Identification of Pbx1, a potential oncogene, as a Notch3 target gene in ovarian cancer

Abstract

Notch3 gene amplification has recently been identified in ovarian cancer but the Notch3 effectors that are involved in the development of ovarian cancer remain elusive. In this study, we have identified Pbx1, a proto-oncogene in hematopoietic malignancy, as a Notch3 target gene. Pbx1 expression is transcriptionally regulated by Notch3 activation, and Notch3/CSL protein complex directly binds to the Pbx1 promoter segment harboring the CSL-binding sequence. The growth-inhibitory effect of gamma-secretase inhibitor could be partially reversed by ectopic Pbx1 expression. Furthermore, functional studies by Pbx1 short hairpin RNA knockdown show that Pbx1 is essential for cell proliferation and tumorigenicity. Taken together, the above findings indicate that Pbx1 is a direct Notch3-regulated gene that mediates the survival signal of Notch3 in ovarian cancer.

Fig. 1. Identification of Pbx1 as a putative Notch3 target gene

A. Upper panel: Western blot was performed to assess Notch3 protein expression in a panel of cancer cell lines. OVCAR3, A2780, and MCF7, which express abundant Notch3 proteins, were selected for microarray analysis. GAPDH protein expression was used as loading control. Of note, the three cell lines with Notch3 overexpression also show Pbx1 overexpression. Lower panel: Affymetrix Genechip analysis was performed to identify genes differentially expressed between GSI-treated (1 µM) versus DMSO-treated cancer cells. dChip software was used to analyze the data. A cut-off ratio of 2 was used to select the GSI-sensitive genes. Columns represent experimental samples (cell lines), while rows represent genes. The expression level of each gene in an individual specimen is shown as a pseudo-color gradient based on the relative expression level normalized for each gene, where red indicates high and green indicates low expression. B. Quantitative real-time PCR was performed to confirm the candidate GSI-sensitive genes identified by Genechip analysis. The genes that can be confirmed by quantitative PCR include Pbx1, glypican-6, histone 4C, histone 4K, and histone 4J. C. Western blot analysis demonstrates that Pbx1 protein is downregulated by either GSI or Notch3 siRNA. In contrast, mock (no transfection) and scramble siRNA do not have significant effects on Pbx1 protein expression. Cells were incubated with GSI or Notch siRNA for 48 hours before assay. D. Western blot analysis of Pbx1 expression in a panel of ovarian tissues. OSE: ovarian surface epithelial cell line; LG: low-grade ovarian serous carcinoma; HG: high-grade ovarian serous carcinoma. * indicates samples with protein degradation. Pbx1a was found to be more susceptible to protein degradation.

Fig. 2. Notch3 induces Pbx1 expression through promoter activation

A. Left Panel: Examples of the conserved CSL binding site in Notch-regulated genes in human. Consensus CSL binding sequence is bolded. The consensus CSL binding site is located at −304 to −292 at Pbx1 promoter. Right Panel: Pbx1 promoter assay shows increased reporter activity in cells transfected with the wild-type Pbx1 promoter construct (pGL3-Pbx1) as compared to cells transfected with vector only control (pGL3). In pGL3-Pbx1 transfected groups, cells co-transduced with both CSL and NICD3 retroviruses demonstrate the highest promoter activity. Cells transduced with vector only virus and pGL3-Pbx1 also showed above background luciferase activity, likely due to the endogenous background expression of CSL and Notch in OSE. In contrast, cells transfected with the mutant Pbx1 promoter construct (pGL3-Pbx1 mut) containing the mutant CSL binding site demonstrate a significantly lower luciferase activity as compared to that of wild-type Pbx1 promoter. B. The pGL3-Pbx1 promoter construct was transfected into 3 cancer cell lines overexpressing Notch3. Pbx1 promoter activity was high in each of these transfected lines, but was significantly reduced by either GSI or Notch3 siRNA.

Fig. 3. Pbx1b is over-expressed in high-grade ovarian serous tumors and its nuclear expression level is positively correlated with Notch3 activation

A. Western blot was performed to determine the specificity of the employed anti-Pbx1b antibody. A predominant protein band corresponding to Pbx1b (~ 38.4 kD) is detected in MPSC-1 transduced with Pbx1b retrovirus, but not in MPSC-1 transduced with control virus or in non-transduced MPSC-1 cells. A single band corresponding to Pbx1b protein can also be detected in ovarian cancer cell lines including OVCAR3 and A2780. GAPDH was used as the loading control. B. Immunohistochemistry of Notch3 and Pbx1b in ovarian high-grade serous carcinoma tissues and in normal ovaries. Top: immunostaining of Notch3 and Pbx1b was performed on the same set of high-grade serous ovarian tumors. Three representative specimens with high Notch3 and Pbx1b expression (case A), medium Notch3 and Pbx1b expression (case B) and undetectable Notch3 and Pbx1b expression (case C) are shown. Normal ovarian surface epithelium (OSE) does not show nuclear Notch3 or Pbx1b immunoreactivity. Bottom: summary of immunohistochemistry result. Data demonstrate a significant trend of Notch3 and Pbx1b co-expression in nucleus (Cochran-Armitage trend test, p< 0.0001).

Fig. 4. Chromatin immunoprecipitation and electrophoretic mobility shift assay of Pbx1 promoter region

A: Chromatin immunoprecipitation (ChIP) assay was performed to determine the CSL binding region at the Pbx1 promoter. Real time PCR was performed on fragmented chromatin precipitated by an anti-V5 antibody from 293T cells previously infected with CSL-V5 virus or control virus. PCR primers were designed to amplify four different regions at the Pbx promoter (−254/−111 bp, −354/−254 bp, −1074/−960 bp, and −1252/−1127 bp). Results were normalized to the total input of chromatin DNA and were presented as the fold change of CSL-V5 infected cells relative to control virus infected cells. Error bars represent standard deviations from triplicate experiments. B: Electrophoretic mobility shift assay (EMSA) was performed on two adjacent regions at the Pbx1 promoter, −254/−111 bp and −354/−254 bp. The −354/−254 bp region contains the CSL consensus binding sequence. Biotin-labeled DNA probe for each promoter region was incubated with nuclear lysates from HEK293 cells transfected with CSL and/or transduced with NICD3 virus. The specificity of CSL binding was determined in a competition reaction in which a 100-fold molar excess of unlabeled DNA probe was added to the binding reaction. The binding complex was resolved by electrophoresis on a 4–12% TBE gel. The position of biotin-labeled probe was detected with avidin-HRP followed by chemiluminescence.

Fig. 5. Constitutive Pbx1a expression rescues cancer cells from GSI-induced growth inhibition

A: Pbx1a cDNA tagged with a V5 epitope was cloned into the pBabe retroviral vector (controlled by a CMV promoter). Cells were transduced with the resulting Pbx1a retrovirus and analyzed by Western blot. Pbx1a protein was detected using an anti-V5 antibody. B: Notch3 expressing cancer cell lines (including OVCAR3, A2780, and MCF7) transduced with Pbx1a or control retrovirus were treated with various concentrations of GSI. The results demonstrate that ectopic Pbx1a expression reduces sensitivity to growth inhibition at 1–2 µM of GSI.

Fig. 6. Pbx1 knockdown by shRNA inhibits cell proliferation

A: Pbx1 shRNA was found to specifically suppress Pbx1 protein expression during the course of the experiment (2–5 days after transfection). Control shRNA or mock transfection did not have such effect. B: Pbx1 shRNA suppresses the growth of tumor cell lines including OVCAR3, A2780, and MCF7 that express both Notch3 and Pbx1 in vitro. In contrast, Pbx1 shRNA does not show significant growth-inhibitory effects on MPSC1, SKOV3 and TOV21G ovarian cancer cells that do not express detectable levels of Pbx1. C: BrdU uptake assay showed a reduction in DNA synthesis in cancer cells treated with Pbx1 shRNA as compared to control shRNA and mock-transfected groups. *: p< 0.001. D: Pbx1 gene knockdown reduces tumorigenecity of A2780 tumors in nude mice. Red bars: average of tumor weight.