Comprehensive transcriptomic analysis of prostate cancer lung metastases.

Metastatic prostate cancer (mPCa) is a widespread disease with high mortality. Unraveling molecular mechanisms of disease progression is of utmost importance. The microenvironment in visceral organs and the skeletal system is of particular interest as a harbinger of metastatic spread. Therefore, we performed a comprehensive transcriptomic analysis of prostate cancer lung metastases with a special focus on differentially expressed genes attributable to the microenvironment. Digital gene expression analysis using the NanoString nCounter analysis system was performed on formalin-fixed, paraffin-embedded (FFPE) tissue from prostate cancer (PCa) lung metastases (n = 24). Data were compared to gene expression data from primary PCa and PCa bone metastases. Bioinformatic analysis was performed using several publicly available tools. In comparison to prostate cancer bone metastases, 209 genes were significantly upregulated, and 100 genes were significantly downregulated in prostate cancer lung metastases. Among the up-regulated genes, the top 10 genes with the most significant P-value were HLA-DPB1, PTPRC, ITGB7, C3, CCL21, CCL5, ITGAM, SERPINA1, MFAP4, ARAP2 and among the down-regulated genes, the top 10 genes with the most significant P-value were FOXC2, TWIST1, CDK14, CHAD, IBSP, EPN3, VIT, HAPLN1, SLC44A4, TBX1. In PCa lung metastases genes associated with immunogenic responses were upregulated while genes associated with epithelial-mesenchymal transition were down-regulated. We also showed that CXCR3/CXCL10 axis plays a significant role in prostate cancer lung metastases in comparison to bone metastases. In this study, we comprehensively explored transcriptomic alterations in PCa lung metastases in comparison to primary PCa and PCa bone metastases. In PCa lung metastases genes associated with immunogenic responses are upregulated while genes associated with epithelial-mesenchymal transition are down-regulated. This points to a more immunogenic phenotype of PCa lung metastases thus potentially making patients more susceptible to immunotherapeutic approaches.

CEACAM6 Promotes Lung Metastasis via Enhancing Proliferation, Migration and Suppressing Apoptosis of Prostate Cancer Cells.

BACKGROUND/AIM: Metastatic prostate cancer (mPCa) results in high morbidity and mortality. Visceral metastases in particular are associated with a shortened survival. Our aim was to unravel the molecular mechanisms that underly pulmonary spread in mPCa. MATERIALS AND METHODS: We performed a comprehensive transcriptomic analysis of PCa lung metastases, followed by functional validation of candidate genes. Digital gene expression analysis utilizing the NanoString technology was performed on mRNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue from PCa lung metastases. The gene expression data from primary PCa and PCa lung metastases were compared, and several publicly available bioinformatic analysis tools were used to annotate and validate the data. RESULTS: In PCa lung metastases, 234 genes were considerably up-regulated, and 78 genes were significantly down-regulated when compared to primary PCa. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) was identified as suitable candidate gene for further functional validation. CEACAM6 as a cell adhesion molecule has been implicated in promoting metastatic disease in several solid tumors, such as colorectal or gastric cancer. We showed that siRNA knockdown of CEACAM6 in PC-3 and LNCaP cells resulted in decreased cell viability and migration as well as enhanced apoptosis. Comprehensive transcriptomic analyses identified several genes of interest that might promote metastatic spread to the lung. CONCLUSION: Functional validation revealed that CEACAM6 might play an important role in fostering metastatic spread to the lung of PCa patients via enhancing proliferation, migration and suppressing apoptosis in PC-3 and LNCaP cells. CEACAM6 might pose an attractive therapeutic target to prevent metastatic disease.

The Gene Expression Landscape of Prostate Cancer BM Reveals Close Interaction with the Bone Microenvironment.

Bone metastatic (BM) prostate cancer (PCa) belongs to the most lethal form of PCa, and therapeutic options are limited. Molecular profiling of metastases contributes to the understanding of mechanisms defining the bone metastatic niche. Our aim was to explore the transcriptional profile of PCa BM and to identify genes that drive progression. Paraffin-embedded tissues of 28 primary PCa and 30 BM were submitted to RNA extraction and analyzed by RNA sequencing using the Nanostring nCounter gene expression platform. A total of 770 cancer-related genes were measured using the Nanostring™ PanCancer progression panel. Gene Ontology (GO), KEGG, Reactome, STRING, Metascape, PANTHER, and Pubmed were used for data integration and gene annotation. We identified 116 differentially expressed genes (DEG) in BM compared to primaries. The most significant DEGs include CD36, FOXC2, CHAD, SPP1, MMPs, IBSP, and PTX3, which are more highly expressed in BM, and ACTG2, MYH11, CNN1, FGF2, SPOCK3, and CHRDL1, which have a lower expression. DEGs functionally relate to extracellular matrix (ECM) proteoglycans, ECM-receptors, cell-substrate adhesion, cell motility as well as receptor tyrosine kinase signaling and response to growth factors. Data integration and gene annotation of 116 DEGs were used to build a gene platform which we termed "Manually Annotated and Curated Nanostring-data Platform". In summary, our results highlight the significance of certain genes in PCa BM to which essential pro-metastatic functions could be ascribed. Data from this study provide a comprehensive platform of genes that are related to PCa BM and provide evidence for further investigations.

Genetic deletion of p66shc and/or cyclophilin D results in decreased pulmonary vascular tone.

AIMS: The pulmonary vascular tone and hypoxia-induced alterations of the pulmonary vasculature may be regulated by the mitochondrial membrane permeability transition pore (mPTP) that controls mitochondrial calcium load and apoptosis. We thus investigated, if the mitochondrial proteins p66shc and cyclophilin D (CypD) that regulate mPTP opening affect the pulmonary vascular tone. METHODS AND RESULTS: Mice deficient for p66shc (p66shc-/-), CypD (CypD-/-), or both proteins (p66shc/CypD-/-) exhibited decreased pulmonary vascular resistance (PVR) compared to wild-type mice determined in isolated lungs and in vivo. In contrast, systemic arterial pressure was only lower in CypD-/- mice. As cardiac function and pulmonary vascular remodelling did not differ between genotypes, we determined alterations of vascular contractility in isolated lungs and calcium handling in pulmonary arterial smooth muscle cells (PASMC) as underlying reason for decreased PVR. Potassium chloride (KCl)-induced pulmonary vasoconstriction and KCl-induced cytosolic calcium increase determined by Fura-2 were attenuated in all gene-deficient mice. In contrast, KCl-induced mitochondrial calcium increase determined by the genetically encoded Mito-Car-GECO and calcium retention capacity were increased only in CypD-/- and p66shc/CypD-/- mitochondria indicating that decreased mPTP opening affected KCl-induced intracellular calcium peaks in these cells. All mouse strains showed a similar pulmonary vascular response to chronic hypoxia, while acute hypoxic pulmonary vasoconstriction was decreased in gene-deficient mice indicating that CypD and p66shc regulate vascular contractility but not remodelling. CONCLUSIONS: We conclude that p66shc specifically regulates the pulmonary vascular tone, while CypD also affects systemic pressure. However, only CypD acts via regulation of mPTP opening and mitochondrial calcium regulation.

Treatment of hepatitis C virus infection with direct-acting antiviral agent-based regimens in Iranian patients with hereditary bleeding disorders.

BACKGROUND: Chronic hepatitis C (CHC) is one of the most important comorbidities in patients with hereditary bleeding disorders (HBD). The present study aimed at evaluating the effectiveness of direct-acting antiviral agent (DAA)-based interferon-free HCV antiviral regimens in patients with HBD. PATIENTS AND METHODS: The present study was performed on the patients with HBD and CHC between 2015 and 2019. Sofosbuvir-based interferon-free regimens with or without ribavirin were prescribed to treat HCV infection. The main endpoint of the study was to determine the sustained virologic response (SVR), assessed 12 weeks after the completion of treatment. RESULTS: A total of 147 patients with a mean age of 41.1 years were enrolled in the study; 4.1% of them were co-infected with HIV, 25.2% had cirrhosis, and 76.9% of them were diagnosed with hemophilia A. HCV genotype-1 includes the largest number (68.1%) of patients. 46.3% of patients were treatment-naïve and others had a treatment history with interferon-based regimens. Out of 147 patients, 15 patients were lost to follow-up during treatment or for SVR evaluation or discontinued treatment. 132 subjects completed treatment and were evaluated for SVR, 12 weeks after the completion of treatment. All of the patients achieved SVR 12 (SVR rate: 100%, 95% CI 97.2-100%). CONCLUSION: Hepatitis C DAA-based regimens are the effective treatments for CHC in patients with HBD, regardless of the treatment modifiers such as previous treatment experience, cirrhosis, HIV co-infection, and HCV genotype.

Analysis of tripartite motif (TRIM) family gene expression in prostate cancer bone metastases.

Tripartite motif (TRIM) family proteins are post-translational protein modifiers with E3-ubiquitin ligase activity, thereby involved in various biological processes. The molecular mechanisms driving prostate cancer (PCa) bone metastasis (BM) are incompletely understood, and targetable genetic alterations are lacking in the majority of cases. Therefore, we aimed to explore the expression and potential functional relevance of 71 TRIM members in bone metastatic PCa. We performed transcriptome analysis of all human TRIM family members and 770 cancer-related genes in 29 localized PCa and 30 PCa BM using Nanostring. KEGG, STRING and Ubibrowser were used for further bioinformatic gene correlation and pathway enrichment analyses. Compared to localized tumors, six TRIMs are under-expressed while nine TRIMs are over-expressed in BM. The differentially expressed TRIM proteins are linked to TNF-, TGFß-, PI3K/AKT- and HIF-1-signaling, and to features such as proteoglycans, platelet activation, adhesion and ECM-interaction based on correlation to cancer-related genes. The identification of TRIM-specific E3-ligase-substrates revealed insight into functional connections to oncogenes, tumor suppressors and cancer-related pathways including androgen receptor- and TGFß signaling, cell cycle regulation and splicing. In summary, this is the first study that comprehensively and systematically characterizes the expression of all TRIM members in PCa BM. Our results describe post-translational protein modification as an important regulatory mechanism of oncogenes, tumor suppressors, and pathway molecules in PCa progression. Therefore, this study may provide evidence for novel therapeutic targets, in particular for the treatment or prevention of BM.

Cracking it - successful mRNA extraction for digital gene expression analysis from decalcified, formalin-fixed and paraffin-embedded bone tissue.

With the advance of precision medicine, the availability of tumor tissue for molecular analysis has become a limiting factor. This is particularly the case for bone metastases which are frequently occurring in cancer types such as prostate cancer. Due to the necessary decalcification process it was long thought that transcriptome analysis will not be feasible from decalcified formalin-fixed, paraffin-embedded (DFFPE) in a large manner. Here we demonstrate that mRNA extraction from DFFPE is feasible, quick, robust and reproducible and that decalcification does not hamper subsequent gene expression analysis. This might assist in implementing transcriptome analysis from DFFPE into every day practice.

Tailored prophylaxis in children with severe hemophilia: A four-year Iranian study.

BACKGROUND: Prophylaxis the current standard care for patients with severe hemophilia should be planned to optimize the replacement therapy and minimize bleeding. We report our single-center experience of tailored prophylaxis in children affected by hemophilia A (HA) and hemophilia B (HB). METHODS: This study was conducted on 55 patients, under 15 years, with HA (PWHA, n: 46) and HB (PWHB, n: 9) between 2015 and 2019. According to the phenotype, three prophylaxis regimens: 25-50 unit/kg once, twice, or three-times a week for PWHA, and two: 30-50 unit/kg once or twice a week for PWHB were administered. Following the occurrence of > 3 joint bleeding, or > 4 soft tissue bleeding, or one spontaneous major bleeding in the last 3 months, the prophylaxis regimen is changed. Annualized bleeding rate (ABR), annualized joint bleeding rate (AJBR), target joints development, inhibitor development, and hemophilia joint health score (HJHS) also were assessed. RESULTS: A mean ± SD of 2520 ± 1045 IU/kg/yr coagulation factor (F) VIII was used to reduce ABR and AJBR from 1.02 ± 1.11 and 0.8 ± 1.3 (in the first year of the study) to 0.27 ± 0.44 (P < 0.001) and 0.19 ± 0.38 (P = 0.004) (at the end of the study) in PWHA, respectively. Furthermore, in PWHB, in the first year of the study, with using 2168 ± 1216 IU/kg coagulation FIX, ABR and AJBR were 0.19 ± 0.39 and 0.06 ± 0.1. At the end of the study, ABR and AJBR were 0.02 ± 0.05 (p = 0.156) and 0.01 ± 0.03 (p = 0.361), respectively. During the study period, the mean number of the target joints and mean HJHS were 0.25 ± 0.57 and 7.6 ± 2.1 for PWHA and 0 and 6.3 ± 1.8 for PWHB, respectively. Finally, 5 PWHA (11 %) did not need dose-escalation in their prophylaxis regimen, whereas 31 (67 %) and 10 (21 %) PWHA needed two and three infusions a week, respectively. In PWHB, 7 (78 %) and 2 (22 %) were adjusted to receive a once and twice weekly regimen, respectively. CONCLUSION: Our results suggest that tailored prophylaxis is an effective strategy to reduce the rate of bleeding and optimize the replacement therapy in children with hemophilia.

Parvovirus 4 in Individuals with Severe Hemophilia A and Matched Control Group.

Background: Hemophilia is a well-known bleeding disorder with worldwide distribution. Replacement therapy, using plasma-derived or recombinant coagulation factors, comprises a gold standard regimen for the treatment. Regardless of the advancements made in viral inactivation methods in the production of plasma-derived coagulation factors, the possibility of transmission of new viral infections remained as a noticeable concern yet. The aim of the current study was to investigate the status of parvovirus 4 (PARV4) in severe hemophilia A, von Willebrand disease (vWD), and healthy control. Materials and Methods: In the current case-control study, 76 patients with hemophilia and vWD and 60 individuals from their family members entered the study. Nested PCR used to determine the presence of PARV4 in study subjects (76 cases). To characterize the PARV4 genotype, positive samples subjected to sequencing and phylogenetic analysis. Results: PARV4 genome detected in 11 (14.47%) patients with bleeding disorders. Among whom, nine patients (14.75%) were with severe hemophilia A and two (13.33%) patients with vWD. Only five healthy controls (8.33%) were positive for PARV4. All PARV4 sequences were found to be genotype 1. Conclusion: PARV4 infection in patients with hemophilia and vWD was higher than the control group. While detection of PARV4 DNA in patients with bleeding disorders may not necessarily reflect a clinical urgency, future investigations are needed to define the clinical significance of PARV4. It seems the detection of the virus immune signature of PARV4 infection, particularly in the context of acute and persistent infections, needs to focus on cellular and tissue targets.

Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing.

Mitochondria play an important role in sensing both acute and chronic hypoxia in the pulmonary vasculature, but their primary oxygen-sensing mechanism and contribution to stabilization of the hypoxia-inducible factor (HIF) remains elusive. Alteration of the mitochondrial electron flux and increased superoxide release from complex III has been proposed as an essential trigger for hypoxic pulmonary vasoconstriction (HPV). We used mice expressing a tunicate alternative oxidase, AOX, which maintains electron flux when respiratory complexes III and/or IV are inhibited. Respiratory restoration by AOX prevented acute HPV and hypoxic responses of pulmonary arterial smooth muscle cells (PASMC), acute hypoxia-induced redox changes of NADH and cytochrome c, and superoxide production. In contrast, AOX did not affect the development of chronic hypoxia-induced pulmonary hypertension and HIF-1α stabilization. These results indicate that distal inhibition of the mitochondrial electron transport chain in PASMC is an essential initial step for acute but not chronic oxygen sensing.

First Report of Prevalence of Blood-Borne Viruses (HBV, HCV, HIV, HTLV-1 and Parvovirus B19) Among Hemophilia Patients in Afghanistan.

Blood-borne viruses including Hepatitis B and C, HIV, HTLV-1 and parvovirus B19 are still a factor of concern, especially for hemophilia patients. Although the safety of the blood supply continues to improve worldwide, the blood supply system in Afghanistan was damaged by many years of conflict and political instability. To date, there are few studies focused on the prevalence of blood-borne viruses in hemophilia patients. This study is first to investigate the prevalence of five blood-borne viruses in Afghanistan hemophilia patients in four cities including Kabul, Herat, Mazar-i-Sharif and Jalal Abad. A total of 80 hemophilia male patients were screening for the presence of five transfusion-transmitted viruses using ELISA and PCR. Data obtained showed 2.5% seropositivity for HBV, 8.75% seropositivity for HCV, and 91.25% seropositivity for parvovirus B19. None of the patients were positive for HIV and HTLV-1 and the prevalence of HCV was higher in older patients rather than younger patients. This finding, the first to report in Afghanistan, shows a high prevalence of parvovirus B19 in Afghanistan hemophilia patients and implementation of highly sensitive screening is necessary.

Inflammatory and immune response genes: A genetic analysis of inhibitor development in Iranian hemophilia A patients.

A major problem of hemophilia A (HA) treatment is the development of factor VIII (FVIII) inhibitor, which usually occurs shortly after initiating replacement therapy. Several studies showed the correlation between inhibitor development and polymorphisms in inflammatory and immune response genes of HA patients; however, literature data are not available to prove this association in Iranian population. The aim of this study was to investigate a possible association between FVIII inhibitor formation and the polymorphisms of 16 inflammatory and immune response genes in Iranian severe HA patients (FVIII activity < 1%). This case-control study was performed on 55 patients with severe HA inhibitors and 45 samples without inhibitors from Iranian Comprehensive Hemophilia Care center. After extraction of whole genomic DNA from blood samples and design of primers for 16 genes, the genotyping was performed by Tetra primer ARMS PCR, and the validation of single nucleotide polymorphisms was determined by DNA sequencing. The data indicated that there was a significant association between inhibitor development, and F13A1 (TT), DOCK2 (CC& CT), and MAPK9 (TT) genotypes. Moreover, a considerably increased inhibitor risk carrying T, C, and T allele for F13A1, DOCK2, and MAPK9 genes was observed in patients with inhibitors, respectively. In contrast, there was no statistically significant difference between the genotypic and allelic frequencies for other genes in patients with inhibitors compared to patients without inhibitors. These results demonstrate that only polymorphisms in F13A1, DOCK2, and MAPK9 genes are associated with the risk of developing FVIII inhibitors in Iranian HA patients.

First detection of human hepegivirus-1 (HHpgV-1) in Iranian patients with hemophilia.

A novel blood-borne virus called the human hepegivirus 1 (HHpgV-1) was recently discovered in hemophilia patients. The present study aimed to investigate the presence of HHpgV-1 in hemophilia patients. A total of 436 serum samples were investigated for the presence of hepatitis C virus (HCV), human pegivirus-1 (HPgV-1), torque teno virus (TTV), and HHpgV-1. Out of the 436 patients, 163 (37.4%), 19 (4.4%), 76 (17.4%), and four (0.9%) patients were positive for HCV, HPgV-1, TTV, and HHpgV-1, respectively. HHpgV-1 patients had a mean viral load of 4.9 ± 0.3 log RNA copies/mL and were co-infected with HCV-1a, HPgV-1, and TTV. Moreover, three HHpgV-1-positive patients exhibited stage F0 liver fibrosis. HCV viral load in HHpgV-1-positive patients was lower than those of HHpgV-1-negative patients. Results also revealed that co-infection of HHpgV-1 with HPgV-1 and HCV may play a protective role in patients with chronic HCV. In conclusion, we detected a low frequency of HHpgV-1 infection in hemophilia patients, and results suggested that HHpgV-1 infection was correlated with the presence of other blood-borne viruses and is likely to also correlate with low HCV viral load and reduced severity of liver disease. Additional studies are required to further investigate the clinical importance of HHpgV-1.

Tagging staphylococcal enterotoxin B (SEB) with TGFaL3 for breast cancer therapy.

Recent research has attempted to direct superantigens towards tumors by means of tumor-targeted superantigen (TTS) strategy. In this study, we explored the antitumor property of TTS by fusing the third loop of transforming growth factor α (TGFαL3) to staphylococcal enterotoxin type B (SEB) and investigated the possibility of the therapeutic application of TGFαL3-SEB as a novel antitumor candidate in mice bearing breast cancer. Treatment was performed through intratumoral and intravenous injection of TGFαL3-SEB. Tumor size/volume, long-term survival, and cytokine secretion were assessed. In addition, the toxicity of each treatment on liver and kidneys was examined. Our results indicated that the relative tumor volume significantly increased in the mice receiving intratumoral TGFaL3-SEB (p < 0.05). Surprisingly, 5 out of the 14 mice were cleared from the tumor thoroughly in 10-25 days after intratumoral administration of TGFaL3-SEB. Quantification of cytokines clearly showed that the mice receiving intratumoral SEB significantly secreted higher interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) compared with the other groups (p < 0.05). The antitumor effect was followed by inhibition of cell proliferation (Ki-67) and micro vascularization (CD31). The highest and lowest levels of tumor necrosis were observed in the intratumoral administration of TGFαL3-SEB (85 %) and PBS (14 %), respectively. Intratumoral injection of TGFαL3-SEB increased the lifespan of the mice so 37.5 % of them could survive for more than 6 months (p < 0.05). Overall, our findings indicated that intratumoral administration of TGFαL3-SEB effectively inhibited the growth of breast tumors through induction of necrosis and suppressing proliferation and angiogenesis without systemic toxicity.

Nanosized tamoxifen-porphyrin-glucose [TPG] conjugate: novel selective anti-breast-cancer agent, synthesis and in vitro evaluations.

Tumor and especially breast cancer is among the most common causes of death worldwide. Finding novel nanosized therapeutic compounds have important role to decrease the chance of death and increase the survival. Cancer cells are highly attractive to glucose [with a nanosize bimolecular structure 1nm] as an energy source more than normal cell and nanosized therapeutics due to possessing different pharmacokinetic and pharmacodynamic have advantageous over classical dosage forms in cancer therapy. The aim of the study was to synthesize Glucosamin-Porphyrin-Tamoxifen [TPG] nanosized complex as a novel selective biocompatible anti breast cancer agent. After the synthesis procedure, this complex was purified and then tested In Vitro on breast cancer cells [MCF-7] in the absence or presence of the red light and found totally successful. The results showed a good anti breast cancer activity mediated by the activation of TNF-α and necrosis/apoptosis pathways for the nanosized complex with no alteration effects on blood PT/APTT and glucose or hexokinase levels/ activity. TPG nanoconjugate seems to be very good opponents to current anti breast cancer drugs and needs to be further investigated in near future.

Detection of hepatitis B virus variants in HBV monoinfected and HBV/HIV coinfected Iranian patients under lamivudine treatment.

Chronic hepatitis B affects nearly 10% of HIV-infected patients. Hepatitis B virus (HBV) infection is a dynamic disease and coinfection with HIV impacts directly on the outcome of HBV infection, considerably complicating its natural history, diagnosis, and management. The aim of this study was to compare two cohorts of HBV monoinfected and HBV/HIV coinfected Iranian patients undergoing long-term lamivudine therapy from the clinical and virological aspects, as well as the frequency of detected mutations in HBV genome. To this end, HBV Pol/S regions from 72 patients were PCR-amplified and directly sequenced. Phylogenetic analysis indicated a 40-times higher risk of coinfection with ayw3 subtype of HBV genotype D rather than ayw2 subtype [P<0.001, odd: 40.66, CI: 95 % (4.69-352.23)]. While no resistance mutation was detected in HBV/HIV coinfected cohort, LAM-resistance mutations (rtM204I/V in YMDD and rtL180M in FLLA polymerase motifs) were identified in 30% (9 out of 30) and 16.66% (5 out of 30) of HBV monoinfected patients (P<0.05). Moreover, several mutations (sP105A, sI110S/L, sS136Y and sP127T/L) with significant differences in the frequency were identified in the S region of both cohorts. Finally, this study found strong correlation between the type of infection (mono or coinfection) and characteristics like patient gender, ALT levels, HBV-DNA levels and HBV subtypes. These results pointed to the importance of determination of HBV variants in the management of patients and suggested that in contrary to HBV monoinfections, LAM may be still an appropriate drug for the treatment of HBV in HBV/HIV coinfected patients; however, further studies to clarify the role of HIV in HBV LAM-resistance mutations are required.