Abstract
Dysregulation of PML, a significant tumor suppressor is linked with cancers of different histological origins, with a decreased expression observed with a higher tumor grade. This necessitates studying the mechanisms to maintain a stable expression of PML. However much less is known about the transcriptional regulation of PML, more so in the context of breast carcinoma. ERβ has emerged as a critical factor in understanding breast cancer, especially since a huge proportion of breast cancers are ERα− and thus insensitive to tamoxifen therapy. This study aims to uncover an unidentified mechanism of PML gene regulation and its stabilization in breast cancer via ERβ signalling and the impact on cellular apoptosis. We found that clinical expression of PML positively correlates with that of ERβ both in normal and breast carcinoma samples and inversely correlates with markers of cellular proliferation, hinting towards a possible mechanistic interdependence. Both mRNA and protein expression of PML were increased in response to ERβ overexpression on multiple human breast cancer cell lines. Mechanistically, luciferase reporter assays and chromatin-immunoprecipitation assays demonstrated that ERβ can interact with the PML promoter via ERE and AP1 sites to enhance its transcription. ERβ induced stable PML expression causes a decline of its target protein Survivin and simultaneously provides a stable docking platform leading to stabilisation of its target Foxo3a, further causing transcriptional upregulation of pro-apoptotic factors p21 and p27. Immunohistochemical analyses of cancer and normal breast tissues and functional assays conducted corroborated the findings. Collectively, our study identifies ERβ signalling as a novel mechanism for PML gene regulation in ERα− breast cancer. It also reveals bi-directional downstream effect in which ‘ERβ-PML-(Foxo3a/Survivin)’ network acts as a therapeutic axis by suppressing cellular survival and promoting cellular apoptosis in breast carcinoma.
Highlights
Introduction Promyelocytic LeukemiaProtein (PML) is an essential component of Promyelocytic LeukemiaProtein (PML) Nuclear bodies (PML-NBs) where it plays a vital role in their formation and stability
All images are taken at ×200 magnification. b Scatter-plot representation of the mean H-scores of ERβ and PML in (i) adjacent normal breast (n = 24), (ii) breast carcinoma (ER+) (n = 35) and (iii) TNBC (n = 19) tissues. c Depiction of correlation coefficient between mean H-scores of ERβ and PML estimated from IHC images of both normal breast and Breast cancer (BCa) tissues combined. d Box-plots depicting distribution of H-scores of ERβ and PML, in normal breast, BCa and TNBC samples. e Comparison of combined average H-scores of ERβ and PML. f Graphical representation of mean ranks of the observed individual H-scores of ERβ and PML as obtained through calculations from M–W U-test
Concomitant loss of ERβ and PML in human breast cancer samples To explore any possible connection between ERβ and PML in BCa we conducted immunohistochemical analyses in BCa (n = 53) [including TNBC (n = 19)] and adjacent normal tissue samples (n = 24)
Summary
Introduction Promyelocytic LeukemiaProtein (PML) is an essential component of PML Nuclear bodies (PML-NBs) where it plays a vital role in their formation and stability. B Scatter-plot representation of the mean H-scores of ERβ and PML in (i) adjacent normal breast (n = 24), (ii) breast carcinoma (ER+) (n = 35) and (iii) TNBC (n = 19) tissues. C Depiction of correlation coefficient (rs) between mean H-scores of ERβ and PML estimated from IHC images of both normal breast and BCa tissues combined. D Box-plots depicting distribution of H-scores of ERβ and PML, in normal breast, BCa and TNBC samples. H Box-plots depicting relative gene expression of ERβ and PML (w.r.t GAPDH) in normal breast and BCa samples. I Depiction of correlation coefficient (rs) between mean relative expressions of ERβ and PML genes as quantified from qRT-PCR data of both normal breast and BCa tissues combined.
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