KSHV-Encoded MicroRNAs: Lessons for Viral Cancer Pathogenesis and Emerging Concepts

The human genome contains microRNAs (miRNAs), small noncoding RNAs that orchestrate a number of physiologic processes through regulation of gene expression. Burgeoning evidence suggests that dysregulation of miRNAs may promote disease progression and cancer pathogenesis. Virus-encoded miRNAs, exhibiting unique molecular signatures and functions, have been increasingly recognized as contributors to viral cancer pathogenesis. A large segment of the existing knowledge in this area has been generated through characterization of miRNAs encoded by the human gamma-herpesviruses, including the Kaposi's sarcoma-associated herpesvirus (KSHV). Recent studies focusing on KSHV miRNAs have led to a better understanding of viral miRNA expression in human tumors, the identification of novel pathologic check points regulated by viral miRNAs, and new insights for viral miRNA interactions with cellular (“human”) miRNAs. Elucidating the functional effects of inhibiting KSHV miRNAs has also provided a foundation for further translational efforts and consideration of clinical applications. This paper summarizes recent literature outlining mechanisms for KSHV miRNA regulation of cellular function and cancer-associated pathogenesis, as well as implications for interactions between KSHV and human miRNAs that may facilitate cancer progression. Finally, insights are offered for the clinical feasibility of targeting miRNAs as a therapeutic approach for viral cancers

Identification and characterization of a novel target of Src tyrosine kinase

Bibliography: p. 177-20

KSHV-Encoded MicroRNAs: Lessons for viral cancer pathogenesis and emerging concepts

The human genome contains microRNAs (miRNAs), small noncoding RNAs that orchestrate a number of physiologic processes through regulation of gene expression. Burgeoning evidence suggests that dysregulation of miRNAs may promote disease progression and cancer pathogenesis. Virus-encoded miRNAs, exhibiting unique molecular signatures and functions, have been increasingly recognized as contributors to viral cancer pathogenesis. A large segment of the existing knowledge in this area has been generated through characterization of miRNAs encoded by the human gamma-herpesviruses, including the Kaposi's sarcomaassociated herpesvirus (KSHV). Recent studies focusing on KSHV miRNAs have led to a better understanding of viral miRNA expression in human tumors, the identification of novel pathologic check points regulated by viral miRNAs, and new insights for viral miRNA interactions with cellular ("human") miRNAs. Elucidating the functional effects of inhibiting KSHV miRNAs has also provided a foundation for further translational efforts and consideration of clinical applications. This paper summarizes recent literature outlining mechanisms for KSHV miRNA regulation of cellular function and cancer-associated pathogenesis, as well as implications for interactions between KSHV and human miRNAs that may facilitate cancer progression. Finally, insights are offered for the clinical feasibility of targeting miRNAs as a therapeutic approach for viral cancers

The GW182 protein colocalizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies

A novel cytoplasmic compartment referred to as GW bodies (GWBs) was initially identified using antibodies specific to a 182-kD protein termed GW182. GW182 was characterized by multiple glycine(G)-tryptophan(W) repeats and an RNA recognition motif (RRM) that bound a subset of HeLa cell messenger RNAs (mRNAs). The function of GWBs was not known; however, more recent evidence suggested similarities between GWBs and cytoplasmic structures that contain hLSm proteins and hDcp1, the human homolog to a yeast decapping enzyme subunit. In this study, we used antibodies to hLSm4 and hDcp1 to show that both of these markers of an mRNA degradation pathway colocalize to the same structures as GW182. Our studies demonstrate that GW182, hLSm4, and hDcp1 are found in the same cytoplasmic structures and suggest that GW182 is involved in the same mRNA processing pathway as hLSm4 and hDcp1

Formation of GW bodies is a consequence of microRNA genesis

GW bodies (GWBs), or mammalian P bodies, proposed to be involved in messenger RNA storage and/or degradation, have recently been linked to RNA interference and microRNA (miRNA) processing. We report that endogenous let-7 miRNA co-precipitates with the GW182 protein complex. In addition, knockdown of two proteins, Drosha and its protein partner DGCR8, which are vital to the generation of mature miRNA, results in the loss of GWBs. Subsequent introduction of short interference RNA specific to lamin A/C is accompanied by reassembly of GWBs and concurrent knockdown of lamin A/C protein. Taken together, these studies show that miRNAs are crucial components in GWB formation

Fusogenic-Oligoarginine Peptide-Mediated Delivery of siRNAs Targeting the CIP2A Oncogene into Oral Cancer Cells

<div><p>Despite a better understanding of the pathogenesis of oral cancer, its treatment outcome remains poor. Thus, there is a need for new therapeutic strategies to improve the prognosis of this disease. RNA interference (RNAi) appears to be a promising therapeutic tool for the treatment of many diseases, including oral cancer. However, an obstacle for RNAi-mediated therapies has been delivery, in particular, the retention of small interfering RNAs (siRNAs) in endosomes and their subsequent degradation in lysosomes, resulting in inefficient gene silencing. Thus, the current study examined the feasibility of designing and utilizing a peptide, termed 599, consisting of a synthetic influenza virus-derived endosome-disruptive fusogenic peptide sequence and a stretch of cationic cell-penetrating nona(D-arginine) residues, to deliver siRNAs into oral cancer cells and induce silencing of the therapeutic target, CIP2A, an oncoprotein overexpressed in various human malignancies including oral cancer. Increasing the 599 peptide-to-siRNA molar ratio demonstrated a higher binding capacity for siRNA molecules and enhanced siRNA delivery into the cytoplasm of oral cancer cells. In fact, quantitative measurements of siRNA delivery into cells demonstrated that a 50∶1 peptide-to-siRNA molar ratio could deliver 18-fold higher amounts of siRNAs compared to cells treated with siRNA alone with no significant long-term cytotoxic effects. Most importantly, the 599 peptide-mediated siRNA delivery promoted significant CIP2A mRNA and protein silencing which resulted in decreased oral cancer cell invasiveness and anchorage-independent growth. Together, these data demonstrate that a chimeric peptide consisting of a fusogenic sequence, in combination with cell-penetrating residues, can be used to effectively deliver siRNAs into oral cancer cells and induce the silencing of its target gene, potentially offering a new therapeutic strategy in combating oral cancer.</p></div

Particle characterization of the 599/siCIP2A complex.

<p>(<b>A</b>) Size distribution and zeta potential of the 599 peptide complexed with siCIP2A at a 50∶1 peptide-to-siRNA molar ratio 20 minutes after formulation in water. (<b>B</b>) Darkfield-based optical microscopy image of the 599 peptide complexed with siCIP2A at a 50∶1 peptide-to-siRNA molar ratio 20 minutes after formulation in water. Scale bar: 10,000 nm.</p

The 599 peptide mediates CIP2A gene silencing and subsequent destabilization of the c-Myc oncoprotein in oral cancer cells.

<p>(<b>A</b>) Real-time PCR analysis of CIP2A mRNA levels in CAL 27 and SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed at a 50∶1 peptide-to-siRNA molar ratio to 100 nM of siRNA targeting CIP2A (siCIP2A) compared with control non-targeting siRNA (siNT). The CIP2A mRNA levels were normalized to 18S rRNA. Data are mean ± SEM of three separate experiments performed in triplicate, where **P<0.01 compared to siNT treated cells (Student’s t test). (<b>B</b>) Western blot analyses of CIP2A and c-Myc protein expression levels in CAL 27 and SCC-25 oral cancer cells 48 hours post-treatment with 599 peptide complexed to either 100 nM of siNT or siCIP2A at a 50∶1 peptide-to-siRNA molar ratio. GAPDH protein levels were monitored to ensure equal loading of samples.</p