Expression analysis and prognostic significance of androgen receptor and cancer stem cell markers in invasive breast cancer with emphasis on metaplastic carcinoma
Date of Award
Doctor of Philosophy in Health Science (PhD)
Biological and Biomedical Sciences
A large body of experimental data supports hierarchical organization of breast cancer (BCa) such that tumor initiation, progression and therapeutic resistance are driven by rare population of cancer stem cells (CSCs). In breast, CSCs have been identified by expression of CD44'/CD24-and ALDH1+ phenotypes. It has been suggested that CSCs exhibit plasticity and transition between epithelial and mesenchymal phenotypes, favoring metastasis. While metaplastic carcinoma of breast (MCa) is a prototype enriched in CSCs and epithelial to mesenchymal transition gene signature, formation of metaplastic elements within the tumor is not well studied. There has been a renewed interest in understanding the biological role of androgens and androgen receptor (AR) in BCa however, prognostic significance of AR in relation to CSC markers across BCa subtypes is not known. To evaluate the potential prognostic value of AR and CSC markers, expression of AR, CD44, CD24 and ALDH1 was determined by immunohistochemistry (IHC) on formalin fixed paraffin embedded (FFPE) sections of 166 stage I-III invasive BCa cases. Expression of AR was observed in 62.7% tumors whereas CD44, CD24 and ALDH1 were expressed in 61.4%, 44% and 30.1% tumors, respectively. AR expression correlated with small tumors, lower grade, ER/PR expression (p < 0.05) and remained an independent prognostic indicator associated with improved outcome (p = 0.016). Significant association was observed between concordant expression of AR and CD24 (p = 0.001) with favorable impact on survival (p = 0.007), whereas expression of CSC phenotypes (CD44+, CD44+/CD24-and ALDH1±) did not correlate with adverse outcome (p > 0.05). However, AR retained the association with better survival even in patients whose tumors expressed CSC markers. Prognostic significance of AR and CSC markers was further evaluated in 197 stage I-III triple negative breast cancer (TNBC) cases. AR expression was found in 18.7% tumors while CD44, CD24 and ALDH1 were expressed in 29.4%, 33% and 13.2% cases, respectively. Lack of AR expression correlated with grade III tumors (p < 0.001), basal markers (CK5, CK14 and nestin), Bc12 (p = 0.04), COX2 (p =0.02) and CD44+/CD24-phenotype (p < 0.001). Significantly adverse overall survival (OS: p = 0.01) and breast cancer specific survival (BCSS: p = 0.04) were observed in tumors lacking AR expression. This work was extended to 29 cases of stage I-III MCa and the analysis revealed that AR was expressed in 41.6% of the evaluable cases, while expression of CD44, CD24 and ALDH1 was found in 44.3%, 51.7% and 20.7% cases, respectively. MCa cases displayed poor OS compared to TNBC cases (p = 0.054) and worst BCSS when TNBC cases were stratified by basal and non-basal subtypes (p =- 0.021). To investigate occurrence of EMT in MCa, a cell line model, AKU-BC42, was established from primary culture of a breast tumor biopsy. AKU-BC42 expressed AR, luminal and basal cytokeratins. In addition, expression of vimentin and low expression of E-cadherin was observed. Expression of mesenchymal stern cell (MSC) markers was assessed by flow cytometry and revealed that CD73, CD90 and CD105 were expressed in 49.4%, 2.9%, 4.7% of cells in AKU-BC42, respectively. AKU-BC42 demonstrated adipogenic, osteogenic and chondrogenic differentiation potential when cultured under defined conditions. Further in vitro and in vivo studies are needed to understand the biological role of AR in TNBC and modulation of EMT in MCa. These studies will provide insight into rationale for AR targeted therapies in these aggressive tumors.
Riaz, N. e. (2020). Expression analysis and prognostic significance of androgen receptor and cancer stem cell markers in invasive breast cancer with emphasis on metaplastic carcinoma (Unpublished doctoral thesis). Aga Khan University, Karachi, Pakistan.