The abundant, nuclear‐retained, metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1) has been associated with a poorly differentiated and aggressive phenotype of mammary carcinomas. This long non‐coding RNA (lncRNA) localizes to nuclear speckles, where it interacts with a subset of splicing factors and modulates their activity. In this study, we demonstrate that oncogenic splicing factor SRSF1 bridges MALAT1 to mutant p53 and ID4 proteins in breast cancer cells. Mutant p53 and ID4 delocalize MALAT1 from nuclear speckles and favor its association with chromatin. This enables aberrant recruitment of MALAT1 on VEGFA pre‐mRNA and modulation of VEGFA isoforms expression. Interestingly, VEGFA‐dependent expression signatures associate with ID4 expression specifically in basal‐like breast cancers carrying TP53 mutations. Our results highlight a key role for MALAT1 in control of VEGFA isoforms expression in breast cancer cells expressing gain‐of‐function mutant p53 and ID4 proteins.

BackgroundAs crucial regulators of the immune response against pathogens, macrophages have been extensively shown also to be important players in several diseases, including cancer. Specifically, breast cancer macrophages tightly control the angiogenic switch and progression to malignancy. ID4, a member of the ID (inhibitors of differentiation) family of proteins, is associated with a stem-like phenotype and poor prognosis in basal-like breast cancer. Moreover, ID4 favours angiogenesis by enhancing the expression of pro-angiogenic cytokines interleukin-8, CXCL1 and vascular endothelial growth factor. In the present study, we investigated whether ID4 protein exerts its pro-angiogenic function while also modulating the activity of tumour-associated macrophages in breast cancer.MethodsWe performed IHC analysis of ID4 protein and macrophage marker CD68 in a triple-negative breast cancer series. Next, we used cell migration assays to evaluate the effect of ID4 expression modulation in breast cancer cells on the motility of co-cultured macrophages. The analysis of breast cancer gene expression data repositories allowed us to evaluate the ability of ID4 to predict survival in subsets of tumours showing high or low macrophage infiltration. By culturing macrophages in conditioned media obtained from breast cancer cells in which ID4 expression was modulated by overexpression or depletion, we identified changes in the expression of ID4-dependent angiogenesis-related transcripts and microRNAs (miRNAs, miRs) in macrophages by RT-qPCR.ResultsWe determined that ID4 and macrophage marker CD68 protein expression were significantly associated in a series of triple-negative breast tumours. Interestingly, ID4 messenger RNA (mRNA) levels robustly predicted survival, specifically in the subset of tumours showing high macrophage infiltration. In vitro and in vivo migration assays demonstrated that expression of ID4 in breast cancer cells stimulates macrophage motility. At the molecular level, ID4 protein expression in breast cancer cells controls, through paracrine signalling, the activation of an angiogenic programme in macrophages. This programme includes both the increase of angiogenesis-related mRNAs and the decrease of members of the anti-angiogenic miR-15b/107 group. Intriguingly, these miRNAs control the expression of the cytokine granulin, whose enhanced expression in macrophages confers increased angiogenic potential.ConclusionsThese results uncover a key role for ID4 in dictating the behaviour of tumour-associated macrophages in breast cancer.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-0990-2) contains supplementary material, which is available to authorized users.

Gain-of-function (GOF) mutations in the TP53 gene lead to acquisition of new functions by the mutated tumor suppressor p53 protein. A number of the over-represented ‘hot spot’ mutations, including the ones in codons 175, 248 or 273, convey GOF phenotypes. Such phenotypes may include resistance to chemotherapeutics or changes in motility and invasiveness. Whereas the prevalent notion is that the acquisition of the p53 GOF phenotype translates into poorer prognosis for the patient, the analysis of a human somatic p53 mutations dataset demonstrated earlier tumor onset, but decreased frequency and altered location of metastases in patients with the p53-R248Q allele. Therefore, the GOF activities of p53-R248Q and p53-D281G were analyzed in triple negative breast cancer MDA-MB-231 and lung adenocarcinoma H1299 cell lines with regard to invasive and metastatic traits. The expression of p53-D281G increased the motility and invasiveness of the lung cancer cells, but not those of the breast cancer cells. In contrast, the expression of p53-R248Q decreased the motility and invasiveness of the breast and lung cancer cells in a p53 transactivation-dependent manner. The intravenous xenotransplantation of MDA-MB-231 cells expressing p53-R248Q into zebrafish embryos resulted in an alteration of the distribution of cancer cells in the body of the fish. In p53-R248Q-expressing H1299 cells a decrease in the expression of TCF8/ZEB1 and N-cadherin was observed, suggesting partial mesenchymal-to-epithelial transition. In the two cell lines expressing p53-R248Q a decrease was noted in the expression of myosin light chain 2, a protein involved in actomyosin-based motility. To the best of our knowledge, the present study is one of only few reports demonstrating the mutated p53 GOF activity resulting in a decrease of a malignant trait in human cancer.

Human p53 protein acts as a transcription factor predominantly in a tetrameric form. Single residue changes, caused by hot-spot mutations of the TP53 gene in human cancer, transform wild-type (wt) p53 tumor suppressor proteins into potent oncoproteins - with gain-of-function, tumor-promoting activity. Oligomerization of p53 allows for a direct interplay between wt and mutant p53 proteins if both are present in the same cells – where a mutant p53's dominant-negative effect known to inactivate wt p53, co-exists with an opposite mechanism – a “dominant-positive” suppression of the mutant p53's gain-of-function activity by wt p53. In this study we determine the oligomerization efficiency of wt and mutant p53 in living cells using FRET-based assays and describe wt p53 to be more efficient than mutant p53 in entering p53 oligomers. The biased p53 oligomerization helps to interpret earlier reports of a low efficiency of the wt p53 inactivation via the dominant-negative effect, while it also implies that the “dominant-positive” effect may be more pronounced. Indeed, we show that at similar wt:mutant p53 concentrations in cells – the mutant p53 gain-of-function stimulation of gene transcription and cell migration is more efficiently inhibited than the wt p53's tumor-suppressive transactivation and suppression of cell migration. These results suggest that the frequent mutant p53 accumulation in human tumor cells does not only directly strengthen its gain-of-function activity, but also protects the oncogenic p53 mutants from the functional dominance of wt p53.