Sterol Regulatory Element-Binding Protein, BbSre1, Controls Oxidative Stress Response, Peroxisome Division, and Lipid Homeostasis in an Insect Fungal Pathogen.

Sterol regulatory element-binding protein, Sre1, regulates sterol biosynthesis, lipid metabolism, hypoxia adaptation, and virulence in some fungi, even though its roles are varied in fungal species. However, few studies report its other functions in fungi. Here, we report novel roles of Sre1 homolog, BbSre1, in the insect fungal pathogen, Beauveria bassiana, that regulates oxidative stress response, peroxisome division, and redox homeostasis. The gene disruption stain showed increased sensitivity to oxidative stress, which was in line with oxidative stress-induced-BbSre1 nuclear import and control of antioxidant and detoxification-involved genes. The gene mutation also inhibited peroxisome division, affected redox homeostasis, and impaired lipid/fatty acid metabolism and sterol biosynthesis, which was verified by downregulation of their associated genes. These data broaden our understanding of role of Sre1, which regulates peroxisome division, antioxidant, and detoxification-involved genes for control of redox homeostasis and oxidative stress response that links to lipid/fatty acid metabolism and sterol biosynthesis.

Two Secretory T2 RNases Act as Cytotoxic Factors Contributing to the Virulence of an Insect Fungal Pathogen.

RNase T2 members are secreted by several pathogens or parasites during infection, playing various roles in pathogen-host interaction. However, functions of those members in biocontrol microbes targeting their hosts are still unknown. Here, we report that an insect fungal pathogen, Beauveria bassiana, produces two secretory RNase T2 members that act as cytotoxic factors, which were examined by insect bioassays using the targeted gene(s) disruption and overexpression strains. Overexpression strains displayed dramatically increased virulence, which was concurrent with few fungal cells and hemocytes in hemocoel, suggesting a cytotoxicity of the overexpressed gene products. In vitro assays using yeast-expressed proteins verified the cytotoxicity of the two members against insect cells, to which the cytotoxic effect was dependent on their RNases enzyme activities and glycosylation modification. Moreover, the excessive humoral immune responses triggered by the two ribonucleases were examined. These results suggested prospects of these two T2 ribonucleases for improvement of biocontrol agents.

Metabolic reprogramming of ovarian cancer involves ACSL1-mediated metastasis stimulation through upregulated protein myristoylation.

As a result of the hostile microenvironment, metabolic alterations are required to enable the malignant growth of cancer cells. To understand metabolic reprogramming during metastasis, we conducted shotgun proteomic analysis of highly metastatic (HM) and non-metastatic (NM) ovarian cancer cells. The results suggest that the genes involved in fatty-acid (FA) metabolism are upregulated, with consequent increases of phospholipids with relatively short FA chains (myristic acid, MA) in HM cells. Among the upregulated proteins, ACSL1 expression could convert the lipid profile of NM cells to that similar of HM cells and make them highly aggressive. Importantly, we demonstrated that ACSL1 activates the AMP-activated protein kinase and Src pathways via protein myristoylation and finally enhances FA beta oxidation. Patient samples and tissue microarray data also suggested that omentum metastatic tumours have higher ACSL1 expression than primary tumours and a strong association with poor clinical outcome. Overall, our data reveal that ACSL1 enhances cancer metastasis by regulating FA metabolism and myristoylation.

Nuclear DLC1 exerts oncogenic function through association with FOXK1 for cooperative activation of MMP9 expression in melanoma.

A Rho GTPase-activating protein (RhoGAP), deleted in liver cancer 1 (DLC1), is known to function as a tumor suppressor in various cancer types; however, whether DLC1 is a tumor-suppressor gene or an oncogene in melanoma remains to be clarified. Here we revealed that high DLC1 expression was detected in most of the melanoma tissues where it was localized in both the nuclei and the cytoplasm. Functional studies unveiled that DLC1 was both required and sufficient for melanoma growth and metastasis. These tumorigenic events were mediated by nuclear-localized DLC1 in a RhoGAP-independent manner. Mechanistically, mass spectrometry analysis identified a DLC1-associated protein, FOXK1 transcription factor, which mediated oncogenic events in melanoma by translocating and retaining DLC1 into the nucleus. RNA-sequencing profiling studies further revealed MMP9 as a direct target of FOXK1 through DLC1-regulated promoter occupancy for cooperative activation of MMP9 expression to promote melanoma invasion and metastasis. Concerted action of DLC1-FOXK1 in MMP9 gene regulation was further supported by their highly correlated expression in melanoma patients' samples and cell lines. Together, our results not only unravel a mechanism by which nuclear DLC1 functions as an oncogene in melanoma but also suggest an unexpected role of RhoGAP protein in transcriptional regulation.

Interference with SMO increases chemotherapy drug sensitivity of A2780/DDP cells by inhibiting the Hh/Gli signaling pathway.

Aberrant activation of the Hedgehog (Hh)/Gli pathway contributes to the tumorigenesis of several human cancers, including ovarian cancers. We investigated the function of SMO on cell growth, drug resistance, and invasive ability in A2780/DDP cells. Moreover, we also tested the levels of the downstream target genes of the Hh/Gli pathway in SMO short hairpin RNA (shRNA) lentivirus-infected A2780/DDP cells. Western blot analysis results revealed that the Hh/Gli pathway was activated in cisplatin-resistant A2780/DDP cells. After infection by SMO shRNA lentivirus, the colony formation rate and invasive rate of cisplatin-resistant A2780/DDP cells were decreased. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that upon transfection with SMO shRNA, cell growth was decreased and drug sensitivity to cisplatin was upregulated. Moreover, interference with SMO decreased the expression of MMP-2, MMP-9, VEGF, and Snail in cisplatin-resistant cells. Thus, the Hh/Gli signaling pathway was aberrantly activated in A2780/DDP cells. The colony formation rate and invasive rate were decreased in SMO shRNA lentivirus-infected A2780/DDP cells. All results showed that inhibiting Hh/Gli signaling may negatively regulate the proliferation, invasion, and metastasis of cisplatin-resistant A2780/DDP cells, as well as increase the sensitivity of A2780/DDP to the chemotherapeutic drug of cisplatin.

FAM83D promotes ovarian cancer progression and its potential application in diagnosis of invasive ovarian cancer.

Although invasive epithelial ovarian cancer (IOC) and low malignant potential ovarian tumour (LMP) are similar, they are associated with different outcomes and treatment strategies. The current accuracy in distinguishing these diseases is unsatisfactory, leading to delays or unnecessary treatments. We compared the molecular signature of IOC and LMP cases by analysing their transcriptomic data and re-clustered them according to these data rather than the pathological dissection. We identified that FAM83D was highly expressed in IOC. To verify the role of FAM83D in the progression and metastasis, we used the isogenic ovarian cancer metastatic models, highly metastatic cells (HM) and non-metastatic cells (NM). Overexpression of FAM83D significantly promoted cell proliferation, migration and spheroid formation. This was consistent with previous data showing that high FAM83D expression is associated with poor prognosis in cancer patients. Moreover, similar to the HM cells, the FAM83D-overexpressing NM cells demonstrated stronger phosphorylation of the epidermal growth factor receptor (EGFR) and c-Raf. This indicates that the action of FAM83D is mediated by the activation of the EGFR pathway. Taken together, this report suggested that FAM83D might be an excellent molecular marker to discriminate between IOC and LMP.

Comprehensive analysis of the GATA transcription factor gene family in breast carcinoma using gene microarrays, online databases and integrated bioinformatics.

Integrated studies of accumulated data can be performed to obtain more reliable information and more feasible measures for investigating the potential diagnostic and prognostic biomarkers of breast cancer and exploring related molecular mechanisms. Our study aimed to explore the GATA family members involved in breast cancer by integrating data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and other online databases. We performed an integrated analysis of published studies from GEO and analyzed clinical data from TCGA and GTEx to evaluate the clinical significance and prognosis values of the GATA family in breast cancer. GATA3 was found to be upregulated and exhibited a favorable value in the diagnosis and prognosis of breast cancer. Through this study, we identified possible GATA3-correlated genes and core pathways that play an important role, which requires further investigation in breast cancer.

Angiotensin II promotes ovarian cancer spheroid formation and metastasis by upregulation of lipid desaturation and suppression of endoplasmic reticulum stress.

BACKGROUND: Angiotensin II (ANGII) and its receptor (AGTR1) have been proposed as significant contributors to metastasis in multiple cancers. Further, high AGTR1 levels are associated with poor epithelial ovarian cancer (EOC) outcomes. However, the mechanistic basis for these effects is unknown. Recent studies have suggested that ovarian cancer metastasis is highly dependent on the formation of multicellular spheroids (MCS). To understand the associations between the ANGII/AGTR1 pathway and cancer outcomes, we evaluated the effects of ANGII on MCS formation by ovarian cancer cells and used a proteomic approach to analyze the mechanistic basis. METHODS: We used the data from the GENT database and immunohistochemistry staining to assess the AGTR1 expression in epithelial ovarian cancer (EOC) patients and to assess its role in cancer progression. Colony formation assay, 3D culture assay, and transwell assays were used to analyze the effect of ANGII on the MCS formation and cell migration. The signaling pathways of AGTR1 and transactivation of epidermal growth factor receptor (EGFR) transactivation were investigated by the western blotting analysis. Xenograft models were used to determine the role of AGTR1 in ovarian cancer metastasis. ANGII release from ovarian cancer cells and ANGII levels in the EOC ascites fluid were measured by immunoassay. A shotgun proteomic approach was used to explore the detail molecular mechanism. Modulation of lipid desaturation and endoplasmic reticulum stress were verified by the in vitro and in vivo functional assays. RESULTS: AGTR1 expression was negatively correlated with EOC prognosis. AGTR1activation significantly enhanced the MCS formation and cell migration. ANGII triggered both of the classical AGTR1 pathway and the EGFR transactivation. ANGII administration increased peritoneal metastasis. In addition, ovarian cancer cells secreted ANGII and enhanced cancer metastasis in a positive feedback manner. Based on the proteomic data, lipid desaturation was activated by induction of stearoyl-CoA desaturase-1 (SCD1), which suggests that inhibition of SCD1 may significantly reduce MCS formation by increasing endoplasmic reticulum stress. CONCLUSIONS: ANGII promotes MCS formation and peritoneal metastasis of EOC cells. AGTR1 activation increases the lipid desaturation via SCD1 upregulation, which ultimately reduces endoplasmic reticulum stress in MCS. This mechanism explained the association between high levels of AGTR1 and poor clinical outcomes in EOC patients.

The Role of G Protein-coupled Receptor Kinases in Cancer.

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. Emerging evidence demonstrates that signaling through GPCRs affects numerous aspects of cancer biology such as vascular remolding, invasion, and migration. Therefore, development of GPCR-targeted drugs could provide a new therapeutic strategy to treating a variety of cancers. G protein-coupled receptor kinases (GRKs) modulate GPCR signaling by interacting with the ligand-activated GPCR and phosphorylating its intracellular domain. This phosphorylation initiates receptor desensitization and internalization, which inhibits downstream signaling pathways related to cancer progression. GRKs can also regulate non-GPCR substrates, resulting in the modulation of a different set of pathophysiological pathways. In this review, we will discuss the role of GRKs in modulating cell signaling and cancer progression, as well as the therapeutic potential of targeting GRKs.

miR-320a regulates high mobility group box 1 expression and inhibits invasion and metastasis in hepatocellular carcinoma.

BACKGROUND & AIMS: Several studies have shown that miR-320a induces apoptosis, inhibits cell proliferation, and affects cell cycle progression as a tumour suppressor in many cancers. However, the involvement of miR-320a in the invasion and metastasis of hepatocellular carcinoma (HCC) is still unknown. METHODS: Endogenous miR-320a and high mobility group box 1 (HMGB1) expressions were assayed by real-time PCR. Luciferase activities were measured using a dual-luciferase reporter assay system. Western blots were used to determine the protein expressions of HMGB1, MMP2, and MMP9. Invasion and metastasis of tumour cells were, respectively, evaluated by the transwell invasion assay and the wound healing assay. RESULTS: The expression of miR-320a was significantly decreased in 24 of 32 (75%) HCC tissues and associated with the invasion and metastasis of HCC. Furthermore, we demonstrated that HMGB1 was a direct target of miR-320a and there was a significant negative correlation between miR-320a and HMGB1 expression in HCC. Ectopic expression or inhibition of miR-320a potently regulated the invasion and metastasis of HCC cells in HMGB1-dependent manner. CONCLUSIONS: Our results showed that miR-320a was involved in the invasion and metastasis by targeting HMGB1 and had an anti-metastasis effect in HCC.

Frequent CpG island methylation: a risk factor in the progression of traditional serrated adenoma of the colorectum.

BACKGROUND: Traditional serrated adenoma (TSA) features a unique serrated configuration because it involves two cell types: tall and short columnar cells. The serrated neoplasia pathway is related to the carcinogenesis of colorectal cancer. CpG island methylator phenotype-high (CIMP-high) is a unique genetic alteration in this pathway. MATERIALS AND METHODS: This study investigated the prevalence and level of methylation and CIMP in 30 TSA cases. The tall and short cells in 28 TSAs were separated by microdissection. Methylation-specific PCR was performed to detect the methylation of MGMT, MLH1, P14, P16, MINT1, MINT2 and MINT31. RESULTS: Overall, 30 cases presented CIMP-high, and the prevalence of CIMP-high was 100% (30/30) in tall cells and 93% (28/30) in short cells. CONCLUSIONS: No significant difference was found between tall and short columnar cells. The relationship between methylation and clinicopathological characters remains to be established.

Differential distribution of U6 (RNU6-1) expression in human carcinoma tissues demonstrates the requirement for caution in the internal control gene selection for microRNA quantification.

Alterations in microRNA (miRNA) expression patterns have been associated with a number of human diseases. Accurate quantitation of miRNA levels is important for their use as biomarkers and in determining their functions. Although the issue of proper miRNA detection was solved with the introduction of standard reverse transcription­quantitative polymerase chain reaction (RT­qPCR) assays, numerous issues with the selection of appropriate internal control genes remain. U6 (RNU6­1) snRNA, the most commonly used internal control gene in miRNA RT­qPCR assays, was shown to be unstable in clinical samples, particularly cancer tissues. Identification of the distribution of U6 in different tissues is the premise of more accurate quantification of miRNAs. However, the distribution of U6 in human carcinoma tissues and corresponding normal tissues is unknown. In the present study, U6 levels were significantly higher in human breast carcinoma tissues compared with the corresponding normal tissues by RT­qPCR. In the carcinoma or corresponding adjacent normal tissues, the expression levels of U6 in epithelial cells were higher than those in the mesenchymal cells. Furthermore, the expression levels of U6 in the carcinoma tissues of the liver and intrahepatic bile ducts were higher than those in the adjacent normal tissues. These results suggest that the expression and distribution of U6 exhibits a high degree of variability among several types of human cells. Therefore, caution is required when selecting U6 as an internal control gene for evaluating expression profiles of miRNAs in patients with carcinoma, particularly carcinoma of the liver and intrahepatic bile ducts.

An unusually large cavernous hemangioma of retropharyngeal space: a rare case.

Hemangiomas rarely occur in the retropharyngeal space with only several cases reported in the current literature. This article reports the hemangiomas of retropharyngeal space. A 55-year-old woman was referred to our institution for dysphagia. Computed tomography and magnetic resonance imaging of the neck and spine revealed a large, well-circumscribed, dense mass that extended from the retropharyngeal space to the sides of the neck. Patient underwent direct excision of the lesion. Complete regression of symptoms was observed after surgery, with no lesions found on routine 24-month follow-up. Although hemangiomas are relatively common in the head and neck, those that originate in the retropharyngeal space are very rarely observed. These benign tumors have the potential to compress adjacent tissues or organs and thereby produce associated symptoms like dysphagia and dyspnea. We present the reported case of larger hemangiomas of the retropharyngeal space and detail their management.

The T-SPOT.TB Test for Diagnosis of Breast Tuberculosis.

OBJECTIVE: To assess the diagnostic value of the T-SPOT.TB test in cases of breast turberculosis (BTB) in China. METHODS: We enrolled 13 female patients with primary BTB as the BTB test group and 10 healthy volunteers as the control group. The 2 groups underwent T-SPOT.TB tests and tuberculin skin tests (TSTs) before receiving a core-needle biopsy or excision biopsy. We then collected and analyzed T-SPOT.TB and TST data. RESULTS: The sensitivity of the T-SPOT.TB test for detection of BTB (84.6%) was significantly greater than that of TST (53.8%) (P <.05); the specificity of each test (80.0% and 60.0%, respectively) for BTB was not significantly different (P >.05). CONCLUSION: The T-SPOT.TB test could be a useful adjunct to current tests for diagnosis of BTB and could be used for early diagnosis of this condition.

Ectopic thyroid papillary carcinoma of nasopharynx associated with adenoid hypertrophy: an unusual presentation.

Ectopic thyroid tissue of nasopharynx is an uncommon phenomenon and papillary thyroid carcinoma arising from the tissue is extremely rare. The authors report a rare case of 16-year-old girl with papillary thyroid carcinoma of nasopharynx. Clinicians were ever confused by adenoid hypertrophy and solved the diagnostic dilemma by adequate examinations. In the case, we mainly emphasize that surgeons should be aware of and actively consider such a possibility of ectopic papillary thyroid carcinoma of nasopharynx in children and adolescents with long-term nasal obstruction, even if thyroid carcinoma is a rare tumor.

Arbutin, an intracellular hydroxyl radical scavenger, protects radiation-induced apoptosis in human lymphoma U937 cells.

Ionizing radiation (IR) can generate reactive oxygen species (ROS). Excessive ROS have the potential to damage cellular macromolecules including DNA, proteins, and lipids and eventually lead to cell death. In this study, we evaluated the potential of arbutin, a drug chosen from a series of traditional herbal medicine by measuring intracellular hydroxyl radical scavenging ability in X-irradiated U937 cells. Arbutin (hydroquinone-ß-D-glucopyranoside), a naturally occurring glucoside of hydroquinone, has been traditionally used to treat pigmentary disorders. However, there are no reports describing the effect of arbutin on IR-induced apoptosis. We confirmed that arbutin can protect cells from apoptosis induced by X-irradiation. The combination of arbutin and X-irradiation could reduce intracellular hydroxyl radical production and prevent mitochondrial membrane potential loss. It also could down-regulate the expression of phospho-JNK, phospho-p38 in whole cell lysate and activate Bax in mitochondria. Arbutin also inhibits cytochrome C release from mitochondria to cytosol. To verify the role of JNK in X-irradiation-induced apoptosis, the cells were pretreated with a JNK inhibitor, and found that JNK inhibitor could reduce apoptosis induced by X-irradiation. Taken together, our data indicate that arbutin plays an anti-apoptotic role via decreasing intracellular hydroxyl radical production, inhibition of Bax-mitochondria pathway and activation of the JNK/p38 MAPK pathway.

Isofraxidin, a potent reactive oxygen species (ROS) scavenger, protects human leukemia cells from radiation-induced apoptosis via ROS/mitochondria pathway in p53-independent manner.

Ionizing radiation (IR) leads to oxidizing events such as excessive reactive oxygen species (ROS) in the exposed cells, resulting in further oxidative damage to lipids, proteins and DNA. To screen the potential radio-protective drug, the intracellular ROS was measured in irradiated U937 cells pretreated with 80 candidate traditional herbal medicine, respectively. Isofraxidin (IF) was one possible radio-protector in these 80 drugs. This study investigated the radio-protective role of IF, a Coumarin compound, in human leukemia cell lines, for the first time. Results indicate that IF protects against IR-induced apoptosis in U937 cells in the time- and concentration- dependent manner. IF decreases IR-induced intracellular ROS generation, especially hydroxyl radicals formation, inhibits IR-induced mitochondrial membrane potential loss and reduces IR-induced high intracellular Ca(2+) levels regardless of ER stress. IF down-regulates the expression of caspase-3, phospho-JNK, phospho-p38 and activates Bax in mitochondria. IF inhibits cytochrome c release from mitochondria to cytosol. IF also moderates IR-induced Fas externalization and caspase-8 activation. IF also exhibits significant protection against IR-induced cell death in other leukemia cell lines such as Molt-4 cells and HL60 cells regardless of p53. Taken together, the data demonstrate that IF protects leukemia cells from radiation-induced apoptosis via ROS/mitochondria pathway in a p53-independent manner.

The relationship between mucin phenotype and clinicopathological factors, proliferative activity and p53 expression in gastric hyperplastic polyps.

AIMS: Gastric hyperplastic polyps (GHPs) are the most common polypoid lesion of the stomach, and their malignant potential has been demonstrated. In the present study, we evaluated the mucin phenotypes of GHPs and investigated the relationships among mucin phenotypes and clinical-pathological factors, proliferative activity and p53 expression in GHPs. METHODS: The CD10, MUC2, MUC5AC and MUC6 expression patterns of 238 GHPs were examined by immunohistochemical staining. The GHP mucin phenotypes were divided into 4 subtypes: the gastric mucin phenotype (G-type), the intestinal mucin phenotype (I-type), the mixed or gastrointestinal mucin phenotype (GI-type) and the unclassified mucin phenotype (U-type). RESULTS: The G and GI types were observed in 58% and 42% of the GHPs, respectively. However, no I or U type GHPs were found in the present study. The GI type was more common in lesions with dysplasia or carcinoma than in polyps without dysplasia or carcinoma (P<0.001). P53-positivity rate and high index of Ki-67 tumors were significantly more common in the GI-type than in the G-type polyps (P<0.001). CONCLUSIONS: The mucin phenotype may serve as a useful marker for the malignant potential of GHPs, and GI-type GHPs should be considered to be a lesion with malignant potential.

BRAF mutation is associated with the unique morphology of traditional serrated adenoma of the colorectum.

Traditional serrated adenoma (TSA) consists of glands with tall cells and short cells. Two kinds of cells alternate to give a unique serrated configuration. The aim of this study was to identify the relationship between the alterations of both Wnt and serrated pathways and the unique morphology of TSAs. The tall and short cells in 28 TSAs were separated by microdissection. Semi-nested polymerase chain reaction was performed to detect the mutations of BRAF, ß-catenin, APC, and KRAS. BRAF mutations were observed in 22 of 28 (78.6%) TSAs, and all mutations occurred at the tall cells. In conclusion, BRAF mutation is associated with the serrated morphology of TSAs. Genetic alterations in both the serrated pathway and the Wnt signaling pathway may both contribute to TSAs.