Jeff Bruce

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. Clin Cancer Res; 19(8); 2154–62. ©2013 AACR.

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. Clin Cancer Res; 17(24); 7539–50. ©2011 AACR.

Fructose and carbonyl metabolites as endogenous toxins

Dietary fructose consumption is one of the environmental factors contributing to the development of obesity and a fatty liver (hepatic steatosis). A two-hit hypothesis has been proposed for progression of hepatic steatosis to the more serious non-alcoholic steatosis (NASH), with the first hit being hepatic steatosis, and the second hit being inflammation and associated oxidative stress caused by reactive oxygen species (ROS) formation. As well, fructose-fed rats develop insulin resistance and serum levels of methylglyoxal, a glycolytic metabolite, are increased. Previously we reported that glyoxal-induced hepatocyte cytotoxicity could be attributed to mitochondrial toxicity as mitochondrial membrane potential was decreased and cytotoxicity was increased several orders of magnitude by low non-cytotoxic doses of H(2)O(2) (hepatocyte inflammation model). In this study, we have assessed the toxicity of fructose towards hepatocytes and investigated the molecular cytotoxic mechanisms involved. Fructose itself was only toxic at 1.5M, whereas 12 mM caused 50% cell death in 2h if the hepatocytes were exposed to a non-cytotoxic dose of H(2)O(2) continuously generated by glucose and glucose oxidase. The cytotoxic mechanism involved oxidative stress as ROS and H(2)O(2) formation preceded cytotoxicity, and cytotoxicity was prevented by radical scavengers, lipid antioxidants and ROS scavengers. It is proposed that the highly potent Fenton derived ROS catalyse the oxidation of fructose and particularly its carbonyl metabolites glycolaldehyde, dihydroxyacetone, glyceraldehyde. The carbon radicals and glyoxal formed compromise the cells resistance to H(2)O(2).

Tumor-derived exosomes and microvesicles in head and neck cancer: Implications for tumor biology and biomarker discovery

Exosomes and microvesicles (MVs) are nanometer‐sized, membranous vesicles secreted from many cell types into their surrounding extracellular space and into body fluids. These two classes of extracellular vesicles are regarded as a novel mechanism through which cancer cells, including virally infected cancer cells, regulate their micro‐environment via the horizontal transfer of bioactive molecules: proteins, lipids, and nucleic acids (DNA, mRNA, micro‐RNAs; oncogenic cargo hence often referred to as oncosomes). In head and neck cancer (HNC), exosomes and MVs have been described in Epstein Barr Virus (EBV)‐associated nasopharyngeal cancer (NPC), as well as being positively correlated with oral squamous cell carcinoma (OSCC) progression. It has therefore been suggested that HNC‐derived vesicles could represent a useful source for biomarker discovery, enriched in tumor antigens and cargo; hence fundamentally important for cancer progression. This current review offers an overall perspective on the roles of exosomes and MVs in HNC biology, focusing on EBV‐associated NPC and OSCC. We also highlight the importance of saliva as a proximal and easily accessible bio‐fluid for HNC detection, and propose that salivary vesicles might serve as an alternative model in the discovery of novel HNC biomarkers.

Novel Insights into Head and Neck Cancer using Next-Generation “Omic” Technologies

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous disease that develops via one of the two primary carcinogenic routes: chemical carcinogenesis through exposure to tobacco and alcohol or virally induced tumorigenesis. Human papillomavirus (HPV)-positive (HPV(+)) and HPV-negative (HPV(-)) HNSCCs represent distinct clinical entities, with the latter associated with significantly inferior outcome. The biologic basis of these different outcomes is an area of intense investigation; their therapeutic regimens are currently also being reevaluated, which would be significantly facilitated by reliable biomarkers for stratification. With the advent of the omics era and accelerated development of targeted therapies, there are unprecedented opportunities to address the challenges in the management of HNSCC. As summarized herein, side-by-side molecular characterization of HPV(+) versus HPV(-) HNSCC has revealed distinct molecular landscapes, novel prognostic signatures, and potentially targetable biologic pathways. In particular, we focus on the evidence acquired from genome-wide omics pertinent to our understanding of the clinical behavior of HNSCC and on insights into personalized treatment opportunities. Integrating, mining, and validating these data toward clinically meaningful outcomes for patients with HNSCC in conjunction with systematic verification of the functional relevance of these findings are critical steps toward the design of personalized therapies.

Nasopharyngeal Cancer: Molecular Landscape

Nasopharyngeal carcinoma (NPC) is a unique epithelial malignancy arising from the superior aspect of the pharyngeal mucosal space, associated with latent Epstein-Barr virus infection in most cases. The capacity to characterize cancer genomes in unprecedented detail is now providing insights into the genesis and molecular underpinnings of this disease. Herein, we provide an overview of the molecular aberrations that likely drive nasopharyngeal tumor development and progression. The contributions of major Epstein-Barr virus-encoded factors, including proteins, small RNAs, and microRNAs, along with their interactions with pathways regulating cell proliferation and survival are highlighted. We review recent analyses that clearly define the role of genetic and epigenetic variations affecting the human genome in NPC. These findings point to the impact of DNA methylation and histone modifications on gene expression programs that promote this malignancy. The molecular interactions that allow NPC cells to evade immune recognition and elimination, which is crucial for the survival of cells expressing potentially immunogenic viral proteins, are also described. Finally, the potential utility of detecting host and viral factors for the diagnosis and prognosis of NPC is discussed. Altogether, the studies summarized herein have greatly expanded our knowledge of the molecular biology of NPC, yet much remains to be uncovered. Emerging techniques for using and analyzing well-annotated biospecimens from patients with NPC will ultimately lead to a greater level of understanding, and enable improvements in precision therapies and clinical outcomes.

MiR-449a promotes breast cancer progression by targeting CRIP2

The identification of prognostic biomarkers and their underlying mechanisms of action remain of great interest in breast cancer biology. Using global miRNA profiling of 71 lymph node-negative invasive ductal breast cancers and 5 normal mammary epithelial tissues, we identified miR-449a to be highly overexpressed in the malignant breast tissue. Its expression was significantly associated with increased incidence of patient relapse, decreased overall survival, and decreased disease-free survival. In vitro, miR-449a promoted breast cancer cell proliferation, clonogenic survival, migration, and invasion. By utilizing a tri-modal in silico approach for target identification, Cysteine-Rich Protein 2 (CRIP2; a transcription factor) was identified as a direct target of miR-449a, corroborated using qRT-PCR, Western blot, and luciferase reporter assays. MDA-MB-231 cells stably transfected with CRIP2 demonstrated a significant reduction in cell viability, migration, and invasion, as well as decreased tumor growth and angiogenesis in mouse xenograft models. Our data revealed that overexpression of miR-449a suppresses CRIP2, which then affects the tumor vasculature, likely via NF-κB/p65 complex-mediated transcription of VEGF. These finding define an oncogenic function of miR-449a in human breast cancer, and highlight the importance of this pathway in driving aggressive behaviour.

MicroRNAs in nasopharyngeal carcinoma

MicroRNAs (miRNAs) provide insight into both the biology and clinical behavior of many human cancers, including nasopharyngeal carcinoma (NPC). The dysregulation of miRNAs in NPC results in a variety of tumor-promoting effects. Furthermore, several miRNAs are prognostic markers for NPC. In addition to cellular miRNAs, NPC samples also often contain miRNAs encoded by Epstein-Barr virus, and these miRNAs may impact NPC biology by targeting both cellular and viral genes. Given their numerous putative roles in NPC development and progression, a thorough understanding of the impact of miRNA dysregulation in NPC is expected to shed light on useful biomarkers and therapeutic targets for the clinical management of this disease. In this review, we describe the efforts to date to identify and characterize such miRNAs in the context of NPC.

Hepatocyte inflammation model for cytotoxicity research: fructose or glycolaldehyde as a source of endogenous toxins

Insulin resistance and hepatotoxicity induced in high fructose fed rats may involve fructose derived endogenous toxins formed by inflammation. Thus fructose was seventy-fold more toxic if hepatocytes were exposed to non-toxic levels of hydrogen peroxide (H2O2) released by inflammatory cells. This was prevented by iron (Fe) chelators, hydroxyl radical scavengers, and increased by Fe, copper (Cu) or catalase inhibition. Fructose or glyceraldehyde/dihydroxyacetone metabolites were oxidized by Fenton radicals to glyoxal. Glyoxal (15μM) cytotoxicity was increased about 200-fold by H2O2. Glycolaldehyde was enzymically formed from glyceraldehyde, the fructokinase/aldolase B product of fructose. Glycolaldehyde cytotoxicity was increased 20-fold by H2O2. The oxidative stress cytotoxicity induced was attributed to the Fenton oxidation of glycolaldehyde forming glycolaldehyde radicals and glyoxal, since cytotoxicity was prevented by aminoguanidine (glyoxal trap) or Fenton inhibitors. Glyoxal was also the Fenton product responsible for glycolaldehyde protein carbonylation as carbonylation was prevented by aminoguanidine or Fenton inhibitors.

Somatic BRCA1/2 Recovery as a Resistance Mechanism After Exceptional Response to Poly (ADP-ribose) Polymerase Inhibition

Purpose Durable and long-term responses to the poly (ADP-ribose) polymerase inhibitor olaparib are observed in patients without BRCA1/2 mutations. However, beyond BRCA1/2 mutations, there are no approved biomarkers for olaparib in high-grade serous ovarian cancer (HGSOC). To determine mechanisms of durable response and resistance to olaparib therapy, we performed an analysis of HGSOC tumors from three patients without germline BRCA1/2 mutations who experienced exceptional responses to olaparib. Patients and Methods We performed integrated exome, low-pass genome, and RNA sequence analysis of tumors at diagnosis and upon relapse from patients with platinum-sensitive HGSOC recurrence who were treated > 5 years with olaparib therapy as a single agent. Results We observed somatic disruption of BRCA1/2 in all three patients at diagnosis, followed by subsequent BRCA recovery upon progression by copy number gain and/or upregulation of the remaining functional allele in two patients. The third patient with ongoing response (> 7 years) had a tumor at diagnosis with biallelic somatic deletion and loss-of-function mutation, thereby lacking a functional allele for recovery of BRCA1 activity and indicating a potential cure. Conclusion Olaparib has durable benefit for patients with ovarian cancer beyond germline BRCA1/2 carriers. These data suggest that biallelic loss of BRCA1/2 in cancer cells may be a potential marker of long-term response to poly (ADP-ribose) polymerase inhibition and that restoration of homologous repair function may be a mechanism of disease resistance.