Citations

Abstract Endocrine therapy, with SERMs and AIs, is the most important treatment modality for the 70% of patients with ER+ early breast cancer. Clinically, there is marked variability between patients in response to treatment and adverse events (AEs) such as musculoskeletal (MS) AEs with AIs. In 649 women with early-stage breast cancer we identified large inter-individual variations in pretreatment and on-anastrozole (A) plasma estrogens as well as plasma A concentrations, suggesting that the approved A dose of 1 mg per day may not be optimal for all patients. We performed a series of genome-wide association studies (GWAS) and identified a genome-wide significant variant SNP associated with concentrations of E2 that created an estrogen response element (ERE), and a significant SNP associated with E1-conjugates, providing potential insights into the variability. Pharmacogenomics (PGx) is the study of the role of germline and somatic genetic variation in individual variability in drug response phenotypes, i.e., disease response and AEs. The scientific goal is to understand mechanisms for the phenotype and the clinical goal is the determination of the right drug at the right dose for the right patient. The Mayo PGx Research Network Center and Breast SPORE and the RIKEN Center for Integrative Medical Science have worked collaboratively with cooperative groups to perform GWAS in women treated with both SERMs and AIs. The PGx paradigm consists of the GWAS as the initial step in a process that goes beyond the identification of associations to study the relationship of the single nucleotide polymorphisms (SNPs) to genes and the relationships of these SNPs and genes to the drug effect and the phenotype under study. Using the phenotype of AI-related MS-AEs, the major reason women stop therapy, a case-control GWAS was performed with patients from MA.27. The GWAS identified a SNP on chromosome (Chr) 14 that was associated with MS-AEs, near a gene (TCL1A), and created an ERE. Importantly, TCL1A expression was related to expression of multiple cytokine receptors, NFκB, and chemokines (related to rheumatoid arthritis) in an E2- and SNP-dependent fashion. These results provide a pharmacogenomic basis for MS-AEs with AIs. Using the phenotype of development of breast cancer in high-risk women receiving SERMs, a case-control GWAS was performed with patients entered on the NSABP P-1 and P-2 trials. The GWAS revealed variant SNPs on Chr 4 near CTSO (deleterious) and Chr 16 in ZNF423 (protective). The OR for differences in risk of breast cancer for participant homozygous for both protective or both deleterious alleles was 5.71. Both CTSO and ZNF423 were shown to participate in E2-dependent induction of BRCA1 expression in a SNP-dependent fashion. Most remarkable was that the presence of a SERM reversed the E2-inducible expression of ZNF423 and BRCA1 in a SNP-dependent fashion. In addition, ZNF423 was shown to be an E2-inducible BRCA1 transcription factor. The deleterious variant SNP near CTSO was found to disrupt an ERE. The GWAS described led to functional genomic studies that produced new knowledge relating to endocrine biology that provide a focus for further research to move towards precision endocrine therapy for women with breast cancer. Citation Format: James N. Ingle. Pharmacogenomics in the Quest for Precision Endocrine Therapy of Breast Cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr ML-1.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Roughly 45,000 patients are diagnosed with the disease each year and of these patients 94% will not survive past 5 years. The most common genetic alteration associated with PDAC is an oncogenic mutation in the KRAS gene, which is believed to initiate the disease. Mutant Kras is generally thought to signal constitutively to its downstream effector proteins, such as Raf/MEK/Erk and PI3K/AKT. However, recently we and others have demonstrated that Epidermal Growth Factor Receptor (EGFR) signaling upstream of mutant Kras is necessary for unlocking its oncogenic potential in the pancreas, distinct from Kras driven tumorigenesis in the lung or colon. Unfortunately, while EGFR inhibition is effective in blocking pancreatic tumorigenesis in mice, it has limited benefit in treating human PDAC patients. Furthermore, it has been shown that wild-type Nras and Hras have indispensable signaling roles in cells expressing oncogenic Kras, and that both the mutant and WT Ras signals are required for cell growth. Here we show that pancreatic cancer cell lines containing oncogenic Kras still require upstream signaling to maintain high levels of both active Erk and AKT although these signals are not necessarily EGFR derived. We have identified a subset of pancreatic cancer cell lines that rely upon IR/IGFR signaling to maintain the high levels of Erk and AKT signaling required for continued cell growth. Additionally, this signaling appears to be independent of oncogenic Kras and instead correlates with the levels of active wild-type Nras and Hras. This data indicates that patients may benefit from the simultaneous inhibition of both the EGFR and IR/IGFR signaling pathways. Citation Format: Kenneth K. Takeuchi, Howard C. Crawford. EGFR and IR/IGFR activation sustains ERK1/2 and AKT signaling independently of mutant Kras in pancreatic cancer cells lines. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B58. doi: 10.1158/1557-3125.RASONC14-B58

Abstract Oncogenic K-RAS activation is a major menace that drives aggressive tumor progression and metastasis in 30% of all human cancer. Currently, there are no effective therapies to treat stage III and IV metastatic human cancers with oncogenic K-RAS hyperactivation that often confer drug resistance, aggressive tumor growth, systemic metastasis, and poor clinical outcome. Therefore, finding novel approaches and new drug targets to inhibit oncogenic K-RAS pathway activation is an urgent goal and the major challenge in cancer therapy and anti-K-RAS-based drug development. Instead of targeting an upstream signaling module such as EGFR/HER2/K-RAS/B-RAF, we targeted the most downstream signaling module in the oncogenic K-RAS signaling pathway called the SIAH-dependent proteolytic machinery. SIAHs are the human homologs of Seven-In-Absentia (SINA), an evolutionarily conserved RING E3 ligase - the most downstream signaling module and a critical “gatekeeper” required for proper RAS signal transduction. Guided by the insights and fundamental principles learned from the Drosophila RAS signal transduction, we conducted preclinical studies to dissect SIAH function in promoting the oncogenic K-RAS-driven tumorigenesis and metastasis in human cancer. We found that (1) SIAH is a new biomarker reflective of oncogenic K-RAS activation in human cancer, and (2) SIAH loss-of-function is highly effective to block tumorigenesis and metastasis against the well-established, end-stage and metastatic pancreatic cancer and triple negative breast cancer (TNBC). These findings demonstrate that SIAH is an attractive and logical new therapeutic target for developing novel and effective anti-K-RAS and anticancer therapy against metastatic human cancer. Through our work, SIAH has emerged as a promising new drug target against oncogenic K-RAS hyperactivation in metastatic human cancer cells. Using anti-SIAH molecules to block oncogenic K-RAS signaling in human cancer is an excellent example of science going “from the bench (basic research in fruit flies) to the bedside (preclinical studies and ultimately clinical trials)”. As a highly evolutionarily conserved E3 ligase that is the most downstream and the most conserved “signaling gatekeeper” in the oncogenic K-RAS signaling network, SIAH is uniquely and strategically positioned to become a great and logical anti-K-RAS drug target. Our preclinical studies have demonstrated that “SIAH-dependent proteolysis” is indeed an Achilles' heel in metastatic human cancer cells. Knowledge gained from our preclinical study has promising translational values. Anti-SIAH-based small molecule inhibitors are likely to aid in expanding our limited arsenal of novel anti-K-RAS-based anticancer therapies. By attacking the oncogenic K-RAS pathway using multi-pronged synergistic inhibitions at upstream (EGFR/HER2 membrane receptors), midstream (K-RAS/B-RAF/MEK/mTOR) and downstream (SIAH E3 ligase) signaling modules in parallel, we will be in a position to control the late-stage, relapsed and metastatic human cancers by shutting down the hyperactivated K-RAS signaling transduction cascades in cancer cells in the future. Citation Format: Minglei Bian, Yang Liao, Vasilena Zheleva, Zena Urban, Monicah Njogu, Justin J. Odanga, Andrew J. Isbell, Roger R. Perry, Richard A. Hoefer, Thomas C. Smyrk, Gloria M. Petersen, Amy H. Tang. Attacking the most downstream “gatekeeper,” the SIAH-dependent proteolytic machinery, in the oncogenic ERBB/K-RAS signaling pathway to block tumorigenesis and control metastasis in human cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A26. doi: 10.1158/1557-3125.RASONC14-A26

Abstract Mounting evidence indicates that oncogenic Ras can modulate cell autonomous inflammatory cytokine production, although the underlying mechanism remains unclear. Here we show that squamous cell carcinoma antigen (SCCA), a member of the Serpin family of serine/cysteine protease inhibitors, is transcriptionally up-regulated by oncogenic Ras via MAPK and the ETS family transcription factor PEA3. Increased SCCA expression leads to inhibition of protein turnover, unfolded protein response (UPR), activation of NF-κB, and is essential for Ras-mediated cytokine production and tumor growth. Analysis of human colorectal and pancreatic tumor samples reveals a positive correlation between Ras mutation, enhanced SCCA expression, and IL-6 expression. These results indicate that SCCA is a Ras-responsive factor that plays an essential role in Ras-associated cytokine production and tumorigenesis. Citation Format: Joseph Catanzaro, Namratha Sheshadri, Ji-An Pan, Yu Sun, Chanjuan Shi, Jinyu Li, Scott Powers, Howard Crawford, Wei-Xing Zong. Oncogenic Ras induces inflammatory cytokine production by upregulating squamous cell carcinoma antigen (SerpinB3). [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A23. doi: 10.1158/1557-3125.RASONC14-A23

Abstract Almost all reported cases of pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer, harbor an oncogenic Kras mutation. Recently, targeting oncogenic Kras to specific cell types in the adult pancreas has shown the acinar compartment to be more susceptible to neoplastic transformation than ductal cells. Acinar to ductal metaplasia (ADM) is a phenomenon where acinar cells undergo a morphological change and begin expressing markers normally found only in ductal cells while losing markers typical of acinar cells. ADM is highly proliferative with the potential to develop into pancreatic intraepithelial neoplasias, a precursor lesion to pancreatic cancer. Acinar cells harvested from mice expressing mutant Kras undergo spontaneous ADM when embedded in 3D collagen cultures, however, we and others have found the epidermal growth factor receptor (EGFR) is required for ADM induced by mutant Kras expression in vitro. Activation of EGFR by treatment of WT collagen embedded acinar cell explants with the EGFR ligand TGF-α has also been shown to induce ADM in a manner requiring Notch pathway activation. Furthermore, it has been demonstrated that expression of Notch2 intracellular domain (N2ICD) is sufficient to induce ADM in WT acinar cells. To directly investigate the relationship of the EGFR, Kras, and Notch signaling pathways in ADM, we examined the effect of Notch signaling in acinar cells harvested from EGFRf/f; Ptf1a+/Cre (EKO) mice, which harbor a pancreas specific deletion of EGFR. Surprisingly, we found that expression of N2ICD failed to induce ADM in acinar cells from EKO mice. We also observed that co-expression of a constitutively active p110α mutant or mutant Kras with N2ICD was able to rescue ADM in acinar cells from EKO mice, though neither was sufficient to drive ADM in the absence of N2ICD. Additionally, treatment of acinar cells from WT mice with either the p110α inhibitor Pik75 or the expression of a dominant negative mutant of the PI3K downstream target Rac1 reduced Erk1/2 phosphorylation, whereas expression of constitutively active p110α increased the level of Erk1/2 phosphorylation. These results demonstrate the importance of EGFR signaling in ADM, and identify the need to determine the mechanism of crosstalk between the PI3K and MAPK pathways in acinar cells. Citation Format: Christopher J. Halbrook, Howard C. Crawford. Notch signaling couples with Kras or PI3K to drive EGFR independent pancreatic acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A09. doi: 10.1158/1557-3125.RASONC14-A09

Abstract Background: In the adjuvant setting, HER2 breast cancer patients treated with trastuzumab along with chemotherapy have superior survival compared to patients treated with chemotherapy alone (Perez EA, et al. J Clin Oncol. 2013 31:2115-22). Functional evidence suggests a role for the FC gamma receptor genes in antibody response. Mice deficient in the FC gamma receptor genes show significantly reduced antitumor effect. Polymorphisms within FCGR2A and FCGR3A are associated with binding affinity of natural killer cells to the IgG1 portion of trastuzumab and a polymorphism in FCGR2B (I232T, T allele) is associated with impaired negative regulatory activity. Association of FC gamma receptor polymorphisms with clinical response among trastuzumab-treated patients is equivocal with both positive (Musolino A, et al. J Clin Oncol. 2008 26:1789-96) and negative associations (Hurvitz SA, et al. Clin Cancer Res. 2012 18:3478-86), a lack of sufficiently powered clinical samples and a lack of cohorts with clearly demonstrated survival benefit to trastuzumab containing regimens. Methods: Genomic DNA was analyzed for FcyRIIIa, FcyRIIa, and FcyRIIb polymorphisms (FCGR3A V158F, FCGR2A H131R and FCGR2B I232T) using Taqman SNP real-time PCR assays (Applied Biosystems) in 1,336 patients from the N9831 clinical trial of adjuvant trastuzumab in HER2+ breast cancer. 1325 patients were evaluable. Patients in Arm A (N=419) received chemotherapy only. Patients in Arms B (N=469) and C (N= 437) were treated with chemotherapy and trastuzumab (sequentially in Arm B and concurrently in Arm C). We performed Kaplan-Meier analyses of disease free survival (DFS) by arm and genotype for each polymorphism. Results: There were only minor differences in demographics and tumor features when comparing the subset with DNA to other N9831 participants who did not provide DNA. Patients who provided DNA in Arms B and C demonstrated superior DFS relative to patients in Arm A who provided DNA (p<0.001). Genotype distributions in the N9831 population for FCGR2A and FCGR3A polymorphisms were in Hardy-Weinberg equilibrium, p>0.05 and FCGR2B, p>0.01. In contrast to previous reports in the metastatic setting, we did not find that trastuzumab-treated patients who had the FCGR3A 158 V and/or FCGR2A 131 H genotypes demonstrated superior DFS to those without those genotypes. Similarly, we did not see an effect of FCGR2B I232T with the exception that I/I patients (p<0.001) apparently derived more benefit, in terms of DFS, from trastuzumab than did T carriers (p=0.81). However, 5-year DFS was not different between these two groups, I/I and T carriers within Arms B/C. Conclusions: Our analysis failed to reveal strong associations between FCGR genotypes and outcome in patients with HER2+ breast cancer treated with trastuzumab. While the present data do not preclude a functional role for FC gamma receptors in the clinical activity of trastuzumab, the results suggest that these naturally occurring polymorphisms may have minor (if any) impact in the adjuvant setting, which is consistent with previous findings by Hurvitz and colleagues. Citation Format: Mark Pegram, Rebecca M. Olson, Nadine Norton, Kathleen Tenner, Karla L. Ballman, Raphael Clynes, Keith L. Knutson, Edith A. Perez. Analysis of the associations of FC gamma receptor polymorphisms (FCGR3A, FCGR2A, and FCGR2B) with outcome in HER2+ breast cancer patients treated with trastuzumab in NCCTG (Alliance) trial N9831. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B123.

Abstract Rational: Poor prognosis epithelial-derived cancers often exhibit morphologic and molecular changes characteristic of an epithelial to mesenchymal transition (EMT). EMT markers are predominantly found in tumors with a basal-like phenotype. Increased expression of the mesenchymal cadherins N-cadherin and/or Cadherin-11 (CDH11) and decreased E-cadherin, have been associated with both EMT and tumor progression. Importantly, CDH11 is a therapeutic target in rheumatoid arthritis (RA), an inflammatory disease with properties often compared with cancer. As CDH11 antibody based therapeutics are in clinical trials for RA and we recently showed that the arthritis drug celecoxib has the structural potential to bind CDH11, there is a strong possibility that, if CDH11can be shown to drive malignant progression rather than simply be associated with it, therapeutic options may be rapidly developed. Results: We show that CDH11 is increased early in breast cancer and ductal carcinoma in-situ. CDH11 knockdown and antibodies effective in RA slowed the growth of basal-like breast tumors. CDH11 attenuation decreased proliferation and colony formation of breast, glioblastoma and prostate cancer cells. The arthritis drug celecoxib, which has characteristics consistent with CDH11 binding, an analogue without COX-2 inhibitory activity and other molecules that target CDH11, preferentially affect CDH11 positive cancer cells. Conclusion: Our work demonstrates that cadherin-11 is important for malignant progression, is a therapeutic target in several aggressive tumors and introduces an antibody, novel small molecules and an approved arthritis drug that inhibit its function with potential for rapid clinical translation. Citation Format: Shahin Assefnia, Sivanesan Dakshanamurthy, Jaime M. Guidry Auvil, Constanze Hampel, Panos Anastasiadis, Bhaskar Kallakury, Aykut Uren, David W. Foley, Milton Brown, Lawrence Shapiro, Michael Brenner, David Haigh, Stephen W. Byers. Cadherin-11, a common therapeutic target in poor prognosis malignancies and rheumatoid arthritis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A045.

Abstract Cancer treatment is complicated when patients develop resistance to standard therapies. Hence, developing strategies to address resistance to chemo therapy and improving therapeutic efficiency is of great importance in treating cancer successfully. Results from various preclinical studies showed that specificity protein (Sp) transcription factors regulate key genes associated with tumorigenesis, angiogenesis, and metastasis in several cancer models. Sp1 and Sp3 modulate the expression of survivin, an inhibitor of apoptosis protein (IAP) and we previously showed that targeting Sp transcription factors induced the response to radiation in pancreatic cancer cells and mice tumors. Research from our laboratory and others demonstrated the anti-cancer activity of a small molecule Tolfenamic Acid (TA) in some preclinical models of cancer including pancreatic cancer. It has been proposed that the anti-cancer activity of TA is associated with modulation of Sp proteins; however the precise underlying mechanisms are not extensively known. In this investigation, we have conducted a molecular profiling analysis in three pancreatic cancer cell lines, Panc1, L3.6 pl and MiaPaCa2 to elucidate the mechanisms involved in the anti-cancer activity of TA. We also evaluated the potential of TA for improving the therapeutic efficacy of chemotherapeutic agents in pancreatic cancer cell lines and mice xenografts using two standard chemotherapeutic agents, 5-Flurouracil (5FU) and gemcitabine (GEM). Human pancreatic cancer cells, Panc1, L3.6pl and MiaPaCa2 were treated with 5FU or GEM or TA for 24-72 h to establish the dose curves. After monitoring the time/dose-response curves, optimized doses were used for the combination treatment. Cells were treated 5FU or GEM or TA or combinations (5FU+TA or GEM+TA) and the cell viability was measured at 48 h post-treatment. The tumor growth inhibitory response of individual and combination treatment was determined in mice xenografts. Nude mice were treated with 5FU (40 mg/kg, 3 times/wk for 2 wk) or GEM (50 mg/kg, 2 times/wk for 2 wk) or TA (50 mg/kg, 3 times/wk for 4 wk) or TA+5FU or TA+GEM as per the established methods. After the termination of the experiment, animals were euthanized; tumors were harvested and measured to determine the changes in tumor weight and volume. Analysis of molecular profiling revealed that Sp1 and survivin are among the top candidates modulated by TA treatment; however TA modulates many genes associated with cancer and cell survival. The combination treatment experiments showed a significant increase in the anti-proliferative response of 5FU or GEM when used along with TA. Mice xenograft experiments also confirmed that TA enhanced the tumor growth inhibitory response of 5FU or GEM. Experiments using drug-resistant cell lines and functional studies to understand the underlying mechanisms of these combination therapies are ongoing. These preclinical studies support the potential of TA for chemo sensitization and induction of the therapeutic response of standard chemotherapeutic agents in pancreatic cancer. Since enhancing the responses of standard therapeutic options is highly beneficial in treating cancer, these findings may lead to advances in developing novel strategies for treating aggressive malignancies like pancreatic cancer. Financial Support: Shirley E. Noland Foundation; UFHCC, Orlando Health, Orlando, FL and Institute for Cancer Research, UNT Health Science Center, Fort Worth, TX. Citation Format: Umesh T. Sankpal, Steven Goodison, Omar Kayaleh, Riyaz Basha. Tolfenamic acid induces the response of chemotherapy in pancreatic cancer: Preclinical study. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A03.

Abstract microRNAs (miRNAs) regulate gene expression and some have been shown to function as either oncogenes or tumor suppressors (oncomirs). Changes in miRNA expression have been observed in many human cancers but their role in endometrioid endometrial cancer (EEC) remains poorly understood. We studied the expression of 365 human miRNAs using Taqman low density arrays in 98 EECs as well as 60 normal endometria. Potentially interesting miRNAs were then validated by qRT-PCR. Expression of highly deregulated miRNAs on malignant cell properties was also examined in vitro using pre-/anti-miRNA transfection. Predicted mRNA targets for the miRNA of interest were also verified. We identified 16 significantly deregulated miRNAs in EEC and 7 of these are reported for the first time. Of these, miR-7, miR-194 and miR-449b knockdown and miR-204 induction repressed migration, invasion and extracellular matrix-adhesion in HEC1A endometrial cancer cells. For the cancer progression dysregulation of the expression of miR204 mediates migration and invasion of endometrial cancer by regulating FOXC1-related miR-204, FOXC1 was determined as its target gene possessing two miR-204-binding sites at the 3’-untranslated region. FOXC1 expression was inversely related to miR-204 expression in EEC. Functional analysis also revealed the involvement of FOXC1 in migration of HEC1A cells. We have identified dysfunctional miRNAs in endometrial cancer and elucidated the crucial role of miR-204-FOXC1 interaction in endometrial cancer progression. This work provides insight into miRNA-based diagnostic and therapeutic approaches for endometrial cancers.