Analysis of oncogenic activities of protein kinase D1 in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer death in the US. The protein kinase D (PKD) family has emerged as a promising target for cancer therapy with PKD1 being most intensively studied; however, its role in HNSCC has not been investigated. METHODS: The expression of PKD was evaluated in human HNSCC by quantitative RT-PCR, Western blot and immunohistochemistry. Cell proliferation, wound healing, and matrigel invasion assays were performed upon siRNA-mediated knockdown of PKD1 in HNSCC cells, and subcutaneous xenograft mouse model was established by implantation of the stable doxycycline (Dox)-inducible PKD1 expression cell lines for analysis of tumorigenic activity in vivo. RESULTS: PKD1 was frequently downregulated in HNSCC cell lines at both transcript and protein levels. In human HNSCC tissues, PKD1 was significantly down-regulated in localized tumors and metastases, and in patient-paired tumor tissues as compared to their normal counterparts, which was in part due to epigenetic modification of the PRKD1 gene. The function of PKD1 in HNSCC was analyzed using stable doxycycline-inducible cell lines that express native or constitutive-active PKD1. Upon induction, the rate of proliferation, survival, migration and invasion of HNSCC cells did not differ significantly between the control and PKD1 overexpressing cells in the basal state, and depletion of endogenous PKD1 did not impact the proliferation of HNSCC cells. However, the median growth rate of the subcutaneous HNSCC tumor xenografts over time was elevated with PKD1 induction, and the final tumor weight was significantly increased in Dox-induced vs. the non-induced tumors. Moreover, induced expression of PKD1 promoted bombesin-induced cell proliferation of HNSCC and resulted in sustained ERK1/2 activation in response to gastrin-releasing peptide or bombesin stimulation, suggesting that PKD1 potentiates GRP/bombesin-induced mitogenic response through the activation of ERK1/2 in HSNCC cells. CONCLUSIONS: Our study has identified PKD1 as a frequently downregulated gene in HNSCC, and functionally, under certain cellular context, may play a role in GRP/bombesin-induced oncogenesis in HNSCC.

Integrative analysis reveals associations between oral microbiota dysbiosis and host genetic and epigenetic aberrations in oral cavity squamous cell carcinoma.

Dysbiosis of the human oral microbiota has been reported to be associated with oral cavity squamous cell carcinoma (OSCC) while the host-microbiota interactions with respect to the potential impact of pathogenic bacteria on host genomic and epigenomic abnormalities remain poorly studied. In this study, the mucosal bacterial community, host genome-wide transcriptome and DNA CpG methylation were simultaneously profiled in tumors and their adjacent normal tissues of OSCC patients. Significant enrichment in the relative abundance of seven bacteria species (Fusobacterium nucleatum, Treponema medium, Peptostreptococcus stomatis, Gemella morbillorum, Catonella morbi, Peptoanaerobacter yurli and Peptococcus simiae) were observed in OSCC tumor microenvironment. These tumor-enriched bacteria formed 254 positive correlations with 206 up-regulated host genes, mainly involving signaling pathways related to cell adhesion, migration and proliferation. Integrative analysis of bacteria-transcriptome and bacteria-methylation correlations identified at least 20 dysregulated host genes with inverted CpG methylation in their promoter regions associated with enrichment of bacterial pathogens, implying a potential of pathogenic bacteria to regulate gene expression, in part, through epigenetic alterations. An in vitro model further confirmed that Fusobacterium nucleatum might contribute to cellular invasion via crosstalk with E-cadherin/ß-catenin signaling, TNFα/NF-κB pathway and extracellular matrix remodeling by up-regulating SNAI2 gene, a key transcription factor of epithelial-mesenchymal transition (EMT). Our work using multi-omics approaches explored complex host-microbiota interactions and provided important insights into genetic and functional basis in OSCC tumorigenesis, which may serve as a precursor for hypothesis-driven study to better understand the causational relationship of pathogenic bacteria in this deadly cancer.

Preclinical evaluation of the AKT inhibitor MK-2206 in nasopharyngeal carcinoma cell lines.

Nasopharyngeal carcinoma (NPC) is endemic to Asia and over 40 % of NPC tissues harbor PIK3CA amplifications. This study characterized the preclinical activity of MK-2206, an oral allosteric inhibitor of AKT in 6 NPC cell lines: C666-1, HK1, HONE-1-EBV, HONE-1, CNE-2 and HNE-1. Exposure to increasing concentrations of MK-2206 resulted in over 95 % of growth inhibition in all NPC cell lines with IC50 values in the low micromolar range. Further experiments were performed in 3 representative NPC cell lines: CNE-2 (harbor PIK3CA mutation and most sensitive to MK-2206), C666-1 (carries PIK3CA amplification), and HONE-1-EBV (least sensitive to MK-2206). MK-2206 induced G0/G1 cycle arrest in all 3 cell lines, but could induce apoptosis only in CNE-2 cells. MK-2206 significantly abrogated AKT signaling in all 3 cell lines by inhibiting the activation of AKT and its downstream effectors (FKHR, GSK3ß and BAD). MK-2206 also reduced mTOR signaling by reducing activation of mTOR and its downstream 4E-BP1 and p70S6 kinase. MAPK activation was observed in HONE-1 and C666-1 cells, but not in CNE-2 cells following exposure to MK-2206. The addition of MK-2206 to cisplatin (but not with paclitaxel) has a supra-additive inhibitory effect on growth in vitro. In summary, MK-2206 can inhibit growth and abrogate AKT and mTOR signaling in NPC cell lines. This agent is currently being evaluated in a phase II study in metastatic NPC.

Activity of the MEK inhibitor selumetinib (AZD6244; ARRY-142886) in nasopharyngeal cancer cell lines.

This study evaluated the preclinical activity of selumetinib (AZD6244, ARRY-142866), an inhibitor of the mitogen-activated protein kinase kinase (MAPKK or MEK1/2) in 6 nasopharyngeal cancer (NPC) cell lines. Selumetinib could achieve up to 90 % inhibition of cell growth with the respective IC(50) values in NPC cell lines as follow: HK1 = 0.04 µM, HK1-LMP1(B95.8) = 0.17 µM, HONE-1-EBV = 0.46 µM, HONE-1 = 1.79 µM, CNE-2 = 2.20 µM and C666-1 > 10 µM. The drug-sensitive cell lines HK1, HK1-LMP1(B95.8) and HONE-1-EBV have higher basal expression of phosphorylated (pi)-MAPK than the less sensitive cell lines. BRAF mutations were not detected in all 6 cell lines. Re-introduction of the EBV genome into HONE-1 cells, generating the HONE-1-EBV cell line, seemed to result in elevated expression of pi-MAPK and sensitivity to selumetinib when compared with the parental HONE-1 cells. At a concentration of 0.5 µM and 5 µM, selumetinib induced apoptosis (as indicated by cleaved PARP expression and caspase 3 induction), and G(0)/G(1) cycle arrest in HONE-1-EBV and HK1-LMP1(B95.8) cells. The combination of selumetinib (at IC(25) concentration) and the EGFR tyrosine kinase inhibitor, gefitinib (at concentrations of 0.1, 3 and 9 µM) resulted in synergistic growth inhibition in HK1-LMP1(B95.8) cells. The combination of selumetinib (at IC(25) concentration) and cisplatin (at concentrations of 0.1, 0.4, 0.8 and 2 µM) resulted in synergistic growth inhibition in HONE-1 and HONE-1-EBV cells. This result suggests that selumetinib alone or in combination with gefitinib or cisplatin maybe a promising strategy against NPC. Further studies are warranted.

PIK3CA, HRAS and PTEN in human papillomavirus positive oropharyngeal squamous cell carcinoma.

BACKGROUND: Recent genomic evidence suggests frequent phosphatidylinositide 3-kinase (PI3K) pathway activation in human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma. Mutations/amplification of the gene encoding p110α catalytic subunit of phosphoinositide 3-kinase (PIK3CA), loss of phosphatase and tensin homolog (PTEN) and HRAS mutations are known to activate PI3K pathway. METHODS AND RESULTS: PIK3CA mutations were identified by Sanger sequencing in 23 of 75 (31%) HPV-positive oropharyngeal carcinomas, including exon 9 (p.E545K [n = 10] and p.E542K [n = 5]) or exon 20 (p.H1047Y, n = 2) mutations. Five rare and one novel (p.R537Q) PIK3CA mutations were identified. HRAS mutation (p.Q61L) was detected in 1 of 62 tested cases. PIK3CA amplification by fluorescence in situ hybridization (FISH) was identified in 4 cases (4/21, 20%), while PTEN loss was seen in 7 (7/21, 33%) cases (chromosome 10 monosomy [n = 4], homozygous deletion [n = 3]). CONCLUSIONS: Overall, genetic alterations that likely lead to PI3K pathway activation were identified in 34 of 75 cases (45%) and did not correlate with disease specific survival. These findings offer a molecular rationale for therapeutic targeting of PI3K pathway in patients with HPV-positive oropharyngeal carcinoma.

The Fanconi anemia pathway: repairing the link between DNA damage and squamous cell carcinoma.

Fanconi anemia (FA) is a rare inherited recessive disease caused by mutations in one of fifteen genes known to encode FA pathway components. In response to DNA damage, nuclear FA proteins associate into high molecular weight complexes through a cascade of post-translational modifications and physical interactions, followed by the repair of damaged DNA. Hematopoietic cells are particularly sensitive to the loss of these interactions, and bone marrow failure occurs almost universally in FA patients. FA as a disease is further characterized by cancer susceptibility, which highlights the importance of the FA pathway in tumor suppression, and will be the focus of this review. Acute myeloid leukemia is the most common cancer type, often subsequent to bone marrow failure. However, FA patients are also at an extreme risk of squamous cell carcinoma (SCC) of the head and neck and gynecological tract, with an even greater incidence in those individuals who have received a bone marrow transplant and recovered from hematopoietic disease. FA tumor suppression in hematopoietic versus epithelial compartments could be mechanistically similar or distinct. Definition of compartment specific FA activities is now critical to assess the effects of today's bone marrow failure treatments on tomorrow's solid tumor development. It is our hope that current therapies can then be optimized to decrease the risk of malignant transformation in both hematopoietic and epithelial cells. Here we review our current understanding of the mechanisms of action of the Fanconi anemia pathway as it contributes to stress responses, DNA repair and squamous cell carcinoma susceptibility.

Hypoxia-targeting by tirapazamine (TPZ) induces preferential growth inhibition of nasopharyngeal carcinoma cells with Chk1/2 activation.

Hypoxia is commonly developed in solid tumors, which contributes to metastasis as well as radio- and chemo-resistance. Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic head and neck cancer prevalent in Southeast Asia with a high incidence rate of 15-30/100,000 persons/year (comparable to that of pancreatic cancer in the US). Previous clinical studies in NPC showed that hypoxia is detected in almost 100% of primary tumors and overexpression of hypoxia markers correlated with poor clinical outcome. Tirapazamine (TPZ) is a synthetic hypoxia-activated prodrug, which preferentially forms cytotoxic and DNA-damaging free radicals under hypoxia, thus selectively eradicate hypoxic cells. Here, we hypothesized that specific hypoxia-targeting by this clinical trial agent may be therapeutic for NPC. Our findings demonstrated that under hypoxia, TPZ was able to induce preferential growth inhibition of NPC cells, which was associated with marked cell cycle arrest at S-phase and PARP cleavage (a hallmark of apoptosis). Examination of S-phase checkpoint regulators revealed that Chk1 and Chk2 were selectively activated by TPZ in NPC cells under hypoxia. Hypoxia-selectivity of TPZ was also demonstrated by preferential downregulation of several important hypoxia-induced markers (HIF-1α, CA IX and VEGF) under hypoxia. Furthermore, we demonstrated that TPZ was equally effective and hypoxia-selective even in the presence of the EBV oncoprotein, LMP1 or the EBV genome. In summary, encouraging results from this proof-of-concept study implicate the therapeutic potential of hypoxia-targeting approaches for the treatment of NPC.

Preclinical evaluation of sunitinib as single agent or in combination with chemotherapy in nasopharyngeal carcinoma.

PURPOSE: Sunitinib is a multi-target receptor tyrosine kinase (RTK) inhibitor against vascular endothelial growth factor receptors, platelet-derived growth factor receptors (PDGFR), c-kit and RET. Several of these RTKs are known to be involved in the progression of nasopharyngeal carcinoma (NPC). Here, we evaluated the preclinical activities of sunitinib in NPC. METHOD: We determined the basal level of total and phosphorylated PDGFR, c-kit and RET by immunoblotting in a panel of five NPC cell lines. The effect of sunitinib on NPC cell proliferation was evaluated by MTT assay. We further studied the effect of sunitinib on NPC cell cycle progression and apoptosis. We investigated the in vitro and in vivo activities of sunitinib as single agent and in combination with cisplatin or docetaxel in NPC cell lines and tumor xenografts. RESULTS: Sunitinib exhibited dose-dependent growth inhibition in all NPC cell lines tested with IC(50) between 2-7.5 µM and maximum inhibition of over 97%. Sunitinib induced apoptosis and cell cycle arrest at G(0)/G(1) phase. In vitro, sunitinib moderately enhanced the growth inhibition of cisplatin or docetaxel. Single agent sunitinib demonstrated significant growth inhibition, reduced microvessel density and caused extensive tumor necrosis in a NPC xenograft model. However, concurrent administration of sunitinib and docetaxel induced severe toxicity in mice without enhanced antitumor effect. CONCLUSIONS: Single agent sunitinib demonstrated potent in vitro and in vivo growth inhibition in NPC. When combined with chemotherapy, sequential instead of concurrent administration schedule should be further explored.

Targeting the PI3K/Akt/mTOR pathway in hepatocellular carcinoma.

Despite recent advances in the understanding of the biologic basis of hepatocellular carcinoma (HCC) development, the clinical management of the disease remains a major challenge. Deregulation of the PI3K/Akt/mTOR pathway, which is a prototypic survival pathway, is increasingly implicated in HCC carcinogenesis. In this article, we detailed the role of this pathway in the pathogenesis of HCC and provide an update on the preclinical and clinical development of various agents targeting this key survival/proliferation pathway, which include various PI3K inhibitors, Akt inhibitors and mTOR inhibitors for HCC. In addition, we highlighted the therapeutic potential of combination strategy for mTOR inhibitors with conventional chemotherapy, in particular, antimicrotubule agents, other molecular targeting agents, as well as radiotherapy.

Whole-genome profiling of nasopharyngeal carcinoma reveals viral-host co-operation in inflammatory NF-κB activation and immune escape.

Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer.

Quantitative proteomics analysis reveals molecular networks regulated by epidermal growth factor receptor level in head and neck cancer.

Epidermal growth factor receptor (EGFR) is overexpressed in up to 90% of head and neck cancer (HNC), where increased expression levels of EGFR correlate with poor prognosis. To date, EGFR expression levels have not predicted the clinical response to the EGFR-targeting therapies. Elucidation of the molecular mechanisms underlying anti-EGFR-induced antitumor effects may shed some light on the mechanisms of HNC resistance to EGFR-targeting therapeutics and provide novel targets for improving the treatment of HNC. Here, we conducted a quantitative proteomics analysis to determine the molecular networks regulated by EGFR levels in HNC by specifically knocking-down EGFR and employing stable isotope labeling with amino acids in cell culture (SILAC). Following data normalization to minimize systematic errors and Western blotting validation, 12 proteins (e.g., p21, stratifin, and maspin) and 24 proteins (e.g., cdc2 and MTA2) were found to be significantly upregulated or downregulated by EGFR knockdown, respectively. Bioinformatic analysis revealed that these proteins were mainly involved in long-chain fatty acid biosynthesis and beta-oxidation, cholesterol biosynthesis, cell proliferation, DNA replication, and apoptosis. Cell cycle analysis confirmed that G(2)/M phase progression was significantly inhibited by EGFR knockdown, a hypothesis generated from network modeling. Further investigation of these molecular networks may not only enhance our understanding of the antitumor mechanisms of EGFR targeting but also improve patient selection and provide novel targets for better therapeutics.

The activity of mTOR inhibitor RAD001 (everolimus) in nasopharyngeal carcinoma and cisplatin-resistant cell lines.

Phosphorylated (pi-) protein kinase B (AKT) is commonly expressed in nasopharyngeal carcinoma (NPC) cell lines and tissues, suggesting the involvement of AKT-mammalian target of rapamycin (mTOR) signaling in NPC carcinogenesis. This study evaluated the activity of an mTOR inhibitor, RAD001 (Everolimus, Novartis Pharma AG, Switzerland), in 5 NPC cell lines (HK1, HONE-1, CNE-1, CNE-2, C666-1), 2 cisplatin-resistant NPC cell lines and their respective parental cell lines (HK1-LMP1, HONE-1-EBV). RAD001 inhibited cell growth in a dose-dependent manner at nanomolar concentrations in all cell lines. HONE-1 was most sensitive to RAD001 (IC(50) = 0.63 nM, 60% maximal inhibition), while Het-1A (a normal esophageal epithelial cell line) was relatively resistant. No consistent relationship between sensitivity to RAD001 and basal expression of pi-mTOR and pi-p70S6 Kinase-1 (p70S6K) was found. Exposure to RAD001 at picomolar concentrations for 48 h resulted in reduction of pi-mTOR and pi-p70S6K1 expression, but increase in pi-AKT (Ser473) expression in HONE-1 and CNE-1 cell lines. RAD001 significantly induced apoptosis in HONE-1 cells, but has no effect on cell cycle progression. RAD001 exerted an additive to synergistic effect on cisplatin-induced growth inhibition in CNE-1 and HONE-1 cells, and could inhibit the growth of both cisplatin-resistant and cisplatin-sensitive NPC cell lines. In summary, combination of RAD001 and cisplatin maybe a useful therapeutic strategy in NPC. AKT upregulation following RAD001 treatment suggests the presence of a feedback loop on AKT signaling in NPC which warrants further investigation.

Preclinical activity of gefitinib in non-keratinizing nasopharyngeal carcinoma cell lines and biomarkers of response.

This study evaluated the preclinical activity and molecular predictors of response to gefitinib (Iressa(R), Astra Zeneca Inc, UK) in nasopharyngeal carcinoma (NPC). The activity of gefitinib was evaluated in four human NPC cell lines--HK1, HONE-1, CNE2, C666-1. A representative gefitinib-sensitive (HK1, IC(50) = 250 nM) and gefitinib-resistant cell line (HONE-1, IC(50) > 15 microM) were selected and compared for expression of epidermal growth factor receptor (EGFR) and related ligands, and activation of downstream proteins. Gefitinib induced G1 cycle arrest, apoptosis and inhibited cell invasion more significantly in HK1 than HONE-1 cells. HK1 expressed higher levels of p-EGFR, lower p-AKT and phospho-signal transducer and activator of transcription 3 (p-STAT3) than other cell lines. EGFR gene was found to be amplified in HK1. Gefitinib at IC(50) concentrations significantly suppressed EGF-induced activation of p-EGFR, phospho-mitogen-activated protein kinase (p-MAPK) and p-STAT3, but p-AKT showed persistent activation in HK1 and HONE-1 cells. There was no difference in EGFR-ligand expression between the 4 NPC cell lines. In NPC samples derived from non-responders to gefitinib, 50% and 60% showed cytoplasmic and nuclear pi-EGFR expression, respectively, and 33% showed p-AKT expression. EGFR or KRAS mutations were not detected. This study suggests that most NPC cell lines are intrinsically resistant to gefitinib (except HK1 cells), and further studies are needed to confirm whether EGFR gene amplification and persistent AKT activation may influence response to gefitinib in NPC.

Understanding Molecular Testing Uptake Across Tumor Types in Eight Countries: Results From a Multinational Cross-Sectional Survey.

PURPOSE: The growth in understanding of molecular biology and genomics has augmented the development of targeted cancer treatments; however, challenges exist in access to molecular testing, an essential precursor to treatment decision-making. We used data from a cross-sectional survey to evaluate the differences in uptake of molecular testing. METHODS: Using the aggregated results of a questionnaire developed and distributed to clinicians by IQVIA, including treatment details and investigations undertaken for patients, we compared proportions of patients receiving molecular testing and targeted treatment by cancer type for the United Kingdom, France, Italy, Germany, Spain, South Korea, Japan, and China. We used multivariable logistic regression methods to understand the effect of country on the odds of receiving a molecular test. RESULTS: There was a total of 61,491 cases. Across countries and cancer types, uptake rates for molecular testing ranged between 2% and 98%, with the greatest differences seen in gastric cancers (range, 23% to 70%), and significant variations were observed for both European and Asian countries. China consistently demonstrated a significantly reduced uptake for all molecular tests assessed; however; uptake of drug treatment in gastric cancers after testing positive for the human epidermal growth factor receptor 2 gene was higher than in some European countries (China, 85%; European range, 8% to 66%). The uptake of epidermal growth factor receptor gene testing was greater in some Asian countries relative to the United Kingdom, where incidence of lung cancer is higher (Japan: odds ratio, 3.1 [95% CI, 2.6 to 3.8]; South Korea: odds ratio, 2.7 [95% CI, 2 to 3.4]). CONCLUSION: We have highlighted inequity in access to molecular testing and subsequent treatments across countries, which warrants improvements.

The Intersection between Oral Microbiota, Host Gene Methylation and Patient Outcomes in Head and Neck Squamous Cell Carcinoma.

The role of oral microbiota in head and neck squamous cell carcinoma (HNSCC) is poorly understood. Here we sought to evaluate the association of the bacterial microbiome with host gene methylation and patient outcomes, and to explore its potential as a biomarker for early detection or intervention. Here we performed 16S rRNA gene amplicon sequencing in sixty-eight HNSCC patients across both tissue and oral rinse samples to identify oral bacteria with differential abundance between HNSCC and controls. A subset of thirty-one pairs of HNSCC tumor tissues and the adjacent normal tissues were characterized for host gene methylation profile using bisulfite capture sequencing. We observed significant enrichments of Fusobacterium and Peptostreptococcus in HNSCC tumor tissues when compared to the adjacent normal tissues, and in HNSCC oral rinses when compared to healthy subjects, while ten other bacterial genera were largely depleted. These HNSCC-related bacteria were discriminative for HNSCC and controls with area under the receiver operating curves (AUCs) of 0.84 and 0.86 in tissue and oral rinse samples, respectively. Moreover, Fusobacterium nucleatum abundance in HNSCC cases was strongly associated with non-smokers, lower tumor stage, lower rate of recurrence, and improved disease-specific survival. An integrative analysis identified that enrichment of F. nucleatum was associated with host gene promoter methylation, including hypermethylation of tumor suppressor genes LXN and SMARCA2, for which gene expressions were downregulated in the HNSCC cohort from The Cancer Genome Atlas. In conclusion, we identified a taxonomically defined microbial consortium associated with HNSCC that may have clinical potential regarding biomarkers for early detection or intervention. Host-microbe interactions between F. nucleatum enrichment and clinical outcomes or host gene methylation imply a potential role of F. nucleatum as a pro-inflammatory driver in initiating HNSCC without traditional risk factors, which warrants further investigation for the underlying mechanisms.

Cucurbitacin I elicits anoikis sensitization, inhibits cellular invasion and in vivo tumor formation ability of nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is an Asian-prevalent head and neck cancer with high invasiveness. Although several important risk factors for NPC development have been identified, there is currently no preventive strategy for NPC, even in endemic regions. Signal transducer and activator of transcription 3 (STAT3) has been implicated in NPC carcinogenesis, which may serve as a potential target for cancer prevention. Here, we examined the chemopreventive potential of Cucurbitacin I, a natural-occurring selective inhibitor of JAK/STAT3, in NPC models. We hypothesized that Cucurbitacin I would prevent NPC invasion and tumor formation. Our data demonstrated that brief exposure of NPC cells to Cucurbitacin I was sufficient to significantly reduce the in vitro clonogenicity and in vivo tumorigenicity of NPC cells. The chemopreventive potential of Cucurbitacin I was further demonstrated by pre-dosing of the animals with Cucurbitacin I prior to tumor inoculation, which was found to be able to suppress tumor growth up to 7 days post-inoculation. The anti-proliferation activity of Cucurbitacin I was accompanied by downregulation of phospho-STAT3 and STAT3 target gene expression (e.g. cyclin D1 and Mcl-1). Cucurbitacin I also reduced the invasiveness of invasive NPC cell lines with elevated STAT3 activation. Furthermore, our data demonstrated for the first time that Cucurbitacin I harbored potent anoikis-sensitization activity (i.e. sensitizing cancer cells to detachment-induced cell death) against human cancer. Taken together, our results suggested that Cucurbitacin I may be a potent chemopreventive agent for NPC with anti-invasion and anoikis-sensitizing activities.

Prediction of radiotherapy response with a 5-microRNA signature-based nomogram in head and neck squamous cell carcinoma.

Radiotherapy is unlikely to benefit all patients with head and neck squamous cell carcinoma (HNSCC). Therefore, novel method is warranted to predict the radiotherapy response. Our study aimed to construct a microRNA (miRNA)-based nomogram to predict clinical outcomes of patients with HNSCC receiving radiotherapy. We screened out 56 differential miRNAs by analyzing 44 paired tumor and adjacent normal samples miRNA expression profiles from The Cancer Genome Atlas (TCGA). A total of 307 patients with HNSCC receiving adjuvant radiotherapy were randomly divided into a training set (n = 154) and a validation set (n = 153). In the training set, we combined the differential miRNA profiles with clinical outcomes, and LASSO regression model was applied to establish a 5-miRNA signature. The prediction accuracy of the 5-miRNA signature was further validated. In addition, target genes of these miRNAs were predicted, and Gene Ontology (GO) analysis as well as KEGG pathway analysis was executed. A 5-miRNA signature including miR-99a, miR-31, miR-410, miR-424, and miR-495 was identified. With a cutoff value of 1.2201 from Youden's index, the training set was divided into high-risk and low-risk groups, and the 5-year overall survival was significantly different (30% vs. 73%, HR 3.65, CI 2.46-8.16; P < 0.0001). Furthermore, our 5-miRNA signature revealed that only low-risk group would benefit from radiotherapy. Then, a nomogram combining 5-miRNA signature with clinical variables to predict radiotherapy response was constructed. The analysis of 108 target genes of these miRNAs revealed some potential mechanisms in HNSCC radiotherapy response for future investigations. In conclusion, the 5-miRNA signature-based nomogram is useful in predicting radiotherapy response in HNSCC and might become a promising tool to optimize radiation strategies.

The RARS-MAD1L1 Fusion Gene Induces Cancer Stem Cell-like Properties and Therapeutic Resistance in Nasopharyngeal Carcinoma.

Purpose: Nasopharyngeal carcinoma (NPC) is the most common head and neck cancer in Southeast Asia. Because local recurrence and distant metastasis are still the main causes of NPC treatment failure, it is urgent to identify new tumor markers and therapeutic targets for advanced NPC.Experimental Design: RNA sequencing (RNA-seq) was applied to look for interchromosome translocation in NPC. PCR, FISH, and immunoprecipitation were used to examine the fusion gene expression at RNA, DNA, and protein levels in NPC biopsies. MTT assay, colony formation assay, sphere formation assay, co-immunoprecipitation, chromatin immunoprecipitation assay, and in vivo chemoresistance assay were applied to explore the function of RARS-MAD1L1 in NPC.Results: We demonstrated that RARS-MAD1L1 was present in 10.03% (35/349) primary NPC biopsies and 10.7% (9/84) in head and neck cancer (HNC) samples. RARS-MAD1L1 overexpression increased cell proliferation, colony formation, and tumorigenicity in vitro, and the silencing of endogenous RARS-MAD1L1 reduced cancer cell growth and colony formation in vitro In addition, RARS-MAD1L1 increased the side population (SP) ratio and induced chemo- and radioresistance. Furthermore RARS-MAD1L1 interacted with AIMP2, which resulted in activation of FUBP1/c-Myc pathway. The silencing of FUBP1 or the administration of a c-Myc inhibitor abrogated the cancer stem cell (CSC)-like characteristics induced by RARS-MAD1L1. The expression of c-Myc and ABCG2 was higher in RARS-MAD1L1-positive HNC samples than in negative samples.Conclusions: Our findings indicate that RARS-MAD1L1 might contribute to tumorigenesis, CSC-like properties, and therapeutic resistance, at least in part, through the FUBP1/c-Myc axis, implying that RARS-MAD1L1 might serve as an attractive target for therapeutic intervention for NPC. Clin Cancer Res; 24(3); 659-73. ©2017 AACR.