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The aim of the present study was to investigate the concentration- and size-dependent effects of non-functionalized polystyrene nanoparticles (PS-NPs) of varying diameters (29 nm, 44 nm, and 72 nm) on specific epigenetic modifications and gene expression profiles related to carcinogenesis in human peripheral blood mononuclear cells (PBMCs) in vitro. This in vitro human-cell-based model is used to investigate the epigenetic effect of various environmental xenobiotics. PBMCs were exposed to PS-NPs at concentrations ranging from 0.001 to 100 µg/mL for 24 h period. The analysis encompassed epigenetic DNA modifications, including levels of 5-methyl-2′-deoxycytidine (5-mdC) and 5-(hydroxymethyl)-2′-deoxycytidine (5-hmdC), as well as the levels of 2′-deoxyuridine (dU) and 5-(hydroxymethyl)-2′-deoxyuridine (5-hmdU) by mass spectrometry methods, methylation in the promoter regions of selected tumor suppressor genes TP53 (P53), CDKN2A (P16), and CDKN1A (P21) and proto-oncogenes (CCND1, BCL2, BCL6), along with the expression profile of the indicated genes by real-time PCR assays. The results obtained revealed no significant changes in global DNA methylation/demethylation levels in PBMCs after short-term exposure to non-functionalized PS-NPs. Furthermore, there were no changes observed in the level of dU, a product of cytosine deamination. However, the level of 5-hmdU, a product of both 5-hmdC deamination and thymine oxidation, was increased at the highest concentrations of larger PS-NPs (72 nm). None of the PS-NPs caused a change in the methylation pattern of the promoter regions of the TP53, CDKN2A, CDKN1A, CCND1, BCL2 and BCL6 genes. However, gene profiling indicated that PS-NPs with a diameter of 29 nm and 44 nm altered the expression of the TP53 gene. The smallest PS-NPs with a diameter of 29 nm increased the expression of the TP53 gene at a concentration of 10 µg/mL, while PS-NPs with a diameter of 44 nm did so at a concentration of 100 µg/mL. An increase in the expression of the CDKN2A gene was also observed when PBMCs were exposed to PS-NPs with 29 nm in diameter at the highest concentration. The observed effect depended on both the concentration and the size of the PS-NPs. [ABSTRACT FROM AUTHOR]

INTRODUCTION. Currently, the choice of tactics in the treatment of esophageal cancer remains an urgent problem. Important factors are: the stage of the disease, the functional state of the patient, as well as the morphological type of tumor. An integrated approach is today the standard for this pathology. However, after radical treatment, a significant number of complications are observed, in particular, the development of leakage of esophagogastroanastomosis. Today, according to world literature, it is generally accepted that the development of anastomotic leakage is associated with the detection of the disease in late stages, the consequences of radiation during combined chemoradiotherapy, and extensive surgical intervention for esophageal cancer.The OBJECTIVE was to improve the results of treatment of patients with malignant neoplasms of the esophagus and esophagogastric junction, through the use of an integrated approach.METHODS AND MATERIALS. During the period of 1988–2022, treated 2784 patients with cancer of the esophagus and esophagogastric junction were treated at the Republican Clinical Oncology Dispensary named after Prof. M. Z. Sigal. The study group included 1,799 patients who received surgical treatment, combined chemoradiotherapy in combination with surgery, and external beam radiation therapy in combination with surgery.RESULTS. Due to changes in treatment tactics for patients with cancer of the esophagus and esophagogastric junction, postoperative mortality after surgical complications decreased from 24.3 % to 6.2 %, and anastomotic leakage in the postoperative period decreased from 27.0 % to 8.1 %.CONCLUSION. Changes in the treatment of patients with cancer of the esophagus and esophagogastric junction made it possible to reduce the development of postoperative complications and increase life expectancy. The molecular genetic approach allows for effective analysis of screening, early diagnosis, treatment and rehabilitation.

Background/Aim: Meningiomas represent the main intracranial primary central nervous system (CNS) tumour in adults worldwide. Oncogenes' over-activation combined with suppressor genes' silencing affect negatively the biological behavior of these neoplasms. This study aimed to explore the impact of p53 suppressor gene expression in meningiomas' clinic-pathological features based on a combination of sophisticated techniques. Materials and Methods: Fifty (n=50) meningiomas were included in the study, comprising a broad spectrum of histopathological subtypes. An immunohistochemistry assay was applied on tissue microarray cores followed by digital image analysis. Results: p53 protein over-expression (high staining intensity levels) was observed in 27/50 (54%) cases, whereas the rest (23/50-/46%) demonstrated moderate to low levels of the protein. p53 over-expression was statistically significantly correlated to the mitotic index of the examined cases (p-value=0.001). Interestingly, the atypical/anaplastic group of histotypes demonstrated the strongest p53 expression rates compared to the others (p-value=0.001). Conclusion: p53 overexpression is observed in a broad spectrum of meningiomas. High expression levels lead to an aggressive biological behavior of the malignancy (combined with increased mitotic rates), especially in atypical and anaplastic sub-types that also have a high recurrence rate. [ABSTRACT FROM AUTHOR]

Purpose: Colorectal cancer (CRC) is the fourth-most common cancer worldwide and the second most common cancer cause of death in the world. The components of the TGFβ-signalling pathway, which are often affected by miRNAs, are involved in the regulation of apoptosis and cell cycle. Therefore, in the current study, the expression of BMP2 gene in CRC tissues at different clinical stages compared to the non-tumour tissues has been assessed. Moreover, the plasma BMP2 protein concentration in the same group of CRC patients has been validated. Due to the constant necessity to conduct further research of the correlation between specific miRNAs and mRNAs in CRC, in silico analysis has been performed to select miRNAs that regulate BMP2 mRNA. Methods: The cDNA samples from tumor and non-tumor tissue were used in a qPCR reaction to determine the mRNA expression of the BMP2 gene and the expression of selected miRNAs. The concentration of BMP2 protein in plasma samples was also measured. Results: It was indicated that BMP2 was downregulated in CRC tissue. Moreover, miR-370 and miR-138 expression showed an upward trend. Decreased BMP2 with accompanied increasing miR-370 and miR-138 expression was relevant to the malignant clinicopathological features of CRC and consequently poor patient prognosis. Conclusion: Our data suggest that miR-370 with its clear expression in plasma samples may be a potential diagnostic marker to determine the severity of the disease in patients at a later stage of colorectal cancer. [ABSTRACT FROM AUTHOR]

Suppressor gene research is one of the main current trends in modern cancer research. Oncogenesis, including colorectal cancer (CRC), is a process strictly dependent on the proper functioning of the suppressor genes and their proteins. The molecular genetic studies of hereditary cases and sporadic forms of cancer conducted for several years have revealed that a significant feature of the occurrence and progression of neoplastic changes is silencing of the expression of tissue-specific suppressor genes. Restoring the proper activity of the respective suppressor proteins plays a key role in anti-cancer prevention and gives hope for the development of effective personalised therapy. This review presents the latest reports on the molecular mechanisms regulating the expression, activity, and function of suppressor genes, which were silenced in tissues and cell lines of colorectal cancer, and restoration of their correct expression inhibited the proliferation and invasion of CRC cells.

Colorectal cancer (CRC) is one of the most prevalent causes of cancer mortality worldwide. Saudi patients are more likely to present CRC at advanced disease stages and at younger ages compared to other populations. The present study evaluated the anti-cancer effects of venoms extracted from indigenous snakes on the growth arrest of colon cancer cells. Walterinnesia aegyptia venom showed a significant cell death proportions against LoVo cells only, whereas Echis coloratus venom-induced apoptosis in LoVo, HTC-116, and HT-29 colon cancer cells. Also, it suppressed the levels of cyclin D1 and survivin, overexpressed p53 and p21 in HCT-116, HT-29, and LoVo cells. Our results provide evidence that snake venoms induce the growth arrest of colon cancer cells via cell cycle arrest, which promotes the direction toward further investigating the innovative therapeutic strategy using biologically synthesized snake venoms. [ABSTRACT FROM AUTHOR]

Suppressor gene research is one of the main current trends in modern cancer research. Oncogenesis, including colorectal cancer (CRC), is a process strictly dependent on the proper functioning of the suppressor genes and their proteins. The molecular genetic studies of hereditary cases and sporadic forms of cancer conducted for several years have revealed that a significant feature of the occurrence and progression of neoplastic changes is silencing of the expression of tissue-specific suppressor genes. Restoring the proper activity of the respective suppressor proteins plays a key role in anti-cancer prevention and gives hope for the development of effective personalised therapy. This review presents the latest reports on the molecular mechanisms regulating the expression, activity, and function of suppressor genes, which were silenced in tissues and cell lines of colorectal cancer, and restoration of their correct expression inhibited the proliferation and invasion of CRC cells. [ABSTRACT FROM AUTHOR]

BackgroundEuploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost.MethodsFluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids.ResultsIn both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region.ConclusionThe inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors.

Our research group has showed that the LIM homeobox transcription factor 1 alpha (LMX1A) is inactivated in gastric cancers. Overexpression of LMX1A inhibits tumor growth. However, the mechanisms remains unclear. Considering LMX1A as a transcription factor, a comparison of RNA-seq between gastric cancer cells (GCCs) and GCCs with LMX1A overexpressed was performed to identify genes transcriptionally activated by LMX1A. Among the potential LMX1A target genes, angiopoietin-like 4 (ANGPTL4) has been reported to be an important tumor suppressor and thus was selected for further validation and research. Both LMX1A and ANGPTL4 showed downregulated expression in gastric cancer samples. More importantly, the expression of LMX1A is positively correlated with ANGPTL4, without including other family members in gastric cancer cell lines. What’s more, knockdown of ANGPTL4 rescued the tumor suppressive phenotype of LMX1A overexpression, which indicated that LMX1A upregulates ANGPTL4 to exert its role. Mechanistically, we found that LMX1A inhibited the expression of the oncogene C-Myc, which is alleviated by ANGPTL4 knockdown. In general, our results showed that LMX1A exerts its tumor suppressive role by activating ANGPTL4 to inhibit C-Myc. [ABSTRACT FROM AUTHOR]

The p53 protein is one of the most important suppressors of neoplastic transformation. It regulates transcription of multiple genes and interacts directly with other proteins. It plays a significant role in the most important processes that take place in the cell, including: DNA repair, cell cycle and programmed cell death -211; apoptosis. Loss of its proper function leads to a disturbance of the mechanisms controlling cell proliferation and survival, which contributes to the development of neoplasms. The TP53 gene is called the guardian of the genome. Its mutations occur in a large percentage of tumors. They most often concern sequences that encode the DNA-binding domain (exons 5-211;8). The TP53 gene, together with the TP63 and TP73 genes, belongs to the oldest evolutionary family of cancer transformation suppressors. Its product, a full length p53 protein, consists of five domains and a flexible consolidator region and functions as a homotetramer. The regulation of p53 activity is caused by MDM2 protein, which contributes to proteasomal degradation of the suppressor. This review deals with the most important aspects of the regulation of cell activity by p53 protein. It describes the structure of p53 protein and the associated therapeutic possibilities. [ABSTRACT FROM AUTHOR]

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I. [ABSTRACT FROM AUTHOR]

Drosophila provides an inexpensive and quantitative platform for measuring whole animal drug response. A complementary approach is virtual screening, where chemical libraries can be efficiently screened against protein target(s). Here, we present a unique discovery platform integrating structure-based modeling with Drosophila biology and organic synthesis. We demonstrate this platform by developing chemicals targeting a Drosophila model of Medullary Thyroid Cancer (MTC) characterized by a transformation network activated by oncogenic dRetM955T. Structural models for kinases relevant to MTC were generated for virtual screening to identify unique preliminary hits that suppressed dRetM955T-induced transformation. We then combined features from our hits with those of known inhibitors to create a ‘hybrid’ molecule with improved suppression of dRetM955T transformation. Our platform provides a framework to efficiently explore novel kinase inhibitors outside of explored inhibitor chemical space that are effective in inhibiting cancer networks while minimizing whole body toxicity. [ABSTRACT FROM AUTHOR]

Cervical cancer is the second most deadly gynecological tumor worldwide. MicroRNAs (miRNAs) play very important roles in tumor oncogenesis and progression. The mechanism of post-transcription regulation of WTX gene is still unknown. A series of differential miRNAs were discovered by microarray analysis comparing three pairs of primary cervical cancer specimens and their relapsed tumors from three patients. Quantitative reverse transcriptase PCR (qRT-PCR), Western Blot (WB) and Immunohistochemistry (IHC) was used to detect the expression of miR-4524b-5p and WTX in cervical cell lines and tissues. The biological function of miR-4524b-5p and WTX was investigated through knockdown and overexpression with inhibitor/siRNA and mimic/plasmid in vitro and in vivo. In this study, we found that miR-4524b-5p is highly expressed in relapsed cervical cancer specimens. Combined in vitro and in vivo experiments, showed that miR-4524b-5p could regulate the migration and invasion ability of cervical cancer. Furthermore, we also found that miR-4524b-5p could regulate the migration and invasion of cervical cancer by targeting WTX and that WTX could regulate the expression of β-catenin. Taken together, our data identified a miR-4524b-5p/WTX/β-catenin regulatory axis for cervical cancer, and miR-4524b-5p may be a potential target for cervical cancer therapy. [ABSTRACT FROM AUTHOR]

Background: There have been few available prognostic biomarkers in gastric cancer. We rigorously assessed the clinical relevance of promoter DNA methylation of Cysteine dioxygenase type 1 (CDO1) gene, a cancer-specific aberration, in human gastric cancer. Methods: Quantitative CDO1 methylation value (TaqMeth V) was initially calculated in 138 gastric cancer patients operated in 2005, and its clinical significance was elucidated. As a subsequent expanded set, 154 gastric cancer patients with pathological stage (pStage) II / III with no postoperative therapy were validated between 2000 and 2010. Results: (1) Median TaqMeth V of CDO1 gene methylation of gastric cancer was 25.6, ranging from 0 to 120.9. As pStage progressed, CDO1 TaqMeth V became higher (p < 0.0001). (2) The optimal cut-off value was determined to be 32.6; gastric cancer patients with high CDO1 gene methylation showed a significantly worse prognosis than those with low CDO1 gene methylation (p < 0.0001). (3) A multivariate cox proportional hazards model identified high CDO1 gene methylation (p = 0.033) as an independent prognostic factor. (4) The results were recapitulated in the expanded set in pStage III, where high CDO1 gene methylation group had a significantly worse prognosis than low CDO1 gene methylation group (p = 0.0065). Hematogenous metastasis was unique in pStage III with high CDO1 gene methylation (p = 0.0075). (5) Anchorage independent growth was reduced in several gastric cancer cell lines due to forced expression of the CDO1 gene, suggesting that abnormal CDO1 gene expression may represent distant metastatic ability. Conclusions: Promoter DNA hypermethylation of CDO1 gene was rigorously validated as an important prognostic biomarker in primary gastric cancer with specific stage. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). Previous studies have identified recurrent nonsense mutations in the HBV large S (LHBs) gene from the liver from HBV core antigen-positive HCC patients. These nonsense mutants have been shown to be oncogenic in mouse xenograft models using a mouse embryonic fibroblast cell line. Here, we expressed in a liver cell line Huh-7 a carboxy terminally truncated protein from a nonsense mutant of the LHBs gene, sW182* (stop codon at tryptophane-182). Although the sW182* protein appeared not to be very stable in the cultured liver cells, we confirmed that the protein can be highly expressed and retained for a prolonged period of time in the hepatocytes in the mouse liver, indicating its stable nature in the physiological condition. In the Huh-7 cells, the sW182* mutant downregulated tumor suppressors p53 and Smad4. This downregulation was reversed by a proteasome inhibitor MG132, implying the involvement of proteasome-based protein degradation in the observed regulation of the tumor suppressors. On the other hand, we found that c-Jun activation domain-binding protein 1 (Jab1) physically interacts with the sW182*, but not wild-type LHBs. RNA interference (RNAi) of Jab1 restored the levels of the downregulated p53 and Smad4. The sW182* mutant inhibited the promoter activity of downstream target genes of the tumor suppressors. Consistently, Jab1 RNAi reversed the inhibition. These results suggest that the LHBs nonsense mutant antagonizes the tumor suppressor pathways through Jab1 in the liver contributing to HCC development. [ABSTRACT FROM AUTHOR]

p53 regulates the expression of hundreds of genes. Recent surprising observations indicate that no single protein-coding gene controls the tumor suppressor effects of p53. This raises the possibility that a subset of these genes, regulated by a p53-induced long noncoding RNA (lncRNA), could control p53’s tumor suppressor function. We propose molecular mechanisms through which lncRNAs could regulate this subset of genes and hypothesize an exciting, direct role of lncRNAs in p53’s genome stability maintenance function. Exploring these mechanisms could reveal lncRNAs as indispensable mediators of p53 and lay the foundation for understanding how other transcription factors could act via lncRNAs. [ABSTRACT FROM AUTHOR]

BAP1 is a tumor suppressor gene important to the development and prognosis of many cancers, especially uveal melanoma (UM). Its role in more common cancers such as breast and colon cancer is largely unknown. We collected the transcriptome profiling data sets from the TCGA uveal melanoma (TCGA-UVM), breast cancer (TCGA-BRCA), and colon cancer (TCGA-COAD) projects to analyze the expression of BAP1. We found that patients with UM and breast cancer, but not colon cancer, who died had a lower level of BAP1 gene expression compared to surviving patients. Importantly, in breast cancer patients, the lowest BAP1 expression levels corresponded to the dead young patients (age at diagnosis < 46). Since the number of cases in TCGA-BRCA was much higher than TCGA-UVM, we obtained highly correlated genes with BAP1 in invasive breast carcinomas. Then, we tested if these genes are also highly correlated with BAP1 in UM and colon cancer. We found that BAP1 is highly positively correlated with RBM15B and USP19 expression in invasive breast carcinoma, UM, and colon adenocarcinoma. All three genes are located in close proximity on the 3p21 tumor suppressor region that is commonly altered in many cancers. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are non-hematopoietic progenitor cells, which can be isolated from different types of tissues including bone marrow, adipose tissue, tooth pulp, and placenta/umbilical cord blood. There isolation from adult tissues circumvents the ethical concerns of working with embryonic or fetal stem cells, whilst still providing cells capable of differentiating into various cell lineages, such as adipocytes, osteocytes and chondrocytes. An important feature of MSCs is the low immunogenicity due to the lack of co-stimulatory molecules expression, meaning there is no need for immunosuppression during allogenic transplantation. The tropism of MSCs to damaged tissues and tumor sites makes them a promising vector for therapeutic agent delivery to tumors and metastatic niches. MSCs can be genetically modified by virus vectors to encode tumor suppressor genes, immunomodulating cytokines and their combinations, other therapeutic approaches include MSCs priming/loading with chemotherapeutic drugs or nanoparticles. MSCs derived membrane microvesicles (MVs), which play an important role in intercellular communication, are also considered as a new therapeutic agent and drug delivery vector. Recruited by the tumor, MSCs can exhibit both pro- and anti-oncogenic properties. In this regard, for the development of new methods for cancer therapy using MSCs, a deeper understanding of the molecular and cellular interactions between MSCs and the tumor microenvironment is necessary. In this review, we discuss MSC and tumor interaction mechanisms and review the new therapeutic strategies using MSCs and MSCs derived MVs for cancer treatment.

Meiotic drivers are selfish alleles that subvert gametogenesis to increase their transmission into progeny. Drivers impose a fitness cost, putting pressure on the genome to evolve suppressors. Here we investigate the wtf gene family from Schizosaccharomyces pombe, previously shown to contain meiotic drivers in wild isolates. We discovered that wtf13 found in lab stocks is a meiotic driver. wtf13 kills spores that do not inherit it by generating both a diffusible poison and a spore-specific antidote. Additionally, we demonstrate that wtf13 is suppressed by another wtf gene, wtf18-2, that arose spontaneously in the lab and makes only an antidote. Wtf18-2 does not act indiscriminately to prevent spore destruction. Instead, it rescues only the spores that inherit wtf18-2. In this way, wtf18-2 selfishly gains a transmission advantage of its own while dampening the drive of wtf13. This establishes a novel paradigm for meiotic drive suppressors and provides insight into the mechanisms and evolution of drive systems. [ABSTRACT FROM AUTHOR]

Background: Large cancer genome studies continue to reveal new players in treatment response and tumorigenesis. The discrimination of functional alterations from the abundance of passenger genetic alterations still poses challenges and determines DNA sequence variant selection procedures. Here we evaluate variant selection strategies that select homozygous variants and rare SNPs and assess its value in detecting tumor cells with DNA repair defects. Methods: To this end we employed a panel of 29 patient-derived head and neck squamous cell carcinoma (HNSCC) cell lines, of which a subset harbors DNA repair defects. Mitomycin C (MMC) sensitivity was used as functional endpoint of DNA crosslink repair deficiency. 556 genes including the Fanconi anemia (FA) and homologous recombination (HR) genes, whose products strongly determine MMC response, were capture-sequenced. Results: We show a strong association between MMC sensitivity, thus loss of DNA repair function, and the presence of homozygous and rare SNPs in the relevant FA/HR genes. Excluding such selection criteria impedes the discrimination of crosslink repair status by mutation analysis. Applied to all KEGG pathways, we find that the association with MMC sensitivity is strongest in the KEGG FA pathway, therefore also demonstrating the value of such selection strategies for exploratory analyses. Variant analyses in 56 clinical samples demonstrate that homozygous variants occur more frequently in tumor suppressor genes than oncogenes further supporting the role of a homozygosity criterion to improve gene function association or tumor suppressor gene identification studies. Conclusion: Together our data show that the detection of relevant genes or of repair pathway defected tumor cells can be improved by the consideration of allele zygosity and SNP allele frequencies. [ABSTRACT FROM AUTHOR]

Two-component signaling systems (TCS) regulate bacterial responses to environmental signals through the process of protein phosphorylation. Specifically, sensor histidine kinases (SK) recognize signals and propagate the response via phosphorylation of a cognate response regulator (RR) that functions to initiate transcription of specific genes. Signaling within a single TCS is remarkably specific and cross-talk between TCS is limited. However, regulation of the flow of information through complex signaling networks that include closely related TCS remains largely unknown. Additionally, many bacteria utilize multi-component signaling networks which provide additional genetic and biochemical interactions that must be regulated for signaling fidelity, input and output specificity, and phosphorylation kinetics. Here we describe the characterization of an NtrC-like RR that participates in regulation of Type-IV pilus-dependent motility of Myxococcus xanthus and is thus named NmpR, trC odulator of ili egulator. A complex multi-component signaling system including NmpR was revealed by suppressor mutations that restored motility to cells lacking PilR, an evolutionarily conserved RR required for expression of pilA encoding the major Type-IV pilus monomer found in many bacterial species. The system contains at least four signaling proteins: a SK with a protoglobin sensor domain (NmpU), a hybrid SK (NmpS), a phospho-sink protein (NmpT), and an NtrC-like RR (NmpR). We demonstrate that ΔpilR bypass suppressor mutations affect regulation of the NmpRSTU multi-component system, such that NmpR activation is capable of restoring expression of pilA in the absence of PilR. Our findings indicate that pilus gene expression in M. xanthus is regulated by an extended network of TCS which interact to refine control of pilus function. [ABSTRACT FROM AUTHOR]

Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, Ras mutations alone are insufficient for tumorigenesis, therefore it is paramount to identify cooperating cancer-relevant signaling pathways. We devised an in vivo near genome-wide, functional screen in Drosophila and discovered multiple novel, evolutionarily-conserved pathways controlling Ras-driven epithelial tumorigenesis. Human gene orthologs of the fly hits were significantly downregulated in thousands of primary tumors, revealing novel prognostic markers for human epithelial tumors. Of the top 100 candidate tumor suppressor genes, 80 were validated in secondary Drosophila assays, identifying many known cancer genes and multiple novel candidate genes that cooperate with Ras-driven tumorigenesis. Low expression of the confirmed hits significantly correlated with the KRASG12 mutation status and poor prognosis in pancreatic cancer. Among the novel top 80 candidate cancer genes, we mechanistically characterized the function of the top hit, the Tetraspanin family member Tsp29Fb, revealing that Tsp29Fb regulates EGFR signaling, epithelial architecture and restrains tumor growth and invasion. Our functional Drosophila screen uncovers multiple novel and evolutionarily conserved epithelial cancer genes, and experimentally confirmed Tsp29Fb as a key regulator of EGFR/Ras induced epithelial tumor growth and invasion. [ABSTRACT FROM AUTHOR]

Background: Annexin A7 (ANXA7) is a member of the multifunctional calcium or phospholipid-binding annexin gene family. While low levels of ANXA7 are associated with aggressive types of cancer, the clinical impact of ANXA7 in prostate cancer remains unclear. Tissue microarrays (TMA) have revealed several new molecular markers in human tumors. Herein, we have identified the prognostic impact of ANXA7 in a prostate cancer using a tissue microarray containing 637 different specimens. Methods: The patients were diagnosed with prostate cancer and long-term follow-up information on progression (median 5.3 years), tumor-specific and overall survival data (median 5.9 years) were available. Expression of Ki67, Bcl-2, p53, CD-10 (neutral endopeptidase), syndecan-1 (CD-138) and ANXA7 were analyzed by immunohistochemistry. Results: A bimodal distribution of ANXA7 was observed. Tumors expressing either high or no ANXA7 were found to be associated with poor prognosis. However, ANXA7 at an optimal level, in between high and no ANXA7 expression, had a better prognosis. This correlated with low Ki67, Bcl-2, p53 and high syndecan-1 which are known predictors of early recurrence. At Gleason grade 3, ANXA7 is an independent predictor of poor overall survival with a p-value of 0.003. Neoadjuvant hormonal therapy, which is known to be associated with overexpression of Bcl-2 and inhibition of Ki67 LI and CD-10, was found to be associated with under-expression of ANXA7. Conclusions: The results of this TMA study identified ANXA7 as a new prognostic factor and indicates a bimodal correlation to tumor progression. [ABSTRACT FROM AUTHOR]

The broadly conserved bacterial signalling molecule cyclic-di-adenosine monophosphate (c-di-AMP) controls osmoresistance via its regulation of potassium (K+) and compatible solute uptake. High levels of c-di-AMP resulting from inactivation of c-di-AMP phosphodiesterase activity leads to poor growth of bacteria under high osmotic conditions. To better understand how bacteria can adjust in response to excessive c-di-AMP levels and to identify signals that feed into the c-di-AMP network, we characterised genes identified in a screen for osmoresistant suppressor mutants of the high c-di-AMP Lactococcus ΔgdpP strain. Mutations were identified which increased the uptake of osmoprotectants, including gain-of-function mutations in a Kup family K+ importer (KupB) and inactivation of the glycine betaine transporter transcriptional repressor BusR. The KupB mutations increased the intracellular K+ level while BusR inactivation increased the glycine betaine level. In addition, BusR was found to directly bind c-di-AMP and repress expression of the glycine betaine transporter in response to elevated c-di-AMP. Interestingly, overactive KupB activity or loss of BusR triggered c-di-AMP accumulation, suggesting turgor pressure changes act as a signal for this second messenger. In another group of suppressors, overexpression of an operon encoding an EmrB family multidrug resistance protein allowed cells to lower their intracellular level of c-di-AMP through active export. Lastly evidence is provided that c-di-AMP levels in several bacteria are rapidly responsive to environmental osmolarity changes. Taken together, this work provides evidence for a model in which high c-di-AMP containing cells are dehydrated due to lower K+ and compatible solute levels and that this osmoregulation system is able to sense and respond to cellular water stress. [ABSTRACT FROM AUTHOR]

To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo. [ABSTRACT FROM AUTHOR]

Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP) is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment. [ABSTRACT FROM AUTHOR]

Competing endogenous RNA (ceRNA) has emerged as an important post-transcriptional mechanism that simultaneously alters expressions of thousands genes in cancers. However, only a few ceRNA genes have been studied for their functions to date. To understand the major biological functions of thousands ceRNA genes as a whole, we designed Model-based Analysis of Competing-endogenous Pathways (MACPath) to infer pathways co-regulated through ceRNA mechanism (cePathways). Our analysis on breast tumors suggested that NGF (nerve growth factor)-induced tumor cell proliferation might be associated with tumor-related growth factor pathways through ceRNA. MACPath also identified indirect cePathways, whose ceRNA relationship is mediated by mediating ceRNAs. Finally, MACPath identified mediating ceRNAs that connect the indirect cePathways based on efficient integer linear programming technique. Mediating ceRNAs are unexpectedly enriched in tumor suppressor genes, whose down-regulation is suspected to disrupt indirect cePathways, such as between DNA replication and WNT signaling pathways. Altogether, MACPath is the first computational method to comprehensively understand functions of thousands ceRNA genes, both direct and indirect, at the pathway level. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are non-hematopoietic progenitor cells, which can be isolated from different types of tissues including bone marrow, adipose tissue, tooth pulp, and placenta/umbilical cord blood. There isolation from adult tissues circumvents the ethical concerns of working with embryonic or fetal stem cells, whilst still providing cells capable of differentiating into various cell lineages, such as adipocytes, osteocytes and chondrocytes. An important feature of MSCs is the low immunogenicity due to the lack of co-stimulatory molecules expression, meaning there is no need for immunosuppression during allogenic transplantation. The tropism of MSCs to damaged tissues and tumor sites makes them a promising vector for therapeutic agent delivery to tumors and metastatic niches. MSCs can be genetically modified by virus vectors to encode tumor suppressor genes, immunomodulating cytokines and their combinations, other therapeutic approaches include MSCs priming/loading with chemotherapeutic drugs or nanoparticles. MSCs derived membrane microvesicles (MVs), which play an important role in intercellular communication, are also considered as a new therapeutic agent and drug delivery vector. Recruited by the tumor, MSCs can exhibit both proand anti-oncogenic properties. In this regard, for the development of new methods for cancer therapy using MSCs, a deeper understanding of the molecular and cellular interactions between MSCs and the tumor microenvironment is necessary. In this review, we discuss MSC and tumor interaction mechanisms and review the new therapeutic strategies using MSCs and MSCs derived MVs for cancer treatment. [ABSTRACT FROM AUTHOR]

Throughout the animal kingdom, the Notch signalling pathway allows cells to acquire diversified cell fates. Notch signals are translated into activation of Notch target genes by CSL transcription factors. In the absence of Notch signals, CSL together with co-repressors functions as a transcriptional repressor. In Drosophila, repression of Notch target genes involves the CSL homologue Suppressor of Hairless (Su(H)) and the Notch (N) antagonist Hairless (H) that together form a repressor complex. Guided by crystal structure, three mutations Su(H)LL, Su(H)LLF and Su(H)LLL were generated that specifically affect interactions with the repressor H, and were introduced into the endogenous Su(H) locus by gene engineering. In contrast to the wild type isoform, these Su(H) mutants are incapable of repressor complex formation. Accordingly, Notch signalling activity is dramatically elevated in the homozygotes, resembling complete absence of H activity. It was noted, however, that heterozygotes do not display a dominant H loss of function phenotype. In this work we addressed genetic interactions the three H-binding deficient Su(H) mutants display in combination with H and N null alleles. We included a null mutant of Delta (Dl), encoding the ligand of the Notch receptor, as well as of Su(H) itself in our genetic analyses. H, N or Dl mutations cause dominant wing phenotypes that are sensitive to gene dose of the others. Moreover, H heterozygotes lack bristle organs and develop bristle sockets instead of shafts. The latter phenotype is suppressed by Su(H) null alleles but not by H-binding deficient Su(H) alleles which we attribute to the socket cell specific activity of Su(H). Modification of the dominant wing phenotypes of either H, N or Dl, however, suggested some lack of repressor activity in the Su(H) null allele and likewise in the H-binding deficient Su(H) alleles. Overall, Su(H) mutants are recessive perhaps reflecting self-adjusting availability of Su(H) protein. [ABSTRACT FROM AUTHOR]

In the African buffalo (Syncerus caffer) population of the Kruger National Park (South Africa) a primary sex-ratio distorter and a primary sex-ratio suppressor have been shown to occur on the Y chromosome. A subsequent autosomal microsatellite study indicated that two types of deleterious alleles with a negative effect on male body condition, but a positive effect on relative fitness when averaged across sexes and generations, occur genome-wide and at high frequencies in the same population. One type negatively affects body condition of both sexes, while the other acts antagonistically: it negatively affects male but positively affects female body condition. Here we show that high frequencies of male-deleterious alleles are attributable to Y-chromosomal distorter-suppressor pair activity and that these alleles are suppressed in individuals born after three dry pre-birth years, likely through epigenetic modification. Epigenetic suppression was indicated by statistical interactions between pre-birth rainfall, a proxy for parental body condition, and the phenotypic effect of homozygosity/heterozygosity status of microsatellites linked to male-deleterious alleles, while a role for the Y-chromosomal distorter-suppressor pair was indicated by between-sex genetic differences among pre-dispersal calves. We argue that suppression of male-deleterious alleles results in negative frequency-dependent selection of the Y distorter and suppressor; a prerequisite for a stable polymorphism of the Y distorter-suppressor pair. The Y distorter seems to be responsible for positive selection of male-deleterious alleles during resource-rich periods and the Y suppressor for positive selection of these alleles during resource-poor periods. Male-deleterious alleles were also associated with susceptibility to bovine tuberculosis, indicating that Kruger buffalo are sensitive to stressors such as diseases and droughts. We anticipate that future genetic studies on African buffalo will provide important new insights into gene fitness and epigenetic modification in the context of sex-ratio distortion and infectious disease dynamics. [ABSTRACT FROM AUTHOR]

Current treatments for glioblastoma multiforme (GBM)—an aggressive form of brain cancer—are minimally effective and yield a median survival of 14.6 months and a two-year survival rate of 30%. Given the severity of GBM and the limitations of its treatment, there is a need for the discovery of novel drug targets for GBM and more personalized treatment approaches based on the characteristics of an individual’s tumor. Most receptor tyrosine kinases—such as EGFR—act as oncogenes, but publicly available data from the Cancer Cell Line Encyclopedia (CCLE) indicates copy number loss in the ERBB4 RTK gene across dozens of GBM cell lines, suggesting a potential tumor suppressor role. This loss is mutually exclusive with loss of its cognate ligand NRG1 in CCLE as well, more strongly suggesting a functional role. The availability of higher resolution copy number data from clinical GBM patients in The Cancer Genome Atlas (TCGA) revealed that a region in Intron 1 of the ERBB4 gene was deleted in 69.1% of tumor samples harboring ERBB4 copy number loss; however, it was also found to be deleted in the matched normal tissue samples from these GBM patients (n = 81). Using the DECIPHER Genome Browser, we also discovered that this mutation occurs at approximately the same frequency in the general population as it does in the disease population. We conclude from these results that this loss in Intron 1 of the ERBB4 gene is neither a de novo driver mutation nor a predisposing factor to GBM, despite the indications from CCLE. A biological role of this significantly occurring genetic alteration is still unknown. While this is a negative result, the broader conclusion is that while copy number data from large cell line-based data repositories may yield compelling hypotheses, careful follow up with higher resolution copy number assays, patient data, and general population analyses are essential to codify initial hypotheses prior to investing experimental resources. [ABSTRACT FROM AUTHOR]

Hsp70 chaperones are well known for their important functions in maintaining protein homeostasis during thermal stress conditions. In many bacteria the Hsp70 homolog DnaK is also required for growth in the absence of stress. The molecular reasons underlying Hsp70 essentiality remain in most cases unclear. Here, we demonstrate that DnaK is essential in the α-proteobacterium Caulobacter crescentus due to its regulatory function in gene expression. Using a suppressor screen we identified mutations that allow growth in the absence of DnaK. All mutations reduced the activity of the heat shock sigma factor σ32, demonstrating that the DnaK-dependent inactivation of σ32 is a growth requirement. While most mutations occurred in the rpoH gene encoding σ32, we also identified mutations affecting σ32 activity or stability in trans, providing important new insight into the regulatory mechanisms controlling σ32 activity. Most notably, we describe a mutation in the ATP dependent protease HslUV that induces rapid degradation of σ32, and a mutation leading to increased levels of the house keeping σ70 that outcompete σ32 for binding to the RNA polymerase. We demonstrate that σ32 inhibits growth and that its unrestrained activity leads to an extensive reprogramming of global gene expression, resulting in upregulation of repair and maintenance functions and downregulation of the growth-promoting functions of protein translation, DNA replication and certain metabolic processes. While this re-allocation from proliferative to maintenance functions could provide an advantage during heat stress, it leads to growth defects under favorable conditions. We conclude that Caulobacter has co-opted the DnaK chaperone system as an essential regulator of gene expression under conditions when its folding activity is dispensable. [ABSTRACT FROM AUTHOR]

Several nucleoporins in the nuclear pore complex (NPC) have been reported to be involved in abiotic stress responses in plants. However, the molecular mechanism of how NPC regulates abiotic stress responses, especially the expression of stress responsive genes remains poorly understood. From a forward genetics screen using an abiotic stress-responsive luciferase reporter (RD29A-LUC) in the sickle-1 (sic-1) mutant background, we identified a suppressor caused by a mutation in NUCLEOPORIN 85 (NUP85), which exhibited reduced expression of RD29A-LUC in response to ABA and salt stress. Consistently, the ABA and salinity induced expression of several stress responsive genes such as RD29A, COR15A and COR47 was significantly compromised in nup85 mutants and other nucleoporin mutants such as nup160 and hos1. Subsequently, Immunoprecipitation and mass spectrometry analysis revealed that NUP85 is potentially associated with HOS1 and other nucleoporins within the nup107-160 complex, along with several mediator subunits. We further showed that there is a direct physical interaction between MED18 and NUP85. Similar to NUP85 mutations, MED18 mutation was also found to attenuate expression of stress responsive genes. Taken together, we not only revealed the involvement of NUP85 and other nucleoporins in regulating ABA and salt stress responses, but also uncovered a potential relation between NPC and mediator complex in modulating the gene expression in plants. [ABSTRACT FROM AUTHOR]

By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70’s ability to function in a wide range of cellular processes—modulating the Hsp70-substrate interaction cycle. [ABSTRACT FROM AUTHOR]

MicroRNAs are biomarkers and potential therapeutic targets for breast cancer. Anacardic acid (AnAc) is a dietary phenolic lipid that inhibits both MCF-7 estrogen receptor α (ERα) positive and MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation with IC50s of 13.5 and 35 μM, respectively. To identify potential mediators of AnAc action in breast cancer, we profiled the genome-wide microRNA transcriptome (microRNAome) in these two cell lines altered by the AnAc 24:1n5 congener. Whole genome expression profiling (RNA-seq) and subsequent network analysis in MetaCore Gene Ontology (GO) algorithm was used to characterize the biological pathways altered by AnAc. In MCF-7 cells, 69 AnAc-responsive miRNAs were identified, e.g., increased let-7a and reduced miR-584. Fewer, i.e., 37 AnAc-responsive miRNAs were identified in MDA-MB-231 cells, e.g., decreased miR-23b and increased miR-1257. Only two miRNAs were increased by AnAc in both cell lines: miR-612 and miR-20b; however, opposite miRNA arm preference was noted: miR-20b-3p and miR-20b-5p were upregulated in MCF-7 and MDA-MB-231, respectively. miR-20b-5p target EFNB2 transcript levels were reduced by AnAc in MDA-MB-231 cells. AnAc reduced miR-378g that targets VIM (vimentin) and VIM mRNA transcript expression was increased in AnAc-treated MCF-7 cells, suggesting a reciprocal relationship. The top three enriched GO terms for AnAc-treated MCF-7 cells were B cell receptor signaling pathway and ribosomal large subunit biogenesis and S-adenosylmethionine metabolic process for AnAc-treated MDA-MB-231 cells. The pathways modulated by these AnAc-regulated miRNAs suggest that key nodal molecules, e.g., Cyclin D1, MYC, c-FOS, PPARγ, and SIN3, are targets of AnAc activity. [ABSTRACT FROM AUTHOR]

The work presents the role of selected microRNA molecules (miRNA) and their participation in intracellular mechanisms determining the phenomenon of carcinogenesis and the progression of neoplastic changes. It focuses on the problems related to the role of miRNA as oncogenes or suppressor genes. The authors review the latest literature on the importance of selected microRNAs in the neoplasmatic process, including cancers of the head and neck region. [ABSTRACT FROM AUTHOR]

Targeted cancer therapy directed at individual targets is often accompanied by the rapid development of drug resistance. The development of a new generation of antitumor drugs involves the search for many targets simultaneously to block or, conversely, restore their activity. In this regard, simultaneous analysis of gene expression in a complex network of interactions, primarily cell cycle control elements, is relevant for the search of specific molecular markers for the differential diagnosis of adenocarcinoma (ADC) and squamous cell lung cancer (SCC), as well as new targets for therapy. In this paper we performed an extended quantitative analysis of the expression of two suppressor genes, CTDSPL and its target RB1, as well as 84 genes of the main participants of the p16-Cdk/cyclin D1-Rb and p53/p21 signaling pathways in the histological types of non-small-cell lung cancer (NSCLC), i.e., ADC and SCC, using the special panel of the Human Cell Cycle Regulation Panel. The expression profile of some genes shows the specificity to the histological type of NSCLC and the presence of metastases. The genes with a significantly increased expression that affect the activity of Rb (cyclins, cyclin-dependent kinases, their activators, inhibitors, etc.) can serve as potential targets for combined therapy of both ADC and SCC. [ABSTRACT FROM AUTHOR]

Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the ‘classical’ (M1) and ‘alternative’ (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans. [ABSTRACT FROM AUTHOR]

In the rice blast fungus Magnaporthe oryzae, the cAMP-PKA pathway regulates surface recognition, appressorium turgor generation, and invasive growth. However, deletion of CPKA failed to block appressorium formation and responses to exogenous cAMP. In this study, we generated and characterized the cpk2 and cpkA cpk2 mutants and spontaneous suppressors of cpkA cpk2 in M. oryzae. Our results demonstrate that CPKA and CPK2 have specific and overlapping functions, and PKA activity is essential for appressorium formation and plant infection. Unlike the single mutants, the cpkA cpk2 mutant was significantly reduced in growth and rarely produced conidia. It failed to form appressoria although the intracellular cAMP level and phosphorylation of Pmk1 MAP kinase were increased. The double mutant also was defective in plant penetration and Mps1 activation. Interestingly, it often produced fast-growing spontaneous suppressors that formed appressoria but were still non-pathogenic. Two suppressor strains of cpkA cpk2 had deletion and insertion mutations in the MoSFL1 transcription factor gene. Deletion of MoSFL1 or its C-terminal 93-aa (MoSFL1ΔCT) was confirmed to suppress the defects of cpkA cpk2 in hyphal growth but not appressorium formation or pathogenesis. We also isolated 30 spontaneous suppressors of the cpkA cpk2 mutant in Fusarium graminearum and identified mutations in 29 of them in FgSFL1. Affinity purification and co-IP assays showed that this C-terminal region of MoSfl1 was essential for its interaction with the conserved Cyc8-Tup1 transcriptional co-repressor, which was reduced by cAMP treatment. Furthermore, the S211D mutation at the conserved PKA-phosphorylation site in MoSFL1 partially suppressed the defects of cpkA cpk2. Overall, our results indicate that PKA activity is essential for appressorium formation and proper activation of Pmk1 or Mps1 in M. oryzae, and phosphorylation of MoSfl1 by PKA relieves its interaction with the Cyc8-Tup1 co-repressor and suppression of genes important for hyphal growth. [ABSTRACT FROM AUTHOR]

Cucumber mosaic virus (CMV) is a model virus for plant–virus protein interaction and mechanism research because of its wide distribution, high-level of replication and simple genome structure. The 2b protein is a multifunctional protein encoded by CMV that suppresses RNA silencing-based antiviral defense and contributes to CMV virulence in host plants. In this report, 12 host proteins were identified as CMV LS2b binding partners using the yeast two-hybrid screen system from the Arabidopsis thaliana cDNA library. Among the host proteins, 30S ribosomal subunit protein S11 (RPS11) was selected for further studies. The interaction between LS2b and full-length RPS11 was confirmed using the yeast two-hybrid system. Bimolecular fluorescence complementation (BIFC) assays observed by confocal laser microscopy and Glutathione S-transferase (GST) pull-down assays were used to verify the interaction between endogenous NbRPS11 and viral CMVLS2b both in vivo and in vitro. TRV-based gene silencing vector was used to knockdown NbRPS11 transcription, and immunoblot analysis revealed a decline in infectious viral RNA replication and a decrease in CMV infection in RPS11 down-regulated Nicotiana benthamiana plants. Thus, the knockdown of RPS11 likely inhibited CMV replication and accumulation. The gene silencing suppressor activity of CMV2b protein was reduced by the RPS11 knockdown. This study demonstrated that the function of viral LS2b protein was remarkably affected by the interaction with host RPS11 protein. [ABSTRACT FROM AUTHOR]

High-risk human papillomaviruses (hr-HPVs) cause cervical cancer, the fourth most common cancer in women worldwide. A HPV-16 candidate therapeutic vaccine, LALF32-51-E7, was developed by fusing a modified E7 protein to a bacterial cell-penetrating peptide (LALF): this elicited both tumour protection and regression in pre-clinical immunization studies. In the current study, we investigated the potential for producing LALF32-51-E7 in a plant expression system by evaluating the effect of subcellular localization and usage of different expression vectors and gene silencing suppressors. The highest expression levels of LALF32-51-E7 were obtained by using a self-replicating plant expression vector and chloroplast targeting, which increased its accumulation by 27-fold compared to cytoplasmic localization. The production and extraction of LALF32-51-E7 was scaled-up and purification optimized by affinity chromatography. If further developed, this platform could potentially allow for the production of a more affordable therapeutic vaccine for HPV-16. This would be extremely relevant in the context of developing countries, where cervical cancer and other HPV-related malignancies are most prevalent, and where the population have limited or no access to preventative vaccines due to their typical high costs. [ABSTRACT FROM AUTHOR]

Background: Prostate cancer (PCa) poses a major health concern in men worldwide. Retinoic Acid Receptor Responder (RARRES1)/ Tazarotene-induced gene-1 (TIG-1) is a putative tumor suppressor gene that exerts its tumor suppressor function via unknown mechanisms. Epigenetic silencing of RARRES1 leads to its loss in several types of cancer, including PCa. Determining the molecular mechanisms that mediate the tumor suppressor role of RARRES1 in PCa is the focus of our study. Findings: Our data indicates that RARRES1 over expression in PCa cell lines represses mitogen-activated protein kinase (MAPK) activation. RARRES1 expression induces the levels of autophagy-related genes, beclin, ATG3 and increases LC3B-II conversion. A significant induction of SIRT1 along with mTOR inhibition is noted on RARRES1 expression. Furthermore, RARRES1 over expression elevates the levels of the antioxidant enzyme, catalase. Our results also indicate that RARRES1 expression inhibits angiogenesis in endothelial cells. Conclusions: In summary, the data presented here indicate that forced expression of RARRES1 in PCa cells (a) induces ER stress and autophagic response; (b) increases SIRT1 levels; and (c) higher levels of anti-oxidant enzymes. Our study also implicates the role of RARRES1 as a novel anti-angiogenic molecule. Overall this study reports the molecular players that RARRES1 modulates to serve as a tumor suppressor molecule. Future studies will help determine the in vivo mechanisms by which RARRES1 may serve as a target for therapeutic intervention both in cancer and in angiogenesis-related disorders. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most common type of malignant bone tumor, often affecting adolescents and children. MicroRNAs (miRNAs) are a group of small, non-protein coding, endogenous RNAs that play critical roles in osteosarcoma tumorigenesis. In our study, we demonstrated that miR-448 expression was downregulated in osteosarcoma tissues and cell lines. Overexpression of miR-448 suppressed osteosarcoma cell proliferation, colony formation and migration. Moreover, we found that EPHA7 was a direct target gene of miR-448 in osteosarcoma cells. We further demonstrated that the EPHA7 expression level was upregulated in osteosarcoma tissues. Interestingly, the expression level of EPHA7 was inversely correlated with the expression level of miR-448 in osteosarcoma tissues. In addition, elevated expression of miR-448 suppressed osteosarcoma cell proliferation and invasion through targeting EPHA7. Taken together, these findings suggest that miR-448 functioned as a tumor suppressor gene in the development of osteosarcoma through targeting EPHA7. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa has one A-type (caa3) and multiple C-type (cbb3) cytochrome c oxidases as well as two quinol oxidases for aerobic respiration. The caa3 oxidase is highly efficient in creating a proton gradient across the cell membrane, but it is not expressed under normal growth conditions and its physiological role has not been investigated. In the present study, a mutant strain deficient in the coxBA-PA0107-coxC genes encoding caa3 exhibited normal growth under any test conditions, but it had low relative fitness under carbon starvation conditions, indicating that the expression of caa3 is advantageous under starvation conditions. A mutant that lacked four terminal oxidase gene clusters except for the cox genes was unable to grow aerobically because of low expression level of caa3. However, suppressor mutants that grew aerobically using caa3 as the only terminal oxidase emerged after aerobic subculturing. Analyses of the suppressor mutants revealed that a mutation of roxS encoding a sensor kinase of a two-component regulator RoxSR was necessary for the aerobic growth in synthetic medium. Two additional mutations in the 5′-flanking region of coxB were necessary for the aerobic growth in LB medium. Although the expression level of caa3 was higher in the suppressor mutants, their growth rates were lower than when the other terminal oxidases were utilized, suggesting that caa3 was not suited for utilization as the only terminal oxidase. Overexpression of the cox genes also inhibited the aerobic growth of the wild-type strain. These results indicate that caa3 is tightly regulated to be expressed only under starvation conditions at low level and it functions in cooperation with other terminal oxidases to facilitate survival in nutrient starvation conditions. [ABSTRACT FROM AUTHOR]

BREVIPEDICELLUS (BP) encodes a class-I KNOTTED1-like homeobox (KNOX) transcription factor that plays a critical role in conditioning a replication competent state in the apical meristem, and it also governs growth and cellular differentiation in internodes and pedicels. To search for factors that modify BP signaling, we conducted a suppressor screen on bp er (erecta) plants and identified a mutant that ameliorates many of the pleiotropic defects of the parent line. Map based cloning and complementation studies revealed that the defect lies in the FILAMENTOUS FLOWER (FIL) gene, a member of the YABBY family of transcriptional regulators that contribute to meristem organization and function, phyllotaxy, leaf and floral organ growth and polarity, and are also known to repress KNOX gene expression. Genetic and cytological analyses of the fil-10 suppressor line indicate that the role of FIL in promoting growth is independent of its previously characterized influences on meristem identity and lateral organ polarity, and likely occurs non-cell-autonomously from superior floral organs. Transcription profiling of inflorescences revealed that FIL downregulates numerous transcription factors which in turn may subordinately regulate inflorescence architecture. In addition, FIL, directly or indirectly, activates over a dozen genes involved in glucosinolate production in part by activating MYB28, a known activator of many aliphatic glucosinolate biosynthesis genes. In the bp er fil-10 suppressor mutant background, enhanced expression of CYP71A13, AMIDASE1 (AMI) and NITRILASE genes suggest that auxin levels can be modulated by shunting glucosinolate metabolites into the IAA biosynthetic pathway, and increased IAA levels in the bp er fil-10 suppressor accompany enhanced internode and pedicel elongation. We propose that FIL acts to oppose KNOX1 gene function through a complex regulatory network that involves changes in secondary metabolites and auxin. [ABSTRACT FROM AUTHOR]

Tumorigenic processes are understood to be driven by epi-/genetic and genomic alterations from single point mutations to chromosomal alterations such as insertions and deletions of nucleotides up to gains and losses of large chromosomal fragments including products of chromosomal rearrangements e.g. fusion genes and proteins. Overall comparisons of copy number alterations (CNAs) presented in 48 clear cell renal cell carcinoma (ccRCC) genomes resulted in ratios of gene losses versus gene gains between 26 ccRCC Fuhrman malignancy grades G1 (ratio 1.25) and 20 G3 (ratio 0.58). Gene losses and gains of 15762 CNA genes were mapped to 795 chromosomal cytoband loci including 280 KEGG pathways. CNAs were classified according to their contribution to Fuhrman tumour gradings G1 and G3. Gene gains and losses turned out to be highly structured processes in ccRCC genomes enabling the subclassification and stratification of ccRCC tumours in a genome-wide manner. CNAs of ccRCC seem to start with common tumour related gene losses flanked by CNAs specifying Fuhrman grade G1 losses and CNA gains favouring grade G3 tumours. The appearance of recurrent CNA signatures implies the presence of causal mechanisms most likely implicated in the pathogenesis and disease-outcome of ccRCC tumours distinguishing lower from higher malignant tumours. The diagnostic quality of initial 201 genes (108 genes supporting G1 and 93 genes G3 phenotypes) has been successfully validated on published Swiss data (GSE19949) leading to a restricted CNA gene set of 171 CNA genes of which 85 genes favour Fuhrman grade G1 and 86 genes Fuhrman grade G3. Regarding these gene sets overall survival decreased with the number of G3 related gene losses plus G3 related gene gains. CNA gene sets presented define an entry to a gene-directed and pathway-related functional understanding of ongoing copy number alterations within and between individual ccRCC tumours leading to CNA genes of prognostic and predictive value. [ABSTRACT FROM AUTHOR]

PTEN is among the most commonly mutated tumor suppressor genes in human cancer. However, studying the role of PTEN in the pathogenesis of cancer has been limited, in part, by the paucity of human cell-based isogenic systems that faithfully model PTEN loss. In an effort to remedy this problem, gene editing was used to correct an endogenous mutant allele of PTEN in two human glioblastoma multiforme (GBM) cell lines– 42MGBA and T98G. PTEN correction resulted in reduced cellular proliferation that was Akt-dependent in 42MGBA cells and Akt-independent in T98G cells. This is the first report of human cancer cell lines in which mutant PTEN has been corrected by gene editing. The isogenic sets of gene edited cell lines reported here will likely prove useful for further study of PTEN mutations in the pathogenesis of cancer, and for the discovery and validation of novel therapeutics targeting the PTEN pathway. [ABSTRACT FROM AUTHOR]