Fecal Metabolome in Hmga1 Transgenic Mice with Polyposis

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The Fecal Metabolome in Hmga1 Transgenic Mice with Polyposis: Evidence for Potential Screen for Early Detection of Precursor Lesions in Colorectal Cancer Michael David Williams, Lingling Xian, Tait Huso, Jeong-Jin Park, David Huso, Leslie M Cope, David R Gang, William F. Siems, Linda Resar, Raymond Reeves, and Herbert H. Hill, Jr. J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00035 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 7, 2016

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Journal of Proteome Research

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The Fecal Metabolome in Hmga1 Transgenic Mice with Polyposis: Evidence for Potential Screen for Early Detection of Precursor Lesions in Colorectal Cancer Authors: Michael D. Williams1, Lingling Xian4, Tait Huso4, Jeong-Jin Park3, David Huso4, Leslie M. Cope5, David R. Gang3, William F. Siems1, Linda Resar4,5,6*, Raymond Reeves2* and Herbert H. Hill Jr.1,*

1. Department of Chemistry, Washington State University, Pullman, WA 99164, USA 2. School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA 3. Institute of Biological Chemistry, Washington State Univ., Pullman, WA 99164 USA 4. Department of Medicine, the Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA 5. Department of Oncology, the Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA 6. Institute for Cellular Engineering, the Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Submitted to: J Proteome Research

January 2016

* Co-Corresponding and senior authors to whom inquiries should be addressed: Herbert H. Hill, Jr., Department of Chemistry, Washington State University, Pullman, WA 99164, email: [email protected] : Tel 509-335-5648 Raymond Reeves, School of Molecular Biosciences, Washington State University, Pullman, WA 99164, email: [email protected] ; Tel: 509-335-1948 Linda Resar, the Johns Hopkins University School of Medicine, Baltimore, MD 21205, email: [email protected] ; Tel: 410-614-0712

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2 ABSTRACT: Because colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, more accessible screening tests are urgently needed to identify early-stage lesions. We hypothesized that highly sensitive, metabolic profile analysis of stool samples will identify metabolites associated with early-stage lesions and could serve as a noninvasive screening test. We therefore applied travelling wave ion mobility mass spectrometry (TWIMMS) coupled with ultra-performance liquid chromatography (UPLC) to investigate metabolic aberrations in stool samples in a transgenic model of pre-malignant polyposis aberrantly expressing the gene encoding the high mobility group A (Hmga1) chromatin remodeling protein. Here, we report for the first time that the fecal metabolome of Hmga1 mice is distinct from that of control mice and includes metabolites previously identified in human CRC. Significant alterations were observed in fatty acid metabolites and metabolites associated with bile acids (hypoxanthine xanthine, taurine) in Hmga1 mice compared to controls. Surprisingly, a marked increase in the levels of distinctive short, arginine-enriched, tetra-peptide fragments was observed in the transgenic mice. Together these findings suggest that specific metabolites are associated with Hmga1-induced polyposis and abnormal proliferation in intestinal epithelium. Although further studies are needed, these data provide a compelling rationale to develop fecal metabolomic analysis as a noninvasive screening tool to detect early precursor lesions to CRC in humans. KEYWORDS: colorectal cancer, high mobility group A1 (HMGA1) chromatin remodeling protein, cancer metabolomics, cancer screening, ion mobility mass spectrometry, INTRODUCTION: Colorectal cancer (CRC) remains a leading cause of cancer death worldwide and the incidence is rising, particularly in developing countries. In the US alone, CRC is the third leading cause of cancer-related deaths among both men and women. In fact, about 50,000 people were expected to die from CRC in the


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3 United States in 2015. Detection of early lesions is key to survival for CRC as the five-year disease-free survival in over 90% in patients presenting with localized tumors, whereas it is 2-fold change) had 2 or more Arg residues (Table 2). Interestingly, the most significant fold change (14.5-fold) occurred in a tetra-peptide with three Arg residues. This suggests that the amino acid arginine plays an important role in distinguishing Hmga1 from control stool and also correlates well with our previous finding that arginine is significantly up-regulated in Hmga1 mouse intestinal tissue21. An additional 26 tetra-peptides were found to contain 1 or more phenylalanine (Phe) residues. Among these Phe-containing tetra-peptides, many with the most significant fold change (2.9 to 7.14-fold) also contained at least one Arg residue. Likewise in our previous study, in addition to arginine and phenylalanine, significant increases in leucine/isoleucine (Leu/Ile) and tyrosine (Tyr) were observed in Hmga1 mouse intestinal tissue. In this study, we found that many of the most highly elevated Arg-containing tetra-peptides (>3-fold; p-values < 0.05) in Hmga1 fecal samples also contained Tyr or Leu/Ile residues. These findings suggest a relationship between these amino acids and the development of hyperproliferation and polyposis driven by Hmga1. The identification of small Arg-enriched peptides was surprising however. Further work is needed to probe the source of these compounds that differentiate Hmga1 from control mouse stool samples. Several possibilities exist, including enhanced protein degradation in the hyperproliferative intestinal epithelium or potential changes in the gut microbiome that could result from Hmga1 and the polyposis.


In

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13 addition, we were not able to definitively identify the specific sequence of amino acid residues within these peptides and further investigation to ascertain their identity is also needed. Hypoxanthine, xanthine, and taurine were all significantly elevated in the Hmga1 stool pellets (Figure 3). Notably, these metabolites were also observed in the intestinal epithelium of the Hmga1 mice21 as compared to control mice and in human CRC samples20 as compared to nonmalignant intestinal epithelium. Hypoxanthine is a product of purine metabolism that is degraded by the enzyme xanthine oxidase reductase (XOR), which is decreased in diverse malignancies, including CRC as well as gastric and breast cancers. Interestingly, decreased XOR protein or gene expression is associated with poor differentiated status, advanced stage and decreased survival in CRC. Although the basis for the elevated hypoxanthine in the Hmga1 stool and abnormal intestinal epithelium is not clear, decreased expression or function of XOR could contribute to this result. Hypoxanthine and xanthine are also both involved in the breakdown of ATP and secretion of uric acid. When ATP is hydrolyzed to release energy, it undergoes two de-phosphorylation cycles, producing ADP, then AMP, releasing 2 phosphates which results in energy for cellular metabolism. AMP is converted to hypoxanthine, then xanthine, and ultimately excreted as uric acid. Increased energy production by ATP breakdown could occur in intestinal epithelium with hyperproliferation and polyposis as observed in our Hmga1 transgenic model. In both this study and in our previous investigations of mouse intestinal tissue,21 we found that fatty acids (FA) were altered in the Hmga1 mice as compared to wildtype controls.

In both intestinal

epithelium and fecal samples, we discovered increases in several FAs, suggesting a link between FA metabolism and the development of pre-malignant polyposis. We also found similar alterations in FA metabolites in localized, primary human CRC samples as compared to nonmalignant, adjacent control intestinal epithelium. Notably, substantial prior literature has documented alterations in FA metabolism



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14 in diverse tumor types.50–62 In our prior study, we also found that FA metabolism alterations increase during the transition from localized tumors to metastatic disease in the human samples.20 Additionally, we observed that several metabolites involved in fatty acid biosynthesis, including phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs), were elevated in the Hmga1 mouse fecal metabolome (Figure 4 & Table 3). Strikingly, we also observed that both PCs and LPCs are elevated in the intestinal epithelial samples from the Hmga1 mice and in primary human CRC samples.21 Increased lipid synthesis is a common feature of cancer cells. While the basis for these specific elevations could not be determined from this study, prior studies have shown that LPC is a lipid-derived second messenger that triggers downstream activation of pathways involved in survival and angiogenesis,

including

phosphatidylinositol

3-kinase/Akt

and

mitogen-activated

protein

kinase/extracellular signal regulated kinase pathways. Both PCs and LPCs are also important for membrane formation and fluidity and could be increased in the setting or cellular proliferation in addition to functioning as bioactive molecules that promote cell growth, angiogenesis and metastasis.63,64

They may also be involved in energy metabolism of malignant cells.50-53

Lysophospholipids (LPLs) have been shown to be important regulatory biomolecules that correlate with disease progression in breast, lung, brain, prostate, and ovarian cancers, as well as in CRC cancer cell lines.51,63,66 Cytosolic phospholipase A2 (cPLA2) enzymes hydrolyze phospholipids to produce lysophospholipids. In addition, functional studies have shown that inhibiting these enzymes will disrupt tumor growth and metastasis, suggesting that cLPA2 and LPLs have important regulatory roles in tumor progression and angiogenesis.54,56,64,67 LPCs are usually present as minor phospholipids in cell membranes that are quickly metabolized by the enzyme lysophosphatidylcholine acyltransferase 1 (LPCAT1), producing PCs. This enzyme is overexpressed in human colorectal adenocarcinomas when compared to normal mucosa,55 which could reflect enhanced enzymatic activity to convert excess LPCs to PCs. In fact, several studies have explored the use of lysophosphatidic acids as biomarkers for cancer


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15 55-56,64

. These include a study of plasma samples from CRC patients in which 18:1-LPC and 18:2-LPC were

identified as potential biomarkers.59 The simultaneous increase in several lysophosphatidylcholine (LPC) species observed in the present study of fecal samples together with the prior study of mouse intestinal epithelium and human CRC suggests a strong association of these metabolites with Hmga1 overexpression. Additional metabolites that were found to significantly differentiate (p

Fecal Metabolome in Hmga1 Transgenic Mice with Polyposis

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