Supplementary Methods and Supplementary Table 1. The supplementary data file contains the supplemental methods section describing the source of the patient samples used in the initial validation of the five-gene signature assay and supplementary table 1 which lists the number of patients with MB with tumor samples assessed per the five-gene signature assay from each clinical study as a second validation set.

The developmental role ofLef-1in ectodermal organs has been characterized usingLef-1murine knockout models. We generated aLef-1conditional over-expression (COEL) mouse to determine the role ofLef-1expression in epithelial structures at later stages of development after endogenous expression switches to the mesenchyme.Lef-1over expression (OE) in the oral epithelium creates a new dental epithelial stem cell niche that significantly increases incisor growth. These data indicate thatLef-1expression is switched off in the dental epithelial at early stages to maintain the stem cell niche and regulate incisor growth. Bioinformatics analyses indicated thatmiR-26bexpression increased coinciding with decreasedLef-1expression in the dental epithelium. We generated a murine model over-expressingmiR-26bthat targets endogenousLef-1expression andLef-1-related developmental mechanisms.miR-26bOE mice have ectodermal organ defects including a lack of incisors, molars, and hair similar to theLef-1null mice.miR-26bOE rescues theLef-1OE phenotype demonstrating a critical genetic and developmental role formiR-26bin the temporal and spatial expression ofLef-1in epithelial tissues.Lef-1expression regulates Wnt signaling and Wnt target genes as well as cell proliferation mechanisms, whilemiR-26bOE reduced the levels of Wnt target gene expression. The extra stem cell compartment in theCOELmice expressedLef-1suggesting thatLef-1is a stem cell factor, which was absent in themiR-26b OE/COELrescue mice. This is the first demonstration of a microRNA OE mouse model that has ectodermal organ defects. These findings demonstrate that the levels ofLef-1are critical for development and establish a role formiR-26bin the regulation of ectodermal organ development through the control ofLef-1expression and an endogenous stem cell niche.

The developmental role ofLef-1in ectodermal organs has been characterized usingLef-1murine knockout models. We generated aLef-1conditional over-expression (COEL) mouse to determine the role ofLef-1expression in epithelial structures at later stages of development after endogenous expression switches to the mesenchyme.Lef-1over expression (OE) in the oral epithelium creates a new dental epithelial stem cell niche that significantly increases incisor growth. These data indicate thatLef-1expression is switched off in the dental epithelial at early stages to maintain the stem cell niche and regulate incisor growth. Bioinformatics analyses indicated thatmiR-26bexpression increased coinciding with decreasedLef-1expression in the dental epithelium. We generated a murine model over-expressingmiR-26bthat targets endogenousLef-1expression andLef-1-related developmental mechanisms.miR-26bOE mice have ectodermal organ defects including a lack of incisors, molars, and hair similar to theLef-1null mice.miR-26bOE rescues theLef-1OE phenotype demonstrating a critical genetic and developmental role formiR-26bin the temporal and spatial expression ofLef-1in epithelial tissues.Lef-1expression regulates Wnt signaling and Wnt target genes as well as cell proliferation mechanisms, whilemiR-26bOE reduced the levels of Wnt target gene expression. The extra stem cell compartment in theCOELmice expressedLef-1suggesting thatLef-1is a stem cell factor, which was absent in themiR-26b OE/COELrescue mice. This is the first demonstration of a microRNA OE mouse model that has ectodermal organ defects. These findings demonstrate that the levels ofLef-1are critical for development and establish a role formiR-26bin the regulation of ectodermal organ development through the control ofLef-1expression and an endogenous stem cell niche.

A new plasmid-based microRNA inhibitor system (PMIS) effectively inhibits microRNA (miR) activity in cells and mice. We generated a novel complex RNA molecule carrying an anti-sense miR seed sequence that effectively knocks down endogenous miRs and mitigates their effects on messenger RNA in the cell. By using a native RNA-based molecule for miR knockdown, the PMIS platform avoids many of the traditional pitfalls associated with miR inhibition using synthetic oligonucleotides. The PMIS approach is more specific and stable, effective at a lower dose, more cost-effective and more importantly it is not toxic to living tissues, in contrast with many traditional miR inhibition strategies. The PMIS-miR complex is loaded into the RISC complex, binds to Ago and Dicer but specific engineering of the molecule inhibits processing of the complex. The PMIS-miR forms a stable complex that inhibits the mature miR from binding its target. We have generated miR knockdown mice that show defects in development. miR inhibition in mice reveals a role for miRs in many developmental pathways and cellular processes. This is the only miR inhibitor that effectively inhibits miRs in vivo. When combined with a bioinformatics approach the PMIS can determine new miR targets and biological processes. The PMIS can be delivered using viral vectors, nanoparticles and lipid-based systems to effectively knockdown miR activity. We have used the PMIS to reprogram cells, define new developmental processes and inhibit cancer cell growth. The PMIS is a new tool that is highly effective for inhibiting miR activity in vivo and in vitro. Importantly, the non-toxic nature of the PMIS molecule makes it promising platform for the delivery of miR inhibiting effects that could have potential as a treatment of human diseases and genetic defects, something that has proven difficult for traditional oligonucleotide approaches to miR inhibition. Support or Funding Information This work was supported by grants from the University of Iowa College of Dentistry, Carver College of Medicine and National Institutes of Health grant DE13941 to B.A.A.

There are many protocols used for regeneration therapies to regenerate tissues and structures. To date, tooth regeneration practices rely heavily on the use of stem cells, however, these can be difficult to obtain and culture. We have previously introduced a new method of cellular reprogramming by over expression of the transcription factor, Pitx2 and microRNA‐200a‐3p . We now demonstrate that over expression of the Paired Box 9 ( Pax9 ) gene and inhibition of miR‐200a using the Plasmid‐based miRNA Inhibitor System ( PMIS‐miR‐200a ), converts oral epithelial cells to progenitor dental mesenchyme cells. Transgenic DNA was introduced using the lentivirus packaging system. The microRNA inhibitor system PMIS‐miR‐200a was constructed by NaturemiRI. A two‐staged approach is used to generate stable oral epithelial cells expressing PAX9 , cells were sorted and after several weeks were transduced with PMIS‐miR‐200a , to inhibit miR‐200a activity. miR‐200a regulates Wnt and TGFß signaling and inhibition of miR‐200a allows for cells to convert to a progeny phenotype in the presence of PAX9 . These cells express early dental mesenchyme and stem cell markers making them suitable for tooth regeneration therapies. Current experiments are using reprogrammed dental epithelial and mesenchyme cells to form a tooth organ. These data demonstrate a new method for cell reprogramming and tissue regeneration. Support or Funding Information Support for this research was provided from grant DE013941 and DE018885 from the National Institute of Dental and Craniofacial Research.

MicroRNAs (miRs) regulate inflammation and BMP antagonists, thus they have potential uses as therapeutic reagents. However, the molecular function of miR-200c in modulating proinflammatory and bone metabolic mediators and osteogenic differentiation is not known. After miR-200c was transduced into a human embryonic palatal mesenchyme (HEPM) (a cell line of preosteoblasts), using lentiviral vectors, the resulting miR-200c overexpression increased osteogenic differentiation biomarkers, including osteocalcin (OCN) transcripts and calcium content. miR-200c expression also down-regulated interleukin (IL)-6, IL-8, and chemokine (C-C motif) ligand (CCL)-5 under lipopolysaccharide (LPS) stimulation and increased osteoprotegerin (OPG) in these cells. miR-200c directly regulates the expression of IL-6, IL-8 and CCL-5 transcripts by binding to their 3'UTRs. A plasmid-based miR-200c inhibitor effectively reduces their binding activities. Additionally, miR-200c delivered using polyethylenimine (PEI) nanoparticles effectively inhibits IL-6, IL-8 and CCL-5 in primary human periodontal ligament fibroblasts and increases the biomarkers of osteogenic differentiation in human bone marrow mesenchymal stem cells (MSCs), including calcium content, ALP, and Runx2. These data demonstrate that miR-200c represses IL-6, IL-8 and CCL-5 and improves osteogenic differentiation. miR-200c may potentially be used as an effective means to prevent periodontitis-associated bone loss by arresting inflammation and osteoclastogenesis and enhancing bone regeneration.

Sox2 marks dental epithelial stem cells (DESCs) in both mammals and reptiles, and in this article we demonstrate several Sox2 transcriptional mechanisms that regulate dental stem cell fate and incisor growth. Conditional Sox2 deletion in the oral and dental epithelium results in severe craniofacial defects, including impaired dental stem cell proliferation, arrested incisor development and abnormal molar development. The murine incisor develops initially but is absorbed independently of apoptosis owing to a lack of progenitor cell proliferation and differentiation. Tamoxifen-induced inactivation of Sox2 demonstrates the requirement of Sox2 for maintenance of the DESCs in adult mice. Conditional overexpression of Lef-1 in mice increases DESC proliferation and creates a new labial cervical loop stem cell compartment, which produces rapidly growing long tusk-like incisors, and Lef-1 epithelial overexpression partially rescues the tooth arrest in Sox2 conditional knockout mice. Mechanistically, Pitx2 and Sox2 interact physically and regulate Lef-1, Pitx2 and Sox2 expression during development. Thus, we have uncovered a Pitx2-Sox2-Lef-1 transcriptional mechanism that regulates DESC homeostasis and dental development.

Abstract Purpose: Distinct molecular subgroups of medulloblastoma, including hedgehog (Hh) pathway–activated disease, have been reported. We identified and clinically validated a five-gene Hh signature assay that can be used to preselect patients with Hh pathway–activated medulloblastoma. Experimental Design: Gene characteristics of the Hh medulloblastoma subgroup were identified through published bioinformatic analyses. Thirty-two genes shown to be differentially expressed in fresh-frozen and formalin-fixed paraffin-embedded tumor samples and reproducibly analyzed by RT-PCR were measured in matched samples. These data formed the basis for building a multi-gene logistic regression model derived through elastic net methods from which the five-gene Hh signature emerged after multiple iterations. On the basis of signature gene expression levels, the model computed a propensity score to determine Hh activation using a threshold set a priori. The association between Hh activation status and tumor response to the Hh pathway inhibitor sonidegib (LDE225) was analyzed. Results: Five differentially expressed genes in medulloblastoma (GLI1, SPHK1, SHROOM2, PDLIM3, and OTX2) were found to associate with Hh pathway activation status. In an independent validation study, Hh activation status of 25 medulloblastoma samples showed 100% concordance between the five-gene signature and Affymetrix profiling. Further, in medulloblastoma samples from 50 patients treated with sonidegib, all 6 patients who responded were found to have Hh-activated tumors. Three patients with Hh-activated tumors had stable or progressive disease. No patients with Hh-nonactivated tumors responded. Conclusions: This five-gene Hh signature can robustly identify Hh-activated medulloblastoma and may be used to preselect patients who might benefit from sonidegib treatment. Clin Cancer Res; 21(3); 585–93. ©2014 AACR.

Pitx2, Wnt/β-catenin signaling, and microRNAs (miRs) play a critical role in the regulation of dental stem cells during embryonic development. In this report, we have identified a Pitx2:β-catenin regulatory pathway involved in epithelial cell differentiation and conversion of mesenchymal cells to amelogenin expressing epithelial cells via miR-200a. Pitx2 and β-catenin are expressed in the labial incisor cervical loop or epithelial stem cell niche, with decreased expression in the differentiating ameloblast cells of the mouse lower incisor. Bioinformatics analyses reveal that miR-200a-3p expression is activated in the pre-ameloblast cells to enhance epithelial cell differentiation. We demonstrate that Pitx2 activates miR-200a-3p expression and miR-200a-3p reciprocally represses Pitx2 and β-catenin expression. Pitx2 and β-catenin interact to synergistically activate gene expression during odontogenesis and miR-200a-3p attenuates their expression and directs differentiation. To understand how this mechanism controls cell differentiation and cell fate, oral epithelial and odontoblast mesenchymal cells were reprogrammed by a two-step induction method using Pitx2 and miR-200a-3p. Conversion to amelogenin expressing dental epithelial cells involved an up-regulation of the stem cell marker Sox2 and proliferation genes and decreased expression of mesenchymal markers. E-cadherin expression was increased as well as ameloblast specific factors. The combination of Pitx2, a regulator of dental stem cells and miR-200a converts mesenchymal cells to a fully differentiated dental epithelial cell type. This pathway and reprogramming can be used to reprogram mesenchymal or oral epithelial cells to dental epithelial (ameloblast) cells, which can be used in tissue repair and regeneration studies.

microRNAs (miR) can act as oncogenes and tumor suppressors and several miRs are associated with cancer development and progression through the modulation of multiple cellular processes. miR26b is downregulated in several cancers and tumors and miR26b directly targets the lymphoid enhancer factor 1 (Lef1)3′UTR and inhibits endogenous Lef1 expression. We report that miR26b expression is associated with human colon cancer through the regulation of LEF1 expression in colon cancer cells. Analyses of multiple colon cancer cell lines revealed an inverse correlation between miR26b and LEF1 expression. Normal human colon cells express low levels of LEF1 and high levels of miR26b; however, human colon cancer cells have decreased miR26b expression and increased LEF1 expression. We demonstrate that miR26b expression is a potent inhibitor of colon cancer cell proliferation and significantly decreases LEF1 expression. The LEF1-regulated genes cyclin D1 and c-Myc were indirectly repressed by miR26b and this was consistent with decreased proliferation. miR26b overexpression in SW480 colon cancer cells also inhibited tumor growth in nude mice and this was due to decreased tumor growth and not apoptosis. Analyses of human colon cancer databases also demonstrated a link between miR26b and LEF1 expression. c-Myc expression is associated with multiple cancers and we propose that miR26b may act as a potential therapeutic agent in reducing cancer cell proliferation through repressing LEF1 activation of c-Myc and cyclin D1 expression. Mol Cancer Ther; 13(7); 1942–51. ©2014 AACR.

Abstract Medulloblastoma (MB), an invasive primitive neuroectodermal tumor of the posterior fossa, is the most common brain tumor in children, comprising ∼20% of childhood and <2% of adult brain tumors. Current standard of care treatment, surgery followed by craniospinal radiation and chemotherapy, can lead to significant long term toxicities, especially in very young patients. At the time of relapse, no standard salvage therapy exists. Therefore, targeted therapies are needed. Several studies have used gene expression profiling to identify distinct molecular subgroups of MB, including one characterized by activated Hedgehog (Hh) signaling. Using available gene expression data, a 5-gene Hh signature that can be assayed in formalin-fixed paraffin-embedded (FFPE) samples by standard RT-PCR was identified. Two sets of matched fresh frozen and FFPE MB specimens were used; one for development of the 5-gene signature and one for its independent validation. Hh activation status was determined in fresh frozen samples by gene expression profiling using the GeneChip human genome U133 Plus 2.0 array (Affymetrix, Santa Clara, CA) and in FFPE samples by RT-PCR analysis. The 5-gene Hh signature was selected from a larger panel of 73 genes that were associated with the Hh subgroup classification, as determined by standard Affymetrix gene expression profiling. Eighteen of these genes shown to be differentially expressed in FFPE were chosen for the RT-PCR gene card that formed the basis of the Elastic Net model building exercise. Based on the expression levels of the 5-gene signature, a predictive model was used to compute a propensity score (0-100%) representative of the Hh activation status of each tumor sample. The median propensity scores for the 17 non-Hh-activated tumors was 0.7% (range: 0.1-3.0%) compared to 87.9% (range: 69.1-97.6%) in the eight Hh-activated tumors. Hh activation status of 25 independent MB samples defined by the 5-gene signature and assayed by RT-PCR were in 100% agreement with the Hh activation status determined by gene expression profiling. In order to determine the predictive value of this assay as a tool to identify patients who might benefit from treatment with a Hh pathway inhibitor, MB samples from patients (n=13) enrolled in recent phase I trials of the Smoothened inhibitor LDE225 were analyzed and correlated with the respective tumor responses. Using the 5-gene signature, all patients (n=4) who responded to LDE225 treatment (PR or CR) were found to have Hh-pathway activated tumors, whereas all patients who did not respond (n=9) were found to have Hh non-activated tumors. These results suggest an association between Hh activation status determined by the 5-gene Hh signature and tumor response to LDE225 treatment. Data from an ongoing phase I/II trial in pediatric patients will enable determination of the predictive value of this patient pre-selection assay. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4818. doi:1538-7445.AM2012-4818

3092 Background: LDE225 is a potent and highly selective inhibitor of Smo, a key regulator protein in the Hedgehog (Hh) signaling pathway, which is linked to the pathogenesis of several human cancers. A first-in-human study to evaluate the safety and tolerability of LDE225 in adult patients with solid tumors is ongoing. The use of hair follicles (HF) as a surrogate tissue to assess the phamacodynamic effect of LDE225 on Hh pathway targets was explored due to the known regulatory role of Hh pathway in hair development. Methods: A total of 190 HFs from 19 healthy subjects was used to optimize a methodology to isolate, amplify, and profile mRNA transcripts. The expression levels of many primary Hh pathway genes including GLI-1, GLI-2, PTCH-1, and PTCH- 2 were reliably detected using quantitative RT- PCR. Results: The optimization of bulb preservation, root disruption, and RNA isolation allowed for a greater RNA yield and quality per hair bulb than previously published. RT-PCR analysis of Hh pathway genes demonstrated low inter- and intrasubject variability (Table). A variance components analysis showed that hair-to-hair variation within a subject was the largest source of total variability compared to intersubject and technical variability. Preliminary results from transcriptome-wide gene expression analysis using U133 Plus 2.0 Arrays (Affymetrix) and pathway mapping suggested that hair follicles can serve as a surrogate tissue that expresses a wide representation of biological pathways associated with neoplasms. Conclusions: An approach for monitoring Hh pathway gene signatures in human hair follicles has been established. Collection of multiple HFs per subject is advised to obtain a more robust measure of gene expression. This method may also be useful for identifying molecular signatures in response to other pharmaceutical drugs or for monitoring known developmental, metabolic or disease pathways. Variability of RT-PCR analysis of Hh genes Gene Mean (CT) Intersubject std (CT) Intersubject CV Inter-HF std (CT) Inter-HF (CT) B2M 22.02 0.91 4.15 1.28 5.82 GLI1 32.57 1.46 4.48 3.10 9.51 GLI2 33.52 1.83 5.45 2.32 6.92 PTCH1 28.13 2.22 7.90 3.87 13.74 PTCH2 31.42 1.35 4.30 3.60 11.44 UBC 21.16 1.58 7.45 1.66 7.83 Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis