UM-Chor1: establishment and characterization of the first validated clival chordoma cell line.

OBJECTIVE Chordomas are rare malignant tumors thought to arise from remnants of the notochord. They can be located anywhere along the axial skeleton but are most commonly found in the clival and sacrococcygeal regions, where the notochord regresses during fetal development. Chordomas are resistant to many current therapies, leaving surgery as the primary method of treatment. Cancer cell lines have been useful for developing new cancer treatments in a laboratory setting that can then be transferred to the clinic, but there are only 4 validated chordoma cell lines available. The objective of this work was to establish chordoma cell lines from surgical tissue in order to expand the library of lines available for laboratory research. METHODS Chordoma tissue from the clivus was processed and sorted by flow cytometry to obtain an isolated population of chordoma cells. These cells were grown in culture and expanded until enough doublings to consider the line established. Identification of a chordoma cell line was made with known markers for chordoma, and the line was observed for ALDH (aldehyde dehydrogenase) subpopulations and tested in serum-free growth conditions as well as in vivo. RESULTS A fifth chordoma cell line, UM-Chor1, was successfully established. This is the first chordoma cell line originating from the clivus. Validation was confirmed by phenotype and positivity for the chordoma markers CD24 and brachyury. The authors also attempted to identify an ALDHhigh cell population in UM-Chor1, UCH1, and UCH2 but did not detect a distinct population. UM-Chor1 cells were able to form spheroids in serum-free culture, were successfully transduced with luciferase, and could be injected parasacrally and grown in NOD/SCID mice. CONCLUSIONS The availability of this novel clival chordoma cell line for in vitro and in vivo research provides an opportunity for developments in treatment against the disease.

Prognostic biomarkers in patients with human immunodeficiency virus-positive disease with head and neck squamous cell carcinoma.

BACKGROUND: We examined the prognostic value of a panel of biomarkers in patients with squamous cell carcinoma of the head and neck (SCCHN) who were human immunodeficiency virus (HIV) positive (HIV-positive head and neck cancer) and HIV negative (HIV-negative head and neck cancer). METHODS: Tissue microarrays (TMAs) were constructed using tumors from 41 disease site-matched and age-matched HIV-positive head and neck cancer cases and 44 HIV-negative head and neck cancer controls. Expression of tumor biomarkers was assessed by immunohistochemistry (IHC) and correlations examined with clinical variables. RESULTS: Expression levels of the studied oncogenic and inflammatory tumor biomarkers were not differentially regulated by HIV status. Among patients with HIV-positive head and neck cancer, laryngeal disease site (P = .003) and Clavien-Dindo classification IV (CD4) counts <200 cells/µL (P = .01) were associated with poor prognosis. Multivariate analysis showed that p16 positivity was associated with improved overall survival (OS; P < .001) whereas increased expression of transforming growth factor-beta (TGF-ß) was associated with poor clinical outcome (P = .001). CONCLUSION: Disease site has significant effect on the expression of biomarkers. Expression of tumor TGF-ß could be a valuable addition to the conventional risk stratification equation for improving head and neck cancer disease management strategies.

Genomic Integration of High-Risk HPV Alters Gene Expression in Oropharyngeal Squamous Cell Carcinoma.

High-risk HPV (hrHPV) is the leading etiologic factor in oropharyngeal cancer. HPV-positive oropharyngeal tumors generally respond well to therapy, with complete recovery in approximately 80% of patients. However, it remains unclear why some patients are nonresponsive to treatment, with 20% of patients recurring within 5 years. In this study, viral factors were examined for possible clues to differences in tumor behavior. Oropharynx tumors that responded well to therapy were compared with those that persisted and recurred. Viral oncogene alternate transcripts were assessed, and cellular sites of viral integration were mapped and sequenced. Effects of integration on gene expression were assessed by transcript analysis at the integration sites. All of the tumors demonstrated active viral oncogenesis, indicated by expression of HPV E6 and E7 oncogenes and alternate E6 splicing. In the responsive tumors, HPV integration occurred exclusively in intergenic chromosome regions, except for one tumor with viral integration into TP63. Each recurrent tumor exhibited complex HPV integration patterns into cancer-associated genes, including TNFRSF13B, SCN2A, SH2B1, UBE2V2, SMOC1, NFIA, and SEMA6D Disrupted cellular transcripts were identified in the region of integration in four of the seven affected genes. IMPLICATIONS: Integration of transcriptionally active hrHPV into cellular intergenic regions associates with tumor behavior by altering gene expression. Mol Cancer Res; 14(10); 941-52. ©2016 AACR.

Identification of Targetable ERBB2 Aberrations in Head and Neck Squamous Cell Carcinoma.

IMPORTANCE: ERBB2 (formerly HER2) is an important drug target in breast cancer, where anti-ERBB2 therapy has been shown to lead to improvements in disease recurrence and overall survival. ERBB2 status in head and neck squamous cell carcinoma (HNSCC) has not been well studied. Identification of ERBB2-positive tumors and characterization of response to ERBB2 therapy could lead to targeted treatment options in HNSCC. OBJECTIVE: To identify ERBB2 aberrations in HNSCCs and investigate the potential for ERBB2-targeted therapy in HNSCCs. DESIGN, SETTING, AND PARTICIPANTS: A retrospective case series of patients with laryngeal (42 tumor specimens) and oral cavity (94 tumor specimens) SCC enrolled in the University of Michigan Head and Neck Specialized Program of Research Excellence was conducted. Publicly available sequencing data (The Cancer Genome Atlas), as well as data from other studies, were reviewed to identify additional mutations and overexpression in ERBB2 in HNSCC. Established HNSCC cell lines were used for follow-up in vitro analysis. The study was conducted from October 1, 2014, to August 30, 2015. INTERVENTIONS: With the use of targeted, amplicon-based sequencing with the Oncomine Cancer Panel, the copy number and mutation status of commonly altered genes in HNSCCs were assessed. Immunohistochemical staining was performed on tissue microarrays of HNSCCs to assess the expression of ERBB2. Western blotting for HNSCC cell line ERBB2 expression and cell survival assays after treatment with ERBB2 inhibitors were performed. MAIN OUTCOMES AND MEASURES: The prevalence of ERBB2 genetic aberrations and ERBB2 overexpression in laryngeal and oral cavity SCCs, prevalence of ERBB2 aberrations in HNSCC in The Cancer Genome Atlas, ERBB2 protein expression in HNSCC cell lines, and response of HNSCC cell lines to targeted ERBB2 inhibitors. RESULTS: Of the 42 laryngeal SCC samples screened by targeted sequencing, 4 (10%) were positive for ERBB2 amplification. Two of these samples showed ERBB2 overexpression on immunohistochemistry. Two of the 94 oral cavity SCC samples (2%) were positive for ERBB2 on immunohistochemistry. Analysis of 288 patients from publicly available HNSCC sequencing data revealed 9 amplifications (3%) in ERBB2. Protein expression was variable across HNSCC cell lines, and a subset of these cell lines showed responsiveness to anti-ERBB2 therapy. CONCLUSIONS AND RELEVANCE: ERBB2 aberrations were identified in a subset of HNSCCs. These tumors may be responsive to targeted therapy against ERBB2. Screening for ERBB2 aberrations and applying targeted therapy in ERBB2-positive patients may be useful in personalized therapy trials, particularly in patients who are refractory to current treatment paradigms.

In vivo Wnt pathway inhibition of human squamous cell carcinoma growth and metastasis in the chick chorioallantoic model.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with poor overall survival. New therapeutic strategies that target specific molecular lesions driving advanced disease are needed. Herein we demonstrate the utility of the chicken chorioallantoic membrane (CAM) assay for in vivo human HNSCC tumor growth and metastasis and the tumor suppressive effects of a new chemotherapeutic agent. METHODS: We tested anti-metastatic effects of a WNT pathway inhibitor, WNT974 (also known as LGK974), which targets porcupine (PORCN) the palmityl-transferase that is essential for secretion of Wnt proteins. CAM assays were performed with 8 HNSCC cell lines: UM-SCC-1, UM-SCC-10A, UM-SCC-10B, UM-SCC-11A, UM-SCC-14A UM-SCC-17A, UM-SCC-17B, UM-SCC-25, and UM-SCC-34. RESULTS: UM-SCC-1 (University of Michigan Squamous Cell Carcinoma cell line) CAM xenografts contain CD44+ and ALDH+ cancer stem cell (CSC) proportions similar to UM-SCC-1 mouse xenografts supporting the applicability of the CAM assay for study of CSCs. Inhibition of WNT signaling by the PORCN inhibitor WNT974 reduced metastatic spread of UM-SCC cells, especially in UM-SCCs with Notch1 deficiency. CONCLUSIONS: Our data demonstrate decreased tumor growth and metastases in tumors from cell lines that showed in vitro responses to WNT974, providing evidence that this agent may have a role in future HNSCC therapy.

Human papillomavirus infection and biomarkers in sinonasal inverted papillomas: clinical significance and molecular mechanisms.

BACKGROUND: The role of human papillomavirus (HPV) in sinonasal inverted papillomas (IPs) is controversial. Determining the prevalence of HPV infection and its impact on the molecular biology of these tumors is critical to characterizing its role in the pathogenesis of IPs. METHODS: A total of 112 paraffin-embedded IPs from 90 patients were studied. A tissue microarray was constructed and stained for p16, p53, epidermal growth factor receptor (EGFR), and cyclin D1. HPV presence and types were determined using PGMY 09/11 primers and integration using HPV 11 detection of integrated papillomavirus sequences by ligation-mediated polymerase chain reaction (DIPS-PCR). RESULTS: HPV was detected in 11 of 90 (12%) patients. HPV 11 was found in 9 samples. HPV 6 and HPV 27 were found in 1 sample each. EGFR staining proportion was higher in HPV-positive IPs vs HPV-negative specimens (56.2% vs 23.6%; p = 0.009). Differences in p16, p53, and cyclin D1 staining were not significant. HPV-positive lesions tend to progress to malignancy (p = 0.064). Three samples were analyzed for integration. Viral integration was found in both malignant tumors but not in the precursor IP. CONCLUSION: Degradation of p53 and p16/cyclin D1 dysregulation are not important mechanisms in low-risk HPV-related IP. The low prevalence of HPV in this series indicates it is not a main etiological factor for IPs; however, when present, low-risk HPV may contribute to the biology of IPs through an increase of EGFR expression and a predisposition for malignant progression by integration into the cellular genome.

Prevention of tumor growth driven by PIK3CA and HPV oncogenes by targeting mTOR signaling with metformin in oral squamous carcinomas expressing OCT3.

Most squamous cell carcinomas of the head and neck (HNSCC) exhibit a persistent activation of the PI3K-mTOR signaling pathway. We have recently shown that metformin, an oral antidiabetic drug that is also used to treat lipodystrophy in HIV-infected (HIV(+)) individuals, diminishes mTOR activity and prevents the progression of chemically induced experimental HNSCC premalignant lesions. Here, we explored the preclinical activity of metformin in HNSCCs harboring PIK3CA mutations and HPV oncogenes, both representing frequent HNSCC alterations, aimed at developing effective targeted preventive strategies. The biochemical and biologic effects of metformin were evaluated in representative HNSCC cells expressing mutated PIK3CA or HPV oncogenes (HPV(+)). The oral delivery of metformin was optimized to achieve clinical relevant blood levels. Molecular determinants of metformin sensitivity were also investigated, and their expression levels were examined in a large collection of HNSCC cases. We found that metformin inhibits mTOR signaling and tumor growth in HNSCC cells expressing mutated PIK3CA and HPV oncogenes, and that these activities require the expression of organic cation transporter 3 (OCT3/SLC22A3), a metformin uptake transporter. Coexpression of OCT3 and the mTOR pathway activation marker pS6 were observed in most HNSCC cases, including those arising in HIV(+) patients. Activation of the PI3K-mTOR pathway is a widespread event in HNSCC, including HPV(-) and HPV(+) lesions arising in HIV(+) patients, all of which coexpress OCT3. These observations may provide a rationale for the clinical evaluation of metformin to halt HNSCC development from precancerous lesions, including in HIV(+) individuals at risk of developing HPV(-) associated cancers.

Human papillomavirus-related oropharyngeal cancer: HPV and p16 status in the recurrent versus parent tumor.

BACKGROUND: Although typically associated with a favorable prognosis, a minority of human papillomavirus (HPV)-related (+) oropharyngeal cancers recur after chemoradiation. We postulated that a minor HPV-negative tumor subfraction may be responsible for recurrences of HPV+ oropharyngeal cancer. METHODS: Paired untreated primary and recurrent tumor specimens were identified for 37 patients with oropharyngeal cancer who received definitive chemoradiotherapy at our institution. Concordance in HPV/p16 expression between primary and recurrent tumors was assessed. RESULTS: Among 31 patients with HPV+/p16+ primary tumors, 30 (97%) retained evidence of both HPV and p16 expression at recurrence (27 HPV+/p16+; 3 HPV+/p16-partial). One (3%) initially HPV+/p16+ patient developed an HPV-negative/p16-negative lung squamous cell carcinoma (SCC), representing either a discordant oropharyngeal cancer metastasis or second primary tumor. CONCLUSION: HPV-related oropharyngeal cancers retain HPV+/p16+ expression at recurrence. Our results fail to provide evidence that a minor HPV-negative tumor subfraction is responsible for biologically aggressive behavior of HPV+ oropharyngeal cancer that recurs after chemoradiation.

Refining risk stratification for locoregional failure after chemoradiotherapy in human papillomavirus-associated oropharyngeal cancer.

BACKGROUND: To determine whether the addition of molecular and imaging biomarkers to established clinical risk factors could help predict locoregional failure (LRF) after chemoradiation in human papillomavirus (HPV)-related (+) oropharyngeal cancer (OPC) and improve patient selection for locoregional treatment de-intensification. METHODS: HPV status was determined for 198 consecutive patients with stage III/IV OPC treated with definitive chemoradiation from 5/2003 to 10/2010. The impact of pre-therapy epidermal growth factor receptor (EGFR) overexpression; imaging biomarkers including primary tumor and nodal maximum standardized uptake values on FDG-PET, gross tumor volumes, and matted nodes; and clinical factors on LRF (including residual disease at adjuvant neck dissection) was assessed. RESULTS: Primary tumors were HPV+ in 184 patients and HPV-negative in 14. EGFR overexpression was related to HPV-negative status and was univariately associated with LRF in the overall population, but was neither retained in the multivariate model after adjustment for HPV status, nor associated with LRF in HPV+ patients. Similarly, imaging biomarkers were univariately associated with LRF, but correlated with T-stage and/or N-stage and did not remain predictive in HPV+ patients after adjustment for T4- and N3-stages, which were the only significant predictors of LRF on multivariate analysis. Among HPV+ patients with non-T4- or N3-stages, only minimal smoking was associated with decreased LRF. CONCLUSIONS: The prognostic impact of EGFR overexpression and imaging biomarkers on LRF was predominantly related to their association with HPV-negative status and T- or N-stage, respectively. Among HPV+ OPC patients treated with uniform chemoradiation, only T4-stage, N3-stage, and smoking contributed to risk-stratification for LRF.

COMMD1 expression is controlled by critical residues that determine XIAP binding.

COMMD {COMM [copper metabolism Murr1 (mouse U2af1-rs1 region 1)] domain-containing} proteins participate in several cellular processes, ranging from NF-kappaB (nuclear factor kappaB) regulation, copper homoeostasis, sodium transport and adaptation to hypoxia. The best-studied member of this family is COMMD1, but relatively little is known about its regulation, except that XIAP [X-linked IAP (inhibitor of apoptosis)] functions as its ubiquitin ligase. In the present study, we identified that the COMM domain of COMMD1 is required for its interaction with XIAP, and other COMMD proteins can similarly interact with IAPs. Two conserved leucine repeats within the COMM domain were found to be critically required for XIAP binding. A COMMD1 mutant which was unable to bind to XIAP demonstrated a complete loss of basal ubiquitination and great stabilization of the protein. Underscoring the importance of IAP-mediated ubiquitination, we found that long-term expression of wild-type COMMD1 results in nearly physiological protein levels as a result of increased ubiquitination, but this regulatory event is circumvented when a mutant form that cannot bind XIAP is expressed. In summary, our findings indicate that COMMD1 expression is controlled primarily by protein ubiquitination, and its interaction with IAP proteins plays an essential role.

COMMD1 promotes the ubiquitination of NF-kappaB subunits through a cullin-containing ubiquitin ligase.

NF-kappaB is a pleiotropic transcription factor involved in multiple processes, including inflammation and oncogenesis. We have previously reported that COMMD1 represses kappaB-dependent transcription by negatively regulating NF-kappaB-chromatin interactions. Recently, ubiquitination of NF-kappaB subunits has been similarly implicated in the control of NF-kappaB recruitment to chromatin. We report here that COMMD1 accelerates the ubiquitination and degradation of NF-kappaB subunits through its interaction with a multimeric ubiquitin ligase containing Elongins B and C, Cul2 and SOCS1 (ECS(SOCS1)). COMMD1-deficient cells demonstrate stabilization of RelA, greater nuclear accumulation of RelA after TNF stimulation, de-repression of several kappaB-responsive genes, and enhanced NF-kappaB-mediated cellular responses. COMMD1 binds to Cul2 in a stimulus-dependent manner and serves to facilitate substrate binding to the ligase by stabilizing the interaction between SOCS1 and RelA. Our data uncover that ubiquitination and degradation of NF-kappaB subunits by this COMMD1-containing ubiquitin ligase is a novel and critical mechanism of regulation of NF-kappaB-mediated transcription.

COMMD proteins, a novel family of structural and functional homologs of MURR1.

MURR1 is a multifunctional protein that inhibits nuclear factor kappaB (NF-kappaB), a transcription factor with pleiotropic functions affecting innate and adaptive immunity, apoptosis, cell cycle regulation, and oncogenesis. Here we report the discovery of a new family of proteins with homology to MURR1. These proteins form multimeric complexes and were identified in a biochemical screen for MURR1-associated factors. The family is defined by the presence of a conserved and unique motif termed the COMM (copper metabolism gene MURR1) domain, which functions as an interface for protein-protein interactions. Like MURR1, several of these factors also associate with and inhibit NF-kappaB. The proteins designated as COMMD or COMM domain containing 1-10 are extensively conserved in multicellular eukaryotic organisms and define a novel family of structural and functional homologs of MURR1. The prototype of this family, MURR1/COMMD1, suppresses NF-kappaB not by affecting nuclear translocation or binding of NF-kappaB to cognate motifs; rather, it functions in the nucleus by affecting the association of NF-kappaB with chromatin.