Differential Roles of Fibroblast Growth Factor Receptors (FGFR) 1, 2 and 3 in the Regulation of S115 Breast Cancer Cell Growth

Fibroblast growth factors (FGFs) regulate the growth and progression of breast cancer. FGF signaling is transduced through FGF receptors 1-4, which have oncogenic or anti-oncogenic roles depending on the ligand and the cellular context. Our aim was to clarify the roles of FGFR1-3 in breast cancer cell growth in vitro and in vivo. Pools of S115 mouse breast cancer cells expressing shRNA against FGFR1, 2 and 3 were created by lentiviral gene transfer, resulting in cells with downregulated expression of FGFR1, FGFR2 or FGFR3 (shR1, shR2 and shR3 cells, respectively) and shLacZ controls. FGFR1-silenced shR1 cells formed small, poorly vascularized tumors in nude mice. Silencing of FGFR2 in shR2 cells was associated with strong upregulation of FGFR1 expression and the formation of large, highly vascularized tumors compared to the control tumors. Silencing FGFR3 did not affect cell survival or tumor growth. Overexpressing FGFR2 in control cells did not affect FGFR1 expression, suggesting that high FGFR1 expression in shR2 cells and tumors was associated with FGFR2 silencing by indirect mechanisms. The expression of FGFR1 was, however, increased by the addition of FGF-8 to starved shLacZ or MCF-7 cells and decreased by the FGFR inhibitor PD173074 in shR2 cells with an elevated FGFR1 level. In conclusion, our results demonstrate that FGFR1 is crucial for S115 breast cancer cell proliferation and tumor growth and angiogenesis, whereas FGFR2 and FGFR3 are less critical for the growth of these cells. The results also suggest that the expression of FGFR1 itself is regulated by FGF-8 and FGF signaling, which may be of importance in breast tumors expressing FGFs at a high level

The relationship between mood and sleep in different female reproductive states

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FGF-induced ERK1/2 activation in sh cells.

<p>Cells were pre-cultured in DC-FBS for 48 h and then treated with A) 25 ng/ml FGF-8b, B) 10 ng/ml FGF-2 or 100 ng/ml FGF-7 or PBS vehicle for indicated time periods. Whole-cell lysates were generated from cells and protein was subjected to SDS-PAGE and immunoblotted with antibodies against p-ERK1/2 and ERK1/2. The intensity of the bands was determined by scanning densitometry and is presented in columns as the p-ERK1/2 expression relative to ERK expression. The experiment was repeated twice with similar results.</p

Regulation of FGFR1 mRNA expression in sh cells and MCF-7 cells.

<p>Expression of total FGFR1 or FGFR1IgIIIc was quantified by qRT-PCR in the cells cultured as follows: A) The cells grown in standard growth medium were treated with PD173074 for 24 h. B) The cells were grown without testosterone (Te) for 5 days and without serum for 48 h. C) The FGF-8b-overexpressing S115 cell lines (FGF8b1 and FGF8b14) and the transfection control cell lines (Mock1 and Mock3) were cultured in 4% DC-FBS in the absence of Te. D) shLacZ cells were grown without Te for 2 days and without serum for 24 h followed by treatment with FGF-8b (25 ng/ml) for 24 h. E) MCF-7 cells were grown in standard growth medium and treated similarly with FGF-8. The experiment was performed as triplicates and repeated twice with similar results. The statistical difference between the vehicle- and FGF-8b-treated cells was determined by independent sample t-test, * <i>P</i><0.05.</p

Growth of sh cells in nude mouse tumors.

<p>1*10⁶ cells were inoculated subcutaneously (s.c.) into male nude mice (n = 5–6). A) Tumor growth was monitored every 3–4 days with a caliper until the end of the experiment. Representative growth curves from one of the two experiments with similar results are shown. Mean ± SE from each group is shown. Statistical significance of the growth rate differences were tested by Repeated Measures ANOVA, * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. B) FGFR inhibitor PD173074 was administered to mice bearing shR2 cell tumors, starting from day 13 (indicated by an arrow), at a dose of 25 mg/kg 5 times/week until the end of the experiment. Mean ± SE from each group is shown. C) FGFR mRNA levels were quantified by qRT-PCR analysis from shLacZ, shR1 and shR2 tumors from a separate experiment, in which the tumors were grown for 8 weeks in nude mice. Statistical difference between mRNA levels were tested by independent sample t-test, * <i>P</i><0.05. D) P-HisH3 immunohistochemical staining of shLacZ, shR1, shR2 and shR3 tumor sections (upper panel). Scale bar = 100 µm. Lower panel shows the number of P-HisH3-positive cells per field. P-HisH3-positive cells were counted in 5–15 fields per tumor section and each group contained six tumor sections. Statistical differences between groups were tested by ANOVA followed by Bonferroni's multiple comparison test, * <i>P</i><0.05.</p

Proliferation and cyclin expression of sh cells <i>in vitro</i>.

<p>A) shLacZ, shR1, shR2 and shR3 cells were grown in full growth medium and cell proliferation was measured at 24 h intervals. Columns represent [³H]-thymidine incorporation, and data are expressed as mean ± SD cpm/well (<i>n</i> = 8). The experiment was repeated once, with similar results. B) Effect of the FGFR inhibitor PD173074 on FGF-8b-induced proliferation in sh cells analyzed by [³H]-thymidine incorporation. sh cells were pre-cultured in DC-FBS for 48 h and then treated with FGF-8b (25 ng/ml) and/or PD173074 or PBS vehicle for 48 h. The inhibitor was added 30 minutes before addition of FGF-8b. Data are expressed as mean ± SD cpm/well (<i>n</i> = 8). The statistical differences between groups were analyzed using one-way ANOVA followed by Bonferroni's multiple comparison test. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. C) Cyclin D1 and cyclin B protein expression in untreated sh cells. Whole-cell lysates were generated from sh cells grown in full growth medium. Protein was subjected to SDS-PAGE and immunoblotted with antibodies against cyclin D1, cyclin B, 1 and β-actin. The experiment was repeated once, with similar results.</p