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1.  Pathogenesis and New Therapeutic Targets of Ovarian Cancer 
Journal of Oncology  2012;2012:867512.
PMCID: PMC3437294  PMID: 22969800
2.  Loss of ARID1A Expression Correlates with Stages of Tumor Progression in Uterine Endometrioid Carcinoma 
ARID1A is a recently identified tumor suppressor that functions in chromatin remodeling. Inactivating mutations of ARID1A and loss of its expression most frequently occur in ovarian clear cell carcinoma, ovarian endometrioid carcinoma, and uterine endometrioid carcinoma. In this study, we performed a detailed immunostaining analysis of ARID1A in 246 cases including benign endometrium and endometrioid carcinoma at different stages of progression. Special attention was paid to recording intratumoral heterogeneity of clonal loss of ARID1A immunoreactivity. All normal endometria (n= 51) and endometrial polyps (n= 14) retained ARID1A expression. Among complex atypical hyperplasia (n= 38), 16% exhibited clonal loss of ARID1A, but none showed complete loss. Among low-grade endometrioid carcinomas (n= 88), 25% exhibited complete loss and 24% exhibited clonal loss. In contrast, 44% of high-grade endometrioid carcinomas (n= 55) showed complete loss of ARID1A and 9% exhibited clonal loss. We found that 19 high-grade carcinomas also contained concurrent low-grade carcinomas. In the high-grade areas, 63% exhibited complete loss and 11% exhibited clonal loss, whereas in the low-grade areas, 37% exhibited complete loss and 42% clonal loss. In 5 of these 19 cases, progressive loss of ARID1A from retention or clonal loss to complete loss was observed between the low-grade and high-grade areas. Overall, the percentage of complete ARID1A loss increased from 0% in complex atypical hyperplasia, to 25% in low-grade endometrioid carcinoma, to 44% in high-grade endometrioid carcinoma. These findings suggest that loss of ARID1A expression, presumably due to mutation, plays an important role in tumor progression of uterine endometrioid carcinoma.
PMCID: PMC3787317  PMID: 24076775
3.  Pathogenesis and the role of ARID1A mutation in endometriosis-related ovarian neoplasms 
Advances in anatomic pathology  2013;20(1):45-52.
Endometriosis-related ovarian neoplasms (ERONs) are a unique group of tumors as they are associated with endometriosis, especially endometriosis presenting as an ovarian endometriotic cyst (endometrioma). ERONs include clear cell carcinoma, endometrioid carcinoma, and seromucinous borderline tumor. A growing body of evidence from both clinicopathological and molecular studies suggests that most, if not all, ERONs develop from endometriotic cyst epithelium through different stages of tumor progression. The endometriotic cyst contains abundant iron-induced reactive oxygen species which are thought to be mutagenic, and chronic exposure of cystic epithelium to this microenvironment facilitates the accumulation of somatic mutations that ultimately result in tumor development. Molecular analyses of ERONs, including genome-wide screens, have identified several molecular genetic alterations that lead to aberrant activation or inactivation of pathways involving ARID1A, PI3K, Wnt, and PP2A. Among all molecular genetic changes identified to date, inactivating mutations of the ARID1A tumor suppressor gene are the most common in ERON. Understanding the molecular changes and pathogenesis involved in the development of ERON is fundamental for future translational studies aimed at designing new diagnostic tests for early detection and identifying critical molecular features for targeted therapeutics.
PMCID: PMC3523307  PMID: 23232571
endometriosis-related ovarian neoplasms; clear cell carcinoma; endometrioid carcinoma; endometrial carcinoma; ARID1A
4.  The diagnostic and biological implications of laminin expression in serous tubal intraepithelial carcinoma 
There is compelling evidence to suggest that serous tubal intraepithelial carcinoma (STIC) is the likely primary site for the development of pelvic high-grade serous carcinomas (HGSCs). Identifying molecules that are upregulated in STIC is important not only to provide biomarkers to assist in the diagnosis of STIC but also to elucidate our understanding of the pathogenesis of HGSC. In this study, we performed RNA sequencing to compare transcriptomes between HGSC and normal fallopian tube epithelium (FTE), and identified LAMC1 encoding laminin γ1 as one of the preferentially upregulated gene associated with HGSC. RT-PCR further validated LAMC1 upregulation in HGSC as compared to normal FTE. Immunohistochemistry was performed on 32 cases of concurrent HGSC and STIC. The latter was diagnosed based on morphology, TP53 mutations, p53 and Ki-67 immunohistochemical pattern. Laminin γ1 immunostaining intensity was found to be significantly higher in STIC and HGSC compared to adjacent FTE in all cases (p< 0.001). In normal FTE, laminin γ1 immunoreactivity was predominantly localized in the basement membrane or on the apical surface of ciliated cells whereas in STIC and HGSC cells, laminin γ1 staining was diffuse and intense throughout the cytoplasm. More importantly, strong laminin γ1 staining was detected in all 13 STICs which lacked p53 immunoreactivity due to null mutations. These findings suggest that the overexpression of laminin γ1 immunoreactivity and alteration of its staining pattern in STICs can serve as a useful tissue biomarker, especially for those STICs that are negative for p53 and have a low Ki-67 labeling index.
PMCID: PMC3500426  PMID: 22892598
ovarian cancer; serous tubal intraepithelial carcinoma; STIC; laminin; LAMC1
5.  Identification of Molecular Pathway Aberrations in Uterine Serous Carcinoma by Genome-wide Analyses 
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.
Whole-exome sequencing was performed on 10 uterine serous carcinomas and the matched normal blood or tissue samples. Somatically acquired sequence mutations were further verified by Sanger sequencing. The most frequent molecular genetic changes were further validated by Sanger sequencing in 66 additional uterine serous carcinomas and in nine serous endometrial intraepithelial carcinomas (the preinvasive precursor of uterine serous carcinoma) that were isolated by laser capture microdissection. In addition, gene copy number was characterized by single-nucleotide polymorphism (SNP) arrays in 23 uterine serous carcinomas, including 10 that were subjected to whole-exome sequencing.
We found frequent somatic mutations in TP53 (81.6%), PIK3CA (23.7%), FBXW7 (19.7%), and PPP2R1A (18.4%) among the 76 uterine serous carcinomas examined. All nine serous carcinomas that had an associated serous endometrial intraepithelial carcinoma had concordant PIK3CA, PPP2R1A, and TP53 mutation status between uterine serous carcinoma and the concurrent serous endometrial intraepithelial carcinoma component. DNA copy number analysis revealed frequent genomic amplification of the CCNE1 locus (which encodes cyclin E, a known substrate of FBXW7) and deletion of the FBXW7 locus. Among 23 uterine serous carcinomas that were subjected to SNP array analysis, seven tumors with FBXW7 mutations (four tumors with point mutations, three tumors with hemizygous deletions) did not have CCNE1 amplification, and 13 (57%) tumors had either a molecular genetic alteration in FBXW7 or CCNE1 amplification. Nearly half of these uterine serous carcinomas (48%) harbored PIK3CA mutation and/or PIK3CA amplification.
Molecular genetic aberrations involving the p53, cyclin E–FBXW7, and PI3K pathways represent major mechanisms in the development of uterine serous carcinoma.
PMCID: PMC3692380  PMID: 22923510
6.  Loss of ARID1A expression is an early molecular event in tumor progression from ovarian endometriotic cyst to clear cell and endometrioid carcinoma 
ARID1A is a recently identified tumor suppressor participating in chromatin remodeling. Somatic inactivating mutations of ARID1A and loss of its expression occur most frequently in ovarian clear cell and endometrioid carcinomas and uterine endometrioid carcinomas. Since endometriosis is thought to be a precursor of most ovarian clear cell and endometrioid carcinomas, we undertook an analysis of ARID1A expression of these tumors arising within an endometriotic cyst (endometrioma).
Our immunohistochemical study set consisted of 47 endometriotic cysts containing clear cell carcinoma in 24 cases, well-differentiated ovarian endometrioid carcinoma in 20 and mixed clear cell and endometrioid carcinoma in 3.
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.
The findings in this study provide cogent evidence that loss of ARID1A function as shown by loss of expression, presumably due to mutations, is an early molecular event, occurring before malignant transformation, in the development of the majority of ovarian clear cell and endometrioid carcinomas arising in endometriomas.
PMCID: PMC3460070  PMID: 22976498
7.  NAC1 is an actin-binding protein that is essential for effective cytokinesis in cancer cells 
Cancer research  2012;72(16):4085-4096.
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.
PMCID: PMC3421062  PMID: 22761335
actin; cytokinesis; NAC1; profilin-1
8.  Ki-67 Labeling Index as an Adjunct in the Diagnosis of Serous Tubal Intraepithelial Carcinoma 
There is mounting evidence that serous tubal intraepithelial carcinoma (STIC) may be the immediate precursor of ovarian high-grade serous carcinoma (HGSC) but the criteria for its diagnosis are not well established as highlighted in a recent study showing that interobserver reproducibility, even among expert gynecologic pathologists, was moderate at best. Given the clinical significance of a diagnosis of STIC in a patient who has no other evidence of ovarian carcinoma, this is a serious issue that we felt needed to be addressed. Although it is not clear, at this time, whether such a patient should or should not be treated, the importance of an accurate and reproducible diagnosis of precursors of ovarian carcinoma cannot be underestimated. We hypothesized that an elevated Ki-67 labeling index may aid the diagnosis of STIC. Accordingly, we compared the Ki-67 index of STIC and HGSC to normal fallopian tube epithelium (FTE) in the same patients and to a control group of patients without carcinoma, matched for age. A total of 41 STICs were analyzed, of which 35 were associated with a concurrent HGSC. In FTE, immunoreactivity for Ki-67 was restricted to a few scattered cells (mean 2.0%). No statistically significant difference was found between patients with and without HGSC (P>0.05). However, both STICs and HGSC had significantly higher Ki-67 indices than normal FTE (P<0.0001). STICs uniformly had an elevated Ki-67 labeling index that ranged from 11.7% to 71.1% (average 35.6%). There was no correlation of the Ki-67 labeling index in the STICs and the associated HGSC, as the labeling index was lower in STIC in 18/35 (51.4%) whereas it was higher in 17/35 (48.6%) (P=0.86). In conclusion, the findings in this study indicate that compared with FTE, STICs have a significantly higher Ki-67 index similar to HGSC. Accordingly, the Ki-67 index can aid the diagnosis of intraepithelial tubal proliferations suspicious for STIC. Therefore, we propose that a Ki-67 index of 10% is a useful diagnostic tool to distinguish STICs from normal FTE.
PMCID: PMC3715095  PMID: 22833080
Ovarian cancer; Serous tubal intraepithelial carcinoma; Ki-67; Serous; STIC
9.  Low-grade serous carcinomas of the ovary contain very few point mutations 
The Journal of pathology  2011;226(3):413-420.
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.
PMCID: PMC3503448  PMID: 22102435
ovarian cancer; exome sequencing; BRAF; KRAS; somatic mutations
10.  Endocervical-type Mucinous Borderline Tumors are Related to Endometrioid Tumors Based on Mutation and Loss of Expression of ARID1A 
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 expression have been studied in gynecological 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 one endometrioid tumor (p<0.01). Mutational analysis was performed on two 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 30–57% of endometrioid and clear cell carcinomas but only rarely in serous tumors. The findings in this study, showing a significantly higher frequency of loss of ARID1A expression in endocervical-type (seromucinous) tumors, presumably due to mutation, compared to the other histologic types suggest that they are molecularly related to endometrioid and clear cell tumors.
PMCID: PMC3501990  PMID: 22653341
ARID1A; ovarian borderline tumor
11.  ARID1A, a factor that promotes formation of SWI/SNF-mediated chromatin remodeling, is a tumor suppressor in gynecologic cancers 
Cancer research  2011;71(21):6718-6727.
ARID1A (BAF250A) promotes the formation of SWI/SNF chromatin remodeling complexes containing BRG1 or BRM. ARID1A has emerged as a candidate tumor suppressor based on its frequent mutations in ovarian clear cell and endometrioid cancers and in uterine endometrioid carcinomas. Here we report that restoring wild-type ARID1A expression in ovarian cancer cells that harbor ARID1A mutations is sufficient to suppress cell proliferation and tumor growth in mice, whereas RNAi-mediated silencing of ARID1A in non-transformed epithelial cells is sufficient to enhance cellular proliferation and tumorigenicity. Gene expression analysis identified several downstream targets of ARID1A including CDKN1A and SMAD3, which are well known p53 target genes. In support of the likelihood that p53 mediates the effects of ARID1A on these genes, we demonstrated that p53 was required and sufficient for their regulation by ARID1A. Further, we showed that CDKN1A (encoding p21) acted in part to mediate growth suppression by ARID1A. Lastly, we obtained evidence that the ARID1A/BRG1 complex interacts directly with p53 and that mutations in the ARID1A and TP53 genes were mutually exclusive in tumor specimens. Our results provide functional evidence in support of the hypothesis that ARID1A is a bona fide tumor suppressor that collaborates with p53 to regulate CDKN1A and SMAD3 transcription and tumor growth in gynecological cancers.
PMCID: PMC3206175  PMID: 21900401
12.  Functional Analysis of In-frame Indel ARID1A Mutations Reveals New Regulatory Mechanisms of Its Tumor Suppressor Functions1 
Neoplasia (New York, N.Y.)  2012;14(10):986-993.
AT-rich interactive domain 1A (ARID1A) has emerged as a new tumor suppressor in which frequent somatic mutations have been identified in several types of human cancers. Although most ARID1A somatic mutations are frame-shift or nonsense mutations that contribute to mRNA decay and loss of protein expression, 5% of ARID1A mutations are in-frame insertions or deletions (indels) that involve only a small stretch of peptides. Naturally occurring in-frame indel mutations provide unique and useful models to explore the biology and regulatory role of ARID1A. In this study, we analyzed indel mutations identified in gynecological cancers to determine how these mutations affect the tumor suppressor function of ARID1A. Our results demonstrate that all in-frame mutants analyzed lost their ability to inhibit cellular proliferation or activate transcription of CDKN1A, which encodes p21, a downstream effector of ARID1A. We also showed that ARID1A is a nucleocytoplasmic protein whose stability depends on its subcellular localization. Nuclear ARID1A is less stable than cytoplasmic ARID1A because ARID1A is rapidly degraded by the ubiquitin-proteasome system in the nucleus. In-frame deletions affecting the consensus nuclear export signal reduce steady-state protein levels of ARID1A. This defect in nuclear exportation leads to nuclear retention and subsequent degradation. Our findings delineate a mechanism underlying the regulation of ARID1A subcellular distribution and protein stability and suggest that targeting the nuclear ubiquitin-proteasome system can increase the amount of the ARID1A protein in the nucleus and restore its tumor suppressor functions.
PMCID: PMC3479842  PMID: 23097632
13.  NAC1 Modulates Sensitivity of Ovarian Cancer Cells to Cisplatin via Altering the HMGB1-Mediated Autophagic Response* 
Oncogene  2011;31(8):1055-1064.
Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, is known to play 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 reported 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 RNAi 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-MA and chloroquine, and siRNAs targeting beclin 1 or Atg5, on the cytotoxicity of cisplatin. Treatment with 3-MA, 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 via high mobility group box1 (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.
PMCID: PMC3275651  PMID: 21743489
NAC1; autophagy; apoptosis; HMGB1; cisplatin; ovarian cancer
14.  Mutation and loss of expression of ARID1A in uterine low-grade endometrioid carcinoma 
ARID1A is a recently identified tumor suppressor gene that is mutated in approximately 50% of ovarian clear cell and 30% of ovarian endometrioid carcinomas. The mutation is associated with loss of protein expression as assessed by immunohistochemistry. In this study, we evaluated ARID1A immunoreactivity in a wide variety of carcinomas in order to determine the prevalence of ARID1A inactivation in carcinomas; mutational analysis of ARID1A was performed in selected cases. Immunoreactivity was not detected (corresponding to inactivation or mutation of ARID1A) in 36 (3.6%) of 995 tumors. Uterine low-grade endometrioid carcinomas demonstrated a relatively high frequency of loss of ARID1A expression, as 15 (26%) of 58 cases were negative. The other tumor that had a relatively high frequency loss of ARID1A expression was gastric carcinoma (11%). Mutational analysis showed 10 (40%) of 25 uterine endometrioid carcinoma, none of 12 uterine serous carcinomas and none of 56 ovarian serous and mucinous carcinomas harbored somatic ARID1A mutations. All mutations in endometrioid carcinomas were nonsense or insertion/deletion mutations and tumors with ARID1A mutations demonstrated complete loss or clonal loss of ARID1A expression. In conclusion, this study is the first large-scale analysis of a wide variety of carcinomas showing that uterine low-grade endometrioid carcinoma is the predominant tumor type harboring ARID1A mutations and frequent loss of ARID1A expression. These findings suggest that the molecular pathogenesis of low-grade uterine endometrioid carcinoma is similar to that of ovarian low-grade endometrioid and clear cell carcinoma, tumors that have previously been shown to have a high frequency of loss of expression and mutation of ARID1A.
PMCID: PMC3077471  PMID: 21412130
ARID1A; BAF250; uterine carcinoma; ovarian carcinoma
15.  Rsf-1 (HBXAP) expression is associated with advanced stage and lymph node metastasis in ovarian clear cell carcinoma 
Ovarian clear cell carcinoma (CCC) is a unique type of ovarian cancer characterized by distinct clinicopathological and molecular features. CCC is considered to be a highly malignant disease because it is resistant to conventional chemotherapy, and, when presented at advanced stages, has a dismal overall survival. Identifying and characterizing biomarkers associated with its malignant behavior is fundamental toward elucidating the mechanisms underlying its aggressive phenotype. In this study, we performed immunohistochemical analysis on 89 CCCs to assess their expression of Rsf-1 (HBXAP), a chromatin remodeling gene frequently amplified and overexpressed in several types of human cancer. We found that 73 (82%) of 89 CCCs expressed Rsf-1 and most importantly, there was a statistically significant correlation between Rsf-1 immunostaining intensity and two disease parameters: advanced stage (p= 0.008) and status of retroperitoneal lymph node metastasis (p= 0.023). However, there was no correlation between Rsf-1 expression and patient age, peritoneal tumor dissemination, or overall survival. In conclusion, a higher expression level of Rsf-1 is associated with advanced clinical stage and lymph node metastasis in CCC. Our data suggest that Rsf-1 participates in tumor progression in CCC, and indicates that the contribution of Rsf-1 to disease aggressiveness deserves further study.
PMCID: PMC3052874  PMID: 21131837
16.  Distinct DNA methylation profiles in ovarian serous neoplasms and their implications in ovarian carcinogenesis 
American journal of obstetrics and gynecology  2010;203(6):584.e1-584.e22.
The purpose is to analyze DNA methylation profiling among different types of ovarian serous neoplasm, a task that has not been performed.
Study Design
The Illumina beads array was used to profile DNA methylation in enriched tumor cells isolated from 75 benign and malignant serous tumor tissues and six tumor-associated stromal cell cultures.
We found significantly fewer hypermethylated genes in high-grade serous carcinomas than in low-grade serous carcinoma and borderline tumors, which in turn had fewer hypermethylated genes than serous cystadenoma. Unsupervised analysis identified that serous cystadenoma, serous borderline tumor and low-grade serous carcinoma tightly clustered together and were clearly different from high-grade serous carcinomas. We also performed supervised analysis to identify differentially methylated genes that may contribute to group separation.
The findings support the view that low-grade and high-grade serous carcinomas are distinctly different with low-grade, but not high-grade serous carcinomas, related to serous borderline tumor and cystadenoma.
PMCID: PMC2993872  PMID: 20965493
ovarian carcinoma; methylation; classification
17.  Amplification of The ch19p13.2 NACC1 Locus in Ovarian High-grade Serous Carcinoma 
Based on digital karyotyping, we have identified a new, discrete amplified region at ch19p13.2 in a high-grade ovarian serous carcinoma. To further characterize this region, we determined the frequency and biological significance of ch19p13.2 amplification by analyzing 341 high-grade serous carcinomas from The Cancer Genome Atlas (TCGA) and found an increased DNA copy number at this locus in 18% of cases. We correlated the DNA and RNA copy number by analyzing the TCGA dataset for all amplified genes and detected 7 genes within ch19p13.2 that were significantly correlated (R ≥0.54) and were, in fact, listed as the top 100 potential “driver” genes at a genome-wide scale. Interestingly, one of the 7 genes, NACC1, encoding NAC1 was previously reported to be involved in the development of tumor recurrence in ovarian serous carcinoma and to play a causal role in the development of paclitaxel resistance. Therefore, we selected NACC1 for validation in an independent cohort. Based on fluorescence in situ hybridization, we found that 35 (20%) of 175 high-grade serous carcinomas had an increased DNA copy number at the NACC1 locus, and those amplified cases were associated with early disease recurrence within 6 months (p= 0.013). A significantly high level of NAC1 protein expression based on immunohistochemistry was detected in amplified tumors as compared to non-amplified tumors (p< 0.005). In summary, our data suggest that amplification at the ch19p13.2 NACC1 locus, leading to NAC1 overexpression, is one of the molecular genetic alterations associated with early tumor recurrence in ovarian cancer.
PMCID: PMC3085564  PMID: 21240255
18.  DNA Damage Response is Prominent in Ovarian High-Grade Serous Carcinomas, Especially Those with Rsf-1 (HBXAP) Overexpression 
Journal of Oncology  2011;2012:621685.
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.
PMCID: PMC3199114  PMID: 22028712
19.  DNA Copy Number Profiles in Affinity-Purified Ovarian Clear Cell Carcinoma 
Advanced ovarian clear cell carcinoma (CCC) is one of the most malignant ovarian malignancies, in part because it tends to be resistant to platinum-based chemotherapy. At present, little is known about the molecular genetic alterations in CCCs except that there are frequent activating mutations in PIK3CA. The purpose of this study is to comprehensively define the genomic changes in CCC based on DNA copy number alterations.
Experimental Design
We performed 250K high-density SNP array analysis in 12 affinity-purified CCCs and 10 CCC cell lines. Discrete regions of amplification and deletion were also analyzed in additional 21 affinity-purified CCCs using quantitative real-time PCR.
The level of chromosomal instability in CCC as defined by the extent of DNA copy number changes is similar to those previously reported in low-grade ovarian serous carcinoma but much less than those in high-grade serous carcinoma. The most remarkable region with DNA copy number gain is at chr20 which harbors a potential oncogene, ZNF217. This is observed in 36% of CCCs but rarely detected in serous carcinomas regardless of grade. In addition, homozygous deletions are detected at CDKN2A/2B and LZTS1 loci. Interestingly, the DNA copy number changes observed in fresh CCC tissues are rarely detected in the established CCC cell lines.
This study provides the first high resolution, genome-wide view of DNA copy number alterations in ovarian CCC. The findings provide a genomic landscape for future studies aimed at elucidating the pathogenesis and developing new target-based therapies for CCCs.
PMCID: PMC2848895  PMID: 20233889
20.  Serous tubal intraepithelial carcinoma upregulates markers associated with high-grade serous carcinomas including Rsf-1 (HBXAP), cyclin E and fatty acid synthase 
Serous tubal intraepithelial carcinoma (STIC) has been proposed as a precursor for many pelvic high-grade serous carcinomas. Our previous analysis of the ovarian cancer genome identified several genes with oncogenic potential that are amplified and/or overexpressed in the majority of high-grade serous carcinomas. Determining whether these genes are upregulated in STICs is important in further elucidating the relationship of STICs to high-grade serous carcinomas and is fundamental in understanding the molecular pathogenesis of high-grade serous carcinomas. In this study, 37 morphologically defined STICs were obtained from 23 patients with stage IIIC/IV high-grade serous carcinomas. Both STICs and the high-grade serous carcinomas were analyzed for expression of Rsf-1, cyclin E, fatty acid synthase (FASN), and mucin-4. In addition, they were examined for expression of established markers including p53, Ki-67 and p16. We found that diffuse nuclear p53 and p16 immunoreactivity was observed in 27 (75%) of 36 and 18 (55%) of 33 STICs, respectively, while an elevated Ki-67 labeling index (≥10%) was detected in 29 (78%) of 37 STICs. Cyclin E nuclear staining was seen in 24 (77%) of 35 STICs while normal tubal epithelial cells were all negative. Increased Rsf-1 and FASN immunoreactivity occurred in 63%, and 62% of STICs, respectively, compared to adjacent normal-appearing tubal epithelium. Interestingly, only one STIC demonstrated increased mucin-4 immunoreactivity. Carcinomas, as compared to STICs, overexpressed p16, Rsf-1, cyclin E and FASN in a higher proportion of cases. In conclusion, STICs express several markers including Rsf-1, cyclin E and FASN in high-grade serous carcinomas. In contrast, mucin-4 immunoreactivity either did not change or was reduced in most STICs. These results suggest that overexpression of Rsf-1, cyclin E and FASN occurs early in tumor progression.
PMCID: PMC2879438  PMID: 20228782
ovarian cancer; tubal intraepithelial carcinoma; HBXAP (Rsf-1)
21.  Defining the cut-point between low- and high-grade ovarian serous carcinomas: A clinicopathologic and molecular genetic analysis 
A two-tier grading system based on nuclear grade divides ovarian serous carcinomas into low- (nuclear grade 1) and high-grade (nuclear grade 3). In most instances the separation is straightforward but at times, the morphologic distinction between them can be difficult. We studied eleven ovarian serous carcinomas with features that were “intermediate” (nuclear grade 2) between low and high grade. All the cases were high stage and had a poor clinical outcome. None of the tumors showed mutations in KRAS, BRAF and ERBB2 genes which characterize most low-grade serous carcinomas. In contrast, 10 (90.9%) of 11 cases contained non-synonymous TP53 mutations characteristic of high-grade serous carcinomas. In summary, the molecular genetic profile and behavior of serous carcinomas with grade 2 nuclei are virtually the same as those of serous carcinomas with grade 3 nuclei, supporting the use of the two-tier grading system for classifying ovarian serous carcinomas.
PMCID: PMC2716424  PMID: 19461510
Ovarian cancer; serous carcinoma; grade; TP53; two-tier grading system
22.  Expression of Fatty Acid Synthase Depends on NAC1 and Is Associated with Recurrent Ovarian Serous Carcinomas 
Journal of Oncology  2010;2010:285191.
Our previous reports demonstrated that NAC1, a BTB/POZ domain-containing nuclear protein, upregulates in recurrent ovarian serous carcinoma and participates in developing drug resistance in cancer cells. The current study applies quantitative proteomics to identify the proteins controlled by NAC1 by comparing the proteomes of SKOV3 cells with and without expression of a dominant negative NAC1 construct, N130. From the proteins that are downregulated by N130 (upregulated by NAC1), we chose to further characterize fatty acid synthase (FASN). Similar to change in protein level, the FASN transcript level in SKOV3 cells was significantly reduced by N130 induction or by NAC1 knockdown. Immunohistochemistry showed that NAC1 and FASN immunointensities in ovarian serous carcinoma tissues had a highly significant correlation (P < .0001). Moreover, we found that recurrent serous carcinomas exhibited higher FASN immunointensities than their matched primary tumors (P < .001). Multivariate analysis showed that an FASN staining score of >1 in serous carcinomas was associated with a worse overall survival time (P < .01). Finally, C93, a new FASN inhibitor, induced massive apoptosis in carboplatin/paclitaxel resistant ovarian cancer cells. In conclusion, we show that NAC1 is essential for FASN expression in ovarian serous carcinomas and the expression of FASN significantly correlates with tumor recurrence and disease aggressiveness. The dependence of drug resistant tumor cells on FASN suggests a potential application of FASN-based therapeutics for recurrent ovarian cancer patients.
PMCID: PMC2873657  PMID: 20508725
23.  Analysis of DNA Copy Number Alterations in Ovarian Serous Tumors Identifies New Molecular Genetic Changes in Low-grade and High-grade Carcinomas 
Cancer research  2009;69(9):4036-4042.
Ovarian serous carcinoma, the most common and lethal type of ovarian cancer, was thought to develop from two distinct molecular pathways. High-grade (HG) serous carcinomas contain frequent TP53 mutations while low-grade (LG) carcinomas arise from serous borderline tumors (SBT) and harbor mutations in KRAS/BRAF/ERBB2 pathway. However, the molecular alterations involved in the progression from SBT to LG carcinoma remain largely unknown. As well, the extent of deletion of tumor suppressors in ovarian serous carcinomas has not been well-studied. To further address these two issues, we assessed DNA copy number changes among affinity-purified tumor cells from 37 ovarian serous neoplasms including SBT, LG and HG tumors using high density 250K SNP arrays. Chromosomal instability index as measured by changes in DNA copy number was significantly higher in HG than in LG serous carcinomas. Hemizygous ch1p36 deletion was common in LG serous carcinomas but was rarely seen in SBT. This region contains several candidate tumor suppressors including miR-34a. In contrast, in HG serous carcinomas, significant numbers of amplifications and deletions including homozygous deletions were identified. Among homozygous deletions, loci containing Rb1, CDKN2A/B, CSMD1, and DOCK4 were most common, being present in 10.6%, 6.4%, 6.4% and 4.3%, respectively, in independent 47 affinity-purified HG serous carcinomas. Except the CDKN2A/B region, these homozygous deletions were not present in either SBT or LG tumors. Our study provides a genome-wide homozygous deletion profiles in HG serous carcinomas, serving as a molecular foundation to study tumor suppressors in ovarian cancer.
PMCID: PMC2782554  PMID: 19383911
24.  Lack of a Y-Chromosomal Complement in the Majority of Gestational Trophoblastic Neoplasms 
Journal of Oncology  2010;2010:364508.
Gestational trophoblastic neoplasms (GTNs) are a rare group of neoplastic diseases composed of choriocarcinomas, placental site trophoblastic tumors (PSTTs) and epithelioid trophoblastic tumors (ETTs). Since these tumors are derivatives of fetal trophoblastic tissue, approximately 50% of GTN cases are expected to originate from a male conceptus and carry a Y-chromosomal complement according to a balanced sex ratio. To investigate this hypothesis, we carried out a comprehensive analysis by genotyping a relatively large sample size of 51 GTN cases using three independent sex chromosome genetic markers; Amelogenin, Protein Kinase and Zinc Finger have X and Y homologues that are distinguishable by their PCR product size. We found that all cases contained the X-chromosomal complement while only five (10%) of 51 tumors harbored the Y-chromosomal complement. Specifically, Y-chromosomal signals were detected in one (5%) of 19 choriocarcinomas, one (7%) of 15 PSTTs and three (18%) of 17 ETTs. The histopathological features of those with a Y-chromosome were similar to those without. Our results demonstrate the presence of a Y-chromosomal complement in GTNs, albeit a low 10% of cases. This shortfall of Y-chromosomal complements in GTNs may reinforce the notion that the majority of GTNs are derived from previous molar gestations.
PMCID: PMC2825661  PMID: 20182630
25.  Functional analysis of 11q13.5 amplicon identifies Rsf-1 (HBXAP) as a gene involved in paclitaxel resistance in ovarian cancer 
Cancer research  2009;69(4):1407-1415.
The chromosome 11q13.5 locus is frequently amplified in several types of human cancer. We have previously shown that 11q13.5 amplification was associated with significantly shorter overall survival in ovarian cancer patients but the molecular mechanisms of how amplification of this locus contributes to disease aggressiveness remain unclear. As ovarian cancer mortality is primarily related to resistance of chemotherapeutic agents, we screened the top six candidate genes within this amplicon for their contribution to drug resistance. Rsf-1 (also known as HBXAP) was found to be the only gene in which gene knockdown sensitized tumor cells to paclitaxel. Rsf-1 has been known to interact with hSNF2H to form an ISWI chromatin remodeling complex. We found that Rsf-1 was upregulated in paclitaxel resistant ovarian cancer cell lines and Rsf-1 immunoreactivity in primary ovarian carcinoma tissues correlated with in vitro paclitaxel resistance. Ectopic expression of Rsf-1 significantly enhanced paclitaxel resistance in ovarian cancer cells. Downregulation of hSNF2H or disruption of hSNF2H and Rsf-1 interaction enhanced paclitaxel sensitivity in tumor cells with Rsf-1 upregulation. Rsf-1 expression altered expression in several genes and activated certain signaling pathways that may contribute to drug resistance. In conclusion, our results suggest that Rsf-1 is the major gene within the 11q13.5 amplicon that contributes to paclitaxel resistance and the formation of the Rsf-1/hSNF2H complex is required for inducing this phenotype.
PMCID: PMC2644345  PMID: 19190325
Ovarian; Cancer; Genomics; Genetics; Pathogenesis

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