A role for FGF2 in visceral adiposity-associated mammary epithelial transformation

Abstract

Obesity is a leading risk factor for post-menopausal breast cancer, and this is concerning as 40% of cancer diagnoses in 2014 were associated with overweight/obesity. Despite this epidemiological link, the underlying mechanism responsible is unknown. We recently published that visceral adipose tissue (VAT) releases FGF2 and stimulates the transformation of skin epithelial cells. Furthermore, obesity is differentially associated with many epithelial cancers, and this mechanistic link could be translational. As FGF2 and FGFR1 are implicated in breast cancer progression, we hypothesize that VAT-derived FGF2 plays a translational role in promoting adiposity-associated mammary epithelial cell transformation. In this brief report, data suggest that FGF2/FGFR1 signaling is a potential mechanistic link in VAT-stimulated transformation of breast epithelial cells.

Keywords: FGF2; Obesity; adiposity; breast; breast cancer; cancer; visceral fat.

Figure 1.

Inhibition of FGFR1 attenuates HuFTF-stimulated transformation of MCF-10A cells. HuFTF significantly stimulated colony formation above the untreated control. Cells were treated with 200 μg/mL of HuFTF protein. HuFTF-stimulated growth in soft agar was significantly attenuated by the FGFR1 Ab. MCF-10A ells were treated with a FGFR1 neutralizing antibody (FGFR1 Ab) at 2 μg/mL and treated with HuFTF from six different donors. The percent of colony formation was calculated as described in Methods, MCF-10A cells were cultured as described in Methods. Data is presented as mean ± six biological replicates. Each biological replicate had three technical replicates. Confidence intervals (CI) were calculated for HuFTF treated MCF-10A cells (95% CI 3.024–7.658) and for HuFTF+Ab (95% CI 0.093–2.929). Statistical significance between HuFTF and control and HuFTF and HuFTF+FGFR1 Ab was determined by unpaired t-test (*p < 0.05).

Figure 2.

FGF2 transforms MCF-10A cells in a concentration-dependent manner. FGF1 and FGF2 significantly stimulated transformation of MCF-10A cells at 10 and 20 ng/mL. FGF18 significantly stimulate transformation at 10 ng/mL but not 20 ng/mL and FGF21 was not statistically significant. MCF-10A cells were cultured as described in Methods, control cells were untreated. Data is presented as mean ± of triplicate values. Statistical significance was determined by one-way ANOVA with multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001). The percent of colony formation was calculated as described in Methods.

Figure 3.

FGF2-tranformed MCF-10A cells are morphologically and functionally distinct from parent MCF-10A cells. (A) MCF-10A cells have epithelial-like morphology and transformed MCF-10A cells have spindle-like morphology. Transformed MCF-10A cells were obtained by treating MCF-10A cells with FGF2 in soft agar. After 14 days, a colony was isolated and cultured in traditional cell culture conditions for several passages, making transformed MCF-10A cells. Untreated transformed MCF-10A cells formed over 44% more colonies in soft agar compared to untreated parent MCF-10A cells. The percent of colony formation was calculated ([colonies counted × 100] 750 cells).

Figure 4.

HuFTF-stimulated transformation of MCF-10A cells is moderately associated with the HuFTF FGF2 concentration and BMI. (A) HuFTF with higher FGF2 concentrations more potently stimulated MCF-10A transformation compared with HuFTF with lower FGF2 concentrations (R² = 0.45). (B) HuFTF from donors with a higher BMI more potently stimulated MCF-10A transformation compared to HuFTF from donors with a lower BMI (R² = 0.64). (C) Higher HuFTF FGF2 concentrations is moderately associated with a higher BMI ((R² = 0.64). The % colony formation, HuFTF FGF2 concentration, and BMI of six HuFTF were used. MCF-10A cells were cultured as described in Methods. Data were analyzed with Linear regression (performed in PRISM).