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Table S1 is the correlation between CBX4 expression and Runx2 expression of CRC patients, Table S2 is correlation between CBX4 expression and clinicopathologic characteristics of CRC patients, and Table S3 is univariate and multivariate analysis for overall survival.

Polycomb chromobox (CBX) proteins participate in the polycomb repressive complex (PRC1) that mediates epigenetic gene silencing and endows PRC1 with distinct oncogenic or tumor suppressor functions in a cell-type–dependent manner. In this study, we report that inhibition of cell migration, invasion, and metastasis in colorectal carcinoma requires CBX4-mediated repression of Runx2, a key transcription factor that promotes colorectal carcinoma metastasis. CBX4 inversely correlated with Runx2 expression in colorectal carcinoma tissues, and the combination of high CBX4 expression and low Runx2 expression significantly correlated with overall survival, more so than either CBX4 or Runx2 expression alone. Mechanistically, CBX4 maintained recruited histone deacetylase 3 (HDAC3) to the Runx2 promoter, which maintained a deacetylated histone H3K27 state to suppress Runx2 expression. This function of CBX4 was dependent on its interaction with HDAC3, but not on its SUMO E3 ligase, its chromodomain, or the PRC1 complex. Disrupting the CBX4–HDAC3 interaction abolished Runx2 inhibition as well as the inhibition of cell migration and invasion. Collectively, our data show that CBX4 may act as a tumor suppressor in colorectal carcinoma, and strategies that stabilize the interaction of CBX4 with HDAC3 may benefit the colorectal carcinoma patients with metastases. Cancer Res; 76(24); 7277–89. ©2016 AACR.

Addtional information on shRNA sequences, qRT-PCR primers, promoter cloning primers, antibodies and ChIP-qPCR primers used in this study.

Purpose: Aspirin has recently been reported to reduce both the incidence and the risk of metastasis in colon cancer. However, there is no evidence at the cellular levels or in the animal models for such an effect of aspirin on cancer metastasis.Experimental Design: MTT assay, colony formation assay, and apoptosis assay were employed to analyze the effects of aspirin on the osteosarcoma cell viability in vitro. The NF-κB activity was measured by the NF-κB p65 luciferase reporter. Western blotting was used to analyze the proteins in cells. The migration and invasion abilities of osteosarcoma cells in vitro were measured by the Transwell assay. Xenograft-bearing mice were used to assess the roles of aspirin in both tumor growth and metastasis of osteosarcoma in vivo (n = 5–8 mice/group). An unpaired Student t test or ANOVA with the Bonferroni post hoc test were used for the statistical comparisons.Results: Aspirin reduced cell viability in a dose- and time-dependent manner in osteosarcoma cell lines, and aspirin synergistically sensitized osteosarcoma cells to cisplatin (DDP) in vitro and in vivo (P < 0.001). Moreover, aspirin markedly repressed the migration and invasion of osteosarcoma cells in vitro (P < 0.001), and dramatically diminished the occurrence of osteosarcoma xenograft metastases to the lungs in vivo (P < 0.001). Mechanistically, aspirin diminishes osteosarcoma migration, invasion, and metastasis through the NF-κB pathway.Conclusions: Aspirin suppresses both the growth and metastasis of osteosarcoma through the NF-κB pathway at the cellular level and in the animal models. Clin Cancer Res; 21(23); 5349–59. ©2015 AACR.

Supplementary Figures S1-6. Figure S1. Aspirin enhances chemosensitivity to DDP in both U2OS and ZOS cells using the clone formation assay; Figure S2. Aspirin promotes the DDP-induced apoptosis in osteosarcoma cell lines; Figure S3. Aspirin inhibits the migration and invasion of OS cells; Figure S4. The orthotopic model of osteosarcoma in vivo; Figure S5. Aspirin has no effect on the migration and invasion in both U2OS and ZOS cells knockdown of p65; Figure S6. The impairment of Aspirin on migration and invasion are rescued in both U2OS and ZOS cells stably overexpressing p65.

Metastasis is the major reason for treatment failure and accounts for cancer-related death in patients with nasopharyngeal carcinoma. However, the genetic alterations and molecular mechanisms that cause nasopharyngeal carcinoma metastasis are elusive. Herein, we performed RNA sequencing in patients with or without metastasis, and found that the early B-cell factor 3 (EBF3) was significantly elevated in the samples with metastasis. Mechanistically, EBF3 promoted metastasis by directly combining with the promoter of Vimentin and transcriptionally upregulating it. In addition, EBF3 was epigenetically silenced by EGR1/EZH2/HDAC9 complexes via sustaining the high level of H3K27-Me3 at its promoter. Clinically, there was a positive correlation between EBF3 and Vimentin in nasopharyngeal carcinoma tissues. Moreover, high expression of EBF3 or Vimentin was correlated with poor overall survival, while the combination of high EBF3 and Vimentin expression was associated with more significant poor prognosis. Therefore, specific agents targeting EBF3 or stabilizing the EGR1/EZH2/HDAC9 complex could be novel therapeutic strategies for cancer metastasis.

Rab22a-NeoF fusion protein has recently been reported as a promising target for osteosarcoma lung metastasis. However, how this fusion protein is regulated in cells remains unknown. Here, using multiple screenings, it is reported that Rab22a-NeoF1 fusion protein is degraded by an E3 ligase STUB1 via the autophagy receptor NDP52-mediated lysosome pathway, which is facilitated by PINK1 kinase. Mechanistically, STUB1 catalyzes the K63-linked ubiquitin chains on lysine112 of Rab22a-NeoF1, which is responsible for the binding of Rab22a-NeoF1 to NDP52, resulting in lysosomal degradation of Rab22a-NeoF1. PINK1 is able to phosphorylate Rab22a-NeoF1 at serine120, which promotes ubiquitination and degradation of Rab22a-NeoF1. Consistently, by upregulating PINK1, Sorafenib and Regorafenib can inhibit osteosarcoma lung metastasis induced by Rab22a-NeoF1. These findings reveal that the lysosomal degradation of Rab22a-NeoF1 fusion protein is targetable for osteosarcoma lung metastasis, proposing that Sorafenib and Regorafenib may benefit cancer patients who are positive for the RAB22A-NeoF1 fusion gene.

BRAF inhibitors (BRAFi) that target BRAF V600E kinase, a driver mutation found in 50% of melanomas, show a significant antitumor response, but the common emergence of acquired resistance remains a challenge. Abnormal expression of RAF isoforms CRAF and ARAF reactivates pERK1/2, which plays crucial roles in the acquisition of resistance of melanoma cells. However, the mechanisms of dysregulation of RAF isoforms in resistant melanoma cells remain unknown. Here, we identified NONO interacted with and stabilized both CRAF and ARAF in melanoma cells, and that NONO was acetylated at 198K by p300 acetyltransferase, which stabilized NONO via antagonizing its ubiquitination/degradation mediated by RNF8. The upregulation of both p300 and NONO promoted the rebound of pERK1/2 and the subsequent resistance of melanoma cells to BRAFi, and the activation of ERK1/2 in turn induced p300 to form a positive feedback loop in resistant melanoma cells. There was a positive correlation between p300 and NONO in resistant melanoma cells and clinical samples, and p300 inhibitor C646 overcame the resistance of resistant melanoma cells to BRAF inhibitors in vitro and in vivo. Our findings reveal that targeting the positive feedback loop of p300-NONO-CRAF/ARAF-pERK1/2 may be excellent strategies to overcome the resistance of BRAF inhibitors for melanoma patients.

Immunologic checkpoint blockade has been proven effective in a variety of malignancies. However, high rates of resistance have substantially hindered its clinical use. Understanding the underlying mechanisms may lead to new strategies for improving therapeutic efficacy. Although a number of signaling pathways have been shown to be associated with tumor cell–mediated resistance to immunotherapy, T cell–intrinsic resistant mechanisms remain elusive. Here, we demonstrated that diacylglycerol kinase alpha (Dgka) mediated T-cell dysfunction during anti–PD-1 therapy by exacerbating the exhaustion of reinvigorated tumor-specific T cells. Pharmacologic ablation of Dgka postponed T-cell exhaustion and delayed development of resistance to PD-1 blockade. Dgka inhibition also enhanced the efficacy of anti–PD-1 therapy. We further found that the expression of DGKA in cancer cells promoted tumor growth via the AKT signaling pathway, suggesting that DGKA might be a target in tumor cells as well. Together, these findings unveiled a molecular pathway mediating resistance to PD-1 blockade and provide a potential therapeutic strategy with combination immunotherapy.

It remains unknown for decades how some of the therapeutic fusion proteins positive in a small percentage of cancer cells account for patient outcome. Here, we report that osteosarcoma Rab22a-NeoF1 fusion protein, together with its binding partner PYK2, is sorted into exosomes by HSP90 via its KFERQ-like motif (RVLFLN142). The exosomal Rab22a-NeoF1 fusion protein facilitates the pulmonary pre-metastatic niche formation by recruiting bone marrow-derived macrophages. The exosomal PYK2 activates RhoA in its negative recipient osteosarcoma cells and induces signal transducer and activator of transcription 3 activation in its recipient macrophages to increase M2 phenotype. Consequently, lung metastases of its recipient osteosarcoma cells are promoted by this exosomal Rab22a-NeoF1 fusion protein, and this event can be targeted by disrupting its interaction with PYK2 using a designed internalizing RGD peptide.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is the fourth-leading cause of cancer-related deaths worldwide. HCC is refractory to many standard cancer treatments and the prognosis is often poor, highlighting a pressing need to identify biomarkers of aggressiveness and potential targets for future treatments. Kinesin family member 2C (KIF2C) is reported to be highly expressed in several human tumors. Nevertheless, the molecular mechanisms underlying the role of KIF2C in tumor development and progression have not been investigated. In this study, we found that KIF2C expression was significantly upregulated in HCC, and that KIF2C up-regulation was associated with a poor prognosis. Utilizing both gain and loss of function assays, we showed that KIF2C promoted HCC cell proliferation, migration, invasion, and metastasis both in vitro and in vivo. Mechanistically, we identified TBC1D7 as a binding partner of KIF2C, and this interaction disrupts the formation of the TSC complex, resulting in the enhancement of mammalian target of rapamycin complex1 (mTORC1) signal transduction. Additionally, we found that KIF2C is a direct target of the Wnt/β-catenin pathway, and acts as a key factor in mediating the crosstalk between Wnt/β-catenin and mTORC1 signaling. Thus, the results of our study establish a link between Wnt/β-catenin and mTORC1 signaling, which highlights the potential of KIF2C as a therapeutic target for the treatment of HCC. Electronic supplementary material The online version of this article (10.1007/s13238-020-00766-y) contains supplementary material, which is available to authorized users.

Purpose: Adults with T-cell lymphoblastic lymphoma (T-LBL) generally benefit from treatment with acute lymphoblastic leukemia (ALL)-like regimens, but approximately 40% will relapse after such treatment. We evaluated the value of CpG methylation in predicting relapse for adults with T-LBL treated with ALL-like regimens. Experimental Design: A total of 549 adults with T-LBL from 27 medical centers were included in the analysis. Using the Illumina Methylation 850K Beadchip, 44 relapse-related CpGs were identified from 49 T-LBL samples by two algorithms: least absolute shrinkage and selector operation (LASSO) and support vector machine–recursive feature elimination (SVM-RFE). We built a four-CpG classifier using LASSO Cox regression based on association between the methylation level of CpGs and relapse-free survival in the training cohort (n = 160). The four-CpG classifier was validated in the internal testing cohort (n = 68) and independent validation cohort (n = 321). Results: The four-CpG–based classifier discriminated patients with T-LBL at high risk of relapse in the training cohort from those at low risk (P < 0.001). This classifier also showed good predictive value in the internal testing cohort (P < 0.001) and the independent validation cohort (P < 0.001). A nomogram incorporating five independent prognostic factors including the CpG-based classifier, lactate dehydrogenase levels, Eastern Cooperative Oncology Group performance status, central nervous system involvement, and NOTCH1/FBXW7 status showed a significantly higher predictive accuracy than each single variable. Stratification into different subgroups by the nomogram helped identify the subset of patients who most benefited from more intensive chemotherapy and/or sequential hematopoietic stem cell transplantation. Conclusions: Our four-CpG–based classifier could predict disease relapse in patients with T-LBL, and could be used to guide treatment decision.

Background Adult patients with T‐cell lymphoblastic lymphoma (T‐LBL) are treated with high‐intensity chemotherapy regimens, but the response rate is still unsatisfactory because of frequent drug resistance. We aimed to investigate the potential mechanisms of drug resistance in adults with T‐LBL. Methods Gene expression microarray was used to identify differential mRNA expression profiles between chemotherapy‐resistant and chemotherapy‐sensitive adult T‐LBL tissues. Real‐time PCR and immunohistochemistry were performed to detect the expression of bromodomain‐containing protein 2 (BRD2) and c‐Myc in fresh‐frozen T‐LBL tissues from 85 adult patients. The Ras pull‐down assay was performed to monitor Ras activation. Chromatin immunoprecipitation assays were used to analyze the binding of E2F transcription factor 1 (E2F1)/BRD2 to the RAS guanyl releasing protein 1 (RasGRP1) promoter region. The drug resistance effect and mechanism of BRD2 were determined by both in vivo and in vitro studies. Results A total of 86 chemotherapy resistance‐related genes in adult T‐LBL were identified by gene expression microarray. Among them, BRD2 was upregulated in chemotherapy‐resistant adult T‐LBL tissues and associated with worse progression‐free survival and overall survival of 85 adult T‐LBL patients. Furthermore, BRD2 suppressed doxorubicin (Dox)‐induced cell apoptosis both in vitro and in vivo. The activation of RasGRP1/Ras/ERK signaling might contribute to the Dox resistance effect of BRD2. Besides, OTX015, a bromodomain and extra‐terminal (BET) inhibitor, reversed the Dox resistance effect of BRD2. Patient‐derived tumor xenograft demonstrated that the sequential use of OTX015 after Dox showed superior therapeutic effects. Conclusions Our data showed that BRD2 promotes drug resistance in adult T‐LBL through the RasGRP1/Ras/ERK signaling pathway. Targeting BRD2 may be a novel strategy to improve the therapeutic efficacy and prolong survival of adults with T‐LBL.

Osteosarcoma is a type of aggressive malignant bone tumour that frequently metastasizes to lungs, resulting in poor prognosis. However, the molecular mechanisms of lung metastasis of osteosarcoma remain poorly understood. Here we identify exon-intron fusion genes in osteosarcoma cell lines and tissues. These fusion genes are derived from chromosomal translocations that juxtapose the coding region for amino acids 1-38 of Rab22a (Rab22a1-38) with multiple inverted introns and untranslated regions of chromosome 20. The resulting translation products, designated Rab22a-NeoFs, acquire the ability to drive lung metastasis of osteosarcoma. The Rab22a1-38 moiety governs the function of Rab22a-NeoFs by binding to SmgGDS-607, a GTP-GDP exchange factor of RhoA. This association facilitates the release of GTP-bound RhoA from SmgGDS-607, which induces increased activity of RhoA and promotes metastasis. Disrupting the interaction between Rab22a-NeoF1 and SmgGDS-607 with a synthetic peptide prevents lung metastasis in an orthotopic model of osteosarcoma. Our findings may provide a promising strategy for a subset of osteosarcoma patients with lung metastases.

The spleen is an important site of hematopoietic stem/progenitor cell (HSPC) preconditioning and tumor-promoting myeloid cell generation in cancer, but the regulatory mechanism remains unclear. Here, we found that PKR-like endoplasmic reticulum kinase (PERK) mediated HSPC reprogramming into committed MDSC precursors in the spleen via PERK–ATF4–C/EBPβ signaling. Pharmacological and genetic inhibition of this pathway in murine and human HSPCs prevented their myeloid descendant cells from becoming MDSCs even with subsequent exposure to tumor microenvironment (TME) factors. In mice, the selective delivery of PERK antagonists to the spleen was not only sufficient but more effective than the tumor-targeted strategy in preventing MDSC activation in the tumor, leading to profound TME reshaping and tumor regression. Clinically, HSPCs in the spleen of cancer patients exhibit increased PERK signaling correlated with enhanced myelopoiesis. Our findings indicate that PERK-mediated HSPC preconditioning plays a crucial role in MDSC generation, suggesting novel spleen-targeting therapeutic opportunities for restraining the tumor-promoting myeloid response at its source.

Ovarian cancer is characterized by aberrant activation of the mitogen-activated protein kinase (MAPK), highlighting the importance of targeting the MAPK pathway as an attractive therapeutic strategy. However, the clinical efficacy of MEK inhibitors is limited due to intrinsic or acquired drug resistance. Here, we established patient-derived ovarian cancer models resistant to MEK inhibitors and demonstrated that resistance to the clinically-approved MEK inhibitor trametinib was associated with enhancer reprogramming. We also showed that enhancer decommissioning induced the downregulation of negative regulators of the MAPK pathway, leading to constitutive ERK activation and acquired resistance to trametinib. Epigenetic compound screening uncovered that HDAC inhibitors could alter the enhancer reprogramming and upregulate the expression of MAPK negative regulators, resulting in sustained MAPK inhibition and reversal of trametinib resistance. Consequently, a combination of HDAC inhibitor and trametinib demonstrated a synergistic anti-tumor effect in vitro and in vivo, including patient-derived xenograft mouse models. These findings demonstrated that enhancer reprogramming of the MAPK regulatory pathway might serve as a potential mechanism underlying MAPK inhibitor resistance and concurrent targeting of epigenetic pathways and MAPK signaling might provide an effective treatment strategy for advanced ovarian cancer.

Personalized immunotherapy targeting tumor-specific antigens (TSAs) could generate efficient and safe antitumor immune response without damaging normal tissues. Although neoantigen vaccines have shown therapeutic effect in clinic trials, precise prediction of neoantigens from tumor mutations is still challenging. The host antitumor immune response selects and activates T cells recognizing tumor antigens. Hence, T cells engineered with T-cell receptors (TCRs) from these naturally occurring tumor antigen-specific T (Tas) cells in a patient will target personal TSAs in his/her tumor. To establish such a personalized TCR-T cell therapy, we comprehensively characterized T cells in tumor and its adjacent tissues by single-cell mRNA sequencing (scRNA-seq), TCR sequencing (TCR-seq) and in vitro neoantigen stimulation. Compared to bystander T cells circulating among tissues, Tas cells were characterized by tumor enrichment, tumor-specific clonal expansion and neoantigen specificity. We found that CXCL13 is a unique marker for both CD4(+) and CD8(+) Tas cells. Importantly, TCR-T cells expressing TCRs from Tas cells showed significant therapeutic effects on autologous patient-derived xenograft (PDX) tumors. Intratumoral Tas cell levels measured by CXCL13 expression precisely predicted the response to immune checkpoint blockade, indicating a critical role of Tas cells in the antitumor immunity. We further identified CD200 and ENTPD1 as surface markers for CD4(+) and CD8(+) Tas cells respectively, which enabled the isolation of Tas cells from tumor by Fluorescence Activating Cell Sorter (FACS) sorting. Overall, our results suggest that TCR-T cells engineered with Tas TCRs are a promising agent for personalized immunotherapy, and intratumoral Tas cell levels determine the response to immunotherapy.

Background: Rab22a-NeoF1 fusion gene containing the 1-38aa of Rab22a (Rab22a1-38) plays a decisive role in driving tumor metastasis by activating RhoA via binding to SmgGDS607. However, its intercellular regulation remains unknown. Methods: The Lys7 (K7) acetylation of Rab22a-NeoF1 was initially identified by mass spectrum. Co-transfection, immunoprecipitation and Western blotting were used to characterize the acetyltransferases and deacetylases responsible for the K7 acetylation of Rab22a-NeoF1, and to define the interaction of proteins. The specificity of K7 acetylation of Rab22a-NeoF1 was determined by its specific anti-K7ac-Rab22a-NeoF1 antibody and its K7R mutant. RhoA-GTP was measured by RhoA activation assay. The migration and invasion were assessed by Transwell assay without and with Matrigel matrix, respectively. The orthotopic osteosarcoma metastasis model in vivo was used to monitor the lung metastases of U2OS/MTX300-Luc stably expressing Vector, Rab22a-NeoF1 or its K7R mutant with or without C646, a relatively specific inhibitor of p300/CBP. The unpaired Student t test was used for the statistical significance. Results: The K7 of Rab22a-NeoF1 is acetylated by p300/CBP while is de-acetylated by both HDAC6 and SIRT1. The K7R mutant of Rab22a-NeoF1 lacks its binding to SmgGDS607 and subsequently lost its promoting functions, such as activation of RhoA, cell migration, invasion and lung metastasis in osteosarcoma in vitro and in vivo, which are also diminished by p300/CBP inhibitor C646. Conclusion:The promoting function of Rab22a-NeoF1 is dependent on its K7 acetylation in osteosarcoma, and targeting this acetylation (e.g., C646) may benefit cancer patients, in particular osteosarcoma patients, who are positive for the Rab22a1-38.

The genetic events occurring in recurrent nasopharyngeal carcinoma (rNPC) are poorly understood. Here, we performed whole-genome and whole-exome sequencing in 55 patients with rNPC and 44 primarily diagnosed NPC (pNPC), with 7 patients having paired rNPC and pNPC samples. Previously published pNPC exome data were integrated for analysis. rNPC and pNPC tissues had similar mutational burdens, however, the number of clonal mutations was increased in rNPC samples. TP53 and three NF-κB pathway components (TRAF3, CYLD, and NFKBIA) were significantly mutated in both pNPC and rNPC. Notably, mutations in TRAF3, CYLD, and NFKBIA were all clonal in rNPC, however, 55.6% to 57.9% of them were clonal in pNPC. In general, the number of clonal mutations in NF-κB pathway–associated genes was significantly higher in rNPC than in pNPC. The NF-κB mutational clonality was selected and/or enriched during NPC recurrence. The amount of NF-κB translocated to the nucleus in samples with clonal NF-κB mutants was significantly higher than that in samples with subclonal NF-κB mutants. Moreover, the nuclear abundance of NF-κB protein was significantly greater in pNPC samples with locoregional relapse than in those without relapse. Furthermore, high nuclear NF-κB levels were an independent negative prognostic marker for locoregional relapse-free survival in pNPC. Finally, inhibition of NF-κB enhanced both radiosensitivity and chemosensitivity in vitro and in vivo. In conclusion, NF-κB pathway activation by clonal mutations plays an important role in promoting the recurrence of NPC. Moreover, nuclear accumulation of NF-κB is a prominent biomarker for predicting locoregional relapse-free survival. Significance: This study uncovers genetic events that promote the progression and recurrence of nasopharyngeal carcinoma and has potential prognostic and therapeutic implications. See related commentary by Sehgal and Barbie, p. 5915

Procollagen C-proteinase enhancer protein (PCOLCE) was originally identified as an enhancer to facilitate the catalysis of procollagens by BMP1. PCOLCE participates in the reconstitution of extracellular and corneal repair. The elevation of PCOLCE in blood indicates that breast cancer has metastasized into the bones. However, direct research on PCOLCE has not been reported. Methods: ECM candidates were identified by RNA-seq analysis from 4 normal and 16 osteosarcoma tissues. The in vitro migration and invasion abilities of osteosarcoma cells were determined by a Transwell assay. A spontaneous metastatic osteosarcoma model was established to assess osteosarcoma metastasis in vivo. The N-linked glycosylated amino acids were identified by PNGase F treatment combined with Western blotting. The mechanism of TWIST1 regulating PCOLCE transcription was elucidated by luciferase, qPCR and ChIP assays. Results: PCOLCE was markedly up-regulated in human osteosarcoma tissues compared to its expression in noncancerous adjacent tissues; high PCOLCE expression in tissues correlated with a poor patient prognosis, and the knockdown of PCOLCE by shRNAs impaired the migration, invasion and lung metastasis of osteosarcoma cells. The overexpression of wild-type PCOLCE, but not its N29Q mutant, promoted migration, invasion and metastasis, indicating that the glycosylation of PCOLCE at Asn29 is necessary for its functions in osteosarcoma. TWIST1, a key transcription factor in metastasis, was also overexpressed in osteosarcoma tissues and positively correlated with either PCOLCE or its potential procollagen substrates, such as COL1A1, COL1A2, COL5A1, COL8A2 and COL10A1. Conclusion: Our findings are the first to provide evidence that PCOLCE plays a critical role in promoting the lung metastasis of osteosarcoma, and this up-regulation of PCOLCE by TWIST1 may lead to a new therapeutic strategy to treat patients with metastatic osteosarcoma.

Bone osteogenic sarcoma has a poor prognosis, as the exact cell of origin and the signaling pathways underlying tumor formation remain undefined. Here, we report an osteogenic tumor mouse model based on the conditional knockout of liver kinase b1 (Lkb1, also known as Stk11) in Cathepsin K–Cre–expressing (Ctsk-Cre–expressing) cells. Lineage-tracing studies demonstrated that Ctsk-Cre could label a population of periosteal cells. The cells functioned as mesenchymal progenitors with regard to markers and functional properties. LKB1 deficiency increased proliferation and osteoblast differentiation of Ctsk+ periosteal cells, while downregulation of mTORC1 activity, using a Raptor genetic mouse model or mTORC1 inhibitor treatment, ameliorated tumor progression of Ctsk-Cre Lkb1fllfl mice. Xenograft mouse models using human osteosarcoma cell lines also demonstrated that LKB1 deficiency promoted tumor formation, while mTOR inhibition suppressed xenograft tumor growth. In summary, we identified periosteum-derived Ctsk-Cre–expressing cells as a cell of origin for osteogenic tumor and suggested the LKB1/mTORC1 pathway as a promising target for treatment of osteogenic tumor.

RNA-binding protein RBM28 (RBM28), as a nucleolar component of spliceosomal small nuclear ribonucleoproteins, is involved in the nucleolar stress response. Whether and how RBM28 regulates tumor progression remains unclear. Here, we report that RBM28 is frequently overexpressed in various types of cancer and that its upregulation is associated with a poor prognosis. Functional and mechanistic assays revealed that RBM28 promotes the survival and growth of cancer cells by interacting with the DNA-binding domain of tumor suppressor p53 to inhibit p53 transcriptional activity. Upon treatment with chemotherapeutic drugs (e.g., adriamycin), RBM28 is translocated from the nucleolus to the nucleoplasm, which is likely mediated via phosphorylation of RBM28 at Ser122 by DNA checkpoint kinases 1 and 2 (Chk1/2), indicating that RBM28 may act as a nucleolar stress sensor in response to DNA damage stress. Our findings not only reveal RBM28 as a potential biomarker and therapeutic target for cancers but also provide mechanistic insights into how cancer cells convert stress signals into a cellular response linking the nucleolus to regulation of the tumor suppressor p53.

Background Gene editing technology has provided researchers with the ability to modify genome sequences in almost all eukaryotes. Gene-edited cell lines are being used with increasing frequency in both bench research and targeted therapy. However, despite the great importance and universality of gene editing, the efficiency of homology-directed DNA repair (HDR) is too low, and base editors (BEs) cannot accomplish desired indel editing tasks. Results and discussion Our group has improved HDR gene editing technology to indicate DNA variation with an independent selection marker using an HDR strategy, which we named Gene Editing through an Intronic Selection marker (GEIS). GEIS uses a simple process to avoid nonhomologous end joining (NHEJ)-mediated false-positive effects and achieves a DsRed positive rate as high as 87.5% after two rounds of fluorescence-activated cell sorter (FACS) selection without disturbing endogenous gene splicing and expression. We re-examined the correlation of the conversion tract and efficiency, and our data suggest that GEIS has the potential to edit approximately 97% of gene editing targets in human and mouse cells. The results of further comprehensive analysis suggest that the strategy may be useful for introducing multiple DNA variations in cells.

BRAF inhibitors (BRAFi) that target BRAF V600E kinase, a driver mutation found in 50% of melanomas, show a significant antitumor response, but the common emergence of acquired resistance remains a challenge. Abnormal expression of RAF isoforms CRAF and ARAF reactivates pERK1/2, which plays crucial roles in the acquisition of resistance of melanoma cells. However, the mechanisms of dysregulation of RAF isoforms in resistant melanoma cells remain unknown. Here, we identified NONO interacted with and stabilized both CRAF and ARAF in melanoma cells, and that NONO was acetylated at 198K by p300 acetyltransferase, which stabilized NONO via antagonizing its ubiquitination/degradation mediated by RNF8. The upregulation of both p300 and NONO promoted the rebound of pERK1/2 and the subsequent resistance of melanoma cells to BRAFi, and the activation of ERK1/2 in turn induced p300 to form a positive feedback loop in resistant melanoma cells. There was a positive correlation between p300 and NONO in resistant melanoma cells and clinical samples, and p300 inhibitor C646 overcame the resistance of resistant melanoma cells to BRAF inhibitors in vitro and in vivo. Our findings reveal that targeting the positive feedback loop of p300-NONO-CRAF/ARAF-pERK1/2 may be excellent strategies to overcome the resistance of BRAF inhibitors for melanoma patients.

Human single-stranded DNA-binding protein 1 (hSSB1) is required for the efficient recruitment of the MRN complex to DNA double-strand breaks and is essential for the maintenance of genome integrity. However, the mechanism by which hSSB1 recruits NBS1 remains elusive. Here, we determined that hSSB1 undergoes SUMOylation at both K79 and K94 under normal conditions and that this modification is dramatically enhanced in response to DNA damage. SUMOylation of hSSB1, which is specifically fine-tuned by PIAS2α, and SENP2, not only stabilizes the protein but also enhances the recruitment of NBS1 to DNA damage sites. Cells with defective hSSB1 SUMOylation are sensitive to ionizing radiation, and global inhibition of SUMOylation by either knocking out UBC9 or adding SUMOylation inhibitors significantly enhances the sensitivity of cancer cells to etoposide. Our findings reveal that SUMOylation, as a novel posttranslational modification of hSSB1, is critical for the functions of this protein, indicating that the use of SUMOylation inhibitors (e.g., 2-D08 and ML-792) may be a new strategy that would benefit cancer patients being treated with chemo- or radiotherapy.

N6-methyladenosin (m6A) is one of the most pervasive modification of mRNA in eukaryotes and the m6A methyltransferases and demethylases play critical roles in many types of cancer. However the role of m6A-binding proteins in cancer remains elusive. Here we report that the down-regulation of YTHDF2 was specifically induced by hypoxia in hepatocellular carcinoma (HCC) cells, and that overexpression of YTHDF2 suppressed cell proliferation, tumor growth and activation of MEK and ERK in HCC cells. Mechanistically, YTHDF2 directly bound the m6A modification site of EGFR 3’-UTR to promote the degradation of EGFR mRNA in HCC cells. This is the first report showing that YTHDF2 may act as a tumor suppressor to repress cell proliferation and growth via destabilizing the EGFR mRNA in HCC.

Exosomes are generated within the multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs) and secreted during the fusion of MVEs with the cell membrane. The mechanisms of exosome biogenesis remain poorly explored. Here we identify that RAB31 marks and controls an ESCRT-independent exosome pathway. Active RAB31, phosphorylated by epidermal growth factor receptor (EGFR), engages flotillin proteins in lipid raft microdomains to drive EGFR entry into MVEs to form ILVs, which is independent of the ESCRT (endosomal sorting complex required for transport) machinery. Active RAB31 interacts with the SPFH domain and drives ILV formation via the Flotillin domain of flotillin proteins. Meanwhile, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing the fusion of MVEs with lysosomes and enabling the secretion of ILVs as exosomes. These findings establish that RAB31 has dual functions in the biogenesis of exosomes: driving ILVs formation and suppressing MVEs degradation, providing an exquisite framework to better understand exosome biogenesis.

Shortening of the 3′ untranslated regions (3′UTR) of mRNA is an important mechanism for oncogene activation. However, 3′UTR alteration events, their pathologic functions, and underlying mechanisms in human urothelial carcinoma of the bladder (UCB) are not clear. Here, we combine RNA sequencing, bioinformatics, and clinical studies in two independent cohorts of patients with UCB to identify a novel RAC1 shorter 3′UTR isoform that is frequently expressed in UCB and is critical in the tumorigenesis and acquisition of a poor prognostic phenotype in patients. Short 3′UTR isoform of RAC1 substantially upregulated RAC1 expression by escaping from miRNA-targeted repression and played an essential oncogenic role in UCB pathogenesis. An important cleavage/polyadenylation factor, cleavage stimulation factor 2 (CSTF2), induced 3′UTR shortening of RAC1 in UCB by mediating slow transcriptional elongation at RAC1. Cotranscriptional recruitment of CSTF2 on the GUAAU motif at proximal polyadenylation site of RAC1 attenuated the recruitment of two transcription factors AFF1 and AFF4, causing the defects in elongation. CSTF2 regulated the tumorigenic functions of the shorter RAC1 isoform in UCB cells, enhancing cell proliferation, migration, and invasion. The combination of high expression of CSTF2 and high usage of RAC1 short-3′UTR isoform may be used as a powerful biomarker to predict poor prognosis in UCB. Our findings also suggest a CSTF2-regulated RAC1-3′UTR shortening program as an exploitable therapeutic strategy for patients with UCB. Significance: These findings demonstrate that the short isoform of RAC1 is critical in UCB tumorigenesis and may have implications for developing new therapeutic strategies to treat this disease. Cancer Res; 78(20); 5848–62. ©2018 AACR.

Rationale: Twist is a key transcription factor for induction of epithelial-mesenchymal transition (EMT), which promotes cell migration, invasion, and cancer metastasis, confers cancer cells with stem cell-like characteristics, and provides therapeutic resistance. However, the functional roles and targeted genes of Twist in EMT and cancer progression remain elusive. Methods: The potential targeted genes of Twist were identified from the global transcriptomes of T47D/Twist cells by microarray analysis. EMT phenotype was detected by western blotting and immunofluorescence of marker proteins. The dual-luciferase reporter and chromatin immunoprecipitation assays were employed to observe the direct transcriptional induction of ROR1 by Twist. A lung metastasis model was used to study the pro-metastatic role of Twist and ROR1 by injecting MDA-MB-231 cells into tail vein of nude mice. Bio-informatics analysis was utilized to measure the metastasis-free survival of breast cancer patients. Results: Twist protein was proved to directly activate the transcription of ROR1 gene, a receptor of Wnt5a in non-canonical WNT signaling pathway. Silencing of ROR1 inhibited EMT process, cell migration, invasion, and cancer metastasis of basal-like breast cancer (BLBC) cells. Knockdown of ROR1 also ameliorated the pro-metastatic effect of Twist. Furthermore, analyses of clinical specimens indicated that high expression of both ROR1 and Twist tightly correlates with poor metastasis-free survival of breast cancer patients. Conclusion: ROR1 is a targeted gene of Twist. Twist/ROR1 signaling is critical for invasion and metastasis of BLBC cells.

Cleavage of transfer (t)RNA and ribosomal (r)RNA are critical and conserved steps of translational control for cells to overcome varied environmental stresses. However, enzymes that are responsible for this event have not been fully identified in high eukaryotes. Here, we report a mammalian tRNA/rRNA-targeting endoribonuclease: SLFN13, a member of the Schlafen family. Structural study reveals a unique pseudo-dimeric U-pillow-shaped architecture of the SLFN13 N′-domain that may clamp base-paired RNAs. SLFN13 is able to digest tRNAs and rRNAs in vitro, and the endonucleolytic cleavage dissevers 11 nucleotides from the 3′-terminus of tRNA at the acceptor stem. The cytoplasmically localised SLFN13 inhibits protein synthesis in 293T cells. Moreover, SLFN13 restricts HIV replication in a nucleolytic activity-dependent manner. According to these observations, we term SLFN13 RNase S13. Our study provides insights into the modulation of translational machinery in high eukaryotes, and sheds light on the functional mechanisms of the Schlafen family., Translation inhibition is a strategy for organisms to overcome various environmental stresses including viral infections. Here the authors show that a tRNA/rRNA-targeting RNase Schlafen13 inhibits protein synthesis by directly digesting cytoplasmic tRNA and rRNA with the ability to restrict viral propagation.

Osteosarcoma is the most common primary malignant bone tumor. However, our understanding of the molecular mechanism underlying its pathogenesis is incomplete. Studies have shown that aberrant expression of NK homeobox (NKX) genes may be involved in the oncogenesis of various cancers. Here, through migration screening assay, we found that a series of NKX genes inhibit the migration of osteosarcoma cells. Among these genes, NKX2-2 is a bona fide tumor suppressor for osteosarcoma. Overexpression of NKX2-2 decreases the migration, invasion, proliferation and colony formation of osteosarcoma cells in vitro and suppresses tumor growth and metastasis in vivo. Moreover, based on the results from both in vitro and in vivo studies, the transcriptional activation domain of NKX2-2 is important for its tumor suppressor function. Mechanistically, we revealed that NKX2-2 acts as a tumor suppressor partially by mediating the transcriptional downregulation of COL5A2, PLAU, SEMA7A and S1PR1 genes. In summary, our studies of NKX2-2 revealed new molecular mechanisms underlying osteosarcoma proliferation and metastasis and may provide a series of potential therapeutic targets for osteosarcoma.

IRS4, a member of the insulin receptor substrate protein family, can induce constitutive PI3K/AKT hyperactivation and cell proliferation even in the absence of insulin or growth factors and promote tumorigenesis, but its regulation has only been explored at the transcriptional level. Methods: Scansite was used to predict the potential protein kinases that may regulate the functions of IRS4, and mass spectrometry was used to identify the E3 ligase for IRS4. The protein interaction was carried out by immunoprecipitation, and protein stability was measured by cycloheximide treatment. In vitro kinase assay was used to determine the phosphorylation of IRS4 by casein kinase 1γ2 (CK1γ2). Colony formation assay and xenograft-bearing mice were employed to assess the cancer cell growth in vitro and in vivo, respectively. Immunohistochemistry was performed to examine protein levels of both IRS4 and CK1γ2 in osteosarcoma specimens and their relationship was evaluated by χ2 test. Two-tailed Student's t-test or the Mann-Whitney U test were used to compare the differences between subgroups. Results: IRS4 was phosphorylated at Ser859 by CK1γ2 in vitro and in vivo, which promoted the polyubiquitination and degradation of IRS4 through the ubiquitin/lysosome pathway by the carboxyl terminus of Hsc70-interacting protein(CHIP). Using osteosarcoma cell lines, the ectopic nonphosphorylated mutant of IRS4 by CK1γ2 triggered higher level of p-Akt and displayed faster cell proliferation and cancer growth in vitro and in nude mice. In addition, a negative correlation in protein levels between CK1γ2 and IRS4 was observed in osteosarcoma cell lines and tissue samples. Conclusions: IRS4, as a new substrate of CHIP, is negatively regulated by CK1γ2 at the posttranslational level, and specific CK1γ2 agonists may be a potentially effective strategy for treating patients with osteosarcoma.

In the original version of [this article][1] ([1][2]), there were three errors in Fig. 6: in Fig. 6A, the GADPH blots were inadvertently duplicated from Fig. 5D; in Fig. 6B, the image for Aspirin/NC was a duplicate of DMSO/sh#2; and in Fig. 6B, the Aspirin/vector image was a duplicate of Aspirin/sh#

Activation of the telomere maintenance mechanism is a key hallmark of cancer. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, which is highly expressed in more than 80% of tumors, including hepatocellular carcinoma (HCC). However, the exact mechanisms by which hTERT is up-regulated in HCCs and promotes tumor growth and progression is not fully understood. The aim of this study was to discover the novel molecular targets that modulate hTERT signaling and HCC growth. In this study, we pulled down and identified RBFOX3 (RNA binding protein fox-1 homolog 3) as a novel hTERT promoter-binding protein in HCC cells using biotin-streptavidin-agarose pull-down and proteomics approach, and validated it as a regulatory factor for hTERT signaling and tumor growth in HCCs. Knockdown of RBFOX3 suppressed the promoter activity and expression of hTERT and consequently inhibited the growth and progression of HCC cells in vitro and in vivo. The suppression of HCC growth mediated by RBFOX3 knockdown could be rescued by hTERT overexpression. Conversely, exogenous overexpression of RBFOX3 activated the promoter activity and expression of hTERT and promoted the growth and progression of HCC cells. Moreover, we found that RBFOX3 interacted with AP-2β to regulate the expression of hTERT. Furthermore, we demonstrated that RBFOX3 expression was higher in the tumor tissues of HCC patients compared to the corresponding paracancer tissues, and was positively correlated with hTERT expression. Kaplan-Meier analysis showed that the HCC patients with high levels of RBFOX3 and hTERT had poor prognosis. Collectively, our data indicate that RBFOX3 promotes HCC growth and progression and predicts a poor prognosis by activating the hTERT signaling, and suggest that the RBFOX3/hTERT pathway may be a potential therapeutic target for HCC patients.

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme that generates NADPH to maintain reduced glutathione (GSH), which scavenges reactive oxygen species (ROS) to protect cancer cell from oxidative damage. In this study, we mainly investigate the potential roles of G6PD in colorectal cancer (CRC) development and chemoresistance. We discover that G6PD is overexpressed in CRC cells and patient specimens. High expression of G6PD predicts poor prognosis and correlated with poor outcome of oxaliplatin-based first-line chemotherapy in patients with CRC. Suppressing G6PD decreases NADPH production, lowers GSH levels, impairs the ability to scavenge ROS levels, and enhances oxaliplatin-induced apoptosis in CRC via ROS-mediated damage in vitro. In vivo experiments further shows that silencing G6PD with lentivirus or non-viral gene delivery vector enhances oxaliplatin anti-tumor effects in cell based xenografts and PDX models. In summary, our finding indicated that disrupting G6PD-mediated NADPH homeostasis enhances oxaliplatin-induced apoptosis in CRC through redox modulation. Thus, this study indicates that G6PD is a potential prognostic biomarker and a promising target for CRC therapy.

Gastric cancer (GC) is one of the most common malignancies worldwide. Due to the low rate of early detection, most GC patients were diagnosed as advance stages and had poor response to chemotherapy. Some studies found that Fumarate hydratase (FH) participated in the DNA damage response and its deficiency was associated with tumorigenesis in some cancers. In this study, we investigated the relationship between FH and cisplatin (CDDP) sensitivity in GC cell lines. We found that FH was the most significant gene which induced by CDDP treatment and the suppression of FH could enhance the cytotoxicity of CDDP. Miconazole Nitrate (MN) could inhibit FH activity and enhance the effect of CDDP in vitro and in vivo. We also investigated the significance of expression of FH in GC tissues. The FH expression, which was higher in GC tissues than in noncancerous tissues, was negatively associated with the prognosis of patients. Together, these results revealed that FH is a reliable indicator for response to CDDP treatment in GC and the inhibition of FH may be a potential strategy to improve the effects of CDDP-based chemotherapy.

Nasopharyngeal carcinoma (NPC) is characterized by a high rate of local invasion and early distant metastasis. Increasing evidence indicates that epigenetic abnormalities play important roles in NPC development. However, the epigenetic mechanisms underlying NPC metastasis remain unclear. Here we investigate aberrantly methylated transcription factors in NPC tissues, and we identify the HOP homeobox HOPX as the most significantly hypermethylated gene. Consistently, we find that HOXP expression is downregulated in NPC tissues and NPC cell lines. Restoring HOPX expression suppresses metastasis and enhances chemosensitivity of NPC cells. These effects are mediated by HOPX-mediated epigenetic silencing of SNAIL transcription through the enhancement of histone H3K9 deacetylation in the SNAIL promoter. Moreover, we find that patients with high methylation levels of HOPX exhibit poor clinical outcomes in both the training and validation cohorts. In summary, HOPX acts as a tumour suppressor via the epigenetic regulation of SNAIL transcription, which provides a novel prognostic biomarker for NPC metastasis and therapeutic target for NPC treatment., HOPX is a transcription factor epigenetically silenced in several cancers. Here the authors, by analysing methylation profiles, identify HOPX as a suppressor of metastasis in nasopharyngeal carcinoma: mechanistically HOPX inhibits SNAIL transcription through deacetylation-mediated silencing.

The poor clinical outcome and prognosis of esophageal squamous cell carcinoma (ESCC) is mainly attributed to its highly invasive and metastatic nature, making it urgent to further elicit the molecular mechanisms of the metastasis of ESCC. The function of each polycomb chromobox (CBX) protein in cancer is cell-type-dependent. Although CBX8 has been reported to promote the esophageal squamous cell carcinoma (ESCC) tumorigenesis, its role in ESCC metastasis has not been explored yet. In this study, we report that the inhibition of cell migration, invasion, and metastasis in ESCC requires CBX8-mediated repression of Snail, a key transcription factor that induces epithelial-to-mesenchymal transition (EMT), and that CBX8 inversely correlated with Snail in the ESCC tissues. Moreover, this novel function of CBX8 is dependent on its binding with the Snail promoter, which in turn suppresses the transcription of Snail. Collectively, CBX8 may play paradoxical roles in ESCC, inhibiting metastasis while promoting cell proliferation.

Aberrant activation of cellular Src (c-Src), a non-receptor tyrosine kinase, could promote cancer progression through activating its downstream signaling pathways. However, the roles of c-Src and phosphorylated-Src (p-Src) in nasopharyngeal carcinoma (NPC) progression are rarely investigated. Herein, we have identified high c-Src concentrations in the serum of NPC patients with distant metastasis using high-throughput protein microarrays. Levels of c-Src in serum and p-Src in human primary NPC samples were unfavorable independent prognostic factors for cancer-specific survival, disease-free survival, and distant metastasis-free survival. Depletion or inactivation of c-Src in NPC cells using sgRNA with CRISPR/Cas9 system or PP2 decreased cell viability, colony formation, migration and invasion in vitro and metastasis in vivo. In contrast, these malignancies could be up-regulated by overexpressed c-Src in a NPC cell line with low-metastasis potential. Furthermore, p-Src was involved in promoting NPC cell metastasis by inducing the epithelial-mesenchymal transition (EMT) process via activating the PI3K/Akt pathway and cytoskeleton remodeling. The p-Src-induced EMT process could be retarded by PP2, which mediated by down-regulating the PI3K/Akt pathway. In conclusion, elevated levels of c-Src in serum and p-Src in primary NPC tissue correlated with poor outcomes of NPC patients. And aberrant activation of c-Src facilitated NPC cells with malignant potential, especially metastasis ability, which mediated by the PI3K/Akt pathway activation and sequentially induced the EMT process. These findings unveiled a promising approach for targeted therapy of advanced NPC.

Molecule interacting with CasL2 (MICAL2), a microtubule-associated monooxygenase, is highly expressed in various cancers and is involved in cancer pathogenesis, but the mechanisms underlying its regulation in carcinogenesis are unclear. In this study, we aim to clarify the mechanism by which MICAL2 participates in colorectal cancer (CRC) and identify novel markers for predicting prognosis of CRC patients. Methods: The value of MICAL2 in CRC prognosis was determined by immunohistochemical analysis of a CRC biopsy array. A short hairpin RNA target MICAL2 (shMICAL2) was designed to knock down MICAL2 expression and observe MICAL2's function on CRC cell growth. mRNA expression array was used to screen target molecules of MICAL2. HCT116 p53+/+ and HCT116 p53-/- cells were used to confirm whether MICAL2 exerts its oncogenic effect through p53. The in vivo effect of MICAL2 on CRC growth was assessed by subcutaneously injecting MICAL2-knockout CRC cells into the dorsal flank of each mouse. Immunofluorescence was used to observe the effect of MICAL2 on p53 cellular location. Reverse-phase nano ESI-LCMS analysis was used to investigate if MICAL2 mediates p53 oxidation. Results: MICAL2 was found to be highly expressed in CRC tissues, and its expression was associated with CRC carcinogenesis and poor patient outcome. MICAL2-knockdown decreased growth and colony formation of CRC cells, which was linked with cell cycle arrest and apoptosis. MICAL2 physically interacted with p53 and retained p53 in the cytoplasm. MICAL2 shortened the half-life of p53, and ectopic MICAL2 expression decreased p53 protein stability through ubiquitin degradation. MICAL2 was also found to oxidize p53 at methionine 40 and 160, which mediated p53 ubiquitin degradation. MICAL2-promoted CRC growth in vivo was confirmed in nude mice. Conclusion: MICAL2 binds to p53, retains p53 in the cytoplasm and oxidizes it at Met 40 and 160, promotes p53 ubiquitination, and decreases p53 function. MICAL2-reduced p53 promotes CRC development.

Background Distant metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC). Although several biomarkers correlate with metastasis and prognosis, the molecular mechanisms of NPC development and progression remain unclear. Methods Quantitative RT-PCR (qRT-PCR), western blotting, cell growth, foci formation, migration and invasion assays, and xenograft mouse models were utilized to examine the expression levels and functions of the CXCL5/CXCR2 axis in NPC. A luciferase reporter assay, western blotting, immunofluorescence, and migration and invasion assays were used to identify and verify the ERK/GSK-3β/Snail signalling pathway. Results CXCL5 was significantly increased in the sera of NPC patients, and high expression levels of CXCL5/CXCR2 in NPC primary tissues indicated poor survival. CXCL5 and CXCR2 were upregulated in NPC cell lines. Ectopic expression of the CXCL5/CXCR2 axis promoted NPC cell migration and invasion in vitro and the formation of lung metastases in vivo. Mechanistically, the dual overexpression of CXCL5 and CXCR2 promoted cell spreading by inducing the epithelial-mesenchymal transition (EMT) through the activation of the ERK/GSK-3β/Snail signalling pathway. Conclusion The CXCL5/CXCR2 axis contributes to the EMT of NPC cells by activating ERK/GSK-3β/Snail signalling, and this axis may be a potential diagnostic marker and therapeutic target for patients with NPC. Electronic supplementary material The online version of this article (10.1186/s13046-018-0722-6) contains supplementary material, which is available to authorized users.

Nasopharyngeal carcinoma (NPC) has a high metastatic clinicopathological feature. As a carcinogen factor, N,N′-Dinitrosopiperazine (DNP) is involved in NPC metastasis, but its precise mechanism has not been fully elucidated. Herein, we showed that DNP promotes NPC metastasis through up-regulating anterior clusterin (CLU). DNP was found to increase CLU, matrix metalloproteinases (MMP) 9 and vascular endothelial growth factor (VEGF) expression and activity, further DNP-increased MMP-9 and VEGF expression was through up-regulating CLU. We also found that DNP increased the binding of CLU with MMP-9 or VEGF. DNP induced the motility and invasion of NPC cell, which was inhibited by siRNA-CLU. The clinical investigation showed that CLU, MMP-9 and VEGF were positively correlated with the tumor-node -metastasis (TNM) classification. These results indicate that DNP may promote NPC tumor metastasis through up-regulating CLU, MMP-9 and VEGF expression. Therefore, DNP-increased CLU expression may be an important factor of NPC-high metastasis, and CLU may serve as a biomarker for NPC metastasis.

Purpose: Aspirin has recently been reported to reduce both the incidence and the risk of metastasis in colon cancer. However, there is no evidence at the cellular levels or in the animal models for such an effect of aspirin on cancer metastasis. Experimental Design: MTT assay, colony formation assay, and apoptosis assay were employed to analyze the effects of aspirin on the osteosarcoma cell viability in vitro. The NF-κB activity was measured by the NF-κB p65 luciferase reporter. Western blotting was used to analyze the proteins in cells. The migration and invasion abilities of osteosarcoma cells in vitro were measured by the Transwell assay. Xenograft-bearing mice were used to assess the roles of aspirin in both tumor growth and metastasis of osteosarcoma in vivo (n = 5–8 mice/group). An unpaired Student t test or ANOVA with the Bonferroni post hoc test were used for the statistical comparisons. Results: Aspirin reduced cell viability in a dose- and time-dependent manner in osteosarcoma cell lines, and aspirin synergistically sensitized osteosarcoma cells to cisplatin (DDP) in vitro and in vivo (P < 0.001). Moreover, aspirin markedly repressed the migration and invasion of osteosarcoma cells in vitro (P < 0.001), and dramatically diminished the occurrence of osteosarcoma xenograft metastases to the lungs in vivo (P < 0.001). Mechanistically, aspirin diminishes osteosarcoma migration, invasion, and metastasis through the NF-κB pathway. Conclusions: Aspirin suppresses both the growth and metastasis of osteosarcoma through the NF-κB pathway at the cellular level and in the animal models. Clin Cancer Res; 21(23); 5349–59. ©2015 AACR.