Upregulation of xCT by KSHV-Encoded microRNAs Facilitates KSHV Dissemination and Persistence in an Environment of Oxidative Stress

Abstract

Upregulation of xCT, the inducible subunit of a membrane-bound amino acid transporter, replenishes intracellular glutathione stores to maintain cell viability in an environment of oxidative stress. xCT also serves as a fusion-entry receptor for the Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS). Ongoing KSHV replication and infection of new cell targets is important for KS progression, but whether xCT regulation within the tumor microenvironment plays a role in KS pathogenesis has not been determined. Using gene transfer and whole virus infection experiments, we found that KSHV-encoded microRNAs (KSHV miRNAs) upregulate xCT expression by macrophages and endothelial cells, largely through miR-K12-11 suppression of BACH-1—a negative regulator of transcription recognizing antioxidant response elements within gene promoters. Correlative functional studies reveal that upregulation of xCT by KSHV miRNAs increases cell permissiveness for KSHV infection and protects infected cells from death induced by reactive nitrogen species (RNS). Interestingly, KSHV miRNAs simultaneously upregulate macrophage secretion of RNS, and biochemical inhibition of RNS secretion by macrophages significantly reduces their permissiveness for KSHV infection. The clinical relevance of these findings is supported by our demonstration of increased xCT expression within more advanced human KS tumors containing a larger number of KSHV-infected cells. Collectively, these data support a role for KSHV itself in promoting de novo KSHV infection and the survival of KSHV-infected, RNS-secreting cells in the tumor microenvironment through the induction of xCT.

Author Summary

Herpesviruses are the most common etiologic agents of cancer in patients with suppressed immune function, and the Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the most common causes of cancer in this setting. KSHV infection of new cell targets is critical for tumor progression, and a better understanding of how viral receptors on the surface of cells are regulated in the tumor microenvironment may lead to new therapies. KSHV encodes unique RNAs called microRNAs (KSHV miRNAs) that regulate a variety of cell functions. In this study, we show that KSHV miRNAs increase the susceptibility of cells to KSHV infection and protect infected cells from death induced by cancer-promoting reactive nitrogen species (RNS). They accomplish this in large part by increasing cell surface expression of a transport protein subunit called xCT. We also show that KSHV miRNAs increase secretion of RNS by infected cells, and that blocking RNS secretion reduces the ability of KSHV to infect cells. Therefore, by regulating xCT and RNS, we find KSHV is able to “fine-tune” cell function in order to maintain a stable population of infected cells which secrete cancer-promoting factors in the local environment. This work has important implications for developing new therapies to target xCT and reduce survival of KSHV-infected tumor cells.