Antonis Giannakakis

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.

Reactive oxygen species and HIF-1 signalling in cancer.

The heterodimeric transcription factor HIF-1 (hypoxia-inducible factor 1) represents the key mediator of hypoxia response. HIF-1 controls numerous genes of pivotal importance for cellular metabolism, angiogenesis, cell cycle regulation and inhibition of apoptosis. HIF-1 overexpression and enhanced transcriptional activity are linked to tumour initiation and progression. Malfunction of the HIF-1 signalling network has been associated with breast, ovarian and prostate cancers. Elevated reactive oxygen species (ROS), also observed in such tumours, have been implicated in HIF-1 signalling. Deciphering the role of ROS in cancer onset and their involvement in signalling networks should prove invaluable for the design of novel anticancer therapeutics.

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.

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.

miRNA genetic alterations in human cancers

MicroRNAs (miRNAs) are endogenous, non-coding, small RNAs, which negatively regulate gene expression in a sequence-specific manner via translational repression and/or mRNA degradation. Their discovery revealed a new and exciting aspect of post-transcriptional gene regulation that is universally involved in cellular homeostasis. Importantly, the advent of miRNAs added another level of complication in the already complex regulatory networks of the cell, undermining that RNA molecules in general, should be considered gene regulators of equal importance with proteins. Recently, the scientific community drew attention to the miRNA field for an additional reason: an increasing line of evidence indicated that miRNA genes are tightly connected with the process of tumorigenesis. Indeed, several miRNAs have already been demonstrated to behave as oncogenes or tumor suppressor genes in many types of cancer. Even though the underlying mechanisms by which miRNAs can destabilize the normal cellular processes, promoting cell transformation and tumor progression, are not well understood, genetic and epigenetic alterations most probably play a critical role. Significant technologic advances facilitated the profiling of the miRNA expression patterns in normal and cancer tissues and discovered an unexpected greater reliability of miRNA expression signatures in classifying cancer types than the respective signatures of protein-coding genes. Along with this extraordinary diagnostic potential, miRNAs have also proved their prognostic value in predicting clinical behaviors of cancer patients. The aim of this review is to describe miRNA expression and how its deregulation is involved in the pathophysiology of human cancers.

Contrasting expression patterns of coding and noncoding parts of the human genome upon oxidative stress

Oxidative stress (OS) is caused by an imbalance between pro- and anti-oxidant reactions leading to accumulation of reactive oxygen species within cells. We here investigate the effect of OS on the transcriptome of human fibroblasts. OS causes a rapid and transient global induction of transcription characterized by pausing of RNA polymerase II (PolII) in both directions, at specific promoters, within 30 minutes of the OS response. In contrast to protein-coding genes, which are commonly down-regulated, this novel divergent, PolII pausing-phenomenon leads to the generation of thousands of long noncoding RNAs (lncRNAs) with promoter-associated antisense lncRNAs transcripts (si-paancRNAs) representing the major group of stress-induced transcripts. OS causes transient dynamics of si-lncRNAs in nucleus and cytosol, leading to their accumulation at polysomes, in contrast to mRNAs, which get depleted from polysomes. We propose that si-lncRNAs represent a novel component of the transcriptional stress that is known to determine the outcome of immediate-early and later cellular stress responses and we provide insights on the fate of those novel mature lncRNA transcripts by showing that their association with polysomal complexes is significantly increased in OS.

Biochemical and molecular analysis of the interaction between ERK2 MAP kinase and hypoxia inducible factor-1α

The mitogen activated protein kinase (MAPK) signaling pathways play significant roles in fundamental cellular processes, such as cell growth and differentiation. It has been shown that the specificity and efficacy of phosphorylation by MAP kinases rely upon distinct MAPK-docking domains (D-domains) found in a wide range of MAPK substrates including the ETS-transcription factor Elk-1. Importantly, the MAPK signaling cascade converges with the hypoxia-induced signaling pathway. The key regulator of hypoxia signaling is the heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1). The α-subunit of HIF-1 (HIF-1α) is a substrate for the ERK2 MAP kinase. Unraveling the interplay of these main signaling systems is a prerequisite for understanding their role in tumor growth, a situation sustained by simultaneous mitogenic and hypoxic signals. In this work, we investigated the molecular cues that direct HIF-1α recognition and phosphorylation by ERK2. We showed that HIF-1α possesses a MAPK docking domain. Utilizing surface plasmon resonance (SPR) methodologies we demonstrated efficient binding between HIF-1α and ERK2, with a K(D) value in the low micromolar range. Although, the D-domain did not contribute to the above interaction significantly, it could act in trans by recruiting ERK2 and conferring responsiveness to poor ERK substrates. These results indicate that, via its conserved D-domain, HIF-1α could serve as a platform for ERK2 in the nucleus of the cell, thus potentially facilitating phosphorylation of other ERK2 substrates. The identification of an ERK2 recognition domain on HIF-1α opens new avenues for the analysis of HIF-1α-related ERK2 signaling and may allow designing of interfering compounds.

Overexpression of SMARCE1 is associated with CD8+ T-cell infiltration in early stage ovarian cancer.

T-lymphocyte infiltration in ovarian tumors has been linked to a favorable prognosis, hence, exploring the mechanism of T-cell recruitment in the tumor is warranted. We employed a differential expression analysis to identify genes over-expressed in early stage ovarian cancer samples that contained CD8 infiltrating T-lymphocytes. Among other genes, we discovered that TTF1, a regulator of ribosomal RNA gene expression, and SMARCE1, a factor associated with chromatin remodeling were overexpressed in first stage CD8+ ovarian tumors. TTF1 and SMARCE1 mRNA levels showed a strong correlation with the number of intra-tumoral CD8+ cells in ovarian tumors. Interestingly, forced overexpression of SMARCE1 in SKOV3 ovarian cancer cells resulted in secretion of IL8, MIP1b and RANTES chemokines in the supernatant and triggered chemotaxis of CD8+ lymphocytes in a cell culture assay. The potency of SMARCE1-mediated chemotaxis appeared comparable to that caused by the transfection of the CXCL9 gene, coding for a chemokine known to attract T-cells. Our analysis pinpoints TTF1 and SMARCE1 as genes potentially involved in cancer immunology. Since both TTF1 and SMARCE1 are involved in chromatin remodeling, our results imply an epigenetic regulatory mechanism for T-cell recruitment that invites deciphering.