Young Woo Seo

The Molecular Mechanism of Noxa-induced Mitochondrial Dysfunction in p53-Mediated Cell Death

Genotoxic stresses stabilize the p53 tumor suppressor protein which, in turn, transactivates target genes to cause apoptosis. Although Noxa, a “BH3-only” member of the Bcl-2 family, was shown to be a target of p53-mediated transactivation and to function as a mediator of p53-dependent apoptosis through mitochondrial dysfunction, the molecular mechanism by which Noxa causes mitochondrial dysfunction is largely unknown. Here we show that two domains (BH3 domain and mitochondrial targeting domain) in Noxa are essential for the release of cytochrome c from mitochondria. Noxainduced cytochrome c release is inhibited by permeability transition pore inhibitors such as CsA or MgCl2, and Noxa induces an ultra-structural change of mitochondria yielding “swollen” mitochondria that are unlike changes induced by tBid. This indicates that Noxa may activate the permeability transition-related pore to release cytochrome c from mitochondria into cytosol. Moreover, Bak-oligomerization, which is an essential event for tBid-induced cytochrome c release in the extrinsic death signaling pathway, is not associated with Noxa-induced cytochrome c release. This finding suggests that the pathway of Noxa-induced mitochondrial dysfunction is distinct from the one of tBid-induced mitochondrial dysfunction. Thus, we propose that there are at least two different pathways of mitochondrial dysfunction; one mediated through Noxa in response to genotoxic stresses and the other through tBid in response to death ligands.

Cisplatin Inactivation of Caspases Inhibits Death Ligand-induced Cell Death in Vitro and Fulminant Liver Damage in Mice

Cisplatin is a platinum-containing chemotherapeutic drug that has been widely used to treat various human cancers. It acts by forming inter- and intracross-links of DNA, which is believed to be a major cause for its therapeutic efficacy. However, little attention has been paid to the effect of cisplatin on death ligand-induced cell death. Here we demonstrate that cisplatin inhibits death ligand-induced cell death in cell lines in a p53-independent manner. This inhibitory effect of cisplatin on cell death is direct, whereby cisplatin forms a complex with caspases leading to their inactivation. The cisplatin-caspase complex is reversed by the addition of reducing agent dithiothreitol, and caspase activity is regained. In addition, cisplatin shows a death-inhibition effect in in vivo animal models of fulminant liver damage induced by Fas activation and lipopolysaccharide-induced liver shock mediated by tumor necrosis factor-α. Together, we demonstrate that cisplatin inhibits cell death induced by death ligands in cell lines and in mice through caspase inactivation.

The Cell Death–Inducing Activity of the Peptide Containing Noxa Mitochondrial-Targeting Domain Is Associated with Calcium Release

DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa is a prodeath domain. Peptide containing MTD causes massive necrosis in vitro through cytosolic calcium increase; it is released from the mitochondria by opening the mitochondrial permeability transition pore. MTD peptide-induced cell death can be inhibited by calcium chelator BAPTA-AM. Moreover, MTD peptide shows the potent tumor-killing activities in mice by joining with tumor-homing motifs.

An Unconventional KITENIN/ErbB4-Mediated Downstream Signal of EGF Upregulates c-Jun and the Invasiveness of Colorectal Cancer Cells

Purpose: EGF-stimulated signaling via EGF receptor (EGFR) is important in colorectal tumorigenesis and drug targeting. However, anti-EGFR therapy is not effective in a subset of patients with colorectal cancer, suggesting that unidentified EGF-stimulated pathways might play roles in colorectal cancer. Previously, we identified KAI1 C-terminal interacting tetraspanin (KITENIN) as a metastasis-enhancing gene and found it to be highly expressed in sporadic colorectal cancer tissues. We recently found that EGF further increases KITENIN-induced elevated AP-1 activity. Here we attempted to clarify this novel EGF-stimulated molecular pathway and its roles in colorectal cancer. Experimental Design: We analyzed how EGF modulates the downstream signaling pathway of oncogenic KITENIN in colorectal cancer cells. Biological alterations following EGF treatment were identified in KITENIN-overexpressed colorectal cancer cells with or without alteration of EGFR activity. Results: We identified the KITENIN/ErbB4–Dvl2–c-Jun axis as a novel downstream signal of EGF that is switched on under elevated KITENIN conditions in an EGFR-independent manner. This unconventional EGF signal upregulates c-Jun and enhances invasion and anchorage-independent growth of colorectal cancer cells. In addition, tumor tissues from metastatic patients with colorectal cancer who showed initial poor responses to cetuximab/chemotherapy expressed higher levels of KITENIN than did responders to therapy. Conclusions: Our results highlight the role of an EGFR-independent EGF signal in mediating the invasiveness and tumorigenesis of colorectal cancer cells. This unconventional pathway might be related to the limited clinical efficacy of anti-EGFR agents in a subset of patients with colorectal cancer. Clin Cancer Res; 20(15); 4115–28. ©2014 AACR.

Homer1 regulates the susceptibility to TRAIL

TRAIL is an apoptotic cell death-inducing ligand that belongs to a TNF superfamily. To identify the regulators that govern the susceptibility to TRAIL, TRAIL-resistant HeLa (TR) cells were established by repeatedly treating HeLa cells with TRAIL. Here we showed that scaffolding protein Homer1 plays a decisive role in regulating the apoptotic susceptibility to TRAIL. TR cells showing the normal susceptibility to FasL and chemotherapeutic agent etoposide expressed the lower protein levels of Homer1 than parental HeLa cells. They showed the delayed activation of caspases-8, Bid cleavage and Bax translocation to mitochondria in response to TRAIL. Reconstitution of Homer1 expression in TR cells significantly restored the susceptibility to TRAIL. In addition, knock-down of Homer1 using interfering shRNA in parental HeLa cells lost the susceptibility to TRAIL. Together, our data indicate that Homer1 plays a critical role in determining the apoptotic susceptibility to TRAIL.

Necrosis-inducing peptide has the beneficial effect on killing tumor cells through neuropilin (NRP-1) targeting.

The therapeutic efficacy of most anti-cancer drugs depends on their apoptosis-inducing abilities. Previously, we showed that a peptide containing the mitochondrial targeting domain (MTD) found in Noxa, a BH-3 only protein of Bcl-2 family, induces necrosis. Here, a fusion peptide of neuropilin-1 (NRP-1) targeting peptide and MTD peptide, designated tumor homing motif 17:MTD (TU17:MTD), was found to induce necrosis in cancer cells in vitro and to cause the regression of tumors when intravenously injected into mice bearing subcutaneous CT26 colorectal carcinoma tumors. The necrosis within tumor tissues was evident upon administering TU17:MTD. TU17:MTD penetrated into tumor cells by targeting to Neuropilin-1, which could be blocked by anti-NRP-1 antibody. The efficacy of TU17:MTD on tumor regression was higher than that of TU17:D(KLAKLAK)2, a fusion peptide of NRP-1 targeting peptide and a pro-apoptotic peptide. The necrotic cell death within tumor tissues was evident at day 1 after administering TU17:MTD systemically. Transplanted subcutaneous substantially reduced in size within two weeks and 5 days, respectively, with no apparent side effects. Together, these results propose that the pro-necrotic peptide MTD may present an alternative approach for development of targeted anti-cancer agents.

Correction: Necrosis-inducing peptide has the beneficial effect on killing tumor cells through neuropilin (NRP-1) targeting [Oncotarget., 7, (2016) (32449-32461)] doi 10.18632/oncotarget.8719

[This corrects the article DOI: 10.18632/oncotarget.8719.].