Figure 6. Overexpression of FoxF1 or FoxF2 promotes tumor growth by decreasing expression of p21^Cip1 and p27^Kip1.

(A) Representative tumors formed 3 weeks after receiving intra-muscular injection of 2 ×10⁵ 76-9 cells transduced with an empty-vector control or FoxF1 overexpression retrovirus (FoxF1-OE). Tumor weights and volumes for mice (n=5) are shown as mean ± SD. (B) Efficiency of FoxF1 overexpression was confirmed by qRT-PCR using total RNA isolated from orthotopic RMS tumors. FoxF1 expression levels were normalized to β-actin mRNA. (C) Overexpression of FoxF1 decreased protein levels of pRb, p21^Cip1 and p27^Kip1 shown by Western blot using of total protein from RMS tumors. Blots were probed with antibodies against phosphorylated Rb (pRb), total Rb, p21^CIP1, p27^Waf1/Cip1, Flag, FoxF1, p53, phospho-p53, or β-actin (left panel). Relative expression levels of these proteins were determined with densitometry and normalized to β-actin (right panel). AU=arbitrary units. (D) Representative tumors formed 3 weeks after receiving intra-muscular injection of 2 ×10⁵ 76-9 cells transduced with an empty-vector control or FoxF2 overexpression retrovirus (FoxF1-OE). Tumor weights and volumes for mice (n=7) are shown as mean ± SD. (E) Efficiency of FoxF1 overexpression was confirmed by qRT-PCR using total RNA isolated from orthotopic RMS tumors (left panel). Overexpression of FoxF2 increased p21^Cip1 mRNA as shown by qRT-PCR using total RNA isolated from rhabdomyosarcoma tumors (right panel). β-actin mRNA was used for normalization. All data represent mean ± SD of three independent determinations using tumor tissue from n=5–7 mice in each group. (F) Overexpression of FoxF1 or FoxF2 increased proliferation of RMS tumor cells in vitro. 76-9 control, FoxF1 OE, or FoxF2 OE cells were plated in triplicates. Cell numbers were measured every 24 hours for 3 days. Each point represents mean±SEM. Data represent mean±s.d. of three independent experiments. A p value <0.05 is shown with (*), a p value <0.01 is shown with (**).