Reconstituted CD74+ NK cells trigger chronic graft versus host disease after allogeneic bone marrow transplantation.

Chronic graft-versus-host disease (cGVHD) is the most common long-term complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The patients with pulmonary cGVHD in particular have a very poor prognosis. NK cells are the first reconstituted lymphocyte subset after allo-HSCT; however, the impact of reconstituted NK cells on cGVHD is unclear. Here, we found allogeneic recipients showed obvious pulmonary cGVHD. Surprisingly, deletion of reconstituted NK cells resulted in maximal relief of pulmonary cGVHD. Mechanistically, reconstituted NK cells with donor profiles modulated the pulmonary inflammatory microenvironment to trigger cGVHD. Reconstituted NK cells secreted IFN-γ and TNF-α to induce CXCL10 production by epithelial cells, which recruited macrophages and CD4+ T cells to the lungs. Then macrophages and CD4+ T cells were activated by the inflammatory microenvironment, thereby mediating lung injury. Through assessment of differences in cellular energy, we found that CD74+ NK cells with high mitochondrial potential and pro-inflammatory activity triggered pulmonary cGVHD. Furthermore, targeted elimination of CD74+ NK cells using the anti-CD74 antibody significantly alleviated pulmonary cGVHD but preserved the CD74- NK cells to exert graft-versus-leukemia (GVL) effects. Data from human samples corroborated our findings in mouse models. Collectively, our results reveal that reconstituted CD74+ NK cells trigger pulmonary cGVHD and suggest that administration of CD74 antibody was a potential therapeutic for patients with cGVHD.

Natural killer cells promote intra-cellular-infected trophoblasts survival via APOD-LRP1 axis.

Natural killer (NK) cells are known for their potent ability to kill stressed cells, whereas host cells infected with intra-cellular bacteria may also be benefit from the selective killing function of NK cells and survive. The mechanism of how NK cells protect host cells infected with intra-cellular bacteria is still unclear. Here, we discovered that decidual NK (dNK) cells cannot only eliminate intra-cellular bacteria which infected trophoblasts, but can also synthesize more lipids and transport lipids to trophoblasts to avoid their apoptosis. Mechanically, NK cells synthesize more lipids accompanied by increasing expression of apolipoprotein APOD. Lipids in NK cells can be delivered to trophoblast cells through APOD, maintaining adequate lipid droplet content and lipid metabolism homeostasis in trophoblasts. Blocking the APOD receptor LRP1 abolished lipid transport from NK cells to trophoblasts, and the reduction of lipid droplets caused by bacterial infection in trophoblast cells could not be restored, culminating in cell apoptosis. Our study provides new evidence for the immune surveillance and protective effect of NK cells on embryos during early pregnancy.

CD158a+ /CD158b+ NK cell imbalance correlates with hypertension in patients with pre-eclampsia.

PROBLEM: Preeclampsia, a pregnancy complication with hypertension and proteinuria, seriously threats the health and lives of the mother and the baby. The pathogenesis of pre-eclampsia remains incompletely understood. The role of peripheral natural killer cells (NK cells) in the pre-eclampsia is unclear. METHOD OF STUDY: Flow cytometry was performed to detect the expression of CD158a (KIR2DL1) and CD158b (KIR2DL2/3) in peripheral NK cells of healthy pregnant women (HP) and patients with pre-eclampsia (PE). Differentially expressed genes (DEGs) in CD158a+ and CD158b+ NK cells were identified by RNA-sequencing and real-time PCR. Protein array analysis was used to identify altered protein levels in the serum of study participants. RESULTS: CD158a+ NK cell numbers were increased in the peripheral blood of patients while the number of CD158b+ NK cells was reduced. In addition, the percentage of CD158a+ NK cells within the peripheral NK subset was positively correlated with systolic blood pressure while the percentage of CD158b+ NK cells was negatively correlated with systolic blood pressure. RNA-seq and real-time PCR showed that the expression of ERAP2 and GCH1, the genes that regulate blood pressure and angiogenesis, was decreased in CD158a+ compared to CD158b+ NK cells. Consistently, the level of proteins involved in angiogenesis was altered in the serum of pre-eclampsia patients compared to healthy individuals. CONCLUSIONS: CD158a+ NK cells increased while CD158b+ NK cells decreased in the peripheral blood of patients with pre-eclampsia compared to healthy individuals. The change in the frequency of CD158a+ /CD158b+ NK cells is related to the increase in blood pressure.

LncRNA DCST1-AS1 Was Upregulated in Endometrial Carcinoma and May Sponge miR-92a-3p to Upregulate Notch1.

INTRODUCTION: The functions of DCST1-AS1 have been investigated in liver cancer, while its role in endometrial carcinoma (EC) remains hardly known. This study aimed to analyze the role of DCST1-AS1 in EC. METHODS: Paired EC and non-tumor tissue samples were obtained from 62 EC patients. These patients were followed up for 5 years since their admission to record their survival conditions. HEC-1 cells were transfected with DCST1-AS1, Notch1 vectors, miRNA negative control or miR-92a-3p mimic. Luciferase activity was measured. QPCR and Western blot were applied to determine the RNA level and protein expression, respectively. The invasion and migration of HEC-1 cells were analyzed by Transwell assay. RESULTS: We in this study found that DCST1-AS1 was upregulated in EC. Survival analysis revealed that high levels of DCST1-AS1 expression predicted poor survival of EC patients. Bioinformatics analysis revealed that miR-92a-3p may bind DCST1-AS1 and the interaction between them was further confirmed by dual-luciferase activity assay. However, overexpression of miR-92a-3p and DCST1-AS1 failed to affect the expression of each other. Moreover, DCST1-AS1 overexpression led to upregulated Notch1 and increased cancer cell invasion and migration rates. Overexpression of miR-92a-3p played an opposite role and attenuated the effects of DCST1-AS1 overexpression. DISCUSSION: DCST1-AS1 is downregulated in EC and may sponge miR-92a-3p, thereby promoting cancer cell invasion and migration.

Progesterone receptor inhibits the proliferation and invasion of endometrial cancer cells by up regulating Krüppel-like factor 9.

BACKGROUND: Krüppel-like factor 9 (KLF9) is one of the most important members of the KLF family, and is abnormally expressed in many tumors. However, the detailed function of KLF9 in endometrial cancer (EC) was barely investigated. METHODS: In this study, a total of 52 paired EC tissues were recruited to detect the KLF9 expression. Then a serial of phenotypic experiments and mechanism researches were performed. RESULTS: The results showed that KLF9 expression was decreased in EC tissues, and the reduced expression of KLF9 is associated with highly metastatic capacity of EC cells. KLF9 could inhibit the proliferation and invasion of EC cells by inhibiting the Wnt/ß-catenin signaling pathway. Progesterone receptor (PR) could bind to KLF9 promoter and a positive correlation between KLF9 and PR expression was witnessed. CONCLUSIONS: Taken together, the reduction of KLF9 induced by PR might participate in the development of EC and targeting KLF9 may provide a novel strategy for EC management.

Prostaglandin E2 triggers cytochrome P450 17α hydroxylase overexpression via signal transducer and activator of transcription 3 phosphorylation and promotes invasion in endometrial cancer.

Prostaglandin E2 (PGE2) is the most common prostaglandin in the human body, meaning that its malfunction impacts on the development of numerous diseases. Prostaglandin E synthase 2 (PTGES2) is involved in the synthesis of PGE2. In the present study, immunohistochemistry of PTGES2 was performed in 152 patients with endometrial cancer and in 66 patients with normal endometria. The results indicate a notable association among increased expression of PTGES2 and age (P=0.0092) and the depth of myometrial invasion (P<0.0001). Reverse transcription-quantitative polymerase chain reaction and western blot analysis demonstrated that cytochrome P450 17α hydroxylase (CYP17), an enzyme for androgen synthesis, is overexpressed following PGE2 stimulation via signal transducer and activator of transcription 3 (STAT3) phosphorylation. ELISA also detected increased androgen (testosterone) secretion. Further invasion of endometrial cancer cells was induced at high androgen levels and when CYP17 was overexpressed. Furthermore, the present study observed that CYP17 is overexpressed via STAT3 phosphorylation in endometrial cancer cells, which grow at a high concentration of PGE2, resulting in increased androgen secretion. Concentrations of estrogen and progesterone were not elevated, while the concentration of androgens was. Overall, a high concentration of androgens caused increased invasion of endometrial cancer cells. A high concentration of androgens, which is initiated by a high expression of PTGES2 and a high concentration of PGE2, is an important promoter of myometrial invasion in endometrial cancer.

Targeting hexokinase 2 inhibition promotes radiosensitization in HPV16 E7-induced cervical cancer and suppresses tumor growth.

In order to improve the sensitivity of cervical cancer cells to irradiation therapy, we targeted hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis, and explore its role in cervical cancer cells. We suppressed HK2 expression and/or function by shRNA and/or metformin and found HK2 inhibition enhanced cells apoptosis with accelerating expression of cleaved PARP and caspase-3. HK2 inhibition also induced much inferior proliferation of cervical cancer cells both in vitro and in vivo with diminishing expression of mTOR, MIB and MGMT. Moreover, HK2 inhibition altered the metabolic profile of cervical cancer cells to one less dependent on glycolysis with a reinforcement of mitochondrial function and an ablation of lactification ability. Importantly, cervical cancer cells contained HK2 inhibition displayed more sensitivity to irradiation. Further results indicated that HPV16 E7 oncoprotein altered the glucose homeostasis of cervical cancer cells into glycolysis by coordinately promoting HK2 expression and its downregulation of glycolysis. Taken together, our findings supported a mechanism whereby targeting HK2 inhibition contributed to suppress HPV16 E7-induced tumor glycolysis metabolism phenotype, inhibiting tumor growth, and induced apoptosis, blocking the cancer cell energy sources and ultimately enhanced the sensitivity of HPV(+) cervical cancer cells to irradiation therapy.

Dicer1 dysfunction promotes stemness and aggression in endometrial carcinoma.

Endometrial carcinoma is one of the most common gynecological malignancies, but the molecular events involved in the development and progression of endometrial carcinoma remain unclear. Dicer1 and cancer stem cells play important roles in cell motility and survival. This study investigated the role of the let-7 family and Dicer1 in the stemness of endometrial carcinoma cells. We profiled Dicer1 expression in clinical samples and explored its relationship with stem cell-associated markers and clinical parameters. We showed that Dicer1 dysfunction leads to the enrichment of tumor stemness features and tumor aggression both in vitro and in vivo. We also identified the mechanism related to this potential tumor-predisposing phenotype: loss of Dicer1 induced abnormal expression of the let-7 family, which comprises well-known tumor suppressors, thus regulating stemness in endometrial carcinoma cells.

ROR1 promotes the proliferation of endometrial cancer cells.

Endometrial cancer (EC) is the most common gynecological malignant tumor. The canonical Wnt/ß-catenin signaling pathway plays a key role in regulating carcinogenesis, and the noncanonical Wnt5a-ROR1 pathway is an important regulator of Wnt signaling. However, the molecular mechanism by which ROR1 influences Wnt signaling in EC is not known. In this study, we found that ROR1 is expressed at higher levels in tumor tissues and blood samples from patients with stage II EC compared with patients with stage I disease. In vitro, human EC cell lines stably overexpressing ROR1 proliferated more rapidly and formed larger colonies than control cells. Consistent with this, overexpression or knockdown of ROR1 increased or decreased, respectively, the percentage of EC cells in M phase of the cell cycle. Elevated levels of ROR1 were associated with increased expression of Wnt5a and of cyclin D1 and c-Myc, two components of the Wnt signaling pathway. Finally, nude mice grew significantly larger tumors after subcutaneous injection of ROR1-overexpressing EC cells compared with control cells. These findings indicate a novel role for ROR1 in promoting EC cell proliferation by upregulating Wnt5a and stimulating the Wnt/ß-catenin signaling pathway.

Prostaglandin E2 (PGE2) promotes proliferation and invasion by enhancing SUMO-1 activity via EP4 receptor in endometrial cancer.

Prostaglandin E2 (PGE2), a derivative of arachidonic acid, has been identified as a tumorigenic factor in many cancers in recent studies. Prostaglandin E synthase 2 (PTGES2) is an enzyme that in humans is encoded by the PTGES2 gene located on chromosome 9, and it synthesizes PGE2 in human cells. In our study, we selected 119 samples from endometrial cancer patients, with 50 normal endometrium tissue samples as controls, in which we examined the expression of PTGES2. Both immunohistochemistry (IHC) and Western blot analyses demonstrated that synthase PTGES2, which is required for PGE2 synthesis, was highly expressed in endometrium cancer tissues compared with normal endometrium. Stable PTGES2-shRNA transfectants were generated in Ishikawa and Hec-1B endometrial cancer cell lines, and transfection efficiencies were confirmed by RT-PCR and Western blot analyses. We found that PGE2 promoted proliferation and invasion of cells in Ishikawa and Hec-1B cells by cell counting kit-8 tests (CCK8) and transwell assays, respectively. PGE2 stimulation enhanced the expression of SUMO-1, via PGE2 receptor subtype 4 (EP4). Further analysis implicated the Wnt/ß-catenin signaling pathway function as the major mediator of EP4 and SUMO-1. The increase in SUMO-1 activity prompted the SUMOlyation of target proteins which may be involved in proliferation and invasion. These findings suggest SUMO-1 and EP4 as two potential targets for new therapeutic or prevention strategies for endometrial cancers.

Tumor-associated macrophage-derived CXCL8 could induce ERα suppression via HOXB13 in endometrial cancer.

PURPOSE: To elucidate the role of tumor-associated macrophage (TAM) in the loss of ERα in endometrial cancer (EC) and the underlying mechanism. MATERIALS AND METHODS: Tissue microarrays and immunohistochemistry assays were performed using endometrial cancer tissue along with coculture, immunofluorescence, invasion assays and ChIP-qPCR using a human endometrial cancer cell line. RESULTS: Compared with normal tissue, an increased number of TAM was found in EC tissue (34.0 ± 2.6 vs. 8.3 ± 1.1, respectively; p < 0.001), which may downregulate ERα (27.4%, p < 0.05 for HEC-1A and 16.9%, p < 0.05 for Ishikawa) and promote EC cell invasion (1.8-fold, p < 0.001 for HEC-1A and 2.0-fold, p < 0.001 for Ishikawa). Furthermore, we found that TAM-derived CXCL8 mediated the loss of ERα and cancer invasion via HOXB13. HOXB13 was highly expressed in the ERα-negative subtype (r = -0.204, p = 0.002) and low expression of ESR1 was associated with a poor prognosis for EC patients (log-rank p < 0.05). CONCLUSION: TAM-secreted CXCL8 downregulated the ERα expression of EC cells via HOXB13, which may be associated with cancer invasion, metastasis and poor prognosis.

Elevated STMN1 promotes tumor growth and invasion in endometrial carcinoma.

Overexpression of stathmin (STMN1) is closely linked to tumor metastases and poor prognosis in endometrial carcinoma (EC). However, the underlying mechanism is little known. In the present study, we investigated the expression of STMN1 in EC. Subsequently, we assessed the role of STMN1 in EC cell proliferation and migration. Our data show that STMN1 is upregulated in EC, and elevated expression of STMN1 is correlated positively with tumor stage and lymph node metastasis. In vitro, forced expression of STMN1 promoted cell invasion and migration. In contrast, knockdown of STMN1 inhibited cell aggressive behaviors. Moreover, the expression and the activity changes of matrix metalloproteinases (MMP)-2/9 were observed in EC cells after the cells being silenced or overexpression of STMN1. In conclusion, STMN1 is an oncogene and it enhances the growth and invasion of EC possibly by mediating the secretion and activation of MMP2 and MMP9 protein.

Mutant p53 induces EZH2 expression and promotes epithelial-mesenchymal transition by disrupting p68-Drosha complex assembly and attenuating miR-26a processing.

The tumor suppressor p53 and the transcriptional repressor Enhancer of Zeste Homolog 2 (EZH2) have both been implicated in the regulation of epithelial-mesenchymal transition (EMT) and tumor metastasis via their impacts on microRNA expression. Here, we report that mutant p53 (mutp53) promotes EMT in endometrial carcinoma (EC) by disrupting p68-Drosha complex assembly. Overexpression of mutp53 has the opposite effect of wild-type p53 (WTp53), repressing miR-26a expression by reducing pri-miR-26a-1 processing in p53-null EC cells. Re-expression of miR-26a in mutp53 EC cells decreases cell invasion and promotes mesenchymal-epithelial transition (MET). Rescuing miR-26a expression also inhibits EZH2, N-cadherin, Vimentin, and Snail expression and induces E-cadherin expression both in vitro and in vivo. Moreover, patients with higher serum miR-26a levels have a better survival rate. These results suggest that p53 gain-of-function mutations accelerate EC tumor progression and metastasis by interfering with Drosha and p68 binding and pri-miR-26a-1 processing, resulting in reduced miR-26a expression and EZH2 overexpression.

Autocrine motility factor promotes epithelial-mesenchymal transition in endometrial cancer via MAPK signaling pathway.

Autocrine motility factor (AMF) as a cytokine and a growth factor, is known to regulate tumor cell growth and motility in the progress of various human malignant tumors, however, its role in endometrial cancer (EC) has not been fully studied. In the present study, using immunohistochemistry, we found that AMF was highly expressed in EC tissues compared with normal endometrial tissues and tissue micrioarray technology showed positive correlation between AMF expression and epithelial-to-mesenchymal transition (EMT) related markers E-cadherin, vimentin and Snail. Next, we detected that silencing of AMF by stable transfection with shRNA induced mesenchymal-to-epithelial transition phenotype in Ishikawa and HEC-1B cells by qRT-PCR, western blotting and immunofluorescence. Gene expression profile revealed that AMF silencing resulted in altered expression of EMT related molecular mediators including Snail and transforming growth factor ß receptor 1, and involvement of mitogen-activated protein kinase (MAPK) signaling pathway. Additionally, we found that EMT related markers were downregulated with pretreatment of the MAPK-specific inhibitor U0126 by western blotting. The present study is the first to support a role for AMF mediating EMT in endometrial cancer through MAPK signaling. Therefore, AMF may provide a potential prognostic and therapeutic target in preventing EC progression.