A HY5-COL3-COL13 regulatory chain for controlling hypocotyl elongation in Arabidopsis.

CONSTANS-LIKE (COL) family members are commonly implicated in light signal transduction during early photomorphogenesis. However, some of their functions remain unclear. Here, we propose a role for COL13 in hypocotyl elongation in Arabidopsis thaliana. We found that COL13 RNA accumulates at high levels in hypocotyls and that a disruption in the COL13 function via a T-DNA insertion or RNAi led to the formation of longer hypocotyls of Arabidopsis seedlings under red light. On the contrary, overexpression of COL13 resulted in the formation of shorter hypocotyls. Using various genetic, genomic, and biochemical assays, we proved that another COL protein, COL3, directly binds to the promoter of COL13, and the promoter region of COL3 was targeted by the transcription factor LONG HYPOCOTYL 5 (HY5), to form an HY5-COL3-COL13 regulatory chain for regulating hypocotyl elongation under red light. Additionally, further study demonstrated that COL13 interacts with COL3, and COL13 promotes the interaction between COL3 and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), suggesting a possible COP1-dependent COL3-COL13 feedback pathway. Our results provide new information regarding the gene network in mediating hypocotyl elongation.

Assessment of multiple pathways involved in the inhibitory effect of HCG22 on oral squamous cell carcinoma progression.

LncRNAs have been proposed to be associated with the tumorigenesis and progression of oral squamous cell carcinoma (OSCC). LncRNA HLA complex group 22 (HCG22) was reported to be lowly expressed and associated with poor prognosis in head and neck squamous cell carcinoma (HNSCC). However, the biological role and related mechanism of HCG22 in OSCC have not been characterized. HCG22 expression in OSCC cells was detected by qRT-PCR. Cell proliferation, invasion, and apoptosis were evaluated by Bromodeoxyuridine (BrdU) proliferation assay, Transwell invasion assay, and flow cytometry analysis, respectively. The protein levels of proliferating cell nuclear antigen (PCNA), E-cadherin, Vimentin, Bcl-2, Bax, protein kinase B (Akt), phosphorylated Akt (p-Akt), mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), and ß-catenin were detected by western blot. Cell growth evaluation was performed using in vitro colony formation assay and in vivo tumor xenograft assay. We found that HCG22 was weakly expressed in OSCC cells. HCG22 overexpression inhibited cell proliferation and invasion and induced apoptosis in OSCC cells. The levels of PCNA, Vimentin, and Bcl-2 were decreased and E-cadherin and Bax expression was elevated in OSCC cells after HCG22 overexpression. Additionally, HCG22 overexpression inhibited the Akt, mTOR and Wnt/ß-catenin pathways. Activation of Akt, mTOR, and Wnt/ß-catenin pathways attenuated the anti-tumor property of HCG22 in OSCC cells. Furthermore, HCG22 overexpression inhibited the growth of OSCC cells in vitro and in vivo. In conclusion, HCG22 exerted anti-tumor property in OSCC by inhibiting the Akt, mTOR, and Wnt/ß-catenin pathways.

A novel ferroptosis-related genes model for prognosis prediction of lung adenocarcinoma.

BACKGROUND: Ferroptosis is a newly discovered form of cell death characterized by iron-dependent lipid peroxidation. This study aims to investigate the potential correlation between ferroptosis and the prognosis of lung adenocarcinoma (LUAD). METHODS: RNA-seq data were collected from the LUAD dataset of The Cancer Genome Atlas (TCGA) database. Based on ferroptosis-related genes, differentially expressed genes (DEGs) between LUAD and paracancerous specimens were identified. The univariate Cox regression analysis was performed to screen key genes associated with the prognosis of LUAD. LUAD patients were divided into the training set and validation set. Then, we screened out key genes and built a prognostic prediction model involving 5 genes using the least absolute shrinkage and selection operator (LASSO) regression with tenfold cross-validation and the multivariate Cox regression analysis. After dividing LUAD patients based on the median level of risk score as cut-off value, the generated prognostic prediction model was validated in the validation set. Moreover, we analyzed the somatic mutations, and estimated the scores of immune infiltration in the high-risk and low-risk groups. Functional enrichment analysis of DEGs was performed as well. RESULTS: High-risk scores indicated the worse prognosis of LUAD. The maximum area under curve (AUC) of the training set and the validation set in this study was 0.7 and 0.69, respectively. Moreover, we integrated the age, gender, and tumor stage to construct the composite nomogram. The charts indicated that the AUC of LUAD cases with the survival time of 1, 3 and 5 years was 0.698, 0.71 and 0.73, respectively. In addition, the mutation frequency of LUAD patients in the high-risk group was significantly higher than that in the low-risk group. Simultaneously, DEGs were mainly enriched in ferroptosis-related pathways by analyzing the functional results. CONCLUSIONS: This study constructs a novel LUAD prognosis prediction model involving 5 ferroptosis-related genes, which can be used as a promising tool for decision-making of clinical therapeutic strategies of LUAD.

EX527, a Sirt-1 inhibitor, induces apoptosis in glioma via activating the p53 signaling pathway.

Sirtuin-1 (Sirt-1), an NAD-dependent deacetylase, promotes tumorigenesis in glioma; however, whether the Sirt-1 specific inhibitor, EX527 exerts antitumor effects and the underlying mechanism in glioma requires further investigation. In the present study, the proliferative and colony formation abilities of two glioma cell lines (U87MG and LN-299) were inhibited by EX527. Treatment with EX527 increased the number of apoptotic cells (Annexin V-fluorescein isothiocyanate/propidium iodide); pretreatment with the caspase inhibitor Z-VAD-FMK suppressed EX527-induced apoptosis, suggesting that EX527 induced caspase-dependent apoptosis. In addition, western blotting revealed that EX527 treatment increased the expression of cleaved-caspase-3, poly (ADP-ribose) polymerase-1, B-cell lymphoma 2 (Bcl-2)-associated-X-protein and Bcl-2-like 11 but decreased that of Bcl-2. p53 is deacetylated by Sirt-1, attenuating its function. Furthermore, EX527 upregulated the expression of p53, acetylated p53 and the p53 target gene p21. This result suggests that EX527 induced cell apoptosis by activating p53 in glioma. Of note, EX527 exhibited antitumor effects on patient-derived glioma cells under three-dimensional culture conditions. Collectively, the results of the present study indicated that EX527 may be used as an effective compound in the treatment of glioma.

Expression levels of TUBB3, ERCC1 and P-gp in ovarian cancer tissues and adjacent normal tissues and their clinical significance.

PURPOSE: To investigate the expressions of class III ß-tubulin (TUBB3), nucleotide excision repair cross-complementary gene 1 (ERCC1) and P-glycoprotein (P-gp) in ovarian cancer tissues and adjacent normal tissues and their clinical significance. METHODS: Ovarian cancer patients undergoing surgical resection at the Department of Oncology of the Affiliated Hospital of Shandong Medical College from March 2012 to May 2016 were enrolled in this study, from which 166 cases of pathologically confirmed cancer tissues and 50 cases of adjacent normal tissues were collected. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the messenger RNA (mRNA) expression levels of TUBB3, ERCC1 and P-gp in ovarian cancer tissues and adjacent normal tissues, and their relationships with ovarian cancer clinical stage and grade of pathological differentiation were analyzed. RESULTS: The expression levels of TUBB3, ERCC1 and P-gp in ovarian cancer tissues were significantly higher than those in adjacent normal tissues (p<0.05). The later the clinical stage of ovarian cancer was, the higher the expression levels of TUBB3, ERCC1 and P-gp were (p<0.05). The lower the pathological differentiation grade of ovarian cancer was, the higher the expression levels of TUBB3, ERCC1 and P-gp were (p<0.05). TUBB3, ERCC1 and P-gb were positively correlated with clinical stage and pathological differentiation grade. CONCLUSION: TUBB3, ERCC1 and P-gp are involved in the occurrence and development of ovarian cancer and can be used as important indexes judging the severity of ovarian cancer, providing a reference for the occurrence and development of the disease in ovarian cancer patients in clinical practice.

Mesenteric lymph nodes contribute to proinflammatory Th17-cell generation during inflammation of the small intestine in mice.

T cells of the small intestine, including Th17 cells, are critically involved in host protection from microbial infection, and also contribute to the pathogenesis of small bowel inflammatory disorders. Accumulating evidence suggests that mesenteric lymph nodes (MLNs) play important roles in gut-tropic T-cell generation, although it is still unclear if MLNs are involved in the pathogenesis of small intestine inflammation. To address this issue, we analyzed the roles of both MLNs and Peyer's patches (PPs) by evaluating MLN- or PP-deficient mice in an experimental model of small intestine inflammation, induced by CD3-specific mAb injection. Interestingly, MLNs, but not PPs, were essential for the pathogenesis of intestinal inflammation, in particular the accumulation and infiltration of CD4(+) T-cell populations, including Th17 cells, from the blood. In addition, CD4(+) T-cell accumulation was dependent on the function of the α4 ß7 integrin. Furthermore, MLN removal led to a significantly reduced number of peripheral α4 ß7 (+) CD4(+) effector memory T cells under normal conditions, suggesting that MLNs may play a role in maintaining the number of gut-tropic CD4(+) effector memory T cells circulating in the blood. Taken together, the present study highlights the important role of MLNs in contributing to the pathogenesis of small intestine inflammation.

OX40 and IL-7 play synergistic roles in the homeostatic proliferation of effector memory CD4⁺ T cells.

T-cell homeostasis preserves the numbers, the diversity and functional competence of different T-cell subsets that are required for adaptive immunity. Naïve CD4(+) T (TN ) cells are maintained in the periphery via the common γ-chain family cytokine IL-7 and weak antigenic signals. However, it is not clear how memory CD4(+) T-cell subsets are maintained in the periphery and which factors are responsible for the maintenance. To examine the homeostatic mechanisms, CFSE-labeled CD4(+) CD44(high) CD62L(low) effector memory T (TEM ) cells were transferred into sublethally-irradiated syngeneic C57BL/6 mice, and the systemic cell proliferative responses, which can be divided distinctively into fast and slow proliferations, were assessed by CFSE dye dilution. We found that the fast homeostatic proliferation of TEM cells was strictly regulated by both antigen and OX40 costimulatory signals and that the slow proliferation was dependent on IL-7. The simultaneous blockade of both OX40 and IL-7 signaling completely inhibited the both fast and slow proliferation. The antigen- and OX40-dependent fast proliferation preferentially expanded IL-17-producing helper T cells (Th17 cells). Thus, OX40 and IL-7 play synergistic, but distinct roles in the homeostatic proliferation of CD4(+) TEM cells.

Homeostatic proliferation of naive CD4+ T cells in mesenteric lymph nodes generates gut-tropic Th17 cells.

Homeostatic proliferation of naive T cells in the spleen and cutaneous lymph nodes supplies memory-phenotype T cells. The "systemic" proliferative responses divide distinctly into fast or slow cell division rates. The fast proliferation is critical for generation of effector memory T cells. Because effector memory T cells are abundant in the lamina propria of the intestinal tissue, "gut-specific" homeostatic proliferation of naive T cells may be important for generation of intestinal effector memory T cells. However, such organ-specific homeostatic proliferation of naive T cells has not yet been addressed. In this study, we examined the gut-specific homeostatic proliferation by transferring CFSE-labeled naive CD4(+) T cells into sublethally irradiated mice and separately evaluating donor cell division and differentiation in the intestine, mesenteric lymph nodes (MLNs), and other lymphoid organs. We found that the fast-proliferating cell population in the intestine and MLNs had a gut-tropic α4ß7(+) Th17 phenotype and that their production was dependent on the presence of commensal bacteria and OX40 costimulation. Mesenteric lymphadenectomy significantly reduced the Th17 cell population in the host intestine. Furthermore, FTY720 treatment induced the accumulation of α4ß7(+)IL-17A(+) fast-dividing cells in MLNs and eliminated donor cells in the intestine, suggesting that MLNs rather than intestinal tissues are essential for generating intestinal Th17 cells. These results reveal that MLNs play a central role in inducing gut-tropic Th17 cells and in maintaining CD4(+) T cell homeostasis in the small intestine.

Y chromosome-linked B and NK cell deficiency in mice.

There are no primary immunodeficiency diseases linked to the Y chromosome, because the Y chromosome does not contain any vital genes. We have established a novel mouse strain in which all males lack B and NK cells and have Peyer's patch defects. By 10 wk of age, 100% of the males had evident immunodeficiencies. Mating these immunodeficient males with wild-type females on two different genetic backgrounds for several generations demonstrated that the immunodeficiency is linked to the Y chromosome and is inherited in a Mendelian fashion. Although multicolor fluorescence in situ hybridization analysis showed that the Y chromosome in the mutant male mice was one third shorter than that in wild-type males, exome sequencing did not identify any significant gene mutations. The precise molecular mechanisms are still unknown. Bone marrow chimeric analyses demonstrated that an intrinsic abnormality in bone marrow hematopoietic cells causes the B and NK cell defects. Interestingly, fetal liver cells transplanted from the mutant male mice reconstituted B and NK cells in lymphocyte-deficient Il2rg(-/-) recipient mice, whereas adult bone marrow transplants did not. Transducing the EBF gene, a master transcription factor for B cell development, into mutant hematopoietic progenitor cells rescued B cell but not NK cell development both in vitro and in vivo. These Y chromosome-linked immunodeficient mice, which have preferential B and NK cell defects, may be a useful model of lymphocyte development.

Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells.

Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs(flox/flox);mb1(cre/)(+):Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

Orbital Electrowetting-on-Dielectric for Droplet Manipulation on Superhydrophobic Surfaces.

Electrowetting-on-dielectric (EWOD), recognized as the most successful electrical droplet actuation method, is essential in diverse applications, ranging from thermal management to microfluidics and water harvesting. Despite significant advances, it remains challenging to achieve repeatability, high speed, and simple circuitry in EWOD-based droplet manipulation on superhydrophobic surfaces. Moreover, its efficient operation typically requires electrode arrays and sophisticated circuit control. Here, a newly observed droplet manipulation phenomenon on superhydrophobic surfaces with orbital EWOD (OEW) is reported. Due to the asymmetric electrowetting force generated on the orbit, flexible and versatile droplet manipulation is facilitated with OEW. It is demonstrated that OEW droplet manipulation on superhydrophobic surfaces exhibits higher speed (up to 5 times faster), enhanced functionality (antigravity), and manipulation of diverse liquids (acid, base, salt, organic, e.g., methyl blue, artificial blood) without contamination, and good durability after 1000 tests. It is envisioned that this robust droplet manipulation strategy using OEW will provide a valuable platform for various processes involving droplets, spanning from microfluidic devices to controllable chemical reactions. The previously unreported droplet manipulation phenomenon and control strategy shown here can potentially upgrade EWOD-based microfluidics, antifogging, anti-icing, dust removal, and beyond.

GASA14 regulates leaf expansion and abiotic stress resistance by modulating reactive oxygen species accumulation.

Gibberellic acid (GA) can regulate many plant developmental processes. GAST1 has been identified as a GA-stimulated transcript, and Arabidopsis GAST-like genes are known to constitute the GASA family. However, the functions of most GASA genes are not clear at present. In this study, the function of GASA14, a member of the GASA family, was investigated. GASA14 expression was upregulated by GA and downregulated by the transcriptional regulators that repress GA responses, the DELLA proteins GAI and RGA. Phenotypic analysis showed that growth of the GASA14 null mutant (gasa14-1) line was retarded, and the growth of the 35S::GASA14 lines were promoted in young plants. Furthermore, seed germination of the gasa14-1 plants showed more sensitivity to paclobutrazol (an inhibitor of GA biosynthesis) than Columbia (Col) plants, suggesting that GASA14 is required for GA-dependent responses. Analysis of the responses of the gasa14-1 and 35S::GASA14 lines to abscisic acid (ABA) and salt revealed that germination and seedling establishment of gasa14-1 were poorer than those of Col plants and that the 35S::GASA14 lines were more resistant to ABA and salt. Further analysis showed that overexpression of GASA14 could suppress reactive oxygen species (ROS) accumulation. Taken together, these results demonstrated that GASA14 regulates leaf expansion and abiotic stress resistance by modulating ROS accumulation. Because GASA14 contains both GASA (GA-stimulated in Arabidopsis) and PRP (proline-rich protein) domains, the PRP domain coding sequence was overexpressed in Col plants and it was found that the growth of the transgenic plants and the responses to ABA and salt were not altered. These results thus suggest that the GASA domain is necessary for the functions of GASA14.

Transcriptional regulations of pollen tube reception are associated with the fertility of the ginger species Zingiber zerumbet and Zingiber corallinum.

Zingiber zerumbet and Zingiber corallinum are economically valuable species in the genus Zingiber. While Z. corallinum is sexually active, Z. zerumbet adopts clonal propagation, although it has the potential for sexual reproduction. It is unclear so far at which step during the sexual reproduction of Z. zerumbet inhibition occurs, and what are the regulatory mechanisms underlying this inhibition. Here, by comparing with the fertile species Z. corallinum using microscopy-based methods, we show that rare differences were observed in Z. zerumbet up to the point when the pollen tubes invaded the ovules. However, a significantly higher percentage of ovules still contained intact pollen tubes 24 h after pollination, suggesting pollen tube rupture was impaired in this species. Further RNA-seq analysis generated accordant results, showing that the transcription of ANX and FER, as well as genes for the partners in the same complexes (e.g., BUPS and LRE, respectively), and those putative peptide signals (e.g., RALF34), were timely activated in Z. corallinum, which ensured the pollen tubes being able to grow, reorient to ovules, and receipt by embryo sacs. In Z. zerumbet, genes for these complexes were cooperatively suppressed, which would result in the maintenance of PT integrity due to the disruption of RALF34-ANX/BUPS signaling in PT and the failure of PT reception by an active synergid due to the insufficiency of the synergid-harbored FER/LRE complex. Taking the results from the cytological and RNA-seq studies together, a model is proposed to illustrate the possible regulation mechanisms in Z. zerumbet and Z. corallinum, in which the regulations for pollen tube rupture and reception are proposed as the barrier for sexual reproduction in Z. zerumbet.

Predictive value of NLR and PLR in response to preoperative chemotherapy and prognosis in locally advanced gastric cancer.

Purpose: Pretreatment neutrophil-to-lymphocyte (NLR) and platelet-to-lymphocyte (PLR) ratios are markers of systemic inflammation. In patients with locally advanced gastric cancer (GC), the utility of these ratios in predicting tumor regression grade (TRG) after neoadjuvant chemotherapy (NCT) remains unclear. Methods: This retrospective study examined 283 locally advanced GC patients who underwent NCT and radical surgery. The receiver operating characteristic (ROC) curve analysis and the Youden index were applied to identify optimal NLR/PLR cutpoints. The Kaplan-Meier method was used to estimate overall survival (OS) and disease-free survival (DFS). Univariate/multivariate analyses were conducted by the logistic regression method. Results: TRG grade proved significantly worse in patients with high values of both NLR and PLR whether in univariate (OR = 3.457; p = 0.044) or multivariate (OR = 6.876; p = 0.028) analysis. The degree of tumor differentiation was an independent predictive factor for TRG (OR = 2.874; p = 0.037) in multivariate analysis. In the subgroup analyses, NLR predicted OS (p = 0.04) and DFS (p = 0.03) in female patients, whereas PLR was predictive of both OS (p = 0.026) and DFS (p = 0.018) in patients with clinical TNM stage 3 disease and dissected lymph node counts <28. PLR similarly predicted OS in patients <65 years old (p = 0.049), those with positive lymph nodes (p = 0.021), or those with moderate or poorly differentiated tumors (p = 0.049). Conclusion: Pretreatment NLR and PLR together serve to independently predict TRG after NCT and surgery in patients with locally advanced GC. Screening for patients with high NLR and PLR values may allow them to benefit upfront from alternatives to NCT.

UM-164, a Dual Inhibitor of c-Src and p38 MAPK, Suppresses Proliferation of Glioma by Reducing YAP Activity.

UM-164 is a dual inhibitor of c-Src and p38 MAPK, and has been a lead compound for targeting triple-negative breast cancer. UM-164 shows stronger binding to the active sites of Src compared with the conventional Src inhibitor Dasatinib. While Dasatinib has displayed some inhibitory effects on glioma growth in clinical trials, whether UM-164 can suppress glioma growth has not been reported. Here we show that UM-164 suppressed the proliferation, migration and spheroid formation of glioma cells, and induced cell cycle arrest in the G1 phase. Moreover, UM-164 triggered YAP translocation to the cytoplasm and reduced the activity of YAP, as evidenced by a luciferase assay. Accordingly, UM-164 markedly decreased the expression levels of YAP target genes CYR61 and AXL. Importantly, ectopic expression of wild-type YAP or YAP-5SA (YAP constitutively active mutant) could rescue the anti-proliferative effect induced by UM-164. Intriguingly, p38 MAPK appears to play a greater role than Src in UM-164-mediated inhibition of YAP activity. Furthermore, the in vitro anti-glioma effect mediated by UM-164 was confirmed in a xenograft glioma model. Together, these findings reveal a mechanism by which UM-164 suppresses the malignant phenotypes of glioma cells and might provide a rationale for UM-164-based anti-glioma clinical trials.

Oleandrin, a cardiac glycoside, induces immunogenic cell death via the PERK/elF2α/ATF4/CHOP pathway in breast cancer.

Chemotherapeutic agents have been linked to immunogenic cell death (ICD) induction that is capable of augmenting anti-tumor immune surveillance. The cardiac glycoside oleandrin, which inhibits Na+/K+-ATPase pump (NKP), has been shown to suppress breast cancer growth via inducing apoptosis. In the present study, we showed that oleandrin treatment triggered breast cancer cell ICD by inducing calreticulin (CRT) exposure on cell surface and the release of high-mobility group protein B1 (HMGB1), heat shock protein 70/90 (HSP70/90), and adenosine triphosphate (ATP). The maturation and activation of dendritic cells (DCs) were increased by co-culturing with the oleandrin-treated cancer cells, which subsequently enhanced CD8+ T cell cytotoxicity. Murine breast cancer cell line EMT6 was engrafted into BALB/c mice, and tumor-bearing mice were administered with oleandrin intraperitoneally every day. Oleandrin inhibited tumor growth and increased tumor infiltrating lymphocytes including DCs and T cells. Furthermore, the differential mRNA expression incurred by oleandrin was investigated by mRNA sequencing and subsequently confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Mechanistically, oleandrin induced endoplasmic reticulum (ER) stress-associated, caspase-independent ICD mainly through PERK/elF2α/ATF4/CHOP pathway. Pharmacological and genetic inhibition of protein kinase R-like ER kinase (PERK) suppressed oleandrin-triggered ICD. Taken together, our findings showed that oleandrin triggered ER stress and induced ICD-mediated immune destruction of breast cancer cells. Oleandrin combined with immune checkpoint inhibitors might improve the efficacy of immunotherapy.

Nuclear Aurora kinase A triggers programmed death-ligand 1-mediated immune suppression by activating MYC transcription in triple-negative breast cancer.

BACKGROUND: Increasing studies have reported that oncogenes regulate components of the immune system, suggesting that this is a mechanism for tumorigenesis. Aurora kinase A (AURKA), a serine/threonine kinase, is involved in cell mitosis and is essential for tumor cell proliferation, metastasis, and drug resistance. However, the mechanism by which AURKA is involved in immune response regulation is unclear. Therefore, this study aimed to investigate the role of AURKA in immune regulation in triple-negative breast cancer (TNBC). METHODS: Peripheral blood mononuclear cells (PBMCs) were co-cultured with TNBC cells. The xCELLigence Real-Time Cell Analyzer-MP system was used to detect the killing efficiency of immune cells on TNBC cells. The expression of immune effector molecules was tested by quantitative real-time polymerase chain reaction (qRT-PCR) to evaluate immune function. Furthermore, to validate AURKA-regulated immune response in vivo, 4T1 murine breast cancer cell line with AURKA overexpression or downregulation was engrafted into BALB/c mice. The distribution and proportion of immune cells in tumors were further evaluated by immunohistochemistry and flow cytometry. RESULTS: Downregulation of AURKA in TNBC cells increased immune response by activating CD8+ T cell proliferation and activity. Nuclear rather than cytoplasmic AURKA-derived programmed death-ligand 1 (PD-L1) expression was independent of its kinase activity. Mechanistic investigations showed that nuclear AURKA increased PD-L1 expression via an MYC-dependent pathway. PD-L1 overexpression mostly reversed AURKA silencing-induced expression of immune effector molecules, including interleukin- (IL-2), interferon-γ (IFN-γ), and perforin. Moreover, AURKA expression was negatively correlated with the enrichment and activity of tumor-infiltrating CD8+ T cells in 4T1 engrafted BALB/c mouse model. CONCLUSIONS: Nuclear AURKA elevated PD-L1 expression via an MYC-dependent pathway and contributed to immune evasion in TNBC. Therapies targeting nuclear AURKA may restore immune responses against tumors.

Oleandrin induces apoptosis via activating endoplasmic reticulum stress in breast cancer cells.

BACKGROUND: Breast cancer is the most common malignant tumor in women. Due to limited treatment outcome and high rate of metastasis, the prognosis is especially poor for triple-negative breast cancer. It is urgent to discover and develop novel agents for treatment of breast cancer. Herein, we investigated the potential mechanisms of Oleandrin's (a cardiac glycoside) cytotoxic activity against breast cancer cells. METHODS: Cell proliferation was assessed by xCELLigence Real-Time Cell Analyzer (RTCA)-MP system. Apoptotic cells were detected by using Annexin V/PI staining and nuclear fragments observation. The effect of oleandrin on ATP1B3 expression and markers of ER stress were determined by western blot. A primary cell sensitivity assay was performed via a collagen gel droplet-embedded culture drug sensitivity method (CD-DST). RESULTS: Oleandrin suppressed cell proliferation and colony formation in the three breast cancer cell lines but did not affect normal mammary epithelial cells. Additionally, the expression of ATP1B3 was higher in the three breast cancer cell lines compared to MCF10A cells. Treatment with oleandrin increased the number of apoptotic cells and led to nuclear pyknosis, fragmentation, and apoptotic body formation in breast cancer cells. Furthermore, oleandrin treatment increased expression of Bax and Bim but decreased that of Bcl-2. Treatment with oleandrin also upregulated the expression of endoplasmic reticulum stress associated proteins, including eIF2α, ATF4, and CHOP, but not PERK. oleandrin treatment also induced the phosphorylation of PERK and eIF2α. Of note, oleandrin exhibited antitumor effects on patient-derived breast cancer cells under three-dimensional culture conditions. CONCLUSIONS: Taken together, our results suggest that oleandrin induces mitochondrial-mediated apoptosis by activating endoplasmic reticulum stress in breast cancer. Moreover, oleandrin may be an effective strategy for the treatment of breast cancer.

The R2R3-MYB transcription factor GhMYB1a regulates flavonol and anthocyanin accumulation in Gerbera hybrida.

Anthocyanins and flavonols have vital roles in flower coloration, plant development, and defense. Because anthocyanins and flavonols share the same subcellular localization and common biosynthetic substrates, these pathways may compete for substrates. However, the mechanism regulating this potential competition remains unclear. Here, we identified GhMYB1a, an R2R3-MYB transcription factor involved in the regulation of anthocyanin and flavonol accumulation in gerbera (Gerberahybrida). GhMYB1a shares high sequence similarity with that of other characterized regulators of flavonol biosynthesis. In addition, GhMYB1a is also phylogenetically grouped with these proteins. The overexpression of GhMYB1a in gerbera and tobacco (Nicotianatabacum) resulted in decreased anthocyanin accumulation and increased accumulation of flavonols by upregulating the structural genes involved in flavonol biosynthesis. We further found that GhMYB1a functions as a homodimer instead of interacting with basic helix-loop-helix cofactors. These results suggest that GhMYB1a is involved in regulating the anthocyanin and flavonol metabolic pathways through precise regulation of gene expression. The functional characterization of GhMYB1a provides insight into the biosynthesis and regulation of flavonols and anthocyanins.

An ETHYLENE INSENSITIVE3-LIKE1 Protein Directly Targets the GEG Promoter and Mediates Ethylene-Induced Ray Petal Elongation in Gerbera hybrida.

Petal morphogenesis has a profound influence on the quality of ornamental flowers. Most current research on petal development focuses on the early developmental stage, and little is known about the late developmental stage. Previously, it was reported that the GEG gene [a gerbera homolog of the gibberellin-stimulated transcript 1 (GAST1) from tomato] negatively regulates ray petal growth during the late stage of development by inhibiting longitudinal cell expansion. To explore the molecular mechanisms of the role of GEG in petal growth inhibition, an ethylene insensitive 3-like 1 (EIL1) protein was identified from a Gerbera hybrida cDNA library by yeast one-hybrid screening. Direct binding between GhEIL1 and the GEG promoter was confirmed by electrophoretic mobility shift and dual-luciferase assays. The expression profiles of GhEIL1 and GEG were correlated during petal development, while a transient transformation assay suggested that GhEIL1 regulates GEG expression and may be involved in the inhibition of ray petal elongation and cell elongation. To study the effect of ethylene on ray petal growth, a hormone treatment assay was performed in detached ray petals. The results showed that petal elongation is limited and promoted by ACC and 1-MCP, respectively, and the expression of GhEIL1 and GEG is regulated and coordinated during this process. Taken together, our research suggests that GhEIL1 forms part of the ethylene signaling pathway and activates GEG to regulate ray petal growth during the late developmental stage in G. hybrida.