Population pharmacokinetics of mirvetuximab soravtansine in patients with folate receptor-α positive ovarian cancer: The antibody-drug conjugate, payload and metabolite.

AIMS: Mirvetuximab soravtansine is a first-in-class antibody-drug conjugate recently approved for the treatment of folate receptor-α positive ovarian cancer. The aim of this study was to develop a population pharmacokinetic model to describe the concentration-time profiles of mirvetuximab soravtansine, the payload (DM4) and a metabolite (S-methyl-DM4). METHODS: Mirvetuximab soravtansine was administered intravenously from 0.15 to 7 mg/kg to 543 patients with predominantly platinum-resistant ovarian cancer in 3 clinical studies, and the plasma drug concentrations were analysed using a nonlinear mixed-effects modelling approach. Stepwise covariate modelling was performed to identify covariates. RESULTS: We developed a semi-mechanistic population pharmacokinetic model that included linear and nonlinear routes for the elimination of mirvetuximab soravtansine and a target compartment for the formation and disposition of the payload and metabolite in tumour cells. The clearance and volume of the central compartment were 0.0153 L/h and 2.63 L for mirvetuximab soravtansine, 8.83 L/h and 3.67 L for DM4, and 2.04 L/h and 6.3 L for S-methyl-DM4, respectively. Body weight, serum albumin and age were identified as statistically significant covariates. Exposures in patients with renal or hepatic impairment and who used concomitant cytochrome P450 (CYP) 3A4 inhibitors were estimated. CONCLUSION: There is no need for dose adjustment due to covariate effects for mirvetuximab soravtansine administered at the recommended dose of 6 mg/kg based on adjusted ideal body weight. Dose adjustment is not required for patients with mild or moderate renal impairment, mild hepatic impairment, or when concomitant weak and moderate CYP3A4 inhibitors are used.

TH17 cells and corticosteroid insensitivity in severe asthma.

Asthma is classically described as having either a type 2 (T2) eosinophilic phenotype or a non-T2 neutrophilic phenotype. T2 asthma usually responds to classical bronchodilation therapy and corticosteroid treatment. Non-T2 neutrophilic asthma is often more severe. Patients with non-T2 asthma or late-onset T2 asthma show poor response to the currently available anti-inflammatory therapies. These therapeutic failures result in increased morbidity and cost associated with asthma and pose a major health care problem. Recent evidence suggests that some non-T2 asthma is associated with elevated TH17 cell immune responses. TH17 cells producing Il-17A and IL-17F are involved in the neutrophilic inflammation and airway remodeling processes in severe asthma and have been suggested to contribute to the development of subsets of corticosteroid-insensitive asthma. This review explores the pathologic role of TH17 cells in corticosteroid insensitivity of severe asthma and potential targets to treat this endotype of asthma.

Clinical and molecular implications of RGS2 promoter genetic variation in severe asthma.

BACKGROUND: Regulator of G protein signaling (RGS) 2 terminates bronchoconstrictive Gαq signaling; murine RGS2 knockout demonstrate airway hyperresponsiveness. While RGS2 promoter variants rs2746071 and rs2746072 associate with a clinical mild asthma phenotype, their impact on human airway smooth muscle (HASM) contractility and asthma severity outcomes is unknown. OBJECTIVE: We sought to determine whether reductions in RGS2 expression seen with these 2 RGS2 promoter variants augment HASM contractility and associate with an asthma severity phenotype. METHODS: We transfected HASM with a range of RGS2-specific small interfering RNA (siRNA) concentrations and determined RGS2 protein expression by Western blot analysis and intracellular calcium flux induced by histamine (a Gαq-coupled H1 receptor bronchoconstrictive agonist). We conducted regression-based genotype association analyses of RGS2 variants from 611 patients from the National Heart, Lung, and Blood Institute Severe Asthma Research Program 3. RESULTS: RGS2-specific siRNA caused dose-dependent increases in histamine-stimulated bronchoconstrictive intracellular calcium signaling (2-way ANOVA, P < .0001) with a concomitant decrease in RGS2 protein expression. RGS2-specific siRNA did not affect Gαq-independent ionomycin-induced intracellular calcium signaling (P = .42). The minor allele frequency of rs2746071 and rs2746072 was 0.46 and 0.28 among African American/non-Hispanic Black patients and was 0.28 and 0.27 among non-Hispanic White patients, among whom these single nucleotide polymorphisms were in stronger linkage disequilibrium (r2 = 0.97). Among non-Hispanic White patients, risk allele homozygotes for rs2746072 and rs2746071 each had nearly 2-fold greater asthma exacerbation rates relative to alternative genotypes with wild-type alleles (Padditive = 2.86 × 10-5/Precessive = 5.22 × 10-6 and Padditive = 3.46 × 10-6/Precessive = 6.74 × 10-7, respectively) at baseline, which was confirmed by prospective longitudinal exacerbation data. CONCLUSION: RGS2 promoter variation associates with a molecular and clinical phenotype characterized by enhanced bronchoconstrictive stimulation in vitro and higher asthma exacerbations rates in non-Hispanic White patients.

Immune receptor recombinations from breast cancer exome files, independently and in combination with specific HLA alleles, correlate with better survival rates.

PURPOSE: Immune characterizations of cancers, including breast cancer, have led to information useful for prognoses and are considered to be important in the future of refining the use of immunotherapies, including immune checkpoint inhibitor therapies. In this study, we sought to extend these characterizations with genomics approaches, particularly with cost-effective employment of exome files. METHODS: By recovery of immune receptor recombination reads from the cancer genome atlas (TCGA) breast cancer dataset, we observed associations of these recombinations with T-cell and B-cell biomarkers and with distinct survival rates. RESULTS: Recovery of TRD or IGH recombination reads was associated with an improved disease-free survival (p = 0.047 and 0.045, respectively). Determination of the HLA types using the exome files allowed matching of T-cell receptor V- and J-gene segment usage with specific HLA alleles, in turn allowing a refinement of the association of immune receptor recombination read recoveries with survival. For example, the TRBV7, HLA-C*07:01 combination represented a significantly worse, disease-free outcome (p = 0.014) compared to all other breast cancer samples. By direct comparisons of distinct TRB gene segment usage, HLA allele combinations revealed breast cancer subgroups, within the entire TCGA breast cancer dataset with even more dramatic survival distinctions. CONCLUSIONS: In sum, the use of exome files for recovery of adaptive immune receptor recombination reads, and the simultaneous determination of HLA types, has the potential of advancing the use of immunogenomics for immune characterization of breast tumor samples.

TcR-α recombinations in renal cell carcinoma exome files correlate with an intermediate level of T-cell exhaustion biomarkers.

Renal cell carcinoma exome-derived, V(D)J recombination reads had an elevated presence and variability, for both TcR-α and -ß, when compared to marginal tissue, reflecting an opportunity to assess tumor immunogenicity by comparison with marginal tissue T cells. PD-1, PD-L2, CTLA4 and FOXP3, all of which are implicated in the evasion of an anti-tumor immune response, had a significantly higher expression for samples representing co-detection of productive TcR-α and -ß recombination reads. Samples representing tumors with productive TcR-α recombination reads but no detectable, productive TcR-ß recombination reads, reflected a 20% survival advantage, and RNASeq data indicated an intermediate level of immune checkpoint gene expression for those samples. These results raise the question of whether relatively high levels of detection of productive TcR-α recombination reads, in comparison with detection of reads representing the TcR-ß gene, identify a microenvironment that has not yet entered a T-cell exhaustion phase and may thereby represent conditions for immune enhancements that do not require anti-immune checkpoint therapies.

TRB-J1 usage, in combination with the HLA-A*01:01 allele, represents an apparent survival advantage for uterine corpus endometrial carcinoma: Comparisons with microscopic assessments of lymphocyte infiltrates.

The opportunity for the highly efficient recovery of immune receptor recombination data from cancer specimens, including the ready assessment of immune receptor V and J usage, raises the issue of establishing precise values of assessing the immune receptor status as opposed to obtaining basic information regarding lymphocyte infiltration, in the cancer setting. In this report, we obtained the lymphocyte infiltration percentages from the cancer digital slide archive representing uterine corpus endometrial carcinoma (UCEC) and correlated these data with recovery of the immune receptor recombination reads from corresponding UCEC exome files. Results indicated a basic correlation of the recovery of productive T-cell receptor beta (TRB) recombination reads with lymphocyte infiltration percentages. However, the recovery of specific immune receptor recombination reads did not indicate the same survival outcomes as microscope detection of lymphocyte infiltrate percentages. To further exploit the value of recovery of the TRB recombination reads from the UCEC exome files, we determined the survival outcomes for combinations of TRB gene segment usage and HLA class I alleles, with the most important result being that the combination of HLA-A*01:01 and TRB-J1 segment usage reflected a strikingly high survival rate. Overall, this report emphasized the increased value of the knowledge of the immune receptor recombinations, in comparison with basic lymphocyte infiltration percentages, in assessing cancer survival rates.

Novel role of Giα2 in cell migration: Downstream of PI3-kinase-AKT and Rac1 in prostate cancer cells.

Tumor cell motility is the essential step in cancer metastasis. Previously, we showed that oxytocin and epidermal growth factor (EGF) effects on cell migration in prostate cancer cells require Giα2 protein. In the current study, we investigated the interactions among G-protein coupled receptor (GPCR), Giα2, PI3-kinase, and Rac1 activation in the induction of migratory and invasive behavior by diverse stimuli. Knockdown and knockout of endogenous Giα2 in PC3 cells resulted in attenuation of transforming growth factor ß1 (TGFß1), oxytocin, SDF-1α, and EGF effects on cell migration and invasion. In addition, knockdown of Giα2 in E006AA cells attenuated cell migration and overexpression of Giα2 in LNCaP cells caused significant increase in basal and EGF-stimulated cell migration. Pretreatment of PC3 cells with Pertussis toxin resulted in attenuation of TGFß1- and oxytocin-induced migratory behavior and PI3-kinase activation without affecting EGF-induced PI3-kinase activation and cell migration. Basal- and EGF-induced activation of Rac1 in PC3 and DU145 cells were not affected in cells after Giα2 knockdown. On the other hand, Giα2 knockdown abolished the migratory capability of PC3 cells overexpressing constitutively active Rac1. The knockdown or knockout of Giα2 resulted in impaired formation of lamellipodia at the leading edge of the migrating cells. We conclude that Giα2 protein acts at two different levels which are both dependent and independent of GPCR signaling to induce cell migration and invasion in prostate cancer cells and its action is downstream of PI3-kinase-AKT-Rac1 axis.

T-cell receptor-ß V and J usage, in combination with particular HLA class I and class II alleles, correlates with cancer survival patterns.

Class I and class II HLA proteins, respectively, have been associated with subsets of V(D)J usage resulting from recombination of the T-cell receptor (TCR) genes. Additionally, particular HLA alleles, in combination with dominant TCR V(D)J recombinations, have been associated with several autoimmune diseases. The recovery of TCR recombination reads from tumor specimen exome files has allowed rapid and extensive assessments of V(D)J usage, likely for cancer resident T-cells, across relatively large cancer datasets. The results from this approach, in this report, have permitted an extensive alignment of TCR-ß VDJ usage and HLA class I and II alleles. Results indicate the correlation of both better and worse cancer survival rates with particular TCR-ß, V and J usage-HLA allele combinations, with differences in median survival times ranging from 7 to 130 months, depending on the cancer and the specific TCR-ß V and J usage/HLA class allele combination.

Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis.

The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant Bid(D60E) and a BH3 defective mutant Bid(G94E) failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis.

Lung tumor exome files with T-cell receptor recombinations: a mouse model of T-cell infiltrates reflecting mutation burdens.

Tumor exomes and RNASeq data were originally intended for obtaining tumor mutations and gene expression profiles, respectively. However, recent work has determined that tumor exome and RNAseq read files contain reads representing T-cell and B-cell receptor (TcR and BcR) recombinations, presumably due to infiltrating lymphocytes. Furthermore, the recovery of immune receptor recombination reads has demonstrated correlations with specific, previously appreciated aspects of tumor immunology. To further understand the usefulness of recovering TcR and BcR recombinations from tumor exome files, we developed a scripted algorithm for recovery of reads representing these recombinations from a previously described mouse model of lung tumorigenesis. Results indicated that exomes representing lung adenomas reveal significantly more TcR recombinations than do exomes from lung adenocarcinomas; and that exome files representing high mutation adenomas, arising from chemical mutagens, have more TcR recombinations than do exome files from low mutation adenomas arising from an activating Kras mutation. The latter results were also consistent with a similar analysis performed on human lung adenocarcinoma exomes. The mouse and human results for obtaining TcR recombination reads from tumor specimen exomes are consistent with human tumor biology results indicating that adenomas and high mutation cancers are sites of high immune activity. The results indicate hitherto unappreciated opportunities for the use of tumor specimen exome files, particularly from experimental animal models, to study the connection between the adenoma stage of tumorigenesis, or high cancer mutation rates, and high level lymphocyte infiltrates.

Assessing microenvironment immunogenicity using tumor specimen exomes: Co-detection of TcR-α/ß V(D)J recombinations correlates with PD-1 expression.

T-cell receptor (TcR) recombinations can be recovered from tumor specimen, whole exome sequences (WXS) files. However, it is not yet clear how these recombinations represent lymphocytes or an anti-tumor immune response. Here we report the identification of productive TcR-ß recombinations in WXS files representing primary and metastatic melanoma. The recombinations are identifiable in about 20% of the cancer genome atlas melanoma samples. This frequency of detection is lower than the frequency of TcR-α VJ recombinations, consistent with the occurrence of biallelic TcR-α recombinations and possibly consistent with the fact that only one junctional recombination is required for TcR-α whereas two recombinations are required to form a TcR-ß gene. Nevertheless, the ratio of productive TcR-ß to unproductive TcR-ß samples, in comparison to the ratio of productive to unproductive TcR-α or TcR-γ positive-samples, is very high. This result indicates that detection of a productive TcR-ß VDJ recombination represents a comparatively high standard for potential antigen binding capacity, when employing a tumor specimen exome file for the assessment. Additionally, PD-1 expression and antigen presentation functions correlated with the co-detection of TcR-α and -ß recombinations (e.g., p < 0.0004), suggesting that co-detection of TcR-α and -ß recombinations represents an anti-melanoma response that has been blunted by the advent of PD-1 expression. We further show that the algorithm for detecting the TcR-ß VDJ recombinations is applicable to exome files generated from mouse tissue, thus providing for opportunities to develop empirical paradigms for interpreting the identification of TcR V(D)J recombinations in tissue resident lymphocytes.

Cyproterone acetate enhances TRAIL-induced androgen-independent prostate cancer cell apoptosis via up-regulation of death receptor 5.

BACKGROUND: Virtually all prostate cancer deaths occur due to obtaining the castration-resistant phenotype after prostate cancer cells escaped from apoptosis and/or growth suppression initially induced by androgen receptor blockade. TNF-related apoptosis-inducing ligand (TRAIL) was an attractive cancer therapeutic agent due to its minimal toxicity to normal cells and remarkable apoptotic activity in tumor cells. However, most localized cancers including prostate cancer are resistant to TRAIL-induced apoptosis, thereby creating a therapeutic challenge of inducing TRAIL sensitivity in cancer cells. Herein the effects of cyproterone acetate, an antiandrogen steroid, on the TRAIL-induced apoptosis of androgen receptor-negative prostate cancer cells are reported. METHODS: Cell apoptosis was assessed by both annexin V/propidium iodide labeling and poly (ADP-ribose) polymerase cleavage assays. Gene and protein expression changes were determined by quantitative real-time PCR and western blot assays. The effect of cyproterone acetate on gene promoter activity was determined by luciferase reporter assay. RESULTS: Cyproterone acetate but not AR antagonist bicalutamide dramatically increased the susceptibility of androgen receptor-negative human prostate cancer PC-3 and DU145 cells to TRAIL-induced apoptosis but no effects on immortalized human prostate stromal PS30 cells and human embryonic kidney HEK293 cells. Further investigation of the TRAIL-induced apoptosis pathway revealed that cyproterone acetate exerted its effect by selectively increasing death receptor 5 (DR5) mRNA and protein expression. Cyproterone acetate treatment also increased DR5 gene promoter activity, which could be abolished by mutation of a consensus binding domain of transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP) in the DR5 gene promoter. Cyproterone acetate increases CHOP expression in a concentration and time-dependent manner and endoplasmic reticulum stress reducer 4-phenylbutyrate could block cyproterone acetate-induced CHOP and DR5 up-regulation. More importantly, siRNA silencing of CHOP significantly reduced cyproterone acetate-induced DR5 up-regulation and TRAIL sensitivity in prostate cancer cells. CONCLUSIONS: Our study shows a novel effect of cyproterone acetate on apoptosis pathways in prostate cancer cells and raises the possibility that a combination of TRAIL with cyproterone acetate could be a promising strategy for treating castration-resistant prostate cancer.

Upregulation of RGS2: a new mechanism for pirfenidone amelioration of pulmonary fibrosis.

BACKGROUND: Pirfenidone was recently approved for treatment of idiopathic pulmonary fibrosis. However, the therapeutic dose of pirfenidone is very high, causing side effects that limit its doses and therapeutic effectiveness. Understanding the molecular mechanisms of action of pirfenidone could improve its safety and efficacy. Because activated fibroblasts are critical effector cells associated with the progression of fibrosis, this study investigated the genes that change expression rapidly in response to pirfenidone treatment of pulmonary fibroblasts and explored their contributions to the anti-fibrotic effects of pirfenidone. METHODS: We used the GeneChip microarray to screen for genes that were rapidly up-regulated upon exposure of human lung fibroblast cells to pirfenidone, with confirmation for specific genes by real-time PCR and western blots. Biochemical and functional analyses were used to establish their anti-fibrotic effects in cellular and animal models of pulmonary fibrosis. RESULTS: We identified Regulator of G-protein Signaling 2 (RGS2) as an early pirfenidone-induced gene. Treatment with pirfenidone significantly increased RGS2 mRNA and protein expression in both a human fetal lung fibroblast cell line and primary pulmonary fibroblasts isolated from patients without or with idiopathic pulmonary fibrosis. Pirfenidone treatment or direct overexpression of recombinant RGS2 in human lung fibroblasts inhibited the profibrotic effects of thrombin, whereas loss of RGS2 exacerbated bleomycin-induced pulmonary fibrosis and mortality in mice. Pirfenidone treatment reduced bleomycin-induced pulmonary fibrosis in wild-type but not RGS2 knockout mice. CONCLUSIONS: Endogenous RGS2 exhibits anti-fibrotic functions. Upregulated RGS2 contributes significantly to the anti-fibrotic effects of pirfenidone.

Regulator of G-protein signaling 2 repression exacerbates airway hyper-responsiveness and remodeling in asthma.

G protein-coupled receptors (GPCRs) are important regulators of cell functions in asthma. We recently reported that regulator of G-protein signaling (RGS) 2, a selective modulator of Gq-coupled GPCRs, is a key regulator of airway hyper-responsiveness (AHR), the pathophysiologic hallmark of asthma. Because RGS2 protein levels in airway cells were significantly lower in patients with asthma compared with patients without asthma, we further investigated the potential pathological importance of RGS2 repression in asthma. The human RGS2 gene maps to chromosome 1q31. We first screened patients with asthma for RGS2 gene promoter single-nucleotide polymorphisms (SNPs) and found significant differences in the distribution of two RGS2 SNPs (A638G, rs2746071 and C395G, rs2746072) between patients with asthma and nonasthmatic subjects. These two SNPs are always associated with each other and have the same higher prevalence in patients with asthma (65%) as compared with nonasthmatic subjects (35%). Point mutations corresponding to these SNPs decrease RGS2 promoter activity by 44%. The importance of RGS2 down-regulation was then determined in an acute IL-13 mouse model of asthma. Intranasal administration of IL-13 in mice also decreased RGS2 expression in lungs by ∼50% and caused AHR. Although naive RGS2 knockout (KO) mice exhibit spontaneous AHR, acute IL-13 exposure further increased AHR in RGS2 KO mice. Loss of RGS2 also significantly enhanced IL-13-induced mouse airway remodeling, including peribronchial smooth muscle thickening and fibrosis, without effects on goblet cell hyperplasia or airway inflammation in mice. Thus, genetic variations and increased inflammatory cytokines can lead to RGS2 repression, which exacerbates AHR and airway remodeling in asthma.

Subtype-specific overexpression of the Rac-GEF P-REX1 in breast cancer is associated with promoter hypomethylation.

INTRODUCTION: The Rac-GEF P-REX1 is a key mediator of ErbB signaling in breast cancer recently implicated in mammary tumorigenesis and metastatic dissemination. Although P-REX1 is essentially undetectable in normal human mammary epithelial tissue, this Rac-GEF is markedly upregulated in human breast carcinomas, particularly of the luminal subtype. The mechanisms underlying P-REX1 upregulation in breast cancer are unknown. Toward the goal of dissecting the mechanistic basis of P-REX1 overexpression in breast cancer, in this study we focused on the analysis of methylation of the PREX1 gene promoter. METHODS: To determine the methylation status of the PREX1 promoter region, we used bisulfite genomic sequencing and pyrosequencing approaches. Re-expression studies in cell lines were carried out by treatment of breast cancer cells with the demethylating agent 5-aza-2'-deoxycitidine. PREX1 gene methylation in different human breast cancer subtypes was analyzed from the TCGA database. RESULTS: We found that the human PREX1 gene promoter has a CpG island located between -1.2 kb and +1.4 kb, and that DNA methylation in this region inversely correlates with P-REX1 expression in human breast cancer cell lines. A comprehensive analysis of human breast cancer cell lines and tumors revealed significant hypomethylation of the PREX1 promoter in ER-positive, luminal subtype, whereas hypermethylation occurs in basal-like breast cancer. Treatment of normal MCF-10A or basal-like cancer cells, MDA-MB-231 with the demethylating agent 5-aza-2'-deoxycitidine in combination with the histone deacetylase inhibitor trichostatin A restores P-REX1 levels to those observed in luminal breast cancer cell lines, suggesting that aberrant expression of P-REX1 in luminal breast cancer is a consequence of PREX1 promoter demethylation. Unlike PREX1, the pro-metastatic Rho/Rac-GEF, VAV3, is not regulated by methylation. Notably, PREX1 gene promoter hypomethylation is a prognostic marker of poor patient survival. CONCLUSIONS: Our study identified for the first time gene promoter hypomethylation as a distinctive subtype-specific mechanism for controlling the expression of a key regulator of Rac-mediated motility and metastasis in breast cancer.

Regulator of G protein signaling 2 is a key modulator of airway hyperresponsiveness.

BACKGROUND: Drugs targeting individual G protein-coupled receptors are used as asthma therapies, but this strategy is limited because of G protein-coupled receptor signal redundancy. Regulator of G protein signaling 2 (RGS2), an intracellular selective inhibitor of multiple bronchoconstrictor receptors, may play a central role in the pathophysiology and treatment of asthma. OBJECTIVE: We defined functions and mechanisms of RGS2 in regulating airway hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. METHODS: Real-time PCR and Western blot were used to determine changes in RGS2 expression in ovalbumin-sensitized/-challenged mice. We also used immunohistochemistry and real-time PCR to compare RGS2 expression between human asthmatic and control subjects. The AHR of RGS2 knockout mice was assessed by using invasive tracheostomy and unrestrained plethysmography. Effects of loss of RGS2 on mouse airway smooth muscle (ASM) remodeling, contraction, intracellular Ca(2+), and mitogenic signaling were determined in vivo and in vitro. RESULTS: RGS2 was highly expressed in human and murine bronchial epithelium and ASM and was markedly downregulated in lungs of ovalbumin-sensitized/-challenged mice. Lung tissues and blood monocytes from asthma patients expressed significantly lower RGS2 protein (lung) and mRNA (monocytes) than from nonasthma subjects. The extent of reduction of RGS2 on human monocytes correlated with increased AHR. RGS2 knockout caused spontaneous AHR in mice. Loss of RGS2 augmented Ca(2+) mobilization and contraction of ASM cells. Loss of RGS2 also increased ASM mass and stimulated ASM cell growth via extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. CONCLUSION: We identified RGS2 as a potent modulator of AHR and a potential novel therapeutic target for asthma.

Chemical complementarity between immune receptor CDR3s and candidate cancer antigens correlating with reduced survival: evidence for outcome mitigation with corticosteroid treatments.

The evaluation of physicochemical characteristics of extensive adaptive immune receptor (IR) recombination sequence collections has led to the discovery of many correlations of those sequences and a variety of diseases, including cancer. In the cancer setting, these evaluations have recently focused on the adaptive IR, complementarity determining region-3 (CDR3) amino acid (AA) sequences, which play a major role in antigen binding. For example, the chemical complementarities of the tumor resident, CDR3 AA sequences and the BRAFV600E mutant, common in melanoma, have proved informative with regard to outcomes. Many of these evaluations led to the conclusion that a high affinity match, efficiently, algorithmically designated as a high chemical complementarity score (CS) for the patient specific, IR CDR3 AA sequences and the cancer antigens, correlated with improved survival outcomes. In this report, the complementarity scoring algorithms were used to investigate the opposite phenomenon, high complementarity chemistry between CRD3 AAs and cancer antigens that correlated with a worse survival, an approach that revealed potential risk stratification biomarkers for lung adenocarcinoma, lung squamous carcinoma, and likely other cancer types. Most importantly, analyses suggested that high IR CDR3 AA-candidate antigen CS, low overall survival results for low grade glioma were mitigated by neoadjuvant corticosteroid treatments. Overall, the analyses of this report, coupled with earlier work establishing the CS approach for identifying likely good outcomes, have the potential to distinguish patients who will benefit from (i) immune activating or (ii) immune augmenting or (iii) even immunosuppressive treatment strategies.Communicated by Ramaswamy H. Sarma.

Phosphatidic acid mediates the targeting of tBid to induce lysosomal membrane permeabilization and apoptosis.

Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling.

Epigenetic regulation of phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 gene expression in prostate cancer cells.

Aberrant up-regulation of P-Rex1 expression plays important roles in cancer progression and metastasis. The present study investigated the regulatory mechanism underlying P-Rex1 gene expression in prostate cancer cells. We showed that P-Rex1 expression was much higher in metastatic prostate cancer cells than in prostate epithelial cells and non-metastatic prostate cancer cells. Histone deacetylase (HDAC) inhibitors or silence of endogenous HDAC1 and HDAC2 markedly elevated P-Rex1 transcription in non-metastatic prostate cancer cells, whereas overexpression of recombinant HDAC1 in metastatic prostate cancer cells suppressed P-Rex1 expression. HDAC inhibitor trichostatin A (TSA) also significantly increased P-Rex1 promoter activity and caused acetylated histones to accumulate and associate with the P-Rex1 promoter. One Sp1 site, essential for basal promoter activity, was identified as critical for the TSA effect. TSA treatment did not alter the DNA-binding activity of Sp1 toward the P-Rex1 promoter; however, it facilitated the dissociation of the repressive HDAC1 and HDAC2 from the Sp1 binding region. Interestingly, HDAC1 association with Sp1 and with the P-Rex1 promoter were much weaker in metastatic prostate cancer PC-3 cells than in non-metastatic prostate cancer cells, and HDAC inhibitors only had very modest stimulatory effects on P-Rex1 promoter activity and P-Rex1 expression in PC-3 cells. Altogether, our studies demonstrate that HDACs could regulate P-Rex1 gene transcription by interaction with Sp1 and by region-specific changes in histone acetylation within the P-Rex1 promoter. Disassociation of HDACs from Sp1 on the P-Rex1 promoter may contribute to aberrant up-regulation of P-Rex1 in cancer.