Trichosanthin Promotes Anti-Tumor Immunity through Mediating Chemokines and Granzyme B Secretion in Hepatocellular Carcinoma.

Trichosanthin (TCS) is a type I ribosome-inactivating protein extracted from the tuberous root of the plant Trichosanthes. TCS shows promising potential in clinical drug abortion, anti-tumor and immunological regulation. However, the molecular mechanisms of its anti-tumor and immune regulation properties are still not well discovered. In the present study, we investigated the anti-tumor activity of TCS in hepatocellular carcinoma (HCC), both in vitro and in vivo. Both HCC cell lines and xenograft tumor tissues showed considerable growth inhibition after they were treated with TCS. TCS provoked caspase-mediated apoptosis in HCC cells and xenograft tumor tissues. The recruitment of CD8+ T cells to HCC tissues and the expression of chemokines, CCL2 and CCL22, were promoted upon TCS treatment. In addition, TCS induced an upregulation of Granzyme B (GrzB), TNF-α and IFN-γ in HCC tissues, which are the major cytotoxic mediators produced by T cells. Furthermore, TCS also resulted in an increase of mannose-6-phosphate receptor (M6PR), the major receptor of GrzB, in HCC tissues. In summary, these results suggest that TCS perhaps increases T-cell immunity via promoting the secretion of chemokines and accelerating the entry of GrzB to HCC cells, which highlights the potential role of TCS in anti-tumor immunotherapy.

Germline-specific dgcr8 knockout in zebrafish using a BACK approach.

Zebrafish is an important model to study developmental biology and human diseases. However, an effective approach to achieve spatial and temporal gene knockout in zebrafish has not been well established. In this study, we have developed a new approach, namely bacterial artificial chromosome-rescue-based knockout (BACK), to achieve conditional gene knockout in zebrafish using the Cre/loxP system. We have successfully deleted the DiGeorge syndrome critical region gene 8 (dgcr8) in zebrafish germ line and demonstrated that the maternal-zygotic dgcr8 (MZdgcr8) embryos exhibit MZdicer-like phenotypes with morphological defects which could be rescued by miR-430, indicating that canonical microRNAs play critical role in early development. Our findings establish that Cre/loxP-mediated tissue-specific gene knockout could be achieved using this BACK strategy and that canonical microRNAs play important roles in early embryonic development in zebrafish.

SKP2 promotes the metastasis of pancreatic ductal adenocarcinoma by suppressing TRIM21-mediated PSPC1 degradation.

Despite significant advances in diagnostic techniques and treatment approaches, the prognosis of pancreatic ductal adenocarcinoma (PDAC) is still poor. Previous studies have reported that S-phase kinase-associated protein 2 (SKP2), a subunit of the SCF E3 ubiquitin ligase complex, is engaged in the malignant biological behavior of some tumor entities. However, SKP2 has not been fully investigated in PDAC. In the present study, it was observed that high expression of SKP2 significantly correlates with decreased survival time. Further experiments suggested that SKP2 promotes metastasis by interacting with the putative transcription factor paraspeckle component 1 (PSPC1). According to the results of coimmunoprecipitation and ubiquitination assays, SKP2 depletion resulted in the polyubiquitination of PSPC1, followed by its degradation. Furthermore, the SKP2-mediated ubiquitination of PSPC1 partially depended on the activity of the E3 ligase TRIM21. In addition, inhibition of the SKP2/PSPC1 axis by SMIP004, a traditional inhibitor of SKP2, impaired the migration of PDAC cells. In summary, this study provides novel insight into the mechanisms involved in PDAC malignant progression. Targeting the SKP2/PSPC1 axis is a promising strategy for the treatment of PDAC.

Golgi phosphoprotein 3 induces autophagy and epithelial-mesenchymal transition to promote metastasis in colon cancer.

In this study, we aimed to investigate whether and how Golgi phosphoprotein 3 (GOLPH3) facilitates colon cancer metastasis via the regulation of autophagy and epithelial-mesenchymal transition (EMT). The role GOLPH3 plays in colon cancer metastasis was analyzed using western blotting, immunohistochemistry, transwell, wound-healing, and zebrafish assays. Autophagy and EMT were assessed via RNA-sequencing (RNA-seq) analysis, mRFP-GFP-LC3 reporter assays, and their related markers. Significant associations were found between colon cancer clinical and pathological stages and poor prognosis. GOLPH3 facilitates colon cancer metastasis, both in vitro and in vivo. RNA-seq analysis of GOLPH3-overexpressing and control cell models revealed that GOLPH3 enhances EMT and autophagy. Moreover, examination of autophagic, epithelial, and mesenchymal markers in GOLPH3-overexpressing, -silenced, and control cell lines revealed that GOLPH3 promotes EMT and autophagy. When autophagy was inhibited, GOLPH3-promoted metastasis and EMT were counteracted in vitro and in vivo. Using RNA-seq, PI3K/Akt signaling was identified as the key downstream pathway on which GOLPH3 acts. Mechanistically, we demonstrated that GOLPH3 stimulates autophagy and induces EMT via the suppression of the phosphorylation of protein kinase B (Akt) at Ser473. In summary, GOLPH3 induces autophagy and EMT, promoting metastasis in colon cancer. Beyond this, and in contrast to conventional perspectives, we discovered that GOLPH3 represses the phosphorylation of Akt at Ser473.

A novel liquidchip platform for simultaneous detection of 70 alleles of DNA somatic mutations on EGFR, KRAS, BRAF and PIK3CA from formalin-fixed and paraffin-embedded slides containing tumor tissue.

BACKGROUND: DNA somatic mutations of EGFR, KRAS, BRAF and PIK3CA in the epidermal growth factor receptor (EGFR) signaling pathway play critical roles in the response or resistance of tumors to targeted therapy with tyrosine kinase inhibitors (EGFR-TKIs). To provide a high-throughput (HTP) clinical testing service for detecting these mutations, we developed a novel platform, SurPlex®-xTAG70plex-EGFR liquidchip. METHODS: This platform was developed based on a universal 100-tag system. The procedures for multiplex PCR, allele specific primer extension (ASPE) and hybridization were optimized and standardized. RESULTS: A total of 70 alleles of somatic mutations of EGFR, KRAS, BRAF and PIK3CA can be detected simultaneously in one reaction from one formalin-fixed and paraffin-embedded (FFPE) slide within one day. Cross-reaction was < 8% between individual amplimers and 70 different ASPE primers. The sensitivity for detecting mutants in the wild-type DNA was 1%-5%. Seventy-three FFPE samples with somatic mutations were used to validate the 70plex. Seventy-one showed a complete match, while two were not detected. CONCLUSIONS: A simple, accurate, sensitive HTP technology was developed and standardized for detecting simultaneously 70 different alleles of EGFR, KRAS, BRAF and PIK3CA gene mutations from FFPE tumor slides.

Trichosanthin cooperates with Granzyme B to restrain tumor formation in tongue squamous cell carcinoma.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is a common type of oral cancer, with a relatively poor prognosis and low post-treatment survival rate. Various strategies and novel drugs to treat TSCC are emerging and under investigation. Trichosanthin (TCS), extracted from the root tubers of Tian-Hua-Fen, has been found to have multiple biological and pharmacological functions, including inhibiting the growth of cancer cells. Granzyme B (GrzB) is a common toxic protein secreted by natural killer cells and cytotoxic T cells. Our group has reported that TCS combined with GrzB might be a superior approach to inhibit liver tumor progression, but data relating to the use of this combination to treat TSCC remain limited. The aim of this study was to examine the effectiveness of TCS on TSCC processes and underlying mechanisms. METHODS: First, we screened the potential antitumor activity of TCS using two types of SCC cell lines. Subsequently, a subcutaneous squamous cell carcinoma xenograft model in nude mice was established. These model mice were randomly divided into four groups and treated as follows: control group, TCS treatment group, GrzB treatment group, and TCS/GrzB combination treatment group. Various tumorigenesis parameters, such as Ki67, PCNA, caspase-3, Bcl-2 and VEGFA, et al., were performed to determine the effects of these treatments on tumor development. RESULTS: Screening confirmed that the SCC25 line exhibited greater sensitivity than the SCC15 line to TCS in vitro studies. TCS or GrzB treatment significantly inhibited tumor growth compared with the inhibition seen in the control group. The TCS/GrzB combination inhibited tumor growth more than either drug alone. TCS treatment inhibited tumor proliferation by downregulating Ki67 and Bcl2 protein expression while accelerating tumor apoptosis. In the TCS/GrzB-treated group, expression of Ki67 was further downregulated, while the level of activated caspase-3 was increased, compared with their expression in either of the single drug treatment groups. CONCLUSION: These results suggest that the TCS/GrzB combination could represent an effective immunotherapy for TSCC.

A novel mutant-enriched liquidchip technology for the qualitative detection of somatic mutations in KRAS gene from both serum and tissue samples.

BACKGROUND: Somatic mutations in the KRAS gene have been reported to confer drug resistance to epidermal growth factor receptor tyrosine kinase inhibitors and some monoclonal antibodies. However, current DNA mutation detection technologies are primarily DNA sequencing-based and not high throughput, nor sensitive enough to meet clinical needs. METHODS: A mutant-enriched PCR method was designed by introducing a unique restriction enzyme site to the PCR product. This allowed the wild-type KRAS sequence to be selectively removed by restriction enzyme digestion before application to the Luminex liquidchip system. RESULTS: A total of 100 copies of mutant KRAS DNA fragment mixed with 1x10(5) copies of the wild-type KRAS DNA could be detected to achieve a sensitivity of 0.1%. This technology is currently used for clinical testing of KRAS somatic mutations for the purpose of pharmacogenomic evaluation. Serum samples from 109 patients with non-small cell lung cancer were tested and 34 mutations were detected (34/109). The formalin-fixed and paraffin-embedded samples from 60 patients with colorectal cancer were tested and 19 mutations were detected (19/60). CONCLUSIONS: A novel, qualitative, sensitive, reliable and high throughput liquidchip technology has been developed for detecting KRAS mutations using clinical serum and formalin-fixed and paraffin-embedded samples.

Extracellular Matrix Remodeling During Palate Development.

The morphogenesis of the mammalian secondary plate is a series of highly dynamic developmental process, including the palate shelves vertical outgrowth, elevation to the horizontal plane and complete fusion in the midline. Extracellular matrix (ECM) proteins not only form the basic infrastructure for palatal mesenchymal cells to adhere via integrins but also interact with cells to regulate their functions such as proliferation and differentiation. ECM remodeling is essential for palatal outgrowth, expansion, elevation, and fusion. Multiple signaling pathways important for palatogenesis such as FGF, TGF ß, BMP, and SHH remodels ECM dynamics. Dysregulation of ECM such as HA synthesis or ECM breakdown enzymes MMPs or ADAMTS causes cleft palate in mouse models. A better understanding of ECM remodeling will contribute to revealing the pathogenesis of cleft palate.

Astrin: A Key Player in Mitosis and Cancer.

Astrin, which is a spindle-associated protein, was found to be closely related to mitotic spindle formation and maintenance. It interacts with other spindle-related proteins to play a key role in maintaining the attachment of the kinetochore-microtubule and integrity of centrosomes and promoting the centriole duplication. In addition, Astrin was quite recently found to be abnormally highly expressed in a variety of cancers. Astrin promotes the development of cancer by participating in various molecular pathways and is considered as a potential prognostic and survival predictor.

Functional Characterization and Expression Analyses Show Differential Roles of Maternal and Zygotic Dgcr8 in Early Embryonic Development.

Dgcr8 is involved in the biogenesis of canonical miRNAs to process pri-miRNA into pre-miRNA. Previous studies have provided evidence that Dgcr8 plays an essential role in different biological processes. However, the function of maternal and zygotic Dgcr8 in early embryonic development remains largely unknown. Recently, we have reported a novel approach for generating germline-specific deletions in zebrafish. This germline knockout model offers an opportunity to investigate into the differential roles of maternal or zygotic Dgcr8. Although germline specific dgcr8 deletion has no influence on gonad development, maternal or zygotic dgcr8 is essential for embryonic development in the offspring. Both maternal dgcr8 (Mdgcr8) and maternal zygotic dgcr8 (MZdgcr8) mutants display multiple developmental defects and die within 1 week. Moreover, MZdcgr8 mutant displays more severe morphogenesis defects. However, when a miR-430 duplex (the most abundantly expressed miRNA in early embryonic stage) is used to rescue the maternal mutant phenotype, the Mdgcr8 embryos could be rescued successfully and grow into adulthood and achieve sexual maturation, whereas the MZdgcr8 embryos are only partially rescued and they all die within 1 week. The differential phenotypes between the Mdgcr8 and MZdgcr8 embryos provide us with an opportunity to study the roles of individual miRNAs during early development.

Genetic Deletion of miR-430 Disrupts Maternal-Zygotic Transition and Embryonic Body Plan.

MiR-430 is considered an important regulator during embryonic development, but genetic loss-of-function study is still lacking. Here we demonstrated that genetic deletion of the miR-430 cluster resulted in developmental defects in cell movement, germ layer specification, axis patterning and organ progenitor formation in zebrafish. Transcriptome analysis indicated that the maternally provided transcripts were not properly degraded whereas the zygotic genome expressed genes were not fully activated in the miR-430 mutants. We further found that a reciprocal regulatory loop exists between miR-430 and maternally provided transcripts: the maternally provided transcripts (Nanog, Dicer1, Dgcr8, and AGOs) are required for miR-430 biogenesis and function, whereas miR-430 is required for the clearance of these maternally provided transcripts. These data provide the first genetic evidence that miR-430 is required for maternal-zygotic transition and subsequent establishment of embryonic body plan.

Developmental protein kinase C hyper-activation results in microcephaly and behavioral abnormalities in zebrafish.

Susceptible genetic polymorphisms and altered expression levels of protein kinase C (PKC)-encoding genes suggest overactivation of PKC in autism spectrum disorder (ASD) development. To delineate the pathological role of PKC, we pharmacologically stimulated its activity during the early development of zebrafish. Results demonstrated that PKC hyper-activation perturbs zebrafish development and induces a long-lasting head size deficit. The anatomical and cellular analysis revealed reduced neural precursor proliferation and newborn neuron formation. ß-Catenin that is essential for brain growth is dramatically degraded. Stabilization of ß-catenin by gsk3ß inhibition partially restores the head size deficit. In addition, the neuropathogenic effect of developmental PKC hyper-activation was further supported by the alterations in the behavioral domain including motor abnormalities, heightened stress reactivity and impaired habituation learning. Taken together, by causally connecting early-life PKC hyper-activation to these neuropathological traits and the impaired neurogenesis, these results suggest that PKC could be a critical pathway in ASD pathogenesis.

Detection of EGFR somatic mutations in non-small cell lung cancer (NSCLC) using a novel mutant-enriched liquidchip (MEL) technology.

We have developed and standardized a novel technology, mutant-enriched liquidchip (MEL), for clinical detection of EGFR mutations. The MEL integrates a mutant-enriched PCR procedure with liquidchip technology for detections of EGFR exon 19 deletions and L858R mutation on both formalin-fixed and paraffin-embedded (FFPE) slides and plasma samples from patients with non-small cell lung cancer (NSCLC). The detection sensitivity was 0.1% of mutant DNA in the presence of its wild-type DNA. The cross-reaction rate was lower than 5%. To evaluate the MEL platform, the EGFR mutation status of 59 patients with advanced NSCLC treated with EGFRTKIs (Tyrosine Kinase Inhibitors) were tested on their FFPE samples. EGFR exon 19 deletions and L858R were detected in 21 patients (21/59) and 76.2% (16/21) of them had partial response to the EGFR-TKIs, while by sequencing method, only 4 (4/59) mutations were detected. Plasma samples from 627 patients with various stages of NSCLC were examined with the MEL and 22% of EGFR exon 19 deletions and L858R were detected. Furthermore, in patients with advanced disease there are more mutations detected in plasma samples than in patients with less advanced disease. In conclusion, the MEL is a sensitive, stable, and robust technology for detecting EGFR DNA mutations from both FFPE and plasma samples from patients with NSCLC and is now routinely used for clinical diagnosis.