Yeung, Li, Patel, in collaboration with Knudson, Bellacosa, Litwin, and Cooper

Our study of colon cancer began with Dr. Al Knudson, inventor of the two-hit hypothesis for cancer, believing that the one-hit state of precancerous cells is an untapped opportunity for cancer development intervention if the transition to the two-hit state can be blocked by exploiting biomarkers of the one-hit state. We studied patients with Familial Adenomatous Polyposis (FAP), because they are virtually certain to develop colon cancer, and because much is known about the causative APC gene. We hypothesized that the inherited heterozygous mutation itself leads to changes in the proteome of morphologically normal crypts and the proteins that changed may represent targets for preventive and therapeutic agents. We determined the differential protein expression of morphologically normal colon crypts of FAP patients versus those of individuals without the mutation, using two-dimensional gel electrophoresis, mass spectrometry and validation by 2D gel Western blotting.

Approximately 13% of 1,695 identified proteins were abnormally expressed in the morphologically normal crypts of APC mutation carriers, indicating that a colon crypt cell under the one-hit state is already abnormal. Many of the expression changes affect pathways consistent with the function of the APC protein, including apoptosis, cell adhesion, cell motility, cytoskeletal organization and biogenesis, mitosis, transcription and oxidative stress response. Thus, heterozygosity for a mutant APC tumor suppressor gene alters the proteome of normal-appearing crypt cells in a gene-specific manner, consistent with a detectable one-hit event. These changes may represent the earliest biomarkers of colorectal cancer development, potentially leading to the identification of molecular targets for cancer prevention.

We continue to seek these candidate biomarkers in the serum, aiming for a more convenient way for early cancer detection.Top

Yeung, Ke, Patel, Li, in collaboration with Tokar, Haluszka, Hoffman, Watson, Weinberg, Nguyen, S.J. Cohen, Meropol, and Litwin

Pancreatic cancer is the fourth leading cause of cancer death in the U.S. Many cases arise from mucinous cystic lesions but most cases diagnosed by current art are not curable. At this time, there are no diagnostic indicators that are consistently reliable, obtainable, and conclusive for diagnosing and risk-stratifying pancreatic cysts. To establish more effective diagnostic biomarkers and to provide deeper understanding about the molecular profile within these cysts, we identified and quantified approximately 500 cyst fluid proteins and correlated the findings to clinical parameters. Pancreatic cyst fluids were collected by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) from 20 patients.

We first performed MALDI-TOF peptidomics with peptides identified by sequencing. Sequencing of more than 350 free peptides showed that exopeptidase activities rendered peptidomics of cyst fluids unreliable. Next we performed 2D gel comparative proteomics of the cyst fluids, identifying all the proteins detectable in the gels. We showed that protein nicking by proteases in the cyst fluids produced hundreds of protein spots from the major proteins, causing 2-dimensional gel proteomics of pancreatic cyst fluids unmanageable.

The proteins in the cyst fluids were ascertained by LC/MS/MS analysis on a highly accurate and stable mass spectrometer. This method uses proteins digested by trypsin into fragments, thus the endogenous proteolysis became minor events by comparison. From just 15 micrograms of protein from less than 40 microliters of cyst fluid per sample, we simultaneously measured traditional pancreatic cancer markers of mucins, amylase, and CEA. The ease of measuring soluble mucins by mass spectrometry sensitively and specifically is a significant progress. We further propose to use of a panel of homologs of these biomarkers, namely, two homologs of amylase, five soluble mucins, five soluble CEA-related cell adhesion molecules (CEACAMs), and four S100 homologs, to facilitate future pancreatic cyst diagnosis and risk-stratification.

The most immediate application of our finding is the ability to analyze dozens of protein biomarkers important to pancreatic cancer simultaneously in less than one drop of pancreatic cyst fluid. This ability may allow earlier diagnosis, or analysis of these biomarkers in situations where a cyst produce too liquid fluid for traditional clinical tests. The second advantage is the ability to eliminate false positive diagnosis of the presence of mucin when stomach mucin contamination is a concern. The mass spectrometry unambiguously distinguishes whether the mucin present is the pancreatic form or another form that can arise from both the stomach and the pancreas.

Our present effort is aimed at developing a mass spectrometry protocol for quantitative assays of these biomarkers that can be a hundred times more sensitive than the "discovery phrase" mass spectrometry we used in above study.Top

Patel BB, He YA, Li XM, and Yeung AT. In collaboration with Frolov A, Vanderveer L, Slater C, Schilder RJ, von Mehren M, Godwin AK

Imatinib mesylate (Gleevec, Novartis, Basel, Switzerland) is a small-molecule tyrosine kinase inhibitor with activity against ABL, BCR-ABL, c-KIT, and PDGFR alpha. Several clinical trials have evaluated the efficacy and safety of imatinib in patients with ovarian carcinoma who have persistent or recurrent disease following front-line platinum/taxane based chemotherapy. However, there is limited pre-clinical and clinical data on the molecular targets and action of imatinib in ovarian cancer. We treated human ovarian cancer cells (A2780) with imatinib mesylate for either 6 or 24 h. We employed a 2D (two-dimensional) gel electrophoresis and mass spectrometry-based proteomics approach to identify protein expression patterns and signaling pathways that were altered in response to imatinib. Cells were analyzed for PDGFR alpha and AKT expression, which were then correlated with imatinib sensitivity.

Using 2D gel electrophoresis of overlapping pH ranges from pH 4 to 11, about 4,000 protein spots could be analyzed reproducibly. Proteins whose levels changed between twofold to 30 fold were grouped according to whether changes were in the same direction at both time points of treatment with respect to the control, or changed their levels only at one of the time points.

We concluded that differentially regulated proteins following imatinib treatment of A2780 cells involved the regulation of actin cytoskeleton, metabolic pathways, cell cycle, cell proliferation, apoptosis, cell junctions, and signal transduction. Thus, exposure of cells to imatinib produces complex changes in the cell that require further investigation.Top