A comprehensive in vivo screen for anti-apoptotic miRNAs indicates broad capacities for oncogenic synergy.

microRNAs (miRNAs) are ~21-22 nucleotide (nt) RNAs that mediate broad post-transcriptional regulatory networks. However, genetic analyses have shown that the phenotypic consequences of deleting individual miRNAs are generally far less overt compared to their misexpression. This suggests that miRNA deregulation may have broader phenotypic impacts during disease situations. We explored this concept in the Drosophila eye, by screening for miRNAs whose misexpression could modify the activity of pro-apoptotic factors. Via unbiased and comprehensive in vivo phenotypic assays, we identify an unexpectedly large set of miRNA hits that can suppress the action of pro-apoptotic genes hid and grim. We utilize secondary assays to validate that a subset of these miRNAs can inhibit irradiation-induced cell death. Since cancer cells might seek to evade apoptosis pathways, we modeled this situation by asking whether activation of anti-apoptotic miRNAs could serve as "second hits". Indeed, while clones of the lethal giant larvae (lgl) tumor suppressor are normally eliminated during larval development, we find that diverse anti-apoptotic miRNAs mediate the survival of lgl mutant clones in third instar larvae. Notably, while certain anti-apoptotic miRNAs can target apoptotic factors, most of our screen hits lack obvious targets in the core apoptosis machinery. These data highlight how a genetic approach can reveal distinct and powerful activities of miRNAs in vivo, including unexpected functional synergies during disease or cancer-relevant settings.

Nonmelanoma Skin Cancer Risk in Patients With Inflammatory Bowel Disease Undergoing Thiopurine Therapy: A Systematic Review of the Literature.

BACKGROUND: Azathioprine and 6-mercaptopurine (thiopurines) are common adjunct treatments for inflammatory bowel disease (IBD). Although thiopurine therapy in organ transplant recipients is known to increase nonmelanoma skin cancers (NMSCs), dermatologic literature yields less data regarding NMSC risk of thiopurine use in IBD. OBJECTIVE: The aim of this study was to systematically review current literature on NMSC risk in patients with IBD using thiopurine therapy. METHODS: Systematic review of PubMed was performed with keywords "inflammatory bowel disease," "ulcerative colitis," "Crohn's disease," "thiopurine," "azathioprine," "6-mercaptopurine," "skin cancer," "non-melanoma," "squamous cell carcinoma," and "basal cell carcinoma." All available publication years were included. Publications were evaluated using PRISMA guidelines. RESULTS: The systematic review yielded 67 articles; 18 met final inclusion criteria. LIMITATIONS: Heterogeneity of study designs limited direct comparisons of thiopurine exposure and NMSC risk. CONCLUSION: Patients with IBD using thiopurines seem to have a moderately increased risk of NMSC that is proportional to therapy duration. Risk of NMSC seems to decrease or return to baseline after discontinuing therapy, although additional data are needed to support this trend. Younger patients with IBD using thiopurines seem to be at greater risk of NMSC. Appreciating NMSC risk in patients with IBD undergoing thiopurine therapy should help direct skin cancer screening recommendations and sun protective measures.

Cutaneous Manifestations of Crohn Disease.

Awareness of the extraintestinal manifestations of Crohn disease is increasing in dermatology and gastroenterology, with enhanced identification of entities that range from granulomatous diseases recapitulating the underlying inflammatory bowel disease to reactive conditions and associated dermatoses. In this review, the underlying etiopathology of Crohn disease is discussed, and how this mirrors certain skin manifestations that present in a subset of patients is explored. The array of extraintestinal manifestations that do not share a similar pathology, but which are often seen in association with inflammatory bowel disease, is also discussed. Treatment and pathogenetic mechanisms, where available, are discussed.

Osteopontin expression in biopsies of calciphylaxis.

BACKGROUND: Calciphylaxis combines features of vascular thrombotic occlusion and endoluminal calcification. In this study we examine the expression of osteopontin as a diagnostic marker and its role in lesional pathogenesis. METHODS: 25 formalin-fixed, paraffin embedded skin biopsies of 20 females and 5 males (mean age of 60 years) with a diagnosis of calciphylaxis were assessed for osteopontin expression. RESULTS: Lower extremities were the most commonly involved areas; however a truncal and genital distribution was also noted in 3 cases. Renal failure was present in 21 of 25 cases. One patient had myeloproliferative disorder and one patient had advanced colon cancer. The dominant pathology was localized to the subcutaneous fat, characterized by mural calcification and luminal thrombosis affecting capillaries, venules, arterioles and small arteries. In 2 cases, a subcutaneous thrombogenic vasculopathy without calcification was noted. Osteopontin expression was confined to the subcutis, being most striking in calcified vessels but also apparent in vessels without calcification, including mineral poor variants of calciphylaxis. CONCLUSION: Calciphylaxis represents a unique calcific thrombogenic vasculopathy, not limited to renal failure. Ectopic osteopontin expression may define a critical and initial event in the calciphylaxis pathogenesis. Therapeutic agents designed to reduce osteopontin expression may be of value in its treatment.

Hypopigmented interface T-cell dyscrasia: a form of cutaneous T-cell dyscrasia distinct from hypopigmented mycosis fungoides.

Hypopigmentation in cutaneous T-cell lymphoproliferative disease should not always be equated with hypopigmented mycosis fungoides (MF). A form of hypopigmented pre-lymphomatous T-cell dyscrasia falling under the designation of the so-called hypopigmented interface variant of T-cell dyscrasia has recently been proposed. The aim of the present study was to establish hypopigmented interface T-cell dyscrasia as its own entity apart from other T-cell dyscrasias and MF using a patient case series. Twenty four cases of hypopigmented interface T-cell dyscrasia were identified in the dermatopathology database of Weill Medical College of Cornell University. There were 17 females and seven males (mean age, 36 years). In children and adolescents, the patients were most commonly of African American extraction. Truncal photo-protected areas manifesting as large solitary patches or multiple smaller macules were characteristic; disease progression to MF occurred in only one patient. The lesions responded to topical steroids and light therapy. The pathology was defined by a cell poor interface associated with degeneration of keratinocytes and melanocytes, and by lymphocytes whose nuclei showed low-grade cerebriform atypia, and which expressed a significant reduction in CD7 and CD62L expression. In 50% of the cases, the implicated cell type was of the CD8 subset. Clonality was not identified. Hypopigmented interface T-cell dyscrasia is a distinct entity separate from and rarely progressive to MF.

Indolent CD8+ lymphoid proliferation of the face with eyelid involvement.

In 2007, Petrella et al described a series of patients with clonally restricted, well-differentiated, nonepidermotropic, CD8-dominant lymphocytic infiltrates localized to the facial area. The clinical course described was indolent. A CD8 variant of primary cutaneous pleomorphic T-cell lymphoma has been proposed; however, there are many dissimilar features. The authors encountered 2 patients with CD8⁺ indolent lymphoid proliferation of the face, localized to the eyelid. Both patients were males in their 30s presenting with localized lesions of the eyelids. The biopsies showed an effacing lymphocytic infiltrate that spanned the sampled dermis. The lymphocytes were well differentiated, exhibiting mild nuclear contour irregularity. The infiltrate was predominated by CD8⁺ lymphocytes demonstrating TIA expression. There was a minimal B-cell component in 1 case, whereas another showed a significant degree of B-cell hyperplasia. They both underwent complete excision without recurrence. CD8⁺ indolent lymphoid proliferation has a reproducible clinical and morphologic presentation that warrants categorization as a distinct form of indolent lymphoproliferative disease, preferentially involving older adults with the most common site being the ear, recognizing that the nose, acral surfaces, and eyelids can also be involved.

CD20+ mycosis fungoides: a report of three cases and review of the literature.

Mycosis fungoides (MF) is the most common of the family of cutaneous T-cell lymphomas, accounting for 65% of all cases of cutaneous T-cell lymphomas. The classic phenotypic profile is one defined by CD4+ T cells showing a reduction in the expression of CD7 and CD62L. There are 3 previous reports describing CD20 expression in MF. The cell surface antigen CD20 is a transmembrane glycosylated phosphoprotein expressed in the early stages of B-cell development before differentiation into plasma cells. Two male patients, aged 14 and 44 years, presented with persistent truncal plaques up to 8 cm of 1 and 4 years duration, respectively. A third patient, an 80-year-old female, presented with a 1-year history of progressive nodules involving the head and neck area. Cases 1 and 2 both responded to topical treatment modalities. The biopsies in cases 1 and 2 showed features typical of plaque stage MF, whereas case 3 was compatible with follicular MF with tumor stage transformation. Phenotypically, the aberrant cell populace demonstrated a CD4+, CD7-, and CD62L- phenotype; at variance with classic MF was the expression of CD20. Although there were a few PAX5-positive staining cells, definitive colocalization studies were negative. Other B-cell markers and heavy chain immunoglobulin rearrangement were not detected. There are a growing number of reports describing T-cell lymphomas and leukemias with CD20 expression. Of the 6 CD20+ MF cases reported in the literature to date, 3 have been associated with a more aggressive clinical course; all but one case have occurred in males.

A potential role for miRNAs in topical drug development.

MicroRNAs (miRNAs) are small, noncoding regulatory RNAs demonstrated to play a role in regulating diverse physiologic and pathologic processes in humans. The understanding of their role in dermatologic disorders has been rapidly expanding, and technological advances in the field of small RNA therapeutics have provided a window into the possibilities for using our understanding of miRNA activities as a stepping-stone to treating a variety of skin diseases. The topical immunomodulator diphenylcyclopropenone (DPCP) has been used for the treatment of skin cancers and alopecia areata and represents one of many drug targets with potential for manipulation of miRNA pathways to enhance clinical efficacy. By exploring the miRNA pathways involved in specific skin diseases and the miRNAs impacted by drug treatments, investigators will discover new ways to treat skin disease and improve preexisting therapies.

Emerging adverse cutaneous drug reactions.

The past several decades have seen the advent and rapidly expanding use of biological agents in the treatment of chronic disease states. As increasingly large pools of patients have been enrolled in treatment protocols using these agents, physicians have become acquainted with both desired and adverse events associated with their use. Dermatologists frequently encounter patients affected by cutaneous drug reactions associated with the use of biological agents, thereby becoming familiar with the full range of side effects reported in the literature. This review discusses these adverse cutaneous effects, their underlying mechanisms, and efforts to predict and minimize their occurrence.

Neurophysiological defects and neuronal gene deregulation in Drosophila mir-124 mutants.

miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation. On the other hand, we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124-expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology.

microRNA control of cell-cell signaling during development and disease.

MicroRNAs (miRNAs) are critical post-transcriptional regulators that may collectively control a majority of animal genes. With thousands of miRNAs identified, a pressing challenge is now to understand their specific biological activities. Many predicted miRNA:target interactions only subtly alter gene activity. It has consequently not been trivial to deduce how miRNAs are relevant to phenotype, and by extension, relevant to disease. We note that the major signal transduction cascades that control animal development are highly dose-sensitive and frequently altered in human disorders. On this basis, we hypothesize that developmental cell signaling pathways represent prime candidates for mediating some of the major phenotypic consequences of miRNA deregulation, especially under gain-of-function conditions. This perspective reviews the evidence for miRNA targeting of the major signaling pathways, and discusses its implications for how aberrant miRNA activity might underlie human disease and cancer.

Functionally distinct regulatory RNAs generated by bidirectional transcription and processing of microRNA loci.

Many microRNA (miRNA) loci exhibit compelling hairpin structures on both sense and antisense strands; however, the possibility that a miRNA gene might produce functional species from its antisense strand has not been examined. We report here that antisense transcription of the Hox miRNA locus mir-iab-4 generates the novel pre-miRNA hairpin mir-iab-8, which is then processed into endogenous mature miRNAs. Sense and antisense iab-4/iab-8 miRNAs are functionally distinguished by their distinct domains of expression and targeting capabilities. We find that miR-iab-8-5p, like miR-iab-4-5p, is also relevant to Hox gene regulation. Ectopic mir-iab-8 can strongly repress the Hox genes Ultrabithorax and abdominal-A via extensive arrays of conserved target sites, and can induce a dramatic homeotic transformation of halteres into wings. We generalize the antisense miRNA principle by showing that several other loci in both invertebrates and vertebrates are endogenously processed on their antisense strands into mature miRNAs with distinct seeds. These findings demonstrate that antisense transcription and processing contributes to the functional diversification of miRNA genes.

Functional screening identifies miR-315 as a potent activator of Wingless signaling.

The existence of vast regulatory networks mediated by microRNAs (miRNAs) suggests broad potential for miRNA dysfunction to contribute to disease. However, relatively few miRNA-target interactions are likely to make detectable contributions to phenotype, and effective strategies to identify these few interactions are currently wanting. We hypothesized that signaling cascades represent critical points of susceptibility to miRNA dysfunction, and we developed a strategy to test this theory by using quantitative cell-based screens. Here we report a screen for miRNAs that affect the Wingless (Wg) pathway, a conserved pathway that regulates growth and tissue specification. This process identified ectopic miR-315 as a potent and specific activator of Wg signaling, an activity that we corroborated in transgenic animals. This miR-315 activity was mediated by direct inhibition of Axin and Notum, which encode essential, negatively acting components of the Wg pathway. Genetic interaction tests substantiated both of these genes as key functional targets of miR-315. The ability of ectopic miR-315 to activate Wg signaling was not a trivial consequence of predicted miRNA-target relationships because other miRNAs with conserved sites in the Axin 3' UTR neither activated Wg outputs nor inhibited an Axin sensor. In summary, activity-based screening can selectively identify miRNAs whose deregulation can lead to interpretable phenotypic consequences.

The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila.

The canonical microRNA (miRNA) pathway converts primary hairpin precursor transcripts into approximately 22 nucleotide regulatory RNAs via consecutive cleavages by two RNase III enzymes, Drosha and Dicer. In this study, we characterize Drosophila small RNAs that derive from short intronic hairpins termed "mirtrons." Their nuclear biogenesis appears to bypass Drosha cleavage, which is essential for miRNA biogenesis. Instead, mirtron hairpins are defined by the action of the splicing machinery and lariat-debranching enzyme, which yield pre-miRNA-like hairpins. The mirtron pathway merges with the canonical miRNA pathway during hairpin export by Exportin-5, and both types of hairpins are subsequently processed by Dicer-1/loqs. This generates small RNAs that can repress perfectly matched and seed-matched targets, and we provide evidence that they function, at least in part, via the RNA-induced silencing complex effector Ago1. These findings reveal that mirtrons are an alternate source of miRNA-type regulatory RNAs.