KSHV is known to possess a complex series of molecular strategies linked to regulation of cell proliferation, induction of cell transformation, and suppression of apoptosis. Indeed, a number of previous single-gene studies have shown that different KSHV-encoded genes harbor many of these functional properties (15
). The LANA protein of KSHV is abundantly expressed in virus-associated malignancies (34
). LANA is not only critical for viral episomal maintenance during latent infection but has also been reported to have transcriptional regulatory properties that allow it to directly modulate the promoters of cellular and viral genes, as well as its own promoters (27
). Another crucial role associated with LANA's oncogenicity is its interaction with an array of transcriptional factors, such as ATF4/CREB2 (43
), c-Jun (10
), p53 (23
), and STAT3 (48
), shown to indirectly regulate downstream target gene expression. Recently, we showed the interaction of LANA with Sp1, resulting in upregulation of the telomerase promoter and potentially contributing to the immortalization of KSHV-infected cells (63
). Additionally, LANA can suppress the functional activities of p53, thereby facilitating KSHV-mediated pathogenesis (57
These reports establish that LANA can modulate various cellular pathways. The results of our pathway-specific gene array analysis of the total RNA from BJAB cells expressing RFP-Vector and RFP-LANA further showed that a number of cell factors were upregulated in LANA-expressing cells, including the inhibitor of apoptosis protein survivin. This suggested that LANA may function as an activator for upregulating survivin expression. As expected, the results further showed that LANA expression resulted in increased levels of survivin mRNA in LANA-expressing HEK 293 and BJAB cells. Additionally, survivin expression was upregulated in KSHV-infected cells (BCBL1, BC3, and JSC) in comparison with its levels in cells not infected with KSHV (HEK 293, Saos-2, and BJAB), and LANA increased survivin expression in a dose-dependent manner in epithelial and B-cell lines. The results suggested that upregulation of survivin was correlated with LANA expression levels.
Generalized transcription activity is a local phenomenon, and every cell coordinates the expression of a vast number of genes important for survival as part of its generalized transcription activity. Survivin gene expression is essential and evolutionarily conserved during late-stage cell division, especially in cytokinesis, which potentially involves cleavage furrow formation (1
). Survivin is also associated with DNA damage and other epigenetic effects (21
). In addition, increased survivin expression leads to uncontrolled cell proliferation and survival, which is a hallmark of cancer (29
). Survivin, an IAP family member, is known to be involved in the control of both cell division and inhibition (3
). In addition, the spliced variant survivin-ΔEx3 may be the cellular homologue of KSHV K7 and functions as a constitutively expressed adaptor protein capable of recruiting activated caspases (65
). Several groups have also independently demonstrated that survivin repression is p53 dependent (30
), but it was not clear if the p53 binding sequence of the survivin promoter was involved in its repression in response to p53 activation. The data described above showed that LANA can bind to Sp1 and p53 in complex with their cis
-acting elements within the survivin promoter. However, the detailed mechanism and significance of LANA interaction with the survivin promoter continues to be explored in our laboratory.
Previous reports showed that the survivin gene promoter contains CG-rich-Sp1 canonical, Sp1-like, cell cycle-dependent element/cell cycle gene homology region, and p53 binding sites (21
). Therefore, we analyzed the promoter region from positions −649 to +1 and tried to determine which responsive elements of the survivin promoter were responsible for interaction and complex formation with LANA. Using a series of survivin promoter-luciferase constructs, we identified GC/Sp1 and p53 elements within the survivin core promoter region that were critical for LANA to enhance survivin transcription. Another study has shown that Sp1 is a major player in regulating survivin gene expression and apoptosis control (39
). EMSA results further revealed that LANA forms a complex with Sp1 or Sp1-like proteins bound to the GC/Sp1 cis
-acting DNA element on the survivin promoter, which results in upregulation of its activity. Interestingly, a recent study has shown that selenium inhibits Sp1 binding to its cis
-acting element and downregulates the survivin promoter activity (19
). In addition, p53 regulated survivin expression by binding directly to the survivin promoter (52
). The LANA/p53 complex was also shown to bind the p53 responsive element on the survivin promoter, indicating that upregulation of survivin expression may occur through alteration of p53 function through its association with LANA. Previous studies have also suggested that LANA contributes to viral persistence and oncogenesis through the promotion of cell survival via targeted disruption of p53 functions (13
). Furthermore, survivin expression also antagonizes p53-induced apoptosis (30
The results of immunohistochemistry analysis revealed that survivin expression was upregulated in KSHV-associated tissue, suggesting that LANA, in addition to other latent proteins, is likely to play an important role in the upregulation of survivin expression in KSHV-infected cells, thus contributing to KSHV-associated tumorigenesis. Furthermore, depletion of survivin by using shRNA decreases the rate of cell proliferation in KSHV-positive lymphoma cells, which strongly suggests that LANA plays an important role in the upregulation of survivin expression and, thus, maintenance of the proliferative state in KSHV-infected cells. The utilization of the survivin pathway by LANA would be at least in part to promote enhanced cellular survival and proliferation, which then, combined with decreased apoptotic activity, may drive polyclonal expansion of cells. These changes, in addition to the acquisition of epigenetic changes and disruption of the cellular processes involved in genome fidelity (57
), are likely to lead to the outgrowth of tumor cells.
In summary, we have shown in this report that the LANA protein of KSHV upregulates survivin expression through the formation of complexes with Sp1 or Sp1-like proteins binding to the Sp1 site within the core survivin promoter region. In addition, LANA also alters p53 function by forming complexes with p53 bound to the p53 binding site on the survivin promoter, thus increasing survivin promoter activity (Fig. ). Survivin expression was also upregulated in KS tissue, suggesting that LANA can contribute to the upregulation of survivin expression in KSHV-positive KS tissue. Furthermore, knockdown of survivin expression by lentivirus-delivered shRNA showed that LANA plays an important role in survivin-mediated cell proliferation. These results show that LANA upregulated survivin expression by complexing with Sp1 or Sp1-like proteins, as well as p53, at specific cis elements on the survivin promoter. This contributes to the enhanced effect of LANA on survivin gene transcription and, therefore, the upregulation of survivin which contributes to cell proliferation. The present study provides an insight into the mechanisms linked to the development of KSHV-associated cancer through the enhancement of a major cellular molecule, survivin, which is known to orchestrate the activities of a range of cellular networks that are important in the development of human cancers.
Hypothetical model showing upregulation of survivin through LANA association with the Sp1 transcription factor and p53 after KSHV infection. RE, responsive element.