Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is diagnosed in approximately 15% of all human breast cancer (BrCa) patients. Currently, no targeted therapies exist for this subtype of BrCa and prognosis remains poor. Our laboratory has previously identified a proliferation/DNA repair/cell cycle gene signature (Tag signature) that is characteristic of human TNBC. We hypothesize that targeting the dysregulated biological networks in the Tag gene signature will lead to the identification of improved combination therapies for TNBC. METHODS: Cross-species genomic analysis was used to identify human breast cancer cell lines that express the Tag signature. Knock-down of the up-regulated genes in the Tag signature by siRNA identified several genes that are critical for TNBC cell growth. Small molecule inhibitors to two of these genes were analyzed, alone and in combination, for their effects on cell proliferation, cell cycle, and apoptosis in vitro and tumor growth in vivo. Synergy between the two drugs was analyzed by the Chou-Talalay method. RESULTS: A custom siRNA screen was used to identify targets within the Tag signature that are critical for growth of TNBC cells. Ribonucleotide reductase 1 and 2 (RRM1 and 2) and checkpoint kinase 1 (CHK1) were found to be critical targets for TNBC cell survival. Combination therapy, to simultaneously attenuate cell cycle checkpoint control through inhibition of CHK1 while inducing DNA damage with gemcitabine, improved therapeutic efficacy in vitro and in xenograft models of TNBC. CONCLUSIONS: This combination therapy may have translational value for patients with TNBC and improve therapeutic response for this aggressive form of breast cancer.

MicroRNA regulation in cancer-associated fibroblasts.

The microenvironment of cancer cells has proven to be a critical component of tumors that strongly influences cancer development and progression into invasive and metastatic disease. Compared to normal tissue, dramatic differences in gene expression occur in multiple cell types that constitute the tumor microenvironment including cancer-associated fibroblasts (CAFs) that are important stromal components of growing tumors. In this review, we present recent advances in understanding how microRNAs are deregulated in cancer-associated fibroblasts (CAFs) and how this affects tumor biology. The microRNA signature of CAFs is discussed with respect to their functional relevance to tumor cells as well as other cell types involved in tumor homeostasis.

Regulation of hypoxia-inducible genes by ETS1 transcription factor.

Hypoxia-inducible factor (HIF-1) regulates the expression of genes that facilitate tumor cell survival by making them more resistant to therapeutic intervention. Recent evidence suggests that the activation of other transcription factors, in cooperation with HIF-1 or acting alone, is involved in the upregulation of hypoxia-inducible genes. Here we report that high cell density, a condition that might mimic the physiologic situation in growing tumor and most probably representing nutritional starvation, upregulates hypoxia-inducible genes. This upregulation can occur in HIF-independent manner since hypoxia-inducible genes carbonic anhydrase 9 (CA9), lysyloxidase like 2 (LOXL2) and n-myc-down regulated 1 (NDRG1)/calcium activated protein (Cap43) can be upregulated by increased cell density under both normoxic and hypoxic conditions in both HIF-1 alpha-proficient and -deficient mouse fibroblasts. Moreover, cell density upregulates the same genes in 1HAEo- and A549 human lung epithelial cells. Searching for other transcription factors involved in the regulation of hypoxia-inducible genes by cell density, we focused our attention on ETS1. As reported previously, members of v-ets erythroblastosis virus E26 oncogene homolog (ETS) family transcription factors participate in the upregulation of hypoxia-inducible genes. Here, we provide evidence that ETS1 protein is upregulated at high cell density in both human and mouse cells. The involvement of ETS1 in the upregulation of hypoxia-inducible genes was further confirmed in a luciferase reporter assay using cotransfection of ETS1 expression vector with NDRG1/Cap43 promoter construct. The downregulation of ETS1 expression with small interfering RNA (siRNA) inhibited the upregulation of CA9 and NDRG1/Cap43 caused by increased cell density. Collectively, our data indicate the involvement of ETS1 along with HIF-1 in regulating hypoxia-inducible genes.

Global expression analysis of cancer/testis genes in uterine cancers reveals a high incidence of BORIS expression.

PURPOSE: Cancer/testis (CT) genes predominantly expressed in the testis (germ cells) and generally not in other normal tissues are aberrantly expressed in human cancers. This highly restricted expression provides a unique opportunity to use these CT genes for diagnostics, immunotherapeutic, or other targeted therapies. The purpose of this study was to identify those CT genes with the greatest incidence of expression in uterine cancers. EXPERIMENTAL DESIGN: We queried the expression of known and putative CT gene transcripts (representing 79 gene loci) using whole genome gene expression arrays. Specifically, the global gene expressions of uterine cancers (n = 122) and normal uteri (n = 10) were determined using expression data from the Affymetrix HG-U133A and HG-U133B chips. Additionally, we also examined the brother of the regulator of imprinted sites (BORIS) transcript by reverse transcription-PCR and quantitative PCR because its transcript was not represented on the array. RESULTS: Global microarray analysis detected many CT genes expressed in various uterine cancers; however, no individual CT gene was expressed in more than 25% of all cancers. The expression of the two most commonly expressed CT genes on the arrays, MAGEA9 (24 of 122 cancers and 0 of 10 normal tissues) and Down syndrome critical region 8 (DSCR8)/MMA1 (16 if 122 cancers and 0 of 10 normal tissues), was confirmed by reverse transcription-PCR methods, validating the array screening approach. In contrast to the relatively low incidence of expression of the other CT genes, BORIS expression was detected in 73 of 95 (77%) endometrial cancers and 24 of 31 (77%) uterine mixed mesodermal tumors. CONCLUSIONS: These data provide the first extensive survey of multiple CT genes in uterine cancers. Importantly, we detected a high frequency of BORIS expression in uterine cancers, suggesting its potential as an immunologic or diagnostic target for these cancers. Given the high incidence of BORIS expression and its possible regulatory role, an examination of BORIS function in the etiology of these cancers is warranted.

Role of ETS transcription factors in the hypoxia-inducible factor-2 target gene selection.

Tumor hypoxia often directly correlates with aggressive phenotype, metastasis progression, and resistance to chemotherapy. Two transcription factors [hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha] are dramatically induced in hypoxic areas and regulate the expression of genes necessary for tumor adaptation to the conditions of low oxygen; however, the relative contribution of these factors is controversial. We used RNA interference-mediated inactivation of HIF-1alpha or HIF-2alpha followed by microarray analysis to identify genes specifically regulated by either HIF-1 or HIF-2 in hypoxia. We found that, in the MCF7 cell line, the vast majority of hypoxia-responsive genes (>80%) were dependent on the presence of HIF-1alpha. However, a small group of genes were preferentially regulated by HIF-2alpha. Promoter analysis for this group of genes revealed that all of them have putative binding sites for ETS family transcription factors, and 10 of 11 HIF-2alpha-dependent genes had at least one potential hypoxia-responsive element (HRE) in proximity to an ETS transcription factor binding site. Knockdown of ELK-1, the most often represented member of ETS family, significantly reduced hypoxic induction of the HIF-2alpha-dependent genes. Physical and functional interaction between ELK-1 and HIF-2alpha were supported by coimmunoprecipitation of these two proteins, luciferase reporter assay using CITED2 promoter, and binding of ELK-1 protein to the promoters of CITED2 and WISP2 genes in proximity to a HRE. These data suggest that the choice of the target genes by HIF-1 or HIF-2 depends on availability and cooperation of HIFs with other factors recognizing their cognate elements in the promoters.

Cancers as wounds that do not heal: differences and similarities between renal regeneration/repair and renal cell carcinoma.

Cancers have been described as wounds that do not heal, suggesting that the two share common features. By comparing microarray data from a model of renal regeneration and repair (RRR) with reported gene expression in renal cell carcinoma (RCC), we asked whether those two processes do, in fact, share molecular features and regulatory mechanisms. The majority (77%) of the genes expressed in RRR and RCC were concordantly regulated, whereas only 23% were discordant (i.e., changed in opposite directions). The orchestrated processes of regeneration, involving cell proliferation and immune response, were reflected in the concordant genes. The discordant gene signature revealed processes (e.g., morphogenesis and glycolysis) and pathways (e.g., hypoxia-inducible factor and insulin-like growth factor-I) that reflect the intrinsic pathologic nature of RCC. This is the first study that compares gene expression patterns in RCC and RRR. It does so, in particular, with relation to the hypothesis that RCC resembles the wound healing processes seen in RRR. However, careful attention to the genes that are regulated in the discordant direction provides new insights into the critical differences between renal carcinogenesis and wound healing. The observations reported here provide a conceptual framework for further efforts to understand the biology and to develop more effective diagnostic biomarkers and therapeutic strategies for renal tumors and renal ischemia.

BRCA1, histone H2AX phosphorylation, and male meiotic sex chromosome inactivation.

In mammalian spermatogenesis, the X and Y chromosomes are transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation, MSCI), forming a condensed chromatin domain termed the sex or XY body. The nucleosomal core histone H2AX is phosphorylated within the XY chromatin domain just prior to MSCI, and it has been hypothesized that this triggers the chromatin condensation and transcriptional repression. Here, we show that the kinase ATR localizes to XY chromatin at the onset of MSCI and that this localization is disrupted in mice with a mutant form of the tumor suppressor protein BRCA1. In the mutant pachytene cells, ATR is usually present at nonsex chromosomal sites, where it colocalizes with aberrant sites of H2AX phosphorylation; in these cells, there is MSCI failure. In rare pachytene cells, ATR does locate to XY chromatin, H2AX is then phosphorylated, a sex body forms, and MSCI ensues. These observations highlight an important role for BRCA1 in recruiting the kinase ATR to XY chromatin at the onset of MSCI and provide compelling evidence that it is ATR that phosphorylates H2AX and triggers MSCI.

BRCA1 may modulate neuronal cell cycle re-entry in Alzheimer disease.

In Alzheimer disease, neuronal degeneration and the presence of neurofibrillary tangles correlate with the severity of cognitive decline. Neurofibrillary tangles contain the antigenic profile of many cell cycle markers, reflecting a re-entry into the cell cycle by affected neurons. However, while such a cell cycle re-entry phenotype is an early and consistent feature of Alzheimer disease, the mechanisms responsible for neuronal cell cycle are unclear. In this regard, given that a dysregulated cell cycle is a characteristic of cancer, we speculated that alterations in oncogenic proteins may play a role in neurodegeneration. To this end, in this study, we examined brain tissue from cases of Alzheimer disease for the presence of BRCA1, a known regulator of cell cycle, and found intense and specific localization of BRCA1 to neurofibrillary tangles, a hallmark lesion of the disease. Analysis of clinically normal aged brain tissue revealed systematically less BRCA1, and surprisingly in many cases with apparent phosphorylated tau-positive neurofibrillary tangles, BRCA1 was absent, yet BRCA1 was present in all cases of Alzheimer disease. These findings not only further define the cell cycle reentry phenotype in Alzheimer disease but also indicate that the neurofibrillary tangles which define Alzheimer disease may have a different genesis from the neurofibrillary tangles of normal aging.

Properties of switch-like bioregulatory networks studied by simulation of the hypoxia response control system.

A complex bioregulatory network could be more easily comprehended if its essential function could be described by a small "core" subsystem, and if its response characteristics were switch-like. We tested this proposition by simulation studies of the hypoxia response control network. We hypothesized that a small subsystem governs the basics of the cellular response to hypoxia and that this response has a sharp oxygen-dependent transition. A molecular interaction map of the network was prepared, and an evolutionarily conserved core subsystem was extracted that could control the activity of hypoxia response promoter elements on the basis of oxygen concentration. The core subsystem included the hypoxia-inducible transcription factor (HIFalpha:ARNT heterodimer), proline hydroxylase, and the von Hippel-Lindau protein. Simulation studies showed that the same core subsystem can exhibit switch-like responses both to oxygen level and to HIFalpha synthesis rate, thus suggesting a mechanism for hypoxia response promoter element-dependent responses common to both hypoxia and growth factor signaling. The studies disclosed the mechanism responsible for the sharp transitions. We show how parameter sets giving switch-like behavior can be found and how this type of behavior provides a foundation for quantitative studies in cells.

Gene expression profiling of microsatellite unstable and microsatellite stable endometrial cancers indicates distinct pathways of aberrant signaling.

Microsatellite instability (MSI) is a molecular phenotype present in approximately 25% of endometrial cancers. We examined the global gene expression profiles of early-stage endometrioid endometrial cancers with and without the MSI phenotype to test the hypothesis that MSI phenotype may determine a unique molecular signature among otherwise similar cancers. Unsupervised principal component analysis of the expression data from these cases indicated two distinct groupings of cancers based on MSI phenotype. A relatively small number of array features (392) at high statistical value (P < 0.001) were identified that drive the instability signature in these cancers; 109 of these transcripts differed by at least 2-fold. These data identify distinct gene expression profiles for MSI and microsatellite stable (MSS) cancers, which suggest that cancers with MSI develop in part by different mechanisms from their similar stable counterparts. In particular, we found evidence that two members of the secreted frizzled related protein family (SFRP1 and SFRP4) were more frequently down-regulated in MSI cancers as compared with MSS cancers. Down-regulation was accompanied by promoter hypermethylation for SFRP1. SFRP1 was hypermethylated in 8 of 12 MSI cancers whereas only 3 of 16 MSS cancers were methylated. The WNT target fibroblast growth factor 18 was found to be up-regulated in MSI cancers. These data classify histologically similar endometrioid endometrial cancers into two distinct groupings with implications affecting therapy and prevention.

Gene expression profiles associated with response to chemotherapy in epithelial ovarian cancers.

PURPOSE: The goal of this study was to determine whether distinct gene expression profiles are associated with intrinsic and/or acquired chemoresistance in epithelial ovarian carcinoma. EXPERIMENTAL DESIGN: Gene expression profiles were generated from 21 primary chemosensitive tumors and 24 primary chemo-resistant tumors using cDNA-based microarrays. Gene expression profiles of both groups of primary tumors were then compared with those of 15 ovarian carcinomas obtained following platinum-based chemotherapy ("post-chemotherapy" tumors). A theme discovery tool was used to identify functional categories of genes involved in drug resistance. RESULTS: Comparison of primary chemosensitive and chemo-resistant tumors revealed differential expression of 85 genes (P < 0.001). Comparison of gene expression profiles of primary chemosensitive tumors and post-chemotherapy tumors revealed more robust differences with 760 genes differentiating the two groups (P < 0.001). In contrast, only 230 genes were differentially expressed between primary chemo-resistant and post-chemotherapy groups (P < 0.001). Common to both gene lists were 178 genes representing transcripts differentially expressed between post-chemotherapy tumors and all primary tumors irrespective of intrinsic chemosensitivity. The gene expression profile of post-chemotherapy tumors compared with that of primary tumors revealed statistically significant overrepresentation of genes encoding extracellular matrix-related proteins. CONCLUSIONS: These data show that gene expression profiling can discriminate primary chemo-resistant from primary chemosensitive ovarian cancers. Gene expression profiles were also identified that correlate with states of intrinsic and acquired chemoresistance and that represent targets for future investigation and potential therapeutic interventions.

Microarray analysis reveals distinct gene expression profiles among different histologic types of endometrial cancer.

Previous studies of oncogene and tumor suppressor gene alterations have suggested that differences exist in the molecular pathogenesis of the various histological types of endometrial cancer. To elucidate further the molecular events involved in endometrial carcinogenesis, we examined global expression patterns of 16 nonendometrioid cancers (13 serous papillary and 3 clear cell), 19 endometrioid cancers, and 7 age-matched normal endometria using cDNA microarrays. Unsupervised analysis of gene expression identified 191 genes that exhibited >2-fold differences (P < 0.001) between the histological groups. Many genes were similarly dysregulated in both nonendometrioid and endometrioid cancers relative to normal endometria. Gene expression differences in only 24 transcripts could distinguish serous from endometrioid cancers, the two most common subgroups. These data provide the basis for investigation of previously unrecognized novel pathways involved in the development of endometrial cancers.

The epigenetic modifier JMJD6 is amplified in mammary tumors and cooperates with c-Myc to enhance cellular transformation, tumor progression, and metastasis.

BACKGROUND: Oncogene overexpression in primary cells often triggers the induction of a cellular safeguard response promoting senescence or apoptosis. Secondary cooperating genetic events are generally required for oncogene-induced tumorigenesis to overcome these biologic obstacles. We employed comparative genomic hybridization for eight genetically engineered mouse models of mammary cancer to identify loci that might harbor genes that enhance oncogene-induced tumorigenesis. RESULTS: Unlike many other mammary tumor models, the MMTV-Myc tumors displayed few copy number variants except for amplification of distal mouse chromosome 11 in 80 % of the tumors (syntenic to human 17q23-qter often amplified in human breast cancer). Analyses of candidate genes located in this region identified JMJD6 as an epigenetic regulatory gene that cooperates with Myc to enhance tumorigenesis. It suppresses Myc-induced apoptosis under varying stress conditions through inhibition of p19ARF messenger RNA (mRNA) and protein, leading to reduced levels of p53. JMJD6 binds to the p19ARF promoter and exerts its inhibitory function through demethylation of H4R3me2a. JMJD6 overexpression in MMTV-Myc cell lines increases tumor burden, induces EMT, and greatly enhances tumor metastasis. Importantly, we demonstrate that co-expression of high levels of JMJD6 and Myc is associated with poor prognosis for human ER+ breast cancer patients. CONCLUSIONS: A novel epigenetic mechanism has been identified for how JMJD6 cooperates with Myc during oncogenic transformation. Combined high expression of Myc and JMJD6 confers a more aggressive phenotype in mouse and human tumors. Given the pleiotropic pro-tumorigenic activities of JMJD6, it may be useful as a prognostic factor and a therapeutic target for Myc-driven mammary tumorigenesis.

Development and Preclinical Application of an Immunocompetent Transplant Model of Basal Breast Cancer with Lung, Liver and Brain Metastases.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is associated with a poor prognosis and for which no targeted therapies currently exist. In order to improve preclinical testing for TNBC that relies primarily on using human xenografts in immunodeficient mice, we have developed a novel immunocompetent syngeneic murine tumor transplant model for basal-like triple-negative breast cancer. The C3(1)/SV40-T/t-antigen (C3(1)/Tag) mouse mammary tumor model in the FVB/N background shares important similarities with human basal-like TNBC. However, these tumors or derived cell lines are rejected when transplanted into wt FVB/N mice, likely due to the expression of SV40 T-antigen. We have developed a sub-line of mice (designated REAR mice) that carry only one copy of the C3(1)/Tag-antigen transgene resulting from a spontaneous transgene rearrangement in the original founder line. Unlike the original C3(1)/Tag mice, REAR mice do not develop mammary tumors or other phenotypes observed in the original C3(1)/Tag transgenic mice. REAR mice are more immunologically tolerant to SV40 T-antigen driven tumors and cell lines in an FVB/N background (including prostate tumors from TRAMP mice), but are otherwise immunologically intact. This transplant model system offers the ability to synchronously implant the C3(1)/Tag tumor-derived M6 cell line or individual C3(1)/Tag tumors from various stages of tumor development into the mammary fat pads or tail veins of REAR mice. C3(1)/Tag tumors or M6 cells implanted into the mammary fat pads spontaneously metastasize at a high frequency to the lung and liver. M6 cells injected by tail vein can form brain metastases. We demonstrate that irradiated M6 tumor cells or the same cells expressing GM-CSF can act as a vaccine to retard tumor growth of implanted tumor cells in the REAR model. Preclinical studies performed in animals with an intact immune system should more authentically replicate treatment responses in human patients.

The in vitro and in vivo effects of re-expressing methylated von Hippel-Lindau tumor suppressor gene in clear cell renal carcinoma with 5-aza-2'-deoxycytidine.

PURPOSE: Clear cell renal carcinoma (ccRCC) is strongly associated with loss of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene is functionally lost through hypermethylation in up to 19% of sporadic ccRCC cases. We theorized that re-expressing VHL silenced by methylation in ccRCC cells, using a hypo-methylating agent, may be an approach to treatment in patients with this type of cancer. We test the ability of two hypo-methylating agents to re-express VHL in cell culture and in mice bearing human ccRCC and evaluate the effects of re-expressed VHL in these models. EXPERIMENTAL DESIGN: Real-time reverse transcription-PCR was used to evaluate the ability of zebularine and 5-aza-2'-deoxycytidine (5-aza-dCyd) to re-express VHL in four ccRCC cell lines with documented VHL gene silencing through hypermethylation. We evaluated if the VHL re-expressed after hypo-methylating agent treatment could recreate similar phenotypic changes in ccRCC cells observed when the VHL gene is re-expressed via transfection in cell culture and in a xenograft mouse model. Finally we evaluate global gene expression changes occurring in our cells, using microarray analysis. RESULTS: 5-Aza-dCyd was able to re-express VHL in our cell lines both in culture and in xenografted murine tumors. Well described phenotypic changes of VHL expression including decreased invasiveness into Matrigel, and decreased vascular endothelial growth factor and glucose transporter-1 expression were observed in the treated lines. VHL methylated ccRCC xenografted tumors were significantly reduced in size in mice treated with 5-aza-dCyd. Mice bearing nonmethylated but VHL-mutated tumors showed no tumor shrinkage with 5-aza-dCyd treatment. CONCLUSION: Hypo-methylating agents may be useful in the treatment of patients having ccRCC tumors consisting of cells with methylated VHL.

Universal Reference RNA as a standard for microarray experiments.

BACKGROUND: Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR), developed with the goal of providing hybridization signal at each microarray probe location (spot). Measuring signal at each spot as the ratio of experimental RNA to reference RNA targets, rather than relying on absolute signal intensity, decreases variability by normalizing signal output in any two-color hybridization experiment. RESULTS: Human, mouse and rat URR (UHRR, UMRR and URRR, respectively) were prepared from pools of RNA derived from individual cell lines representing different tissues. A variety of microarrays were used to determine percentage of spots hybridizing with URR and producing signal above a user defined threshold (microarray coverage). Microarray coverage was consistently greater than 80% for all arrays tested. We confirmed that individual cell lines contribute their own unique set of genes to URR, arguing for a pool of RNA from several cell lines as a better configuration for URR as opposed to a single cell line source for URR. Microarray coverage comparing two separately prepared batches each of UHRR, UMRR and URRR were highly correlated (Pearson's correlation coefficients of 0.97). CONCLUSION: Results of this study demonstrate that large quantities of pooled RNA from individual cell lines are reproducibly prepared and possess diverse gene representation. This type of reference provides a standard for reducing variation in microarray experiments and allows more reliable comparison of gene expression data within and between experiments and laboratories.

BRCA1-mediated repression of select X chromosome genes.

Recently BRCA1 has been implicated in the regulation of gene expression from the X chromosome. In this study the influence of BRCA1 on expression of X chromosome genes was investigated. Complementary DNA microarrays were used to compare the expression levels of X chromosome genes in 18 BRCA1-associated ovarian cancers to those of the 13 "BRCA1-like" and 14 "BRCA2-like" sporadic tumors (as defined by previously reported expression profiling). Significance was determined using parametric statistics with P < 0.005 as a cutoff. Forty of 178 total X-chromosome transcripts were differentially expressed between the BRCA1-associated tumors and sporadic cancers with a BRCA2-like molecular profile. Thirty of these 40 genes showed higher mean expression in the BRCA1-associated samples including all 11 transcripts that mapped to Xp11. In contrast, four of 178 total X chromosome transcripts showed significant differential expression between BRCA1-associated and sporadic tumors with a BRCA1-like molecular profile. All four mapped to Xp11 and showed higher mean expression in BRCA1-associated tumors. Re-expression of BRCA1 in HCC1937 BRCA1-deficient breast cancer cell resulted in the repression of 21 transcripts. Eleven of the 21 (54.5%) transcripts mapped to Xp11. However, there was no significant overlap between these Xp11 genes and those found to be differentially expressed between BRCA1-associated and sporadic ovarian cancer samples. These results demonstrate that BRCA1 mediates the repression of several X chromosome genes, many of which map to the Xp11 locus.

Expression of GATA3 in MDA-MB-231 triple-negative breast cancer cells induces a growth inhibitory response to TGFß.

Transforming growth factor (ß1TGFß1) can promote proliferation in late stage cancers but acts as a tumor suppressor in normal epithelial cells and in early stage cancers. Although, the TGFß pathway has been shown to play a key role in tumorigenesis and metastasis, only a limited number of models have been developed to understand this process. Here, we present a novel model system to discern this paradoxical role of TGFß1 using the MDA-MB-231 (MB-231) cell line. The MB-231 triple-negative breast cancer cell line has been extensively characterized and has been shown to continue to proliferate and undergo epithelial-to-mesenchymal transition (EMT) upon TGFß1 stimulation. We have previously shown by microarray analysis that expression of GATA3 in MB-231 cells results in reprogramming of these cells from a basal to a luminal subtype associated with a reduction of metastasis and tumorigenesis when implanted as xenografts. We now demonstrate that GATA3 overexpression in these cells results in a reduction of TGFß1 response, reversal of EMT, and most importantly, restoration of sensitivity to the inhibitory effects on proliferation of TGFß1. Microarray analysis revealed that TGFß1 treatment resulted in reduction of several cell cycle effectors in 231-GATA3 cells but not in control cells. Furthermore, our microarray analysis revealed a significant increase of BMP5 in 231-GATA3 cells. We demonstrate that combined treatment of MB-231 control cells with TGFß1 and BMP5 results in a significant reduction of cellular proliferation. Thus, this model offers a means to further investigate potentially novel mechanisms involved in the switch in response to TGFß1 from tumor promoter to tumor suppressor through the reprogramming of a triple-negative breast cancer cell line by the GATA3 transcription factor.

Impact of ischemia and procurement conditions on gene expression in renal cell carcinoma.

PURPOSE: Previous studies have shown that ischemia alters gene expression in normal and malignant tissues. There are no studies that evaluated effects of ischemia in renal tumors. This study examines the impact of ischemia and tissue procurement conditions on RNA integrity and gene expression in renal cell carcinoma. EXPERIMENTAL DESIGN: Ten renal tumors were resected without renal hilar clamping from 10 patients with renal clear cell carcinoma. Immediately after tumor resection, a piece of tumor was snap frozen. Remaining tumor samples were stored at 4°C, 22°C, and 37°C and frozen at 5, 30, 60, 120, and 240 minutes. Histopathologic evaluation was conducted on all tissue samples, and only those with greater than 80% tumor were selected for further analysis. RNA integrity was confirmed by electropherograms and quantitated using RNA integrity number index. Altered gene expression was assessed by paired, two-sample t test between the zero time point and aliquots from various conditions obtained from the same tumor. RESULTS: One hundred and forty microarrays were conducted. Some RNA degradation was observed 240 minutes after resection at 37°C. The expression of more than 4,000 genes was significantly altered by ischemia times or storage conditions. The greatest gene expression changes were observed with longer ischemia time and warmer tissue procurement conditions. CONCLUSION: RNA from kidney cancer remains intact for up to 4 hours post surgical resection regardless of storage conditions. Despite excellent RNA preservation, time after resection and procurement conditions significantly influence gene expression profiles. Meticulous attention to preacquisition variables is of paramount importance for accurate tumor profiling.

Differential genetic expression in large versus small clear cell renal cell carcinoma: results from microarray analysis.

PURPOSE: Tumor growth and progression requires multiple steps and genetic alterations. The molecular events that occur as tumors increase in size are unknown. Patients with von Hippel-Lindau (VHL) provide a unique opportunity to study molecular alterations during tumor growth as these patients develop multiple bilateral renal tumors. To better characterize biologic events associated with tumor growth, we evaluated the alterations in gene expression in large versus small renal tumors removed from the same kidney of the same individuals. EXPERIMENTAL DESIGN: We reviewed pathology reports from patients who underwent partial nephrectomies at the National Cancer Institute for multiple tumors. We identified 11 patients who fulfilled the following inclusion criteria: 1) The patient must have had a surgical resection of more than one solid tumor from the same kidney during the same operation; 2) Among the solid tumors at least one must have been greater than 3 cm in the largest dimension and at least one less than 2 cm; 3) the nuclear Furhman grade for both larger and smaller solid tumors was identical; 4) a portion of each tumor was procured and snap frozen after surgical removal; 5) Hematoxylin and eosin staining of the frozen sample confirmed clear cell carcinoma to be present in at least 80% of the section.Affymetrix platform and protocol for gene expression arrays were used. RNA from the frozen large and small tumor samples was extracted using Trizol-Chlorophorm method. The RNA was then reverse transcribed, labeled, fragmented, and hybridized on to an Affymetrix U133 Plus 2.0 array that contains 54,000 probe sets representing 24,568 genes. Analysis included unsupervised clustering and chromosomal analysis. The paired t-test was performed to compare gene expression levels in small and large tumors. P<0.01 was considered statistically significant. RESULTS: Gene expression profiles were assessed for 22 tumors (11 patients). Upon unsupervised clustering the pairs with larger tumor volume difference clustered separately from pairs with smaller volume difference. Chromosomal analysis revealed few consistent changes other than reduced expression of chromosome 3p25 among all tumors. Paired t-test showed 860 differentially expressed genes in the T1b vs T1a group, a number far greater than expected due to chance alone. When analyzed by gene function, most differences were observed in genes involved in DNA replication and in cytokine signaling. CONCLUSIONS: This study demonstrates that as tumors increase in size there is an increasing difference in gene expression. Unsupervised clustering analysis confirms that as the volume difference increases there are a distinct set of genes that are regulated either as a response to a tumor's growth or as an early event that causes the tumor to grow. While we did not observe chromosomal instability, we did note differences in expression of individual transcripts as tumors grew larger.