Development of a colon cancer GEMM-derived orthotopic transplant model for drug discovery and validation.

PURPOSE: Effective therapies for KRAS-mutant colorectal cancer (CRC) are a critical unmet clinical need. Previously, we described genetically engineered mouse models (GEMM) for sporadic Kras-mutant and non-mutant CRC suitable for preclinical evaluation of experimental therapeutics. To accelerate drug discovery and validation, we sought to derive low-passage cell lines from GEMM Kras-mutant and wild-type tumors for in vitro screening and transplantation into the native colonic environment of immunocompetent mice for in vivo validation. EXPERIMENTAL DESIGN: Cell lines were derived from Kras-mutant and non-mutant GEMM tumors under defined media conditions. Growth kinetics, phosphoproteomes, transcriptomes, drug sensitivity, and metabolism were examined. Cell lines were implanted in mice and monitored for in vivo tumor analysis. RESULTS: Kras-mutant cell lines displayed increased proliferation, mitogen-activated protein kinase signaling, and phosphoinositide-3 kinase signaling. Microarray analysis identified significant overlap with human CRC-related gene signatures, including KRAS-mutant and metastatic CRC. Further analyses revealed enrichment for numerous disease-relevant biologic pathways, including glucose metabolism. Functional assessment in vitro and in vivo validated this finding and highlighted the dependence of Kras-mutant CRC on oncogenic signaling and on aerobic glycolysis. CONCLUSIONS: We have successfully characterized a novel GEMM-derived orthotopic transplant model of human KRAS-mutant CRC. This approach combines in vitro screening capability using low-passage cell lines that recapitulate human CRC and potential for rapid in vivo validation using cell line-derived tumors that develop in the colonic microenvironment of immunocompetent animals. Taken together, this platform is a clear advancement in preclinical CRC models for comprehensive drug discovery and validation efforts.

The dual PI3K/mTOR inhibitor NVP-BEZ235 induces tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer.

PURPOSE: To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC). EXPERIMENTAL DESIGN: PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM) model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined. RESULTS: In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC(50) = 9.0-14.3 nM). Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01) vs. a 43% decrease (p = 0.008) in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003), no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013). CONCLUSIONS: These studies provide the preclinical rationale for studies examining the efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type CRC.

Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment.

Most genetically engineered mouse (GEM) models for colon cancer are based on tissuewide or germline gene modification, resulting in tumors predominantly of the small intestine. Several of these models involve modification of the adenomatous polyposis coli (Apc) gene and are excellent models for familial cancer predisposition syndromes. We have developed a stochastic somatic mutation model for sporadic colon cancer that presents with isolated primary tumors in the distal colon and recapitulates the entire adenoma-carcinoma-metastasis axis seen in human colon cancer. Using this model, we have analyzed tumors that are either solely mutant in the Apc gene or in combination with another colon cancer-associated mutant gene, the Kras G12D allele. Because of the restricted location in the distal colon, the natural history of the tumors can be analyzed by serial colonoscopy. As the mammalian target of rapamycin (mTOR) pathway is a critical component of the complex signaling network in colon cancer, we used this model to assess the efficacy of mTOR blockade through rapamycin treatment of mice with established tumors. After treatment, Apc mutant tumors were more than 80% smaller than control tumors. However, tumors that possessed both Apc and Kras mutations did not respond to rapamycin treatment. These studies suggest that mTOR inhibitors should be further explored as potential colorectal cancer therapies in patients whose tumors do not have activating mutations in KRAS.

Comprehensive proteome analysis of an Apc mouse model uncovers proteins associated with intestinal tumorigenesis.

Tumor-derived proteins may occur in the circulation as a result of secretion, shedding from the cell surface, or cell turnover. We have applied an in-depth comprehensive proteomic strategy to plasma from intestinal tumor-bearing Apc mutant mice to identify proteins associated with tumor development. We used quantitative tandem mass spectrometry of fractionated mouse plasma to identify differentially expressed proteins in plasma from intestinal tumor-bearing Apc mutant mice relative to matched controls. Up-regulated proteins were assessed for the expression of corresponding genes in tumor tissue. A subset of proteins implicated in colorectal cancer were selected for further analysis at the tissue level using antibody microarrays, Western blotting, tumor immunohistochemistry, and novel fluorescent imaging. We identified 51 proteins that were elevated in plasma with concordant up-regulation at the RNA level in tumor tissue. The list included multiple proteins involved in colon cancer pathogenesis: cathepsin B and cathepsin D, cullin 1, Parkinson disease 7, muscle pyruvate kinase, and Ran. Of these, Parkinson disease 7, muscle pyruvate kinase, and Ran were also found to be up-regulated in human colon adenoma samples. We have identified proteins with direct relevance to colorectal carcinogenesis that are present both in plasma and in tumor tissue in intestinal tumor-bearing mice. Our results show that integrated analysis of the plasma proteome and tumor transcriptome of genetically engineered mouse models is a powerful approach for the identification of tumor-related plasma proteins.

Mass spectrometry-based study of the plasma proteome in a mouse intestinal tumor model.

Early detection of cancer can greatly improve prognosis. Identification of proteins or peptides in the circulation, at different stages of cancer, would greatly enhance treatment decisions. Mass spectrometry (MS) is emerging as a powerful tool to identify proteins from complex mixtures such as plasma that may help identify novel sets of markers that may be associated with the presence of tumors. To examine this feature we have used a genetically modified mouse model, Apc(Min), which develops intestinal tumors with 100% penetrance. Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified total plasma proteome (TPP) and plasma glycoproteome (PGP) profiles in tumor-bearing mice. Principal component analysis (PCA) and agglomerative hierarchial clustering analysis revealed that these protein profiles can be used to distinguish between tumor-bearing Apc(Min) and wild-type control mice. Leave-one-out cross-validation analysis established that global TPP and global PGP profiles can be used to correctly predict tumor-bearing animals in 17/19 (89%) and 19/19 (100%) of cases, respectively. Furthermore, leave-one-out cross-validation analysis confirmed that the significant differentially expressed proteins from both the TPP and the PGP were able to correctly predict tumor-bearing animals in 19/19 (100%) of cases. A subset of these proteins was independently validated by antibody microarrays using detection by two color rolling circle amplification (TC-RCA). Analysis of the significant differentially expressed proteins indicated that some might derive from the stroma or the host response. These studies suggest that mass spectrometry-based approaches to examine the plasma proteome may prove to be a valuable method for determining the presence of intestinal tumors.