USP11-dependent selective cIAP2 deubiquitylation and stabilization determine sensitivity to Smac mimetics

Given their crucial role in apoptosis suppression, inhibitor of apoptosis proteins (IAPs) have recently become attractive targets for cancer therapy. Here, we report that cellular IAP2 (cIAP2) is specifically stabilized in several cancer cell lines, leading to resistance to Smac mimetics, such as BV6 and birinapant. In particular, our results showed that cIAP2 depletion, but not cIAP1 depletion, sensitized cancer cells to Smac mimetic-induced apoptosis. Ubiquitin-specific protease 11 (USP11) is a deubiquitylase that directly stabilizes cIAP2. USP11 overexpression is frequently found in colorectal cancer and melanoma and is correlated with poor survival. In our study, cancer cell lines expressing high levels of USP11 exhibited strong resistance to Smac mimetic-induced cIAP2 degradation. Furthermore, USP11 downregulation sensitized these cells to apoptosis induced by TRAIL and BV6 and suppressed tumor growth in a xenograft model. Finally, the TNFα/JNK pathway induced USP11 expression and maintained cIAP2 stability, suggesting an alternative TNFα-dependent cell survival pathway. Collectively, our data suggest that USP11-stabilized cIAP2 may serve as a barrier against IAP-targeted clinical approaches.Apoptosis is an inherent cell death program that is crucial for various physiological processes such as development, the immune response, and tumorigenesis.¹ This process is finely tuned by numerous cellular signaling pathways involving hundreds of pro-apoptotic and anti-apoptotic factors.^{2, 3} Inhibitor of apoptosis proteins (IAPs) are a conserved protein family containing the baculoviral IAP repeat (BIR) domain.⁴ There are eight human IAP proteins, including cellular IAP1 (cIAP1/BIRC2), cIAP2/BIRC3, X chromosome-linked IAP (XIAP/BIRC4), and melanoma IAP (ML-IAP/BIRC7).⁵ IAPs such as XIAP can exert their anti-apoptotic function through the BIR domain, which directly interacts with caspases.⁵ In addition, several IAPs contain a RING domain with E3 ubiquitin ligase activities, which are crucial for apoptosis suppression. In particular, the E3 ligase activities of cIAP1/2 are necessary to regulate tumor necrosis factor receptor (TNFR) signaling.⁶ Upon TNFR activation, cIAP1/2 is recruited to TNFR through TNFα receptor-associated factor 2 (TRAF2), leading to K63-linked polyubiquitylation of receptor interacting protein kinase 1 (RIPK1), which is essential for NF-κB-mediated cell survival.⁷ The lack of RIPK1 polyubiquitylation via cIAP1/2 depletion or the presence of CYLD deubiquitylase triggers TNFR complex IIa formation, thereby inducing caspase-8-dependent apoptosis.⁸ In addition, cIAP1/2 prevents the formation of the RIPK1-containing death complex ripoptosome in response to several stimuli including CD95, TNFα-related apoptosis-inducing ligand (TRAIL), genotoxic stress, and Toll-like receptor (TLR) activation.^{9, 10, 11, 12, 13}IAPs are frequently overexpressed in various human cancers, and their expression is associated with chemoresistance and poor clinical outcome.⁶ Therefore, inhibiting IAP function is an attractive strategy to treat cancer through the induction of apoptosis.^{5, 14} Upon apoptotic stimuli, IAPs are inhibited by the second mitochondria-derived activator of caspases (Smac),⁵ and this discovery led to the development of Smac mimetic peptides using the IAP binding motif containing four amino acids (Ala-Val-Pro-Ile). These peptides were shown to sensitize cells to apoptotic stimuli and efficiently suppress tumor growth in a xenograft model.^{15, 16} Subsequently, a number of small-molecule compounds mimicking the Smac mimetic peptide (Smac mimetics) were developed with improved pharmacological properties and IAP-binding affinity. Interestingly, Smac mimetics, such as BV6 and compound A, were found to induce autoubiquitylation and degradation of cIAP1/2.^{17, 18} Furthermore, cIAP1/2 depletion with Smac mimetics activates the non-canonical NF-κB pathway to induce autocrine TNFα production, which is essential for Smac mimetic-induced apoptotic cell death.^{18, 19}Because cIAP1 and cIAP2 show functional redundancy in TNFα-mediated survival, the depletion of both proteins is usually required for effective induction of cell death upon TNFα treatment.^{20, 21} However, there are several reports showing that cIAP2 expression, but not cIAP1 expression, renders cells resistant to Smac mimetic-induced cell death.^{20, 21} For example, cIAP2 upregulation via phosphoinositide 3-kinase (PI3K) upon compound 3 treatment in certain cell lines was shown to facilitate apoptosis evasion.²² In addition, treatment with compounds A and C led to cIAP1 dimerization, without cIAP2 dimerization, resulting in the autoubiquitylation and subsequent degradation of cIAP1. These findings may explain why cIAP1 is degraded more efficiently than cIAP2 upon treatment with Smac mimetics.²³ Alternatively, because cIAP2 degradation requires cIAP1, cIAP2 may become more stable when cIAP1 is depleted using Smac mimetics.²⁴ Direct cIAP deubiquitylation by OTUB1 or USP19 has been suggested to be responsible for cIAP stabilization;^{25, 26} however, these previous studies did not focus on the difference in stabilization between cIAP1 and cIAP2 and only provided general deubiquitylation-dependent mechanisms.^{25, 26}While several studies have supported hypotheses for how cIAP2 survives in the presence of Smac mimetics, numerous independent studies have also shown that cIAP2 can be efficiently degraded by Smac mimetics in various cell lines.^{27, 28, 29, 30, 31, 32} These observations suggest the existence of other factors that specifically regulate cIAP2 stability upon Smac mimetic treatment. In this study, we propose a new mechanism involving USP11-mediated cIAP2 regulation. We found that the differential destabilization of cIAP1 and cIAP2 is dependent on the presence of the cIAP2-specific deubiquitylase USP11. Mechanistically, USP11 can protect cIAP2 from Smac mimetic-mediated degradation, rendering cell lines with high USP11 expression unresponsive to Smac mimetic treatment. However, USP11 downregulation sensitized these cells to TNFα- or TRAIL-induced apoptosis in the presence of Smac mimetic and further suppressed tumor growth in a xenograft model. Corroborating these data, USP11 overexpression was observed in colon cancer and melanoma patients with poor clinical outcome. Finally, the TNFα/c-Jun N-terminal kinase (JNK) pathway induced USP11 expression, which was necessary for cIAP2 protein stabilization and its anti-apoptotic function. Thus, the identification of cIAP2-specific deubiquitylation indicates that more elaborate strategies should be developed for pharmaceutical therapies targeting cIAPs.     

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Targets Down Syndrome Candidate Region 1 (DSCR1/RCAN1) to control Neuronal Differentiation

Pituitary adenylate cyclase-activating peptide (PACAP) is a neurotrophic peptide involved in a wide range of nervous functions, including development, differentiation, and survival, and various aspects of learning and memory. Here we report that PACAP induces the expression of regulator of calcineurin 1 (RCAN1, also known as DSCR1), which is abnormally expressed in the brains of Down syndrome patients. Increased RCAN1 expression is accompanied by activation of the PKA-cAMP response element-binding protein pathways. EMSA and ChIP analyses demonstrate the presence of a functional cAMP response element in the RCAN1 promoter. Moreover, we show that PACAP-dependent neuronal differentiation is significantly disturbed by improper RCAN1 expression. Our data provide the first evidence of RCAN1, a Down syndrome-related gene, as a novel target for control of the neurotrophic function of PACAP.     

An Outbreak of Trichinellosis by Consumption of Raw Soft-Shelled Turtle Meat in Korea

Trichinellosis transmission to humans via the consumption of reptile meat is rare worldwide. In Korea, however, 2 such outbreaks, possibly via consumption of soft-shelled turtle meat, have occurred in 2 successive years. In 17 August 2014, 6 patients were admitted to Wonju Severance Christian Hospital complaining of myalgia, fever, and headache. Eosinophilia was the indication of the initial laboratory results, and they were eventually diagnosed as trichinellosis by ELISA. All of the patients worked at the same company and had eaten raw soft-shelled turtle meat at a company dinner 10 days prior to their admission. They were treated with albendazole for 2 weeks, upon which all of their symptoms disappeared. This is the 8th report on human trichinellosis in Korea, and the second implicating raw soft-shelled turtle meat.     

Comorbid Gastric Adenocarcinoma and Gastric and Duodenal Strongyloides stercoralis Infection: A Case Report

Strongyloides stercoralis can cause systemic infection, termed strongyloidiasis, and gastrointestinal ulcer disease in immunocompromised patients. However, to our knowledge, there are no reported cases of comorbid gastric adenocarcinoma and S. stercoralis infection. Here, we report a case of an 81-year-old Korean man who presented with S. stercoralis infection coexisting with early gastric adenocarcinoma (T1aN0M0). S. stercoralis eggs, rhabditiform larvae, and adult females were observed in normal gastric and duodenal crypts. They were also observed in atypical glands representative of adenocarcinoma and adenoma. Preliminary laboratory tests revealed mild neutrophilic and eosinophilic leukocytosis. A routine stool test failed to detect rhabditiform larvae in the patient’s fecal sample; however, S. stercoralis was identified by PCR amplification and 18S rRNA sequencing using genomic DNA extracted from formalin-fixed paraffin-embedded tissues. Postoperatively, the patient had a persistent fever and was treated with albendazole for 7 days, which alleviated the fever. The patient was followed-up by monitoring and laboratory testing for 4 months postoperatively, and no abnormalities were observed thus far. The fact that S. stercoralis infection may be fatal in immunocompromised patients should be kept in mind when assessing high-risk patients.