Validating organoid-derived human intestinal monolayers for personalized therapy in cystic fibrosis.

Highly effective drugs modulating the defective protein encoded by the CFTR gene have revolutionized cystic fibrosis (CF) therapy. Preclinical drug-testing on human nasal epithelial (HNE) cell cultures and 3-dimensional human intestinal organoids (3D HIO) are used to address patient-specific variation in drug response and to optimize individual treatment for people with CF. This study is the first to report comparable CFTR functional responses to CFTR modulator treatment among patients with different classes of CFTR gene variants using the three methods of 2D HIO, 3D HIO, and HNE. Furthermore, 2D HIO showed good correlation to clinical outcome markers. A larger measurable CFTR functional range and access to the apical membrane were identified as advantages of 2D HIO over HNE and 3D HIO, respectively. Our study thus expands the utility of 2D intestinal monolayers as a preclinical drug testing tool for CF.

Impacts of opium addiction on patterns of angiographic findings in patients with acute coronary syndrome.

Opium is one of the most abused substances in the Middle East. The effects of opium use on coronary artery disease (CAD) are a matter of debate. This study aimed to assess the association between opium use and angiographic findings as well as the complexity of CAD in patients with acute coronary syndrome (ACS) diagnosis. In this case-control study, all patients admitted for coronary angiography from 2019 to 2020 were evaluated. After applying the eligibility criteria, they were categorized into two groups opium and non-opium based on their history of opium use. Both groups were matched regarding the demographic features. The prevalence, location, and severity of obstruction of the vessels were compared between the non-opium and opium groups. The SYNTAX score was also calculated and compared between the two groups. The scores ≤ 22 are considered low risk and the higher scores are a non-low risk. P value < 0.05 is considered significant. A total of 170 patients with a mean age of 61.59 ± 9.07 years were finally enrolled in our study. Regarding the severity of vascular involvement, there was a significant difference between the non-opium and opium groups in LAD (P = 0.025), and PLV (P = 0.018) vessels. From the location points of view of obstructive coronary artery involved segments, only in the PDA (P = 0.006), and LCX (P = 0.004) vessels, a significant difference was observed. Moreover, 47.1% of opium and 30.6% of non-opium use group were in the non-low risk SYNTAX score classification which is a statistically significant difference between these two groups (P value = 0.048). Opium, as an independent risk factor for cardiovascular diseases, can have specific effects on angiographic findings in patients with acute coronary syndrome. Likewise, the complexity of CAD in opium users who undergo percutaneous coronary intervention is significantly higher.

Genetic evidence supports the development of SLC26A9 targeting therapies for the treatment of lung disease.

Over 400 variants in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) are CF-causing. CFTR modulators target variants to improve lung function, but marked variability in response exists and current therapies do not address all CF-causing variants highlighting unmet needs. Alternative epithelial ion channel/transporters such as SLC26A9 could compensate for CFTR dysfunction, providing therapeutic targets that may benefit all individuals with CF. We investigate the relationship between rs7512462, a marker of SLC26A9 activity, and lung function pre- and post-treatment with CFTR modulators in Canadian and US CF cohorts, in the general population, and in those with chronic obstructive pulmonary disease (COPD). Rs7512462 CC genotype is associated with greater lung function in CF individuals with minimal function variants (for which there are currently no approved therapies; p = 0.008); and for gating (p = 0.033) and p.Phe508del/ p.Phe508del (p = 0.006) genotypes upon treatment with CFTR modulators. In parallel, human nasal epithelia with CC and p.Phe508del/p.Phe508del after Ussing chamber analysis of a combination of approved and experimental modulator treatments show greater CFTR function (p = 0.0022). Beyond CF, rs7512462 is associated with peak expiratory flow in a meta-analysis of the UK Biobank and Spirometa Consortium (p = 2.74 × 10-44) and provides p = 0.0891 in an analysis of COPD case-control status in the UK Biobank defined by spirometry. These findings support SLC26A9 as a therapeutic target to improve lung function for all people with CF and in individuals with other obstructive lung diseases.

CFTR interactome mapping using the mammalian membrane two-hybrid high-throughput screening system.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein-protein interactions of wild-type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient-specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient-derived intestinal organoids has a significant effect on CFTR functional expression.

Antithetic hTERT Regulation by Androgens in Prostate Cancer Cells: hTERT Inhibition Is Mediated by the ING1 and ING2 Tumor Suppressors.

The human telomerase is a key factor during tumorigenesis in prostate cancer (PCa). The androgen receptor (AR) is a key drug target controlling PCa growth and regulates hTERT expression, but is described to either inhibit or to activate. Here, we reveal that androgens repress and activate hTERT expression in a concentration-dependent manner. Physiological low androgen levels activate, while, notably, supraphysiological androgen levels (SAL), used in bipolar androgen therapy (BAT), repress hTERT expression. We confirmed the SAL-mediated gene repression of hTERT in PCa cell lines, native human PCa samples derived from patients treated ex vivo, as well as in cancer spheroids derived from androgen-dependent or castration resistant PCa (CRPC) cells. Interestingly, chromatin immuno-precipitation (ChIP) combined with functional assays revealed a positive (pARE) and a negative androgen response element (nARE). The nARE was narrowed down to 63 bp in the hTERT core promoter region. AR and tumor suppressors, inhibitor of growth 1 and 2 (ING1 and ING2, respectively), are androgen-dependently recruited. Mechanistically, knockdown indicates that ING1 and ING2 mediate AR-regulated transrepression. Thus, our data suggest an oppositional, biphasic function of AR to control the hTERT expression, while the inhibition of hTERT by androgens is mediated by the AR co-repressors ING1 and ING2.

Sex Differences in Diabetes- and TGF-ß1-Induced Renal Damage.

While females are less affected by non-diabetic kidney diseases compared to males, available data on sex differences in diabetic nephropathy (DN) are controversial. Although there is evidence for an imbalance of sex hormones in diabetes and hormone-dependent mechanisms in transforming growth factor ß1 (TGF-ß1) signaling, causes and consequences are still incompletely understood. Here we investigated the influence of sex hormones and sex-specific gene signatures in diabetes- and TGF-ß1-induced renal damage using various complementary approaches (a db/db diabetes mouse model, ex vivo experiments on murine renal tissue, and experiments with a proximal tubular cell line TKPTS). Our results show that: (i) diabetes affects sex hormone concentrations and renal expression of their receptors in a sex-specific manner; (ii) sex, sex hormones and diabetic conditions influence differences in expression of TGF-ß1, its receptor and bone morphogenetic protein 7 (BMP7); (iii) the sex and sex hormones, in combination with variable TGF-ß1 doses, determine the net outcome in TGF-ß1-induced expression of connective tissue growth factor (CTGF), a profibrotic cytokine. Altogether, these results suggest complex crosstalk between sex hormones, sex-dependent expression pattern and profibrotic signals for the precise course of DN development. Our data may help to better understand previous contradictory findings regarding sex differences in DN.

Genetic association and transcriptome integration identify contributing genes and tissues at cystic fibrosis modifier loci.

Cystic Fibrosis (CF) exhibits morbidity in several organs, including progressive lung disease in all patients and intestinal obstruction at birth (meconium ileus) in ~15%. Individuals with the same causal CFTR mutations show variable disease presentation which is partly attributed to modifier genes. With >6,500 participants from the International CF Gene Modifier Consortium, genome-wide association investigation identified a new modifier locus for meconium ileus encompassing ATP12A on chromosome 13 (min p = 3.83x10(-10)); replicated loci encompassing SLC6A14 on chromosome X and SLC26A9 on chromosome 1, (min p<2.2x10(-16), 2.81x10(-11), respectively); and replicated a suggestive locus on chromosome 7 near PRSS1 (min p = 2.55x10(-7)). PRSS1 is exclusively expressed in the exocrine pancreas and was previously associated with non-CF pancreatitis with functional characterization demonstrating impact on PRSS1 gene expression. We thus asked whether the other meconium ileus modifier loci impact gene expression and in which organ. We developed and applied a colocalization framework called the Simple Sum (SS) that integrates regulatory and genetic association information, and also contrasts colocalization evidence across tissues or genes. The associated modifier loci colocalized with expression quantitative trait loci (eQTLs) for ATP12A (p = 3.35x10(-8)), SLC6A14 (p = 1.12x10(-10)) and SLC26A9 (p = 4.48x10(-5)) in the pancreas, even though meconium ileus manifests in the intestine. The meconium ileus susceptibility locus on chromosome X appeared shifted in location from a previously identified locus for CF lung disease severity. Using the SS we integrated the lung disease association locus with eQTLs from nasal epithelia of 63 CF participants and demonstrated evidence of colocalization with airway-specific regulation of SLC6A14 (p = 2.3x10(-4)). Cystic Fibrosis is realizing the promise of personalized medicine, and identification of the contributing organ and understanding of tissue specificity for a gene modifier is essential for the next phase of personalizing therapeutic strategies.

Improving imputation in disease-relevant regions: lessons from cystic fibrosis.

Does genotype imputation with public reference panels identify variants contributing to disease? Genotype imputation using the 1000 Genomes Project (1KG; 2504 individuals) displayed poor coverage at the causal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) locus for the International CF Gene Modifier Consortium. Imputation with the larger Haplotype Reference Consortium (HRC; 32,470 individuals) displayed improved coverage but low sensitivity of variants clinically relevant for CF. A hybrid reference that combined whole genome sequencing (WGS) from 101 CF individuals with the 1KG imputed a greater number of single-nucleotide variants (SNVs) that would be analyzed in a genetic association study (r2 ≥ 0.3 and MAF ≥ 0.5%) than imputation with the HRC, while the HRC excelled in the lower frequency spectrum. Using the 1KG or HRC as reference panels missed the most common CF-causing variants or displayed low imputation accuracy. Designs that incorporate population-specific WGS can improve imputation accuracy at disease-specific loci, while imputation using public data sets can omit disease-relevant genotypes.

A novel crosstalk between the tumor suppressors ING1 and ING2 regulates androgen receptor signaling.

The androgen receptor (AR) is a transcriptional factor that has a pivotal role in the development of normal and also cancerous prostate. Therefore, analyzing AR signaling is essential to understand cancerogensis and proliferation of prostate cancer (PCa). Inhibitor of growth 1 (ING1) and ING2 are tumor suppressors with reduced expression in many cancer types. There are also indications of misregulation of ING1 and ING2 in PCa. However, the roles of ING1 and ING2 in PCa and AR signaling are poorly understood. Here, we show that surprisingly the ING1b knockdown (KD) represses AR-mediated transactivation on AR key target genes in the human LNCaP PCa cells. This is associated with growth reduction of LNCaP cells by ING1 KD. In line with this, using Ing1 knockout (KO) mice, we provide further evidence that ING1 deficiency downregulates prostate-specific AR target genes in vivo. Further analyses suggest that KD of ING1b results in induction of both cellular senescence and the cell cycle inhibitor p16 INK4a . The unexpected finding that the ING1 KD results in growth inhibition was further analyzed and can be explained by a compensatory mechanism through enhanced levels of ING2 protein in ING1-deficient condition. Accordingly, the data suggest that ING2 interacts with AR and hampers the AR transcriptional activation, causes growth arrest, and induces cellular senescence. The data further suggest that ING2 upregulates p16 INK4a , which is a novel target for ING2. Taken together, our data suggest that ING2 is a novel corepressor for AR. ING2 levels are increased upon downregulation of ING1 expression indicating a compensatory mechanism and suggests a novel crosstalk between ING1 and ING2 tumor suppressors to inhibit AR signaling and induce cellular senescence in PCa cells. KEY MESSAGE: • The tumor suppressors ING1 and 2 are dysregulated in human prostate cancer. • ING1 deficiency reduces AR-mediated gene expression in vitro and in vivo. • ING2, like ING1, inhibits AR-mediated transactivation and prostate cancer cell growth. • ING1 regulates ING2. • ING1 and ING2 crosstalk with each other to inhibit AR signaling in prostate cancer.

The tumor suppressor ING1b is a novel corepressor for the androgen receptor and induces cellular senescence in prostate cancer cells.

The androgen receptor (AR) signaling is critical for prostate cancer (PCa) progression to the castration-resistant stage with poor clinical outcome. Altered function of AR-interacting factors may contribute to castration-resistant PCa (CRPCa). Inhibitor of growth 1 (ING1) is a tumor suppressor that regulates various cellular processes including cell proliferation. Interestingly, ING1 expression is upregulated in senescent primary human prostate cells; however, its role in AR signaling in PCa was unknown. Using a proteomic approach by surface-enhanced laser desorption ionization-mass spectrometry (SELDI-MS) combined with immunological techniques, we provide here evidence that ING1b interacts in vivo with the AR. The interaction was confirmed by co-immunoprecipitation, in vitro GST-pull-down, and quantitative intracellular colocalization analyses. Functionally, ING1b inhibits AR-responsive promoters and endogenous key AR target genes in the human PCa LNCaP cells. Conversely, ING1b knockout (KO) mouse embryonic fibroblasts (MEFs) exhibit enhanced AR activity, suggesting that the interaction with ING1b represses the AR-mediated transcription. Also, data suggest that ING1b expression is downregulated in CRPCa cells compared with androgen-dependent LNCaP cells. Interestingly, its ectopic expression induces cellular senescence and reduces cell migration in both androgen-dependent and CRPCa cells. Intriguingly, ING1b can also inhibit androgen-induced growth in LNCaP cells in a similar manner as AR antagonists. Moreover, ING1b upregulates different cell cycle inhibitors including p27(KIP1), which is a novel target for ING1b. Taken together, our findings reveal a novel corepressor function of ING1b on various AR functions, thereby inhibiting PCa cell growth.

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target.

BACKGROUND: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. METHODS: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ(2) tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. RESULTS: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. CONCLUSIONS: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.

Docosahexaenoic acid inhibits the growth of hormone-dependent prostate cancer cells by promoting the degradation of the androgen receptor.

Epidemiological and preclinical data have demonstrated the preventative effects of ω-3 polyunsaturated fatty acids, including docosahexaenoic acid (DHA), on prostate cancer. However, there are inconsistencies in these previous studies and the underlying mechanisms remain to be elucidated. In the present study, the androgen receptor (AR), which is a transcription factor involved in cell proliferation and prostate carcinogenesis, was identified as a target of DHA. It was revealed that DHA inhibited hormone­dependent growth of LNCaP prostate cancer cells. Reverse transcription-quantitative polymerase chain reaction analysis revealed that treatment with DHA caused no alteration in the transcribed mRNA expression levels of the AR gene. However, immunoblotting revealed that this treatment reduces the protein expression level of the AR. The androgen­induced genes were subsequently repressed by treatment with DHA. It was demonstrated that DHA exhibits no effect on the translation process of the AR, however, it promotes the proteasome­mediated degradation of the AR. Therefore, the present study provided a novel mechanism by which DHA exhibits an inhibitory effect on growth of prostate cancer cells.

Supraphysiological androgen levels induce cellular senescence in human prostate cancer cells through the Src-Akt pathway.

BACKGROUND: Prostate cancer (PCa) is the second leading cause of cancer mortality of men in Western countries. The androgen receptor (AR) and AR-agonists (androgens) are required for the development and progression of the normal prostate as well as PCa. However, it is discussed that in addition to their tumor promoting activity, androgens may also exhibit tumor suppressive effects. A biphasic growth response to androgens a growth-promoting and -inhibition has been observed that suggests that administration of supraphysiological androgen levels mediates growth reduction in AR expressing PCa cells. METHODS: Detection of senescence markers, three dimensional interphase fluorescence in situ hybridization (3D-iFISH), qRT-PCR, Western blotting, detection of GFP fusions, prostatectomy, ex vivo culturing. RESULTS: Here, we describe that supraphysiological levels of androgens induce cell cycle arrest and markers of cellular senescence in human PCa cells, which may in part explain the growth inhibitory role of androgens. The expression of the senescence associated beta galactosidase is observed by treatment with the natural androgen DHT or the less metabolized synthetic androgen R1881. The induction of senescence marker was detected in human PCa cell lines as well as in human primary PCa tissue derived from prostatectomy treated ex vivo. Using interphase FISH (iFISH) suggests that the androgen-induced cellular senescence is associated with localizing the genomic E2F1 locus to senescence associated heterochromatic foci. Analysis of different signaling pathways in LNCaP cells suggest that the p16-Rb-E2F1 pathway is essential for the induction of cellular senescence since treatment with siRNA directed against p16 reduces the level of androgen-induced cellular senescence. Based on the rapid induction of androgen-mediated cellular senescence we identified the Src-PI3K-Akt-signaling pathway and autophagy being in part involved in androgen regulation. CONCLUSIONS: Taken together, our data suggest that AR-agonists at supraphysiological levels mediate induction of cellular senescence in human PCa cells, which may have a protective anti-cancer role. These results provide also new insights for understanding androgen-mediated regulation of PCa growth.

Common Mediterranean fever gene mutations in the Azeri Turkish population of Iran.

Familial Mediterranean fever (FMF) is an autosomal recessive disorder primarily affecting the Mediterranean populations. It is characterized by recurrent attacks of fever and inflammation of serosal membranes and gradual development of nephropathic amyloidosis. More than 70 disease-associated mutations have been identified in the Mediterranean fever gene (MEFV) responsible for FMF. The aim of this study was to determine the mutation carrier rate in the Iranian Azeri Turkish population. A cohort of 200 unrelated healthy individuals was screened for the five most common MEFV mutations (M694V, V726A, M680I, M694I, and E148Q) using the amplification refractory mutation system for the first four and by polymerase chain reaction-restriction-digestion testing for E148Q. Genotyping revealed that the carrier rate in the Azeri Turkish population was 25.5%, with E148Q being the most common mutation (11.5%) followed by V726A (1.75%). The remaining common mutations were not found in this cohort. Our data indicate that the FMF carrier rate and E148Q mutation frequency are high in the Iranian Azeri Turkish population.

TNF-alpha gene polymorphisms in Iranian Azeri Turkish patients with Behcet's Disease.

Genetic factors that predispose individuals to Behcet's disease (BD) are considered to play an important role in the development of the disease. The serum level of tumor necrosis factor (TNF) is elevated in patients with BD, and a dramatic response to anti-TNF-alpha antibody treatment further supports the role of TNF in BD. We investigated the distribution of TNF-alpha promoter -1031T/C and -308G/A polymorphisms in 53 BD patients of Iranian Azeri Turks and 79 matched healthy controls, via the PCR-RFLP technique. The frequency of the TNF-alpha -1031C allele was significantly higher in Behcet's patients than in healthy controls (p < 0.0001, OR = 3.08; 95% CI = 1.73-5.47), whereas the frequency of the TNF-alpha -308A allele was similar in the two compared groups. The frequency of CG haplotype was significantly higher (p < 0.0001, OR = 3.42; 95% CI = 1.89-6.18), and that of the TA haplotype was significantly lower in BD patients than in healthy controls. These results suggest that TNF-alpha is a susceptibility gene for BD in patients from Iranian Azeri Turk ethnic group.

HPV typing in women with cervical precancerous and cancerous lesions in northwestern Iran.

BACKGROUND/AIMS: The role of human papillomavirus (HPV) in the etiology of cervical cancer is now well established. This investigation was designed to study the prevalence of the four most common high-risk HPVs in the archival tissues with precancerous and cancerous lesions from patients from northwestern Iran. METHODS: 133 formalin-fixed paraffin-embedded tissue specimens were tested for HPV DNA by using GP5+/6+-based general PCR and two type-specific PCRs. RESULTS: In total, 84 (64%) out of 131 amplifiable samples were positive for HPV DNA. The most prevalent oncogenic HPV was type 16 (67.6%) followed by types 31 (22.8%), 18 (7.6%) and 33 (1%). Multiple HPV infections were present in 20 (15.3%) of the 131 samples. Notably, of these 20 cases with multiple infections, 15 were from patients with invasive cervical cancer. CONCLUSIONS: The multiplicity of HPV genotypes was noted in invasive cervical carcinoma samples, along with rather different circulating HPV types in the study population. Hence, relevant HPV typing information in cervical carcinoma is very important for planning more efficient screening programs and for further HPV vaccine design.