Christine M. Komarck

COMMD1 promotes the ubiquitination of NF‐κB subunits through a cullin‐containing ubiquitin ligase

NF‐κB is a pleiotropic transcription factor involved in multiple processes, including inflammation and oncogenesis. We have previously reported that COMMD1 represses κB‐dependent transcription by negatively regulating NF‐κB–chromatin interactions. Recently, ubiquitination of NF‐κB subunits has been similarly implicated in the control of NF‐κB recruitment to chromatin. We report here that COMMD1 accelerates the ubiquitination and degradation of NF‐κB subunits through its interaction with a multimeric ubiquitin ligase containing Elongins B and C, Cul2 and SOCS1 (ECSSOCS1). COMMD1‐deficient cells demonstrate stabilization of RelA, greater nuclear accumulation of RelA after TNF stimulation, de‐repression of several κB‐responsive genes, and enhanced NF‐κB‐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‐κB subunits by this COMMD1‐containing ubiquitin ligase is a novel and critical mechanism of regulation of NF‐κB‐mediated transcription.

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.

Nonendemic HPV-Positive Nasopharyngeal Carcinoma: Association With Poor Prognosis

PURPOSE To investigate the relationship between human papillomavirus (HPV) and Epstein-Barr virus (EBV) in nonendemic nasopharyngeal carcinoma (NPC) and assess the prognostic implications of viral status. METHODS AND MATERIALS Paraffin-embedded tumor specimens from 62 patients with primary NPC diagnosed between 1985 and 2011 were analyzed for EBV and high-risk HPV. EBV status was determined by the use of in situ hybridization for EBV encoded RNA. HPV status was assessed with p16 immunohistochemistry and multiplex polymerase chain reaction MassArray for determination of HPV type. Proportional hazards models were used to compare the risk of death among patients as stratified by viral status. RESULTS Of 61 evaluable tumors, 26 (43%) were EBV-positive/HPV-negative, 18 (30%) were HPV-positive/EBV-negative, and 17 (28%) were EBV/HPV-negative. EBV and HPV infection was mutually exclusive. HPV positivity was significantly correlated with World Health Organization grade 2 tumors, older age, and smoking (all P<.001). The racial distribution of the study population was 74% white, 15% African American, and 11% Asian/Middle Eastern. Among HPV-positive patients, 94% were white. At a median follow-up time of 7 years, HPV-positive and EBV/HPV-negative tumors exhibited worse outcomes than did EBV-positive tumors, including decreased overall survival (hazard ratio [HR] 2.98, P=.01; and HR 3.89, P=.002), progression-free survival (HR 2.55, P=.02; and HR 4.04, P<.001), and locoregional control (HR 4.01, P=.03; and HR 6.87, P=.001). CONCLUSION In our Midwestern population, high-risk HPV infection may play an etiologic role in the development of nonendemic, EBV-negative NPC. Compared with EBV-positive NPC, HPV-positive and EBV/HPV-negative NPC are associated with worse outcomes. A larger confirmatory study is needed to validate these findings.

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.

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. Cancer Prev Res; 8(3); 197–207. ©2015 AACR.

Human Papillomavirus Infection and Biomarkers in Sinonasal Inverted Papillomas: Clinical Significance and Molecular Mechanisms

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.

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.

Epidermal growth factor receptor, p16, cyclin D1, and p53 staining patterns for inverted papilloma

The aim of this study was better characterize the staining patterns of inverted papilloma (IP) with and without carcinoma by performing immunohistochemistry for p16, epidermal growth factor receptor (EGFR), p53, and cyclin D1 antibodies in a large patient cohort.

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.

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

BackgroundHead 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.MethodsWe 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.ResultsUM-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.ConclusionsOur 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.