Recent insights into the role of NF-kappaB in ovarian carcinogenesis

The NF-κBs are a family of ubiquitously expressed transcription factors that have been described to be responsible for the establishment of an inflammatory response. Studies in the past decade have also demonstrated this family's role in the initiation and progression of hematological and solid tumors. Recently, research has uncovered a specific role for NF-κBs in the development and maintenance of ovarian cancer.

sents with nonspecific symptoms, such as bloating or abdominal discomfort, which can all be mistaken for a more benign condition, and because there is currently no means for early detection, patients with EOC are often diagnosed with latestage disease (International Federa tion of Gynecology and Obstetrics (FIGO) stage III or IV). On initial diagnosis, patients undergo complete surgical debulking (resection whose only goal is to make subsequent therapy more effective) followed by combina tion chemotherapy usually consisting of carboplatin and paclitaxel. Approximately 80% of patients respond to this treatment regimen, which has been the standard for more than 10 years [2]. However, 60 to 80% of the res pon ders present with recurrent disease between 6 months and 2 years after treatment. Unfortunately, disease recurrence is characterized by chemoresistance, resulting in disease progression and death. As a result, the 5year survival rate for patients diagnosed with latestage disease is only 15 to 20% [3].

NF-κB in ovarian cancer initiation and progression
Chronic inflammation has been associated with tumor initiation and progression. In the ovary, carcinogenesis has been linked to inflammatory processes, such as repeated ovulation, endometriosis and pelvic infections [4,5]. The molecular link between inflammation and cancer is nuclear factor κ light chain enhancer of activated B cells (NFκB) [6]. The NFκB family of proteins, which has five members (Table 1), controls several key processes that are required for tumor develop ment and progression, such as: activation of antiapoptotic genes and genes involved in the progression of cell cycle [7,8]; secretion of factors such as tumor necrosis factor (TNF) α and inter leukin (IL)6, which enhances cell growth [6]; promo tion of a proangiogenic environment through enhanced production of IL8 and vascular endothelial growth factor [9]; and creation of a microenvironment that may prevent immune surveillance [10] (Figure 1).
A correlation between NFκB activation and EOC clinical profile has been described. Guo et al. [11] demonstrated that the expression of NFκB p65 in EOC tumors is mainly nuclear and that the levels correlate with poor differentiation and late FIGO stage. Moreover, they showed that patients who were positive for NFκB p65 subunit staining had lower cumulative survival rates and lower median survival (20% and 24 months, respec tively) than patients that were negative (46.2% and 39 months, respectively). The correlation between NFκB activation status (that is, levels of NFκB p65 and RelB) and poor clinical outcome in EOC patients was corro bor ated in more recent studies by two other independent groups [12,13].
In addition to these correlation studies [1113], the in vitro activation or specific inhibition of the NFκB pathway using either smallmolecule inhibitors or short interfering RNA (siRNA) was recently shown to affect the growth behavior of EOC cells. Using the ligand TNFlike weak inducer of apoptosis (TWEAK) to activate NFκB

Abstract
The NF-κBs are a family of ubiquitously expressed transcription factors that have been described to be responsible for the establishment of an inflammatory response. Studies in the past decade have also demonstrated this family's role in the initiation and progression of hematological and solid tumors. Recently, research has uncovered a specific role for NF-κBs in the development and maintenance of ovarian cancer.
in the highly metastatic human EOC cell line HO 8910PM, Dai et al. [14] showed that although TWEAK induced nuclear translocation of NFκB p65 does not enhance cell growth, treatment with TWEAK for 6 hours can significantly enhance adhesion and promote the migration and invasion capacity of these cells [14]. These effects were inhibited when cells were treated with TWEAK in the presence of the NFκB inhibitor pyrro lidine dithiocarbamate.
In another study, which showed that microRNA9 (miR9) could control the levels of NFκB1, the authors [15] showed that a decrease in NFκB1 levels, as a result of overexpressing miR9 or by using the siRNA expres sion vector pSilencer/siNFκB1, is associated with a reduction in cell growth and colony formation by the human EOC line ES2.
In a more recent study using several EOC cell lines, Hernandez et al. [16] showed that inhibition of the NF κB pathway through specific inhibition of inhibitor of NFκB kinase β (IKKβ) can decrease the percentage of viable CAOV3, IGROV1 and A2780 cells. In addition, they showed that blocking IKKβ activity through either smallmolecule inhibition or siRNA can inhibit anchorageindependent growth and the capacity of the cells to invade through a basement membrane. More importantly, the authors [16] identified the network of genes controlled by the IKKβNFκB pathway in CAOV3 cells. Using the highly specific IKKβ smallmolecule inhibitor ML120b or IKKβ siRNA to decrease IKKβ expression, gene expression microarray results showed that the IKKβNFκB pathway controls genes associated with EOC cell proliferation, adhesion, invasion, angiogenesis and the creation of a proinflammatory micro environment.

NF-κB signaling and ovarian cancer stem cells
Our group has identified a subpopulation of EOC cells that is responsive to the pathway involving Tolllike receptor 4 (TLR4) and NFκB [17]. Treatment with the chemotherapy agent paclitaxel, which is a known TLR4 ligand, induced NFκB activation, leading to enhanced cell proliferation. NFκB is constitutively active in these cells, resulting in constitutive secretion of proinflam matory cytokines [17], and this is brought about by consti tutive IKKβ activity [18]. These cells also express the cancer stem cell marker CD44 and are in fact the ovarian cancer stem cells (OCSCs) [19].
The CD44 + OCSCs are resistant to chemotherapeutic agents, and this resistance is partly regulated by the NFκB pathway [19]. In our most recent study [20], we showed that the NFκB inhibitor Eriocalyxin B can sensitize these cells to TNFα and Fasmediated apop tosis. The CD44+ OCSCs can also serve as tumor vascular progenitors in vitro and in vivo, an effect also regulated by the NFκB pathway [21].

Conclusions
Research in the past 5 years has unraveled the multiple mechanisms that enable NFκB to support ovarian carcinogenesis, including that this pathway confers some of the properties of OCSCs. These findings highlight the clinical potential for NFκB inhibitors to prevent recurrence and improve survival in EOC patients. The fact that the main effectors of this pathway significantly correlate with disease activity suggests the feasibility of choosing patients that may benefit from targeting this molecular pathway.
Abbreviations EOC, epithelial ovarian cancer cells; IKKα, inhibitor of NF-κB kinase α; IKKβ, inhibitor of NF-κB kinase β; IκB, inhibitor of NFκB; IL, interleukin; NF-κB, Nuclear factor kappa-light-chain-enhancer of activated B cells; OCSC, ovarian cancer stem cells; shRNA, short hairpin RNA; siRNA, small interfering RNA; TNFα, tumor necrosis factor α. Figure 1. The NF-κB pathway. In unstimulated cells, the NF-κB subunits p65 and p50 are sequestered in the cytoplasm by IκB. Ligand binding to receptors (such as TNFα and TLR4) leads to the activation of the IKK complex, which then phosphorylates IκB. Phosphorylated IκB is then ubiquitinated and degraded by the proteasome system, leading to the release of p65 and p50. The heterodimer then translocates to the nucleus and initiates the expression of genes that regulate proliferation, cell death, invasion, migration and immune regulation. This is the canonical pathway; there is also a non-canonical pathway involving other NF-κB family members. Most of the studies on EOC have looked at the members of the canonical pathway.