Gli1 promotes cell survival and is predictive of a poor outcome in ERalpha-negative breast cancer

Abstract

Gli1 is a transcription factor and oncogene with documented roles in the progression of several cancer types, including cancers of the skin and pancreas. The contribution of Gli1 to the progression of breast cancer is less established. In order to investigate the functional impact of Gli1 in breast cancer, expression of Gli1 and its contribution to cell growth was assessed in breast cancer cell lines. These in vitro results were compared to expression of Gli1, determined by immunohistochemistry, in 171 breast cancers. In these cancers, the association of Gli1 with expression of estrogen receptor alpha (ERalpha) and progesterone receptor (PR), ErbB2, p53, the rate of proliferation, and clinicopathologic parameters and outcome was assessed. Expression of Gli1 and ERalpha mRNA was strongly correlated in ERalpha-positive cell lines (r = 0.999). Treatment with estrogen increased expression of Gli1 in 2 of 3 ERalpha-positive cell lines; this increase was prevented by treatment with the ERalpha-specific antagonist MPP. Silencing of Gli1 by shRNA markedly reduced the survival of two ERalpha-negative cell lines, but caused only a modest reduction in ERalpha-positive cell lines. In breast cancer tissues, cancers with nuclear localization of Gli1 had a higher ERalpha (P=0.027) and lower p53 expression (P=0.017) than those without nuclear localization of Gli1. However, nuclear localization of Gli1 was predictive of a poorer cancer-specific survival in ERalpha-negative, including triple negative, cancers (P = 0.005), but not ERalpha-positive cancers. In conclusion, we demonstrate a positive association between expression of Gli1 and ERalpha; however, our data indicate a greater functional effect of Gli1 in ERalpha-negative cancers.

Fig. 1

Estrogen, ERα, and Gli1 in breast cancer cell lines. a Expression of Gli1 and ERα were measured by quantitative RT–PCR (QRT) and expression was normalized to expression in MCF10A cells. A positive correlation was determined by Pearson's correlation co-efficient (r = 0.999, P < 0.001). b MCF7 cells were treated with a range of concentrations of 17β-estradiol for 48 h. Gli1 expression was measured by QRT and expression was normalized to the vehicle control. c MCF7, MDA-MB-361 and T47D ERα-positive cell lines were treated with 1 nM 17β-estradiol (E2) for 48 h and expression of Gli1 was measured by QRT. Expression is normalized to the vehicle control. The data are the mean and standard error of three experiments performed in triplicate. Asterisks indicate significance, P = 0.018 for MCF7 and P = 0.010 for MDA-MB-361. d MCF7 cells were treated with the ERα-specific antagonist, MPP, at 1 μM for 1 h prior to the addition of E2 (1 nM) for 48 h. Gli1 expression was measured by QRT. Expression is normalized to the vehicle control. Asterisks indicate significance, P = 0.021). The data are the mean and standard error of three experiments performed in triplicate

Fig. 2

Growth of Gli1-silenced breast cancer cells. a Gli1 expression was reduced by lentiviral transduction of shRNA directed to Gli1 (sh1, sh2). A non-targeting (NT) shRNA was a control. QRT was performed to confirm a reduction in Gli1 expression in the ERα-positive MCF7 and MDA-MB-361 (361) cells and the ERα-negative MDA-MB-231 (231) and Hs578T cells. The mean expression and standard deviation are presented. b Gli1 expression was also verified by western blot analysis using a rabbit polyclonal antibody. β-actin is a loading control. F9 cells are a teratocarcinoma cell line with a very high expression of Gli1 and are a positive control. The asterisks mark a non-specific immunoreactive band. The absence of an immunore-active band to β-actin in F9 cells in the blot for Hs578T cells is due to technical issues, but does not interfere in the interpretation of the results. c NT control and Gli1 silenced cells were grown until the NT cells approached confluence, which was 7 days for MCF7 cells, 9 days for MDA-MB-361 cells, and 5 days for both MDA-MB-231 and Hs578T cells. Growth was measured by MTT assay. Data are normalized to the NT control and are the mean and standard error of three experiments performed in triplicate. Significance is indicated by asterisks (P < 0.05)

Fig. 3

Gli1 in ERα-positive and ERα-negative breast cancers. a–d There was variability in the staining pattern of infiltrating ductal carcinomas of the breast for Gli1. In some cancers, expression was both cytoplasmic and nuclear (arrowheads) (a). In others, expression was primarily or exclusively cytoplasmic (b), or primarily nuclear (arrowheads) (c). In 8 cancers, there was no nuclear or cytoplasmic Gli1 expression (d). For a–d, magnification is 200×, scale bar = 40 μM. e Cancers with Gli1 nuclear staining (Gli1-positive, n = 53) and without nuclear staining (Gli1-negative, n = 118) were compared for the percentages of cells positive for ERα, PR, p53, Ki-67 and membrane expression of ErbB2 (immunoscore). Asterisks indicate statistical significance for ERα (P = 0.027) and p53 (P = 0.017). f A comparison of the percentage of cancers which were Gli1-positive among ERα-positive (n = 89) and ERα-negative (n = 60) breast cancers was significantly different (P = 0.033). g A comparison of tumor size and the number of cases with lymph node metastases or in clinical stage I at the time of diagnosis was not different between Gli1-positive and Gli1-negative cancers. h The Kaplan–Meier estimate was used to compare cause-specific survival (i.e., death due to cancer) in women with Gli1-positive (n = 45) and Gli1-negative (n = 94) cancers. The log-rank test indicated there was no significant difference

Fig. 4

Nuclear localization of Gli1 in ERα-positive breast cancers. a In ERα-positive breast cancers, there was no significant difference in expression of ERα, PR, p53, Ki67 or ErbB2 in Gli1-positive (n = 34) and Gli1-negative (n = 55) cancers. b In ERα-positive breast cancers, a comparison of tumor size and the number of cases with lymph node metastases or in clinical stage I at the time of diagnosis was not different between Gli1-positive and Gli1-negative cancers. c In ERα-positive breast cancers, the Kaplan–Meier estimate was used to compare cause-specific survival (i.e., death due to cancer) in women with Gli1-positive (n = 30) and Gli1-negative (n = 46) cancers. The log-rank test indicated there was no significant difference. d Cause-specific survival was compare in only those ERα-positive breast cancers which were treated with tamoxifen. There was no difference in survival in Gli1-positive (n = 15) versus Gli1-negative cancers (n = 28)

Fig. 5

Nuclear localization of Gli1 in ERα-negative breast cancers. a In ERα-negative breast cancers, there was no significant difference in expression of ERα, PR, p53, Ki67 or ErbB2 in Gli1-positive (n = 13) and Gli1-negative (n = 47) cancers. b In ERα-negative breast cancers, a comparison of tumor size and the number of cases with lymph node metastases or in clinical stage I at the time of diagnosis was not different between Gli1-positive and Gli1-negative cancers. c In ERα-negative breast cancers, the Kaplan–Meier estimate was used to compare cause-specific survival (i.e., death due to cancer) in women with Gli1-positive (n = 10) and Gli1-negative (n = 41) cancers. The log-rank test indicated a significant difference (P = 0.005). d Cause-specific survival was compare in triple negative breast cancers. There was a statistically significant difference in survival in Gli1-positive (n = 7) versus Gli1-negative cancers (n = 27) (P = 0.003)