miR-196b target screen reveals mechanisms maintaining leukemia stemness with therapeutic potential.

We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1) is a direct miR-196b target whose repression enhanced an embryonic stem cell-like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2-containing SCF E3-ubiquitin ligase complex increased p27Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia.

Regulatory landscape of the Hox transcriptome.

Precise regulation of Hox gene activity is essential to achieve proper control of animal embryonic development and to avoid generation of a variety of malignancies. This is a multilayered process, including complex polycistronic transcription, RNA processing, microRNA repression, long noncoding RNA regulation and sequence-specific translational control, acting together to achieve robust quantitative and qualitative Hox protein output. For many such mechanisms, the Hox cluster gene network has turned out to serve as a paradigmatic model for their study. In this review, we discuss current knowledge of how the different layers of post-transcriptional regulation and the production of a variety of noncoding RNA species control Hox output, and how this shapes formation of developmental systems that are reproducibly patterned by complex Hox networks.

The polarity protein Scrib mediates epidermal development and exerts a tumor suppressive function during skin carcinogenesis.

BACKGROUND: The establishment and maintenance of polarity is vital for embryonic development and loss of polarity is a frequent characteristic of epithelial cancers, however the underlying molecular mechanisms remain unclear. Here, we identify a novel role for the polarity protein Scrib as a mediator of epidermal permeability barrier acquisition, skeletal morphogenesis, and as a potent tumor suppressor in cutaneous carcinogenesis. METHODS: To explore the role of Scrib during epidermal development, we compared the permeability of toluidine blue dye in wild-type, Scrib heterozygous and Scrib KO embryonic epidermis at E16.5, E17.5 and E18.5. Mouse embryos were stained with alcian blue and alizarin red for skeletal analysis. To establish whether Scrib plays a tumor suppressive role during skin tumorigenesis and/or progression, we evaluated an autochthonous mouse model of skin carcinogenesis in the context of Scrib loss. We utilised Cre-LoxP technology to conditionally deplete Scrib in adult epidermis, since Scrib KO embryos are neonatal lethal. RESULTS: We establish that Scrib perturbs keratinocyte maturation during embryonic development, causing impaired epidermal barrier formation, and that Scrib is required for skeletal morphogenesis in mice. Analysis of conditional transgenic mice deficient for Scrib specifically within the epidermis revealed no skin pathologies, indicating that Scrib is dispensable for normal adult epidermal homeostasis. Nevertheless, bi-allelic loss of Scrib significantly enhanced tumor multiplicity and progression in an autochthonous model of epidermal carcinogenesis in vivo, demonstrating Scrib is an epidermal tumor suppressor. Mechanistically, we show that apoptosis is the critical effector of Scrib tumor suppressor activity during skin carcinogenesis and provide new insight into the function of polarity proteins during DNA damage repair. CONCLUSIONS: For the first time, we provide genetic evidence of a unique link between skin carcinogenesis and loss of the epithelial polarity regulator Scrib, emphasizing that Scrib exerts a wide-spread tumor suppressive function in epithelia.

Autonomous and nonautonomous roles of Hedgehog signaling in regulating limb muscle formation.

Muscle progenitor cells migrate from the lateral somites into the developing vertebrate limb, where they undergo patterning and differentiation in response to local signals. Sonic hedgehog (Shh) is a secreted molecule made in the posterior limb bud that affects patterning and development of multiple tissues, including skeletal muscles. However, the cell-autonomous and non-cell-autonomous functions of Shh during limb muscle formation have remained unclear. We found that Shh affects the pattern of limb musculature non-cell-autonomously, acting through adjacent nonmuscle mesenchyme. However, Shh plays a cell-autonomous role in maintaining cell survival in the dermomyotome and initiating early activation of the myogenic program in the ventral limb. At later stages, Shh promotes slow muscle differentiation cell-autonomously. In addition, Shh signaling is required cell-autonomously to regulate directional muscle cell migration in the distal limb. We identify neuroepithelial cell transforming gene 1 (Net1) as a downstream target and effector of Shh signaling in that context.

Patched 1 is a crucial determinant of asymmetry and digit number in the vertebrate limb.

The vertebrate hedgehog receptor patched 1 (Ptc1) is crucial for negative regulation of the sonic hedgehog (Shh) pathway during anterior-posterior patterning of the limb. We have conditionally inactivated Ptc1 in the mesenchyme of the mouse limb using Prx1-Cre. This results in constitutive activation of hedgehog (Hh) signalling during the early stages of limb budding. Our data suggest that variations in the timing and efficiency of Cre-mediated excision result in differential forelimb and hindlimb phenotypes. Hindlimbs display polydactyly (gain of digits) and a molecular profile similar to the Gli3 mutant extra-toes. Strikingly, forelimbs are predominantly oligodactylous (displaying a loss of digits), with a symmetrical, mirror-image molecular profile that is consistent with re-specification of the anterior forelimb to a posterior identity. Our data suggest that this is related to very early inactivation of Ptc1 in the forelimb perturbing the gene regulatory networks responsible for both the pre-patterning and the subsequent patterning stages of limb development. These results establish the importance of the downstream consequences of Hh pathway repression, and identify Ptc1 as a key player in limb patterning even prior to the onset of Shh expression.

Fgf-dependent Etv4/5 activity is required for posterior restriction of Sonic Hedgehog and promoting outgrowth of the vertebrate limb.

Crosstalk between the fibroblast growth factor (FGF) and Sonic Hedgehog (Shh) pathways is critical for proper patterning and growth of the developing limb bud. Here, we show that FGF-dependent activation of the ETS transcription factors Etv4 and Etv5 contributes to proximal-distal limb outgrowth. Surprisingly, blockage of Etv activity in early distal mesenchyme also resulted in ectopic, anterior expansion of Shh, leading to a polydactylous phenotype. These data indicate an unexpected function for an FGF/Etv pathway in anterior-posterior patterning. FGF activity in the limb is not only responsible for maintaining posterior-specific Shh expression, but it also acts via Etvs to prevent inappropriate anterior expansion of Shh. This study identifies another level of genetic interaction between the orthogonal axes during limb development.

Pax9 and Jagged1 act downstream of Gli3 in vertebrate limb development.

From early in limb development the transcription factor Gli3 acts to define boundaries of gene expression along the anterior-posterior (AP) axis, establishing asymmetric patterns required to provide positional information. As limb development proceeds, posterior mesenchyme expression of Sonic hedgehog (Shh) regulates Gli3 transcription and post-translational processing to specify digit number and identity. The molecular cascades dependent on Gli3 at later stages of limb development, which link early patterning events with final digit morphogenesis, remain poorly characterised. By analysing the transcriptional consequences of loss of Gli3 in the anterior margin of the E11.5 and E12.5 limb bud in the polydactylous mouse mutant extra-toes (Gli3(Xt/Xt)), we have identified a number of known and novel transcripts dependent on Gli3 in the limb. In particular, we demonstrated that the genes encoding the paired box transcription factor Pax9, the Notch ligand Jagged1 and the cell surface receptor Cdo are dependent on Gli3 for correct expression in the anterior limb mesenchyme. Analysis of expression in compound Shh;Gli3 mutant mouse embryos and in both in vitro and in vivo Shh signaling assays, further defined the importance of Shh regulated processing of Gli3 in controlling gene expression. In particular Pax9 regulation by Shh and Gli3 was shown to be context dependent, with major differences between the limb and somite revealed by Shh bead implantation experiments in the chick. Jagged1 was shown to be induced by Shh in the chick limb and in a C3H10T1/2 cell based signaling assay, with Shh;Gli3 mutant analysis indicating that expression is dependent on Gli3 derepression. Our data have also revealed that perturbation of early patterning events within the Gli3(Xt/Xt) limb culminates in a specific delay of anterior chondrogenesis which is subsequently realised as extra digits.

DLC1 is unlikely to be a primary target for deletions on chromosome arm 8p22 in head and neck squamous cell carcinoma.

Allelic imbalance on chromosome arm 8p is common in head and neck squamous cell carcinoma (HNSCC). DLC1, a tumour suppressor gene inactivated in liver carcinogenesis and encoding a Rho GTPase activating protein (RhoGAP) maps to one of the deleted regions (8p21.3-22). In order to determine whether inactivation of DLC1 is involved in HNSCC, we have screened tumour cell lines for DLC1 mutations and expression. Pathological mutations were not identified in any of the 17 cell lines tested. Seven polymorphisms were identified; 13 of the 17 of cell lines were homozygous for all seven polymorphisms compared to only 2 of 17 controls suggesting a loss of heterozygosity in a majority of the cell lines. DLC1 expression was observed in all 11 HNSCC cell lines tested, thus excluding the possibility of transcriptional silencing of DLC1 by promoter hypermethylation. Overall, our data suggest that hemizygous deletions of the DLC1 locus are frequent in HNSCCs but this gene is unlikely to be primary target for inactivation on this chromosomal arm.