Kenneth E. Hung

EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib

UNLABELLEDnBRAF mutations occur in 10-15% of colorectal cancers (CRCs) and confer adverse outcome. While RAF inhibitors such as vemurafenib (PLX4032) have proven effective in BRAF mutant melanoma, they are surprisingly ineffective in BRAF mutant CRCs, and the reason for this disparity remains unclear. Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to vemurafenib, and P-ERK suppression was not sustained in response to treatment. Although transient inhibition of phospho-ERK by vemurafenib was observed in CRC, rapid ERK re-activation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRCs expressed higher levels of phospho-EGFR than BRAF mutant melanomas, suggesting that CRCs are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells and markedly improved efficacy in vitro and in vivo. These findings support evaluation of combined RAF and EGFR inhibition in BRAF mutant CRC patients.nnnSIGNIFICANCEnBRAF valine 600 (V600) mutations occur in 10% to 15% of colorectal cancers, yet these tumors show a surprisingly low clinical response rate (~5%) to selective RAF inhibitors such as vemurafenib, which have produced dramatic response rates (60%–80%) in melanomas harboring the identical BRAF V600 mutation. We found that EGFR-mediated MAPK pathway reactivation leads to resistance to vemurafenib in BRAF-mutant colorectal cancers and that combined RAF and EGFR inhibition can lead to sustained MAPK pathway suppression and improved efficacy in vitro and in tumor xenografts.

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.

Cathepsin B Expression and Survival in Colon Cancer: Implications for Molecular Detection of Neoplasia

Background and Aims: Proteases play a critical role in tumorigenesis and are upregulated in colorectal cancer and neoplastic polyps. In animal models, cathepsin B (CTSB)–activatable imaging agents show high enzyme activity within intestinal tumors. Methods: We conducted a prospective cohort study of 558 men and women with colon cancer with tumors that were accessible for immunohistochemical assessment. We used Cox proportional hazards models, stratified by stage, to compute colon cancer–specific and overall mortality according to tumoral expression of CTSB. Results: Among 558 participants, 457 (82%) had tumors that expressed CTSB (CTSB positive) and 101 (18%) had tumors that did not express CTSB (CTSB negative). CTSB expression was not associated with disease stage (P = 0.19). After a median follow-up of 11.6 years, there were 254 total and 155 colon cancer–specific deaths. Compared with participants with CTSB-negative tumors, participants with CTSB-positive tumors experienced a multivariate hazard ratio for colon cancer–specific mortality of 1.99 (95% confidence interval, 1.19-3.34) and overall mortality of 1.71 (95% confidence interval, 1.16-2.50). CTSB expression was independently associated with KRAS (P = 0.01) and BRAF mutation (P = 0.04), but not microsatellite instability status, CpG island methylator phenotype status, PIK3CA mutation, LINE-1 methylation, TP53 expression, or PTGS2 (cyclooxygenase-2) expression. Among 123 individuals with adenomas, 91% expressed CTSB. Conclusions: As assessed by immunohistochemistry, CTSB is expressed in the vast majority of colon cancers, independent of stage, and is significantly associated with higher risk of colon cancer–specific and overall mortality. Impact: These results support the potential of CTSB a target for image detection of neoplastic lesions in humans. Cancer Epidemiol Biomarkers Prev; 19(11); 2777–85. ©2010 AACR.

A mouse plasma peptide atlas as a resource for disease proteomics

We present an in-depth analysis of mouse plasma leading to the development of a publicly available repository composed of 568 liquid chromatography-tandem mass spectrometry runs. A total of 13,779 distinct peptides have been identified with high confidence. The corresponding approximately 3,000 proteins are estimated to span a 7 logarithmic range of abundance in plasma. A major finding from this study is the identification of novel isoforms and transcript variants not previously predicted from genome analysis.

Low-volume, high-throughput sandwich immunoassays for profiling plasma proteins in mice: identification of early-stage systemic inflammation in a mouse model of intestinal cancer.

Mouse models of human cancers may provide a valuable resource for the discovery of cancer biomarkers. We have developed a practical strategy for profiling specific proteins in mouse plasma using low‐volume sandwich‐immunoassays. We used this method to profile the levels of 14 different cytokines, acute‐phase reactants, and other cancer markers in plasma from mouse models of intestinal tumors and their wild‐type littermates, using as little as 1.5μl of diluted plasma per assay. Many of the proteins were significantly and consistently up‐regulated in the mutant mice. The mutant mice could be distinguished nearly perfectly from the wild‐type mice based on the combined levels of as few as three markers. Many of the proteins were up‐regulated even in the mutant mice with few or no tumors, suggesting the presence of a systemic host response at an early stage of cancer development. These results have implications for the study of host responses in mouse models of cancers and demonstrate the value of a new low‐volume, high‐throughput sandwich‐immunoassay method for sensitively profiling protein levels in cancer.

In Vivo Optical Molecular Imaging of Matrix Metalloproteinase Activity following Celecoxib Therapy for Colorectal Cancer

We present an optical molecular imaging approach to measure the efficacy of the cyclooxygenase-2 (COX-2) inhibitor celecoxib on tumor growth rate through its effect on matrix metalloproteinase (MMP) activity. A xenograft model of colorectal cancer was generated in nude mice, which were then randomized to receive celecoxib versus vehicle. MMP activity was measured by an enzyme-activatable optical molecular probe. A novel genetically engineered mouse (GEM) model of colorectal cancer was also used to assess celecoxibs effect on MMP activity, which was measured by quantitative fluorescence colonoscopy. Subcutaneously implanted xenograft tumors were 84% (SD 20.2%) smaller in volume in the treatment group versus the control group. Moreover, treated animals exhibited only a 7.6% (SEM 9%) increase in MMP activity versus 106% (SEM 8%) for untreated animals. There was an apparent linear relationship (r = .91) between measured MMP activity and tumor growth rate. Finally, in the GEM model experiment, treated murine tumors remained relatively unchanged in volume and MMP activity; however, untreated tumors grew significantly and showed an increase in MMP activity. This method may provide for the improved identification of patients for whom COX-2 inhibition therapy is indicated by allowing one to balance the patients cardiovascular risk with the cancers responsiveness to celecoxib.