Neuropilin-1 promotes the oncogenic Tenascin-C/integrin β3 pathway and modulates chemoresistance in breast cancer cells

Abstract

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.

Modeling Neoadjuvant chemotherapy resistance in vitro increased NRP‐1 and HER2 expression and converted MCF7 breast cancer subtype

“…Instead, NRP‐1 plays a key role in promoting resistance in these cells. The same inverse relationship between NRP‐1 and BCRP/ABCG2 expression was observed in resistant MCF7 cells and the previous resistance model in BT‐474 cells (Naik et al, ). Down‐regulation of BCRP/ABCG2 expression in resistant MDA‐MB‐231 cells can be a consequence of PTEN overexpression and down‐regulation of p‐AKT similar to that reported in chronic myeloid leukaemia cells (Huang et al, ).…”

“…In this study, we identified distinct mechanisms of resistance in the two different subtypes of breast cancer cells, as illustrated in Figure . We have already reported that recombinant overexpression of NRP‐1 in BT‐474 (BT‐474 NRP‐1) breast cancer cells induced the TNC/integrin β3/FAK/NF‐κB p65 survival axis (Naik et al, ). In this study, we showed that resistance to NAC in MDA‐MB‐231 cells caused an increase in NRP‐1 levels and hence increased the same survival axis, indicating that NRP‐1 up‐regulation is involved in MDA‐MB‐231 cell survival and resistance to NAC.…”

“…Reaction mixture for real‐time PCR was prepared using Fast SYBR Supermix (Applied Biosystems, USA); 15 ng of cDNA and 100 μM of specific primers were designed using Primer Express Software (Primer Express, ; Applied Biosystems, USA). Primers for tenascin C ( TNC ) amplification and the reference genes, actin and GUSB, were listed in the additional file of our previous publication (Naik et al, ). The 7500 Fast Real‐Time PCR System (Applied Biosystems, USA) was used to quantify the expression of TNC under the following optimized conditions: enzyme activation at 95°C for 20 s followed by 40 cycles of denaturing at 95°C for 3 s and annealing/extension at 63.4°C for 30 s (primers concentration and annealing/extension were optimized before targets quantification).…”

Extracellular Matrix in the Tumor Microenvironment and Its Impact on Cancer Therapy

“…Especially under hypoxic conditions, CAFs produce high levels of TGFβ, which induces stem celllike properties in tumor cells including increased resistance to chemotherapy (Tang et al, 2018). Finally, the complex ECM produced by CAFs interferes with therapeutic response by forming a shielding barrier and providing prompts for protective signaling, e.g., via interaction with integrins and cadherins (Eke et al, 2010;Naci et al, 2012;McGrail et al, 2015;Jakubzig et al, 2018;Naik et al, 2018). Based on these observations, it should be possible to significantly improve drug distribution and response to therapy by depleting CAFs in the tumor stroma.…”

Epithelium morphogenesis and oviduct development are regulated by significant increase of expression of genes after long-term in vitro primary culture – a microarray assays

“…TNC and ITGB3 interaction has been described e.g. in breast cancer cells, where it resulted in FAK/Akt-473 pathway activation and cell migration [31]. In this case, such interaction may indicate tube forming activity in OECs, as integrin-mediated signaling pathway is known to be involved in epithelial apicobasal polarization -a crucial event in tubal development [6].…”