Integrated Genomic and Functional microRNA Analysis Identifies miR-30-5p as a Tumor Suppressor and Potential Therapeutic Nanomedicine in Head and Neck Cancer.

PURPOSE: To identify deregulated and inhibitory miRNAs and generate novel mimics for replacement nanomedicine for head and neck squamous cell carcinomas (HNSCC). EXPERIMENTAL DESIGN: We integrated miRNA and mRNA expression, copy number variation, and DNA methylation results from The Cancer Genome Atlas (TCGA), with a functional genome-wide screen. RESULTS: We reveal that the miR-30 family is commonly repressed, and all 5 members sharing these seed sequence similarly inhibit HNSCC proliferation in vitro. We uncover a previously unrecognized inverse relationship with overexpression of a network of important predicted target mRNAs deregulated in HNSCC, that includes key molecules involved in proliferation (EGFR, MET, IGF1R, IRS1, E2F7), differentiation (WNT7B, FZD2), adhesion, and invasion (ITGA6, SERPINE1). Reexpression of the most differentially repressed family member, miR-30a-5p, suppressed this mRNA program, selected signaling proteins and pathways, and inhibited cell proliferation, migration, and invasion in vitro. Furthermore, a novel miR-30a-5p mimic formulated into a targeted nanomedicine significantly inhibited HNSCC xenograft tumor growth and target growth receptors EGFR and MET in vivo. Significantly decreased miR-30a/e family expression was related to DNA promoter hypermethylation and/or copy loss in TCGA data, and clinically with decreased disease-specific survival in a validation dataset. Strikingly, decreased miR-30e-5p distinguished oropharyngeal HNSCC with poor prognosis in TCGA (P = 0.002) and validation (P = 0.007) datasets, identifying a novel candidate biomarker and target for this HNSCC subset. CONCLUSIONS: We identify the miR-30 family as an important regulator of signal networks and tumor suppressor in a subset of HNSCC patients, which may benefit from miRNA replacement nanomedicine therapy.

Genomic and Transcriptomic Characterization Links Cell Lines with Aggressive Head and Neck Cancers.

Cell lines are important tools for biological and preclinical investigation, and establishing their relationship to genomic alterations in tumors could accelerate functional and therapeutic discoveries. We conducted integrated analyses of genomic and transcriptomic profiles of 15 human papillomavirus (HPV)-negative and 11 HPV-positive head and neck squamous cell carcinoma (HNSCC) lines to compare with 279 tumors from The Cancer Genome Atlas (TCGA). We identified recurrent amplifications on chromosomes 3q22-29, 5p15, 11q13/22, and 8p11 that drive increased expression of more than 100 genes in cell lines and tumors. These alterations, together with loss or mutations of tumor suppressor genes, converge on important signaling pathways, recapitulating the genomic landscape of aggressive HNSCCs. Among these, concurrent 3q26.3 amplification and TP53 mutation in most HPV(-) cell lines reflect tumors with worse survival. Our findings elucidate and validate genomic alterations underpinning numerous discoveries made with HNSCC lines and provide valuable models for future studies.

Nanocomplex-based TP53 gene therapy promotes anti-tumor immunity through TP53- and STING-dependent mechanisms.

Loss or mutation of TP53 has been linked to alterations in anti-tumor immunity as well as dysregulation of cell cycle and apoptosis. We explored immunologic effects and mechanisms following restoration of wild-type human TP53 cDNA in murine oral cancer cells using the therapeutic nanocomplex scL-53. We demonstrated scL-53 induces dose-dependent expression of TP53 and induction of apoptosis and immunogenic cell death. We further demonstrated both TP53-dependent and independent induction of tumor cell immunogenicity through the use of blocking mAbs, nanocomplex loaded with DNA plasmid with or without TP53 cDNA, empty nanocomplex and siRNA knockdown techniques. TP53-independent immune modulation was observed following treatment with nanocomplex loaded with DNA plasmid lacking TP53 cDNA and abrogated in STING-deficient tumor cells, supporting the presence of a cytoplasmic DNA sensing, STING-dependent type-I IFN response. Cooperatively, TP53- and STING-dependent alterations sensitized tumor cells to CTL-mediated lysis, which was further enhanced following reversal of adaptive immune resistance with PD-1 mAb. In vivo, combination scL-53 and PD-1 mAb resulted in growth control or rejection of established tumors that was abrogated in mice depleted of CD8+ cells or in STING deficient mice. Cumulatively, this work demonstrates 1) a direct anti-tumor effects of functional TP53; 2) non-redundant TP53- and STING-dependent induction of tumor cell immunogenicity following scL-53 treatment; and 3) that adaptive immune resistance following scL-53 treatment can be reversed with PD-based immune checkpoint blockade, resulting in the rejection or control of syngeneic murine tumors. These data strongly support the clinical combination of scL-53 and immune checkpoint blockade.

Conditional deletion of nonmuscle myosin II-A in mouse tongue epithelium results in squamous cell carcinoma.

To investigate the contribution of nonmuscle myosin II-A (NM II-A) to early cardiac development we crossed Myh9 floxed mice and Nkx2.5 cre-recombinase mice. Nkx2.5 is expressed in the early heart (E7.5) and later in the tongue epithelium. Mice homozygous for deletion of NM II-A (A(Nkx)/A(Nkx)) are born at the expected ratio with normal hearts, but consistently develop an invasive squamous cell carcinoma (SCC) of the tongue (32/32 A(Nkx)/A(Nkx)) as early as E17.5. To assess reproducibility a second, independent line of Myh9 floxed mice derived from a different embryonic stem cell clone was tested. This second line also develops SCC indistinguishable from the first (15/15). In A(Nkx)/A(Nkx) mouse tongue epithelium, genetic deletion of NM II-A does not affect stabilization of TP53, unlike a previous report for SCC. We attribute the consistent, early formation of SCC with high penetrance to the role of NM II in maintaining mitotic stability during karyokinesis.

Tapping microRNA regulation networks through integrated analysis of microRNA-mRNA high-throughput profiles.

Understanding the biological relevance and context of microRNA (miRNA) regulation of target mRNAs is difficult to ascertain because an individual miRNA aids simultaneously in the regulation of hundreds of mRNAs in a cell. With the increasing availability of large public datasets that profile both mRNA and miRNA expression levels from the same samples, it is possible to apply robust statistical methods to identify global negative correlations in miRNA and target mRNA expression. Using a dataset from The Cancer Genome Atlas as a case study, we show how to use linear regression analysis followed by permutation-based false discovery rate to assign high statistical power to pair-wise negative correlations of miRNA and mRNA expression. Used in conjunction with available prediction tools or other target databases, a high confidence dataset of global miRNA-mRNA interactions can be generated. We also describe further methods to prioritize identified interactions by integrating with mutation, copy number variation, methylation, or survival data to support observations and provide context. Finally, we discuss methods to experimentally validate selected novel targets.

Caloric restriction augments radiation efficacy in breast cancer.

Dietary modification such as caloric restriction (CR) has been shown to decrease tumor initiation and progression. We sought to determine if nutrient restriction could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (IR) and alter the molecular profile of triple-negative breast cancer (TNBC), which displays a poor prognosis. In two murine models of TNBC, significant tumor regression is noted with IR or diet modification, and a greater regression is observed combining diet modification with IR. Two methods of diet modification were compared, and it was found that a daily 30% reduction in total calories provided more significant tumor regression than alternate day feeding. At the molecular level, tumors treated with CR and IR showed less proliferation and more apoptosis. cDNA array analysis demonstrated the IGF-1R pathway plays a key role in achieving this physiologic response, and multiple members of the IGF-1R pathway including IGF-1R, IRS, PIK3ca and mTOR were found to be downregulated. The innovative use of CR as a novel therapeutic option has the potential to change the biology of tumors and enhance the opportunity for clinical benefit in the treatment of patients with TNBC.

Signaling Networks of Activated Oncogenic and Altered Tumor Suppressor Genes in Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) arises from the upper aerodigestive tract and is the six most common cancers worldwide. HNSCC is associated with high morbidity and mortality, as standard surgery, radiation, and chemotherapy can cause significant disfigurement and only provide 5-year survival rates of ~50-60%. The heterogeneity of HNSCC subsets with different potentials for recurrence and metastasis challenges the traditional pathological classification system, thereby increasing demand for the development of new diagnostic, prognostic, and therapeutic tools based on global molecular signatures of HNSCC. Historically, using classical biological techniques, it has been extremely difficult and time-consuming to survey hundreds or thousands of genes in a given disease. However, the development of high throughput technologies and high-powered computation throughout the last two decades has enabled us to investigate hundreds or thousands of genes simultaneously. Using high throughput technologies, our laboratory has identified the gene signatures and protein networks, which significantly affect HNSCC malignant phenotypes, including TP53/p63/p73 family members, IL-1/TNF-ß/NF-κB, PI3K/AKT/mTOR, IL-6/IL-6R/JAK/STAT3, EGFR/MAPK/AP1, HGF/cMET/EGR1, and TGFß/TGFßR/TAK1/SMAD pathways. This review summarizes the results from high-throughput technological assays conducted on HNSCC samples, including microarray, DNA methylation, miRNA profiling, and protein array, using primarily experimental data and conclusions generated in our own laboratory. The use of bioinformatics and integrated analyses of data sets from different platforms, as well as meta-analysis of large datasets pulled from multiple publicly available studies, provided significantly higher statistical power to extract biologically relevant information. The data suggested that the heterogeneity of HNSCC genotype and phenotype are much more complex than we previously thought. Understanding of global molecular signatures and disease classification for specific subsets of HNSCC will be essential to provide accurate diagnoses for targeted therapy and personalized treatment, which is an important effort toward improving patient outcomes.

MicroRNA-203 regulates caveolin-1 in breast tissue during caloric restriction.

Caloric restriction has been shown to increase lifespan in several organisms and to delay onset of age-related diseases. The transcriptional response to caloric restriction has been studied for mRNAs, while the microRNA signature following caloric restriction remains unexplored. Here, we characterize the microRNA expression in mouse breast tissue before and after caloric restriction, reporting several changes in the microRNA expression profile. In particular, miR-203 is found to be highly induced by caloric restriction, and we demonstrate that caveolin-1 as well as p63 are direct targets of miR-203 in vivo during caloric restriction. Using tissue culture models, we suggest that this regulation is important in both mouse and human. In conclusion, we show that the microRNA response induced by caloric restriction can regulate important factors in processes such as longevity and aging and is an integral and important component of the cellular response to caloric restriction.

Cellular stress induced alterations in microRNA let-7a and let-7b expression are dependent on p53.

Genotoxic stressors, such as radiation, induce cellular damage that activates pre-programmed repair pathways, some of which involve microRNAs (miRNA) that alter gene expression. The let-7 family of miRNA regulates multiple cellular processes including cell division and DNA repair pathways. However, the role and mechanism underlying regulation of let-7 genes in response to stress have yet to be elucidated. In this study we demonstrate that let-7a and let-7b expression decreases significantly following exposure to agents that induce stress including ionizing radiation. This decrease in expression is dependent on p53 and ATM in vitro and is not observed in a p53(-/-) colon cancer cell line (HCT116) or ATM(-/-) human fibroblasts. Chromatin Immunoprecipitation (ChIP) analysis showed p53 binding to a region upstream of the let-7 gene following radiation exposure. Luciferase transient transfections demonstrated that this p53 binding site is necessary for radiation-induced decreases in let-7 expression. A radiation-induced decrease in let-7a and let-7b expression is also observed in radiation-sensitive tissues in vivo and correlates with altered expression of proteins in p53-regulated pro-apoptotic signaling pathways. In contrast, this decreased expression is not observed in p53 knock-out mice suggesting that p53 directly repress let-7 expression. Exogenous expression of let-7a and let-7b increased radiation-induced cytotoxicity in HCT116 p53(+/+) cells but not HCT116 p53(-/-) cells. These results are the first demonstration of a mechanistic connection between the radiation-induced stress response and the regulation of miRNA and radiation-induced cytotoxicity and suggest that this process may be a molecular target for anticancer agents.

Hydrolysis of bulged nucleotides in hybrids formed by RNA and imidazole-derivatized oligo-2'-O-methylribonucleotides.

In order to enhance the efficacy of small antisense molecules, we examined a series of antisense oligonucleotides derivatized with functional groups designed to enable them to hydrolyze their RNA target. Solid phase synthetic methods were used to prepare imidazole-derivatized antisense oligo-2'-O-methylribonucleotides. Upon binding, these oligonucleotides create internal bulged bases in the target RNA that serve as sites for hydrolysis. We observed that an oligonucleotide derivatized with a side chain containing two imidazole groups was capable of hydrolyzing 58% of its RNA target when incubated with the target for 48 hours at 37°C and physiological pH.

Ionizing radiation-induced oxidative stress alters miRNA expression.

BACKGROUND: MicroRNAs (miRNAs) are small, highly conserved, non-coding RNA that alter protein expression and regulate multiple intracellular processes, including those involved in the response to cellular stress. Alterations in miRNA expression may occur following exposure to several stress-inducing anticancer agents including ionizing radiation, etoposide, and hydrogen peroxide (H(2)O(2)). METHODOLOGY/PRINCIPAL FINDINGS: Normal human fibroblasts were exposed to radiation, H(2)O(2), or etoposide at doses determined by clonogenic cell survival curves. Total RNA was extracted and miRNA expression was determined by microarray. Time course and radiation dose responses were determined using RT-PCR for individual miRNA species. Changes in miRNA expression were observed for 17 miRNA species following exposure to radiation, 23 after H(2)O(2) treatment, and 45 after etoposide treatment. Substantial overlap between the miRNA expression changes between agents was observed suggesting a signature miRNA response to cell stress. Changes in the expression of selected miRNA species varied in response to radiation dose and time. Finally, production of reactive oxygen species (ROS) increased with increasing doses of radiation and pre-treatment with the thiol antioxidant cysteine decreased both ROS production and the miRNA response to radiation. CONCLUSIONS: These results demonstrate a common miRNA expression signature in response to exogenous genotoxic agents including radiation, H(2)O(2), and etoposide. Additionally, pre-treatment with cysteine prevented radiation-induced alterations in miRNA expression which suggests that miRNAs are responsive to oxidative stress. Taken together, these results imply that miRNAs play a role in cellular defense against exogenous stress and are involved in the generalized cellular response to genotoxic oxidative stress.

Allosteric inhibition of the HIV-1 Rev/RRE interaction by a 3'-methylphosphonate modified antisense oligo-2'-O-methylribonucleotide.

The HIV-1 Rev response element (RRE), a highly structured RNA sequence consisting of five stemloops, is found in all spliced and partially spliced human immunodeficiency virus (HIV) mRNA transcripts. The RRE interacts with HIV-encoded Rev protein, which facilitates exit of the transcripts from the nucleus to the cytoplasm. Because the Rev/RRE interaction is critical to virus function, it is considered a potential target for therapeutic drugs. We have investigated the interactions of antisense oligonucleotides with stem-loop II, a region that contains the high-affinity binding site for Rev. Oligo-2'-O-methylribonucleotides terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were targeted to the 5'- or 3'-side of stem-loop IIB, which is adjacent to the Rev binding site. Thermal denaturation experiments showed that oligonucleotides of this type form highly stable duplexes with complementary single-stranded RNA. Gel electrophoretic mobility shift assays (EMSA) showed that the oligonucleotides bound with high affinity and specificity at 37 degrees C to RRE stem-loop II RNA with apparent dissociation constants, K(D), in the low nM range. A 16-mer, 2-1mp, whose K(D) is 46 nM, competitively inhibited binding of Rev peptide to RRE stem-loop II RNA as shown by EMSA experiments. When transfected into HEK 293T cells, 2-1mp inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) by 60% at a concentration of 300 nM oligonucleotide. These results are consistent with a mechanism by which 2-1mp blocks access of Rev to the RRE/CAT transcript thus preventing nuclear export and subsequent translation.

Chimeric RNase H-competent oligonucleotides directed to the HIV-1 Rev response element.

Chimeric oligo-2'-O-methylribonucleotides containing centrally located patches of contiguous 2'-deoxyribonucleotides and terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were prepared. The oligonucleotides were targeted to the 3'-side of HIV Rev response element (RRE) stem-loop IIB RNA, which is adjacent to the high affinity Rev protein binding site and is critical to virus function. Thermal denaturation experiments showed that chimeric oligonucleotides form very stable duplexes with a complementary single-stranded RNA, and gel electrophoretic mobility shift assays (EMSA) showed that they bind with high affinity and specificity to RRE stem-loop II RNA (K(D) approximately 200 nM). The chimeric oligonucleotides promote RNase H-mediated hydrolysis of RRE stem-loop II RNA and have half-lives exceeding 24h when incubated in cell culture medium containing 10% fetal calf serum. One of the chimeric oligonucleotides inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) approximately 60% at a concentration of 300 nM in HEK 293T cells co-transfected with p-RRE/CAT and p-Rev mammalian expression vectors.