Kaposin-B enhances the PROX1 mRNA stability during lymphatic reprogramming of vascular endothelial cells by Kaposi's sarcoma herpes virus.
ABSTRACT: Kaposi's sarcoma (KS) is the most common cancer among HIV-positive patients. Histogenetic origin of KS has long been elusive due to a mixed expression of both blood and lymphatic endothelial markers in KS tumor cells. However, we and others discovered that Kaposi's sarcoma herpes virus (KSHV) induces lymphatic reprogramming of blood vascular endothelial cells by upregulating PROX1, which functions as the master regulator for lymphatic endothelial differentiation. Here, we demonstrate that the KSHV latent gene kaposin-B enhances the PROX1 mRNA stability and plays an important role in KSHV-mediated PROX1 upregulation. We found that PROX1 mRNA contains a canonical AU-rich element (ARE) in its 3'-untranslated region that promotes PROX1 mRNA turnover and that kaposin-B stimulates cytoplasmic accumulation of the ARE-binding protein HuR through activation of the p38/MK2 pathway. Moreover, HuR binds to and stabilizes PROX1 mRNA through its ARE and is necessary for KSHV-mediated PROX1 mRNA stabilization. Together, our study demonstrates that kaposin-B plays a key role in PROX1 upregulation during lymphatic reprogramming of blood vascular endothelial cells by KSHV.
Project description:Kaposi's sarcoma associated herpesvirus (KSHV) is etiologically associated with endothelial Kaposi's sarcoma (KS) and B-cell proliferative primary effusion lymphoma (PEL), common malignancies seen in immunocompromised HIV-1 infected patients. The progression of these cancers occurs by the proliferation of cells latently infected with KSHV, which is highly dependent on autocrine and paracrine factors secreted from the infected cells. Glutamate and glutamate receptors have emerged as key regulators of intracellular signaling pathways and cell proliferation. However, whether they play any role in the pathological changes associated with virus induced oncogenesis is not known. Here, we report the first systematic study of the role of glutamate and its metabotropic glutamate receptor 1 (mGluR1) in KSHV infected cell proliferation. Our studies show increased glutamate secretion and glutaminase expression during de novo KSHV infection of endothelial cells as well as in KSHV latently infected endothelial and B-cells. Increased mGluR1 expression was detected in KSHV infected KS and PEL tissue sections. Increased c-Myc and glutaminase expression in the infected cells was mediated by KSHV latency associated nuclear antigen 1 (LANA-1). In addition, mGluR1 expression regulating host RE-1 silencing transcription factor/neuron restrictive silencer factor (REST/NRSF) was retained in the cytoplasm of infected cells. KSHV latent protein Kaposin A was also involved in the over expression of mGluR1 by interacting with REST in the cytoplasm of infected cells and by regulating the phosphorylation of REST and interaction with ?-TRCP for ubiquitination. Colocalization of Kaposin A with REST was also observed in KS and PEL tissue samples. KSHV infected cell proliferation was significantly inhibited by glutamate release inhibitor and mGluR1 antagonists. These studies demonstrated that elevated glutamate secretion and mGluR1 expression play a role in KSHV induced cell proliferation and suggest that targeting glutamate and mGluR1 is an attractive therapeutic strategy to effectively control the KSHV associated malignancies.
Project description:Lymphatic endothelial cells (LECs) are differentiated from blood vascular endothelial cells (BECs) during embryogenesis and this physiological cell fate specification is controlled by PROX1, the master regulator for lymphatic development. When Kaposi sarcoma herpes virus (KSHV) infects host cells, it activates the otherwise silenced embryonic endothelial differentiation program and reprograms their cell fates. Interestingly, previous studies demonstrated that KSHV drives BECs to acquire a partial lymphatic phenotype by upregulating PROX1 (forward reprogramming), but stimulates LECs to regain some BEC-signature genes by downregulating PROX1 (reverse reprogramming). Despite the significance of this KSHV-induced bidirectional cell fate reprogramming in KS pathogenesis, its underlying molecular mechanism remains undefined. Here, we report that IL3 receptor alpha (IL3R?) and NOTCH play integral roles in the host cell type-specific regulation of PROX1 by KSHV. In BECs, KSHV upregulates IL3R? and phosphorylates STAT5, which binds and activates the PROX1 promoter. In LECs, however, PROX1 was rather downregulated by KSHV-induced NOTCH signal via HEY1, which binds and represses the PROX1 promoter. Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1. Upon co-activation of IL3R? and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs. Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.
Project description:The predominant tumor cell of Kaposi's Sarcoma (KS) is the spindle cell, a cell of endothelial origin that expresses markers of lymphatic endothelium. In culture, Kaposi's Sarcoma-associated herpesvirus (KSHV) infection of blood endothelial cells drives expression of lymphatic endothelial cell specific markers, in a process that requires activation of the gp130 receptor and the JAK2/STAT3 and PI3K/AKT signaling pathways. While expression of each of the KSHV major latent genes in endothelial cells failed to increase expression of lymphatic markers, the viral homolog of human IL-6 (vIL-6) was sufficient for induction and requires the JAK2/STAT3 and PI3K/AKT pathways. Therefore, activation of gp130 and downstream signaling by vIL-6 is sufficient to drive blood to lymphatic endothelial cell differentiation. While sufficient, vIL-6 is not necessary for lymphatic reprogramming in the context of viral infection. This indicates that multiple viral genes are involved and suggests a central importance of this pathway to KSHV pathogenesis.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. Most KS tumor cells are latently infected with KSHV and are of endothelial origin. While PEL-derived cell lines maintain KSHV indefinitely, all KS tumor-derived cells to date have lost viral genomes upon ex vivo cultivation. To study KSHV latency and tumorigenesis in endothelial cells, we generated telomerase-immortalized human umbilical vein endothelial (TIVE) cells. TIVE cells express all KSHV latent genes 48 h postinfection, and productive lytic replication could be induced by RTA/Orf50. Similar to prior models, infected cultures gradually lost viral episomes. However, we also obtained, for the first time, two endothelial cell lines in which KSHV episomes were maintained indefinitely in the absence of selection. Long-term KSHV maintenance correlated with loss of reactivation in response to RTA/Orf50 and complete oncogenic transformation. Long-term-infected TIVE cells (LTC) grew in soft agar and proliferated under reduced-serum conditions. LTC, but not parental TIVE cells, formed tumors in nude mice. These tumors expressed high levels of the latency-associated nuclear antigen (LANA) and expressed lymphatic endothelial specific antigens as found in KS (LYVE-1). Furthermore, host genes, like those encoding interleukin 6, vascular endothelial growth factor, and basic fibroblast growth factor, known to be highly expressed in KS lesions were also induced in LTC-derived tumors. KSHV-infected LTCs represent the first xenograft model for KS and should be of use to study KS pathogenesis and for the validation of anti-KS drug candidates.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is necessary for KS, a highly vascularized tumor predominated by endothelial-derived spindle cells that express markers of lymphatic endothelium. Following KSHV infection of TIME cells, an immortalized human dermal microvascular endothelial cell (DMVEC) line, expression of many genes specific to lymphatic endothelium, including VEGFR3, podoplanin, LYVE-1, and Prox-1, is significantly increased. Increases in VEGFR3 and podoplanin protein are also demonstrated following latent infection. Examination of cytokine secretion showed that KSHV infection significantly induces hIL-6 while strongly inhibiting secretion of IL-8, a gene product that is decreased by differentiation of blood to lymphatic endothelial cells. These studies support the hypotheses that latent KSHV infection of blood endothelial cells drives their differentiation to lymphatic endothelial cells.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of several AIDS-related cancers, including the endothelial cell (EC) neoplasm Kaposi's sarcoma (KS). KSHV-infected ECs secrete abundant host-derived pro-inflammatory molecules and angiogenic factors that contribute to tumorigenesis. The precise contributions of viral gene products to this secretory phenotype remain to be elucidated, but there is emerging evidence for post-transcriptional regulation. The Kaposin B (KapB) protein is thought to contribute to the secretory phenotype in infected cells by binding and activating the stress-responsive kinase MK2, thereby selectively blocking decay of AU-rich mRNAs (ARE-mRNAs) encoding pro-inflammatory cytokines and angiogenic factors. Processing bodies (PBs) are cytoplasmic ribonucleoprotein foci in which ARE-mRNAs normally undergo rapid 5' to 3' decay. Here, we demonstrate that PB dispersion is a feature of latent KSHV infection, which is dependent on kaposin protein expression. KapB is sufficient to disperse PBs, and KapB-mediated ARE-mRNA stabilization could be partially reversed by treatments that restore PBs. Using a combination of genetic and chemical approaches we provide evidence that KapB-mediated PB dispersion is dependent on activation of a non-canonical Rho-GTPase signaling axis involving MK2, hsp27, p115RhoGEF and RhoA. PB dispersion in latently infected cells is likewise dependent on p115RhoGEF. In addition to PB dispersion, KapB-mediated RhoA activation in primary ECs caused actin stress fiber formation, increased cell motility and angiogenesis; these effects were dependent on the activity of the RhoA substrate kinases ROCK1/2. By contrast, KapB-mediated PB dispersion occurred in a ROCK1/2-independent manner. Taken together, these observations position KapB as a key contributor to viral reprogramming of ECs, capable of eliciting many of the phenotypes characteristic of KS tumor cells, and strongly contributing to the post-transcriptional control of EC gene expression and secretion.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with endothelial Kaposi's sarcoma (KS) in immunocompromised individuals. KS lesion cells exhibit many similarities to neuroendocrine (NE) cancers, such as highly vascular and red/purple tumor lesions, spindle-shaped cells, an insignificant role for classic oncogenes in tumor development, the release of bioactive amines, and indolent growth of the tumors. However, the mechanistic basis for the similarity of KS lesion endothelial cells to neuroendocrine tumors remains unknown. Next-generation sequencing and bioinformatics analysis in the present study demonstrate that endothelial cells latently infected with KSHV express several neuronal and NE genes. De novo infection of primary dermal endothelial cells with live and UV-inactivated KSHV demonstrated that viral gene expression is responsible for the upregulation of five selected NE genes (adrenomedullin 2 [ADM2], histamine receptor H1 [HRH1], neuron-specific enolase [NSE] [ENO2], neuronal protein gene product 9.5 [PGP9.5], and somatostatin receptor 1 [SSTR1]). Immunofluorescence and immunohistochemistry examinations demonstrated the robust expression of the NE genes HRH1 and NSE/ENO2 in KSHV-infected KS tissue samples and KS visceral tissue microarrays. Further analysis demonstrated that KSHV latent open reading frame K12 (ORFK12) gene (kaposin A)-mediated decreased host REST/NRSF (RE1-silencing transcription factor/neuron-restrictive silencer factor) protein, a neuronal gene transcription repressor protein, is responsible for NE gene expression in infected endothelial cells. The NE gene expression observed in KSHV-infected cells was recapitulated in uninfected endothelial cells by the exogenous expression of ORFK12 and by the treatment of cells with the REST inhibitor X5050. When the neuroactive ligand-activating receptor HRH1 and inhibitory SSTR1 were knocked out by CRISPR, HRH1 knockout (KO) significantly inhibited cell proliferation, while SSTR1 KO induced cell proliferation, thus suggesting that HRH1 and SSTR1 probably counteract each other in regulating KSHV-infected endothelial cell proliferation. These results demonstrate that the similarity of KS lesion cells to neuroendocrine tumors is probably a result of KSHV infection-induced transformation of nonneuronal endothelial cells into cells with neuroendocrine features. These studies suggest a potential role of neuroendocrine pathway genes in the pathobiological characteristics of KSHV-infected endothelial cells, including a potential mechanism of escape from the host immune system by the expression of immunologically privileged neuronal-site NE genes, and NE genes could potentially serve as markers for KSHV-infected KS lesion endothelial cells as well as novel therapeutic targets to control KS lesions.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates several cellular pathways for its survival advantage during its latency in the infected human host. Here, we demonstrate that KSHV infection upregulates the expression of genes related to neuronal and neuroendocrine (NE) functions that are characteristic of NE tumors, both in vitro and in KS patient tissues and the heterogeneity of neuroendocrine receptors having opposing roles in KSHV-infected cell proliferation. Induction of NE genes by KSHV could also provide a potential survival advantage, as the expression of proteins at immunologically privileged sites such as neurons on endothelial cells may be an avenue to escape host immune surveillance functions. The NE gene products identified here could serve as markers for KSHV-infected cells and could potentially serve as therapeutic targets to combat KSHV-associated KS.
Project description:Kaposi's Sarcoma Herpesvirus (KSHV) is present in the main tumor cells of Kaposi's Sarcoma (KS), the spindle cells, which are of endothelial origin. KSHV is also associated with two B-cell lymphomas, Primary Effusion Lymphoma (PEL) and Multicentric Castleman's Disease. In KS and PEL, KSHV is primarily latent in the infected cells, expressing only a few genes. Although KSHV infection is required for KS and PEL, it is unclear how latent gene expression contributes to their formation. Proliferation of cancer cells occurs despite multiple checkpoints intended to prevent dysregulated cell growth. The first of these checkpoints, caused by shortening of telomeres, results in replicative senescence, where cells are metabolically active, but no longer divide. We found that human dermal lymphatic endothelial cells (LECs) are more susceptible to KSHV infection than their blood-specific endothelial cell counterparts and maintain KSHV latency to higher levels during passage. Importantly, KSHV infection of human LECs but not human BECs promotes their continued proliferation beyond this first checkpoint of replicative senescence. The latently expressed viral cyclin homolog is essential for KSHV-induced bypass of senescence in LECs. These data suggest that LECs may be an important reservoir for KSHV infection and may play a role during KS tumor development and that the viral cyclin is a critical oncogene for this process.
Project description:Immunosuppression therapy following organ transplantation is a significant factor in the development and progression of Kaposi's sarcoma-associated herpesvirus (KSHV)-induced posttransplant Kaposi's sarcoma (KS). Switching from cyclosporine to the mTOR inhibitor rapamycin is reported to promote KS regression without allograft rejection. Examining the underlying molecular basis for this clinical observation, we find that KSHV infection selectively upregulates mTOR signaling in primary human lymphatic endothelial cells (LECs), but not blood endothelial cells (BECs), and sensitizes LECs to rapamycin-induced apoptosis. Viral transcriptome analysis revealed that while infected BECs display conventional latency, KSHV-infected LECs support a radically different program involving widespread deregulation of both latent and lytic genes. ORF45, a lytic gene selectively expressed in infected LECs, is required for mTOR activation and critical for rapamycin sensitivity. These studies reveal the existence of a unique herpesviral gene expression program corresponding to neither canonical latency nor lytic replication, with important pathogenetic and therapeutic consequences.
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi's sarcoma (KS), which is one of the most common HIV-associated neoplasms. The endothelium is the thin layer of squamous cells where vascular blood endothelial cells (BECs) line the interior surface of blood vessels and lymphatic endothelial cells (LECs) are in direct contact with lymphatic vessels. The KS lesions contain a prominent compartment of neoplastic spindle morphology cells that are closely related to LECs. Furthermore, while KSHV can infect both LECs and BECs in vitro, its infection activates genetic programming related to lymphatic endothelial cell fate, suggesting that lymphangiogenic pathways are involved in KSHV infection and malignancy. Here, we report for the first time that viral interferon regulatory factor 3 (vIRF3) is readily detected in over 40% of KS lesions and that vIRF3 functions as a proangiogenic factor, inducing hypersprouting formation and abnormal growth in a LEC-specific manner. Mass spectrometry analysis revealed that vIRF3 interacted with histone deacetylase 5 (HDAC5), which is a signal-responsive regulator for vascular homeostasis. This interaction blocked the phosphorylation-dependent cytosolic translocation of HDAC5 and ultimately altered global gene expression in LECs but not in BECs. Consequently, vIRF3 robustly induced spindle morphology and hypersprouting formation of LECs but not BECs. Finally, KSHV infection led to the hypersprouting formation of LECs, whereas infection with a ?vIRF3 mutant did not do so. Collectively, our data indicate that vIRF3 alters global gene expression and induces a hypersprouting formation in an HDAC5-binding-dependent and LEC-specific manner, ultimately contributing to KSHV-associated pathogenesis.IMPORTANCE Several lines of evidences indicate that KSHV infection of LECs induces pathological lymphangiogenesis and that the results resemble KS-like spindle morphology. However, the underlying molecular mechanism remains unclear. Here, we demonstrated that KSHV vIRF3 is readily detected in over 40% of various KS lesions and functions as a potent prolymphangiogenic factor by blocking the phosphorylation-dependent cytosolic translocation of HDAC5, which in turn modulates global gene expression in LECs. Consequently, vIRF3-HDAC5 interaction contributes to virus-induced lymphangiogenesis. The results of this study suggest that KSHV vIRF3 plays a crucial role in KSHV-induced malignancy.