HER2-targeting CAR-T cells show highly efficient anti-tumor activity against glioblastoma both in vitro and in vivo.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. Current treatment options for GBM include surgical resection, radiation, and chemotherapy, which predominantly slow cancer growth and reduce symptoms, resulting in a 5-year survival rate of no more than 10%. Chimeric antigen receptor (CAR) T-cell therapy is a new class of cellular immunotherapy that has made great progress in treating malignant tumors. Human epidermal growth factor receptor 2 (HER2) is overexpressed in GBM and may provide a potential therapeutic target for GBM treatment. In this study, we constructed third-generation CAR-T cells targeting the HER2 antigen in GBM. HER2-CAR-T cells showed effective anti-tumor activity both in vitro and in vivo. Furthermore, HER2-specific CAR-T cells exhibited strong cytotoxicity and cytokine-secreting abilities against GBM cells in vitro. Anti-HER2 CAR-T cells also exhibited increased cytotoxicity with increasing effector-to-target ratios. Anti-HER2 CAR-T cells delivered via peritumoral injection successfully stunted tumor progression in vivo. Moreover, peritumoral intravenous administration of anti-HER2 CAR-T cells resulted in therapeutic improvement against GBM cells compared with intravenous administration. In conclusion, our study shows that HER2 CAR-T cells represent an emerging immunotherapy for treating GBM.

Ablation of the GDP-fucose transporter suppresses lung cancer cell proliferation and migration by reducing expression of PD-L1.

Fucosylation, an important post-translational modification, is closely related to the development of tumors. In the microenvironment of lung cancer, expression of PD-L1 and fucosylation is abnormally upregulated. However, the correlation between PD-L1 expression and its fucosylation in lung adenocarcinoma (LUAD) remains unclear. The GDP-fucose transporter (GFT) is a key molecule in cellular fucosylation. To explore the correlation between fucosylation and PD-L1 expression, we knocked out the GFT-encoding gene SLC35C1 in mouse Lewis lung adenocarcinoma cells and in human H1299 lung adenocarcinoma cells. Loss of SLC35C1 impaired the phosphorylation of EGFR and its downstream target ERK. Moreover, loss of SLC35C1 up-regulated the expression of ß-TrCP, a PD-L1 E3 ligase, thereby promoting the ubiquitination of PD-L1 and its subsequent degradation. The down-regulated expression of PD-L1 leads to a decline in lung cancer cell proliferation and migration. These results suggest that fucosylation partially influences LUAD tumorigenesis by regulating PD-L1 expression.

Serum CXCL8 and CXCR2 as diagnostic biomarkers for noninvasive screening of cervical cancer.

BACKGROUND: Cervical cancer (CC) is the fourth most frequently diagnosed cancer and the fourth leading cause of cancer-related death in women. Identifying new biomarkers for the early detection of CC is an essential requirement in this field. CXCL8 was originally discovered because of its role in inflammation by binding to CXCR1 and CXCR2; however, it is now known to play an important role in cancer. In this study, we aimed to evaluate the expression levels of potential biomarkers (CXCL8, CXCR1, and CXCR2) and to explore their diagnostic potential in CC. METHODS: The expression levels of serum CXCL8, CXCR1, and CXCR2 were investigated by kit method on Immulite-1000 in 30 healthy volunteers, 30 precancerous patients and 70 CC patients. RESULTS: The results indicated that the expression of CXCL8 and CXCR2 was significantly higher in the serum of CC patients than in healthy volunteers, similar to the well-established tumor marker (squamous-cell cancerantigen [SCC]). Receiver operating characteristic analyses showed that the combination of CXCL8, CXCR2, and SCC had the highest diagnostic sensitivity and area under the curve value. Meanwhile, the positive predictive value and negative predictive value were not very low. Moreover, high concentrations of CXCL8 and CXCR2 are associated with an increased risk of CC. CONCLUSIONS: In conclusion, our data demonstrated that combined serum CXCL8, CXCR2, and SCC measurements are helpful for CC diagnosis and can be used as potential biomarkers for the early detection of CC. Cytokines, such as CXCL8 and CXCR2, can be easily measured in most university hospital laboratories and in some private laboratories with a routine test.

Core fucosylation and its roles in gastrointestinal glycoimmunology.

Glycosylation is a common post-translational modification in eukaryotic cells. It is involved in the production of many biologically active glycoproteins and the regulation of protein structure and function. Core fucosylation plays a vital role in the immune response. Most immune system molecules are core fucosylated glycoproteins such as complements, cluster differentiation antigens, immunoglobulins, cytokines, major histocompatibility complex molecules, adhesion molecules, and immune molecule synthesis-related transcription factors. These core fucosylated glycoproteins play important roles in antigen recognition and clearance, cell adhesion, lymphocyte activation, apoptosis, signal transduction, and endocytosis. Core fucosylation is dominated by fucosyltransferase 8 (Fut8), which catalyzes the addition of α-1,6-fucose to the innermost GlcNAc residue of N-glycans. Fut8 is involved in humoral, cellular, and mucosal immunity. Tumor immunology is associated with aberrant core fucosylation. Here, we summarize the roles and potential modulatory mechanisms of Fut8 in various immune processes of the gastrointestinal system.

Loss of core fucosylation suppressed the humoral immune response in Salmonella typhimurium infected mice.

BACKGROUND: The humoral immune response is pivotal to protect the host from Salmonella typhimurium (S. typhimurium) infection. Previously, we found that core fucosylation catalyzed by core fucosyltransferase (Fut8) could regulate the immune responses. However, the role of core fucosylation during S. typhimurium infection remains unclear. METHODS: To demonstrate the role of Fut8 in S. typhimurium infection, we infected Fut8+/+ and Fut8-/- mice using S. typhimurium. The production of antiserum against the S. typhimurium was detected. The expression of T and B cell activation-related genes during S. typhimurium infection was analyzed. The role of core fucosylation on CD4+ T-B cell interaction and B cell generation was investigated during S. typhimurium infection. The production of sIgA was compared between Fut8+/+ and Fut8-/- mice. RESULTS: Compared to Fut8+/+ mice, the number of S. typhimurium colonized in the cecum was markedly increased in Fut8-/- mice. The production of the IgG and sIgA specific for S. typhimurium was significantly decreased in Fut8-/- mice. Moreover, loss of Fut8 decreased the induction of Th2-type cytokines from splenic cells of Fut8-/- mice during S. typhimurium infection. In addition, we found that the core fucosylation regulated the interaction between B and T cells in the lipid raft formation. CONCLUSION: Core fucosylation plays important roles in host defence against S. typhimurium infection.

Loss of core fucosylation enhances the anticancer activity of cytotoxic T lymphocytes by increasing PD-1 degradation.

As an immune checkpoint, programmed cell death 1 (PD-1) and its ligand (PD-L1) pathway plays a crucial role in CD8+ cytotoxic T lymphocytes (CTL) activation and provides antitumor responses. The N-glycans of PD-1 and PD-L1 are highly core fucosylated, which are solely catalyzed by the core fucosyltransferase (Fut8). However, the precise biological mechanisms underlying effects of core fucosylation of PD-1 and PD-L1 on CTL activation have not been fully understood. In this study, we found that core fucosylation was significantly upregulated in lung adenocarcinoma. Compared to those of Fut8+/+ OT-I mice, the lung adenocarcinoma formation induced by urethane was markedly reduced in Fut8-/- OT-I mice. De-core fucosylation of PD-1 compromised its expression on Fut8-/- CTL, resulted in enhanced Fut8-/- CTL activation and cytotoxicity, leading to more efficient tumor eradication. Indeed, loss of core fucosylation significantly enhanced the PD-1 ubiquitination and in turn led to the degradation of PD-1 in the proteasome. Our current work indicates that inhibition of core fucosylation is a unique strategy to reduce PD-1 expression for the antilung adenocarcinoma immune therapy in the future.

FoxO1 controls the expansion of pre-B cells by regulating the expression of interleukin 7 receptor α chain and its signal pathway.

Forkhead box O1 (FoxO1) has a crucial role in the early B cell development. To understand the functional importance of FoxO1 gene in the early B cell expansion, we established a FoxO1 knockdown model using 70Z/3 pre-B cell line. The FoxO1 knockdown 70Z/3 cells (70Z/3-KD cells) showed the down-regulated expression of interleukin 7 receptor α chain (IL-7Rα). Moreover, the signaling via IL-7Rα was significantly attenuated in the 70Z/3-KD cells, and this alteration was fully rescued by re-expression of FoxO1 gene. Compared to the mock cells, loss of FoxO1 reduced the growth rates in the 70Z/3-KD cells, and was fully rescued by reintroduction of FoxO1 gene. The expansion of pre-B cells (CD45R+CD43- fraction) was also reduced by the knockdown of FoxO1 gene. Indeed, FoxO1 induces accumulation in the p27-mediated G0/G1 phase arrest in 70Z/3 cells. FoxO1 bound to the Il7ra locus specifically and regulate the IL-7Rα transcription. In conclusion, FoxO1 regulates the expansion of pre-B cells by regulating the expression of IL-7Rα and its signal transduction.

Ablation of core fucosylation attenuates the signal transduction via T cell receptor to suppress the T cell development.

Precise glycosylation plays a crucial and distinctive role in thymic T cell development. The core fucosylation is dramatically up-regulated at the transition from CD4-CD8- (DN) to CD4+CD8+ (DP) in the thymic development. Ablation of core fucosylation in T cells did reduce the size of the thymus due to a significant loss of CD4+ SP, CD8+ SP and DP thymocytes in core fucosyltransferase (Fut8) knockout (Fut8-/-) mice. T cell receptors (TCRs) are heavily core fucosylated glycoproteins. Loss of core fucosylation of TCR contributed to the reduced phosphorylation of ZAP70 (pZAP70) in Fut8-/- DP cells was observed. Compare to the Fut8+/+OT-II DP thymocytes, pZAP70 was significantly reduced in Fut8-/- OT-II DP thymocytes with OVA323-339 stimulation. Also, the pZAP70 of Fut8+/+OT-I DP thymocytes with OVA257-264 stimulation was remarkably attenuated by treatment of the fucosidase. Upon anti-CD3/CD28 Abs stimulation, the increased apoptosis was found in Fut8-/- thymocytes compared with Fut8+/+ thymocytes. Moreover, the TCRhiCD69hi (post-positive selection thymocytes) was markedly depleted in the Fut8-/- thymus without any stimulation. The expression of CD5 was significantly down-regulated on the DP cells in the Fut8-/- thymus. Our results therefore demonstrate that ablation of core fucosylation results in the abnormal T cell development due to the attenuated signaling via TCR.

Forced FoxO1:S249V expression suppressed glioma cell proliferation through G2/M cell cycle arrests and increased apoptosis.

OBJECTIVE: Forkhead box O1 (FoxO1) plays a crucial role in the development of many tumors. Cyclin D kinase (CDK) 1 could influence the nuclear export and activity of FoxO1 through phosphorylation of serine (S)249. However, the effects of S249 phosphorylation in the development of glioma remain unclear. The aim of the present study is to assess the function of FoxO1:S249V mutant, which was converted S249 phosphorylation site into valine (V) residues in the glioma development. METHODS: FoxO1-knockdown U251 glioma cells (U251-KD cells) were established by infection of retrovirus particles with FoxO1 siRNA and FoxO1 restored cells (FoxO1:S249V) were obtained by re-introduction of FoxO1:S249V cDNA. We detected mRNA expression by real-time PCR, and cell cycle arrest and apoptosis by flow cytometric assay, and cell proliferation by BrdU assay and CCK-8 assay. The protective effects of FoxO1:S249V were detected by the xenograft tumor formation assay. RESULTS: The FoxO1 mRNA expression was significantly decreased in the glioma specimens (n = 24). The U251-KD cells showed downregulation of p27 and Bim, while the phosphorylation of CDK1 was upregulated. FoxO1:S249V cells inhibited the phosphorylation of S249, and induced G2/M cell cycle arrest, following reduced cell growth and increased apoptosis. Moreover, FoxO1:S249V expression effectively inhibits the glioma growth. CONCLUSION: Our findings suggest that the forced FoxO1:S249V suppressed the cell growth through G2/M cell cycle arrests and increased apoptosis in glioma.

The requirement of SEPT2 and SEPT7 for migration and invasion in human breast cancer via MEK/ERK activation.

Septins are a novel class of GTP-binding cytoskeletal proteins evolutionarily conserved from yeast to mammals and have now been found to play a contributing role in a broad range of tumor types. However, their functional importance in breast cancer remains largely unclear. Here, we demonstrated that pharmaceutical inhibition of global septin dynamics would greatly suppress proliferation, migration and invasiveness in breast cancer cell lines. We then examined the expression and subcellular distribution of the selected septins SEPT2 and SEPT7 in breast cancer cells, revealing a rather variable localization of the two proteins with cell cycle progression. To determine the role of both septins in mediating malignant behavior of cancer cells, we used RNA silencing to specifically deplete endogenous SEPT2 or SEPT7 in highly invasive breast cancer cell line MDA-MB-231. Our findings showed that SEPT2/7 depletion had virtually identical inhibitory effects on cellular proliferation, apoptosis, migration and invasion. Moreover, the opposite performance in migration and invasion was observed after enforced expression of SEPT2/7 in the same cell line. We further demonstrated MEK/ERK activation, but not other MAPKs and AKT, was positively correlated with the protein levels of SEPT2 and SEPT7. Additionally, in SEPT2/7-overexpressing cells, the MEK specific inhibitor U0126 was able to correct the high active status of MEK/ERK while normalizing the increased invasive behaviors of these cells. Taken together, these results strongly suggest that SEPT2 and SEPT7 are involved in breast carcinogenesis and may serve as valuable therapeutic targets for breast cancer.