Fibroblast growth factor receptor 1-transformed mammary epithelial cells are dependent on RSK activity for growth and survival

Abstract

Fibroblast growth factor receptor 1 (FGFR1) is frequently amplified and highly expressed in lobular carcinomas of the breast. In this report, we evaluated the biological activity of FGFR1 in a wide range of in vitro assays. Conditional activation of FGFR1 in the nontransformed MCF10A human mammary cell line, MCF10A, resulted in cellular transformation marked by epidermal growth factor-independent cell growth, anchorage-independent cell proliferation and survival, loss of cell polarity, and epithelial-to-mesenchymal transition. Interestingly, small-molecule or small interfering RNA inhibition of ribosomal S6 kinase (RSK) activity induced death of the FGFR1-transformed cells, but not of the parental MCF10A cell line. The dependence of FGFR1-transformed cells on RSK activity was further confirmed in cell lines derived from mouse and human lobular carcinomas that possess high FGFR1 activity. Taken together, these results show the transforming activity of FGFR1 in mammary epithelial cells and identify RSK as a critical component of FGFR1 signaling in lobular carcinomas, thus implicating RSK as a candidate therapeutic target in FGFR1-expressing tumors.

Figure 1.

FGFR1 expression in lobular carcinoma. A, analysis of FGFR1 expression in ductal and lobular carcinomas via gene expression microarray. Box plots and two-sample t statistics of the normalized log ratios of gene expression levels for ILCs and IDCs were generated using JMP 7.0. B, representative confocal images from lobular carcinoma tissue microarrays BR805 and BR809 stained with an antibody to FGFR1 (red). Nuclei were stained with DAPI (blue). Bar, 50 μm.

Figure 2.

iFGFR1 activation in MCF10A cells induces multiple phenotypes characteristic of breast tumor cells. A, time course of signaling following iFGFR1 activation. MCF10A cells expressing iFGFR1 were incubated in starvation medium lacking EGF and insulin for 24 h and then stimulated for the indicated times. Lysates were immunoblotted with phospho-specific antibodies as indicated. Tubulin was probed as a loading control. B, EGF-independent cell growth. MCF-10A cells expressing iFGFR1 were incubated in growth medium ± EGF and ± dimerizer (D) on day 0 and cell numbers were plotted for each day indicated. C, histogram of soft agar colony formation. WT MCF10A cells or MCF10A cells expressing iFGFR1 were mixed with agarose in growth medium and plated in six-well plates with and without the iFGFR1 dimerizer. Colonies in the entire well were counted using a dissecting microscope. Columns, mean of triplicate wells for each sample from a representative experiment; bars, SD. D, escape from anoikis. Cells were cultured in suspension in poly-HEMA–coated plates for 42 h and apoptosis was measured using the Cell Death ELISA kit. Results are plotted on histogram as arbitrary units of absorbance at 405 nm. Greater absorbance indicates greater apoptosis and histone release. Columns, mean of triplicate wells for each sample from a representative experiment; bars, SD.

Figure 3.

iFGFR1 activation induces altered three-dimensional morphogenesis, EMT, and cell polarity disruption. A, MCF-10A cells expressing iFGFR1 were cultured in Matrigel as described in Materials and Methods. Top, 10-d-old iFGFR1 structures were grown in the absence (−D) or presence of dimerizer (+D). Bright-field images of structures are shown. Bar, 100 μm. Bottom, representative confocal images of day 10 iFGFR1 structures −D/+D stained with antibodies detecting cleaved caspase-3 (red). DAPI (blue) marked the nuclei. Bar, 50 μm. B, top, representative confocal images of the day 10 iFGFR1 structures −D/+D stained with antibodies detecting E-cadherin (green), and DAPI (blue) was used to mark nuclei. Bar, 20 μm. Bottom, immunoblotting was performed on protein extracts from three-dimensional cultures of MCF10A cells expressing iFGFR1 −D/+D. C, representative confocal images of the day 10 iFGFR1 structures −D/+D stained with antibodies detecting β1-integrin (green), and DAPI (blue) was used to label nuclei. Bar, 50 μm. D, cell invasion. Cells expressing iFGFR1 were seeded in Transwell invasion chamber −D/+D and incubated for 18 h, and the invading cells were counted. For assays with the FGFR inhibitor PD173074, cells were incubated with inhibitor for 15 min before plating. Experiments were repeated a minimum of three times.

Figure 4.

RSK activity is critical for iFGFR1-dependent cell growth and survival. A, growth curve of MCF-10A cells expressing iFGFR1 in growth medium with or without dimerizer and CMK, a selective RSK inhibitor. B, histogram of anoikis in cells cultured in medium with or without dimerizer and CMK. Apoptosis was measured using the Cell Death ELISA kit. Results are plotted as arbitrary units of absorbance at 405 nm. Greater absorbance indicates greater apoptosis and histone release. Columns, mean of triplicate wells for each sample from a representative experiment; bars, SD. C, phase-contrast images of MCF10A cells expressing iFGFR1 cultured on Matrigel with and without dimerizer and RSK inhibitor. Representative bright-field images of structures were taken on day 10. D, top, Western blot of RSK1 and RSK2 expression in iFGFR1 MCF10A cells following RSK1- and RSK2-specific siRNA transfection; bottom, anoikis in cells with and without RSK siRNA. Apoptosis was measured using the Cell Death ELISA kit. Results are plotted as arbitrary units of absorbance at 405 nm. Columns, mean of triplicate wells for each sample from a representative experiment; bars, SD.

Figure 5.

RSK activity is essential for the growth and survival of MDA-MB-134 cells. A, Western blot of extracts of 13 breast cancer cell lines was probed with an antibody to FGFR1. Antitubulin was used as loading control. B, Western blot of immunoprecipitated FGFR1 from three breast cancer cell lines revealed by an antibody phospho-FGFR Tyr^653/654. C, soft agar colony formation of MCF-10A cells expressing activated iFGFR1 or MDA-MB-134 cells with and without the RSK inhibitor CMK. Colonies in the entire well were counted using a dissecting microscope. Columns, mean of triplicate wells for each sample from a representative experiment; bars, SD. D, phase-contrast images of MDA-MB-134, T47D, and HS578T cells grown on Matrigel with or without the RSK inhibitor CMK. Bar, 100 μm.

Figure 6.

A murine lobular carcinoma cell line expressing FGFR1 is sensitive to RSK inhibition. A, Western blot of extracts of cell lines derived from seven tumors that arose in mice deficient for p53 or p53 and E-cadherin using antibodies to E-cadherin, tubulin, or phospho-FGFR (Tyr^653/654). B, immunofluorescence of tumor sections from WEP7 and WEP4 probed with anti-FGFR1 antibody (red) and DAPI (blue) to stain nuclei. Bar, 20 μm. C, top, Western blot of WEP4 and WEP7 tumor cell lines transfected with specific RSK siRNA using antibodies to RSK1 and RSK2; bottom, phase-contrast images of WEP7 and WEP4 cells grown on Matrigel with or without RSK inhibitor or RSK-specific siRNAs. D, top, images of WEP7 cells grown on Matrigel for 6 d and incubated with RSK inhibitor for an additional 6 d. Cleaved caspase-3 antibodies (red) and DAPI staining for nuclei (blue) are shown. Bottom, anoikis in WEP7 cells with and without RSK inhibitor. Apoptosis was measured using the Cell Death ELISA kit. Results are plotted as arbitrary units of absorbance at 405 nm. Columns, mean of triplicate from a representative experiment; bars, SD.