«Cancer Therapeutic Targets», 2016
Target: nuclear factor kappa B (NFkB)
Nuclear factor kappa B (NFkB) is a group of structurally related transcription factors, including RelA (p65), RelB, c-Rel, NF-kB1 (p50 and precursor p105), and NF-kB2 (p52 and precursor p100) (Youn et al. 2009). They form homodimers and heterodimers with different combinations when binding to their consensus DNA elements to regulate gene transcription at the promoters and enhancers. NFkB is active in many cellular processes and plays a key role in regulating innate and adaptive immune response, inflammation, proliferation, and cell death. Aberrant activation of NFkB and the signaling pathways that regulate its activity contributes to the carcinogenesis in most cancer sites and can be linked to tumor resistance to chemotherapy and radiotherapy (Baud and Karin 2009).
Biology of NF-kB
NFkB consists of p65:p50 and RelB:p52 heterodimers which differentially lead to the activation of canonical and noncanonical NF-kB pathways. IkBα is a major inhibitor of NF-kB that binds to Rel proteins in the cytoplasm and masks the nuclear translocation signal of NF-kB components, thereby blocking NF-kB translocation to the nucleus. The IkB kinase (IKK) complex containing IKKα, IKKβ, and IKKγ (NF-kB essential modifier: NEMO) is a major activator for canonical NF-kB signaling (Ghosh and Karin 2002). IKK specifically phosphorylates serine 32 and 36 of IkBα. The phosphorylation induces IkBα protein degradation through the S26 proteasome ubiquitination machinery, allowing the freed NF-kB to move to the nucleus and function as a transcription factor. IKKα and IKKβ contain a kinase domain, a leucine zipper (LZ), and a helix-loop-helix (HLH) motif and form homodimers and heterodimers through their motifs. The two are highly conserved serine/threonine kinases and share many kinase substrates, although IKKβ is more active in phosphorylating IkBα than IKKα. IKKγ is a regulatory subunit. In addition, IKKα phosphorylates the C-terminal region of p100 to induce p100 processing and generate p52 (Dejardin et al. 2002; Senftleben et al. 2001). Sequentially, RelB:p52 heterodimers translocate from the cytoplasm to the nucleus. The two major NF-kB pathways highly integrate as well (Basak et al. 2007; Saccani et al. 2003).
Role of NF-kB in cancer
Rank on a 0–10 scale: 8/9.
NF-kB regulates the expression of many genes encoding proteins involved in immune and inflammatory responses, cell death, cell-cycle regulation, cell proliferation, and cell migration. Deregulated NF-kB activity has an important impact on tumor development. NF-kB’s ability to regulate genes that inhibit apoptosis and necrosis promotes cell survival of tumor cells. Resistance to chemoand radiation therapy has also been linked to an increase in NF-kB activity (Baud and Karin 2009). NF-kB is one of the most important drivers of tumorigenesis in primary multiple myeloma cells, and inhibition of NF-kB results in a decrease in the expression of known anti-apoptotic NF-kB target genes. In solid tumors constitutively active NFkB has been linked to breast, cervical, prostate, renal, lung, colon, liver, pancreatic, esophageal, gastric, laryngeal, thyroid, parathyroid, bladder, and ovarian cancers, melanoma, cylindroma, squamous cell carcinoma (skin, head, and neck), oral carcinoma, endometrial carcinoma, retinoblastoma, and astrocytoma/glioblastoma (Baud and Karin 2009). Recently, Meylan et al. have shown elevated NF-kB activity is linked to activated RAS mutation and p53 loss in lung cancers (Meylan et al. 2009). Inhibition of NF-kB signaling in vivo resulted in significant reduction in tumor development providing support for the development of NF-kB inhibitory drugs as targeted therapies for the treatment of patients with defined mutations in Kras and p53.
Microenvironmental inflammation is important for tumor development, particularly in colon, lung, and breast cancers. IKKβ, required for NF-kB activation, is a critical regulator of inflammatory cytokine production (Karin and Greten 2005), and it links NF-kB activity to chronic inflammation. Repetitive exposure to tobacco smoke promotes tumor development both in carcinogen-treated mice and in transgenic mice undergoing sporadic Kras mutation in lung epithelial cells. NF-kB activity and IKKβ-/NF-kB-dependent production of cytokines IL-6 and TNFa are elevated in induced lung carcinomas (Vallabhapurapu and Karin 2009). IKKβ ablation in myeloid cells abrogates enhanced pneumocyte proliferation and reduces lung carcinogenesis. Similarly, deletion of IKKβ in myeloid cells reduced tumorigenesis in a mouse model of colitis-associated colon cancer (Greten et al. 2004).
Assessing aberrant NFkB activity and the consequences of therapeutically targeting NFkB is challenging as multiple combinations of NFkB dimers and activated pathways require confirmation of specificity of the compound. Gene expression profiling has been used to distinguish NFkB-dependent activated B-cell-like (ABC) and primary mediastinal B-cell lymphoma (PMBL) diffuse large B-cell lymphoma (DLBCL) from germinal center B-cell-like (GCB) DLBCL (Takahashi et al. 2010). In solid tumors, biopsies are required to assess activation of NFkB proteins by immunohistochemistry or expression of specific NFkB target genes by QPCR. Target gene assessment, however, may be cancer site specific. In colorectal cancer, MSX1, CXCL1, THBS2, CCK5, and TNC have been evaluated by both gene expression and immunohistochemistry (Horst et al. 2009). Gene expression of a different set of markers, BCL2, BLC-XL, cIAP1 and cIAP2, and TRAF1 and TRAF2, was used to measure the effects of bortezomib and 5-fluorouracil or radiation therapy for the treatment of locally advanced or metastatic rectal cancer (O’Neil et al. 2010). A detailed review of methods for detection of NFkB activity in cancer cells is described in Mauro et al. (2009). Whether these methods can be extended to the clinic will have to be evaluated.
Diagnostic, prognostic, and predictive
Diagnosing is limited to gene profiling and assessment of the activation of NFkB proteins. Diagnosis is most often made by analysis of tissue from biopsies. Elevated NFkB activity, especially in chemoor radioresistant cancers, is considered a poor prognostic marker. For multiple myeloma and activated B-cell lymphoma, diagnosis of NFkB dependency is predictive of a positive response to the proteasome inhibitor bortezomib (Staudt and Dave 2005). In colorectal adenocarcinomas, T2, T3, and T4, immunohistochemical detection of (a) proteins that mark the tumor cells in the invading front in contrast to the tumor center and (b) proteins that distinguish stromal cells in the invading front (Horst et al. 2009) are diagnostic for NFkB-dependent cancer. For head and neck squamous cell carcinoma specimens, inhibitor of differentiation 1 (Id1) is overexpressed along with activated p65 and NFkB target gene survivin, contributing to apoptosis resistance (Lin et al. 2010). Clinical trials targeting NFkB in solid tumors have not been forthcoming for assigning a predictive value (Russo et al. 2010).
Therapeutics: cancer prevention
Natural products and synthetic antiinflammatory agents that target NFkB are being investigated as safe and inexpensive methods for cancer prevention, particularly in medium to high-risk individuals. Nonsteroidal antiinflammatory drugs (NSAIDs) such as aspirin, selective COX-2 inhibitors, and sulindac can inhibit NFkB activity by targeting multiple points in the pathway. The use of aspirin for preventing cancer is best documented for colon cancer. Randomized trials show that long-term use of aspirin can prevent colorectal carcinogenesis (Chan and Giovannucci 2010). The high dose required for protection against colon cancer may result in unwanted side effects such as gastric ulcers. Clinical trials also showed that the selective COX-2 inhibitors, celecoxib and rofecoxib, prevented adenoma recurrence (Chan and Giovannucci 2010). Unfortunately, there was an increased risk of cardiovascular events in patients with a history of atherosclerotic heart disease. In addition to the effects of NSAIDs on COX-2, a downstream target of NFkB, these agents may also regulate NFkB activity by sequestering RelA in the cytoplasm or by inhibiting IKK activity. Collectively, the data suggest that NSAIDs that are targeting NFkB and COX-2 could be potential chemoprevention agents against lung, prostate, and esophageal cancers in addition to colon cancer (Brown et al. 2008).
Many natural products affect NFkB activity, either directly or indirectly, and due to their low toxicity, they are good candidates for chemoprevention. Curcumin is a polyphenol derived from turmeric (Curuma longa). In the laboratory, curcumin inhibits NFkB activity in ovarian, breast, head and neck, lung, and prostate cancer cell lines (Brown et al. 2008). In the clinic, curcumin is well tolerated (Sharma et al. 2004) and has some biological activity in phase II pancreatic cancer trials (Dhillon et al. 2008). Resveratrol is a polyphenol derived from red grapes and berries. Like curcumin, a significant amount of in vitro data has shown that resveratrol inhibits the growth of multiple cancer cell lines including the breast, prostate, thyroid, head and neck, ovarian, and cervical. Resveratrol appears to regulate cancer cell growth by inhibition of IKK and suppression of NFkB activity (Brown et al. 2008). Resveratrol also inhibits IKK activity in animal models of colitis (Zikri et al. 2009). Data from in vivo preclinical trials shows that resveratrol can prevent tumor growth or carcinogenesis in several cancer sites including the breast, skin, prostate, gastrointestinal, and lung (Bishayee 2009). Clinical trials to date, which are mostly risk assessment studies, show that resveratrol-rich products may be beneficial for cancer prevention. Ongoing clinical trials with pure resveratrol will provide toxicity and efficacy dosage for this chemopreventive agent (Bishayee 2009). A third chemopreventive agent that regulates NFkB activity is epigallocatechin 3-gallate (EGCG), the major polyphenol found in green tea. In animal studies, green tea polyphenols in the drinking water resulted in a delay of primary tumor incidence and tumor burden in a mouse model of prostate cancer that correlated with a substantial reduction in NFkB activity. The data from human studies suggest that green tea polyphenols may provide greater efficacy for preventing prostate cancer than for treating cancer patients (Khan et al. 2009). There are multiple ongoing clinical trials to access the effects of green tea on prostate, lung, bladder, esophageal, breast, and head and neck cancer (see www.clinicaltrials.gov for an updated list of trials). Two other natural products known to inhibit NFkB activity, dietary isothiocyanates, from watercress, and sulforaphanes, from crucifiers (Cheung and Kong 2010), are also being evaluated in clinical trials against various cancers (www.clinicaltrials.com). Other natural products known to block NFkB activity include catechins, silymarin, caffeic acid phenethyl ester (CAPE), sanguinarine, anethole, emodin, piceatannol, capsaicin, ursolic acid, betulinic acid, flavopiridol, oleandrin (Dorai and Aggarwal 2004), parthenolide, kambekaurin (Brown et al. 2008), and freeze-dried black raspberries (Huang et al. 2002).
Therapeutics: cancer treatment
Activated or aberrant regulation of NFkB has been detected not only in lymphoid cancer but in many solid tumors as well (Karin 2009). Often NFkB activation is the result of activating one or more of the upstream components of the NFkB signaling pathway, all of which are possible targets for therapeutic intervention. The primary targets include the IKKs, the 26s proteasome, CK2, and PPAR-γ (Brown et al. 2008). IKKβ is the most active target of the IKK complex and many inhibitors of IKKβ have been developed; however, long-term inhibition of IKKβ may impair the immune system (Youn et al. 2009).
Bortezomib, which targets the ubiquitin-proteasome pathway, inhibits proteasome degradation of IkB, thereby inhibiting NFkB. While bortezomib has been approved to treat multiple myeloma because it delays progression of the disease, it is not clear if NFkB is the only target of bortezomib (Staudt and Dave 2005). For solid tumors, phase II trials with bortezomib showed low to no objective responses for metastatic renal, neuroendocrine, NSCLC, metastatic colorectal, metastatic melanoma, sarcomas, metastatic breast, SCLC, metastatic urothelial, and castration resistant metastatic prostate cancer (Russo et al. 2010). Preclinical data predicted that combinations with bortezomib would sensitize tumors to chemoor radiotherapy. Unfortunately, in clinical trials, combining bortezomib with cytotoxic drugs including docetaxel, carboplatin, or paclitaxel against hormone refractory prostate, advanced breast, metastatic gastrointestinal cancer, or advanced NSCLC proved to be no more effective than using bortezomib alone (Russo et al. 2010).
The results from current clinical trials have provided new insights into the role of NFkB in cancer and are generating guidelines for new compounds in the pipeline. New drugs in preclinical studies target multiple components of the NFkB pathway. IKK inhibitors include BMS-345541, which targets both IKKα and IKKβ; BAY 11-7085, an irreversible inhibitor of IkBα phosphorylation; MLN120B; and PS-1145. MLN4924 targets the proteasome by inhibiting neddylation of βTrCP. Selective estrogen receptor modulators (SERMS) regulate estrogen receptor ligand activation of NFkB. Peroxisome proliferator-activated receptor (PPAR), a transcription factor that regulates proliferation and inflammation, can be targeted by NSAIDs and antidiabetic agents such as thiazolidinediones (TZDs). Casein kinase II, (CK2), a kinase with multiple substrates in the NFkB pathway, can be targeted by dimethylamino-4,5,6,7-tetrabromo-benzimidazole (DMAT) and by apigenin, a natural plant flavone (Brown et al. 2008; Staudt and Dave 2005). An extensive list of other natural products that affect NFkB activity can be found in Luqman and Pezzuto (2010). Antimalarial quinacrine inhibits both basal and activated NFkB and doing so restores tumor suppressor p53 activity (Gurova et al. 2005).
Proteasome inhibitor bortezomib has shown the most promise in treating patients with multiple myeloma and is in clinical trials in combinations with other drugs for solid tumors (Russo et al. 2010). However, more studies are required before bortezomib will be approved for treatment of solid tumors. Similarly, more information is needed for inhibitors of IKK, and caution needs to be taken with long-term use of NFkB inhibitors for prevention. Complications due to suppression of the immune response and their effects on inflammation (Karin 2009) need to be avoided. On the other hand, targeting NFkB for cancer prevention is more promising than for treatment, possibly because the pre-cancer cells have not become addicted to elevated NFkB activity and because carcinogenesis is driven by microenvironment-associated inflammation which can be attenuated by these compounds (Grivennikov et al. 2010).
Because NFkB is over activated in many cancers, it remains an attractive target for cancer therapy. Preclinical studies need to incorporate what has been learned from clinical trials. These studies need to monitor the effects of NFkB inhibition on the immune system and on inflammation. Also more focus is needed on downstream targets of NFkB, including IL-6 and STAT3 (Karin 2009). Finally, biomarkers of efficacy and of off-target effects need to be identified and incorporated in all trials.
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