Date of Award

2012-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Jianying Zhang

Abstract

p90/CIP2A is a recently characterized oncoprotein which promotes cancer cell proliferation through the inhibition of c-myc-associated PP2A phosphatase activity, from which it derived the name as cancerous inhibitor of PP2A (CIP2A). Although p90/CIP2A has been found to be overexpressed in various cancer types, from solid tumors to hematological malignancies, the function of this protein is still limited to its ability to regulate the stability of c-myc. The recent study showing p90/CIP2A is able to regulate the phosphorylation of protein kinase B (PKB) in response to the treatment of chemotherapy drugs implied p90/CIP2A may have more functions. In order to investigate the function of p90/CIP2A in cancer progression, we examined the expression level of p90/CIP2A in 72 lung cancer tissue specimen and 63 normal human lung tissues by immunohistochemistry, where results demonstrated that p90/CIP2A is significantly overexpressed in lung cancer (84.7% in lung cancer v.s. 11.1% in normal lung tissue). In addition, we also found that p90/CIP2A is overexpressed in primary lung cancer cell lines. Functional analysis of the p90/CIP2A in lung cancer cell lines, either by knock-down or overexpression, confirmed that p90/CIP2A is important for the cell proliferation.

To characterize the function of p90/CIP2A in tumor formation, we used three approaches to interrogate the role of p90/CIP2A in lung cancer. First, we used phosphoproteome array to compare the phosphorylation status of 72 key signaling molecules, and found that c-Jun N-terminal kinase (JNK) was the molecules most significantly altered by the change of p90/CIP2A, which implied a possible target utilized by p90/CIP2A to promote cell proliferation. Second, proteomic approach has been used to identify the proteome change in response to the expression level change of p90/CIP2A. We identified 49 differentially expressed protein spots on 2D-gel in which 47 proteins are identified by mass spectrometry. These proteins belong to different functional groups. A majority of the proteins are associated with metabolism, signal transduction and transcription and translational control. Furthermore, among these 47 differentially expressed proteins, 30 proteins have been well documented to be associated with cancer, of which 12 proteins are up-regulated and 18 proteins are down-regulated after knock-down of p90/CIP2A. To investigate whether these proteins are associated with any known cancer-related pathway, differentially expressed proteins are searched against the database using Pathway Studio bioinformatic software. The predicted network showed that four possible transcription factors (c-myc, ESR-1, ETS-1 and CREB) may be involved in the regulation of the differentially expressed proteins. Treatment with serum induced less phosphorylation of CREB in cells transduced with p90/CIP2A shRNA than the cells tranduced with control shRNA. However, treatment with KCl and forskolin did not have a significant difference between these two groups. Furthermore, we found that the phosphorylation of the CREB upstream kinase AKT is also down-regulated, while other kinases p38 and p90RSK is unaffected. This preliminary data implied that p90/CIP2A may target AKT-CREB pathway to promote cell proliferation in a c-myc independent manner. At last, we applied gas chromatography-mass spectrometry (GC/MS)-based metabolomic approach to identify the differential metabolites in response to either the loss or overexpression of p90/CIP2A. Seventy-six metabolites were identified, most of which were catergorized to nucleic acid metabolism, carbohydrate metabolism and fatty acid metabolism. By using Significant Analysis of Microarray (SAM) and independent component analysis (ICA), we identified three metabolite: D-glucose, L-proline and 1,2,3-propanoic acid, which can be the biomarkers to differentiate the overexpression of p90/CIP2A to the loss of p90/CIP2A. Furthermore, we found that the depletion of p90/CIP2A would cause the accumulation of glucose inside the cell, while the overexpression would promote glucose utilization. The quantification of glucose in a dynamic way showed that p90/CIP2A does not have an effect on glucose uptake, instead, it regulates the glucose metabolism through the regulation of hexokinase activity. Our data strongly suggested that the role of p90/CIP2A in promoting cell proliferation is not only replied on c-myc, but also in a c-myc-independent manner.

Language

en

Provenance

Received from ProQuest

File Size

130 pages

File Format

application/pdf

Rights Holder

Bo Peng

Download

Included in

Biology Commons

Share

COinS