An architecturally rational hemostat for rapid stopping of massive bleeding on anticoagulation therapy.

Hemostatic devices are critical for managing emergent severe bleeding. With the increased use of anticoagulant therapy, there is a need for next-generation hemostats. We rationalized that a hemostat with an architecture designed to increase contact with blood, and engineered from a material that activates a distinct and undrugged coagulation pathway can address the emerging need. Inspired by lung alveolar architecture, here, we describe the engineering of a next-generation single-phase chitosan hemostat with a tortuous spherical microporous design that enables rapid blood absorption and concentrated platelets and fibrin microthrombi in localized regions, a phenomenon less observed with other classical hemostats without structural optimization. The interaction between blood components and the porous hemostat was further amplified based on the charged surface of chitosan. Contrary to the dogma that chitosan does not directly affect physiological clotting mechanism, the hemostat induced coagulation via a direct activation of platelet Toll-like receptor 2. Our engineered porous hemostat effectively stopped the bleeding from murine liver wounds, swine liver and carotid artery injuries, and the human radial artery puncture site within a few minutes with significantly reduced blood loss, even under the anticoagulant treatment. The integration of engineering design principles with an understanding of the molecular mechanisms can lead to hemostats with improved functions to address emerging medical needs.

ß-catenin/CBP activation of mTORC1 signaling promotes partial epithelial-mesenchymal states in head and neck cancer.

Head and neck cancers, which include oral squamous cell carcinoma (OSCC) as a major subsite, exhibit cellular plasticity that includes features of an epithelial-mesenchymal transition (EMT), referred to as partial-EMT (p-EMT). To identify molecular mechanisms contributing to OSCC plasticity, we performed a multiphase analysis of single cell RNA sequencing (scRNAseq) data from human OSCC. This included a multiresolution characterization of cancer cell subgroups to identify pathways and cell states that are heterogeneously represented, followed by casual inference analysis to elucidate activating and inhibitory relationships between these pathways and cell states. This approach revealed signaling networks associated with hierarchical cell state transitions, which notably included an association between ß-catenin-driven CREB-binding protein (CBP) activity and mTORC1 signaling. This network was associated with subpopulations of cancer cells that were enriched for markers of the p-EMT state and poor patient survival. Functional analyses revealed that ß-catenin/CBP induced mTORC1 activity in part through the transcriptional regulation of a raptor-interacting protein, chaperonin containing TCP1 subunit 5 (CCT5). Inhibition of ß-catenin-CBP activity through the use of the orally active small molecule, E7386, reduced the expression of CCT5 and mTORC1 activity in vitro, and inhibited p-EMT-associated markers and tumor development in a murine model of OSCC. Our study highlights the use of multiresolution network analyses of scRNAseq data to identify targetable signals for therapeutic benefit, thus defining an underappreciated association between ß-catenin/CBP and mTORC1 signaling in head and neck cancer plasticity.

Inhibition of LSD1 Attenuates Oral Cancer Development and Promotes Therapeutic Efficacy of Immune Checkpoint Blockade and YAP/TAZ Inhibition.

Lysine-specific demethylase 1 (LSD1) is a histone demethylase that contributes to the etiology of oral squamous cell carcinoma (OSCC) in part by promoting cancer stem cell phenotypes. The molecular signals regulated by LSD1, or acting with LSD1, are poorly understood, particularly in the development of OSSC. In this study, we show that conditional deletion of the Lsd1 gene or pharmacologic inhibition of LSD1 in the tongue epithelium leads to reduced development of OSCC following exposure to the tobacco carcinogen 4NQO. LSD1 inhibition attenuated proliferation and clonogenic survival and showed an additive effect when combined with the YAP inhibitor Verteporfin. Interestingly, LSD1 inhibition upregulated the expression of PD-L1, leading to immune checkpoint inhibitor therapy responses. IMPLICATIONS: Collectively, our studies reveal a critical role for LSD1 in OSCC development and identification of tumor growth targeting strategies that can be combined with LSD1 inhibition for improved therapeutic application.

Novel peptide (RATH) mediated delivery of peptide nucleic acids for antiviral interventions.

The peptide nucleic acid (PNA) is a chimeric molecule with the nucleobases connected by peptide bonds. This chimeric nature gives the PNA certain therapeutic advantages over natural antisense nucleic acid molecules. The PNA probes are known for its better and stronger complementation with target nucleic acids. However, cellular delivery of PNA is a major hurdle due to the charge-neutral nature of the PNA. For cellular delivery of PNA, peptide-PNA conjugates are used. This approach may face some practical limitation in terms of PNA antisense activity. In this study, we propose a novel RATH-2 peptide-based non-covalent PNA delivery mechanism. We observed RATH-2 shows a favorable molecular interaction with PNA at 16:1 (peptide:PNA) molar ratio resulting in co-centric nanoparticle formation. With this combination, we could achieve as high as 93% cellular delivery of the PNA. The proposed non-covalent RATH:PNA delivery model showed endocytic entrapment free delivery of PNA. The study further demonstrated the therapeutic application of PNA with in vitro antiviral intervention model. Using RATH-2 non-covalent PNA delivery system, we could inhibit 69.5% viral load. The present study demonstrates a cell-penetrating peptide:PNA interaction can lead to nanoparticle formations that facilitated cellular delivery of PNA.Key points• A novel cell-penetrating peptide (RATH-2) was identified for non-covalent delivery of PNA.• RATH-2 and PNA formed co-centric nanoparticles at appropriate molar combination.• PNA delivered through the RATH-2 inhibited the viral gene expression and reduced the viral load.

LOXL2 promotes aggrecan and gender-specific anabolic differences to TMJ cartilage.

In the United States, 5-12% of adults have at least one symptom of temporomandibular joint (TMJ) disorders, including TMJ osteoarthritis (TMJ-OA). However, there is no chondroprotective agent that is approved for clinical application. We showed that LOXL2 is elevated in the regenerative response during fracture healing in mice and has a critical role in chondrogenic differentiation. Indeed, LOXL2 is an anabolic effector that attenuates pro-inflammatory signaling in OA cartilage of the TMJ and knee joint, induces chondroprotective and regenerative responses, and attenuates NF-kB signaling. The specific goal of the study was to evaluate if adenoviral delivery of LOXL2 is anabolic to human and mouse TMJ condylar cartilage in vivo and evaluate the protective and anabolic effect on cartilage-specific factors. We employed two different models to assess TMJ-OA. In one model, clinical TMJ-OA cartilage from 5 different samples in TMJ-OA cartilage plugs were implanted subcutaneously in nude mice. Adenovirus LOXL2 -treated implants showed higher mRNA levels of LOXL2, ACAN, and other anabolic genes compared to the adenovirus-Empty-treated implants. Further characterization by RNA-seq analysis showed LOXL2 promotes proteoglycan networks and extracellular matrix in human TMJ-OA cartilage implants in vivo. In order to evaluate if LOXL2-induced functional and sex-linked differences, both male and female four-month-old chondrodysplasia (Cho/+) mice, which develop progressive TMJ-OA due to a point mutation in the Col11a1 gene, were subjected to intraperitoneal injection with Adv-RFP-LOXL2 every 2 weeks for 12 weeks. The data showed that adenovirus delivery of LOXL2 upregulated LOXL2 and aggrecan (Acan), whereas MMP13 expression was slightly downregulated. The fold change expression of Acan and Runx2 induced by Adv-RFP-LOXL2 was higher in females compared to males. Interestingly, Adv-RFP-LOXL2 injection significantly increased Rankl expression in male but there was no change in females, whereas VegfB gene expression was increased in females, but not in males, as compared to those injected with Adv-RFP-Empty in respective groups. Our findings indicate that LOXL2 can induce specifically the expression of Acan and other anabolic genes in two preclinical models in vivo. Further, LOXL2 has beneficial functions in human TMJ-OA cartilage implants and promotes gender-specific anabolic responses in Cho/+ mice with progressive TMJ-OA, suggesting its merit for further study as an anabolic therapy for TMJ-OA.

ß-Catenin/CBP inhibition alters epidermal growth factor receptor fucosylation status in oral squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is a major driver of head and neck cancer, a devastating malignancy with a major sub-site in the oral cavity manifesting as oral squamous cell carcinoma (OSCC). EGFR is a glycoprotein receptor tyrosine kinase (RTK) whose activity is upregulated in >80% OSCC. Current anti-EGFR therapy relies on the use of cetuximab, a monoclonal antibody against EGFR, although it has had only a limited response in patients. Here, we uncover a novel mechanism regulating EGFR activity, identifying a role of the nuclear branch of the Wnt/ß-catenin signaling pathway, the ß-catenin/CBP axis, in control of post-translational modification of N-glycans on the EGFR. Genomic and structural analyses reveal that ß-catenin/CBP signaling represses fucosylation on the antennae of N-linked glycans on EGFR. By employing nUPLC-MS/MS, we determined that malignant human OSCC cells harbor EGFR with a paucity of N-glycan antennary fucosylation, while indolent cells display higher levels of fucosylation at sites N420 and N579. Additionally, treatment with either ICG-001 or E7386, which are both small molecule inhibitors of ß-catenin/CBP signaling, leads to increased transcriptional expression of fucosyltransferases FUT2 and FUT3, with a concomitant increase in EGFR N-glycan antennary fucosylation. In order to discover which fucosylated glycan epitopes are involved in the observed effect, we performed in-depth characterization of multiply-fucosylated N-glycans via tandem mass spectrometry analysis of the EGFR tryptic glycopeptides. Data are available via ProteomeXchange with identifier PXD017060. We propose that ß-catenin/CBP signaling promotes EGFR oncogenic activity in OSCC by inhibiting its N-glycan antennary fucosylation through transcriptional repression of FUT2 and FUT3.

Lysyl Oxidase-Like 2 Protects against Progressive and Aging Related Knee Joint Osteoarthritis in Mice.

BACKGROUND: The goal of this study was to determine if adenovirus-delivered LOXL2 protects against progressive knee osteoarthritis (OA), assess its specific mechanism of action; and determine if the overexpression of LOXL2 in transgenic mice can protect against the development of OA-related cartilage damage and joint disability. METHODS: Four-month-old Cho/+ male and female mice were intraperitoneally injected with either Adv-RFP-LOXL2 or an empty vector twice a month for four months. The proteoglycan levels and the expression of anabolic and catabolic genes were examined by immunostaining and qRT-PCR. The effect of LOXL2 expression on signaling was tested via the pro-inflammatory cytokine IL1ß in the cartilage cell line ATDC5. Finally; the OA by monosodium iodoacetate (MIA) injection was also induced in transgenic mice with systemic overexpression of LOXL2 and examined gene expression and joint function by treadmill tests and assessment of allodynia. RESULTS: The adenovirus treatment upregulated LOXL2; Sox9; Acan and Runx2 expression in both males and females. The Adv-RFP-LOXL2 injection; but not the empty vector injection increased proteoglycan staining and aggrecan expression but reduced MMP13 expression. LOXL2 attenuated IL-1ß-induced phospho-NF-κB/p65 and rescued chondrogenic lineage-related genes in ATDC5 cells; demonstrating one potential protective mechanism. LOXL2 attenuated phospho-NF-κB independent of its enzymatic activity. Finally; LOXL2-overexpressing transgenic mice were protected from MIA-induced OA-related functional changes; including the time and distance traveled on the treadmill and allodynia. CONCLUSION: Our study demonstrates that systemic LOXL2 adenovirus or LOXL2 genetic overexpression in mice can protect against OA. These findings demonstrate the potential for LOXL2 gene therapy for knee-OA clinical treatment in the future.

Functional and genomic analyses reveal therapeutic potential of targeting ß-catenin/CBP activity in head and neck cancer.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy characterized by tumor heterogeneity, locoregional metastases, and resistance to existing treatments. Although a number of genomic and molecular alterations associated with HNSCC have been identified, they have had limited impact on the clinical management of this disease. To date, few targeted therapies are available for HNSCC, and only a small fraction of patients have benefited from these treatments. A frequent feature of HNSCC is the inappropriate activation of ß-catenin that has been implicated in cell survival and in the maintenance and expansion of stem cell-like populations, thought to be the underlying cause of tumor recurrence and resistance to treatment. However, the therapeutic value of targeting ß-catenin activity in HNSCC has not been explored. METHODS: We utilized a combination of computational and experimental profiling approaches to examine the effects of blocking the interaction between ß-catenin and cAMP-responsive element binding (CREB)-binding protein (CBP) using the small molecule inhibitor ICG-001. We generated and annotated in vitro treatment gene expression signatures of HNSCC cells, derived from human oral squamous cell carcinomas (OSCCs), using microarrays. We validated the anti-tumorigenic activity of ICG-001 in vivo using SCC-derived tumor xenografts in murine models, as well as embryonic zebrafish-based screens of sorted stem cell-like subpopulations. Additionally, ICG-001-inhibition signatures were overlaid with RNA-sequencing data from The Cancer Genome Atlas (TCGA) for human OSCCs to evaluate its association with tumor progression and prognosis. RESULTS: ICG-001 inhibited HNSCC cell proliferation and tumor growth in cellular and murine models, respectively, while promoting intercellular adhesion and loss of invasive phenotypes. Furthermore, ICG-001 preferentially targeted the ability of subpopulations of stem-like cells to establish metastatic tumors in zebrafish. Significantly, interrogation of the ICG-001 inhibition-associated gene expression signature in the TCGA OSCC human cohort indicated that the targeted ß-catenin/CBP transcriptional activity tracked with tumor status, advanced tumor grade, and poor overall patient survival. CONCLUSIONS: Collectively, our results identify ß-catenin/CBP interaction as a novel target for anti-HNSCC therapy and provide evidence that derivatives of ICG-001 with enhanced inhibitory activity may serve as an effective strategy to interfere with aggressive features of HNSCC.

Measurement of lysyl oxidase activity from small tissue samples and cell cultures.

Increasing interest in the multifunctional lysyl oxidase family of proteins is evident from the growth in the number of new publications each year. The enzymes have unique properties of strong affinities to extracellular matrix components, relative insolubility in typical buffers, low catalytic rates, and often low abundance. Here we provide detailed protocols to extract and assay lysyl oxidase enzymes from tissue samples, cell culture cell layers, and media. Buffer conditions and procedures are optimized based on the characteristics mentioned above, while avoiding the use of radioactive substrates. Peroxidase/Amplex Red-based coupled reactions have proven to be the most useful in this context under specified conditions, and permit calculation of specific enzyme activities in absolute amounts of nanomoles of product/unit time/mg protein.

Inhibition of LSD1 epigenetically attenuates oral cancer growth and metastasis.

Lysine-specific demethylase 1 (LSD1) is a nuclear histone demethylase and a member of the amine oxidase (AO) family. LSD1 is a flavin-containing AO that specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 through an FAD-dependent oxidative reaction. LSD1 is inappropriately upregulated in lung, liver, brain and esophageal cancers, where it promotes cancer initiation, progression, and metastasis. However, unlike other lysine-specific demethylases, the role and specific targets of LSD1 in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. We show that LSD1 protein expression was increased in malignant OSCC tissues in a clinical tissue microarray, and its expression correlated with progressive tumor stages. In an orthotopic oral cancer mouse model, LSD1 overexpression in aggressive HSC-3 cells promoted metastasis whereas knockdown of LSD1 inhibited tumor spread, suggesting that LSD1 is a key regulator of OSCC metastasis. Pharmacological inhibition of LSD1 using a specific small molecule inhibitor, GSK-LSD1, down-regulated EGF signaling pathway. Further, GSK-LSD1 attenuates CTGF/CCN2, MMP13, LOXL4 and vimentin expression but increased E-cadherin expression in pre-existing, patient-derived tonsillar OSCC xenografts. Similarly, GSK-LSD1 inhibited proliferation and CTGF expression in mesenchymal cells, including myoepithelial cells and osteosarcoma cells. In addition, gene set enrichment analysis revealed that GSK-LSD1 increased p53 expression and apoptosis while inhibiting c-myc, ß-catenin and YAP-induced oncogenic transcriptional networks. These data reveal that aberrant LSD1 activation regulates key OSCC microenvironment and EMT promoting factors, including CTGF, LOXL4 and MMP13.

Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis.

BACKGROUND: Lysyl oxidase like-2 (LOXL2) is a copper-dependent amine oxidase. Our previous studies showed that LOXL2 is elevated during mouse fracture healing. The goal of this study was to evaluate the potential of LOXL2 to act as an anabolic agent in cartilage affected by osteoarthritis (OA). METHODS: LOXL2 was visualized in tissues from human knee and hip joints and temporomandibular joints (TMJ) by immunofluorescence. The activity of LOXL2 in human articular and TMJ chondrocytes was assessed by cell-based assays, microarray analysis, and RT-qPCR, and LOXL2-mediated activation of NF-κB and extracellular signal-related kinase (ERK) signaling pathways was measured by western blotting. To examine LOXL2-induced effect in vivo, we implanted Matrigel-imbedded human chondrocytes into nude mice and exposed them to exogenous LOXL2 for 6 weeks. Finally, LOXL2-induced effects on collagen type 2 α1 (COL2A1) and phospho-SMAD2/3 were evaluated by immunofluorescence analysis. RESULTS: LOXL2 staining was detected in damaged regions of human TMJ, hip and knee joints affected by OA. Stimulation with transforming growth factor (TGF)-ß1 upregulated LOXL2 expression, while pro-inflammatory cytokines IL-1ß and TNF-α downregulated LOXL2, in human chondrocytes. Viral transduction of LOXL2 in OA chondrocytes increased the mRNA levels of chondroitin sulfate proteoglycan (CSPG4), aggrecan (ACAN), sex determining region Y-box containing gene 9 (SOX9), and COL2A1 but reduced the levels of extracellular matrix (ECM)-degrading enzymes matrix metalloproteinase (MMP)1, MMP3, and MMP13. Further, forced expression of LOXL2 promoted chondrogenic lineage-specific gene expression, increased the expression of COL2A1 in the presence of TNF-α, and inhibited chondrocyte apoptosis. LOXL2 expression also inhibited IL-1ß-induced phospho-NF-κB/p65 and TGF-ß1-induced ERK1/2 phosphorylation. Matrigel constructs of human chondrocytes from the knee joint and TMJ implanted in nude mice showed anabolic responses after LOXL2 transduction, including increased expression of SOX9, ACAN, and COL2A1. Finally, immunofluorescence staining revealed co-localization of LOXL2 with SOX9 in the nuclei of cells in the implants, decreased phospho-SMAD2/3, and increased COL2A1 staining. CONCLUSION: Our results suggest that although LOXL2 is upregulated in cartilage affected by OA, this may be a protective response that promotes anabolism while inhibiting specific catabolic responses in the pathophysiology of OA.

TGF-ß1- and CCN2-Stimulated Sirius Red Assay for Collagen Accumulation in Cultured Cells.

A simple method for the determination of relative levels of insoluble collagen accumulation in fibroblast cultures is presented. Confluent cell cultures are provided with sodium ascorbate which is then permissive for collagen deposition. At intervals, cultures are fixed and stained successively with sirius red and then crystal Violet to, respectively, assess for relative changes in collagen accumulation in response to factors such as TGF-ß1 or matricellular CCN2 and changes in DNA content as an index of changes in cell density.

Role for the Aryl Hydrocarbon Receptor and Diverse Ligands in Oral Squamous Cell Carcinoma Migration and Tumorigenesis.

UNLABELLED: Over 45,000 new cases of oral and pharyngeal cancers are diagnosed and account for over 8,000 deaths a year in the United States. An environmental chemical receptor, the aryl hydrocarbon receptor (AhR), has previously been implicated in oral squamous cell carcinoma (OSCC) initiation as well as in normal tissue-specific stem cell self-renewal. These previous studies inspired the hypothesis that the AhR plays a role in both the acquisition and progression of OSCC, as well as in the formation and maintenance of cancer stem-like cells. To test this hypothesis, AhR activity in two oral squamous cell lines was modulated with AhR prototypic, environmental and bacterial AhR ligands, AhR-specific inhibitors, and phenotypic, genomic and functional characteristics were evaluated. The data demonstrate that: (i) primary OSCC tissue expresses elevated levels of nuclear AhR as compared with normal tissue, (ii) AhR mRNA expression is upregulated in 320 primary OSCCs, (iii) AhR hyperactivation with several ligands, including environmental and bacterial ligands, significantly increases AhR activity, ALDH1 activity, and accelerates cell migration, (iv) AhR inhibition blocks the rapid migration of OSCC cells and reduces cell chemoresistance, (v) AhR knockdown inhibits tumorsphere formation in low adherence conditions, and (vi) AhR knockdown inhibits tumor growth and increases overall survival in vivo These data demonstrate that the AhR plays an important role in development and progression of OSCC, and specifically cancer stem-like cells. Prototypic, environmental, and bacterial AhR ligands may exacerbate OSCC by enhancing expression of these properties. IMPLICATIONS: This study, for the first time, demonstrates the ability of diverse AhR ligands to regulate AhR activity in oral squamous cell carcinoma cells, as well as regulate several important characteristics of oral cancer stem cells, in vivo and in vitro Mol Cancer Res; 14(8); 696-706. ©2016 AACR.

Lysyl oxidase propeptide stimulates osteoblast and osteoclast differentiation and enhances PC3 and DU145 prostate cancer cell effects on bone in vivo.

Lysyl oxidase pro-enzyme is secreted by tumor cells and normal cells as a 50 kDa pro-enzyme into the extracellular environment where it is cleaved into the ~30 kDa mature enzyme (LOX) and 18 kDa pro-peptide (LOX-PP). Extracellular LOX enzyme activity is required for normal collagen and elastin extracellular cross-linking and maturation of the extracellular matrix. Extracellular LOX-PP acts as a tumor suppressor and can re-enter cells from the extracellular environment to induce its effects. The underlying hypothesis is that LOX-PP has the potential to promote bone cell differentiation, while inhibiting cancer cell effects in bone. Here we investigate the effect of LOX-PP on bone marrow cell proliferation and differentiation towards osteoblasts or osteoclasts, and LOX-PP modulation of prostate cancer cell conditioned media-induced alterations of proliferation and differentiation of bone marrow cells in vitro. Effects of overexpression of rLOX-PP in DU145 and PC3 prostate cancer cell lines on bone structure in vivo after intramedullary injections were determined. Data show that prostate cancer cell conditioned media inhibited osteoblast differentiation in bone marrow-derived cells, which was reversed by rLOX-PP treatment. Prostate cancer conditioned media stimulated osteoclast differentiation which was further enhanced by rLOX-PP treatment. rLOX-PP stimulated osteoclast differentiation by inhibiting OPG expression, up-regulating CCN2 expression, and increasing osteoclast fusion. In vivo studies indicate that rLOX-PP expression by PC3 cells implanted into the tibia of mice further enhanced PC3 cell ability to resorb bone, while rLOX-PP expression in DU145 cells resulted in non-significant increases in net bone formation. rLOX-PP enhances both osteoclast and osteoblast differentiation. rLOX-PP may serve to enhance coupling interactions between osteoclasts and osteoblasts helping to maintain a normal bone turnover in health, while contributing to bone abnormalities in disease.

Determination of cell uptake pathways for tumor inhibitor lysyl oxidase propeptide.

The lysyl oxidase propeptide (LOX-PP) is derived from pro-lysyl oxidase (Pro-LOX) by extracellular biosynthetic proteolysis. LOX-PP inhibits breast and prostate cancer xenograft tumor growth and has tumor suppressor activity. Although, several intracellular targets and molecular mechanisms of action of LOX-PP have been identified, LOX-PP uptake pathways have not been reported. Here we demonstrate that the major uptake pathway for recombinant LOX-PP (rLOX-PP) is PI3K-dependent macropinocytosis in PWR-1E, PC3, SCC9, MDA-MB-231 cell lines. A secondary pathway appears to be dynamin- and caveola dependent. The ionic properties of highly basic rLOX-PP provide buffering capacity at both high and low pHs. We suggest that the buffering capacity of rLOX-PP, which serves to limit endosomal acidification, sustains PI3K-dependent macropinocytosis in endosomes which in turn is likely to facilitate LOX-PP endosomal escape into the cytoplasm and its observed interactions with cytoplasmic targets and nuclear uptake.

A YAP/TAZ-Regulated Molecular Signature Is Associated with Oral Squamous Cell Carcinoma.

UNLABELLED: Oral squamous cell carcinoma (OSCC) is a prevalent form of cancer that develops from the epithelium of the oral cavity. OSCC is on the rise worldwide, and death rates associated with the disease are particularly high. Despite progress in understanding the mutational and expression landscape associated with OSCC, advances in deciphering these alterations for the development of therapeutic strategies have been limited. Further insight into the molecular cues that contribute to OSCC is therefore required. Here, we show that the transcriptional regulators YAP (YAP1) and TAZ (WWTR1), which are key effectors of the Hippo pathway, drive protumorigenic signals in OSCC. Regions of premalignant oral tissues exhibit aberrant nuclear YAP accumulation, suggesting that dysregulated YAP activity contributes to the onset of OSCC. Supporting this premise, we determined that nuclear YAP and TAZ activity drives OSCC cell proliferation, survival, and migration in vitro, and is required for OSCC tumor growth and metastasis in vivo. Global gene expression profiles associated with YAP and TAZ knockdown revealed changes in the control of gene expression implicated in protumorigenic signaling, including those required for cell cycle progression and survival. Notably, the transcriptional signature regulated by YAP and TAZ significantly correlates with gene expression changes occurring in human OSCCs identified by The Cancer Genome Atlas (TCGA), emphasizing a central role for YAP and TAZ in OSCC biology. IMPLICATIONS: This study defines a YAP/TAZ-regulated transcriptional program in OSCC and reveals novel roles for nuclear YAP/TAZ activity in the onset and progression of this cancer.

Orthotopic non-metastatic and metastatic oral cancer mouse models.

Oral cancer is characterized by high morbidity and mortality with a predisposition to metastasize to different tissues, including lung, liver, and bone. Despite progress in the understanding of mutational profiles and deregulated pathways in oral cancer, patient survival has not significantly improved over the past decades. Therefore, there is a need to establish in vivo models that recapitulate human oral cancer metastasis to evaluate therapeutic potential of novel drugs. Here we report orthotopic tongue cancer nude mouse models to study oral cancer growth and metastasis using human metastatic (UMSCC2) and non-metastatic (CAL27) cell lines, respectively. Transduction of these cell lines with lentivirus expressing red fluorescent protein (DsRed) followed by injection into tongues of immunodeficient mice generated orthotopic tongue tumors that could be monitored for growth and metastasis by fluorescence measurement with an in vivo Imaging System (IVIS 200). The growth rates of CAL27-DsRed induced tumors were higher than UMSCC2-DsRed tumors after day 15, while UMSCC2-DsRed tumors revealed metastasis beginning on day 21. Importantly, UMSCC2 tumors metastasized to a number of tissues including the submandibular gland, lung, kidney, liver, and bone. Further, immunohistochemical analyses of tongue tumors induced by CAL27 and UMSCC2 cells revealed elevated expression of components of protumorigenic pathways deregulated in human cancers, including Cyclin D1, PCNA, Ki-67, LSD1, LOXL2, MT-MMP1, DPAGT1, E-cadherin, OCT4A, and H3K4me1/2. These orthotopic mouse models are likely to be useful tools for gaining insights into the activity and mechanisms of novel oral cancer drug candidates.