Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43-depleted human iPSC-derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

Tackling neurodegenerative diseases with genomic engineering: A new stem cell initiative from the NIH.

The iPSC Neurodegenerative Disease Initiative (iNDI) is the largest-ever iPSC genome engineering project. iNDI will model more than 100 mutations associated with Alzheimer's disease and related dementias (ADRD) in isogenic iPSC lines. Resulting cell lines and phenotypic datasets will be broadly shared.

The GENDULF algorithm: mining transcriptomics to uncover modifier genes for monogenic diseases.

Modifier genes are believed to account for the clinical variability observed in many Mendelian disorders, but their identification remains challenging due to the limited availability of genomics data from large patient cohorts. Here, we present GENDULF (GENetic moDULators identiFication), one of the first methods to facilitate prediction of disease modifiers using healthy and diseased tissue gene expression data. GENDULF is designed for monogenic diseases in which the mechanism is loss of function leading to reduced expression of the mutated gene. When applied to cystic fibrosis, GENDULF successfully identifies multiple, previously established disease modifiers, including EHF, SLC6A14, and CLCA1. It is then utilized in spinal muscular atrophy (SMA) and predicts U2AF1 as a modifier whose low expression correlates with higher SMN2 pre-mRNA exon 7 retention. Indeed, knockdown of U2AF1 in SMA patient-derived cells leads to increased full-length SMN2 transcript and SMN protein expression. Taking advantage of the increasing availability of transcriptomic data, GENDULF is a novel addition to existing strategies for prediction of genetic disease modifiers, providing insights into disease pathogenesis and uncovering novel therapeutic targets.

αvß6-Fyn Kinase Promotes Epithelial Phenotype in SYF Cells.

The 5-year survival rate for patients with oral cancer remains at 50%, in large part due the high rate of post-treatment recurrence. In this study, we transfected epithelial-specific integrin αvß6 and Fyn-kinase, a member of the Src-family kinases, into embryonic murine fibroblasts. In oral cancer, expression of αvß6 is neo-expressed. Using a variety of in vitro assays, including cell migration and multicellular spheroid formation, we determined that these embryonic fibroblasts expressing αvß6 and Fyn-kinase were able to acquire an epithelial phenotype. This is in direct contrast to human oral SCC, where expression of αvß6 with Fyn-kinase promotes epithelial to mesenchymal transition. This demonstrates that signaling pathways can be species-specific.

Multicellular Spheroids as a Model for Hypoxia-induced EMT.

Oral cancer is aggressive and invasive. The 5-year survival rate is around 50% and has not improved in over 50 years. One-third of oral cancer patients develop local and/or regional tumor recurrence following treatment. We continue to use our multicellular spheroid (MCS) model to better understand how the extracellular matrix contributes to epithelial to mesenchymal transition and how hypoxia contributes to the progression of oral squamous cell carcinoma (SCC).

Regulation of Multicellular Spheroids by MAPK and FYN Kinase.

Understanding of the biology of oral squamous cell carcinoma (SCC) has not progressed significantly in the past 60 years, with 5-year survival remaining at approximately 50%. The epidemic of Human Papilloma Virus and its associated SCC warrants a renewed emphasis on fully understanding this disease. We previously used the 3-dimensional multicellular spheroid (MCS) model system to evaluate SCC behavior more accurately. In this study, we determined that SCC growth in MCS approximates epithelial to mesenchymal transition. Organization of an MCS requires the full-length ß6 integrin subunit and its maintenance requires mitogen-activated protein kinase (MAPK). Limiting FYN kinase activation results in the down-regulation of E-cadherin, ß-catenin and an increase in expression of N-cadherin and SNAIL. These results indicate that the microenvironment and growth patterns in an MCS are complex and require MAPK and FYN kinase.

Gingival pustules and sterile diffuse sclerosing osteomyelitis as a feature of synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome.

Synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome represents the rare co-occurrence of sterile inflammatory osteoarticular disease in association with a variety of cutaneous manifestations. Oral involvement is uncommon. The etiology of SAPHO is complex and is likely the combined result of infectious, genetic, and immunologic factors. Due to diverse clinical presentations, SAPHO is difficult to diagnose. Here, we describe the case of a 74-year-old man, who had a history of SAPHO syndrome and presented with gingival pustules and sterile diffuse sclerosing osteomyelitis of the mandible. This is the first case report describing neutrophilic mucositis as a feature of SAPHO.

Differential diagnosis and management of oral ulcers.

The diagnosis and treatment of oral lesions is often challenging due to the clinician's limited exposure to the conditions that may cause the lesions and their similar appearances. While many oral ulcers are the result of chronic trauma, some may indicate an underlying systemic condition such as a gastrointestinal dysfunction, malignancy, immunologic abnormality, or cutaneous disease. Correctly establishing a definitive diagnosis is of major importance to clinicians who manage patients with oral mucosal disease. Some of these diseases are infectious; however, most are chronic, symptomatic, and desquamative. Treatment and management requires an understanding of the immunopathologic nature of the lesion. This review will address how to differentiate and diagnose varying types of oral ulcers and provide a treatment strategy.

Differential spheroid formation by oral cancer cells.

Squamous cell carcinomas (SCC) make up 96% of all oral cancers. Most laboratory SCC studies grow cells as a monolayer, which does not accurately represent the disease in vivo. We used a more relevant multicellular spheroid (MCS) model to study this disease. The SCC9ß6KDFyn cell line, which expresses full-length ß6 and a kinase dead Fyn formed the largest MCS. Cell adhesive properties are dynamic and N-cadherin was increased in the largest MCS. c-Raf mediates the survival of tumor cells and was consistently expressed both in monolayers and in the MCS by SCC9ß6D1 cells which lack the ß6 cytoplasmic tail and, do not activate Fyn. SCC9ß6KDFyn cells also express high levels of c-Raf when grown as spheroids in which Fyn suppression stimulates MCS formation. Tumor microenvironment and growth patterns modulate cell behavior and suppression of Fyn kinase may promote MCS growth.

The cytoplasmic extension of the integrin ß6 subunit regulates epithelial-to-mesenchymal transition.

Prognosis for oral cancer patients has not improved in over 60 years due to invasion and recurrence. To understand the invasive behavior of this tumor, we evaluated the role of the αvß6 integrin. Invasive oral SCC cells express the αvß6 integrin, which contains an 11-amino-acid extension on its ß-subunit unique to the integrin family. We determined that this ß6 cytoplasmic extension regulates the composition of the intermediate filament network and the organization of signaling structures called focal contacts. The auto-phosphorylation of FAK, which is localized to focal contacts, was also regulated by the ß6-cytoplasmic tail, as were the transcription factors Notch and STAT3. Lastly, we also determined that activation of MAPK required the full-length ß6 integrin. Together these results indicate that the signaling critical to epithelial-to-mesenchymal transition (EMT) is regulated by the ß6 integrin cytoplasmic domain.

Wnt signaling regulates pulp volume and dentin thickness.

Odontoblasts, cementoblasts, ameloblasts, and osteoblasts all form mineralized tissues in the craniofacial complex, and all these cell types exhibit active Wnt signaling during postnatal life. We set out to understand the functions of this Wnt signaling, by evaluating the phenotypes of mice in which the essential Wnt chaperone protein, Wntless was eliminated. The deletion of Wls was restricted to cells expressing Osteocalcin (OCN), which in addition to osteoblasts includes odontoblasts, cementoblasts, and ameloblasts. Dentin, cementum, enamel, and bone all formed in OCN-Cre;Wls(fl/fl) mice but their homeostasis was dramatically affected. The most notable feature was a significant increase in dentin volume and density. We attribute this gain in dentin volume to a Wnt-mediated misregulation of Runx2. Normally, Wnt signaling stimulates Runx2, which in turn inhibits dentin sialoprotein (DSP); this inhibition must be relieved for odontoblasts to differentiate. In OCN-Cre;Wls(fl/fl) mice, Wnt pathway activation is reduced and Runx2 levels decline. The Runx2-mediated repression of DSP is relieved and odontoblast differentiation is accordingly enhanced. This study demonstrates the importance of Wnt signaling in the homeostasis of mineralized tissues of the craniofacial complex.

Expression of EMMPRIN modulates mediators of tumor invasion in oral squamous cell carcinoma.

Squamous cell carcinoma (SCC) accounts for 96 percent of all intraoral malignancies. The five-year survival rate is 50 percent and has not improved in 60 years. During SCC progression, subsets of SCC cells undergo an epithelial-to-mesenchymal transition (EMT) to become highly invasive. The extracellular matrix metalloproteinase inducer (EMMPRIN) contributes to EMT by activating local matrix metalloproteinases (MMPs). In this study, we found that EMMPRIN modulates the invasive phenotype and may be a potential therapeutic target.

Revisiting epithelial-to-mesenchymal transition through adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) has a 5-year survival rate of 90%. The 15-year survival rate drops to 10% due to recurrence and invasion. ACC has three subtypes: cribriform, tubular, and solid. The cribriform subtype has the best prognosis and the solid subtype has the worst prognosis. By immunohistochemistry of tissue sections, we showed that the solid form expresses αvß6 integrin and tenascin-C, which are known promoters of epithelial-to-mesenchymal transition (EMT). We also defined two ACC cell lines with the characteristics of the cribriform and solid subtype. The SACC83 cells grow in basaloid-like clusters and express high levels of E-cadherin. In contrast, the ACCh cells are more myoepithelial-like and express high levels of vimentin and of αvß6 integrin. The ACCh cells are highly invasive and this behavior is dependent upon the αvß6 integrin function. Our results suggest that the transition from the cribriform to solid form may occur through EMT.

Stem cell markers as predictors of oral cancer invasion.

We previously showed that within primary tumors there exist subpopulations of cells expressing stem cell markers. Using immunofluorescence and western blotting, we examined the expression of stem cell markers tumor-rejection antigen 1-60 (TRA1-60) and octamer-binding transcription factor 3/ 4 (OCT3/4) to determine their relationship with cell invasiveness. Six human oral cancer cell lines were examined and a direct correlation was found between expression of these stem cell markers and invasion. Poor expression of E-cadherin and increased expression of N-cadherin was also found in TRA1-60- and OCT3/4- expressing cells. Phosphorylation of the major signaling molecule mitogen activated protein kinase (MAPK) was greatest in the TRA1-60- and OCT3/4- expressing cells. These results suggest that expression of specific stem cell markers in tumors may help guide a clinician's choice of treatment.

Alphavbeta3 suppresses the RhoA-LIMK1 pathway in K1735 melanoma.

Mucocutaneous melanoma has a five-year survival rate of less than 10 percent. The alphavbeta3 integrin promotes invasion, which requires actin reorganization by cofilin. The authors previously showed that cofilin and alphavbeta3 promote invasion. K1735 melanoma has several clones, each with different levels of alphavbeta3. The authors found that expression of alphavbeta3 suppresses activation of RhoA thus inhibiting LIMK1 phosphorylation of cofilin. This indicates that alphavbeta3 integrin suppresses the RhoA/ ROCK/LIMK1 pathway.

EMMPRIN expression in oral SCC is regulated by FYN kinase.

This study shows that the expression of the extracellular matrix metalloproteinase inducer (EMMPRIN) in oral squamous cell carcinoma cells (SCC) depends upon activation of the Src Family kinaseFyn; and that EMMPRIN and ß6 form a complex that requires active Fyn and the full length ß6 integrin cytoplasmic domain. Fyn is also important for matrix remodeling as it regulates both matrix type 1 metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-1 and -2 (TIMP1/2). The tumor promoter/suppressor caveolin-1, which associates with MT1-MMP, also requires FYN activation for expression. Lastly, EMMPRIN expression can act as a readout for the mitogen-activated protein kinase (MAPK) pathway, since when MAPK is blocked, so is the expression of EMMPRIN. In oral cancer, the activation of FYN occurs post ß6 integrin ligand binding. That the activation of FYN drives EMMPRIN expression and several important pathways associated with invasive oral SCC is now demonstrated.

Expression of Fyn kinase modulates EMT in oral cancer cells.

Oral squamous cell carcinoma (SCC) is an aggressive tumor with a poor 5-year survival rate. Oral SCC can undergo epithelial to mesenchymal transition (EMT). We previously showed that the epithelial integrin alphavbeta6 complexes with Fyn kinase in oral SCC to promote EMT. Using immunofluorescence microscopy and Western blotting, we evaluated whether the expression of specific markers of EMT were influenced by modulating serum concentration (ie. growth factors). The SCC cultures were grown under contrasting levels of serum. In low serum (1%), Fyn promoted EMT; whereas suppression of Fyn kinase promoted the epithelial phenotype. However, when the SCC cells were grown in 10% serum, activation of Fyn had the reverse effect. Lastly, cell migration was evaluated under low serum conditions (1% FBS). Activation of Fyn promoted SCC cell migration and its suppression thwarted SCC migration toward FN. These results indicate that the activation of Fyn kinase as well as local growth factor concentration modulate EMT in oral SCC.

Identification of {alpha}v{beta}6-positive stem cells in oral squamous cell carcinoma.

Oral squamous cell carcinoma (SCC) is composed of a heterogeneous population of cells which range anywhere from epithelial to mesenchymal in phenotype. Several oral cancer specimens with antibodies to TRA160, a marker of pluripotent cells, were screened. Compared with the well differentiated lesions, pluripotent cells were more numerous in specimens from poorly differentiated tumors. In vitro, the expression of TRA160 was much greater in invasive oral SCC9beta6 cells compared with the poorly invasive SCC9SN or SCC9beta6D1 cells, which express a truncated beta6. In vitro, pluripotent cells were instrumental in aggressively closing an experimental wound assay. Lastly, TRA-1-60+/beta6+ tumor cells which formed vascular-like structures in vivo were identified. SCC9beta6 cells formed interconnecting channels, whereas SCC9SN cells did not in an in vitro Matrigel angiogenesis assay. The results of this study clearly demonstrated the differential distribution of pluripotent stem cells in oral SCC and that the beta6 integrin may be an important regulatory component of the pluripotent phenotype.

The role of the integrin alpha v beta6 in regulating the epithelial to mesenchymal transition in oral cancer.

In this study, we evaluated whether the forced expression of beta6 integrin would modulate the epithelial to mesenchymal transition (EMT). When the full length beta6 integrin was expressed in poorly invasive squamous cell carcinoma SCC9 cells, the resulting SCC9/6 cells acquired a fibroblast-like morphology, increased expression of the mesenchymal marker vimentin and reduced expression of the epithelial markers keratin and E-cadherin. SCC9beta6D1 cells, which express a truncated form of beta6 subunit lacking the C-terminal 11 amino acids (AA), retained their epithelial morphology and did not alter vimentin or E-cadherin expression. This suggests that the full-length beta6 subunit can induce EMT in oral SCC cells. We previously showed that expression of beta6 increases both MMP-3 activation and tenascin-C expression and we now show that both molecules are MEK dependent. These results also demonstrate that the terminal 11 AA of beta6 contain information important for establishing an epithelial to mesenchymal transition.

Modulation of EGFR by oral squamous cell carcinoma cell lines.

Oral cancer is the sixth most frequent cancer worldwide. Prognosis for these patients remains poor. Recently, the epidermal growth factor receptor has been targeted as an adjunct to radiotherapy and surgery with limited success. The authors now present data suggesting that the alphanubeta6 integrin, which is a marker for aggressive oral cancer, may regulate epidermal growth factor receptor expression. The authors suggest perhaps targeting both alphanubeta6 and EGFR may provide additional benefits.