Higuchi Biosciences Center - University of Kansas

Fall 2008 Science Talks
December 5, 2008

C1. Determining the Active Site Reduction Potential in Phosphatase of Regenerating Liver-1 (PRL-1), A Phosphatase Implicated in Metastatic Cancer, by High-Resolution NMR Spectroscopy
Andria L. Skinner and Jennifer S. Laurence
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047

PRL-1 was identified as an immediate early response gene in damaged liver tissue but has since been shown to be essential during development of many cellular tissues. More importantly, this protein has been found to promote cancer and trigger metastasis when over-expressed. While many drugs exist for the treatment of cancer, there are no current treatments for cancers that have metastasized, making this enzyme a novel therapeutic target. PRL-1 belongs to the protein tyrosine phosphatase (PTPase) family and plays an important role in maintaining appropriate tyrosine phosphorylation levels in the cell. Although the exact role that PRL-1 plays in both controlled and aberrant growth processes is not well understood, the PTPase activity of PRL-1 is required for normal mitotic progression and cellular motility. A deeper understanding of PRL-1 regulation is needed in order to elucidate the mechanism by which PRL-1 promotes cancer and metastasis.

Like other PTPases, PRL-1 is redox regulated such that oxidation inhibits its activity, specifically by disulfide bond formation at the active site. Cellular redox environments differ with compartment, tissue type, stage of the cell cycle and age. PRL-1 localizes to different subcellular locations in different cells, and as such, PRL-1 activity may differ depending on its cellular localization. To understand the function of PRL-1 in different cellular environments, we are using NMR to determine the reduction potential of PRL-1’s active site disulfide bond.

Complete oxidation of WT-PRL-1 leads to inactivation and rapid precipitation, which precludes its use in this study. As such, site-directed mutagenesis was used to produce a PRL-1 variant amenable to large changes in redox conditions. SDS-PAGE, NMR, MS and the pNPP phosphatase activity assay were used to characterize the resulting mutants. PRL-1-C98A was determined to be a stable oxidized form of PRL-1 that is virtually identical in structure to wild type. This variant was used to determine the reduction potential of the active site disulfide bond in PRL-1, which was measured by monitoring peak volume changes in the HSQC NMR spectrum upon the addition of DTT.

The active site reduction potential of PRL-1 was determined to be -345.6 ± 8.0 mV. Because inside the cell, the reduction potential normally varies between -170 mV to -240 mV depending on the stage of the cell cycle, we conclude that PRL-1 is predominantly oxidized inside the cell and the implications of this finding in terms of PRL-1’s biological role in both controlled and aberrant growth processes will be discussed.

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C2. Developing Novel Datamining Algorithms for Cancer Research — Applications in Genomics, Proteomics, and Drug Discovery
Jianwen Fang¹ and Jerzy W. Grzymala-Busse²
¹Applied Bioinformatics Laboratory, ²Department of Electrical Engineering and Computer Science, University of Kansas, Lawrence, KS 66047

Recently emerging biotechnologies bring in new promises to more precise preventive interventions, earlier diagnostic testing, and more potent treatments with weaker side effects for cancer. However, for these promises to be realized, advanced computational data analysis is critical. Unprecedented amounts of data generated using these technologies require sophisticated new tools for analyzing and making sense of data in order to enhance our understanding of the trends and patterns of these diseases, and, ultimately, to improve the clinical care by applying the resulting knowledge.

Here we report our recent work on developing novel software tools for the data mining and knowledge discovery in cancer research. We have developed algorithms that can be used to discover underlying characteristic patterns distinguishing one class of subjects from another, e.g., it may differentiate between tumors and normal cells or make distinction between different tumors. We have successfully applied the novel algorithms in various areas including genomics, proteomics, and drug discovery.

The long-term objective of the project is to develop a software suite that can be used to perform data mining and knowledge discovery for cancer research. (Partially Supported by the NIH grant number P20 RR016475)

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C3. Musashi and APC: A Link Between Wnt Signaling and Differentiation of Colonic Epithelium
Erick Spears and Kristi Neufeld
Department of Molecular Biosciences, University of Kansas, Lawrence, KS

Colorectal cancer is the second leading cause of cancer related deaths in the United States. Approximately 80% of all colon cancers are associated with a mutation in the adenomatous polyposis coli (APC) gene. These two observations have led us to investigate the intracellular role of normal APC and how truncation of APC, the most common type of carcinogenic mutation, leads to the formation of precancerous polyps in the intestines of mice and humans. The studies presented here are particularly concerned with the potential role of APC in maintenance of homeostasis in the intestinal epithelium. We have identified a regulatory relationship between APC and a sequence specific RNA binding protein involved in stem cell maintenance, Mushashi-1 (MSI). Previous studies have shown that Apc loss-of-function in the mouse intestine leads to a significant loss of differentiation in the intestinal epithelium (Sansom et al., 2004, Genes & Dev. 18: 1385-1390). These mice also had a greater than twelve-fold increase in Msi-1 mRNA in intestinal epithelium indicating that APC regulates MSI expression. Our cell culture experiments indicate double-negative feedback between MSI and APC. Our studies of the mechanisms involved in this reciprocal regulation indicate that MSI is a novel Wnt target gene. Over-expression of a stabilized ?-catenin, known to upregulate Wnt target expression, in cultured colonocytes leads to increased MSI expression. Interestingly, this ?-catenin over-expression also resulted in increased APC expression, likely to compensate for the upregulation of Wnt target expression. Our data indicate that the link between APC and colonocytes differentiation observed in vivo may be attributable to the double-negative feedback between MSI and APC. Thus, we hypothesize that the product of a novel Wnt target gene, MSI, interacts with APC to maintain intestinal homeostasis. Furthermore, deregulation of this double-negative feedback system by loss of APC may play a central role in colorectal carcinogenesis.

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C4. Nuclear APC in DNA repair
Zeineldin, M, and Neufeld, KL.
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045

Mutation of the Adenomatous polyposis coli gene (Apc) is an early step in progression of about 80% of colorectal cancers. The product of the Apc gene, APC, is a large protein implicated in many cellular functions including cellular proliferation, migration, adhesion, and chromosome segregation. APC shuttles between the cytoplasm and nucleus using several nuclear localization signals (NLS) and nuclear export sequences (NES). Our lab has a long standing interest in nuclear functions of APC. To facilitate our studies, we have developed mice that are defective in nuclear import of APC because of mutations in two nuclear localization signals in APC [APC(mNLS)]. Others have suggested that APC participates in long-patch base excision repair (LP-BER). In the nucleus, oxidation, reduction or alkylation of DNA leads to modified bases which are repaired using BER. Using our mouse model as well as cultured colon cell lines, we have further explored the potential role of nuclear APC in DNA repair and examined the regulatory mechanisms that control APC stability. We will provide evidence that APC(mNLS) mouse embryonic fibroblasts (MEFs) have a reduced ability to repair DNA damaged by alkylating agents compared to MEFs with wild-type APC. In addition, we have evidence to suggest that hsp70 participates in stabilization of APC by preventing it from being degraded through the ubiquitin-proteasome pathway.

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C5. Intralymphatic drug delivery of cisplatin for treatment of breast cancer and head and neck cancer: synthesis, characterization, pharmacokinetics and anti-cancer activity in rodents
Shuang Cai¹, Yumei Xie¹, Taryn R. Bagby¹, Benjamin J. Johnston², Mark S. Cohen², M. Laird Forrest¹
¹Department of Pharmaceutical Chemistry, University of Kansas
²Department of Surgery, University of Kansas Medical Center

Breast cancers and head and neck cancers spread initially to regional lymph nodes prior to systemic spread; adequate evaluation and treatment of the axillary lymph nodes is paramount to treatment of early stage disease. An intralymphatically-targeted chemotherapy treating metastatic breast cancer and head and neck cancer was developed, which may significantly reduce side effects and increase the effectivity of cisplatin based chemotherapy. Cisplatin was conjugated to hyaluronan (HA), which is a highly biocompatible and nonimmunogenic polymer, with a conjugation degree of 20-30 w/w%. Cisplatin-HA conjugates demonstrate antiproliferative efficacy similar to standard cisplatin formulations in breast cancer cells in vitro. IC50 values of cisplatin-HA were determined to be 3 µg/mL, 7 µg/mL and 7 µg/mL, respectively, using cell lines MDA-MB-468LN, MDA-MB-231 and MCF-7. Drug-release characteristics of cisplatin-HA were determined using atomic absorption spectrometry, demonstrating sustained release of cisplatin (t1/2 of 10 hours at pH 7.4) from the HA carriers. Pharmacokinetic studies of cisplatin were conducted in rats. The area-under-the-curve of cisplatin in the axially lymph nodes after injection with HA-cisplatin increased 74% compared to normal cisplatin. The intralymphatic delivery model also exhibits sustained release kinetics, allowing higher plasma AUC and lower Cmax levels, which could translate into lower organ toxicities (eg. renal and neural). Localization of HA conjugates in lymph nodes was determined by in vivo fluorescent imaging using Texas Red tagged HA carriers. Long term toxicology studies (up to 30 days) at a dose of 3.3 mg/kg indicated that the intralymphatic delivery model demonstrated less renal toxicity during days 18 to 30 after drug administration compared to IV therapy. Pathology studies demonstrated that animals with HA-cisplatin treatment showed milder degenerative changes in livers and less congestion and necrosis in kidneys. A lymphatic metastasis model was established for breast cancer by orthotopic implantation of MDA-MB-468LN, a human breast cancer cell line with extensive lymph node metastasis, in female nude mice. A similar tumor model was established for head and neck cancer by orthotopic implantation of MDA-1986, a head and neck cancer cell line, in female nude mice. After lymphatic chemotherapy (1/wk × 2 wks), breast cancer tumor progression was delayed for two weeks during ten weeks of treatment compared to conventional treatment. Head and neck cancers (1/wk × 3 wks) were completely cured for 60% of the animals. Partial response was observed for the remaining animals.

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Higuchi Biosciences Center
University of Kansas
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Lawrence, KS 66047-2535
785-864-5183
hbc@ku.edu