SPOREs : Organ-Specific SPORE Programs : Gastrointestinal (GI)

Project Summaries

Project 1: Prognostic and Predictive Factors in Outcomes of Patients with Colorectal Cancer: A Population-Based Study

The molecular characteristics of colorectal cancer clearly influence prognosis and predict response to therapy. This project was motivated by the belief that better information on prognostic and predictive factors would make it possible to tailor therapy to maximize benefits and reduce cost, and by a desire to understand racial disparities in colorectal cancer mortality. The specific aims of the study are:

1. To determine which patient, treatment and molecular characteristics of colon tumors are independent predictors of prognosis.
2. To determine interactions between tumor characteristics and treatment factors and response to therapy.
3. To determine whether racial differences in tumor characteristics are responsible for the worse five year survival from colorectal cancer among blacks.

The study takes advantage of data collected in a prospective, population-based study that has obtained exceptionally detailed information on patient characteristics, treatment, outcomes as well as tumor blocks on 1000 newly-diagnosed colorectal cancer patients (600 whites and 400 blacks) drawn at random from a diverse, mixed-race, 22 county area in North Carolina (CanCORS). We have evaluated molecular characteristics including expression of protein products of genes involved in cell cycle, cell growth, apoptosis, and cellular adhesion. These markers were evaluated using tissue microarrays (TMAs). In addition, the study used DNA from microdissected blocks to evaluate loss of heterogeneity on various chromosomes, and microsatellite instability.

The parent CanCORS study has completed baseline interviews on 1019 subjects, with a 19% refusal rate. Of the 1019 subjects 755 consented to path review and we have obtained blocks from 544 subjects with additional blocks pending. We have constructed 33 TMAs and have extracted tumor and normal DNA from 460 patients. The genetic and immunohistochemical markers are shown below:

Genetic markers

Protein markers/pathways

*MDM2 Stopping rules applied

Data from this project will shed light on how patient and treatment factors interact with molecular characteristics of tumors to influence colorectal cancer prognosis. We will complete accrual of tissue blocks and process them for genetic and IHC analysis in year 5. These data will be merged with CANCORS clinical records, and then the data analyses will be completed. Because of the design of the project, by the end of year five the vast majority of the laboratory work will have been accomplished. Long-term outcome (patient survival) is the critical information needed for final analysis, and that is just beginning to become available. After full discussion within the Executive Committee of the SPORE and with the External Advisory Committee, we elected to not include this additional work as a primary project in the SPORE renewal application. The work will continue to be performed with institutional resources as we consider completion of this project to be of very high priority. We expect many additional publications to come from this further analysis that will provide critically important information on various molecular parameters and outcome in colorectal cancer in this population based analysis.

Publications
Keku TO, Qu P, Peacock J, Shen XJ, Tepper J, Ibrahim JG, Sandler RS. Statistical strategies to improve the efficiency of molecular studies of colorectal cancer prognosis. Statistics in Medicine. (submitted 2008)

Yeh JJ, Rubinas T, Routh ED, Peacock J, Martin TD, Shen XJ, Sandler RS, Keku TO, Der CJ. KRAS and BRAF Mutational Activation but not ERK Activation may correlate with Sensitivity to MEK Inhibitor Treatment in Colorectal Cancer Cell Lines. Cancer Research (submitted 2008)

Penland SK, Keku TO, Torrice C, He X, Krishnamurthy J, Hoadley KA, Woosley JT, Thomas NE, Perou CM, Sandler RS, Sharpless NE. RNA expression analysis of formalin-fixed paraffin- embedded tumors. Lab Invest. 2007;87:383-391.

Project 2: Molecular Changes in the NF-κB Pathway in Response to Chemoradiation Therapy in Rectal Cancer

We have reported that the transcription factor NF-κB provides a powerful anti-apoptotic mechanism through its ability to transcriptionally regulate genes encoding proteins that suppress apoptosis. NF-κB activation is basally detected in colorectal tumor tissue and is strongly activated in tumor cells following radiation or chemotherapy exposure. We hypothesized that radiation-induced NF-κB activation and associated downstream transcriptional responses will occur in colorectal tumors and will correlate with a decreased therapeutic response by providing an anti-apoptotic signal. The specific aims were to:

1. determine whether radiation-induced activation of NF-κB and associated induced genes occurs in pre-operative radio-chemotherapy and determine whether this responses correlates with clinical response
2. determine the ability of PS-341 to modulate NF-κB-dependent responses and to measure the toxicity of PS-341 delivered with pre-operative radio-chemotherapy
3. determine whether new approaches to inhibiting NF-κB will improve therapeutic responses in experimental colorectal tumors and to analyze the efficacy of different therapies in association with NF-κB inhibitors.

Aims 1 and 2 involved clinical trials in patients with rectal adenocarcinoma. Despite slow accrual, valuable information has been gleaned and several publications are in progress and the trials are virtually completed.

Specific Aim 1. This clinical study is unique, in that there are virtually no studies (in any disease) that evaluate the induction of gene expression by radiation in-vivo. This is critically important in understanding radiation response since radiation is given as a 5-6 week course, and the molecular profile existing before therapy is not the pattern that will drive response.

Twenty-five patients were enrolled and all but three patients underwent tumor biopsies at baseline, 2-6 hours after their initial RT fraction, and 24 hours after the initial RT fraction. Three main sets of assays have been completed on these biopsies. The first was immunohistochemistry for three NF-κB subunits, p50, p52 and p65. P50 and p65 are considered the ‘canonical’ NF-κB, while p52 often forms homodimers and has been found by us to be potentially important in several cancer types including hepatocellular carcinoma (O’Neil et al, Oncology- funded as a SPORE developmental project).

Based on the existing literature at the time this project began, we expected to observe relatively infrequent baseline activation of NF-κB, and our hypothesis was that RT-induced activation would interfere with the pro-apoptotic effects of irradiation. However, an important observation is that the majority of cases were positive at baseline for p50/p65 by IHC. This finding negatively affected our ability to assess radiation-induced changes. Therefore, we went back to our pathology database through the Tissue Procurement Core and collected another 50 archived cases of initial biopsies. The majority of these were also positive for p50/p65 at baseline. We are now in the process of analyzing our data to determine whether this baseline NF-κB activation correlates with response to radiotherapy and local recurrence-free survival. Because our results were contrary to the literature, we evaluated TMAs that were created for another SPORE project, for NF-κB expression. In this group the majority of tumors expressed p50 and p65 at baseline, suggesting frequent constitutive activation of NF-κB. In this sample NF-κB nuclear reactivity by IHC was associated with worse overall survival, and correlated statistically with activation of the RAF-MEK-ERK pathway using an antibody against phosphorylated ERK. This finding was presented at ASCO 2008, and a manuscript is in preparation for submission to Clinical Cancer Research.

The function of NF-κB was also analyzed in our samples by RT-PCR for message of 10 well-described transcriptional targets of NF-κB. We were able to observe changes in several of these NF-κB targets induced by RT. In particular, the inhibitor of apoptosis family member cIAP-2 was significantly increased by RT at 24 hours, as was the inflammatory mediator IL-8. A strong trend was also seen for the apoptosis inhibitor TRAF-1 and for Bcl-2. These findings suggest that NF-κB is indeed activated by RT in human tumors, and that NF-κB activity can be monitored in vivo. Based on this result, a larger study is warranted, perhaps as an inter-SPORE collaboration.

We also tried to define the genome-wide mRNA changes associated with a single fraction of radiotherapy at two time-points compared with baseline. We were able to perform time series arrays on 19 patients. A very surprising finding is that essentially no genes were significantly changed across all samples at either the early time-point (2-6 hours) or at 24 hours. This finding could be caused by heterogeneity of gene expression between patients. We are in the process of testing other published gene signatures of response to RT against our microarray database, including the signature derived from the German preoperative versus postoperative chemoradiation study.

Specific Aim 2. This clinical trial aimed to assess both the safety and the biologic effect (on NF-κB specifically) of the addition of the proteasome inhibitor bortezomib to standard 5-FU and radiotherapy for rectal cancer. Initial enrollment was slow, so we made biopsies optional to enhance enrollment, after discussion with our patient advocates (patients described the complexity of the study as a major factor in their refusing participation). We exceeded MTD at the second planned dose cohort (standard fraction RT, 5FU 225 mg/m2 daily and bortezomib 1.0 mg/m2 twice weekly 4/5 weeks), and we are in the process of enrolling the last three patients at the first dose level (bortezomib 0.7 mg/m2). This study will be written up for publication after one more patient completes therapy.

Specific Aim 3. We recognize that the use of a proteasome inhibitor to block NF-κB activation presents a number of specificity issues. Thus, we have begun studies aimed at the application of specific IKK inhibitors (blocking the kinase controlling NF-κB activation). We have obtained a highly specific IKKβ inhibitor and have shown that this compound has strong cytotoxic activity in certain hematologic malignancies. Additionally we have shown that both IKKα and IKKβ control NF-κB activation downstream of doxorubicin treatment of cells. This work is in press. Additionally we have found that rapamycin can block NF-κB activation under certain circumstances and we are presently testing its effect on NF-κB activation by certain chemotherapies:

Publications:
Duncan EA, Goetz CA, Stein SJ, Mayo KJ, Skaggs BJ, Ziegelbauer K, Sawyers CL, Baldwin AS. IkappaB kinase beta inhibition induces cell death in Imatinib-resistant and T315I Dasatinib-resistant BCR-ABL+ cells. Mol Cancer Ther. 2008 Feb;7(2):391-7.

W. Wilson and A. Baldwin. 2008. Maintenance of constitutive IkappaB kinase (IKK) activity by glycogen synthase kinase-3 alpha/beta in pancreatic cancer. Cancer Research, in press.

B. Bednarski, X Ding, A. Baldwin, and H.J. Kim Molecular Cancer Therapeutics, In press

H. Dan, M. Cooper, P. Cogswell, and A. Baldwin (in press, Genes and Development).

Adli M, Baldwin AS. IKK-i/IKKepsilon controls constitutive, cancer cell-associated NF-kappaB activity via regulation of Ser-536 p65/RelA phosphorylation. J Biol Chem. 2006 Sep 15;281(37):2676-84.

Basseres DS, Baldwin AS. Nuclear factor-kappaB and inhibitor of kappaB kinase pathways in oncogenic initiation and progression. Oncogene. 2006 Oct 30;25(51):6817-30.

Dave SH, Tilstra JS, Matsuoka K, Li F, Karrasch T, Uno JK, Sepulveda AR, Jobin C, Baldwin AS, Robbins PD, Plevy SE. Amelioration of chronic murine colitis by peptide-mediated transduction of the IkappaB kinase inhibitor NEMO binding domain peptide. J Immunol. 2007 Dec 1;179(11):7852-9.

Duncan EA, Anest V, Cogswell P, Baldwin AS. The kinases MSK1 and MSK2 are required for epidermal growth factor-induced, but not tumor necrosis factor-induced, histone H3 Ser10 phosphorylation. J Biol Chem. 2006 May 5;281(18):12521-5

Kim HJ, Hawke N, Baldwin AS. NF-kappaB and IKK as therapeutic targets in cancer. Cell Death Differ. 2006 May;13(5):738-47.

Steinbrecher KA, Harmel-Laws E, Sitcheran R, Baldwin AS. Loss of epithelial RelA results in deregulated intestinal proliferative/apoptotic homeostasis and susceptibility to inflammation. J Immunol. 2008 Feb 15;180(4):2588-99.

Steinbrecher KA, Wilson W, 3rd, Cogswell PC, Baldwin AS. Glycogen synthase kinase 3beta functions to specify gene-specific, NF-kappaB-dependent transcription. Mol Cell Biol. 2005 Oct;25(19):8444-55.

Project 3: Investigation of ERBB signaling in Colorectal Cancers during Liver Metastasis

The aims during the previous grant cycle were:

1. to identify markers of target inhibition by EGFR inhibitors;
2. to investigate EGFR independent mechanisms of CRC growth and
3. to develop alternative or synergistic therapeutic targets.

We have achieved the original aims and will build upon these results by developing a new target against CRC that should have much greater efficacy in the clinic than EGFR. More specifically, using a panel of human CRCs, we identified expression of the EGFR ligand genes amphiregulin (AREG) and epiregulin (EREG) as markers of CRCs likely to respond to EGFR inhibitor therapy (Caudle et al. submitted). These ligands are much more highly expressed in the tumor than in normal colonic mucosa, and even more highly expressed in liver metatases. Concurrently, we showed that expression of the same genes also correlates with EGFR inhibition sensitivity in the ApcMin mouse model of human CRC, and further showed with the mouse model that EGFR dependency of CRCs is a constitutional characteristic indicating that molecular markers, like expression of AREG and EREG, can identify those cancers that are likely to respond to EGFR inhibitor treatment before therapeutic intervention (Yu et al. submitted). Gene expression signatures in mice containing Areg and Ereg also led to the identification of ERBB3 as a target likely to be more important than EGFR in CRC development (Lee et al. submitted).

Importantly, to correlate our findings in snap frozen tumors and cell lines to clinical trial specimens we are collaborating with the Cancer and Leukemia Group B (CALGB). We have approval from the CALGB to study banked patient samples from CALGB clinical trial 80203 which randomized untreated metastatic colorectal cancer pts to FOLFOX or FOLFIRI ± cetuximab (independent of EGFR status.). We have developed and verified methods to extract mRNA from paraffin embedded samples and correlating IHC staining for the receptors and ligands of the family. We intend also to correlate epiregulin and amphiregulin expression to cetuximab response and to validate our findings of increased expression of Her2 and Her3.

These results, described in detail in the Project 3 write up, form the foundation for renewal of this project. In addition to these key findings, research supported in whole or part by this grant resulted in the following 21 peer-reviewed publications and manuscripts under review.

Publications
Lee D, Pearsall RS, Das S, Dey SK, Godfrey VL,Threadgill DW. 2004. Epiregulin is not essential for development of intestinal tumors but is required for protection from intestinal damage. Molecular and Cellular Biology 24:8907-8916.

Threadgill DW. 2005. Metastatic potential as a heritable trait. Nature Genetics 37:1026-1027.

Fujimoto H, Wislez M, Zhang J, Iwanaga K, Dackor J, Hanna AE, Kalyankrishna S, Cody DD, Price RE, Sato M, Shay JW, Minna JD, Peyton M, Tang X, Massarelli E, Herbst R, Threadgill DW, Wistuba I, Kurie JM. 2005. High expression of ErbB family members and their ligands in lung adenocarcinomas that are sensitive to inhibition of epidermal growth factor receptor. Cancer Research 65:11478-11485. NOTE: This paper resulted from an inter-SPORE collaboration with the Lung SPORE at MD Anderson Cancer Center.

Bissahoyo A, Pearsall RS, Hanlon KE, Amann V, Hicks D, Godfrey VL, Threadgill DW. 2005. Azoxymethane is a genetic background-dependent colorectal tumor initiator and promoter in mice: effects of dose, route, and diet. Toxicological Sciences 88:340-345.

Genther Williams SM, Disbrow G, Schlegel R, Lee D, Threadgill DW, Lambert PF. 2005. Requirement of epidermal growth factor receptor for hyperplasia induced by E5, a high risk human papillomavirus oncogene. Cancer Research: 65:6534-6542.

Hatch SB, Lightfoot HM Jr, Garwacki CP, Moore DT, Calvo BF, Woosley JT, Sciarrotta J, Funkhouser WK, Farber RA. 2005. Microsatellite instability testing in colorectal carcinoma: choice of markers affects sensitivity of detection of mismatch repair-deficient tumors. Clin Cancer Res 11:2180-2187.

Franklin JL, Yoshiura K, Dempsey PJ, Bogatcheva G, Jeyakumar L, Meise S, Pearsall RS, Threadgill DW, Coffey RJ. 2005. Identification of MAGI-3 as a transforming growth factor-a binding protein. Experimental Cell Research 303:457-470. NOTE: This paper resulted from an inter-SPORE collaboration with the GI SPORE at Vanderbilt University.

Syed HA, Threadgill DW. 2006. Enhanced oligonucleotide microarray labeling and hybridization. BioTechniques 41:685-686.

Alexander AD, Orcutt RP, Henry JC, Baker J, Bissahoyo AC, Threadgill DW. 2006. Quantitative PCR assays for mouse enteric flora reveal strain-dependent differences in composition that are influenced by the microenvironment. Mammalian Genome 17:1093-1104.

Uronis JM, Herfarth HH, Rubinas TC, Bissahoyo AC, Hanlon K, Threadgill DW. 2007. Flat colorectal cancers are genetically determined and progress to invasive cancer without going through a polypoid stage. Cancer Research 67:11594-11600.

Kaiser S, Park Y, Franklin J, Halberg RB, Yu M, Jessen WJ, Freudenberg J, Chen X, Haigis K, Jegga A, Kong S, Sakthivel B, Xu H, Reichling T, Azhar M, Roberts RB, Bissahoyo AC, Gonzales F, Bloom G, Eschrich S, Carter SL, Aronow J, Kleimeyer J, Kleimeyer M, Ramaswamy V, Settle SH, Boone B, Levy S, Graff JM, Doetschman T, Groden JM, Dove WF, Threadgill DW, Yeatman T, Coffey RJ, Aronow BJ. 2007. Recapitulation of embryonic colon transcriptional patterns by mouse colon tumor models and human colon cancer. Genome Biology 8:R131.1-R131.26. NOTE: This paper resulted from an inter-SPORE collaboration with the GI SPORE at Vanderbilt University and an inter-program collaboration with the Mouse Models of Human Cancer Consortium (UNC, Vanderbilt, and Ohio State).

Huang H, Eversley CD, Threadgill DW, Zou F. 2007. Bayesian multiple quantitative trait loci mapping for complex traits using markers of the entire genome. Genetics 176:2529-2540.

Chi Y-Y, Ibrahim JG, Bissahoyo AC, Threadgill DW. 2007. Baysian hierarchical modeling for time course microarray experiments. Biometrics 63:496-504.

Abate-Shen C, Brown PH, Colburn NH, Gerner EW, Green JE, Lipkin M, Nelson WG, Threadgill DW. 2008. The untapped potential of genetically-engineered mouse models in chemoprevention research: opportunities and challenges. Cancer Prevention Research 1:161-166. NOTE: This paper resulted from an inter-program collaboration with the Mouse Models of Human Cancer Consortium (UNC, Columbia University, and NCI).

Radiloff D, Rinella E, Threadgill DW. 2008. Modeling cancer patient populations in mice: complex genetics and environmental factors. Drug Discovery Today: Disease Models 4:83-88.

Barrick CJ, Yu M, Chao H-H, Threadgill DW. 2008. Chronic pharmacologic inhibition of EGFR leads to cardiac dysfunction in C57BL/6J mice. Toxicology and Applied Pharmacology 228:315-325.

Lee T-C, Threadgill DW. 2008. Generation and validation of mice carrying a conditional allele of the epidermal growth factor receptor. genesis: J of Genet and Develop, in press.

Submitted
Caudle AS, Caskey LS, Rubinas TC, Moore DT, Earp HS, Calvo BF
. Increased amphiregulin and epiregulin expression in colorectal tumors may explain clinical response to EGFR-targeted antibody therapy.

Lee DK, Yu M, Kim H, Paniccia C, Kurie JM, Threadgill DW. Tumor-specific apoptosis caused by deletion of the ERBB3 pseudo-kinase in the intestinal epithelium. (revision under consideration by the Journal of Clinical Investigation). NOTE: This manuscript resulted from an inter-SPORE collaboration with the Lung SPORE at MD Anderson Cancer Center.

Uronis JM, Threadgill DW. Murine models of colorectal cancer.

Alexander AD, Orcutt R, Bissahoyo AC, Hanlon K, Threadgill DW. Gastrointestinal flora alters progression but not penetrance of azoxymethane-induced colorectal cancers.

Yu M, Lee T-C, Threadgill DW. A constitutional molecular signature for EGFR-dependent colorectal cancers.

Project 4: Targeting the RAS>ERK Pathway for Colorectal Cancer Treatment

Specific Aims

1. To determine the contribution of mutated Ras to colorectal carcinoma tumorigenicity
2. To determine if Ras mutation status alone determines the susceptibility of colorectal carcinomas to growth inhibition by inhibitors of Raf and MEK
3. To determine in a prospective collection of patient-derived colorectal carcinomas, a gene expression profile of clinical relevance in predicting sensitivity to anti-Ras and anti-Raf or anti-MEK therapeutic strategies

Aim 1. We showed that interfering RNA (RNAi) suppression of mutant KRAS suppressed the anchorage-independent and tumorigenic growth of colon (CRC) and pancreatic (PDAC) carcinoma cell lines. These results validate KRAS mutation, despite being an early event in CRC and PDAC tumor progression, as a clinically useful drug target for treatment of these cancers. The recent discovery of mutations in BRAF, a key downstream effector of Ras, in CRC suggests that Ras activation of the Raf-MEK-ERK pathway will be important in CRC. Therefore, we used constitutive RNAi to silence mutant BRAF in CRC cell lines. We found that mutant BRAF function was necessary for the anchorage-independent and tumorigenic growth of BRAF mutation positive CRC as well as melanoma cell lines. However, surprisingly, the anchorage-dependent growth of CRCs did not require mutant BRAF function, whereas BRAF mutant melanoma cells underwent apoptosis when mutant BRAF was suppressed. This latter result emphasizes the importance of cell context in evaluating the role of specific Ras effector signaling pathways in different cancers.

Aim 2. We utilized three highly selective and potent inhibitors of the MEK1 and MEK2 serine/threonine kinases to determine if (1) KRAS mutation status was a useful genetic determinant to identify CRC and PDAC patients who will be responsive to MEK inhibitor therapy and to determine if ERK1/ERK2 protein kinase activity provided an accurate biomarker for MEK inhibitor anti-tumor activity. In one study we used the U0126 and CI-1040 (PD184352) MEK inhibitors on a panel of 15 colorectal carcinoma (CRC) tumor cell lines that harbor mutant KRAS or BRAF. First, we found a correlation between BRAF but not KRAS mutation status and ERK activation. Second, elevated ERK activation, regardless of KRAS or BRAF mutation status, did not correlate with sensitivity to MEK inhibitor- mediated growth inhibition. However, MEK inhibitor treatment impaired the anchorage-independent growth of nearly all KRAS/BRAF mutants, and not wild type, CRC cells. Third, MEK inhibitor reduction of ERK activity did not correlate with inhibition of anchorage-independent growth. Finally, we validated our cell line observations and found that although ERK activation did correlate with BRAF mutation status, it did not correlate with KRAS mutation status in patient CRC tissues. These results suggest that KRAS or BRAF mutation status, but not ERK activation, may be useful biomarkers for MEK inhibitor sensitivity. In a second study we evaluated another MEK inhibitor (AZD6244/ARRY-142886) that is currently in clinical evaluation, for related questions in CRC, as well as PDAC cell lines. We found that BRAF may be a reliable biomarker for AZD6244 sensitivity. However, ERK activity may not be a reliable biomarker since we found that ERK inhibition did not correlate with the ability of AZD6244 to inhibit tumor growth. These results prompted our use of microarray analyses to identify a gene expression profile to better predict MEK inhibitor sensitivity. This analysis identified a gene set that suggests that activation of the phosphatidylinositol 3-kinase pathway may contribute to MEK inhibitor insensitivity.

Aim 3. These studies have utilized a CRC tumor microarray comprised of 190 patient samples generated by Project #1 of the SPORE. We found that ERK activation correlated with BRAF but not KRAS mutational activation. Thus, we conclude that KRAS mutation status will not provide a reliable genetic marker to identify patients who may respond to anti-MEK therapy. We have used immunohistochemical staining analyses to evaluate ERK activity in normal and tumor colonic tissue. The surprising observation from this study was that we found that ERK activity is elevated in normal colonic epithelial cells, and reduced in a subset of tumors. This result raises the concern that MEK inhibitor treatment may show toxicity for normal colonic tissue. Thus, while our studies do support a therapeutic value in targeting the Raf-MEK-ERK cascade for CRC and PDAC treatment, we also suggest that targeting additional Ras effector pathways will be necessary for effective inhibition of aberrant Ras signaling. This prompted our focus on the RalGEF-Ral small GTPase effector pathway. Our studies have validated the importance of this pathway in both PDAC and CRC growth. Additionally, we have identified two candidate pharmacologic approaches to block Ral GTPase signaling in PDAC. These observations provide the foundation for our future studies to perform further preclinical evaluation of inhibitors of geranylgeranyltransferase-I and Aurora-A kinase using state-of-the art mouse models for PDAC, with the goal of initiating clinical trials for PDAC.

Publications

1. Baines, A.T., Lim, K.H., Shields, J.M., Lambert, J.M., Counter, C.M., Der, C.J. and Cox, A.D. (2006). Use of retrovirus expression of interfering RNA to determine the contribution of activated K-Ras and Raf effector expression to human tumor cell growth. Methods Enzymol., 407, 556-574.
2. Hao, H., Muniz-Medina, V.M., Mehta, H., Thomas, N.E., Klazak, V., Der, C.J. and Shields, J.M. (2007). Differential roles of mutant B-Raf signaling in melanoma and colorectal carcinoma cell growth. Mol. Cancer Res., 6, 2220-2229.
3. Campbell, P.M., Groehler, A.L., Lee, K.M., Ouellette, M.M., Khazak, V. and Der, C.J. (2007). K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling. Cancer Res, 67, 2098-2106.
4. Yeh, J.J., Rubinas, T., Routh, E.D., Peacock, J., Martin, T.D., Shen, X.J., Sandler, R.S., Kim, H.J., Keku, T.O. and Der, C.J. KRAS and BRAF mutational activation but not ERK1/2 activation may correlate with sensitivity to MEK inhibitor treatment in colorectal cancer cell lines. Cancer Res., revised manuscript under review.
5. Shields, J.M., Thomas, N.E., Cregger, M., Berger, A.J., Leslie, M., Torrice, C., Hao, H., Penland, S., Arbiser, J., Scott, G., Zhou, T., Bar-Eli, M., Bear, J.E., Der, C.J., Kaufmann, W., Rimm, D.L. and Sharpless, N.E. (2007). Lack of ERK mitogen-activated protein kinase activation and signaling demonstrates a new type of melanoma. Cancer Res, 67, 1502-1512.
6. Campbell, P.M., Lee, K.M., Ouellette, M.M., Kim, H.J., Groehler, A.L., Khazak, V. and Der, C.J. (2008). Ras-driven transformation of human nestin-positive pancreatic epithelial cells. Methods Enzymol., 439, 451-465.
7. Lim, K.-H., Baines, A.T., Fiordalisi, J.J., Shipitsin, M., Feig, L.A., Cox, A.D., Der, C.J. and Counter, C.M. (2005). Activation of RalA is critical for Ras-induced tumorigenesis of human cells. Cancer Cell, 7, 533-545.
8. Lim, K.H., O’Hayer, K., Adams, S.J., Kendall, S.D., Campbell, P.M., Der, C.J. and Counter, C.M. (2006). Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells. Curr, Biol, 16, 2385-2394.
9. Lim, K.H., Brady, D.C., Der, C.J., Cox, A.D., and Counter, C.M. Aurora-A promotes Ras oncogenesis through phosphorylation and relocalization of RalA. Mol. Cell. Biol., under review.
10. Reiner, D.J., Gonzalez-Perez, V., Der, C.J. and Cox, A.D. Use of C. elegans to evaluate inhibitors of Ras function in vivo. Methods Enzymol., 439, 425-449.
11. Roberts, P.J. and Der, C.J. (2007). Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene, 26, 3291-3310.
12. Yeh, J.J. and Der, C.J. (2007). Targeting signal transduction in pancreatic cancer treatment. Expert Opin. Targeted Therapy, 11, 673-694.

Project 5: Pharmacogenomic Dissection of Metastatic Colorectal Cancer.

Project 5’s hypothesis is that evaluation of genomic changes in patients with clinically resistant tumors, will allow for improved patient selection for therapy and will define new therapeutic targets and drug combinations. The specific aims are to:

1. Discover somatic mutations and copy number changes associated with clinical resistance in resected colorectal liver metastases.
2. Define genomic changes associated with clinical resistance in prospectively ascertained colorectal liver metastases.
3. Determine the clinical impact of ‘resistance’ genomic alterations in patients with metastatic colorectal cancer.

We have performed an extensive genomic analysis of 50 cancer chemotherapy genes in the germline of 120 European American, African American, and Asian American individuals.43 We identified 904 variants in 50 genes, including 139 coding variants. A substantial number of variants (38%) had not been previously reported in the public databases, highlighting that resequencing is still of high value in the current informatics age. We have also been performing resequencing of chemotherapy candidate genes in previously untreated gastrointestinal tumors. Our initial effort for irinotecan, gemcitabine, and bevacizumab candidate genes has identified 18-37 genetic variants per gene, of which 4-6 are novel somatic mutations. These variants are then either expressed in heterologous expression systems (if in a coding region) or in a reporter construct for promoter activity (if in a regulatory region).

We have performed initial candidate gene copy analysis for a number of genes of relevance to GI cancer therapy, including TYMS (5FU). DNA copy number was determined using TaqMan® real-time quantitative PCR assays. Nucleic acids from tumor and normal tissue were obtained from the GI SPORE Tissue Procurement/Pathology core. TYMS amplification was not found in any of the normal controls (CEPH cells from unrelated donors) or normal tissues (colonic mucosa or liver tissue adjacent to tumor). However, amplification (> 2 copies) of TYMS was observed in 7% (3/41) of colorectal liver metastasis from patients who had not been treated with anticancer therapy in the previous 6 months. The incidence of TYMS amplification nearly doubled (13%; 7/55) in colorectal liver metastases from patients who received FOLFOX therapy prior to liver resection.

Advances in genomic technology now allow for very dense assessment of gene copy number across the human genome. We have used high density Nimblegen microarray-based assessment of DNA copy number, using up to 3,500,000 probes across the genome . This work demonstrates that the informatics and statistical analysis infrastructure is in place to use high density copy number assessment in this project.

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