Showing papers by "Ie Ming Shih published in 2012"

TP53 mutations in serous tubal intraepithelial carcinoma and concurrent pelvic high-grade serous carcinoma--evidence supporting the clonal relationship of the two lesions.

Abstract: The proposal that serous tubal intraepithelial carcinoma (STIC) is the precursor of ovarian high-grade serous carcinoma (HGSC) is based on several lines of investigation [1,2]. First, STICs are found in approximately 10–15% of Fallopian tubes removed prophylactically from women at high risk of developing ovarian carcinoma because of a germline BRCA mutation. Second, STICs are detected in 50–60% of cases of sporadic (without germline mutations of BRCA) ovarian, tubal,and so-called primary peritoneal HGSCs [3,4]. Third, STICs frequently up-regulate oncogene products, such as cyclin E1, Rsf-1, and fatty acid synthase, that are also overexpressed in HGSC [5]. Fourth, STICs have relatively shorter telomeres compared with concurrent ovarian HGSC, as occurs with precursor lesions in other sites. Finally, in a small series of five cases, STICs and concurrent ovarian HGSCs, the same TP53mutations were detected in STICs and HGSCs [12], indicating a potential clonal relationship. Besides exploiting the presence of TP53 mutations in STIC and HGSC as a method of showing a clonal relationship, detection of TP53 mutations in tissue specimens has utility in confirming the histological diagnosis of STIC since it has been reported that TP53 mutations occur in over 95% of ovarian HGSCs [6,7]. For histological diagnosis, however, detection of mutated TP53 is not practical and therefore immunohistochemical detection of p53 protein has been used as a surrogate marker. There have been only a few studies correlating p53 expression with TP53 mutation in ovarian HGSC [8–11] and none that we are aware of in STICs. Accordingly, we undertook the present study of STICs with concurrent pelvic HGSCs in order to (1) confirm a clonal relationship of STIC with HGSC in a relatively large series of cases, and (2) clarify the relationship of immunohistochemical expression of p53 protein with the mutational status of the TP53 gene.

A fluorescence light-up Ag nanocluster probe that discriminates single-nucleotide variants by emission color.

Abstract: Rapid and precise screening of small genetic variations, such as single-nucleotide polymorphisms (SNPs), among an individual’s genome is still an unmet challenge at point-of-care settings. One crucial step toward this goal is the development of discrimination probes that require no enzymatic reaction and are easy to use. Here we report a new type of fluorescent molecular probe, termed a chameleon NanoCluster Beacon (cNCB), that lights up into different colors upon binding SNP targets. NanoCluster Beacons (NCBs) are collections of a small number of Ag atoms templated on single-stranded DNA that fluoresce strongly when placed in proximity to particular DNA sequences, termed enhancers. Here we show the fluorescence emission color of a NCB can change substantially (a shift of 60–70 nm in the emission maximum) depending upon the alignment between the silver nanocluster and the DNA enhancer sequence. Chameleon NCBs exploit this color shift to directly detect SNPs, based on the fact that different SNPs produce a...

Identification of Molecular Pathway Aberrations in Uterine Serous Carcinoma by Genome-wide Analyses

Abstract: Background Uterine cancer is the fourth most common malignancy in women, and uterine serous carcinoma is the most aggressive subtype. However, the molecular pathogenesis of uterine serous carcinoma is largely unknown. We analyzed the genomes of uterine serous carcinoma samples to better understand the molecular genetic characteristics of this cancer.

Low-grade serous carcinomas of the ovary contain very few point mutations

Abstract: It has been well established that ovarian low-grade and high-grade serous carcinomas are fundamentally different types of tumours. While the molecular genetic features of ovarian high-grade serous carcinomas are now well known, the pathogenesis of low-grade serous carcinomas, apart from the recognition of frequent somatic mutations involving KRAS and BRAF, is largely unknown. In order to comprehensively analyse somatic mutations in low-grade serous carcinomas, we applied exome sequencing to the DNA of eight samples of affinity-purified, low-grade, serous carcinomas. A remarkably small number of mutations were identified in seven of these tumours: a total of 70 somatic mutations in 64 genes. The eighth case displayed mixed serous and endometrioid features and a mutator phenotype with 783 somatic mutations, including a nonsense mutation in the mismatch repair gene, MSH2. We validated representative mutations in an additional nine low-grade serous carcinomas and 10 serous borderline tumours, the precursors of ovarian low-grade, serous carcinomas. Overall, the genes showing the most frequent mutations were BRAF and KRAS, occurring in 10 (38%) and 5 (19%) of 27 low-grade tumours, respectively. Except for a single case with a PIK3CA mutation, other mutations identified in the discovery set were not detected in the validation set of specimens. Our mutational analysis demonstrates that point mutations are much less common in low-grade serous tumours of the ovary than in other adult tumours, a finding with interesting scientific and clinical implications.

Loss of ARID1A expression is an early molecular event in tumor progression from ovarian endometriotic cyst to clear cell and endometrioid carcinoma.

Abstract: Objectives ARID1A is a recently identified tumor suppressor participating in chromatin remodeling Somatic inactivating mutations of ARID1A and loss of its expression occur frequently in ovarian clear cell and endometrioid carcinomas and in uterine endometrioid carcinomas Because endometriotic epithelium is thought to be the cell of origin of most ovarian clear cell and endometrioid carcinomas, we undertook an analysis of ARID1A expression of these tumors arising within an endometriotic cyst (endometrioma) Materials and Methods Our immunohistochemical study set consisted of 47 endometriotic cysts containing clear cell carcinoma in 24 cases, well-differentiated ovarian endometrioid carcinoma in 20 cases, and mixed clear cell and endometrioid carcinoma in 3 cases Results ARID1A loss was observed in 31 (66%) of 47 carcinomas; and therefore, these cases were informative for determining the temporal sequence of loss of ARID1A expression in tumor progression In 16 of the 47 cases, ARID1A immunoreactivity was retained in both the endometriotic cyst and the carcinoma; and thus, these cases were not informative All of the 31 informative cases showed loss of ARID1A immunoreactivity in the carcinoma and in the endometriotic cyst epithelium in direct continuity with the carcinoma but not in the cyst epithelium that was not adjacent to the tumor Conclusions Loss of ARID1A function as shown by loss of expression, presumably due to mutations, is an early molecular event in the development of most ovarian clear cell and endometrioid carcinomas arising in endometriomas

Validation of an algorithm for the diagnosis of serous tubal intraepithelial carcinoma

Abstract: It has been reported that the diagnosis of serous tubal intraepithelial carcinoma (STIC) is not optimally reproducible based only on histologic assessment. Recently, we reported that the use of a diagnostic algorithm that combines histologic features and coordinate immunohistochemical expression of p53 and Ki-67 substantially improves reproducibility of the diagnosis. The goal of the current study was to validate this algorithm by testing a group of 6 gynecologic pathologists who had not participated in the development of the algorithm (3 faculty, 3 fellows) but who were trained in its use by referring to a website designed for that purpose. They then reviewed a set of microscopic slides, which contained 41 mucosal lesions of the fallopian tube. Overall consensus (≥4 of 6 pathologists) for the 4 categories of STIC, serous tubal intraepithelial lesion (our atypical intermediate category), p53 signature, and normal/reactive was achieved in 76% of lesions with no consensus in 24%. Combining diagnoses into 2 categories (STIC vs. non-STIC) resulted in overall consensus in 93% with no consensus in 7%. The kappa value for STIC vs. non-STIC among all 6 observers was also high at 0.67 and did not significantly differ whether for faculty (κ=0.66) or fellows (κ=0.60). These findings confirm the reproducibility of this algorithm by a group of gynecologic pathologists who were trained on a website for that purpose. Accordingly, we recommend its use in research studies. Before applying it in routine clinical practice, the algorithm should be evaluated by general surgical pathologists in the community setting.

NAC1 modulates sensitivity of ovarian cancer cells to cisplatin by altering the HMGB1-mediated autophagic response

Abstract: Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, is known to have important roles in proliferation and growth of tumor cells and in chemotherapy resistance. Yet, the mechanisms underlying how NAC1 contributes to drug resistance remain largely unclear. We report here that autophagy was involved in NAC1-mediated resistance to cisplatin, a commonly used chemotherapeutic drug in the treatment of ovarian cancer. We found that treatment with cisplatin caused an activation of autophagy in ovarian cancer cell lines, A2780, OVCAR3 and SKOV3. We further demonstrated that knockdown of NAC1 by RNA interference or inactivation of NAC1 by inducing the expression of a NAC1 deletion mutant that contains only the BTB/POZ domain significantly inhibited the cisplatin-induced autophagy, resulting in increased cisplatin cytotoxicity. Moreover, inhibition of autophagy and sensitization to cisplatin by NAC1 knockdown or inactivation were accompanied by induction of apoptosis. To confirm that the sensitizing effect of NAC1 inhibition on the cytotoxicity of cisplatin was attributed to suppression of autophagy, we assessed the effects of the autophagy inhibitors 3-methyladenosine and chloroquine, and small interfering RNAs (siRNAs) targeting beclin 1 or Atg5 on the cytotoxicity of cisplatin. Treatment with 3-methyladenosine, chloroquine or beclin 1 and Atg5-targeted siRNA also enhanced the sensitivity of SKOV3, A2780 and OVCAR3 cells to cisplatin, indicating that suppression of autophagy indeed renders tumor cells more sensitive to cisplatin. Regulation of autophagy by NAC1 was mediated by the high-mobility group box 1 (HMGB1), as the functional status of NAC1 was associated with the expression, translocation and release of HMGB1. The results of our study not only revealed a new mechanism determining cisplatin sensitivity but also identified NAC1 as a novel regulator of autophagy. Thus, the NAC1-mediated autophagy may be exploited as a new target for enhancing the efficacy of cisplatin against ovarian cancer and other types of malignancies.

Ovarian Cancer Is an Imported Disease: Fact or Fiction?

Abstract: The cell of origin of ovarian cancer has been long debated. The current paradigm is that epithelial ovarian cancer (EOC) arises from the ovarian surface epithelium (OSE). OSE is composed of flat, nondescript cells more closely resembling the mesothelium lining the peritoneal cavity, with which it is continuous, rather than the various histologic types of ovarian carcinoma (serous, endometrioid, and clear cell carcinoma), which have a Mullerian phenotype. Accordingly, it has been argued that the OSE undergoes a process termed "metaplasia" to account for this profound morphologic transformation. Recent molecular and clinicopathologic studies not only have failed to support this hypothesis but also have provided evidence that EOC stems from Mullerian-derived extraovarian cells that involve the ovary secondarily, thereby calling into question the very existence of primary EOC. This new model of ovarian carcinogenesis proposes that fallopian tube epithelium (benign or malignant) implants on the ovary to give rise to both high-grade and low-grade serous carcinomas, and that endometrial tissue implants on the ovary and produces endometriosis, which can undergo malignant transformation into endometrioid and clear cell carcinoma. Thus, ultimately EOC is not ovarian in origin but rather is secondary, and it is logical to conclude that the only true primary ovarian neoplasms are germ cell and gonadal stromal tumors analogous to tumors in the testis. If this new model is confirmed, it has profound implications for the early detection and treatment of "ovarian cancer."

Endocervical-type Mucinous Borderline Tumors are Related to Endometrioid Tumors Based on Mutation and Loss of Expression of ARID1A

Abstract: Nongastrointestinal-type mucinous borderline tumors have been described as displaying endocervical and serous differentiation and hence have been termed "endocervical-type" mucinous borderline tumors, "mixed-epithelial papillary cystadenoma of borderline malignancy of mullerian type," or "atypical proliferative seromucinous tumors." A striking feature of these tumors is their frequent association with endometriosis, which has been reported in a third to a half of cases. This is an unusual finding, as pure endocervical and serous tumors are not usually associated with endometriosis. ARID1A is a recently identified tumor suppressor, which frequently loses its expression and is mutated in endometrium-related carcinomas including ovarian clear cell, ovarian endometrioid, and uterine endometrioid carcinomas. Although ARID1A mutations and their expression have been studied in gynecologic cancer, the expression pattern of ARID1A has not been investigated in ovarian atypical proliferative (borderline) tumors. In this study, we analyzed ARID1A expression in serous, gastrointestinal-type and endocervical-type (seromucinous) mucinous, and endometrioid atypical proliferative (borderline) tumors using immunohistochemistry and performed mutational analysis in selected cases. We observed loss of ARID1A staining in 8 (33%) of 24 seromucinous tumors. In contrast, ARID1A staining was retained in all the other 32 tumors except in 1 endometrioid tumor (P<0.01). Mutational analysis was performed on 2 representative seromucinous tumors, which showed complete loss of ARID1A. Both tumors harbored somatic inactivating ARID1A mutations. Previous studies have reported loss of expression and/or mutation of ARID1A in 30% to 57% of endometrioid and clear cell carcinomas but only rarely in serous tumors. In summary, these tumors often contain endocervical-type mucinous epithelium, but they typically display papillary architecture, unlike most endocervical neoplasms, and their immunophenotype is different from both endocervical and serous tumors. Moreover, they frequently contain ciliated cells, endometrial-type cells, cells with abundant eosinophilic cytoplasm, and hobnail-shaped cells, all of which can be found in endometrioid tumors. The loss of expression of ARID1A and the presence of inactivating mutations of the ARID1A gene further link this tumor to endometrioid and clear cell tumors, as does the frequent association with endometriosis. Accordingly, we suggest designating these tumors "atypical proliferative (borderline) papillary mullerian tumors" as this designation more accurately reflects their clinicopathologic, immunohistochemical, and molecular genetic features.

Defining NOTCH3 Target Genes in Ovarian Cancer

Abstract: NOTCH3 gene amplification plays an important role in the progression of many ovarian and breast cancers, but the targets of NOTCH3 signaling are unclear. Here we report the use of an integrated systems biology approach to identify direct target genes for NOTCH3. Transcriptome analysis showed that suppression of NOTCH signaling in ovarian and breast cancer cells led to downregulation of genes in pathways involved in cell cycle regulation and nucleotide metabolism. ChIP-on-chip analysis defined promoter target sequences, including a new CSL binding motif (N1) in addition to the canonical CSL binding motif, that were occupied by the NOTCH3/CSL transcription complex. Integration of transcriptome and ChIP-on-chip data demonstrated that the ChIP target genes overlapped significantly with the NOTCH-regulated transcriptome in ovarian cancer cells. From the set of genes identified we determined that the mitotic apparatus organizing protein DLGAP5 (HURP/DLG7) was a critical target. Both the N1 motif and the canonical CSL binding motif were essential to activate DLGAP5 transcription. DLGAP5 silencing in cancer cells suppressed tumorigenicity and inhibited cellular proliferation by arresting the cell cycle at the G2/M phase. In contrast, enforced expression of DLGAP5 partially counteracted the growth inhibitory effects of a pharmacological or RNAi-mediated inhibition in cancer cells. Our findings define direct target genes of NOTCH3 and highlight DLGAP5 in the tumor-promoting function of NOTCH3.

Prognostic and therapeutic impact of the chromosome 20q13.2 ZNF217 locus amplification in ovarian clear cell carcinoma.

Abstract: BACKGROUND: The goal of this study was to examine the clinical significance of ZNF217 amplification and assess whether ZNF217 could be a potential therapeutic target in ovarian clear cell carcinoma (OCCC). METHODS: ZNF217 expression and amplification in OCCC was assessed by immunohistochemistry, fluorescence in situ hybridization, and clinical data collected via a retrospective chart review. ZNF217 gene knockdown using silencing RNA (siRNA) was used to assess ZNF217 functions in OCCC cell lines. RESULTS: Gene amplification was identified in 12 of 60 (20.0%) OCCCs. ZNF217 copy number correlated significantly with ZNF217 protein expression (r = 0.341; P<.01). ZNF217 amplification correlated significantly with shorter progression-free (P = .0042) and overall (P = .0199) survival. There were nonsignificant trends between high ZNF217 protein expression and poor progression-free (P = .2594) and overall (P = .2199) survival. Multivariate analysis revealed ZNF217 gene amplification to be an independent prognostic factor for progression-free and overall survival after standard platinum agent-based chemotherapy (P = .0339 and P = .031, respectively). Profound growth inhibition and apoptosis were observed in ZNF217 siRNA-treated cancer cells with gene amplification compared with cancer cells with ZNF217 moderate expression without ZNF217 gene amplification or with low ZNF217 expression. CONCLUSION: These findings indicate that ZNF217 overexpression is critical to growth and survival of OCCCs with ZNF217 gene amplification. Furthermore, they suggest that ZNF217 siRNA-induced phenotypes depend on amplification status of OCCCs. Therefore, ZNF217-targeted therapy may benefit OCCC patients with ZNF217 amplification. Cancer 2011. © 2011 American Cancer Society.

The diagnostic and biological implications of laminin expression in serous tubal intraepithelial carcinoma.

Abstract: There is compelling evidence to suggest that seroustubal intraepithelial carcinoma (STIC) is the likely primary sitefor the development of many pelvic high-grade serous carcino-mas (HGSCs). Identifying molecules that are upregulated inSTIC is important not only to provide biomarkers to assist inthe diagnosis of STIC but also to elucidate our understanding ofthe pathogenesis of HGSC. In this study, we performed RNAsequencing to compare transcriptomes between HGSC and nor-mal fallopian tube epithelium (FTE), and we identified LAMC1encoding laminin g1 as one of the preferentially upregulated genesassociated with HGSC. Reverse transcription polymerase chainreaction further validated LAMC1 upregulation in HGSC ascompared with normal FTE. Immunohistochemical analysis wasperformed on 32 cases of concurrent HGSC and STIC. The latterwas diagnosed on the basis of morphology, TP53 mutations, andp53 and Ki-67 immunohistochemical patterns. Laminin g1im-munostaining intensity was found to be significantly higher inSTIC and HGSC compared with adjacent FTE in all cases(P<0.001). In normal FTE, laminin g1 immunoreactivity waspredominantly localized in the basement membrane or on theapical surface of ciliated cells, whereas in STIC and HGSC cells,laminin g1 staining was diffuse and intense throughout the cyto-plasm. More importantly, strong laminin g1 staining was detectedin all 13 STICs, which lacked p53 immunoreactivity because ofnull mutations. These findings suggest that the overexpression oflaminin g1 immunoreactivity and alteration of its staining patternin STICs can serve as a useful tissue biomarker, especially forthose STICs that are negative for p53 and have a low Ki-67 la-beling index.Key Words: ovarian cancer, serous tubal intraepithelial carci-noma, STIC, laminin, LAMC1(Am J Surg Pathol 2012;36:1826–1834)

Ki-67 labeling index as an adjunct in the diagnosis of serous tubal intraepithelial carcinoma.

Abstract: There is mounting evidence that serous tubal intraepithelial carcinoma (STIC) is the immediate precursor of ovarian high-grade serous carcinoma (HGSC) (1–4) but the criteria for its diagnosis are not well established. A recent study, in fact, showed that interobserver reproducibility, even among expert gynecologic pathologists, was moderate at best (5). As more women with a family history of ovarian carcinoma undergo risk-reducing salpingo oophorectomy and as the SEE-FIM protocol, which allows more thorough evaluation of the fallopian tubes (6), becomes more widely used, this problem will become increasingly important. Pathologists will encounter not only STICs but other tubal abnormalities that may either be precursors of STICs or benign mimickers. In view of the difficulties in establishing a diagnosis on the basis of morphology alone, we have undertaken a number of studies to determine the most sensitive and specific methods of diagnosing STICs. We first investigated alterations in p53, because somatic mutation of TP53 is the most common molecular genetic change of HGSC. TP53 mutational status was correlated with p53 immunoreactivity and clinicopathologic characteristics (7). We found that STICs harbor TP53 mutations in the majority of the cases (91.3%) and these were the identical mutations found in the associated HGSC. Positive immunostaining for p53, however, depended on whether the mutations were missense mutations that resulted in strong staining or nonsense mutations that resulted in a truncated protein not recognized by the p53 antibody and therefore there was complete absence of staining. For these latter cases, in particular, we reasoned that the Ki-67 labeling index might be a useful adjunct to morphology and p53 immunostaining, because Mib1 staining would provide a sensitive and potentially quantifiable method of assessing proliferation in these lesions. Some previous studies have reported an increase in proliferation in STIC (range from 10% to 95%) compared with epithelium of the normal fallopian tube, but the range of the index in normal fallopian tube epithelium (FTE) has not been systematically evaluated so far (8,9). We therefore carried out the present study to determine the baseline Ki-67 proliferation index using Ki-67 staining in normal FTE compared with STICs.

Identification of PBX1 Target Genes in Cancer Cells by Global Mapping of PBX1 Binding Sites

Abstract: PBX1 is a TALE homeodomain transcription factor involved in organogenesis and tumorigenesis. Although it has been shown that ovarian, breast, and melanoma cancer cells depend on PBX1 for cell growth and survival, the molecular mechanism of how PBX1 promotes tumorigenesis remains unclear. Here, we applied an integrated approach by overlapping PBX1 ChIP-chip targets with the PBX1-regulated transcriptome in ovarian cancer cells to identify genes whose transcription was directly regulated by PBX1. We further determined if PBX1 target genes identified in ovarian cancer cells were co-overexpressed with PBX1 in carcinoma tissues. By analyzing TCGA gene expression microarray datasets from ovarian serous carcinomas, we found co-upregulation of PBX1 and a significant number of its direct target genes. Among the PBX1 target genes, a homeodomain protein MEOX1 whose DNA binding motif was enriched in PBX1-immunoprecipicated DNA sequences was selected for functional analysis. We demonstrated that MEOX1 protein interacts with PBX1 protein and inhibition of MEOX1 yields a similar growth inhibitory phenotype as PBX1 suppression. Furthermore, ectopically expressed MEOX1 functionally rescued the PBX1-withdrawn effect, suggesting MEOX1 mediates the cellular growth signal of PBX1. These results demonstrate that MEOX1 is a critical target gene and cofactor of PBX1 in ovarian cancers.

Mutant BRAF Induces DNA Strand Breaks, Activates DNA Damage Response Pathway, and Up-Regulates Glucose Transporter-1 in Nontransformed Epithelial Cells

Abstract: Although the oncogenic functions of activating BRAF mutations have been clearly demonstrated in human cancer, their roles in nontransformed epithelial cells remain largely unclear. Investigating the cellular response to the expression of mutant BRAF in nontransformed epithelial cells is fundamental to the understanding of the roles of BRAF in cancer pathogenesis. In this study, we used two nontransformed cyst108 and RK3E epithelial cell lines as models in which to compare the phenotypes of cells expressing BRAF WT and BRAF V600E . We found that transfection of the BRAF V600E , but not the BRAF WT , expression vector suppressed cellular proliferation and induced apoptosis in both cell types. BRAF V600E generated reactive oxygen species, induced DNA double-strand breaks, and caused subsequent DNA damage response as evidenced by an increased number of pCHK2 and γH2AX nuclear foci as well as the up-regulation of pCHK2, p53, and p21. Because BRAF and KRAS (alias Ki-ras) mutations have been correlated with GLUT1 up-regulation, which encodes glucose transporter-1, we demonstrated here that expression of BRAF V600E , but not BRAF WT , was sufficient to up-regulate GLUT1. Taken together, our findings provide new insights into mutant BRAF-induced oncogenic stress that is manifested by DNA damage and growth arrest by activating the pCHK2-p53-p21 pathway in nontransformed cells, while it also confers tumor-promoting phenotypes such as the up-regulation of GLUT1 that contributes to enhanced glucose metabolism that characterizes tumor cells.

Genome-wide identification of significant aberrations in cancer genome

Abstract: Somatic Copy Number Alterations (CNAs) in human genomes are present in almost all human cancers. Systematic efforts to characterize such structural variants must effectively distinguish significant consensus events from random background aberrations. Here we introduce Significant Aberration in Cancer (SAIC), a new method for characterizing and assessing the statistical significance of recurrent CNA units. Three main features of SAIC include: (1) exploiting the intrinsic correlation among consecutive probes to assign a score to each CNA unit instead of single probes; (2) performing permutations on CNA units that preserve correlations inherent in the copy number data; and (3) iteratively detecting Significant Copy Number Aberrations (SCAs) and estimating an unbiased null distribution by applying an SCA-exclusive permutation scheme. We test and compare the performance of SAIC against four peer methods (GISTIC, STAC, KC-SMART, CMDS) on a large number of simulation datasets. Experimental results show that SAIC outperforms peer methods in terms of larger area under the Receiver Operating Characteristics curve and increased detection power. We then apply SAIC to analyze structural genomic aberrations acquired in four real cancer genome-wide copy number data sets (ovarian cancer, metastatic prostate cancer, lung adenocarcinoma, glioblastoma). When compared with previously reported results, SAIC successfully identifies most SCAs known to be of biological significance and associated with oncogenes (e.g., KRAS, CCNE1, and MYC) or tumor suppressor genes (e.g., CDKN2A/B). Furthermore, SAIC identifies a number of novel SCAs in these copy number data that encompass tumor related genes and may warrant further studies. Supported by a well-grounded theoretical framework, SAIC has been developed and used to identify SCAs in various cancer copy number data sets, providing useful information to study the landscape of cancer genomes. Open–source and platform-independent SAIC software is implemented using C++, together with R scripts for data formatting and Perl scripts for user interfacing, and it is easy to install and efficient to use. The source code and documentation are freely available at http://www.cbil.ece.vt.edu/software.htm .

Dysfunction of Nucleus Accumbens-1 Activates Cellular Senescence and Inhibits Tumor Cell Proliferation and Oncogenesis

Abstract: Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, has emerging roles in cancer. We report here that NAC1 acts as a negative regulator of cellular senescence in transformed and nontransformed cells, and dysfunction of NAC1 induces senescence and inhibits its oncogenic potential. We show that NAC1 deficiency markedly activates senescence and inhibits proliferation in tumor cells treated with sublethal doses of γ-irradiation. In mouse embryonic fibroblasts from NAC1 knockout mice, following infection with a Ras virus, NAC1−/− cells undergo significantly more senescence and are either nontransformed or less transformed in vitro and less tumorigenic in vivo when compared with NAC1+/+ cells. Furthermore, we show that the NAC1-caused senescence blunting is mediated by ΔNp63, which exerts its effect on senescence through p21, and that NAC1 activates transcription of ΔNp63 under stressful conditions. Our results not only reveal a previously unrecognized function of NAC1, the molecular pathway involved and its impact on pathogenesis of tumor initiation and development, but also identify a novel senescence regulator that may be exploited as a potential target for cancer prevention and treatment. Cancer Res; 72(16); 4262–75. ©2012 AACR.

NAC1 Is an Actin-Binding Protein That Is Essential for Effective Cytokinesis in Cancer Cells

Abstract: NAC1 is a transcriptional co-repressor protein that is essential to sustain cancer cell proliferation and migration. However, the underlying molecular mechanisms of NAC1 function in cancer cells remain unknown. In this study, we show that NAC1 functions as an actin monomer binding protein. The conserved BTB protein interaction domain in NAC1 is the minimal region for actin binding. Disrupting NAC1 complex function by dominant negative or siRNA strategies reduced cell retraction and abscission during late stage cytokinesis, causing multinucleation in cancer cells. In Nac1-deficient murine fibroblasts, restoring NAC1 expression was sufficient to partially avert multinucleation. We found that siRNA-mediated silencing of the actin binding protein profilin-1 in cancer cells caused a similar multinucleation phenotype and that NAC1 modulated the binding of actin to profillin-1. Taken together, our results indicate that the NAC1/actin/profilin-1 complex is crucial for cancer cell cytokinesis, with a variety of important biological and clinical implications.

The Role of Forkhead Box Q1 Transcription Factor in Ovarian Epithelial Carcinomas

Abstract: The role of the forkhead box Q1 (FOXQ1) transcription factor in cancer pathogenesis has recently emerged. Overexpression of FOXQ1 has been found in a variety of human cancers, and its upregulation has been associated with poor prognosis in colorectal, breast, and non-small cell lung carcinomas. However, the molecular mechanism underlying how FOXQ1 contributes to ovarian epithelial carcinomas remains unclear. To this end, we analyzed gene expression levels in ovarian cancer tissues and cell lines and demonstrated a higher expression level of FOXQ1 in epithelial ovarian cancer cells than that in normal epithelial cells. We then used a human ovarian cancer cell line, SKOV3, which expressed a higher level of FOXQ1, as a cell model to investigate the biological effects of FOXQ1 by using RNA interference. Silencing of FOXQ1 expression using a shRNA knockdown approach affected the expression of several cell cycle regulators, leading to suppressed cell proliferation, reduced cell motility/invasion, and upregulation of epithelial cell markers and the downregulation of mesenchymal cell markers. Taken together, these results suggest that FOXQ1 expression is essential to maintain cell proliferation, motility/invasion, and epithelial-mesenchymal transition phenotypes in ovarian cancer cells.

Mapping DNA quantity into electrophoretic mobility through quantum dot nanotethers for high-resolution genetic and epigenetic analysis.

Abstract: Newly discovered nanoparticle properties have driven the development of novel applications and uses We report a new observation where the electrophoretic mobility of a quantum dot/DNA nanoassembly can be precisely modulated by the degree of surface DNA conjugation By using streptavidin-coated quantum dots (QDs) as nanotethers to gather biotin-labeled DNA into electrophoretic nanoassemblies, the QD surface charge is modulated and transformed into electrophoretic mobility shifts using standard agarose gel electrophoresis Typical fluorescent assays quantify based on relative intensity However, this phenomenon uses a novel approach that accurately maps DNA quantity into shifts in relative band position This property was applied in a QD-enabled nanoassay called quantum dot electrophoretic mobility shift assay (QEMSA) that enables accurate quantification of DNA targets down to 11-fold (9%) changes in quantity, beyond what is achievable in qPCR In addition to these experimental findings, an analytical model is presented to explain this behavior Finally, QEMSA was applied to both genetic and epigenetic analysis of cancer First, it was used to analyze copy number variation (CNV) of the RSF1/HBXAP gene, where conventional approaches for CNV analysis based on comparative genomic hybridization (CGH), microarrays, and qPCR are unable to reliably differentiate less than 2-fold changes in copy number Then, QEMSA was used for DNA methylation analysis of the p16/CDK2A tumor suppressor gene, where its ability to detect subtle changes in methylation was shown to be superior to that of qPCR

DNA Damage Response is Prominent in Ovarian High-Grade Serous Carcinomas, Especially Those with Rsf-1 (HBXAP) Overexpression.

Abstract: DNA damage commonly occurs in cancer cells as a result of endogenous and tumor microenvironmental stress. In this study, we applied immunohistochemistry to study the expression of phosphorylated Chk2 (pChk2), a surrogate marker of the DNA damage response, in high grade and low grade of ovarian serous carcinoma. A phospho-specific antibody specific for threonine 68 of Chk2 was used for immunohistochemistry on a total of 292 ovarian carcinoma tissues including 250 high-grade and 42 low-grade serous carcinomas. Immunostaining intensity was correlated with clinicopathological features. We found that there was a significant correlation between pChk2 immunostaining intensity and percentage of pChk2 positive cells in tumors and demonstrated that high-grade serous carcinomas expressed an elevated level of pChk2 as compared to low-grade serous carcinomas. Normal ovarian, fallopian tube, ovarian cyst, and serous borderline tumors did not show detectable pChk2 immunoreactivity. There was no significant difference in pChk2 immunoreactivity between primary and recurrent high-grade serous carcinomas. In high-grade serous carcinomas, a significant correlation (P < 0.0001) in expression level (both in intensity and percentage) was found between pChk2 and Rsf-1 (HBXAP), a gene involved in chromatin remodeling that is amplified in high-grade serous carcinoma. Our results suggest that the DNA damage response is common in high-grade ovarian serous carcinomas, especially those with Rsf-1 overexpression, suggesting that Rsf-1 may be associated with DNA damage response in high-grade serous carcinomas.

Pathogenesis and New Therapeutic Targets of Ovarian Cancer

Abstract: Ovarian cancer remains the most lethal gynecologic malignancy, largely due to the lack of early detection tools and effective therapeutic interventions. Anti-tumor agents targeting critical molecular pathways hold promise for improving survival in these patients and understanding the critical molecular pathways involved in the pathogenesis of ovarian cancer development is central to the development of such agents. For example, using genome-wide DNA copy number analysis, investigators have identified amplification in a genomic locus (ch11q13.5) harboring a chromatin remodeling gene, RSF1, encoding Rsf-1 in high-grade ovarian serous carcinomas. Recent studies have shown that excessive Rsf-1 expression attributes to genomic instability and alters gene expression profiles to favor tumor growth and survival, especially in the presence of cytotoxic agents. The article entitled “DNA damage response is prominent in ovarian high-grade serous carcinomas, especially those with Rsf-1 (HBXAP) overexpression” reported by M. Kushirsagar et al. in this special issue provides new evidence that Rsf-1 overexpression was correlated with DNA damages which was observed more frequently in high-grade ovarian serous carcinoma. This finding should have several biological and clinical implications for the future studies of Rsf-1 in human cancer.

Detecting aberrant signal transduction pathways from high-throughput data using GIST algorithm

Abstract: It is biologically important to integrate high-throughput data to identify aberrant signal transduction pathways in cancer research. The high-throughput data acquired from The Cancer Genome Atlas (TCGA) Project offer a comprehensive picture of the genomic and transcriptional changes across hundreds of tumor samples. In this paper we propose a novel method, namely Gibbs sampler to Infer Signal Transduction pathways (GIST), to detect aberrant pathways that are highly associated with biological phenotypes or clinical information. GIST endeavors to estimate the edge probability by using a Markov Chain Monte Carlo (MCMC) method (i.e., a Gibbs sampling strategy). Through the sampling process, GIST is able to infer the correct signal transduction direction because the sampled edge probabilities are jointly determined by gene expression data and network topology. We first tested the efficacy of the GIST algorithm on yeast data and successfully uncovered several biologically meaningful signaling pathways. A case study on TCGA ovarian cancer data was further designed, aiming to unravel diverse signaling pathways associated with the development of ovarian cancer. The experimental results demonstrated the feasibility of applying GIST to identify and prioritize important signaling pathways in ovarian cancer for further biological validation.

Abstract 5536:TP53andMED12mutations in uterine smooth muscle tumors

Abstract: Most uterine smooth muscle tumors can be broadly subdivided into two groups: benign leiomyoma (including variant subtypes) and leiomyosarcoma based on cytologic atypia, mitotic activity and coagulative tumor cell necrosis. A small number defies classification into unequivocally benign and unequivocally malignant categories. These tumors, classified as “atypical leiomyoma” and “STUMP” (Smooth muscle Tumors of Uncertain Malignant Potential), generally behave in a benign fashion. Although p53 overexpression, as determined by immunohistochemical staining, has been reported in the atypical group and the leiomyosarcomas, it is uncertain whether they harbor TP53 mutations. In addition, a recent study has reported mutations in the mediator complex 12 (MED12) gene in the majority of benign uterine leiomyomas. We therefore performed a mutational analysis of uterine smooth muscle tumors to determine their TP53 and MED12 mutation status, in order to better understand their molecular genetic characteristics. A total of 9 uterine conventional leiomyomas, 27 atypical leiomyomas and 12 leiomyosarcomas from 40 patients were analyzed. DNA samples were sequenced and TP53 (from exons 4 to 9) and MED12 (exon 2) mutations were analyzed. Clinico-pathological data were collected. TP53 mutations were detected in 17 (63%) of 27 atypical leiomyomas and 8 (67%) of 12 leiomyosarcomas. MED12 mutations were identified in 6 (67%) of 9 conventional leiomyomas, 7 (26%) of 27 atypical leiomyomas and 1 (8%) of 12 leiomyosarcomas. Follow-up was available in 30 patients. None of the patients with atypical leiomyoma had recurrence or died of her disease (average follow-up 23 months). Seven patients with leiomyosarcoma, 4 of whom had TP53 mutations, died of their disease (average follow-up 42 months). The preliminary findings of this study indicate that the presence of TP53 mutations in uterine smooth muscle tumors is not sufficient to drive malignant behavior. Moreover, based on the presence of a MED12 mutation in one case of leiomyosarcoma, we postulate that at least some leiomyosarcomas develop from benign leiomyomas. Future correlated molecular genetic and clinicopathologic studies with a larger number of cases and long-term follow-up information are necessary to confirm this important finding. 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 5536. doi:1538-7445.AM2012-5536

The Role of Forkhead Box Q1 Transcription Factor in Ovarian

Abstract: The role of the forkhead box Q1 (FOXQ1) transcription factor in cancer pathogenesis has recently emerged. Overexpression of FOXQ1 has been found in a variety of human cancers, and its upregulation has been associated with poor prognosis in colorectal, breast, and non-small cell lung carcinomas. However, the molecular mechanism underlying how FOXQ1 contributes to ovarian epithelial carcinomas remains unclear. To this end, we analyzed gene expression levels in ovarian cancer tissues and cell lines and demonstrated a higher expression level of FOXQ1 in epithelial ovarian cancer cells than that in normal epithelial cells. We then used a human ovarian cancer cell line, SKOV3, which expressed a higher level of FOXQ1, as a cell model to investigate the biological effects of FOXQ1 by using RNA interference. Silencing of FOXQ1 expression using a shRNA knockdown approach affected the expression of several cell cycle regulators, leading to suppressed cell proliferation, reduced cell motility/invasion, and upregulation of epithelial cell markers and the downregulation of mesenchymal cell markers. Taken together, these results suggest that FOXQ1 expression is essential to maintain cell proliferation, motility/invasion, and epithelial-mesenchymal transition phenotypes in ovarian cancer cells.

Accurate identification of significant aberrations in contaminated cancer genome

Abstract: Somatic Copy Number Alterations (CNAs) are quite common in human cancers. Identifying CNAs and Significant Copy number Aberrations (SCAs) in cancer genomes is a critical task in searching for cancer-associated genes. The advanced genomic technologies, such as SNP array technology, facilitate copy number study at a genome-wide scale with high resolution. However, in reality, due to normal tissue contamination, the observed intensity signals are actually the mixture of copy number signals contributed from both tumor cells and normal cells. This genetic heterogeneity could significantly affect the subsequent copy number analysis and SCAs detection. In order to accurately identify significant aberrations in contaminated cancer genome, we devise an approach including two major steps. We first use a statistical method, Bayesian Analysis of Copy number Mixtures (BACOM) to estimate the normal tissue contamination fraction and recover the “true” copy number profile. Then, based on the recovered profiles, we detect SCAs using Genome-wide Identification of Significant Aberrations in Cancer Genome (SAIC). We comprehensively evaluate the performance of the proposed algorithm on a large number of simulation data. The results show that the algorithm has higher detection power than peer methods including the most popular GISTIC. We then apply the method to the real copy number data of Glioblastoma Multiforme and successfully identified majority of SCAs reported by GISTIC, and some novel SCAs that contain some cancer-associated genes.