The evaluation of hyaluronan metabolic enzymes as a therapeutic target in the treatment of breast cancer stem cells
2017-02-26T22:39:16Z (GMT) by
Breast cancer is the most common cancer in women and remains a leading cause of cancer-related mortality attributable to disease recurrence and metastases highlighting the need for improved therapeutics. Recently, a shift in the cancer biology paradigm has acknowledged that a small subset of cells, termed Cancer Stem Cells (CSCs), play a role in poor patient outcome. CSCs reside within a hyaluronan-rich component of the tumor microenvironment (TME). Hyaluronan (HA) accumulation is associated with an aggressive phenotype in breast cancer and its anabolic enzymes, the HA synthases (HAS), have recently been implicated in CSC plasticity. Although characterised in heterogeneous tumors, there is a gap in knowledge surrounding the role HA metabolism may play in the maintenance of the CSC subpopulation, its niche and ultimately disease outcome. The primary objective for this thesis was therefore to ascertain if breast cancer cell subpopulations display varying HA metabolic potentials. Additionally, we aim to demonstrate that modulation of HA metabolism impacts the renewal and maintenance of CSCs thus highlighting this process as a potential therapeutic target. Cell subpopulations isolated from basal and luminal breast cancer cell lines using FACS (isolation was based on CD44/CD24 and ALDEFLUOR expression) were analysed for mRNA levels of HA metabolic enzymes (HAS1-3) and (Hyal1-3) using qRT-PCR. Cell-associated and liberated HA synthesised by each subpopulation was quantitated using HA-ELISA where molecular weight was characterised by size exclusion chromatography. Following a modified Boyden chamber assay, HA metabolic enzymes associated with invasive subpopulations was determined using immunohistochemistry. Finally, the impact of HA synthesis modulation was assessed; transfectant cell lines were generated reducing or increasing endogenous levels of HAS2 in basal and luminal breast cancer cell lines. Response to chemical modulation via exposure to 4-MU, DoN or AICAR was examined. Cells were monitored for proliferation and HA production in adherent and non-adherent cultures. Changes in subpopulation frequency were analysed using FACS. CSCs (CD44+/CD24 /low) preferentially expressed HAS2 and Hyal2 translating to the production of high levels HA and an invasive phenotype. CSCs liberated large quantities of 520kDa HA (48pg/cell/24h) and retained a thick 26-54µM HA-dependent glycocalyx while non-CSCs (CD44+/CD24+) liberated 37pg/cell/24h of 220kDa HA and did not retain a detectable glycocalyx. Modulation of HAS2 expression resulted in reciprocal changes in CSC frequency; HAS2-overexpression led to a 7-fold increase whilst antisense HAS2-transfected cells demonstrated a 7,500-fold reduction in CSCs. These changes coincided with concomitant fluctuations in non-CSC subpopulations. Chemical inhibition of HAS aligned with these results; 4-MU and DoN were able to chemically-reduce HA synthesis by 20% and 88% respectively and diminish the capacity to maintain growth in non-adherent conditions resulting in a reduction in CSC frequency. Additionally, a direct correlation between increased cell death and the expression of a CD44+/CD24-/low/HAS2+/HYAL2+ (Hyaluronan Metabolic Signature; HMS) was observed. Overall, we have characterised a unique HMS that provides CSCs with an innately aggressive phenotype which may contribute to the CSC niche. We have demonstrated that HAS2 directly impacts the CSC subpopulation where a reduction in HA synthesis either through genetic or chemical intervention results in a decrease in the CSC subpopulation thereby substantiating HA metabolism as a valid therapeutic target.