Exosomal long noncoding RNA MLETA1 promotes tumor progression and metastasis by regulating the miR-186-5p/EGFR and miR-497-5p/IGF1R axes in non-small cell lung cancer.

BACKGROUND: Lung cancer is the most common and deadliest cancer worldwide, and approximately 90% of all lung cancer deaths are caused by tumor metastasis. Tumor-derived exosomes could potentially promote tumor metastasis through the delivery of metastasis-related molecules. However, the function and underlying mechanism of exosomal long noncoding RNA (lncRNA) in lung cancer metastasis remain largely unclear. METHODS: Cell exosomes were purified from conditioned media by differential ultracentrifugation and observed using transmission electron microscopy, and the size distributions were determined by nanoparticle tracking analysis. Exosomal lncRNA sequencing (lncRNA-seq) was used to identify long noncoding RNAs. Cell migration and invasion were determined by wound-healing assays, two-chamber transwell invasion assays and cell mobility tracking. Mice orthotopically and subcutaneously xenografted with human cancer cells were used to evaluate tumor metastasis in vivo. Western blot, qRT‒PCR, RNA-seq, and dual-luciferase reporter assays were performed to investigate the potential mechanism. The level of exosomal lncRNA in plasma was examined by qRT‒PCR. MS2-tagged RNA affinity purification (MS2-TRAP) assays were performed to verify lncRNA-bound miRNAs. RESULTS: Exosomes derived from highly metastatic lung cancer cells promoted the migration and invasion of lung cancer cells with low metastatic potential. Using lncRNA-seq, we found that a novel lncRNA, lnc-MLETA1, was upregulated in highly metastatic cells and their secreted exosomes. Overexpression of lnc-MLETA1 augmented cell migration and invasion of lung cancer. Conversely, knockdown of lnc-MLETA1 attenuated the motility and metastasis of lung cancer cells. Interestingly, exosome-transmitted lnc-MLETA1 promoted cell motility and metastasis of lung cancer. Reciprocally, targeting lnc-MLETA1 with an LNA suppressed exosome-induced lung cancer cell motility. Mechanistically, lnc-MLETA1 regulated the expression of EGFR and IGF1R by sponging miR-186-5p and miR-497-5p to facilitate cell motility. The clinical datasets revealed that lnc-MLETA1 is upregulated in tumor tissues and predicts survival in lung cancer patients. Importantly, the levels of exosomal lnc-MLETA1 in plasma were positively correlated with metastasis in lung cancer patients. CONCLUSIONS: This study identifies lnc-MLETA1 as a critical exosomal lncRNA that mediates crosstalk in lung cancer cells to promote cancer metastasis and may serve as a prognostic biomarker and potential therapeutic target for lung cancer diagnosis and treatment.

Ubiquitin-specific peptidase 5 facilitates cancer stem cell-like properties in lung cancer by deubiquitinating ß-catenin.

BACKGROUND: Lung cancer has the highest mortality rate in the world, and mounting evidence suggests that cancer stem cells (CSCs) are associated with poor prognosis, recurrence, and metastasis of lung cancer. It is urgent to identify new biomarkers and therapeutic targets for targeting lung CSCs. METHODS: We computed the single-sample gene set enrichment analysis (ssGSEA) of 1554 Reactome gene sets to identify the mRNA expression-based stemness index (mRNAsi)-associated pathways using the genome-wide RNA sequencing data of 509 patients from The Cancer Genome Atlas (TCGA) cohort of lung adenocarcinoma (LUAD). Phenotypic effects of ubiquitin-specific peptidase 5 (USP5) on the CSC-like properties and metastasis were examined by in vitro sphere formation assay, migration assay, invasion assay, and in vivo xenografted animal models. Cycloheximide chase assay, co-immunoprecipitation assay, and deubiquitination assay were performed to confirm the effect of USP5 on the deubiquitination of ß-catenin. RESULTS: We demonstrated that USP5 expression were positively correlated with the stemness-associated signatures and poor outcomes in lung cancer specimens. Silencing of endogenous USP5 reduced CSC-like characteristics, epithelial-mesenchymal transition (EMT), and metastasis in vitro and in vivo. Furthermore, USP5 interacted with ß-catenin, which resulted in deubiquitination, stabilization of ß-catenin, and activation of Wnt/ß-catenin pathway. Accordingly, expression of USP5 was positively correlated with the enrichment score of the Wnt/TCF pathway signature in human lung cancer. Silencing of ß-catenin expression suppressed USP5-enhancing sphere formation. Targeting USP5 with the small molecule WP1130 promoted the degradation of ß-catenin, and showed great inhibitory effects on sphere formation, migration, and invasion. Finally, we identified a poor-prognosis subset of tumors characterized by high levels of USP5, Wnt signaling score, and Stemness score in both TCGA-LUAD and Rousseaux_2013 datasets. CONCLUSIONS: These findings reveal a clinical evidence for USP5-enhanced Wnt/ß-catenin signaling in promoting lung cancer stemness and metastasis, implying that targeting USP5 could provide beneficial effects to improve lung cancer therapeutics.

MiR-455-5p suppresses PDZK1IP1 to promote the motility of oral squamous cell carcinoma and accelerate clinical cancer invasion by regulating partial epithelial-to-mesenchymal transition.

BACKGROUND: Lymph node and distant metastasis contribute to poor outcomes in patients with oral squamous cell carcinoma (OSCC). The mechanisms regulating cancer migration and invasion play a key role in OSCC. METHODS: We determined migration and invasion ability of OSCC by wound-healing assay, two-chamber transwell invasion assay and cell mobility tracking and evaluated tumor metastasis in vivo. Western blot (WB), qRT-PCR, RNA-seq, dual-luciferase reporter assays and nuclear/cytoplasmic fractionation were performed to investigate the potential mechanism. Immunohistochimical (IHC) staining determined vimentin and PDZK1IP1 expression in OSCC tissues. RESULTS AND CONCLUSION: In this study, we determined that miR-455-5p was associated with lymph node metastasis and clinical invasion, leading to poor outcomes in patients with OSCC. MiR-455-5p promoted oral cancer cell migration and invasion and induced epithelial-to-mesenchymal transition (EMT). We also identified a new biomarker, PDZK1IP1 (MAP17), that was targeted by miR-455-5p. PDZK1IP1 knockdown led to migration, metastasis, EMT, and increased transforming growth factor-ß signaling in OSCC. In addition, miR-455-5p overexpression and PDZK1IP1 inhibition promoted collective OSCC cell migration. According to data from the Cancer Genome Atlas database and the NCKU-OrCA-40TN data set, miR-455-5p and PDZK1IP1 are positively and negatively correlated, respectively, with partial EMT score. High miR-455-5p expression was associated with high vimentin levels and low MAP17 H-scores. The patients with low MAP17 expression had higher rates of disease recurrence than did patients with high MAP17 expression, especially for patients with clinical invasion risk factors and low MAP17 expression. These results suggest that miR-455-5p suppresses PDZK1IP1 expression and mediates OSCC progression. MiR-455-5p and PDZK1IP1 may therefore serve as key biomarkers and be involved in regulating partial EMT in OSCC cells. PDZK1IP1 expression may also serve as an independent factor that impacts outcomes in patients with clinical risk factors for recurrence.

CASZ1 promotes migration, invasion, and metastasis of lung cancer cells by controlling expression of ITGAV.

CASZ1, a zinc finger transcription factor with two isoforms, is known to play important roles in cardiac and neural development. The abnormal expression of CASZ1 is also frequently found in a variety of tumors but has different effects on different tumors; for example, it acts as a tumor suppressor in neuroblastoma but promotes cancer metastasis in ovarian cancer. However, the effect of CASZ1 in lung cancer, the most lethal cancer, remains unclear. Here, we found that the expression of CASZ1 in lung cancer is positively associated with cancer metastasis and poor prognosis. The overexpression of CASZ1b promotes lung cancer cell migration, invasion, and epithelial-mesenchymal transition and is associated with poor prognosis in lung cancer patients. The knockdown of CASZ1 resulted in the suppression of epithelial-mesenchymal transition, migration, and invasion of lung cancer cells and reduced metastasis in vivo. The results of an RNA-sequencing analysis of CASZ1-silenced cells showed that CASZ1 considerably affected the integrin-mediated pathways. CASZ1 bound to the ITGAV promoter and transcriptionally regulated ITGAV expression. Our findings demonstrate that CASZ1 plays an oncogenic role in lung cancer and that CASZ1 promotes lung cancer migration, invasion and metastasis is mediated by ITGAV.

A novel DNA aptamer targeting lung cancer stem cells exerts a therapeutic effect by binding and neutralizing Annexin A2.

Cancer remains one of the leading causes of death worldwide. Cancer stem cells (CSCs) are the underlying reason for tumor recurrence, progression, and therapeutic resistance. Aptamers are synthetic single-stranded oligonucleotides that can specifically bind to various molecular targets. Here, we aim to develop an effective aptamer-based biomarker and therapeutic tool that targets CSCs for cancer therapy. We perform whole-cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) to screen DNA aptamers that specifically bound to lung CSCs, modeled by E-cadherin-silenced A549 cells. We develop a CSC-specific aptamer (AP-9R) specifically recognizing lung CSCs with high affinity and identify Annexin A2, a Ca2+-dependent membrane-binding protein, as its target. Annexin A2 expression was upregulated in lung CSCs and involved in cancer stemness. The expression of Annexin A2 was associated with signatures of stemness and metastasis, as well as poor clinical outcomes, in lung cancer in silico. Moreover, AP-9R decreased Annexin A2 expression and suppressed CSC properties in CSCs in vitro and in vivo. The present findings suggest that Annexin A2 is a CSC marker and regulator, and the CSC-specific aptamer AP-9R has potential theranostic applications for lung cancer.

α-Catulin promotes cancer stemness by antagonizing WWP1-mediated KLF5 degradation in lung cancer.

Background: The cytoskeletal linker protein α-Catulin has been shown to be important for tumor progression in various cancers. However, its role in the regulation of cancer stemness remains unclear. Methods: Phenotypic effects of α-Catulin on the cancer stem cell (CSC)-like properties and metastasis were examined by in vitro sphere formation assay, migration assay, invasion assay, and in vivo xenografted animal models. Yeast two-hybrid assay, co-immunoprecipitation assay, and cycloheximide chase assay were performed to confirm the effect of α-Catulin on the WWP1-mediated degradation of KLF5. CPTAC and TCGA database were analyzed to determine the clinical association of α-Catulin, KLF5, and stemness-associated signatures in lung adenocarcinoma. Results: We report that α-Catulin increases cancer stem-like properties in non-small cell lung cancer (NSCLC). The expression of α-Catulin is elevated in tumor spheres compared to sphere-derived adherent cells and promotes the acquisition of cancer stemness characteristics in vitro and in vivo. Mechanistically, the interaction of α-Catulin and the C-terminal region of Kruppel-like transcription factor KLF5 results in the inhibition of WWP1-mediated degradation of KLF5. Accordingly, increased protein expression of KLF5 is observed in clinical specimens of lung adenocarcinoma with high expression of α-Catulin compared to specimens with low α-Catulin-expression. Knockdown of KLF5 abrogates α-Catulin-driven cancer stemness. α-Catulin is known to interact with integrin-linked kinase (ILK). Notably, an ILK inhibitor disrupts the α-Catulin-KLF5 interaction, promotes the degradation of KLF5, and decreases α-Catulin-driven cancer stemness. Importantly, we identify a CTNNAL1/ILK/KLF5 three-gene signature for predicting poor overall survival in patients with lung adenocarcinoma. Conclusions: These findings reveal a molecular basis of α-Catulin-enhanced KLF5 signaling and highlight a role for α-Catulin in promoting cancer stemness.

Omental Macrophagic "Crown-like Structures" Are Associated with Poor Prognosis in Advanced-Stage Serous Ovarian Cancer.

The tumor microenvironment is a well-recognized framework in which immune cells present in the tumor microenvironment promote or inhibit cancer formation and development. A crown-like structure (CLS) has been reported as a dying or dead adipocyte surrounded by a 'crown' of macrophages within adipose tissue, which is a histologic hallmark of the inflammatory process in this tissue. CLSs have also been found to be related to formation, progression and prognosis of some types of cancer. However, the presence of CLSs in the omentum of advanced-stage high-grade serous ovarian carcinoma (HGSOC) has not been thoroughly investigated. By using CD68, a pan-macrophage marker, and CD163, an M2-like polarization macrophage marker, immunohistochemistry (IHC) was performed to identify tumor-associated macrophages (TAMs) and CLSs. This retrospective study analyzed 116 patients with advanced-stage HGSOC who received complete treatment and had available clinical data from July 2008 through December 2016 at National Cheng Kung University Hospital (NCKUH) (Tainan, Taiwan). Based on multivariate Cox regression analysis, patients with omental CD68+ CLSs had poor OS (median survival: 24 vs. 38 months, p = 0.001, hazard ratio (HR): 2.26, 95% confidence interval (CI): 1.41-3.61); patients with omental CD163+ CLSs also had poor OS (median survival: 22 vs. 36 months, HR: 2.14, 95%CI: 1.33-3.44, p = 0.002). Additionally, patients with omental CD68+ or CD163+ CLSs showed poor PFS (median survival: 11 vs. 15 months, HR: 2.28, 95%CI: 1.43-3.64, p = 0.001; median survival: 11 vs. 15 months, HR: 2.17, 95%CI: 1.35-3.47, respectively, p = 0.001). Conversely, the density of CD68+ or CD163+ TAMs in ovarian tumors was not associated with patient prognosis in advanced-stage HGSOC in our cohort. In conclusion, we, for the first time, demonstrate that the presence of omental CLSs is associated with poor prognosis in advanced-stage HGSOC.

DNA methylation maintains the CLDN1-EPHB6-SLUG axis to enhance chemotherapeutic efficacy and inhibit lung cancer progression.

The loss of cancer-cell junctions and escape from the primary-tumor microenvironment are hallmarks of metastasis. A tight-junction protein, Claudin 1 (CLDN1), is a metastasis suppressor in lung adenocarcinoma. However, as a metastasis suppressor, the underlying molecular mechanisms of CLDN1 has not been well studied. Methods: The signaling pathway regulated by CLDN1 was analyzed by Metacore software and validated by immunoblots. The effect of the CLDN1-EPHB6-ERK-SLUG axis on the formation of cancer stem-like cells, drug resistance and metastasis were evaluated by sphere assay, aldefluor assay, flow cytometry, migration assay, cytotoxicity, soft agar assay, immunoprecipitation assay and xenograft experiments. Furthermore, the methylation-specific PCR, pyrosequencing assay, chromatin immunoprecipitation and reporter assay were used to study the epigenetic and RUNX3-mediated CLDN1 transcription. Finally, the molecular signatures of RUNX3/CLDN1/SLUG were used to evaluate the correlation with overall survival by using gene expression omnibus (GEO) data. Results: We demonstrated that CLDN1 repressed cancer progression via a feedback loop of the CLDN1-EPHB6-ERK1/2-SLUG axis, which repressed metastasis, drug resistance, and cancer stemness, indicating that CLDN1 acts as a metastasis suppressor. CLDN1 upregulated the cellular level of EPHB6 and enhanced its activation, resulting in suppression of ERK1/2 signaling. Interestingly, DNA hypermethylation of the CLDN1 promoter abrogated SLUG-mediated suppression of CLDN1 in low-metastatic cancer cells. In contrast, the histone deacetylase inhibitor trichostatin A or vorinostat facilitated CLDN1 expression in high-metastatic cancer cells and thus increased the efficacy of chemotherapy. Combined treatment with cisplatin and trichostatin A or vorinostat had a synergistic effect on cancer-cell death. Conclusions: This study revealed that DNA methylation maintains CLDN1 expression and then represses lung cancer progression via the CLDN1-EPHB6-ERK1/2-SLUG axis. Because CLDN1 enhances the efficacy of chemotherapy, CLDN1 is not only a prognostic marker but a predictive marker for lung adenocarcinoma patients who are good candidates for chemotherapy. Forced CLDN1 expression in low CLDN1-expressing lung adenocarcinoma will increase the chemotherapy response, providing a novel therapeutic strategy.

Monocarboxylate Transporter 4 Is a Therapeutic Target in Non-small Cell Lung Cancer with Aerobic Glycolysis Preference.

Targeting metabolic reprogramming is an emerging strategy in cancer therapy. However, clinical attempts to target metabolic reprogramming have been proved to be challenging, with metabolic heterogeneity of cancer being one of many reasons that causes treatment failure. Here, we stratified non-small cell lung cancer (NSCLC) cells, mainly lung adenocarcinoma, based on their metabolic phenotypes and demonstrated that the aerobic glycolysis-preference NSCLC cell subtype was resistant to the OXPHOS-targeting inhibitors. We identified that monocarboxylate transporter 4 (MCT4), a lactate transporter, was highly expressed in the aerobic glycolysis-preference subtype with function supporting the proliferation of these cells. Glucose could induce the expression of MCT4 in these cells through a ΔNp63α and Sp1-dependent pathway. Next, we showed that knockdown of MCT4 increased intracellular lactate concentration and induced a reactive oxygen species (ROS)-dependent cellular apoptosis in the aerobic glycolysis-preference NSCLC cell subtype. By scanning a panel of monoclonal antibodies with MCT4 neutralizing activity, we further identified a MCT4 immunoglobulin M (IgM) monoclonal antibody showing capable anti-proliferation efficacy on the aerobic glycolysis-preference NSCLC cell subtype. Our findings indicate that the metabolic heterogeneity is a critical factor for NSCLC therapy and manipulating the expression or function of MCT4 can be an effective strategy in targeting the aerobic glycolysis-preference NSCLC cell subtype.

Low Galectin-3 Expression Level in Primary Tumors Is Associated with Metastasis in T1 Lung Adenocarcinoma.

BACKGROUND AND OBJECTIVE: Although nodal and distant metastasis is rare in T1 lung adenocarcinoma, it is related to poor clinical prognosis. Association between galectin-3 (Gal-3) expression level, and clinical outcome of T1 lung adenocarcinoma has not been clarified. METHODS: From January 2009 to December 2014, 74 patients with surgically resected T1 lung adenocarcinoma were enrolled in this retrospective cohort study. Patient outcomes were followed up until December 2019. Gal-3 expression level in primary tumors was assessed immunohistochemically and evaluated based on the staining intensity and percentage. Patient characteristics and correlation between Gal-3 expression level and clinical outcomes were reviewed. RESULTS: Low Gal-3 expression was associated with increased metastatic events (p = 0.03), especially distant metastasis (p = 0.007), and mortality rate (p = 0.04). Kaplan-Meier analysis revealed that high Gal-3 expression level was associated with favorable recurrence-free survival in T1 lung adenocarcinoma (log-rank p = 0.048) and T1a (≤ 2 cm, American Joint Committee on Cancer (AJCC) 7th edition) lung adenocarcinoma (log-rank p = 0.043). Gal-3 expression along with tumor size showed a larger area under curve (AUC) than tumor size alone for predicting metastatic events (AUC = 0.747 vs. 0.681) and recurrence (AUC = 0.813 vs. 0.766) in T1a lung adenocarcinoma in the receiver-operating characteristic curve. CONCLUSION: Low Gal-3 expression level in primary tumors was remarkably associated with increased metastatic events and reduced recurrence-free survival in T1 lung adenocarcinoma. We suggest that Gal-3 expression level in addition to tumor size may potentially be stronger than tumor size alone in predicting metastasis in T1a lung adenocarcinoma patients.

MicroRNA-150-5p promotes cell motility by inhibiting c-Myb-mediated Slug suppression and is a prognostic biomarker for recurrent ovarian cancer.

Treatment of ovarian cancer (OvCa) remains challenging owing to its high recurrence rates. Detachment of cancer cells into the peritoneal fluid plays a key role in OvCa relapse, but how this occurs remains incompletely understood. Here we examined global miRNA expression profiles of paired primary/recurrent OvCa specimens and identified a novel biomarker, microRNA-150-5p (miR-150-5p), that was significantly upregulated in 16 recurrent OvCa tissues compared with their matched primary specimens. Analyses of cohorts from two other groups confirmed that expression of miR-150-5p was associated with early relapse and poor survival of OvCa patients. Inhibition of miR-150-5p significantly inhibited the migration and invasion of OvCa cells and induced a mesenchymal-epithelial transition (MET) phenotype. We demonstrated that the proto-oncogene, MYB, is an miR-150-5p target in OvCa cells and that the miR-150-5p/c-Myb/Slug axis plays important roles in regulating epithelial-mesenchymal transition (EMT) in OvCa cells. Expression of MYB was significantly correlated with good clinical outcome in OvCa and was negatively correlated with Slug expression in late-stage clinical specimens. These results suggest that miR-150-5p upregulation mediates the progression of recurrent OvCa by targeting the c-Myb/Slug pathway. Inhibition of miR-150-5p may serve as a new therapeutic strategy for preventing recurrence of OvCa.

A DNA Aptamer Targeting Galectin-1 as a Novel Immunotherapeutic Strategy for Lung Cancer.

Galectin-1 (Gal-1) is a pleiotropic homodimeric ß-galactoside-binding protein with a single carbohydrate recognition domain. It has been implicated in several biological processes that are important during tumor progression. Several lines of evidence have indicated that Gal-1 is involved in cancer immune escape and induces T cell apoptosis. These observations all emphasized Gal-1 as a novel target for cancer immunotherapy. Here, we developed a novel Gal-1-targeting DNA aptamer (AP-74 M-545) and demonstrated its antitumor effect by restoring immune function. AP-74 M-545 binds to Gal-1 with high affinity. AP-74 M-545 targets tumors in murine tumor models but suppresses tumor growth only in immunocompetent C57BL/6 mice, not in immunocompromised non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. Immunohistochemistry revealed increased CD4+ and CD8+ T cells in AP-74 M-545-treated tumor tissues. AP-74 M-545 suppresses T cell apoptosis by blocking the binding of Gal-1 to CD45, the main receptor and apoptosis mediator of Gal-1 on T cells. Collectively, our data suggest that the Gal-1 aptamer suppresses tumor growth by blocking the interaction between Gal-1 and CD45 to rescue T cells from apoptosis and restores T cell-mediated immunity. These results indicate that AP-74 M-545 may be a potential strategy for cancer immunotherapy.

4(1H)-quinolone derivatives overcome acquired resistance to anti-microtubule agents by targeting the colchicine site of ß-tubulin.

Developing new therapeutic strategies to overcome drug resistance of cancer cells is an ongoing endeavor. From among 2 million chemicals, we identified ethyl 4-oxo-2-phenyl-1,4-dihydroquinoline-6-carboxylate (AS1712) as a low-toxicity inhibitor of lung cancer cell proliferation and xenograft tumor growth. We show that AS1712 is active against broad cancer cell lines and is able to bind in the colchicine-binding pocket of ß-tubulin, thereby inhibiting microtubule assembly and, consequently, inducing mitotic arrest and apoptosis. Our cell-based structure-activity relationship study identified a new lead compound, RJ-LC-15-8, which had a greater anti-proliferative potency for H1975 cells than did AS1712, while maintaining a similar mechanism of action. Notably, AS1712 and RJ-LC-15-8 overcame P-glycoprotein efflux pump and ß-tubulin alterations that lead to acquired resistance against microtubule-targeting drugs of cancer cells. AS1712 and RJ-LC-15-8 may be lead compounds that overcome acquired resistance to microtubule-targeting agents of cancer cells.

The dual PI3K/mTOR inhibitor BEZ235 restricts the growth of lung cancer tumors regardless of EGFR status, as a potent accompanist in combined therapeutic regimens.

BACKGROUND: Lung cancer is the most common cause of cancer-related mortality worldwide despite diagnostic improvements and the development of targeted therapies, notably including epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). The phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) signaling has been shown to contribute to tumorigenesis, tumor progression, and resistance to therapy in most human cancer types, including lung cancer. Here, we explored the therapeutic effects of co-inhibition of PI3K and mTOR in non-small-cell lung cancer (NSCLC) cells with different EGFR status. METHODS: The antiproliferative activity of a dual PI3K/mTOR inhibitor BEZ235 was examined by the WST-1 assay and the soft agar colony-formation assay in 2 normal cell lines and 12 NSCLC cell lines: 6 expressing wild-type EGFR and 6 expressing EGFR with activating mutations, including exon 19 deletions, and L858R and T790 M point mutations. The combination indexes of BEZ235 with cisplatin or an EGFR-TKI, BIBW2992 (afatinib), were calculated. The mechanisms triggered by BEZ235 were explored by western blotting analysis. The anti-tumor effect of BEZ235 alone or combined with cisplatin or BIBW2992 were also studied in vivo. RESULTS: BEZ235 suppressed tumor growth in vitro and in vivo by inducing cell-cycle arrest at G1 phase, but without causing cell death. It also reduced the expression of cyclin D1/D3 by regulating both its transcription and protein stability. Moreover, BEZ235 synergistically enhanced cisplatin-induced apoptosis in NSCLC cells by enhancing or prolonging DNA damage and BIBW2992-induced apoptosis in EGFR-TKI-resistant NSCLC cells containing a second TKI-resistant EGFR mutant. CONCLUSIONS: The dual PI3K/mTOR inhibition by BEZ235 is an effective antitumor strategy for enhancing the efficacy of chemotherapy or targeted therapy, even as a monotherapy, to restrict tumor growth in lung cancer treatment.

Hypoxia-induced Slug SUMOylation enhances lung cancer metastasis.

BACKGROUND: The Slug-E-cadherin axis plays a critical role in non-small-cell lung cancers (NSCLCs) where aberrant upregulation of Slug promotes cancer metastasis. Now, the post-translational modifications of Slug and their regulation mechanisms still remain unclear in lung cancer. Hence, exploring the protein linkage map of Slug is of great interest for investigating the scenario of how Slug protein is regulated in lung cancer metastasis. METHODS: The Slug associated proteins, Ubc9 and SUMO-1, were identified using yeast two-hybrid screening; and in vitro SUMOylation assays combined with immunoprecipitation and immunoblotting were performed to explore the detail events and regulations of Slug SUMOylation. The functional effects of SUMOylation on Slug proteins were examined by EMSA, reporter assay, ChIP assay, RT-PCR, migration and invasion assays in vitro, tail vein metastatic analysis in vivo, and also evaluated the association with clinical outcome of NSCLC patients. RESULTS: Slug protein could interact with Ubc9 and SUMO-1 and be SUMOylated in cells. Amino acids 130-212 and 33-129 of Slug are responsible for its binding to Ubc9 and protein inhibitor of activated STAT (PIAS)y, respectively. SUMOylation could enhance the transcriptional repression activity of Slug via recruiting more HDAC1, resulting in reduced expression of downstream Slug target genes and enhanced lung cancer metastasis. In addition, hypoxia could increase Slug SUMOylation through attenuating the interactions of Slug with SENP1 and SENP2. Finally, high expression Slug and Ubc9 levels were associated with poor overall survival among NSCLC patients. CONCLUSIONS: Ubc9/PIASy-mediated Slug SUMOylation and subsequent HDAC1 recruitment may play a crucial role in hypoxia-induced lung cancer progression, and these processes may serve as therapeutic targets for NSCLC.

Growth-regulated oncogene-α from oral submucous fibrosis fibroblasts promotes malignant transformation of oral precancerous cells.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common human malignancy and is usually preceded by the oral precancerous lesions. Oral submucous fibrosis (OSF) is one of the oral precancerous lesions with high incidence of malignant transformation. In addition to cancer cells, cancer-associated fibroblasts in the tumor microenvironment are correlated with cancer progression, but the role of fibroblasts from OSF in tumorigenesis and progression is still unknown. Growth-regulated oncogene-α (GRO-α), a member of CXC chemokine family, is related to tumorigenesis in several cancers. In this study, we would like to explore the role of GRO-α from OSF-associated fibroblasts in oral cancer progression. METHODS: We isolated primary culture fibroblasts of normal, precancerous, and tumor tissues from patients with OSCC accompanied with OSF. A cytokine array was used to screen cytokine secretions in the conditioned media of the fibroblasts. A wound healing migration assay, WST-1 cell proliferation assay, rhodamine-phalloidin staining, and soft agar colony formation assay were used to investigate the effects of GRO-α on a dysplastic oral keratinocyte cell line (DOK) cell migration, growth, and anchorage-independent growth. RESULTS: GRO-α was identified to be increased in conditioned media of OSF-associated fibroblasts. GRO-α promotes DOK cells proliferation, migration, and anchorage-independent growth through enhancing the EGFR/ERK signaling pathway, F-actin rearrangement, and stemness properties, respectively. Moreover, GRO-α neutralizing antibodies downregulated the conditioned medium-induced cell proliferation and migration of DOK. CONCLUSION: GRO-α from OSF-associated fibroblasts paracrinally promotes oral malignant transformation and significantly contributes to OSCC development.

Phosphoproteomics Reveals HMGA1, a CK2 Substrate, as a Drug-Resistant Target in Non-Small Cell Lung Cancer.

Although EGFR tyrosine kinase inhibitors (TKIs) have demonstrated good efficacy in non-small-cell lung cancer (NSCLC) patients harboring EGFR mutations, most patients develop intrinsic and acquired resistance. We quantitatively profiled the phosphoproteome and proteome of drug-sensitive and drug-resistant NSCLC cells under gefitinib treatment. The construction of a dose-dependent responsive kinase-substrate network of 1548 phosphoproteins and 3834 proteins revealed CK2-centric modules as the dominant core network for the potential gefitinib resistance-associated proteins. CK2 knockdown decreased cell survival in gefitinib-resistant NSCLCs. Using motif analysis to identify the CK2 core sub-network, we verified that elevated phosphorylation level of a CK2 substrate, HMGA1 was a critical node contributing to EGFR-TKI resistance in NSCLC cell. Both HMGA1 knockdown or mutation of the CK2 phosphorylation site, S102, of HMGA1 reinforced the efficacy of gefitinib in resistant NSCLC cells through reactivation of the downstream signaling of EGFR. Our results delineate the TKI resistance-associated kinase-substrate network, suggesting a potential therapeutic strategy for overcoming TKI-induced resistance in NSCLC.

Daxx inhibits hypoxia-induced lung cancer cell metastasis by suppressing the HIF-1α/HDAC1/Slug axis.

Hypoxia is a major driving force of cancer invasion and metastasis. Here we show that death domain-associated protein (Daxx) acts to negatively regulate hypoxia-induced cell dissemination and invasion by inhibiting the HIF-1α/HDAC1/Slug pathway. Daxx directly binds to the DNA-binding domain of Slug, impeding histone deacetylase 1 (HDAC1) recruitment and antagonizing Slug E-box binding. This, in turn, stimulates E-cadherin and occludin expression and suppresses Slug-mediated epithelial-mesenchymal transition (EMT) and cell invasiveness. Under hypoxic conditions, stabilized hypoxia-inducible factor (HIF)-1α downregulates Daxx expression and promotes cancer invasion, whereas re-expression of Daxx represses hypoxia-induced cancer invasion. Daxx also suppresses Slug-mediated lung cancer metastasis in an orthotopic lung metastasis mouse model. Using clinical tumour samples, we confirmed that the HIF-1α/Daxx/Slug pathway is an outcome predictor. Our results support that Daxx can act as a repressor in controlling HIF-1α/HDAC1/Slug-mediated cancer cell invasion and is a potential therapeutic target for inhibition of cancer metastasis.

Purine-Type Compounds Induce Microtubule Fragmentation and Lung Cancer Cell Death through Interaction with Katanin.

Microtubule targeting agents (MTAs) constitute a class of drugs for cancer treatment. Despite many MTAs have been proven to significantly improve the treatment outcomes of various malignancies, resistance has usually occurred. By selection from a two million entry chemical library based on the efficacy and safety, we identified purine-type compounds that were active against lung small cell lung cancer (NSCLC). The purine compound 5a (GRC0321) was an MTA with good effects against NSCLC. Lung cancer cells H1975 treated with 5a could induce microtubule fragmentation, leading to G2/M cell cycle arrest and intrinsic apoptosis. Compound 5a directly targeted katanin and regulated the severing activity of katanin, which cut the cellular microtubules into short pieces and activated c-Jun N-terminal kinases (JNK). The microtubule fragmenting effect of 5a is a unique mechanism in MTAs. It might overcome the resistance problems that most of the MTAs have faced.