Phase transition of tensin-1 during the focal adhesion disassembly and cell division.

Biomolecular condensates are nonmembranous structures that are mainly formed through liquid-liquid phase separation. Tensins are focal adhesion (FA) proteins linking the actin cytoskeleton to integrin receptors. Here, we report that GFP-tagged tensin-1 (TNS1) proteins phase-separate to form biomolecular condensates in cells. Live-cell imaging showed that new TNS1 condensates are budding from the disassembling ends of FAs, and the presence of these condensates is cell cycle dependent. TNS1 condensates dissolve immediately prior to mitosis and rapidly reappear while postmitotic daughter cells establish new FAs. TNS1 condensates contain selected FA proteins and signaling molecules such as pT308Akt but not pS473Akt, suggesting previously unknown roles of TNS1 condensates in disassembling FAs, as the storage of core FA components and the signaling intermediates.

Identification of Tensin-3 as a MALT1 substrate that controls B cell adhesion and lymphoma dissemination.

The protease MALT1 promotes lymphocyte activation and lymphomagenesis by cleaving a limited set of cellular substrates, most of which control gene expression. Here, we identified the integrin-binding scaffold protein Tensin-3 as a MALT1 substrate in activated human B cells. Activated B cells lacking Tensin-3 showed decreased integrin-dependent adhesion but exhibited comparable NF-κB1 and Jun N-terminal kinase transcriptional responses. Cells expressing a noncleavable form of Tensin-3, on the other hand, showed increased adhesion. To test the role of Tensin-3 cleavage in vivo, mice expressing a noncleavable version of Tensin-3 were generated, which showed a partial reduction in the T cell-dependent B cell response. Interestingly, human diffuse large B cell lymphomas and mantle cell lymphomas with constitutive MALT1 activity showed strong constitutive Tensin-3 cleavage and a decrease in uncleaved Tensin-3 levels. Moreover, silencing of Tensin-3 expression in MALT1-driven lymphoma promoted dissemination of xenografted lymphoma cells to the bone marrow and spleen. Thus, MALT1-dependent Tensin-3 cleavage reveals a unique aspect of the function of MALT1, which negatively regulates integrin-dependent B cell adhesion and facilitates metastatic spread of B cell lymphomas.

Protein kinase A is a functional component of focal adhesions.

Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and the actin cytoskeleton and also transmit signals that regulate cell adhesion, cytoskeletal dynamics, and cell migration. While many of these signals are rooted in reversible tyrosine phosphorylation, phosphorylation of FA proteins on Ser/Thr residues is far more abundant yet its mechanisms and consequences are far less understood. The cAMP-dependent protein kinase (protein kinase A; PKA) has important roles in cell adhesion and cell migration and is both an effector and regulator of integrin-mediated adhesion to the ECM. Importantly, subcellular localization plays a critically important role in specifying PKA function. Here, we show that PKA is present in isolated FA-cytoskeleton complexes and active within FAs in live cells. Furthermore, using kinase-catalyzed biotinylation of isolated FA-cytoskeleton complexes, we identify 53 high-stringency candidate PKA substrates within FAs. From this list, we validate tensin-3 (Tns3)-a well-established molecular scaffold, regulator of cell migration, and a component of focal and fibrillar adhesions-as a novel direct substrate for PKA. These observations identify a new pathway for phospho-regulation of Tns3 and, importantly, establish a new and important niche for localized PKA signaling and thus provide a foundation for further investigation of the role of PKA in the regulation of FA dynamics and signaling.

Testis-specific serine kinase 6 (TSSK6) is abnormally expressed in colorectal cancer and promotes oncogenic behaviors.

Cancer testis antigens (CTAs) are a collection of proteins whose expression is normally restricted to the gamete but abnormally activated in a wide variety of tumors. The CTA, Testis-specific serine kinase 6 (TSSK6), is essential for male fertility in mice. The functional relevance of TSSK6 to cancer, if any, has not previously been investigated. Here we find that TSSK6 is frequently anomalously expressed in colorectal cancer and patients with elevated TSSK6 expression have reduced relapse-free survival. Depletion of TSSK6 from colorectal cancer cells attenuates anchorage-independent growth, invasion, and growth in vivo. Conversely, overexpression of TSSK6 enhances anchorage independence and invasion in vitro as well as in vivo tumor growth. Notably, ectopic expression of TSSK6 in semi-transformed human colonic epithelial cells is sufficient to confer anchorage independence and enhance invasion. In somatic cells, TSSK6 co-localizes with and enhances the formation of paxillin and tensin-positive foci at the cell periphery, suggesting a function in focal adhesion formation. Importantly, TSSK6 kinase activity is essential to induce these tumorigenic behaviors. Our findings establish that TSSK6 exhibits oncogenic activity when abnormally expressed in colorectal cancer cells. Thus, TSSK6 is a previously unrecognized intervention target for therapy, which could exhibit an exceptionally broad therapeutic window.

Thrombomodulin Regulates PTEN/AKT Signaling Axis in Endothelial Cells.

BACKGROUND: We recently demonstrated that deletion of thrombomodulin gene from endothelial cells results in upregulation of proinflammatory phenotype. In this study, we investigated the molecular basis for the altered phenotype in thrombomodulin-deficient (TM-/-) cells. METHODS: Different constructs containing deletions or mutations in the cytoplasmic domain of thrombomodulin were prepared and introduced to TM-/- cells. The phenotype of cells expressing different derivatives of thrombomodulin and tissue samples of thrombomodulin-knockout mice were analyzed for expression of distinct regulatory genes in established signaling assays. RESULTS: The phosphatase and tensin homolog were phosphorylated and its recruitment to the plasma membrane was impaired in TM-/- cells, leading to hyperactivation of AKT (protein kinase B) and phosphorylation-dependent nuclear exclusion of the transcription factor, forkhead box O1. The proliferative/migratory properties of TM-/- cells were enhanced, and cells exhibited hypersensitivity to stimulation by angiopoietin 1 and vascular endothelial growth factor. Reexpression of wild-type thrombomodulin in TM-/- cells normalized the cellular phenotype; however, thrombomodulin lacking its cytoplasmic domain failed to restore the normal phenotype in TM-/- cells. Increased basal permeability and loss of VE-cadherin were restored to normal levels by reexpression of wild-type thrombomodulin but not by a thrombomodulin construct lacking its cytoplasmic domain. A thrombomodulin cytoplasmic domain deletion mutant containing 3-membrane-proximal Arg-Lys-Lys residues restored the barrier-permeability function of TM-/- cells. Enhanced phosphatase and tensin homolog phosphorylation and activation of AKT and mTORC1 (mammalian target of rapamycin complex 1) were also observed in the liver of thrombomodulin-KO mice. CONCLUSIONS: These results suggest that the cytoplasmic domain of thrombomodulin interacts with the actin cytoskeleton and plays a crucial role in regulation of phosphatase and tensin homolog/AKT signaling in endothelial cells.

Stabilizing Immature Dendritic Spines in the Auditory Cortex: A Key Mechanism for mTORC1-Mediated Enhancement of Long-Term Fear Memories.

Mammalian target of rapamycin (mTOR) pathway has emerged as a key molecular mechanism underlying memory processes. Although mTOR inhibition is known to block memory processes, it remains elusive whether and how an enhancement of mTOR signaling may improve memory processes. Here we found in male mice that the administration of VO-OHpic, an inhibitor of the phosphatase and tensin homolog (PTEN) that negatively modulates AKT-mTOR pathway, enhanced auditory fear memory for days and weeks, while it left short-term memory unchanged. Memory enhancement was associated with a long-lasting increase in immature-type dendritic spines of pyramidal neurons into the auditory cortex. The persistence of spine remodeling over time arose by the interplay between PTEN inhibition and memory processes, as VO-OHpic induced only a transient immature spine growth in the somatosensory cortex, a region not involved in long-term auditory memory. Both the potentiation of fear memories and increase in immature spines were hampered by rapamycin, a selective inhibitor of mTORC1. These data revealed that memory can be potentiated over time by the administration of a selective PTEN inhibitor. In addition to disclosing new information on the cellular mechanisms underlying long-term memory maintenance, our study provides new insights on the molecular processes that aid enhancing memories over time.SIGNIFICANCE STATEMENT The neuronal mechanisms that may help improve the maintenance of long-term memories are still elusive. The inhibition of mammalian-target of rapamycin (mTOR) signaling shows that this pathway plays a crucial role in synaptic plasticity and memory formation. However, whether its activation may strengthen long-term memory storage is unclear. We assessed the consequences of positive modulation of AKT-mTOR pathway obtained by VO-OHpic administration, a phosphatase and tensin homolog inhibitor, on memory retention and underlying synaptic modifications. We found that mTOR activation greatly enhanced memory maintenance for weeks by producing a long-lasting increase of immature-type dendritic spines in pyramidal neurons of the auditory cortex. These results offer new insights on the cellular and molecular mechanisms that can aid enhancing memories over time.

Tissue Usage Preference and Intrinsically Disordered Region Remodeling of Alternative Splicing Derived Proteoforms in the Heart.

A computational analysis of mass spectrometry data was performed to uncover alternative splicing derived protein variants across chambers of the human heart. Evidence for 216 non-canonical isoforms was apparent in the atrium and the ventricle, including 52 isoforms not documented on SwissProt and recovered using an RNA sequencing derived database. Among non-canonical isoforms, 29 show signs of regulation based on statistically significant preferences in tissue usage, including a ventricular enriched protein isoform of tensin-1 (TNS1) and an atrium-enriched PDZ and LIM Domain 3 (PDLIM3) isoform 2 (PDLIM3-2/ALP-H). Examined variant regions that differ between alternative and canonical isoforms are highly enriched with intrinsically disordered regions. Moreover, over two-thirds of such regions are predicted to function in protein binding and RNA binding. The analysis here lends further credence to the notion that alternative splicing diversifies the proteome by rewiring intrinsically disordered regions, which are increasingly recognized to play important roles in the generation of biological function from protein sequences.

MiR-29a-3p mediates phosphatase and tensin homolog and inhibits osteoarthritis progression.

Despite substantial progress in clinical trials of osteoarthritis (OA) gene therapy, the prevalence of OA is still on the rise. MiRNAs have a potential biomarker and therapeutic target for OA. OA cartilage and chondrosarcoma cells were studied to determine the role of miR-29a-3p and PTEN. OA cartilage and human chondrosarcoma cells (SW1353) were obtained. miR-29a-3p and PTEN signature expression was determined by RT-qPCR. The binding relationship between miR-29a-3p and PTEN was investigated by dual-luciferase reporter gene and western blot assay. TUNEL, immunohistochemistry, CCK-8, and flow cytometry were utilized to determine the proliferation and apoptosis of SW1353 cells. This study indicated downregulation of miR-29a-3p expression and upregulation of PTEN expression in human OA primary chondrocytes or OA tissue samples, compared with the normal cartilage cells or tissues. PTEN expression was negatively correlated with miR-29a-3p expression, and miR-29a-3p targeted PTEN mechanistically. miR-29a-3p reduced SW1353 cell activity and proliferation and promoted cell apoptosis. However, the aforementioned effects could be reversed by downregulating PTEN. miR-29a-3p can stimulate chondrocyte proliferation and inhibit apoptosis by inhibiting PTEN expression.

A critical role of the endothelial S-phase kinase-associated protein 2/phosphatase and tensin homologue axis in angiogenesis and psoriasis.

BACKGROUND: Psoriasis is a common chronic skin disorder. Pathologically, it features abnormal epidermal proliferation, infiltrating inflammatory cells and increased angiogenesis in the dermis. Aberrant expression of E3 ubiquitin ligase and a dysregulated protein ubiquitination system are implicated in the pathogenesis of psoriasis. OBJECTIVES: To examine the potential role of S-phase kinase-associated protein 2 (Skp2), an E3 ligase and oncogene, in psoriasis. METHODS: Gene expression and protein levels were evaluated with quantitative reverse transcriptase polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence staining of skin samples from patients with psoriasis vulgaris and an imiquimod (IMQ)-induced mouse model, as well as from cultured endothelial cells (ECs). Protein interaction, substrate ubiquitination and degradation were examined using co-immunoprecipitation, Western blotting and a cycloheximide chase assay in human umbilical vein ECs. Angiogenesis was measured in vitro using human dermal microvascular ECs (HDMECs) for BrdU incorporation, migration and tube formation. In vivo angiogenesis assays included chick embryonic chorioallantoic membrane, the Matrigel plug assay and quantification of vasculature in the mouse lesions. Skp2 gene global knockout (KO) mice and endothelial-specific conditional KO mice were used. RESULTS: Skp2 was increased in skin samples from patients with psoriasis and IMQ-induced mouse lesions. Immunofluorescent double staining indicated a close association of Skp2 expression with excessive vascularity in the lesional dermal papillae. In HDMECs, Skp2 overexpression was enhanced, whereas Skp2 knockdown inhibited EC proliferation, migration and tube-like structure formation. Mechanistically, phosphatase and tensin homologue (PTEN), which suppresses the phosphoinositide 3-kinase/Akt pathway, was identified to be a novel substrate for Skp2-mediated ubiquitination. A selective inhibitor of Skp2 (C1) or Skp2 small interfering RNA significantly reduced vascular endothelial growth factor-triggered PTEN ubiquitination and degradation. In addition, Skp2-mediated ubiquitination depended on the phosphorylation of PTEN by glycogen synthase kinase 3ß. In the mouse model, Skp2 gene deficiency alleviated IMQ-induced psoriasis. Importantly, tamoxifen-induced endothelial-specific Skp2 KO mice developed significantly ameliorated psoriasis with diminished angiogenesis of papillae. Furthermore, topical use of the Skp2 inhibitor C1 effectively prevented the experimental psoriasis. CONCLUSIONS: The Skp2/PTEN axis may play an important role in psoriasis-associated angiogenesis. Thus, targeting Skp2-driven angiogenesis may be a potential approach to treating psoriasis.

Tensin-3 is involved in osteogenic versus adipogenic fate of human bone marrow stromal cells.

BACKGROUND: The tightly controlled balance between osteogenic and adipogenic differentiation of human bone marrow-derived stromal cells (BMSCs) is critical to maintain bone homeostasis. Age-related osteoporosis is characterized by low bone mass with excessive infiltration of adipose tissue in the bone marrow compartment. The shift of BMSC differentiation from osteoblasts to adipocytes could result in bone loss and adiposity. METHODS: TNS3 gene expression during osteogenic and adipogenic differentiation of BMSCs was evaluated by qPCR and Western blot analyses. Lentiviral-mediated knockdown or overexpression of TNS3 was used to assess its function. The organization of cytoskeleton was examined by immunofluorescent staining at multiple time points. The role of TNS3 and its domain function in osteogenic differentiation were evaluated by ALP activity, calcium assay, and Alizarin Red S staining. The expression of Rho-GTP was determined using the RhoA pull-down activation assay. RESULTS: Loss of TNS3 impaired osteogenic differentiation of BMSCs but promoted adipogenic differentiation. Conversely, TNS3 overexpression hampered adipogenesis while enhancing osteogenesis. The expression level of TNS3 determined cell shape and cytoskeletal reorganization during osteogenic differentiation. TNS3 truncation experiments revealed that for optimal osteogenesis to occur, all domains proved essential. Pull-down and immunocytochemical experiments suggested that TNS3 mediates osteogenic differentiation through RhoA. CONCLUSIONS: Here, we identify TNS3 to be involved in BMSC fate decision. Our study links the domain structure in TNS3 to RhoA activity via actin dynamics and implicates an important role for TNS3 in regulating osteogenesis and adipogenesis from BMSCs. Furthermore, it supports the critical involvement of cytoskeletal reorganization in BMSC differentiation.

The Role of the PTEN Tumor Suppressor Gene and Its Anti-Angiogenic Activity in Melanoma and Other Cancers.

Human malignant melanoma and other solid cancers are largely driven by the inactivation of tumor suppressor genes and angiogenesis. Conventional treatments for cancer (surgery, radiation therapy, and chemotherapy) are employed as first-line treatments for solid cancers but are often ineffective as monotherapies due to resistance and toxicity. Thus, targeted therapies, such as bevacizumab, which targets vascular endothelial growth factor, have been approved by the US Food and Drug Administration (FDA) as angiogenesis inhibitors. The downregulation of the tumor suppressor, phosphatase tensin homolog (PTEN), occurs in 30-40% of human malignant melanomas, thereby elucidating the importance of the upregulation of PTEN activity. Phosphatase tensin homolog (PTEN) is modulated at the transcriptional, translational, and post-translational levels and regulates key signaling pathways such as the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways, which also drive angiogenesis. This review discusses the inhibition of angiogenesis through the upregulation of PTEN and the inhibition of hypoxia-inducible factor 1 alpha (HIF-1-α) in human malignant melanoma, as no targeted therapies have been approved by the FDA for the inhibition of angiogenesis in human malignant melanoma. The emergence of nanocarrier formulations to enhance the pharmacokinetic profile of phytochemicals that upregulate PTEN activity and improve the upregulation of PTEN has also been discussed.

The molecular and clinical role of Tensin 1/2/3 in cancer.

Tensin 1 was originally described as a focal adhesion adaptor protein, playing a role in extracellular matrix and cytoskeletal interactions. Three other Tensin proteins were subsequently discovered, and the family was grouped as Tensin. It is now recognized that these proteins interact with multiple cell signalling cascades that are implicated in tumorigenesis. To understand the role of Tensin 1-3 in neoplasia, current molecular evidence is categorized by the hallmarks of cancer model. Additionally, clinical data involving Tensin 1-3 are reviewed to investigate the correlation between cellular effects and clinical phenotype. Tensin proteins commonly interact with the tumour suppressor, DLC1. The ability of Tensin to promote tumour progression is directly correlated with DLC1 expression. Members of the Tensin family appear to have tumour subtype-dependent effects on oncogenesis; despite numerous data evidencing a tumour suppressor role for Tensin 2, association of Tensins 1-3 with an oncogenic role notably in colorectal carcinoma and pancreatic ductal adenocarcinoma is of potential clinical relevance. The complex interplay between these focal adhesion adaptor proteins and signalling pathways are discussed to provide an up to date review of their role in cancer biology.

Abnormal glomerular basement membrane maturation impairs mesangial cell differentiation during murine postnatal nephrogenesis.

Although mesangial cell-glomerular basement membrane (GBM) connections play a key role in maintaining the glomerular capillary loop structure, information remains limited about how these connections are formed during glomerulogenesis. We have previously shown that weakened podocyte-GBM interactions owing to tensin 2 (Tns2) deficiency lead to abnormal GBM maturation during postnatal glomerulogenesis. Here, we investigated whether abnormal GBM maturation affected mesangial cell-GBM connections and mesangial cell differentiation. Histological analysis of the outer cortical glomeruli in Tns2-deficient mice revealed that GBM materials overproduced by stressed immature podocytes accumulated in the mesangium and interrupted the formation of mesangial cell-GBM connections, resulting in fewer capillary loops compared with that of normal glomeruli. In addition, expression of α-smooth muscle actin, an immature mesangial cell marker, persisted in mesangial cells of Tns2-deficient outer cortical glomeruli even after glomerulogenesis was completed, resulting in mesangial expansion. Furthermore, analysis of mouse primary mesangial cells revealed that mesangial cell differentiation depended on the type of extracellular matrix components to which the cells adhered, suggesting the participation of mesangial cell-GBM connections in mesangial cell differentiation. These findings suggest that abnormal GBM maturation affects mesangial cell differentiation by impairing mesangial cell-GBM connections.NEW & NOTEWORTHY Mesangial cell-glomerular basement membrane (GBM) connections play an important role in maintaining the structural integrity of the glomerular tuft. However, information remains scarce about how GBM maturation affects the formation of these connections during glomerular development. Here, we show that abnormal GBM maturation due to tensin 2 deficiency affects mesangial cell differentiation by impairing mesangial cell-GBM connections during postnatal glomerulogenesis.

Yes-Associated Protein Nuclear Translocation Is Regulated by Epidermal Growth Factor Receptor Activation Through Phosphatase and Tensin Homolog/AKT Axis in Glioblastomas.

Gliomas are the most common and lethal primary brain tumors in adults. Glioblastomas, the most frequent and aggressive form of gliomas, represent a therapeutic challenge as no curative treatment exists to date, and the prognosis remains extremely poor. Recently, the transcriptional cofactors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) belonging to the Hippo pathway have emerged as a major determinant of malignancy in solid tumors, including gliomas. However, the mechanisms involved in its regulation, particularly in brain tumors, remain ill-defined. In glioblastomas, EGFR represents one of the most altered oncogenes affected by chromosomal rearrangements, mutations, amplifications, and overexpression. In this study, we investigated the potential link between epidermal growth factor receptor (EGFR) and the transcriptional cofactors YAP and TAZ by in situ and in vitro approaches. We first studied their activation on tissue microarray, including 137 patients from different glioma molecular subtypes. We observed that YAP and TAZ nuclear location was highly associated with isocitrate dehydrogenase 1/2 (IDH1/2) wild-type glioblastomas and poor patient outcomes. Interestingly, we found an association between EGFR activation and YAP nuclear location in glioblastoma clinical samples, suggesting a link between these 2 markers contrary to its ortholog TAZ. We tested this hypothesis in patient-derived glioblastoma cultures by pharmacologic inhibition of EGFR using gefinitib. We showed an increase of S397-YAP phosphorylation associated with decreased AKT phosphorylation after EGFR inhibition in phosphatase and tensin homolog (PTEN) wild-type cultures, unlike PTEN-mutated cell lines. Finally, we used bpV(HOpic), a potent PTEN inhibitor, to mimic the effect of PTEN mutations. We found that the inhibition of PTEN was sufficient to revert back the effect induced by Gefitinib in PTEN-wild-type cultures. Altogether, to our knowledge, these results show for the first time the regulation of pS397-YAP by the EGFR-AKT axis in a PTEN-dependent manner.

Tensins - emerging insights into their domain functions, biological roles and disease relevance.

Tensins are a family of focal adhesion proteins consisting of four members in mammals (TNS1, TNS2, TNS3 and TNS4). Their multiple domains and activities contribute to the molecular linkage between the extracellular matrix and cytoskeletal networks, as well as mediating signal transduction pathways, leading to a variety of physiological processes, including cell proliferation, attachment, migration and mechanical sensing in a cell. Tensins are required for maintaining normal tissue structures and functions, especially in the kidney and heart, as well as in muscle regeneration, in animals. This Review discusses our current understanding of the domain functions and biological roles of tensins in cells and mice, as well as highlighting their relevance to human diseases.

Exosomes derived from bone marrow mesenchymal stem cells pretreated with decellularized extracellular matrix enhance the alleviation of osteoarthritis through miR-3473b/phosphatase and tensin homolog axis.

BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative articular disease for which there is no effective treatment. Progress has been made in mesenchymal stem cell (MSC)-based therapy in OA, and the efficacy has been demonstrated to be a result of paracrine exosomes from MSCs. Decellularized extracellular matrix (dECM) provides an optimum microenvironment for the expansion of MSCs. In the present study, we aimed to investigate whether exosomes isolated from bone marrow mesenchymal stem cells (BMSCs) with dECM pretreatment (dECM-BMSC-Exos) enhance the amelioration of OA. METHODS: Exosomes from BMSCs with or without dECM pretreatment were isolated. We measured and compared the effect of the BMSC-Exo and dECM-BMSC-Exo on interleukin (IL)-1ß-induced chondrocytes by analyzing proliferation, anabolism and catabolism, migration and apoptosis in vitro. The in vivo experiment was performed by articular injection of exosomes into DMM mice, followed by histological evaluation of cartilage. MicroRNA sequencing of exosomes was performed on BMSC-Exo and dECM-BMSC-Exo to investigate the underlying mechanism. The function of miR-3473b was validated by rescue studies in vitro and in vivo using antagomir-3473b. RESULTS: IL-1ß-treated chondrocytes treated with dECM-BMSC-Exos showed enhanced proliferation, anabolism, migration and anti-apoptosis properties compared to BMSC-Exos. DMM mice injected with dECM-BMSC-Exo showed better cartilage regeneration than those injected with BMSC-Exo. Interestingly, miR-3473b was significantly elevated in dECM-BMSC-Exos and was found to mediate the protective effect in chondrocytes by targeting phosphatase and tensin homolog (PTEN), which activated the PTEN/AKT signaling pathway. CONCLUSIONS: dECM-BMSC-Exo can enhance the alleviation of osteoarthritis via promoting migration, improving anabolism and inhibiting apoptosis of chondrocytes by upregulating miR-3473b, which targets PTEN.

The transcription factor zinc fingers and homeoboxes 2 alleviates NASH by transcriptional activation of phosphatase and tensin homolog.

BACKGROUND AND AIMS: NASH, which is a common clinical condition predisposing to advanced liver diseases, has become a worldwide epidemic. A large and growing unmet therapeutic need for this condition reflects incomplete understanding of its pathogenesis. In the current study, we identified a transcription factor, zinc fingers and homeoboxes 2 (ZHX2), in hepatocytes as a protective factor against steatohepatitis. APPROACH AND RESULTS: We found that hepatic ZHX2 was significantly suppressed in NASH models and steatotic hepatic cells. Hepatocyte-specific ablation of ZHX2 exacerbated NASH-related phenotypes in mice, including lipid accumulation, enhanced inflammation, and hepatic fibrosis. Conversely, hepatocyte-specific overexpression of ZHX2 significantly alleviated the progression of NASH in an experimental setting. Integrated analysis of transcriptomic profiling and chromatin immunoprecipitation sequencing data demonstrated that the phosphatase and tensin homolog (PTEN) was a target gene of ZHX2 in hepatocyte. ZHX2 bound to the promoter of PTEN gene and subsequently promoted the transcription of PTEN, which mediated the beneficial role of ZHX2 against NASH. CONCLUSIONS: The current findings demonstrate a protective role of ZHX2 against NASH progression by transcriptionally activating PTEN. These findings shed light on the therapeutic potential of targeting ZHX2 for treating NASH and related metabolic disorders.

Tumor-derived exosomes induce immunosuppressive macrophages to foster intrahepatic cholangiocarcinoma progression.

BACKGROUND AND AIMS: Macrophages are prominent components of solid tumors and exhibit distinct functions in different tumor microenvironments. Exosomes are emerging as necessary mediators of the cross-talk between tumor cells and the microenvironment. However, the underlying mechanisms of exosomes involving into crosstalk between tumor cells and macrophages during disease progression of intrahepatic cholangiocarcinoma (ICC) have not been yet fully realized. APPROACH AND RESULTS: We found that the macrophages of ICC tumor tissues up-regulated the expression levels of immunosuppressive molecule programmed death-ligand 1 (PD-L1). Increased PD-L1+ macrophages in tumor tissues effectively suppressed T-cell immunity and correlated with poor survival rates in patients with ICC. High-throughput RNA-sequencing analysis that was performed to identify differential levels of microRNAs (miRNAs) between exosomes derived from ICC cells and primary human intrahepatic biliary epithelial cells revealed that miR-183-5p was increased in ICC cell-derived exosomes. Exosomal miR-183-5p inhibited phosphatase and tensin homolog (PTEN) expression, to subsequently affect the elevations on both phosphorylated AKT and PD-L1 expression in macrophages. Furthermore, macrophages that treated with ICC cell-derived exosomes significantly suppressed T-cell immunity in vitro and contributed to the growth and progression of ICC in vivo, which were reversible through blockages on PD-L1 of these macrophages. Finally, clinical data showed that up-regulated levels of plasma exosomal miR-183-5p correlated with poor prognosis of patients with ICC after curative resection. CONCLUSIONS: Tumor-derived exosomal miR-183-5p up-regulates PD-L1-expressing macrophages to foster immune suppression and disease progression in ICC through the miR-183-5p/PTEN/AKT/PD-L1 pathway. Exosomal miR-183-5p is a potential predictive biomarker for ICC progression and a potential target for development of therapeutic strategies against immune tolerance feature of ICC.

Therapeutic efficacy of FASN inhibition in preclinical models of HCC.

BACKGROUND AND AIMS: Aberrant activation of fatty acid synthase (FASN) is a major metabolic event during the development of HCC. We evaluated the therapeutic efficacy of TVB3664, a FASN inhibitor, either alone or in combination, for HCC treatment. APPROACH AND RESULTS: The therapeutic efficacy and the molecular pathways targeted by TVB3664, either alone or with tyrosine kinase inhibitors or the checkpoint inhibitor anti-programmed death ligand 1 antibody, were assessed in human HCC cell lines and multiple oncogene-driven HCC mouse models. RNA sequencing was performed to elucidate the effects of TVB3664 on global gene expression and tumor metabolism. TVB3664 significantly ameliorated the fatty liver phenotype in the aged mice and AKT-induced hepatic steatosis. TVB3664 monotherapy showed moderate efficacy in NASH-related murine HCCs, induced by loss of phosphatase and tensin homolog and MET proto-oncogene, receptor tyrosine kinase (c-MET) overexpression. TVB3664, in combination with cabozantinib, triggered tumor regression in this murine model but did not improve the responsiveness to immunotherapy. Global gene expression revealed that TVB3664 predominantly modulated metabolic processes, whereas TVB3664 synergized with cabozantinib to down-regulate multiple cancer-related pathways, especially the AKT/mammalian target of rapamycin pathway and cell proliferation genes. TVB3664 also improved the therapeutic efficacy of sorafenib and cabozantinib in the FASN-dependent c-MYC-driven HCC model. However, TVB3664 had no efficacy nor synergistic effects in FASN-independent murine HCC models. CONCLUSIONS: This preclinical study suggests the limited efficacy of targeting FASN as monotherapy for HCC treatment. However, FASN inhibitors could be combined with other drugs for improved effectiveness. These combination therapies could be developed based on the driver oncogenes, supporting precision medicine approaches for HCC treatment.

PTEN in Immunity.

The tumor suppressor PTEN (Phosphatase and Tensin homolog deleted on Chromosome 10) executes critical biological functions that limit cellular growth and proliferation. PTEN inhibits activation of the proto-oncogenic PI3K pathway and is required during embryogenesis and to suppress tumor formation and cancer progression throughout life. The critical role that PTEN plays in restraining cellular growth has been validated through the generation of a number of animal models whereby PTEN inactivation invariably leads to tumor formation in a cell-autonomous fashion. However, the increasing understanding of the mechanisms through which the immune system contributes to suppressing tumor progression has highlighted how, in a cell non-autonomous fashion, cancer-associated mutations can indirectly enhance oncogenesis by evading immune cell recognition. Here, in light of the essential role of PTEN in the regulation of immune cell development and function, and based on recent findings showing that PTEN loss can promote resistance to immune checkpoint inhibitors in various tumor types, we re-evaluate our understanding of the mechanisms through which PTEN functions as a tumor suppressor and postulate that this task is achieved through a combination of cell autonomous and non-autonomous effects. We highlight some of the critical studies that have delineated the functional role of PTEN in immune cell development and blood malignancies and propose new strategies for the treatment of PTEN loss-driven diseases.