Elucidation of Dysregulated Pathways Associated With Hypoxia in Oestrogen Receptor-Negative Breast Cancer

Purpose: Carbonic anhydrase IX (CAIX) is a well-established prognostic marker in breast cancer (BC). Nevertheless, this prognostic value is yet to be confirmed in BC subtypes. This study aims to investigate the prognostic effects of CAIX in oestrogen receptor (ER)-negative (ER-) BCs and to establish pathways related to cytoplasmic CAIX expression in ER- and lymph node-negative BCs.

Methods: Immunohistochemistry was performed to identify the prognostic role of CAIX protein expression in ER- tissue microarrays (TMAs) (n = 191). CAIX-positive samples (n = 37) were transcriptionally profiled by TempO-Seq and analysed by STRING. Real-time quantitative PCR (RT-qPCR) analysis was used to validate differentially expressed genes.

Results: Overexpression of cytoplasmic CAIX was an independent predictor of recurrence free survival, disease-free survival and overall survival in ER- cohort. RNA transcriptomic analysis identified 10 significant genes in ER- cohort and 3 genes in the node-negative group. The STRING database demonstrated a significant interaction between MUCL1 and GALNT6, which were linked with extracellular matrix organisation, degradation of the extracellular matrix and disease of glycosylation pathways. In the node-negative group, SPNS2 is mainly involved in the sphingolipid de novo biosynthesis pathway. A significant correlation between cytoplasmic SphK1 and cytoplasmic hypoxia-inducible factor-1α was observed. Among the 10 genes, 7 genes (SERHL2, GALNT6, MUCL1, MMP7, PITX2, CEACAM6 and SPNS2) were selected, and their expression was quantitatively assessed by RT-qPCR. The PCR data of these genes showed that SERHL2, GALNT6, MUCL1, PITX2, and SPNS2 mRNA levels were expressed in MDA-MB-231 BC cell lines at variable levels of hypoxic exposure.

Conclusion: Cytoplasmic CAIX was independently associated with poor prognosis in ER- BC. Gene expression profiles shed light on the pathways and genes associated with hypoxia in ER- BC. In node-negative patients, SPNS2 was of particular interest.