Jia Huang

Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer

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 ≈15% and at least ≈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.

miR-210 links hypoxia with cell cycle regulation and is deleted in human epithelial ovarian cancer.

Tumor growth results in hypoxia. Understanding the mechanisms of gene expression reprogramming under hypoxia may provide important clues to cancer pathogenesis. We studied miRNA genes that are regulated by hypoxia in ovarian cancer cell lines by TaqMan miRNA assay containing 157 mature miRNAs. MiR-210 was the most prominent miRNA consistently stimulated under hypoxic conditions. We provide evidence for the involvement of the HIF signaling pathway in miR-210 regulation. Biocomputational analysis and in vitro assays demonstrated that e2f transcription factor 3 (e2f3), a key protein in cell cycle, is regulated by miR-210. E2F3 was further confirmed to be downregulated at the protein level upon induction of miR-210. Importantly, we found remarkably high frequency of miR-210 gene copy deletions in ovarian cancer patients (64%, n=114) and that gene copy number correlates with miR-210 expression levels. Taken together, our results indicate that miR-210 plays a crucial role in tumor onset as a key regulator of the hypoxia response and provide evidence for a link between hypoxia and the regulation of cell cycle.

Distinct Genomic Profiles in Hereditary Breast Tumors Identified by Array-Based Comparative Genomic Hybridization

Mutations in BRCA1 and BRCA2 account for a significant proportion of hereditary breast cancers. Earlier studies have shown that inherited and sporadic tumors progress along different somatic genetic pathways and that global gene expression profiles distinguish between these groups. To determine whether genomic profiles similarly discriminate among BRCA1, BRCA2, and sporadic tumors, we established DNA copy number profiles using comparative genomic hybridization to BAC-clone microarrays providing <1 Mb resolution. Tumor DNA was obtained from BRCA1 (n = 14) and BRCA2 (n = 12) mutation carriers, as well as sporadic cases (n = 26). Overall, BRCA1 tumors had a higher frequency of copy number alterations than sporadic breast cancers (P = 0.00078). In particular, frequent losses on 4p, 4q, and 5q in BRCA1 tumors and frequent gains on 7p and 17q24 in BRCA2 tumors distinguish these from sporadic tumors. Distinct amplicons at 3q27.1-q27.3 were identified in BRCA1 tumors and at 17q23.3-q24.2 in BRCA2 tumors. A homozygous deletion on 5q12.1 was found in a BRCA1 tumor. Using a set of 169 BAC clones that detect significantly (P < 0.001) different frequencies of copy number changes in inherited and sporadic tumors, these could be discriminated into separate groups using hierarchical clustering. By comparing DNA copy number and RNA expression for genes in these regions, several candidate genes affected by up- or down-regulation were identified. Moreover, using support vector machines, we correctly classified BRCA1 and BRCA2 tumors (P < 0.0000004 and 0.00005, respectively). Further validation may prove this tumor classifier to be useful for selecting familial breast cancer cases for further mutation screening, particularly, as these data can be obtained using archival tissue.

Integrative Genomic Analysis of Protein Kinase C (PKC) Family Identifies PKCι as a Biomarker and Potential Oncogene in Ovarian Carcinoma

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.

Integrative genomic analysis of phosphatidylinositol 3'-kinase family identifies PIK3R3 as a potential therapeutic target in epithelial ovarian cancer.

Purpose: 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. Experimental Design: 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 p55γ protein in ovarian cancer was analyzed on tissue arrays. Small interfering RNA was used to study the function of PIK3R3 in ovarian cancer. Results: 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 p55γ 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. Conclusion: We propose that PIK3R3 may serve as a potential therapeutic target in human ovarian cancer.

Frequent genetic abnormalities of the PI3K/AKT pathway in primary ovarian cancer predict patient outcome

Identification and characterization of underlying genetic aberrations could facilitate diagnosis and treatment of ovarian cancer. Copy number analysis using array Comparative Genomic Hybridization (aCGH) on 93 primary ovarian tumors identified PI3K/AKT pathway as the most frequently altered cancer related pathway. Furthermore, survival analyses to correlate gene copy number and mutation data with patient outcome showed that copy number gains of PIK3CA, PIK3CB, and PIK3R4 in these tumors were associated with decreased survival. To confirm these findings at the protein level, immunohistochemistry (IHC) for PIK3CA product p110α and p‐Akt was performed on tissue microarrays from 522 independent serous ovarian cancers. Overexpression of either of these two proteins was found to be associated with decreased survival. Multivariant analysis from these samples further showed that overexpression of p‐AKT and/or p110α is an independent prognostic factor for these tumors. siRNAs targeting altered PI3K/AKT pathway genes inhibited proliferation and induced apoptosis in ovarian cancer cell lines. In addition, the effect of the siRNAs in different cell lines seemed to correlate with the particular genetic alterations that the cell line carries. These results strongly support the utilization of PI3K pathway inhibitors in ovarian cancer. They also suggest identifying the specific component in the PI3K pathway that is genetically altered has the potential to help select the most effective therapy. Both mutation as well as copy number changes can be used as predictive markers for this purpose.

Transcriptional Regulation of PIK3CA Oncogene by NF-κB in Ovarian Cancer Microenvironment

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-κB pathway. These results offer a new mechanism through which the tumor microenvironment directly activates oncogenic pathways in tumor cells.

Estrogen Receptor Status Could Modulate the Genomic Pattern in Familial and Sporadic Breast Cancer

Purpose: Familial breast cancer represents 5% to 10% of all breast tumors. Mutations in the two known major breast cancer susceptibility genes, BRCA1 and BRCA2, account for a minority of familial breast cancer, whereas families without mutations in these genes (BRCAX group) account for 70% of familial breast cancer cases. Experimental Design: To better characterize and define the genomic differences between the three classes of familial tumors and sporadic malignancies, we have analyzed 19 BRCA1, 24 BRCA2, and 31 BRCAX samples from familial breast cancer patients and 19 sporadic breast tumors using a 1-Mb resolution bacterial artificial chromosome array-based comparative genomic hybridization. Results: We found that BRCA1/2 tumors showed a higher genomic instability than BRCAX and sporadic cancers. There were common genomic alterations present in all breast cancer groups, such as gains of 1q and 16p or losses of 8ptel-p12 and 16q. We found that the presence/absence of the estrogen receptor (ER) may play a crucial role in driving tumor development through distinct genomic pathways independently of the tumor type (sporadic or familial) and mutation status (BRCA1 or BRCA2). ER− tumors presented higher genomic instability and different altered regions than ER+ ones. Conclusions: According to our results, the BRCA gene mutation status (mainly BRCA1) would contribute to the genomic profile of abnormalities by increasing or modulating the genome instability.

Distinct MHC gene expression patterns during progression of melanoma

Abnormal expression of major histocompatibility complex (MHC) molecules in melanoma has been reported previously. However, the MHC molecule expression patterns in different growth phases of melanoma and the underlying mechanisms are not well understood. Here, we demonstrate that in vertical growth phase (VGP) melanomas, MHC genes are subject to increased rates of DNA copy number gains, accompanied by increased expression, in comparison to normal melanocytes. In contrast, MHC expression in metastatic melanomas drastically decreased compared to VGP melanomas, despite still prevalent DNA copy number gains. Subsequent investigations found that the master transactivator of MHC genes, CIITA, was also significantly downregulated in metastatic melanomas when compared to VGP melanomas. This could be one of the mechanisms accounting for the discrepancy between DNA copy number and expression level in metastatic melanomas, a potentially separate mechanism of gene regulation. These results infer a dynamic role of MHC function in melanoma progression. We propose potential mechanisms for the overexpression of MHC molecules in earlier stages of melanoma as well as for its downregulation in metastatic melanomas.

MULTIPLE INITIAL CULTURE CONDITIONS ENHANCE THE ESTABLISHMENT OF CELL LINES FROM PRIMARY OVARIAN CANCER SPECIMENS

SummaryTo increase the efficiency of stable cell line establishment from primary ovarian cancer specimens, we simultaneously initiated cultures under, multiple conditions, varying extracellular matrices and the inclusion of supplements (e.g., serum or serum albumin), while minimizing exposure to xenogeneic antigens (e.g., fetal calf serum). Primary cultures were initiated from 30 specimens; cell lines were established from 10 of these for a success rate of 33%. In some instances, multiple cell lines were established from the same specimen. Five lines were characterized extensively with respect to growth properties, antigen expression, and genomic alterations. Although these lines are all low-passage, marked heterogeneity was observed, even between lines derived from the same specimen. The culture approach outlined herein will facilitate generation of reagents useful for many aspects of ovarian cancer biology.