PML controls angiogenesis and EMT to limit metastasis in MDAMB231 cells

Breast cancer is a heterogeneous disease that underlies the divergent role of some regulatory factors on tumor biology. Here we studied PML, a factor with opposing context-dependent roles in cancer. We chose to Knock Down (KD) PML in MDAMB231 and MCF7 breast cancer cell lines that represent aggressive-mesenchymal (claudin–low) and non-aggressive, epithelial (luminal A) subtype respectively. We examined tumor cell physiology in vitro and by xenografting and sought to correlate the PML-KD effects with transcriptional changes. In vitro assays showed that PML loss exacerbated the aggressive properties of MDAMB231 cells only, in correlation with deregulation of cell-cycle and adhesion related genes. PML-KD xenografts had increased local growth and metastatic ability with a preference for the lung. We propose that in tumor xenografts, PML loss facilitates long-term maintenance of tumor initiating cells and unleashes a hypoxia-angiogenic switch. Thus, PML expression may serve as a useful breast cancer biomarker. ABSTRACT PML is the core protein of the cognate nuclear particles, called PML nuclear bodies (NBs), that regulate a large number of highly divergent and often opposing functions. Being originally shown to act as a tumor suppressor PML has been recently implicated in tumor survival and metastases. Using two different Breast Cancer cell types, MDAMB231 and MCF7 we observed highly cell type and growth context dependent regulation. In line with our transcriptional profiling, PML deficient MDAMB231 but not MCF7 cells show increased cell cycle and decreased cell adhesion related features consistent with a highly aggressive propensity. In agreement with this, PML KD tumor xenografts grew faster and had an increased metastatic activity with a preference for lung and to a lesser degree the liver. PML deficient primary and lung metastatic tumors expressed increased levels of the mesenchymal TWIST2 protein. Moreover both TWIST2 and SNAIL were shown to interact with different PML isotypes implying a possible role of PML in regulating their abundance and/or activity. Notably, PML loss in MDAMB231 cells led to an enhanced hypoxia-angiogenesis phenotype and metastasis of in vivo growing cells which aquare a gene expression profile resembling the earlier described lung metastasis signature. We propose that PML limits the in vivo emergence of aggressive and metastatic tumor phenotype. Mechanistically this is linked to the long term maintenance of tumor initiating cells, potentiation of their mesenchymal phenotype and a hypoxia-angiogenic switch. we show that constitutive PML loss enhances cell migration in vitro & metastatic aggressiveness in vivo of TNBC cells. Transcriptional profiling showed enrichment in cell cycle and adhesion related gene categories deregulated by PML ablation. Moreover cells derived from in vivo primary tumors or lung metastasis stably maintain an in vivo aggressive phenotype and transcriptionally resemble the earlier described lung metastasis signature (LMS)[14] . We found that PML loss deregulates the hypoxia signaling pathway and enhances in vivo tumor angiogenesis. In addition we found that PML interacts and functionally affects the activity of EMT mediators, such as TWIST2. Thus, PML may impede results suggest that PML loss facilitated in the acquisition of a hypoxic phenotype that is intensified upon in vivo cell growth