| Abstract |
Breast cancer is one of the most prevalent causes of cancer-related deaths in women worldwide. Breast tumours exhibit striking phenotypic and functional heterogeneity during the multistep course of tumourigenesis. In fact, this variability is mirrored in sustaining proliferation signalling, mutational frequencies of oncogenes and tumour suppressors, invasiveness, metastatic potential, energy metabolism, immune evasion and response to cytotoxic therapies. This heterogeneity (inter/intra-tumoural) stems from stochastic genetic and epigenetic alterations that lead to hereditary phenotypic and functional differences among the cancer cells. Despite successful treatment regimens of breast cancer, heterogeneity enhances the robustness of tumours and is associated to poor therapeutic responses and bad prognosis. Therefore, understanding the molecular networks governing breast cancer heterogeneity is required for improving the efficacy of existing agents and for developing novel strategies for personalized treatment.
The ProMyelocytic Leukemia (PML) protein is usually characterized as a tumour suppressor factor because of its implication in cell cycle arrest, apoptosis and senescence. Accordingly, PML expression is commonly lost in primary breast cancer tissue samples. However, recent reports suggest that PML may exert pro-survival functions. Notably, PML expression is elevated in chronic myelogenic leukemia (CML), gloioblastomas and in some cases of triple negative breast cancer. The oncogenic activities of PML are related to cell cycle regulation of normal and cancer stem cells as well as the maintenance of their self-renewal through metabolic pathways such as fatty acid oxidation. Consequently, PML has a dual role in tumourigenesis acting as a tumour suppressor or promoter in cell specific contexts.
The aim of this study was to elucidate the genetic and epigenetic mechanisms via which PML regulates cell proliferation and self-renewal pathways in breast cancer. Here, we show that inducible PMLIV expression inhibits cell proliferation and impairs cell cycle progression of the triple negative breast cancer (TNBC) cells, MDA-MB-231. Transcriptomic profiling identified a large number of PMLIV deregulated genes associated with various cell processes. Among them, cell cycle and division related genes and their cognitive regulators are highly ranked. Notably, PMLIV overexpression (OE) differentially expressed genes significantly overlap with the targets of FOXM1 transcription factor. We showed that PML is a potent FOXM1 repressor since it interacts with FOXM1 primarily via its DNA binding domain and downregulates FOXM1 expression at mRNA and protein level. In parallel, PML modulates the activity of FOXO3, a factor opposing some of the FOXM1 activities, to promote cell survival and stress resistance. Our findings suggest that PML affects the
balance of FOXO3 and FOXM1 transcriptional programs by acting on discrete gene subsets. We propose that high PMLIV levels may affect simultaneously diverse pathways by targeting the regulatory axis, FOXO3-FOXM1 that interlinks cell proliferation (FOXM1) to apoptosis, cell cycle arrest and pro-survival signalling (FOXO3). We also found that PMLIV affects as well other transcription factors such as NFYA, E2F, TBP that have crucial roles in transcription and cell proliferation. Collectively, our study suggests that the cell cycle arrest caused by PMLIV ectopic expression is mediated by the activity of multiple transcription factors, frequently with overlapping functions, along with changes in the epigenetic state of MDA-MB-231 cells. Preliminary experiments of PMLIV OE in another breast cancer cell type, T47D, demonstrated that PMLIV induction results in cell growth arrest but also triggers unique cell responses compared to MDA-MD-231 cells.
We also investigated the effect of PML by culturing breast cancer cells under conditions that favour growth of aggregates (tumour spheres) enriched in stem like activity. PMLIV OE decreased the tumour sphere forming efficiency of MDA-MB-231 cells indicating that PML represses the self-renewal capacity of breast cancer stem cells. In order to isolate distinct mammary epithelial subpopulations and to study the impact of PML on them, MDA-MB-231 cells were sorted based on their EpCAM/CD24 expression pattern. Interestingly, the different cell subpopulations had discrete gene signatures in terms of epithelial differentiation, EMT, proliferation and stem markers, as well as variable self-renewal ability as measured by serial in vitro sphere formation and by in vivo xenografting. Interestingly, all cell subsets were similarly sensitive to PMLIV OE. Overall, our preliminary data support the notion that contrary to earlier reports, highly tumourigenic cell subsets fall in more than one EpCAM/CD24 biotype that they all inhibited by PMLIV in a different degree.
Taken together, our study provides insights into the regulation of tumour growth by PML in breast cancer. We have defined FOXO3 and FOXM1 as PML’s discriminating partners that dictate PML’s biological output in ΜDA-MB-231 cells and signalling context and revealed a novel and potentially targetable PML-FOXO3-FOXM1 axis implicated in breast cancer proliferation. In addition, more evidence for PML’s context-depended function comes from our preliminary observations in luminal B like, T47D cells. Our initial results suggest that in the latter epithelial cancer cell line, stem cell like activity measured by sphere formation was much less affected compared to the TNBC cells. Our comparative studies show that PML deregulates both common and distinct gene sets in these cell lines that represent different breast cancer molecular and biological subtypes. We propose that PML favors both growth
inhibition and pro-survival processes at variable levels, dictated by other concurrent genetic or epigenetic cancer cell states that may account for its disparate effects in cancer.
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