Interactions of Melanoma Cells with Distal Keratinocytes Trigger Metastasis via Notch Signaling Inhibition of MITF.

The most critical stage in initiation of melanoma metastasis is the radial to vertical growth transition, yet the triggers of this transition remain elusive. We suggest that the microenvironment drives melanoma metastasis independently of mutation acquisition. Here we examined the changes in microenvironment that occur during melanoma radial growth. We show that direct contact of melanoma cells with the remote epidermal layer triggers vertical invasion via Notch signaling activation, the latter serving to inhibit MITF function. Briefly, within the native Notch ligand-free microenvironment, MITF, the melanocyte lineage master regulator, binds and represses miR-222/221 promoter in an RBPJK-dependent manner. However, when radial growth brings melanoma cells into contact with distal differentiated keratinocytes that express Notch ligands, the activated Notch intracellular domain impairs MITF binding to miR-222/221 promoter. This de-repression of miR-222/221 expression triggers initiation of invasion. Our findings may direct melanoma prevention opportunities via targeting specific microenvironments.

Schistosomal MicroRNAs Isolated From Extracellular Vesicles in Sera of Infected Patients: A New Tool for Diagnosis and Follow-up of Human Schistosomiasis.

BACKGROUND: Schistosomiasis traditionally has been diagnosed by detecting eggs in stool or urine. However, the sensitivity of these examinations is limited, especially in travelers with a low worm burden. Serologic tests have a greater sensitivity, but their results remain positive regardless of treatment and thus cannot be used for follow-up of patients. We hypothesized that detection of worm microRNAs (miRNAs) in serum can overcome the drawbacks of the existing diagnostic methods. METHODS AND RESULTS: Twenty-six returning travelers with schistosomiasis (based on positive results of serologic tests or detection of ova) and 17 healthy controls were included in the study. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) amplification of miRNA extracted directly from 500 µL of serum had limited sensitivity and specificity. However, qRT-PCR analysis of RNA extracted from 200 µL of serum extracellular vesicles detected 4 schistosomal miRNAs; the sensitivity and specificity of the 2 highest expressed miRNAs (bantam and miR-2c-3p) were 86% and 84%, respectively. In 7 patients with posttreatment serum available for analysis, we observed outcomes ranging from a reduction in the schistosomal miRNA level to full recovery from disease. CONCLUSIONS: qRT-PCR of pathogen miRNAs isolated from extracellular vesicles in sera from infected individuals may provide a new tool for diagnosing schistosomiasis in patients with a low parasite burden. This assay could also be used for evaluating the outcome of therapy, as well as disease-control programs.

Therapeutic inhibitory RNA in head and neck cancer via functional targeted lipid nanoparticles.

Currently there are no specific therapies addressing the distinctive biology of human papillomavirus (HPV)-induced cancer approved for clinical use. Short interfering RNA (siRNA) has much potential for therapeutic manipulation of HPV E6/E7 oncoproteins. Lipid-based nanoparticles (LNPs) can be utilized for systemic transportation and delivery of siRNA at target site. We recently developed a recombinant protein linker that enables uniform conjugation of targeting antibodies to the LNPs. Herein, we demonstrate the therapeutic efficacy of anti-E6/E7 siRNA delivered via targeted LNPs (tLNPs) in a xenograft HPV-positive tumor model. We show that anti-epidermal growth factor receptor (EGFR) antibodies, anchored to the LNPs as targeting moieties, facilitate cargo delivery but also mediate anti-tumor activity. Treatment with siE6 via tLNPs resulted in 50% greater reduction of tumor volume compared to treatment with siControl encapsulated in isoLNPs (coated with isotype control antibodies). We demonstrate superior suppression of HPV oncogenes and higher induction of apoptosis by the tLNPs both in vitro and in vivo. Altogether, the coupling of inhibitory siE6 with anti-EGFR antibodies, that further elicited anti-tumor effects, successfully restricted tumor progression. This system that combines potent siRNA and therapeutically functional tLNPs can be modulated against various cancer models.

Ultrasound targeting of Q-starch/miR-197 complexes for topical treatment of psoriasis.

Psoriasis is a common, worldwide autoinflammatory, incurable skin disease. miR-197 has therapeutic potential for psoriasis since it can down-regulate the expression of both IL-22RA1 and IL-17RA, subunits of the receptors of IL-22 and IL-17, respectively, which are key cytokines in the disease. Although miR-197 has the potential to treat the disease, several inherent physical barrier properties of the skin challenge miRNA's delivery to the target skin cells. In the present study, we evaluated a therapeutic approach that combines the use of ultrasound (US) as a means to enhance skin permeability with quaternized starch (Q-starch) as an miRNA delivery carrier. This resulted in decreased expression of the miR-197 target proteins and in a significant reduction in the psoriatic activity markers. Our results demonstrate the potential of combinations of US and Q-starch/miR-197 complexes for the topical skin treatment of psoriasis.

The crosstalk between IL-22 signaling and miR-197 in human keratinocytes.

The interaction between the immune system and epithelial cells is tightly regulated. Aberrations of this balance may result in inflammatory diseases such as psoriasis, inflammatory bowel disease and rheumatoid arthritis. IL-22 is produced by Th17, Th22 and Th1 cells. Putative targets for IL-22 are cells in the skin, kidney, digestive and respiratory systems. The highest expression of IL-22 receptor is found in the skin. IL-22 plays an important role in the pathogenesis of T cell-mediated inflammatory diseases such as psoriasis, inflammatory bowel disease and rheumatoid arthritis. Recently, we found that miR-197 is down regulated in psoriatic lesions. In the present work we show that miR-197 over expression inhibits keratinocytes proliferation induced by IL-22 and keratinocytes migration. In addition, we found that IL-22 activates miR-197 expression through the binding of phosphorylated STAT3 to sequences in the putative promoter of miR-197. Finally we found that IL-22 receptor subunit IL22RA1 is a direct target of miR-197. Hence, we identified a novel feedback loop controlling IL-22 signaling, in which IL-22 induces miR-197, which in turn, negatively regulates IL-22 receptor and attenuates the biological outcome of such signaling. Regulation of this pathway may be important in inflammatory skin disorders such a psoriasis and in wound healing.

MiRNA expression in psoriatic skin: reciprocal regulation of hsa-miR-99a and IGF-1R.

BACKGROUND: Psoriasis is a complex disease at the cellular, genomic and genetic levels. The role of microRNAs in skin development was shown in a keratinocyte-specific Dicer knockout mouse model. Considering that two main characteristics of psoriasis are keratinocytes hyperproliferation and abnormal skin differentiation, we hypothesized that aberrant microRNA expression contributes to the psoriatic phenotype. Here, we describe the differential expression of miRNAs in psoriatic involved and uninvolved skin as compared to normal skin, revealing an additional aspect of this complex disorder. METHODOLOGY/PRINCIPAL FINDINGS: Expression arrays were used to compare microRNA expression in normal skin versus psoriatic involved and uninvolved skin. Fourteen differentially expressed microRNAs were identified, including hsa-miR-99a, hsa-miR-150, hsa-miR-423 and hsa-miR-197. The expression of these microRNAs was reevaluated by qPCR. IGF-1R, which is involved in skin development and the pathogenesis of psoriasis, is a predicted target of hsa-miR-99a. In an in situ hybridization assay, we found that IGF-1R and miR-99a are reciprocally expressed in the epidermis. Using a reporter assay, we found that IGF-1R is targeted by hsa-miR-99a. Moreover, over expression of miR-99a in primary keratinocytes down-regulates the expression of the endogenous IGF-1R protein. Over expression of miR-99a also inhibits keratinocyte proliferation and increases Keratin 10 expression. These findings suggest that overexpression of hsa-miR-99a in keratinocytes drives them towards differentiation. In primary keratinocytes grown in high Ca(++), miR-99a expression increases over time. Finally, we found that IGF1 increases the expression of miR-99a. CONCLUSIONS/SIGNIFICANCE: We identified several microRNAs that are expressed differentially in normal and psoriatic skin. One of these miRNAs is miR-99a that regulates the expression of IGF-1R. Moreover, miR-99a seems to play a role in the differentiation of keratinocytes. We suggest that miR-99a is one of the regulators of the IGF-1R signaling pathway in keratinocytes. Activation of IGF1 signaling results in elevation of miR-99a which represses the expression of IGF-1R.