Dae-Seok Kim

Reversal of Drug Resistance in Breast Cancer Cells by Transglutaminase 2 Inhibition and Nuclear Factor-κB Inactivation

Induction of transglutaminase 2 (TGase 2) by epidermal growth factor (EGF) in human breast cancer cells increases their oncogenic potential and chemoresistance. The role of TGase 2 in the development of these tumor-related phenotypes remains to be elucidated, but it has been shown that expression of a dominant-negative form of TGase 2 reverses EGF-mediated chemoresistance in breast cancer cells. We examined several different breast cancer cell lines, representing both EGF receptor (EGFR)-positive and EGFR-negative breast cancers, and found that doxorubicin-resistant cells had a higher level of TGase 2 compared with doxorubicin-sensitive cells independent of the EGFR expression level. TGase 2 inhibition increased the chemosensitivity of drug-resistant cells, concomitant with a decrease in nuclear factor-kappaB (NF-kappaB) activity. Increasing the level of TGase 2 in drug-sensitive cells by transient transfection reduced the level of inhibitory subunit alpha of NF-kappaB (IkappaBalpha) and increased NF-kappaB activity in these cells. Inhibition of TGase 2 in drug-resistant cells by RNA interference increased the levels of IkappaBalpha, and this correlated with a shift in the accumulation of NF-kappaB from the nucleus to the cytosol. We recently showed that TGase 2 activated NF-kappaB through polymerization and depletion of free IkappaBalpha during inflammation. Therefore, increased expression of TGase 2 and subsequent activation of NF-kappaB may contribute to drug resistance in breast cancer cells independently of EGF signaling.

Transglutaminase 2 mediates polymer formation of I-κBα through C-terminal glutamine cluster

Recently we reported that transglutaminase 2 (TGase 2) activates nuclear factor-κB (NF-κB) independently of I-κB kinase (IKK) activation, by inducing cross-linking and protein polymer formation of inhibitor of nuclear factor-κBα (I-κBα). TGase 2 catalyzes covalent isopeptide bond formation between the peptide bound-glutamine and the lysine residues. Using matrix-assisted laser desorption ionization time-of-flight mass spectra analysis of I-κBα polymers cross-linked by TGase 2, as well as synthetic peptides in an in vitro competition assay, we identified a glutamine cluster at the C terminus of I-κBα (amino acids 266–268) that appeared to play a key role in the formation of I-κBα polymers. Although there appeared to be no requirement for specific lysine residues, we found a considerably higher preference for the use of lysine residues at positions 21, 22, and 177 in TGase 2-mediated cross-linking of I-κBα. We demonstrated that synthetic peptides encompassing the glutamine cluster at amino acid positions 266–268 reversed I-κBα polymerization in vitro. Furthermore, the depletion of free I-κBα in EcR/TG cells was completely rescued in vivo by transfection of mutant I-κBαs in glutamine sites (Q266G, Q267G, and Q313G) as well as in a lysine site (K177G). These findings provide additional clues into the mechanism by which TGase 2 contributes to the inflammatory process via activation of NF-κB.

Glucosamine is an effective chemo-sensitizer via transglutaminase 2 inhibition

Aberrant increases of transglutaminase 2 (TGase 2) in tumors contribute to drug resistance. The role of TGase 2 in cancer pathogenesis was unknown until we showed that TGase 2 activates NF-kappaB in the absence of kinase-dependent phosphorylation. It appears that increased expression of TGase 2 is responsible for the constitutive activation of NF-kappaB in cancer cells. We have demonstrated that TGase 2 inhibition using siRNA, cystamine or R2 peptide promotes cell death in drug-resistant cancer cells through NF-kappaB inactivation. Therefore, a safe and effective small molecule for TGase 2 inhibition is being sought in the development of therapeutics for malignant cancers. By screening for TGase inhibitors in a natural compound library, we found that glucosamine has a TGase 2 inhibitory effect in vitro. Glucosamine also recovered the depletion of I-kappaBalpha via TGase 2 inhibition, which resulted in a decrease of NF-kappaB activity in EcR293/TG cells. Furthermore, glucosamine efficiently promoted cell death via inhibiting TGase 2-mediated NF-kappaB activation in drug-resistant breast cancer cells. These results suggest that glucosamine, as a TGase 2 inhibitor, might be an attractive novel target for treatment of malignant cancers.

Transglutaminase 2 inhibition found to induce p53 mediated apoptosis in renal cell carcinoma

Renal cell carcinoma (RCC), the predominant form of kidney cancer, is characterized by high resistance to radiation and chemotherapy. This study shows that expression of protein cross‐linking enzyme transglutaminase 2 (TGase 2) is markedly increased in 7 renal cell carcinoma (RCC) cell lines in comparison to HEK293 and other cancer cell lines, such as NCI 60. However, the key role of TGase 2 in RCC was not clear. The down‐regulation of TGase 2 was found to stabilize p53 expression, thereby inducing a 3‐ to 10‐fold increase in apoptosis for 786‐O, A498, CAKI‐1, and ACHN cell lines by DAPI staining. MEF cells from TGase 2–/– mice showed stabilized p53 under apoptotic stress to compare to MEFs from wild‐type mice. TGase 2 directly cross links the DNA binding domain of p53, leading to p53 depletion via autophagy in RCC. TGase 2 and p53 expression showed an inverse relationship in RCC cells. This finding implies that induced expression of TGase 2 promotes tumor cell survival through p53 depletion in RCC.—Ku, B.M., Kim, D.‐S., Kim, K.‐H., Yoo, B.C., Kim, S.‐H., Gong, Y.‐D., Kim, S.‐Y., Transglutaminase 2 inhibition found to induce p53 mediated apoptosis in renal cell carcinoma. FASEB J. 27, 3487–3495 (2013). www.fasebj.org

Transglutaminase 2 gene ablation protects against renal ischemic injury by blocking constant NF-κB activation.

Transglutaminase 2 knockout (TGase2(-/-)) mice show significantly reduced inflammation with decreased myofibroblasts in a unilateral ureteral obstruction (UUO) model, but the mechanism remains to be clarified. Nuclear factor-κB (NF-κB) activation plays a major role in the progression of inflammation in an obstructive nephropathy model. However, the key factors extending the duration of NF-κB activation in UUO are not known. In several inflammatory diseases, we and others recently found that TGase 2 plays a key role in extending NF-κB activation, which contributes to the pathogenesis of disease. In the current study, we found that NF-κB activity in mouse embryogenic fibroblasts (MEFs) from TGase2(-/-) mice remained at the control level while the NF-κB activity of wild-type (WT) MEFs was highly increased under hypoxic stress. Using the obstructive nephropathy model, we found that NF-κB activity remained at the control level in TGase2(-/-) mouse kidney tissues, as measured by COX-2 expression, but was highly increased in WT tissues. We conclude that TGase 2 gene ablation reduces the duration of NF-κB activation in ischemic injury.

Depletion of nucleophosmin via transglutaminase 2 cross-linking increases drug resistance in cancer cells

It has been suggested that nucleophosmin has an anti-apoptotic function via Bax binding. We found that nucleophosmin is a substrate of transglutaminase 2 (TGase 2) in cancer cells. Increased expression of TGase 2 expression is highly associated with drug resistance, and polymerization of nucleophosmin by TGase 2 also can be correlated with the drug resistance of cancer cells. In the present study, an accumulation of nucleophosmin in cytosol was detected when doxorubicin was treated to cancer cells, and it was found, moreover, that an increase of cytosolic nucleophosmin can result in drug-induced apoptosis. Nucleophosmin was polymerized by TGase 2, and the polymerization was inhibited with the TGase 2 inhibitor, cystamine, in vitro. The nucleophosmin level in the cytosolic cell fraction was reduced when TGase 2 was expressed, and the reduced nucleophosmin level was rescued by cystamine treatment. Moreover, nucleophosmin cross-linked by TGase 2 was eradicated in MCF7 cells via the ubiquitin-proteasomal pathway. In parallel with this nucleophosmin-level restoration, the pro-apoptotic Bax protein level was increased. Therefore, depletion of cytosolic nucleophosmin by TGase 2 can decrease Bax protein stability and lead to anti-apoptosis. Drug-resistant cancer cells became sensitive to doxorubicin treatment when nucleophosmin was expressed in cytosol. Taking these results together, it can be concluded that TGase 2 inhibits accumulation of cytosolic nucleophosmin through polymerization, which results in drug resistance in cancer cells.

Transglutaminase 2 as an independent prognostic marker for survival of patients with non-adenocarcinoma subtype of non-small cell lung cancer

BackgroundExpression of transglutaminase 2 (TGase 2) is related to invasion and resistance to chemotherapeutic agents in several cancer cells. However, there has been only limited clinical validation of TGase 2 as an independent prognostic marker in cancer.MethodsThe significance of TGase 2 expression as an invasive/migratory factor was addressed by in vitro assays employing down-regulation of TGase 2. TGase 2 expression as a prognostic indicator was assessed in 429 Korean patients with early-stage non-small cell lung cancer (NSCLC) by immunohistochemical staining.ResultsTGase 2 expression increased the invasive and migratory properties of NSCLC cells in vitro, which might be related to the induction of MMP-9. In the analysis of the immunohistochemical staining, TGase 2 expression in tumors was significantly correlated with recurrence in NSCLC (p = 0.005) or in the non-adenocarcinoma subtype (p = 0.031). Additionally, a multivariate analysis also showed a significant correlation between strong TGase 2 expression and shorter disease-free survival (DFS) in NSCLC (p = 0.029 and HR = 1.554) and in the non-adenocarcinoma subtype (p = 0.030 and HR = 2.184). However, the correlation in the adenocarcinoma subtype was not significant.ConclusionsTGase 2 expression was significantly correlated with recurrence and shorter DFS in NSCLC, especially in the non-adenocarcinoma subtype including squamous cell carcinoma.

A New Regulatory Mechanism of NF-κB Activation by I-κBβ in Cancer Cells

Transglutaminase 2 (TGase 2) catalyzes covalent isopeptide bond formation between glutamine and lysine residues. Recently, we reported that TGase 2 activates nuclear factor-kappa B (NF-kappaB) by depleting inhibitor of NF-kappaBalpha (I-kappaBalpha) levels via polymer formation. Furthermore, TGase 2 expression synergistically increases NF-kappaB activity with canonical pathway. The major I-kappaB proteins such as I-kappaBalpha and I-kappaBbeta resemble each other in both primary sequence and tertiary structure. However, I-kappaBbeta does not degrade fully, while I-kappaBalpha degrades immediately in response to most stimuli. We found that I-kappaBbeta does not contain any of the previously identified TGase 2 target sites. In this study, both an in vitro cross-linking assay and a TGase 2 transfection assay revealed that I-kappaBbeta is independent from TGase 2-mediated polymerization. Furthermore, increased I-kappaBbeta expression reversed NF-kappaB activation in cancer cells, compensating for the loss of I-kappaBalpha via TGase 2 polymerization.

I-κBα depletion by transglutaminase 2 and μ-calpain occurs in parallel with the ubiquitin-proteasome pathway

Transglutaminase 2 (TGase2) is a calcium-dependent, cross-linking enzyme that catalyzes iso-peptide bond formation between peptide-bound lysine and glutamine residues. TGase 2 can activate NF-kappaB through the polymerization-mediated depletion of I-kappaBalpha without IKK activation. This NF-kappaB activation mechanism is associated with drug resistance in cancer cells. However, the polymers cannot be detected in cells, while TGase 2 over-expression depletes free I-kappaBalpha, which raises the question of how the polymerized I-kappaBalpha can be metabolized in cells. Among proteasome, lysosome and calpain systems, calpain inhibition was found to effectively increase the accumulation of I-kappaBalpha polymers in MCF7 cells transfected with TGase 2, and induced high levels of I-kappaBalpha polymers as well in MDA-MB-231 breast cancer cells that naturally express a high level of TGase 2. Inhibition of calpain also boosted the level of I-kappaBalpha polymers in HEK-293 cells in case of TGase 2 transfection either with I-kappaBalpha or I-kappaBalpha mutant (S32A, S36A). Interestingly, the combined inhibition of calpain and the proteasome resulted in an increased accumulation of both I-kappaBalpha polymers and I-kappaBalpha, concurrent with an inhibition of NF-kappaB activity in MDA-MB-231 cells. This suggests that mu-calpain proteasome-dependent I-kappaBalpha polymer degradation may contribute to cancer progression through constitutive NF-kappaB activation.

Proteomic analysis of high-molecular-weight protein polymers in a doxorubicin-resistant breast-cancer cell line

We recently reported that increased transglutaminase 2 (TGase 2) expression correlates with increased resistance to the cancer drug doxorubicin in breast‐cancer cell lines. Interestingly, high‐molecular‐weight (HMW) proteins also increased with increased TGase 2 expression in the drug‐resistant cell lines. TGase 2 is likely to be responsible for the formation of HMW proteins, because TGase 2 catalyzes cross‐linking between proteins. Although the role of the HMW proteins is unclear, we demonstrated that TGase 2 inhibition increases drug sensitivity in breast‐cancer cells. Herein we find that TGase 2 inhibition by cystamine dramatically reduces the level of HMW proteins. Identification of the HMW proteins may suggest the mechanism of cancer drug resistance associated with aberrant TGase 2 function. To explore the identities of HMW proteins, we performed in‐gel tryptic digestions of unresolved HMW proteins and analyzed the resulting peptides using LC‐MALDI‐MS/MS. Most of the identified proteins were associated with gene regulation, such as polyadenylate‐binding proteins, translation initiation factors, and ribonucleoproteins. This finding suggests that TGase 2 may participate in gene regulation, in addition to its role in cell adhesion.