A NOVEL FUNCTION FOR AURORA B KINASE IN THE REGULATION OF P53 BY PHOSPHORYLATION

The mitotic kinase Aurora B plays a pivotal role in mitosis and cytokinesis and governs the spindle assembly checkpoint which ensures correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in breast and other cancers and may be an important molecular target for chemotherapy. Tumor suppressor p53 is the guardian of the genome and an important negative regulator of the cell cycle. Previously, it was unknown whether Aurora B and p53 had mutual regulation during the cell cycle. A small molecule specific inhibitor of Aurora B, AZD1152, gave us an indication that Aurora B negatively impacted p53 during interphase and mitosis. Here, we show the antineoplastic activity of AZD1152 in six human breast cancer cell lines, three of which overexpress HER2. AZD1152 specifically inhibited Aurora B kinase activity, thereby causing mitotic catastrophe, polyploidy and apoptosis, which in turn led to apoptotic death. Further, AZD1152 administration efficiently suppressed tumor growth in orthotopic and metastatic breast cancer cell xenograft models. Notably, it was found that the protein level of Aurora B kinase declined after inhibition of Aurora B kinase activity. Investigation of the underlying mechanism suggested that AZD1152 accelerated the protein turnover of Aurora B by enhancing its ubiquitination. As a consequence of inhibition of Aurora B, p53 levels were increased in tissue culture and murine models. This hinted at a possible direct interaction between p53 and Aurora B. Indeed, it was found that p53 and Aurora B exist in complex and interact directly during interphase and at the centromere in mitosis. Further, Aurora B was shown to phosphorylate p53 at several serine/threonine residues in the DNA binding domain and these events caused downregulation of p53 levels via ubiquitination mediated by Mdm2. Importantly, phosphorylation of threonine 211 was shown to reduce p53’s transcriptional activity while other phosphorylation sites did not. On a functional level, Aurora B was shown to reduce p53’s capacity to mediate apoptosis in response to the DNA damaging agent, cisplatin. These results define a novel mechanism for p53 inactivation by Aurora B and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise p53’s tumor suppressor function.

THE ROLE OF CYCLIN E IN PKC IOTA-DRIVEN EARLY OVARIAN TUMORIGENESIS

Among the gynecologic malignancies, epithelial ovarian tumors are the leading cause of death. For the past few decades, the only treatment has involved surgical resection of the tumor and/or general chemotherapies. In an attempt to improve treatment options, we have shown that several oncogenes that are overexpressed in ovarian cancer, PI3K, PKCiota, and cyclin E, all of which have been shown to lead to a poor prognosis and decreased survival, converge into a single pathway that could potentially be targeted therapeutically. Because of the ability of either PKCiota or cyclin E overexpression to independently induce anchorage-independent growth, a hallmark of cancer, we hypothesized that targeting PKCiota expression in ovarian cancer cells could induce a reversion of the transformed phenotype through down regulation of cyclin E. To test this hypothesis, we first established a correlation between PKCiota and cyclin E in a panel of 20 ovarian cancer cell lines. To show that PKCiota is upstream of cyclin E, PKCiota was stably knocked down using RNAi in IGROV cells (epithelial ovarian cancer cell line of serous histology). The silencing of PKCiota resulted in decreased expression of cell cycle drivers, such as cyclin D1/E and CDK2/4, and an increase in p27. These alteration in the regulators of the cell cycle resulted in a decrease in both proliferation and anchorage-independent growth, which was specifically through cyclin E, as determined by a rescue experiment. We also found that the mechanism of cyclin E regulation by PKCiota was at the level of degradation rather than transcription. Using two inhibitors to PI3K, we found that both the active form of PKCiota and total cyclin E levels decreased, implying that the PKCiota/cyclin E pathway is downstream from PI3K. In conclusion, we have identified a novel pathway in epithelial ovarian tumorigenesis (PI3K à PKCiota à Cyclin E à anchorage-independent growth), which could potentially be targeted therapeutically.

Dissecting the Interaction between p53 and TRIM24

Dissecting the Interaction of p53 and TRIM24 Aundrietta DeVan Duncan Supervisory Professor, Michelle Barton, Ph.D. p53, the “guardian of the genome”, plays an important role in multiple biological processes including cell cycle, angiogenesis, DNA repair and apoptosis. Because it is mutated in over 50% of cancers, p53 has been widely studied in established cancer cell lines. However, little is known about the function of p53 in a normal cell. We focused on characterizing p53 in normal cells and during differentiation. Our lab recently identified a novel binding partner of p53, Tripartite Motif 24 protein (TRIM24). TRIM24 is a member of the TRIM family of proteins, defined by their conserved RING, B-box, and coiled coil domains. Specifically, TRIM24 is a member of the TIF1 subfamily, which is characterized by PHD and Bromo domains in the C-terminus. Between the Coiled-coil and PHD domain is a linker region, 437 amino acids in length. This linker region houses important functions of TRIM24 including it’s site of interaction with nuclear receptors. TRIM24 is an E3-ubiquitin ligase, recently discovered to negatively regulate p53 by targeting it for degradation. Though it is known that Trim24 and p53 interact, it is not known if the interaction is direct and what effect this interaction has on the function of TRIM24 and p53. My study aims to elucidate the specific interaction domains of p53 and TRIM24. To determine the specific domains of p53 required for interaction with TRIM24, we performed co-immuoprecipitation (Co-IP) with recombinant full-length Flag-tagged TRIM24 protein and various deletion constructs of in vitro translated GST-p53, as well as the reverse. I found that TRIM24 binds both the carboxy terminus and DNA binding domain of p53. Furthermore, my results show that binding is altered when post-translational modifications of p53 are present, suggesting that the interaction between p53 and TRIM24 may be affected by these post-translational modifications. To determine the specific domains of TRIM24 required for p53 interaction, we performed GST pull-downs with in vitro translated, Flag-TRIM24 protein constructs and recombinant GST-p53 protein purified from E. coli. We found that the Linker region is sufficient for interaction of p53 and TRIM24. Taken together, these data indicate that the interaction between p53 and TRIM24 does occur in vitro and that interaction may be influenced by post-translational modifications of the proteins.

THE ROLE OF RECEPTOR TYROSINE KINASE AXL IN PANCREATIC DUCTAL ADENOCARCINOMA AND ITS REGULATION BY HEMATOPOIETIC PROGENITOR KINASE 1

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies with less than 5% of five year survival rate. New molecular markers and new therapeutic targets are urgently needed for patients with PDA. Oncogenic receptor tyrosine kinase Axl has been reported to be overexpressed in many types of human malignancies, including diffuse glioma, melanoma, osteosarcoma, and carcinomas of lung, colon, prostate, breast, ovary, esophagus, stomach, and kidney. However, the expression and functions of Axl in PDA are unclear. We hypothesized that Axl contributes to the development and progression of PDA. We examined Axl expression in 54 human PDA samples and their paired benign pancreatic tissue by immunohistochemistry, we found that Axl was overexpressed in 70% of stage II PDAs, but only 22% of benign ducts (P=0.0001). Axl overexpression was associated with higher frequencies of distant metastasis and was an independent prognostic factor for both poor overall and recurrence-free survivals in patients with stage II PDA (p = 0.03 and 0.04). Axl silencing by shRNA in pancreatic cancer cell lines, panc-28 and Panc-1, decreased tumor cell migration and invasion and sensitized PDA cells to apoptosis stimuli such as γ-irradiation and serum starvation. In addition, we found that Axl-mediated Akt and NF-κB activation and up regulation of MMP2 were involved in the invasion, migration and survival of PDA cells. Thus, we demonstrate that Axl plays an important role in the development and progression of PDA. Targeting Axl signaling pathway may represent a new approach for the treatment of PDA. To understand the molecular mechanisms of Axl overexpression in PDA, we found that Axl expression was down-regulated by hematopoietic progenitor kinase 1 (HPK1), a newly identified tumor suppressor in PDA. HPK1 is lost in over 95% of PDAs. Restoration of HPK1 in PDA cells down-regulated Axl expression. HPK1-mediated Axl degradation was inhibited by leupeptin, baflomycin A1, and monensin, suggesting that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway. HPK1 interacted with and phosphorylated dynamin, a critical component of endocytosis pathway. Overexpression of dominant negative form of dynamin blocked the HPK1-mediated Axl degradation. Therefore we concluded that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway and loss of HPK1 expression may contribute to Axl overexpression in PDAs.

Targeting of {\it HER\/}-{\it 2\/}/{\it neu\/} with E1A gene therapy

Amplification or overexpression of HER-2/neu has been demonstrated in human cancers of the ovary, breast, lung and correlated with chemoresistance and poor clinic prognosis. We have previously found that the adenovirus type 5 early region 1A (E1A) gene product can repress the overexpression and suppress the tumorigenic potential of HER-2/neu-overexpressing cancer cells. In addition, E1A has been reported to induce apoptosis and inhibit the metastatic potential of tumor cells. Therefore, E1A could be considered as a tumor suppressor gene in HER-2/neu-overexpressing cancer cells. To develop an efficient HER-2/neu-targeting gene therapy with E1A, adenoviral vector or cationic liposome was used to introduce E1A into human ovarian, breast and lung cancer cells. Successful therapeutic effects were achieved.^ A replication-deficient adenovirus containing the E1A gene, Ad.E1A(+), was used to infect HER-2/neu-overexpressing human ovarian cancer cell line. Ovarian cancer growth in vitro and colony formation in soft agarose were greatly inhibited.^ To examine tumor suppressor function of E1A in breast cancer, we introduced E1A in vitro by adenovirus into both HER-2/neu-overexpressing and low-expressing human breast cancer cell lines. In HER-2/neu-overexpressing cells, E1A greatly inhibited tumor cell growth in vitro and colony formation in soft agarose. However, in low HER-2/neu expressing cancer cell lines, E1A could only reduce colony formation in soft agarose but had no significant effect on cell growth in monolayer, indicating different effects of E1A in these two types of cancer cells. To test the local therapeutic efficacy of E1A, we used either adenovirus- or liposome-mediated E1A gene delivery systems in an orthotopic breast cancer animal model.^ To test the therapeutic efficacy of systemically-delivered E1A in vivo lung cancer, we treated mice bearing intratracheal lung cancer by i.v. tail injections of Ad.E1A(+). As a result, Ad.E1A(+) suppressed HER-2/neu overexpression and inhibited intratracheal lung cancer growth. However, no significant tumor suppression effect of Ad.E1A(+) was observed in mice bearing HER-2/neu low expressing cell line when the same therapeutic procedure was followed. (Abstract shortened by UMI.) ^

Anti-PSMA antibody-coupled gold nanorods detection by optical and electron microscopies

While cancer is one of the greatest challenges to public health care, prostate cancer was chosen as cancer model to develop a more accurate imaging assessment than those currently available. Indeed, an efficient imaging technique which considerably improves the sensitivity and specificity of the diagnostic and predicting the cancer behavior would be extremely valuable. The concept of optoacoustic imaging using home-made functionalized gold nanoparticles coupled to an antibody targeting PSMA (prostate specific membrane antigen) was evaluated on different cancer cell lines to demonstrate the specificity of the designed platform. Two commonly used microscopy techniques (indirect fluorescence and scanning electron microscopy) showed their straightforwardness and versatility for the nanoparticle binding investigations regardless the composition of the investigated nanoobjects. Moreover most of the research laboratories and centers are equipped with fluorescence microscopes, so indirect fluorescence using Quantum dots can be used for any active targeting nanocarriers (polymers, ceramics, metals, etc.). The second technique based on backscattered electron is not only limited to gold nanoparticles but also suits for any study of metallic nanoparticles as the electronic density difference between the nanoparticles and binding surface stays high enough. Optoacoustic imaging was finally performed on a 3D cellular model to assess and prove the concept of the developed platform.

Binding of Metallocenes to Short Oligonucleotides

Cancer is one of the most severe and widespread diseases and an ideal treatment has not yet been found. In the last decades, cisplatinum was commonly applied in cancer therapy with very good results. However, serious side effects and resistant tumors necessitated the development of new antineoplastic agents, such as metallocenes dihalides. These are metal-based compounds exhibiting two cyclopentadienyl ligands and a cis-dihalide motif. They resemble the cis-chloro configuration of cisplatinum, which propounds a similar mode of action. Metallocenes comprising one of the transition metals titanium, molybdenum, vanadium, niobium, and zirconium as the metal center have been shown to be effective against several cancer cell lines. Evidence for the accumulation of metallocenes in the nucleus implied that DNA is one of the major targets. Although several studies reported adduct formation of metallocenes with nuclear DNA, as yet substantial information about the general binding pattern and the binding to higher-order structures is lacking. Mass spectrometry can fill this gap as it constitutes a powerful technique to investigate the formation of organometallic adducts. Presented data demonstrate that the two agents titanocene dichloride and molybdenocene dichloride bind to single-stranded DNA and RNA. Distinct fragment ions formed upon collision-induced dissociation help to unravel preferential binding sites within the oligonucleotides. Moreover, adducts with duplexes and quadruplexes shed light on the molecular mechanism of action.

Tandem Mass Spectrometric Analysis of Metallocene Adducts with Short Oligonucleotides

Metallocene dichlorides constitute a remarkable class of antineoplastic agents that are highly effective against several cancer cell lines. They were shown to accumulate in the DNA-rich region, which suggests DNA as the primary target. These compounds exhibit two cyclopentadienyl ligands and two labile halide ligands, resulting in a bent sandwich structure. The cis-dihalide motif is structurally related to the cis-chloro configuration of cisplatin and similar modes of action can thus be assumed. Cisplatin binds to two neighboring guanine nucleobases in DNA and consequently, distorts the double-helix, thereby inhibiting DNA replication and transcription. Platinum is classified as a soft Lewis acid and binds preferentially to the nitrogen atoms within the nucleobases. The metallocene dichlorides investigated in this study comprise the metal centers Ti, V, Nb, Mo, Hf, and W, which are classified as hard or intermediate Lewis acids, and thus, favor binding to the phosphate oxygen. Although several studies reported adduct formation of metallocene dichlorides with nucleic acids, substantial information about the adduct composition, the binding pattern, and the nucleobase selectivity has not been provided yet. ESI-MS analyses gave evidence for the formation of metallocene adducts (M = Ti, V, Mo, and W) with single-stranded DNA homologues at pH 7. No adducts were formed with Nb and Hf at neutral pH, albeit adducts with Nb were observed at a low pH. MS2 data revealed considerable differences of the adduct compositions. The product ion spectra of DNA adducts with hard Lewis acids (Ti, V) gave evidence for the loss of metallocene ligands and only moderate backbone fragmentation was observed. By contrast, adducts with intermediate Lewis acids (Mo, W) retained the hydroxy ligands. Preliminary results are in good agreement with the Pearson concept and DFT calculations. Since the metallodrugs were not lost upon CID, the nucleobase selectivity, stoichiometry, and binding patterns can be elucidated by means of tandem mass spectrometry.

NFkappaB and STAT binding elements participate in regulation of MUC1 expression in normal and transformed mammary epithelial cells

The MUC1 gene encodes a transmembrane mucin glycoprotein that is overexpressed in several cancers of epithelial origin, including those of breast, pancreas, lung, ovary, and colon. Functions of MUC1 include protection of mucosal epithelium, modulation of cellular adhesion, and signal transduction. Aberrantly increased expression of MUC1 in cancer cells promotes tumor progression through adaptation of these functions. Some regulatory elements participating in MUC1 transcription have been described, but the mechanisms responsible for overexpression are largely unknown. A region of MUC1 5′ flanking sequence containing two conserved potential cytokine response elements, an NFκB site at −589/−580 and a STAT binding element (SBE) at −503/−495, has been implicated in high level expression in breast and pancreatic cancer cell lines. Persistent stimulation by proinflammatory cytokines may contribute to increased MUC1 transcription by tumor cells. ^ T47D breast cancer cells and normal human mammary epithelial cells (HMEC) were used to determine the roles of the κB site and SBE in basal and stimulated expression of MUC1. Treatment of T47D cells and HMEC with interferon-γ (IFNγ) alone enhanced MUC1 expression at the level of transcription, and the effect of IFNγ was further stimulated by tumor necrosis factor-α (TNFα). MUC1 responsiveness to these cytokines was modest in T47D cells but clearly evident in HMEC. Transient transfection of T47D cells with mutant MUC1 promoter constructs revealed that the κB site at −589/−580 and the SBE at −503/−495 and were required for cooperative stimulation by TNFα and IFNγ. Electrophoretic mobility shift assays (EMSA) revealed that the synergy was mediated not by cooperative binding of transcription factors but by the independent actions of STAT1α and NFκB p65 on their respective binding sites. Independent mutations in the κB site and SBE abrogated cytokine responsiveness and reduced basal MUC1 promoter activity by 45–50%. However, only the κB site appeared to be constitutively activated in T47D cells, in part by NFκB p65. These findings implicate two cytokine response elements in the 5 ′ flanking region of MUC1, specifically a κB site and a STAT binding element, in overexpression of MUC1 in breast cancer cells. ^

Role of the transcription factor activator protein-2alpha in prostate carcinogenesis

Activator protein 2α (AP-2) is a transcription factor known to play a crucial role in the progression of malignant melanoma, colorectal carcinoma, and breast cancer. Several AP-2 target genes are known to be deregulated in prostate cancer, therefore, we hypothesize that loss AP-2 expression plays a causal role in prostate carcinogenesis. Immunofluorescent staining for AP-2 of 30 radical prostatectomy specimens demonstrated that while AP-2 was highly expressed in normal prostate epithelium, its expression was lost in most cases of high grade prostatic intraepithelial neoplasia (PIN), and all cases of prostate cancer studied. Additional analyses demonstrated that AP-2 was associated with normal luminal differentiation and it was not expressed in the basal cell layer. In cell lines, AP-2 was strongly expressed in immortalized normal prostate epithelial cells, whereas low expression was observed in the LNCaP, LNCaP-LN3, and PC3M-LN4 prostate cancer cell lines. Transfection of the highly tumorigenic and metastatic cell line PC3M-LN4 with the AP-2 gene significantly decreased tumor growth in the prostate of nude mice (p = 0.032) and inhibited metastases to the lymph nodes. Moreover, transfection of the low tumorigenic, low metastatic cell line LNCaP-LN3 with full length AP-2; resulted in complete inhibition of tumor incidence in the AP-2 transfectants (0/19) vs. neo control (10/16). A potential mechanism for this loss of tumorigenicity was the modulation of gene expression in prostate cancer cells that mimicked the normal phenotype. Analysis of differential expression between neo control- and AP-2-transfected cells in vitro and in tumors demonstrated low VEGF expression in AP-2 transfectants. We further demonstrated that AP-2 acted as a transcriptional repressor of the VEGF promoter by binding to a GC-rich region located between −88 and −66. This region contains an AP-2 consensus element overlapping two Sp1 consensus elements. We found that Sp3 and AP-2 bound to this region in a mutually exclusive manner to promote activation or repression. Increased VEGF expression has been observed in high grade PIN and in prostate cancer. Here we provide evidence that this early molecular change could be a result of loss of AP-2 expression in the prostatic epithelium. ^

Tumor heterogeneity dictates sensitivity to gefitinib (Iressa(TM)/ZD1839) in solid tumor models

The epidermal growth factor receptor (EGFR) and its ligands are overexpressed in many human tumors, including bladder and pancreas, correlating with a more aggressive tumor phenotype and poor patient prognosis. We initiated the present study to characterize the heterogeneity of gefitinib responsiveness in a panel of human bladder and pancreatic cancer cell lines in order to identify the biological characteristics of EGFR-dependent proliferation that could be used to prospectively identify drug-sensitive tumors. A second objective was to elucidate how to best exploit these results by utilizing gefitinib in combination therapy. To these ends, we examined the effects of the EGFR antagonist gefitinib on proliferation and apoptosis in a panel of 18 human bladder cancer cell lines and 9 human pancreatic cancer cell lines. Our data confirmed the existence of marked heterogeneity in Iressa responsiveness with less than half of the cell lines displaying significant growth inhibition by clinically relevant concentrations of the drug. Gefitinib responsiveness was found to be p27 kip1 dependent as DNA synthesis was restored following exposure to p27siRNA. Unfortunately, Iressa responsiveness was not closely linked to surface EGFR or TGF-α expression in the bladder cancer cells, however, cellular TGF-α expression correlated directly with Iressa sensitivity in the pancreatic cancer cell lines. These findings provide the potential for prospectively identifying patients with drug-sensitive tumors. ^ Further studies aimed at exploiting gefitinib-mediated cell cycle effects led us to investigate if gefitinib-mediated TRAIL sensitization correlated with increased p27kip1 accumulation. We observed that increased TRAIL sensitivity following gefitinib exposure was not dependent on p27 kip1 expression. Additional studies initiated to examine the role(s) of Akt and Erk signaling demonstrated that exposure to PI3K or MEK inhibitors significantly enhanced TRAIL-induced apoptosis at concentrations that block target phosphorylation. Furthermore, combinations of TRAIL and the PI3K or MEK inhibitors increased procaspase-8 processing above levels observed with TRAIL alone, indicating that the effects were exerted at the level of caspase-8 activation, considered the earliest step in the TRAIL pathway. ^

Maximizing efficacy of the hypomethylating drug 5-aza-2'-deoxycytidine in human leukemia

5-aza-2'-deoxycytidine (DAC) is a cytidine analogue that strongly inhibits DNA methylation, and was recently approved for the treatment of myelodysplastic syndromes (MDS). To maximize clinical results with DAC, we investigated its use as an anti-cancer drug. We also investigated mechanisms of resistance to DAC in vitro in cancer cell lines and in vivo in MDS patients after relapse. We found DAC sensitized cells to the effect of 1-β-D-Arabinofuranosylcytosine (Ara-C). The combination of DAC and Ara-C or Ara-C following DAC showed additive or synergistic effects on cell death in four human leukemia cell lines in vitro, but antagonism in terms of global methylation. RIL gene activation and H3 lys-9 acetylation of short interspersed elements (Alu). One possible explanation is that hypomethylated cells are sensitized to cell killing by Ara-C. Turning to resistance, we found that the IC50 of DAC differed 1000 fold among and was correlated with the dose of DAC that induced peak hypomethylation of long interspersed nuclear elements (LINE) (r=0.94, P<0.001), but not with LINE methylation at baseline (r=0.05, P=0.97). Sensitivity to DAC did not significantly correlate with sensitivity to another hypomethylating agent 5-azacytidine (AZA) (r=0.44, P=0.11). The cell lines most resistant to DAC had low dCK, hENT1, and hENT2 transporters and high cytosine deaminase (CDA). In an HL60 leukemia cell line, resistance to DAC could be rapidly induced by drug exposure, and was related to a switch from monoallelic to biallelic mutation of dCK or a loss of wild type DCK allele. Furthermore, we showed that DAC induced DNA breaks evidenced by histone H2AX phosphorylation and increased homologous recombination rates 7-10 folds. Finally, we found there were no dCK mutations in MDS patients after relapse. Cytogenetics showed that three of the patients acquired new abnormalities at relapse. These data suggest that in vitro spontaneous and acquired resistance to DAC can be explained by insufficient incorporation of drug into DNA. In vivo resistance to DAC is likely due to methylation-independent pathways such as chromosome changes. The lack of cross resistance between DAC and AZA is of potential clinical relevance, as is the combination of DAC and Ara-C. ^

ARD1 stabilization of TSC2 suppresses tumorigenesis via the mTOR signaling pathway

Mammalian target of rapamycin (mTOR) plays an important role in regulating various cellular functions, and the tuberous sclerosis 1 (TSC1)/TSC2 complex serves as a major repressor of the mTOR pathway. Here we demonstrated that arrest-defective protein 1 (ARD1) physically interacts with, acetylates, and stabilizes TSC2, thereby reducing mTOR activity. The inhibition of mTOR by ARD1 suppresses cell proliferation and increases autophagy, which further impairs tumorigenicity. Correlation between the levels of ARD1 and TSC2 was found in multiple tumor types, suggesting the physiological importance of ARD1 in stabilizing TSC2. Moreover, evaluation of loss of heterozygosity (LOH) at Xq28 revealed allelic loss in 31% of tested breast cancer cell lines and tumor samples. Together, our findings suggest that ARD1 functions as a negative regulator of the mTOR pathway and that dysregulation of the ARD1/TSC2/mTOR axis may contribute to cancer development. To further explore the signaling pathway of ARD1, we provided evidence showing the phosphorylation of ARD1 by IKKβ, which mediated the destabilization of ARD1. Future work may be needed to study the biological effect of this post-translational modification. ^

Statistical Methods for Differential Expressions of Genes Detected in Multiple-Condition Experiment of Microarray

Most studies of differential gene-expressions have been conducted between two given conditions. The two-condition experimental (TCE) approach is simple in that all genes detected display a common differential expression pattern responsive to a common two-condition difference. Therefore, the genes that are differentially expressed under the other conditions other than the given two conditions are undetectable with the TCE approach. In order to address the problem, we propose a new approach called multiple-condition experiment (MCE) without replication and develop corresponding statistical methods including inference of pairs of conditions for genes, new t-statistics, and a generalized multiple-testing method for any multiple-testing procedure via a control parameter C. We applied these statistical methods to analyze our real MCE data from breast cancer cell lines and found that 85 percent of gene-expression variations were caused by genotypic effects and genotype-ANAX1 overexpression interactions, which agrees well with our expected results. We also applied our methods to the adenoma dataset of Notterman et al. and identified 93 differentially expressed genes that could not be found in TCE. The MCE approach is a conceptual breakthrough in many aspects: (a) many conditions of interests can be conducted simultaneously; (b) study of association between differential expressions of genes and conditions becomes easy; (c) it can provide more precise information for molecular classification and diagnosis of tumors; (d) it can save lot of experimental resources and time for investigators.^

Regulation of Net1A subcellular localization by the small GTPase Rac1

Activation of Rho family small G proteins is thought to be a critical event in breast cancer development and metastatic progression. Rho protein activation is stimulated by a family of enzymes known as guanine nucleotide exchange factors (Rho GEFs). The neuroepithelioma transforming gene 1 (Net1) is a Rho GEF specific for the RhoA subfamily that is overexpressed in primary breast tumors and breast cancer cell lines. Net1 isoform expression is also required for migration and invasion of breast cancer cells in vitro. These data indicate that Net1 may be a critical regulator of metastatic progression in breast cancer. Net1 activity is negatively regulated by sequestration in the nucleus, and relocalization of Net1 outside the nucleus is required to stimulate RhoA activation, actin cytoskeletal reorganization, and oncogenic transformation. However, regulatory mechanisms controlling the extranuclear localization of Net1 have not been identified. In this study, we have addressed the regulation of Net1A isoform localization by Rac1. Specifically, co-expression of constitutively active Rac1 with Net1A stimulates the relocalization of Net1A from the nucleus to the plasma membrane in breast cancer cells, and results in Net1A activation. Importantly, Net1A localization is also driven by endogenous Rac1 activity. Net1A relocalizes outside the nucleus in cells spreading on collagen, and when endogenous Rac1 expression was silenced by siRNA, Net1A remained nuclear in spreading cells. These data indicate that Rac1 controls the localization of the Net1A isoform and suggests a physiological role for Net1A in breast cancer cell adhesion and motility.