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.

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.

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.

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.

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.

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.

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.

Fibronectin regulates anoikis resistance via cell aggregate formation.

The loss of cell-matrix interactions induces apoptosis, known as anoikis. For successful distant metastasis, circulating tumor cells (CTCs) that have lost matrix attachment need to acquire anoikis resistance in order to survive. Cell aggregate formation confers anoikis resistance, and CTC clusters are more highly metastatic compared to single cells; however, the molecular mechanisms underlying this aggregation are not well understood. In this study, we demonstrated that cell detachment increased cell aggregation and upregulated fibronectin (FN) levels in lung and breast cancer cells, but not in their normal counterparts. FN knockdown decreased cell aggregation and increased anoikis. In addition, cell detachment induced cell-cell adhesion proteins, including E-cadherin, desmoglein-2, desmocollin-2/3, and plakoglobin. Interestingly, FN knockdown decreased the levels of desmoglein-2, desmocollin-2/3, and plakoglobin, but not E-cadherin, suggesting the involvement of desmosomal junction in cell aggregation. Accordingly, knockdown of desmoglein-2, desmocollin-2, or plakoglobin reduced cell aggregation and increased cell sensitivity to anoikis. Previously, we reported that NADPH oxidase 4 (Nox4) upregulation is important for anoikis resistance. Nox4 inhibition by siRNA or apocynin decreased cell aggregation and increased anoikis with the downregulation of FN, and, consequently, decreased desmoglein-2, desmocollin-2/3, or plakoglobin. The coexpression of Nox4 and FN was found to be significant in lung and breast cancer patients, based on cBioPortal data. In vivo mouse lung metastasis model showed that FN knockdown suppressed lung metastasis and thus enhanced survival. FN staining of micro tissue array revealed that FN expression was positive for human lung cancer (61%) and breast cancer (58%) patients. Furthermore, the expression levels of FN, desmoglein-2, desmocollin-2, and plakoglobin were significantly correlated with the poor survival of lung and breast cancer patients, as per the Kaplan-Meier plotter analysis. Altogether, our data suggest that FN upregulation and enhanced desmosomal interactions are critical for cell aggregation and anoikis resistance upon cell detachment.

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.

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.

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.

FAM188B Expression Is Critical for Cell Growth via FOXM1 Regulation in Lung Cancer.

Although family with sequence similarity 188 member B (FAM188B) is known to be a member of a novel putative deubiquitinase family, its biological role has not been fully elucidated. Here, we demonstrate the oncogenic function of FAM188B via regulation of forkhead box M1 (FOXM1), another oncogenic transcription factor, in lung cancer cells. FAM188B knockdown induced the inhibition of cell growth along with the downregulation of mRNA and protein levels of FOXM1. FAM188B knockdown also resulted in downregulation of Survivin and cell cycle-related proteins, which are direct targets of FOXM1. Interestingly, FOXM1 co-immunoprecipitated with FAM188B, and the levels of FOXM1 ubiquitination increased with FAM188B knockdown but decreased with FAM188B overexpression. In addition, in vivo xenograft of FAM188B siRNA (siFAM188B) RNA-treated cells resulted in the retardation of tumor growth compared with that in the control. Furthermore, protein levels of FAM188B and FOXM1 were elevated in the human lung cancer tissues, and FAM188B expression was negatively correlated with the overall survival of lung cancer patients. These results indicate that FAM188B exerts its oncogenic effects by regulating FOXM1 deubiquitination and thus its stability. Therefore, FAM188B might be a potential therapeutic target to control lung cancer progression.

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.

SEZ6L2 Is an Important Regulator of Drug-Resistant Cells and Tumor Spheroid Cells in Lung Adenocarcinoma.

Many lung cancer deaths result from relapses in distant organs, such as the brain or bones, after standard chemotherapy. For cancer cells to spread to other organs, they must survive as circulating tumor cells (CTCs) in blood vessels. Thus, reducing distant recurrence after chemotherapy requires simultaneously inhibiting drug resistance and CTC survival. Here, we investigated the molecular pathways and genes that are commonly altered in drug-resistant lung cancer cells and lung tumor spheroid (TS) cells. First, RNA sequencing was performed in drug-resistant cells and TS cells originating from H460 and A549 lung cancer cells. Bioinformatic pathway analysis showed that cell cycle-related pathways were downregulated in drug-resistant cells, and cholesterol biosynthesis-related pathways were upregulated in TS cells. Seizure-related 6 homolog-like 2 (SEZ6L2) was selected as a gene that was commonly upregulated in both drug-resistant cells and TS cells, and that showed elevated expression in samples from lung adenocarcinoma patients. Second, the protein expression of SEZ6L2 was analyzed by flow cytometry. The proportions of SEZ6L2 positive cells among both drug-resistant cells and TS cells was increased. Finally, as SEZ6L2 is a transmembrane protein with an extracellular region, the function of SEZ6L2 was disrupted by treatment with an anti-SEZ6L2 antibody. Treatment with the anti-SEZ6L2 antibody reduced drug resistance and TS formation. Overall, our data showed that SEZ6L2 plays an important role in drug resistance and TS formation and may be a therapeutic target for reducing distant recurrence of lung adenocarcinoma.

Isoharringtonine Induces Apoptosis of Non-Small Cell Lung Cancer Cells in Tumorspheroids via the Intrinsic Pathway.

Lung cancer is the major cause of cancer-associated death worldwide, and development of new therapeutic drugs is needed to improve treatment outcomes. Three-dimensional (3D) tumorspheroids offer many advantages over conventional two-dimensional cell cultures due to the similarities to in vivo tumors. We found that isoharringtonine, a natural product purified from Cephalotaxus koreana Nakai, significantly inhibited the growth of tumorspheroids with NCI-H460 cells in a dose-dependent manner and induced apoptotic cell death in our 3D cell culture system. On the other hand, A549 tumorspheroids displayed low sensitivity to isoharringtonine-induced apoptosis. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor known to regulate proliferation and apoptosis of cancer cells. We observed that knockdown of NR4A1 dramatically increased isoharringtonine-induced cancer cell death in A549 tumorspheroids by activating the intrinsic apoptosis pathway. Furthermore, treatment with combined isoharringtonine and iNR4A1 significantly inhibited multivulva formation in a Caenorhabditis elegans model and tumor development in a xenograft mouse model. Taken together, our data suggest that isoharringtonine is a potential natural product for treatment of non-small cell lung cancers, and inhibition of NR4A1 sensitizes cancer cells to anti-cancer treatment.

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.

Endothelial cells under therapy-induced senescence secrete CXCL11, which increases aggressiveness of breast cancer cells.

The effects of senescence associated secretory phenotype (SASP) from therapy-induced senescent endothelial cells on tumor microenvironment (TME) remains to be clarified. Here, we investigated effects of ionizing radiation (IR)- and doxorubicin-induced senescent HUVEC on TME. MDA-MB-231 cancer cells treated with conditioned medium (CM) from senescent HUVEC or co-cultured with senescent HUVEC significantly increased cancer cell proliferation, migration, and invasion. We found that CXCL11 plays a principal role in the senescent CM-induced aggressive activities of MDA-MB-231 cells. When we treated HUVEC with a neutralizing anti-CXCL11 antibody or CXCL11 SiRNA, or treated MDA-MB-231 cells with CXCR3 SiRNA, we observed synergistic diminution of the ability of the HUVEC SASP to alter the migration and spheroid invasion of cancer cells. ERK activation was involved in the HUVEC SASP-induced aggressive activity of MDA-MB-231 cells. Finally, we observed the in vivo effect of CXCL11 from the senescent HUVEC in tumor-bearing mice. Together, our results demonstrate that SASP from endothelial cells experiencing therapy-induced senescence promotes the aggressive behavior of cancer cells, and that CXCL11 can potentially be targeted to prevent the adverse effects of therapy-induced senescent endothelial cells on the tumor microenvironment.

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.

Ginsenoside Rp1, A Ginsenoside Derivative, Augments Anti-Cancer Effects of Actinomycin D via Downregulation of an AKT-SIRT1 Pathway.

Novel strategies for overcoming multidrug resistance are urgently needed to improve chemotherapy success and reduce side effects. Ginsenosides, the main active components of Panax ginseng, display anti-cancer properties and reverse drug resistance; however, the biological pathways mediating this phenomenon remain incompletely understood. This study aimed to evaluate the anti-cancer effects of ginsenoside Rp1, actinomycin D (ActD), and their co-administration in drug-resistant cells and murine xenograft model of colon cancer, and explore the underlying mechanisms. ActD increased expression and activity of SIRT1 in drug-resistant LS513 colon cancer, OVCAR8-DXR ovarian cancer, and A549-DXR lung cancer cells, but not in ActD-sensitive SW620 colon cancer cells. Inhibition of SIRT1, either pharmacologically, with EX527 or through siRNA, stimulated p53 acetylation and apoptosis in LS513 cells when treated with ActD. ActD also increased AKT activation in drug-resistant cells. Inhibition of AKT abrogated ActD-induced upregulation of SIRT1, suggesting that the AKT-SIRT1 pathway is important in ActD resistance. Rp1 inhibited both ActD-induced AKT activation and SIRT1 upregulation and re-sensitized the cells to ActD. Synergistic antitumor effects of Rp1 with ActD were also observed in vivo. Our results suggest that combining Rp1 with chemotherapeutic agents could circumvent drug resistance and improve treatment efficacy.

Deacetylation of CHK2 by SIRT1 protects cells from oxidative stress-dependent DNA damage response.

Growing evidence indicates that metabolic signaling pathways are interconnected to DNA damage response (DDR). However, factors that link metabolism to DDR remain incompletely understood. SIRT1, an NAD+-dependent deacetylase that regulates metabolism and aging, has been shown to protect cells from DDR. Here, we demonstrate that SIRT1 protects cells from oxidative stress-dependent DDR by binding and deacetylating checkpoint kinase 2 (CHK2). We first showed that essential proteins in DDR were hyperacetylated in Sirt1-deficient cells and that among them, the level of acetylated CHK2 was highly increased. We found that Sirt1 formed molecular complexes with CHK2, BRCA1/BRCA2-associated helicase 1 (BACH1), tumor suppressor p53-binding protein 1 (53BP1), and H2AX, all of which are key factors in response to DNA damage. We then demonstrated that CHK2 was normally inhibited by SIRT1 via deacetylation but dissociated with SIRT1 under oxidative stress conditions. This led to acetylation and activation of CHK2, which increased cell death under oxidative stress conditions. Our data also indicated that SIRT1 deacetylated the K235 and K249 residues of CHK2, whose acetylation increased cell death in response to oxidative stress. Thus, SIRT1, a metabolic sensor, protects cells from oxidative stress-dependent DDR by the deacetylation of CHK2. Our findings suggest a crucial function of SIRT1 in inhibiting CHK2 as a potential therapeutic target for cancer treatment.