Circulating plasma and peripheral blood mononuclear (PBMCs) cells provide an informative snapshot of the systemic physiological state. Moreover, they provide a non-invasively accessible compartment to identify biomarkers for personalized medicine in advanced breast cancer. The role of Neuropilin-1 (NRP-1) and its interacting molecules in breast tumor tissue was correlated with cancer progression; however, the clinical impact of their systemic levels was not extensively evaluated. In this cross-sectional study, we found that circulating and tumor tissue expression of NRP-1 and circulating placental growth factor (PlGF) increase in advanced nodal and metastatic breast cancer compared with locally advanced disease. Tumor tissue expression of NRP-1 and PlGF is also upregulated in triple negative breast cancer (TNBC) compared to other subtypes. Conversely, in PBMCs, NRP-1 and its interacting molecules SEMA4A and SNAI1 are significantly downregulated in breast cancer patients compared to healthy controls, indicating a protective role. Moreover, we report differential PBMC expression profiles that correlate inversely with disease stage (SEMA4A, SNAI1, PLXNA1 and VEGFR3) and can differentiate between the TNBC and non-TNBC tumor subtypes (VEGFR3 and PLXNA1). This work supports the importance of NRP-1-associated molecules in circulation to characterize poor prognosis breast cancer and emphasizes on their role as favorable drug targets.

BackgroundNeuropilin-1 (NRP-1), a non-tyrosine kinase glycoprotein receptor, is associated with poor prognosis breast cancer, however transcriptomic changes triggered by NRP-1 overexpression and its association with chemoresistance in breast cancer have not yet been explored.MethodsBT-474 NRP-1 variant cells were generated by stable overexpression of NRP-1 in the BT-474 breast cancer cell line. RNA sequencing and qRT-PCR were conducted to identify differentially expressed genes. The role of an upregulated oncogene, Tenascin C (TNC) and its associated pathway was investigated by siRNA-mediated knockdown. Resistant variants of the control and BT-474 NRP-1 cells were generated by sequential treatment with four cycles of Adriamycin/Cyclophosphamide (4xAC) followed by four cycles of Paclitaxel (4xAC + 4xPAC).ResultsNRP-1 overexpression increased cellular tumorigenic behavior. RNA sequencing identified upregulation of an oncogene, Tenascin-C (TNC) and downregulation of several tumor suppressors in BT-474 NRP-1 cells. Additionally, protein analysis indicated activation of the TNC-associated integrin β3 (ITGB3) pathway via focal adhesion kinase (FAK), Akt (Ser473) and nuclear factor kappa B (NF-kB) p65. siRNA-mediated TNC knockdown ablated the migratory capacity of BT-474 NRP-1 cells and inactivated FAK/Akt473 signaling. NRP-1 overexpressing cells downregulated breast cancer resistance protein (BCRP/ABCG2). Consequently, sequential treatment with Adriamycin/Cyclophosphamide (AC) cytotoxic drugs to generate resistant cells indicated that BT-474 NRP-1 cells increased sensitivity to treatment by inactivating NRP-1/ITGB3/FAK/Akt/NF-kB p65 signaling compared to wild-type BT-474 resistant cells.ConclusionsWe thus report a novel mechanism correlating high baseline NRP-1 with upregulated TNC/ITGB3 signaling, but decreased ABCG2 expression, which sensitizes BT-474 NRP-1 cells to Adriamycin/Cyclophosphamide. The study emphasizes on the targetability of the NRP-1/ITGB3 axis and its potential as a predictive biomarker for chemotherapy response.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4446-y) contains supplementary material, which is available to authorized users.

Circulating proteins hold a potential benefit as biomarkers for precision medicine. Previously, we showed that systemic levels of neuropilin-1 (NRP-1) and its associated molecules correlated with poor-prognosis breast cancer. To further identify the role of NRP-1 and its interacting molecules in correspondence with patients' response to neoadjuvant chemotherapy (NAC), we conducted a comparative study on blood and tissue samples collected from a cohort of locally advanced breast cancer patients, before and after neoadjuvant chemotherapy (NAC). From a panel of tested proteins and genes, we found that the levels of plasma NRP-1, placenta growth factor (PlGF) and immune cell expression of the transcription factor SNAI1 before and after NAC were significantly different. Paired t -test analysis of 22 locally advanced breast cancer patients showed that plasma NRP-1 levels were increased significantly ( p = 0.018) post-NAC in patients with pathological partial response (pPR). Kaplan–Meier analysis indicated that patients who received NAC cycles and their excised tumors remained with high levels of NRP-1 had a lower overall survival compared with patients whose tissue NRP-1 decreased post-NAC (log-rank p = 0.049). In vitro validation of the former result showed an increase in the secreted and cellular NRP-1 levels in resistant MDA-MB-231 cells to the most common NAC regimen Adriyamicin/cyclophosphamide+Paclitaxel (AC+PAC). In addition, NRP-1 knockdown in MDA-MB-231 cells sensitized the cells to AC and more profoundly to PAC treatment and the cells sensitivity was proportional to the expressed levels of NRP-1. Unlike NRP-1, circulating PlGF was significantly increased ( p = 0.014) in patients with a pathological complete response (pCR). SNAI1 expression in immune cells showed a significant increase ( p = 0.018) in patients with pCR, consistent with its posited protective role. We conclude that increased plasma and tissue NRP-1 post-NAC correlate with pPR and shorter overall survival, respectively. These observations support the need to consider anti-NRP-1 as a potential targeted therapy for breast cancer patients who are identified with high NRP-1 levels. Meanwhile, the increase in both PlGF and SNAI1 in pCR patients potentially suggests their antitumorigenic role in breast cancer that paves the way for further mechanistic investigation to validate their role as potential predictive markers for pCR in breast cancer.

Background and Purpose: Patients with locally advanced breast cancer usually receive third-generation neoadjuvant chemotherapy (NAC). Although NAC treatment improved the overall survival, patients' response varies, some acquire resistance and others exhibit a conversion in their breast cancer molecular subtype. We aimed to identify the molecular changes involved in NAC resistance attempting to find new therapeutic targets in different breast cancer subtypes. Experimental Approach: We modelled NAC treatments used in clinical practice and generated resistant cell lines in vitro. The resistant cells were generated by consecutive treatment with four cycles of doxorubicin (adriamycin)/cyclophosphamide (4xAC) followed by an additional four cycles of paclitaxel (4xAC + 4xPAC). Key Results: Our data revealed distinct mechanisms of resistance depending on breast cancer subtype and drugs used. MDA-MB-231 cells resistant to 4xAC + 4xPAC activated neuropilin-1/TNC/integrin β3/FAK/NF-κB p65 axis and displayed a decrease in breast cancer resistance protein (BCRP/ABCB2). However, MCF7 cells resistant to 4xAC treatments induced HER2 expression, which converted MCF7 subtype from luminal A to luminal B HER2 type, up-regulated neuropilin-1, oestrogen receptor-α, and EGFR, and activated PI3K/Akt/NF-κB p65 axis. However, MCF7 cells resistant to 4xAC + 4xPAC exhibited down-regulation of the survival axis and up-regulated BCRP/ABCG2. Co-immunoprecipitation demonstrated a novel interaction between HER2 and neuropilin-1 driving the resistance features.Conclusions and Implications: The concurrent increase in neuropilin-1 and HER2 upon resistance and the inverse relationship between neuropilin-1 and BCRP/ABCG2 suggest that, in addition to HER2, neuropilin-1 status should be assessed in patients undergoing NAC, and as a potential drug target for refractory breast cancer.

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Breast cancer (BC) metastasis remains a leading cause of female mortality. Neuropilin-1 (NRP-1) is a glycoprotein receptor that plays ligand-dependent roles in BC. Clinical studies indicate its correlation with metastatic disease; however, its functional role in BC metastasis remains uncertain. CRISPR-Cas9 was used to knockout the NRP-1 gene in MDA-MB-231 BC cells, and the effects on metastasis were determined using an orthotopic mouse engraftment model. NRP-1 expression in knockout cells was rescued using a recombinant cDNA with a silent mutation in the sgRNA target-adjacent PAM sequence. Differentially expressed genes between NRP-1 knockout and control cells were determined using whole-transcriptome sequencing and validated using real-time PCR. NRP-1KO cells showed a pronounced reduction in the metastasis to the lungs. KEGG pathway analysis of the transcriptome data revealed that PI3K and ECM receptor interactions were among the top altered pathways in the NRP-1KO cells. In addition, reduction in metastasis enhancers proteins, Integrin-β3 and Tenascin-C, and genes CCL20 and FN1 and upregulation of metastasis suppressor genes, ACVRL and GPX3 in NRP-1KO were detected. These findings provide evidence for a functional role for NRP-1 in BC metastasis, supporting further exploration of NRP-1 and the identified genes as targets in treating metastatic BC.

The Arabian tahr/ibex is a small species of goat-antelope-like animal, indigenous to the United Arab Emirates and with a larger population in Oman. Although the animal was discovered in 1894, its numbers continue to decline, and the animal has been classified on the IUCN red list as an endangered species. Little is known about the male reproduction, specifically, extending and cryopreserving semen. This provides a crucial tool for the ex situ conservation of this endangered species. The objective of this study was to evaluate sperm characteristics of the Arabian ibex after dilution in 2 commercial extenders, namely Bioxcell® and Triladyl® based on multiple qualitative parameters. Semen was collected using an electroejaculator from males (n = 4, ~4 years old) during the reproductive season (February–April). Ejaculates were divided into 2 aliquots and extended 1 : 2 in Bioxcell® or Trilidayl® at 37°C. Diluted semen was evaluated for motility with computer-assisted semen analysis (Sperm Vision®, Minitube, Verona, WI, USA). Morphologically, samples were examined with eosin and nigrosin stain under light microscopy, in addition to structural or ultrastructural evaluation with the use of scanning (SEM) or transmission (TEM) electron microscopy. Viability tests were carried out with a LIVE/DEAD® sperm viability kit (Invitrogen, Carlsbad, CA, USA); samples were examined using a fluorescent microscope (Olympus BX51) or with a flow cytometer (FACSAriaTM III, FACSDiva software, BD) with an average flow of 20 000 sperm/event. Sperm samples were liquefied (37°C/5 min), washed with 0.9% NaCl, centrifuged (2000 rpm for 3 min), and filtered using 40-µm nylon mesh (BD, USA) before FACS analysis. Our data showed that semen diluted with Triladyl® had higher motility compared with samples extended with Bioxcell® (60 v. 41%). While eosin and nigrosin staining showed very low morphological abnormalities with both extenders (less than 2%), SEM and TEM assessment demonstrated slightly high incidents of cracked tails, ruptured membrane, and broken midpeace with samples diluted with Bioxcell®. Viability test showed that samples extended with Triladyl® had higher percentages of live sperm (33%) compared with Bioxcell® (21%). Similar viability numbers were obtained with FACSAriaTM III. These results indicate that diluting Arabian ibex semen with Triladyl® would provide improved motility, morphology, and viability compared with Bioxcell®. These findings would be extremely useful for an ex situ conservation program of the Arabian tahr/ibex.