KSHV MicroRNAs Mediate Cellular Transformation and Tumorigenesis by Redundantly Targeting Cell Growth and Survival Pathways

<div><p>Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several human cancers, including Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease, malignancies commonly found in HIV-infected patients. While KSHV encodes diverse functional products, its mechanism of oncogenesis remains unknown. In this study, we determined the roles KSHV microRNAs (miRs) in cellular transformation and tumorigenesis using a recently developed KSHV-induced cellular transformation system of primary rat mesenchymal precursor cells. A mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Remarkably, the oncogenicity of the mutant virus was fully restored by genetic complementation with the miR cluster or several individual pre-miRs, which rescued cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. Genomic analysis identified common targets of KSHV miRs in diverse pathways with several cancer-related pathways preferentially targeted. These works define for the first time an essential viral determinant for KSHV-induced oncogenesis and identify NF-κB as a critical pathway targeted by the viral miRs. Our results illustrate a common theme of shared functions with hierarchical order among the KSHV miRs.</p></div

Multiple KSHV miRs rescue cellular transformation and tumorigenesis of the Mut virus.

<p>(A) Formation of colonies in softagar medium plated with MM cells infected by WT, REV and Mut viruses and Mut virus complemented by individual KSHV pre-miRs (MutKi), miR cluster (MutCl) or vector control (MutVt). (B–C) Tumor incidences over time (B) and Kaplan-Meier survival curves (C) of nude mice inoculated with MM cells infected by WT virus or Mut virus complemented by individual KSHV pre-miRs or vector control. Tumor volume of 0.2 cm³ was used as a threshold for tumor incidence. Tumor analyses were performed at 10 weeks following inoculation of the cells or when the volumes reached 1 cm³.</p

KSHV miRs promote cellular proliferation by regulating cell cycle and inhibiting apoptosis.

<p>(A–D) Growth curves (A), cell cycle profiles (B), floating cells (C), and annexin V-positive adherent cells (D) in cultures of MM cells infected by different KSHV recombinant viruses. (E–G) Cell growth (E), annexin V-positive adherent cells (F), and cell cycle profiles (G) of cultures of MM cells infected by the Mut virus complemented by individual KSHV pre-miRs (MutKi), miR cluster (MutCl) or vector control (MutVt). Cell cycle and apoptosis were analyzed at day 5 post-seeding. All statistical analyses were performed by comparing other cells with the MutVt cells.</p

MiR-K1 targeting of IκBα is essential and sufficient for KSHV subversion of cell cycle and apoptosis pathways.

<p>(A) Expression of IκBα protein in cells with and without the expression of miR-K1 measured by Western-blotting. Cells analyzed were WT cells (WT), Mut cells (Mut), Rev cells (Rev), and Mut cells complemented with miR-K1 (MutK1), miR cluster (MutCl and vector control (MutVt). (B) Sequence alignment of miR-K1 with rat IκBα 3′UTR WT reporter and its mutant reporter containing a mutation in the putative miR-K1 targeting site, and the corresponding human IκBα 3′UTR sequence. (C) Suppression of IκBα 3′UTR WT reporter activity but not its mutant reporter activity by KSHV miR-K1. 293 cells were cotransfected with the IκBα 3′UTR WT reporter or its mutant reporter together with a miR-K1 mimic or a scrambled control and a β-galatosidase expression construct for 48 h and measured for relative luciferase activities. (D) Derepression of the inhibitory effect of miR-K1 on IκBα 3′UTR WT reporter but not its mutant reporter in WT cells by a miR-K1 suppressor. (E-F) Cell cycle profiles (E) and apoptosis(F) in Mut cells, Mut cells complemented with miR-K1 or vector control with knock down of IκBα using a specific siRNA or a scrambled control. (G–I) Expression of IκBα in WT cells(G) is sufficient to cause a shift in cell cycle profile (H) or apoptosis rate (I) to that resembling Mut cells. Statistical analyses were performed by comparing other cells with Mut cells transfected with scrambled siRNA (E–F) or WT cells transfected with control vector (H–I).</p

Expression of KSHV miRs in MM cells infected by WT, Mut and Rev viruses, as well as MM cells infected by Mut virus complemented by pre-miRs (Ki), miR cluster (Cl) or vector control (V).

<p>MiRs were detected by quantitative real-time reverse transcription PCR (RT-qPCR).</p

A model illustrating the regulation of cellular transformation by KSHV miRs.

<p>A model illustrating the regulation of cellular transformation by KSHV miRs.</p

Top enriched pathways in WT cells compared to Mut cells.

<p>Top enriched pathways in WT cells compared to Mut cells.</p

Gene expression profiling analysis of KSHV miRs.

<p>(A) Unsupervised clustering of gene expression profiles of WT cells, and Mut cells complemented with the miR cluster (Cl) or individual pre-miRs (Ki). Note that the Mut cells condition was subtracted from all the MutKi cells to eliminate the effect of other unrelated viral genes (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003857#s4" target="_blank">Materials and Methods</a> for details). (B) Top 20 most enriched pathways in WT cells, and Mut cells complemented with the miR cluster or individual pre-miRs compared to Mut cells complemented with the vector control. The color scale represents the GSEA normalized enrichment score. The expression fold changes of all genes of these cells are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003857#ppat.1003857.s016" target="_blank">Table S5</a>. (C) Signature genes that are positively or negatively correlated with tumorigenicity identified by Anova. (D) Principle components (PCs) obtained from the expression data of MutKi cells and the corresponding percentages of explained expression variances. Note that >95% of expression variances of the 16,501 genes in MutKi cells can be explained using only 8 PCs. (E) Lasso fitting to determine the linear combinatory effect of individual pre-miRs to the overall expression pattern of Mut cells complemented with the miR cluster. The x-axis denotes the Lasso iteration and the y-axis represents the coefficients or predicted effects for each MutKi. The Lasso reached the converged predictions at the 30th iteration.</p

Top networks of signature genes associated with tumorigenicity induced by KSHV miRs.

<p>Top networks of signature genes associated with tumorigenicity induced by KSHV miRs.</p

Multiple KSHV miRs target the IκBα and NF-κB pathway.

<p>(A) IκBα expression in MM cells infected by Mut virus complemented by individual KSHV pre-miRs (MutKi) or vector control (MutVt). Cell grown to confluency were examined for IκBα protein expression by Western-blotting. (B) NF-κB reporter activity in MM cells infected by Mut virus complemented by individual KSHV pre-miRs or vector control. Cells were transfected with a NF-κB reporter construct or it's a mutant reporter together with a β-galatosidase expression construct for 48 h and measured for relative luciferase activities. (C) Deletion of either the miR cluster or vFLIP from the KSHV genome is sufficient to abolish KSHV activation of the NF-κB pathway. Uninfected cells or cells infected by WT and mutants of miRNA cluster (Mut) or vFLIP (ΔvFLIP) were transfected with either NF-κB WT luciferase reporter or NF-κB mutant luciferase reporter together with a β-galactosidase construct for 48 h. Cells were collected and measured for relative luciferase activities. Statistical analyses were performed by comparing other cells with Mut cells (B) or WT cells (C).</p