Search

Immunohistochemistry for ZBP-89 and Ki67 in WT and VC:Zfp148F/F mice

Percentage of APC, CTNNB1 and ZNF148 mutations in CRC (TCGA)

In colorectal cancer, APC-mediated induction of unregulated cell growth involves posttranslational mechanisms that prevent proteasomal degradation of proto-oncogene β-catenin (CTNNB1) and its eventual translocation to the nucleus. However, about 10% of colorectal tumors also exhibit increased CTNNB1 mRNA. Here, we show in colorectal cancer that increased expression of ZNF148, the gene coding for transcription factor ZBP-89, correlated with reduced patient survival. Tissue arrays showed that ZBP-89 protein was overexpressed in the early stages of colorectal cancer. Conditional deletion of Zfp148 in a mouse model of Apc-mediated intestinal polyps demonstrated that ZBP-89 was required for polyp formation due to induction of Ctnnb1 gene expression. Chromatin immunoprecipitation (ChIP) and EMSA identified a ZBP-89–binding site in the proximal promoter of CTNNB1. Reciprocally, siRNA-mediated reduction of CTNNB1 expression also decreased ZBP-89 protein. ChIP identified TCF DNA binding sites in the ZNF148 promoter through which Wnt signaling regulates ZNF148 gene expression. Suppression of either ZNF148 or CTNNB1 reduced colony formation in WNT-dependent, but not WNT-independent cell lines. Therefore, the increase in intracellular β-catenin protein initiated by APC mutations is sustained by ZBP-89–mediated feedforward induction of CTNNB1 mRNA. Cancer Res; 76(23); 6877–87. ©2016 AACR.

Supplemental Methods, Figure Legends and References for Supplemental Methods

ZBP-89 Probe Sets:BFCOL1 and BERF1

Pancreatic fluid from a patient with a post operative pancreatic fistula was used to isolate human alpha-amylase by means of acarbose affinity chromatography. Amylase thermostability was measured in 4 solutions: (1) EDTA-dialyzed; (2) dialyzed solution plus 0.15 mmol/l (1.0 g/dl) human serum albumin; (3) dialyzed solution plus 0.25 mmol/l (1.0 mg/dl) calcium ions; and (4) dialyzed solution with both human serum albumin and calcium ions. Amylase activity was measured at predetermined times in samples heated to 60 degrees C. Thermostability was characterized by t1/2, the time to 50% initial amylase enzyme activity. In the dialyzed solution t1/2 was 0.75 +/- 0.19 min. This rose to 1.62 +/- 0.34 min with added human serum albumin, and to 8.24 +/- 0.13 min with added calcium ions. The combination of human serum albumin and calcium ions resulted in a synergistic increase of t1/2 to 180 +/- 26 min. These findings support our contention that human serum albumin, calcium ions and possibly other body fluid constituents must be considered in any utility involving amylase thermostability as a clinically relevant diagnostic marker.