A Gene Expression Signature to Predict Nucleotide Excision Repair Defects and Novel Therapeutic Approaches.

Nucleotide excision repair (NER) resolves DNA adducts, such as those caused by ultraviolet light. Deficient NER (dNER) results in a higher mutation rate that can predispose to cancer development and premature ageing phenotypes. Here, we used isogenic dNER model cell lines to establish a gene expression signature that can accurately predict functional NER capacity in both cell lines and patient samples. Critically, none of the identified NER deficient cell lines harbored mutations in any NER genes, suggesting that the prevalence of NER defects may currently be underestimated. Identification of compounds that induce the dNER gene expression signature led to the discovery that NER can be functionally impaired by GSK3 inhibition, leading to synergy when combined with cisplatin treatment. Furthermore, we predicted and validated multiple novel drugs that are synthetically lethal with NER defects using the dNER gene signature as a drug discovery platform. Taken together, our work provides a dynamic predictor of NER function that may be applied for therapeutic stratification as well as development of novel biological insights in human tumors.

BRCT-domain protein BRIT1 influences class switch recombination.

DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs.

Mass Transfer Analysis for Achieving High-Rate Lithium-Air Batteries.

Lithium-air batteries (LABs) have aroused worldwide interest due to their high energy density as a promising next-generation battery technology. From a practical standpoint, one of the most pressing issues currently in LABs is their poor rate performance. Accelerating the mass transfer rate within LABs is a crucial aspect for enhancing their rate capability. In this Perspective, we have meticulously analyzed the ion and oxygen transport processes to provide readers with a comprehensive understanding of the mass transfer within LABs. Following this, we have discussed potential misconceptions in the existing literature and propose our recommendations for improving the rate performance of LABs. This Perspective provides a deep insight into the mass transfer process in LABs and offers promising strategies for developing other high-rate metal-O2 batteries.

A Rotating Cathode with Periodical Changes in Electrolyte Layer Thickness for High-Rate Li‒O2 Batteries.

Li-O2 batteries (LOBs) possess the highest theoretical gravimetric energy density among all types of secondary batteries, but they are still far from practical applications. The poor rate performance resulting from the slow mass transfer is one of the primary obstacles in LOBs. To solve this issue, a rotating cathode with periodic changes in the electrolyte layer thickness is designed, decoupling the maximum transfer rate of Li+ and O2. During rotation, the thinner electrolyte layer on the cathode facilitates the O2 transfer, and the thicker electrolyte layer enhances the Li+ transfer. As a result, the rotating cathode enables the LOBs to undergo 58 cycles at 2.5 mA cm-2 and discharge stably even at a high current density of 7.5 mA cm-2. Besides, it also makes the batteries exhibit a large discharge capacity of 6.8 mAh cm-2, and the capacity decay is much slower with increasing current density. Notably, this rotating electrode holds great promise for utilization in other electrochemical cells involving gas-liquid-solid triple-phase interfaces, suggesting a viable approach to enhance the mass transfer in such systems.

BRIT1 regulates p53 stability and functions as a tumor suppressor in breast cancer.

In humans, the gene encoding the BRCA1 C terminus-repeat inhibitor of human telomerase expression 1 (BRIT1) protein is located on chromosome 8p23.1, a region implicated in the development of several malignancies, including breast cancer. Previous studies by our group and others suggested that BRIT1 might function as a novel tumor suppressor. Thus, identifying the molecular mechanisms that underlie BRIT1's tumor suppressive function is important to understand cancer etiology and to identify effective therapeutic strategies for BRIT1-deficient tumors. We thus investigated the role of BRIT1 as a tumor suppressor in breast cancer by using genetic approaches. We discovered that BRIT1 functions as a post-transcriptional regulator of p53 expression. BRIT1 regulates p53 protein stability through blocking murine double minute 2-mediated p53 ubiquitination. To fully demonstrate the role of BRIT1 as a tumor suppressor, we depleted BRIT1 in normal breast epithelial cells. We found that knockdown of BRIT1 caused the oncogenic transformation of normal mammary epithelial cells. Furthermore, ectopic expression of BRIT1 effectively suppressed breast cancer cell proliferation and colony formation in vitro and tumor growth in vivo. Taken together, our study provides new insights into the biological functions of BRIT1 as a tumor suppressor in human breast cancer.

BRIT1/MCPH1 is essential for mitotic and meiotic recombination DNA repair and maintaining genomic stability in mice.

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1(-/-) mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1(-/-) mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to gamma-irradiation. BRIT1(-/-) MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1(-/-) mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2's function and as a result leads to infertility and genomic instability in mice.

Analysis of the value and safety of thyroid-stimulating hormone in the clinical efficacy of patients with thyroid cancer.

BACKGROUND: Thyroid cancer (TC) is a common malignant tumor in the endocrine system. In recent years, the incidence and recurrence rates of TC have been raising due to increasing work pressure and irregular lifestyles. Thyroid-stimulating hormone (TSH) is a specific parameter for thyroid function screening. This study aims to explore the clinical value of TSH in regulating the progression of TC, so as to find a breakthrough for the early diagnosis and treatment of TC. AIM: To explore the value and safety of TSH in the clinical efficacy of patients with TC. METHODS: 75 patients with TC admitted to the Department of Thyroid and Breast Surgery of our hospital from September 2019 to September 2021 were selected as the observation group, and 50 healthy subjects were selected as the control group during the same period. The control group was treated with conventional thyroid replacement therapy, and the observation group was treated with TSH suppression therapy. The soluble interleukin (IL)-2 receptor (sIL-2R), IL-17, IL-35 levels, free triiodothyronine (FT3), free tetraiodothyronine (FT4), CD3+, CD4+, CD8+, CD44V6, and tumor supplied group of factor (TSGF) levels were observed in the two groups. The occurrence of adverse reactions was compared between the two groups. RESULTS: After treatment with different therapies, the levels of FT3, FT4, CD3+, and CD4+ in the observation group and the control group were higher than those before treatment, while the levels of CD8+, CD44V6, and TSGF were lower than those before treatment, and the differences were statistically significant (P < 0.05). More importantly, the levels of sIL-2R and IL-17 in the observation group were lower than those in the control group after 4 wk of treatment, while the levels of IL-35 were higher than those in the control group, and the differences were statistically significant (P < 0.05). The levels of FT3, FT4, CD3 +, and CD4 + in the observation group were higher than those in the control group, and the levels of CD8+, CD44V6, and TSGF were lower than those in the control group. There was no significant difference in the overall incidence rate of adverse reactions between the two groups (P > 0.05). CONCLUSION: TSH suppression therapy can improve the immune function of patients with TC, lower the CD44V6 and TSGF levels, and improve serum FT3 and FT4 levels. It demonstrated excellent clinical efficacy and a good safety profile.

Identification of Hypoxia-Associated Signature in Colon Cancer to Assess Tumor Immune Microenvironment and Predict Prognosis Based on 14 Hypoxia-Associated Genes.

Purpose: Colon cancer is the main malignant tumor of the digestive tract. Hypoxia is highly related to the occurrence, progression and tumor immune microenvironment (TIME) of cancer. The aim of this study was to identify a hypoxia-associated signature with high accuracy for predicting the prognosis and TIME of colon cancer. Methods: Download colon cancer data from the GEO and TCGA databases. A novel hypoxia risk model was identified to predict the prognosis of colon cancer patients. Subsequently, GSEA, TIME and mutation analysis were performed in the hypoxia high and low risk score groups. Finally, the signature gene ANKZF1 was selected for functional verification at the cellular level. Results: A novel hypoxia risk model was identified. The risk score was significantly associated with poorer overall survival in colon cancer, and could be used as an independent prognostic factor for colon cancer. GSEA analysis found that the processes related to stimulate tumor proliferation and anti-apoptosis were significantly enriched in the hypoxia high risk score group. The expression of immunosuppressive cells and most immune checkpoints in the high risk score group was significantly higher than that in the low risk score group. In vitro cell experiments showed that knockdown the expression of ANKZF1 could inhibit the proliferation, migration and invasion of colon cancer cells. Conclusion: Hypoxia plays an important role in evaluating the TIME and predicting the prognosis of colon cancer.

Hydrogen-Bond-Assisted Solution Discharge in Aprotic Li-O2 Batteries.

Surface discharge mechanism induced cathode passivation is a critical challenge that blocks the full liberation of the ultrahigh theoretical energy density in aprotic Li-O2 batteries. Herein, a facile and universal concept of hydrogen-bond-assisted solvation is proposed to trigger the solution discharge process for averting the shortcomings associated with surface discharge. 2,5-Di-tert-butylhydroquinone (DBHQ), an antioxidant with hydroxyl groups, is introduced as an exemplary soluble catalyst to promote solution discharge by hydrogen-bond-assisted solvation of O2 - and Li2 O2 (OH···O). Thus, a Li-O2 battery with 50 × 10-3 m DBHQ delivers an extraordinary discharge capacity of 18 945 mAh g-1 (i.e., 9.47 mAh cm-2 ), even surpassing the capacity endowed by the state-of-the-art reduction mediator of 2,5-di-tert-butyl-1,4-benzoquinone. Besides, an ultrahigh Li2 O2 yield of 97.1% is also achieved due to the depressed reactivity of the reduced oxygen-containing species (O2 - , LiO2 , and Li2 O2 ) by the solvating and antioxidative abilities of DBHQ. Consequently, the Li-O2 battery with DBHQ exhibits excellent cycling lifetime and rate capability. Furthermore, the generalizability of this approach of hydrogen-bond-assisted solution discharge is verified by other soluble catalysts that contain OH or NH groups, with implications that could bring Li-O2 batteries one step closer to being a viable technology.

Autophagy-related genes contribute to malignant progression and have a clinical prognostic impact in colon adenocarcinoma.

Autophagy has an important role in regulating tumor cell survival. However, the roles of autophagy-related genes (ARGs) during colon adenocarcinoma (COAD) progression and their prognostic value have remained elusive. The present study aimed to identify the correlation between ARGs and the progression of COAD, as well as the prognostic significance of ARGs. The transcriptome profiles and the corresponding clinicopathological information of patients with COAD were downloaded from The Cancer Genome Atlas and Genotype-Tissue Expression databases. A list of ARGs was obtained from the Human Autophagy Database and bioinformatics analysis was performed to investigate the functions of these ARGs. Statistical analyses of these genes were performed to identify independent prognostic markers. The selected prognostic markers were then validated in 15 patients with COAD via immunohistochemistry. Differentially expressed ARGs between normal and tumor tissues were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that the differentially expressed ARGs were mainly enriched in toxoplasmosis and pathways in cancer. The ATG4B, DAPK1 and SERPINA1 genes were determined to be associated with COAD progression. In addition, a risk signature was proposed that may serve as an independent prognostic marker. In conclusion, ATG4B, DAPK1 and SERPINA1 are crucial participants in tumorigenesis of COAD. The present study may promote the development of novel treatment strategies for COAD.

Long Noncoding RNA DLX6-AS1 Regulates the Growth and Aggressiveness of Colorectal Cancer Cells Via Mediating the miR-26a/EZH2 Axis.

Objective: To understand the regulation of long noncoding RNA DLX6-AS1-mediated miR-26a/EZH2 axis in the growth of colorectal cancer (CRC) cells. Methods: The expression of DLX6-AS1, miR-26a, and EZH2 was detected in CRC tissues by quantitative reverse transcription-polymerase chain reaction. The CRC HT-29 cell line was selected for transfection and subjected to observe the growth by MTT and colony formation assays, cell cycle by flow cytometry, and migration and invasion by wound healing and Transwell assays, respectively. Finally, the expression of cycle- and metastasis-related proteins was detected by Western blotting. Results: DLX6-AS1 and EZH2 were increased, with a decreased miR-26a in CRC tissues, showing significant negative correlations between DLX6-AS1 and miR-26a, and between miR-26a and EZH2. CRC patients at advanced stage or with lymphatic metastasis had higher DLX6-AS1 expression. Dual-luciferase reporter gene assay uncovered the targeting correlations between DLX6-AS1 and miR-26a, or miR-26a and EZH2. After transfection of DLX6-AS1 siRNA or EZH2 siRNA, the growth and metastasis of CRC cells were suppressed, arresting the cells in G0/G1 phase, with a magnificent reduction in the ratio of cells in S phase or G2/M phase; meanwhile, Cyclin D1, Vimentin, and MMP9 expressions decreased evidently, whereas E-cadherin expression was upregulated. Changes above were fully reversed after transfection of miR-26a inhibitor, whereas si-EZH2 transfection abolished the positive role of miR-26a inhibitor on growth of CRC cells. Conclusion: Silencing DLX6-AS1 may block the malignant features of CRC cells by inhibiting the expression of EZH2 through upregulation of miR-26a. Thus, it is critical to the development and progression of CRC.

Replication stress response defects are associated with response to immune checkpoint blockade in nonhypermutated cancers.

Treatment with immune checkpoint blockade (ICB) has resulted in durable responses for a subset of patients with cancer, with predictive biomarkers for ICB response originally identified largely in the context of hypermutated cancers. Although recent clinical data have demonstrated clinical responses to ICB in certain patients with nonhypermutated cancers, previously established ICB response biomarkers have failed to accurately identify which of these patients may benefit from ICB. Here, we demonstrated that a replication stress response (RSR) defect gene expression signature, but not other proposed biomarkers, is associated with ICB response in 12 independent cohorts of patients with nonhypermutated cancer across seven tumor types, including those of the breast, prostate, kidney, and brain. Induction or suppression of RSR deficiencies was sufficient to modulate response to ICB in preclinical models of breast and renal cancers. Mechanistically, we found that despite robust activation of checkpoint kinase 1 signaling in RSR-deficient cancer cells, aberrant replication origin firing caused exhaustion of replication protein A, resulting in accumulation of immunostimulatory cytosolic DNA. We further found that deficient RSR coincided with increased intratumoral dendritic cells in both mouse cancer models and human tumors. Together, this work demonstrates that the RSR defect gene signature can accurately identify patients who may benefit from ICB across numerous nonhypermutated tumor types, and pharmacological induction of RSR defects may further expand the benefits of ICB to more patients.

BRIT1 dysfunction confers synergistic inhibition of hepatocellular carcinoma by targeting poly (ADP-ribose) polymerases and PI3K.

BRIT1 has emerged as a novel key player in homologous recombination (HR). It is located in 8p23, a locus frequently deleted in hepatocellular carcinoma (HCC). Previously, we found that BRIT1-deficiency triggered genomic instability and tumor formation in our mouse model. Here we aim to determine whether BRIT1 aberrations are implicated in HCC and, if so, whether they can be used for targeted therapy with PARP inhibitors and other agents. We analyzed HCC samples for BRIT1 alterations at DNA, RNA and protein levels. BRIT1 was found deleted and/or downregulated in ~30% of HCC samples; BRIT1 mutant K659fsX10 identified in HCC abolished DNA repair function. Notably, BRIT1 deletion was correlated with poor survival and high recurrence of HCC. To determine the role of BRIT1 deficiency in potentiating the drug response, we subsequently generated BRIT1-deficient HCC cells, determined their HR defects, and assessed their response to the PARPi olaparib and PI3K inhibitor in vitro and in mice. BRIT1-deficient HCC cells were HR defective and hypersensitive to olaparib alone or in combination with PI3K inhibitor BEZ235, both in vitro and in vivo. The cytotoxicity of olaparib alone or in combination with BEZ235 was largely alleviated by ectopic BRIT1. We also found that BEZ235 markedly enhanced the production of poly (ADP-ribose) and the level of double-strand breaks (DSB) and single-strand breaks (SSB) in BRIT1-deficient cells. In summary, our results identify BRIT1 deficiency as a potential driver for HCC development, and BRIT1 status is critical to sensitivity to treatment with olaparib and/or BEZ235. PI3K inhibition induces substantial DNA damage and makes cells more dependent on PARP activity in the context of BRIT1 deficiency, thus, BRIT1 depletion facilitates enhancing synthetic lethality of PARP inhibitors and PI3K inhibitors in HCC. This study provides a new mechanistic foundation for significantly expanding the application of PARPi in HCC therapy.

Proteome Instability Is a Therapeutic Vulnerability in Mismatch Repair-Deficient Cancer.

Deficient DNA mismatch repair (dMMR) induces a hypermutator phenotype that can lead to tumorigenesis; however, the functional impact of the high mutation burden resulting from this phenotype remains poorly explored. Here, we demonstrate that dMMR-induced destabilizing mutations lead to proteome instability in dMMR tumors, resulting in an abundance of misfolded protein aggregates. To compensate, dMMR cells utilize a Nedd8-mediated degradation pathway to facilitate clearance of misfolded proteins. Blockade of this Nedd8 clearance pathway with MLN4924 causes accumulation of misfolded protein aggregates, ultimately inducing immunogenic cell death in dMMR cancer cells. To leverage this immunogenic cell death, we combined MLN4924 treatment with PD1 inhibition and found the combination was synergistic, significantly improving efficacy over either treatment alone.

LncRNA LINC00461 Promotes Colorectal Cancer Progression via miRNA-323b-3p/NFIB Axis.

BACKGROUND: LncRNA LINC00461 has been reported to play crucial regulatory roles in a variety of biological processes, including cell migration, cell invasion and cancer progression. However, its biological role in colorectal cancer (CRC) is completely unknown. The aim of our study was to explore the function of LINC00461 on CRC cells and the underlying mechanism. MATERIALS AND METHODS: CRC tumor tissues and cell lines derived from hospital and corporation. The expression level of LINC00461 in CRC tissues and cell lines were analyzed by quantitative real-time PCR (qRT-PCR). The effect of LINC00461 on cell proliferation, colony formation, migration and invasion were detected by CCK-8 assay, colony formation and transwell assay, respectively. In addition, cell apoptosis was analyzed by flow cytometry, and the role of LINC00461 on tumor growth was investigated by tumor xenografts in nude mice. The targets of LINC00461 were predicted by starBase v3.0 and confirmed by a dual-luciferase reporter system. The expression level of transcription factors of nuclear factor I B (NFIB), p21 and CDK2 was determined by Western blot or qRT-PCR. The NFIB expression levels in CRC tissues and mice tumors were analyzed by immunofluorescence assay (IHC). RESULTS: We found that the expression of LINC00461 was significantly overexpressed in CRC tissues and different cell lines, and the high level of LINC00461 expression was associated with poor overall survival. Downregulation of LINC00461 expression significantly suppressed the proliferation, migration and invasion of CRC cells and promoted cell apoptosis. We also found that LINC00461 could directly interact with miR-323b-3p. In addition, LINC00461 significantly increased the expression NFIB and CDK2, but, p21 was inhibited. Finally, we found that the growth of tumors in nude mice was suppressed upon LINC00461 deletion. CONCLUSION: We demonstrated that LINC00461 may play an oncogenic role in CRC cells through NFIB signaling pathway by targeting miR-323b-3p. Our report showed that LINC00461 may be a prognostic biomarker and candidate therapeutic target for CRC.

N-glycosylation affects the adhesive function of E-Cadherin through modifying the composition of adherens junctions (AJs) in human breast carcinoma cell line MDA-MB-435.

E-cadherin mediates calcium-dependent cell-cell adhesion between epithelial cells. The ectodomain of human E-cadherin contains four potential N-glycosylation sites at Asn residues 554, 566, 618, and 633. In this study, the role of N-glycosylation in E-cadherin-mediated cell-cell adhesion was investigated by site-directed mutagenesis. In MDA-MB-435 cells, all four potential N-glycosylation sites of human E-cadherin were N-glycosylated. Removal of N-glycan at Asn-633 dramatically affected E-cadherin stability. In contrast, mutant E-cadherin lacking the other three N-glycans showed similar protein stability in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn-554 and Asn-566 were found to affect E-cadherin-mediated calcium-dependent cell-cell adhesion, and removal of either of the two N-glycans caused a significant decrease in calcium-dependent cell-cell adhesion accompanied with elevated cell migration. Analysis of the composition of adherens junctions (AJs) revealed that removal of N-glycans on E-cadherin resulted in elevated tyrosine phosphorylation level of beta-catenin and reduced beta- and alpha-catenins at AJs. These findings demonstrate that N-glycosylation may affect the adhesive function of E-cadherin through modifying the composition of AJs.

Phosphatidylinositol 3-kinase/protein kinase B pathway stabilizes DNA methyltransferase I protein and maintains DNA methylation.

DNA methylation, which affects gene expression and chromatin stability, is catalyzed by DNA methyltransferases (DNMTs) of which DNMT1 possesses most abundant activity. PI3K/PKB pathway is an important pathway involved in cell proliferation, viability, and metabolism and often disrupted in cancer. Here we investigated the impact of PKB on DNMT1 and DNA methylation. Positive correlation between PKB-Ser473-phosphorylation and DNMT1 protein level in 17 human cell lines (p<0.01) and in 27 human bladder cancer tissues (p<0.05) was found. With activator, inhibitor, siRNA and constitutively active or dominant-negative plasmids of PKB, we found that PKB increased the protein level of DNMT1 without coordinate mRNA change, which was specific rather than due to cell-cycle change. PKB enhanced DNMT1 protein stability independent of de novo synthesis of any protein, which was attributed to down-regulation of N-terminal-120-amino-acids-dependent DNMT1 degradation via ubiquitin-proteasome pathway. Gsk3beta inhibitor rescued the decrease of DNMT1 by PKB inhibition, suggesting that Gsk3beta mediated the stabilization of DNMT1 by PKB. Then role of PKB regulating DNMT1 was investigated. Inhibition of PKB caused observable DNA hypomethylation and chromatin decondensation and DNMT1 overexpression partially reversed cell growth inhibition by PKB inhibition. In conclusion, our results suggested that PKB enhanced DNMT1 stability and maintained DNA methylation and chromatin structure, which might contribute to cancer cell growth.

Cyclophilin J PPIase Inhibitors Derived from 2,3-Quinoxaline-6 Amine Exhibit Antitumor Activity.

Cyclophilin J (CyPJ), also called peptidylprolyl isomerase like 3, has been identified as a novel member of the cyclophilin family. Our previous research has resolved the three-dimensional structure of CyPJ and demonstrated the peptidylprolyl cis-trans isomerase (PPIase) activity of CyPJ, which can be inhibited by the common immunosuppressive drug cyclosporine A (CsA). Importantly, CyPJ is upregulated in hepatocellular carcinoma (HCC) and promotes tumor growth; CyPJ inhibition by CsA- or siRNA-based knockdown results in a remarkable suppression of HCC. These findings suggest that CyPJ may be a potential therapeutic target for HCC, and discovery of relevant inhibitors may facilitate development of a novel CyPJ-based targeting therapy. However, apart from the common inhibitor CsA, CyPJ has yet to be investigated as a target for cancer therapy. Here, we report structure-based identification of novel small molecule non-peptidic CyPJ inhibitors and their potential as antitumor lead compounds. Based on computer-aided virtual screening, in silico, and subsequently surface plasmon resonance analysis, 19 potential inhibitors of CyPJ were identified and selected for further evaluation of PPIase CyPJ inhibition in vitro. Thirteen out of 19 compounds exhibited notable inhibition against PPIase activity. Among them, the compound ZX-J-19, with a quinoxaline nucleus, showed potential for tumor inhibition; thus, we selected it for further structure-activity optimization. A total of 22 chemical derivatives with 2,3-substituted quinoxaline-6-amine modifications were designed and successfully synthesized. At least 2 out of the 22 derivatives, such as ZX-J-19j and ZX-J-19l, demonstrated remarkable inhibition of tumor cell growth, comparable to CsA but much stronger than 5-fluorouracil. These results indicate that these two small molecules represent novel potential lead compounds for CyPJ-based antitumor drug development.

Cadherins Associate with Distinct Stem Cell-Related Transcription Factors to Coordinate the Maintenance of Stemness in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer with poor prognosis and is enriched in cancer stem cells (CSCs). However, it is not completely understood how the CSCs were maintained in TNBC. In this study, by analyzing The Cancer Genome Atlas (TCGA) provisional datasets and several small-size breast datasets, we found that cadherins (CDHs) 2, 4, 6, and 17 were frequently amplified/overexpressed in 47% of TNBC while E-cadherin (CDH1) was downregulated/mutated at 10%. The alterations of CDH2/4/6/17 were strongly associated with the elevated levels of several stem cell-related transcription factors (SC-TFs) including FOXM1, MCM2, WWTR1, SNAI1, and SOX9. CDH2/4/6/17-enriched genes including FOXM1 and MCM2 were also clustered and regulated by NFY (nuclear transcription factor Y) and/or EVI1/MECOM. Meanwhile, these SC-TFs including NFYA were upregulated in TNBC cells, but they were downregulated in luminal type of cells. Furthermore, small compounds might be predicted via the Connectivity Map analysis to target TNBC with the alterations of CDH2/4/6/17 and SC-TFs. Together with the important role of these SC-TFs in the stem cell regulation, our data provide novel insights into the maintenance of CSCs in TNBC and the discovery of these SC-TFs associated with the alterations of CDH2/4/6/17 has an implication in targeted therapy of TNBC.

Positive expression of E-cadherin suppresses cell adhesion to fibronectin via reduction of alpha5beta1 integrin in human breast carcinoma cells.

E-cadherin mainly mediated the epithelial cell-cell adhesion, and integrin signaling can modulate the signaling pathway of E-cadherin in the different levels. Up to now, however, it is still unclear that whether E-cadherin could interfere with cell-matrix interaction, a typical adhesion through integrins. In this study we investigated the effects of E-cadherin on cell-matrix adhesion and alpha5beta1 integrin expression in human breast carcinoma cells. It was found that either mRNA or protein level of alpha5 and beta1 subunits of integrin decreased in E-cad-231 compared with Mock-231. Furthermore, the promoter activity of alpha5 gene was inhibited in E-cad-231 compared with Mock-231. Consistently, phosphorylated focal adhesion kinase, a closer key downstream protein kinase of integrin signaling, were also down-regulated in E-cad-231. Furthermore, distribution of beta-catenin was observed and data showed beta-catenin was accumulated in the nucleus in Mock-231, while disappeared from the nucleus and mainly accumulated near the cell surface membrane in E-cad-231. LiCl, a molecule that can inhibit the GSK-3beta activity and down-regulate beta-catenin degradation, could inversely stimulate expression of alpha5 and beta1 integrin. Taken together, these results indicated that positive expression of E-cadherin inhibits the cell adhesion to extracellular matrix mediated by alpha5beta1 integrin signaling.