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Background: Our recent analyses of miRNA expression signatures showed that miR-1 and miR-133a were significantly reduced in several types of cancer. Interestingly, miR-1 and miR-133a are located on the same chromosomal locus in the human genome. We examined the functional significance of miR-1 and miR-133a in prostate cancer (PCa) cells and identified the novel molecular targets regulated by both miR-1 and miR-133a.Methods and Results: The expression levels of miR-1 and miR-133a were significantly downregulated in PCa compared with non-PCa tissues. Restoration of miR-1 or miR-133a in PC3 and DU145 cells revealed significant inhibition of proliferation, migration, and invasion. Molecular target identification by genome-wide gene expression analysis and luciferase reporter assay showed that purine nucleoside phosphorylase (PNP) was directly regulated by both miRNAs. Silencing of the PNP gene inhibited proliferation, migration, and invasion in both PC3 and DU145 cells. Immunohistochemistry detected positive staining of PNP in PCa specimens.Conclusions: Downregulation of miR-1 and miR-133a was a frequent event in PCa and both function as tumour suppressors. The PNP is a novel target gene of both miRNAs and potentially functions as an oncogene. Therefore, identification of novel molecular networks regulated by miRNAs may provide new insights into the underlying causes of PCa oncogenesis. [ABSTRACT FROM AUTHOR]

Background: Previously reported results from phase III KEYNOTE-048 demonstrated similar or improved overall survival (OS) with pembrolizumab or pembrolizumab-chemotherapy versus cetuximab-chemotherapy (EXTREME) in Japanese patients with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). We report results in Japanese patients from KEYNOTE-048 after 5 years of follow-up., Methods: Patients with R/M HNSCC of the oropharynx, oral cavity, hypopharynx, or larynx were randomly assigned 1:1:1 to pembrolizumab, pembrolizumab-chemotherapy, or EXTREME. Primary endpoints were OS and progression-free survival. Efficacy was evaluated in the programmed cell death ligand 1 (PD-L1) combined positive score (CPS) ≥ 20, PD-L1 CPS ≥ 1, and total Japanese populations., Results: In Japan, 67 patients were enrolled (pembrolizumab, n = 23; pembrolizumab-chemotherapy, n = 25; EXTREME, n = 19). Median follow-up was 71.0 months (range, 61.2-81.5); data cutoff, February 21, 2022. 5-year OS rates with pembrolizumab versus EXTREME were 35.7% versus 12.5% (hazard ratio [HR] 0.38; 95% CI 0.13-1.05), 23.8% versus 12.5% (HR 0.70; 95% CI 0.34-1.45), and 30.4% versus 10.5% (HR 0.54; 95% CI 0.27-1.07) in the PD-L1 CPS ≥ 20, CPS ≥ 1, and total Japanese populations, respectively. 5-year OS rates with pembrolizumab-chemotherapy versus EXTREME were 20.0% versus 14.3% (HR 0.79; 95% CI 0.27-2.33), 10.5% versus 14.3% (HR 1.18; 95% CI 0.56-2.48), and 8.0% versus 12.5% (HR 1.11; 95% CI 0.57-2.16) in the PD-L1 CPS ≥ 20, CPS ≥ 1, and total Japanese populations, respectively., Conclusion: After 5 years of follow-up, pembrolizumab and pembrolizumab-chemotherapy showed long-term clinical benefits; results further support these treatments as first-line options for Japanese patients with R/M HNSCC., Clinical Trial Registration: NCT02358031., (© 2024. The Author(s).)

Traditional therapeutic approaches for malignant melanoma, have proved to be limited and/or ineffective, especially with respect to their role in improving patient survival and tumor recurrence. In this regard, immunotherapy has been demonstrated to be a promising therapeutic alternative, boosting antitumor responses through the modulation of cell signaling pathways involved in the effector mechanisms of the immune system, particularly, the so-called "immunological checkpoints". Clinical studies on the efficacy and safety of immunotherapeutic regimens, alone or in combination with other antitumor approaches, have increased dramatically in recent decades, with very encouraging results. Hence, this review will discuss the current immunotherapeutic regimens used to treat malignant melanoma, as well as the molecular and cellular mechanisms involved. In addition, current clinical studies that have investigated the use, efficacy, and adverse events of immunotherapy in melanoma will also be discussed., Competing Interests: Declaration of Competing Interest The authors certify that they have NO conflict of interest, affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. Nijiro Nohata is an employee of MSD K.K., a subsidiary of Merck & Co., Inc. and reports personal fees from MSD K.K. outside this study., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Here, we report the results of the Japanese subgroup of the phase 3 KEYNOTE-048 study of pembrolizumab alone, pembrolizumab plus platinum and 5-fluorouracil (pembrolizumab-chemotherapy), or cetuximab plus platinum and 5-fluorouracil (EXTREME) in previously untreated recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC)., Methods: Primary end points were overall survival (OS) and progression-free survival (PFS). Efficacy was evaluated in patients with PD-L1 combined positive score (CPS) ≥ 20 and ≥ 1 and the total Japanese subgroup (n = 67)., Results: At data cutoff (25 February 2019), pembrolizumab led to longer OS versus EXTREME in the PD-L1 CPS ≥ 20 subgroup (median, 28.2 vs. 13.3 months; HR, 0.29 [95% CI 0.09-0.89]) and to similar OS in the total Japanese (23.4 vs. 13.6 months; HR, 0.51 [95% CI 0.25-1.05]) and CPS ≥ 1 subgroups (22.6 vs. 15.8 months; HR, 0.66 [95% CI 0.31-1.41]). Pembrolizumab-chemotherapy led to similar OS versus EXTREME in the PD-L1 CPS ≥ 20 (median, 18.1 vs. 15.8 months; HR, 0.72 [95% CI 0.23-2.19]), CPS ≥ 1 (12.6 vs. 15.8 months; HR, 1.19 [95% CI 0.55-2.58]), and total Japanese subgroups (12.6 vs. 13.3 months; unadjusted HR, 1.10 [95% CI 0.55-2.22]). Median PFS was similar for pembrolizumab and pembrolizumab-chemotherapy versus EXTREME in all subgroups. Grades 3-5 treatment-related adverse events occurred in 5 (22%), 19 (76%), and 17 (89%) patients receiving pembrolizumab, pembrolizumab-chemotherapy, and EXTREME, respectively. One patient receiving pembrolizumab-chemotherapy died because of treatment-related pneumonitis., Conclusion: These results support the use of first-line pembrolizumab and pembrolizumab-chemotherapy for Japanese patients with R/M HNSCC. Clinical trial registry ClinicalTrials.gov, NCT02358031., (© 2022. Merck & Co., Inc., Rahway, NJ, USA and its affiliates.)

The microRNA (miR) miR-874, a potential tumour suppressor, causes cell death via target gene suppression in various cancer types. Mevalonate pathway inhibition also causes cell death in breast cancer. However, the relationship between the mevalonate pathway and miR-874-induced apoptosis or its association with the tumour suppressor p53 has not been elucidated. We identified phosphomevalonate kinase (PMVK), a key mevalonate pathway enzyme, and sterol regulatory element-binding factor 2 (SREBF2), the master cholesterol biosynthesis regulator, as direct miR‑874 targets. Next-generation sequencing analysis revealed a significant miR-874-mediated downregulation of PMVK and SREBF2 gene expression and p53 pathway enrichment. Luciferase reporter assays showed that miR-874 directly regulated PMVK and SREBF2. miR-874-induced apoptosis was p53 dependent, and single-cell RNA sequencing analysis demonstrated that miR-874 transfection resulted in apoptosis and p53 pathway activation. Downregulation of PMVK expression also caused cell cycle arrest and p53 pathway activation, which was rescued by geranylgeranyl pyrophosphate (GGPP) supplementation. Analysis of The Cancer Genome Atlas (TCGA) database indicated a negative correlation between miR-874 and PMVK expression and between miR-874 and SREBF2 expression. These findings suggest that miR-874 suppresses the mevalonate pathway by targeting SREBF2 and PMVK, resulting in GGPP depletion, which activates the p53 pathway and promotes cycle arrest or apoptosis., (© 2022. The Author(s).)

Advanced-stage oral squamous cell carcinoma (OSCC) patients are treated with combination therapies, such as surgery, radiation, chemotherapy, and immunotherapy. However, OSCC cells acquire resistance to these treatments, resulting in local recurrence and distant metastasis. The identification of genes involved in drug resistance is essential for improving the treatment of this disease. In this study, we applied chromatin immunoprecipitation sequencing (ChIP-Seq) to profile active enhancers. For that purpose, we used OSCC cell lines that had been exposed to cetuximab for a prolonged period. In total, 64 chromosomal loci were identified as active super-enhancers (SE) according to active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) ChIP-Seq. In addition, a total of 131 genes were located in SE regions, and 34 genes were upregulated in OSCC tissues by TCGA-OSCC analysis. Moreover, high expression of four genes ( C9orf89 ; p = 0.035, CENPA ; p = 0.020, PISD ; p = 0.0051, and TRAF2 ; p = 0.0075) closely predicted a poorer prognosis for OSCC patients according to log-rank tests. Increased expression of the four genes (mRNA Z-score ≥ 0) frequently co-occurred in TCGA-OSCC analyses. The high and low expression groups of the four genes showed significant differences in prognosis, suggesting that there are clear differences in the pathways based on the underlying gene expression profiles. These data indicate that potential stratified therapeutic strategies could be used to overcome resistance to drugs (including cetuximab) and further improve responses in drug-sensitive patients.

Background: Understanding the intricate signaling network involved in triple-negative breast cancer (TNBC) represents a challenge for developing novel therapeutic approaches. Here, we aim to provide novel mechanistic insights on the function of the S100A8/A9-RAGE system in TNBC., Methods: TNM plot analyzer, Kaplan-Meier plotter, Meta-analysis, GEPIA2 and GOBO publicly available datasets were used to evaluate the clinical significance of S100A8/A9 and expression levels of S100A8/A9, RAGE and Filamin family members in breast cancer (BC) subtypes. METABRIC database and Cox proportional hazard model defined the clinical impact of high RAGE expression in BC patients. Multiple bioinformatics programs identified the main enriched pathways within high RAGE expression BC cohorts. By lentiviral system, TNBC cells were engineered to overexpress RAGE. Western blotting, immunofluorescence, nucleus/cytoplasm fractionation, qRT-PCR, gene silencing and luciferase experiments were performed to identify signal transduction mediators engaged by RAGE upon stimulation with S100A8/A9 in TNBC cells. Proliferation, colony formation and transwell migration assays were carried out to evaluate the growth and migratory capacity of TNBC cells. Statistical analysis was performed by ANOVA and independent t-tests., Results: We found a remarkable high expression of S100A8 and S100A9 in BC, particularly in HER2-positive and TNBC, with the latter associated to worst clinical outcomes. In addition, high RAGE expression correlated with a poor overall survival in BC. Next, we determined that the S100A8/A9-RAGE system triggers FAK activation by engaging a cytoskeleton mechanosensing complex in TNBC cells. Through bioinformatics analysis, we identified the Hippo pathway as the most enriched in BC patients expressing high RAGE levels. In accordance with these data, we demonstrated the involvement of S100A8/A9-RAGE-FAK signaling in the control of Hippo/YAP activities, and we established the crucial contribution of RAGE-FAK-YAP circuitry in the growth and migratory effects initiated by S100A8/A9 in TNBC cells., Conclusions: The present study provides novel mechanistic insights on RAGE actions in TNBC. Moreover, our findings suggest that RAGE-FAK-YAP transduction pathway could be exploited as a druggable system halting the aggressive TNBC subtype., (© 2022. The Author(s).)

Based on our original RNA sequence-based microRNA (miRNA) signatures of head and neck squamous cell carcinoma (HNSCC), it was revealed that the expression levels of miR-1-3p, miR-206, miR-133a-3p, and miR-133b were significantly suppressed in cancer specimens. Seed sequences of miR-1-3p/miR-206 and miR-133a-3p/miR-133b are identical. Interestingly, miR-1-3p/miR-133a-3p and miR-206/miR-133b are clustered in the human genome. We hypothesized that the genes coordinately controlled by these miRNAs are closely involved in the malignant transformation of HNSCC. Our in silico analysis identified a total of 28 genes that had putative miR-1-3p/miR-133a-3p and miR-206/miR-133b binding sites. Moreover, their expression levels were upregulated in HNSCC tissues. Multivariate Cox regression analyses showed that expression of PFN2 and PSEN1 were independent prognostic factors for patients with HNSCC (p < 0.05). Notably, four miRNAs (i.e., miR-1-3p, miR-206, miR-133a-3p, and miR-133b) directly bound the 3′untranslated region of PFN2 and controlled expression of the gene in HNSCC cells. Overexpression of PFN2 was confirmed in clinical specimens, and its aberrant expression facilitated cancer cell migration and invasion abilities. Our miRNA-based strategy continues to uncover novel genes closely involved in the oncogenesis of HNSCC.

Our recent research has revealed that passenger strands of certain microRNAs (miRNAs) function as tumor-suppressive miRNAs in cancer cells, e.g., miR-101-5p, miR-143-5p, miR-144-5p, miR-145-3p, and miR-150-3p. Thus, they are important in cancer pathogenesis. Analysis of the miRNA expression signature of breast cancer (BrCa) showed that the expression levels of two miRNAs derived from pre-miR-99a (miR-99a-5p and miR-99a-3p) were suppressed in cancerous tissues. The aim of this study was to identify oncogenic genes controlled by pre-miR-99a that are closely involved in the molecular pathogenesis of BrCa. A total of 113 genes were identified as targets of pre-miR-99a regulation (19 genes modulated by miR-99a-5p, and 95 genes regulated by miR-99a-3p) in BrCa cells. Notably, FAM64A was targeted by both of the miRNAs. Among these targets, high expression of 16 genes (C5orf22, YOD1, SLBP, F11R, C12orf49, SRPK1, ZNF250, ZNF695, CDK1, DNMT3B, TRIM25, MCM4, CDKN3, PRPS, FAM64A, and DESI2) significantly predicted reduced survival of BrCa patients based upon The Cancer Genome Atlas (TCGA) database. In this study, we focused on FAM64A and investigated the relationship between FAM64A expression and molecular pathogenesis of BrCa subtypes. The upregulation of FAM64A was confirmed in BrCa clinical specimens. Importantly, the expression of FAM64A significantly differed between patients with Luminal-A and Luminal-B subtypes. Our data strongly suggest that the aberrant expression of FAM64A is involved in the malignant transformation of BrCa. Our miRNA-based approaches (identification of tumor-suppressive miRNAs and their controlled targets) will provide novel information regarding the molecular pathogenesis of BrCa.

Small cell lung cancer (SCLC) is a highly aggressive cancer, and patients who become refractory to first-line treatment have a poor prognosis. The development of effective treatment regimens is urgently needed. In this study, we identified a gene expression signature of SCLC after treatment failure using SCLC clinical specimens (GEO accession number: GSE162102). A total of 1,136 genes were significantly upregulated in SCLC tissues. These upregulated genes were subjected to KEGG pathway analysis, and "cell cycle", "Fanconi anemia", "alcoholism", "systemic lupus erythematosus", "oocyte meiosis", "homologous recombination", "DNA replication", and "p53 signaling" were identified as the enriched pathways among the genes. We focused on the cell cycle pathway and investigated the clinical significance of four genes associated with this pathway: minichromosome maintenance ( MCM ) 2, MCM4 , MCM6 , and MCM7 . The overexpression of these MCM genes was confirmed in SCLC clinical specimens. Knockdown assays using siRNAs targeting each of these four MCM genes showed significant attenuation of cancer cell proliferation. Moreover, siRNA-mediated knockdown of each MCM gene enhanced the cisplatin sensitivity of SCLC cells. Our SCLC molecular signature based on SCLC clinical specimens after treatment failure will provide useful information to identify novel molecular targets for this disease.

Lung adenocarcinoma (LUAD) is the most aggressive cancer and the prognosis of these patients is unfavorable. We revealed that the expression levels of both strands of miR-99a ( miR-99a-5p and miR-99a-3p ) were significantly suppressed in several cancer tissues. Analyses of large The Cancer Genome Atlas (TCGA) datasets showed that reduced miR-99a-5p or miR-99a-3p expression is associated with worse prognoses in LUAD patients (disease-free survival (DFS): p = 0.1264 and 0.0316; overall survival (OS): p = 0.0176 and 0.0756, respectively). Ectopic expression of these miRNAs attenuated LUAD cell proliferation, suggesting their tumor-suppressive roles. Our in silico analysis revealed 23 putative target genes of pre- miR-99a in LUAD cells. Among these targets, high expressions of 19 genes were associated with worse prognoses in LUAD patients (OS: p < 0.05). Notably, FAM64A was regulated by both miR-99a-5p and miR-99a-3p in LUAD cells, and its aberrant expression was significantly associated with poor prognosis in LUAD patients (OS: p = 0.0175; DFS: p = 0.0276). FAM64A knockdown using siRNAs suggested that elevated FAM64A expression contributes to cancer progression. Aberrant FAM64A expression was detected in LUAD tissues by immunostaining. Taken together, our miRNA-based analysis might be effective for identifying prognostic and therapeutic molecules in LUAD.

d-3-Phosphoglycerate dehydrogenase (PHGDH) conducts an important step in the synthesis of serine. Importantly, the PHGDH gene is often amplified in certain cancers. Our previous studies revealed that PHGDH gene amplification was associated with poor overall survival in clear cell renal cell carcinoma (ccRCC) and that metabolic reprogramming of serine synthesis through PHGDH recruitment allowed ccRCC cells to survive in unfavorable environments. There have been no investigations of the role of PHGDH expression in bladder cancer (BC). In this investigation, we examined the clinical importance of PHDGH in BC. Furthermore, we asked whether PHGDH expression could be exploited for BC therapy. Finally, we investigated the regulatory mechanisms that modulated the expression of PHGDH. Using data from The Cancer Genome Atlas, we found that patients with high-grade BC had significantly higher PHGDH expression levels than did those with low-grade BC. In addition, patients with high PHGDH expression did not survive as long as those with low expression. PHGDH downregulation by si-RNAs or an inhibitor in BC cell lines significantly inhibited proliferative ability and induced apoptosis. Furthermore, combined treatment using a PHGDH inhibitor and gemcitabine/cisplatin achieved synergistic tumor suppression compared to use of a single agent both in vitro as well as in vivo. Mechanistic analyses of PHGDH regulation showed that PHGDH expression might be associated with DNA copy number and hypomethylation in BC. These findings suggest novel therapeutic strategies could be used in BC. Finally, our data enhance our understanding of the role of PHGDH in BC., (© 2020 Kagoshima University. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Triple-negative breast cancer (TNBC) is an aggressive breast tumor subtype that currently lacks targeted treatment options. The role played by the insulin-like growth factor-1 (IGF-1) and its cognate receptor IGF-1R in TNBC has been reported. Nevertheless, the molecular mechanisms by which the IGF-1/IGF-1R system may contribute to TNBC progression still remains to be fully understood. By computational analysis of the vast cancer genomics information in public databases (TCGA and METABRIC), we obtained evidence that high IGF-1 or IGF-1R levels correlate with a worse clinical outcome in TNBC patients. Further bioinformatics analysis revealed that both the focal adhesion and the Hippo pathways are enriched in TNBC harboring an elevated expression of IGF-1 or IGF-1R. Mechanistically, we found that in TNBC cells, the IGF-1/IGF-1R system promotes the activation of the FAK signal transduction pathway, which in turn regulates the nuclear accumulation of YAP (yes-associated protein/yes-related protein) and the expression of its target genes. At the biological level, we found that the IGF-1/IGF-1R-FAK-YAP network cascade triggers the growth potential of TNBC cells, as evaluated in different experimental systems. Overall, our results suggest that the IGF-1/IGF-1R/FAK/YAP axis may contribute to the progression of the aggressive TNBC subtype.

Analysis of microRNA (miRNA) regulatory networks is useful for exploring novel biomarkers and therapeutic targets in cancer cells. The Cancer Genome Atlas dataset shows that low expression of both strands of pre-miR-101 (miR-101-5p and miR-101-3p) significantly predicted poor prognosis in clear cell renal cell carcinoma (ccRCC). The functional significance of miR-101-5p in cancer cells is poorly understood. Here, we focused on miR-101-5p to investigate the antitumor function and its regulatory networks in ccRCC cells. Ectopic expression of mature miRNAs or siRNAs was investigated in cancer cell lines to characterize cell function, ie, proliferation, apoptosis, migration, and invasion. Genome-wide gene expression and in silico database analyses were undertaken to predict miRNA regulatory networks. Expression of miR-101-5p caused cell cycle arrest and apoptosis in ccRCC cells. Downstream neighbor of son (DONSON) was directly regulated by miR-101-5p, and its aberrant expression was significantly associated with shorter survival in propensity score-matched analysis (P = .0001). Knockdown of DONSON attenuated ccRCC cell aggressiveness. Several replisome genes controlled by DONSON and their expression were closely associated with ccRCC pathogenesis. The antitumor miR-101-5p/DONSON axis and its modulated replisome genes might be a novel diagnostic and therapeutic target for ccRCC., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Aberrantly expressed microRNA (miRNA) are known to disrupt intracellular RNA networks in cancer cells. Exploring miRNA-dependent molecular networks is a major challenge in cancer research. In this study, we performed RNA-sequencing of breast cancer (BrCa) clinical specimens to identify tumor-suppressive miRNA in BrCa. In total, 64 miRNA were identified as candidate tumor-suppressive miRNA in BrCa cells. Analysis of our BrCa signature revealed that several miRNA duplexes (guide strand/passenger strand) derived from pre-miRNA were downregulated in BrCa tissues (e.g. miR-99a-5p/-3p, miR-101-5p/-3p, miR-126-5p/-3p, miR-143-5p/-3p, and miR-144-5p/-3p). Among these miRNA, we focused on miR-101-5p, the passenger strand of pre-miR-101, and investigated its tumor-suppressive roles and oncogenic targets in BrCa cells. Low expression of miR-101-5p predicted poor prognosis in patients with BrCa (overall survival rate: P = 0.0316). Ectopic expression of miR-101-5p attenuated aggressive phenotypes, e.g. proliferation, migration, and invasion, in BrCa cells. Finally, we identified seven putative oncogenic genes (i.e. High Mobility Group Box 3, Epithelial splicing regulatory protein 1, GINS complex subunit 1 (GINS1), Tumor Protein D52, Serine/Arginine-Rich Splicing Factor Kinase 1, Vang-like protein 1, and Mago Homolog B) regulated by miR-101-5p in BrCa cells. The expression of these target genes was associated with the molecular pathogenesis of BrCa. Furthermore, we explored the oncogenic roles of GINS1, whose function had not been previously elucidated, in BrCa cells. Aberrant expression of GINS1 mRNA and protein was observed in BrCa clinical specimens, and high GINS1 expression significantly predicted poor prognosis in patients with BrCa (overall survival rate: P = 0.0126). Knockdown of GINS1 inhibited the malignant features of BrCa cells. Thus, identification of tumor-suppressive miRNA and molecular networks controlled by these miRNA in BrCa cells may be an effective strategy for elucidation of the molecular pathogenesis of this disease., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

G protein-coupled receptors (GPCRs) are the largest gene family of cell membrane-associated molecules mediating signal transmission, and their involvement in key physiological functions is well-established. The ability of GPCRs to regulate a vast array of fundamental biological processes, such as cardiovascular functions, immune responses, hormone and enzyme release from endocrine and exocrine glands, neurotransmission, and sensory perception ( e.g. vision, odor, and taste), is largely due to the diversity of these receptors and the layers of their downstream signaling circuits. Dysregulated expression and aberrant functions of GPCRs have been linked to some of the most prevalent human diseases, which renders GPCRs one of the top targets for pharmaceutical drug development. However, the study of the role of GPCRs in tumor biology has only just begun to make headway. Recent studies have shown that GPCRs can contribute to the many facets of tumorigenesis, including proliferation, survival, angiogenesis, invasion, metastasis, therapy resistance, and immune evasion. Indeed, GPCRs are widely dysregulated in cancer and yet are underexploited in oncology. We present here a comprehensive analysis of GPCR gene expression, copy number variation, and mutational signatures in 33 cancer types. We also highlight the emerging role of GPCRs as part of oncocrine networks promoting tumor growth, dissemination, and immune evasion, and we stress the potential benefits of targeting GPCRs and their signaling circuits in the new era of precision medicine and cancer immunotherapies., (© 2019 Wu et al.)

Aberrant activation of the PI3K-mTOR signaling pathway occurs in >80% of head and neck squamous cell carcinomas (HNSCC), and overreliance on this signaling circuit may in turn represent a cancer-specific vulnerability that can be exploited therapeutically. mTOR inhibitors (mTORi) promote tumor regression in genetically defined and chemically induced HNSCC animal models, and encouraging results have been recently reported. However, the mTOR-regulated targets contributing to the clinical response have not yet been identified. Here, we focused on EIF4E-BP1 ( 4E-BP1 ), a direct target of mTOR that serves as key effector for protein synthesis. A systematic analysis of genomic alterations in the PIK3CA -mTOR pathway in HNSCC revealed that 4E-BP1 is rarely mutated, but at least one 4E-BP1 gene copy is lost in over 35% of the patients with HNSCC, correlating with decreased 4E-BP1 protein expression. 4E-BP1 gene copy number loss correlated with poor disease-free and overall survival. Aligned with a tumor-suppressive role, 4e-bp1/2 knockout mice formed larger and more lesions in models of HNSCC carcinogenesis. mTORi treatment or conditional expression of a mutant 4E-BP1 that cannot be phosphorylated by mTOR was sufficient to disrupt the translation-initiation complex and prevent tumor growth. Furthermore, CRISPR/Cas9-targeted 4E-BP1 HNSCC cells resulted in reduced sensitivity to mTORi in vitro and in vivo . Overall, these findings indicate that in HNSCC, mTOR persistently restrains 4E-BP1 via phosphorylation and that mTORi can restore the tumor-suppressive function of 4E-BP1. Our findings also support 4E-BP1 expression and phosphorylation status as a mechanistic biomarker of mTORi sensitivity in patients with HNSCC. SIGNIFICANCE: These findings suggest that EIF4E-BP1 acts as a tumor suppressor in HNSCC and that 4E-BP1 dephosphorylation mediates the therapeutic response to mTORi, providing a mechanistic biomarker for future precision oncology trials., (©2019 American Association for Cancer Research.)

Background: Focal adhesion kinase (FAK) is a cytoplasmatic protein tyrosine kinase that associates with both integrins and growth factor receptors toward the adhesion, migration and invasion of cancer cells. The G-protein coupled estrogen receptor (GPER) has been involved in the stimulatory action of estrogens in breast tumor. In this study, we have investigated the engagement of FAK by GPER signaling in triple negative breast cancer (TNBC) cells., Methods: Publicly available large-scale database and patient data sets derived from "The Cancer Genome Atlas" (TCGA; www.cbioportal.org ) were used to assess FAK expression in TNBC, non-TNBC tumors and normal breast tissues. MDA-MB 231 and SUM159 TNBC cells were used as model system. The levels of phosphorylated FAK, other transduction mediators and target genes were detected by western blotting analysis. Focal adhesion assay was carried out in order to determine the focal adhesion points and the formation of focal adhesions (FAs). Luciferase assays were performed to evaluate the promoters activity of c-FOS, EGR1 and CTGF upon GPER activation. The mRNA expression of the aforementioned genes was measured by real time-PCR. Boyden chamber and wound healing assays were used in order to evaluate cell migration. The statistical analysis was performed by ANOVA., Results: We first determined by bioinformatic analysis that the mRNA expression levels of the gene encoding FAK, namely PTK2, is higher in TNBC respect to non-TNBC and normal breast tissues. Next, we found that estrogenic GPER signaling triggers Y397 FAK phosphorylation as well as the increase of focal adhesion points (FAs) in TNBC cells. Besides, we ascertained that GPER and FAK activation are involved in the STAT3 nuclear accumulation and gene expression changes. As biological counterpart, we show that FAK inhibition prevents the migration of TNBC cells upon GPER activation., Conclusions: The present data provide novel insights regarding the action of FAK in TNBC. Moreover, on the basis of our findings estrogenic GPER signaling may be considered among the transduction mechanisms engaging FAK toward breast cancer progression.

Continuous activation of hypoxia-inducible factor (HIF) is important for progression of renal cell carcinoma (RCC) and acquired resistance to antiangiogenic multikinase and mTOR inhibitors. Recently, HIF2α antagonists PT2385 and PT2399 were developed and are being evaluated in a phase I clinical trial for advanced or metastatic clear cell RCC (ccRCC). However, resistance to HIF2α antagonists would be expected to develop. In this study, we identified signals activated by HIF2α deficiency as candidate mediators of resistance to the HIF2α antagonists. We established sunitinib-resistant tumor cells in vivo and created HIF2α-deficient variants of these cells using CRISPR/Cas9 technology. Mechanistic investigations revealed that a regulator of the serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), was upregulated commonly in HIF2α-deficient tumor cells along with the serine biosynthesis pathway itself. Accordingly, treatment with a PHGDH inhibitor reduced the growth of HIF2α-deficient tumor cells in vivo and in vitro by inducing apoptosis. Our findings identify the serine biosynthesis pathway as a source of candidate therapeutic targets to eradicate advanced or metastatic ccRCC resistant to HIF2α antagonists. Cancer Res; 77(22); 6321-9. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: Despite recent advancements, metastatic castration-resistant prostate cancer (CRPC) is not considered curative. Novel approaches for identification of therapeutic targets of CRPC are needed., Methods: Next-generation sequencing revealed 945-1248 miRNAs from each lethal mCRPC sample. We constructed miRNA expression signatures of CRPC by comparing the expression of miRNAs between CRPC and normal prostate tissue or hormone-sensitive prostate cancer (HSPC). Genome-wide gene expression studies and in silico analyses were carried out to predict miRNA regulation and investigate the functional significance and clinical utility of the novel oncogenic pathways regulated by these miRNAs in prostate cancer (PCa)., Results: Based on the novel miRNA expression signature of CRPC, miR-145-5p and miR-145-3p were downregulated in CRPC. By focusing on miR-145-3p, which is a passenger strand and has not been well studied in previous reports, we showed that miR-145-3p targeted 4 key molecules, i.e., MELK, NCAPG, BUB1, and CDK1, in CPRC. These 4 genes significantly predicted survival in patients with PCa., Conclusions: Small RNA sequencing for lethal CRPC and in silico analyses provided novel therapeutic targets for CRPC.

We adopted into RNA-sequencing technologies to construct the microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC). Our signature revealed that a total of 160 miRNAs (44 upregulated and 116 downregulated) were aberrantly expressed in cancer tissues. Expression of miR-150-5p (guide strand miRNA) and miR-150-3p (passenger strand miRNA) were significantly silenced in cancer tissues, suggesting both miRNAs act as antitumor miRNAs in HNSCC cells. Ectopic expression of mature miRNAs, miR-150-5p and miR-150-3p inhibited cancer cell aggressiveness. Low expression of miR-150-5p and miR-150-3p predicted significantly shorter overall survival in patients with HNSCC (P = 0.0091 and P = 0.0386) by Kaplan-Meier survival curves analyses. We identified that integrin α3 (ITGA3), integrin α6 (ITGA6), and tenascin C (TNC) were coordinately regulated by these miRNAs in HNSCC cells. Knockdown assays using siRNAs showed that ITGA3, ITGA6 and TNC acted as cancer promoting genes in HNSCC cells. Moreover, ITGA3, ITGA6, and TNC alterations were associated with significantly poorer overall survival (P = 0.0177, P = 0.0237, and P = 0.026, respectively). Dual strands of pre-150 (miR-150-5p and miR-150-3p) functioned as antitumor miRNAs based on the miRNA expression signature of HNSCC. Identification of antitumor miR-150-mediated RNA networks may provide novel insights into pathogenesis of HNSCC.

Previous studies showed that five miRNAs (miR-885-5p, miR-1274, miR-210-3p, miR-224 and miR-1290) were upregulated the most in clear cell renal cell carcinoma (ccRCC). Our focus was to understand from a clinical standpoint the functional consequences of upregulating miR-210-3p. Towards this, we utilized the CRISPR/Cas9 gene editing system to deplete miR-210-3p in RCC cell lines (786-o, A498 and Caki2) and characterized the outcomes. We observed that miR-210-3p depletion dramatically increased tumorigenesis, including altering the morphology of A498 and Caki2 cells in a manner characteristic of epithelial-mesenchymal transition (EMT). These results were corroborated by in vivo xenograft studies, which showed enhanced growth of tumors from miR-210-3p-depleted A498 cells. We identified Twist-related protein 1 (TWIST1) as a key target of miR-210-3p. Analysis of the ccRCC patient data in The Cancer Genome Atlas database showed a negative correlation between miR-210-3p and TWIST1 expression. High TWIST1 and low miR-210-3p expression associated with poorer overall and disease-free survival as compared to low TWIST1 and high miR-210-3p expression. These findings suggest that renal cell carcinoma progression is promoted by TWIST1 suppression mediated by miR-210-3p.

The G-protein-coupled receptor (GPCR) family is the largest family of cell-surface receptors involved in signal transduction. Aberrant expression of GPCRs and G proteins are frequently associated with prevalent human diseases, including cancer. In fact, GPCRs represent the therapeutic targets of more than a quarter of the clinical drugs currently on the market. MiRNAs (miRNAs) are also aberrantly expressed in many human cancers, and they have significant roles in the initiation, development and metastasis of human malignancies. Recent studies have revealed that dysregulation of miRNAs and their target genes expression are associated with cancer progression. The emerging information suggests that miRNAs play an important role in the fine tuning of many signaling pathways, including GPCR signaling. We summarize our current knowledge of the individual functions of miRNAs regulated by GPCRs and GPCR signaling-associated molecules, and miRNAs that regulate the expression and activity of GPCRs, their endogenous ligands and their coupled heterotrimeric G proteins in human cancer.

Patients with lung adenocarcinoma may benefit from recently developed molecular targeted therapies. However, analogous advanced treatments are not available for patients with lung squamous cell carcinoma (lung SCC). The survival rate of patients with the advanced stage of lung SCC remains poor. Exploration of novel lung SCC oncogenic pathways might lead to new treatment protocols for the disease. Based on this concept, we have identified microRNA- (miRNA) mediated oncogenic pathways in lung SCC. It is well known that miR-145-5p (the guide strand) functions as a tumor suppressor in several types of cancer. However, the impact of miR-145-3p (the passenger strand) on cancer cells is still ambiguous. Expression levels of miR-145-5p and miR-145-3p were markedly reduced in cancer tissues, and ectopic expression of these miRNAs inhibited cancer cell aggressiveness, suggesting that both miR-145-3p as well as miR-145-5p acted as antitumor miRNAs. We identified seven putative target genes (MTDH, EPN3, TPD52, CYP27B1, LMAN1, STAT1 and TXNDC12) that were coordinately regulated by miR-145-5p and miR-145-3p in lung SCC. Among the seven genes, we found that metadherin (MTDH) was a direct target of these miRNAs. Kaplan-Meier survival curves showed that high expression of MTDH predicted reduced survival of lung SCC patients. We investigated pathways downstream from MTDH by using genome-wide gene expression analysis. Our data showed that several anti-apoptosis and pro-proliferation genes were involved in pathways downstream from MTDH in lung SCC. Taken together, both strands of miR-145, miR-145-5p and miR-145-3p are functional and play pivotal roles as antitumor miRNAs in lung SCC.

Molecular targeted therapy is a standard treatment for patients with advanced renal cell carcinoma (RCC). Sunitinib is one of the most common molecular-targeted drugs for metastatic RCC. Molecular mechanisms of sunitinib resistance in RCC cells is still ambiguous. The microRNA (miRNA) expression signature of patients with sunitinib failure in RCC was constructed using a polymerase chain reaction (PCR)-based array. Several miRNAs that were aberrantly expressed in RCC tissues from patients treated with sunitinib were identified in this analysis. MicroRNA-101 (miR- 101) was markedly suppressed in sunitinib treated RCC tissues. Restoration of miR-101 significantly inhibited cell migration and invasion in Caki-1 and 786-O cells. Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) was directly suppressed by miR-101 in RCC cells, and overexpression of UHRF1 was confirmed in sunitinib-treated RCC tissues. The pathways of nucleotide excision repair and mismatch repair were significantly suppressed by knockdown of UHRF1. Our findings showed that antitumor miR-101- mediated UHRF1 pathways may be suppressed by sunitinib treatment.

Objectives: The role of long non-coding RNA (lncRNA) expression in human head and neck squamous cell carcinoma (HNSCC) is still poorly understood. In this study, we aimed at establishing the onco-lncRNAome profiling of HNSCC and to identify lncRNAs correlating with prognosis and patient survival., Materials and Methods: The Atlas of Noncoding RNAs in Cancer (TANRIC) database was employed to retrieve the lncRNA expression information generated from The Cancer Genome Atlas (TCGA) HNSCC RNA-sequencing data. RNA-sequencing data from HNSCC cell lines were also considered for this study. Bioinformatics approaches, such as differential gene expression analysis, survival analysis, principal component analysis, and Co-LncRNA enrichment analysis were performed., Results: Using TCGA HNSCC RNA-sequencing data from 426 HNSCC and 42 adjacent normal tissues, we found 728 lncRNA transcripts significantly and differentially expressed in HNSCC. Among the 728 lncRNAs, 55 lncRNAs were significantly associated with poor prognosis, such as overall survival and/or disease-free survival. Next, we found 140 lncRNA transcripts significantly and differentially expressed between Human Papilloma Virus (HPV) positive tumors and HPV negative tumors. Thirty lncRNA transcripts were differentially expressed between TP53 mutated and TP53 wild type tumors. Co-LncRNA analysis suggested that protein-coding genes that are co-expressed with these deregulated lncRNAs might be involved in cancer associated molecular events. With consideration of differential expression of lncRNAs in a HNSCC cell lines panel (n=22), we found several lncRNAs that may represent potential targets for diagnosis, therapy and prevention of HNSCC., Conclusion: LncRNAs profiling could provide novel insights into the potential mechanisms of HNSCC oncogenesis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Angiogenesis, the formation of new blood vessels by remodeling and growth of pre-existing vessels, is a highly orchestrated process that requires a tight balance between pro-angiogenic and anti-angiogenic factors and the integration of their corresponding signaling networks. The family of Rho GTPases, including RhoA, Rac1, and Cdc42, play a central role in many cell biological processes that involve cytoskeletal changes and cell movement. Specifically for Rac1, we have shown that excision of Rac1 using a Tie2-Cre animal line results in embryonic lethality in midgestation (embryonic day (E) 9.5), with multiple vascular defects. However, Tie2-Cre can be also expressed during vasculogenesis, prior to angiogenesis, and is active in some hematopoietic precursors that can affect vessel formation. To circumvent these limitations, we have now conditionally deleted Rac1 in a temporally controlled and endothelial-restricted fashion using Cdh5(PAC)-iCreERT2 transgenic mice. In this highly controlled experimental in vivo system, we now show that Rac1 is required for embryonic vascular integrity and angiogenesis, and for the formation of superficial and deep vascular networks in the post-natal developing retina, the latter involving a novel specific function for Rac1 in vertical blood vessel sprouting. Aligned with these findings, we show that RAC1 is spatially involved in endothelial cell migration, invasion, and radial sprouting activities in 3D collagen matrix in vitro models. Hence, Rac1 and its downstream molecules may represent potential anti-angiogeneic therapeutic targets for the treatment of many human diseases that involve aberrant neovascularization and blood vessel overgrowth., (Copyright © 2016. Published by Elsevier Inc.)

Our recent study of the microRNA (miRNA) expression signature of hypopharyngeal squamous cell carcinoma (HSCC) revealed that microRNA-504 (miR-504) is significantly downregulated in HSCC tissues, suggesting that this miRNA is a candidate tumor suppressor. However, several previous reports indicated that miR-504 has an oncogenic function through targeting TP53. The aim of this study was to investigate the functional significance of miR-504 in cancer cells and to identify novel targets regulated by this miRNA in HSCC cells. First, we confirmed the downregulation of miR-504 in HSCC clinical specimens (P<0.0001) by qPCR. Using two sources of miR-504 to restore function, we observed significant inhibition of cancer cell proliferation in head and neck SCC (HNSCC) cell lines (FaDu, SAS and HSC3) and HCT116 colon carcinoma cells (p53+/+ and p53-/-). In HNSCC cells, induction of cell cycle arrest was observed by miR-504 transfection. To identify the molecular targets of miR-504, we performed gene expression analysis of miR-504 transfectants and in silico database analyses. Our data showed that cell cycle-related genes (RB1, CDK6, CDC23 and CCND1) were candidate target genes of miR-504. In HSCC clinical specimens, the expression of cyclin-dependent kinase 6 (CDK6) was significantly higher in cancer tissues compared to non-cancer tissues (P=0.0004). A significant inverse correlation between CDK6 and miR-504 expression was found (r=-0.43, P=0.0039). Expression of miR-504 inhibited CDK6 expression in HNSCC cells. Loss of tumor-suppressive miR-504 enhanced HSCC cell proliferation through targeting CDK6. The identification of novel tumor-suppressive miR-504-mediated molecular pathways and targets provide new insights into HSCC oncogenesis.

Current genome-wide microRNA (miRNA) expression signature analysis using deep sequencing technologies can drive the discovery of novel cancer pathways regulated by oncogenic and/or tumor suppressive miRNAs. We determined the genome-wide miRNA expression signature in bladder cancer (BC) by deep sequencing technology. A total of ten small RNA libraries were sequenced (five BCs and five samples of histologically normal bladder epithelia (NBE)), and 13,190,619 to 18,559,060 clean small RNA reads were obtained. A total of 933 known miRNAs and 17 new miRNA candidates were detected in this analysis. Among the known miRNAs, a total of 60 miRNAs were significantly downregulated in BC compared with NBE. We also found that several miRNAs, such as miR-1/133a, miR-206/133b, let-7c/miR-99a, miR-143/145 and miR-195/497, were located close together at five distinct loci and constituted clustered miRNAs. Among these clustered miRNAs, we focused on the miR-195/497 cluster because this clustered miRNA had not been analyzed in BC. Transfection of mature miR-195 or miR-497 in two BC cell lines (BOY and T24) significantly inhibited cancer cell proliferation, migration and invasion, suggesting that the miR-195/497 cluster functioned as tumor suppressors in BC. Regarding the genes targeted by the miR-195/497 cluster, the TargetScan algorithm showed that 6,730 genes were putative miR-195/497 targets, and 113 significantly enriched signaling pathways were identified in this analysis. The "Pathways in cancer" category was the most enriched, involving 104 candidate target genes. Gene expression data revealed that 27 of 104 candidate target genes were actually upregulated in BC clinical specimens. Luciferase reporter assays and Western blotting demonstrated that BIRC5 and WNT7A were directly targeted by miR-195/497. In conclusion, aberrant expression of clustered miRNAs was identified by deep sequencing, and downregulation of miR-195/497 contributed to BC progression and metastasis. Tumor suppressive miRNA-mediated cancer pathways provide new insights into the potential mechanisms of BC oncogenesis.

Our recent studies of microRNA (miRNA) expression signatures indicated that microRNA-29a (miR-29a) was significantly downregulated in several types of human cancers, suggesting that miR-29a may be a putative tumor-suppressive miRNA in human cancers. The aim of this study was to investigate the functional significance of miR-29a in cervical squamous cell carcinoma (SCC) and to identify novel miR-29a-regulated cancer pathways and target genes involved in cervical SCC oncogenesis and metastasis. Restoration of miR-29a in cervical cancer cell lines (CaSKi, HeLa, ME180 and Yumoto) revealed that this miRNA significantly inhibited cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that heat-shock protein 47 (HSP47), a member of the serpin superfamily of serine proteinase inhibitors and a molecular chaperone involved in the maturation of collagen molecules, was a potential target of miR-29a regulation. Luciferase reporter assays showed that miR-29a directly regulated HSP47. Moreover, silencing of the HSP47 gene significantly inhibited cell migration and invasion in cancer cells and the expression of HSP47 was upregulated in cancer tissues and cervical intraepithelial neoplasia (CIN), as demonstrated by immunostaining. Downregulation of miR-29a was a frequent event in cervical SCC and miR-29a acted as a tumor suppressor by directly targeting HSP47. Recognition of tumor-suppressive miRNA-regulated molecular targets provides new insights into the potential mechanisms of cervical SCC oncogenesis and metastasis and suggests novel therapeutic strategies for treatment of this disease.

Cervical cancer is one of the most common cancers in women. More than 275,100 women die from cervical cancer each year. Cervical squamous cell carcinoma (cervical SCC), one of the most frequent types of cervical cancers, is associated with high-risk human papilloma virus (HPV), although HPV infection alone may not be enough to induce malignant transformation. MicroRNAs (miRNAs), a class of small non-coding RNAs, regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. A growing body of evidence suggests that miRNAs contribute to cervical SCC progression, development and metastasis. miRNA expression signatures in SCC (hypopharyngeal SCC and esophageal SCC) revealed that miR-218 expression was significantly reduced in cancer tissues compared with adjacent non-cancerous epithelium, suggesting that miR-218 is a candidate tumor suppressor. The aim of this study was to investigate the functional significance of miR-218 in cervical SCC and to identify novel miR‑218-mediated cancer pathways in cervical SCC. Restoration of miR-218 significantly inhibited cancer cell migration and invasion in both HPV-positive and HPV-negative cervical SCC cell lines. These data indicated that miR-218 acts as a tumor suppressor in cervical SCC. Our in silico analysis showed that miR-218 appeared to be an important modulator of tumor cell processes through suppression of many targets, particularly those involved in focal adhesion signaling pathways. Gene expression data indicated that LAMB3, a laminin protein known to influence cell differentiation, migration, adhesion, proliferation and survival, was upregulated in cervical SCC clinical specimens, and silencing studies demonstrated that LAMB3 functioned as an oncogene in cervical SCC. The identification of novel tumor-suppressive miR-218-mediated molecular pathways has provided new insights into cervical SCC oncogenesis and metastasis.

MicroRNAs (miRNAs) are endogenous short non-coding RNA molecules that regulate gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner. Bioinformatic analyses predict that miRNAs regulate more than 30% of protein coding genes. To date, 1921 human mature miRNAs have been registered in miRBase release 18.0 (http://microrna.sanger.ac.uk/). A growing body of evidence suggests that miRNAs are aberrantly expressed in many human carcinomas and that they play key roles in the initiation, development and metastasis of human cancers, including head and neck squamous cell carcinoma (HNSCC). In this review, eight genome-wide miRNA expression profiles were used to selected aberrantly expressed miRNAs (up-regulated and down-regulated miRNAs) in HNSCC clinical specimens including our miRNA profiles of hypopharyngeal and maxillary sinus squamous cell carcinoma. We discuss recent findings on the aberrant expression of miRNAs and their contribution to human HNSCC oncogenesis., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

microRNAs (miRNAs) have key roles in human tumorigenesis, tumor progression and metastasis. miRNAs are aberrantly expressed in many human cancers and can function as tumor suppressors or oncogenes that target many cancer-related genes. This study seeks to identify novel miRNA-regulated molecular pathways in prostate cancer (PCa). The miRNA expression signature in clinical specimens of PCa showed that 56 miRNAs were significantly downregulated in PCa compared with non-PCa tissues. We focused on the top four downregulated miRNAs (miR-187, miR-205, miR-222 and miR-31) to investigate their functional significance in PCa cells. Expression levels of these four miRNAs were validated in PCa specimens (15 PCa tissues and 17 non-PCa tissues) to confirm that they were significantly reduced in these PCa tissues. Gain-of-function analysis demonstrated that miR-222 and miR-31 inhibited cell proliferation, invasion and migration in PCa cell lines (PC3 and DU145), suggesting that miR-222 and miR-31 may act as tumor suppressors in PCa. Genome-wide gene expression analysis using miR-222 or miR-31 transfectants to identify the pathways they affect showed that many cancer-related genes are regulated by these miRNAs in PC3 cells. Identification and categorization of the molecular pathways regulated by tumor suppressive miRNAs could provide new information about the molecular mechanisms of PCa tumorigenesis.

Recent our microRNA (miRNA) expression signature revealed that expression of microRNA-218 (miR-218) was reduced in cancer tissues, suggesting a candidate of tumor suppressor in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to investigate the functional significance of miR-218 and its mediated moleculer pathways in HNSCC. Restoration of miR-218 in cancer cells led to significant inhibition of cell migration and invasion activities in HNSCC cell lines (FaDu and SAS). Genome-wide gene expression analysis of miR-218 transfectants and in silico database analysis showed that focal adhesion pathway was a promising candidate of miR-218 target pathways. The laminins are an important and biologically active part of the basal lamina, the function of that are various such as influencing cell differentiation, migration and adhesion as well as proliferation and cell survival. Interestingly, all components of laminin-332 (LAMA3, LAMB3 and LAMC2) are listed on the candidate genes in focal adhesion pathway. Furthermore, we focused on LAMB3 which has a miR-218 target site and gene expression studies and luciferase reporter assays showed that LAMB3 was directly regulated by miR-218. Silencing study of LAMB3 demonstrated significant inhibition of cell migration and invasion. In clinical specimens with HNSCC, the expression levels of laminin-332 were significantly upregulated in cancer tissues compared to adjacent non-cancerous tissues. Our analysis data showed that tumor suppressive miR-218 contributes to cancer cell migration and invasion through regulating focal adhesion pathway, especially laminin-332. Tumor suppressive miRNA-mediated novel cancer pathways provide new insights into the potential mechanisms of HNSCC oncogenesis.