A New Systemic Disease Mouse Model for Glioblastoma Capable of Single-Tumour-Cell Detection.

BACKGROUND: Glioblastoma is characterised by extensive infiltration into the brain parenchyma, leading to inevitable tumor recurrence and therapeutic failure. Future treatments will need to target the specific biology of tumour recurrence, but our current understanding of the underlying mechanisms is limited. Significantly, there is a lack of available methods and models that are tailored to the examination of tumour recurrence. METHODS: NOD-SCID mice were orthotopically implanted with luciferase-labelled donor U87MG or MU20 glioblastoma cells. Four days later, an unlabelled recipient tumor was implanted on the contralateral side. The mice were euthanised at a humane end-point and tissue and blood samples were collected for ex vivo analyses. RESULTS: The ex vivo analyses of the firefly-labelled MU20 tumours displayed extensive invasion at the primary tumour margins, whereas the firefly-labelled U87MG tumours exhibited expansive phenotypes with no evident invasions at the tumour margins. Luciferase signals were detected in the contralateral unlabelled recipient tumours for both the U87MG and MU20 tumours compared to the non-implanted control brain. Remarkably, tumour cells were uniformly detected in all tissue samples of the supratentorial brain region compared to the control tissue, with single tumour cells detected in some tissue samples. Circulating tumour cells were also detected in the blood samples of most of the xenografted mice. Moreover, tumour cells were detected in the lungs of all of the mice, a probable event related to haematogenous dissemination. Similar results were obtained when the U87MG cells were alternatively labelled with gaussian luciferase. CONCLUSIONS: These findings describe a systemic disease model for glioblastoma which can be used to investigate recurrence biology and therapeutic efficacy towards recurrence.

Signaling pathways underlying TGF-ß mediated suppression of IL-12A gene expression in monocytes.

Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine essential for multiple biological processes, including the regulation of inflammatory and immune responses. One of the important functions of TGF-ß is the suppression of the proinflammatory cytokine interleukin-12 (IL-12), which is crucial for mounting an anti-tumorigenic response. Although the regulation of the IL-12p40 subunit (encoded by the IL-12B gene) of IL-12 has been extensively investigated, the knowledge of IL-12p35 (encoded by IL-12A gene) subunit regulation is relatively limited. This study investigates the molecular regulation of IL-12A by TGF-ß-activated signaling pathways in THP-1 monocytes. Our study identifies a complex regulation of IL-12A gene expression by TGF-ß, which involves multiple cellular signaling pathways, such as Smad2/3, NF-κB, p38 and JNK1/2. Pharmacological inhibition of NF-κB signaling decreased IL-12A expression, while blocking the Smad2/3 signaling pathway by overexpression of Smad7 and inhibiting JNK1/2 signaling with a pharmacological inhibitor, SP600125, increased its expression. The elucidated signaling pathways that regulate IL-12A gene expression potentially provide new therapeutic targets to increase IL-12 levels in the tumor microenvironment.

A New Era of Integration between Multiomics and Spatio-Temporal Analysis for the Translation of EMT towards Clinical Applications in Cancer.

Epithelial-mesenchymal transition (EMT) is crucial to metastasis by increasing cancer cell migration and invasion. At the cellular level, EMT-related morphological and functional changes are well established. At the molecular level, critical signaling pathways able to drive EMT have been described. Yet, the translation of EMT into efficient diagnostic methods and anti-metastatic therapies is still missing. This highlights a gap in our understanding of the precise mechanisms governing EMT. Here, we discuss evidence suggesting that overcoming this limitation requires the integration of multiple omics, a hitherto neglected strategy in the EMT field. More specifically, this work summarizes results that were independently obtained through epigenomics/transcriptomics while comprehensively reviewing the achievements of proteomics in cancer research. Additionally, we prospect gains to be obtained by applying spatio-temporal multiomics in the investigation of EMT-driven metastasis. Along with the development of more sensitive technologies, the integration of currently available omics, and a look at dynamic alterations that regulate EMT at the subcellular level will lead to a deeper understanding of this process. Further, considering the significance of EMT to cancer progression, this integrative strategy may enable the development of new and improved biomarkers and therapeutics capable of increasing the survival and quality of life of cancer patients.

Simultaneously targeting extracellular vesicle trafficking and TGF-ß receptor kinase activity blocks signaling hyperactivation and metastasis.

Metastasis is the leading cause of cancer-related deaths. Transforming growth factor beta (TGF-ß) signaling drives metastasis and is strongly enhanced during cancer progression. Yet, the use of on-target TGF-ß signaling inhibitors in the treatment of cancer patients remains unsuccessful, highlighting a gap in the understanding of TGF-ß biology that limits the establishment of efficient anti-metastatic therapies. Here, we show that TGF-ß signaling hyperactivation in breast cancer cells is required for metastasis and relies on increased small extracellular vesicle (sEV) secretion. Demonstrating sEV's unique role, TGF-ß signaling levels induced by sEVs exceed the activity of matching concentrations of soluble ligand TGF-ß. Further, genetic disruption of sEV secretion in highly-metastatic breast cancer cells impairs cancer cell aggressiveness by reducing TGF-ß signaling to nearly-normal levels. Otherwise, TGF-ß signaling activity in non-invasive breast cancer cells is inherently low, but can be amplified by sEVs, enabling invasion and metastasis of poorly-metastatic breast cancer cells. Underscoring the translational potential of inhibiting sEV trafficking in advanced breast cancers, treatment with dimethyl amiloride (DMA) decreases sEV secretion, TGF-ß signaling activity, and breast cancer progression in vivo. Targeting both the sEV trafficking and TGF-ß signaling by combining DMA and SB431542 at suboptimal doses potentiated this effect, normalizing the TGF-ß signaling in primary tumors to potently reduce circulating tumor cells, metastasis, and tumor self-seeding. Collectively, this study establishes sEVs as critical elements in TGF-ß biology, demonstrating the feasibility of inhibiting sEV trafficking as a new therapeutic approach to impair metastasis by normalizing TGF-ß signaling levels in breast cancer cells.

Dynamics of transforming growth factor ß signaling and therapeutic efficacy.

Transforming growth factor ß (TGFß) is a multifunctional cytokine, and its signalling responses are exerted via integrated intracellular pathways and complex regulatory mechanisms. Due to its high potency, TGFß signalling is tightly controlled under normal circumstances, while its dysregulation in cancer favours metastasis. The recognised potential of TGFß as a therapeutic target led to emerging development of anti-TGFß reagents with preclinical success, yet these therapeutics failed to recapitulate their efficacy in experimental settings. In this review, possible reasons for this inconsistency are discussed, addressing the knowledge gap between theoretical and actual behaviours of TGFß signalling. Previous studies on oncogenic cells have demonstrated the spatiotemporal heterogeneity of TGFß signalling intensity. Under feedback mechanisms and exosomal ligand recycling, cancer cells may achieve cyclic TGFß signalling to facilitate dissemination and colonisation. This challenges the current presumption of persistently high TGFß signalling in cancer, pointing to a new direction of research on TGFß-targeted therapeutics.

Activin and BMP Signalling in Human Testicular Cancer Cell Lines, and a Role for the Nucleocytoplasmic Transport Protein Importin-5 in Their Crosstalk.

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-ß, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-ß, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-ß superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-ß signalling pathway outcomes in TGCTs.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Discovery and exploration of widespread infection of mycoviruses in Phomopsis vexans, the causal agent of phomopsis blight of eggplant in China.

Phomopsis vexans, which causes Phomopsis blight of eggplant, has been reported worldwide. To study the biocontrol of this disease, 162 leaf and fruit samples of eggplant Phomopsis blight were collected from Hunan, Hubei, Jiangxi, Sichuan, Zhejiang, Fujian, Guangdong and Anhui Provinces from 2017 to 2019. Eighty-seven pathogenic fungus isolates were identified as P. vexans. The following studies were conducted: screening of sporulation medium, spore morphology analysis, mycovirus detection and identification of novel mycoviruses in these isolates. The results showed that eggplant tissue medium was the most suitable medium for rapid sporulation, and all isolates had mycoviruses consisting of mainly mixed infections. The genome of these mycoviruses varied from 1-15 kb. Five novel mycoviruses infecting P. vexans were obtained, including "Phomopsis vexans fusarivirus 1" (PvFV1), "Phomopsis vexans ourmia-like virus 1" (PvOLV1), "Phomopsis vexans endornavirus 2" (PvEV2), "Phomopsis vexans partitivirus 1" (PvPV1) and "Phomopsis vexans victorivirus L1" (PvVVL1). Thus, PvVVL1 displays a unique genome structure, and this is the first report of a victorivirus consisting of two segments and of a deltapartitivirus infecting the fungus host.

Fast Quantitation of TGF-ß Signaling Using Adenoviral Reporter.

The transforming growth factor-ß (TGF-ß) is a multifunctional cytokine critical for embryogenesis and tissue homeostasis. Alterations in TGF-ß signaling pathway are observed in several types of malignant tumors and often related with cancer progression and metastasis. TGF-ß signaling is transduced across the plasma membrane after ligand-receptor binding and consequent phosphorylation of the intracellular effectors SMAD2/3 by TGF-ß receptors. Phosphorylated SMAD2/3 accumulates in the nucleus after complex formation with SMAD4 to act as transcription factors and regulate the expression of genes critically associated with cell proliferation and differentiation. Traditional methodologies used to assess TGF-ß signaling pathway lack accuracy and/or show poor scalability, limiting in vitro experiments and almost excluding their use in vivo. Here, we describe a fast method to quantitate TGF-ß signaling pathway activity in vitro and in vivo by using adenoviral reporters. Its implementation in vitro allows quantitating cell response to TGF-ß at concentrations as low as pictograms/mL. Additionally, the use of an in vivo imaging system (IVIS) enables quantitating and monitoring TGF-ß signaling pathway activity over time during cancer progression, eliminating the requirement of animal euthanasia at multiple time points for this purpose. Importantly, this protocol has been consistently used in different models and effectively led to the visualization and measurement of TGF-ß activity levels. Improving the sensitivity, specificity, and scalability of methods focused on characterizing this and other molecular pathways will result in a better understanding of their biology in physiological and pathological processes.

Cancer associated-fibroblast-derived exosomes in cancer progression.

To identify novel cancer therapies, the tumor microenvironment (TME) has received a lot of attention in recent years in particular with the advent of clinical successes achieved by targeting immune checkpoint inhibitors (ICIs). The TME consists of multiple cell types that are embedded in the extracellular matrix (ECM), including immune cells, endothelial cells and cancer associated fibroblasts (CAFs), which communicate with cancer cells and each other during tumor progression. CAFs are a dominant and heterogeneous cell type within the TME with a pivotal role in controlling cancer cell invasion and metastasis, immune evasion, angiogenesis and chemotherapy resistance. CAFs mediate their effects in part by remodeling the ECM and by secreting soluble factors and extracellular vesicles. Exosomes are a subtype of extracellular vesicles (EVs), which contain various biomolecules such as nucleic acids, lipids, and proteins. The biomolecules in exosomes can be transmitted from one to another cell, and thereby affect the behavior of the receiving cell. As exosomes are also present in circulation, their contents can also be explored as biomarkers for the diagnosis and prognosis of cancer patients. In this review, we concentrate on the role of CAFs-derived exosomes in the communication between CAFs and cancer cells and other cells of the TME. First, we introduce the multiple roles of CAFs in tumorigenesis. Thereafter, we discuss the ways CAFs communicate with cancer cells and interplay with other cells of the TME, and focus in particular on the role of exosomes. Then, we elaborate on the mechanisms by which CAFs-derived exosomes contribute to cancer progression, as well as and the clinical impact of exosomes. We conclude by discussing aspects of exosomes that deserve further investigation, including emerging insights into making treatment with immune checkpoint inhibitor blockade more efficient.

CSK-homologous kinase (CHK/MATK) is a potential colorectal cancer tumour suppressor gene epigenetically silenced by promoter methylation.

Hyperactivation of SRC-family protein kinases (SFKs) contributes to the initiation and progression of human colorectal cancer (CRC). Since oncogenic mutations of SFK genes are rare in human CRC, we investigated if SFK hyperactivation is linked to dysregulation of their upstream inhibitors, C-terminal SRC kinase (CSK) and its homolog CSK-homologous kinase (CHK/MATK). We demonstrate that expression of CHK/MATK but not CSK was significantly downregulated in CRC cell lines and primary tumours compared to normal colonic tissue. Investigation of the mechanism by which CHK/MATK expression is down-regulated in CRC cells uncovered hypermethylation of the CHK/MATK promoter in CRC cell lines and primary tumours. Promoter methylation of CHK/MATK was also observed in several other tumour types. Consistent with epigenetic silencing of CHK/MATK, genetic deletion or pharmacological inhibition of DNA methyltransferases increased CHK/MATK mRNA expression in CHK/MATK-methylated colon cancer cell lines. SFKs were hyperactivated in CHK/MATK-methylated CRC cells despite expressing enzymatically active CSK, suggesting loss of CHK/MATK contributes to SFK hyperactivation. Re-expression of CHK/MATK in CRC cell lines led to reduction in SFK activity via a non-catalytic mechanism, a reduction in anchorage-independent growth, cell proliferation and migration in vitro, and a reduction in tumour growth and metastasis in a zebrafish embryo xenotransplantation model in vivo, collectively identifying CHK/MATK as a novel putative tumour suppressor gene in CRC. Furthermore, our discovery that CHK/MATK hypermethylation occurs in the majority of tumours warrants its further investigation as a diagnostic marker of CRC.

Transglutaminase-2, RNA-binding proteins and mitochondrial proteins selectively traffic to MDCK cell-derived microvesicles following H-Ras-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) describes an evolutionary conserved morphogenic process defined by loss of epithelial characteristics and acquisition of mesenchymal phenotype, and altered patterns of intercellular communication, leading to functional changes in cell migration and invasion. In this regard, we have previously reported that oncogenic H-Ras induced EMT in Madin-Darby Canine Kidney (MDCK) cells (21D1 cells) trigger changes in the protein distribution pattern in cells, exosomes, and soluble protein factors (secretome) which modulate the tumor microenvironment. Here, we report that shed microvesicles (also termed microparticles/ectosomes) secreted from MDCK cells following oncogenic H-Ras-induced EMT (21D1-sMVs) are biochemically distinct from exosomes and parental MDCK-sMVs. The protein spectra of RNA-binding proteins and mitochondrial proteins in 21D1-sMVs differ profoundly compared to those of exosomes, likewise proteins associated with suppression of anoikis. We show that 21D1-sMVs promote cell migration, confer anchorage-independent growth, and induce EMT in parental MDCK cells. An unexpected and novel finding was the selective sorting of tissue transglutaminase-2 (TGM2) into 21D1-sMVs; there was no evidence of TGM2 in MDCK-sMVs. Prior treatment of 21D1-sMVs with neutralizing anti-TGM2 or anti-FN1 antibodies attenuates the invasive capability of fibroblasts. These finding suggest that microvesicle-associated TGM2 may play an important contributory role in the EMT process and warrants further investigation.

Complete nucleotide sequence of a novel partitivirus infecting the plant-pathogenic fungus Phomopsis vexans.

Here, we report the molecular characterization of a novel partitivirus from Phomopsis vexans strain PvHZ002, a plant-pathogenic fungus infecting eggplant. The virus was designated "Phomopsis vexans partitivirus 1" (PvPV1). PvPV1 contains two dsRNA segments, dsRNA1 and dsRNA2, which are 1,662 bp and 1,628 bp long, respectively. Each segment contains a single open reading frame, putatively encoding RNA-dependent RNA polymerase (dsRNA 1) and capsid protein (dsRNA 2). A homology search and phylogenetic analysis showed that PvPV1 clustered with viruses of the genus Deltapartitivirus of the family Partitiviridae.

On-Target Anti-TGF-ß Therapies Are Not Succeeding in Clinical Cancer Treatments: What Are Remaining Challenges?

Metastasis is the leading cause of death for cancer patients. During cancer progression, the initial detachment of cells from the primary tumor and the later colonization of a secondary organ are characterized as limiting steps for metastasis. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are opposite dynamic multistep processes that enable these critical events in metastasis by altering the phenotype of cancer cells and improving their ability to migrate, invade and seed at distant organs. Among the molecular pathways that promote tumorigenesis in late-stage cancers, transforming growth factor-ß (TGF-ß) is described as an EMT master inducer by controlling different genes and proteins related to cytoskeleton assembly, cell-cell attachment and extracellular matrix remodeling. Still, despite the successful outcomes of different TGF-ß pharmacological inhibitors in cell culture (in vitro) and animal models (in vivo), results in cancer clinical trials are poor or inconsistent at least, highlighting the existence of crucial components in human cancers that have not been properly explored. Here we review most recent findings to provide perspectives bridging the gap between on-target anti-TGF-ß therapies in vitro and in pre-clinical models and the poor clinical outcomes in treating cancer patients. Specifically, we focus on (i) the dual roles of TGF-ß signaling in cancer metastasis; (ii) dynamic signaling; (iii) functional differences of TGF-ß free in solution vs. in exosomes; (iv) the regulatory effects of tumor microenvironment (TME) - particularly by cancer-associated fibroblasts - on TGF-ß signaling pathway. Clearly identifying and establishing those missing links may provide strategies to revitalize and clinically improve the efficacy of TGF-ß targeted therapies.

Reactivation of BMP signaling by suboptimal concentrations of MEK inhibitor and FK506 reduces organ-specific breast cancer metastasis.

TGFß-SMAD3 signaling is a major driving force for cancer metastasis, while BMP-SMAD1/5 signaling can counteract this response. Analysis of gene expression profiles revealed that an increased TGFß-SMAD3 and a reduced BMP-SMAD1/5 targeted gene expression signature correlated with shortened distant metastasis free survival and overall survival of patients. At molecular levels, we discovered that TGFß abolished BMP-induced SMAD1/5 activation in the highly-invasive breast cancer MDA-MB-231 cells, but to a less extent in the non-invasive cancer and normal breast cells. This suggests an inverse correlation between BMP signaling and invasiveness of tumor cells and TGFß signaling acts in a double whammy fashion in driving cancer invasion and metastasis. Sustained ERK activation by TGFß was specifically observed in MDA-MB-231 cells, and MEK inhibitor (MEKi) treatment restored BMP-SMAD1/5 signaling while not affecting SMAD2/3 activation. FK506 potently activated BMP, but not TGFß signaling in breast cancer cells. MEKi or FK506 alone inhibited MDA-MB-231 extravasation in a zebrafish xenograft cancer model. Importantly, when administrated at suboptimal concentrations MEKi and FK506 strongly synergized in promoting BMP-SMAD1/5 signaling and inhibiting cancer cell extravasation. Furthermore, this combination of suboptimal concentrations treatment in a mouse tumor model resulted in real-time reduction of BMP-SMAD1/5 signaling in live tumors, and consequently potently inhibited tumor self-seeding, liver and bone metastasis, but not lung and brain metastasis. Mechanistically, it is the first time to identify BMP-SMAD1/5 signaling as an underlying molecular driver for organ-specific metastasis. Combining of MEKi and FK506, or their analogues, may be explored for clinical development of breast cancer.

Therapeutic Reversal of Radiotherapy Injury to Pro-fibrotic Dysfunctional Fibroblasts In Vitro Using Adipose-derived Stem Cells.

Cancer patients often require radiotherapy (RTx) to enhance their survival. Unfortunately, RTx also damages nearby healthy non-cancer tissues, leading to progressive fibrotic soft-tissue injury, consisting of pain, contracture, tissue-breakdown, infection, and lymphoedema. Mechanisms underlying the clinically observed ability of fat grafting to ameliorate some of these effects, however, are poorly understood. It was hypothesized that RTx significantly alters fibroblast cell function and the paracrine secretome of adipose-derived stem cells (ADSC) may mitigate these changes. METHODS: To investigate cellular changes resulting in the fibrotic side-effects of RTx, cultured normal human dermal fibroblasts (NHDF) were irradiated (10Gy), then studied using functional assays that reflect key fibroblast functions, and compared with unirradiated controls. RNA-Seq and targeted microarrays (with specific examination of TGFß) were performed to elucidate altered gene pathways. Finally, conditioned-media from ADSC was used to treat irradiated fibroblasts and model fat graft surgery. RESULTS: RTx altered NHDF morphology, with cellular functional changes reflecting transition into a more invasive phenotype: increased migration, adhesion, contractility, and disordered invasion. Changes in genes regulating collagen and MMP homeostasis and cell-cycle progression were also detected. However, TGFß was not identified as a key intracellular regulator of the fibroblast response. Finally, treatment with ADSC-conditioned media reversed the RTx-induced hypermigratory state of NHDF. CONCLUSIONS: Our findings regarding cellular and molecular changes in irradiated fibroblasts help explain clinical manifestations of debilitating RTx-induced fibrosis. ADSC-secretome-mediated reversal indicated that these constituents may be used to combat the devastating side-effects of excessive unwanted fibrosis in RTx and other human fibrotic diseases.

Deubiquitinase Activity Profiling Identifies UCHL1 as a Candidate Oncoprotein That Promotes TGFß-Induced Breast Cancer Metastasis.

PURPOSE: Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification of highly active DUBs in TNBC may lead to novel therapies. EXPERIMENTAL DESIGN: Using DUB activity probes, we profiled global DUB activities in 52 breast cancer cell lines and 52 patients' tumor tissues. To validate our findings in vivo, we employed both zebrafish and murine breast cancer xenograft models. Cellular and molecular mechanisms were elucidated using in vivo and in vitro biochemical methods. A specific inhibitor was synthesized, and its biochemical and biological functions were assessed in a range of assays. Finally, we used patient sera samples to investigate clinical correlations. RESULTS: Two DUB activity profiling approaches identified UCHL1 as being highly active in TNBC cell lines and aggressive tumors. Functionally, UCHL1 promoted metastasis in zebrafish and murine breast cancer xenograft models. Mechanistically, UCHL1 facilitates TGFß signaling-induced metastasis by protecting TGFß type I receptor and SMAD2 from ubiquitination. We found that these responses are potently suppressed by the specific UCHL1 inhibitor, 6RK73. Furthermore, UCHL1 levels were significantly increased in sera of patients with TNBC, and highly enriched in sera exosomes as well as TNBC cell-conditioned media. UCHL1-enriched exosomes stimulated breast cancer migration and extravasation, suggesting that UCHL1 may act in a paracrine manner to promote tumor progression. CONCLUSIONS: Our DUB activity profiling identified UCHL1 as a candidate oncoprotein that promotes TGFß-induced breast cancer metastasis and may provide a potential target for TNBC treatment.

USP26 regulates TGF-ß signalling by deubiquitinating and stabilizing SMAD7; not applicable in glioblastoma.

Comment on "USP26 regulates TGF-ß signaling by deubiquitinating and stabilizing SMAD7" by Kit Leng Lui et al.

Cancer-associated fibroblast-derived Gremlin 1 promotes breast cancer progression.

BACKGROUND: Bone morphogenetic proteins (BMPs) have been reported to maintain epithelial integrity and to antagonize the transforming growth factor ß (TGFß)-induced epithelial to mesenchymal transition. The expression of soluble BMP antagonists is dysregulated in cancers and interrupts proper BMP signaling in breast cancer. METHODS: In this study, we mined the prognostic role of BMP antagonists GREMLIN 1 (GREM1) in primary breast cancer tissues using in-house and publicly available datasets. We determined which cells express GREM1 RNA using in situ hybridization (ISH) on a breast cancer tissue microarray. The effects of Grem1 on the properties of breast cancer cells were assessed by measuring the mesenchymal/stem cell marker expression and functional cell-based assays for stemness and invasion. The role of Grem1 in breast cancer-associated fibroblast (CAF) activation was measured by analyzing the expression of fibroblast markers, phalloidin staining, and collagen contraction assays. The role of Grem1 in CAF-induced breast cancer cell intravasation and extravasation was studied by utilizing xenograft zebrafish breast cancer (co-) injection models. RESULTS: Expression analysis of clinical breast cancer datasets revealed that high expression of GREM1 in breast cancer stroma is correlated with a poor prognosis regardless of the molecular subtype. The large majority of human breast cancer cell lines did not express GREM1 in vitro, but breast CAFs did express GREM1 both in vitro and in vivo. Transforming growth factor ß (TGFß) secreted by breast cancer cells, and also inflammatory cytokines, stimulated GREM1 expression in CAFs. Grem1 abrogated bone morphogenetic protein (BMP)/SMAD signaling in breast cancer cells and promoted their mesenchymal phenotype, stemness, and invasion. Moreover, Grem1 production by CAFs strongly promoted the fibrogenic activation of CAFs and promoted breast cancer cell intravasation and extravasation in co-injection xenograft zebrafish models. CONCLUSIONS: Our results demonstrated that Grem1 is a pivotal factor in the reciprocal interplay between breast cancer cells and CAFs, which promotes cancer cell invasion. Targeting Grem1 could be beneficial in the treatment of breast cancer patients with high Grem1 expression.

Molecular characterization of a novel mycovirus from the plant pathogenic fungus Colletotrichum gloeosporioides.

In this study, we report the molecular characterization of a novel mycovirus in a phytopathogenic fungus of the species Colletotrichum gloeosporioides, which we named "Colletotrichum gloeosporioides RNA virus 1" (CgRV1). The virus has a dsRNA genome of 2,975 bp and possesses two non-overlapping open reading frames (ORFs 1 and 2). The smaller ORF1 encodes a protein of unknown function, and the larger ORF2 encodes the RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis based on the RdRp sequence showed that CgRV1 clustered with and is closely related to unclassified mycoviruses that are distinct from members of the family Partitiviridae.