The Oncolytic Activity of Zika Viral Therapy in Human Neuroblastoma In Vivo Models Confers a Major Survival Advantage in a CD24-dependent Manner.

Neuroblastoma is the most common extracranial tumor, accounting for 15% of all childhood cancer-related deaths. The long-term survival of patients with high-risk tumors is less than 40%, and MYCN amplification is one of the most common indicators of poor outcomes. Zika virus (ZIKV) is a mosquito-borne flavivirus associated with mild constitutional symptoms outside the fetal period. Our published data showed that high-risk and recurrent neuroblastoma cells are permissive to ZIKV infection, resulting in cell type-specific lysis. In this study, we assessed the efficacy of ZIKV as an oncolytic treatment for high-risk neuroblastoma using in vivo tumor models. Utilizing both MYCN-amplified and non-amplified models, we demonstrated that the application of ZIKV had a rapid tumoricidal effect. This led to a nearly total loss of the tumor mass without evidence of recurrence, offering a robust survival advantage to the host. Detection of the viral NS1 protein within the tumors confirmed that a permissive infection preceded tissue necrosis. Despite robust titers within the tumor, viral shedding to the host was poor and diminished rapidly, correlating with no detectable side effects to the murine host. Assessments from both primary pretreatment and recurrent posttreatment isolates confirmed that permissive sensitivity to ZIKV killing was dependent on the expression of CD24, which was highly expressed in neuroblastomas and conferred a proliferative advantage to tumor growth. Exploiting this viral sensitivity to CD24 offers the possibility of its use as a prognostic target for a broad population of expressing cancers, many of which have shown resistance to current clinical therapies. SIGNIFICANCE: Sensitivity to the tumoricidal effect of ZIKV on high-risk neuroblastoma tumors is dependent on CD24 expression, offering a prognostic marker for this oncolytic therapy in an extensive array of CD24-expressing cancers.

Chaperonin containing TCP-1 (CCT/TRiC) is a novel therapeutic and diagnostic target for neuroblastoma.

Chaperonin containing TCP1 (CCT/TRiC) is a multi-subunit protein folding complex that enables the cancer phenotype to emerge from the mutational landscape that drives oncogenesis. We and others linked increased expression of CCT subunits to advanced tumor stage and invasiveness that inversely correlates with cancer patient outcomes. In this study, we examined the expression of the second CCT subunit, CCT2, using genomic databases of adult and pediatric tumors and normal tissues, and found that it was highly expressed in pediatric cancers, showing a significant difference compared to normal tissues. Histologic staining confirmed that CCT subunits are highly expressed in tumor tissues, which was exemplified in neuroblastoma. Using two neuroblastoma cells, MYCN-amplified, IMR-32 cells, and non-amplified, SK-N-AS cells, we assessed baseline levels for CCT subunits and found expressions comparable to the highly invasive triple-negative breast cancer (TNBC) cell line, MDA-MB-231. Exogenous expression of CCT2 in both SK-N-AS and IMR-32 cells resulted in morphological changes, such as larger cell size and increased adherence, with significant increases in the CCT substrates, actin, and tubulin, as well as increased migration. Depletion of CCT2 reversed these effects and reduced cell viability. We evaluated CCT as a therapeutic target in IMR-32 cells by testing a novel peptide CCT inhibitor, CT20p. Treatment with CT20p induced cell death in these neuroblastoma cells. The use of CCT2 as a biological indicator for detection of neuroblastoma cells shed in blood was examined by spiking IMR-32 cells into human blood and using an anti-CCT2 antibody for the identification of spiked cancer cells with the CellSearch system. Results showed that using CCT2 for the detection of neuroblastoma cells in blood was more effective than the conventional approach of using epithelial markers like cytokeratins. CCT2 plays an essential role in promoting the invasive capacity of neuroblastoma cells and thus offers the potential to act as a molecular target in the development of novel therapeutics and diagnostics for pediatric cancers.

CD24 Expression Dampens the Basal Antiviral State in Human Neuroblastoma Cells and Enhances Permissivity to Zika Virus Infection.

Zika virus (ZIKV) exhibits distinct selectivity for infection of various cells and tissues, but how host cellular factors modulate varying permissivity remains largely unknown. Previous studies showed that the neuroblastoma cell line SK-N-AS (expressing low levels of cellular protein CD24) was highly restricted for ZIKV infection, and that this restriction was relieved by ectopic expression of CD24. We tested the hypothesis that CD24 expression allowed ZIKV replication by suppression of the antiviral response. SK-N-AS cells expressing an empty vector (termed CD24-low cells) showed elevated basal levels of phosphorylated STAT1, IRF-1, IKKE, and NFκB. In response to exogenously added type I interferon (IFN-I), CD24-low cells had higher-level induction of antiviral genes and activity against two IFN-I-sensitive viruses (VSV and PIV5-P/V) compared to SK-N-AS cells with ectopic CD24 expression (termed CD24-high cells). Media-transfer experiments showed that the inherent antiviral state of CD24-low cells was not dependent on a secreted factor such as IFN-I. Transcriptomics analysis revealed that CD24 expression decreased expression of genes involved in intracellular antiviral pathways, including IFN-I, NFκB, and Ras. Our findings that CD24 expression in neuroblastoma cells represses intracellular antiviral pathways support the proposal that CD24 may represent a novel biomarker in cancer cells for susceptibility to oncolytic viruses.

MicroRNA-211 Modulates the DUSP6-ERK5 Signaling Axis to Promote BRAFV600E-Driven Melanoma Growth In Vivo and BRAF/MEK Inhibitor Resistance.

MicroRNAs (miRs) are important posttranscriptional regulators of cell fate in both normal and disease states. miR-211 has previously been shown to be a direct regulator of metabolism in BRAFV600E-mutant melanoma cells in vitro. Here, we report that miR-211 expression promotes the aggressive growth of BRAFV600E-mutant melanoma xenografts in vivo. miR-211 promoted proliferation through the posttranscriptional activation of extracellular signal-regulated kinase (ERK) 5 signaling, which has recently been implicated in the resistance to BRAF and MAPK/ERK kinase inhibitors. We therefore examined whether miR-211 similarly modulated melanoma resistance to the BRAF inhibitor vemurafenib and the MAPK/ERK kinase inhibitor cobimetinib. Consistent with this model, miR-211 expression increased melanoma cell resistance to both the inhibitors, and this resistance was associated with an increased ERK5 phosphorylation. miR-211 mediates these effects by directly inhibiting the expression of DUSP6, an ERK5 pathway-specific phosphatase and now shown to be an miR-211 target gene. These results dissect the role of the miR-211-DUSP6-ERK5 axis in melanoma tumor growth and suggest a mechanism for the development of drug-resistant tumors and a target for overcoming resistance.

The Killing of Human Neuroblastoma Cells by the Small Molecule JQ1 Occurs in a p53-Dependent Manner.

BACKGROUND: MYCN amplification is a prognostic biomarker associated with poor prognosis of neuroblastoma in children. The overall survival of children with MYCN-amplified neuroblastoma has only marginally improved within the last 20 years. The Bromodomain and Extra-Terminal motif (BET) inhibitor, JQ1, has been shown to downregulate MYCN in neuroblastoma cells. OBJECTIVE: To determine if JQ1 downregulation of MYCN in neuroblastomas can offer a target- specific therapy for this, difficult to treat, pediatric cancer. METHODS: Since MYCN-amplified neuroblastoma accounts for as much as 40 to 50 percent of all high-risk cases, we compared the effect of JQ1 on both MYCN-amplified and non-MYCN-amplified neuroblastoma cell lines and investigated its mechanism of action. RESULTS: In this study, we show that JQ1 can specifically target MYCN for downregulation, though this effect is not specific to only MYCN-amplified cells. And although we can confirm that the loss of MYCN alone can induce apoptosis, the exogenous rescue of MYCN expression can abrogate much of this cytotoxicity. More fascinating, however, was the discovery that the JQ1-induced knockdown of MYCN, which led to the loss of the human double minute 2 homolog (HDM2) protein, also led to the accumulation of tumor protein 53 (also known as TP53 or p53), which ultimately induced apoptosis. Likewise, the knockdown of p53 also blunted the cytotoxic effects of JQ1. CONCLUSION: These data suggest a mechanism of action for JQ1 cytotoxicity in neuroblastomas and offer a possible prognostic target for determining its efficacy as a therapeutic.

Zika virus as an oncolytic treatment of human neuroblastoma cells requires CD24.

Neuroblastoma is the second most common childhood tumor. Survival is poor even with intensive therapy. In a search for therapies to neuroblastoma, we assessed the oncolytic potential of Zika virus. Zika virus is an emerging mosquito-borne pathogen unique among flaviviruses because of its association with congenital defects. Recent studies have shown that neuronal progenitor cells are likely the human target of Zika virus. Neuroblastoma has been shown to be responsive to infection. In this study, we show that neuroblastoma cells are widely permissive to Zika infection, revealing extensive cytopathic effects (CPE) and producing high titers of virus. However, a single cell line appeared poorly responsive to infection, producing undetectable levels of non-structural protein 1 (NS1), limited CPE, and low virus titers. A comparison of these poorly permissive cells to highly permissive neuroblastoma cells revealed a dramatic loss in the expression of the cell surface glycoprotein CD24 in poorly permissive cells. Complementation of CD24 expression in these cells led to the production of detectable levels of NS1 expression after infection with Zika, as well as dramatic increases in viral titers and CPE. Complementary studies using the Zika virus index strain and a north African isolate confirmed these phenotypes. These results suggest a possible role for CD24 in host cell specificity by Zika virus and offer a potential therapeutic target for its treatment. In addition, Zika viral therapy can serve as an adjunctive treatment for neuroblastoma by targeting tumor cells that can lead to recurrent disease and treatment failure.

Nanoparticle delivery of curcumin induces cellular hypoxia and ROS-mediated apoptosis via modulation of Bcl-2/Bax in human neuroblastoma.

In this study, several formulations of nanoceria and dextran-nanoceria with curcumin, each demonstrated to have anti-cancer properties, were synthesized and applied as treatment for human childhood neuroblastoma. The anti-cancer activities of these formulations were explored in neuroblastoma models of both MYCN-amplified and non-amplified cell lines. Ceria nanoparticles, coated with dextran and loaded with curcumin, were found to induce substantial cell death in neuroblastoma cells (up to a 2-fold and a 1.6-fold decrease in cell viability for MYCN-upregulated and normal expressing cell lines, respectively; *p < 0.05) while producing no or only minor toxicity in healthy cells (no toxicity at 100 µM; **p < 0.01). This formulation evokes prolonged oxidative stress, stabilizing HIF-1α, and inducing caspase-dependent apoptosis (up to a 2.4-fold increase over control; *p < 0.05). Overall, nano-therapeutic treatments showed a more pronounced effect in MYCN-amplified cells, which are traditionally more resistant to drug therapies. These results represent a very promising alternative to small molecule drug therapies for robust cancers.

The long non-coding RNA GAS5 differentially regulates cell cycle arrest and apoptosis through activation of BRCA1 and p53 in human neuroblastoma.

The long non-coding RNA GAS5 has been shown to modulate cancer proliferation in numerous human cancer systems and has been correlated with successful patient outcome. Our examination of GAS5 in neuroblastoma has revealed robust expression in both MYCN-amplified and non-amplified cell lines. Knockdown of GAS5 In vitro resulted in defects in cell proliferation, apoptosis, and induced cell cycle arrest. Further analysis of GAS5 clones revealed multiple novel splice variants, two of which inversely modulated with MYCN status. Complementation studies of the variants post-knockdown of GAS5 indicated alternate phenotypes, with one variant (FL) considerably enhancing cell proliferation by rescuing cell cycle arrest and the other (C2) driving apoptosis, suggesting a unique role for each in neuroblastoma cancer physiology. Global sequencing and ELISA arrays revealed that the loss of GAS5 induced p53, BRCA1, and GADD45A, which appeared to modulate cell cycle arrest in concert. Complementation with only the FL GAS5 clone could rescue cell cycle arrest, stabilizing HDM2, and leading to the loss of p53. Together, these data offer novel therapeutic targets in the form of lncRNA splice variants for separate challenges against cancer growth and cell death.

The Long Noncoding RNA SPRIGHTLY Regulates Cell Proliferation in Primary Human Melanocytes.

The long noncoding RNA SPRIGHTLY (formerly SPRY4-IT1), which lies within the intronic region of the SPRY4 gene, is up-regulated in human melanoma cells compared to melanocytes. SPRIGHTLY regulates a number of cancer hallmarks, including proliferation, motility, and apoptosis. To better understand its oncogenic role, SPRIGHTLY was stably transfected into human melanocytes, which resulted in increased cellular proliferation, colony formation, invasion, and development of a multinucleated dendritic-like phenotype. RNA sequencing and mass spectrometric analysis of SPRIGHTLY-expressing cells revealed changes in the expression of genes involved in cell proliferation, apoptosis, chromosome organization, regulation of DNA damage responses, and cell cycle. The proliferation marker Ki67, minichromosome maintenance genes 2-5, antiapoptotic gene X-linked inhibitor of apoptosis, and baculoviral IAP repeat-containing 7 were all up-regulated in SPRIGHTLY-expressing melanocytes, whereas the proapoptotic tumor suppressor gene DPPIV/CD26 was down-regulated, followed by an increase in extracellular signal-regulated kinase 1/2 phosphorylation, suggesting an increase in mitogen-activated protein kinase activity. Because down-regulation of DPPIV is known to be associated with malignant transformation in melanocytes, SPRIGHTLY-mediated DPPIV down-regulation may play an important role in melanoma pathobiology. Together, these findings provide important insights into how SPRIGHTLY regulates cell proliferation and anchorage-independent colony formation in primary human melanocytes.

MicroRNA 211 Functions as a Metabolic Switch in Human Melanoma Cells.

MicroRNA 211 (miR-211) negatively regulates genes that drive invasion of metastatic melanoma. Compared to normal human melanocytes, miR-211 expression is significantly reduced or absent in nonpigmented melanoma cells and lost during human melanoma progression. To investigate the molecular mechanism of its tumor suppressor function, miR-211 was ectopically expressed in nonpigmented melanoma cells. Ectopic expression of miR-211 reduced hypoxia-inducible factor 1α (HIF-1α) protein levels and decreased cell growth during hypoxia. HIF-1α protein loss was correlated with the downregulation of a miR-211 target gene, pyruvate dehydrogenase kinase 4 (PDK4). We present evidence that resumption of miR-211-mediated downregulation of PDK4 in melanoma cells causes inhibition of invasion by nonpigmented melanomas via HIF-1α protein destabilization. Thus, the tumor suppressor miR-211 acts as a metabolic switch, and its loss is expected to promote cancer hallmarks in human melanomas. Melanoma, one of the deadliest forms of skin cancer, kills nearly 10,000 people in the United States per year. We had previously shown that a small noncoding RNA, termed miR-211, suppresses invasion and the growth of aggressive melanoma cells. The results presented here support the hypothesis that miR-211 loss in melanoma cells causes abnormal regulation of energy metabolism, which in turn allows cancer cells to survive under low oxygen concentrations-a condition that generally kills normal cells. These findings highlight a novel mechanism of melanoma formation: miR-211 is a molecular switch that is turned off in melanoma cells, raising the hope that in the future we might be able to turn the switch back on, thus providing a better treatment option for melanoma.

Long noncoding RNAs as putative biomarkers for prostate cancer detection.

Prostate cancer is one of the leading causes of mortality among US males. There is an urgent unmet need to develop sensitive and specific biomarkers for the early detection of prostate cancer to reduce overtreatment and accompanying morbidity. We identified a group of differentially expressed long noncoding RNAs in prostate cancer cell lines and patient samples and further characterized six long noncoding RNAs (AK024556, XLOC_007697, LOC100287482, XLOC_005327, XLOC_008559, and XLOC_009911) in prostatic adenocarcinoma tissue samples (Gleason score >6.0) and compared them with matched normal (healthy) tissues. Interestingly, these markers were also successfully detected in patient urine samples and were found to be up-regulated when compared with normal (healthy) urine. AK024556 (SPRY4-IT1) was highly up-regulated in human prostate cancer cell line PC3 but not in LNCaP, and siRNA knockdown of SPRY4-IT1 in PC3 cells inhibited cell proliferation and invasion and increased cell apoptosis. Chromogenic in situ hybridization assay was developed to detect long noncoding RNAs in primary prostatic adenocarcinoma tissue samples, paving the way for clinical diagnostics. We believe that these results will set the stage for more extensive studies to develop novel long noncoding RNA-based diagnostic assays for early prostate cancer detection and will help to distinguish benign prostate cancer from precancerous lesions.

The functional characterization of long noncoding RNA SPRY4-IT1 in human melanoma cells.

Expression of the long noncoding RNA (lncRNA) SPRY4-IT1 is low in normal human melanocytes but high in melanoma cells. siRNA knockdown of SPRY4-IT1 blocks melanoma cell invasion and proliferation, and increases apoptosis. To investigate its function further, we affinity purified SPRY4-IT1 from melanoma cells and used mass spectrometry to identify the protein lipin 2, an enzyme that converts phosphatidate to diacylglycerol (DAG), as a major binding partner. SPRY4-IT1 knockdown increases the accumulation of lipin2 protein and upregulate the expression of diacylglycerol O-acyltransferase 2 (DGAT2) an enzyme involved in the conversion of DAG to triacylglycerol (TAG). When SPRY4-IT1 knockdown and control melanoma cells were subjected to shotgun lipidomics, an MS-based assay that permits the quantification of changes in the cellular lipid profile, we found that SPRY4-IT1 knockdown induced significant changes in a number of lipid species, including increased acyl carnitine, fatty acyl chains, and triacylglycerol (TAG). Together, these results suggest the possibility that SPRY4-IT1 knockdown may induce apoptosis via lipin 2-mediated alterations in lipid metabolism leading to cellular lipotoxicity.

Genome-wide methylated CpG island profiles of melanoma cells reveal a melanoma coregulation network.

Metastatic melanoma is a malignant cancer with generally poor prognosis, with no targeted chemotherapy. To identify epigenetic changes related to melanoma, we have determined genome-wide methylated CpG island distributions by next-generation sequencing. Melanoma chromosomes tend to be differentially methylated over short CpG island tracts. CpG islands in the upstream regulatory regions of many coding and noncoding RNA genes, including, for example, TERC, which encodes the telomerase RNA, exhibit extensive hypermethylation, whereas several repeated elements, such as LINE 2, and several LTR elements, are hypomethylated in advanced stage melanoma cell lines. By using CpG island demethylation profiles, and by integrating these data with RNA-seq data obtained from melanoma cells, we have identified a co-expression network of differentially methylated genes with significance for cancer related functions. Focused assays of melanoma patient tissue samples for CpG island methylation near the noncoding RNA gene SNORD-10 demonstrated high specificity.

The prodomain of the Bordetella two-partner secretion pathway protein FhaB remains intracellular yet affects the conformation of the mature C-terminal domain.

Two-partner secretion (TPS) systems use ß-barrel proteins of the Omp85-TpsB superfamily to transport large exoproteins across the outer membranes of Gram-negative bacteria. The Bordetella FHA/FhaC proteins are prototypical of TPS systems in which the exoprotein contains a large C-terminal prodomain that is removed during translocation. Although it is known that the FhaB prodomain is required for FHA function in vivo, its role in FHA maturation has remained mysterious. We show here that the FhaB prodomain is required for the extracellularly located mature C-terminal domain (MCD) of FHA to achieve its proper conformation. We show that the C-terminus of the prodomain is retained intracellularly and that sequences within the N-terminus of the prodomain are required for this intracellular localization. We also identify sequences at the C-terminus of the MCD that are required for release of mature FHA from the cell surface. Our data support a model in which the intracellularly located prodomain affects the final conformation of the extracellularly located MCD. We hypothesize that maturation triggers cleavage and degradation of the prodomain.

Epigenetic regulation of microRNA genes and the role of miR-34b in cell invasion and motility in human melanoma.

Invasive melanoma is the most lethal form of skin cancer. The treatment of melanoma-derived cell lines with 5-aza-2'-deoxycytidine (5-Aza-dC) markedly increases the expression of several miRNAs, suggesting that the miRNA-encoding genes might be epigenetically regulated, either directly or indirectly, by DNA methylation. We have identified a group of epigenetically regulated miRNA genes in melanoma cells, and have confirmed that the upstream CpG island sequences of several such miRNA genes are hypermethylated in cell lines derived from different stages of melanoma, but not in melanocytes and keratinocytes. We used direct DNA bisulfite and immunoprecipitated DNA (Methyl-DIP) to identify changes in CpG island methylation in distinct melanoma patient samples classified as primary in situ, regional metastatic, and distant metastatic. Two melanoma cell lines (WM1552C and A375 derived from stage 3 and stage 4 human melanoma, respectively) were engineered to ectopically express one of the epigenetically modified miRNA: miR-34b. Expression of miR-34b reduced cell invasion and motility rates of both WM1552C and A375, suggesting that the enhanced cell invasiveness and motility observed in metastatic melanoma cells may be related to their reduced expression of miR-34b. Total RNA isolated from control or miR-34b-expressing WM1552C cells was subjected to deep sequencing to identify gene networks around miR-34b. We identified network modules that are potentially regulated by miR-34b, and which suggest a mechanism for the role of miR-34b in regulating normal cell motility and cytokinesis.

Epigenetic regulation of microRNA-375 and its role in melanoma development in humans.

To identify epigenetically regulated miRNAs in melanoma, we treated a stage 3 melanoma cell line WM1552C, with 5AzadC and/or 4-PBA. Several hypermethylated miRNAs were detected, one of which, miR-375, was highly methylated and was studied further. Minimal CpG island methylation was observed in melanocytes, keratinocytes, normal skin, and nevus but hypermethylation was observed in patient tissue samples from primary, regional, distant, and nodular metastatic melanoma. Ectopic expression of miR-375 inhibited melanoma cell proliferation, invasion, and cell motility, and induced cell shape changes, strongly suggesting that miR-375 may have an important function in the development and progression of human melanomas.

The melanoma-upregulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion.

The identification of cancer-associated long noncoding RNAs (lncRNAs) and the investigation of their molecular and biological functions are important to understand the molecular biology of cancer and its progression. Although the functions of lncRNAs and the mechanisms regulating their expression are largely unknown, recent studies are beginning to unravel their importance in human health and disease. Here, we report that a number of lncRNAs are differentially expressed in melanoma cell lines in comparison to melanocytes and keratinocyte controls. One of these lncRNAs, SPRY4-IT1 (GenBank accession ID AK024556), is derived from an intron of the SPRY4 gene and is predicted to contain several long hairpins in its secondary structure. RNA-FISH analysis showed that SPRY4-IT1 is predominantly localized in the cytoplasm of melanoma cells, and SPRY4-IT1 RNAi knockdown results in defects in cell growth, differentiation, and higher rates of apoptosis in melanoma cell lines. Differential expression of both SPRY4 and SPRY4-IT1 was also detected in vivo, in 30 distinct patient samples, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic melanoma. The elevated expression of SPRY4-IT1 in melanoma cells compared to melanocytes, its accumulation in cell cytoplasm, and effects on cell dynamics, including increased rate of wound closure on SPRY4-IT1 overexpression, suggest that the higher expression of SPRY4-IT1 may have an important role in the molecular etiology of human melanoma.

The regulation of miRNA-211 expression and its role in melanoma cell invasiveness.

The immediate molecular mechanisms behind invasive melanoma are poorly understood. Recent studies implicate microRNAs (miRNAs) as important agents in melanoma and other cancers. To investigate the role of miRNAs in melanoma, we subjected human melanoma cell lines to miRNA expression profiling, and report a range of variations in several miRNAs. Specifically, compared with expression levels in melanocytes, levels of miR-211 were consistently reduced in all eight non-pigmented melanoma cell lines we examined; they were also reduced in 21 out of 30 distinct melanoma samples from patients, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic. The levels of several predicted target mRNAs of miR-211 were reduced in melanoma cell lines that ectopically expressed miR-211. In vivo target cleavage assays confirmed one such target mRNA encoded by KCNMA1. Mutating the miR-211 binding site seed sequences at the KCNMA1 3'-UTR abolished target cleavage. KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels. Two different melanoma cell lines ectopically expressing miR-211 exhibited significant growth inhibition and reduced invasiveness compared with the respective parental melanoma cell lines. An shRNA against KCNMA1 mRNA also demonstrated similar effects on melanoma cells. miR-211 is encoded within the sixth intron of TRPM1, a candidate suppressor of melanoma metastasis. The transcription factor MITF, important for melanocyte development and function, is needed for high TRPM1 expression. MITF is also needed for miR-211 expression, suggesting that the tumor-suppressor activities of MITF and/or TRPM1 may at least partially be due to miR-211's negative post transcriptional effects on the KCNMA1 transcript. Given previous reports of high KCNMA1 levels in metastasizing melanoma, prostate cancer and glioma, our findings that miR-211 is a direct posttranscriptional regulator of KCNMA1 expression as well as the dependence of this miRNA's expression on MITF activity, establishes miR-211 as an important regulatory agent in human melanoma.

Protein-coding and non-coding gene expression analysis in differentiating human keratinocytes using a three-dimensional epidermal equivalent.

The epidermal compartment is complex and organized into several strata composed of keratinocytes (KCs), including basal, spinous, granular, and cornified layers. The continuous process of self-renewal and barrier formation is dependent on a homeostatic balance achieved amongst KCs involving proliferation, differentiation, and cell death. To determine genes responsible for initiating and maintaining a cornified epidermis, organotypic cultures comprised entirely of stratified KCs creating epidermal equivalents (EE) were raised from a submerged state to an air/liquid (A/L) interface. Compared to the array profile of submerged cultures containing KCs predominantly in a proliferative (relatively undifferentiated) state, EEs raised to an A/L interface displayed a remarkably consistent and distinct profile of mRNAs. Cultures lifted to an A/L interface triggered the induction of gene groups that regulate proliferation, differentiation, and cell death. Next, differentially expressed microRNAs (miRNAs) and long non-coding (lncRNA) RNAs were identified in EEs. Several differentially expressed miRNAs were validated by qRT-PCR and Northern blots. miRNAs 203, 205 and Let-7b were up-regulated at early time points (6, 18 and 24 h) but down-regulated by 120 h. To study the lncRNA regulation in EEs, we profiled lncRNA expression by microarray and validated the results by qRT-PCR. Although the differential expression of several lncRNAs is suggestive of a role in epidermal differentiation, their biological functions remain to be elucidated. The current studies lay the foundation for relevant model systems to address such fundamentally important biological aspects of epidermal structure and function in normal and diseased human skin.

Natural-host animal models indicate functional interchangeability between the filamentous haemagglutinins of Bordetella pertussis and Bordetella bronchiseptica and reveal a role for the mature C-terminal domain, but not the RGD motif, during infection.

Bacteria of the Bordetella genus cause respiratory tract infections. Both broad host range (e.g. Bordetella bronchiseptica) and human-adapted (e.g. Bordetella pertussis) strains produce a surface-exposed and secreted protein called filamentous haemagglutinin (FHA) that functions in adherence and immunomodulation. Previous studies using B. pertussis and cultured mammalian cells identified several FHA domains with potential roles in host cell interactions, including an Arg-Gly-Asp (RGD) triplet that was reported to bind integrins on epithelial cells and monocytes to activate host signalling pathways. We show here that, in contrast to our previous report, the fhaB genes of B. pertussis and B. bronchiseptica are functionally interchangeable, at least with regard to the various in vitro and in vivo assays investigated. This result is significant because it indicates that information obtained studying FHA using B. bronchiseptica and natural-host animal models should apply to B. pertussis FHA as well. We also show that the C-terminus of mature FHA, which we name the MCD, mediates adherence to epithelial and macrophage-like cells and is required for colonization of the rat respiratory tract and modulation of the inflammatory response in mouse lungs. We could not, however, detect a role for the RGD in any of these processes.