Transcriptional Regulation of Chemokine Expression in Ovarian Cancer

The increased expression of pro-inflammatory and pro-angiogenic chemokines contributes to ovarian cancer progression through the induction of tumor cell proliferation, survival, angiogenesis, and metastasis. The substantial potential of these chemokines to facilitate the progression and metastasis of ovarian cancer underscores the need for their stringent transcriptional regulation. In this Review, we highlight the key mechanisms that regulate the transcription of pro-inflammatory chemokines in ovarian cancer cells, and that have important roles in controlling ovarian cancer progression. We further discuss the potential mechanisms underlying the increased chemokine expression in drug resistance, along with our perspective for future studies.


Introduction
Chemokines are a family of cytokines that induce chemotaxis of target cells. Though they were originally discovered for their ability to induce leukocyte migration into the infected or injured sites, more recently, it became clear that they could also promote cancer progression [1][2][3][4][5][6][7][8][9]. In addition to inducing tumor cell proliferation, angiogenesis and metastasis, chemokines and their receptors regulate tumor cell differentiation and survival. Currently, the human chemokine network includes more than OPEN ACCESS 45 known chemokines and 20 chemokine receptors. Based on the number and spacing of conserved N-terminal cysteine residues that form disulfide bonds, chemokines are divided into four groups: (X)C, CC, CXC, and CX3C [10][11][12].
Epithelial ovarian cancer (EOC) is among the leading causes of cancer death in women. Since most ovarian cancers relapse and become drug-resistant, the survival rates remain low. Progression of ovarian cancer (OC) has been associated with the increased expression and release of pro-inflammatory chemokines, which contribute to ovarian cancer development through their induction of tumor cell proliferation, survival, migration, and angiogenesis [13][14][15]. The chemokine expression by ovarian cancer cells is controlled at several levels that include transcriptional regulation, post-transcriptional regulation and regulation of mRNA stability, translation, and mechanisms regulating the cytokine intracellular storage, transport, and release. Table 1 summarizes chemokines produced by ovarian cancer cells. Several excellent reviews have addressed the physiological and cellular functions of these chemokines in ovarian cancer [9,16,17]. Thus, in this review, we focus instead on the main mechanisms that regulate transcription of these chemokines in ovarian cancer cells.

Chemokine Regulation by NFțB and Epigenetic Acetylation
Chemokines are regulated at the transcriptional level by binding of transcription factors and repressors to gene promoter and enhancer regions. The transcription factors that control the expression of most inflammatory chemokines include the nuclear factor-țB (NFțB), activator protein-1 (AP-1) and the signal transducers and activators of transcription (STAT) family. The NFțB activity is constitutively increased in aggressive ovarian cancers, and inhibition of NFțB signaling suppresses angiogenesis and tumorigenicity of ovarian cancer cells and increases their sensitivity to chemotherapy and apoptosis [37][38][39][40]. The underlying mechanisms likely involve the NFțB-regulated chemokine expression, since several studies have demonstrated that the expression of CCL2, CXCL1, CXCL2, and IL-8/CXCL8 is mediated by NFțB in ovarian cancer cells [28][29][30]41].
The increased activity of NFțB in ovarian cancer cells is mediated by enzymes of the IțB kinase (IKK) complex, which phosphorylate the NFțB inhibitory protein, IțBĮ, resulting in IțBĮ proteasomal degradation and nuclear translocation of NFțB subunits [42][43][44][45]. In addition to phosphorylating IțBĮ, IKKs can also phosphorylate the NFțB subunits, particularly p65 [46]. While the cytoplasmic degradation of IțBĮ, resulting in the nuclear translocation of NFțB subunits, represents a general step in NFțB activation, the specificity of NFțB-regulated responses is mediated by the subunit composition of NFțB complexes and their post-translational modifications [47,48].
In addition to transcription factor binding to promoter sequences, chemokine expression is regulated by epigenetic modifications that include histone modifications as well as post-translational modifications of transcription factors, particularly the p65 subunit of NFțB. It is believed that while histone acetylation and acetylation of transcription factors induced by histone acetyl transferases (HATs) generally promotes transcriptional activation, hypoacetylation induced by histone deacetylase (HDAC) activity is associated with transcriptional repression. Since hypoacetylation of tumor suppressor genes by HDACs has been linked to tumor development, HDACs inhibitors are now being evaluated for their therapeutic effects in cancer, including ovarian cancer [49][50][51]. Clinical studies using HDAC inhibitors in the treatment of ovarian cancer are summarized in the recent elegant review by Khabele [52]. Numerous studies have shown that HDACs regulate chemokine expression in different cell types [53][54][55][56][57][58]; however, their role in the regulation of chemokine expression in ovarian cancer has yet to be documented.

Chemokine Modulation by Hypoxia and Metabolism
Ovarian cancer tissues and ascites are characterized by decreased oxygen content, which stabilizes the Į-subunit of the transcription factor hypoxia-inducible factor-1 (Hif-1) [59]. Hif-1 responds to hypoxia by increasing the transcription of genes that promote survival in low-oxygen conditions, thus promoting angiogenesis and oncogenesis. Indeed, the increased expression of Hif-1 has been detected in epithelial ovarian cancer, and correlates with poor prognosis [60][61][62]. Hypoxia induces IL-8 [30], CXCL12 [63], and CCL28 [24] expression in ovarian cancer cells. The seminal study by Xu et al. [30] demonstrated that hypoxic conditions increase the IL-8 expression in ovarian cancer cells by increasing NFțB and AP-1 binding to IL-8 promoter. The mechanisms of how hypoxia increases the NFțB-dependent IL-8 transcription involve activation of the transforming growth factor beta-activated kinase 1 (TAK1), resulting in increased IKK activation, and p65 NFțB recruitment to the IL-8 promoter [64,65]. In addition, hypoxia induces a direct binding of Hif-1Į to the hypoxia-response element (HRE) located next to the NFțB binding site in human IL-8 promoter, resulting in the increased IL-8 expression [66].
One of the consequences of Hif-1 activation is the increased expression of glycolytic genes, resulting in increased aerobic glycolysis, glucose consumption, and lactic acid production (Warburg effect) [67][68][69]. The high rate of glucose consumption and lactic acid production contributes to the acidification of the tumor environment and cancer progression. Xu et al. showed that acidic pH increases the IL-8 transcription by enhancing the binding of AP-1 and NFțB to IL-8 promoter in ovarian cancer cells [70]. In addition, in endothelial cells, lactate was shown to activate the NFțB-dependent IL-8 transcription by inducing degradation of IțBĮ [71]. The role of lactate and other metabolites of the glycolytic pathway in the regulation of pro-angiogenic chemokine expression in ovarian cancer cells is yet to be investigated, especially since recent studies have indicated high levels of aerobic glycolysis and lactate production in ovarian tumors [72,73].
While hyperglycemia and obesity are thought to be contributing factors to cancer development and progression, caloric restriction has been associated with reduced cancer incidence [74][75][76][77]. During reduced calorie intake or exercise, the body switches to obtaining energy from fatty acid oxidation, which results in ketone bodies production. Intriguingly, the recent study by Shimazu et al. [78] has demonstrated that the ketone body ȕ-hydroxybutyrate (ȕOHB) is an endogenous and specific inhibitor of HDACs, and that administration of exogenous ȕOHB increases histone acetylation, correlating with changes in transcription. Since HDACs regulate chemokine transcription by both deacetylating histones and p65 NFțB [53][54][55][56][57][58], it will be important to analyze whether ȕOHB and other HDAC inhibitors regulate chemokine expression in ovarian cancer cells, and whether this is modulated by the metabolic state.

Chemokine Modulation by Chemotherapeutic Interventions
There is growing evidence that the increased chemokine expression by tumor cells modulates not only cancer development but also cancer responsiveness and resistance to chemotherapy [79]. A major contributor to the acquired chemoresistance of ovarian cancer cells is the increased expression of NFțB-dependent chemokines that is induced by the platinum-based drugs carboplatin and cisplatin, and by the mitotic inhibitors docetaxel and paclitaxel [29, [80][81][82][83]. The mechanisms responsible for the increased IL-8 expression induced by paclitaxel in ovarian cancer cells involve increased expression of toll-like receptors (TLRs) and increased p65 NFțB binding to IL-8 promoter [80,83].
Bortezomib (BZ) is the first FDA approved proteasome inhibitor, which has shown a limited effectiveness in ovarian cancer treatment as a single agent [84][85][86][87]. However, BZ has been considered in combination with cisplatin, since BZ prevents the cisplatin-induced degradation of cisplatin influx transporter, resulting in enhanced cisplatin uptake and tumor cell killing [88,89]. We have recently shown that BZ increases expression of IL-8 and CCL2 in ovarian cancer cells, while it does not affect expression of other NFțB-dependent genes. The responsible mechanisms involve a gene specific and IKKȕ-dependent recruitment of S536 phosphorylated p65 NFțB to IL-8 and CCL2 promoters, suggesting that anti-inflammatory therapy targeting IKKȕ might increase the BZ effectiveness in ovarian cancer treatment [41]. Since approximately 50% of women diagnosed with ovarian cancer die from chemoresistant metastatic disease, understanding the molecular mechanisms by which chemotherapeutic interventions increase the chemokine expression in ovarian cancer cells should lead to the development of more effective combination strategies.

Chemokine Transcriptional Regulation in Ovarian Cancer Cells
Chemokines listed in Table 1 have all been identified in ovarian cancer cells and tissues. Various online databases can be used to assess putative transcription factor binding sites. For this review, we have obtained chemokine promoter sequences from the NCBI database and used the Alggen promoter-mapping program to search for the transcription factor binding sites [90,91]. All found putative binding sites are listed in Tables 2-5; the binding sites that have been experimentally confirmed are highlighted in bold and labeled with an asterisk. Below, we limit discussion of the transcriptional mechanisms only to the chemokines that have been experimentally confirmed in ovarian cancer cells. While the first insights into the chemokine transcriptional regulation were obtained by using in vitro electrophoretic mobility shift assays (EMSA) or overexpression experiments, chromatin immunoprecipitations (ChIP) generally provides a more realistic picture about the transcription factor binding to endogenous promoter sequences in living cells.

CCL2
CCL2 (MCP-1) is an important determinant of macrophage infiltration in ovarian tumors [92,93]. Although CCL2 has been originally thought to have an inhibitory effect on ovarian cancer progression [94][95][96], recent studies have indicated that CCL2 increases invasion of ovarian cancer cells and resistance to chemotherapy [97,98]. The putative transcription factor binding sites identified in human CCL2 promoter are listed in Table 2. Experimental studies demonstrated binding of NFțB, STAT1, STAT3, AP-1, and Hif-1Į to the CCL2 promoter in OC cells (Figure 1).
Even though the NFțB binding site is located in the distal regulatory region of human CCL2 promoter (Figure 1), several studies have demonstrated p65 NFțB involvement in the regulation of CCL2 expression in OC cells [27,41,99]. In addition, CCL2 expression is regulated by IKKȕ-dependent recruitment of the transcription factor EGR-1, and inhibition of IKKȕ activity decreases p65 and EGR-1 promoter recruitment and CCL2 expression [41]. Interestingly, the NFțB binding site in human CCL2 promoter has the same nucleotide sequence as the NFțB site in human IL-8/CXCL8 promoter. Curiously, both CCL2 and IL-8 are increased by paclitaxel [83] and bortezomib [41], indicating that the paclitaxel and BZ-induced CCL2 (and IL-8) increase is promoter specific.

Activity of the transcription factors STAT-1 and STAT-3 is also constitutively increased in OC cells,
where it promotes cell motility and invasiveness [100]. Phosphorylation of STAT3 at tyrosine residues 705 and 727 increases its transcriptional activity [101]. In OC cells, IL-6 [102] and M-CSF [103] induce phosphorylation and activation of STAT3, and increase the CCL2 expression. In addition to NFțB and STAT transcription factors, studies in other cell types indicated that the CCL2 expression is positively regulated by AP-1 and Hif-1Į [104][105][106][107].
Though no transcription factors have been reported to be involved in the negative regulation of CCL2 in OC cells, studies involving other cell types have reported negative regulators of CCL2. Specifically, NFțB p50/p50 homodimers, HDAC1, and the transcription factors Nrf2 and SMRT have been suggested to suppress the CCL2 expression in hepatic cells and adipocytes [108][109][110].

CXCL1
CXCL1 (GRO-Į) contributes to ovarian cancer progression by inducing endothelial and epithelial cell proliferation and migration [25,26]. The putative transcription factor binding sites identified in human CXCL1 promoter are listed in Table 3. Experimental studies have demonstrated binding of the transcription factors p65 NFțB, AP-2, CCAAT displacement protein (CDP), and the stimulating protein-1 (SP-1) to the CXCL1 promoter in human cells (Figure 2). In ovarian cancer cells, though, the CXCL1 gene expression was found to be regulated mainly by NFțB pathway, specifically by the p65 DNA binding [25,27,28,111,112].
In addition to the positive regulation by p65 NFțB, AP-2 and SP-1, studies using human melanocytes have indicated that the CXCL1 expression is negatively controlled by the transcriptional repressors CDP and the poly(ADPribose) polymerase-1 (PARP-1) [113,114]. The exact mechanisms of how CDP and PARP-1 inhibit the CXCL1 expression are not fully understood; however, they likely involve displacement of trans-activating factors that bind to CXCL1 promoter, resulting in transcriptional repression.

CXCL2
The putative transcription factor binding sites identified in human CXCL2 (GRO-ȕ) promoter are listed in Table 4. However, experimental studies have demonstrated only binding of NFțB, AP-1, and STAT3 to human CXCL2 promoter (Figure 3). In ovarian cancer cells, the CXCL2 expression is dependent on IțBĮ [28] and IKKȕ [44]. In addition, the CXCL2 expression in OC cells is induced by TNF, and is inhibited by overexpression of the tumor suppressor p53 [115].
Adjacent to the NFțB site in the IL-8 promoter are C/EBP and Hif-1 binding sites (Figure 4). Even though the direct involvement of C/EBP and Hif-1 in the IL-8 regulation in ovarian cancer cells has yet to be demonstrated, the up-regulation of IL-8 expression by hypoxia in ovarian cancer cells has been well documented [30,134].
Transcription of IL-8 is also regulated by the transcription factor AP-1 that consists of Fos, FosB, Jun, and Jun-B subunits. Activation of AP-1 mediates the increased IL-8 expression in hypoxia, paclitaxel, and lysophosphatidic acid (LPA) treated OC cells [30, 80,135]. Interestingly, a recent study has shown that the stress hormones norepinephrine and epinephrine enhance the IL-8 expression by a FosB-dependent mechanism [136]. Table 5 lists all putative transcription factor binding sites identified in the human CXCL8/IL-8 promoter.
Although studies from other cell types have shown that the IL-8 expression is negatively regulated by the NFțB repressing factor NRF, nuclear receptor corepressor (NCoR), the silencing mediator for retinoic acid and thyroid hormone receptor SMRT, and HDACs [54,[137][138][139], the potential involvement of these corepressors in OC cells has yet to be demonstrated. Considering the important role these corepressors play in the IL-8 regulation, it will be important to elucidate their function in ovarian cancer setting.

Conclusions and Perspectives
As we continue to improve our understanding of the mechanisms regulating chemokine expression in ovarian cancer cells, our knowledge will contribute to the development of new therapeutic strategies targeting the increased chemokine expression in chemoresistant metastatic ovarian cancer. Several important questions remain to be answered: What are the specific molecular targets and mechanisms responsible for the chemokine expression induced by chemotherapeutic drugs and hypoxia? What is the role of HDACs and other transcriptional repressors in regulating the chemokine expression in ovarian cancer cells? What is the role of the metabolic state of ovarian cancer cells in regulating the chemokine expression? Answers to these questions may open new avenues for therapeutic approaches for treating ovarian cancer.