Gene expression analysis of murine normal mammary gland fibroblasts (mNFs) after perturbing the expression of ASPA (silencing and over-expression)

Metabolic reprogramming is a hallmark of cancer, and the field has predominantly focused on investigating metabolic alterations in tumour cells. However, the relevance, mechanism and consequences of metabolic adaptations in stromal cells remains largely unexplored. Here, we identify Aspartoacylase (ASPA) as a metabolic enzyme consistently repressed in tumour stroma and cancer-associated fibroblasts (CAFs). Loss of ASPA is associated with the accumulation of its substrate, N-acetylaspartate (NAA), and we corroborate the relevance of this metabolite in the control of macrophage polarization. Importantly, we report an unprecedented reciprocal crosstalk of ASPA with TGFß signalling. TGFß suppresses ASPA expression, whereas ASPA restrains TGFß-dependent myofibroblast conversion and the generation of aggressive pro-tumoral responses in fibroblasts. Analyses of human specimens revealed a strong negative prognostic value for ASPA in different tumour types, associated with pro-tumoral macrophages and TGFß signalling levels. Our findings unveil ASPA expression in fibroblasts as a previously unknown gatekeeper of TGFß responses and activation in cancer, and shed light into the intratumoral metabolic crosstalk that governs the process of cancer progression. Overall design: RNA-seq profiling of murine mammary normal fibroblasts (mNF1) after silencing ASPA with two independent shRNAs. Scramble shRNA (shC) and two individual shRNA targeting Aspa (Sh3-ASPA and Sh4-ASPA), under doxycyclin control, were transfected in mNF1, and selection was performed using Blasticidin at 10 µg/mL final concentration. Stable clones were cultured in subconfluency in full DMEM (10% FBS) and treated with 300ng/mL of doxycyclin for 5 days, and RNA was extracted. In addition, RNA-seq profiling of mNF1 control and over-expressing a wild-type form of ASPA, after treatment with vehicle or TGF-beta1 , on duplicates. mNF1 were stably transfected with a doxycyclin-inducible cDNA for ASPA and selected using Blasticidin at 10 µg/mL final concentration. Stable clones were cultured in full DMEM (10%FBS) and treated with Vehicle (control) or with 300ng/mL of doxycyclin for 5 days to induce ectopic ASPA expression (mNF1-ASPA). Cells were then exposed to vehicle (Vh) or to 10 ng/mL of TGF-beta1 for 24 h, and RNA extracted.