Sarcoma care in the era of precision medicine.

Sarcoma subtype classification is currently mainly based upon histopathological morphology. Molecular analyses have emerged as an efficient addition to the diagnostic workup and sarcoma care. Knowledge about the sarcoma genome increases, and genetic events that can either support a histopathological diagnosis or suggest a differential diagnosis are identified, as well as novel therapeutic targets. In this review, we present diagnostic, therapeutic, and prognostic molecular markers that are, or might soon be, used clinically. For sarcoma diagnostics, there are specific fusions highly supportive or pathognomonic for a diagnostic entity-for instance, SYT::SSX in synovial sarcoma. Complex karyotypes also give diagnostic information-for example, supporting dedifferentiation rather than low-grade central osteosarcoma or well-differentiated liposarcoma when detected in combination with MDM2/CDK4 amplification. Molecular treatment predictive sarcoma markers are available for gastrointestinal stromal tumor (GIST) and locally aggressive benign mesenchymal tumors. The molecular prognostic markers for sarcomas in clinical practice are few. For solitary fibrous tumor, the type of NAB2::STAT6 fusion is associated with the outcome, and the KIT/PDGFRA pathogenic variant in GISTs can give prognostic information. With the exploding availability of sequencing technologies, it becomes increasingly important to understand the strengths and limitations of those methods and their context in sarcoma diagnostics. It is reasonable to believe that most sarcoma treatment centers will increase the use of massive-parallel sequencing soon. We conclude that the context in which the genetic findings are interpreted is of importance, and the interpretation of genomic findings requires considering tumor histomorphology.

TERT promoter mutation is an objective clinical marker for disease progression in chondrosarcoma.

Chondrosarcomas are the second most common malignant bone tumor. Activating promoter mutations in telomerase reverse transcriptase (TERT) was recently described by us and others as a frequent mutation in high-grade chondrosarcoma. In this study, we investigate the prognostic significance of TERT promoter mutations in 241 chondrosarcomas from 190 patients collected over 24 years (1994-2017). The TERT promoter was sequenced after microdissection of 135 chondrosarcomas from 106 patients in addition to data from our previous cohort. The TERT promoter mutation at -124 C > T was found in 45% of all patients and was significantly associated (p > 0,001) with higher tumor grade, shorter metastasis-free survival, and disease-specific survival. Additionally, TERT promoter-mutated tumors were associated with a more aggressive metastatic pattern. Shorter survival was observed in patients with wild-type primary tumors who developed a mutated metastasis indicative of tumor progression. Primary tumor genetic heterogeneity and altering mutational status between nonsynchronous metastatic lesions suggests that chondrosarcoma is a multiclonal disease progressing through a branching evolution. Conclusion: TERT promoter mutation seems to be a central event in chondrosarcoma progression with association to metastatic disease and disease-related mortality. As an easily analyzed marker, there is future potential to utilize TERT promoter mutation status as a prognostic marker and investigate telomerase-targeted therapy in chondrosarcomas.

Classical swine fever virus infection suppresses claudin-1 expression to facilitate its replication in PK-15 cells.

Classical swine fever (CSF) is one of the most epidemic viral diseases in swine industry. The causative pathogen is CSF virus (CSFV), a small enveloped RNA virus of Flaviviridae family. Claudin-1 was reported to be involved in the infections of a number of viruses, including many from Flaviviridae family, but no studies have investigated the role of porcine claudin-1 during CSFV infection in PK-15 cells. In this study, on the one hand, we demonstrated that CSFV infection reduced the claudin-1 expression at both mRNA and protein levels; on the other hand, CSFV infection was enhanced after claudin-1 knockdown, but inhibited by claudin-1 overexpression in a dose-dependent manner. Furthermore, negative correlation was demonstrated between the claudin-1 expression and CSFV titer. In conclusion, claudin-1 might be a barrier for CSFV infection in PK-15 cells, while CSFV bypasses the barrier through lysosome mediated degradation of claudin-1, which could be repressed by bafilomycin A1. Although the elaborate mechanisms how claudin-1 plays its roles in CSFV infection require further investigations, this study may advance our understanding of the molecular host-pathogen interaction mechanisms underlying CSFV infection and suggests enhancement of porcine claudin-1 as a potential preventive or therapeutic strategy for CSF control.

Decrease of morphine-CPP by sinomenine via mediation of tyrosine hydroxylase, NMDA receptor subunit 2B and opioid receptor in the zebrafish brain.

To study the effects of sinomenine on conditioned place preference (CPP) zebrafish induced by morphine and expression levels of intracephalic tyrosine hydroxylase (TH), NMDA receptor subunit 2B (NR2B), µ-opioid receptor (zfmor) and δ-opioid receptors (zfdor1 and zfdor2), morphine (40mg/kg) was administrated to zebrafish and the effect of CPP was detected in these zebrafish treated with sinomenine. The expression of TH and NR2B was detected by immunohistochemistry; and the mRNA expression of opioid receptors zfmor, zfdor1 and zfdor2 in the zebrafish brain was assayed by RT-qPCR. In the CPP test, morphine induced significant behavioral alteration, while pretreatment with sinomenine or methadone, resulted in decreased activity time in the morphine-paired compartment significantly. Morphine also increased the integral optical density value of TH- and NR2B-positive cells in the zebrafish brain, and reduced the amount of opioid receptors. However, the compound sinomenine could attenuate these effects. These findings demonstrate that sinomenine (80mg/kg) decreased the CPP effects of zebrafish induced by morphine significantly, downregulated expression of TH and NR2B, and upregulated µ-opioid (zfmor) and δ-opioid (zfdor1 and zfdor2) receptor expression in the CPP zebrafish brains.

High-throughput screening identified mitoxantrone to induce death of hepatocellular carcinoma cells with autophagy involvement.

The use of highly efficient high-throughput screening (HTS) platform has recently gained more attention as a plausible approach to identify de novo therapeutic application potential of conventional anti-tumor drugs for cancer treatments. In this study, we used hepatocellular carcinoma (HCC) cells as models to identify cytotoxic compounds by HTS. To identify cytotoxic compounds for potential HCC treatments, 3271 compounds from three well established small molecule libraries were screened against HCC cell lines. Thirty-two small molecules were identified from the primary screen to induce cell death. Particularly, mitoxantrone (MTX), which is an established antineoplastic drug, significantly and specifically inhibited the growth and proliferation of HCC cells in vitro. Mechanistic studies of LC3-II, p62 and phosphorylation of p70S6K in HepG2 cells revealed that MTX treatment induced mTOR-dependent autophagy activation, which was further confirmed by the autophagic flux assay using lysosomal inhibitor chloroquine (CQ). In the combined treatment of MTX and CQ, where autophagy was inhibited by CQ, the elevations of cleaved Caspase-3 and PARP were observed, indicating the enhanced apoptosis in HepG2 cells. Taken together, we hypothesize that MTX-induced autophagy plays an pro-survival role in HCC treatment. Combined treatment with autophagy inhibitor may combat the chemo-resistance of HCC to MTX treatment and therefore deserves future clinical investment.

Expression of miR-133a-5p and ROCK2 in Heart in Methamphetamine-Induced Rats and Intervention of Rhynchophylline.

INTRODUCTION: Rhynchophylline, as a traditional Chinese medicine, was used for the treatment of drug addiction. OBJECTIVE: To investigate miRNAs expression profile in the rat hearts of methamphetamine dependence and the intervention mechanisms of rhynchophylline. MATERIALS AND METHODS: This study detected the expression profile of miRNAs in the methamphetamine-induced rat hearts by microarray and verified the expression of miR-133a-5P and Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) protein. RESULTS: After conditioned place preference training, methamphetamine significantly increased the time spent in the drug-paired compartment, while rhynchophylline and MK-801 could reduce the time. Cluster analysis results of miRNAs showed that compared with the control group, the expression of miR-133a-5p in methamphetamine-induced rat hearts was decreased significantly; rhynchophylline could significantly increase the expression of miR-133a-5p. The result was verified by real-time polymerase chain reaction. The results of target gene predictive software and related research showed that ROCK2 protein may be the target gene of miR-133a-5p. The immunohistochemistry results of heart tissues showed that the expression of ROCK2 protein was significantly upregulated in the methamphetamine group and downregulate in the rhynchophylline group; the difference between the MK-801 group and the methamphetamine group was not significant. The result of western blot was consistent with the immunohistochemistry. CONCLUSION: The active ingredient of Chinese herbal medicine rhynchophylline can effectively inhibit the formation of methamphetamine-dependent conditional place preference (CPP) effect in rats to some extent. MiR-133a-5p may participate in the cardioprotective effects of CPP rats by targeting ROCK2.

Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

hTERT promoter mutations in chondrosarcomas associate with progression and disease-related mortality.

Chondrosarcomas are malignant skeletal tumors with chondroid differentiation. Prognosis is largely dependent on histological grading, which suffer from significant interobserver variability. Telomerase activity and abundant telomerase reverse transcriptase (hTERT) expression has previously been associated with chondrosarcoma grade and metastasis. We therefore analyzed the hTERT promoter in clinicopathologically well-characterized chondrosarcomas (grade 1-3) from 87 patients. Using Sanger sequencing we identified an activating -124 C > T mutation in 23 cases (26%). Promoter mutations were significantly associated with increased histological grade (8% of grade 1, 32% of grade 2 and 46% of grade 3, P = 0.002), suggesting a role in tumor progression. In four chondrosarcomas where the histopathological grade was heterogenous, the hTERT mutation was only identified in the higher-grade areas. Additionally, hTERT promoter mutations were significantly associated with worse metastasis-free survival (P = 0.018), chondrosarcoma-specific survival (P = 0.022) and older patient age (P = 0.003). These data suggest that hTERT promoter mutations are common in high grade conventional chondrosarcomas. Granted that additional studies can confirm these findings; hTERT promoter analysis could potentially serve as an adjuvant prognostic marker in routine chondrosarcoma grading. This study reinforces the rationale of telomerase targeted therapy in a subset of chondrosarcomas.

SUMO-modified insulin-like growth factor 1 receptor (IGF-1R) increases cell cycle progression and cell proliferation.

Increasing number of studies have shown nuclear localization of the insulin-like growth factor 1 receptor (nIGF-1R) in tumor cells and its links to adverse clinical outcome in various cancers. Any obvious cell physiological roles of nIGF-1R have, however, still not been disclosed. Previously, we reported that IGF-1R translocates to cell nucleus and modulates gene expression by binding to enhancers, provided that the receptor is SUMOylated. In this study, we constructed stable transfectants of wild type IGF1R (WT) and triple-SUMO-site-mutated IGF1R (TSM) using igf1r knockout mouse fibroblasts (R-). Cell clones (R-WT and R-TSM) expressing equal amounts of IGF-1R were selected for experiments. Phosphorylation of IGF-1R, Akt, and Erk upon IGF-1 stimulation was equal in R-WT and R-TSM. WT was confirmed to enter nuclei. TSM did also undergo nuclear translocation, although to a lesser extent. This may be explained by that TSM heterodimerizes with insulin receptor, which is known to translocate to cell nuclei. R-WT proliferated substantially faster than R-TSM, which did not differ significantly from the empty vector control. Upon IGF-1 stimulation G1-S-phase progression of R-WT increased from 12 to 38%, compared to 13 to 20% of R-TSM. The G1-S progression of R-WT correlated with increased expression of cyclin D1, A, and CDK2, as well as downregulation of p27. This suggests that SUMO-IGF-1R affects upstream mechanisms that control and coordinate expression of cell cycle regulators. Further studies to identify such SUMO-IGF-1R dependent mechanisms seem important.

The Nucleus-Localized Epidermal Growth Factor Receptor Is SUMOylated.

The epidermal growth factor receptor (EGFR) plays important roles in normal and cancer cell growth. The EGFR has principally two different signaling pathways: the canonical kinase route induced at the plasma membrane resulting in an intracellular phosphorylation cascade via MAPKs and PI3K and the more recently discovered pathway by which the receptor functions as a transcriptional co-activator inside the cell nucleus. Full length EGFR translocates to the inner nuclear membrane, via the endoplasmic reticulum, through association with the sec61ß translocon. The c-myc (MYC) and cyclin D1 (CNND1) genes represent two target genes for nuclear EGFR (nEGFR). Here we show that EGFR is SUMOylated and that the SUMO-1-modified receptors are almost unexceptionally nuclear. Co-immunoprecipitation experiments suggest that EGFR is multi-SUMOylated. Using two mass spectrometry-based strategies (matrix-assisted laser desorption ionization time of flight and electrospray ionization liquid chromatography with tandem mass spectrometry), lysine 37 was identified as a SUMO-1-modified residue by both methods. A lysine 37 site mutant (K37R) was transfected into EGFR deficient cells. Total SUMOylation of EGFR was not altered in the K37R-transfected cells, confirming the presence of other SUMOylation sites. To gain preliminary insight into the possible functional role of EGFR SUMOylation, we compared the effect of expression of the wild-type EGFR with the K37R mutant on promoter activity and expression of CMYC and CNND1. Our results indicate that SUMO-1 modification may affect the transcriptional activity of EGFR, which might have additional impact on, e.g., cancer progression.

Local control of nuclear calcium signaling in cardiac myocytes by perinuclear microdomains of sarcolemmal insulin-like growth factor 1 receptors.

RATIONALE: The ability of a cell to independently regulate nuclear and cytosolic Ca(2+) signaling is currently attributed to the differential distribution of inositol 1,4,5-trisphosphate receptor channel isoforms in the nucleoplasmic versus the endoplasmic reticulum. In cardiac myocytes, T-tubules confer the necessary compartmentation of Ca(2+) signals, which allows sarcomere contraction in response to plasma membrane depolarization, but whether there is a similar structure tunneling extracellular stimulation to control nuclear Ca(2+) signals locally has not been explored. OBJECTIVE: To study the role of perinuclear sarcolemma in selective nuclear Ca(2+) signaling. METHODS AND RESULTS: We report here that insulin-like growth factor 1 triggers a fast and independent nuclear Ca(2+) signal in neonatal rat cardiac myocytes, human embryonic cardiac myocytes, and adult rat cardiac myocytes. This fast and localized response is achieved by activation of insulin-like growth factor 1 receptor signaling complexes present in perinuclear invaginations of the plasma membrane. The perinuclear insulin-like growth factor 1 receptor pool connects extracellular stimulation to local activation of nuclear Ca(2+) signaling and transcriptional upregulation through the perinuclear hydrolysis of phosphatidylinositol 4,5-biphosphate inositol 1,4,5-trisphosphate production, nuclear Ca(2+) release, and activation of the transcription factor myocyte-enhancing factor 2C. Genetically engineered Ca(2+) buffers--parvalbumin--with cytosolic or nuclear localization demonstrated that the nuclear Ca(2+) handling system is physically and functionally segregated from the cytosolic Ca(2+) signaling machinery. CONCLUSIONS: These data reveal the existence of an inositol 1,4,5-trisphosphate-dependent nuclear Ca(2+) toolkit located in direct apposition to the cell surface, which allows the local control of rapid and independent activation of nuclear Ca(2+) signaling in response to an extracellular ligand.

Polysaccharide of Alocasia cucullata Exerts Antitumor Effect by Regulating Bcl-2, Caspase-3 and ERK1/2 Expressions during Long-Time Administration.

OBJECTIVE: To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism. METHODS: B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR. RESULTS: In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells. CONCLUSIONS: Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.

Comprehensive Genomic Profiling Alters Clinical Diagnoses in a Significant Fraction of Tumors Suspicious of Sarcoma.

PURPOSE: Tumor classification is a key component in personalized cancer care. For soft-tissue and bone tumors, this classification is currently based primarily on morphology assessment and IHC staining. However, these standard-of-care methods can pose challenges for pathologists. We therefore assessed how whole-genome and whole-transcriptome sequencing (WGTS) impacted tumor classification and clinical management when interpreted together with histomorphology. EXPERIMENTAL DESIGN: We prospectively evaluated WGTS in routine diagnostics of 200 soft-tissue and bone tumors suspicious for malignancy, including DNA and RNA isolation from the tumor, and DNA isolation from a peripheral blood sample or any non-tumor tissue. RESULTS: On the basis of specific genomic alterations or absence of presumed findings, WGTS resulted in reclassification of 7% (13/197) of the histopathologic diagnoses. Four cases were downgraded from low-grade sarcomas to benign lesions, and two cases were reclassified as metastatic malignant melanomas. Fusion genes associated with specific tumor entities were found in 30 samples. For malignant soft-tissue and bone tumors, we identified treatment relevant variants in 15% of cases. Germline pathogenic variants associated with a hereditary cancer syndrome were found in 22 participants (11%). CONCLUSIONS: WGTS provides an important dimension of data that aids in the classification of soft-tissue and bone tumors, correcting a significant fraction of clinical diagnoses, and identifies molecular targets relevant for precision medicine. However, genetic findings need to be evaluated in their morphopathologic context, just as germline findings need to be evaluated in the context of patient phenotype and family history.

Host miR-129-5p reverses effects of ginsenoside Rg1 on morphine reward possibly mediated by changes in B. vulgatus and serotonin metabolism in hippocampus.

Morphine addiction is closely associated with dysbiosis of the gut microbiota. miRNAs play a crucial role in regulating intestinal bacterial growth and are involved in the development of disease. Ginsenoside Rg1 exhibits an anti-addiction effect and significantly improves intestinal microbiota disorders. In pseudo-germfree mice, supplementation with Bacteroides vulgatus (B. vulgatus) synergistically enhanced Rg1 to alleviate morphine addiction. However, it is currently unknown the relationship between fecal miRNAs in morphine-exposed mice and their potential modulation of gut microbiome, as well as their role in mediating the resistance of ginsenoside Rg1 to drug addiction. Here, we studied the fecal miRNA abundance in mice treated with morphine to explore the different miRNAs expressed, their association with B. vulgatus and their role in the amelioration of morphine reward by ginsenoside Rg1. Our results indicated ginsenoside Rg1 attenuated the significant increase in miR-129-5p expression observed in the feces of morphine-treated mice. The miR-129-5p, specifically, inhibited the growth of B. vulgatus by modulating the transcript of the site-tag BVU_RS11835 and increased the levels of 5-hydroxytryptophan and indole-3-carboxaldehyde in vitro. Subsequently, we noticed that oral administration of synthetic miR-129-5p increased 5-HT levels in the hippocampus and inhibited the reversal effect of ginsenoside Rg1 both on the relative abundance of B. vulgatus in the feces and CPP effect induced by morphine exposure. In short, Ginsenoside Rg1 might play an indirect role in remodeling the B. vulgatus against morphine reward by suppressing miR-129-5p expression. These results highlight the role of miR-129-5p and B. vulgatus in morphine reward and the anti-morphine addiction of ginsenoside Rg1.

Long non-coding RNA LOC103222771 promotes infection of porcine reproductive and respiratory syndrome virus in Marc-145 cells by downregulating Claudin-4.

Porcine reproductive and respiratory syndrome (PRRS) is an important swine disease caused by infection of porcine reproductive and respiratory syndrome virus (PRRSV), which leads to huge loss in swine industry. How to effectively control PRRS is challenging. Long non-coding RNA (lncRNA) are key regulator of viral infections and anti-virus immunological responses, therefore, further understanding of lncRNAs will aid to identification of novel regulators of viral infections and better design of prevention and control strategies to viral infection related diseases and immune disorders. We demonstrated that PRRSV infection upregulated the expression of lncRNA LOC103222771 in Marc-145 cells and porcine alveolar macrophage cells (PAMs) and that LOC103222771 is mainly located in cytoplasm. Knockdown of LOC103222771 could inhibit the PRRSV infection in Marc-145 cells. RNA-seq analysis and subsequent validation revealed increased expression of Claudin-4 (CLDN4) in Marc-145 when LOC103222771 was specifically downregulated，suggesting that LOC103222771 might be an upstream regulator of CLDN4, an important component of tight junctions for establishment of the paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. We and others showed that Downregulation of CLDN4 could boost the infection of PRRSV. Collectively, LOC103222771/CLDN4 signal axis might be a novel mechanism of PRRSV pathogenesis, implying a potential therapeutic target against PRRSV infection.

RACK1 promotes porcine reproductive and respiratory syndrome virus infection in Marc-145 cells through ERK1/2 activation.

Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease that spreads rapidly among pigs and seriously threatens the pig industry. Activation of ERK1/2 is a hallmark of most viral infections. RACK1 interacts with a variety of kinases and membrane receptors that closely associated with viral infections and the development and progression of cancer. However, no studies have clearly defined whether RACK1 can regulate PRRSV infection through ERK1/2 activation. In our study, using RT-qPCR, immunoblotting, indirect fluorescent staining, siRNA knockdown and protein overexpression techniques, we found that downregulation of cellular RACK1 inhibited ERK1/2 activation and subsequently suppressed PRRSV infection, while overexpression of RACK1 enhanced ERK1/2 activation and PRRSV infection. Bioinformatic and Co-immunoprecipitation experimental analysis revealed that cellular RACK1 could interact with viral N protein to exert its function. We elaborated that RACK1 promoted PRRSV replication in Marc-145 cells through ERK1/2 activation. Our study provides new insights into regulating the innate antiviral immune responses during PRRSV infection and contributes to further understanding of the molecular mechanisms underlying PRRSV replication.

Nuclear HER3 is associated with favorable overall survival in uveal melanoma.

HER3 is a member of the epidermal growth factor receptor (EGFR) family and is expressed in several types of cancer. Both the cytoplasmic and nuclear appearances of the receptor have been reported. Here, we investigate the expression and subcellular distribution of HER3 in uveal melanoma (UM) cells and tissues and its potential impact on clinical outcome of patients. Paraffin-embedded samples from 128 consecutive UM patients, enucleated without alternative treatment on UM diagnosis, were evaluated for HER3 using immunohistochemistry. Immunoreactivity was scored for frequency, intensity of positive cells, and subcellular distribution. The results were correlated with the established clinicopathological parameters using univariate and multivariate statistical analyses. HER3 expression was shown in 70% of the cases (89/128). This contrasts with the other EGFR family receptors (EGFR, HER2 and HER4) that are infrequently expressed in UM. Surprisingly, HER3 was found to be localized solely in the cell nuclei in 56 cases. The remaining 33 HER3 positive cases showed diffuse distribution (cytoplasmic ± nuclear). Nuclear HER3 was independently correlated with a more favorable overall survival (p = 0.043 and hazard ratio = 0.618) compared to cases with diffuse and/or no HER3. Nuclear localization of HER3 was also confirmed in fresh UM material and in UM cell lines. In conclusion, HER3 is frequently localized solely in the cell nuclei in UM and as such it predicts a more favorable overall survival.

High Expression of TGF-ß1 Contributes to Hepatocellular Carcinoma Prognosis via Regulating Tumor Immunity.

Background: Transforming growth factor-beta (TGF-ß) signaling is essential in initialization and progression of hepatocellular carcinoma (HCC). Therefore, a treatment targeting TGF-ß pathway may be a promising option for HCC control. Methods: First, publicly available RNA-seq datasets and clinical characteristics of 374 HCC patients in The Cancer Genome Atlas (TCGA) database were downloaded. Then, Cox regression analysis and LASSO analysis were used to construct a prognostic model for TGF-ß family genes. The area under the curve (AUC) of the risk signature was calculated to evaluate the predictive power of the model. Cox regression analysis was applied to predict whether TGF-ß1 can be an independent prognosis factor for HCC. Next, hazard ratio and survival analyses were performed to investigate the correlation between TGF-ß1 expression and survival time. Furthermore, differential expression level of TGF-ß1 in HCC tissues and cells was determined. In addition, Gene Set Enrichment Analysis (GSEA) identified the top significantly activated and inhibited signal pathways related to high expression of TGF-ß1. Finally, the CIBERSORT tool was adopted to correlate the tumor-infiltrating immune cells (TICs) with TGF-ß1 expression in HCC cohorts. Results: Cox regression analysis and LASSO analysis revealed that seven TGF-ß family members (including TGF-ß1) could be used as prognostic factors for HCC. Interestingly, TGF-ß1 was demonstrated to be an independent prognostic factor of HCC. RT-qPCR and immunofluorescence staining confirmed the high expression of TGF-ß1 in HCC cell lines and tissues, which is significantly related to pathological classifications, poor prognosis, and short survival time. Finally, GSEA and CIBERSORT analyses suggested that TGF-ß1 may interact with various immune cells and influence the prognosis of HCC patients through Tregs and γδ T cells. Conclusion: We established a novel prognostic prediction method to predict the risk scores of TGF-ß genes in HCC prognosis. TGF-ß1 is highly expressed in HCC cell lines and tissues, correlates to poor prognosis, and thus can be used as a potential biomarker to predict HCC prognosis. We showed that TGF-ß1 may play its roles in HCC prognosis by modulating the immune microenvironment of tumor cells. Our data may shed more light on better understanding the role of TGF-ß1 in HCC prognosis.

Ginsenoside Rg1 mitigates morphine dependence via regulation of gut microbiota, tryptophan metabolism, and serotonergic system function.

BACKGROUND: Morphine dependence, a devastating neuropsychiatric condition, may be closely associated with gut microbiota dysbiosis. Ginsenoside Rg1 (Rg1), an active ingredient extracted from the roots of Panax ginseng C.A. Meyer, has potential health-promoting effects on the nervous system. However, its role in substance use disorders remains unclear. Here, we explored the potential modulatory roles of Rg1 in protection against morphine dependence. METHODS: Conditioned place preference (CPP) was used for establishing a murine model of morphine dependence. 16S rRNA gene sequencing and metabolomics were performed for microbial and metabolite analysis. Molecular analysis was tested for evaluating the host serum and brain responses. RESULTS: Rg1 prevented morphine-induced CPP in mice. The 16S rRNA gene-based microbiota analysis demonstrated that Rg1 ameliorated morphine-induced gut microbiota dysbiosis, specifically for Bacteroidetes. Moreover, Rg1 also inhibited gut microbiota-derived tryptophan metabolism and reduced the serotonin, 5-hydroxytryptamine receptor 1B (5-HTR1B), and 5-hydroxytryptamine receptor 2 A (5-HTR2A) levels. However, the Rg1-induced amelioration of CPP was not observed in mice when their gut microbiome was depleted by non-absorbable antibiotics. Subsequently, gavage with Bacteroides vulgatus increased the abundance of Bacteroidetes. B. vulgatus supplementation synergistically enhanced Rg1-alleviated morphine-induced CPP in mice with microbiome knockdown. Co-treatment with B. vulgatus and Rg1 produced suppressive effects against morphine dependency by inhibiting tryptophan metabolism and reducing the serotonin and 5-HTR1B/5-HTR2A levels. CONCLUSIONS: The gut microbiota-tryptophan metabolism-serotonin plays an important role in gut-brain signaling in morphine disorders, which may represent a novel approach for drug dependence treatment via manipulation of the gut microbial composition and tryptophan metabolite.

Over-accumulation of nuclear IGF-1 receptor in tumor cells requires elevated expression of the receptor and the SUMO-conjugating enzyme Ubc9.

The insulin-like growth factor 1 receptor (IGF-1R) plays crucial roles in tumor cell growth and is overexpressed in many cancers. IGF-1R's trans-membrane kinase signaling pathways have been well characterized. Very recently, we showed that SUMOylation mediates nuclear translocation of the IGF-1R, and that nuclear IGF-1R (nIGF-1R) binds to enhancer regions and activates transcription. We identified three lysine residues in the ß-subunit of the receptor and that mutation of these blocks nuclear translocation and gene activation. Furthermore, accumulation of nIGF-1R was proven strongly dependent on the specific SUMO-conjugating enzyme Ubc9. Here we show that nIGF-1R originates solely from the cell membrane and that phosphorylation of the core tyrosine residues of the receptor kinase is crucial for nuclear accumulation. We also compared the levels of nIGF-1R, measured as nuclear/membrane ratios, in tumor and normal cells. We found that the breast cancer cell line MCF-7 has 13-fold higher amounts of nIGF-1R than breast epithelial cells (IME) which showed only a small amount of nIGF-1R. In comparison, the total expression of IGF-1R was only 3.7- higher in MCF-7. Comparison of several other tumor and normal cell lines showed similar tumor cell over-accumulation of nIGF-1R, exceeding the total receptor expression substantially. Ectopic overexpression (>10-fold) of the receptor increased nIGF-1R in IME cells but not to that high level as in wild type MCF-7. The levels of Ubc9 were higher in all tumor cell lines, compared to the normal cells, and this probably contributes to over-accumulation of nIGF-1R. Over-accumulation of nIGF-1R may contribute to deregulated gene expression and therewith play a pathophysiological role in cancer cells.