RBBP9: a tumor-associated serine hydrolase activity required for pancreatic neoplasia

Abstract

Pancreatic cancer is one of the most lethal malignancies. To discover functionally relevant modulators of pancreatic neoplasia, we performed activity-based proteomic profiling on primary human ductal adenocarcinomas. Here, we identify retinoblastoma-binding protein 9 (RBBP9) as a tumor-associated serine hydrolase that displays elevated activity in pancreatic carcinomas. Whereas RBBP9 is expressed in normal and malignant tissues at similar levels, its elevated activity in tumor cells promotes anchorage-independent growth in vitro as well as pancreatic carcinogenesis in vivo. At the molecular level, RBBP9 activity overcomes TGF-beta-mediated antiproliferative signaling by reducing Smad2/3 phosphorylation, a previously unknown role for a serine hydrolase in cancer biology. Conversely, loss of endogenous RBBP9 or expression of mutationally inactive RBBP9 leads to elevated Smad2/3 phosphorylation, implicating this serine hydrolase as an essential suppressor of TGF-beta signaling. Finally, RBBP9-mediated suppression of TGF-beta signaling is required for E-cadherin expression as loss of the serine hydrolase activity leads to a reduction in E-cadherin levels and a concomitant decrease in the integrity of tumor cell-cell junctions. These data not only define a previously uncharacterized serine hydrolase activity associated with epithelial neoplasia, but also demonstrate the potential benefit of functional proteomics in the identification of new therapeutic targets.

Fig. 1.

Activity-based proteomics identified RBBP9 as a pancreatic cancer-associated serine hydrolase. (A) Schematic of the ABPP-MudPIT method. Primary tumor specimens are homogenized and fractionated to yield soluble and insoluble proteomes. Isolated proteomes are chemically labeled with active site-directed probes conjugated to biotin. Avidin beads permit enrichment of biotinylated probe-labeled active hydrolases and MS-based identification of individual hydrolase activities. (B) (Upper) Sequence coverage of RBBP9. Residues in red were identified by automated database searching of the peptides enriched from the tumor proteome that correspond to RBBP9; residues in gray were not observed. (Lower) Tandem mass spectrum of a representative RBBP9 peptide identified by LC/LC MS/MS. (C) (Upper) Immunohistochemical analysis of RBBP9 distribution in pancreatic ductal adenocarcinoma specimens shows strong expression in the neoplastic ductal epithelium. Stromal (S) and tumor (T) subcompartments are labeled. (Lower) Serial sections stained with hematoxylin and eosin. (D) Role of RBBP9 in anchorage-independent growth. Pancreatic carcinoma cells with a stable knockdown of RBBP9 show a significant reduction in colony growth on soft agar. A representative experiment is shown. n = 3 independent experiments. *P < 0.05, as compared to control cells expressing a nonsilencing shRNA, paired t test.

Fig. 2.

RBBP9 promotes tumor cell proliferation during pancreatic neoplasia. Requirement for RBBP9 in pancreatic tumorigenesis is shown; carcinoma cells (FG or FGM) with a stable knockdown of RBBP9 (SH) developed significantly smaller tumors following orthotopic implantation into the tail of the pancreas in mice, as compared to tumors derived from implanted control cells that express a nonsilencing shRNA (N/S). (A) FGM tumors were visualized by intravital fluorescence imaging (tumor boundary is demarcated in yellow). (Scale bar: 10 mm.) (B) FG tumor burden was assessed following resection. *P < 0.05 compared to tumors with a nonsilencing shRNA, paired t test (n = 8 mice/condition). (C) Loss of RBBP9 led to a significant reduction in tumor cell proliferation as determined by immunofluorescence analysis of the Ki67-positive cells in sections from the FG orthotopic tumors. (Scale bar: 100 μm.) (D) Quantitation of the Ki67-positive cells following analysis of Ki67 signal in 10 high-power fields (hpf’s) per tumor; tumors from 3 mice/condition were analyzed. Data are presented as the mean total pixel area in all hpf’s analyzed for that condition. *P < 0.05 compared to cells expressing a nonsilencing shRNA.

Fig. 3.

RBBP9 serine hydrolase activity constrains TGF-β-mediated and basal Smad2/3 phosphorylation. (A) (Upper) Primary sequence alignment of RBBP9 orthologs. The canonical serine hydrolase motif is boxed. A conserved serine residue (putative nucleophillic serine) is highlighted in red. (Lower) Gel-based activity proteomics of GST-purified proteins demonstrated that Ser-75 is required for RBBP9 serine hydrolase activity. Total levels of the purified proteins were visualized using Sypro Ruby gel stain. Results represent three similar experiments. (B) (Left) Effect of RBBP9 serine hydrolase activity on cellular proliferation rate. Data are presented as a percentage of the proliferation rate of control cells and are presented as the mean of three separate experiments ± SEM. *P < 0.05 compared to proliferation of control cells. (Right) Change in cell proliferation rate as a result of TGF-β treatment in cells with different levels of RBBP9 activity. Data are presented as the change (%) in proliferation rate of cell lines treated with TGF-β1 compared to vehicle-treated control cells and the impact of wild-type (RBBP9) or catalytically inactive RBBP9 (S75A) expression on TGF-β-driven antiproliferative signaling. Data are presented as the mean of three separate experiments ± SEM. *P < 0.05 compared to proliferation of control cells treated with vehicle. (C) Immunofluorescence analysis demonstrates that RBBP9 (green) is primarily localized external to the nucleus (blue) in pancreatic carcinoma cells. (D) Effect of RBBP9 activity on TGF-β1-mediated phosphorylation of Smad2/3. Results are representative of at least three separate experiments. (E) qPCR analysis of TGF-β target gene expression. Data are presented as transcript levels of PAI-1 (Left) and E-cadherin (Right) in FG carcinoma cells (control) and in cells expressing the wild-type (RBBP9) or catalytically inactive enyzme (S75A) treated with TGF-β1 (+) or vehicle (−) for 24 h. *P < 0.05 compared to control cells that received vehicle (n = 3).

Fig. 4.

RBBP9 promotes E-cadherin expression and tumor cell cohesiveness in pancreatic neoplasia. (A) qPCR analysis of E-cadherin transcript levels in pancreatic carcinoma cells that express different levels of wild-type or catalytically inactive RBBP9. Data are presented as the fold difference in E-cadherin transcript levels relative to control cells expressing empty vector. *P < 0.05 compared to control cells (n = 3). (B) Immunoblot analysis of E-cadherin abundance in FG pancreatic carcinoma cell lines that stably express different levels of RBBP9 serine hydrolase. (C) Immunofluorescence analysis of E-cadherin in confluent pancreatic carcinoma monolayers. N/S, FG carcinoma cells that stably express a nonsilencing shRNA; SH, FG cells that stably express a RBBP9-targeted shRNA construct. (D) Immunoblot analysis of β-catenin levels from E-cadherin immunoprecipitates (Top) or whole cell lysates (Middle) from FG cells that express different levels of wild-type or catalytically inactive RBBP9. (E) Immunohistochemical analysis of serial sections from well-differentiated ductal adenocarcinoma: RBBP9 (Left), E-cadherin (Center), and H&E (Right). Neoplastic ducts (D) and stroma (S) are labeled. (Scale bar: 50 μm.)