CD8+ Tumor-Infiltrating Lymphocyte Abundance Is a Positive Prognostic Indicator in Nasopharyngeal Cancer.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) are immune cell populations found within tumors, critical in the antigen-specific host immune response. In this study, we aimed to elucidate the prognostic significance of CD3+, CD4+, and CD8+ TILs in nasopharyngeal cancer (NPC). EXPERIMENTAL DESIGN: Immune cell infiltration was quantified in NPC samples (n = 50) using RNA-sequencing (RNA-seq) data based on rearranged T-cell receptor (TCR) reads and the Estimation of Stromal and Immune cells in malignant tumors using expression data (ESTIMATE) immune score tool. The differential abundances of TIL subset populations were also characterized through IHC staining of formalin-fixed, paraffin-embedded samples from a training cohort (n = 35), which was a subset of the RNA-seq cohort (n = 50). RESULTS: In the RNA-seq cohort, patients with higher rearranged TCR reads experienced superior 5- and 10-year overall survival (OS; P < 0.001), and disease-free survival (DFS; P < 0.001). Similarly, patients with higher ESTIMATE immune scores experienced superior 5- and 10-year OS (P = 0.024) and DFS (P = 0.007). In the training cohort, high abundances of CD8+ TILs were significantly associated with improved 5- and 10-year OS (P = 0.003) and DFS (P = 0.005). These findings were corroborated in an independent validation cohort (n = 84), and combined analysis of the training and validation cohorts [n = 119 (35+84)], which further demonstrated improved 5- and 10-year survival in terms of locoregional control (P < 0.001) and distant metastasis (P = 0.03). CONCLUSIONS: Taken together, our study highlights the prognostic value of CD8+ TILs in NPC, and the potential of future investigations into cellular-based immunotherapies employing CD8+ lymphocytes.

Inferring gene expression from cell-free DNA fragmentation profiles.

Profiling of circulating tumor DNA (ctDNA) in the bloodstream shows promise for noninvasive cancer detection. Chromatin fragmentation features have previously been explored to infer gene expression profiles from cell-free DNA (cfDNA), but current fragmentomic methods require high concentrations of tumor-derived DNA and provide limited resolution. Here we describe promoter fragmentation entropy as an epigenomic cfDNA feature that predicts RNA expression levels at individual genes. We developed 'epigenetic expression inference from cell-free DNA-sequencing' (EPIC-seq), a method that uses targeted sequencing of promoters of genes of interest. Profiling 329 blood samples from 201 patients with cancer and 87 healthy adults, we demonstrate classification of subtypes of lung carcinoma and diffuse large B cell lymphoma. Applying EPIC-seq to serial blood samples from patients treated with PD-(L)1 immune-checkpoint inhibitors, we show that gene expression profiles inferred by EPIC-seq are correlated with clinical response. Our results indicate that EPIC-seq could enable noninvasive, high-throughput tissue-of-origin characterization with diagnostic, prognostic and therapeutic potential.

Identification of a novel 12p13.3 amplicon in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer commonly occurring in southern China. To decipher the molecular basis of this cancer, we performed high-resolution array CGH analysis on eight tumour lines and 10 primary tumours to identify the genes involved in NPC tumorigenesis. In this study, multiple regions of gain were consistently found at 1q21-q24, 7q11-12, 7q21-22., 11q13, 12p13, 12q13, 19p13 and 19q13. Importantly, a 2.1 Mb region at 12p13.31 was highly amplified in a NPC xenograft, xeno-2117. By FISH mapping, we have further delineated the amplicon to a 1.24 region flanked by RP11-319E16 and RP11-433J6. Copy number gains of this amplicon were confirmed in 21/41 (51%) primary tumours, while three cases (7.3%) showed high copy number amplification. Among the 13 genes within this amplicon, three candidate genes, lymphotoxin beta receptor (LTbetaR), tumour necrosis factor receptor superfamily memeber 1A (TNFRSF1R) and FLJ10665, were specifically over-expressed in the NPC xenograft with 12p13.3 amplification. However, only LTbetaR was frequently over-expressed in primary tumours. LTbetaR is a member of the TNF family of receptors, which can modulate NF-kappaB signalling pathways. Over-expression of LTbetaR in nasopharyngeal epithelial cells resulted in an increase of NF-kappaB activity and cell proliferation. In vivo study showed that suppression of LTbetaR by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification. These findings implied that LTbetaR is a potential NPC-associated oncogene within the 12p13.3 amplicon and that its alteration is important in NPC tumorigenesis.

Significance of Plk1 regulation by miR-100 in human nasopharyngeal cancer.

Polo-like kinase 1 (Plk1) is a critical regulator of many stages of mitosis; increasing evidence indicates that Plk1 overexpression correlates with poor clinical outcome, yet its mechanism of regulation remains unknown. Hence, a detailed evaluation was undertaken of Plk1 expression in human nasopharyngeal cancer (NPC), the cellular effects of targeting Plk1 using siRNA in combination with ionizing radiation (RT) and potential upstream microRNAs (miRs) that might regulate Plk1 expression. Using immunohistochemistry, Plk1 was observed to be overexpressed in 28 of 40 (70%) primary NPC biopsies, which in turn was associated with a higher likelihood of recurrence (p = 0.018). SiPlk1 significantly inhibited Plk1 mRNA and protein expression, and decreased Cdc25c levels in NPC cell lines. This depletion resulted in cytotoxicity of C666-1 cells, enhanced by the addition of RT, mediated by G2/M arrest, increased DNA double-strand breaks, apoptosis, and caspase activation. Immunofluorescence demonstrated that the G2/M arrest was associated with aberrant spindle formation, leading to mitotic arrest. In vivo, transfection of C666-1 cells and systemic delivery of siPlk1 decreased tumour growth. MicroRNA-100 (miR-100) was predicted to target Plk1 mRNA, which was indeed underexpressed in C666-1 cells, inversely correlating with Plk1 expression. Using luciferase constructs containing the 3'-UTR of Plk1 sequence, we document that miR-100 can directly target Plk1. Hence, our data demonstrate for the first time that underexpressed miR-100 leads to Plk1 overexpression, which in turn contributes to NPC progression. Targeting Plk1 will cause mitotic catastrophe, with significant cytotoxicity both in vitro and in vivo, underscoring the important therapeutic opportunity of Plk1 in NPC.

Therapeutic efficacy of seliciclib in combination with ionizing radiation for human nasopharyngeal carcinoma.

PURPOSE: Seliciclib is a small-molecule cyclin-dependent kinase inhibitor, which has been reported to induce apoptosis and cell cycle arrest in EBV-negative nasopharyngeal carcinoma cell lines. Because most nasopharyngeal carcinoma patients harbor EBV, we proceeded to evaluate the cytotoxic effects of seliciclib in EBV-positive nasopharyngeal carcinoma models. EXPERIMENTAL DESIGN: Cytotoxicity of seliciclib was investigated in the EBV-positive cell line C666-1 and the C666-1 and C15 xenograft models. Caspase activities and cell cycle analyses were measured by flow cytometry. Efficacy of combined treatment of seliciclib with radiation therapy was also evaluated. RESULTS: Seliciclib caused significant cytotoxicity in the C666-1 cells in a time- and dose-dependent manner, with accumulation of cells in both sub-G(1) and G(2)-M phases, indicative of apoptosis and cell cycle arrest, respectively. Caspase-2, -3, -8, and -9 activities were all increased, with caspase-3 being the most significantly activated at 48 h after treatment. These cells also showed a reduction of Mcl-1 mRNA and protein levels. Combined treatment of seliciclib with radiation therapy showed a synergistic interaction with enhanced cytotoxicity in C666-1 cells and delayed repair of double-strand DNA breaks. For in vivo models, significant delays in tumor growth were observed for both C666-1 and C15 tumors, which were associated with enhanced apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry analyses. CONCLUSIONS: Seliciclib enhanced the antitumor efficacy of radiation therapy in EBV-positive nasopharyngeal carcinoma, characterized by G(2)-M arrest, and apoptosis, associated with an induction in caspase activity. This process is mediated by reduction in Mcl-1 expression and by attenuation of double-strand DNA break repair.

Robust global micro-RNA profiling with formalin-fixed paraffin-embedded breast cancer tissues.

Global micro-RNA (miR) profiling of human malignancies is increasingly performed, but to date, the majority of such analyses have used frozen tissues. However, formalin fixation is the standard and routine histological practice for optimal preservation of cellular morphology. To determine whether miR analysis of formalin-fixed tissues is feasible, quantitative real-time PCR (qRT-PCR) profiling of miR expression in 40 archival formalin-fixed paraffin-embedded (FFPE) breast lumpectomy specimens were performed. Taqman Low Density Arrays (TLDAs) were used to assess the expression level of 365 miRs in 34 invasive ductal carcinomas and in 6 normal comparators derived from reduction mammoplasties. Its technical reproducibility was high, with intra-sample correlations above 0.9 and with 92.8% accuracy in differential expression comparisons, indicating such global profiling studies to be technically and biologically robust. The TLDA data were confirmed using conventional single-well qRT-PCR analysis, showing a strong and statistically significant concordance between these two methods. Paired frozen and FFPE breast cancer samples from the same patients showed a similar level of robust correlation of at least 0.94. Compared with normal breast samples, a panel of miRs was consistently dysregulated in breast cancer, including earlier-reported breast cancer-related miRs, such as upregulated miR-21, miR-155, miR-191, and miR-196a, and downregulated miR-125b and miR-221. Additional novel miR sequences of potential biological relevance were also uncovered. These results show the validity and utility of conducting global miR profiling using FFPE samples, thereby offering enormous opportunities to evaluate archival banks of such materials, linked to clinical databases, to rapidly acquire greater insight into the clinically relevant role for miRs in human malignancies.

Efficacy of systemically administered mutant vesicular stomatitis virus (VSVDelta51) combined with radiation for nasopharyngeal carcinoma.

PURPOSE: Nasopharyngeal carcinoma (NPC) is a malignancy of the head and neck region that is associated with EBV latency. Curative treatments for NPC achieve modest survival rates, underscoring a need to develop novel therapies. We evaluated the therapeutic potential of a mutant vesicular stomatitis virus (VSVDelta51) as single treatment modality or in combination with ionizing radiation (RT) in NPC. EXPERIMENTAL DESIGN: MTS assay was used to assess cell viability in vitro; apoptosis was measured using propidium iodide staining and caspase activation. In vivo experiments were conducted using tumor-bearing nude mice with or without local RT (4 Gy). Apoptosis was assessed in excised tumor sections with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. RESULTS: Our data showed that NPC cells are exquisitely sensitive to VSVDelta51 oncolysis, which correlated with the presence of EBV. Efficacy of VSVDelta51 against NPC cells was further augmented when combined with RT. A single systemic injection of VSVDelta51 achieved 50% survival in treated mice, which increased to 83% when combined with local tumor RT. In addition to induction of apoptosis, an antiangiogenic effect of VSVDelta51 was observed in vivo, suggesting a novel tumoricidal mechanism for VSVDelta51. This virus also prevented growth of NPC sphere-forming cells in vitro, showing potential utility in targeting NPC-initiating cells. CONCLUSIONS: Our data represent the first report showing that EBV-positive NPC cells are exquisitely sensitive to VSVDelta51 oncolysis and documenting the successful utilization of this combinatorial regimen as a novel curative therapeutic strategy for NPC.

Identification of a novel homozygous deletion region at 6q23.1 in medulloblastomas using high-resolution array comparative genomic hybridization analysis.

PURPOSE: The aim of this study is to comprehensively characterize genome copy number aberrations in medulloblastomas using high-resolution array comparative genomic hybridization. EXPERIMENTAL DESIGN: High-density genomic arrays containing 1,803 BAC clones were used to define recurrent chromosomal regions of gains or losses throughout the whole genome of medulloblastoma. A series of 3 medulloblastoma cell lines and 16 primary tumors were investigated. RESULTS: The detected consistent chromosomal aberrations included gains of 1q21.3-q23.1 (36.8%), 1q32.1 (47.4%), 2p23.1-p25.3 (52.6%), 7 (57.9%), 9q34.13-q34.3 (47.4%), 17p11.2-q25.3 (89.5%), and 20q13.31-q13.33 (42.1%), as well as losses of 3q26.1 (57.9%), 4q31.23-q32.3 (42.1%), 6q23.1-25.3 (57.9%), 8p22-23.3 (79%), 10q24.32-26.2 (57.9%), and 16q23.2-q24.3 (63.2%). One of the most notable aberrations was a homozygous deletion on chromosome 6q23 in the cell line DAOY, and single copy loss on 30.3% primary tumors. Further analyses defined a 0.887 Mbp minimal region of homozygous deletion at 6q23.1 flanked by markers SHGC-14149 (6q22.33) and SHGC-110551 (6q23.1). Quantitative reverse transcription-PCR analysis showed complete loss of expression of two genes located at 6q23.1, AK091351 (hypothetical protein FLJ34032) and KIAA1913, in the cell line DAOY. mRNA levels of these genes was reduced in cell lines D283 and D384, and in 50% and 70% of primary tumors, respectively. CONCLUSION: Current array comparative genomic hybridization analysis generates a comprehensive pattern of chromosomal aberrations in medulloblastomas. This information will lead to a better understanding of medulloblastoma tumorigenesis. The delineated regions of gains or losses will indicate locations of medulloblastoma-associated genes. A 0.887 Mbp homozygous deletion region was newly identified at 6q23.1. Frequent detection of reduced expression of AK091351 and KIAA1913 genes implicates them as suppressors of medulloblastoma tumorigenesis.

Developing a prognostic micro-RNA signature for human cervical carcinoma.

Cervical cancer remains the third most frequently diagnosed and fourth leading cause of cancer death in women worldwide. We sought to develop a micro-RNA signature that was prognostic for disease-free survival, which could potentially allow tailoring of treatment for cervical cancer patients. A candidate prognostic 9-micro-RNA signature set was identified in the training set of 79 frozen specimens. However, three different approaches to validate this signature in an independent cohort of 87 patients with formalin-fixed paraffin-embedded (FFPE) specimens, were unsuccessful. There are several challenges and considerations associated with developing a prognostic micro-RNA signature for cervical cancer, namely: tumour heterogeneity, lack of concordance between frozen and FFPE specimens, and platform selection for global micro-RNA expression profiling in this disease. Our observations provide an important cautionary tale for future miRNA signature studies for cervical cancer, which can also be potentially applicable to miRNA profiling studies involving other types of human malignancies.

Identification of a microRNA signature associated with risk of distant metastasis in nasopharyngeal carcinoma.

PURPOSE: Despite significant improvement in locoregional control in the contemporary era of nasopharyngeal carcinoma (NPC) treatment, patients still suffer from a significant risk of distant metastasis (DM). Identifying those patients at risk of DM would aid in personalized treatment in the future. MicroRNAs (miRNAs) play many important roles in human cancers; hence, we proceeded to address the primary hypothesis that there is a miRNA expression signature capable of predicting DM for NPC patients. METHODS AND RESULTS: The expression of 734 miRNAs was measured in 125 (Training) and 121 (Validation) clinically annotated NPC diagnostic biopsy samples. A 4-miRNA expression signature associated with risk of developing DM was identified by fitting a penalized Cox Proportion Hazard regression model to the Training data set (HR 8.25; p < 0.001), and subsequently validated in an independent Validation set (HR 3.2; p = 0.01). Pathway enrichment analysis indicated that the targets of miRNAs associated with DM appear to be converging on cell-cycle pathways. CONCLUSIONS: This 4-miRNA signature adds to the prognostic value of the current "gold standard" of TNM staging. In-depth interrogation of these 4-miRNAs will provide important biological insights that could facilitate the discovery and development of novel molecularly targeted therapies to improve outcome for future NPC patients.

Molecular cytogenetic characterization of nasopharyngeal carcinoma cell lines and xenografts by comparative genomic hybridization and spectral karyotyping.

Nasopharyngeal carcinoma (NPC) cell lines and xenografts represent valuable models for functional and therapeutic studies on this common malignancy in Southeast Asia. The karyotypic information in most NPC cell lines and xenografts, however, remains largely unclear to date. We have characterized the chromosomal aberrations in six commonly used human NPC cell lines and xenografts using the molecular cytogenetic technique of comparative genomic hybridization (CGH). Genomic imbalances identified in cell lines were further correlated with structural abnormalities indicated from spectral karyotyping (SKY) analysis. CGH revealed consistent overrepresentations of 8q (six out of six cases) with a smallest overlapping region identified on 8q21.1 approximately q22. Other common gains included 7p (4/6 cases), 7q (4/6 cases), 12q (4/6), and 20q (4/6 cases), where minimal overlapping regions were suggested on 7p15 approximately p14, 7q11.2 approximately q21, and 12q22 approximately q24.1. Common losses were detected on 3p12 approximately p21 (4/6 cases) and 11q14 approximately qter (4/6 cases). Although SKY analysis on cell lines revealed predominantly unbalanced rearrangements, reciprocal translocations that involved chromosome 2 [i.e., t(1;2), t(2;3), and t(2;4)] were suggested. Furthermore, SKY examination illustrated additional breakpoints on a number of apparently balanced chromosomes. These breakpoints included 3p21, 3q26, 5q31, 6p21.1 approximately p25, 7p14 approximately p22, and 8q22. Our finding of regional gains and losses and breakpoints represents information that may contribute to NPC studies in vitro.

Detection of oncogene amplifications in medulloblastomas by comparative genomic hybridization and array-based comparative genomic hybridization.

OBJECT: Few studies have been conducted to investigate the genomic survey of oncogene amplification in medulloblastoma. Low frequency of N-myc, C-myc, and epidermal grow factor receptor (EGFR) gene amplification (< 10%) has been reported in medulloblastoma. Previous comparative genomic hybridization (CGH) study of primary medulloblastomas has revealed chromosomal amplification on 2p21, 3p, 5p15.3, 7q, 8q24, 11q22.3, and 17q. The aim of this study was to detect common oncogenes involved in medulloblastoma tumorigenesis. METHODS: The authors studied a series of 14 samples by performing CGH and array-based CGH. The CGH analysis detected nonrandom losses on 8p, 17p, 16q, 8q, and 1p, whereas gains were found on 17q, 12q, 7q, and 1p. Array-based CGH was conducted to investigate amplification of 58 oncogenes throughout the genome of these samples. Gene amplifications identified for the first time included PGY1 at 7q21.1, MDM2 at 12q14.3-q15, and ERBB2 at 17q21.2. The highest frequencies of oncogene gain were detected in D17S1670 (61.5%), PIK3CA (46.2%), PGY1 (38.5%), MET (38.5%), ERBB2 (38.5%), and CSE1L (38.5%). The gain in gene copy numbers was confirmed in 34 additional archival medulloblastoma cases by using fluorescence in situ hybridization analysis. CONCLUSIONS: This is the first genome-wide survey of multiple oncogene amplifications involved in the development of medulloblastoma. Gains of several candidate oncogenes such as D17S1670, ERBB2, PIK3CA, PGY1, MET, and CSE1L were frequently detected. These genes may be used as molecular markers and therapeutic targets of medulloblastomas.

MicroRNA-193b enhances tumor progression via down regulation of neurofibromin 1.

Despite improvements in therapeutic approaches for head and neck squamous cell carcinomas (HNSCC), clinical outcome has remained disappointing, with 5-year overall survival rates hovering around 40-50%, underscoring an urgent need to better understand the biological bases of this disease. We chose to address this challenge by studying the role of micro-RNAs (miRNAs) in HNSCC. MiR-193b was identified as an over-expressed miRNA from global miRNA profiling studies previously conducted in our lab, and confirmed in HNSCC cell lines. In vitro knockdown of miR-193b in FaDu cancer cells substantially reduced cell proliferation, migration and invasion, along with suppressed tumour formation in vivo. By integrating in silico prediction algorithms with in vitro experimental mRNA profilings, plus mRNA expression data of clinical specimens, neurofibromin 1 (NF1) was identified to be a target of miR-193b. Concordantly, miR-193b knockdown decreased NF1 transcript and protein levels significantly. Luciferase reporter assays confirmed the direct interaction of miR-193b with NF1. Moreover, p-ERK, a downstream target of NF1 was also suppressed after miR-193b knockdown. FaDu cells treated with a p-ERK inhibitor (U0126) phenocopied the reduced cell proliferation, migration and invasion observed with miR-193b knockdown. Finally, HNSCC patients whose tumours expressed high levels of miR-193b experienced a lower disease-free survival compared to patients with low miR-193b expression. Our findings identified miR-193b as a potentially novel prognostic marker in HNSCC that drives tumour progression via down-regulating NF1, in turn leading to activation of ERK, resulting in proliferation, migration, invasion, and tumour formation.

Hemochromatosis enhances tumor progression via upregulation of intracellular iron in head and neck cancer.

INTRODUCTION: Despite improvements in treatment strategies for head and neck squamous cell carcinoma (HNSCC), outcomes have not significantly improved; highlighting the importance of identifying novel therapeutic approaches to target this disease. To address this challenge, we proceeded to evaluate the role of iron in HNSCC. EXPERIMENTAL DESIGN: Expression levels of iron-related genes were evaluated in HNSCC cell lines using quantitative RT-PCR. Cellular phenotypic effects were assessed using viability (MTS), clonogenic survival, BrdU, and tumor formation assays. The prognostic significance of iron-related proteins was determined using immunohistochemistry. RESULTS: In a panel of HNSCC cell lines, hemochromatosis (HFE) was one of the most overexpressed genes involved in iron regulation. In vitro knockdown of HFE in HNSCC cell lines significantly decreased hepcidin (HAMP) expression and intracellular iron level. This in turn, resulted in a significant decrease in HNSCC cell viability, clonogenicity, DNA synthesis, and Wnt signalling. These cellular changes were reversed by re-introducing iron back into HNSCC cells after HFE knockdown, indicating that iron was mediating this phenotype. Concordantly, treating HNSCC cells with an iron chelator, ciclopirox olamine (CPX), significantly reduced viability and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression. CONCLUSIONS: Our data identify HFE as potentially novel prognostic marker in HNSCC that promotes tumour progression via HAMP and elevated intracellular iron levels, leading to increased cellular proliferation and tumour formation. Hence, these findings suggest that iron chelators might have a therapeutic role in HNSCC management.

Potentially prognostic miRNAs in HPV-associated oropharyngeal carcinoma.

PURPOSE: Deregulation of miRNAs is associated with almost all human malignancies. Human papillomavirus (HPV)-associated oropharyngeal carcinoma (OPC) has a significantly more favorable outcome compared with HPV-negative OPCs; however, the underlying mechanisms are not well understood. Hence, the objectives of this study were to determine whether miRNA expression differed as a function of HPV status and to assess whether such miRNAs provide prognostic value beyond HPV status. METHODS: Global miRNA profilings were conducted on 88 formalin-fixed and paraffin-embedded (FFPE) OPC biopsies (p16-positive: 56; p16-negative: 32), wherein the expression levels of 365 miRNAs plus 3 endogenous controls were simultaneously measured using quantitative real-time (qRT)-PCR. Seven FFPE specimens of histologically normal tonsils were used as controls. RESULTS: Overall, 224 miRNAs were expressed in more than 80% of the investigated samples, with 128 (57%) being significantly differentially expressed between tumor versus normal tissues (P < 0.05). Upregulated miR-20b, miR-9, and miR-9* were significantly associated with HPV/p16-status. Three miRNA sets were significantly associated with overall survival (miR-107, miR-151, miR-492; P = 0.0002), disease-free survival (miR-20b, miR-107, miR-151, miR-182, miR-361; P = 0.0001), and distant metastasis (miR-151, miR-152, miR-324-5p, miR-361, miR492; P = 0.0087), which retained significance even after adjusting for p16 status. The associated biologic functions of these miRNAs include immune surveillance, treatment resistance, invasion, and metastasis. CONCLUSION: We have identified several miRNAs, which associate with HPV status in OPC; furthermore, three candidate prognostic sets of miRNAs seem to correlate with clinical outcome, independent of p16 status. Furthermore, evaluations will offer biologic insights into the mechanisms underlying the differences between HPV-positive versus HPV-negative OPC.

MicroRNA-196b regulates the homeobox B7-vascular endothelial growth factor axis in cervical cancer.

The down-regulation of microRNA-196b (miR-196b) has been reported, but its contribution to cervical cancer progression remains to be investigated. In this study, we first demonstrated that miR-196b down-regulation was significantly associated with worse disease-free survival (DFS) for cervical cancer patients treated with combined chemo-radiation. Secondly, using a tri-modal approach for target identification, we determined that homeobox-B7 (HOXB7) was a bona fide target for miR-196b, and in turn, vascular endothelial growth factor (VEGF) was a downstream transcript regulated by HOXB7. Reconstitution of miR-196b expression by transient transfection resulted in reduced cell growth, clonogenicity, migration and invasion in vitro, as well as reduced tumor angiogenesis and tumor cell proliferation in vivo. Concordantly, siRNA knockdown of HOXB7 or VEGF phenocopied the biological effects of miR-196b over-expression. Our findings have demonstrated that the miR-196b/HOXB7/VEGF pathway plays an important role in cervical cancer progression; hence targeting this pathway could be a promising therapeutic strategy for the future management of this disease.

MiR-218 suppresses nasopharyngeal cancer progression through downregulation of survivin and the SLIT2-ROBO1 pathway.

Nasopharayngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignancy most common in East Asia and Africa. Here we report frequent downregulation of the microRNA miR-218 in primary NPC tissues and cell lines where it plays a critical role in NPC progression. Suppression of miR-218 was associated with epigenetic silencing of SLIT2 and SLIT3, ligands of ROBO receptors that have been previously implicated in tumor angiogenesis. Exogenous expression of miR-218 caused significant toxicity in NPC cells in vitro and delayed tumor growth in vivo. We used an integrated trimodality approach to identify targets of miR-218 in NPC, cervical, and breast cell lines. Direct interaction between miR-218 and the 3'-untranslated regions (UTR) of mRNAs encoding ROBO1, survivin (BIRC5), and connexin43 (GJA1) was validated in a luciferase-based transcription reporter assay. Mechanistic investigations revealed a negative feedback loop wherein miR-218 regulates NPC cell migration via the SLIT-ROBO pathway. Pleotropic effects of miR-218 on NPC survival and migration were rescued by enforced expression of miR-218-resistant, engineered isoforms of survivin and ROBO1, respectively. In clinical specimens of NPC (n=71), ROBO1 overexpression was significantly associated with worse overall (P=0.04, HR=2.4) and nodal relapse-free survival (P=0.008, HR=6.0). Our findings define an integrative tumor suppressor function for miR-218 in NPC and further suggest that restoring miR-218 expression in NPC might be useful for its clinical management.

Significance of dysregulated metadherin and microRNA-375 in head and neck cancer.

PURPOSE: Despite recent improvements in local control of head and neck cancers (HNC), distant metastasis remains a major cause of death. Hence, further understanding of HNC biology, and in particular, the genes/pathways driving metastasis is essential to improve outcome. EXPERIMENTAL DESIGN: Quantitative reverse transcriptase PCR (qRT-PCR) was used to measure the expression of miR-375 and metadherin (MTDH) in HNC patient samples. Targets of miR-375 were confirmed using qRT-PCR, Western blot analysis, and luciferase assays. Phenotypic effects of miR-375 reexpression and MTDH knockdown were assessed using viability (MTS), clonogenic survival, cell migration/invasion, as well as in vivo tumor formation assays. The prognostic significance of miR-375 or MTDH in nasopharyngeal carcinoma (NPC) was determined by comparing low versus high expression groups. RESULTS: MiR-375 expression was significantly reduced (P = 0.01), and conversely, MTDH was significantly increased (P = 0.0001) in NPC samples. qRT-PCR, Western blots, and luciferase assays corroborated MTDH as a target of miR-375. Reexpression of miR-375 and siRNA knockdown of MTDH both decreased cell viability and clonogenic survival, cell migration/invasion, as well as in vivo tumor formation. NPC patients whose tumors expressed high levels of MTDH experienced significantly lower survival and, in particular, higher distant relapse rates (5-year distant relapse rates: 26% vs. 5%; P = 0.005). CONCLUSIONS: Dysregulation of miR-375 and MTDH may represent an important oncogenic pathway driving human HNC progression, particularly distant metastases, which is now emerging as a major cause of death for HNC patients. Hence, targeting this pathway could potentially be a novel therapeutic strategy by which HNC patient outcome could be improved.

Comprehensive MicroRNA profiling for head and neck squamous cell carcinomas.

PURPOSE: The objective of this study is to investigate the significance of microRNAs (miRNA) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: A global miRNA profiling was done on 51 formalin-fixed archival HNSCC samples using quantitative reverse transcription-PCR approach, correlated with patients' clinical parameters. Functional characterization of HNSCC-associated miRNAs was conducted on three HNSCC cell lines. Cell viability and proliferation were investigated using MTS and clonogenic assays, respectively; cell cycle analyses were assessed using flow cytometry. RESULTS: Thirty-eight of the 117 (33%) consistently detected miRNAs were significantly differentially expressed between malignant versus normal tissues. Concordant with previous reports, overexpression of miR-21, miR-155, let-7i, and miR-142-3p and underexpression of miR-125b and miR-375 were detected. Upregulation of miR-423, miR-106b, miR-20a, and miR-16 as well as downregulation of miR-10a were newly observed. Exogenous overexpression of miR-375 in HNSCC cell lines reduced proliferation and clonogenicity and increased cells in sub-G(1). Similar cellular effects were observed in knockdown studies of the miR-106b-25 cluster but with accumulation of cells in G(1) arrest. No major difference was detected in miRNA profiles among laryngeal, oropharyngeal, or hypopharyngeal cancers. miR-451 was found to be the only significantly overexpressed miRNA by 4.7-fold between nonrelapsed and relapsed patients. CONCLUSION: We have identified a group of aberrantly expressed miRNAs in HNSCC and showed that underexpression of miR-375 and overexpression of miR-106b-25 cluster might play oncogenic roles in this disease. Further detailed examinations of miRNAs will provide opportunities to dissect the complex molecular abnormalities driving HNSCC progression.

4q loss is potentially an important genetic event in MM tumorigenesis: identification of a tumor suppressor gene regulated by promoter methylation at 4q13.3, platelet factor 4.

In this study, we have elucidated the chromosomal imbalances in the multistep pathogenesis and delineated several critical tumor suppressor gene (TSG) loci in multiple myeloma (MM). By using comparative genomic hybridization, allelotyping, and multicolor interphase fluorescence in situ hybridization, 5 MM cell lines and bone marrow CD138+ plasma cells from 88 Chinese patients with monoclonal gammopathy of undetermined significance (MGUS) and early and advanced stages of MM were investigated. In all MGUS and MM samples, chromosome copy number abnormalities were detected. A higher number of chromosomal imbalances and specific genetic alterations are involved in MGUS to MM transition (-6q, +3p, and +1p) and MM progression (+2p and +9q). In addition to -13q, we first found high frequencies (42% to 46%) of -4q involving high percentages (70% to 74%) of clonal plasma cells in both MGUS and MM, suggesting that inactivation of TSG in this region is also a potentially critical genetic event in MM tumorigenesis. By high-resolution allelotyping, we defined a common deletion region on 4q13.3 and found that a candidate TSG, platelet factor 4, was frequently silenced by promoter hypermethylation in MM (15 of 28) and MM cell lines (5 of 5). These data have opened up a new approach in the molecular targeting therapy and provide novel insights into MM tumorigenesis.