Tissue-specific requirement of sodium channel and clathrin linker 1 (Sclt1) for ciliogenesis during limb development.

Primary cilia have essential roles as signaling centers during development and adult homeostasis. Disruption of ciliary structure or function causes congenital human disorders called ciliopathies. Centriolar distal appendage (DAP) proteins are important for anchoring cilia to the membrane. However, the exact functions of DAP during in vivo ciliogenesis and animal development remain poorly understood. Here, we showed that the DAP component sodium channel and clathrin linker 1 (Sclt1) mutant mice had abnormal craniofacial and limb development with postnatal lethality. In mutant embryos, most of the affected tissues had defects in DAP recruitment to the basal body and docking to the membrane that resulted in reduced ciliogenesis and disrupted hedgehog (Hh) signaling in limb bud mesenchymal cells. However, limb digit formation and ciliogenesis in Sclt1 mutant mice were differentially affected between the fore- and hindlimb buds. The forelimbs developed normally in Sclt1 mutants, but the hindlimbs had preaxial polydactyly. Heterozygous loss of Cep83, another core DAP component, in Sclt1 mutant mice, caused forelimb and hindlimb polydactyly. These findings revealed the tissue-specific differential requirement of DAPs. Taken together, these results indicated that during limb development the ciliary base components, DAPs, play an essential role in ciliogenesis and Hh signaling in vivo in a position-dependent manner.

TGF-ß downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade.

Transforming growth factor (TGF)-ß signaling is increasingly recognized as a key driver in cancer. In progressive cancer tissues, TGF-ß promotes tumor formation, and its increased expression often correlates with cancer malignancy. In this study, we utilized adenoviruses expressing short hairpin RNAs against TGF-ß1 and TGF-ß2 to investigate the role of TGF-ß downregulation in cancer cell death. We found that the downregulation of TGF-ß increased the phosphorylation of several SAPKs, such as p38 and JNK. Moreover, reactive oxygen species (ROS) production was also increased by TGF-ß downregulation, which triggered Akt inactivation and NOX4 increase-derived ROS in a cancer cell-type-specific manner. We also revealed the possibility of substantial gene fluctuation in response to TGF-ß downregulation related to SAPKs. The expression levels of Trx and GSTM1, which encode inhibitory proteins that bind to ASK1, were reduced, likely a result of the altered translocation of Smad complex proteins rather than from ROS production. Instead, both ROS and ROS-mediated ER stress were responsible for the decrease in interactions between ASK1 and Trx or GSTM1. Through these pathways, ASK1 was activated and induced cytotoxic tumor cell death via p38/JNK activation and (or) induction of ER stress.

Survivin silencing and TRAIL expression using oncolytic adenovirus increase anti-tumorigenic activity in gemcitabine-resistant pancreatic cancer cells.

In this study, we demonstrated that survivin downregulation with TRAIL expression greatly enhanced the cytotoxic death of pancreatic cancer cells after gemcitabine treatment. Using real-time RT-PCR, we analyzed five survivin shRNAs to identify the best target sequence for suppression of human survivin, with the goal of treating gemcitabine-resistant pancreatic cancer cells. Survivin shRNA 5, corresponding to target 5, showed the greatest reduction in survivin mRNA levels. Furthermore, combined treatment with survivin shRNA-expressing adenovirus with gemcitabine plus TRAIL decreased uncleaved PARP and increased consequent PARP cleavage, which was correlated with the greatest levels of survivin downregulation and cell death. These results indicate that survivin functions as a common mediator of gemcitabine- and TRAIL-induced cell death. Using a nude mouse model implanted with MiaPaCa-2 pancreatic cancer cells, we observed tumor regression induced by an oncolytic adenovirus expressing survivin shRNA and TRAIL plus gemcitabine. Together, our findings provide a strong rationale for treating pancreatic cancer patients with both gemcitabine and oncolytic adenovirus armed with survivin shRNA and TRAIL.

Silencing Daxx increases the anti-tumor activity of a TRAIL/shRNA Bcl-xL-expressing oncolytic adenovirus through enhanced viral replication and cellular arrest.

We previously showed that an increase of cellular Bcl-xL mediates acquired resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and knockdown of Bcl-xL expression greatly sensitized TRAIL-induced cytotoxicity. Here, we show that Daxx downregulation increases the anti-tumorigenic activity through enhancement of viral replication and cellular arrest with combination of TRAIL/shBcl-xL-induced apoptosis. This study was conducted to determine the effect of Daxx downregulation on the anti-tumorigenesis induced by oncolytic adenovirus arming TRAIL or TRAIL/shRNA of Bcl-xL genes. Unlike the enhanced cancer cell death induced by exogenous TRAIL or TRAIL plus shRNA of Bcl-xL, oncolytic adenovirus expressing TRAIL or TRAIL plus shRNA of Bcl-xL did not show much enhanced cancer cell death compared to oncolytic adenovirus itself. On the other hand, enhanced cytotoxic cell death and viral replication was observed after infection with oncolytic adenovirus expressing TRAIL plus shRNA of Bcl-xL and shRNA of Daxx at the same construct. Then we realized that enhanced adenoviral replication through Daxx downregulation was caused by increased adenoviral E1A protein expression and Daxx downregulation also stimulated cellular arrest through p21/p53 accumulation. Taken all together, we have shown here that Daxx downregulation should be essentially needed for the increase of anti-tumor activity through enhancement of viral replication and cellular arrest with the combination of TRAIL/shBcl-xL-induced apoptosis and oncolytic adenovirus.

Ratio of phosphorylated HSP27 to nonphosphorylated HSP27 biphasically acts as a determinant of cellular fate in gemcitabine-resistant pancreatic cancer cells.

Gemcitabine has been used most commonly as an anticancer drug to treat advanced pancreatic cancer patients. However, intrinsic or acquired resistance of pancreatic cancer to gemcitabine was also developed, which leads to very low five-year survival rates. Here, we investigated whether cellular levels of HSP27 phosphorylation act as a determinant of cellular fate with gemcitabine. In addition we have demonstrated whether HSP27 downregulation effectively could overcome the acquisition of gemcitabine resistance by using transcriptomic analysis. We observed that gemcitabine induced p38/HSP27 phosphorylation and caused acquired resistance. After acquisition of gemcitabine resistance, cancer cells showed higher activity of NF-κB. NF-κB activity, as well as colony formation in gemcitabine-resistant pancreatic cancer cells, was significantly decreased by HSP27 downregulation and subsequent TRAIL treatment, showing that HSP27 was a common network mediator of gemcitabine/TRAIL-induced cell death. After transcriptomic analysis, gene fluctuation after HSP27 downregulation was very similar to that of pancreatic cancer cells susceptible to gemcitabine, and then in opposite position to that of acquired gemcitabine resistance, which makes it possible to downregulate HSP27 to overcome the acquired gemcitabine resistance to function as an overall survival network inhibitor. Most importantly, we demonstrated that the ratio of phosphorylated HSP27 to nonphosphorylated HSP27 rather than the cellular level of HSP27 itself acts biphasically as a determinant of cellular fate in gemcitabine-resistant pancreatic cancer cells.

Establishment of a mouse melanoma model system for the efficient infection and replication of human adenovirus type 5-based oncolytic virus.

Due to poor adenoviral infectivity and replication in mouse tumor cell types compared with human tumor cell types, use of human-type adenoviral vectors in mouse animal model systems was limited. Here, we demonstrate enhanced infectivity and productive replication of adenovirus in mouse melanoma cells following introduction of both the Coxsackievirus and adenovirus receptor (CAR) and E1B-55K genes. Introduction of CAR into B16BL6 or B16F10 cells increased the infectivity of GFP-expressing adenovirus; however, viral replication was unaffected. We demonstrated a dramatic increase of adenoviral replication (up to 100-fold) in mouse cells via E1B-55K expression and subsequent viral spreading in mouse tissue. These results reveal for the first time that human adenovirus type 5 (Ad5)-based oncolytic virus can be applied to immunocompetent mouse with the introduction of CAR and E1B-55K to syngenic mouse cell line.