Naomi Nakayama

Sequence mutations and amplification of PIK3CA and AKT2 genes in purified ovarian serous neoplasms

Sequence mutations and gene amplifications lead to activation of the PIK3CA-AKT2 signaling pathway and have been reported in several types of neoplasms including ovarian cancer. Analysis of such genetic alterations, however, is usually complicated by contamination of normal cell DNA, artifacts associated with formalin-fixed tissues and the sensitivity of the techniques employed. In this study, we analyzed the sequence mutations in PIK3CA and AKT2 genes using purified tumor cells that were isolated from high-grade ovarian serous carcinomas and serous borderline tumors (SBTs) and assessed gene amplification using a dual-color FISH on tissue microarrays. Somatic sequence mutations in the kinase domain of AKT2 were not detected in any of the 65 ovarian tumors analyzed. Mutations of PIK3CA were rare, occurring only in one (2.3%) of 44 high-grade serous carcinomas and in only one (4.8%) of 21 SBTs. Dual-color FISH demonstrated that PIK3CA and AKT2 were not amplified in SBTs but amplified in 13.3% and 18.2% high-grade carcinomas, respectively. High-level amplification (>3 fold) was more frequently observed in AKT2 than in PIK3CA. Unlike mutations in ERBB2, KRAS and BRAF which are mutually exclusive in SBTs, co-amplification of PIK3CA and AKT2 was present in five high-grade carcinomas including the OVCAR3 cells. Amplification in either of the genes occurred in 27% high-grade serous carcinomas. In conclusion, the methods we employed provide unambiguous evidence that somatic sequence mutations of PIK3CA and ATK2 are rare in ovarian serous tumors but amplification of both genes may play an important role in the development of high-grade ovarian serous carcinoma.

Amplicon profiles in ovarian serous carcinomas.

Ovarian serous carcinoma is the most common and lethal type of ovarian cancer and its molecular etiology remains poorly understood. As an ongoing effort to elucidate the pathogenesis of ovarian serous carcinomas, we assessed the DNA copy number changes in 33 high‐grade serous carcinomas and 10 low‐grade serous tumors by using a genome‐wide technique, single nucleotide polymorphism array, performed on affinity‐purified tumor cells from fresh surgical specimens. Compared to low‐grade tumors, high‐grade serous carcinomas showed widespread DNA copy number changes. The most frequent alterations were in loci harboring candidate oncogenes: cyclin E1 (CCNE1), AKT2, Notch3 and PIK3CA as well as in novel loci, including 12p13, 8q24, 12p13 and 12q15. Seven amplicons were selected for dual color fluorescence in situ hybridization analysis in ∼90 high‐grade serous carcinomas and 26 low‐grade serous tumors, and a high level of DNA copy number gain (amplification) was found in CCNE1, Notch3, HBXAP/Rsf‐1, AKT2, PIK3CA and chr12p13 occurring in 36.1%, 7.8%, 15.7%, 13.6%, 10.8% and 7.3% of high‐grade serous carcinomas. In contrast, we did not observe high level of ERBB2 amplification in any of the samples. Low‐grade tumors did not show DNA copy number gain in any of the loci, except in 2 (8%) of 24 low‐grade tumors showing low copy number gain in the Notch3 locus. Taken together, our results provide the first comprehensive analysis of DNA copy number changes in highly pure ovarian serous carcinoma. These findings may have important biological and clinical implications.

A BTB/POZ protein, NAC-1, is related to tumor recurrence and is essential for tumor growth and survival

Recent studies have suggested an oncogenic role of the BTB/POZ-domain genes in hematopoietic malignancy. The aim of this study is to identify and characterize BTB/POZ-domain genes in the development of human epithelial cancers, i.e., carcinomas. In this study, we focused on ovarian carcinoma and analyzed gene expression levels using the serial analysis of gene expression (SAGE) data in all 130 deduced BTB/POZ genes. Our analysis reveals that NAC-1 is significantly overexpressed in ovarian serous carcinomas and several other types of carcinomas. Immunohistochemistry studies in ovarian serous carcinomas demonstrate that NAC-1 is localized in discrete nuclear bodies (tentatively named NAC-1 bodies), and the levels of NAC-1 expression correlate with tumor recurrence. Furthermore, intense NAC-1 immunoreactivity in primary tumors predicts early recurrence in ovarian cancer. Both coimmunoprecipitation and double immunofluorescence staining demonstrate that NAC-1 molecules homooligomerize through the BTB/POZ domain. Induced expression of the NAC-1 mutant containing only the BTB/POZ domain disrupts NAC-1 bodies, prevents tumor formation, and promotes tumor cell apoptosis in established tumors in a mouse xenograft model. Overexpression of full-length NAC-1 enhanced tumorigenicity of ovarian surface epithelial cells and NIH 3T3 cells in athymic nu/nu mice. In summary, NAC-1 is a tumor recurrence-associated gene with oncogenic potential, and the interaction between BTB/POZ domains of NAC-1 proteins is critical to form the discrete NAC-1 nuclear bodies and essential for tumor cell proliferation and survival.

KRAS or BRAF mutation status is a useful predictor of sensitivity to MEK inhibition in ovarian cancer

This study examined the status of KRAS and BRAF mutations, in relation to extracellular signal-regulated protein kinase (ERK) activation in 58 ovarian carcinomas to clarify the clinicopathological and prognostic significance of KRAS/BRAF mutations. Somatic mutations of either KRAS or BRAF were identified in 12 (20.6%) out of 58 ovarian carcinomas. The frequency of KRAS/BRAF mutations in conventional serous high-grade carcinomas (4.0% : 1/25) was significantly lower than that in the other histological type (32.3% : 10/31). Phosphorylated ERK1/2 (p-ERK1/2) expression was identified in 18 (38.2%) out of 45 ovarian carcinomas. KRAS/BRAF mutation was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage I, II (P<0.001), and p-ERK1/2 (P<0.001). No significant correlations between KRAS/BRAF mutations or p-ERK1/2 expression and overall survival were found in patients with ovarian carcinoma treated with platinum and taxane chemotherapy (P=0.2460, P=0.9339, respectively). Next, to clarify the roles of ERK1/2 activation in ovarian cancers harbouring KRAS or BRAF mutations, we inactivated ERK1/2 in ovarian cancer cells using CI-1040. Cl-1040 is a compound that selectively inhibits MAP kinase kinase (MEK), an upstream regulator of ERK1/2, and thus prevents ERK1/2 activation. Profound growth inhibition and apoptosis were observed in CI-1040-treated cancer cells with mutations in either KRAS or BRAF in comparison with the ovarian cancer cells containing wild-type sequences. This was evident in both in vitro and in vivo studies. The findings in this study indicate that an activated ERK1/2 pathway is critical to tumour growth and survival of ovarian cancers with KRAS or BRAF mutations. Furthermore, they suggest that the CI-1040-induced phenotypes depend on the mutational status of KRAS and BRAF in ovarian cancers. Therefore, ovarian cancer patients with KRAS or BRAF mutations may benefit from CI-1040 treatment.

Homozygous deletion of MKK4 in ovarian serous carcinoma

Analysis of deleted chromosomal regions in tumors has historically led to the identification of tumor suppressor genes. In this study, we used digital karyotyping, a genome-wide, high-resolution technology, to search for chromosomal deletions in ovarian serous carcinoma, the most lethal gynecological malignancy in women. Five purified ovarian serous carcinomas were analyzed by digital karyotyping and small interstitial deletions at chromosome 17p were identified in two tumor samples. Aligning these two deletions identified an overlapping region that spanned 2.4 Mb which harbored a candidate tumor suppressor gene, mitogen-activated protein kinase kinase-4 (MKK4). Dual-color FISH analysis confirmed homozygous deletion of the MKK4 locus in both samples and RT-PCR demonstrated that both carcinomas lacked MKK4 transcript expression. Loss of heterozygosity of 17p occurred in 24 (86%) of 28 high-grade serous carcinomas including both cases with homozygous MKK4 deletion. Additionally, downregulation of MKK4 expression was found in 96 (75%) of 128 ovarian serous carcinomas as compared to benign ovarian tissues. These findings suggest that homozygous deletion or reduced expression of MKK4 may contribute to the development of ovarian serous carcinoma.

NAC-1 Controls Cell Growth and Survival by Repressing Transcription of Gadd45GIP1, a Candidate Tumor Suppressor

Cancer mortality and morbidity are primarily related to recurrent tumors, and characterization of recurrence-associated genes should illuminate fundamental properties of tumor progression and provide new therapeutic targets. We have previously identified NAC-1, a member of the BTB/POZ gene family and a transcription repressor, as a gene associated with recurrent ovarian carcinomas after chemotherapy. We further showed that homodimerization of NAC-1 proteins is essential for tumor growth and survival. In this study, we applied serial analysis of gene expression and identified growth arrest and DNA-damage-inducible 45-gamma interacting protein (Gadd45GIP1) as one of the downstream genes negatively regulated by NAC-1. NAC-1 knockdown in both SKOV3 and HeLa cells that expressed abundant endogenous NAC-1 induced Gadd45GIP1 expression transcriptionally; on the other hand, engineered expression of NAC-1 in NAC-1-negative RK3E and HEK293 cells suppressed endogenous Gadd45GIP1 expression. In NAC-1-expressing tumor cells, induction of dominant negative NAC-1 conferred a growth-inhibitory effect that can be partially reversed by Gadd45GIP1 knockdown. Induced Gadd45GIP1 expression resulted in growth arrest in SKOV3 and HeLa cells both in vitro and in vivo. In summary, NAC-1 contributes to tumor growth and survival by at least inhibiting Gadd45GIP1 expression, which has a tumor suppressor effect in cancer cells.

Expression of the Bric-a-Brac Tramtrack Broad Complex Protein NAC-1 in Cervical Carcinomas Seems to Correlate with Poorer Prognosis

Purpose: Recent studies have suggested a novel oncogenic role of a bric-a-brac tramtrack broad complex (also known as POZ) domain gene, NAC-1, in ovarian carcinomas. The aim of this study was to clarify the functional role of NAC-1 in human cervical carcinomas. Experimental Design: NAC-1 expression in cervical cancer was assessed by immunohistochemistry, and data on clinical variables were collected by retrospective chart review. NAC-1 gene knockdown using small interfering RNA and a NAC-1 gene transfection system were used to asses NAC-1 function in cervical cancer in vivo. Results: Immunohistochemical and gene expression analysis revealed that NAC-1 is significantly overexpressed in cervical adenocarcinomas and adenosquamous carcinomas compared with squamous cell carcinomas. Patients with squamous cell carcinomas positive for NAC-1 expression who received radiotherapy had significantly shorter overall survival than peers whose tumors did not express NAC-1, and multivariate analysis showed that NAC-1 expression was an independent prognostic factor for overall survival after radiotherapy. Overexpressions of the NAC-1 gene stimulated cell proliferation in cervical carcinoma cells of the TCS, CaSki, and HeLa P3 lines, which do not have endogenous NAC-1 expression. NAC-1 gene knockdown inhibited cell growth and induced apoptosis in HeLa, HeLa TG, and ME180 cells, all of which overexpressed NAC-1. Conclusions: Our findings suggest that NAC-1 may play an important role in cervical carcinomas; moreover, these findings provide a rationale for future development of NAC-1–based therapy for cervical carcinomas that overexpress this candidate oncogene.

Phase I first-in-human study of TAK-285, a novel investigational dual HER2/EGFR inhibitor, in cancer patients

Background:This phase I first-in-human study was conducted in Japanese patients to investigate the safety, pharmacokinetics (PKs), and determine the maximum tolerated dose (MTD) of oral TAK-285, a novel dual erbB protein kinase inhibitor that specifically targets human epidermal growth factor receptor (EGFR) and HER2.Methods:The TAK-285 dose was escalated until MTD was determined. A second patient cohort received TAK-285 at the MTD for at least 4 weeks.Results:In all, 26 patients received TAK-285 at doses ranging from 50 to 400 mg once daily (q.d.) or twice daily (b.i.d.); 20 patients made up the dose escalation cohort and the remaining 6 patients were the repeated administration cohort. TAK-285 was well tolerated. Dose-limiting toxicities noted in two patients who received 400 mg b.i.d. were grade 3 increases in aminotransferases and grade 3 decreased appetite. Consequently, the MTD was determined to be 300 mg b.i.d. Absorption of TAK-285 was rapid after oral dosing, and plasma exposure at steady-state increased in a dose-proportional fashion for doses ranging from 50 to 300 mg b.i.d. A partial response was observed for one patient with parotid cancer who received 300 mg b.i.d.Conclusion:The toxicity profile and PK properties of oral TAK-285 warrant further evaluation.

Low-grade Serous Carcinoma of the Ovary Displaying a Macropapillary Pattern of Invasion

Invasive micropapillary serous carcinoma (MPSC) also designated “low-grade serous carcinoma” (LGSC) of the ovary is characterized by small micropapillae that infiltrate underlying tissue (ovarian stroma). On occasion these tumors in addition to the micropapillae contain large macropapillae lined by bland epithelium. In rare cases, the entire tumor is composed of macropapillae. In these cases, the question of whether this is an invasive carcinoma or an unusual type of adenofibroma has been raised. The goal of this study was to describe this unusual macropapillary pattern of invasion in LGSC. Cases of LGSC containing macropapillae were retrieved from the files of the Johns Hopkins Hospital. In addition to a detailed morphologic analysis, the mutational status of KRAS and BRAF in the macropapillary, noninvasive, and invasive MPSC components was analyzed by nucleotide sequencing. There were 14 cases containing macropapillae (11 cases of LGSC, 2 cases of atypical proliferative serous tumor (APST) with microinvasion, and 1 case of APST with a focus of LGSC with macropapillae in perivaginal soft tissue). In 3 cases, extraovarian metastases contained macropapillae. Molecular analysis of the primary tumor components (macropapillary, noninvasive, and invasive MPSC and/or APST) was performed in 7 cases and of a lymph node metastasis with macropapillae in 1 case. The identical KRAS mutation was detected in all of the analyzed components of the primary ovarian tumors in 4 cases. In one of these cases, macropapillae in the lymph node metastasis contained a KRAS mutation identical to the primary tumor. The BRAF mutation identified in 1 case was identical in all components of the ovarian tumor. The identical mutations in the macropapillae and the other tumor components in each case indicate that they are clonally related. The finding of macropapillae within lymph nodes supports the interpretation that the macropapillary component is another manifestation of invasion in LGSC. The recognition of this pattern is important, especially in cases when a tumor is composed entirely of macropapillae.

Expression of a BTB/POZ Protein, NAC1, Is Essential for the Proliferation of Normal Cyclic Endometrial Glandular Cells and Is Up-regulated by Estrogen

Purpose: The purpose of this study was to investigate the expression and localization of NAC1, a member of the BTB/POZ gene family in the human cyclic endometrium. Experimental Design: NAC1 expression in normal cyclic endometrium was assessed by immunohistochemistry, and data on clinical variables were collected by retrospective chart review. To elucidate the molecular mechanisms of NAC1 expression in the normal endometrium endometrial carcinoma cell lines (Ishikawa, HHUA; ER+, PR+) and primary cultured normal endometria were tested in a sex steroid induction assay and a NAC1 knockdown assay using siRNA. Results: Expression of NAC1 in glandular cells was significantly higher in the early and mid proliferative phases than in the other menstrual phases. Both NAC1 RNA and protein expression were up-regulated by treatment with 10 nmol/L 17β-Estradiol (E2) in Ishikawa, HHUA and primary cultured normal endometrial cells. The estrogen receptor antagonist ICI 182,780 significantly attenuated E2-induced NAC1 expression. NAC1 gene knockdown inhibited cell growth and induced apoptosis in Ishikawa, HHUA, and normal endometria, all of which expressed NAC1. Furthermore, NAC1 siRNA significantly abrogated estrogen-driven cellular proliferation in Ishikawa, HHUA, and primary cultured normal endometrial cells, whereas the control siRNA had no effect on cell growth in any of these cells. Conclusions: These findings suggest that NAC1 is functionally involved in E2-induced cell growth of the normal endometrial glandular cells. Because NAC1 is thought to have oncogenic potential, the current findings may provide new insight into the mechanism of estrogen induced endometrial carcinogenesis.