Richard B. Halberg

Transcriptional recapitulation and subversion of embryonic colon development by mouse colon tumor models and human colon cancer

BackgroundThe expression of carcino-embryonic antigen by colorectal cancer is an example of oncogenic activation of embryonic gene expression. Hypothesizing that oncogenesis-recapitulating-ontogenesis may represent a broad programmatic commitment, we compared gene expression patterns of human colorectal cancers (CRCs) and mouse colon tumor models to those of mouse colon development embryonic days 13.5-18.5.ResultsWe report here that 39 colon tumors from four independent mouse models and 100 human CRCs encompassing all clinical stages shared a striking recapitulation of embryonic colon gene expression. Compared to normal adult colon, all mouse and human tumors over-expressed a large cluster of genes highly enriched for functional association to the control of cell cycle progression, proliferation, and migration, including those encoding MYC, AKT2, PLK1 and SPARC. Mouse tumors positive for nuclear β-catenin shifted the shared embryonic pattern to that of early development. Human and mouse tumors differed from normal embryonic colon by their loss of expression modules enriched for tumor suppressors (EDNRB, HSPE, KIT and LSP1). Human CRC adenocarcinomas lost an additional suppressor module (IGFBP4, MAP4K1, PDGFRA, STAB1 and WNT4). Many human tumor samples also gained expression of a coordinately regulated module associated with advanced malignancy (ABCC1, FOXO3A, LIF, PIK3R1, PRNP, TNC, TIMP3 and VEGF).ConclusionCross-species, developmental, and multi-model gene expression patterning comparisons provide an integrated and versatile framework for definition of transcriptional programs associated with oncogenesis. This approach also provides a general method for identifying pattern-specific biomarkers and therapeutic targets. This delineation and categorization of developmental and non-developmental activator and suppressor gene modules can thus facilitate the formulation of sophisticated hypotheses to evaluate potential synergistic effects of targeting within- and between-modules for next-generation combinatorial therapeutics and improved mouse models.

Development of a Fluorescent Multiplex Assay for Detection of MSI-High Tumors

Determining whether a tumor exhibits microsatellite instability (MSI) is useful in identifying patients with hereditary non-polyposis colorectal cancer and sporadic gastrointestinal cancers with defective DNA mismatch repair (MMR). The assessment of MSI status aids in establishing a clinical prognosis and may be predictive of tumor response to chemotherapy. A reference panel of five markers was suggested for MSI analysis by a National Cancer Institute (NCI) workshop in 1997 that has helped to standardize testing. But this panel of markers has limitations resulting from the inclusion of dinucleotide markers, which are less sensitive and specific for detection of tumors with MMR deficiencies compared to other types of markers that are currently available. This study demonstrates that mononucleotides are the most sensitive and specific markers for detection of tumors with defects in MMR and identifies an optimal panel of markers for detection of MSI-H tumors. A set of 266 mono-, di-, tetra- and penta-nucleotide repeat microsatellite markers were used to screen for MSI in colorectal tumors. The best markers for detection of MSI-H tumors were selected for a MSI Multiplex System, which included five mononucleotide markers: BAT-25, BAT-26, NR-21, NR-24 and MONO-27. In addition, two pentanucleotide markers were added to identify sample mix-ups and/or contamination. We classified 153 colorectal tumors using the new MSI Multiplex System and compared the results to those obtained with a panel of 10 microsatellite markers combined with immunohistochemical (IHC) analysis. We observed 99% concordance between the two methods with nearly 100% accuracy in detection of MSI-H tumors. Approximately 5% of the MSI-H tumors had normal levels of four MMR proteins and as a result would have been misclassified based solely on IHC analysis, emphasizing the importance of performing MSI testing. The new MSI Multiplex System offers several distinct advantages over other methods of MSI testing in that it is both extremely sensitive and specific and amenable to high-throughput analysis. The MSI Multiplex System meets the new recommendations proposed at the recent 2002 NCI workshop on HNPCC and MSI testing and overcomes problems inherent to the original five-marker panel. The use of a single multiplex fluorescent MSI assay reduces the time and costs involved in MSI testing with increased reliability and accuracy and thus should facilitate widespread screening for microsatellite instability in tumors of patients with gastrointestinal cancers.

Clusterin as a biomarker in murine and human intestinal neoplasia

Early detection of colorectal cancer is critical for the management of this disease. Biomarkers for early detection of several cancers have been developed and applied clinically in recent years. We have sought to discover candidate biomarkers without the restricted choice of markers placed on microarrays, and without the biological complications of genetic and environmental heterogeneity. We have compared by cDNA subtraction two genetically matched sets of mice, one developing multiple intestinal neoplasia (C57BL/6J-ApcMin) and the other tumor-free (C57BL/6J). One prominent candidate biomarker, clusterin, was then subjected to a series of validation steps. In situ hybridization and immunohistochemistry were used to analyze clusterin expression at a cellular level on a series of murine intestinal and human colonic neoplasms. Elevated clusterin expression was characterized within certain regions of murine and human tumors regardless of tumor stage, location, or mode of initiation. The cells showing high clusterin levels generally lacked differentiation markers and adenomatous polyposis coli antigen. Tumor cells undergoing apoptosis expressed low levels of clusterin. Its specific expression patterns and correlation with cellular events during tumorigenesis make it a useful diagnostic tool in the mouse and a potential contributor to the set of biomarkers for early detection of human colon cancer.

The Mom1 AKR intestinal tumor resistance region consists of Pla2g2a and a locus distal to D4Mit64

The Mom1 (Modifier of Min-1) region of distal chromosome 4 was identified during a screen for polymorphic modifiers of intestinal tumorigenesis in ApcMin/+ mice. Here, we demonstrate that the Mom1AKR allele consists of two genetic components. These include the secretory phospholipase Pla2g2a, whose candidacy as a Mom1 resistance modifier has now been tested with several transgenic lines. A second region, distal to Pla2g2a, has also been identified using fine structure recombinants. Pla2g2aAKR transgenic mice demonstrate a modest resistance to tumorigenesis in the small intestine and a very robust resistance in the large intestine. Moreover, the tumor resistance in the colon of Pla2g2aAKR animals is dosage-dependent, a finding that is consistent with our observation that Pla2g2a is expressed in goblet cells. By contrast, mice carrying the distal Mom1 modifier demonstrate a modest tumor resistance that is confined to the small intestine. Thus, the phenotypes of these two modifier loci are complementary, both in their quantitative and regional effects. The additive effects and tight linkage of these modifiers may have been necessary for the initial identification of the Mom1 region.

Intestinal adenomagenesis involves core molecular signatures of the epithelial–mesenchymal transition

The epithelial–mesenchymal transition (EMT) occurs commonly during carcinoma invasion and metastasis, but not during early tumorigenesis. Microarray data demonstrated elevation of vimentin, a mesenchymal marker, in intestinal adenomas from ApcMin/+ (Min) mice. We have tested the involvement of EMT in early tumorigenesis in mammalian intestines by following EMT-associated markers. Elevated vimentin RNA expression and protein production were detected within neoplastic cells in murine intestinal adenomas. Similarly, vimentin protein was detected in both adenomas and invasive adenocarcinomas of the human colon, but not in the normal colonic epithelium or in hyperplastic polyps. Expression of E-cadherin varied inversely with vimentin. In addition, the expression of fibronectin was elevated while that of E-cadherin decreased. Canonical E-cadherin suppressors, such as Snail, were not elevated in the same tumor. Elevated vimentin expression in the adenoma was not correlated with persistent Ras signaling, but was strongly correlated with reduced proliferation indices, active Wnt signaling, and TGF-β signaling, as demonstrated by its dependence on Smad3. We designate our observations of expression of only some of the canonical features of EMT as “truncated EMT”. These unexpected observations are interpreted as reflecting the involvement of a core of the EMT system during the tissue remodeling of early tumorigenesis.

Mice expressing activated PI3K rapidly develop advanced colon cancer

Aberrations in the phosphoinositide 3-kinase (PI3K) signaling pathway play a key role in the pathogenesis of numerous cancers by altering cellular growth, metabolism, proliferation, and apoptosis. Mutations in the catalytic domain of PI3K that generate a dominantly active kinase are commonly found in human colorectal cancers and have been thought to drive tumor progression but not initiation. However, the effects of constitutively activated PI3K upon the intestinal mucosa have not been previously studied in animal models. Here, we show that the expression of a dominantly active form of the PI3K protein in the mouse intestine results in hyperplasia and advanced neoplasia. Mice expressing constitutively active PI3K in the epithelial cells of the distal small bowel and colon rapidly developed invasive adenocarcinomas in the colon that spread into the mesentery and adjacent organs. The histologic characteristics of these tumors were strikingly similar to invasive mucinous colon cancers in humans. Interestingly, these tumors formed without a benign polypoid intermediary, consistent with the lack of aberrant WNT signaling observed. Together, our findings indicate a noncanonical mechanism of colon tumor initiation that is mediated through activation of PI3K. This unique model has the potential to further our understanding of human disease and facilitate the development of therapeutics through pharmacologic screening and biomarker identification.

PIK3CA and APC Mutations are Synergistic in the Development of Intestinal Cancers

Human colorectal cancers are known to possess multiple mutations, though how these mutations interact in tumor development and progression has not been fully investigated. We have previously described the FCPIK3ca* murine colon cancer model, which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K results in hyperplasia and invasive mucinous adenocarcinomas. These cancers form via a non-canonical mechanism of tumor initiation that is mediated through activation of PI3K and not through aberrations in WNT signaling. Since the Adenomatous Polyposis Coli (APC) gene is mutated in the majority of human colon cancers and often occurs simultaneously with PIK3CA mutations, we sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine. In this study, we have generated mice in which the expression of a constitutively active PI3K and the loss of APC occur simultaneously in the distal small intestine and colon. Here, we demonstrate that expression of a dominant active PI3K synergizes with loss of APC activity resulting in a dramatic change in tumor multiplicity, size, morphology and invasiveness. Activation of the PI3K pathway is not able to directly activate WNT signaling through the nuclear localization of CTNNB1 (β-catenin) in the absence of aberrant WNT signaling. Alterations at the transcriptional level, including increased CCND1, may be the etiology of synergy between these activated pathways.

Long-lived Min Mice Develop Advanced Intestinal Cancers through a Genetically Conservative Pathway

C57BL/6J mice carrying the Min allele of Adenomatous polyposis coli (Apc) develop numerous adenomas along the entire length of the intestine and consequently die at an early age. This short lifespan would prevent the accumulation of somatic genetic mutations or epigenetic alterations necessary for tumor progression. To overcome this limitation, we generated F(1) Apc(Min/+) hybrids by crossing C57BR/cdcJ and SWR/J females to C57BL/6J Apc(Min/+) males. These hybrids developed few intestinal tumors and often lived longer than 1 year. Many of the tumors (24-87%) were invasive adenocarcinomas, in which neoplastic tissue penetrated through the muscle wall into the mesentery. In a few cases (3%), lesions metastasized by extension to regional lymph nodes. The development of these familial cancers does not require chromosomal gains or losses, a high level of microsatellite instability, or the presence of Helicobacter. To test whether genetic instability might accelerate tumor progression, we generated Apc(Min/+) mice homozygous for the hypomorphic allele of the Nijmegen breakage syndrome gene (Nbs1(DeltaB)) and also treated Apc(Min/+) mice with a strong somatic mutagen. These imposed genetic instabilities did not reduce the time required for cancers to form nor increase the percentage of cancers nor drive progression to the point of distant metastasis. In summary, we have found that the Apc(Min/+) mouse model for familial intestinal cancer can develop frequent invasive cancers in the absence of overt genomic instability. Possible factors that promote invasion include age-dependent epigenetic changes, conservative somatic recombination, or direct effects of alleles in the F(1) hybrid genetic background.

Discovery and validation of colonic tumor-associated proteins via metabolic labeling and stable isotopic dilution

The unique biology of a neoplasm is reflected by its distinct molecular profile compared with normal tissue. To understand tumor development better, we have undertaken a quantitative proteomic search for abnormally expressed proteins in colonic tumors from ApcMin/+ (Min) mice. By raising pairs of Min and wild-type mice on diets derived from natural-abundance or 15N-labeled algae, we used metabolic labeling to compare protein levels in colonic tumor versus normal tissue. Because metabolic labeling allows internal control throughout sample preparation and analysis, technical error is minimized as compared with in vitro labeling. Several proteins displayed altered expression, and a subset was validated via stable isotopic dilution using synthetic peptide standards. We also compared gene and protein expression among tumor and nontumor tissue, revealing limited correlation. This divergence was especially pronounced for species showing biological change, highlighting the complementary perspectives provided by transcriptomics and proteomics. Our work demonstrates the power of metabolic labeling combined with stable isotopic dilution as an integrated strategy for the identification and validation of differentially expressed proteins using rodent models of human disease.

Use of mononucleotide repeat markers for detection of microsatellite instability in mouse tumors

Tumors lacking DNA mismatch repair activity (MMR) from patients with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) or those with sporadic colorectal cancer can be identified by the presence of high levels of instability in repetitive sequences known as microsatellites (MSI). The assessment of MSI phenotype in human tumors helps to establish a clinical diagnosis and is accomplished with a reference panel of five mononucleotide repeats. By contrast, detection of MSI in mouse tumors has proven to be problematic and lack of a uniform set of markers for classification of MSI has impeded comparison of results between studies. We tested for MSI in intestinal tumors from MMR‐deficient mice with four mononucleotide repeats with polyA24‐37 tracts and three new markers with extended polyA59‐67 tracts. All seven markers were sensitive to MSI in MMR‐deficient tumors, but those with extended mononucleotide tracts displayed larger deletions, which were easily distinguishable from the germline alleles. With a panel of the five most sensitive and specific mononucleotide repeats, a high level of MSI was detected in 100% of MMR‐deficient tumors, but not in tumors with MMR activity. This novel panel is an improvement over existing combinations of mono‐ and dinucleotide repeat markers and should facilitate MSI screening and standardize results from different studies.