Genome-wide CRISPR knockout screening identified G protein pathway suppressor 2 as a novel tumor suppressor for uveal melanoma metastasis.

PURPOSE: Uveal melanoma (UM) is the most common intraocular malignant tumor in adults. Due to the lack of effective treatments for metastatic UM, the survival of UM has not changed over the past 3 decades. Therefore, it is important to identify essential genes regulating the metastasis of UM. METHODS: In this study, a genome-wide CRISPR knockout screen in an orthotopic mouse model of UM was performed to identify the regulatory genes conferring the metastatic phenotype. Loss-of-function analyses were performed to explore the function of G protein pathway suppressor 2 (GPS2) in UM metastasis in vitro and in vivo. RNA sequencing was performed to investigate the molecular mechanism underlying the function of GPS2 as a tumor suppressor in UM. RESULTS: Among the highest-ranking genes, we found several validated tumor suppressors, such as SHPRH, GPS2, PRPH2, and hsa-mir-1229; GPS2 was chosen as the candidate gene for further studies. GPS2 was lower expressed in the tumor tissues of UM patients. Furthermore, knocking-down GPS2 promoted the proliferation and metastatic abilities of UM cells both in vivo and in vitro. Finally, analysis of the transcriptome data revealed that silencing GPS2 upregulates oncogenic signaling pathways MAPK and PI3K-Akt, and in the meantime downregulates tumor suppressor signaling pathway Slit/Robo in UM cells. CONCLUSION: Altogether, our study proved that the GPS2 gene functions as a tumor suppressor and might be a novel potential therapeutic target for UM treatment.

Golden 2-like transcription factor contributes to the major QTL against rice black-streaked dwarf virus disease.

KEY MESSAGE: A major resistance QTL was identified on chromosome 6 in rice variety Wuke; both overexpression and knockdown experiments confirmed that OsGLK1 is the candidate gene for association with Rice black-streaked dwarf virus disease. Rice black-streaked dwarf virus disease is one of the most destructive rice viral diseases in China and East Asia. Progress has been limited in RBSDVD resistance breeding due to inadequate knowledge on the underlying functional genes. In this study, a major QTL for RBSDV (rice black-streaked dwarf virus) independent of SBPH (small brown planthopper) resistance was mapped in a 1.8 Mb interval on chromosome 6 by using an F2:3 population originated from resistant rice variety Wuke. Representative transcripts within this region were analysed and three genes showing amino acid sequence variation in functional domains were selected for transformation. Overexpression experiments showed that one gene exhibited significant enhanced resistance compared to control lines, encoding protein involving Myb domain and probable transcription factor Golden 2-like1 (GLK1). Furthermore, OsGLK1 knockdown rice lines were investigated and the resistance ability was significantly declined without this gene compared to the wild type. Taken together, both overexpression and knockdown experiments strongly suggested that OsGLK1 plays an important role for RBSDV resistance and contributes to the major QTL. The study paves the way for elucidating the molecular mechanism underlying RBSDVD resistance and the molecular markers associated with OsGLK1 may be used for marker-assisted selection.

Generation of triacyl lipopeptide-modified glycoproteins by metabolic glycoengineering as the neoantigen to boost anti-tumor immune response.

The lack of tumor specific antigens (TSA) and the immune tolerance are two major obstacles for the immunotherapy of cancer. Current immune checkpoint inhibitors (ICIs) show clinical responses in only limited subsets of cancer patients, which, to some extent, depends on the mutation load of tumor cells that may generate neoantigens. Here, we aimed to generate a neoantigen MDP to exhibit stronger anti-tumor efficacy. Methods: In this study, we utilized chemically modified sialic acid precursor tetra acetyl-N-azidoacetyl-mannosamine (AC4ManNAZ) to engineer the glycoproteins on the membranes of tumor cells for the covalent ligation of hapten adjuvant Pam3CSK4 in vivo, which eventually generated a neoantigen, i.e., ManNAZ-DBCO-Pam3CSK4 (MDP), on tumor cells. The high labeling efficiency, relatively specific biodistribution in tumor tissues and the anti-tumor efficacy were confirmed in the syngeneic murine models of the breast cancer and the lung cancer. Results: The generation of MDP neoantigen in tumor-bearing mice significantly evoked both the humoral and the T-cell-dependent antitumor immune responses, resulting in a strong inhibition on the growth of the breast cancer and the lung cancer allografts and significantly prolonged survival of tumor-bearing mice. Interestingly, MDP neoantigen was able to dramatically increase the sensitivity of cancer cells to ICIs and greatly enhance the anti-tumor efficacy in the murine models of both breast cancer and the lung cancer, which showed no or low responses to the immunotherapy with anti-PD1 antibody alone. Conclusions: We developed a simple metabolic glycoengineering method to artificially generate neoantigens on tumor cells to enhance tumor cell immunogenicity, which is able to significantly improve the response and the clinical outcome of ICIs.

Serotonin/HTR1E signaling blocks chronic stress-promoted progression of ovarian cancer.

Rationale: Psychological stress has been linked to cancer development and resistance to therapy by many epidemiological and clinical studies. Stress-induced immunosuppressive microenvironment by stress hormones, in particular glucocorticoids, has been extensively studied. However, the impacts of other stress-related neurotransmitters, such as serotonin (5-hydroxytryptamine, 5-HT), on cancer development just start to be revealed. Here, we aimed to identify novel neurotransmitters involved in stress-induced growth and dissemination of ovarian cancer (OC) and reveal the major underlying signaling pathway and the therapeutic significance. Methods: Through a genome-wide CRISPR/Cas9 knockout screen in the murine orthotopic model of ovarian carcinoma (OC), we identified candidate genes regulating the peritoneal dissemination of OC. Among them, we picked out HTR1E, one member of 5-HT receptor family specifically expressed in the ovary and endometrium in addition to brain. The correlation of HTR1E expression with OC progression was analyzed in OC patient specimen by quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry (IHC). Gain-of-function and loss-of-function analyses were performed to explore the functions of 5-HT/HTR1E signaling in OC growth and dissemination in vitro and in vivo. In addition, we investigated the therapeutic values of HTR1E specific agonist and small molecular inhibitors against HTR1E downstream factor SRC in a stressed murine OC xenograft model. Results: In OC patients, the HTR1E expression is dramatically decreased in peritoneal disseminated OC cells, which correlates with poor clinical outcome. Silence of HTR1E in OC cells greatly promotes cell proliferation and epithelial mesenchymal transition (EMT) by the activation of SRC-mediated downstream signaling pathways. Furthermore, chronic stress results in significantly decreased serotonin in the ovary and the enhanced OC growth and peritoneal dissemination in mice, which can be strongly inhibited by specific HTR1E agonist or the SRC inhibitor. Conclusions: We discovered the essential role of serotonin/HTR1E signaling in preventing the chronic psychological stress-promoted progression of OC, suggesting the potential therapeutic value of the HTR1E specific agonist and the SRC inhibitor for OC patients who are suffering from psychological stress.

Functional Analysis of OsCIPK17 in Rice Grain Filling.

We used mutant cipk17 and Nipponbare in field experiments to analyze agronomic traits, photosynthetic parameters, transcriptome, and gene expression. The results demonstrated cytoplasmic localization of OsCIPK17, while GUS allogeneic (A. thaliana) tissue-staining and quantitative analysis showed the gene was expressed in many organs, including flower buds; furthermore, it was involved in root, stem, and leaf growth. Compared to Nipponbare plants, grain filling rate and final grain weight decreased in plants of the knockout mutant owing to a delay in attainment of maximum grain filling rate. Photosystem II (PSII) efficiency was also reduced. Enrichment analysis showed that the functions of differentially expressed genes (DEGs) focused on nucleoside-, nucleotide-, and lipid-binding, as well as hydrolase, transferase, and phosphorylase activities. Signaling pathways mainly included starch and sucrose metabolism, as well as photosynthesis. Additionally, some DEGs were verified by fluorescence analysis. The results showed that knockout of OsCIPK17 affected photosynthesis and starch-, sucrose-, and amino acid metabolism-related gene expression; furthermore, the mutation reduced PSII utilization efficiency, it blocked the synthesis and metabolism of starch and sucrose, and affected the formation and transport of assimilates, thereby reducing final grain weight.