Discovery of Small and Bifunctional Molecules Targeting PD-L1/CD73 for Cancer Dual Immunotherapy.

In this work, a series of bifunctional PD-L1/CD73 (cluster of differentiation 73) small-molecule inhibitors were designed and synthesized. Among them, CC-5 showed the strongest PD-L1 inhibitory effects with an IC50 of 6 nM and potent anti-CD73 activity with an IC50 of 0.773 µM. The high PD-L1/CD73 inhibitory activity of CC-5 was further confirmed by SPR assays with KD of 182 nM for human PD-L1 and 101 nM for CD73, respectively. Importantly, CC-5 significantly suppressed tumor growth in a CT26 and B16-F10 tumor model with TGI of 64.3% and 39.6%, respectively. Immunohistochemical (IHC) and flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) indicated that CC-5 exerted anticancer effects via activating the tumor immune microenvironment. Collectively, CC-5 represents the first dual PD-L1/CD73 inhibitor worthy of further research as a bifunctional immunotherapeutic agent.

[Clinical and genetic analysis of a patient with Loeys-Dietz syndrome due to variant of TGFBR2 gene].

OBJECTIVE: To explore the genetic basis of a patient with clinically suspected Loeys-Dietz syndrome (LDS). METHODS: A child who had presented at Beijing Anzhen Hospital in September 2018 was selected as the study subject. Clinical data and family history of the patient were collected, along with peripheral blood samples of the proband and his parents. Whole exome sequencing (WES) was carried out through next-generation sequencing. RESULTS: Candidate variants were searched through bioinformatic analysis focusing on genes associated with hereditary aortic aneurysms. Candidate variant was verified by Sanger sequencing. The patient was found to have cardiovascular abnormalities including early-onset aortic dilatation and coarctation, and LDS syndrome was suspected. WES revealed that he has harbored a heterozygous c.1526G>T missense variant of the TGFBR2 gene. The same variant was not found in either parent and was predicted as likely pathogenic (PM1+PM2_Supporting+ PM6+PP3+PP4) based on the guidelines from the American College for Medical Genetics and Genomics (ACMG). CONCLUSION: The TGFBR2 c.1526G>T variant probably underlay the LDS in this patient and was unreported previously in China. Above finding has enriched the mutational spectrum of the TGFBR2 gene associated with the LDS and provided a basis for the genetic counseling for the patient.

Circ_0069094 regulates malignant phenotype and paclitaxel resistance in breast cancer cells via targeting the miR-136-5p/YWHAZ axis.

BACKGROUND: Breast cancer is one of the most malignant cancers. Increasing evidence suggests that circular RNAs (circRNAs) are involved in breast cancer progression through sponging microRNA (miRNA). However, the underlying molecular mechanisms of circ_0069094 in breast cancer are unclear. This study aimed to reveal the effect of the circ_0069094/miR-136-5p/tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) pathway on the malignant progression of breast cancer. METHODS: The quantitative real-time polymerase chain reaction and western blot were used to assess the expression of circRNA/miRNA/mRNA. The functional effects of circ_0069094 on the cell processes of breast cancer were investigated by cell counting kit-8, colony-forming assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry, and transwell invasion assay. The interactions among circ_0069094, miR-136-5p, and YWHAZ were assessed by dual-luciferase reporter assay. A xenograft experiment was performed to determine the effects of circ_0069094 on tumor formation. RESULTS: Circ_0069094 was overexpressed in paclitaxel (PTX)-resistant breast cancer tissues and cells, and the silencing of circ_0069094 decreased tumor growth, cell proliferation and cell invasion while increasing PTX sensitivity and cell apoptosis in PTX-resistant cells. In addition, miR-136-5p was a target of circ_0069094, and miR-136-5p inhibition abolished circ_0069094 knockdown-induced effects in PTX-resistant cells. MiR-136-5p expression was decreased in PTX-resistant breast cancer tissues and cells, and the overexpression of miR-136-5p suppressed the malignant behaviors of breast cancer cells by targeting YWHAZ. Importantly, circ_0069094 regulated YWHAZ expression in breast cancer by targeting miR-136-5p. CONCLUSION: Circ_0069094 silencing improved PTX sensitivity in breast cancer progression through competitively sponging miR-136-5p.

Circ-TFF1 Facilitates Breast Cancer Development via Regulation of miR-338-3p/FGFR1 Axis.

Some circular RNAs (circRNAs) have been verified to act as essential regulators in the progression of breast cancer (BC). We aimed to investigate the role of circRNA trefoil factor 1 (circ-TFF1) in BC progression. The expression of circ-TFF1, microRNA-338-3p (miR-338-3p) and fibroblast growth factor receptor 1 (FGFR1) mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was evaluated by methylthiazolyldiphenyl-tetrazolium bromide (MTT), colony formation, and 5-Ethynyl-2'-deoxyuridine (EDU) assays. Cell apoptosis and invasion were assessed by flow cytometry and transwell assay, respectively. Cellular glycolysis, including glucose consumption, lactate production, and ATP/ADP ratio, was detected by commercial kits. All protein levels were measured by western blot assay. The relationship between miR-338-3p and circ-TFF1 or FGFR1 was predicted by online bioinformatics tool and verified by dual-luciferase reporter assay. Xenograft tumor model was established to verify the function of circ-TFF1 in vivo. Circ-TFF1 was overexpressed in BC tissues and cells. Circ-TFF1 knockdown inhibited cell proliferation, invasion and glycolysis and induced apoptosis in BC cells. Circ-TFF1 acted as a sponge of miR-338-3p, and the effects of circ-TFF1 knockdown on BC cell proliferation, apoptosis, invasion, and glycolysis were abolished by miR-338-3p inhibition. FGFR1 was confirmed to be a target gene of miR-338-3p, and miR-338-3p played a tumor-suppressive role in BC by targeting FGFR1. Moreover, circ-TFF1 regulated FGFR1 expression by targeting miR-338-3p. Additionally, circ-TFF1 knockdown hampered tumorigenesis in vivo. Circ-TFF1 knockdown suppressed BC progression by regulating miR-338-3p/FGFR1 axis, providing a promising therapeutic target for BC.

Clinical significance of proliferation, apoptosis and senescence of nasopharyngeal cells by the simultaneously blocking EGF, IGF-1 receptors and Bcl-xl genes.

BACKGROUND: In previous work, we constructed short hairpin RNA (shRNA) expression plasmids that targeted human EGF and IGF-1 receptors messenger RNA, respectively, and demonstrated that these vectors could induce apoptosis of human nasopharyngeal cell lines (CNE2) and inhibit ligand-induced pAkt and pErk activation. METHOD: We have constructed multiple shRNA expression vectors of targeting EGFR, IGF1R and Bcl-xl, which were transfected to the CNE2 cells. The mRNA expression was assessed by RT-PCR. The growth of the cells, cell cycle progression, apoptosis of the cells, senescent tumor cells and the proteins of EGFR, IGF1R and Bcl-xl were analyzed by MTT, flow cytometry, cytochemical therapy or Western blot. RESULTS: In group of simultaneously blocking EGFR, IGF1R and Bcl-xl genes, the mRNA of EGFR, IGF1R and Bcl-xl expression was decreased by (66.66±3.42)%, (73.97±2.83)% and (64.79±2.83)%, and the protein expressions was diminished to (67.69±4.02)%, (74.32±2.30)%, and (60.00±3.34)%, respectively. Meanwhile, the cell apoptosis increased by 65.32±0.18%, 65.16±0.25% and 55.47±0.45%, and senescent cells increased by 1.42±0.15%, 2.26±0.15% and 3.22±0.15% in the second, third and fourth day cultures, respectively. CONCLUSIONS: Simultaneously blocking EGFR, IGF1R and Bcl-xl genes is capable of altering the balance between proliferating versus apoptotic and senescent cells in the favor of both of apoptosis and senescence and, therefore, the tumor cells regression.