Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Background: As 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. Methods: We 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. Results: We 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. Conclusions: These results uncover a key role for ID4 in dictating the behaviour of tumour-associated macrophages in breast cancer

The mutant p53-ID4 complex controls VEGFA isoforms by recruiting lncRNA MALAT1

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 the key role for MALAT1 in control of VEGFA isoforms expression in breast cancer cells expressing gain-of-function mutant p53 and ID4 proteins

Additional file 1: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Figure S3 Growth curve of MDA-MB-468 cells depleted (si-ID4) or not (si-SCR) of ID4 expression by siRNA transfection (a). Cells were transfected for 16 hours, and then equal numbers of cells were plated and counted at the indicated time points. Efficiency of ID4 depletion at 48 hours and 72 hours was evaluated by Western blotting (b). (PDF 4554 kb

Additional file 3: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Figure S1. Comparison of ID4 mRNA expression in basal-like breast cancer (BLBC) and triple-negative breast cancer (TNBC) versus all other breast cancer subtypes (Others) in the indicated representative datasets [19, 22, 60]. (PDF 143 kb

Additional file 6: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Figure S2. Predictive power of ID4 mRNA expression for overall survival (OS) was evaluated by Kaplan-Meier analysis on the TCGA cohort in BLBCs showing high or low CD68 (a and b) or macrophage signature (MacSig) (c and d) levels. Macrophage signature is composed of eight widely used markers for the mononuclear phagocyte system (CD14, CD105, CD11b, CD68, CD93, CD33, IL4R and CD163 [37]). e Evaluation of association between ID4 or CD68 and the pathological variables T, N, G and TP53 status in the BLBCs from the TCGA cohort. (PDF 4464 kb

Additional file 5: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Table S3 mRNAs modulated in an ID4-dependent manner in differentiated HL60 cells cultured with conditioned medium from control (CM EV) or ID4-overexpressing (CM ID4) MDA-MB-468 cells. The presence of HIF-1 consensus sequences on promoters was evaluated using the LASAGNA-Search web tool (http://biogrid-lasagna.engr.uconn.edu/lasagna_search/). The presence of putative binding sites for miR-107, miR-15b and miR-195 on 3′-UTR or coding (CDS) sequences of mRNAs was evaluated using the miRWalk analysis tool ( http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/ ) by selecting the following databases: (1) 3′-UTR analysis = miRWalk, miRanda, miRDB, miRNAMap, Pictar2, RNA22, RNAhybrid, TargetScan; and (2) CDS analysis = miRWalk, miRanda, RNA22, RNAhybrid, TargetScan. (DOCX 22 kb

Additional file 8: of Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

Figure S5 a Expression of miR-107, miR-15b and miR-195 in differentiated HL60 cells cultured with CM from control (CM EV) or ID4-overexpressing (CM ID4) MDA-MB-468 cells. b–e Expression of miR-15b and miR-195 in HL60 and U937 cells cultured with CM from control (si-SCR) or ID4-depleted (si-ID4) BC cells. f miR-107, miR-15b and miR-195 expression evaluated by RT-qPCR in differentiated U937 cells cultured with CM from MDA-MB-468 cells depleted or not of VEGFA expression. VEGFA interference efficiency is shown in Fig. 3i. g Expression levels of miR-15b and miR-195 in differentiated U937 cells cultivated in RPMI medium (CTR) or CM from MDA-MB-468 cells for the indicated time points. h and i HIF1A mRNA (h) and protein (i) expression evaluated, respectively, by RT-qPCR and immunofluorescence in differentiated U937 cells transfected with control mimic or miR-107 mimic and cultured in the presence of CM from MDA-MB-468 cells for 48 hours. (PDF 2150 kb