Investigating the role of Neuropilin-1 in Triple-Negative Breast Cancer metastasis and chemoresistance

Overview

Triple-negative breast cancers (TNBC) are negative for Estrogen Receptor, Progesterone Receptor and HER2 expression, are clinically aggressive and are unresponsive to the available hormonal or targeted drugs used for other breast cancer subtypes, so that TNBC patients rely mainly on chemotherapy. TNBC accounts for 15-20% of all invasive breast cancer and patients have increased risk of recurrence, mortality and early metastatic progression. Thus, there is an urgent clinical need to develop improved treatment strategies for TNBC. Neuropilin-1 (NRP1) is a cell surface co-receptor that acts as a hub for activation of multiple growth-inducing pathways involved in progression, therapy resistance and metastasis of several cancers. We have determined NRP1 to be: A) a strong marker of poor prognosis in breast cancer patients, B) elevated in TNBC patient samples and cell lines within the Claudin-low subtype of TNBC, an aggressive subtype enriched in mesenchymal and cancer stem cell characteristics, and C) required for the growth and proliferation of Claudin-low cell lines and Claudin-low tumours in mice. We have identified elevated NRP1 expression in the stem cell-like and mammosphere-forming populations of immortalized mammary epithelial cells, observed that NRP1 is critical for the colony forming and mammary gland reconstitution abilities of mammary stem cells, and identified NRP1 as a downstream target of FOXC2, a key inducer of the mesenchymal phenotype. We hypothesise that NRP1 is a key driver of mammary cell ‘stemness’ and confers aggressive cancer stem cell-like properties to Claudin-low TNBC, and that its inhibition will impair invasion, metastasis and chemotherapy resistance of Claudin-low TNBC.

Approaches/skills and techniques

1) Validate the direct transcriptional regulation of NRP1 by FOXC2 in CLDNlow tumour models and its role in mediating mesenchymal and cancer stem cell properties. Migration, invasion, mammosphere culture and flow cytometry will be used to determine whether NRP1 regulates mesenchymal and cancer stem cell properties, including expression of CD24 and CD44, in claudin-low cells. Transcriptional regulation of NRP1 by FOXC2 will be investigated using CHIP-sequencing.

2b) Test the efficacy of NRP1 inhibition in combination with chemotherapies for Claudin-low TNBC. Informed by outcomes of Aim 2a, we will work with medical oncology colleagues to design pre-clinical trials of prioritised NRP1 inhibitor plus chemotherapy combinations to most closely recapitulate current clinical practice for treatment of TNBC. The most promising 2 combinations will be tested in a clinically relevant series of Claudin-low tumour models in mice, with mice randomised into 3 groups: 1) NRP1 inhibitor + Chemotherapy; 2) NRP1 inhibitor alone; and 3) Chemotherapy alone.

2a) Identify additive or synergistic NRP1 / chemotherapy combinations in vitro. Combination studies combining various standard of care chemotherapies (Doxorubicin, Docetaxel, Fluorouracil, Paclitaxel and Cyclophosphamide) with an NRP1 inhibitor will be performed using 3D Matrigel cell culture models analysed for growth and morphology by the IncuCyte 3D tumour spheroid and cell viability assays. NRP1 siRNA knockdown cell lines will be similarly treated with chemotherapies and effects compared to NRP1 inhibitor treatment. The most promising drug combinations will then be tested in Aim 2B.

Outcomes

This work will establish the mechanisms underlying NRP1 over-expression in Claudin-low tumours, its functional role in promoting aggressive characteristics associated with this TNBC subtype, and the efficacy of NRP1 inhibition alongside current standard of care TNBC therapies.

Keywords

• breast cancer
• cancer
• metastasis
• chemotherapy
• therapy resistance
• therapy

Contact

Contact the supervisor for more information.