الملخص الإنجليزي
Breast cancer is a heterogeneous disease with various pathological features and molecular profiling. Patients with locally advanced disease are treated with preoperative cycles of neoadjuvant chemotherapy (NAC) regardless of their molecular subtype and thus their response varies. Hence, we aimed to validate appropriate biomarkers involved in tumor progression, response or resistance to NAC as new therapeutic targets to promote a more precise management of patients who don't respond to the commonly used regimens. To achieve the main aim of the study we followed two different routes; clinical and in vitro translational studies.
Neuropilin-1 (NRP-1) is a multifunctional glycoprotein receptor expressed in several cancers and acts as a ligand for several molecules such as vascular endothelial growth factors (VEGF), class III/V1 semaphorins and transforming growth factor β (TGFβ). However, NRP-1 systematic levels and its association with breast cancer characteristics were not yet fully investigated. A cross-sectional study was conducted to validate NRP-1 and its associated molecules as potential biomarkers. Blood and tissue samples of breast cancer patients and age matched healthy individuals were used to measure the levels of NRP-1 and its co-receptors; placenta growth factor (PlGF),VEGF and TGFβ in circulating plasma and peripheral blood mononuclear (PBMCs) cells. The main findings of this study revealed that both plasma NRP-1 and PlGF levels were increased significantly in patients with advanced nodal and metastatic disease. In addition, in tissue sections, the expression of NRP-1 and PlGF increased in triple negative subtype compared to other breast cancer molecular subtypes. Conversely, in PBMCs, NRP-1 and its interacting molecules; class V1 semaphorin (SEMA4A) and EMT regulator, Zinc finger protein SNAI1 (SNAI1) were 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 triple negative breast cancer (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.
To understand the relationship of NRP-1 and PlGF with patients' response to chemotherapy, plasma and tissue samples were collected before and after NAC and compared with patients' response and survival data. The results revealed that elevated NRP-1 levels in the tissue post NAC were significantly correlated with poor patients' survival. Similarly, patients who remained with high plasma levels of NRP- 1 post NAC responded partially to the treatment and those who were presented with low levels of NRP-1 responded completely, which indicates that NRP-1 can be a potential pharmacodynamic biomarker for breast cancer. We found that SNAI1 expression in PBMCs in patients who received NAC was significantly increased, especially in patients who showed a complete pathological response to the treatment but did not increase in those who had a partial response, which indicates that SNAI1 might be a good candidate to be used as a predictive marker for a complete pathological response.
In an in vitro translational study, the role of NRP-1 in triggering NAC resistance was also determined by generating resistant variant cells to the commonly used drugs in neoadjuvant setting. Our data revealed distinct mechanisms of resistance depending on breast cancer subtype and drugs used. Adriamycin/cyclophosphamide (4xAC) and Paclitaxel (4xAC+4xPAC) resistant MDA-MB-231 and 4xAC resistant BT-474 cells activated NRP-1/Integrin β3/FAK/NF-κBp65 axis. In turn, resistant MCF7 cells to Adriamycin/cyclophosphamide (4xAC), induced HER-2 expression which converted MCF7 subtype from being a luminal A to luminal B-HER-2 type and upregulated NRP-1, ER-α, and EGFR and activated PI3K/Akt/NF-κBp65 survival axis. Unlike resistant MDA-MB-231 cells, resistant MCF7 and BT-474 cells upregulated BCRP/ABCG2 only upon Paclitaxel treatment. Co-Immunoprecipitation demonstrated a novel interaction between HER-2 and NRP-1 which drives the resistance features. NRP-1 transfected BT-474 (BT-474 NRP-1) cells sensitized the cells to chemotherapy and the resistant cells inactivated NRP-1/Integrin β3/FAK/NF- κBp65 survival axis.
Additionally, the knockout of NRP-1 in MDA-MB-231 cells sensitized them to the AC and PAC treatment, validating our clinical data in which, patients with low levels of NRP-1 responded better to NAC than patients with higher NRP-1 levels.
This study pin pointed the significance of circulating NRP-1-associated molecules (PlGF, SNAI1, SEMA4A, VEGFR3, PLXNA1) to characterize poor prognosis breast cancer, predicts response and survival and thus, emphasizes on their role as favorable drug targets to increase the treatment success of refractory breast cancer. The study highlighted the role of NRP-1 in driving the mechanisms of acquired resistance to NAC in different breast cancer subtypes.
