Schlafen 11 further sensitizes BRCA-deficient cells to PARP inhibitors through single-strand DNA gap accumulation behind replication forks.

The preferential response to PARP inhibitors (PARPis) in BRCA-deficient and Schlafen 11 (SLFN11)-expressing ovarian cancers has been documented, yet the underlying molecular mechanisms remain unclear. As the accumulation of single-strand DNA (ssDNA) gaps behind replication forks is key for the lethality effect of PARPis, we investigated the combined effects of SLFN11 expression and BRCA deficiency on PARPi sensitivity and ssDNA gap formation in human cancer cells. PARPis increased chromatin-bound RPA2 and ssDNA gaps in SLFN11-expressing cells and even more in cells with BRCA1 or BRCA2 deficiency. SLFN11 was co-localized with chromatin-bound RPA2 under PARPis treatment, with enhanced recruitment in BRCA2-deficient cells. Notably, the chromatin-bound SLFN11 under PARPis did not block replication, contrary to its function under replication stress. SLFN11 recruitment was attenuated by the inactivation of MRE11. Hence, under PARPi treatment, MRE11 expression and BRCA deficiency lead to ssDNA gaps behind replication forks, where SLFN11 binds and increases their accumulation. As ovarian cancer patients who responded (progression-free survival >2 years) to olaparib maintenance therapy had a significantly higher SLFN11-positivity than short-responders (<6 months), our findings provide a mechanistic understanding of the favorable responses to PARPis in SLFN11-expressing and BRCA-deficient tumors. It highlight the clinical implications of SLFN11.

Non-classical Monocytes Enhance the Efficacy of Immune Checkpoint Inhibitors on Colon Cancer in a Syngeneic Mouse Model.

BACKGROUND/AIM: The response rate to immune checkpoint inhibitors (ICIs) is approximately 10%-30% and only in a few cancer types. In the present study, we determined whether non-classical monocytes (NCMs) could enhance ICI efficacy in colon cancer using a syngeneic mouse model. MATERIALS AND METHODS: The MC38 C57BL/6 mouse colon cancer model was used. Cells collected from the bone marrow of C57BL/6 mice were cultured, and NCMs were fractionated by cell sorting and administered via the tail veins to the mice implanted with MC38 cells. The anti-mouse PD-L1 antibody was administered three times, and tumor volume and overall survival were observed. RESULTS: More tumors were eradicated and more complete response occurred, after cotreatment with ICIs and NCMs than after treatment with ICIs alone. Moreover, no efficacy was observed when NCMs were administered alone. CONCLUSION: NCMs enhance ICI efficacy. The underlying mechanisms and clinical applications will be studied in the future.

Novel quantitative immunohistochemical analysis for evaluating PD-L1 expression with phosphor-integrated dots for predicting the efficacy of patients with cancer treated with immune checkpoint inhibitors.

Introduction: Programmed cell death ligand 1 (PD-L1) expression in tumor tissues is measured as a predictor of the therapeutic efficacy of immune checkpoint inhibitors (ICIs) in many cancer types. PD-L1 expression is evaluated by immunohistochemical staining using 3,3´-diaminobenzidine (DAB) chronogenesis (IHC-DAB); however, quantitative and reproducibility issues remain. We focused on a highly sensitive quantitative immunohistochemical method using phosphor-integrated dots (PIDs), which are fluorescent nanoparticles, and evaluated PD-L1 expression between the PID method and conventional DAB method. Methods: In total, 155 patients with metastatic or recurrent cancer treated with ICIs were enrolled from four university hospitals. Tumor tissue specimens collected before treatment were subjected to immunohistochemical staining with both the PID and conventional DAB methods to evaluate PD-L1 protein expression. Results: PD-L1 expression assessed using the PID and DAB methods was positively correlated. We quantified PD-L1 expression using the PID method and calculated PD-L1 PID scores. The PID score was significantly higher in the responder group than in the non-responder group. Survival analysis demonstrated that PD-L1 expression evaluated using the IHC-DAB method was not associated with progression-free survival (PFS) or overall survival (OS). Yet, PFS and OS were strikingly prolonged in the high PD-L1 PID score group. Conclusion: Quantification of PD-L1 expression as a PID score was more effective in predicting the treatment efficacy and prognosis of patients with cancer treated with ICIs. The quantitative evaluation of PD-L1 expression using the PID method is a novel strategy for protein detection. It is highly significant that the PID method was able to identify a group of patients with a favorable prognosis who could not be identified by the conventional DAB method.

Monocyte subsets associated with the efficacy of anti­PD­1 antibody monotherapy.

Immune checkpoint inhibitors (ICIs) are among the most notable advances in cancer immunotherapy; however, reliable biomarkers for the efficacy of ICIs are yet to be reported. Programmed death (PD)-ligand 1 (L1)-expressing CD14+ monocytes are associated with shorter overall survival (OS) time in patients with cancer treated with anti-PD-1 antibodies. The present study focused on the classification of monocytes into three subsets: Classical, intermediate and non-classical. A total of 44 patients with different types of cancer treated with anti-PD-1 monotherapy (pembrolizumab or nivolumab) were enrolled in the present study. The percentage of each monocyte subset was investigated, and the percentage of cells expressing PD-L1 or PD-1 within each of the three subsets was further analyzed. Higher pretreatment classical monocyte percentages were correlated with shorter OS (r=-0.32; P=0.032), whereas higher non-classical monocyte percentages were correlated with a favorable OS (r=0.39; P=0.0083). PD-L1-expressing classical monocytes accounted for a higher percentage of the total monocytes than non-classical monocytes with PD-L1 expression. In patients with non-small cell lung cancer (NSCLC), a higher percentage of PD-L1-expressing classical monocytes was correlated with shorter OS (r=-0.60; P=0.012), which is similar to the observation for the whole patient cohort. Comparatively, higher percentages of non-classical monocytes expressing PD-L1 were significantly associated with better OS, especially in patients with NSCLC (r=0.60; P=0.010). Moreover, a higher percentage of non-classical monocytes contributed to prolonged progression-free survival in patients with NSCLC (r=0.50; P=0.042), with similar results for PD-L1-expressing non-classical monocytes. The results suggested that the percentage of monocyte subsets in patients with cancer before anti-PD-1 monotherapy may predict the treatment efficacy and prognosis. Furthermore, more classical monocytes and fewer non-classical monocytes, especially those expressing PD-L1, are involved in shortening OS time, which may indicate the poor efficiency of anti-PD-1 treatment approaches.

KLC1-ROS1 Fusion Exerts Oncogenic Properties of Glioma Cells via Specific Activation of JAK-STAT Pathway.

Here, we investigated the detailed molecular oncogenic mechanisms of a novel receptor tyrosine kinase (RTK) fusion, KLC1-ROS1, with an adapter molecule, KLC1, and an RTK, ROS1, discovered in pediatric glioma, and we explored a novel therapeutic target for glioma that possesses oncogenic RTK fusion. When wild-type ROS1 and KLC1-ROS1 fusions were stably expressed in the human glioma cell lines A172 and U343MG, immunoblotting revealed that KLC1-ROS1 fusion specifically activated the JAK2-STAT3 pathway, a major RTK downstream signaling pathway, when compared with wild-type ROS1. Immunoprecipitation of the fractionated cell lysates revealed a more abundant association of the KLC1-ROS1 fusion with JAK2 than that observed for wild-type ROS1 in the cytosolic fraction. A mutagenesis study of the KLC1-ROS1 fusion protein demonstrated the fundamental roles of both the KLC1 and ROS1 domains in the constitutive activation of KLC1-ROS1 fusion. Additionally, in vitro assays demonstrated that KLC1-ROS1 fusion upregulated cell proliferation, invasion, and chemoresistance when compared to wild-type ROS1. Combination treatment with the chemotherapeutic agent temozolomide and an inhibitor of ROS1, JAK2, or a downstream target of STAT3, demonstrated antitumor effects against KLC1-ROS1 fusion-expressing glioma cells. Our results demonstrate that KLC1-ROS1 fusion exerts oncogenic activity through serum-independent constitutive activation, resulting in specific activation of the JAK-STAT pathway. Our data suggested that molecules other than RTKs may serve as novel therapeutic targets for RTK fusion in gliomas.

Current-Voltage Characteristics and Solvent Dissociation of Bipolar Membranes in Organic Solvents.

In this work, the chronopotentiometric responses, pH changes, and current-voltage (I-V) characteristics of bipolar membrane (BPM)/LiCl-organic solvent systems were measured and compared with those of the BPM/LiCl-water system. Monohydric alcohols, polyhydric alcohols, and amides were used as organic solvents. The chronopotentiograms and pH changes supported that the organic solvents can dissociate into cations and anions at the BPM interface. It is found that amides cannot dissociate easily at the BPM compared with alcohols. The I-V characteristics showed that both the viscosity and acid-base property of organic solvents substantially influences the dissociation behaviors in addition to the autoprotolysis constant and relative permittivity of the solvents.

A protein kinase D inhibitor suppresses AKT on T cells and antagonizes cancer immunotherapy by anti-PD-1.

Antibodies that block the interaction between PD-1 and PD-1 ligands (anti-PD-1) are in clinical use for the treatment of cancer, yet their efficacy is limited. Pre-approved therapies that enhance the effect of anti-PD-1 in combination are beneficial. Small-molecule inhibitors that attenuate T cell receptor signaling are reported to prevent T cell exhaustion and induce memory T cells with stem cell potential, resulting in a durable effector T cell response in combination with anti-PD-1. In search of such targets, we focused on protein kinase D (PKD), which is suggested to be suppressive in both tumor growth and TCR signaling. We report that CRT0066101, a PKD inhibitor (PKDi), suppressed the growth of mouse tumors at a sub-micromolar concentration in vitro. Despite its inhibitory effects on tumors, a single treatment of tumor-bearing mice with PKDi did not inhibit, but rather accelerated tumor growth, and reversed the therapeutic effect of anti-PD-1. Mice treated with PKDi showed reduced T cell infiltration and defects in the generation of effector T cells, compared to those treated with anti-PD-1, suggesting that PKDi inhibited ongoing antitumor responses. Mechanistically, PKDi inhibited phosphorylation of AKT, a primary checkpoint that is reactivated by anti-PD-1. In conclusion, PKD is fundamentally required for T cell reactivation by anti-PD-1; therefore, inhibition of PKD is not appropriate for combination therapy with anti-PD-1. On the other hand, a single dose of PKDi was shown to strongly suppress experimental autoimmunity in mice, indicating that PKDi could be useful for the treatment of immune-related adverse events that are frequently reported in anti-PD-1 therapy.

Regulatory roles of fibronectin and integrin α5 in reorganization of the actin cytoskeleton and completion of adipogenesis.

Cellular differentiation is characterized by changes in cell morphology that are largely determined by actin dynamics. We previously showed that depolymerization of the actin cytoskeleton triggers the differentiation of preadipocytes into mature adipocytes as a result of inhibition of the transcriptional coactivator activity of megakaryoblastic leukemia 1 (MKL1). The extracellular matrix (ECM) influences cell morphology via interaction with integrins, and reorganization of the ECM is associated with cell differentiation. Here we show that interaction between actin dynamics and ECM rearrangement plays a key role in adipocyte differentiation. We found that depolymerization of the actin cytoskeleton precedes disruption and degradation of fibrillar fibronectin (FN) structures at the cell surface after the induction of adipogenesis in cultured preadipocytes. A FN matrix suppressed both reorganization of the actin cytoskeleton into the pattern characteristic of adipocytes and terminal adipocyte differentiation, and these inhibitory effects were overcome by knockdown of integrin α5 (ITGα5). Peroxisome proliferator-activated receptor γ was required for down-regulation of FN during adipocyte differentiation, and MKL1 was necessary for the expression of ITGα5. Our findings suggest that cell-autonomous down-regulation of FN-ITGα5 interaction contributes to reorganization of the actin cytoskeleton and completion of adipocyte differentiation.

Isolation and characterization of neural stem/progenitor cells in the subventricular zone of the naked mole-rat brain.

BACKGROUND: The naked mole-rat (NMR) is the longest-lived rodent with a maximum lifespan of more than 37 years and shows a negligible senescence phenotype, suggesting that tissue stem cells of NMRs are highly capable of maintaining homeostasis. However, the properties of NMR tissue stem cells, including neural stem cells (NSCs), are largely unclear. METHODS: Neural stem/progenitor cells (NS/PCs) were isolated from the subventricular zone of the neonate NMR brain (NMR-NS/PCs) and cultured in neurosphere and adherent culture conditions. Expression of NSC markers and markers of neurons, astrocytes, and oligodendrocytes was analyzed by immunocytochemistry. In adherent culture conditions, the proliferation rate and cell cycle of NMR-NS/PCs were assessed and compared with those of NS/PCs from mice (mouse-NS/PCs). The DNA damage response to γ-irradiation was analyzed by immunocytochemistry and reverse transcription-quantitative PCR. RESULTS: NMR-NS/PCs expressed several NSC markers and differentiated into neurons, astrocytes, and oligodendrocytes. NMR-NS/PCs proliferated markedly slower than mouse-NS/PCs, and a higher percentage of NMR-NS/PCs than mouse-NS/PCs was in G0/G1 phase. Notably, upon γ-irradiation, NMR-NS/PCs exhibited a faster initiation of the DNA damage response and were less prone to dying than mouse-NS/PCs. CONCLUSIONS: NMR-NS/PCs were successfully isolated and cultured. The slow proliferation of NMR-NS/PCs and their resistance to DNA damage may help to prevent stem cell exhaustion in the brain during the long lifespan of NMRs. Our findings provide novel insights into the mechanism underlying delayed aging of NMRs. Further analysis of NMR tissue stem cells may lead to the development of new strategies that can prevent aging in humans.

Epigenetic suppression of SLFN11 in germinal center B-cells during B-cell development.

BACKGROUND: SLFN11 has recently been reported to execute cancer cells harboring replicative stress induced by DNA damaging agents. However, the roles of SLFN11 under physiological conditions remain poorly understood. Germinal center B-cells (GCBs) undergo somatic hypermutations and class-switch recombination, which can cause physiological genotoxic stress. Hence, we tested whether SLFN11 expression needs to be suppressed in GCBs during B-cell development. OBJECTIVE: To clarify the expression profile of SLFN11 in different developmental stages of B-cells and B-cell-derived cancers. METHODS: We analyzed the expression of SLFN11 by mining cell line databases for different stages of normal B-cells and various types of B-cell-derived cancer cell lines. We performed dual immunohistochemical staining for SLFN11 and B-cell specific markers in normal human lymphatic tissues. We tested the effects of two epigenetic modifiers, an EZH2 inhibitor, tazemetostat (EPZ6438) and a histone deacetylase inhibitor, panobinostat (LBH589) on SLFN11 expression in GCB-derived lymphoma cell lines. We also examined the therapeutic efficacy of these drugs in combination with cytosine arabinoside and the effects of SLFN11 on the efficacy of cytosine arabinoside in SLFN11-overexpressing cells. RESULTS: SLFN11 mRNA level was found low in both normal GCBs and GCB-DLBCL (GCB like-diffuse large B-cell lymphoma). Immunohistochemical staining showed low SLFN11 expression in GCBs and high SLFN11 expression in plasmablasts and plasmacytes. The EZH2 and HDAC epigenetic modifiers upregulated SLFN11 expression in GCB-derived lymphoma cells and made them more susceptible to cytosine arabinoside. SLFN11 overexpression further sensitized GCB-derived lymphoma cells to cytosine arabinoside. CONCLUSIONS: The expression of SLFN11 is epigenetically suppressed in normal GCBs and GCB-derived lymphomas. GCB-derived lymphomas with low SLFN11 expression can be treated by the combination of epigenetic modifiers and cytosine arabinoside.

2-Nitroimidazoles induce mitochondrial stress and ferroptosis in glioma stem cells residing in a hypoxic niche.

Under hypoxic conditions, nitroimidazoles can replace oxygen as electron acceptors, thereby enhancing the effects of radiation on malignant cells. These compounds also accumulate in hypoxic cells, where they can act as cytotoxins or imaging agents. However, whether these effects apply to cancer stem cells has not been sufficiently explored. Here we show that the 2-nitroimidazole doranidazole potentiates radiation-induced DNA damage in hypoxic glioma stem cells (GSCs) and confers a significant survival benefit in mice harboring GSC-derived tumors in radiotherapy settings. Furthermore, doranidazole and misonidazole, but not metronidazole, manifested radiation-independent cytotoxicity for hypoxic GSCs that was mediated by ferroptosis induced partially through blockade of mitochondrial complexes I and II and resultant metabolic alterations in oxidative stress responses. Doranidazole also limited the growth of GSC-derived subcutaneous tumors and that of tumors in orthotopic brain slices. Our results thus reveal the theranostic potential of 2-nitroimidazoles as ferroptosis inducers that enable targeting GSCs in their hypoxic niche.

The insulin-PI3K-Rac1 axis contributes to terminal adipocyte differentiation through regulation of actin cytoskeleton dynamics.

Adipocyte differentiation is accompanied by a pronounced change in the actin cytoskeleton characterized by the reorganization of filamentous (F)-actin stress fibers into cortical F-actin structures. We previously showed that depolymerization of F-actin stress fibers induced by inactivation of RhoA-ROCK (Rho-associated kinase) signaling acts as a trigger for adipocyte differentiation. The relevance and underlying mechanism of the formation of cortical F-actin structures from depolymerized actin during adipocyte differentiation have remained unclear, however. We have now examined the mechanistic relation between actin dynamics and adipogenic induction. Transient exposure to the actin-depolymerizing agent latrunculin A (LatA) supported the formation of adipocyte-associated cortical actin structures and the completion of terminal adipocyte differentiation in the presence of insulin, whereas long-term exposure to LatA prevented such actin reorganization as well as terminal adipogenesis. Moreover, these effects of insulin were prevented by inhibition of phosphatidylinositol 3-kinase (PI3K)-Rac1 signaling and the actin-related protein 2/3 (Arp2/3) complex which is a critical component of the cortical actin networks. Our findings thus suggest that the insulin-PI3K-Rac1 axis leads to the formation of adipocyte-associated cortical actin structures which is essential for the completion of adipocyte differentiation.

IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma.

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.

ROCK Inhibition Induces Terminal Adipocyte Differentiation and Suppresses Tumorigenesis in Chemoresistant Osteosarcoma Cells.

Tumors comprise heterogeneous cell types including cancer stem cells (CSC), progenitor cells, and differentiated cells. Chemoresistance is a potential cause of relapse and a key characteristic of CSC, but the development of novel therapeutic approaches for targeting these cells has been limited. We previously established osteosarcoma-initiating (OSi) cells by introducing the gene for c-Myc into bone marrow stromal cells of Ink4a/Arf knockout mice. These OSi cells are composed of two distinct clones: highly tumorigenic cells (AX cells), similar to bipotent committed osteochondral progenitor cells, and tripotent cells of low tumorigenicity (AO cells), similar to mesenchymal stem cells. Here we show that depolymerization of the actin cytoskeleton induces terminal adipocyte differentiation and suppresses tumorigenesis in chemoresistant OSi cells. In contrast to AX cells, AO cells were highly resistant to conventional chemotherapeutic agents such as doxorubicin and were thus identified as chemoresistant cells. Inhibition of Rho-associated coiled-coil containing protein kinase (ROCK) elicited terminal adipocyte differentiation in chemoresistant AO cells through negative regulation of the transcriptional coactivator megakaryoblastic leukemia 1 associated with actin depolymerization. The clinically administered ROCK inhibitor fasudil significantly suppressed growth in vitro and tumorigenicity in vivo of chemoresistant AO cells as well as of OSi cells. Our findings thus suggest a new therapeutic strategy based on the induction of trans-terminal differentiation via modulation of actin cytoskeleton dynamics for therapy-resistant osteosarcoma stem cells. SIGNIFICANCE: These findings suggest that induction of trans-terminal differentiation through regulation of actin dynamics is a potential novel therapeutic approach for targeting chemoresistant stem-like tumor cells.

Development of a functional thyroid model based on an organoid culture system.

The low turnover rate of thyroid follicular cells and the lack of a long-term thyroid cell culture system have hampered studies of thyroid carcinogenesis. We have now established a thyroid organoid culture system that supports thyroid cell proliferation in vitro. The established mouse thyroid organoids performed thyroid functions including thyroglobulin synthesis, iodide uptake, and the production and release of thyroid hormone. Furthermore, transplantation of the organoids into recipient mice resulted in the formation of normal thyroid-like tissue capable of iodide uptake and thyroglobulin production in vivo. Finally, forced expression of oncogenic NRAS (NRASQ61R) in thyroid organoids established from p53 knockout mice and transplantation of the manipulated organoids into mouse recipients generated a model of poorly differentiated thyroid cancer. Our findings suggest that this newly developed thyroid organoid culture system is a potential research tool for the study of thyroid physiology and pathology including thyroid cancer.

Activation of Transforming Growth Factor Beta 1 Signaling in Gastric Cancer-associated Fibroblasts Increases Their Motility, via Expression of Rhomboid 5 Homolog 2, and Ability to Induce Invasiveness of Gastric Cancer Cells.

BACKGROUND & AIMS: Fibroblasts that interact with cancer cells are called cancer-associated fibroblasts (CAFs), which promote progression of different tumor types. We investigated the characteristics and functions of CAFs in diffuse-type gastric cancers (DGCs) by analyzing features of their genome and gene expression patterns. METHODS: We isolated CAFs and adjacent non-cancer fibroblasts (NFs) from 110 gastric cancer (GC) tissues from patients who underwent gastrectomy in Japan from 2008 through 2016. Cells were identified using specific markers of various cell types by immunoblot and flow cytometry. We selected pairs of CAFs and NFs for whole-exome and RNA sequencing analyses, and compared expression of specific genes using quantitative reverse transcription PCR. Protein levels and phosphorylation were compared by immunoblot and immunofluorescence analyses. Rhomboid 5 homolog 2 (RHBDF2) was overexpressed from a transgene in fibroblasts or knocked down using small interfering RNAs. Motility and invasiveness of isolated fibroblasts and GC cell lines (AGS, KATOIII, MKN45, NUGC3, NUGC4, OCUM-2MD3 and OCUM-12 cell lines) were quantified by real-time imaging analyses. We analyzed 7 independent sets of DNA microarray data from patients with GC and associated expression levels of specific genes with patient survival times. Nude mice were given injections of OCUM-2MD3 in the stomach wall; tumors and metastases were collected and analyzed by immunohistochemistry. RESULTS: Many of the genes with increased expression in CAFs compared with NFs were associated with transforming growth factor beta 1 (TGFB1) activity. When CAFs were cultured in extracellular matrix, they became more motile than NFs; DGC cells incubated with CAFs were also more motile and invasive in vitro than DGC cells not incubated with CAFs. When injected into nude mice, CAF-incubated DGC cells invaded a greater number of lymphatic vessels than NF-incubated DGC cells. We identified RHBDF2 as a gene overexpressed in CAFs compared with NFs. Knockdown of RHBDF2 in CAFs reduced their elongation and motility in response to TGFB1, whereas overexpression of RHBDF2 in NFs increased their motility in extracellular matrix. RHBDF2 appeared to regulate oncogenic and non-canonical TGFB1 signaling. Knockdown of RHBDF2 in CAFs reduced cleavage of the TGFB receptor 1 (TGFBR1) by ADAM metallopeptidase domain 17 (ADAM17 or TACE) and reduced expression of genes that regulate motility. Incubation of NFs with in interleukin 1 alpha (IL1A), IL1B or tumor necrosis factor, secreted by DGCs, increased fibroblast expression of RHBDF2. Simultaneous high expression of these cytokines in GC samples was associated with shorter survival times of patients. CONCLUSIONS: In CAFs isolated from human DGCs, we observed increased expression of RHBDF2, which regulates TGFB1 signaling. Expression of RHBDF2 in fibroblasts is induced by inflammatory cytokines (such as IL1A, IL1B, and tumor necrosis factor) secreted by DGCs. RHBDF2 promotes cleavage of TGFBR1 by activating TACE and motility of CAFs in response to TGFB1. These highly motile CAFs induce DGCs to invade extracellular matrix and lymphatic vessels in nude mice.

Simvastatin-Induced Apoptosis in Osteosarcoma Cells: A Key Role of RhoA-AMPK/p38 MAPK Signaling in Antitumor Activity.

Osteosarcoma is the most common type of primary bone tumor, novel therapeutic agents for which are urgently needed. To identify such agents, we screened a panel of approved drugs with a mouse model of osteosarcoma. The screen identified simvastatin, which inhibited the proliferation and migration of osteosarcoma cells in vitro Simvastatin also induced apoptosis in osteosarcoma cells in a manner dependent on inhibition of the mevalonate biosynthetic pathway. It also disrupted the function of the small GTPase RhoA and induced activation of AMP-activated protein kinase (AMPK) and p38 MAPK, with AMPK functioning upstream of p38 MAPK. Inhibitors of AMPK or p38 MAPK attenuated the induction of apoptosis by simvastatin, whereas metformin enhanced this effect of simvastatin by further activation of AMPK. Although treatment with simvastatin alone did not inhibit osteosarcoma tumor growth in vivo, its combination with a fat-free diet induced a significant antitumor effect that was enhanced further by metformin administration. Our findings suggest that simvastatin induces apoptosis in osteosarcoma cells via activation of AMPK and p38 MAPK, and that, in combination with other approaches, it holds therapeutic potential for osteosarcoma. Mol Cancer Ther; 16(1); 182-92. ©2016 AACR.

Tumour resistance in induced pluripotent stem cells derived from naked mole-rats.

The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype-ARF suppression-induced senescence (ASIS)-that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR.

The EGF Receptor Promotes the Malignant Potential of Glioma by Regulating Amino Acid Transport System xc(-).

Extracellular free amino acids contribute to the interaction between a tumor and its microenvironment through effects on cellular metabolism and malignant behavior. System xc(-) is composed of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. Here, we show that the EGFR interacts with xCT and thereby promotes its cell surface expression and function in human glioma cells. EGFR-expressing glioma cells manifested both enhanced antioxidant capacity as a result of increased cystine uptake, as well as increased glutamate, which promotes matrix invasion. Imaging mass spectrometry also revealed that brain tumors formed in mice by human glioma cells stably overexpressing EGFR contained higher levels of reduced glutathione compared with those formed by parental cells. Targeted inhibition of xCT suppressed the EGFR-dependent enhancement of antioxidant capacity in glioma cells, as well as tumor growth and invasiveness. Our findings establish a new functional role for EGFR in promoting the malignant potential of glioma cells through interaction with xCT at the cell surface. Cancer Res; 76(10); 2954-63. ©2016 AACR.