Triple knockdown of CDC37, HSP90-alpha and HSP90-beta diminishes extracellular vesicles-driven malignancy events and macrophage M2 polarization in oral cancer.

Evidence has been accumulating to indicate that extracellular vesicles (EVs), including exosomes, released by cancer cells can foster tumour progression. The molecular chaperones - CDC37, HSP90α and HSP90ß play key roles in cancer progression including epithelial-mesenchymal transition (EMT), although their contribution to EVs-mediated cell-cell communication in tumour microenvironment has not been thoroughly examined. Here we show that triple depletion of the chaperone trio attenuates numerous cancer malignancy events exerted through EV release. Metastatic oral cancer-derived EVs (MEV) were enriched with HSP90α HSP90ß and cancer-initiating cell marker CD326/EpCAM. Depletion of these chaperones individually induced compensatory increases in the other chaperones, whereas triple siRNA targeting of these molecules markedly diminished the levels of the chaperone trio and attenuated EMT. MEV were potent agents in initiating EMT in normal epithelial cells, a process that was attenuated by the triple chaperone depletion. The migration, invasion, and in vitro tumour initiation of oral cancer cells were significantly promoted by MEV, while triple depletion of CDC37/HSP90α/ß reversed these MEV-driven malignancy events. In metastatic oral cancer patient-derived tumours, HSP90ß was significantly accumulated in infiltrating tumour-associated macrophages (TAM) as compared to lower grade oral cancer cases. HSP90-enriched MEV-induced TAM polarization to an M2 phenotype, a transition known to support cancer progression, whereas the triple chaperone depletion attenuated this effect. Mechanistically, the triple chaperone depletion in metastatic oral cancer cells effectively reduced MEV transmission into macrophages. Hence, siRNA-mediated knockdown of the chaperone trio (CDC37/HSP90α/HSP90ß) could potentially be a novel therapeutic strategy to attenuate several EV-driven malignancy events in the tumour microenvironment. ABBREVIATIONS: CDC37: cell division control 37; EMT: epithelial-mesenchymal transmission; EV: extracellular vesicles; HNSCC: head and neck squamous cell carcinoma; HSP90: heat shock protein 90; TAM: tumour-associated macrophage.

Extracellular Vesicles Enriched with Moonlighting Metalloproteinase Are Highly Transmissive, Pro-Tumorigenic, and Trans-Activates Cellular Communication Network Factor (CCN2/CTGF): CRISPR against Cancer.

Matrix metalloproteinase 3 (MMP3) plays multiple roles in extracellular proteolysis as well as intracellular transcription, prompting a new definition of moonlighting metalloproteinase (MMP), according to a definition of protein moonlighting (or gene sharing), a phenomenon by which a protein can perform more than one function. Indeed, connective tissue growth factor (CTGF, aka cellular communication network factor 2 (CCN2)) is transcriptionally induced as well as cleaved by MMP3. Moreover, several members of the MMP family have been found within tumor-derived extracellular vesicles (EVs). We here investigated the roles of MMP3-rich EVs in tumor progression, molecular transmission, and gene regulation. EVs derived from a rapidly metastatic cancer cell line (LuM1) were enriched in MMP3 and a C-terminal half fragment of CCN2/CTGF. MMP3-rich, LuM1-derived EVs were disseminated to multiple organs through body fluid and were pro-tumorigenic in an allograft mouse model, which prompted us to define LuM1-EVs as oncosomes in the present study. Oncosome-derived MMP3 was transferred into recipient cell nuclei and thereby trans-activated the CCN2/CTGF promoter, and induced CCN2/CTGF production in vitro. TRENDIC and other cis-elements in the CCN2/CTGF promoter were essential for the oncosomal responsivity. The CRISPR/Cas9-mediated knockout of MMP3 showed significant anti-tumor effects such as the inhibition of migration and invasion of tumor cells, and a reduction in CCN2/CTGF promoter activity and fragmentations in vitro. A high expression level of MMP3 or CCN2/CTGF mRNA was prognostic and unfavorable in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers. These data newly demonstrate that oncogenic EVs-derived MMP is a transmissive trans-activator for the cellular communication network gene and promotes tumorigenesis at distant sites.

Antiparkinson Drug Benztropine Suppresses Tumor Growth, Circulating Tumor Cells, and Metastasis by Acting on SLC6A3/DAT and Reducing STAT3.

Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. The properties of three-dimensional (3D) tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a 3D tumoroid-based screening system. We screened six pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with a matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-κB, and ß-catenin, and the properties of cancer stem cells/cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited the tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors.

A Reporter System Evaluates Tumorigenesis, Metastasis, ß-catenin/MMP Regulation, and Druggability.

Cancer invasion, metastasis, and therapy resistance are the crucial phenomena in cancer malignancy. The high expression of matrix metalloproteinase 9 (MMP9) is a biomarker as well as a causal factor of cancer invasiveness and metastatic activity. However, a regulatory mechanism underlying MMP9 expression in cancer is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming an important clue. In the present study, we aimed (i) to develop a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional tumoroid model and Mmp9 promoter and (ii) to examine pharmacological actions of anticancer medications using this reporter system. High expression and genetic amplification of MMP9 were found in colon cancer cases. We found that proximal promoter sequences of MMP9 in murine and human contained conserved binding sites for transcription factors ß-catenin/TCF/LEF, glucocorticoid receptor (GR), and nuclear factor kappa-B (NF-κB). The murine Mmp9 promoter (-569 to +19) was markedly activated in metastatic colon cancer cells and additionally activated by tumoroid formation and by ß-catenin signaling stimulator lithium chloride. The Mmp9 promoter-driven fluorescent reporter cells enabled the monitoring of activities of MMP9/gelatinase, tumorigenesis, invasion, and metastasis in syngeneic transplantation experiments. We also demonstrated pharmacological actions as follows: dexamethasone and hydrocortisone, steroidal medications binding to GR, inhibited the Mmp9 promoter but did not inhibit tumorigenesis. On the contrary, antimetabolite 5-fluorouracil, a gold standard for colon cancer chemotherapy, inhibited tumoroid formation but did not inhibit Mmp9 promoter activity. Notably, antimalaria medication artesunate inhibited both tumorigenesis and the Mmp9 promoter in vitro, potentially through inhibition of ß-catenin/TCF/LEF signaling. Thus, this novel reporter system enabled monitoring tumorigenesis, invasiveness, metastasis, key regulatory signalings such as ß-catenin/MMP9 axis, and druggability. Impact Statement Cancer invasion and metastasis have been shown to be driven by matrix metalloproteinase 9 (MMP9), whose expression mechanism is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming important. We established a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional (3D) tumoroid model and Mmp9 promoter. Using this reporter system, we demonstrated pharmacological actions of anticancer medications such as antimetabolite 5-fluorouracil (5-FU) and antimalaria medication artesunate (ART), which inhibited both tumorigenesis and ß-catenin/MMP regulatory signaling. Our study impacts the translational fields of oncology, drug discovery, and organoid model.

Carcinogenic epithelial-mesenchymal transition initiated by oral cancer exosomes is inhibited by anti-EGFR antibody cetuximab.

Overexpression and increased signaling from the epidermal growth factor receptor (EGFR) often changes oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed the roles of OSCC-derived extracellular vesicles (EVs), including exosomes in the trafficking of cetuximab and in epithelial-mesenchymal transition (EMT) of epithelial cells. OSCC cells abundantly expressed EGFR, which was secreted from cells with OSCC-EVs upon EGF stimulations. The OSCC-EGFR-EVs were then able to enter into and transform epithelial cells leading to increased mesenchymal traits with increased vimentin and spindle-like shapes. EGF priming of OSCC cells further increased this EMT-initiating effect of the OSCC-EVs. The internalization and pro-EMT effects of the OSCC-EVs were largely blocked by cetuximab. Thus, OSCC-derived EVs transform normal epithelial cells into a mesenchymal phenotype and anti-EGFR therapeutic antibody cetuximab inhibits such a carcinogenic effect of the OSCC-EVs.

Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells.

The ATP-binding cassette transporter G1 (ABCG1) is a cholesterol lipid efflux pump whose role in tumor growth has been largely unknown. Our transcriptomics revealed that ABCG1 was powerfully expressed in rapidly metastatic, aggregative colon cancer cells, in all the ABC transporter family members. Coincidently, genetic amplification of ABCG1 is found in 10-35% of clinical samples of metastatic cancer cases. Expression of ABCG1 was further elevated in three-dimensional tumoroids (tumor organoids) within stemness-enhancing tumor milieu, whereas depletion of ABCG1 lowered cellular aggregation and tumoroid growth in vitro as well as hypoxia-inducible factor 1α in cancer cells around the central necrotic areas in tumors in vivo. Notably, depletion of ABCG1 triggered the intracellular accumulation of extracellular vesicles (EVs) and regression of tumoroids. Collectively, these data suggest that ABCG1 plays a crucial role in tumorigenesis in metastatic cancer and that depletion of ABCG1 triggers tumor regression with the accumulation of EVs and their derivatives and cargos, implicating a novel ABCG1-targeting therapeutic strategy by which redundant and toxic substances may be accumulated in tumors leading to their regression.

Anti-EGFR antibody cetuximab is secreted by oral squamous cell carcinoma and alters EGF-driven mesenchymal transition.

Genetic amplification, overexpression, and increased signaling from the epidermal growth factor receptor (EGFR) are often found in oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed effects of cetuximab in control of EGF-driven malignant traits of OSCC cells. EGF stimulation promoted progression level of mesenchymal traits in OSCC cells, which were attenuated by cetuximab but incompletely. We pursued a potential mechanism underlying such incomplete attenuation of OSCC malignant traits. Cetuximab promoted secretion of EGFR-EVs by OSCC cells and failed to inhibit EGF-driven secretion of EGFR-EVs. Cetuximab was also found to be robustly secreted with the EGFR-EVs by the OSCC cells. Thus, EGF promotes the level of mesenchymal traits of OSCC cells and secretion of EGFR-EVs, which involve cetuximab resistance.

The intranuclear PEX domain of MMP involves proliferation, migration, and metastasis of aggressive adenocarcinoma cells.

Members of matrix metalloproteinase (MMP) family promote cancer cell migration, invasion, and metastasis through alteration of the tumor milieu, intracellular signaling pathways, and transcription. We examined gene expression signatures of colon adenocarcinoma cell lines with different metastatic potentials and found that rapidly metastatic cells powerfully expressed genes encoding MMP3 and MMP9. The non-proteolytic PEX isoform and proteolytic isoforms of MMPs were significantly expressed in the metastatic cells in vitro. Knockdown of MMP3 attenuated cancer cell migration and invasion in vitro and lung metastasis in vivo. Profound nuclear localization of MMP3/PEX was found in tumor-stroma marginal area. In contrast, MMP9 was localized in central area of subcutaneous tumors. Overexpression of the PEX isoform of MMP3 promoted proliferation and migration of the rapidly metastatic cells in vitro. Taken together, the non-proteolytic PEX isoform of MMPs locating in cell nuclei involves proliferation, migration, and subsequent metastasis of aggressive adenocarcinoma cells.

HSP-enriched properties of extracellular vesicles involve survival of metastatic oral cancer cells.

Cancer cells often secrete extracellular vesicles (EVs) that carry heat shock proteins (HSPs) with roles in tumor progression. Oral squamous cell carcinoma (OSCC) belongs to head and neck cancers (HNC) whose lymph-node-metastases often lead to poor prognosis. We have examined the EV proteome of OSCC cells and found abundant secretion of HSP90-enriched EVs in lymph-node-metastatic OSCC cells. Double knockdown of HSP90α and HSP90ß, using small interfering RNA significantly reduced the survival of the metastatic OSCC cells, although single knockdown of each HSP90 was ineffective. Elevated expression of these HSP90 family members was found to correlate with poor prognosis of HNC cases. Thus, elevated HSP90 levels in secreted vesicles are potential prognostic biomarkers and therapeutic targets in metastatic OSCC.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment.

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

Genome-wide screening of DNA methylation associated with lymph node metastasis in esophageal squamous cell carcinoma.

Lymph node metastasis (LNM) of esophageal squamous cell carcinoma (ESCC) is well-known to be an early event associated with poor prognosis in patients with ESCC. Recently, tumor-specific aberrant DNA methylation of CpG islands around the promoter regions of tumor-related genes has been investigated as a possible biomarker for use in early diagnosis and prediction of prognosis. However, there are few DNA methylation markers able to predict the presence of LNM in ESCC. To identify DNA methylation markers associated with LNM of ESCC, we performed a genome-wide screening of DNA methylation status in a discovery cohort of 67 primary ESCC tissues and their paired normal esophageal tissues using the Illumina Infinium HumanMethylation450 BeadChip. In this screening, we focused on differentially methylated regions (DMRs) that were associated with LNM of ESCC, as prime candidates for DNA methylation markers. We extracted three genes, HOXB2, SLC15A3, and SEPT9, as candidates predicting LNM of ESCC, using pyrosequencing and several statistical analyses in the discovery cohort. We confirmed that HOXB2 and SEPT9 were highly methylated in LNM-positive tumors in 59 ESCC validation samples. These results suggested that HOXB2 and SEPT9 may be useful epigenetic biomarkers for the prediction of the presence of LNM in ESCC.

Intracellular MMP3 Promotes HSP Gene Expression in Collaboration With Chromobox Proteins.

Matrix metalloproteinases (MMPs) are crucial factors in tumor progression, inflammatory/immune responses and tissue development/regeneration. Of note, it has been known that MMPs promote genome instability, epithelial-mesenchymal transition, invasion, and metastasis in tumor progression. We previously reported that human MMP3 could translocate into cellular nuclei and control transcription in human chondrosarcoma-derived cells and in articular cartilage (Eguchi et al. [2008] Mol Cell Biol 28(7):2391-2413); however, further transcriptional target genes and cofactors of intranuclear MMP3 have not been uncovered. In this paper, we used transcriptomics analysis in order to examine novel transcriptional target genes regulated by intracellular MMP3. We found that mRNA levels of HSP family members (HSP70B', HSP72, HSP40/DNAJ, and HSP20/CRYAB) are upregulated by the intracellular MMP3 overload. Bioinformatic analysis predicted several transcription factors that possibly interact with MMP3. Among these factors, heat shock factor 1 (HSF1) cooperated with the MMP3 to activate the HSP70B' gene promoter in reporter gene assays, while a dominant negative HSF1 blocked the role for MMP3 in the trans-activation. The hemopexin-like repeat (PEX) domain of the human MMP3 was essential for transcriptional induction of the HSP70B' gene. In addition, chromobox proteins CBX5/HP1α and CBX3/HP1γ cooperated with the PEX domain in induction of HSP70B' mRNA. Taken together, this study newly clarified that intracellular MMP3 cooperate with CBXs/HP1s in transcriptional promotion of HSP genes. J. Cell. Biochem. 118: 43-51, 2017. © 2016 Wiley Periodicals, Inc.

A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516.

Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-ß promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-ßreceptor I (TGFßR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-ß-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFßR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. The half maximal inhibitory concentration (IC50) of SB-525334 and SU9516 were 0.31 µM and 1.21 µM, respectively, while IC50 of SB431542 was 2.38 µM. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.

Structure of a New Palatal Plate and the Artificial Tongue for Articulation Disorder in a Patient with Subtotal Glossectomy.

A palatal augmentation prosthesis (PAP) is used to facilitate improvement in the speech and swallowing functions of patients with tongue resection or tongue movement disorders. However, a PAP's effect is limited in cases where articulation disorder is severe due to wide glossectomy and/or segmental mandibulectomy. In this paper, we describe speech outcomes of a patient with an articulation disorder following glossectomy and segmental mandibulectomy. We used a palatal plate (PP) based on a PAP, along with an artificial tongue (KAT). Speech improvement was evaluated by a standardized speech intelligibility test consisting of 100 syllables. The speech intelligibility score was significantly higher when the patient wore both the PP and KAT than when he wore neither (p＝0.013). The conversational intelligibility score was significantly improved with the PP and KAT than without PP and KAT (p＝0.024). These results suggest that speech function can be improved in patients with hard tissue defects with segmental mandibulectomy using both a PP and a KAT. The nature of the design of the PP and that of the KAT will allow these prostheses to address a wide range of tissue defects.

miR-544a induces epithelial-mesenchymal transition through the activation of WNT signaling pathway in gastric cancer.

The epithelial-mesenchymal transition (EMT) contributes to cancer progression, as well as the development of normal organs, wound healing and organ fibrosis. We established a cell-based reporter system for identifying EMT-inducing microRNAs (miRNAs) with a gastric cancer (GC) cell line, MKN1, transfected with a reporter construct containing a promoter sequence of VIM in the 5' upstream region of the TurboRFP reporter gene. Function-based screening using this reporter system was performed with a 328-miRNA library, and resulted in the identification miR-544a as an EMT-inducing miRNA. Although miR-544a is already known to be involved in the regulation of CDH1, the mechanism by which EMT occurs remains poorly understood. Herein, we demonstrated that overexpression of miR-544a induces VIM, SNAI1 and ZEB1 expression, and reduces CDH1 expression, resulting in an EMT phenotype. In addition, we found that CDH1 and AXIN2, which are related to the degradation and the translocation of ß-catenin, are direct targets of miR-544a. Subsequently, the reduction of CDH1 and AXIN2 by miR-544a induced the nuclear import of ß-catenin, suggesting that miR-544a may activate the WNT signaling pathway through the stabilization of ß-catenin in nucleus. Our findings raise the possibility that inhibition of miR-544a may be a therapeutic target of metastatic GC.

miR-634 Activates the Mitochondrial Apoptosis Pathway and Enhances Chemotherapy-Induced Cytotoxicity.

Some tumor-suppressing miRNAs target multiple oncogenes concurrently and therefore may be useful as cancer therapeutic agents. Further, such miRNAs may be useful to address chemotherapeutic resistance in cancer, which remains a primary clinical challenge in need of solutions. Thus, cytoprotective processes upregulated in cancer cells that are resistant to chemotherapy are a logical target for investigation. Here, we report that overexpression of miR-634 activates the mitochondrial apoptotic pathway by direct concurrent targeting of genes associated with mitochondrial homeostasis, antiapoptosis, antioxidant ability, and autophagy. In particular, we show how enforced expression of miR-634 enhanced chemotherapy-induced cytotoxicity in a model of esophageal squamous cell carcinoma, where resistance to chemotherapy remains clinically problematic. Our findings illustrate how reversing miR-634-mediated cytoprotective processes may offer a broadly useful approach to improving cancer therapy.

Expression and clinical significance of connective tissue growth factor in advanced head and neck squamous cell cancer.

Connective tissue growth factor (CTGF) has been reported to play critical roles in the tumorigenesis of several human malignancies. This study was performed to evaluate CTGF protein expression in head and neck squamous cell carcinoma (HNSCC). Surgical specimens from 76 primary HNSCC were obtained with written informed consents and the expression level of CTGF was immunohistochemically evaluated. The cytoplasmic immunoreactivity of CTGF in cancer cells was semiquantitatively classified into low and high expression. Among all 76 cases with or without neoadjuvant therapy, low CTGF showed significantly longer (P = 0.0282) overall survival (OS), but not disease-free survival (DFS) than high CTGF. Although low CTGF in patients with stage I, II and III did not result in any significant difference of the OS and DFS, stage IV HNSCC patients with low CTGF showed significantly longer OS (P = 0.032) and DFS (P = 0.0107) than those with high CTGF. These differences in stage IV cases were also confirmed using multivariate analyses. These results suggest that low CTGF in stage IV HNSCC is an independent prognostic factor, despite with or without neoadjuvant therapy.

The impact of miRNA-based molecular diagnostics and treatment of NRF2-stabilized tumors.

UNLABELLED: NF-E2-related factor 2 (NRF2) is a master transcriptional regulator that integrates cellular stress responses and is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1) at the posttranslational level. In human cancers, aberrantly stabilized NRF2, either by mutation of NRF2 or KEAP1, plays a vital role in chemoresistance and tumor cell growth through the transcriptional activation of target genes, suggesting that targeted inhibition of NRF2 is a potential therapy for NRF2-stabilized tumors. MicroRNAs (miRNA) are endogenous small noncoding RNAs that can negatively regulate gene expression by interfering with the translation or stability of target transcripts. Moreover, tumor-suppressor miRNAs have been suggested to be useful for cancer treatment. Here, a reporter-coupled miRNA library screen identified four miRNAs (miR-507, -634, -450a, and -129-5p) that negatively regulate the NRF2-mediated oncogenic pathway by directly targeting NRF2. Importantly, downregulation of these miRNAs, in addition to the somatic mutation of NRF2 or KEAP1, is associated with stabilized NRF2 and poor prognosis in esophageal squamous cell carcinoma (ESCC). Furthermore, administration of a miR-507 alone or in combination with cisplatin inhibited tumor growth in vivo. Thus, these findings reveal that miRNA-based therapy is effective against NRF2-stabilized ESCC tumors. IMPLICATIONS: This study determines the potential of miRNA-based molecular diagnostics and therapeutics in NRF2-stablized tumors.

miR-655 Is an EMT-suppressive microRNA targeting ZEB1 and TGFBR2.

Recently, the epithelial-to-mesenchymal transition (EMT) has been demonstrated to contribute to normal and disease processes including cancer progression. To explore EMT-suppressive microRNAs (miRNAs), we established a cell-based reporter system using a stable clone derived from a pancreatic cancer cell line, Panc1, transfected with a reporter construct containing a promoter sequence of CDH1/E-cadherin in the 5' upstream region of the ZsGreen1 reporter gene. Then, we performed function-based screening with 470 synthetic double-stranded RNAs (dsRNAs) mimicking human mature miRNAs using the system and identified miR-655 as a novel EMT-suppressive miRNA. Overexpression of miR-655 not only induced the upregulation of E-cadherin and downregulation of typical EMT-inducers but also suppressed migration and invasion of mesenchymal-like cancer cells accompanied by a morphological shift toward the epithelial phenotype. In addition, we found a significant correlation between miR-655 expression and a better prognosis in esophageal squamous cell carcinoma (ESCC). Moreover, ZEB1 and TGFBR2, which are essential components of the TGF-b signaling pathway, were identified as direct targets of miR-655, suggesting that the activation of the TGF-b-ZEB1-E-cadherin axis by aberrant downregulation of miR-655 may accelerate cancer progression.

The tumor-suppressive miR-497-195 cluster targets multiple cell-cycle regulators in hepatocellular carcinoma.

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression and commonly deregulated in carcinogenesis. To explore functionally crucial tumor-suppressive (TS)-miRNAs in hepatocellular carcinoma (HCC), we performed integrative function- and expression-based screenings of TS-miRNAs in six HCC cell lines. The screenings identified seven miRNAs, which showed growth-suppressive activities through the overexpression of each miRNA and were endogenously downregulated in HCC cell lines. Further expression analyses using a large panel of HCC cell lines and primary tumors demonstrated four miRNAs, miR-101, -195, -378 and -497, as candidate TS-miRNAs frequently silenced in HCCs. Among them, two clustered miRNAs miR-195 and miR-497 showed significant growth-suppressive activity with induction of G1 arrest. Comprehensive exploration of their targets using Argonute2-immunoprecipitation-deep-sequencing (Ago2-IP-seq) and genome-wide expression profiling after their overexpression followed by pathway analysis, revealed a significant enrichment of cell cycle regulators. Among the candidates, we successfully identified CCNE1, CDC25A, CCND3, CDK4, and BTRC as direct targets for miR-497 and miR-195. Moreover, target genes frequently upregulated in HCC in a tumor-specific manner, such as CDK6, CCNE1, CDC25A and CDK4, showed an inverse correlation in the expression of miR-195 and miR-497, and their targets. These results suggest the molecular pathway regulating cell cycle progression to be integrally altered by downregulation of miR-195 and miR-497 expression, leading to the aberrant cell proliferation in hepatocarcinogenesis.