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cDNA microarray technology can be used to establish associations between characteristic gene expression patterns and molecular responses to drug therapy. In this study, we used cDNA microarrays of 1694 cancer-related genes to monitor the gene expression consequences of the treatment of HCT116 colon cancer cells with the topoisomerase I inhibitor camptothecin (CPT). To obtain a more homogeneous cellular response, we synchronized the cells in S-phase using aphidicolin (APH) before CPT treatment. Brief incubation with 20 and 1000 nM CPT caused reversible and irreversible G(2) arrest, respectively, and the patterns of gene expression change (with reference to untreated controls) were strikingly different at the two concentrations. Thirty-three genes, mainly divided into three groups, showed characteristic changes in the first 20 h as a consequence of treatment. Northern blots performed for five of these genes (each under eight experimental conditions) were quite consistent with the microarray results (average correlation coefficient, 0.86). Several p53-activated stress response genes were up-regulated after treatment with 1000 nM CPT or prolonged exposure to APH, but it seemed that the up-regulation did not directly cause cell cycle arrest because the up-regulation induced by prolonged treatment with APH did not prevent cell cycle progression after removal of APH. In contrast, cell cycle-dependent up-regulation of a group of mitosis-related genes was delayed or blocked after CPT treatments. The interrupted up-regulation of this group of genes was directly associated with G(2) arrest. In addition, we observed down-regulation of gene expression in cells that were recovering from cell cycle delay. The observations reported here suggest a fundamental difference at the gene expression level between the molecular mechanism of reversible G(2) delay that follows mild DNA damage and the mechanism of permanent G(2) arrest that follows more extensive DNA damage.

Driver genes are directly responsible for oncogenesis and identifying them is essential in order to fully understand the mechanisms of cancer. However, it is difficult to delineate them from the larger pool of genes that are deregulated in cancer (ie, passenger genes). In order to address this problem, we developed an approach called TRIAngulating Gene Expression (TRIAGE through clinico-genomic intersects). Here, we present a refinement of this approach incorporating a new scoring methodology to identify putative driver genes that are deregulated in cancer. TRIAGE triangulates - or integrates -three levels of information: gene expression, gene location, and patient survival. First, TRIAGE identifies regions of deregulated expression (ie, expression footprints) by deriving a newly established measure called the Local Singular Value Decomposition (LSVD) score for each locus. Driver genes are then distinguished from passenger genes using dual survival analyses. Incorporating measurements of gene expression and weighting them according to the LSVD weight of each tumor, these analyses are performed using the genes located in significant expression footprints. Here, we first use simulated data to characterize the newly established LSVD score. We then present the results of our application of this refined version of TRIAGE to gene expression data from five cancer types. This refined version of TRIAGE not only allowed us to identify known prominent driver genes, such as MMP1, IL8, and COL1A2, but it also led us to identify several novel ones. These results illustrate that TRIAGE complements existing tools, allows for the identification of genes that drive cancer and could perhaps elucidate potential future targets of novel anticancer therapeutics.

Because abnormal erythroid differentiation is the most common manifestation of the myelodysplastic syndromes (MDS), it was hypothesized that erythroid gene expression may be used to illustrate myelodysplastic transcription patterns. Ten normal bone marrow aspirates (NBM) were first analyzed using an erythroid-focused cDNA array to define steady-state transcription levels. Proliferation and differentiation gene subsets were identified by statistically significant differences between NBM and erythroleukemia gene expression. Next, cDNAs from 5 separate MDS aspirates were studied: refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation (RAEB-T), and RAEB-T/secondary MDS. A distinct pattern of significantly increased proliferation-associated and reduced differentiation-associated gene activity was established for MDS.

To investigate the transcriptional program underlying thyroid hormone (T3)-induced cell proliferation, cDNA microarrays were used to survey the temporal expression profiles of 4,400 genes. Of 358 responsive genes identified, 88% had not previously been reported to be transcriptionally or functionally modulated by T3. Partitioning the genes into functional classes revealed the activation of multiple pathways, including glucose metabolism, biosynthesis, transcriptional regulation, protein degradation, and detoxification in T3-induced cell proliferation. Clustering the genes by temporal expression patterns provided further insight into the dynamics of T3 response pathways. Of particular significance was the finding that T3 rapidly repressed the expression of key regulators of the Wnt signaling pathway and suppressed the transcriptional downstream elements of the beta-catenin-T-cell factor complex. This was confirmed biochemically, as beta-catenin protein levels also decreased, leading to a decrease in the transcriptional activity of a beta-catenin-responsive promoter. These results indicate that T3-induced cell proliferation is accompanied by a complex coordinated transcriptional reprogramming of many genes in different pathways and that early silencing of the Wnt pathway may be critical to this event.

Infection with high-risk human papillomaviruses (HPV) is a major risk factor for development of cervical cancer. Expression of the HPV E6 and E7 oncoproteins increases in differentiating keratinocytes, resulting in inactivation of the p53 and retinoblastoma proteins, two important transcriptional regulators. We used cDNA microarrays to examine global alterations in gene expression in differentiating cervical keratinocytes after infection with retroviruses encoding HPV type 16 (HPV-16) E6 and E7. Expression of 80 cellular genes (approximately 4% of the genes on the array) was altered reproducibly by E6 and/or E7. Cluster analysis classified these genes into three functional groups: (i) interferon (IFN)-responsive genes, (ii) genes stimulated by NF-κB, and (iii) genes regulated in cell cycle progression and DNA synthesis. HPV-16 E6 or a dominant negative p53 protein downregulated multiple IFN-responsive genes. E6 decreased expression of IFN-α and -β, downregulated nuclear STAT-1 protein, and decreased binding of STAT-1 to the IFN-stimulated response element. E7 alone was less effective; however, coexpression of E6 and E7 downregulated IFN-responsive genes more efficiently than E6. The HPV-16 E6 protein also stimulated expression of multiple genes known to be inducible by NF-κB and AP-1. E6 enhanced expression of functional components of the NF-κB signal pathway, including p50, NIK, and TRAF-interacting protein, and increased binding to NF-κB and AP-1 DNA consensus binding sites. Secretion of interleukin-8, RANTES, macrophage inflammatory protein 1α, and 10-κDa IFN-γ-inducible protein were increased in differentiating keratinocytes by E6. Thus, high-level expression of the HPV-16 E6 protein in differentiating keratinocytes directly alters expression of genes that influence host resistance to infection and immune function.

Background Neoadjuvant chemotherapy for early breast cancer leads to significant clinical response rates of 70-90%, with only 10-20% complete pathologic responses (pCR). The biological and clinical factors that determine the degree of pCR are incompletely understood and most investigated are drug choice, proliferation index, intrinsic subtypes and host-therapy interactions. Mounting evidence indicates that the patient's immune system contributes to tumor regression and can be modulated by therapies. Investigations using immunohistochemical approaches confirmed that immune cell infiltrate on pre-treatment or post-treatment tumor biopsies are associated with greater frequency of pCR. The cell types most frequently observed with this association are effector tumor infiltrating lymphocytes (TILs) such as cytotoxic T cells, natural killer cells and B cells suggesting a strong link between infiltrating immune cell abundance and anti-tumor immunogenicity. More recently, we and others have shown that the relative abundance of TIL in breast cancer can be quantified by intratumoral transcript levels of coordinately expressed, immune cell-specific genes. Through expression microarray analysis, we recently discovered three separate immune gene signatures, or metagenes, that appear to reflect the relative abundance of distinct tumor-infiltrating leukocyte populations. These metagenes, referred as the B/P (B-cell/Plasma cell), T/NK (T-cell/Natural Killer cell) and M/D (Monocyte/Dendritic cell) immune metagenes, were significantly associated with the distant metastasis-free survival of patients with highly proliferative cancer of the Basal-like, HER2-Enriched and Luminal B subtypes, in particular. Aim Given the histopathological evidence that TIL abundance is predictive of neoadjuvant treatment efficacy, we evaluated the therapy-predictive potential of the prognostic immune metagenes. We hypothesized that the pre-chemotherapy immune gene signatures would be significantly predictive of tumor responses. Methods In a multi-institutional, meta-cohort analysis of 701 breast tumor patients receiving neoadjuvant chemotherapy, gene expression profiles of tumor biopsies were investigated to determine the relationships between immune metagenes, tumor proliferative capacity, and molecular breast tumor subtypes. Results We used multiple logistic regression to investigate the predictive value of the immune signatures. In a univariate analysis the B/P, T/NK and M/D immune metagenes were statistically significantly associated with excellent pathologic response as defined by residual cancer burden scores ((B/P (odds ratio (95% CI) 1.6 (1.3, 1.9), T/NK 1.6 (1.2, 2.0), M/D 1.7 (1.3, 2.1)). In multivariate analysis, B/P and M/D metagenes remained significant after adjustment for intrinsic subtype and proliferation (B/P 1.25 (1.01, 1.54), M/D 1.4 (1.03, 1.90). Conclusions Gene expression signatures of infiltrating immune cells carry both prognostic and therapy-predictive value. Furthermore, our work highlights a less described role for myeloid derived antigen presenting cells that could explain the variability of pathologic response to neoadjuvant chemotherapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-05.

The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.

The Juneau gold belt in northern southeastern Alaska is composed of a disparate assemblage of lithotectonic terranes ranging in age from Paleozoic and perhaps older to Cretaceous. Four progressive deformational events (D 1 -D 4 ) associated with metamorphism and contractional tectonism began in mid-Cretaceous time, and continued well into the Tertiary. Structures associated with these events are overprinted by Eocene vein systems (D 5 ) that contain gold mineralization. The final recognized deformation event (D 6 ) formed brittle contractional structures and strike-slip faults. The geometry of deformed sedimentary and volcanic clasts outside of shear zones indicates that a flattening style of strain developed during contraction. Clast orientations, associated mineral lineations, folds, and asymmetric fabrics are interpreted to indicate flattening and top-to-the-west shear associated with D 1 -D 4 . The distinct D 5 faulting and fluid-flow event occurred over a short time period (56.5-52.8 Ma) and produced a series of economically important auriferous quartz vein deposits that characterize the 160-km-long Juneau gold belt. Gold vein mineralization may have been initiated by changes in the far-field stress regime and/or rapid exhumation. Structural relations are interpreted to indicate that the deformation regime initiated as contractional and ultimately developed into transpressional.

THE large-scale migration of fluids through the continental crust has been well documented, but there is no consensus regarding the timing of fluid migration relative to orogenic episodes, or rates of crustal dewatering1. Here we present40Ar/39Ar dates for muscovites from quartz veins along a major shear zone in southeast Alaska, which show that the veins were emplaced in the early Eocene, during the late stages of orogenic deformation. Hydrother-mal activity took place for only about 1 Myr and along a distance of at least 200 km. The fluids were generated by metamorphic reactions in subducted crust along the North American plate margin, and were apparently trapped in the crust by the low permeabilities accompanying a convergent tectonic regime until 56 Myr ago. The rapid dewatering event coincided with a change in plate motion at 56–55 Myr, which caused a shift from convergent to partly transcurrent tectonics. We suggest that this change in tectonic regime led to increased crustal permeabilities and hence the possibility of large-scale fluid migration.

Mineral resources have been important to the Alaskan economy for hundreds of years. The Indians, Eskimos, and Aleuts used gold, copper, and other metals for jewelry, utensils, and weapons and as items of trade. The Russians, who arrived in 1728, showed only minor interest in minerals, focusing instead on furs. Nonetheless, records show that they mined iron ore on the Kenai Peninsula in 1793 and located gold there in 1834. In addition, the Russians were aware of copper-rich occurrences in the Copper River basin. The mining industry grew rapidly in Alaska after the United States purchased the region from Russia in 1867. By 1870, gold was being mined near Windham Bay and near Sitka, in southeastern Alaska. The first major hard-rock gold mining began at the Alaska-Juneau, Perseverance, and Treadwell mines near present-day Juneau following the discovery of placer gold near tidewater in 1880. Significant placer mining operations for gold soon spread northward into districts such as Fairban ks, Nome, Iditarod, and Circle. The exploitation of mineral resources, particularly gold, influenced the settlement and development of much of the state. Major cities such as Nome, Fairbanks, and Juneau, the capital, were originally built around early mining camps. By1996, more than 33 million oz of gold, as well as significant amounts of copper, lead, zinc, silver, and platinum-group elements, had been produced. In addition, there has been minor production of nickel. tin, mercury, and uranium Alaska is presently experiencing a renaissance in mining on federal, state, private, and native lands. From 1990 to 1996, the minerals industly had an annual value of approximately $600 million, with a record 1 billion in 1996. Nearly 3,500 year-round jobs were directly attributed to the minerals industq. Looking forward into the twenty-fi rst centu1y, the outlook is even better. Major mines such as Heel Dog are in production. Recently completed exploration at the Red Dog sedimenta1y exhalative deposit has significantly expanded the reserve base and major mine expansion is contemplated. The Nixon Fork gold skarn deposit was put into production in 1995, with the first ore poured in November. The Greens Creek mine resumed production in late 1996, following renewed exploration and development of the volcanogenic massive sulfide deposit. The Fort Knox gold mine, near Fairbanks, is under construction and is scheduled to yield ore by late 1996. In southwest Alaska exploration success has yielded a significant gold resource at Donlin Creek. In addition, the Kensington-Jualin project is

Loss of tumor suppressors by both epigenetic silencing and genetic deletion contributes to the development and progression of breast cancer. SOX7 is an important developmental transcription factor and its downregulation has been reported in tumor tissues and cell lines of prostate, colon and lung cancers. Our current study demonstrates that SOX7 mRNA and protein expression are downregulated in breast cancer tissues and cell lines compared to adjacent normal tissues and nontumorigenic cells, respectively. Further, we show that both hypermethylation of the SOX7 promoter and microRNAs contribute to SOX7 downregulation. SOX7 silencing by shRNAs in nontumorigenic immortal breast cells leads to increased proliferation and invasion, and they form structures resembling that of breast cancer cells in a three-dimensional culture system. Conversely, ectopic SOX7 expression inhibits proliferation, and invasion of breast cancer cells in vitro and tumor growth in vivo. Importantly, we discovered that SOX7 transcript levels positively correlated with clinical outcome of 674 breast cancer patients. Collectively, our data suggest that SOX7 acts as a tumor suppressor in breast cancer. SOX7 expression is likely regulated by multiple mechanisms and potentially serves as a prognostic marker for breast cancer patients. Citation Format: Daniel B. Stovall, Meimei Wan, Lance D. Miller, Paul Cao, Qiang Zhang, Martha Stampfer, Wennuan Liu, Jianfeng Xu, Guangchao Sui. The role and regulation of SOX7 in breast cancer. [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 B025.

As a polycomb group protein, Yin Yang 1 (YY1) plays an important role in cancer epigenetics. YY1 regulates both gene expression and protein modifications, including histone acetylation and methylation. We recently reported a proliferative role of YY1 and its cytoplasmic presence in breast cancer. In the current study, we demonstrated that YY1 promotes AKT phosphorylation at S473, a marker of AKT activation. The immunostaining signal of YY1 and AKT(S473) phosphorylation positively correlated and colocalized in breast cancer tissues and cells. The oncogene protein binding (OPB) domain (G201-S226) of YY1 bound to AKT and was necessary for YY1 in promoting AKT phosphorylation. This novel function of YY1 is independent of its transcriptional activity because YY1 promoted AKT phosphorylation in an in vitro setting using purified proteins and YY1 mutants deficient in binding to DNA retained this activity. Our further studies revealed that YY1 interacted with the PH domain of AKT where PIP3 binds and its role in promoting AKT phosphorylation was abolished when we depleted mSIN1, a specific component of mTORC2 that mediates AKT(S473) phosphorylation. Thus, we discovered a PI3K-independent mechanism of mTORC2-mediated AKT activation stimulated by YY1 binding to its PH domain, which supports the proliferative role of YY1 in mammary oncogenesis. Citation Format: Qiang Zhang, Meimei Wan, David A. Horita, Lance D. Miller, Steven J. Kridel, Timothy E. Kute, George Kulik, Guangchao Sui. Yin Yang 1 promotes AKT activation through direct protein binding. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A25.

BACKGROUND: Gene expression signatures indicative of tumor proliferative capacity and tumor-immune cell interactions have emerged as principal biology-driven predictors of breast cancer outcomes. How these signatures relate to one another in biological and prognostic contexts remains to be clarified. RESULTS: To investigate the relationship between proliferation and immune gene signatures, we analyzed an integrated dataset of 1,954 clinically annotated breast tumor expression profiles randomized into training and test sets to allow two-way discovery and validation of gene-survival associations. Hierarchical clustering revealed a large cluster of distant metastasis-free survival-associated genes with known immunological functions that further partitioned into three distinct immune metagenes likely reflecting B cells and/or plasma cells; T cells and natural killer cells; and monocytes and/or dendritic cells. A proliferation metagene allowed stratification of cases into proliferation tertiles. The prognostic strength of these metagenes was largely restricted to tumors within the highest proliferation tertile, though intrinsic subtype-specific differences were observed in the intermediate and low proliferation tertiles. In highly proliferative tumors, high tertile immune metagene expression equated with markedly reduced risk of metastasis whereas tumors with low tertile expression of any one of the three immune metagenes were associated with poor outcome despite higher expression of the other two metagenes. CONCLUSIONS: These findings suggest that a productive interplay among multiple immune cell types at the tumor site promotes long-term anti-metastatic immunity in a proliferation-dependent manner. The emergence of a subset of effective immune responders among highly proliferative tumors has novel prognostic ramifications., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Among U.S. women, breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths. Both epigenetic silencing and genetic deletion of tumor suppressors are critical for the development and progression of breast cancer. The SOX (Sex-determining region Y-box) genes encode a family of over 20 transcription factors, including SOX7. The SOX7 gene is located on chromosome arm 8p and is deleted in a number of human cancers, including breast cancer. Previously, SOX7 has been demonstrated to be down-regulated in human tissues of prostate and colon cancers and its ectopic expression inhibits proliferation and increase apoptosis in prostate and colon cancer cell lines. However, the functional role of SOX7 in breast cancer has not yet been reported. Our studies revealed a significant correlation between SOX7 transcript levels and clinical outcome in gene array data from an international meta-cohort of 674 breast cancer patients. Consistently, we observed that SOX7 mRNA levels and protein expression are down-regulated in multiple breast cancer tissues and breast cancer cell lines compared to adjacent normal tissue and non-tumorigenic cells, respectively. Further, we have observed that the SOX7 promoter is hyper-methylated in breast cancer cell lines compared to non-tumorigenic breast cells, and that inhibition of SOX7 methylation results in an increase in SOX7 mRNA. Importantly, shRNA-mediated silencing of SOX7 conferred a growth advantage to non-tumorigenic breast cells, while induced ectopic SOX7 expression inhibited proliferation and migration of breast cancer cells. Overall, our data suggest that SOX7 may act as a novel tumor suppressor in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4001. doi:1538-7445.AM2012-4001

We developed an analytic strategy that correlates gene expression and clinical outcomes as a means to identify novel candidate oncogenes operative in breast cancer. This analysis, followed by functional characterization, resulted in the identification of Jumonji Domain Containing 6 (JMJD6) protein as a novel driver of oncogenic properties in breast cancer. Through microarray informatics, Cox proportional hazards regression was used to analyze the correlation between gene expression and distant metastasis-free survival (DMFS) of patients in 14 independent breast cancer cohorts. JMJD6 emerged as a top candidate gene robustly associated with poor patient survival. Immunohistochemistry, siRNA-mediated silencing, and forced overexpression of JMJD6 in cell-based assays elucidated molecular mechanisms of JMJD6 action in breast cancer progression and shed light on the clinical breast cancer subtypes relevant to JMJD6 action. JMJD6 was expressed at highest levels in tumors associated with worse outcomes, including ER- and basal-like, Claudin-low, Her2-enriched, and ER+ Luminal B tumors. High nuclear JMJD6 protein was associated with ER negativity, advanced grade, and poor differentiation in tissue microarrays. Separation of ER+/LN- patients that received endocrine monotherapy indicated that JMJD6 is predictive of poor outcome in treatment-specific subgroups. In breast cancer cell lines, loss of JMJD6 consistently resulted in suppressed proliferation but not apoptosis, whereas forced stable overexpression increased growth. In addition, knockdown of JMJD6 in invasive cell lines, such as MDA-MB231, decreased motility and invasion, whereas overexpression in MCF-7 cells slightly promoted motility but did not confer invasive growth. Microarray analysis showed that the most significant transcriptional changes occurred in cell-proliferation genes and genes of the TGF-β tumor-suppressor pathway. High proliferation was characterized by constitutively high cyclin E protein levels. The inverse relation of JMJD6 expression with TGF-β 2 could be extrapolated to the breast cancer cohorts, suggesting that JMJD6 may affect similar pathways in primary breast cancer. JMJD6 is a novel biomarker of tumor aggressiveness with functional implications in breast cancer growth and migration.

Extensive experimental and clinical literature attests to the importance of iron in tumor cell growth. For example, high levels of dietary iron have been linked epidemiologically to the increased development of tumors in humans; in animal models, levels of dietary iron affect tumor growth. However, pathways that underlie the apparent demand for iron by tumors remain largely uncharacterized. We recently discovered that an iron efflux pathway plays a role in breast cancer growth and metastasis: expression of the iron efflux pump ferroportin and its regulator hepcidin were predictive of metastasis-free survival in multiple independent breast cancer cohorts. To determine the optimal “iron gene regulatory signature,” we used microarray datasets comprising 674 breast cancer cases to systematically investigate how expression of genes related to iron metabolism is linked to breast cancer prognosis. Of 61 genes involved in iron regulation, 49% were statistically significantly associated with distant metastasis-free survival (DMFS). Optimal risk stratification was achieved with a model comprising 16 genes, which we term the iron regulatory gene signature (IRGS). Multivariable analysis revealed that the IRGS contributes information not captured by conventional prognostic indicators (hazard ratio 1.61; 95% CI 1.16–2.24; p=0.004). The IRGS successfully stratified homogeneously treated patients, including ER+ patients treated with tamoxifen monotherapy, both with (p=0.006) and without (p=0.03) lymph node metastases. To test whether multiple pathways were embedded within the IRGS, we evaluated the performance of two gene dyads with known roles in iron biology in ER+ patients treated with tamoxifen monotherapy (n=371). For both dyads, gene combinations that minimized intracellular iron content (anti-import: TFRCLow/HFEHigh; or pro-export: FPHigh/HAMPLow) were associated with favorable prognosis (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C64.

e21027 Background: In Vitro Diagnostic Multivariate Index Assays (IVDMIA) are diagnostic tools that produce a diagnostic, prognostic and predictive index. The development of a gene expression (GE)-...

502 Background: ER-positive (ER+) breast cancer includes all of the intrinsic molecular subtypes, although the luminal subtypes predominate. In this study, we evaluated the prognostic ability and b...

We report herein that trefoil factor 3 (TFF3) is oncogenic and mediates anti-estrogen resistance in human mammary carcinoma. Forced expression of TFF3 in mammary carcinoma cells increased cell proliferation and survival, enhanced anchorage-independent growth, and promoted migration and invasion. Moreover, forced expression of TFF3 increased tumor size in xenograft models. Conversely, depletion of endogenous TFF3 with small interfering RNA (siRNA) decreased the oncogenicity and invasiveness of mammary carcinoma cells. Neutralization of secreted TFF3 by antibody promoted apoptosis, decreased cell growth in vitro, and arrested mammary carcinoma xenograft growth. TFF3 expression was significantly correlated to decreased survival of estrogen receptor (ER)-positive breast cancer patients treated with tamoxifen. Forced expression of TFF3 in mammary carcinoma cells increased ER transcriptional activity, promoted estrogen-independent growth, and produced resistance to tamoxifen and fulvestrant in vitro and to tamoxifen in xenograft models. siRNA-mediated depletion or antibody inhibition of TFF3 significantly enhanced the efficacy of antiestrogens. Increased TFF3 expression was observed in tamoxifen-resistant (TAMR) cells and antibody inhibition of TFF3 in TAMR cells improved tamoxifen sensitivity. Functional antagonism of TFF3 therefore warrants consideration as a novel therapeutic strategy for mammary carcinoma.

OBJECTIVE. Aldehyde dehydrogenase-1 (ALDH1) expression characterizes a subpopulation of cells with enhanced tumor initiating and differentiating properties in some cancers. We have examined the association of ALDH1 with chemoresistance and whether downregulation of ALDH1 sensitizes cells to chemotherapy in models of ovarian cancer. METHODS. Microarray profiling was performed on SKOV3ip1 and the taxane-resistant SKOV3TRip2 cell lines. Primary ovarian cancer xenografts with and without cisplatin exposure were examined for selection of ALDH1-positive cells. Small interfering RNA (siRNA) was used to downregulate ALDH1 in vitro, and in vivo by incorporation into neutral DOPC liposomes, for evaluation of chemosensitization in an orthotopic model of ovarian cancer. RESULTS. Microarray analysis found 29 genes upregulated and 18 genes downregulated by more than 10-fold when comparing the taxane-resistant SKOV3TRip2 ovarian cancer line compared to its parental SKOV3ip1 line. Included among these was a 92.7-fold higher ALDH1 signature. Increased expression and activity of ALDH1 was confirmed by Western blot and the ALDEFLUOR assay (58% of cells ALDH1-active). In primary ovarian cancer xenografts in NOD-Scid mice, cisplatin treatment resulted in an increase in ALDH1-positive cells, from a baseline of 1% to 38% with therapy (p CONCLUSIONS. ALDH1 expression is associated with taxane and cisplatin chemoresistance in ovarian cancer cell lines. ALDH1 expression can be induced by cisplatin treatment in vivo, and targeting ALDH1 sensitizes resistant cell lines to chemotherapy. This enzyme may be important for identification and targeting the chemoresistant population in ovarian cancer. 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 4288.

Background: Cancer/testis-X antigens are a multigene family that are predominantly expressed in human germ line cells, with little or no expression in somatic adult tissues, but become aberrantly activated in various malignancies. Some such CT-X antigens represent ideal targets for cancer immunotherapy and have already been used in clinical testing. In contrast to melanomas, bladder, lung, ovarian and hepatocellular carcinomas which show higher levels of CT-X antigen expression, the reports in breast cancers have been inconclusive to date and a comprehensive gene expression and clinicopathological analysis has yet to be performed.Material and Methods: Using sequencing data as well as nine publicly available gene expression data sets, we analyzed the expression of Cancer/testis-X antigens in more than 1900 primary breast cancers. Complementary analysis was performed on three tissue microarrays comprising a total of 201 primary breast carcinomas and 53 brain metastases. Clinical information on the ER-, PR-, HER2, Ki67, p53, EGFR and basal markers was available for statistical analysis.Results: A significantly higher expression of Cancer/testis-X antigens was found in ER negative breast carcinomas over different data sets with a concordant gene expression pattern of several Cancer/testis-X antigens. Members of the MAGEA family and NY-ESO-1/CTAG1B were consistently the most prevalent. Immunohistochemical analyses confirmed a significant correlation of MAGEA family and NY-ESO-1/CTAG1B with ER negative (pValue < 0.0001), PR negative (pValue < 0.01) and Ki67 staining (pValue < 0.0001). Many of these tumors were also positive for basal markers.Discussion: Previous studies of Cancer/testis-X antigens in breast have focused on ER positive cancers, in smaller subsets and provided inconclusive results. Using comprehensive gene expression data sets and tissue microarrays, we have demonstrated a significant association of MAGEA family and NY-ESO-1/CTAG1B with ER/PR negative breast cancer. Since these cancers represent a subgroup for which less therapeutic modalities are available, we propose the use of MAGEA and NY-ESO-1/CTAG1B cancer vaccines in the adjuvant setting as an approach to restricting tumor growth and metastases. Clinical trails using MAGEA and NY-ESO-1/CTAG1B are warranted. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3139.

CDKN1C (encoding tumor suppressor p57KIP2) is a cyclin-dependent kinase (CDK) inhibitor whose family members are often transcriptionally downregulated in human cancer via promoter DNA methylation. In this study, we show that CDKN1C is repressed in breast cancer cells mainly through histone modifications. In particular, we show that CDKN1C is targeted by histone methyltransferase EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3), and can be strongly activated by inhibition of EZH2 in synergy with histone deacetylase inhibitor. Consistent with the overexpression of EZH2 in a variety of human cancers including breast cancer, CDKN1C in these cancers is downregulated, and breast tumors expressing low levels of CDKN1C are associated with a poor prognosis. We further show that assessing both EZH2 and CDKN1C expression levels as a measurement of EZH2 pathway activity provides a more predictive power of disease outcome than that achieved with EZH2 or CDKN1C alone. Taken together, our study reveals a novel epigenetic mechanism governing CDKN1C repression in breast cancer. Importantly, as a newly identified EZH2 target with prognostic value, it has implications in patient stratification for cancer therapeutic targeting EZH2-mediated gene repression.

Successful tumor development and progression involves the complex interplay of both pro- and anti-oncogenic signaling pathways. Genetic components balancing these opposing activities are likely to require tight regulation, because even subtle alterations in their expression may disrupt this balance with major consequences for various cancer-associated phenotypes. Here, we describe a cassette of cancer-specific genes exhibiting precise transcriptional control in solid tumors. Mining a database of tumor gene expression profiles from six different tissues, we identified 48 genes exhibiting highly restricted levels of gene expression variation in tumors (n = 270) compared to nonmalignant tissues (n = 71). Comprising genes linked to multiple cancer-related pathways, the restricted expression of this “Poised Gene Cassette” (PGC) was robustly validated across 11 independent cohorts of ∼1,300 samples from multiple cancer types. In three separate experimental models, subtle alterations in PGC expression were consistently associated with significant differences in metastatic and invasive potential. We functionally confirmed this association in siRNA knockdown experiments of five PGC genes (p53CSV, MAP3K11, MTCH2, CPSF6, and SKIP), which either directly enhanced the invasive capacities or inhibited the proliferation of AGS cancer cells. In primary tumors, similar subtle alterations in PGC expression were also repeatedly associated with clinical outcome in multiple cohorts. Taken collectively, these findings support the existence of a common set of precisely controlled genes in solid tumors. Since inducing small activity changes in these genes may prove sufficient to potently influence various tumor phenotypes such as metastasis, targeting such precisely regulated genes may represent a promising avenue for novel anti-cancer therapies., Author Summary Successful carcinogenesis involves the integration of both pro- and anti-oncogenic pathways. We postulated that genes critical for balancing these opposing pathways are likely to be precisely controlled in tumors, since even subtle alterations in their activity might cause substantial alterations in tumor growth and survival. Using a novel genomic approach, we identified a 48-gene “Poised Gene Cassette” (PGC) showing tight regulation specifically in human cancers but not in corresponding nonmalignant tissues. We show, using a wide variety of in vitro and in vivo approaches, that small alterations in PGC expression are consistently associated with significant differences in experimental metastasis and patient survival, and we demonstrate a direct functional role for five PGC genes (p53CSV, MAP3K11, MTCH2, CPSF6 and SKIP) in cancer invasion. Our findings support the existence of a novel class of ultrasensitive genes that may regulate various cancer-associated phenotypes such as metastasis. Such precisely controlled genes could represent appealing drug targets, since even partial alterations in their activity should prove sufficient to induce potent effects on tumors. Besides cancer, our analytical approach is quite generalizable and likely to be applicable to other disease conditions.

The effects of intravenous injection of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF) were investigated in cynomolgus monkeys (Macaca fascicularis). Injection of 20 micrograms/kg of LPS from E. coli (serotype 055:B5) into cynomolgus monkeys fed a monkey chow diet caused a twofold increase in plasma triglyceride and a 25% reduction in plasma cholesterol 48 h after injection. Similar results were found with injection of recombinant human TNF at a dose of 20 micrograms/kg into chow-fed animals. However, injection of the same dose of LPS or TNF into animals fed an atherogenic diet containing saturated fat and cholesterol resulted in a 2.4- to 5-fold increase in plasma triglyceride concentrations and no significant change in plasma cholesterol levels. The fall in plasma cholesterol levels observed in chow-fed animals was associated with a 57% decrease in the cholesteryl ester (CE) content in low density lipoprotein (LDL) and 35% decrease in CE in high density lipoprotein (HDL) in LPS-injected animals, and a decrease of 33% in CE concentration of LDL and 41% in CE of HDL in animals injected with TNF. In animals fed the atherogenic diet containing saturated fat and cholesterol, the injection of both LPS and TNF also resulted in a significant decrease in the CE content of LDL and HDL. However, the plasma total cholesterol levels did not change in the animals fed saturated fat and cholesterol because the decrease in CE content of LDL and HDL was offset by an increase in very low density lipoprotein (VLDL)-CE.(ABSTRACT TRUNCATED AT 250 WORDS)

This paper presents an investigation of V-ribbed belt (Micro-V TM ) wet friction and friction-induced slip noise in Accessory Belt Drive Systems (ABDS). A set of experiments was conducted by using an industry standard test rig and a standard material friction tester. A theoretical analysis and model are presented to elaborate the results. The tribological property of the interface is characterised to be unsteady mixed lubrication with water, which possesses the feature of negative slope of friction with velocity. The friction-induced dynamic instability and noises are also investigated. It is shown that the noise is directly associated with the negative slope of the friction-velocity curve.

509 Background: The majority of early-stage BC express estrogen receptors (ER) and receive tamoxifen in the adjuvant setting. Yet up to 40% of these patients will relapse on tamoxifen and develop incurable metastatic disease. Recent evidence from three large randomised controlled trials exploring the role of AI in the adjuvant setting shows a benefit from the novel strategy, however the optimal sequence and duration of AI/tamoxifen treatment is unknown. Therefore, it is vital that we learn to identify those women at higher risk of tamoxifen resistance. Our aim was to identify genes that could predict for this subset of women. Methods: We determined the gene expression profiles from 105 tamoxifen-only treated ER positive early stage BC (training set) using Affymetrix U133 A and B chips. Within this group, 30 (29%) patients developed distant recurrence at a median time to relapse of 3.8 years (yrs) and 75 (71%) remained disease free at a median of 5.7 yrs of follow-up. The independent validation set consist...

There are currently a number of competing techniques for low-level processing of oligonucleotide array data. The choice of technique has a profound effect on subsequent statistical analyses, but there is no method to assess whether a particular technique is appropriate for a specific data set, without reference to external data. We analyzed coregulation between genes in order to detect insufficient normalization between arrays, where coregulation is measured in terms of statistical correlation. In a large collection of genes, a random pair of genes should have on average zero correlation, hence allowing a correlation test. For all data sets that we evaluated, and the three most commonly used low-level processing procedures including MAS5, RMA and MBEI, the housekeeping-gene normalization failed the test. For a real clinical data set, RMA and MBEI showed significant correlation for absent genes. We also found that a second round of normalization on the probe set level improved normalization significantly throughout. Previous evaluation of low-level processing in the literature has been limited to artificial spike-in and mixture data sets. In the absence of a known gold-standard, the correlation criterion allows us to assess the appropriateness of low-level processing of a specific data set and the success of normalization for subsets of genes.

Estrogens and their receptors are important in human development, physiology and disease. In this study, we utilized an integrated genome-wide molecular and computational approach to characterize the interaction between the activated estrogen receptor (ER) and the regulatory elements of candidate target genes. Of around 19,000 genes surveyed in this study, we observed 137 ER-regulated genes in T-47D cells, of which only 89 were direct target genes. Meta-analysis of heterogeneous in vitro and in vivo datasets showed that the expression profiles in T-47D and MCF-7 cells are remarkably similar and overlap with genes differentially expressed between ER-positive and ER-negative tumors. Computational analysis revealed a significant enrichment of putative estrogen response elements (EREs) in the cis-regulatory regions of direct target genes. Chromatin immunoprecipitation confirmed ligand-dependent ER binding at the computationally predicted EREs in our highest ranked ER direct target genes, NRIP1, GREB1 and ABCA3. Wider examination of the cis-regulatory regions flanking the transcriptional start sites showed species conservation in mouse-human comparisons in only 6% of predicted EREs. Only a small core set of human genes, validated across experimental systems and closely associated with ER status in breast tumors, appear to be sufficient to induce ER effects in breast cancer cells. That cis-regulatory regions of these core ER target genes are poorly conserved suggests that different evolutionary mechanisms are operative at transcriptional control elements than at coding regions. These results predict that certain biological effects of estrogen signaling will differ between mouse and human to a larger extent than previously thought.

Background Resistance to thyroid hormone (RTH) is caused by mutations of the thyroid hormone receptor β (TRβ) gene. To understand the transcriptional program underlying TRβ mutant-induced phenotypic expression of RTH, cDNA microarrays were used to profile the expression of 11,500 genes in a mouse model of human RTH.

lnterleukin-2 (IL-2) has direct pluripotent effects on cells with immune and inflammatory function. Which of these effects has a critical role in mediating tumor regression remains enigmatic. In this study, we compared early changes in transcriptional profiles of circulating mononuclear cells with those occurring within the microenvironment of melanoma metastases following systemic IL-2 administration. The results suggest that the immediate effects of IL-2 administration on the tumor microenvironment is transcriptional activation of genes predominantly associated with monocyte cell function; minimal effects were noted on migration, activation and proliferation of T cells. However, production of chemokines and markers of adhesion and migration within few hours of IL-2 administration may be responsible for a secondary recruitment of immune cells to the tumor site later. Our results suggest that IL-2 induces inflammation at tumor sites with three predominant secondary effects: activation of antigen-presenting monocytes; massive production of chemoattractants that may recruit other immune cells to the tumor (including MIG and PARC, which are specific for T cells); and activation of cytolytic mechanisms in monocytes (calgranulin, grancalcin) and NK cells (NKG5, NK4).

Gold-bearing quartz vein systems in the Juneau gold belt formed within a 160-km- long by 5- to 8-km-wide zone along the western margin of the Coast Mountains, Alaska. Vein systems are spatially associated with shear zones adjacent to terrane-bounding, mid-Cretaceous thrust faults. Analysis of vein orientations and sense of shear data define a stress configuration with greatest and least principal axes oriented subhorizontally with northeast-southwest trends and subverticaly, respectively. This local stress configuration is compatible with the far-field plate configuration during Eocene time. Isotopic ages of vein formation indicate that fluid cycling occurred between 56.5 and ≥52.8 Ma, and are consistent with a genetic link between veining and a change in plate motion in early Eocene time. Veining was also synchronous with the latter stages of rapid exhumation and voluminous plutonism immediately inboard of the gold belt. We propose a model in which interacting tectonic events facilitated fault-valve action and vein development along now-exhumed shear zones.

DNA microarrays make possible the rapid and comprehensive assessment of the transcriptional activity of a cell, and as such have proven valuable in assessing the molecular contributors to biological processes and in the classification of human cancers. The major challenge in using this technology is the analysis of its massive data output, which requires computational means for interpretation and a heightened need for quality data. The optimal analysis requires an accounting and control of the many sources of variance within the system, an understanding of the limitations of the statistical approaches, and the ability to make sense of the results through intelligent database interrogation. Expression array technology Expression genomics is an approach that examines gene expression in a comprehensive and massively parallel fashion. The core technology in expression genomics is microarrays, whereby thousands of DNA probes are immobilized on a solid surface and hybridized against fluorophore-labeled cDNA or cRNA targets from template RNA sources. The two major platforms for microarrays are spotted arrays, where the probes are mechanically deposited onto modified glass slides by contact or ink jet printing, and in situ arrays, where oligo probes are synthesized in silico (e.g., via photolithographic synthesis as in Affymetrix GeneChip arrays [Affymetrix, Santa Clara, CA]). For a comparative review of the two platforms, we refer the reader to Harrington et al. (2000).

Yin Yang 1 (YY1) is highly expressed in various types of cancers and regulates tumorigenesis through multiple pathways. In the present study, we evaluated YY1 expression levels in breast cancer cell lines, a breast cancer TMA, and two gene arrays. We observed that, compared with normal samples, YY1 is generally overexpressed in breast cancer cells and tissues. In functional studies, depletion of YY1 inhibited the clonogenicity, migration, invasion, and tumor formation of breast cancer cells, but did not affect the clonogenicity of nontumorigenic cells. Conversely, ectopically expressed YY1 enhanced the migration and invasion of nontumorigenic MCF-10A breast cells. In both a monolayer culture condition and a three-dimensional Matrigel system, silenced YY1 expression changed the architecture of breast cancer MCF-7 cells to that resembling MCF-10A cells, whereas ectopically expressed YY1 in MCF-10A cells had the opposite effect. Furthermore, we detected an inverse correlation between YY1 and p27 expression in both breast cancer cells and xenograft tumors with manipulated YY1 expression. Counteracting the changes in p27 expression attenuated the effects of YY1 alterations on these cells. In addition, YY1 promoted p27 ubiquitination and physically interacted with p27. In conclusion, our data suggest that YY1 is an oncogene and identify p27 as a new target of YY1.

SummaryThe ability to derive a whole-genome map of transcription-factor binding sites (TFBS) is crucial for elucidating gene regulatory networks. Herein, we describe a robust approach that couples chromatin immunoprecipitation (ChIP) with the paired-end ditag (PET) sequencing strategy for unbiased and precise global localization of TFBS. We have applied this strategy to map p53 targets in the human genome. From a saturated sampling of over half a million PET sequences, we characterized 65,572 unique p53 ChIP DNA fragments and established overlapping PET clusters as a readout to define p53 binding loci with remarkable specificity. Based on this information, we refined the consensus p53 binding motif, identified at least 542 binding loci with high confidence, discovered 98 previously unidentified p53 target genes that were implicated in novel aspects of p53 functions, and showed their clinical relevance to p53-dependent tumorigenesis in primary cancer samples.

Introduction The impact of interactions between the two estrogen receptor (ER) subtypes, ERα and ERβ, on gene expression in breast cancer biology is not clear. The goal of this study was to examine transcriptomic alterations in cancer cells co-expressing both receptors and the association of gene expression signatures with disease outcome. Methods Transcriptional effects of ERβ overexpression were determined in a stably transfected cell line derived from ERα-positive T-47D cells. Microarray analysis was carried out to identify differential gene expression in the cell line, and expression of key genes was validated by quantitative polymerase chain reaction. Microarray and clinical data from patient samples were then assessed to determine the in vivo relevance of the expression profiles observed in the cell line. Results A subset of 14 DNA replication and cell cycle-related genes was found to be specifically downregulated by ERβ. Expression profiles of four genes, CDC2, CDC6, CKS2, and DNA2L, were significantly inversely correlated with ERβ transcript levels in patient samples, consistent with in vitro observations. Kaplan-Meier analysis revealed better disease outcome for the patient group with an expression signature linked to higher ERβ expression as compared to the lower ERβ-expressing group for both disease-free survival (p = 0.00165) and disease-specific survival (p = 0.0268). These findings were further validated in an independent cohort. Conclusion Our findings revealed a transcriptionally regulated mechanism for the previously described growth inhibitory effects of ERβ in ERα-positive breast tumor cells and provide evidence for a functional and beneficial impact of ERβ in primary breast tumors.

Background Tumor-infiltrating leukocytes can either limit cancer growth or facilitate its spread. Diagnostic strategies that comprehensively assess the functional complexity of tumor immune infiltrates could have wide-reaching clinical value. In previous work we identified distinct immune gene signatures in breast tumors that reflect the relative abundance of infiltrating immune cells and exhibited significant associations with patient outcomes. Here we hypothesized that immune gene signatures agnostic to tumor type can be identified by de novo discovery of gene clusters enriched for immunological functions and possessing internal correlation structure conserved across solid tumors from different anatomic sites. Methods We assembled microarray expression datasets encompassing 5,295 tumors of the breast, colon, lung, ovarian and prostate. Unsupervised clustering methods were used to determine number and composition of gene clusters within each dataset. Immune-enriched gene clusters (signatures) identified by gene ontology enrichment were analyzed for internal correlation structure and conservation across tumors then compared against expression profiles of: 1) flow-sorted leukocytes from peripheral blood and 2) >300 cancer cell lines from solid and hematologic cancers. Cox regression analysis was used to identify signatures with significant associations with clinical outcome. Results We identified nine distinct immune-enriched gene signatures conserved across all five tumor types. The signatures differentiated specific leukocyte lineages with moderate discernment overall, and naturally organized into six discrete groups indicative of admixed lineages. Moreover, seven of the signatures exhibit minimal and uncorrelated expression in cancer cell lines, suggesting that these signatures derive predominantly from infiltrating immune cells. All nine immune signatures achieved statistically significant associations with patient prognosis (p

Meeting abstracts Overlapping immune signatures are observed among cancers with a better prognostic connotation and those with an increased likelihood to respond to immunotherapeutic approaches [[1][1], [2][2]]. Such signatures qualitatively overlap with those detected during other conditions of

The abundance and functional orientation of tumor-infiltrating effector cells has long been observed to predict for reduced incidence of clinical metastasis and cancer-specific death. Using bioinformatics to mine large breast tumor microarray datasets, we and others have identified prognostic immune gene signatures, or metagenes. Robust evidence indicates that these metagenes are: 1) positively correlated with distant metastasis-free survival (DMFS) of patients, 2) comprised of genes that regulate immune cell-specific biology, and 3) reflective of the relative abundance of discernible populations of tumor infiltrating leukocytes. In recent work we have leveraged the statistical associations between the immune metagenes and the DMFS of breast cancer patients to explore the underlying phenotypes that differ in their ability to potentiate long-term, immune-mediated tumor rejection. Using a tumor classification model that combines the prognostic attributes of three distinct immune metagenes, termed the B/P, T/NK and M/D metagenes, we have identified molecular subtypes of breast cancer that either permit or prohibit prognostication by the immune metagenes. On this basis, we have delineated the phenotypic attributes of breast cancer that distinguish two novel immunogenic tumor subtypes, which we have defined as: immune benefit-enabled (IBE) and immune benefit-disabled (IBD). Phenotypically, IBE tumors comprise of Basal-like tumors and highly-proliferative HER2-Enriched and Luminal-B subtypes, while IBD tumors comprise of Claudin-Low, Luminal-A, and low-proliferative HER2-Enriched and Luminal-B tumors. Prognostically, IBE tumors (n = 666) can be stratified by the immune metagene model into prognostic subgroups with high statistical significance (P