Dysregulated expression of Fau and MELK is associated with poor prognosis in breast cancer

Abstract

Introduction: Programmed cell death through apoptosis plays an essential role in the hormone-regulated physiological turnover of mammary tissue. Failure of this active gene-dependent process is central both to the development of breast cancer and to the appearance of the therapy-resistant cancer cells that produce clinical relapse. Functional expression cloning in two independent laboratories has identified Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed gene (Fau) as a novel apoptosis regulator and candidate tumour suppressor. Fau modifies apoptosis-controller Bcl-G, which is also a key target for candidate oncoprotein maternal embryonic leucine zipper kinase (MELK).

Methods: We have used RNA interference to downregulate Fau and Bcl-G expression, both simultaneously and independently, in breast cancer cells in vitro to determine the importance of their roles in apoptosis. Expression of Fau, Bcl-G and MELK was measured by quantitative RT-PCR in breast cancer tissue and in matched breast epithelial tissue from the same patients. Expression data of these genes obtained using microarrays from a separate group of patients were related to patient survival in Kaplan-Meier analyses.

Results: siRNA-mediated downregulation of either Fau or Bcl-G expression inhibited apoptosis, and the inhibition produced by combining the two siRNAs was consistent with control of Bcl-G by Fau. Fau expression is significantly reduced in breast cancer tissue and this reduction is associated with poor patient survival, as predicted for a candidate breast cancer tumour suppressor. In addition, MELK expression is increased in breast cancer tissue and this increase is also associated with poor patient survival, as predicted for a candidate oncogene. Bcl-G expression is reduced in breast cancer tissue but decreased Bcl-G expression showed no correlation with survival, indicating that the most important factors controlling Bcl-G activity are post-translational modification (by Fau and MELK) rather than the rate of transcription of Bcl-G itself.

Conclusions: The combination of in vitro functional studies with the analysis of gene expression in clinical breast cancer samples indicates that three functionally interconnected genes, Fau, Bcl-G and MELK, are crucially important in breast cancer and identifies them as attractive targets for improvements in breast cancer risk prediction, prognosis and therapy.

Figure 1

Downregulation of Fau inhibits UV-induced apoptosis of T-47D breast cancer cells. T-47D cells were transfected with one of two different siRNAs to Fau or with a negative control (NC) (Ambion code 4611) siRNA using RNAiFect (Qiagen). After 120 hours samples were collected for real-time RT-PCR analysis, and trypsinized cells were either exposed to UV light (40 J/m²; closed bars) or were mock-irradiated (open bars), and then replated in fresh medium. (a) Real-time RT-PCR analysis of Fau transcript levels. Data, expressed relative to the house-keeping gene ALAS1, are the mean ± standard error of the mean. *P < 0.05 versus NC siRNA (one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test); n = 3. (b) The proportion of apoptotic cells was determined 48 hours post UV exposure by acridine orange staining and fluorescence microscopy. *P < 0.05, **P < 0.01 versus NC siRNA (UV-irradiated; one-way ANOVA with Bonferroni's multiple comparison test); n = 3. (c) Short-term cell viability was determined 48 hours post UV exposure by dye exclusion. *P < 0.01 versus NC siRNA (UV-irradiated; one-way ANOVA with Bonferroni's multiple comparison test); n = 3. (d) Long-term cell viability after UV irradiation was determined in further cultures by measuring colony formation; colonies were counted 3 weeks post UV exposure. *P < 0.05, **P < 0.01 versus NC siRNA (UV-irradiated; one-way ANOVA with Bonferroni's multiple comparison test); n = 4.

Figure 2

Reduced Fau transcript levels in breast cancer are associated with poor patient survival. (a) RNA was isolated from tumour tissue and adjacent normal tissue (n = 21 patients), was reverse transcribed, and Fau and ALAS1 transcript levels were determined using Taqman assays and real-time PCR using a relative standard curve protocol (cDNA from T-47D cells as standard). The Fau/ALAS1 transcript ratio is reduced in tumour tissue. *P = 0.002; paired Student's t test. Inset: scattergram of data. (b) Fau transcript levels are also reduced in tumour tissue (closed bars) versus normal tissue (open bars) in subsets of patients with grade II and grade III disease. *P < 0.05; n = 7 and **P < 0.001; n = 12; one-way analysis of variance with Bonferroni's multiple comparison test. (c) Kaplan–Meier survival curve showing reduced overall breast-cancer-specific survival in invasive breast cancer with lower Fau expression levels (P = 0.006).

Figure 3

Increased MELK transcript levels in breast cancer are associated with poor patient survival. (a) RNA was isolated from tumour and adjacent normal tissue (n = 11 patients), was reverse transcribed, and MELK and ALAS1 transcript levels were determined using Taqman assays and real-time PCR using a relative standard curve protocol (cDNA from T-47D cells as standard). The MELK/ALAS1 transcript ratio is elevated in tumour tissue. *P = 0.022; paired Student's t test on log-transformed data. Inset: scattergram of data. (b) Kaplan–Meier survival curve showing significantly reduced overall breast-cancer-specific survival in invasive breast cancer with higher MELK expression levels (P = 0.001).

Figure 4

siRNA-mediated knockdown of Fau and Bcl-G expression attenuates UV-induced apoptosis in breast cancer cells. siRNA-mediated knockdown of Fau and Bcl-G expression, either alone or in combination, attenuates UV-induced apoptosis of T-47D breast cancer cells. T-47D cells were transfected with siRNA to Fau (FAU2), Bcl-G or negative control (NC). To observe the effects of combined knockdown, Fau plus Bcl-G siRNAs were co-transfected (Fau + Bcl-G); the controls for this group were cells transfected with double the amount of NC siRNA (2× NC). At 120 hours post transfection, samples were collected for the determination of Fau and Bcl-G transcript levels, and cells were either exposed to UV light (40 J/m²) or were mock-irradiated. (a) Real-time RT-PCR analysis of Fau (left-hand panel) and Bcl-G (right-hand panel) transcript levels. Data, expressed relative to the housekeeping gene ALAS1, are the mean ± standard error of the mean. *P < 0.05 versus NC siRNA, ^P < 0.05 versus 2× NC siRNA (Bonferroni's multiple comparison test); n = 5. (b) At 48 hours post UV exposure, the proportion of apoptotic cells (left-hand panel) was determined by a CaspaTag assay and fluorescence microscopy, and the short-term cell viability (right-hand panel) was determined by dye exclusion. Data for mock-irradiated (light bars) and UV-treated (dark bars) cells are the mean ± standard error of the mean. *P < 0.05 versus NC siRNA (UV-irradiated), ^P < 0.05 versus 2× NC siRNA (UV-irradiated) (Bonferroni's multiple comparison test); n = 5. Note that knockdown of either Fau or Bcl-G alone or in combination attenuates apoptosis induction by UV and that the extent of inhibition is similar for all three treatments. (c) Bcl-G transcript levels are reduced in ductal carcinoma of the breast. RNA was isolated from tumour and adjacent normal tissue (n = 18 patients), was reverse transcribed, and Bcl-G and ALAS1 transcript levels were determined using Taqman assays and real-time PCR using a relative standard curve protocol (cDNA from T-47D cells as standard). The Bcl-G/ALAS1 transcript ratio is reduced in tumour tissue. *P = 0.0021; paired Student's t test. Inset: scattergram of data.(d) Expression of Bcl-G was analysed in a cohort of 99 breast carcinoma patients and was correlated with survival data, as previously reported [27]. Kaplan–Meier survival curve showing no significant correlation between total Bcl-G expression levels and patient survival.