ITGAM-macrophage modulation as a potential strategy for treating neutrophilic Asthma: insights from bioinformatics analysis and in vivo experiments.

Identification of molecular biomarkers associated with neutrophilic asthma (NA) phenotype may inform the discovery of novel pathobiological mechanisms and the development of diagnostic markers. Three mRNA transcriptome datasets extracted from induced sputum of asthma patients with various inflammatory types were used to screen for macrophage-related molecular mechanisms and targets in NA. Furthermore, the predicted targets were also validated on an independent dataset (N = 3) and animal model (N = 5). A significant increase in total cells, neutrophils and macrophages was observed in bronchoalveolar lavage (BAL) fluid of NA mice induced by ovalbumin/freund's adjuvant, complete (OVA/CFA). And we also found elevated levels of neutrophil and macrophage infiltration in NA subtype in external datasets. NA mice had increased secretion of IgE, IL-1ß, TNF-α and IL-6 in serum and BAL fluid. MPO, an enzyme present in neutrophils, was also highly expressed in NA mice. Then, weighted gene co-expression network analysis (WGCNA) identified 684 targets with the strongest correlation with NA, and we obtained 609 macrophage-related specific differentially expressed genes (DEGs) in NA by integrating macrophage-related genes. The top 10 genes with high degree values were obtained and their mRNA levels and diagnostic performance were then determined by RT-qPCR and receiver operator characteristic (ROC) analysis. Statistically significant correlations were found between macrophages and all key targets, with the strongest correlation between ITGAM and macrophages in NA. Double-Immunofluorescence staining further confirmed the co-localization of ITGAM and F4/80 in NA. ITGAM was identified as a critical target to distinguish NA from healthy/non-NA individuals, which may provide a novel avenue to further uncover the mechanisms and therapy of NA.

The role of smoking and alcohol in mediating the effect of gastroesophageal reflux disease on lung cancer: A Mendelian randomization study.

Observational studies have suggested a positive association between gastroesophageal reflux disease and lung cancer, but due to the existence of confounders, it remains undetermined whether gastroesophageal reflux disease (GERD) has a causal association with lung cancer. Therefore, Mendelian randomization (MR) analyses were applied to investigate the relationship between the two conditions. Two-sample Mendelian randomization analysis was utilized with summary genetic data from the European Bioinformatics Institute (602,604 individuals) and International Lung Cancer Consortium, which provides information on lung cancer and its histological subgroups. Furthermore, we used two-step Mendelian randomization and multivariable Mendelian randomization to estimate whether smoking initiation (311,629 cases and 321,173 controls) and alcohol intake frequency (n = 462,346) mediate any effect of gastroesophageal reflux disease on lung cancer risk. The Mendelian randomization analyses indicated that gastroesophageal reflux disease was associated with and significantly increased the risk of lung cancer (ORIVW = 1.35, 95% CI = 1.18-1.54; p = 1.36 × 10-5). Smoking initiation and alcohol intake frequency mediated 35% and 3% of the total effect of gastroesophageal reflux disease on lung cancer, respectively. The combined effect of these two factors accounted for 60% of the total effect. In conclusion, gastroesophageal reflux disease is associated with an increased risk of lung cancer, and interventions to reduce smoking and alcohol intake may reduce the incidence of lung cancer.

Immunoregulatory protein B7-H3 regulates cancer stem cell enrichment and drug resistance through MVP-mediated MEK activation.

B7-H3 is a tumor-promoting glycoprotein that is expressed at low levels in most normal tissues, but is overexpressed in various human cancers which is associated with disease progression and poor patient outcome. Although numerous publications have reported the correlation between B7-H3 and cancer progression in many types of cancers, mechanistic studies on how B7-H3 regulates cancer malignancy are rare, and the mechanisms underlying the role of B7-H3 in drug resistance are almost unknown. Here we report a novel finding that upregulation of B7-H3 increases the breast cancer stem cell population and promotes cancer development. Depletion of B7-H3 in breast cancer significantly inhibits the cancer stem cells. By immunoprecipitation and mass spectrometry, we found that B7-H3 is associated with the major vault protein (MVP) and activates MEK through MVP-enhancing B-RAF and MEK interaction. B7-H3 expression increases stem cell population by binding to MVP which regulates the activation of the MAPK kinase pathway. Depletion of MVP blocks the activation of MEK induced by B7-H3 and dramatically inhibits B7-H3 induced stem cells. This study reports novel functions of B7-H3 in regulating breast cancer stem cell enrichment. The novel mechanism for B7-H3-induced stem cell propagation by regulating MVP/MEK signaling axis independent of the classic Ras pathway may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

miR-125b regulates differentiation and metabolic reprogramming of T cell acute lymphoblastic leukemia by directly targeting A20.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy. Although it has been reported that overexpression of miR-125b leads to T-ALL development, the underlying mechanisms of miR-125b action are still unclear. The goal of this study is to delineate the role of miR-125b in T-ALL development. We found that miR-125b is highly expressed in undifferentiated leukemic T cells (CD4-negative) while its expression is low in differentiated T cells (CD4-positive). Overexpression of miR-125b increased the CD4-negative population in T cells, whereas depletion of miR-125b by miR-125b-sponge decreased the CD4-negative cell population. We identified that A20 (TNFAIP3) is a direct target of miR-125b in T cells. Overexpression of miR-125b also increased glucose uptake and oxygen consumption in T cells through targeting A20. Furthermore, restoration of A20 in miR-125b-overexpressing cells decreased the CD4-negative population in T cell leukemia, and decreased glucose uptake and oxygen consumption to the basal level of T cells transfected with vector. In conclusion, our data demonstrate that miR-125b regulates differentiation and reprogramming of T cell glucose metabolism via targeting A20. Since both de-differentiation and dysregulated glucose metabolism contribute to the development of T-cell leukemia, these findings provide novel insights into the understanding and treatment of T-ALL.

Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1α.

B7-H3 is a member of B7 family of immunoregulatory transmembrane glycoproteins expressed by T cells. While B7-H3 overexpression is associated with poor outcomes in multiple cancers, it also has immune-independent roles outside T cells and its precise mechanistic contributions to cancer are unclear. In this study, we investigated the role of B7-H3 in metabolic reprogramming of cancer cells in vitro and in vivo We found that B7-H3 promoted the Warburg effect, evidenced by increased glucose uptake and lactate production in B7-H3-expressing cells. B7-H3 also increased the protein levels of HIF1α and its downstream targets, LDHA and PDK1, key enzymes in the glycolytic pathway. Furthermore, B7-H3 promoted reactive oxygen species-dependent stabilization of HIF1α by suppressing the activity of the stress-activated transcription factor Nrf2 and its target genes, including the antioxidants SOD1, SOD2, and PRX3. Metabolic imaging of human breast cancer xenografts in mice confirmed that B7-H3 enhanced tumor glucose uptake and tumor growth. Together, our results illuminate the critical immune-independent contributions of B7-H3 to cancer metabolism, presenting a radically new perspective on B7 family immunoregulatory proteins in malignant progression. Cancer Res; 76(8); 2231-42. ©2016 AACR.

Rig-I regulates NF-κB activity through binding to Nf-κb1 3'-UTR mRNA.

Retinoic acid inducible gene I (RIG-I) senses viral RNAs and triggers innate antiviral responses through induction of type I IFNs and inflammatory cytokines. However, whether RIG-I interacts with host cellular RNA remains undetermined. Here we report that Rig-I interacts with multiple cellular mRNAs, especially Nf-κb1. Rig-I is required for NF-κB activity via regulating Nf-κb1 expression at posttranscriptional levels. It interacts with the multiple binding sites within 3'-UTR of Nf-κb1 mRNA. Further analyses reveal that three distinct tandem motifs enriched in the 3'-UTR fragments can be recognized by Rig-I. The 3'-UTR binding with Rig-I plays a critical role in normal translation of Nf-κb1 by recruiting the ribosomal proteins [ribosomal protein L13 (Rpl13) and Rpl8] and rRNAs (18S and 28S). Down-regulation of Rig-I or Rpl13 significantly reduces Nf-κb1 and 3'-UTR-mediated luciferase expression levels. These findings indicate that Rig-I functions as a positive regulator for NF-κB signaling and is involved in multiple biological processes in addition to host antivirus immunity.

Heat shock factor 1 (HSF1) controls chemoresistance and autophagy through transcriptional regulation of autophagy-related protein 7 (ATG7).

Heat shock factor 1 (HSF1), a master regulator of heat shock responses, plays an important role in tumorigenesis. In this study we demonstrated that HSF1 is required for chemotherapeutic agent-induced cytoprotective autophagy through transcriptional up-regulation of autophagy-related gene ATG7. Interestingly, this is independent of the HSF1 heat shock response function. Treatment of cancer cells with the FDA-approved chemotherapeutic agent carboplatin induced autophagy and growth inhibition, which were significantly increased upon knockdown of HSF1. Mechanistic studies revealed that HSF1 regulates autophagy by directly binding to ATG7 promoter and transcriptionally up-regulating its expression. Significantly, breast cancer patient sample study revealed that a higher ATG7 expression level is associated with poor patient survival. This novel finding was further confirmed by analysis of two independent patient databases, demonstrating a prognostic value of ATG7. Furthermore, a strong positive correlation was observed between levels of HSF1 and ATG7 in triple-negative breast cancer patient samples, thus validating our in vitro findings. This is the first study identifying a critical role for HSF1 in controlling cytoprotective autophagy through regulation of ATG7, which is distinct from the HSF1 function in the heat shock response. This is also the first study demonstrating a prognostic value of ATG7 in breast cancer patients. These findings strongly argue that combining chemotherapeutic agents with autophagy inhibition by repressing HSF1/ATG7 axis represents a promising strategy for future cancer treatment.

miR-125b functions as a key mediator for snail-induced stem cell propagation and chemoresistance.

Chemoresistance is a major obstacle in cancer treatment. Our previous studies have shown that miR-125b plays an important role in chemoresistance. Here we report a novel mechanism that up-regulation of miR-125b through Wnt signaling by Snail enriches cancer stem cells. Overexpression of Snail dramatically increases the expression of miR-125b through the Snail-activated Wnt/ß-catenin/TCF4 axis. Snail confers chemoresistance by repressing Bak1 through up-regulation of miR-125b. Restoring the expression of Bak1 or depleting miR-125b re-sensitizes Snail-expressing cancer cells to Taxol, indicating that miR-125b is critical in Snail-induced chemoresistance. Moreover, overexpression of miR-125b significantly increases the cancer stem cell population (CD24-CD44+), while depletion of miR-125b or rescue of the expression of Bak1 increases the non-stem cell population (CD24+CD44+) in Snail-overexpressing cells. These findings strongly support that miR-125b functions as a key mediator in Snail-induced cancer stem cell enrichment and chemoresistance. This novel mechanism for Snail-induced stem cell propagation and chemoresistance may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

Receptor tyrosine kinase ErbB2 translocates into mitochondria and regulates cellular metabolism.

It is well known that ErbB2, a receptor tyrosine kinase, localizes to the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and cellular ATP levels were also decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to the ErbB2-targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 has an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics.

Overcoming trastuzumab resistance in breast cancer by targeting dysregulated glucose metabolism.

Trastuzumab shows remarkable efficacy in treatment of ErbB2-positive breast cancers when used alone or in combination with other chemotherapeutics. However, acquired resistance develops in most treated patients, necessitating alternate treatment strategies. Increased aerobic glycolysis is a hallmark of cancer and inhibition of glycolysis may offer a promising strategy to preferentially kill cancer cells. In this study, we investigated the antitumor effects of trastuzumab in combination with glycolysis inhibitors in ErbB2-positive breast cancer. We found that trastuzumab inhibits glycolysis via downregulation of heat shock factor 1 (HSF1) and lactate dehydrogenase A (LDH-A) in ErbB2-positive cancer cells, resulting in tumor growth inhibition. Moreover, increased glycolysis via HSF1 and LDH-A contributes to trastuzumab resistance. Importantly, we found that combining trastuzumab with glycolysis inhibition synergistically inhibited trastuzumab-sensitive and -resistant breast cancers in vitro and in vivo, due to more efficient inhibition of glycolysis. Taken together, our findings show how glycolysis inhibition can dramatically enhance the therapeutic efficacy of trastuzumab in ErbB2-positive breast cancers, potentially useful as a strategy to overcome trastuzumab resistance.

B7-H3 silencing increases paclitaxel sensitivity by abrogating Jak2/Stat3 phosphorylation.

In many types of cancer, the expression of the immunoregulatory protein B7-H3 has been associated with poor prognosis. Previously, we observed a link between B7-H3 and tumor cell migration and invasion, and in present study, we have investigated the role of B7-H3 in chemoresistance in breast cancer. We observed that silencing of B7-H3, via stable short hairpin RNA or transient short interfering RNA transfection, increased the sensitivity of multiple human breast cancer cell lines to paclitaxel as a result of enhanced drug-induced apoptosis. Overexpression of B7-H3 made the cancer cells more resistant to the drug. Next, we investigated the mechanisms behind B7-H3-mediated paclitaxel resistance and found that the level of Stat3 Tyr705 phosphorylation was decreased in B7-H3 knockdown cells along with the expression of its direct downstream targets Mcl-1 and survivin. The phosphorylation of Janus kinase 2 (Jak2), an upstream molecule of Stat3, was also significantly decreased. In contrast, reexpression of B7-H3 in B7-H3 knockdown and low B7-H3 expressing cells increased the phosphorylation of Jak2 and Stat3. In vivo animal experiments showed that B7-H3 knockdown tumors displayed a slower growth rate than the control xenografts. Importantly, paclitaxel treatment showed a strong antitumor activity in the mice with B7-H3 knockdown tumors, but only a marginal effect in the control group. Taken together, our data show that in breast cancer cells, B7-H3 induces paclitaxel resistance, at least partially by interfering with Jak2/Stat3 pathway. These results provide novel insight into the function of B7-H3 and encourage the design and testing of approaches targeting this protein and its partners.

MicroRNA-125b confers the resistance of breast cancer cells to paclitaxel through suppression of pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) expression.

Paclitaxel (Taxol) is an effective chemotherapeutic agent for treatment of cancer patients. Despite impressive initial clinical responses, the majority of patients eventually develop some degree of resistance to Taxol-based therapy. The mechanisms underlying cancer cells resistance to Taxol are not fully understood. MicroRNA (miRNA) has emerged to play important roles in tumorigenesis and drug resistance. However, the interaction between the development of Taxol resistance and miRNA has not been previously explored. In this study we utilized a miRNA array to compare the differentially expressed miRNAs in Taxol-resistant and their Taxol-sensitive parental cells. We verified that miR-125b, miR-221, miR-222, and miR-923 were up-regulated in Taxol-resistant cancer cells by real-time PCR. We further investigated the role and mechanisms of miR-125b in Taxol resistance. We found that miR-125b was up-regulated in Taxol-resistant cells, causing a marked inhibition of Taxol-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to Taxol in cancer cells. Moreover, we demonstrated that the pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) is a direct target of miR-125b. Down-regulation of Bak1 suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol. Restoring Bak1 expression by either miR-125b inhibitor or re-expression of Bak1 in miR-125b-overexpressing cells recovered Taxol sensitivity, overcoming miR-125-mediated Taxol resistance. Taken together, our data strongly support a central role for miR-125b in conferring Taxol resistance through the suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming Taxol resistance in a number of different tumor histologies.

Warburg effect in chemosensitivity: targeting lactate dehydrogenase-A re-sensitizes taxol-resistant cancer cells to taxol.

BACKGROUND: Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells. RESULTS: Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells. CONCLUSION: LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.

Demethylating Agents in the Treatment of Cancer.

Gene silencing resulting from aberrant DNA methylation can lead to tumorigenesis. Therefore, drugs that inhibit or interfere with DNA methylation have been used to reactivate and induce silenced gene re-expression in malignancies. Two demethylating agents, azacitidine and decitabine, are approved for the treatment of myelodysplastic syndromes (MDS) by the U.S. Food and Drug Administration (FDA), and are now considered the standard of care in MDS. In this review, we discuss clinical data, including clinical benefits and toxicities, which led to the approval of azacitidine and decitabine. We also summarize findings from clinical trials that used these two demethylating agents in the treatment of solid tumors. Lastly, we discuss some limitations in the use of azacitidine and decitabine in cancer therapy.

Rig-I-/- mice develop colitis associated with downregulation of G alpha i2.

RIG-I (retinoid acid-inducible gene-I), a putative RNA helicase with a cytoplasmic caspase-recruitment domain (CARD), was identified as a pattern-recognition receptor (PRR) that mediates antiviral immunity by inducing type I interferon production. To further study the biological function of RIG-I, we generated Rig-I(-/-) mice through homologous recombination, taking a different strategy to the previously reported strategy. Our Rig-I(-/-) mice are viable and fertile. Histological analysis shows that Rig-I(-/-) mice develop a colitis-like phenotype and increased susceptibility to dextran sulfate sodium-induced colitis. Accordingly, the size and number of Peyer's patches dramatically decreased in mutant mice. The peripheral T-cell subsets in mutant mice are characterized by an increase in effector T cells and a decrease in naive T cells, indicating an important role for Rig-I in the regulation of T-cell activation. It was further found that Rig-I deficiency leads to the downregulation of G protein alpha i2 subunit (G alpha i2) in various tissues, including T and B lymphocytes. By contrast, upregulation of Rig-I in NB4 cells that are treated with ATRA is accompanied by elevated G alpha i2 expression. Moreover, G alpha i2 promoter activity is increased in co-transfected NIH3T3 cells in a Rig-I dose-dependent manner. All these findings suggest that Rig-I has crucial roles in the regulation of G alpha i2 expression and T-cell activation. The development of colitis may be, at least in part, associated with downregulation of G alpha i2 and disturbed T-cell homeostasis.

CCDC98 targets BRCA1 to DNA damage sites.

Breast cancer-1 (BRCA1) participates in the DNA damage response. However, the mechanism by which BRCA1 is recruited to DNA damage sites remains elusive. Recently, we have demonstrated that a ubiquitin-binding protein, RAP80, is required for DNA damage-induced BRCA1 translocation. Here we identify another component, CCDC98, in the BRCA1-RAP80 complex. CCDC98 mediates BRCA1's association with RAP80. Moreover, CCDC98 controls both DNA damage-induced formation of BRCA1 foci and BRCA1-dependent G2/M checkpoint activation. Together, our results demonstrate that CCDC98 is a BRCA1 binding partner that mediates BRCA1 function in response to DNA damage.