Cross-species oncogenomics offers insight into human muscle-invasive bladder cancer.

BACKGROUND: In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. RESULTS: Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. CONCLUSION: Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

The Proteasome Inhibitor Ixazomib Inhibits the Formation and Growth of Pulmonary and Abdominal Osteosarcoma Metastases in Mice.

Osteosarcoma is the most common form of primary bone cancer. Over 20% of osteosarcoma patients present with pulmonary metastases at diagnosis, and nearly 70% of these patients fail to respond to treatment. Previous work revealed that human and canine osteosarcoma cell lines are extremely sensitive to the therapeutic proteasome inhibitor bortezomib in vitro. However, bortezomib has proven disappointingly ineffective against solid tumors including sarcomas in animal experiments and clinical trials. Poor tumor penetration has been speculated to account for the inconsistency between in vitro and in vivo responses of solid tumors to bortezomib. Here we show that the second-generation proteasome inhibitor ixazomib, which reportedly has enhanced solid tumor penetration compared to bortezomib, is toxic to human and canine osteosarcoma cells in vitro. We used experimental osteosarcoma metastasis models to compare the efficacies of ixazomib and bortezomib against primary tumors and metastases derived from luciferase-expressing KRIB or 143B human osteosarcoma cell lines in athymic mice. Neither proteasome inhibitor reduced the growth of primary intramuscular KRIB tumors, however both drugs inhibited the growth of established pulmonary metastases created via intravenous inoculation with KRIB cells, which were significantly better vascularized than the primary tumors. Only ixazomib slowed metastases from KRIB primary tumors and inhibited the growth of 143B pulmonary and abdominal metastases, significantly enhancing the survival of mice intravenously injected with 143B cells. Taken together, these results suggest ixazomib exerts better single agent activity against osteosarcoma metastases than bortezomib. These data provide hope that incorporation of ixazomib, or other proteasome inhibitors that penetrate efficiently into solid tumors, into current regimens may improve outcomes for patients diagnosed with metastatic osteosarcoma.

Inactivation of Zeb1 in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure.

Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like (Grhl1-3) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, Grhl2, is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. Grhl2-null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of Grhl2-null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors Ovol1 and Ovol2, which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of Zeb1 expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in Zeb1-/- mice is evident, with rescue of both palatal and facial fusion seen in Grhl2-/-;Zeb1-/- embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.

Stage-dependent therapeutic efficacy in PI3K/mTOR-driven squamous cell carcinoma of the skin.

Cutaneous squamous cell carcinoma (SCC) is a recurrent cancer that is prevalent in predisposed subjects such as immunosuppressed patients and patients being treated for other malignancies. Model systems to trial therapies at different stages of SCC development are lacking, therefore precluding efficient therapeutic interventions. Here, we have disrupted the expression of the tumor suppressor GRHL3 to induce loss of PTEN and activation of the PI3K/mTOR signaling pathway in mice and human skin, promoting aggressive SCC development. We then examined the potential for targeting PI3K/mTOR and an oncogenic driver miR-21, alone and in combination, for the prevention and treatment of SCC during the initiation, promotion/progression and establishment stages. Treatment with PI3K/mTOR inhibitors completely prevented tumor initiation, and these inhibitors significantly delayed the course of papilloma progression to malignancy. However, established SCC did not undergo any growth regression, indicating that this therapy is ineffective in established cancers. Mechanistically, the resistant SCCs displayed increased miR-21 expression in mice and humans where antagonists of miR-21 rescued expression levels of GRHL3/PTEN, but the combination of miR-21 antagonism with PI3K/mTOR inhibition resulted in acquired SCC resistance in part via c-MYC and OCT-4 upregulation. In conclusion, our data provide molecular evidence for the efficacy of targeting oncogenic drivers of SCC during the initiation and promotion stages and indicate that combination therapy may induce an aggressive phenotype when applied in the establishment stage.

Grainyhead-like 3 (Grhl3) deficiency in brain leads to altered locomotor activity and decreased anxiety-like behaviors in aged mice.

The highly conserved Grainyhead-like (Grhl) family of transcription factors, comprising three members in vertebrates (Grhl1-3), play critical regulatory roles during embryonic development, cellular proliferation, and apoptosis. Although loss of Grhl function leads to multiple neural abnormalities in numerous animal models, a comprehensive analysis of Grhl expression and function in the mammalian brain has not been reported. Here they show that only Grhl3 expression is detectable in the embryonic mouse brain; particularly within the habenula, an organ known to modulate repressive behaviors. Using both Grhl3-knockout mice (Grhl3-/- ), and brain-specific conditional deletion of Grhl3 in adult mice (Nestin-Cre/Grhl3flox/flox ), they performed histological expression analyses and behavioral tests to assess long-term effects of Grhl3 loss on motor co-ordination, spatial memory, anxiety, and stress. They found that complete deletion of Grhl3 did not lead to noticeable structural or cell-intrinsic defects in the embryonic brain; however, aged Grhl3 conditional knockout (cKO) mice showed enlarged lateral ventricles and displayed marked changes in motor function and behaviors suggestive of decreased fear and anxiety. They conclude that loss of Grhl3 in the brain leads to significant alterations in locomotor activity and decreased self-inhibition, and as such, these mice may serve as a novel model of human conditions of impulsive behavior or hyperactivity. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 775-788, 2017.

Two Ancient Gene Families Are Critical for Maintenance of the Mammalian Skin Barrier in Postnatal Life.

The skin barrier is critical for mammalian survival in the terrestrial environment, affording protection against fluid loss, microbes, toxins, and UV exposure. Many genes indispensable for barrier formation in the embryo have been identified, but loss of these genes in adult mice does not induce barrier regression. We describe a complex regulatory network centered on two ancient gene families, the grainyhead-like (Grhl) transcription factors and the protein cross-linking enzymes (tissue transglutaminases [Tgms]), which are essential for skin permeability barrier maintenance in adult mice. Embryonic deletion of Grhl3 induces loss of Tgm1 expression, which disrupts the cornified envelope, thus preventing permeability barrier formation leading to neonatal death. However, gene deletion of Grhl3 in adult mice does not disrupt the preformed barrier, with cornified envelope integrity maintained by Grhl1 and Tgm5, which are up-regulated in response to postnatal loss of Grhl3. Concomitant deletion of both Grhl factors in adult mice induced loss of Tgm1 and Tgm5 expression, perturbation of the cornified envelope, and complete permeability barrier regression that was incompatible with life. These findings define the molecular safeguards for barrier function that accompany the transition from intrauterine to terrestrial life.

Identification of a Novel Proto-oncogenic Network in Head and Neck Squamous Cell Carcinoma.

BACKGROUND: The developmental transcription factor Grainyhead-like 3 (GRHL3) plays a critical tumor suppressor role in the mammalian epidermis through direct regulation of PTEN and the PI3K/AKT/mTOR signaling pathway. GRHL3 is highly expressed in all tissues derived from the surface ectoderm, including the oral cavity, raising a question about its potential role in suppression of head and neck squamous cell carcinoma (HNSCC). METHODS: We explored the tumor suppressor role of Grhl3 in HNSCC using a conditional knockout (Grhl3 (∆/-) /K14Cre (+) ) mouse line (n = 26) exposed to an oral chemical carcinogen. We defined the proto-oncogenic pathway activated in the HNSCC derived from these mice and assessed it in primary human HNSCC samples, normal oral epithelial cell lines carrying shRNA to GRHL3, and human HNSCC cell lines. Data were analyzed with two-sided chi square and Student's t tests. RESULTS: Deletion of Grhl3 in oral epithelium in mice did not perturb PTEN/PI3K/AKT/mTOR signaling, but instead evoked loss of GSK3B expression, resulting in stabilization and accumulation of c-MYC and aggressive HNSCC. This molecular signature was also evident in a subset of primary human HNSCC and HNSCC cell lines. Loss of Gsk3b in mice, independent of Grhl3, predisposed to chemical-induced HNSCC. Restoration of GSK3B expression blocked proliferation of normal oral epithelial cell lines carrying shRNA to GRHL3 (cell no., Day 8: Scramble ctl, 616±21.8 x 10(3) vs GRHL3-kd, 1194±44 X 10(3), P < .001; GRHL3-kd vs GRHL3-kd + GSK3B, 800±98.84 X 10(3), P = .003) and human HNSCC cells. CONCLUSIONS: We defined a novel molecular signature in mammalian HNSCC, suggesting new treatment strategies targeting the GRHL3/GSK3B/c-MYC proto-oncogenic network.

Grainyhead-like 3 regulation of endothelin-1 in the pharyngeal endoderm is critical for growth and development of the craniofacial skeleton.

Craniofacial development is a highly conserved process that requires complex interactions between neural crest cells (NCCs) and pharyngeal tissues derived from all three germ layers. Signals emanating from the pharyngeal endoderm drive differentiation of NCCs into craniofacial cartilage, and disruption of this process underpins several human craniofacial defects (CFD). Here, we demonstrate that morpholino (MO)-mediated knockdown in zebrafish of the highly conserved transcription factor grainyhead-like 3 (grhl3), which is selectively expressed in the pharyngeal endoderm, leads to severe hypoplasia of the lower jaw cartilages. Phylogenetic analysis of conserved grhl-binding sites in gene regulatory regions identified endothelin-1 (edn1) as a putative direct grhl3 target gene, and this was confirmed by chromatin precipitation (ChIP) assays in zebrafish embryos. Injection of sub-phenotypic concentrations of MOs targeting both grhl3 and edn1 induced jaw abnormalities, and injection of edn1 mRNA into grhl3-morphants rescued both pharyngeal expression of the downstream effectors of edn1, and jaw cartilage formation. This study sheds new light on the role of endodermal endothelin-1 in vertebrate jaw development, and highlights potential new genetic defects that could underpin human CFD.

Midbrain-hindbrain boundary patterning and morphogenesis are regulated by diverse grainy head-like 2-dependent pathways.

The isthmic organiser located at the midbrain-hindbrain boundary (MHB) is the crucial developmental signalling centre responsible for patterning mesencephalic and metencephalic regions of the vertebrate brain. Formation and maintenance of the MHB is characterised by a hierarchical program of gene expression initiated by fibroblast growth factor 8 (Fgf8), coupled with cellular morphogenesis, culminating in the formation of the tectal-isthmo-cerebellar structures. Here, we show in zebrafish that one orthologue of the transcription factor grainy head-like 2 (Grhl2), zebrafish grhl2b plays a central role in both MHB maintenance and folding by regulating two distinct, non-linear pathways. Loss of grhl2b expression induces neural apoptosis and extinction of MHB markers, which are rescued by re-expression of engrailed 2a (eng2a), an evolutionarily conserved target of the Grhl family. Co-injection of sub-phenotypic doses of grhl2b and eng2a morpholinos reproduces the apoptosis and MHB marker loss, but fails to substantially disrupt formation of the isthmic constriction. By contrast, a novel direct grhl2b target, spec1, identified by phylogenetic analysis and confirmed by ChIP, functionally cooperates with grhl2b to induce MHB morphogenesis, but plays no role in apoptosis or maintenance of MHB markers. Collectively, these data show that MHB maintenance and morphogenesis are dissociable events regulated by grhl2b through diverse transcriptional targets.

Targeting of the tumor suppressor GRHL3 by a miR-21-dependent proto-oncogenic network results in PTEN loss and tumorigenesis.

Despite its prevalence, the molecular basis of squamous cell carcinoma (SCC) remains poorly understood. Here, we identify the developmental transcription factor Grhl3 as a potent tumor suppressor of SCC in mice, and demonstrate that targeting of Grhl3 by a miR-21-dependent proto-oncogenic network underpins SCC in humans. Deletion of Grhl3 in adult epidermis evokes loss of expression of PTEN, a direct GRHL3 target, resulting in aggressive SCC induced by activation of PI3K/AKT/mTOR signaling. Restoration of Pten expression completely abrogates SCC formation. Reduced levels of GRHL3 and PTEN are evident in human skin, and head and neck SCC, associated with increased expression of miR-21, which targets both tumor suppressors. Our data define the GRHL3-PTEN axis as a critical tumor suppressor pathway in SCC.

Proteinase-activated receptor-2 (PAR2) and mouse osteoblasts: regulation of cell function and lack of specificity of PAR2-activating peptides.

1. Using synthetic proteinase-activated receptor-2 (PAR(2))-activating peptides (PAR(2)APs) corresponding to the tethered ligand domain of the extracellular N-terminus of PAR(2) to mimic the actions of activating proteinases and using primary cultures of calvarial osteoblasts derived from both wild-type (WT) and PAR(2)-null (KO) mice, we investigated the potential role of PAR(2) in regulating osteoblast function. 2. Primary calvarial osteoblasts from WT and KO mice were evaluated for their growth kinetics and mineralization in the absence of PAR(2) agonists and for their responses in a variety of functional assays to the PAR(2)APs Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH(2)) and 2-furoyl-Leu-Ile-Gly-Arg-Leu-Orn-amide (2-fLIGRLO-NH(2)), as well as to trypsin. 3. In contrast with WT cells, PAR(2)-KO osteoblasts did not exhibit increased collagen Type I mRNA expression in response to SLIGRL-NH(2). When grown in serum-containing medium, KO cells increased in number more rapidly than WT cells, an effect that could be attributed to decreased apoptosis rather than increased proliferation. Surprisingly, in both WT and KO osteoblasts, the two PAR(2)APs induced mobilization of intracellular calcium stores. Similarly, the PAR(2)APs inhibited serum deprivation-induced apoptosis and parathyroid hormone-, 1,25-dihydroxyvitamin D(3)- or interleukin-11-induced mineralization in WT and KO cells. 4. We conclude that PAR(2) plays a role in osteoblast survival and collagen Type I mRNA induction and that osteoblasts can respond to the PAR(2)APs via both PAR(2)-dependent and -independent mechanisms.