Substituted Syndecan-2-Derived Mimetic Peptides Show Improved Antitumor Activity over the Parent Syndecan-2-Derived Peptide.

We previously showed that a synthetic peptide (S2-P) corresponding to a portion of the human syndecan-2 (SDC2) sequence can bind to the pro-domain of matrix metalloproteinase-7 (MMP-7) to inhibit colon cancer activities. Since S2-P had a relatively weak binding affinity for the MMP-7 pro-domain, we herein modified the amino acid sequence of S2-P to improve the anticancer potential. On the basis of the interaction structure of S2-P and MMP-7, four peptides were generated by replacing amino acids near Tyr 51, which is critical for the interaction. The SDC2-mimetic peptides harboring an Ala-to-Asp substitution at the C-terminal side of Tyr 51 (S2-D) or with an Ala-to-Phe substitution at the N-terminal side of Tyr 51 and an Ala-to-Asp substitution at the C-terminal side of Tyr 51 (S2-FE) showed improved interaction affinities for the MMP-7 pro-domain. Compared to S2-P, S2-FE was better able to inhibit the SDC2-MMP-7 interaction, the cell surface localization of MMP-7, the gelatin degradation activity of MMP-7, and the cancer activities (cell migration, invasion, and colony-forming activity) of human HCT116 colon cancer cells in vitro. In vivo, S2-FE inhibited the primary tumor growth and lung metastasis of CT26 mouse colon cancer cells in a xenograft mouse model. Together, these data suggest that S2-FE could be useful therapeutic anticancer peptides for colon cancer.

Sulfated syndecan 1 is critical to preventing cellular senescence by modulating fibroblast growth factor receptor endocytosis.

Cellular senescence can be triggered by various intrinsic and extrinsic stimuli. We previously reported that silencing of 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2) induces cellular senescence through augmented fibroblast growth factor receptor 1 (FGFR1) signaling. However, the exact molecular mechanism connecting heparan sulfation and cellular senescence remains unclear. Here, we investigated the potential involvement of heparan sulfate proteoglycans (HSPGs) in augmented FGFR1 signaling and cellular senescence. Depletion of several types of HSPGs revealed that cells depleted of syndecan 1 (SDC1) exhibited typical senescence phenotypes, and those depleted of PAPSS2-, SDC1-, or heparan sulfate 2-O sulfotransferase 1 (HS2ST1) showed decreased FGFR1 internalization along with hyperresponsiveness to and prolonged activation of fibroblast growth factor 2 (FGF2)-stimulated FGFR1- v-akt murine thymoma viral oncogene homolog (AKT) signaling. Clathrin- and caveolin-mediated FGFR1 endocytosis contributed to cellular senescence through the FGFR1-AKT-p53-p21 signaling pathway. Dynasore treatment triggered senescence phenotypes, augmented FGFR1-AKT-p53-p21 signaling, and decreased SDC1 expression. Finally, the replicatively and prematurely senescent cells were characterized by decreases of SDC1 expression and FGFR1 internalization, and an increase in FGFR1-AKT-p53-p21 signaling. Together, our results demonstrate that properly sulfated SDC1 plays a critical role in preventing cellular senescence through the regulation of FGFR1 endocytosis.

AP2M1 Supports TGF-ß Signals to Promote Collagen Expression by Inhibiting Caveolin Expression.

The extracellular matrix (ECM) is important for normal development and disease states, including inflammation and fibrosis. To understand the complex regulation of ECM, we performed a suppressor screening using Caenorhabditis elegans expressing the mutant ROL-6 collagen protein. One cuticle mutant has a mutation in dpy-23 that encodes the µ2 adaptin (AP2M1) of clathrin-associated protein complex II (AP-2). The subsequent suppressor screening for dpy-23 revealed the lon-2 mutation. LON-2 functions to regulate body size through negative regulation of the tumor growth factor-beta (TGF-ß) signaling pathway responsible for ECM production. RNA-seq analysis showed a dominant change in the expression of collagen genes and cuticle components. We noted an increase in the cav-1 gene encoding caveolin-1, which functions in clathrin-independent endocytosis. By knockdown of cav-1, the reduced TGF-ß signal was significantly restored in the dpy-23 mutant. In conclusion, the dpy-23 mutation upregulated cav-1 expression in the hypodermis, and increased CAV-1 resulted in a decrease of TßRI. Finally, the reduction of collagen expression including rol-6 by the reduced TGF-ß signal influenced the cuticle formation of the dpy-23 mutant. These findings could help us to understand the complex process of ECM regulation in organism development and disease conditions.

Rab8 and Rabin8-Mediated Tumor Formation by Hyperactivated EGFR Signaling via FGFR Signaling.

The epidermal growth factor receptor (EGFR) signaling is important for normal development, such as vulval development in Caenorhabditis elegans, and hyperactivation of the EGFR is often associated with cancer development. Our previous report demonstrated the multivulva (Muv) phenotype, a tumor model in C. elegans (jgIs25 strain) by engineering LET-23/EGFR with a TKI-resistant human EGFR T790-L858 mutant. Because Rab proteins regulate vesicle transport, which is important for receptor signaling, we screened the RNAi in the jgIs25 strain to find the Rabs critical for Muv formation. Herein, we show that rab-8 RNAi and the rab-8 (-/-) mutation effectively reduce Muv formation. We demonstrate that RABN-8, an ortholog of Rabin8, known as a GEF for Rab8, is also required for Muv formation by promoting the secretion of EGL-17/FGF from vulval precursor cells. In addition, FGFR inhibitors decreased Muv formation mediated by mutant EGFR. Our data suggest that Rab8 and Rabin8 mediate Muv formation through FGF secretion in the EGFR-TKI-resistant nematode model. Furthermore, FGFR-TKIs more effectively inhibit the growth of lung cancer cell lines in H1975 (EGFR T790M-L858R; EGFR-TKI-resistant) than H522 (wild-type EGFR) and H1650 (EGFR exon 19 deletion; EGFR-TKI-sensitive) cells, suggesting that FGFR-TKIs could be used to control cancers with EGFR-TKI-resistant mutations.

Pygenic Acid A (PA) Sensitizes Metastatic Breast Cancer Cells to Anoikis and Inhibits Metastasis In Vivo.

Metastasis is the main cause of cancer-related deaths. Anoikis is a type of apoptosis caused by cell detachment, and cancer cells become anoikis resistant such that they survive during circulation and can successfully metastasize. Therefore, sensitization of cancer cells to anoikis could prevent metastasis. Here, by screening for anoikis sensitizer using natural compounds, we found that pygenic acid A (PA), a natural compound from Prunella vulgaris, not only induced apoptosis but also sensitized the metastatic triple-negative breast cancer cell lines, MDA-MB-231 cells (human) and 4T1 cells (mouse), to anoikis. Apoptosis protein array and immunoblotting analysis revealed that PA downregulated the pro-survival proteins, including cIAP1, cIAP2, and survivin, leading to cell death of both attached and suspended cells. Interestingly, PA decreased the levels of proteins associated with anoikis resistance, including p21, cyclin D1, p-STAT3, and HO-1. Ectopic expression of active STAT3 attenuated PA-induced anoikis sensitivity. Although PA activated ER stress and autophagy, as determined by increases in the levels of characteristic markers, such as IRE1α, p-elF2α, LC3B I, and LC3B II, PA treatment resulted in p62 accumulation, which could be due to PA-induced defects in autophagy flux. PA also decreased metastatic characteristics, such as cell invasion, migration, wound closure, and 3D growth. Finally, lung metastasis of luciferase-labeled 4T1 cells decreased following PA treatment in a syngeneic mouse model when compared with the control. These data suggest that PA sensitizes metastatic breast cancer cells to anoikis via multiple pathways, such as inhibition of pro-survival pathways and activation of ER stress and autophagy, leading to the inhibition of metastasis. These findings suggest that sensitization to anoikis by PA could be used as a new therapeutic strategy to control the metastasis of breast cancer.

Anti-Tumor Effects of Sodium Meta-Arsenite in Glioblastoma Cells with Higher Akt Activities.

Glioblastoma is a type of aggressive brain tumor that grows very fast and evades surrounding normal brain, lead to treatment failure. Glioblastomas are associated with Akt activation due to somatic alterations in PI3 kinase/Akt pathway and/or PTEN tumor suppressor. Sodium meta-arsenite, KML001 is an orally bioavailable, water-soluble, and trivalent arsenical and it shows antitumoral effects in several solid tumor cells via inhibiting oncogenic signaling, including Akt and MAPK. Here, we evaluated the effect of sodium meta-arsenite, KML001, on the growth of human glioblastoma cell lines with different PTEN expression status and Akt activation, including PTEN-deficient cells (U87-MG and U251) and PTEN-positive cells (LN229). The growth-inhibitory effect of KML001 was stronger in U87-MG and U251 cells, which exhibited higher Akt activity than LN229 cells. KML001 deactivated Akt and decreased its protein levels via proteasomal degradation in U87-MG cells. KML001 upregulated mutant PTEN levels via inhibition of its proteasomal degradation. KML001 inhibited cell growth more effectively in active Akt-overexpressing LN229 cells than in mock-expressing LN229 cells. Consistent with these results, KML001 sensitized PTEN-deficient cells more strongly to growth inhibition than it did PTEN-positive cells in prostate and breast cancer cell lines. Finally, we illustrated in vivo anti-tumor effects of KML001 using an intracranial xenograft mouse model. These results suggest that KML001 could be an effective chemotherapeutic drug for the treatment of glioblastoma cancer patients with higher Akt activity and PTEN loss.

Anti-cancer effect of doxorubicin is mediated by downregulation of HMG-Co A reductase via inhibition of EGFR/Src pathway.

Doxorubicin is a widely used DNA damage-inducing anti-cancer drug. However, its use is limited by its dose-dependent side effects, such as cardiac toxicity. Cholesterol-lowering statin drugs increase the efficacy of some anti-cancer drugs. Cholesterol is important for cell growth and a critical component of lipid rafts, which are plasma membrane microdomains important for cell signaling. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMG-CR) is a critical enzyme in cholesterol synthesis. Here, we show that doxorubicin downregulated HMG-CR protein levels and thus reduced levels of cholesterol and lipid rafts. Cholesterol addition attenuated doxorubicin-induced cell death, and cholesterol depletion enhanced it. Reduction of HMG-CR activity by simvastatin, a statin that acts as an HMG-CR inhibitor, or by siRNA-mediated HMG-CR knockdown enhanced doxorubicin cytotoxicity. Doxorubicin-induced HMG-CR downregulation was associated with inactivation of the EGFR-Src pathway. Furthermore, a high-cholesterol-diet attenuated the anti-cancer activity of doxorubicin in a tumor xenograft mouse model. In a multivulva model of Caenorhabditis elegans expressing an active-EGFR mutant, doxorubicin decreased hyperplasia more efficiently in the absence than in the presence of cholesterol. These data indicate that EGFR/Src/HMG-CR is a new pathway mediating doxorubicin-induced cell death and that cholesterol control could be combined with doxorubicin treatment to enhance efficacy and thus reduce side effects.

Dynamic and coordinated single-molecular interactions at TM4SF5-enriched microdomains guide invasive behaviors in 2- and 3-dimensional environments.

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.

Regulation of anoikis resistance by NADPH oxidase 4 and epidermal growth factor receptor.

BACKGROUND: Normal cells are sensitive to anoikis, which is a cell detachment-induced apoptosis. However, cancer cells acquire anoikis resistance that is essential for successful metastasis. This study aimed to demonstrate the function and potential mechanism of NADPH oxidase 4 (NOX4) and EGFR activation in regulating anoikis resistance in lung cancer. METHODS: Cells were cultured either in the attached or suspended condition. Cell viability was measured by cell counting and live and dead cell staining. Expression levels of NOX4 and EGFR were measured by PCR and immunoblotting. Reactive oxygen species (ROS) levels were measured by flow cytometry. Effects of NOX4 overexpression or NOX4 knockdown by si-NOX4 on anoikis sensitivity were explored. Levels of NOX4 and EGFR in lung cancer tissues were evaluated by IHC staining. RESULTS: NOX4 was upregulated but EGFR decreased in suspended cells compared with attached cells. Accordingly, ROS levels were increased in suspended cells, resulting in the activation of Src and EGFR. NOX4 knockdown decreased activation of Src and EGFR, and thus sensitised cells to anoikis. NOX4 overexpression increased EGFR levels and attenuated anoikis. NOX4 expression is upregulated and is positively correlated with EGFR levels in the lung cancer patient tissues. CONCLUSIONS: NOX4 upregulation confers anoikis resistance by ROS-mediated activation of EGFR and Src, and by maintaining EGFR levels, which is critical for cell survival.

Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis.

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function.

Discoidin domain receptor 1 is a novel transcriptional target of ZEB1 in breast epithelial cells undergoing H-Ras-induced epithelial to mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) process allows carcinoma cells to dissociate from the primary tumor thereby facilitating tumor cell invasion and metastasis. Ras-dependent hyperactive signaling is commonly associated with tumorigenesis, invasion, EMT, and metastasis. However, the downstream effectors by which Ras regulates EMT remain ill defined. In this study, we show that the H-Ras pathway leads to mesenchymal-like phenotypic changes in human breast epithelial cells by controlling the ZEB1/microRNA-200c axis. Moreover, H-Ras suppresses the expression of the discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine kinase, via ZEB1, thus identifying ZEB1 as a novel transcriptional repressor of DDR1. Mutation studies on the putative promoter of the DDR1 gene revealed that bipartite Z- and E-box elements play a key role in transcriptional repression of DDR1 in Hs578T and MDA-MB-231 breast carcinoma cell lines by ZEB1. Furthermore, we found an inverse correlation between ZEB1 and DDR1 expression in various cancer cell lines and in human breast carcinoma tissues. Consistently, overexpression of DDR1 reduced the invasive phenotype of mesenchymal-like triple-negative breast cancer cells in 3D cultures and in vivo. Thus, ZEB1's role in maintenance of EMT in breast carcinoma cells is mediated in part by its ability to suppress DDR1 expression and consequently contribute to the activation of the invasive phenotype. Taken together, our results unveil a novel H-Ras/ZEB1/DDR1 network that contributes to breast cancer progression in triple-negative breast cancers.

TRIM22 induces cellular senescence by targeting PHLPP2 in hepatocellular carcinoma.

The ubiquitin-proteasome system is a vital protein degradation system that is involved in various cellular processes, such as cell cycle progression, apoptosis, and differentiation. Dysregulation of this system has been implicated in numerous diseases, including cancer, vascular disease, and neurodegenerative disorders. Induction of cellular senescence in hepatocellular carcinoma (HCC) is a potential anticancer strategy, but the precise role of the ubiquitin-proteasome system in cellular senescence remains unclear. In this study, we show that the E3 ubiquitin ligase, TRIM22, plays a critical role in the cellular senescence of HCC cells. TRIM22 expression is transcriptionally upregulated by p53 in HCC cells experiencing ionizing radiation (IR)-induced senescence. Overexpression of TRIM22 triggers cellular senescence by targeting the AKT phosphatase, PHLPP2. Mechanistically, the SPRY domain of TRIM22 directly associates with the C-terminal domain of PHLPP2, which contains phosphorylation sites that are subject to IKKß-mediated phosphorylation. The TRIM22-mediated PHLPP2 degradation leads to activation of AKT-p53-p21 signaling, ultimately resulting in cellular senescence. In both human HCC databases and patient specimens, the levels of TRIM22 and PHLPP2 show inverse correlations at the mRNA and protein levels. Collectively, our findings reveal that TRIM22 regulates cancer cell senescence by modulating the proteasomal degradation of PHLPP2 in HCC cells, suggesting that TRIM22 could potentially serve as a therapeutic target for treating cancer.

Identification of annexin II as a novel secretory biomarker for breast cancer.

Early prediction of metastatic breast cancer is important for improvement of prognosis and survival rate. The present study aimed to identify secreted protein biomarkers for detection of invasive breast cancer. To this end, we performed a comparative proteomic analysis by a combination of 2DE and MALDI-TOF MS analysis of conditioned media from invasive H-Ras MCF10A human breast epithelial cells and noninvasive MCF10A and N-Ras MCF10A cells. We identified a list of 25 proteins that were strongly detected in media of H-Ras MCF10A and focused on annexin II, which was shown to be involved in cell motility. Invasive triple-negative human breast carcinoma cells, Hs578T, and MDA-MB-231, showed increased levels of annexin II in media, demonstrating that secretion of annexin II correlated well with the invasive phenotype of cells. We demonstrated a crucial role of annexin II in breast cell invasion/migration and actin cytoskeleton reorganization required for filopodia formation. Annexin II levels in the plasma samples and breast cancer tissues of breast cancer patients were significantly higher than those of normal groups, providing a clinical relevance to our in vitro findings. Taken together, we identified annexin II as a novel secretory biomarker candidate for invasive breast cancer, especially estrogen receptor-negative breast cancer.

Pharmacological anti-tumor effects of natural Chamaecyparis obtusa (siebold & zucc.) endl. Leaf extracts on breast cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Chamaecyparis obtusa (C. obtusa, cypress species) is a plant that grows mainly in the temperate Northern Hemisphere and has long been used as a traditional anti-inflammatory treatment in East Asia. C. obtusa contains phytoncides, flavonoids, and terpenes, which have excellent anti-cancer effects and have been reported to prevent the progression of various cancers. However, the detailed mechanisms underlying the anti-cancer effects of C. obtusa extracts are unknown. AIM OF THE STUDY: We sought to confirm the anti-cancer effects of C. obtusa leaf extracts and to reveal the mechanism of action, with the possibility of its application in the treatment or prevention of cancer. MATERIAL &METHODS: The cytotoxicity of C. obtusa leaf extracts was confirmed using an MTT assay. Intracellular changes in protein levels were measured by immunoblotting, and mRNA levels were measured with qRT-PCR. Wound healing assay and transwell migration assay were used to evaluate the metastatic potential of breast cancer cells. The extract-induced apoptosis was observed using IncuCyte Annexin V Red staining analysis. A syngeneic breast cancer mouse model was established by injecting 4T1-Luc mouse breast cancer cells into the fat pad of female BALB/c mice, and the extract was administered orally. Luciferin solution was injected intraperitoneally to assess primary tumor development and metastasis by bioluminescence. RESULTS: C. obtusa leaf extracts were extracted with boiling water, 70% EtOH, and 99% EtOH. Among the extracts, the 99% EtOH extract of C. obtusa leaf (CO99EL) most clearly inhibited the tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (pY-STAT3) in MDA-MB-231 breast cancer cells at a concentration of 25 and 50 µg/mL. In addition, CO99EL strongly inhibited not only endogenous pY-STAT3 levels but also IL-6-induced STAT3 activation in various types of cancer cells, including breast cancer. CO99EL inhibited metastatic potential by downregulating the expression of N-cadherin, fibronectin, TWIST, MMP2, and MMP9 in MDA-MB-231 breast cancer cells. CO99EL also induced apoptotic cell death by increasing cleaved caspase-3 and decreasing anti-apoptotic proteins Bcl-2 and Bcl-xL. In an in vivo syngeneic breast cancer mouse model, 100 mg/kg CO99EL suppressed tumor growth and induced apoptosis of cancer cells. Moreover, CO99EL significantly inhibited lung metastasis from primary breast cancer. CONCLUSIONS: Our study demonstrated that 100 mg/kg CO99EL has potent anti-tumor effects against breast cancer, thus suggesting that 100 mg/kg CO99EL has potential applications in the treatment and prevention of breast cancer.

ICSBP-induced PD-L1 enhances osteosarcoma cell growth.

Background: Interferon (IFN) consensus sequence binding protein (ICSBP) is a transcription factor induced by IFN-γ. We previously reported that ICSBP expression promotes osteosarcoma progression by enhancing transforming growth factor-ß signaling. In cancer cells, programmed death-ligand 1 (PD-L1) contributes to immune escape and may also be involved in tumor progression. Because IFN-γ induces the expression of both ICSBP and PD-L1, we explored the association between ICSBP and PD-L1 expression in terms of osteosarcoma progression. Methods: Three osteosarcoma cell lines (Saos2, U2OS, and 143B) were employed. Gene expression was measured by qRT-PCR, and protein levels were assessed by immunoblotting. PD-L1 expression was evaluated in cells overexpressing ICSBP and in ICSBP knockdown cells. The effects of PD-L1 expression on cell growth were examined by MTS assays, Incucyte analysis, soft agar assays, and three-dimensional (3D) culture. Cell cycle and apoptosis were evaluated by FACS analysis of cells stained with propidium iodide (PI) and annexin V/PI, respectively. The antitumor effects of PD-L1 knockdown without or with doxorubicin treatment were evaluated in vivo in nude mice bearing ICSBP-overexpressing 143B cell xenograft. The clinical relevance of PD-L1 and ICSBP expression was evaluated immunohistochemically using a human osteosarcoma microarray and through analysis of publicly available data using Gene Expression Profiling Interactive Analysis2. Results: ICSBP overexpression upregulated PD-L1 expression in all three cell lines, whereas ICSBP knockdown decreased the PD-L1 expression. PD-L1 knockdown attenuated the cell growth and reduced colony-forming capacity in both soft agar assays and 3D culture. PD-L1 knockdown increased apoptosis and induced G2/M arrest, which was associated with decreased expression of survivin, cyclin-dependent kinase 4 (CDK4), cyclin E, and cyclin D1 expression and increased the expression of p27, phosphorylated Cdc2, and phosphorylated Wee1. PD-L1 knockdown decreased the growth of tumor xenografts and increased the doxorubicin sensitivity of ICSBP-overexpressing 143B cells both in vitro and in vivo. PD-L1 was expressed in human osteosarcoma tissues, and its expression was moderately correlated with that of ICSBP in osteosarcoma patients. Conclusion: ICSBP regulates PD-L1 expression in osteosarcoma cells, and PD-L1 knockdown combined with doxorubicin treatment could represent a strategy for controlling osteosarcoma expressing ICSBP.

Shed syndecan-2 enhances colon cancer progression by increasing cooperative angiogenesis in the tumor microenvironment.

Although shed syndecan-2 potentiated the tumorigenic activities of colon cancer cells, how shed syndecan-2 increases this tumorigenic potential remains unclear. Using an orthotopic mouse model of colon cancer, we show that shed syndecan-2 increases colon cancer progression by cooperatively promoting angiogenesis. Co-administration with a synthetic peptide of shed syndecan-2 (S2LQ) enhanced the survival and tumor engraftment of luciferase-expressing CT26 colon adenocarcinoma cells orthotopically implanted into the cecum of BALB/c mice. Intravenous injection of S2LQ further enhanced the growth of orthotopic tumors in the cecum, with increases in the tissue infiltration of macrophages and the formation of blood vessels, mainly in peripheral layers of the tumor facing the stroma. Furthermore, S2LQ stabilized HIF1α and enhanced the VEGF expression in human colon cancer cell lines, and increased the migration of RAW 264.7 murine macrophage cells and bone marrow-derived macrophages. Finally, S2LQ increased the tube formation of vascular endothelial cells in vitro. Together, these data demonstrate that shed syndecan-2 enhances tumorigenic activity by increasing the crosstalk of cancer cells with tumor-associated macrophages and endothelial cells to enhance angiogenesis for colon cancer progression in the tumor microenvironment.

Loss of phospholipase Cγ1 suppresses hepatocellular carcinogenesis through blockade of STAT3-mediated cancer development.

Phospholipase C gamma 1 (PLCγ1) plays an oncogenic role in several cancers, alongside its usual physiological roles. Despite studies aimed at identifying the effect of PLCγ1 on tumors, the pathogenic role of PLCγ1 in the tumorigenesis and development of hepatocellular carcinoma (HCC) remains unknown. To investigate the function of PLCγ1 in HCC, we generated hepatocyte-specific PLCγ1 conditional knockout (PLCγ1f/f ; Alb-Cre) mice and induced HCC with diethylnitrosamine (DEN). Here, we identified that hepatocyte-specific PLCγ1 deletion effectively prevented DEN-induced HCC in mice. PLCγ1f/f ; Alb-Cre mice showed reduced tumor burden and tumor progression, as well as a decreased incidence of HCC and less marked proliferative and inflammatory responses. We also showed that oncogenic phenotypes such as repressed apoptosis, and promoted proliferation, cell cycle progression and migration, were induced by PLCγ1. In terms of molecular mechanism, PLCγ1 regulated the activation of signal transducer and activator of transcription 3 (STAT3) signaling. Moreover, PLCγ1 expression is elevated in human HCC and correlates with a poor prognosis in patients with HCC. Our results suggest that PLCγ1 promotes the pathogenic progression of HCC, and PLCγ1/STAT3 axis was identified as a potential therapeutic target pathway for HCC.

Reprogramming of T cell-derived small extracellular vesicles using IL2 surface engineering induces potent anti-cancer effects through miRNA delivery.

T cell-derived small extracellular vesicles (sEVs) exhibit anti-cancer effects. However, their anti-cancer potential should be reinforced to enhance clinical applicability. Herein, we generated interleukin-2-tethered sEVs (IL2-sEVs) from engineered Jurkat T cells expressing IL2 at the plasma membrane via a flexible linker to induce an autocrine effect. IL2-sEVs increased the anti-cancer ability of CD8+ T cells without affecting regulatory T (Treg ) cells and down-regulated cellular and exosomal PD-L1 expression in melanoma cells, causing their increased sensitivity to CD8+ T cell-mediated cytotoxicity. Its effect on CD8+ T and melanoma cells was mediated by several IL2-sEV-resident microRNAs (miRNAs), whose expressions were upregulated by the autocrine effects of IL2. Among the miRNAs, miR-181a-3p and miR-223-3p notably reduced the PD-L1 protein levels in melanoma cells. Interestingly, miR-181a-3p increased the activity of CD8+ T cells while suppressing Treg cell activity. IL2-sEVs inhibited tumour progression in melanoma-bearing immunocompetent mice, but not in immunodeficient mice. The combination of IL2-sEVs and existing anti-cancer drugs significantly improved anti-cancer efficacy by decreasing PD-L1 expression in vivo. Thus, IL2-sEVs are potential cancer immunotherapeutic agents that regulate both immune and cancer cells by reprogramming miRNA levels.

Interferon consensus sequence binding protein-induced cell proliferation is mediated by TGF-ß signaling and p38 MAPK activation.

Interferon consensus sequence binding protein (ICSBP), also known as interferon regulatory factor (IRF)-8, is a member of the interferon (IFN)-γ regulatory transcription factors. Studies have suggested a connection between TGF-ß signaling and IRFs. Thus, we investigated the effect of ICSBP on transforming growth factor (TGF)-ß signaling in HL-60, an acute promyelocytic leukemia cell line. Stable expression of ICSBP in HL-60 cells resulted in strong induction of TGF-ß receptor expression and activation of non-Smad as well as Smad pathways. ICSBP expression also augmented cell growth. ICSBP knockdown with small interfering RNA (siRNA) attenuated cell growth and decreased TGF-ß receptor I (TGF-ßRI) expression. In addition, reduction of TGF-ßRI using siRNA or pharmacological inhibitor reduced growth of ICSBP-expressing cells. ICSBP expression also led to increased phosphorylation and activation of Akt and p38 MAPK. However, p38 MAPK, but not PI3K-Akt, inhibition abrogated ICSBP-mediated proliferation. Furthermore, siRNA knockdown of either ICSBP or TGF-ßRI resulted in decreased p38 activation. Intriguingly, TGF-ß-activated kinase 1 (TAK-1), which phosphorylates p38, was activated in ICSBP-expressing cells and its activity was reduced by TGF-ßRI inhibition. Finally, siRNA knockdown of ICSBP or TGF-ßRI reduced TAK-1 phosphorylation. This study identifies a novel role for ICSBP in regulating cell growth via TGF-ß receptor upregulation and subsequent activation of the TGF-ß receptor/TAK-1/p38 pathway.

FAM188B Downregulation Sensitizes Lung Cancer Cells to Anoikis via EGFR Downregulation and Inhibits Tumor Metastasis In Vivo.

Anoikis is a type of apoptosis induced by cell detachment from the extracellular matrix (ECM), which removes mislocalized cells. Acquisition of anoikis resistance is critical for cancer cells to survive during circulation and, thus, metastasize at a secondary site. Although the sensitization of cancer cells to anoikis is a potential strategy to prevent metastasis, the mechanism underlying anoikis resistance is not well defined. Although family with sequence similarity 188 member B (FAM188B) is predicted as a new deubiquitinase (DUB) member, its biological function has not been fully studied. In this study, we demonstrated that FAM188B knockdown sensitized anoikis of lung cancer cell lines expressing WT-EGFR (A549 and H1299) or TKI-resistant EGFR mutant T790M/L858R (H1975). FAM188B knockdown using si-FAM188B inhibited the growth of all three human lung cancer cell lines cultured in both attachment and suspension conditions. FAM188B knockdown resulted in EGFR downregulation and thus decreased its activity. FAM188B knockdown decreased the activities of several oncogenic proteins downstream of EGFR that are involved in anoikis resistance, including pAkt, pSrc, and pSTAT3, with little changes to their protein levels. Intriguingly, si-FAM188B treatment increased EGFR mRNA levels but decreased its protein levels, which was reversed by treatment with the proteasomal inhibitor MG132, indicating that FAM188B regulates EGFR levels via the proteasomal pathway. In addition, cells transfected with si-FAM188B had decreased expression of FOXM1, an oncogenic transcription factor involved in cell growth and survival. Moreover, FAM188B downregulation reduced metastatic characteristics, such as cell adhesion, invasion, and migration, as well as growth in 3D culture conditions. Finally, tail vein injection of si-FAM188B-treated A549 cells resulted in a decrease in lung metastasis and an increase in mice survival in vivo. Taken together, these findings indicate that FAM188B plays an important role in anoikis resistance and metastatic characteristics by maintaining the levels of various oncogenic proteins and/or their activity, leading to tumor malignancy. Our study suggests FAM188B as a potential target for controlling tumor malignancy.