American Association for Cancer Research
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Figure S4 from JMJD6 Shapes a Pro-tumor Microenvironment via ANXA1-Dependent Macrophage Polarization in Breast Cancer

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journal contribution
posted on 2023-03-29, 14:00 authored by Bianca Cioni, Silvia Ratti, Annamaria Piva, Irene Tripodi, Matteo Milani, Francesca Menichetti, Tiziana Langella, Laura Botti, Loris De Cecco, Claudia Chiodoni, Daniele Lecis, Mario P. Colombo

Jmjd6-mediated ANXA1 secretion promotes M2 macrophage differentiation in vitro and ex vivo. A) Mean fluorescence intensity (MFI) of M1 and M2 markers in BMDM differentiated into M0, M1 and M2.** p<0,001, *** means p<0,0001, **** p<0,00001 without conditioned medium. B) Real time PCR showing the expression M1 (CD40 and NOS2) and M2 (CD206 and ARG1) markers in BMDMs differentiated into M0, M1 and M2 macrophages without CM. Datapoints show technical replicates.** p<0,001, *** p<0,0001. C) Gating strategies for FACS analysis on ex vivo mouse samples of Figure 6D. CD45+ leukocytes were gated on live cells, excluding dead cells and debris. In CD45+ population, myeloid cells were analyzed by the expression of CD11b, Gr-1, Ly6C and F4/80 markers. Monocyte-like myleloid-derived immunosuppressive cells (M-MDSC) were identified as Ly6Chigh Gr-1int expression. Granulocyte-like myeloid-derived immunosuppressive cells (G-MDSC) were identified as Gr-1high Ly6Cint cells. M2 macrophages were identified as CD206+ cells in the F4/80 gate. G-MDSC were identified as Gr-1high Ly6Cint cells. M2 macrophages were identified as CD206+ cells in the F4/80 gate.



Breast cancer is the most common type of cancer in women worldwide, with the luminal subtype being the most widespread. Although characterized by better prognosis compared with other subtypes, luminal breast cancer is still considered a threatening disease due to therapy resistance, which occurs via both cell- and non–cell-autonomous mechanisms. Jumonji domain-containing 6, arginine demethylase and lysine hydroxylase (JMJD6) is endowed with a negative prognostic value in luminal breast cancer and, via its epigenetic activity, it is known to regulate many intrinsic cancer cell pathways. So far, the effect of JMJD6 in molding the surrounding microenvironment has not been explored.Here, we describe a novel function of JMJD6 showing that its genetic inhibition in breast cancer cells suppresses lipid droplet formation and ANXA1 expression, via estrogen receptor alpha and PPARα modulation. Reduction of intracellular ANXA1 results in decreased release in the tumor microenvironment (TME), ultimately preventing M2-type macrophage polarization and tumor aggressiveness. Our findings identify JMJD6 as a determinant of breast cancer aggressiveness and provide the rationale for the development of inhibitory molecules to reduce disease progression also through the remodeling of TME composition.

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