Severe liver disease resembling PSC in mice with K5-Cre mediated deletion of Krüppel-like factor 5 (Klf5).

Chronic cholestatic liver diseases including primary sclerosing cholangitis (PSC) present a complex spectrum with regards to the cause, age of manifestation and histopathological features. Current treatment options are severely limited primarily due to a paucity of model systems mirroring the disease. Here, we describe the Keratin 5 (K5)-Cre; Klf5fl/fl mouse that spontaneously develops severe liver disease during the postnatal period with features resembling PSC including a prominent ductular reaction, fibrotic obliteration of the bile ducts and secondary degeneration/necrosis of liver parenchyma. Over time, there is an expansion of Sox9+ hepatocytes in the damaged livers suggestive of a hepatocyte-mediated regenerative response. We conclude that Klf5 is required for the normal function of the hepatobiliary system and that the K5-Cre; Klf5fl/fl mouse is an excellent model to probe the molecular events interlinking damage and regenerative response in the liver.

GLI1-induced mammary gland tumours are transplantable and maintain major molecular features.

Increased expression of GLI1, the main Hedgehog signalling pathway effector, is related to unfavourable prognosis and progressive disease of certain breast cancer subtypes. We used conditional transgenic mice induced to overexpress GLI1 in the mammary epithelium either alone or in combination with deletion of one Trp53 allele to address the role of elevated GLI1 expression in breast tumour initiation and progression. Induced GLI1 expression facilitates mammary gland tumour formation and this was further increased upon heterozygous deletion of Trp53. The GLI1-induced primary tumours were of different murine molecular subtypes, including Normal-likeEx , Class8Ex , Claudin-LowEx and Erbb2-likeEx . The gene expression profiles of some of the tumours correlated well with the PAM50 subtypes for human breast cancer. Whole-exome sequencing revealed somatic mutation profiles with only little overlap between the primary tumours. Orthotopically serially transplanted GLI1-induced tumours maintained the main morphological characteristics of the primary tumours for ≥10 generations. Independent of Trp53 status and molecular subtype, the serially transplanted GLI1-induced tumours were able to grow both in the absence of transgenic GLI1 expression and in the presence of the GLI1 inhibitor GANT61. These data suggest that elevated GLI1 expression has a determinant role in tumour initiation; however, additional genetic events are required for tumour progression.

Differential requirement of SUFU in tissue development discovered in a hypomorphic mouse model.

Suppressor of Fused (SUFU) is an essential negative regulator of the Hedgehog (HH) pathway and involved in GLI transcription factor regulation. Due to early embryonic lethality of Sufu-/- mice, investigations of SUFU's role later in development are limited to conditional, tissue-specific knockout models. In this study we developed a mouse model (SufuEx456(fl)/Ex456(fl)) with hypomorphic features where embryos were viable up to E18.5, although with a spectrum of developmental defects of varying severity, including polydactyly, exencephaly and omphalocele. Development of certain tissues, like the skeleton, was more affected than that of others such as skin, which remained largely normal. Interestingly, no apparent changes in the dorso-ventral patterning of the neural tube at E9.0 could be seen. Thus, this model provides an opportunity to globally study SUFU's molecular function in organogenesis beyond E9.5. Molecularly, SufuEx456(fl)/Ex456(fl) embryos displayed aberrant mRNA splicing and drastically reduced levels of Sufu wild-type mRNA and SUFU protein in all tissues. As a consequence, at E9.5 the levels of all three different GLI proteins were reduced. Interestingly, despite the reduction of GLI3 protein levels, the critical ratio of the GLI3 full-length transcriptional activator versus GLI3 truncated repressor remained unchanged compared to wild-type embryos. This suggests that the limited amount of SUFU protein present is sufficient for GLI processing but not for stabilization. Our data demonstrate that tissue development is differentially affected in response to the reduced SUFU levels, providing novel insight regarding the requirements of different levels of SUFU for proper organogenesis.

Lgr6 labels a rare population of mammary gland progenitor cells that are able to originate luminal mammary tumours.

The mammary gland is composed of a complex cellular hierarchy with unusual postnatal plasticity. The identities of stem/progenitor cell populations, as well as tumour-initiating cells that give rise to breast cancer, are incompletely understood. Here we show that Lgr6 marks rare populations of cells in both basal and luminal mammary gland compartments in mice. Lineage tracing analysis showed that Lgr6+ cells are unipotent progenitors, which expand clonally during puberty but diminish in adulthood. In pregnancy or following stimulation with ovarian hormones, adult Lgr6+ cells regained proliferative potency and their progeny formed alveoli over repeated pregnancies. Oncogenic mutations in Lgr6+ cells resulted in expansion of luminal cells, culminating in mammary gland tumours. Conversely, depletion of Lgr6+ cells in the MMTV-PyMT model of mammary tumorigenesis significantly impaired tumour growth. Thus, Lgr6 marks mammary gland progenitor cells that can initiate tumours, and cells of luminal breast tumours required for efficient tumour maintenance.

Stromal Hedgehog signalling is downregulated in colon cancer and its restoration restrains tumour growth.

A role for Hedgehog (Hh) signalling in the development of colorectal cancer (CRC) has been proposed. In CRC and other solid tumours, Hh ligands are upregulated; however, a specific Hh antagonist provided no benefit in a clinical trial. Here we use Hh reporter mice to show that downstream Hh activity is unexpectedly diminished in a mouse model of colitis-associated colon cancer, and that downstream Hh signalling is restricted to the stroma. Functionally, stroma-specific Hh activation in mice markedly reduces the tumour load and blocks progression of advanced neoplasms, partly via the modulation of BMP signalling and restriction of the colonic stem cell signature. By contrast, attenuated Hh signalling accelerates colonic tumourigenesis. In human CRC, downstream Hh activity is similarly reduced and canonical Hh signalling remains predominantly paracrine. Our results suggest that diminished downstream Hh signalling enhances CRC development, and that stromal Hh activation can act as a colonic tumour suppressor.

Isolation and Fluorescence-Activated Cell Sorting of Mouse Keratinocytes Expressing ß-Galactosidase.

During the past decade, the rapid development of new transgenic and knock-in mouse models has propelled epidermal stem-cell research into "fast-forward mode". It has become possible to identify and visualize defined cell populations during normal tissue maintenance, and to follow their progeny during the processes of homeostasis, wound repair, and tumorigenesis. Moreover, these cells can be isolated using specific labels, and characterized in detail using an array of molecular and cell biology approaches. The bacterial enzyme, ß-galactosidase (ß-gal), the product of the LacZ gene, is one of the most commonly used in vivo cell labels in genetically-engineered mice. The protocol described in this chapter provides a guideline for the isolation of viable murine epidermal cells expressing ß-gal, which can then be subjected to further characterization in vivo or in vitro.

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development.

Skp1-Cul1-F-box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma-associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F-box and leucine-rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17-mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17-Sufu axis in the pathogenesis of medulloblastoma.

FNDC4 acts as an anti-inflammatory factor on macrophages and improves colitis in mice.

FNDC4 is a secreted factor sharing high homology with the exercise-associated myokine irisin (FNDC5). Here we report that Fndc4 is robustly upregulated in several mouse models of inflammation as well as in human inflammatory conditions. Specifically, FNDC4 levels are increased locally at inflamed sites of the intestine of inflammatory bowel disease patients. Interestingly, administration of recombinant FNDC4 in the mouse model of induced colitis markedly reduces disease severity compared with mice injected with a control protein. Conversely, mice lacking Fndc4 develop more severe colitis. Analysis of binding of FNDC4 to different immune cell types reveals strong and specific binding to macrophages and monocytes. FNDC4 treatment of bone marrow-derived macrophages in vitro results in reduced phagocytosis, increased cell survival and reduced proinflammatory chemokine expression. Hence, treatment with FNDC4 results in a state of dampened macrophage activity, while enhancing their survival. Thus, we have characterized FNDC4 as a factor with direct therapeutic potential in inflammatory bowel disease and possibly other inflammatory diseases.

Dynamics of Lgr6⁺ Progenitor Cells in the Hair Follicle, Sebaceous Gland, and Interfollicular Epidermis.

The dynamics and interactions between stem cell pools in the hair follicle (HF), sebaceous gland (SG), and interfollicular epidermis (IFE) of murine skin are still poorly understood. In this study, we used multicolor lineage tracing to mark Lgr6⁺ -expressing basal cells in the HF isthmus, SG, and IFE.We show that these Lgr6⁺ cells constitute long-term self-renewing populations within each compartment in adult skin. Quantitative analysis of clonal dynamics revealed that the Lgr6⁺ progenitor cells compete neutrally in the IFE, isthmus, and SG, indicating population asymmetry as the underlying mode of tissue renewal. Transcriptional profiling of Lgr6⁺ and Lgr6⁺ cells did not reveal a distinct Lgr6⁺ -associated gene expression signature, raising the question of whether Lgr6⁺ expression requires extrinsic niche signals. Our results elucidate the interrelation and behavior of Lgr6⁺ populations in the IFE, HF, and SG and suggest population asymmetry as a common mechanism for homeostasis in several epithelial skin compartments.

Suppressor of Fused Plays an Important Role in Regulating Mesodermal Differentiation of Murine Embryonic Stem Cells In Vivo.

The hedgehog (Hh) signaling pathway plays fundamental roles during embryonic development and tumorigenesis. Previously, we have shown that ablation of the tumor suppressor and negative regulator, Suppressor of fused (Sufu), within this pathway causes embryonic lethality around E9.5 in the mouse. In this study, we examine how lack of Sufu influences early cell fate determination processes. We established embryonic stem cell (ESC) lines from preimplantation Sufu(-/-) and wild-type mouse embryos and show that these ESCs express the typical pluripotency markers, alkaline phosphatase, SSEA-1, Oct4, Sox2, and Nanog. We demonstrate that these ESCs express all core Hh pathway components and that glioma-associated protein (Gli)1 mRNA levels are increased in Sufu(-/-) ESCs. Upon spontaneous differentiation of Sufu(-/-) ESCs into embryoid bodies (EBs) in vitro, the Hh pathway is strongly upregulated as indicated by an increase in both Gli1 and patched1 (Ptch1) gene expression. Interestingly, developing Sufu(-/-) EBs were smaller than their wild-type counterparts and showed decreased expression of the ectodermal markers, Fgf5 and Sox1. In vivo teratoma formation revealed that Sufu(-/-) ESCs have a limited capacity for differentiation as the resulting tumors lacked the mesodermal derivatives, cartilage and bone. However, Sufu(-/-) ESCs were able to develop into chondrocytes and osteocytes in vitro, which suggests a differential response of ESCs compared with in vivo conditions. Our findings suggest a regulatory function of the Hh signaling pathway in early mesodermal cell fate determination and emphasize the role of Sufu as a key molecule in this process.

A conditional transgenic mouse line for targeted expression of the stem cell marker LGR5.

LGR5 is a known marker of embryonic and adult stem cells in several tissues. In a mouse model, Lgr5+ cells have shown tumour-initiating properties, while in human cancers, such as basal cell carcinoma and colon cancer, LGR5 expression levels are increased: however, the effect of increased LGR5 expression is not fully understood. To study the effects of elevated LGR5 expression levels we generated a novel tetracycline-responsive, conditional transgenic mouse line expressing human LGR5, designated TRELGR5. In this transgenic line, LGR5 expression can be induced in any tissue depending on the expression pattern of the chosen transcriptional regulator. For the current study, we used transgenic mice with a tetracycline-regulated transcriptional transactivator linked to the bovine keratin 5 promoter (K5tTA) to drive expression of LGR5 in the epidermis. As expected, expression of human LGR5 was induced in the skin of double transgenic mice (K5tTA;TRELGR5). Inducing LGR5 expression during embryogenesis and early development resulted in macroscopically and microscopically detectable phenotypic changes, including kink tail, sparse fur coat and enlarged sebaceous glands. The fur and sebaceous gland phenotypes were reversible upon discontinued expression of transgenic LGR5, but this was not observed for the kink tail phenotype. There were no apparent phenotypic changes if LGR5 expression was induced at three weeks of age. The results demonstrate that increased expression of LGR5 during embryogenesis and the neonatal period alter skin development and homeostasis.

ß-Catenin-Gli1 interaction regulates proliferation and tumor growth in medulloblastoma.

BACKGROUND: The Wnt/beta-catenin and the Hedgehog (Hh) pathway interact in various cell types while eliciting opposing or synergistic cellular effects. Both pathways are known as exclusive drivers of two distinct molecular subtypes of medulloblastoma (MB). In sonic hedgehog (Shh)-driven MB, activation of Wnt signaling has been shown to suppress tumor growth by either beta-catenin-dependent or -independent inhibition of Shh signaling. However, mechanistic insight in how beta-catenin inhibits the Hh pathway is not known. FINDINGS: Here we show that beta-catenin stabilization by the glycogen synthase kinase 3 inhibitor lithium chloride (LiCl) reduced growth of primary hedgehog-driven MB tumor spheres from patched heterozygous mice (Ptch(+/-)) in vitro. LiCl treatment of MB spheres down-regulated the Hh target Gli1, whereas the repressive Gli3 protein (Gli3R) was increased. Mechanistically, we show by co-immunoprecipitation and proximity ligation assay that stabilized beta-catenin physically interacts with Gli1, leading to Gli1 sequestration and inhibition of its transcriptional activity. Reduction of Hh signaling upon LiCl stimulation resulted in reduced proliferation, sphere self renewal, a G2/M arrest and induction of a senescent-like state, indicated by p21 upregulation and by increased staining of senescence-associated beta-galactosidase (SA-betaGal). Moreover, LiCl treatment of subcutaneously transplanted MB cells significantly reduced tumor initiation defined as "tumor take". Although tumor progression was similar, LiCl-treated tumors showed decreased mitotic figures and phospho-histone H3 staining. CONCLUSION: We propose that beta-catenin stabilization increases its physical interaction with Gli1, leading to Gli1 degradation and inhibition of Hh signaling, thereby promoting tumor cell senescence and suppression of "tumor take" in mice.

Think inside the BOCs: a mechanism underlying medulloblastoma progression.

Approximately one-third of medulloblastoma cases are associated with genetic lesions of Hedgehog (Hh) signaling pathway components. In this issue of Developmental Cell, Mille et al. (2014) show that the Hh coreceptor Boc functions specifically in the progression of early- to advanced-stage medulloblastoma by promoting Cyclin D1-dependent DNA damage and genomic instability.

Regulated splicing of the α6 integrin cytoplasmic domain determines the fate of breast cancer stem cells.

Although the α6ß1 integrin has been implicated in the function of breast and other cancer stem cells (CSCs), little is known about its regulation and relationship to mechanisms involved in the genesis of CSCs. We report that a CD44(high)/CD24(low) population, enriched for CSCs, is comprised of distinct epithelial and mesenchymal populations that differ in expression of the two α6 cytoplasmic domain splice variants: α6A and α6B. α6Bß1 expression defines the mesenchymal population and is necessary for CSC function, a function that cannot be executed by α6A integrins. The generation of α6Bß1 is tightly controlled and occurs as a consequence of an autocrine vascular endothelial growth factor (VEGF) signaling that culminates in the transcriptional repression of a key RNA-splicing factor. These data alter our understanding of how α6ß1 contributes to breast cancer, and they resolve ambiguities regarding the use of total α6 (CD49f) expression as a biomarker for CSCs.

Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation.

Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

Tumor necrosis factor receptor signaling in keratinocytes triggers interleukin-24-dependent psoriasis-like skin inflammation in mice.

Psoriasis is a common chronic inflammatory skin disease with a prevalence of about 2% in the Caucasian population. Tumor necrosis factor (TNF) plays an essential role in the pathogenesis of psoriasis, but its mechanism of action remains poorly understood. Here we report that the development of psoriasis-like skin inflammation in mice with epidermis-specific inhibition of the transcription factor NF-κB was triggered by TNF receptor 1 (TNFR1)-dependent upregulation of interleukin-24 (IL-24) and activation of signal transducer and activator of transcription 3 (STAT3) signaling in keratinocytes. IL-24 was strongly expressed in human psoriatic epidermis, and pharmacological inhibition of NF-κB increased IL-24 expression in TNF-stimulated human primary keratinocytes, suggesting that this mechanism is relevant for human psoriasis. Therefore, our results expand current views on psoriasis pathogenesis by revealing a new keratinocyte-intrinsic mechanism that links TNFR1, NF-κB, ERK, IL-24, IL-22R1, and STAT3 signaling to disease initiation.

3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer related death. It is lethal in nearly all patients, due to an almost complete chemoresistance. Most if not all drugs that pass preclinical tests successfully, fail miserably in the patient. This raises the question whether traditional 2D cell culture is the correct tool for drug screening. The objective of this study is to develop a simple, high-throughput 3D model of human PDAC cell lines, and to explore mechanisms underlying the transition from 2D to 3D that might be responsible for chemoresistance. METHODS: Several established human PDAC and a KPC mouse cell lines were tested, whereby Panc-1 was studied in more detail. 3D spheroid formation was facilitated with methylcellulose. Spheroids were studied morphologically, electron microscopically and by qRT-PCR for selected matrix genes, related factors and miRNA. Metabolic studies were performed, and a panel of novel drugs was tested against gemcitabine. RESULTS: Comparing 3D to 2D cell culture, matrix proteins were significantly increased as were lumican, SNED1, DARP32, and miR-146a. Cell metabolism in 3D was shifted towards glycolysis. All drugs tested were less effective in 3D, except for allicin, MT100 and AX, which demonstrated effect. CONCLUSIONS: We developed a high-throughput 3D cell culture drug screening system for pancreatic cancer, which displays a strongly increased chemoresistance. Features associated to the 3D cell model are increased expression of matrix proteins and miRNA as well as stromal markers such as PPP1R1B and SNED1. This is supporting the concept of cell adhesion mediated drug resistance.

GLI1 regulates a novel neuropilin-2/α6ß1 integrin based autocrine pathway that contributes to breast cancer initiation.

The characterization of cells with tumour initiating potential is significant for advancing our understanding of cancer and improving therapy. Aggressive, triple-negative breast cancers (TNBCs) are enriched for tumour-initiating cells (TICs). We investigated that hypothesis that VEGF receptors expressed on TNBC cells mediate autocrine signalling that contributes to tumour initiation. We discovered the VEGF receptor neuropilin-2 (NRP2) is expressed preferentially on TICs, involved in the genesis of TNBCs and necessary for tumour initiation. The mechanism by which NRP2 signalling promotes tumour initiation involves stimulation of the α6ß1 integrin, focal adhesion kinase-mediated activation of Ras/MEK signalling and consequent expression of the Hedgehog effector GLI1. GLI1 also induces BMI-1, a key stem cell factor, and it enhances NRP2 expression and the function of α6ß1, establishing an autocrine loop. NRP2 can be targeted in vivo to retard tumour initiation. These findings reveal a novel autocrine pathway involving VEGF/NRP2, α6ß1 and GLI1 that contributes to the initiation of TNBC. They also support the feasibility of NRP2-based therapy for the treatment of TNBC that targets and impedes the function of TICs.

Smoothing out drug resistance.

Smoothened inhibitors have emerged as successful treatment alternatives for Hedgehog pathway-dependent tumors, but they are linked with the appearance of drug resistance. In this issue of Cancer Cell, Kim and colleagues overcome such resistance by combining approved drugs itraconazole and arsenic trioxide, thus opening a path toward new treatment options.