Heparanase: A Potential Marker of Worse Prognosis in Estrogen Receptor Positive Breast Cancer

Introduction

Heparanase enzyme mediates ECM remodelling by specially cleaving heparan sulfate side chains from Heparan sulphate proteoglycans and releasing a multitude of bioactive molecules.We have previously shown that heparanase promotes tumor growth of primary breast tumors. To gain better insight into the role of heparanse in breast cancer prognosis, we evaluated heparanase protein and gene expression status and investigated its impact on disease free survival in a pooled analysis and in a large adjuvant phase III trial. The involvement of heparenase and its effect on drug treatment on tumorigenesis were further evaluated in vitro.

Material and Methods

Using pooled analysis of gene-expression data from over 7,000 breast cancer patients, we assessed the association of heparanase with prognosis. We further validated the heparanase prognostic effect in hormone receptor positive (HR+) tumors using primary tumors tissue microarrays from the breast international group (BIG) 2-98, phase III, prospective randomized trial. Immunohistochemistry analysis of heparanase was performed blinded to clinical outcome. Association between heparanase and clinical outcome was assessed using univariate and multivariate Cox modeling.

Furthermore, we tested the effect of different chemotherapy drugs treatment on several breast cell lines in the absence or presence of active heparenase. Cell survival and apoptosis were determined by MTT assay and flow cytometry assessment (Annexin-V -PI), respectively. Finally, we applied surprisal analysis of flow cytometry-based proteomic data on MCF7 cells following treatment with 5-Fluorouracil (5-FU) in the absence or presence of recombinant active heparanase enzyme.

Results and discussion

Our pooled analysis showed that heparanase was elevated in triple negative and HER2+ breast cancers in comparison to luminal cancers (Kruskal–Wallis test p<10e^-10), however heparanase was associated with worse prognosis only in HR+ tumors (Log rank p<10e^-10) while in the basal and HER2+ subtypes it did not affect outcome.

These results were confirmed by analysis of the BIG 2-98 phase 3 trial that showed after median follow-up of 10.1 years, that heparanase protein staining by IHC was associated with increased risk of recurrence in HR+ breast tumors (Log rank p=0.004).

Results obtained from in vitro experiments showed that survival of MCF-7 cells after treatment with 5-FU was increased, while the proportion of apoptotic death was decreased in presence of active heparanase. Finally, surprisal analysis of flow cytometry-based proteomic data showed different effects of 5-FU treatment on MCF7 cells in the absence or presence of active heparanase.

Conclusion

Heparanase is associated with worse outcome in HR+ breast cancer.