Protein and chemotherapy profiling of extracellular vesicles harvested from therapeutic induced senescent triple negative breast cancer cells

E L Kavanagh,E Collins,P O'Gorman,M H Z Guang,A Mccann,M Halasz,A Mcgoldrick,M J Higgins,P Fitzpatrick,L C Gubbins,P Dowling,S Lindsay,A Blanco-Fernández,M Henry,K Weiner-Gorzel

doi:10.1038/oncsis.2017.82

E L Kavanagh, E Collins + Show 13 more

Open Access

https://doi.org/10.1038/oncsis.2017.82

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Abstract

Triple negative breast cancer (TNBC) is an aggressive subtype with relatively poor clinical outcomes and limited treatment options. Chemotherapy, while killing cancer cells, can result in the generation of highly chemoresistant therapeutic induced senescent (TIS) cells that potentially form stem cell niches resulting in metastases. Intriguingly, senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Our aim was to profile EVs harvested from TIS TNBC cells compared with control cells to identify a potential mechanism by which TIS TNBC cells maintain survival in the face of chemotherapy. TIS was induced and confirmed in Cal51 TNBC cells using the chemotherapeutic paclitaxel (PTX) (Taxol). Mass spectrometry (MS) analysis of EVs harvested from TIS compared with control Cal51 cells was performed using Ingenuity Pathway Analysis and InnateDB programs. We demonstrate that TIS Cal51 cells treated with 75 nM PTX for 7 days became senescent (senescence-associated β-galactosidase (SA-β-Gal) positive, Ki67-negative, increased p21 and p16, G2/M cell cycle arrest) and released significantly more EVs (P=0.0002) and exosomes (P=0.0007) than non-senescent control cells. Moreover, TIS cells displayed an increased expression of the multidrug resistance protein 1/p-glycoprotein. MS analysis demonstrated that EVs derived from senescent Cal51 cells contained 142 proteins with a significant increased fold change compared with control EVs. Key proteins included ATPases, annexins, tubulins, integrins, Rabs and insoluble senescence-associated secretory phenotype (SASP) factors. A fluorescent analogue of PTX (Flutax-2) allowed appreciation of the removal of chemotherapy in EVs from senescent cells. Treatment of TIS cells with the exosome biogenesis inhibitor GW4869 resulted in reduced SA-β-Gal staining (P=0.04). In summary, this study demonstrates that TIS cells release significantly more EVs compared with control cells, containing chemotherapy and key proteins involved in cell proliferation, ATP depletion, apoptosis and the SASP. These findings may partially explain why cancer senescent cells remain viable despite chemotherapeutic challenge.

Highlights

In Ireland, ~ 2883 women will be diagnosed annually with breast cancer, representing the highest incidence of all female cancers in Ireland, with 10–15% described as triple negative breast cancer (TNBC).[1]
In light of the ability of chemotherapy to induce viable therapeutic induced senescent (TIS) cancer cells, and the documented preponderance of extracellular vesicle (EV) release from senescent compared with non-senescent cells, the overall aim of this study was to investigate the chemotherapy and protein content of EVs derived from TIS cancer cells and determine whether the resultant profiles may partially explain why cancer senescent cells remain viable despite chemotherapeutic challenge
TIS cells release significantly more EVs than control cells EVs were isolated from cells by differential centrifugation unless otherwise stated

Summary

Introduction

In Ireland, ~ 2883 women will be diagnosed annually with breast cancer, representing the highest incidence of all female cancers in Ireland, with 10–15% described as triple negative breast cancer (TNBC).[1] TNBC is characterised by its aggressive behaviour and lack of expression of the oestrogen, progesterone and HER2 receptors This results in TNBCs not being amenable to endocrine and trastuzumab-based therapies, with conventional cytotoxic therapies such as the chemotherapeutic paclitaxel (PTX) (Taxol) the only option.[2] TNBC is further characterised by early onset and poor prognosis,[3] with TNBC tumours being at a higher risk for subsequent tumour recurrence[4] owing to its chemoresistant biology. When TNBC relapses, it usually presents as distant metastases, which tend to have central nervous system involvement, rather than local, more treatable metastases.[5].

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