[Show abstract][Hide abstract] ABSTRACT: The Phosphatidylinositol 3'-kinase is a key regulator in various cancer-associated signal transduction pathways. Genetic alterations of its catalytic subunit alpha, PIK3CA, have been identified in ovarian cancer. Our in vivo data suggests that PIK3CA activation is one of the early genetic events in ovarian cancer. However, its role in malignant transformation of ovarian surface epithelium (OSE) is largely unclear.
Using the Müllerian inhibiting substance type II receptor (MISIIR) promoter, we generated transgenic mice that expressed activated PIK3CA in the Müllerian epithelium. Overexpression of PIK3CA in OSE induced remarkable hyperplasia, but was not able to malignantly transform OSE in vivo. The consistent result was also observed in primary cultured OSEs. Although enforced expression of PIK3CA could not induce OSE anchorage-independent growth, it significantly increased anchorage-independent growth of OSE transformed by mutant K-ras.
While PIK3CA activation may not be able to initiate OSE transformation, we conclude that activation of PIK3CA may be an important molecular event contributing to the maintenance of OSE transformation initiated by oncogenes such as K-ras.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNA) are approximately 22-nucleotide noncoding RNAs that negatively regulate protein-coding gene expression in a sequence-specific manner via translational inhibition or mRNA degradation. Our recent studies showed that miRNAs exhibit genomic alterations at a high frequency and their expression is remarkably deregulated in ovarian cancer, strongly suggesting that miRNAs are involved in the initiation and progression of this disease. In the present study, we performed miRNA microarray to identify the miRNAs associated with chemotherapy response in ovarian cancer and found that let-7i expression was significantly reduced in chemotherapy-resistant patients (n = 69, P = 0.003). This result was further validated by stem-loop real-time reverse transcription-PCR (n = 62, P = 0.015). Both loss-of-function (by synthetic let-7i inhibitor) and gain-of-function (by retroviral overexpression of let-7i) studies showed that reduced let-7i expression significantly increased the resistance of ovarian and breast cancer cells to the chemotherapy drug, cis-platinum. Finally, using miRNA microarray, we found that decreased let-7i expression was significantly associated with the shorter progression-free survival of patients with late-stage ovarian cancer (n = 72, P = 0.042). This finding was further validated in the same sample set by stem-loop real-time reverse transcription-PCR (n = 62, P = 0.001) and in an independent sample set by in situ hybridization (n = 53, P = 0.049). Taken together, our results strongly suggest that let-7i might be used as a therapeutic target to modulate platinum-based chemotherapy and as a biomarker to predict chemotherapy response and survival in patients with ovarian cancer.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that function as negative gene regulators. miRNA deregulation is involved in the initiation and progression of human cancer; however, the underlying mechanism and its contributions to genome-wide transcriptional changes in cancer are still largely unknown. We studied miRNA deregulation in human epithelial ovarian cancer by integrative genomic approach, including miRNA microarray (n = 106), array-based comparative genomic hybridization (n = 109), cDNA microarray (n = 76), and tissue array (n = 504). miRNA expression is markedly down-regulated in malignant transformation and tumor progression. Genomic copy number loss and epigenetic silencing, respectively, may account for the down-regulation of approximately 15% and at least approximately 36% of miRNAs in advanced ovarian tumors and miRNA down-regulation contributes to a genome-wide transcriptional deregulation. Last, eight miRNAs located in the chromosome 14 miRNA cluster (Dlk1-Gtl2 domain) were identified as potential tumor suppressor genes. Therefore, our results suggest that miRNAs may offer new biomarkers and therapeutic targets in epithelial ovarian cancer.
Full-text · Article · Jun 2008 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: PIK3CA upregulation, amplification and mutation have been widely reported in ovarian cancers and other tumors, which strongly suggests that PIK3CA is a promising therapeutic target. However, to date the mechanisms underlying PIK3CA regulation and activation in vivo is still unclear. During tumorigenesis, host-tumor interactions may play a critical role in editing the tumor. Here, we report a novel mechanism through which the tumor microenvironment activates the PIK3CA oncogene. We show that PIK3CA upregulation occurs in non-proliferating tumor regions in vivo. We identified and characterized the PIK3CA 5' upstream transcriptional regulatory region and confirmed that PIK3CA is transcriptionally regulated through NF-kappaB pathway. These results offer a new mechanism through which the tumor microenvironment directly activates oncogenic pathways in tumor cells.
[Show abstract][Hide abstract] ABSTRACT: Cancer is a disease involving multistep dynamic changes in the genome. However, studies to date on the cancer genome have
focused most heavily on protein-coding genes, and our knowledge on alterations of the functional noncoding sequences in cancer
is largely absent. MicroRNAs (miRNAs) are ~22 nucleotide (nt) noncoding RNAs, which regulate gene expression in a sequence-specific
manner via translational inhibition or mRNA degradation. Mounting evidence shows that miRNAs may play an important role in
tumor development, and a better understanding of their alteration in cancer genome and oncogenic property should contribute
to the diagnosis and treatment of cancer.
No preview · Article · Feb 2008 · Advances in Experimental Medicine and Biology
[Show abstract][Hide abstract] ABSTRACT: The phosphatidylinositol 3'-kinase (PI3K) family plays a key regulatory role in various cancer-associated signal transduction pathways. Here, we investigated the genomic alterations and gene expression of most known PI3K family members in human epithelial ovarian cancer.
The DNA copy number of PI3K family genes was screened by a high-resolution array comparative genomic hybridization in 89 human ovarian cancer specimens. The mRNA expression level of PI3K genes was analyzed by microarray retrieval approach, and further validated by real-time reverse transcription-PCR. The expression of p55gamma protein in ovarian cancer was analyzed on tissue arrays. Small interfering RNA was used to study the function of PIK3R3 in ovarian cancer.
In ovarian cancer, 6 of 12 PI3K genes exhibited significant DNA copy number gains (>20%), including PIK3CA (23.6%), PIK3CB (27.0%), PIK3CG (25.8%), PIK3R2 (29.2%), PIK3R3 (21.3%), and PIK3C2B (40.4%). Among those, only PIK3R3 had significantly up-regulated mRNA expression level in ovarian cancer compared with normal ovary. Up-regulated PIK3R3 mRNA expression was also observed in liver, prostate, and breast cancers. The PIK3R3 mRNA expression level was significantly higher in ovarian cancer cell lines (n = 18) than in human ovarian surface epithelial cells (n = 6, P = 0.002). Overexpression of p55gamma protein in ovarian cancer was confirmed by tissue array analysis. In addition, we found that knockdown of PIK3R3 expression by small interfering RNA significantly increased the apoptosis in cultured ovarian cancer cell lines.
We propose that PIK3R3 may serve as a potential therapeutic target in human ovarian cancer.
Full-text · Article · Sep 2007 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are approximately 22 nt non-coding RNAs, which regulate gene expression in a sequence-specific manner via translational inhibition or messenger RNA (mRNA) degradation. Since the discovery of their fundamental mechanisms of action, the field of miRNAs has opened a new era in the understanding of small noncoding RNAs. By molecular cloning and bioinformatic approaches, miRNAs have been identified in viruses, plants and animals. miRNAs are predicted to negatively target up to one-third of human mRNAs. Cancer is a complex genetic disease caused by abnormalities in gene structure and expression. Previous studies have heavily focused on protein-coding genes; however, accumulating evidence is revealing an important role of miRNAs in cancer. Epigenetics is defined as mitotically and/or meiotically heritable changes in gene expression that are not accompanied by changes in DNA sequence. Given the critical roles of miRNAs and epigenetics in cancer, characterizing the epigenetic regulation of miRNAs will provide novel opportunities for the development of cancer biomarkers and/or the identification of new therapeutic targets in the foreseeable future.
Preview · Article · Mar 2007 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: LINE-1s, or L1s, are highly abundant retrotransposons comprising 17% of the human genome. Most L1s are retrotransposition defective; nonetheless, there are approximately 100 full-length L1s potentially capable of retrotransposition in the diploid genome. L1 retrotransposition may be detrimental to the host and thus needs to be controlled. Previous studies have identified sense and antisense promoters in the 5' UTR of full-length human L1. Here we show that the resulting bidirectional transcripts can be processed to small interfering RNAs (siRNAs) that suppress retrotransposition by an RNA interference (RNAi) mechanism. We thus provide evidence that RNAi triggered by antisense transcripts may modulate human L1 retrotransposition efficiently and economically. L1-specific siRNAs are among the first natural siRNAs reported in mammalian systems. This work may contribute to understanding the regulatory role of abundant antisense transcripts in eukaryotic genomes.
No preview · Article · Oct 2006 · Nature Structural & Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are endogenous noncoding RNAs, which negatively regulate gene expression. To determine genomewide miRNA DNA copy number abnormalities in cancer, 283 known human miRNA genes were analyzed by high-resolution array-based comparative genomic hybridization in 227 human ovarian cancer, breast cancer, and melanoma specimens. A high proportion of genomic loci containing miRNA genes exhibited DNA copy number alterations in ovarian cancer (37.1%), breast cancer (72.8%), and melanoma (85.9%), where copy number alterations observed in >15% tumors were considered significant for each miRNA gene. We identified 41 miRNA genes with gene copy number changes that were shared among the three cancer types (26 with gains and 15 with losses) as well as miRNA genes with copy number changes that were unique to each tumor type. Importantly, we show that miRNA copy changes correlate with miRNA expression. Finally, we identified high frequency copy number abnormalities of Dicer1, Argonaute2, and other miRNA-associated genes in breast and ovarian cancer as well as melanoma. These findings support the notion that copy number alterations of miRNAs and their regulatory genes are highly prevalent in cancer and may account partly for the frequent miRNA gene deregulation reported in several tumor types.
Full-text · Article · Jun 2006 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The protein kinase C (PKC) family plays a key regulatory role in a wide range of cellular functions as well as in various cancer-associated signal transduction pathways. Here, we investigated the genomic alteration and gene expression of most known PKC family members in human ovarian cancer. The DNA copy number of PKC family genes was screened by a high-resolution array-based comparative genomic hybridization in 89 human ovarian cancer specimens. Five PKC genes exhibited significant DNA copy number gains, including PKCiota (43.8%), PKCbeta1 (37.1%), PKCgamma (27.6%), PKCzeta (22.5%), and PKCtheta (21.3%). None of the PKC genes exhibited copy number loss. The mRNA expression level of PKC genes was analyzed by microarray retrieval approach. Two of the amplified PKC genes, PKCiota and PKCtheta, were significantly up-regulated in ovarian cancer compared with normal ovary. Increased PKCiota expression correlated with tumor stage or grade, and PKCiota overexpression was seen mostly in ovarian carcinoma but not in other solid tumors. The above results were further validated by real-time reverse transcription-PCR with 54 ovarian cancer specimens and 24 cell lines; overexpression of PKCiota protein was also confirmed by tissue array and Western blot. Interestingly, overexpressed PKCiota did not affect ovarian cancer cell proliferation or apoptosis in vitro. However, decreased PKCiota expression significantly reduced anchorage-independent growth of ovarian cancer cells, whereas overexpression of PKCiota contributed to murine ovarian surface epithelium transformation in cooperation with mutant Ras. We propose that PKCiota may serve as an oncogene and a biomarker of aggressive disease in human ovarian cancer.
[Show abstract][Hide abstract] ABSTRACT: Epithelial ovarian cancer is the most frequent cause of gynecologic malignancy-related mortality in women. To identify genes up-regulated in ovarian cancer, PCR-select cDNA subtraction was done and Drosophila Eyes Absent Homologue 2 (EYA2) was isolated as a promising candidate. The transcriptional coactivator eya controls essential cellular functions during organogenesis of Drosophila. EYA2 mRNA was found to be up-regulated in ovarian cancer by real-time reverse transcription-PCR, whereas its protein product was detected in 93.6% of ovarian cancer specimens by immunohistochemistry (n = 140). EYA2 was amplified in 14.8% of ovarian carcinomas, as detected by array-based comparative genomic hybridization (n = 88). Most importantly, EYA2 overexpression was significantly associated with short overall survival in advanced ovarian cancer (n = 99, P = 0.0361). EYA2 was found to function as transcriptional activator in ovarian cancer cells by Gal4 assay and to promote tumor growth in vivo in xenograft models. Therefore, this study suggests an important role of EYA2 in ovarian cancer and its potential application as a therapeutic target.
[Show abstract][Hide abstract] ABSTRACT: RNA interference (RNAi) is widely used for functional studies and has been proposed as a potential therapeutic agent. Current
RNAi systems are largely efficient, but have limitations including transient effect, the need for viral handling and potential
insertional mutations. Here, we describe a simple L1 retrotransposon-based system for the delivery of small interfering RNA
(siRNA) and stable silencing in human cells. This system demonstrated long-term siRNA expression and significant reduction
in both exogenous and endogenous gene expression by up to 90%. Further characterization indicated that retrotransposition
occurred in a controlled manner such that essentially only one RNAi-cassette was integrated into the host genome and was sufficient
for strong interference. Our system provides a novel strategy for stable gene silencing that is easy and efficient, and it
may have potential applications for ex vivo and in vivo molecular therapy.
Preview · Article · Feb 2005 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylinositol 3-kinase (PI3-kinase) is a novel intracellular transducer involved in a wide range of cancer-associated signaling pathways, which comprises various isoforms and splice variants with distinct biologic activities and clinical implications. Especially, the class Ia PI3-kinase 110 kD catalytic subunit alpha (PIK3CA) is the most important isoform in tumorigenesis and possibly, tumor angiogenesis. Several strategies have been developed to block PI3-kinase for cancer therapy; however, the approach to target specific PI3-kinase isoform has not been explored to date. In the present study, we show that RNA interference (RNAi) through small interfering (siRNA) sequences targeting PIK3CA has potential applications in isoform-specific "knock-down" of PI3-kinase. This strategy provides a novel tool to study the function of various PI3-kinase isoforms and may contribute to isoform-specific targeting of PI3-kinase in human cancer.
Full-text · Article · Jan 2005 · Cancer biology & therapy
[Show abstract][Hide abstract] ABSTRACT: LINE‐1s (long interspersed nuclear elements‐1) are abundant non‐LTR retrotransposons that comprise 17% of the human genome. The 5′ untranslated region (5′UTR)
of human L1 (L1Hs) houses a poorly understood internal promoter. Here we report that mutations at a putative runt‐domain transcription
factor (RUNX) site (+83 to +101) in the 5′UTR decreased L1Hs transcription and retrotransposition in cell culture‐based assays.
Exogenous expression of RUNX3, but not the other two RUNX family members, RUNX1 and RUNX2, increased L1Hs transcription and
retrotransposition, which were otherwise decreased by siRNAs targeting RUNX3 and a dominant negative RUNX. Further more, the
specific interaction between RUNX3 and its binding site was demonstrated by an electrophoretic mobility shift assay (EMSA)
using an anti‐RUNX3 antibody. Interestingly, RUNX3 may also regulate the antisense promoter activity of L1Hs 5′UTR via another
putative RUNX site (+526 to +508), as revealed by site‐directed mutations and exogenous expression of RUNX factors. Our results
indicate an important role for RUNX3 in L1Hs retrotransposition as well as transcription from its 5′UTR in both sense and
antisense directions, and they should contribute to our understanding of the mechanism underlying L1Hs retrotransposition
and its impact on the expression of adjacent cellular genes.
Preview · Article · Sep 2003 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: The gene of phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) has been implicated as an oncogene in ovarian cancer [L. Shayesteh et al., Nat. Genet., 21: 99-102, 1999]. In this study, we examined the expression of PIK3CA mRNA and its p110alpha protein product in human ovarian carcinoma and investigated its role in regulating angiogenesis via vascular endothelial growth factor (VEGF). PIK3CA mRNA was detected in 66.6% of stage I and 93.9% of advanced stage ovarian cancer specimens and in all 17 ovarian cancer cell lines. PIK3CA mRNA levels were significantly higher in invasive carcinomas compared with benign and low malignant potential neoplasms (P = 0.007), but no significant difference was seen between early and advanced stage carcinomas (P = 0.812). Strong expression of immunoreactive p110alpha was detected in tumor cells and/or stroma endothelium. PIK3CA expression in vivo positively correlated, both at the mRNA and the protein level, with the expression of VEGF as well as with the extent of microvascular development. Furthermore, PIK3CA mRNA overexpression positively correlated with increased proliferation and decreased apoptosis of tumor cells in vivo. In vitro, PIK3CA expression positively correlated with the expression of VEGF in ovarian cancer cells, whereas the phosphatidylinositol 3'-kinase inhibitor Ly294002 reduced both the constitutive and inducible expression of hypoxia-inducible factor-1alpha at the mRNA and protein levels and abrogated VEGF up-regulation by glucose starvation. Furthermore, Ly294002 suppressed cell proliferation and, at higher doses, induced marked apoptosis in ovarian cancer cells. Collectively, these data strongly indicate that PIK3CA supports ovarian cancer growth through multiple and independent pathways affecting cell proliferation, apoptosis and angiogenesis, and plays an important role in ovarian cancer progression.
[Show abstract][Hide abstract] ABSTRACT: Vascular remodeling in host tissues surrounding growing tumors is implicated in the successful development of tumor neovasculature. Cooperation between vascular endothelial growth factor (VEGF) and angiopoietins (Angs) is considered to be critical in this context. However, the mechanisms regulating the coordinated expression of these molecules remain, to date, elusive. In this study, we used a murine ovarian cancer angiogenesis model induced by overexpression of VEGF, as well as 52 human ovarian cancer specimens and 36 established cancer cell lines to characterize the expression and regulation of Ang-2 in the context of tumor angiogenesis. Using a combination of immunohistochemistry, laser capture microdissection and real-time quantitative reverse transcription-PCR, we showed that tumor-derived VEGF significantly up-regulated the expression of Ang-2 in host stroma endothelial cells, resulting in markedly increased Ang-2/Tie-2 mRNA copy number ratio in vivo. In vitro experiments showed that VEGF directly up-regulated Ang-2, which is mediated via VEGF receptor-2/flk-1/KDR pathway, in cultured endothelial cells through transcriptional activation rather than the enhanced mRNA stability. In human ovarian cancer, Ang-2 was primarily expressed in stroma endothelial cells and detectable in tumor cells of only 12% tumor specimens; however, it was not detected in the majority of established ovarian cancer cell lines. In addition, a significant correlation was observed between VEGF and Ang-2 mRNA expression (P < 0.01) but not between VEGF and Ang-1 or Tie-2 in human ovarian cancer specimens. In the mouse ovarian cancer model, up-regulation of Ang-2 in host stroma endothelial cells was significantly associated with pericyte loss and instability of the host vasculature surrounding the tumor. Our study suggests a novel mechanism by which tumor-derived VEGF interacts with Angs/Tie-2 system in host stroma endothelial cells and induces in a paracrine manner the remodeling of host vasculature to support angiogenesis during tumor growth.
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) carries out multifaceted functions in tumor development, and it exists as at least five isoforms with distinct biologic activities and clinical implications. Several strategies have been developed to block VEGF for cancer therapy; however, the approach to target-specific VEGF isoform(s) has not been explored to date. In the present study, we show that DNA vector-based RNA interference (RNAi), in which RNAi sequences targeting murine VEGF isoforms are inserted downstream of an RNA polymerase III promoter, has potential applications in isoform-specific "knock-down" of VEGF. Large molecular weight VEGF isoforms were specifically reduced in vitro in the presence of isoform-specific RNAi constructs. Additionally, H1 promoter may be superior to U6 promoter when used for vector-based RNAi of VEGF isoforms. This strategy provides a novel tool to study the function of various VEGF isoforms and may contribute to VEGF isoform-specific treatment in cancer.
No preview · Article · May 2003 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: The first tissue-specific angiogenic molecule, endocrine gland-derived vascular endothelial growth factor (EG-VEGF), was identified recently in human ovary, raising hopes of developing tumor type-specific angiogenesis inhibitors. In the present study, we analyzed the expression of EG-VEGF mRNA in normal human tissues and ovarian neoplasms by quantitative real-time reverse transcription-PCR. EG-VEGF mRNA was expressed in all ovarian neoplasms examined. No significant difference was identified among benign, low malignant potential neoplasms or stage I ovarian cancer, all of which exhibited 2-fold lower mRNA levels compared with normal premenopausal ovaries. EG-VEGF mRNA levels further decreased in late stage compared with early stage carcinomas (P < 0.05) and were consistently lower in laser capture microdissected tumor islets compared with surrounding stroma. EG-VEGF was undetectable by reverse transcription-PCR in 17 established epithelial ovarian cancer cell lines or in cultured human ovarian surface epithelial cells, whereas it was detected in peripheral blood as well as tumor-infiltrating T lymphocytes. Finally, in contrast to VEGF, EG-VEGF mRNA levels did not correlate with clinical outcome in advanced ovarian carcinoma. These results suggest that EG-VEGF is most likely derived from nonepithelial components of ovarian carcinomas and may play a marginal role in promoting angiogenesis in advanced ovarian carcinoma. We postulate that EG-VEGF-targeted antiangiogenic therapy may prove useful in early stage but not in advanced stage ovarian carcinoma.
No preview · Article · Jan 2003 · Clinical Cancer Research