Lang Bai

Epidermal growth factor receptor-mediated tissue transglutaminase overexpression couples acquired tumor necrosis factor-related apoptosis-inducing ligand resistance and migration through c-FLIP and MMP-9 proteins in lung cancer cells.

Acquired chemoresistance not only blunts anticancer therapy but may also promote cancer cell migration and metastasis. Our previous studies have revealed that acquired tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance in lung cancer cells is associated with Akt-mediated stabilization of cellular caspase 8 and Fas-associated death domain (FADD)-like apoptosis regulator-like inhibitory protein (c-FLIP) and myeloid cell leukemia 1 (Mcl-1). In this report, we show that cells with acquired TRAIL resistance have significantly increased capacities in migration and invasion. By gene expression screening, tissue transglutaminase (TGM2) was identified as one of the genes with the highest expression increase in TRAIL-resistant cells. Suppressing TGM2 dramatically alleviated TRAIL resistance and cell migration, suggesting that TGM2 contributes to these two phenotypes in TRAIL-resistant cells. TGM2-mediated TRAIL resistance is likely through c-FLIP because TGM2 suppression significantly reduced c-FLIP but not Mcl-1 expression. The expression of matrix metalloproteinase 9 (MMP-9) was suppressed when TGM2 was inhibited, suggesting that TGM2 potentiates cell migration through up-regulating MMP-9 expression. We found that EGF receptor (EGFR) was highly active in the TRAIL-resistant cells, and suppression of EGFR dramatically reduced TGM2 expression. We further determined JNK and ERK, but not Akt and NF-κB, are responsible for EGFR-mediated TGM2 expression. These results identify a novel pathway that involves EGFR, MAPK (JNK and ERK), and TGM2 for acquired TRAIL resistance and cell migration in lung cancer cells. Because TGM2 couples TRAIL resistance and cell migration, it could be a molecular target for circumventing acquired chemoresistance and metastasis in lung cancer.

Acquired Activation of the Akt/Cyclooxygenase-2/Mcl-1 Pathway Renders Lung Cancer Cells Resistant to Apoptosis

Acquired apoptosis resistance plays an important role in acquired chemoresistance in cancer cells during chemotherapy. Our previous observations demonstrated that acquired tumor necrosis factor-related apoptosis-inducing ligand resistance in lung cancer cells was associated with Akt-mediated stabilization of cellular FLICE-like inhibitory protein (c-FLIP) and Mcl-1. In this report, we determined that these cells also have acquired resistance to apoptosis induced by chemotherapeutics such as cisplatin and doxorubicin (Adriamycin), which was detected in vitro in cell cultures and in vivo in xenografted tumors. We further found that cyclooxygenase-2 (COX-2) is dramatically overexpressed in cells with acquired apoptosis resistance. COX-2 seems to be a crucial mediator in acquired apoptosis resistance because suppressing COX-2 activity with a chemical inhibitor or reducing COX-2 protein expression level with COX-2 small interfering RNA dramatically alleviated resistance to therapeutic-induced apoptosis. Inhibiting Akt markedly suppressed COX-2 expression, suggesting COX-2 is a downstream effector of this cell survival kinase-mediated apoptosis resistance. Furthermore, the expression of Mcl-1 but not c-FLIP was significantly reduced when COX-2 was suppressed, and knockdown of Mcl-1 substantially sensitized the cells to apoptosis. Our results establish a novel pathway that consists of Akt, COX-2, and Mcl-1 for acquired apoptosis resistance, which could be a molecular target for circumventing acquired chemoresistance in lung cancer.

Receptor-interacting Protein 1 Increases Chemoresistance by Maintaining Inhibitor of Apoptosis Protein Levels and Reducing Reactive Oxygen Species through a microRNA-146a-mediated Catalase Pathway

Background: Whether RIP1 directly contributes to chemotherapy response in cancer has not been determined. Results: RIP1 knockdown resulted in miR-146a-mediated catalase reduction, ROS induction, IAP degradation, and increased cisplatin cytotoxicity. Conclusion: RIP1 blunts the anticancer activity of cisplatin by releasing miR-146a-mediated catalase suppression. Significance: Our results establish a chemoresistant role for RIP1, and intervention within the RIP1-mediated pathway may be exploited for chemosensitization. Although receptor-interacting protein 1 (RIP1) is well known as a key mediator in cell survival and death signaling, whether RIP1 directly contributes to chemotherapy response in cancer has not been determined. In this report, we found that, in human lung cancer cells, knockdown of RIP1 substantially increased cytotoxicity induced by the frontline anticancer therapeutic drug cisplatin, which has been associated with robust cellular reactive oxygen species (ROS) accumulation and enhanced apoptosis. Scavenging ROS dramatically protected RIP1 knockdown cells against cisplatin-induced cytotoxicity. Furthermore, we found that, in RIP1 knockdown cells, the expression of the hydrogen peroxide-reducing enzyme catalase was dramatically reduced, which was associated with increased miR-146a expression. Inhibition of microRNA-146a restored catalase expression, suppressed ROS induction, and protected against cytotoxicity in cisplatin-treated RIP1 knockdown cells, suggesting that RIP1 maintains catalase expression to restrain ROS levels in therapy response in cancer cells. Additionally, cisplatin significantly triggered the proteasomal degradation of cellular inhibitor of apoptosis protein 1 and 2 (c-IAP1 and c-IAP2), and X-linked inhibitor of apoptosis (XIAP) in a ROS-dependent manner, and in RIP1 knockdown cells, ectopic expression of c-IAP2 attenuated cisplatin-induced cytotoxicity. Thus, our results establish a chemoresistant role for RIP1 that maintains inhibitor of apoptosis protein (IAP) expression by release of microRNA-146a-mediated catalase suppression, where intervention within this pathway may be exploited for chemosensitization.

Low-dose gamma-irradiation inhibits IL-6 secretion from human lung fibroblasts that promotes bronchial epithelial cell transformation by cigarette-smoke carcinogen

Despite decades of research in defining the health effects of low-dose (<100 mGy) ionizing photon radiation (LDR), the relationship between LDR and human cancer risk remains elusive. Because chemical carcinogens modify the tumor microenvironment, which is critical for cancer development, we investigated the role and mechanism of LDR in modulating the response of stromal cells to chemical carcinogen-induced lung cancer development. Secretion of proinflammatory cytokines such as interleukin-6 (IL-6), CXCL1 and CXCL5 from human lung fibroblasts was induced by cigarette-smoke carcinogen benzo[a]pyrene diol epoxide (BPDE), which was inhibited by a single dose of LDR. The activation of NF-κB, which is important for BPDE-induced IL-6 secretion, was also effectively suppressed by LDR. In addition, conditioned media from BPDE-treated fibroblasts activated STAT3 in the immortalized normal human bronchial epithelial cell line Beas-2B, which was blocked with an IL-6 neutralizing antibody. Conditioned medium from LDR-primed and BPDE-treated fibroblast showed diminished capacity in activating STAT3. Furthermore, IL-6 enhanced BPDE-induced Beas-2B cell transformation in vitro. These results suggest that LDR inhibits cigarette smoke-induced lung carcinogenesis by suppressing secretion of cytokines such as IL-6 from fibroblasts in lung tumor-prone microenvironment.

MUC1 Contributes to BPDE-Induced Human Bronchial Epithelial Cell Transformation through Facilitating EGFR Activation

Although it is well known that epidermal growth factor receptor (EGFR) is involved in lung cancer progression, whether EGFR contributes to lung epithelial cell transformation is less clear. Mucin 1 (MUC1 in human and Muc1 in animals), a glycoprotein component of airway mucus, is overexpressed in lung tumors; however, its role and underlying mechanisms in early stage lung carcinogenesis is still elusive. This study provides strong evidence demonstrating that EGFR and MUC1 are involved in bronchial epithelial cell transformation. Knockdown of MUC1 expression significantly reduced transformation of immortalized human bronchial epithelial cells induced by benzo[a]pyrene diol epoxide (BPDE), the active form of the cigarette smoke (CS) carcinogen benzo(a)pyrene (BaP)s. BPDE exposure robustly activated a pathway consisting of EGFR, Akt and ERK, and blocking this pathway significantly increased BPDE-induced cell death and inhibited cell transformation. Suppression of MUC1 expression resulted in EGFR destabilization and inhibition of the BPDE-induced activation of Akt and ERK and increase of cytotoxicity. These results strongly suggest an important role for EGFR in BPDE-induced transformation, and substantiate that MUC1 is involved in lung cancer development, at least partly through mediating carcinogen-induced activation of the EGFR-mediated cell survival pathway that facilitates cell transformation.

IKKβ-mediated nuclear factor-κB activation attenuates smac mimetic–induced apoptosis in cancer cells

Smac mimetics (SM) have been recently reported to kill cancer cells through the extrinsic apoptosis pathway mediated by autocrine tumor necrosis factor (TNF). SM also activates nuclear factor-κB (NF-κB). However, how SM induces NF-κB and the role of NF-κB in SM-induced cancer cell death has not been well elucidated. We found that effective blockage of NF-κB had no detectable effect on SM compound 3 (SMC3)–induced TNF secretion, suggesting that the induction of TNF by SMC3 is independent of NF-κB. Conversely, SMC3-induced NF-κB activation was found to be mediated by autocrine TNF because this effect of SMC3 was effectively inhibited when TNF was blocked with either a TNF neutralizing antibody or TNF small interfering RNA. In addition, although SMC3 dramatically reduced c-IAP1 level, it had marginal effect on c-IAP2 expression, TNF-induced RIP modification, NF-κB activation, and downstream antiapoptosis NF-κB target expression. Furthermore, blocking NF-κB by targeting IKKβ or RelA substantially potentiated SMC3-induced cytotoxicity, suggesting that the NF-κB pathway inhibits SMC3-induced apoptosis in cancer cells. Our results show that through TNF autocrine, SM induces an IKKβ-mediated NF-κB activation pathway that protects cancer cells against SM-induced apoptosis, and thus, NF-κB blockage could be an effective approach for improving the anticancer value of SM. [Mol Cancer Ther 2009;8(6):1636–45]

A Superoxide-Mediated Mitogen-Activated Protein Kinase Phosphatase-1 Degradation and c-Jun NH2-Terminal Kinase Activation Pathway for Luteolin-Induced Lung Cancer Cytotoxicity

Although luteolin is identified as a potential cancer therapeutic and preventive agent because of its potent cancer cell-killing activity, the molecular mechanisms by which its cancer cell cytotoxicity is achieved have not been well elucidated. In this report, luteolin-induced cellular signaling was systematically investigated, and a novel pathway for luteolins lung cancer killing was identified. The results show that induction of superoxide is an early and crucial step for luteolin-induced apoptotic and nonapoptotic death in lung cancer cells. The c-Jun N-terminal kinase (JNK) was potently activated after superoxide accumulation. Suppression of superoxide completely blocked luteolin-induced JNK activation, which was well correlated to alleviation of luteolins cytotoxicity. Although luteolin slightly stimulated the JNK-activating kinase mitogen-activated protein kinase kinase 7, the latter was not dependent on superoxide. We further found that luteolin triggers a superoxide-dependent rapid degradation of the JNK-inactivating phosphatase mitogen-activated protein kinase phosphatase-1 (MKP-1). Introduction of a degradation-resistant MKP-1 mutant effectively attenuated luteolin-induced JNK activation and cytotoxicity, suggesting that inhibition of the JNK suppressor MKP-1 plays a major role in luteolin-induced lung cancer cell death. Taken together, our results unveil a novel pathway consisting of superoxide, MKP-1, and JNK for luteolins cytotoxicity in lung cancer cells, and manipulation of this pathway could be a useful approach for applying luteolin for lung cancer prevention and therapy.

Attenuating Smac Mimetic Compound 3-induced NF-κB Activation by Luteolin Leads to Synergistic Cytotoxicity in Cancer Cells

Smac mimetics are potential anticancer therapeutics selectively killing cancer cells through autocrine tumor necrosis factor (TNF)‐mediated apoptosis pathway. Our recent study reveal that the Smac mimetic compound 3 (SMC3)‐activated NF‐κB protects cancer cells against apoptosis, thus blunting SMC3s anticancer activity. Based on our previous observations that the nutrient flavonoid luteolin potently blocks TNF‐induced NF‐κB activation in cancer cells, we investigated if the combination of SMC3 and luteolin would achieve a synergistic anticancer activity. The results show that luteolin had no effect on autocrine TNF but it effectively blocked SMC3‐induced nuclear factor kappa B (NF‐κB) activation and expression of anti‐apoptotic NF‐κB targets. When SMC3 and luteolin were combined in treating cancer cells derived from lung and liver tumors, the activation of TNF‐dependent apoptosis was markedly sensitized and a synergistic cytotoxic effect was achieved. In addition, the SMC3 and luteolin co‐treatment had marginal effect on immortalized normal human bronchial epithelial cells. The results suggest that combination of SMC3 and luteolin is an effective approach for improving the anticancer value of SMC3, which has implications in cancer prevention and therapy. J. Cell. Biochem. 108: 1125–1131, 2009.

Abstract B27: RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis

In the presence of ample oxygen, cancer cells prefer glycolysis to mitochondrial respiration for energy supply. This metabolic alteration, known as the Warburg effect or aerobic glycolysis, has been shown to contribute to cancer cell proliferation. However, how this glucose metabolism pathway is precisely regulated remains elusive. Receptor-interacting protein 1 (RIP1) is an important cellular signaling molecule that plays a pivotal role in the regulation of cell death, survival and proliferation. However, the role of RIP1 in cell metabolism is unclear. To investigate the function of RIP1 in lung cancer cell proliferation, we established stable RIP1 knockdown lung cancer cell lines using lentivius-mediated shRNA expression. RIP1 knockdown inhibited the proliferation of lung cancer cells, which was associated with DNA damage-induced p53 activation. Compared with control cells, the RIP1 knockdown cells had higher glucose consumption and lactate production, indicating a higher glycolytic rate. The increased glycolysis lowered the cellular level of nicotinamide adenine dinucleotide (NAD+), a cofactor essential for DNA damage repair. By microarray profiling and promoter reporter assays, we determined that RIP1 positively regulates the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a key factor involved in the regulation of mitochondrial function. The downregulation of PGC-1α reduced the expression of components of electron transfer chain that impaired oxidative phosphorylation, which underlays the increased glycolysis in RIP1 knockdown cells. Moreover, RIP1 knockdown cells were more sensitive to both DNA damage anticancer agents and glycolysis inhibitors. Our data suggest that while aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis may cause cytostasis. Thus, maintenance of glycolysis by RIP1 is critical to cancer cell energy homeostasis and DNA integrity, which may be exploited for anticancer therapy. Citation Format: Wenshu Chen, Qiong Wang, Lang Bai, Weijie Chen, Xia Wang, Carmen S. Tellez, Shuguang Leng, Mabel T. Padilla, Toru Nyunoya, Steven A. Belinsky, Yong Lin. RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B27.

Abstract 393: Low-dose gamma-irradiation inhibits IL-6 secretion from human lung fibroblasts to suppress bronchial epithelial cell transformation by cigarette smoke carcinogen

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The relationship between low-dose (<100 mSv) ionizing radiation (LDR) and human cancer risk is controversial. While microenvironment consisting of stromal cells, cytokines and growth factors is modulated by carcinogens to favor carcinogenesis, how LDR impacts this oncogenic process is not well understood. In this study, we investigated the role and mechanism of low-dose ionizing radiation in modulating the response of human lung fibroblasts to carcinogen stimulation and the impacts of which on bronchial epithelial cell transformation. Using a human cytokine antibody array, we found that secretion of proinflammatory cytokines IL-6, CXCL1 and CXCL5 from human lung fibroblasts was induced by cigarette smoke carcinogen benzo[a]pyrene diol epoxide (BPDE), which was inhibited by a low dose of γ-ray irradiation. BPDE induced IL-6 secretion from lung fibroblasts in a dose-dependent manner, which was suppressed when the NF-κB and ERK pathways were blocked. LDR effectively inhibited NF-κB but not ERK activated by BPDE, suggesting that NF-κB is the target for LDR in fibroblasts. In addition, conditioned media from BPDE-treated fibroblasts activated STAT3 in the immortalized human bronchial epithelial cell line Beas-2B, which was blocked with an IL-6 neutralizing antibody as well as by pretreating fibroblasts with LDR. Furthermore, IL-6 significantly enhanced BPDE-induced Beas-2B cell transformation in vitro. These results suggest that LDR inhibits cigarette smoke carcinogen-induced lung carcinogenesis through suppressing secretion of protumorigenic cytokines such as IL-6 from fibroblasts in the tumor-prone microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 393. doi:1538-7445.AM2012-393