Combination of Antiretroviral Drugs Zidovudine and Efavirenz Impairs Tumor Growths in a Mouse Model of Cancer.

LINE1 retrotransposons, which are thought to be the remnants of ancient integrations of retrovirus-like elements, are aberrantly (re)activated in many cancer cells. Due to LINE1-induced alterations in target gene expression and/or chromosomal rearrangements, they may be important drivers of tumorigenesis. Moreover, LINE1 encoded proteins, Open Reading Frame (ORF)1 and ORF2, may have pro-oncogenic potential through inductors of oncogenic transcription factors or inhibitors of cell cycle suppressors. The current study therefore aimed to investigate in vitro and in vivo anti-tumorigenic effects of two well-known antiretroviral drugs, zidovudine, a nucleoside analogue inhibitor of RT (NRTI), and efavirenz, a non-nucleoside RT inhibitor (NNRTI). Our data demonstrate that both drugs in clinically relevant doses significantly reduced the proliferation of murine and human cancer cell lines, as well as growth of tumors in a murine subcutaneous model. Intriguingly, we found that the combination of both zidovudine and efavirenz almost entirely blocked tumorigenesis in vivo. Because both drugs are FDA-approved agents and the combination was very well tolerated in mice, the combination therapy as presented in our paper might be an opportunity to treat colorectal tumors and metastasis to the liver in an inexpensive way.

Prognostic Role of FGFR3 Expression Status and Tumor-Related MicroRNAs Level in Association with PD-L1 Expression in Primary Luminal Non-Muscular Invasive Bladder Carcinoma.

BACKGROUND: bladder cancer is one of the most common urinary tract malignancies. Establishment of robust predictors of disease progression and outcome is important for personalizing treatment of non-muscular invasive bladder carcinoma (NMIBC). In this study we evaluated association of PD-L1 expression with other prognostic biomarkers, such as expression of miRNA-145 and miRNA-200a, FGFR3 gene expression, and mutation status in tissue specimens of the luminal subtype of newly diagnosed high and low grade NMIBC. METHODS: twenty patients with primary luminal NMIBC were enrolled in the study. Tumor grade and risk level were determined in accordance with European Organization for Research and Treatment of Cancer (EORTC) guidelines and World Health Organization (WHO) classification. Neoplasm molecular subtype and PD-L1 expression level were assessed by immunohistochemistry. We used real-time PCR to evaluate the expression of microRNAs and FGFR3. We detected FGFR3 hotspot mutations in codons 248 and 249 by Sanger sequencing. RESULTS: high grade primary luminal NMIBC showed comparatively higher expression of PD-L1 and microRNA-145 than a low grade tumor, whereas the latter had a higher FGFR3 expression and hotspot mutation rate. The tumor grade (HR = 571.72 [11.03-2.96] p = 0.002), PD-L1 expression (HR = 2.33 [0.92-1.92] p = 0.012), and FGFR3 expression (HR = 0.08 [0.17-0.42] p = 0.003) were associated with relapse-free survival. CONCLUSIONS: tumor grade in association with PD-L1 and FGFR3 expression can be considered as a complex predictor for primary luminal NMIBC progression.

Large-Scale Profiling of Signaling Pathways Reveals a Distinct Demarcation between Normal and Extended Liver Resection.

Despite numerous studies addressing normal liver regeneration, we still lack comprehensive understanding of the biological processes underlying failed liver regeneration. Therefore, we analyzed the activity of 271 intracellular signaling pathways (ISPs) by genome wide profiling of differentially expressed RNAs in murine liver tissue biopsies after normal hepatectomy (nHx; 68% of liver removed) and extended hepatectomy (eHx; 86% of liver removed). Comprehensive, genome-wide transcriptome profiling using RNAseq was performed in liver tissue obtained from mice (sham, nHx, and eHx) harvested 1, 8, 16, 32, and 48 h after operation (n = 3 per group) and the OncoFinder toolkit was used for an unsupervised, unbiased identification of intracellular signaling pathways (ISP) activity. We observed that the normal regenerative process requires a transient activation and silencing of approximately two dozen of ISPs. After nHx, the Akt Pathway represented with 13 branches, the Chromatin Pathway and the DDR Pathways dominated. After eHx, the ATM main pathway and two of its branches (Cell Survival; G2_M Checkpoint Arrest) dominated, as well as the Hypoxia Pathways. Further, 14 ISPs demonstrated a strong inverse regulation, with the Hedgehog and the Brca1 Main Pathways as chief activators after nHx, and the ATM Pathway (G2_M Checkpoint Arrest) as the dominating constraining response after eHx.

Exploration of the Transcriptional Landscape of ALPPS Reveals the Pathways of Accelerated Liver Regeneration.

Background and Aims: ALPPS (associating liver partition and portal vein ligation for staged hepatectomy), a novel 2-staged hepatectomy, dramatically accelerates liver regeneration and thus enables extensive liver tumor resection. The signaling networks underlying the ALPPS-induced accelerated regeneration process are largely unknown. Methods: We performed transcriptome profiling (TP) of liver tissue obtained from a mouse model of ALPPS, standard hepatectomy (68% model), and additional control surgeries (sham, PVL and Tx). We also performed TP using human liver biopsies (n = 5) taken from the occluded lobe and the future liver remnant (FLR) during the first step of ALPPS surgery (4-5 h apart). We used Oncofinder computational tools, which covers 378 ISPs, for unsupervised, unbiased quantification of ISP activity. Results: Gene expression cluster analysis revealed an ALPPS specific signature: the IGF1R Signaling Pathway (Cell survival), the ILK Pathway (Induced cell proliferation), and the IL-10 Pathway (Stability determination) were significantly enriched, whereas the activity of the Interferon Pathway (Transcription) was reduced (p < 0.05). Further, the PAK- and ILK-associated ISPs were activated at an earlier time point, reflecting significant acceleration of liver regeneration (p < 0.001). These pathways, which were also recovered in human liver biopsies, control cell growth and proliferation, inflammatory response, and hypoxia-related processes. Conclusions: ALPPS is not a straightforward addition of portal vein ligation (PVL) plus transection-it is more. The early stages of normal and accelerated liver regeneration are clearly discernible by a significantly increased and earlier activation of a small number of signaling pathways. Compounds mimicking these responses may help to improve the ALPPS method and further reduce the hospitalization time of the patient.

Correction: Nikitin, D., et al. Retroelement-Linked Transcription Factor Binding Patterns Point to Quickly Developing Molecular Pathways in Human Evolution. Cells 2019, 8, 130.

In the article 'Retroelement-Linked Transcription Factor Binding Patterns Point to Quickly Developing Molecular Pathways in Human Evolution,' a number of transcription factor binding sites (TFBS) mapped on all retroelement classes were incorrectly calculated as sum of TFBS numbers separately mapped on LINEs, SINEs and LTR retrotransposons/endogenous retroviruses (LR/ERVs) [...].

Pathway Based Analysis of Mutation Data Is Efficient for Scoring Target Cancer Drugs.

Despite the significant achievements in chemotherapy, cancer remains one of the leading causes of death. Target therapy revolutionized this field, but efficiencies of target drugs show dramatic variation among individual patients. Personalization of target therapies remains, therefore, a challenge in oncology. Here, we proposed molecular pathway-based algorithm for scoring of target drugs using high throughput mutation data to personalize their clinical efficacies. This algorithm was validated on 3,800 exome mutation profiles from The Cancer Genome Atlas (TCGA) project for 128 target drugs. The output values termed Mutational Drug Scores (MDS) showed positive correlation with the published drug efficiencies in clinical trials. We also used MDS approach to simulate all known protein coding genes as the putative drug targets. The model used was built on the basis of 18,273 mutation profiles from COSMIC database for eight cancer types. We found that the MDS algorithm-predicted hits frequently coincide with those already used as targets of the existing cancer drugs, but several novel candidates can be considered promising for further developments. Our results evidence that the MDS is applicable to ranking of anticancer drugs and can be applied for the identification of novel molecular targets.

Retroelement-Linked Transcription Factor Binding Patterns Point to Quickly Developing Molecular Pathways in Human Evolution.

BACKGROUND: Retroelements (REs) are transposable elements occupying ~40% of the human genome that can regulate genes by providing transcription factor binding sites (TFBS). RE-linked TFBS profile can serve as a marker of gene transcriptional regulation evolution. This approach allows for interrogating the regulatory evolution of organisms with RE-rich genomes. We aimed to characterize the evolution of transcriptional regulation for human genes and molecular pathways using RE-linked TFBS accumulation as a metric. Methods: We characterized human genes and molecular pathways either enriched or deficient in RE-linked TFBS regulation. We used ENCODE database with mapped TFBS for 563 transcription factors in 13 human cell lines. For 24,389 genes and 3124 molecular pathways, we calculated the score of RE-linked TFBS regulation reflecting the regulatory evolution rate at the level of individual genes and molecular pathways. Results: The major groups enriched by RE regulation deal with gene regulation by microRNAs, olfaction, color vision, fertilization, cellular immune response, and amino acids and fatty acids metabolism and detoxication. The deficient groups were involved in translation, RNA transcription and processing, chromatin organization, and molecular signaling. Conclusion: We identified genes and molecular processes that have characteristics of especially high or low evolutionary rates at the level of RE-linked TFBS regulation in human lineage.

Temporary portal vein embolization is as efficient as permanent portal vein embolization in mice.

BACKGROUND: Temporary portal vein embolization may be a safe alternative to permanent portal vein embolization. Such a new approach could be applied in living-related liver transplantation to increase graft volume before procurement. The impact of temporary portal vein embolization on occluded liver after recanalization, however, has never been assessed. Using a mouse model of temporary portal vein embolization, we investigated (1) the efficiency of temporary portal vein embolization in inducing nonoccluded liver hypertrophy and (2) the regeneration potential and functional recovery of embolized liver after recanalization. METHODS: Selected portal vein branches were occluded using gelfoam powder (temporary portal vein embolization) or embospheres (permanent portal vein embolization), n = 5/group. Magnetic resonance volumetry and angiography were used to determine volumes of the liver lobe and portal vein branch recanalization. In order to assess the functional and regenerative capacity of occluded liver lobes, nonoccluded lobes were resected 14 days (timespan of complete portal vein recanalization) after temporary portal vein embolization or permanent portal vein embolization. Subsequently, RNA sequencing was performed to compare the signaling pathways of early liver regeneration among the groups. RESULTS: Hypertrophy of nonoccluded lobes 30 days after temporary portal vein embolization and permanent portal vein embolization was similar (103 ± 26% and 129 ± 13%, P = .11). Temporary occluded lobes increased their volumes after nonoccluded lobes resection, reaching similar liver-to-body-weight ratios and similar functional capacity after 7 days compared with partial hepatectomy controls (4 ± 1% vs 4 ± 1%, P = .22). Partial hepatectomy activated similar signaling pathways in temporary occluded and native liver. CONCLUSION: Temporary portal vein embolization induces hypertrophy of contralateral liver lobes similarly to permanent portal vein embolization in mice. This experimental work suggests that temporary portal vein embolization may be considered as a possibility in living liver donation, because regenerative and functional capacities are preserved in the embolized liver after recanalization in mice.

Hedgehog pathway mediates early acceleration of liver regeneration induced by a novel two-staged hepatectomy in mice.

BACKGROUND & AIMS: ALPPS, a novel two-staged approach for the surgical removal of large/multiple liver tumors, combines portal vein ligation (PVL) with parenchymal transection. This causes acceleration of compensatory liver growth, enabling faster and more extensive tumor removal. We sought to identify the plasma factors thought to mediate the regenerative acceleration following ALPPS. METHODS: We compared a mouse model of ALPPS against PVL and additional control surgeries (n=6 per group). RNA deep sequencing was performed to identify candidate molecules unique to ALPPS liver (n=3 per group). Recombinant protein and a neutralizing antibody combined with appropriate surgeries were used to explore candidate functions in ALPPS (n=6 per group). Indian hedgehog (IHH/Ihh) levels were assessed in human ALPPS patient plasma (n=6). RESULTS: ALPPS in mouse confirmed significant acceleration of liver regeneration relative to PVL (p<0.001). Ihh mRNA, coding for a secreted ligand inducing hedgehog signaling, was uniquely upregulated in ALPPS liver (p<0.001). Ihh plasma levels rose 4h after surgery (p<0.01), along with hedgehog pathway activation and subsequent cyclin D1 induction in the liver. When combined with PVL, Ihh alone was sufficient to induce ALPPS-like acceleration of liver growth. Conversely, blocking Ihh markedly inhibited the accelerating effects of ALPPS. In the small cohort of ALPPS patients, IHH tended to be elevated early after surgery. CONCLUSIONS: Ihh and hedgehog pathway activation provide the first mechanistic insight into the acceleration of liver regeneration triggered by ALPPS surgery. The accelerating potency of recombinant Ihh, and its potential effect in human ALPPS may lead to a clinical role for this protein. LAY SUMMARY: ALPPS, a novel two-staged hepatectomy, accelerates liver regeneration, thereby helping to treat patients with otherwise unresectable liver tumors. The molecular mechanisms behind this accelerated regeneration are unknown. Here, we elucidate that Indian hedgehog, a secreted ligand important for fetal development, is a crucial mediator of the regenerative acceleration triggered by ALPPS surgery.

Large-scale profiling of signalling pathways reveals an asthma specific signature in bronchial smooth muscle cells.

BACKGROUND: Bronchial smooth muscle (BSM) cells from asthmatic patients maintain in vitro a distinct hyper-reactive ("primed") phenotype, characterized by increased release of pro-inflammatory factors and mediators, as well as hyperplasia and/or hypertrophy. This "primed" phenotype helps to understand pathogenesis of asthma, as changes in BSM function are essential for manifestation of allergic and inflammatory responses and airway wall remodelling. OBJECTIVE: To identify signalling pathways in cultured primary BSMs of asthma patients and non-asthmatic subjects by genome wide profiling of differentially expressed mRNAs and activated intracellular signalling pathways (ISPs). METHODS: Transcriptome profiling by cap-analysis-of-gene-expression (CAGE), which permits selection of preferentially capped mRNAs most likely to be translated into proteins, was performed in human BSM cells from asthmatic (n=8) and non-asthmatic (n=6) subjects and OncoFinder tool were then exploited for identification of ISP deregulations. RESULTS: CAGE revealed >600 RNAs differentially expressed in asthma vs control cells (p≤0.005), with asthma samples showing a high degree of similarity among them. Comprehensive ISP activation analysis revealed that among 269 pathways analysed, 145 (p<0.05) or 103 (p<0.01) are differentially active in asthma, with profiles that clearly characterize BSM cells of asthmatic individuals. Notably, we identified 7 clusters of coherently acting pathways functionally related to the disease, with ISPs down-regulated in asthma mostly targeting cell death-promoting pathways and up-regulated ones affecting cell growth and proliferation, inflammatory response, control of smooth muscle contraction and hypoxia-related signalization. CONCLUSIONS: These first-time results can now be exploited toward development of novel therapeutic strategies targeting ISP signatures linked to asthma pathophysiology.

Small RNA profiling reveals deregulated phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/Akt pathway in bronchial smooth muscle cells from asthmatic patients.

BACKGROUND: Aberrant expression of small noncoding RNAs (sncRNAs), microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) in particular, define several pathologic processes. Asthma is characterized by airway hyperreactivity, chronic inflammation, and airway wall remodeling. Asthma-specific miRNA profiles were reported for bronchial epithelial cells, whereas sncRNA expression in asthmatic bronchial smooth muscle (BSM) cells is almost completely unexplored. OBJECTIVE: We sought to determine whether the primary BSM sncRNA expression profile is altered in asthmatic patients and identify targets of differentially expressed sncRNAs. METHODS: Small RNA sequencing was used for sncRNA profiling in BSM cells (from 8 asthmatic and 6 nonasthmatic subjects). sncRNA identification and differential expression analysis was performed with iMir software. Experimentally validated miRNA targets were identified by using Ingenuity Pathway Analysis, and putative piRNA targets were identified by using miRanda software. RESULTS: BSM cells from asthmatic patients showed abnormal expression of 32 sncRNAs (26 miRNAs, 5 piRNAs, and 1 small nucleolar RNA). Target prediction for deregulated miRNAs and piRNAs revealed experimentally validated and predicted mRNA targets expressed in the BSM cells. Thirty-eight of these mRNAs represent major targets for deregulated miRNAs and might play important roles in the pathophysiology of asthma. Interestingly, 6 of these mRNAs were previously associated with asthma, considered as novel therapeutic targets for treatment of this disease, or both. Signaling pathway analysis revealed involvement of 38 miRNA-targeted mRNAs in increased cell proliferation through phosphatase and tensin homolog and phosphoinositide 3-kinase/Akt signaling pathways. CONCLUSIONS: BSM cells of asthmatic patients are characterized by aberrant sncRNA expression that recapitulates multiple pathologic phenotypes of these cells.

Proteolytic Activity Present in House-Dust-Mite Extracts Degrades ENA-78/CXCL5 and Reduces Neutrophil Migration.

Background. Bronchial smooth muscle cells (BSMC) are a major source of proinflammatory and proangiogenic cytokines and chemokines, including VEGF and CXC-chemokines. CXC-chemokines act primarily on neutrophils, mediating their recruitment to and activation at the site of inflammation. In humans, house-dust mite (HDM) allergens can cause asthmatic exacerbations and trigger an inflammatory response through protease-dependent mechanisms. Objective. We investigated the effect HDM extract on the release of pro-angiogenic and proinflammatory cytokines from BSMC. Methods. Human primary BSMC were stimulated with HDM extract in the absence or presence of fetal calf serum (FCS). Twenty angiogenic cytokines were detected by a specific antibody array and modified protein levels were confirmed by ELISA. Neutrophil migration was measured using a 96-well Boyden chamber. Results. ENA-78/CXCL5 protein levels in conditioned medium of BSMC stimulated with HDM extract were significantly reduced (n = 10, P < 0.05) but restored in the presence of 5% FCS. HDM extracts did not affect ENA-78/CXCL5 mRNA levels. Recombinant ENA-78/CXCL5 was degraded after incubation with HDM extracts (n = 7, P < 0.05) but restored after the addition of the serine protease AEBSF. Neutrophil migration towards recombinant ENA-78/CXCL5 was also reduced in the presence of HDM extract. Conclusion. HDM proteases degrade ENA-78/CXCL5. Thus exposure to HDM allergens may alter ENA-78/CXCL5 levels in the lungs and may affect angiogenesis and the inflammatory response in the airways of asthma patients.

Tiotropium sustains the anti-inflammatory action of olodaterol via the cyclic AMP pathway.

Mesenchymal cells (fibroblasts) of the airway wall respond to cholinergic stimulation by releasing pro-inflammatory and chemotactic cytokines and may thus contribute to chronic inflammation of the lung. Here, we studied the anti-inflammatory potential of olodaterol, a long acting ß2-adrenergic receptor agonist, and tiotropium, a long-acting muscarinic receptor antagonist, and whether they interact at the level of the cyclic AMP dependent signaling pathway. Pulmonary fibroblasts of asthmatic (n = 9) and non-asthmatic (n = 8) subjects were stimulated with the muscarinic receptor agonist carbachol and interleukin-1ß (IL-1 beta) in presence or absence of tiotropium or olodaterol alone, or their combination. We also measured cAMP levels and phosphorylation of the cAMP response element binding protein (CREB). As single components, carbachol, olodaterol and tiotropium did not affect IL-6 and IL-8 release. Carbachol concentration-dependently enhanced the production of IL-1ß-induced IL-6 and IL-8, which was blocked by the simultaneous addition of tiotropium. The combination of olodaterol plus tiotropium further reduced IL-6 and IL-8 release. Olodaterol induced cAMP and the phosphorylation of CREB, an effect counteracted by carbachol, but rescued by tiotropium. We conclude that olodaterol plus tiotropium cooperate to decrease the inflammatory response in pulmonary fibroblasts in vitro.

Noncanonical WNT-5A signaling regulates TGF-ß-induced extracellular matrix production by airway smooth muscle cells.

Transforming growth factor ß (TGF-ß), a key mediator of fibrotic responses, is increased in asthma and drives airway remodeling by inducing expression of extracellular matrix (ECM) proteins. We investigated the molecular mechanisms underlying TGF-ß-induced ECM expression by airway smooth muscle cells and demonstrate a novel link between TGF-ß and Wingless/integrase 1 (WNT) signaling in ECM deposition. Airway smooth muscle expresses abundant WNT ligands, with the noncanonical WNT-5A being the most profoundly expressed. Interestingly, WNT-5A shows ∼2-fold higher abundance in airway smooth muscle cells isolated from individuals with asthma than individuals without asthma. WNT-5A is markedly induced in response to TGF-ß (4-16-fold; EC50 0.3 ng/ml) and is required for collagen and fibronectin expression by airway smooth muscle. WNT-5A engages noncanonical WNT signaling pathways, as inhibition of Ca(2+) and c-Jun N-terminal kinase (JNK) signaling attenuated this TGF-ß response, whereas the canonical WNT antagonist Dickkopf 1 (DKK-1) did not. Accordingly, WNT-5A induced JNK phosphorylation and nuclear translocation of nuclear factor of activated T cells c1 (NFATc1). Furthermore, silencing of the WNT-5A receptors Frizzled 8 (FZD8) and RYK attenuated TGF-ß-induced ECM expression. Collectively, these findings demonstrate that noncanonical WNT-5A signaling is activated by and necessary for TGF-ß-induced ECM production by airway smooth muscle cells, which could have significance in asthma pathogenesis.

Cell density and serum exposure modify the function of the glucocorticoid receptor C/EBP complex.

The glucocorticoid receptor (GR) is a major control factor for proliferation, differentiation, and inflammation. Our knowledge about the GR is focused on its function as a transcription regulator. However, cells do not always respond to steroids in the same way or develop resistance. The mechanism underlying such a modified steroid response is not well understood, and may depend on the microenvironment of the cells or on the stage of their differentiation. Therefore, we studied the effect of cell density and inflammatory conditions on the expression, compartmentalization, activation, and the anti-proliferative function of the GR in primary human lung fibroblast cultures. In subconfluent cells the GR was located perinuclear, while in confluent cells it was ubiquitously expressed. Serum stimulation up-regulated the level of GR mRNA and protein under all conditions. In subconfluent cells dexamethasone activated the nuclear accumulation and DNA binding of the GR persistently, while in confluent cells its activity declined after 6 hours. In subconfluent cells, but not in confluent cells, the GR interacted with a 42-kD, but not the 30-kD C/EBP-alpha isoprotein, which resulted in an up-regulation of p21((Waf1/Cip1)) expression and suppression of proliferation. In confluent cells, glucocorticoids induced p27((Kip1)) expression via p38 mitogen-activated protein kinase and a 52-kD C/EBP-beta isoprotein. However, p27((Kip1)) did not mediate the antiproliferative effect of glucocorticoids, but simultaneous inhibition of p21((Waf1/Cip1)) and p27((Kip1)) unlocked contact inhibition in confluent cells. Our results indicate that cell density and serum exposure alter the localization and function of the GR.