Novel approaches to target NF-B and other signaling pathways in cancer stem cells

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  • Advances in Biological Regulation 56 (2014) 108e115Contents lists available at ScienceDirect

    Advances in Biological Regulationjournal homepage: www.elsevier .com/locate/ jb iorNovel approaches to target NF-kB and othersignaling pathways in cancer stem cells

    Tamami Ukaji, Kazuo Umezawa*

    Department of Molecular Target Medicine Screening, Aichi Medical University School of Medicine,Nagakute 480-1195, JapanKeywords:NF-kBCancer stem cellsDHMEQ* Corresponding author. Tel./fax: 81 561 61195E-mail address: (K 2014 Elsevier Ltd. All rights reserveda b s t r a c t

    Recently cancer tissue is considered to consist of large number ofbalk cancer cells and a small number of cancer stem cells. Aftersurgery, radiotherapy, or chemotherapy, most cancer cells areremoved, but if there are still very small number of cancer stemcells left. They may form the similar tumor again. So removal ofcancer stem cells is considered to be important for future cancertherapy. In one hand, NF-kB is the transcription factor that pro-motes expressions of various inflammatory cytokines andapoptosis inhibitory proteins. Cancer cells often possess constitu-tively activated NF-kB that often provides excess survival andtherapeutic resistance in cancer cells. We have discovered DHMEQas a specific inhibitor of NF-kB. This compound was found to bemore active in cancer stem cells than in balk cancer cells. In breastcancer cells both PI3K-Akt and NF-kB pathways appear in thesurvival of cancer stem cells.

    2014 Elsevier Ltd. All rights reserved.Introduction

    NF-kB is the transcription factor that promotes expressions of various inflammatory cytokines andinhibitor of apoptosis proteins. Human solid cancer and leukemia cells often possess activated NF-kB.Then, it enhances viability and secretion of pro-metastatic proteins in cancer cells increasing themalignancy. Moreover, it also activates secretion of inflammatory cytokines and growth factors in the9.. Umezawa).


  • T. Ukaji, K. Umezawa / Advances in Biological Regulation 56 (2014) 108e115 109environmental cells around cancer tissues. These effects in the cancer micro-environment shouldactivate the growth of cancer. Moreover, NF-kB promotes expressions of matrix metallo-proteases(MMPs), vascular endothelial growth factor (VEGF)-C, and various chemokines and the receptors toactivate metastasis. Thus, it is an attractive target for suppression of cancer metastasis.

    We have been screening cellular signal transduction inhibitors of lowmolecular weight from natureand by molecular design. We started screening of transcription factor NF-kB inhibitors about 15 yearsago. As a result, we discovered dehydroxymethylepoxyquinomicin (DHMEQ) by modification of thestructure of an antibioticepoxyquinomicin as an inhibitor of NF-kB (Ishikawa et al., 2009; Matsumotoet al., 2000). In the present review, we describe discovery and especially the selective inhibitory effectof DHMEQ on cancer stem cells.

    Cellular role of NF-kB and mechanism of activation

    NF-kB is the transcription factor that binds to the kB sequence of DNA. Structurally, it is a hetero-dimer consisting of 2 Rel family proteins including p65, cRel, RelB, p50, and p52. There are canonicaland noncanonical signaling pathways for NF-kB activation. The canonical NF-kB consists of mainly p65and p50 and noncanonical one of RelB and p52 (Beinke and Ley, 2004). The canonical pathway ismainly involved in natural immunity, most inflammations, and cancer progression, while non ca-nonical one is involved in B-lymphocyte maturation and autoimmune diseases. Recently, noncanonicalNF-kB is getting more important than before for lymphoma and breast carcinoma. NF-kB promotes thetranscription of many cytokines such as interleukin (IL)-1a and b, IL-2, IL-6, IL-8, IL-10 (which oftensuppresses inflammation), IL-12, IL-17, and TNF-a. These proteins are essential for proper immunefunctions in the physiological state, but their over-expression often causes inflammation in thepathological state (Li and Verma, 2002). NF-kB also promotes expressions of cell-adhesion moleculessuch as E-selectin, ICAM-1, and VCAM-1. These molecules are important for the proper control of celldistribution. But their over-expression in endothelial cells accumulates macrophages and may causeinflammation such as arteriosclerosis. Adhesion molecules are also involved in the progression ofmetastasis of cancer cells. NF-kB is also essential for the osteoclast differentiation to promote theexpression of NFATc1, the most important transcription factor. NF-kB increases the expression of anti-apoptotic proteins such as inhibitor of apoptosis proteins (IAPs), Bcl-xL, survivin, Bfl1 and FLIP, XIAP,and Bim. These proteins are essential for the tissue stability, but too much expression especially incancer cells is considered to increase the malignancy. NF-kB activates expressions of many pro-metastasis proteins including MMP-3 and 9, urokinase-type plasminogen activator, MCP-1, MIP-1,endoglin, IGFBP, PDGF B chain, thrombospondin,VEGF-C, HIF-1a, cathepsin B, collagenase-1, endo-thelin, and galectin-3 (selected from

    NF-kB is usually inactive in the cytoplasmwithout stimulation. The canonical NF-kB is activated byvarious ligands through their receptors and signaling proteins TRAF2 or TRAF6. TRAF2 is activated byextracellular signals such as TNF-a, IL-1, lipopolysaccharide (LPS), lipopeptides, and tumor promoterphorbol esters. TRAF2 activates IKK, which induces the phosohorylation of IkB. IkB is an inhibitoryprotein that is bound to NF-kB. The phosphorylation of IkB induces its release from its complex withNF-kB and results in itsubiquitination and degradation by proteasomes. Liberated NF-kBmolecules thathave NLS regions then enter the nucleus whereby they bind to the B site of DNA (Fig. 1). In one hand,various ligands and their cell surface receptors activate TRAF6 instead of TRAF2 to activate NF-kB. Thesereceptors include the IL-1 receptor, Toll-like receptors 4 (TLR4) (activated by LPS), TLR2 (activated bylipopeptide), and receptor activator of NF-kB (RANK). In the case of the noncanonical pathway, theinhibitory protein p100 is degraded into the active component p52 when the activating signal comes(Kulms and Schwarz, 2006).

    Discovery of NF-kB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) and mechanism of inhibition

    In the course of our search for inhibitors of the NF-kB function, we designed newNF-kB inhibitors oflow molecular weight with reference to the structure of epoxyquinomicin C (Matsumoto et al., 2000).Epoxyquinomicin C shows very little toxicity in animals. In one hand, related compounds such aspanepoxydone (Erkel et al., 1996) and cycloepoxydone (Gehrt et al., 1998) inhibit NF-kB; however,

  • Fig. 1. Typical signaling pathways for NF-kB activation.

    T. Ukaji, K. Umezawa / Advances in Biological Regulation 56 (2014) 108e115110epoxyquinomicin C does not inhibit NF-kB. However, after the removal of the protruding hydrox-ymethyl moietythe designed compound dehydroxymethylepoxyquinomicin (DHMEQ, Fig. 2a), wasfound to inhibit NF-kB activity. DHMEQ ameliorates inflammation in a collagen-induced rheumatoidarthritis model in mice when administered by the intraperitoneal (IP) route (Matsumoto et al., 2000).Thus, we found a new NF-kB inhibitor active in vivo.

    Racemic DHMEQ can be synthesized from a simple chemical 2,5-dimethoxyaniline in 5 steps andcan be separated into each enantiomer practically by lipase. Lipase reacts with racemic dihexanoyl-DHMEQ to give ()-DHMEQ and monohexanoyl-()-DHMEQ that can be easily removed by differ-ence of solubility. ()-DHMEQ is about 10 times more effective than ()-DHMEQ in inhibiting NF-kB.Racemic DHMEQ is much easier to prepare than ()-DHMEQ. So ()-DHMEQ is mainly used for cellularexperiments, while racemic DHMEQ for animal experiments.

    As to the mechanism of DHMEQ, firstly we reported that it inhibited the nuclear translocation ofNF-kB (Ariga et al., 2002). However, more recently, we found that DHMEQ directly binds to the Rel-family proteins to inhibit their DNA-binding activity (11). Thus, ()-DHMEQ inhibits NF-kB at thefinal signaling process (Fig. 1). ()-DHMEQ binds to p65 covalently with 1:1 stoichiometry as revealedby surface plasmon resonance (SPR) and MALDI-TOF mass spectrum (MS) analyses. MS analysis of thechymotrypsin-digested peptide suggested the binding of ()-DHMEQ to a specific cysteine residue. Inthe case of p65, DHMEQ only binds to the Cys38 residue, which is located close to the DNA (Fig. 2b), butnot to other cysteines such as Cys120. The specific binding to Cys38 can also be shown in cultured cellsby using mutation analysis (Yamamoto et al., 2008). Observation of an adduct in MALDI-TOF MSsuggests that the ()-DHMEQ-cysteine binding is a covalent one. The formation of ()-DHMEQ-cysteine covalent binding in the protein was supported by chemical synthesis of the adduct (Kozawaet al., 2009). Because ()-DHMEQ binds to the cysteine residue covalently in an NF-kB molecule, theinhibitory effect is irreversible (Shimada et al., 2010). Treatment of cells with ()-DHMEQ for only15 min induced dormancy of the cells on activation of NF-kB by LPS ((Yamamoto et al., 2008), Fig. 2c).All Rel family proteins possess specific cysteine residues essential for their DNA binding as shown inFig. 2d. ()-DHMEQ binds to p65, cRel, RelB, and p50, but not to p52 at the specific cysteine residues.()-DHMEQ is the first NF-kB inhibitor shown by MALDI-TOF MS analysis to covalently bind to aspecific cysteine. In inhibition of noncanonical NF-kB, we have recently found that ()-DHMEQ inhibitsnot only DNA-binding of RelB, but also its interaction to importin-5 as shown in Fig. 2e. It also inducesinstability of RelB (Takeiri et al., 2012).

    Thus, ()-DHMEQ specifically binds to acysteine residuein both the canonical and the noncanonicalNF-kB components (Takeiri et al., 2012; Yamamoto et al., 2008), as shown in Fig. 2f. It is likely that

  • a







    Fig. 2. a. Molecular design of ()-DHMEQ. ()-DHMEQ was designed based on the structure of epoxyquinomicin C. ()-DHMEQ isabout 10 times more effective than ()-DHMEQ. Racemic DHMEQ is shown as DHMEQ throughout the text. b. Structure of the p65/p50-DNA complex. Cysteine residues are shown in red in p65. ()-DHMEQ covalently binds to Cys38. c. Irreversible inhibition ofNF-kB by ()-DHMEQ in cultured cells. When ()-DHMEQ is added for 15 min and washed out, even after 8 h LPS cannot activateNF-kB. d. Sequence homology in Rel family proteins. The cysteines in red are conserved, and considered to be essential for their DNAbinding. e. ()-DHMEQ covalently binds to RelBto cause inhibition of DNA binding. Moreover, interaction to importin a5 is inhibitedand the cellular stability of RelB decreases after the binding. f. ()-DHMEQ inhibits both canonical and noncanonical NF-kB. g.Suppression of inflammation and cancer growth in vivo by administration of DHMEQ in various animal models. (For interpretation ofthe references to color in this figure legend, the reader is referred to the web version of this article.)

    T. Ukaji, K. Umezawa / Advances in Biological Regulation 56 (2014) 108e115 111

  • T. Ukaji, K. Umezawa / Advances in Biological Regulation 56 (2014) 108e115112DHMEQ can enter into aspecific pocket viaa key and lock mechanism to bind limitedly to this cysteineresidue. These findings may explain the highly selectiveNF-kB inhibition and the low toxic effect ofDHMEQ in cells and in animals.

    Anti-inflammatory and anticancer activities of DHMEQ

    ()-DHMEQ inhibits ligand-induced activation ofmouse bonemarrow-derivedmacrophages (BMM).It blocks the expression of iNOS and COX-2 and secretions ofmany inflammatory cytokines including IL-6andTNF-a.Moreover, althoughBMMincorporatesoxidizedLDL togive rise to foamcells, the ()-DHMEQ-treated bone marrow cells do not take up oxidized LDL (Suzuki et al., 2009). Excess osteoclast differen-tiation often causes bone destruction in rheumatoid arthritis andmultiple myeloma. RANKL/Rank is themain ligand/receptor system that induces osteoclast differentiation from macrophages. RANKL inducesactivation of NF-kB andthen NFATc1, which is the most important transcription factor for the osteoclastdifferentiation. Itwasnot knownhowNF-kBacts in this differentiation. ()-DHMEQwas shown to inhibitthe RANKL-induced NFATc1 expression to inhibit osteoclast differentiation (Takatsuna et al., 2005).Moreover, subcutaneous administration of DHMEQ inhibitsthe expression of NFATc1 even in vivo in themouse rheumatoidmodel (Kubota et al., 2007). DHMEQ shows anti-inflammatory effects on rheumatoidarthritis, inflammatory injury of the kidney induced by unilateral urethral obstruction in rats and anti-Thy1.1-induced glomerulonephritis in rats. In addition, it ameliorates cancer cachexia induced by loadingof prostate carcinoma in mice, possibly by lowering the blood level of IL-6 (Kuroda et al., 2005). It alsosuppresses diabetes-induced retinal inflammation mediated by the rennin-angiotensin system in mice(Nagai et al., 2007). Amniotic apoptosis is essential for the onsetof delivery. Amniotic apoptosis is inducedby the TNF-a pathway via the TNF-a receptor 1 expressed in the amniotic epithelial cells, which wasinhibited byDHMEQ inmice (Kobayashi et al., 2010). Then, DHMEQmay be useful for prevention of babyloss. It also inhibits allergic inflammation and airway remodeling in a murine asthma model (Shimizuet al., 2012). DHMEQ also effectively inhibits the mixed lymphocyte reaction in cultures of mousespleen cells, and IP administration of DHMEQ inhibits graft rejection and increases the graft survival inheart transplantation experiments (Ueki et al., 2006). Donor (Takahashi et al., 2010) or recipient(Watanabe et al., 2013) pretreatment with DHMEQ improves islet transplantation in mice. Kidneytransplantation is also improved by administration of DHMEQ in mice (Kono et al., 2013).

    Since 2003, there have been many reports in which DHMEQ inhibited the cancer growth in animalmodels. Particularly, DHMEQ has been used for the suppression of aggressive cancer in animal modelsincluding hormone-refractory solid cancers and lymphomas. DHMEQ suppresses the subcutaneousgrowth of JCA-1 tumor cells in nude mice (Kikuchi et al., 2003). DHMEQ inhibits the tumor growth ofhighly malignant breast carcinoma MDA-MB-231 cells in SCID mice (Matsumoto et al., 2005). Thyroidcarcinoma is often resistant to chemotherapy and possesses constitutively activated NF-kB. Adminis-tration of DHMEQ inhibited the growth of thyroid carcinoma in mice (Starenki et al., 2004). Recently, IPadministration showed anticancer activity in the mouse model of glioblastoma (Fukushima et al.,2012). Adult T-cell leukemia (ATL) is extremely resistant to chemotherapy. DHMEQ shows anticanceractivity against ATL tumors in mice (Watanabe et al., 2005). DHMEQ also displays potent anticanceractivity against the growth of multiple myeloma cells in mice (Tatetsu et al., 2005).

    Fig. 2g shows suppression of inflammation and cancer in vivo. No toxicity has been observed in anyof the animal experiments reported above with DHMEQ. In these animal experiments DHMEQ wasgivenmainly via peritoneal cavity. DHMEQ is usually given tomice at 2e12mg/kg for IP administration.The LD50 value of DHMEQ for acute toxicity in mice is around 200 mg/kg by IP administration.Consequently, DHMEQ can inhibit inflammation and cancer growth at a dose about 20e100 timeslower than its LD50 value. According to the unique distribution of DHMEQ in the body after the IPadministration, Umezawa has proposed that suppression of inflammatory cell activity in the peritonealcavity may cause suppression of various inflammation and cancer, as shown in Fig. 3 (Umezawa, 2011).

    Importance of targeting cancer stem cells for the suppression of cancer

    Recently cancer tissue is considered to consist of large number of bulk cancer cells and a smallnumber of cancer stem cells. Cancer stem cells were originally discovered in acute myeloid leukemia

  • Fig. 3. A. Importance of cancer stem cells in the progression of cancer. B. Effect of DHMEQ on soft agar colony formation in PC3 bulkcancer cells (BCs) and cancer initiating cells (CICs)DHMEQ is more active in the cancer stem cells than in the bulk cancer cells.Modified from McCubrey et al., Advances in Enzyme Regulation 2012 (35). C. Importance of NF-kB and PI3K-Akt pathways for thesurvival of cancer stem cells.

    T. Ukaji, K. Umezawa / Advances in Biological Regulation 56 (2014) 108e115 113

  • T. Ukaji, K. Umezawa / Advances in Biological Regulation 56 (2014) 108e115114cells, but they have more recently found in almost all types of cancer including breast, prostate, he-patocellular, cervical, colorectal, and gastrointestinal carcinomas (Fitzgerald and McCubrey, 2014;Jhanwar-Uniyal et al., 2013; McCubrey et al., 2012, 2014).

    After surgery, radiotherapy, or chemotherapy, most cancer cells are removed, but if there are stillvery small number of cancer stem cells left, theymay form the similar tumor again (Fig. 3A). So removalof cancer stem cells is considered to be important for future cancer therapy.

    Cancer stem cells are also called as cancer initiating cells or tumor initiating cells, while bulk cancercells as proliferating cancer cells. Cancer stem cells often grow slowly than bulk cancer cells, and theyare often drug-resistant.

    Suppression of cancer stem cell growth in cultured cells and in animal experiment

    McCubrey et al. reported that DHMEQ reduced the soft agar colony formation ability of bothprostate carcinoma bulk cancer (PC3BC) and cancer initiating (PC3CIC) cells (McCubrey et al., 2012).However, DHMEQ appeared to be more effective in reducing the soft agar colony formation of PC3CICmore than PC3BC cells. As shown in Fig. 3B, DHMEQ reduced the soft agar colony formation of PC3CICand PC3BC 13.9-fold and 3.2-fold, respectively, at the highest dose examined (McCubrey et al., 2012).Similar to the results observed with DHMEQ, rapamycin, an mTOR inhibitor, inhibited the soft agarcolony formation ability of PC3CIC more than PC3BC cells. Rapamycin reduced the soft agar colonyformation of PC3CIC and PC3BC 12.1-fold and 2.8-fold, respectively, at the highest dose examined.

    In human breast cancers, breast cancer stem cells are enriched in the CD44(high)/CD24(low) cellpopulation, whereas the CD44(low)/CD24(high)cells represent a more differentiated phenotype withlimited stem cellelike potential. Breast cancer stem cells are resistant to anoikis, and they expandunder anchorage-independent conditions. Goto and co-workers prepared breast cancer stem cells byseparation with surface markers, and those cancer stem cell population was found to possess highertumorigenicity and NF-kB activity compared with bulk cancer cell population (Murohashi et al., 2010).DHMEQ alone inhibited the tumor growth of purified cancer stem cells inmice (Murohashi et al., 2010).Moreover, they found that the NF-kB and PI3K-Akt pathways are essential signaling pathways in breastcancer cells (Hinohara et al., 2012). They found that heregulin (HRG), a ligand for ErbB3, induced softagar colony formation of a breast cancer stem cell-enriched population. HRG-induced colony formationwas reduced by treatment with inhibitors for phosphatidyl inositol 3-kinase (PI3K) or DHMEQ and by adominant-negative inhibitor protein for NF-kB. Moreover, the overexpression of dominant negativeNF-kB inhibitor in breast cancer cells inhibited tumorigenesis in NOD/SCID mice. Furthermore, theexpression of IL8, a regulator of self-renewal in breast cancer stem cell-enriched populations, wasinduced by HRG through the activation of the PI3K and NF-kB pathway. Thus, they concluded that HRG/ErbB3 signaling appears to maintain soft agar colony formation through PI3K and NF-kB pathways inhuman breast cancer. PI3K inhibitor and NF-kB inhibitor including DHMEQ would be candidates oftherapeutic agents suppressing cancer stem cells.


    Thisworkwas supported inpart bygrants fromtheprogramsGrants-in-Aids in ScientificResearch (B,Project number 23310163) of theMinistry of Education, Culture, Sports, Science and Technology (MEXT)(B 23310163 and C 26350975) of Japan, and from the MEXT-supported Program for the StrategicResearch Foundation at PrivateUniversities,which is forAichiMedical University 2011-2015 (S1101027).


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    Novel approaches to target NF-B and other signaling pathways in cancer stem cellsIntroductionCellular role of NF-B and mechanism of activationDiscovery of NF-B inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) and mechanism of inhibitionAnti-inflammatory and anticancer activities of DHMEQImportance of targeting cancer stem cells for the suppression of cancerSuppression of cancer stem cell growth in cultured cells and in animal experiment



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