Kenneth S. McCarty

Comprehensive genome and transcriptome analysis of the 11q13 amplicon in human oral cancer and synteny to the 7F5 amplicon in murine oral carcinoma

11q13 amplification occurs in a wide variety of tumors, including almost half of oral squamous cell carcinomas (OSCC) where it has been correlated with a poor outcome. In this study, we compiled 3.6 Mb of DNA sequence in the 11q13 amplicon core and refined the physical map of the amplicon. In the process, we determined the genomic structure and normal tissue expression patterns of two recently identified genes, TAOS2/TMEM16A and MRGF, which reside in the amplicon core. We then quantified DNA copy number and mRNA expression of all genes in the 11q13 amplicon in cell lines and primary tumors from OSCC. With the exception of FGF3, FGF4, FGF19, and MRGF, all genes were overexpressed in most tumors with genomic amplification. Furthermore, we found that the expression of genes in the amplicon appeared to be highly coordinated, making it difficult to determine which gene or genes are driving amplification. However, in nonamplified primary tumors, three genes, TAOS2/TMEM16A, OCIM, and TPCN2, are frequently overexpressed, whereas CCND1 and EMS1 are not. These results suggest that in addition to CCND1 and EMS1, other important genes also may be target genes driving 11q13 amplification. We hypothesize that 11q13 amplification may be driven by a cassette of genes that provide growth or metastatic advantage to cancer cells. This is supported by the finding that the human 11q13 amplicon core is syntenic to mouse chromosomal band 7F5, which is frequently amplified in chemically induced murine OSCC. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

Magnetic resonance imaging detection and wire localization of an ‘occult’ breast cancer

SummaryAn occult breast cancer was detected and wire localization performed using magnetic resonance imaging.

Breast cancer-derived M543V mutation in helix 12 of estrogen receptor α inverts response to estrogen and SERMs

We have isolated from human breast cancers several mutations in the Helix 12 component of activation function 2 (AF-2) in the estrogen receptor alpha (ERα). We used a novel approach to detect changes in the hormone-binding domain of ERα, based on the evidence that antiestrogens, such as 4-hydroxytamoxifen (ZOHT) and ICI 182,780, block the function of ERα by binding and folding the AF-2 transcriptional domain in a way that inhibits its association with coactivator proteins. We have identified a Helix 12 mutation, M543V, which leads to greater ERα transcription with ZOHT and other antiestrogens (including 1,1-dichloro-2,2,3-triarylcyclopropanes, DTACs) than with 17-β estradiol (E2). We also found an independent mutation at the same position, M543I, which did not show this inverted ligand phenotype. In comparison to further Helix 12 mutations made in vitro, it appears that relative hydrophobicity of the amino acid side chains on the inner face of Helix 12 is key to maintaining the transcriptionally active, agonist conformation with bound E2. This active conformation can be induced, resulting in increased transcription, by adding excess p160 coactivator AIB1 in transcriptional assays with E2-bound receptors, while the ZOHT-bound receptors were not further activated by AIB1. Other experiments show that the cross talk between ERα and AP-1 protein from AP-1-binding sites is not dependent on Helix 12 integrity. We show that two alleles containing a proline substitution in Helix 12 that inactivate AF-2 function of ERα at EREs have little negative effect on function through AP-1 elements, supporting a prominent role for the N-terminal AF-1 of ERα in AP-1/ERα transcriptional cross talk.

Functional Mutations of Estrogen Receptor Protein: Assay for Detection

Antiestrogens block the function of estrogen receptor (ER) by binding and misfolding the AF-2 transcriptional activation region in the ligand-binding domain, inhibiting or altering its association with coactivator proteins. We describe a novel assay uniquely configured to identify aberrations in this function that may lead to antiestrogen resistance. The identification of mutations of ER that affect its function is important to current breast cancer therapies. Standard methods to detect these mutations are cumbersome and the number of described mutations is limited, reflecting this difficulty. Conventional ER analysis in the clinic demonstrates the presence of antigenic determinants of the receptor protein or estrogenic ligand binding without reflection on the critical ability of the liganded receptor to interact with transcription cofactors. Here, we describe the use of estrogenic regulation of a site-specific recombinase activity, measuring deletion of a color marker gene via FLP-ER fusion proteins, to detect functional changes in ER protein folding that affects the site where cofactors interact. The assay provides a method to readily detect single amino acid changes in ER, some with biologically important consequences. Without such a functional assay as described, phenotypic changes are likely to remain undetected and under-evaluated. It is probable that some human tumors have antihormone resistance resulting from ER mutations that either block antihormone binding or transmit antihormone binding as a positive transcriptional signal via cofactor interaction. An assay to evaluate functional ER will lead to better predictive tests of treatment modalities.

Abstract 3270: Environmental stress results in an earlier onset of tumors in a HER2/Neu breast cancer model

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction Possible environmental effects on the development of HER2+ breast cancer have received relatively little research attention. Here we used the well-established MMTV-neu mouse model, which overexpresses mammary ErbB2 (HER2-neu) and stochastically develops primary tumors over 6-12 months, to test the hypothesis that chronic exposure to environmental stressors may result in an earlier onset of tumors. Experimental Procedures Female mice (FVB/N-Tg(MMTVneu)202Mul/J) were purchased from Jackson Labs at age 4-5 weeks, housed in standard cages in an isolation chamber in a controlled-access animal room, and randomly assigned to one of four experimental exposure conditions in a factorial design that was maintained for the course of the study: 1) Restraint stress (RS) 90 min - 1x/wk (n=24); 2) Social disruption stress (SD) (via cage mate switching across home cages) 2x/wk (n=23); 3) RSS 4) home cage control (HC)(n=20). Mice were visually inspected and palpated for tumor development 1x/wk (blinded as to treatment group). All mammary glands from each mouse were processed for histologic evaluation one week following positive tumor palpation. All palpated tumors were histologically confirmed by a pathologists evaluation of H&E sections (blinded as to treatment group). Results Consistent with the study hypothesis, the results indicated that mice chronically exposed to the two environmental stressors (RSS SD = 32.3 +/− 0.8 wk), suggesting a possible additive pattern of stress responses. Consistent with stress effects early in the process of tumorigenesis, evidence of mammary hyperplasia in the H&E sections was also significantly (p<0.04) more frequent in mice exposed to both stressors (RS&SD) compared with the control group (HC). Body weights increased significantly over the entire course of the study and did not differ across groups, suggesting that the group differences in tumor development were not due to a failure to thrive. Conclusions Exposure to environmental stressors may speed the development of tumors in this transgenic model of HER2+ breast cancer. These novel findings suggest the importance of further investigation to determine the mechanisms responsible for these effects of exposure to environmental stressors. (Support: [CA120795][1]) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3270. [1]: /lookup/external-ref?link_type=GEN&access_num=CA120795&atom=%2Fcanres%2F70%2F8_Supplement%2F3270.atom