Hedgehog signaling and response to cyclopamine differ in epithelial and stromal cells in benign breast and breast cancer

Abstract

The hedgehog pathway regulates epithelial-mesenchymal interactions, differentiation, proliferation and survival during development. Stimulation of hedgehog signaling induces carcinogenesis or promotes cell survival in cancers of multiple organs. Using real-time, quantitative PCR, laser capture microdissection, and immunohistochemistry, distinctive patterns of expression of the hedgehog pathway members patched 1 (PTCH1), smoothened, GLI1, GLI2 and the 3 hedgehog ligands were identified for epithelial cells and stromal fibroblasts in benign breast and breast cancer. Hedgehog ligands were expressed at higher levels in some cancer epithelial cell lines compared to noncancerous epithelial cells. Correspondingly, expression of GLI1, a transcription factor and transcriptional product of hedgehog signaling, was increased 8-fold in cancer epithelial cell lines; however, PTCH1, also a transcriptional target of hedgehog signaling in many cell types, was not increased. GLI1 protein and mRNA, and PTCH1 and sonic hedgehog (SHH) proteins were elevated in 3 of 10 breast cancers; however, PTCH1 transcripts were not consistently increased. Hedgehog-mediated transcription, as indicated by a reporter of GLI-dependent promoter activity and by expression of GLI1 transcripts, was reduced by the hedgehog pathway inhibitor cyclopamine in both MDA-MB-435 cancer epithelial cells and MCF10AT epithelial cells, a cell line derived from benign breast. However, cyclopamine reduced viability of cancer epithelial cell lines, including MDA-MB-435, but did not specifically affect fibroblasts or epithelial cells from benign breast, including MCF10AT. Treatment with sonic hedgehog ligand diminished the cyclopamine-induced reduction in GLI-dependent promoter activity in MCF10AT and MDA-MB-435 and viability of MDA-MB-435. These results demonstrate modulation of GLI-mediated transcription in both cancer and benign-derived epithelial cells by cyclopamine and sonic hedgehog, and further suggest that hedgehog signaling contributes to the survival of only the cancer epithelial cells. Determination as to whether the increase in GLI1 and SHH expression in breast cancer indicates a significant increase in hedgehog signaling will require further evaluation.

Figure 1

Expression of hedgehog pathway members in cultured breast cells. IHH, SHH, DHH, PTCH1, SMO, GLI1, GLI2 and mRNA expression was examined by real-time, quantitative PCR. The cell lines analyzed included epithelial cells derived from benign breast (HMEC, MCF10A and MCF10AT) and from breast cancer (MCF7, T47D, MDA-MB-231 and MDA-MB-435), and fibroblasts from benign breast (NAF1—3) and breast cancer (CAF1—3). IHH was detected in all cells, both fibroblasts and epithelial cells. DHH and SHH were expressed at relatively higher levels in some cancer epithelial cell lines than in epithelial cells derived from benign breast. PTCH1, SMO and GLI1 were detected in all cell lines examined. There was no significant difference in PTCH1 and SMO in benign-versus cancer-derived epithelial cells. SMO was higher in fibroblasts than epithelial cells, both benign- and cancer-derived (p = 0.0001), whereas, there was no difference in PTCH1 in fibroblasts and epithelial cells. Mean expression of GLI1 was 8-fold higher in cancer epithelial cells than HMEC (p = 0.0003) and was higher in all fibroblasts than all epithelial cells (p = 0.0003). GLI2 was markedly higher in fibroblasts than epithelial cells (p = 0.0001). Results are relative to expression in HMEC and are presented as the mean and standard deviation.

Figure 2

Expression of hedgehog pathway members in breast tissues. (A) Epithelium was isolated by laser capture microdissection from 10 breast cancers and corresponding histologically normal (i.e., benign) breast, while benign- and cancer-associated stroma was isolated from 5 and 3 cases, respectively. Expression of pathway members was assessed by RTQ. GLI1 was significantly higher in cancer than benign epithelium (p = 0.04), and significantly higher in cancer- than benign-associated stroma (p = 0.04). GLI1 transcripts were higher in stroma than epithelium, in general (p < 0.001). There was no significant difference in expression of PTCH1 or SMO in benign versus cancer epithelium or in stroma versus epithelium in general. However, expression of PTCH1 and SMO were higher in benign- than cancer-associated stroma (p = 0.05, 0.02, respectively). Results are relative to expression of endogenous ribosomal protein, large, P0 (RPLP0). (B) Immunohistochemical staining for GLI1, PTCH1 and SHH was semi-quantified by an immunoscore that incorporated the intensity and percentage of cells that stained. Overall, mean immunoscores for GLI1, PTCH1 and SHH were increased 1.8-, 1.9- and 2.9-fold, respectively, in cancer epithelium compared to normal epithelium. Mean GLI1, PTCH1 and SHH immunoscores were lower in stroma than epithelium. The boundary of the boxes closest to zero indicates the 25^th percentile, the boundary farthest from zero indicates the 75^th percentile, and error bars above and below the box indicate the 90^th and 10^th percentiles (calculated only for benign and cancer epithelium due to sample size). The thin line within the box marks the median, and the thick line marks the mean.

Figure 3

Cyclopamine reduced viability and increased apoptosis of cancer epithelial cells. (A) Growth was measured by MTS assay after culture in 5, 10 or 20 μM cyclopamine or 20 μM tomatidine for 96 hours. The quantity of viable cells was reduced in MDA-MD-435 (435), T47D, MDA-MB-231 (231), MCF7 and HMEC in response to cyclopamine (p < 0.001, Anova, for each). Asterisks indicate concentrations with significant reduction (p < 0.05, Tukey). Tomatidine at 20 μM also significantly inhibited viability of MDA-MB-231 (231), MCF7 cells and HMEC, but not MDA-MB-435 (435) or T47D cells. Viability of the remaining cell lines was not reduced by cyclopamine or tomatidine. Values are normalized to the vehicle control and are the mean and standard error of replicate cultures. For each culture condition, n = 9 (435, T47D, 231, MCF7, HMEC, MCF10A, MCF10AT), n = 6 (NAF1, NAF4, NAF5, CAF2, CAF4) or n = 3 (CAF1). Some fibroblast cultures (NAF4, NAF5, CAF4) were different from those used for RTQ analysis. (B) MDA-MB-435 cells were treated with 2, 5, 10 and 20 μM cyclopamine for 48 and 96 hours. Early apoptotic cells were identified by FITC-conjugated Annexin V and absence of PI staining. Late apoptotic cells were labeled with FITC-conjugated Annexin V and PI. Proliferation was determined by BrdU incorporation. Early apoptotic cells were increased to a greater extent than late apoptotic cells at 48 hours (p < 0.001 and p = 0.003, Anova, respectively), whereas at 96 hours the increase in late apoptotic cells (p = 0.02, Anova) was more significant than the increase in early apoptotic cells (p = 0.07, Anova). Asterisks indicate concentrations with a significant increase in apoptosis (p < 0.05, Tukey). C) In these culture conditions, there was no decrease in cyclopamine-induced BrdU incorporation at 48 or 96 hours. The data are normalized to the vehicle control and are the mean and standard error of three separate experiments performed in duplicate.

Figure 4

Cyclopamine-induced inhibition of GLI-dependent promoter activity correlated with a reduction in GLI1 transcription, but not PTCH1 transcription or decreased viability. (A) MDA-MB-435, MDA-MB-231, MCF10AT and MCF10A, representing a range of sensitivity to cyclopamine-induced cell death, were transfected with a GLI-luciferase reporter construct and treated with 2–20 μM cyclopamine for 30 hours. Cyclopamine significantly inhibited reporter activity in MDA-MB-435 (p = 0.001) and MCF10AT (p < 0.001), but not MDA-MB-231 or MCF10A cells. These results demonstrate hedgehog transcriptional activity in MDA-MB-435 and MCF10AT cells that can be reduced by cyclopamine. GLI-reporter activity is normalized to the vehicle control. Asterisks indicate concentrations that induced significant reduction (p < 0.05, Tukey). The data are the mean and standard error of three separate experiments performed in triplicate. (B and C) GLI1 transcripts, but not PTCH1 transcripts, measured by RTQ are reduced in MCF10AT (p = 0.005) and MDA-MB-435 (p < 0.001) after treatment with 10 μM cyclopamine. Significance is indicated by asterisks. Data are normalized to the vehicle control and are the mean and standard error of three (MCF10AT) or five (MDA-MB-435) separate experiments performed in duplicate.

Figure 5

Hedgehog signaling and viability were modulated by cyclopamine and SHH. (A and B) MDA-MB-435 and MCF10AT cells were treated with 10 μM cyclopamine alone or with 400, 800 or 1200 ng/ml recombinant, N-terminal SHH (ShhN). ShhN significantly increases GLI-mediated transcriptional activity in both cell lines (p < 0.001, MDA-MB-435; p < 0.008, MCF10AT; Anova), confirming regulatable hedgehog signaling. Asterisks indicate concentrations that induced a significant increase (p < 0.05, Tukey). Results are normalized to 10 μM cyclopamine with vehicle and are the mean and standard error of three separate experiments performed in triplicate. (C) Treatment of MDA-MB-435 with 5 μM cyclopamine and ShhN lessened the effect of cyclopamine on cell viability (MTS assay). The asterisk indicates the concentration with a significant increase (p < 0.05, Tukey). Results are normalized to 5 μM cyclopamine with vehicle and are the mean and standard error of three separate experiments performed in triplicate.