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.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.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.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.
Fibrotic lung disease is characterized by distorted lung architecture and severe loss of respiratory function secondary to alveolar epithelial cell (AEC) hyperplasia, enhanced extracellular matrix (ECM) deposition and fibroblast proliferation. Repetitive epithelial injuries with impaired alveolar wound healing and altered AEC gene expression represent a trigger mechanism for development of fibrosis. To reveal gene regulatory networks in lung fibrosis, we compared gene expression profiles of freshly isolated AEC obtained from mice 14 days after saline or bleomycin (BM) instillation using whole genome microarray analysis. Several genes of the Wnt signaling pathway, in particular WISP-1, a member of the CCN family, were highly regulated. WISP-1 protein expression was demonstrated in proliferating AEC in BM-treated lungs by immunofluorescence. When analyzing all six CCN family members, WISP-1 was upregulated the most 14 days after BM challenge, as analyzed by qRT-PCR. To elucidate WISP-1 function, cultured primary mouse AEC were stimulated with WISP-1 and demonstrated a 230% increase in proliferation, analyzed by 3H-thymidine incorporation. This was mediated through enhanced phosphorylation, but not expression of protein kinase B (PKB/Akt), as detected by immunoblot. Finally, increased expression of WISP-1 was detected in lung homogenates and isolated AEC from IPF patients, using qRT-PCR. Immunohistochemical analysis of WISP-1 and Ki67 verified the existence of hyperplastic and proliferative AEC expressing WISP-1 in vivo . Our study thus identifies WISP-1 as a novel regulator of AEC injury and repair, and suggests that WISP-1 is a key mediator in pulmonary fibrosis.
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.
Idiopathic pulmonary fibrosis (IPF) is a devastating disease, characterized by severe loss of respiratory function due to enhanced extracellular matrix (ECM) deposition and (myo)fibroblast proliferation. Repetitive epithelial injuries with impaired alveolar wound healing represent a trigger mechanism for development of fibrosis, but molecular mechanisms linking alveolar epithelial cell (AEC) repair with development of fibrosis are poorly understood. Using whole genome microarrays, we report altered gene expression profiles of AEC derived from mice with lung fibrosis. In particular, we demonstrate that WISP1 (CCN4/Wnt-inducible signalling protein) RNA and protein expression is highly upregulated in experimental lung fibrosis and lungs of IPF patients. WISP1 protein expression localized to hyperplastic AEC in vitro and in vivo. WISP1 treatment of primary AEC resulted in increased AEC proliferation, mediated by enhanced phosphorylation, but not expression, of protein kinase B (PKB/Akt). WISP1 induced epithelial-to-mesenchymal transition (EMT) and the expression of the profibrotic markers Spp1, Pai1, and Mmp7 in primary AEC, as assessed by qRT-PCR and immunhistochemistry. Depletion of WISP1 (using neutralizing antibodies) in vivo resulted in marked attenuation of bleomycin-induced lung fibrosis and improved survival in vivo, as assessed by histological analysis, flat-panel volumetric computed tomography, lung function, and collagen abundance (Sircoll assay). WISP1 depletion also reduced profibrotic marker gene expression, collagen deposition, and EMT. In sum, our study uncovered WISP1 as a novel regulator of profibrotic pathomechansims in hyperplastic AEC in vitro and in vivo, and presents WISP1 as a potential tool for therapeutic intervention in pulmonary fibrosis.
Breast cancer (BCa) is the most common cancer affecting women worldwide. Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in ~20‑25% of invasive ductal breast carcinomas and is associated with the more aggressive phenotype. Herceptin, a humanized antibody against HER2, is a standard therapy in HER2‑overexpressing cases. Approximately one‑third of patients relapse despite treatment. Therefore numerous studies have investigated the molecular mechanisms associated with Herceptin resistance. An interaction between HER2 signalling and steroid hormone receptor signalling pathways has been previously investigated, but the effect of this relationship on Herceptin resistance has never been studied. The present study analysed an impact of the steroid hormone, progesterone (PG), on Herceptin‑dependent cell growth inhibition. Results indicated that Herceptin‑inhibited proliferation of breast cancer cell lines overexpressing HER2 (BT474 and MCF/HER2) in 3D culture is abolished by PG. Furthermore, results demonstrated that PG led to the activation of HER2/HER3‑mediated signalling. Moreover, PG treatment induced a shift of Herceptin‑dependent cell cycle arrest in G1 phase towards S and G2 phases with concomitant upregulation of cyclin‑dependent kinase 2 (CDK2) and downregulation of CDK inhibitor p27Kip1. These results demonstrate for the first time PG involvement in the failure of Herceptin treatment in vitro. The present observations suggest that cross‑talk between PG‑ and HRG/HER2‑initiated signalling pathways may lead to the acquisition of resistance to Herceptin in patients with BCa.
Deregulation of fibroblast growth factor receptors (FGFRs) signaling, as a result of FGFR amplification, chromosomal translocation, or mutations, is involved in both initiation and progression of a wide range of human cancers. Clinical data demonstrating the dependence of cancer cells on FGFRs signaling clearly indicate these receptors as the molecular targets of anti-cancer therapies. Despite the increasing number of tyrosine kinase inhibitors (TKIs) being investigated in clinical trials, acquired resistance to these drugs poses a serious therapeutic problem. In this study, we focused on a novel pan-FGFR inhibitor—CPL304110, currently being investigated in phase I clinical trials in adults with advanced solid malignancies. We analyzed the sensitivity of 17 cell lines derived from cancers with aberrant FGFR signaling, i.e. non-small cell lung cancer, gastric and bladder cancer to CPL304110. In order to explore the mechanism of acquired resistance to this FGFR inhibitor, we developed from sensitive cell lines their variants resistant to CPL304110. Herein, for the first time we revealed that the process of acquired resistance to the novel FGFR inhibitor was associated with increased expression of MET in lung, gastric, and bladder cancer cells. Overexpression of MET in NCI-H1703, SNU-16, RT-112 cells as well as treatment with HGF resulted in the impaired response to inhibition of FGFR activity. Moreover, we demonstrated that cells with acquired resistance to FGFR inhibitor as well as cells overexpressing MET displayed enhanced migratory abilities what was accompanied with increased levels of Pyk2 expression. Importantly, inhibition of both MET and Pyk2 activity restored sensitivity to FGFR inhibition in these cells. Our results demonstrate that the HGF/MET-Pyk2 signaling axis confers resistance to the novel FGFR inhibitor, and this mechanism is common for lung, gastric, and bladder cancer cells. Our study suggests that targeting of MET/Pyk2 could be an approach to overcome resistance to FGFR inhibition.
