Asanprakit, Wichitra
(2021)
The role of polymeric immunoglobulin receptor in breast cancer.
PhD thesis, University of Nottingham.
Abstract
Introduction
Neoadjuvant chemotherapy (NAC) is used with increasing frequency as the primary treatment for breast cancer. A pathological complete response (pCR), which contributes to survival benefit is achieved in only 25-30% of patients with little benefit and significant morbidity in the remainder. Therefore, predicting the patients who are likely to achieve a pCR before commencing NAC is desirable. A pilot study of 10 large and locally advanced breast cancer biopsy specimens, using next generation RNA sequencing and validated by real-time quantitative polymerase chain reaction (real- time qPCR) and Western blotting, demonstrated that the expression of polymeric immunoglobulin receptor (PIGR) was significantly increased in breast cancers achieving a pCR compared with non-pCR. PIGR has a major role in mucosal immunity as a transporter of polymeric immunoglobulin across the epithelial cells. Increased PIGR expression has been reported in many cancers and is related to patient outcome and response to chemotherapy. The role of PIGR in breast cancer was expanded in this thesis.
Methods
This thesis consists of preclinical and clinical studies. The preclinical study was performed using MCF7 and MDA-MB468 breast cancer cell lines. The basal level of PIGR mRNA and protein expression in MCF7 and MDA-MB468 cells was evaluated by real-time qPCR and Western blotting. MCF7/PIGR and MDA-MB468/PIGR stable cell lines, overexpressing the PIGR gene, were generated using a lentiviral vector with tetracycline dependent induction of expression. Cell viability, cell proliferation and chemo-sensitivity of PIGR transduced cells were evaluated and compared with un-transduced cells to determine the effect of PIGR overexpression on cell phenotype. The effect of PIGR expression in breast cancer cells on macrophage polarisation was investigated by treatment of bone marrow derived macrophages (BMDMs) with MDA-MB468/PIGR conditioned media. Real-time qPCR was performed to evaluate the expression of M1 (interleukin [IL]-1β and inducible nitric oxide synthase [iNOS]) and M2 (IL-10 and arginase [ARG] 1) macrophage markers which demonstrated polarisation of macrophages. On the other hand, M1, M2 macrophage conditioned media and recombinant human cytokines: interferon (IFN)-γ, IL-1β, tumour necrosis factor (TNF)-α, IL-10 and transforming growth factor (TGF)-β were used to determine the role of macrophages and cytokines on PIGR expression in breast cancer cell lines. For the clinical study, PIGR mRNA expression was determined by the RNA in situ hybridization assay using formalin-fixed paraffin- embedded specimens of pre-treatment breast cancer core biopsy from 46 women with breast cancer who received NAC. The expression of PIGR and its associations with pCR and overall survival was examined.
Results
The levels of PIGR mRNA and protein expression were significantly higher in MDA- MB468 cells than in MCF7 cells (380-fold, p <0.0001). However, the differential expression of PIGR in these two cell lines did not lead to significant differences in chemo-sensitivity. Viral overexpression of PIGR was also not found to change any of the parameters measured in either cell line. There was no significant difference in M1 and M2 markers between BMDMs treated with conditioned media of MDA-MB468/PIGR with or without induction of PIGR expression. In contrast, M1 macrophage conditioned media induced a striking dose-dependent increase in PIGR mRNA expression in MDA-MB468 cells, up to 20-fold in 100% conditioned media. IFN-γ and IL-1β were able to increase PIGR expression in MDA-MB468 cells while TNF-α, IL-10 and TGF-β were not. IL-1β mRNA expression increased significantly in M1 macrophages, however, IFN-γ mRNA expression was not detected. The role of IL-1β secreted from M1 macrophages in increasing expression of PIGR was confirmed by IL-1 receptor blockade indicating that IL-1β is the M1 macrophage cytokine that enhances PIGR expression in breast cancer cells. The study of PIGR expression in breast cancer tissues from the patients demonstrated that PIGR mRNA expression is lower in breast cancer compared with normal breast tissue (median H score: 0 vs.120, p <0.0001). There was no statistically significant difference in the proportion achieving a pCR in PIGR positive compared with PIGR negative tumours (22.2% vs. 32.1%, p = 0.466). Five-year overall survival of the patients with PIGR positive and negative tumours was 88.9% and 78.3%, respectively. However, overall survival was not significantly different (p = 0.406).
Conclusions
PIGR did not affect cellular behaviour and chemo-sensitivity of these breast cancer cell lines and did not polarise macrophages. Nevertheless IL-1β, which is the M1 macrophage cytokine, enhanced PIGR expression in breast cancer cells. These results imply that increased PIGR expression in breast cancer in vivo may reflect the polarisation state of tumour-associated immune cells. However, the expression of PIGR did not appear to be associated with a pCR after NAC and did not correlate with overall survival in this group of breast cancer patients.
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