Human Mammary Fibroblasts

Global Histone Deacetylation and TGF-β-Smad2/3 Signaling Activation are Induced in Gln-starved HMFs in a Class I HDAC-dependent Fashion

The TME shapes carcinoma-associated fibroblasts (CAFs) function through glutamine scarcity which influences tumor growth by interfering with metabolic pathways such as transforming growth factor-β (TGF-β) and mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling. Mezawa et al. investigated how glutamine deprivation leads to TGF-β signaling activation through class I HDAC activity and mTORC1 suppression in human mammary fibroblasts (HMFs) and ultimately drives their transformation into myCAFs and cancer progression.

They studied the effects of glutamine deprivation on TGF-β signaling and histone modification by culturing HMFs under glutamine-starved conditions in vitro. The total histone acetylation levels of H3 and H4 diminished by 65.7% and 53.8% respectively in glutamine-starved HMFs compared to their glutamine-supplemented counterparts (Fig. 1A). The histone deacetylation process reflects myCAF features observed in breast cancer which indicates that a lack of glutamine triggers TGF-β signaling activation which serves as a myCAF signature. The ratio of phosphorylated Smad2 to Smad2/3 rose 3.6 times which signifies enhanced TGF-β signaling (Fig. 1B). The mRNA levels of TGFB1, TGFB2, SERPIN1, and SMAD7 TGF-β genes increased except for ACTA2 as shown in Figure 1C. 1C). HDAC inhibitors such as trichostatin A (TSA) caused dose-dependent increases in acH3/H3 and acH4/H4 levels while decreasing pSmad2/Smad2/3 ratios (Fig. 1D). 1D). SAHA and entinostat which are class I HDAC inhibitors demonstrated effectiveness in reducing pSmad2/Smad2/3 elevation under glutamine-deficient conditions (Fig. 1E). 1E). The deficiency of glutamine leads to histone deacetylation and TGF-β signaling through class I HDAC activation.

Fig. 1. Glutamine (Gln) starvation induces global histone deacetylation and transforming growth factor-β (TGF-β)-Smad2/3 signaling activation in human mammary fibroblasts (HMFs) in a class I histone deacetylase-dependent fashion (Mezawa Y, Wang T, et al., 2023).

Morphologic Changes and Alterations in the Expression of CAF Markers after the TNC Treatment

Tenascin-C (TNC), an extracellular matrix glycoprotein, is highly expressed in breast cancer by both cancer and stromal cells. TNC expression is linked to worse outcomes in breast cancer, influencing tumor progression by promoting cancer cell proliferation, migration, and invasion.

Katoh et al. investigates how TNC affects the transformation of human mammary fibroblasts (HMFs) into cancer-associated fibroblasts (CAFs). After 4 days of TNC (10 μg/mL) treatment, HMFs mainly exhibited a spindle shape on plastic and formed more clusters on glass, similar to smooth muscle cells (Fig. 2A). TNC significantly increased α-SMA and calponin expression, indicating differentiation into myofibroblasts, but not high-molecular-weight caldesmon (Fig. 2B and C). The upregulation of platelet-derived growth factor receptor-β suggested potential smooth muscle cell differentiation. There were no significant changes in fibroblast-activation protein α, neural/glial antigen 2, prolyl-4-hydoroxylase, podoplanin, or S100A4 expressions. TNC likely induces differentiation into myofibroblasts rather than other CAF subsets. Immunofluorescence showed that treated HMFs developed prominent stress fibers with positive α-SMA/calponin staining, while untreated cells had scant fibers (Fig. 2D).

Fig. 2. Morphologic changes and the altered expression of cancer-associated fibroblast (CAF) markers in human mammary fibroblasts (HMFs) after the tenascin-C (TNC) treatment (Katoh D, Kozuka Y, et al., 2020).