR2 in breast cancer (BCa) remains largely unexplored. We have recently showed in vitro that FGFR2-mediated signaling alters PR activity and response to anti-ER treatment. Herein, prognostic significance of FGFR2 in BCa was evaluated in relation to both ER/PR protein status and a molecular signature designed to reflect PR transcriptional activity. FGFR2 was examined in 353 BCa cases using immunohistochemistry and Nanostring-based RNA quantification. FGFR2 expression was higher in ER+PR+ and ER+PR- compared to ER&minus, PR&minus, cases (p &lt, 0.001). Low FGFR2 was associated with higher grade (p &lt, 0.001), higher Ki67 proliferation index (p &lt, 0.001), and worse overall and disease-free survival (HR = 2.34 (95% CI: 1.26&ndash, 4.34), p = 0.007 and HR = 2.22 (95% CI: 1.25&ndash, 3.93), p = 0.006, respectively). The poor prognostic value of low FGFR2 was apparent in ER+PR+, but not in ER+PR&minus, patients, and it did not depend on the expression level of PR-dependent genes. Despite the functional link between FGFR2 and ER/PR revealed by preclinical studies, the data showed a link between FGFR2 expression and poor prognosis in BCa patients.

// Dominika Piasecka 1, 4 , Kamila Kitowska 1 , Dominika Czaplinska 2 , Kamil Mieczkowski 1 , Magdalena Mieszkowska 1 , Lukasz Turczyk 1 , Andrzej C. Skladanowski 1 , Anna J. Zaczek 2 , Wojciech Biernat 3 , Radzislaw Kordek 4 , Hanna M. Romanska 4 , Rafal Sadej 1 1 Department of Molecular Enzymology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Poland 2 Department of Cell Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Poland 3 Department of Pathomorphology, Medical University of Gdansk, Poland 4 Department of Pathology, Medical University of Lodz, Poland Correspondence to: Rafal Sadej, email: rsadej@gumed.edu.pl Hanna M. Romanska, email: hanna.romanska@gmail.com Keywords: progesterone receptor, FGFR2, breast cancer Received: April 12, 2016 Accepted: November 07, 2016 Published: November 12, 2016 ABSTRACT We have recently demonstrated that, fibroblast growth factor 2 (FGFR2), signalling via ribosomal S6 kinase 2 (RSK2), promotes progression of breast cancer (BCa). Loss of progesterone receptor (PR), whose activity in BCa cells can be stimulated by growth factor receptors (GFRs), is associated with poor patient outcome. Here we showed that FGF7/FGFR2 triggered phosphorylation of PR at Ser294, PR ubiquitination and subsequent receptor`s degradation via the 26S proteasome pathway in BCa cells. We further demonstrated that RSK2 mediated FGF7/FGFR2-induced PR downregulation. In addition, a strong synergistic effect of FGF7 and progesterone (Pg), reflected in the enhanced anchorage-independent growth and cell migration, was observed. Analysis of clinical material demonstrated that expression of PR inversely correlated with activated RSK (RSK-P) ( p = 0.016). Patients with RSK-P(+)/PR(–) tumours had 3.629-fold higher risk of recurrence ( p = 0.002), when compared with the rest of the cohort. Moreover, RSK-P(+)/PR(–) phenotype was shown as an independent prognostic factor ( p = 0.006). These results indicate that the FGF7/FGFR2-RSK2 axis promotes PR turnover and activity, which may sensitize BCa cells to stromal stimuli and contribute to the progression toward steroid hormone negative BCa.

We have previously demonstrated that fibroblast growth factor receptor 2 (FGFR2) activates ribosomal s6 kinase 2 (RSK2) in mammary epithelial cells and that this pathway promotes in vitro cell growth and migration. Potential clinical significance of FGFR2 and RSK2 association has never been investigated. Herein, we have undertaken an evaluation of a possible relationship between FGFR2/RSK2 interdependence and disease outcome in breast cancer (BCa) patients. The clinical analysis was complemented by an in vitro investigation of an involvement of RSK2 in the regulation of FGFR2 function. Primary tumour samples from 152 stage I–III BCa patients were examined for FGFR2 and RSK2 gene and protein expression. FGFR2 showed a positive correlation with RSK2 at both protein (p = 0.003) and messenger RNA (mRNA) (p = 0.001) levels. Lack of both FGFR2 and activated RSK (RSK-P) significantly correlated with better disease-free survival (DFS) (p = 0.01). Patients with tumours displaying immunoreactivity for either or both FGFR2 and RSK-P had 4.89-fold higher risk of recurrence when compared to the FGFR2/RSK-P-negative subgroup. FGFR2-RSK2 interactions were verified by co-immunoprecipitation and internalization assays in HB2 mammary epithelial cell line (characterized by high endogenous FGFR2 and RSK2 expression). In vitro analyses revealed that FGFR2 and RSK2 formed an indirect complex and that activated RSK exerted a significant impact on fibroblast growth factor 2 (FGF2)-triggered internalization of FGFR2. Our results suggest that the FGFR2-RSK2 signalling pathway is involved in pathophysiology of BCa and evaluation of FGFR2/RSK-P expression may be useful in disease prognostication. Electronic supplementary material The online version of this article (doi:10.1007/s13277-016-5266-9) contains supplementary material, which is available to authorized users.

Introduction. Triple-negative breast cancer (TNBC), representing over 15% of all breast cancers, has a poorer prognosis than other subtypes. There is no effective targeted treatment available for the TNBC sufferers. Ribosomal S6 kinases (RSKs) have been previously proposed as drug targets for TNBC based on observations that 85% of these tumors express activated RSKs. Materials and methods. Herein we examined an involvement of RSK1 (p90 ribosomal S6 kinase 1) in a regulation of TNBC growth and metastatic spread in an animal model, which closely imitates human disease. Mice were inoculated into mammary fat pad with 4T1 cells or their RSK1-depleted variant. We examined tumor growth and formation of pulmonary metastasis. Boyden chamber, wound healing and soft agarose assays were performed to evaluate cells invasion, migration and anchorage-independent growth. Results. We found that RSK1 promoted tumor growth and metastasis in vivo . After 35 days all animals inoculated with control cells developed tumors while in the group injected with RSK1-negative cells, there were 75% tumor-bearing mice. Average tumor mass was estimated as 1.16 g and 0.37 g for RSK1-positive vs. -negative samples, respectively (p < 0.0001). Quantification of the macroscopic pulmonary metastases indicated that mice with RSK1-negative tumors developed approximately 85% less metastatic foci on the lung surface (p < 0.001). This has been supported by in vitro data presenting that RSK1 promoted anchorage-independent cell growth and migration. Moreover, RSK1 knock-down corresponded with decreased expression of cell cycle regulating proteins, i.e. cyclin D3, CDK6 and CDK4. Conclusions. We provide evidence that RSK1 supports tumor growth and metastatic spread in vivo as well as in vitro migration and survival in non-adherent conditions. Further studies of RSK1 involvement in TNBC progression may substantiate our findings, laying the foundations for development of anti-RSK1-based therapeutic strategies in the management of patients with TNBC.