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The acquisition of tamoxifen resistance is a major therapeutic problem in breast cancer. We developed a tamoxifen-resistant MCF-7 (TRM-7) cell line to elucidate the molecular mechanisms and factors associated with acquisition of such resistance. We showed that phosphorylation of STAT3 at tyrosine 705 (Y705) and RANTES expression are increased in response to tamoxifen in human breast cancer cells. On the basis of these results, we hypothesize that upregulated STAT3 phosphorylation and RANTES may be correlated with the development of drug resistance. Here, we showed that STAT3 and RANTES contribute to the maintenance of drug resistance. STAT3 phosphorylation is constitutively retained via a RANTES autocrine loop, which in turn upregulates anti-apoptotic signals in TRM-7 cells. STAT3–RANTES autocrine signaling affected expression of anti-apoptotic BCL-2 family genes and prevented TRM-7 cells from undergoing programmed cell death by inhibiting PARP and caspase-9 cleavage. Subsequently, blockade of STAT3 and RANTES in TRM-7 cells resulted in reduction of anti-apoptotic signals, which was rescued by exogenous RANTES treatment; drug resistance was also restored. Taken together, our results suggested that STAT3–RANTES autocrine signaling is essential for maintenance of drug resistance and inhibition of programmed cell death. These mechanisms of STAT3–RANTES autocrine signaling suggest a novel strategy for management of patients with tamoxifen-resistant tumors. Mol Cancer Res; 11(1); 31–42. ©2012 AACR.

Active c-Src non-receptor tyrosine kinase localizes to the plasma membrane via N-terminal lipid modification. Membranous c-Src causes cancer initiation and progression. Even though transmembrane 4 L six family member 5 (TM4SF5), a tetraspan(in), can be involved in this mechanism, the molecular and structural influence of TM4SF5 on c-Src remains unknown. Methods: Here, we investigated molecular and structural details by which TM4SF5 regulated c-Src devoid of its N-terminus and how cell-penetrating peptides were able to interrupt c-Src activation via interference of c-Src-TM4SF5 interaction in hepatocellular carcinoma models. Results: The TM4SF5 C-terminus efficiently bound the c-Src SH1 kinase domain, efficiently to the inactively-closed form. The complex involved protein tyrosine phosphatase 1B able to dephosphorylate Tyr530. The c-Src SH1 domain alone, even in a closed form, bound TM4SF5 to cause c-Src Tyr419 and FAK Y861 phosphorylation. Homology modeling and molecular dynamics simulation studies predicted the directly interfacing residues, which were further validated by mutational studies. Cell penetration of TM4SF5 C-terminal peptides blocked the interaction of TM4SF5 with c-Src and prevented c-Src-dependent tumor initiation and progression in vivo. Conclusions: Collectively, these data demonstrate that binding of the TM4SF5 C-terminus to the kinase domain of inactive c-Src leads to its activation. Because this binding can be abolished by cell-penetrating peptides containing the TM4SF5 C-terminus, targeting this direct interaction may be an effective strategy for developing therapeutics that block the development and progression of hepatocellular carcinoma.

Nonalcoholic fatty liver disease is a chronic condition involving steatosis, steatohepatitis and fibrosis, and its progression remains unclear. Although the tetraspanin transmembrane 4 L six family member 5 (TM4SF5) is involved in hepatic fibrosis and cancer, its role in nonalcoholic steatohepatitis (NASH) progression is unknown. We investigated the contribution of TM4SF5 to liver pathology using transgenic and KO mice, diet- or drug-treated mice, in vitro primary cells, and in human tissue. TM4SF5-overexpressing mice exhibited nonalcoholic steatosis and NASH in an age-dependent manner. Initially, TM4SF5-positive hepatocytes and liver tissue exhibited lipid accumulation, decreased Sirtuin 1 (SIRT1), increased sterol regulatory-element binding proteins (SREBPs) and inactive STAT3 via suppressor of cytokine signaling (SOCS)1/3 upregulation. In older mice, TM4SF5 promoted inflammatory factor induction, SIRT1 expression and STAT3 activity, but did not change SOCS or SREBP levels, leading to active STAT3-mediated ECM production for NASH progression. A TM4SF5-associated increase in chemokines promoted SIRT1 expression and progression to NASH with fibrosis. Suppression of the chemokine CCL20 reduced immune cell infiltration and ECM production. Liver tissue from high-fat diet- or CCl4 -treated mice and human patients exhibited TM4SF5-dependent steatotic or steatohepatitic livers with links between TM4SF5-mediated SIRT1 modulation and SREBP or SOCS/STAT3 signaling axes. TM4SF5-mediated STAT3 activation in fibrotic NASH livers increased collagen I and laminin γ2. Both collagen I α1 and laminin γ2 suppression resulted in reduced SIRT1 and active STAT3, but no change in SREBP1 or SOCS, and abolished CCl4 -mediated mouse liver damage. TM4SF5-mediated signaling pathways that involve SIRT1, SREBPs and SOCS/STAT3 promoted progression to NASH. Therefore, TM4SF5 and its downstream effectors may be promising therapeutic targets to treat nonalcoholic fatty liver disease. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mechanosensory feedback from the digestive tract to the brain is critical for limiting excessive food and water intake, but the underlying gut–brain communication pathways and mechanisms remain poorly understood1–12. Here we show that, in mice, neurons in the parabrachial nucleus that express the prodynorphin gene (hereafter, PBPdyn neurons) monitor the intake of both fluids and solids, using mechanosensory signals that arise from the upper digestive tract. Most individual PBPdyn neurons are activated by ingestion as well as the stimulation of the mouth and stomach, which indicates the representation of integrated sensory signals across distinct parts of the digestive tract. PBPdyn neurons are anatomically connected to the digestive periphery via cranial and spinal pathways; we show that, among these pathways, the vagus nerve conveys stomach-distension signals to PBPdyn neurons. Upon receipt of these signals, these neurons produce aversive and sustained appetite-suppressing signals, which discourages the initiation of feeding and drinking (fully recapitulating the symptoms of gastric distension) in part via signalling to the paraventricular hypothalamus. By contrast, inhibiting the same population of PBPdyn neurons induces overconsumption only if a drive for ingestion exists, which confirms that these neurons mediate negative feedback signalling. Our findings reveal a neural mechanism that underlies the mechanosensory monitoring of ingestion and negative feedback control of intake behaviours upon distension of the digestive tract. A population of neurons in the parabrachial nucleus that expresses prodynorphin monitors ingestion using mechanosensory signals from the upper digestive tract, and mediates negative feedback control of intake when the digestive tract is distended.

Transmembrane 4 L6 family member 5 (TM4SF5) is a tetraspanin that has four transmembrane domains and can be N-glycosylated and palmitoylated. These posttranslational modifications of TM4SF5 enable homophilic or heterophilic binding to diverse membrane proteins and receptors, including growth factor receptors, integrins, and tetraspanins. As a member of the tetraspanin family, TM4SF5 promotes protein-protein complexes for the spatiotemporal regulation of the expression, stability, binding, and signaling activity of its binding partners. Chronic diseases such as liver diseases involve bidirectional communication between extracellular and intracellular spaces, resulting in immune-related metabolic effects during the development of pathological phenotypes. It has recently been shown that, during the development of fibrosis and cancer, TM4SF5 forms protein-protein complexes with amino acid transporters, which can lead to the regulation of cystine uptake from the extracellular space to the cytosol and arginine export from the lysosomal lumen to the cytosol. Furthermore, using proteomic analyses, we found that diverse amino acid transporters were precipitated with TM4SF5, although these binding partners need to be confirmed by other approaches and in functionally relevant studies. This review discusses the scope of the pathological relevance of TM4SF5 and its binding to certain amino acid transporters., Cell biology: troubles with trafficking Interactions between a membrane protein and transporters that move amino acids in and out of cells may play a prominent role in various diseases. The transmembrane protein TM4SF5 is known to regulate the activity of diverse signaling pathways, and has been linked to various liver diseases. Jung Weon Lee and colleagues at Seoul National University review the recently identified interplay between TM4SF5 and transporter proteins that regulate trafficking of the amino acids cysteine and arginine. Through its interactions with cysteine transporters, TM4SF5 appears to contribute to hormetic production of damaging chemicals that can cause the tissue scarring underlying idiopathic pulmonary fibrosis. Evidence also suggests that TM4SF5-facilitated arginine trafficking may contribute to liver cancer recurrence. This protein may also interact with other prominent transporters, and the authors conclude that TM4SF5 may affect other, still undefined pathological processes.

Transmembrane 4 L six family member 5 (TM4SF5) functions as a sensor for lysosomal arginine levels and activates the mammalian target of rapamycin complex 1 (mTORC1). While the mTORC1 signaling pathway plays a key role in adipose tissue metabolism, the regulatory function of TM4SF5 in adipocytes remains unclear. In this study we aimed to establish a TM4SF5 knockout (KO) mouse model and investigated the effects of TM4SF5 KO on mTORC1 signaling–mediated autophagy and mitochondrial metabolism in adipose tissue. TM4SF5 expression was higher in inguinal white adipose tissue (iWAT) than in brown adipose tissue and significantly upregulated by a high-fat diet (HFD). TM4SF5 KO reduced mTORC1 activation and enhanced autophagy and lipolysis in adipocytes. RNA sequencing analysis of TM4SF5 KO mouse iWAT showed that the expression of genes involved in peroxisome proliferator–activated receptor α signaling pathways and mitochondrial oxidative metabolism was upregulated. Consequently, TM4SF5 KO reduced adiposity and increased energy expenditure and mitochondrial oxidative metabolism. TM4SF5 KO prevented HFD-induced glucose intolerance and inflammation in adipose tissue. Collectively, the results of our study demonstrate that TM4SF5 regulates autophagy and lipid catabolism in adipose tissue and suggest that TM4SF5 could be therapeutically targeted for the treatment of obesity-related metabolic diseases.

Transmembrane 4 L six family member 5 (TM4SF5) is involved in nonalcoholic steatosis and further aggravation of liver disease. However, its mechanism for regulating FA accumulation is unknown. We investigated how TM4SF5 in hepatocytes affected FA accumulation during acute FA supply. TM4SF5-expressing hepatocytes and mouse livers accumulated less FAs, compared with those of TM4SF5 deficiency or inactivation. Binding of TM4SF5 to SLC27A2 increased gradually upon acute FA treatment, whereas TM4SF5 constitutively bound SLC27A5. Suppression of either SLC27A2 or SLC27A5 in hepatocytes expressing TM4SF5 differentially modulated initial and maximal FA uptake levels for a fast turnover of fatty acid. Altogether, TM4SF5 negatively modulates FA accumulation into hepatocytes via association with the transporters for an energy homeostasis, when FA are supplied acutely.

Although immune checkpoint inhibitors have emerged as a breakthrough in cancer therapy, a monotherapy approach is not sufficient. Here, we report an immune checkpoint inhibitor-modified nanoparticle for an in situ-assembled tumor vaccine that can activate immune systems in the tumor microenvironment and prevent the long-term recurrence of tumors. Adjuvant-loaded nanoparticles were prepared by entrapping imiquimod (IQ) in photoresponsive polydopamine nanoparticles (IQ/PNs). The surfaces of IQ/PNs were then modified with anti-PDL1 antibody (PDL1Ab-IQ/PNs) for in situ assembly with inactivated tumor cells and immune checkpoint blocking of PDL1 (programmed cell death 1 ligand 1). The presence of anti-PDL1 antibodies on IQ/PNs increased the binding of nanoparticles to CT26 cancer cells overexpressing PDL1. Subsequent near-infrared (NIR) irradiation induced a greater photothermal anticancer effect against cells treated with PDL1Ab-IQ/PNs than cells treated with plain PNs or unmodified IQ/PNs. To mimic the tumor microenvironment, we cocultured bone marrow-derived dendritic cells with CT26 cells treated with various nanoparticle formulations and NIR irradiated. This coculture study revealed that NIR-inactivated, PDL1Ab-IQ/PN-bound CT26 cells induced maturation of dendritic cells to the greatest extent. Following a single intravenous administration of different nanoparticle formulations in CT26 tumor-bearing mice, PDL1Ab-IQ/PNs showed greater tumor tissue accumulation than unmodified nanoparticles. Subsequent NIR irradiation of mice treated with PDL1Ab-IQ/PNs resulted in tumor ablation. In addition to primary tumor ablation, PDL1Ab-IQ/PNs completely prevented the growth of a secondarily challenged CT26 tumor at a distant site, producing 100% survival for up to 150 days. A long-term protection study revealed that treatment with PDL1Ab-IQ/PNs followed by NIR irradiation inhibited the growth of distant, secondarily challenged CT26 tumors 150 days after the first tumor inoculation. Moreover, increased infiltration of T cells was observed in tumor tissues treated with PDL1Ab-IQ/PNs and NIR-irradiated, and T cells isolated from splenocytes of mice in which tumor recurrence was prevented showed active killing of CT26 cells. These results suggest that PDL1Ab-IQ/PNs in conjunction with NIR irradiation induce a potent, in situ-assembled, all-in-one tumor vaccine with adjuvant-containing nanoparticle-bound, inactivated tumor cells. Such in situ nanoadjuvant-assembled tumor vaccines can be further developed for long-term prevention of tumor recurrence without the need for chemotherapy.

Robust and efficient enzymes are essential modules for metabolic engineering and synthetic biology strategies across biological systems to engineer whole-cell biocatalysts. By condensing an acyl-CoA and an alcohol, alcohol acyltransferases (AATs) can serve as an interchangeable metabolic module for microbial biosynthesis of a diverse class of ester molecules with broad applications as flavors, fragrances, solvents, and drop-in biofuels. However, the current lack of robust and efficient AATs significantly limits their compatibility with heterologous precursor pathways and microbial hosts. Through bioprospecting and rational protein engineering, we identified and repurposed chloramphenicol acetyltransferases (CATs) from mesophilic prokaryotes to function as robust and efficient AATs compatible with at least 21 alcohol and 8 acyl-CoA substrates for microbial biosynthesis of linear, branched, saturated, unsaturated and/or aromatic esters. By plugging the best engineered CAT (CATec3 Y20F) into the gram-negative mesophilic bacteriumEscherichia coli, we demonstrated that the recombinant strain could effectively convert various alcohols into desirable esters, for instance, achieving a titer of 13.9 g/L isoamyl acetate with 95% conversion by fed-batch fermentation. The recombinantE. coliwas also capable of simulating the ester profile of roses with high conversion (> 97%) and titer (> 1 g/L) from fermentable sugars at 37°C. Likewise, a recombinant gram-positive, cellulolytic, thermophilic bacteriumClostridium thermocellumharboring CATec3 Y20F could produce many of these esters from recalcitrant cellulosic biomass at elevated temperatures (>50°C) due to the engineered enzyme’s remarkable thermostability. Overall, the engineered CATs can serve as a robust and efficient platform for designer ester biosynthesis from renewable and sustainable feedstocks.

Transmembrane 4 L six family member 5 (TM4SF5) translocates intracellularly and promotes cell migration, but how subcellular TM4SF5 traffic is regulated to guide cellular migration is unknown. We investigated the influences of the extracellular environment and intracellular signaling on the TM4SF5 traffic with regard to migration directionality. Cell adhesion to fibronectin (FN) but not poly-l-lysine enhanced the traffic velocity and straightness of the TM4SF5WT (but not palmitoylation-deficient mutant TM4SF5 Pal - ) toward the leading edges, depending on tubulin acetylation. Acetylated-microtubules in SLAC2B-positive cells reached mostly the juxtanuclear regions, but reached-out toward the leading edges upon SLAC2B suppression. TM4SF5 expression caused SLAC2B not to be localized at the leading edges. TM4SF5 colocalization with HDAC6 depended on paxillin expression. The trimeric complex consisting of TM4SF5, HDAC6, and SLAC2B might, thus, be enriched at the perinuclear cytosols toward the leading edges. More TM4SF5WT translocation to the leading edges was possible when acetylated-microtubules reached the frontal edges following HDAC6 inhibition by paxillin presumably at new cell-FN adhesions, leading to persistent cell migration. Collectively, this study revealed that cell-FN adhesion and microtubule acetylation could control intracellular traffic of TM4SF5 vesicles to the leading edges via coordinated actions of paxillin, SLAC2B, and HDAC6, leading to TM4SF5-dependent cell migration.

Cancer metastasis involves diverse cellular functions via bidirectional communications between intracellular and extracellular spaces. To achieve development of the anti-metastatic drugs, one needs to consider the efficacy and mode of action (MOA) of the drug candidates to block the metastatic potentials of cancerous cells. Rather than under two-dimensional environment, investigation of the metastatic potentials under three-dimensional environment would be much pharmaceutically beneficent, since it can mimic the in vivo tumor lesions in cancer patients, leading to allowance of drug candidates analyzed in the 3D culture systems to lower failure rates during the anti-metastatic drug development. Here we have reviewed on the analyses of metastatic potentials of certain cancer models in 3D culture systems surrounded with extracellular matrix proteins, which could be supported by TM4SF5- and/or EMT-mediated actions. We particularly focused the initial events of the cancer metastasis, such as invasive outgrowth and dissemination from the cancer cell masses, spheroids, embedded in the 3D gel culture systems. This review summarizes the significance of tetraspanin TM4SF5 and Snail1 that are related to EMT in the metastatic potentials explored in the 3D gel systems.

Thermoregulatory behavior is a basic motivated behavior for body temperature homeostasis. Despite its fundamental importance, a forebrain region or defined neural population required for this process has yet to be established. Here, we show that Vgat-expressing neurons in the lateral hypothalamus (LHVgat neurons) are required for diverse thermoregulatory behaviors. The population activity of LHVgat neurons is increased during thermoregulatory behavior and bidirectionally encodes thermal punishment and reward (P&R). Although this population also regulates feeding and caloric reward, inhibition of parabrachial inputs selectively impaired thermoregulatory behaviors and encoding of thermal stimulus by LHVgat neurons. Furthermore, two-photon calcium imaging revealed a subpopulation of LHVgat neurons bidirectionally encoding thermal P&R, which is engaged during thermoregulatory behavior, but is largely distinct from caloric reward-encoding LHVgat neurons. Our data establish LHVgat neurons as a required neural substrate for behavioral thermoregulation and point to the key role of the thermal P&R-encoding LHVgat subpopulation in thermoregulatory behavior.

Here we show the roles of transmembrane 4 L six family member 5 (TM4SF5) in the progression of nonalcoholic steatosis (or NAFL) to steatohepatitis (NASH). The overexpression of TM4SF5 caused nonalcoholic steatosis and NASH in an age-dependent manner. Initially, TM4SF5-positive hepatocytes and livers exhibited lipid accumulation, decreased SIRT1, increased SREBPs levels, and inactive STAT3 via SOCS1/3 upregulation. In older animals, TM4SF5 under an inflammatory environment increased SIRT1 expression and STAT3 activity with no significant change to SOCSs and SREBPs levels, leading to active STAT3-mediated fibrotic extracellular matrix (ECM) production. Liver tissues from clinical human patients with NAFL or NASH also showed such a TM4SF5-SIRT1-STAT3-ECM relationship correlated with fibrosis score and age. Ligand-independent and TM4SF5-mediated STAT3 activity led to collagen I and laminins/laminin γ2 expression in hepatic stellate cells and hepatocytes, respectively. Laminin γ2 suppression abolished CCl4-mediated liver damage and ECM production and reduced SIRT1 and active-STAT3, but did not alter SREBP1 or SOCSs levels. These findings suggest that TM4SF5, CCL20, SIRT1, and/or laminin γ2 may be promising therapeutic targets against liver disease.

By comparing imatinib-sensitive and -resistant chronic myeloid leukemia (CML) cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase-dependent apoptosis at low concentrations accompanied by loss of mitochondrial membrane potential (MMP) and increase of cytosolic free Ca2+ levels. Interestingly, at higher concentrations, TMQ0153 induced necroptotic cell death with accumulation of ROS, both preventable by N-acetyl-L-cysteine (NAC) pretreatment. At necroptosis-inducing concentrations, we observed increased ROS and decreased ATP and GSH levels, concomitant with protective autophagy induction. Inhibitors such as bafilomycin A1 (baf-A1) and siRNA against beclin 1 abrogated autophagy, sensitized CML cells against TMQ0153 and enhanced necroptotic cell death. Importantly, TMQ153-induced necrosis led to cell surface exposure of calreticulin (CRT) and ERp57 as well as the release of extracellular ATP and high mobility group box (HMGB1) demonstrating the capacity of this compound to release immunogenic cell death (ICD) markers. We validated the anti-cancer potential of TMQ0153 by in vivo inhibition of K562 microtumor formation in zebrafish. Taken together, our findings provide evidence that cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models.

Lysyl-tRNA synthetase (KRS) functions canonically in cytosolic translational processes. However, KRS is highly expressed in colon cancer, and localizes to distinct cellular compartments upon phosphorylations (i.e., the plasma membranes after T52 phosphorylation and the nucleus after S207 phosphorylation), leading to probably alternative noncanonical functions. It is unknown how other subcellular KRSs crosstalk with environmental cues during cancer progression. Here, we demonstrate that the KRS-dependent metastatic behavior of colon cancer spheroids within 3D gels requires communication between cellular molecules and extracellular soluble factors and neighboring cells. Membranous KRS and nuclear KRS were found to participate in invasive cell dissemination of colon cancer spheroids in 3D gels. Cancer spheroids secreted GAS6 via a KRS-dependent mechanism and caused the M2 polarization of macrophages, which activated the neighboring cells via secretion of FGF2/GROα/M-CSF to promote cancer dissemination under environmental remodeling via fibroblast-mediated laminin production. Analyses of tissues from clinical colon cancer patients and Krs-/+ animal models for cancer metastasis supported the roles of KRS, GAS6, and M2 macrophages in KRS-dependent positive feedback between tumors and environmental factors. Altogether, KRS in colon cancer cells remodels the microenvironment to promote metastasis, which can thus be therapeutically targeted at these bidirectional KRS-dependent communications of cancer spheroids with environmental cues.

Coumarins are natural compounds with antioxidant, anti-inflammatory and anti-cancer potential known to modulate inflammatory pathways. Here, non-toxic biscoumarin OT52 strongly inhibited proliferation of non-small cell lung cancer cells with KRAS mutations, inhibited stem-like characteristics by reducing aldehyde dehydrogenase expression and abrogated spheroid formation capacity. This cytostatic effect was characterized by cell cycle arrest and onset of senescence concomitant with endoplasmic reticulum and Golgi stress, leading to metabolic alterations. Mechanistically, this cellular response was associated with the novel capacity of biscoumarin OT52 to inhibit STAT3 transactivation and expression of its target genes linked to proliferation. These results were validated by computational docking of OT52 to the STAT3 DNA-binding domain. Combination treatments of OT52 with subtoxic concentrations of Bcl-xL and Mcl-1-targeting BH3 protein inhibitors triggered synergistic immunogenic cell death validated in colony formation assays as well as in vivo by zebrafish xenografts.

Summary Chronic injury to hepatocytes results in inflammation, steatohepatitis, fibrosis, and nonalcoholic fatty liver disease (NAFLD). The tetraspanin TM4SF5 is implicated in fibrosis and cancer. We investigate the role of TM4SF5 in communication between hepatocytes and macrophages (MΦs) and its possible influence on the inflammatory microenvironment that may lead to NAFLD. TM4SF5 induction in differentiated MΦs promotes glucose uptake, glycolysis, and glucose sensitivity, leading to M1-type MΦ activation. Activated M1-type MΦs secrete pro-inflammatory interleukin-6 (IL-6), which induces the secretion of CCL20 and CXCL10 from TM4SF5-positive hepatocytes. Although TM4SF5-dependent secretion of these chemokines enhances glycolysis in M0 MΦs, further chronic exposure reprograms MΦs for an increase in the proportion of M2-type MΦs in the population, which may support diet- and chemical-induced NAFLD progression. We suggest that TM4SF5 expression in MΦs and hepatocytes is critically involved in modulating the inflammatory environment during NAFLD progression.

Transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in hepatocellular carcinoma tissues and enhances migration in two-dimensional environments. Here, we investigated how TM4SF5 is involved in diverse pro-metastatic phenotypes in in vivo-like three-dimensional (3D) extracellular matrix gels. TM4SF5-positive cells aggressively formed invasive foci in 3D Matrigel, depending on TM4SF5-mediated signaling activity, cytoskeletal organization, and matrix metallopeptidase (MMP) 2-mediated extracellular remodeling, whereas TM4SF5-null cells did not. The TM4SF5-null cells did, however, form invasive foci in 3D Matrigel following inhibition of Rho-associated protein kinase or addition of collagen I, suggesting that collagen I compensated for TM4SF5 expression. Similarly, TM4SF5-positive cells expressing vascular endothelial-cadherin formed network-like vasculogenic mimicry in 3D Matrigel and collagen I mixture gels, whereas TM4SF5-negative cells in the mixture gels displayed the network structures only upon further treatment with epidermal growth factor. The foci formation also required MMP2-mediated remodeling of the extracellular matrix. Co-cultures exhibited TM4SF5-positive or cancer-associated fibroblasts at the outward edges of TM4SF5-null cell clusters. Compared with TM4SF5-null cells, TM4SF5-positive cells in 3D collagen gels showed a more invasive outgrowth with dramatic invadopodia. These observations suggest that TM4SF5 plays roles in the promotion of diverse metastatic properties with fewer environmental requirements than TM4SF5-negative cells.

Membrane proteins sense extracellular cues and transduce intracellular signaling to coordinate directionality and speed during cellular migration. They are often localized to specific regions, as with lipid rafts or tetraspanin-enriched microdomains; however, the dynamic interactions of tetraspanins with diverse receptors within tetraspanin-enriched microdomains on cellular surfaces remain largely unexplored. Here, we investigated effects of tetraspan(in) TM4SF5 (transmembrane 4 L6 family member 5)-enriched microdomains (T5ERMs) on the directionality of cell migration. Physical association of TM4SF5 with epidermal growth factor receptor (EGFR) and integrin α5 was visualized by live fluorescence cross-correlation spectroscopy and higher-resolution microscopy at the leading edge of migratory cells, presumably forming TM4SF5-enriched microdomains. Whereas TM4SF5 and EGFR colocalized at the migrating leading region more than at the rear, TM4SF5 and integrin α5 colocalized evenly throughout cells. Cholesterol depletion and disruption in TM4SF5 post-translational modifications, including N-glycosylation and palmitoylation, altered TM4SF5 interactions and cellular localization, which led to less cellular migration speed and directionality in 2- or 3-dimensional conditions. TM4SF5 controlled directional cell migration and invasion, and importantly, these TM4SF5 functions were dependent on cholesterol, TM4SF5 post-translational modifications, and EGFR and integrin α5 activity. Altogether, we showed that TM4SF5 dynamically interacted with EGFR and integrin α5 in migratory cells to control directionality and invasion.-Kim, H.-J., Kwon, S., Nam, S. H., Jung, J. W., Kang, M., Ryu, J., Kim, J. E., Cheong, J.-G., Cho, C. Y., Kim, S., Song, D.-G., Kim, Y.-N., Kim, T. Y., Jung, M.-K., Lee, K.-M., Pack, C.-G., Lee, J. W. Dynamic and coordinated single-molecular interactions at TM4SF5-enriched microdomains guide invasive behaviors in 2- and 3-dimensional environments.

// Jin-Gyu Cheong 1 , Dae-Geun Song 1, 2 , Haeng Eun Song 1 , Fedor Berditchevski 3 , Seo Hee Nam 4 , Jae Woo Jung 4 , Hye-Jin Kim 1 , Ji Eon Kim 1 , Somi Kim 1 , Jihye Ryu 1 , Chang Yun Cho 1 , Kyung-Min Lee 1 , Jung Weon Lee 1, 4 1 Department of Pharmacy, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea 2 Systems Biotechnology Research Center, Korea Institute of Science and Technology (KIST), Gangneung-si, Gangwon-do 25451, Republic of Korea 3 Institute of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK 4 Interdisciplinary Program in Genetic Engineering, Seoul National University, Seoul 08826, Republic of Korea Correspondence to: Jung Weon Lee, email: jwl@snu.ac.kr Keywords: 3D cell culture, migration, proliferation, spheroids, transmembrane 4 L six family Received: August 20, 2016 Accepted: December 27, 2016 Published: January 25, 2017 ABSTRACT The transmembrane 4 L six family proteins TM4SF1, TM4SF4, and TM4SF5 share 40-50% overall sequence identity, but their C-terminus identity is limited. It may be likely that the C-termini of the members are important and unique for own regulatory functions. We thus examined how the TM4SF5 C-terminus affected cellular functions differentially from other family members. Using colon cancer cells expressing wildtype (WT), C-terminus-deleted, or chimeric mutants, diverse cellular functions were explored in 2-dimensional (2D) and 3-dimensional (3D) condition. The C-termini of the proteins were relatively comparable with respect to 2D cell proliferation, although each C-terminal-deletion mutant exhibited increased proliferation relative to the WT. Using chimeric constructs, we found that the TM4SF5 C-terminus was critical for regulating the diverse metastatic functions of TM4SF5, and could positively replace the C-termini of other family members. Replacement of the TM4SF1 or TM4SF4 C-terminus with that of TM4SF5 increased spheroids growth, transwell migration, and invasive dissemination from spheroids in 3D collagen gels. TM4SF5-mediated effects required its extracellular loop 2 linked to the C-terminus via the transmembrane domain 4, with causing c-Src activation. Altogether, the C-terminus of TM4SF5 appears to mediate pro-migratory roles, depending on a structural relay from the second extracellular loop to the C-terminus.

The impact of liver disease on whole-body glucose homeostasis is largely attributed to dysregulated release of secretory proteins in response to metabolic stress. The molecular cues linking liver to whole-body glucose metabolism remain elusive. We found that expression of G protein α-13 (Gα13) was decreased in the liver of mice and humans with diabetes. Liver-specific deletion of the Gna13 gene in mice resulted in systemic glucose intolerance. Comparative secretome analysis identified inter-α-trypsin inhibitor heavy chain 1 (ITIH1) as a protein secreted by liver that was responsible for systemic insulin resistance in Gna13-deficient mice. Liver expression of ITIH1 positively correlated with surrogate markers for diabetes in patients with impaired glucose tolerance or overt diabetes. Mechanistically, a decrease in hepatic Gα13 caused ITIH1 oversecretion by liver through induction of O-GlcNAc transferase expression, facilitating ITIH1 deposition on the hyaluronan surrounding mouse adipose tissue and skeletal muscle. Neutralization of secreted ITIH1 ameliorated glucose intolerance in obese mice. Our findings demonstrate systemic insulin resistance in mice resulting from liver-secreted ITIH1 downstream of Gα13 and its reversal by ITIH1 neutralization.

Reactive oxygen species (ROS) regulate cell fate, although signaling molecules that regulate ROS hormesis remain unclear. Here we show that transmembrane 4 L six family member 5 (TM4SF5) in lung epithelial cells induced the alternatively spliced CD44v8-10 variant via an inverse ZEB2/epithelial splicing regulatory proteins (ESRPs) linkage. TM4SF5 formed complexes with the cystine/glutamate antiporter system via TM4SF5- and CD44v8-10-dependent CD98hc plasma-membrane enrichment. Dynamic TM4SF5 binding to CD98hc required CD44v8-10 under ROS-generating inflammatory conditions. TM4SF5 and CD44v8-10 upregulated cystine/glutamate antiporter activity and intracellular glutathione levels, leading to ROS modulation for cell survival. Tm4sf5-null mice exhibited attenuated bleomycin-induced pulmonary fibrosis with lower CD44v8-10 and ESRPs levels than wild-type mice. Primary mouse alveolar epithelial cells (AECs) revealed type II AECs (AECII), but not type I, to adapt the TM4SF5-mediated characteristics, suggesting TM4SF5-mediated AECII survival following AECI injury during idiopathic pulmonary fibrosis (IPF). Thus, the TM4SF5-mediated CD44v8-10 splice variant could be targeted against IPF.

Fibrosis is characterized by the increased accumulation of extracellular matrix (ECM), which drives abnormal cell proliferation and progressive organ dysfunction in many inflammatory and metabolic diseases. Studies have shown that halofuginone, a racemic halogenated derivative, inhibits glutamyl-prolyl-transfer RNA-synthetase (EPRS)-mediated fibrosis. However, the mechanism by which this occurs is unclear. We explored the mechanistic aspects of how EPRS could develop liver fibrotic phenotypes in cells and animal models. Treatment with TGF-β1 up-regulated fibronectin and collagen I levels in LX2 hepatic stellate cells. This effect was inhibited in prolyl-transfer RNA synthetase (PRS)-suppressed LX2 cells. Using the promoter luciferase assay, TGF-β1-mediated collagen I, α1 chain transcription and γ2 basal laminin transcription in LX2 cells were down-regulated by EPRS suppression, suggesting that EPRS may play roles in ECM production at transcriptional levels. Furthermore, signal transducer and activator of transcription (STAT) signaling activation was involved in the effects of TGF-β1 on ECM expression in a PRS-dependent manner. This was mediated via a protein-protein complex formation consisting of TGF-β1 receptor, EPRS, Janus kinases, and STAT6. Additionally, ECM expression in fibrotic livers overlapped with EPRS expression along fibrotic septa regions and was positively correlated with STAT6 activation in carbon tetrachloride-treated mice. This was less obvious in livers of Eprs-/+ mice. These findings suggest that, during fibrosis development, EPRS plays roles in nontranslational processes of ECM expression via intracellular signaling regulation upon TGF-β1 stimulation.-Song, D.-G., Kim, D., Jung, J. W., Nam, S. H., Kim, J. E., Kim, H.-J., Kim, J. H., Lee, S.-J., Pan, C.-H., Kim, S., Lee, J. W. Glutamyl-prolyl-tRNA synthetase induces fibrotic extracellular matrix via both transcriptional and translational mechanisms.

Human 8-oxoguanine DNA glycosylase 1 (OGG1) is the major DNA repair enzyme that plays a key role in excision of oxidative damaged DNA bases such as 8-oxoguainine (8-oxoG). Recent studies suggest another function of OGG1, namely that it may be involved in the endotoxin- or oxidative stress-induced inflammatory response. In this study, we investigated the role of OGG1 in the inflammatory response. OGG1 expression is increased in the organs of endotoxin-induced or myelin oligodendrocyte glycoprotein (MOG)-immunized mice and immune cells, resulting in induction of the expression of pro-inflammatory mediators at the transcriptional levels. Biochemical studies showed that signal transducer and activator of transcription 1 (STAT1) plays a key role in endotoxin-induced OGG1 expression and inflammatory response. STAT1 regulates the transcriptional activity of OGG1 through recruiting and binding to the gamma-interferon activation site (GAS) motif of the OGG1 promoter region, and chromatin remodeling by acetylation and dimethylation of lysine-14 and -4 residues of histone H3. In addition, OGG1 acts as a STAT1 coactivator and has transcriptional activity in the presence of endotoxin. The data presented here identifies a novel mechanism, and may provide new therapeutic strategies for the treatment of endotoxin-mediated inflammatory diseases.

CD133 is a surface marker of liver cancer stem cells. Transmembrane 4 L six family member 5 (TM4SF5) promotes sphere growth and circulation. However, it is unknown how CD133 and TM4SF5 cross-talk with each other for cancer stem cell properties. Here, we investigated the significance of inter-relationships between CD133, TM4SF5, CD44, and protein tyrosine phosphatase receptor type F (PTPRF) in a three-dimensional (3D) sphere growth system. We found that CD133 upregulated TM4SF5 and CD44, whereas TM4SF5 and CD44 did not affect CD133 expression. Signaling activity following CD133 phosphorylation caused TM4SF5 expression and sphere growth. TM4SF5 bound to CD133 and promoted c-Src activity for CD133 phosphorylation as a positive feedback loop, leading to CD133-mediated sphere growth that was inhibited by TM4SF5 inhibition or suppression. TM4SF5 also bound PTPRF and promoted paxillin phosphorylation. Decreased sphere growth upon CD133 suppression was recovered by TM4SF5 expression and partially by PTPRF suppression. TM4SF5 inhibition enhanced PTPRF levels and abolished PTPRF suppression-mediated sphere growth. Altogether, CD133-induced TM4SF5 expression and function were important for liver cancer sphere growth and may be a promising target to block metastasis.

Mitochondrial dysfunction may play a key role in the progression of steatosis to nonalcoholic steatohepatitis (NASH); however, the molecular mechanism that controls the structure and function of mitochondria in NASH is not clearly understood. Here, we demonstrated that RORα is a regulator of expression of Bnip3 and PGC-1α, and thereby enhances mitochondrial quality. First, we observed that liver-specific RORα knockout mice (RORα-LKO) were more susceptible to high-fat diet-induced NASH compared with control, probably due to mitochondrial dysfunction. Concordantly, mitochondrial fission in response to nutrient stimuli was abolished with downregulation of Bnip3 and phospho-Drp1 in the hepatocytes of RORα-LKO. RORα enhanced oxygen consumption rate and expression of genes associated with mitochondrial quality control. Finally, we observed the positive correlation of the expression levels of Bnip3 and PGC-1α with those of RORα in patients with steatohepatitis. Together, we demonstrated that RORα mediates mitochondrial quality under nutrient-overloaded conditions and propose RORα as a potential therapeutic target in treatment of NASH.

Objectives The membrane glycoprotein TM4SF5 (transmembrane 4 L6 family member 5), which is similar to the tetraspanins, is highly expressed in different cancers and causes epithelial–mesenchymal transition (EMT). TM4SF5 interacts with other membrane proteins during its pro-tumorigenic roles, presumably at tetraspanin-enriched microdomains (TEMs/TERMs). Here, we explored TM4SF5-mediated resistance against the clinically important EGFR kinase inhibitors, with regards to cooperation with other membrane proteins, particularly the insulin-like growth factor 1 receptor (IGF1R). Materials and methods Using cancer cells including NSCLC with TM4SF5 overexpression or IGF1R suppression in either normal 2 dimensional (2D), 3D aqueous spheroids, or 3D collagen I gels systems, the sensitivity to tyrosine kinase inhibitors (TKIs) were evaluated. Results and conclusion We found that TM4SF5 and IGF1R transcriptionally modulated one another, with each protein promoting the expressions of the other. Expression of TM4SF5 in gefitinib-sensitive HCC827 cells caused resistance to erlotinib and gefitinib, but not to sorafenib [a platelet derived growth factor receptor (PDGFR) inhibitor]; whereas suppression of IGF1R from gefitinib-resistant NCI-H1299 cells caused enhanced sensitization to the inhibitors. Expression of TM4SF5 and IGF1R in the drug-sensitive cells promoted signaling activities of extracellular signal-regulated kinases (ERKs), protein kinase B (Akt), and S6 kinase (S6K), and resulted in a higher residual EGFR activity, even after EGFR kinase inhibitor treatment. Complex formation between TM4SF5 and IGF1R was observed, and also included EGFR, dependent on TM4SF5 expression. The TM4SF5-mediated drug resistance was further confirmed in an aqueous 3D spheroid system or upon being embedded in 3D extracellular matrix (ECM)-surrounded gel systems. Collectively, these data suggest that anti-TM4SF5 reagents may be combined with the EGFR kinase inhibitors to enhance the efficacy of chemotherapies against NSCLC.

// Seo Hee Nam 1 , Doyeun Kim 2 , Mi-Sook Lee 2 , Doohyung Lee 2 , Tae Kyoung Kwak 2 , Minkyung Kang 2,3 , Jihye Ryu 2 , Hye-Jin Kim 2 , Haeng Eun Song 2 , Jungeun Choi 1 , Gyu-Ho Lee 2 , Sang-Yeob Kim 4 , Song Hwa Park 2 , Dae Gyu Kim 2 , Nam Hoon Kwon 2 , Tai Young Kim 2 , Jean Paul Thiery 5,6,7 , Sunghoon Kim 1,2 and Jung Weon Lee 1,2 1 Interdisciplinary Program in Genetic Engineering, Seoul National University, Seoul, Republic of Korea 2 Department of Pharmacy, Medicinal Bioconvergence Research Center, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, Republic of Korea 3 Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea 4 Department of Medicine, University of Ulsan, College of Medicine, Seoul, Republic of Korea 5 Cancer Science Institute, National University of Singapore, Singapore 6 Institute of Molecular and Cell Biology, A*STAR, Singapore 7 Department of Biochemistry, School of Medicine, National University of Singapore, Singapore Correspondence to: Jung Weon Lee, email: // Keywords : 3D culture; cancer metastasis; cell-ECM adhesion; lysyl-tRNA synthetase; signal transduction Received : January 15, 2015 Accepted : April 30, 2015 Published : May 12, 2015 Abstract The adhesion properties of cells are involved in tumor metastasis. Although KRS at the plasma membrane is shown important for cancer metastasis, additionally to canonical roles of cytosolic KRS in protein translation, how KRS and its downstream effectors promote the metastatic migration remains unexplored. Disseminative behaviors (an earlier metastatic process) of colon cancer cell spheroids embedded in 3D collagen gels were studied with regards to cell adhesion properties, and relevance in KRS -/+ knocked-down animal and clinical colon cancer tissues. Time-lapse imaging revealed KRS-dependent cell dissemination from the spheroids, whereas KRS-suppressed spheroids remained static due to the absence of outbound movements supported by cell-extracellular matrix (ECM) adhesion. While keeping E-cadherin at the outward disseminative cells, KRS caused integrin-involved intracellular signaling for ERK/c-Jun, paxillin, and cell-ECM adhesion-mediated signaling to modulate traction force for crawling movement. KRS-suppressed spheroids became disseminative following ERK or paxillin re-expression. The KRS-dependent intracellular signaling activities correlated with the invasiveness in clinical colon tumor tissues and in KRS -/+ knocked-down mice tissues. Collectively, these observations indicate that KRS at the plasma membrane plays new roles in metastatic migration as a signaling inducer, and causes intracellular signaling for cancer dissemination, involving cell-cell and cell-ECM adhesion, during KRS-mediated metastasis.

Tumor metastasis involves circulating and tumor-initiating capacities of metastatic cancer cells. Epithelial-mesenchymal transition (EMT) is related to self-renewal capacity and circulating tumor cell (CTC) characteristics for tumor metastasis. Although tumor metastasis is a life-threatening, complicated process that occurs through circulation of tumor cells, mechanistic aspects of self-renewal and circulating capacities have been largely unknown. Hepatic transmembrane 4 L six family member 5 (TM4SF5) promotes EMT for malignant growth and migration, so it was rationalized that TM4SF5, as a hepatocellular carcinoma (HCC) biomarker, might be important for metastatic potential. Here, self-renewal capacity by TM4SF5 was mechanistically explored using hepatocarcinoma cells with or without TM4SF5 expression, and we explored whether they became CTCs using mouse liver-orthotopic model systems. We found that TM4SF5-dependent sphere growth correlated with CD24−, aldehyde dehydrogenase (ALDH) activity, as well as a physical association between CD44 and TM4SF5. Interaction between TM4SF5 and CD44 was through their extracellular domains with N-glycosylation modifications. TM4SF5/CD44 interaction activated proto-oncogene tyrosine-protein kinase Src (c-Src)/signal transducer and activator of transcription 3 (STAT3)/Twist-related protein 1 (Twist1)/B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi1) signaling for spheroid formation, whereas disturbing the interaction, expression, or activity of any component in this signaling pathway inhibited spheroid formation. In serial xenografts using 200∼5,000 cells per injection, TM4SF5-positive tumors exhibited subpopulations with locally increased CD44 expressions, supporting for tumor cell differentiation. TM4SF5-positive, but not TM4SF5- or CD44-knocked-down, cells were identified circulating in blood 4-6 weeks after orthotopic liver injection using in vivo laser scanning endomicroscopy. Anti-TM4SF5 reagent blocked their metastasis to distal intestinal organs. Conclusion: TM4SF5 promotes self-renewal and CTC properties supported by TM4SF5+/CD44+(TM4SF5-bound)/ALDH+/CD24− markers during HCC metastasis. (Hepatology 2015;61:1978-1997)

The impact of liver disease on whole-body glucose homeostasis is largely attributed to dysregulated release of secretory proteins in response to metabolic stress. The molecular cues linking liver to whole-body glucose metabolism remain elusive. We found that expression of G protein α-13 (Gα

The transmembrane four L6 family member 5 (TM4SF5) protein is a novel molecular target for the prevention and treatment of hepatocellular carcinoma. TM4SF5 is highly expressed in liver, colon, esophageal, and pancreatic cancers and is implicated in tumor progression. Here, we screened monoclonal antibodies that specifically bound to the extracellular loop 2 (EC2) of TM4SF5 from a phage-displayed murine antibody (single-chain variable fragment; scFv) library. We constructed and characterized chimeric antibodies, Ab27 and Ab79, of scFv fused with Fc domain of human IgG1. The affinity (K-D) of Ab27 and Ab79 for soluble EC2 was approximately 9.2 nM and 16.9 nM, respectively, as determined by surface plasmon resonance analysis. Ab27 and Ab79 efficiently bound to native TM4SF5 on the cell surface were internalized into the cancer cells, leading to a decrease in cell surface TM4SF5. Ab27 and Ab79 inhibited the proliferation and invasion of TM4SF5-positive liver and colon cancer cells and reduced FAK and c-Src phosphorylation. Ab27 and Ab79 also enhanced anoikis sensitivity and reduced survivin. Ab27 mediated antibody-dependent cell-mediated cytotoxicity in vitro. Ab27 and Ab79 efficiently inhibited tumor growth in a liver cancer xenograft model. These results strongly support the further development of Ab27 as a novel anti-cancer agent in the clinic.

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.

The epithelial-mesenchymal transition (EMT) is one mechanism by which cells with mesenchymal features can be generated and is a fundamental event in morphogenesis. Recently, invasion and metastasis of cancer cells from the primary tumor are now thought to be initiated by the developmental process termed the EMT, whereby epithelial cells lose cell polarity and cell-cell interactions, and gain mesenchymal phenotypes with increased migratory and invasive properties. The EMT is believed to be an important step in metastasis and is implicated in cancer progression, although the influence of the EMT in clinical specimens has been debated. This review presents the recent results of two cell surface proteins, the functions and underlying mechanisms of which have recently begun to be demonstrated, as novel regulators of the molecular networks that induce the EMT and cancer progression.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that conjugate specific amino acids to their cognate tRNAs for protein synthesis. Besides their catalytic activity, recent studies have uncovered many additional functions of these enzymes through their interactions with diverse cellular factors. Among human ARSs, cytosolic lysyl-tRNA synthetase (KRS) is often highly expressed in cancer cells and tissues, and facilitates cancer cell migration and invasion through the interaction with the 67kDa laminin receptor on the plasma membrane. Specific modulation of this interaction by small molecule inhibitors has revealed a new way to control metastasis. Here, we summarize the pro-metastatic functions of KRS and their patho-physiological implications.