Ranjan J. Perera

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-211s 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 miRNAs expression on MITF activity, establishes miR-211 as an important regulatory agent in human melanoma.

MicroRNAs in the Search for Understanding Human Diseases

MiroRNAs (miRNAs) are double-stranded, noncoding RNA molecules (with an average size of 22bp) that serve as post-transcriptional regulators of gene expression in higher eukaryotes. miRNAs play an important role in development and other cellular processes by hybridizing with complementary target mRNA transcripts, preventing their translation and thereby destabilizing the target transcripts. Though hundreds of miRNAs have been discovered in a variety of organisms, little is known about their cellular function. They have been implicated in the regulation of developmental timing and pattern formation, restriction of differentiation potential, regulation of insulin secretion, resistance to viral infection, and in genomic rearrangements associated with carcinogenesis or other genetic disorders, such as fragile X syndrome. Recent evidence suggests that the number of unique miRNA genes in humans exceeds 1000, and may be as high as 20 000. It is estimated that 20–30% of all human mRNAs are miRNA targets.During the last few years, special attention has been given to miRNAs as candidate drug targets for cancer, diabetes mellitus, obesity, and viral diseases.

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.

Identification of a novel lncRNA in gluteal adipose tissue and evidence for its positive effect on preadipocyte differentiation

Peripheral lower body fat is associated with lower cardiometabolic risk. Physiological differences in gluteal compared with abdominal subcutaneous (sc) adipocyte functions are known but the molecular basis for depot differences in adipocyte function is poorly understood. Our goal is to identify novel gene regulatory pathways that underlie the heterogeneity of human fat distribution.

Vascular Smooth Muscle LRP6 Limits Arteriosclerotic Calcification in Diabetic LDLR−/− Mice by Restraining Noncanonical Wnt Signals

RATIONALE Wnt signaling regulates key aspects of diabetic vascular disease. OBJECTIVE We generated SM22-Cre;LRP6(fl/fl);LDLR(-/-) mice to determine contributions of Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6) in the vascular smooth muscle lineage of male low-density lipoprotein receptor-null mice, a background susceptible to diet (high-fat diet)-induced diabetic arteriosclerosis. METHODS AND RESULTS As compared with LRP6(fl/fl);LDLR(-/-) controls, SM22-Cre;LRP6(fl/fl);LDLR(-/-) (LRP6-VKO) siblings exhibited increased aortic calcification on high-fat diet without changes in fasting glucose, lipids, or body composition. Pulse wave velocity (index of arterial stiffness) was also increased. Vascular calcification paralleled enhanced aortic osteochondrogenic programs and circulating osteopontin (OPN), a matricellular regulator of arteriosclerosis. Survey of ligands and Frizzled (Fzd) receptor profiles in LRP6-VKO revealed upregulation of canonical and noncanonical Wnts alongside Fzd10. Fzd10 stimulated noncanonical signaling and OPN promoter activity via an upstream stimulatory factor (USF)-activated cognate inhibited by LRP6. RNA interference revealed that USF1 but not USF2 supports OPN expression in LRP6-VKO vascular smooth muscle lineage, and immunoprecipitation confirmed increased USF1 association with OPN chromatin. ML141, an antagonist of cdc42/Rac1 noncanonical signaling, inhibited USF1 activation, osteochondrogenic programs, alkaline phosphatase, and vascular smooth muscle lineage calcification. Mass spectrometry identified LRP6 binding to protein arginine methyltransferase (PRMT)-1, and nuclear asymmetrical dimethylarginine modification was increased with LRP6-VKO. RNA interference demonstrated that PRMT1 inhibits OPN and TNAP, whereas PRMT4 supports expression. USF1 complexes containing the histone H3 asymmetrically dimethylated on Arg-17 signature of PRMT4 are increased with LRP6-VKO. Jmjd6, a demethylase downregulated with LRP6 deficiency, inhibits OPN and TNAP expression, USF1: histone H3 asymmetrically dimethylated on Arg-17 complex formation, and transactivation. CONCLUSIONS LRP6 restrains vascular smooth muscle lineage noncanonical signals that promote osteochondrogenic differentiation, mediated in part via USF1- and arginine methylation-dependent relays.

Positive-negative selection for homologous recombination in Arabidopsis.

In plants gene knock-outs and targeted mutational analyses are hampered by the inefficiency of homologous recombination. We have developed a strategy to enrich for rare events of homologous recombination in Arabidopsis using combined positive and negative selection. The T-DNA targeting construct contained two flanking regions of the target alcohol dehydrogenase gene as homologous sequences, and neomycin phosphotransferase and cytosine deaminase as positive and negative markers, respectively. A root explant transformation procedure was used to obtain transgenic calli. Among 6250 transformants isolated by positive selection, 39 were found to be resistant to negative selection as well. Of these 39, at least one had undergone homologous recombination correlated with a unidirectional transfer of information. Although the ADH locus was not changed, our data demonstrate that a homologous recombination event can be selected by positive negative selection in plants.

The role of microRNAs and long non-coding RNAs in the pathology, diagnosis, and management of melanoma

Melanoma is frequently lethal and its global incidence is steadily increasing. Despite the rapid development of different modes of targeted treatment, durable clinical responses remain elusive. A complete understanding of the molecular mechanisms that drive melanomagenesis is required, both genetic and epigenetic, in order to improve prevention, diagnosis, and treatment. There is increased appreciation of the role of microRNAs (miRNAs) in melanoma biology, including in proliferation, cell cycle, migration, invasion, and immune evasion. Data are also emerging on the role of long non-coding RNAs (lncRNAs), such as SPRY4-IT1, BANCR, and HOTAIR, in melanomagenesis. Here we review the data on the miRNAs and lncRNAs implicated in melanoma biology. An overview of these studies will be useful for providing insights into mechanisms of melanoma development and the miRNAs and lncRNAs that might be useful biomarkers or future therapeutic targets.

Epigenetic regulation of miRNA genes and their role in human melanomas.

Melanoma is a leading cause of death from cancers in the USA. While exposure to UV radiation has long been identified as a primary risk factor for melanoma, molecular mechanisms directly linking UV radiation to the development of melanoma, especially metastatic melanoma, are poorly understood. Besides abnormality in several signal transduction pathways important for normal melanocyte development, a number of ncRNAs, including miRNAs, are emerging as important causal factors to melanoma initiation and progression. The recent discovery of altered patterns of epigenetic regulation in ncRNA genes adds further complexity. Since miRNA precursor genes are usually nested within other protein-coding genes, the abnormal regulation of these protein-coding genes by epigenetic mechanisms is expected to cause aberrant regulation of the miRNA target genes. We discuss recent findings that link epigenetic regulation of ncRNA genes to melanoma, and speculate on a possible connection between UV irradiation and epigenetic regulation that might be important for this disease.

MicroRNA 211 Functions as a Metabolic Switch in Human Melanoma Cells

ABSTRACT 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.