Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA <em>In Situ</em> Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Case Western Reserve University

Protein tyrosine phosphatase receptor-type T (PTPRT) is the most frequently mutated tyrosine phosphatase in human cancers. However, the cell signaling pathways regulated by PTPRT largely remain to be elucidated. Here, we show that paxillin is a direct substrate of PTPRT and that PTPRT specifically regulates paxillin phosphorylation at tyrosine residue 88 (Y88) in colorectal cancer (CRC) cells. We engineered CRC cells homozygous for a paxillin Y88F knock-in mutant and found that these cells exhibit significantly reduced cell migration and impaired anchorage-independent growth, fail to form xenograft tumors in nude mice, and have decreased phosphorylation of p130CAS, SHP2, and AKT. PTPRT knockout mice that we generated exhibit increased levels of colonic paxillin phosphorylation at residue Y88 and are highly susceptible to carcinogen azoxymethane-induced colon tumor, providing critical in vivo evidence that PTPRT normally functions as a tumor suppressor. Moreover, similarly increased paxillin pY88 is also found as a common feature of human colon cancers. These studies reveal an important signaling pathway that plays a critical role in colorectal tumorigenesis.

Identification and functional characterization of paxillin as a target of protein tyrosine phosphatase receptor T.

DNA methyltransferase 1 (DNMT1) is the primary enzyme that maintains DNA methylation. We describe a previously unknown mode of regulation of DNMT1 protein stability through the coordinated action of an array of DNMT1-associated proteins. DNMT1 was destabilized by acetylation by the acetyltransferase Tip60, which triggered ubiquitination by the E3 ligase UHRF1, thereby targeting DNMT1 for proteasomal degradation. In contrast, DNMT1 was stabilized by histone deacetylase 1 (HDAC1) and the deubiquitinase HAUSP (herpes virus-associated ubiquitin-specific protease). Analysis of the abundance of DNMT1 and Tip60, as well as the association between HAUSP and DNMT1, suggested that during the cell cycle the initiation of DNMT1 degradation was coordinated with the end of DNA replication and the need for DNMT activity. In human colon cancers, the abundance of DNMT1 correlated with that of HAUSP. HAUSP knockdown rendered colon cancer cells more sensitive to killing by HDAC inhibitors both in tissue culture and in tumor xenograft models. Thus, these studies provide a mechanism-based rationale for the development of HDAC and HAUSP inhibitors for combined use in cancer therapy.

DNMT1 stability is regulated by proteins coordinating deubiquitination and acetylation-driven ubiquitination.

Hyperphosphorylation at the Y705 residue of signal transducer and activator of transcription 3 (STAT3) is implicated in tumorigenesis of leukemia and some solid tumors. However, its role in the development of colorectal cancer is not well defined. To rigorously test the impact of this phosphorylation on colorectal tumorigenesis, we engineered a STAT3 Y705F knock-in to interrupt STAT3 activity in HCT116 and RKO colorectal cancer cells. These STAT3 Y705F mutant cells fail to respond to cytokine stimulation and grow slower than parental cells. These mutant cells are also greatly diminished in their abilities to form colonies in culture, to exhibit anchorage-independent growth in soft agar, and to grow as xenografts in nude mice. These observations strongly support the premise that STAT3 Y705 phosphorylation is crucial in colorectal tumorigenesis. Although it is generally believed that STAT3 functions as a transcription factor, recent studies indicate that transcription-independent functions of STAT3 also play an important role in tumorigenesis. We show here that wild-type STAT3, but not STAT3 Y705F mutant protein, associates with phospholipase Cγ1 (PLCγ1). PLCγ1 is a central signal transducer of growth factor and cytokine signaling pathways that are involved in tumorigenesis. In STAT3 Y705F mutant colorectal cancer cells, PLCγ1 activity is reduced. Moreover, overexpression of a constitutively active form of PLCγ1 rescues the transformation defect of STAT3 Y705F mutant cells. In aggregate, our study identifies previously unknown cross-talk between STAT3 and the PLCγ signaling pathways that may play a critical role in colorectal tumorigenesis.

Cross-talk between phospho-STAT3 and PLCγ1 plays a critical role in colorectal tumorigenesis.

Obesity is a risk factor for many human diseases. However, the underlying molecular causes of obesity are not well understood. Here, we report that protein tyrosine phosphatase receptor T (PTPRT) knockout mice are resistant to high-fat diet-induced obesity. Those mice avoid many deleterious side effects of high-fat diet-induced obesity, displaying improved peripheral insulin sensitivity, lower blood glucose and insulin levels. Compared to wild type littermates, PTPRT knockout mice show reduced food intake. Consistently, STAT3 phosphorylation is up-regulated in the hypothalamus of PTPRT knockout mice. These studies implicate PTPRT-modulated STAT3 signaling in the regulation of high-fat diet-induced obesity.

PTPRT regulates high-fat diet-induced obesity and insulin resistance.

Protein tyrosine phosphatase receptor T (PTPRT) is frequently mutated in a variety of human cancers including colorectal cancer. Here we report that PTPRT knockout increases the size of mouse colon tumors in the Apcmin+/- genetic background, suggesting that inactivation of PTPRT promotes tumor progression. We previously demonstrated that PTPRT dephosphorylates paxillin at tyrosine-Y88 residue. Consistently, phosphorylation of Y88 paxillin (pY88) is up-regulated in colon tumors derived from Apcmin+/- Ptprt-/- mice. An important downstream effector of pY88 paxillin is the oncogene Akt. Here, we show that pY88 paxillin impacts the Akt pathway by regulating the interaction between p130cas and the p85 regulatory subunit of PI3-Kinase. Additionally, while pY88 paxillin is a substrate of the tumor suppressor phosphatase PTPRT, the corresponding kinase has not been previously identified. In this study, we demonstrate that the oncogenic kinase Src directly phosphorylates paxillin at Y88. Moreover, colorectal cancer cells that express high levels of pY88 paxillin are sensitive to dasatinib treatment, suggesting that pY88 paxillin may serve as a predictive biomarker for Src family kinase inhibitors.

Regulation of paxillin-p130-PI3K-AKT signaling axis by Src and PTPRT impacts colon tumorigenesis.

It is unclear whether and how whole-genome sequencing (WGS) data can be used to implement genomic medicine. Our objective is to retrospectively evaluate whether WGS can facilitate improving prevention and care for patients with susceptibility to cancer syndromes.

Yiqing Zhao

Department of Genetics and Genome Sciences,
Case Comprehensive Cancer Center

Case Western Reserve University

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Yiqing Zhao

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Genetics and Genome Sciences,Case Comprehensive Cancer Center

Case Western Reserve University

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Department of Genetics and Genome Sciences,
Case Comprehensive Cancer Center