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. Author manuscript; available in PMC: 2021 Mar 18.
Published in final edited form as: Oncogene. 2020 May 11;39(23):4603–4618. doi: 10.1038/s41388-020-1317-1

Viral interleukin-6 encoded by an oncogenic virus promotes angiogenesis and cellular transformation by enhancing STAT3-mediated epigenetic silencing of caveolin 1

Wan Li 1,2,3, Qingxia Wang 1, Xiaoyu Qi 1, Yuanyuan Guo 4, Hongmei Lu 5, Yuheng Chen 1, Zhongmou Lu 1, Qin Yan 1, Xiaofei Zhu 6, Jae U Jung 7, Giovanna Tosato 8, Shou-Jiang Gao 9, Chun Lu 1,2,3
PMCID: PMC7970339  NIHMSID: NIHMS1676903  PMID: 32393833

Abstract

Kaposi’s sarcoma (KS) caused by oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) is a highly angiogenic and invasive vascular tumor and the most common AIDS-associated cancer. KSHV-encoded viral interleukin-6 (vIL-6) is implicated in the development of KSHV-induced malignancies; however, the mechanisms underlying vIL-6-induced angiogenesis and tumorigenesis remain undefined. Here, we show that vIL-6 promotes angiogenesis, cell proliferation, and invasion by downregulating caveolin 1 (CAV1) that plays a pivotal and versatile role in multiple cancer-associated processes. Mechanistically, vIL-6 signaling led to the phosphorylation and acetylation of STAT3 that targeted DNA methyltransferase 1 (DNMT1) in a sequential manner. Specifically, the vIL-6-induced phosphorylated form of STAT3 transcriptionally activated DNMT1 expression. Furthermore, vIL-6-induced acetylated form of STAT3 interacted with DNMT1 to form a transcription factor complex that bound to and methylated the CAV1 promoter, leading to CAV1 expression silencing. In fact, downregulation of CAV1 expression resulted in the activation of AKT signaling, promoting cell invasion, and growth transformation induced by KSHV. Finally, genetic deletion of vIL-6 from the KSHV genome abolished KSHV-induced cellular transformation and impaired angiogenesis. Our results reveal that vIL-6 epigenetically silences CAV1 expression to promote angiogenesis and tumorigenesis by regulating the formation of STAT3-DNMT1 complex. These novel findings define a mechanism by which KSHV inhibits the CAV1 pathway and establish the scientific basis for targeting this pathway to treat KSHV-associated cancers.

Introduction

Kaposi’s sarcoma (KS), an angiogenic tumor of endothelial cells, is the most common cancer in HIV/AIDS patients characterized by abnormal spindle cell proliferation and increased blood vessels [1]. As a causative agent of KS, KS-associated herpesvirus (KSHV) is an oncogenic virus, which is also associated with primary effusion lymphoma (PEL), a subset of multicentric Castleman’s disease (MCD), and KSHV-associated inflammatory cytokine syndrome [2].

As a gamma-2 herpesvirus, KSHV has a large double-stranded DNA genome and expresses over 90 open reading frames (ORFs). Like other herpesviruses, KSHV life cycle contains latent and lytic phases [3]. KSHV uses these two modes of infection to establish lifelong persistence resulting in the induction of pathogenesis in the host. Numerous KSHV latent and lytic genes encode pro-oncogenic protein products, including latency-associated nuclear antigen (LANA), viral cyclin (vCyclin), viral FLICE inhibitory protein (vFLIP), kaposin, viral interferon regulatory factors, viral G protein-coupled receptor (vGPCR), viral Bcl-2, and viral interleukin-6 (vIL-6) [4]. These proteins contribute to KSHV-induced cellular transformation, angiogenesis and tumorigenesis, and thus are the areas of intensive research.

One of KSHV lytic proteins is vIL-6, a homolog of human interleukin-6, which is encoded by KSHV ORF-K2. vIL-6 is also expressed at low but functional levels during latency [5]. Importantly, vIL6 is detectable in the sera and/or tumor tissues of patients with KS, PEL, and MCD [6], implying that vIL-6 plays a crucial role in the pathogenesis of KSHV-associated malignancies. Indeed, vIL-6 has been demonstrated to drive the expression of vascular endothelial growth factor and induce the transformation of NIH3T3 cells [7]. In addition, vIL-6 transgenic mice develop IL-6-dependent MCD-like disease [8]. Our previous studies also showed that vIL-6 could enhance cell proliferation, angiogenesis and tumorigenesis [9, 10]. vIL-6 also promotes endothelial cell migration [11]. A recent report showed that vIL-6 gene regulated tumor metastasis and expression of B cell markers in a murine xenograft model [12]. Despite these intensive studies, the underlying mechanism by which vIL-6 manipulates the critical host factors to promote angiogenesis and tumorigenesis remains unclear.

Caveolae are small flask-shaped plasma membrane invaginations of 50–100 nm diameter ubiquitously present in most cell types. They have been described to participate in membrane lipid homeostasis, cell proliferation, endocytosis, transcellular transport, and signal transduction [13]. The formation and stabilization of caveolae depend on caveolin proteins, which consists of three isoforms (caveolin 1–3) [14]. Among them, caveolin 1 (CAV1), which is a 21–24-kDa scaffolding protein, was first identified as a protein component of caveolae membrane coats in 1992 [15]. Then, a further study analyzed Cav1(−/−) null mice and demonstrated the fundamental role of CAV1 in the formation of caveolae, paving the way for delineating the functions of CAV1 in orchestrating multiple signaling pathways [16]. Apart from being a component of caveolae, CAV1 is also located in cells outside of caveolae and contributes to diverse cellular processes, ranging from signal transduction, cholesterol trafficking and efflux, to senescence, and cell cycle [17]. It is noticeable that CAV1 plays a pivotal and versatile role in multiple cancer-associated processes, including angiogenesis, multidrug resistance, cell death and survival, cell migration and metastasis, tumor growth, and cellular transformation [18]. However, the impact of CAV1 on tumor progression remains controversies. Upregulation of CAV1, which has been observed in tumors, including bladder, leukemia liver, lung, colon, prostate, and kidney, favored cell survival and metastasis. By striking contrast, the loss of CAV1 in small cell lung cancer, breast cancer, and ovarian tumors was correlated with poor clinical outcome [18, 19]. Accumulating evidence indicates that the role of CAV1 in cancer progression depends on tumor type and stage. In well-differentiated tumors, CAV1 is likely to act as a tumor promoter. Conversely, in poorly differentiated tumors, CAV1 probably functions as a tumor suppressor. However, it remains unclear whether CAV1 is linked to the development of KS.

In this study, we have observed vIL-6 downregulation of CAV1, which is an essential mediator of vIL-6-induced angiogenesis, cellular transformation, and cell invasion. Both phosphorylation and acetylation of STAT3 induced by vIL-6 contributed to DNA methyltransferase 1 (DNMT1)-mediated epigenetic silencing of CAV1. Overall, our study reveals that CAV1 can serve as a potential therapeutic target for KSHV-related malignancies.

Results

vIL-6 downregulates CAV1 to promote cell proliferation, invasion, and angiogenesis

We transduced HUVECs with lentiviral vIL-6 and found that the expression of CAV1, which has been proved to play a pivotal role in multiple cancer-associated processes [18], is decreased in vIL-6-transduced HUVECs compared with the control vector-transduced cells (Fig. 1a). Further, KSHV infection also inhibited CAV1 expression in HUVECs (Fig. 1b). To examine whether there is a causal link between the ectopic expression of vIL-6 and downregulation of CAV1 in the presence of the KSHV genome, we generated a KSHV mutant with a deletion of the vIL-6 coding sequence (ORF-K2 of KSHV) using a two-step “scarless” homologous recombination procedure previously described [20, 21]. PCR detection confirmed the deletion of vIL-6 gene in the recombinant BACmid, and RT-qPCR did not detect vIL-6 mRNA transcription in HUVECs infected by mutant virus (Fig. S1), confirming deletion of the vIL-6 coding sequence (ORF-K2) from the KSHV genome. Furthermore, deletion of vIL-6 gene from the KSHV genome restored CAV1 protein expression in HUVECs (Fig. 1b). We also generated primary rat embryonic metanephric mesenchymal (MM) cells infected by KSHV wild-type virus (KMM) [22] and vIL-6 gene K2_Mut virus. Consistently, CAV1 expression was reduced in KMM cells, and the loss of vIL-6 gene restored CAV1 expression (Fig. 1c). Most importantly, less CAV1-positive cells were observed in KS lesions compared with normal skin tissues (Fig. 1d; Fig. S2).

Fig. 1. CAV1 is downregulated in vIL-6-transduced cells, KSHV-infected cells, and KS lesions.

Fig. 1

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a Western blotting analysis of CAV1 in HUVECs transduced with lentiviral vIL-6 (vIL-6) or its control vector lentiviral pHAGE (pHAGE) for 48 h. vIL-6 was detected using the antibody against the Flag-tag. GAPDH was used as a loading control. b Western blotting analysis of CAV1 in HUVECs treated with PBS (PBS) or infected by KSHV wild-type virus (KSHV_WT) (MOI of 3) and a mutant virus with a deletion of KSHV ORF-K2 (K2_Mut) (MOI of 3) for 48 h. c Western blotting analysis of CAV1 in MM cells treated with PBS (PBS) or infected by KSHV wild-type virus (KSHV_WT) (MOI of 3) and a mutant virus with a deletion of KSHV ORF-K2 (K2_Mut) (MOI of 3) for 48 h. d Hematoxylin and eosin (H&E) staining and immunohistochemical staining of KSHV LANA, CAV1 in normal skin, skin KS of patient #1 (Skin KS1), skin KS of patient #2 (Skin KS2), and skin KS of patient #3 (Skin KS3). Magnification, ×200, ×400.

To determine whether CAV1 regulates the oncogenic phenotypes of vIL-6, we overexpressed CAV1 in vIL-6-transduced HUVECs (Fig. 2a). At first, we quantified cell viability and proliferation using Cell Counting Kit-8 assay (CCK-8). We found that overexpression of CAV1 diminished vIL-6-induced cell proliferation (Fig. 2b). Wound-healing assay was adopted to measure cell migratory and proliferation rates. We showed that overexpression of CAV1 inhibited vIL-6-induced cell motility and proliferation (Fig. 2c, d). In addition, the plate colony formation assay, another assay to examine cell proliferation, further confirmed the suppressive effect of CAV1 on cell proliferation (Fig. 2e). We also investigated the functional consequence of CAV1 overexpression on vIL-6-induced cell invasion by using the transwell migration and matrigel invasion assays. We found that overexpression of CAV1 repressed vIL-6-induced cell migration and invasion (Fig. 2f, g). To explore whether CAV1 mediated vIL-6-induced angiogenesis, we transduced endothelial cell line with vIL-6, and then performed chick chorioallantoic membrane (CAM) assay, one of the earliest in vivo models used for the assessment of angiogenic property. We observed that overexpression of CAV1 blocked vIL-6-induced angiogenesis (Fig. 2h, i). These data suggest that vIL-6 downregulates CAV1 to promote cell proliferation, invasion, and angiogenesis.

Fig. 2. Downregulation of CAV1 contributes to vIL-6-induced cell proliferation, invasion, and angiogenesis.

Fig. 2

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a Lentiviral vIL-6- or its control pHAGE-infected HUVECs were transduced with lentivirus expressing CAV1, and then immunoblotted with antibodies against CAV1, Flag-tag and His-tag. Tubulin was used as a loading control. b CCK-8 assay of HUVECs treated as in (a). c Wound-healing assay of HUVECs treated as in (a). The representative images were captured at 0 and 24 h after being scratched. Magnification, ×100. d Quantification of wound-healing described in (c). e Plate colony formation assay of HUVECs treated as in (a). The number of colonies was counted at 2 weeks post seeding. f, g Transwell migration and matrigel invasion analyses of HUVECs treated as in (a). The migrated and invaded HUVECs were collected at 6 and 12 h post seeding. h Lentiviral vIL-6- or its control pHAGE-infected endothelial cell line were transduced with lentivirus expressing CAV1, and then were subjected to chicken chorioallantoic membranes (CAMs) assay. Representative images are shown. Magnification, ×100. Scar bars, 40 μm. i Quantification of CAM assay described in (h). Data were shown as mean ± SD. *P < 0.05, **P < 0.01, and ***P <0.001, Student’s t test.

vIL-6 inhibits CAV1 by increasing DNMT1 to promote hypermethylation of the CAV1 promoter

We next sought to dissect the mechanism of vIL-6 downregulation of CAV1. We first examined whether vIL-6 had any effect on CAV1 transcription. RT-qPCR showed that transduction of vIL-6 in HUVECs inhibited CAV1 mRNA expression (Fig. 3a). Downregulation of CAV1 mRNA was also observed in KSHV-infected HUVECs (Fig. 3b). Hypermethylation of the CAV1 promoter is a mechanism of decreased CAV1 expression in multiple cancers [23, 24]. We then asked whether vIL-6 prevented CAV1 transcription by regulating CAV1 promoter methylation. Methylation-specific PCR showed the presence of increased DNA methylation density at the CAV1 promoter in both vIL-6-transduced and KSHV-infected HUVECs cells (Fig. 3c, d). Further, treatment with the CpG methylation inhibitor, 5-aza, partially restored CAV1 expression suppressed by vIL-6 and KSHV (Fig. 3e, f).

Fig. 3. The methylation of CAV1 promoter is enhanced in vIL-6-transduced cells and KSHV-infected cells.

Fig. 3

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a qPCR analysis of CAV1 mRNA expression in HUVECs transduced with lentiviral vIL-6 (vIL-6) or its control vector lentiviral pHAGE (pHAGE) for 48 h. b qPCR analysis of CAV1 mRNA expression in HUVECs treated with PBS (PBS) or infected by KSHV wild-type virus (KSHV_WT) (MOI of 3) for 48 h. c Methylation-specific PCR assay of DNA methylation status of CAV1 promoter in HUVECs transduced with lentiviral vIL-6 (vIL-6) or its control vector lentiviral pHAGE (pHAGE) for 48 h. d Methylation-specific PCR assay of DNA methylation status of CAV1 promoter in HUVECs treated with PBS (PBS) or infected by KSHV wild-type virus (KSHV_WT) (MOI of 3) for 48 h. e Lentiviral vIL-6- or its control pHAGE-infected HUVECs were treated with 5-aza (10 μM) for 6 d, and then were immunoblotted with antibodies against CAV1 and Flag-tag. GAPDH was used as a loading control. f HUVECs treated with PBS (PBS) or infected by KSHV wild-type virus (KSHV_WT) (MOI of 3) for 6 h were treated with 5-aza (10 μM) for 6 d, and then immunoblotted with antibodies against CAV1. GAPDH was used as a loading control. Data were shown as mean ± SD. *P <0.05 and ***P < 0.001, Student’s t test.

DNA methylation is generally mediated by methyltransferase 1 (DNMT1). Western blotting showed that DNMT1 protein was upregulated in vIL-6-transduced cells and KSHV-infected cells (Fig. 4a, b), which is consistent with previous studies [25, 26]. Importantly, deletion of vIL-6 from the KSHV genome caused a decrease of DNMT1 protein (Fig. 4b). To examine whether DNMT1 directly causes aberrant methylation of CAV1 promoter in vIL-6-transduced cells, we performed chromatin immunoprecipitation (ChIP) assay. We found that DNMT1 bound to the CAV1 promoter (Fig. 4c), and that this binding was prominently decreased in K2_mut virus-infected cells compared with KSHV-infected cells (Fig. S3). Knockdown of DNMT1 in vIL-6-transduced cells using a mixture of lentivirus-mediated short hairpins RNAs (shRNAs) not only inhibited vIL-6-induced hypermethylation of CAV1 promoter, mRNA transcription, and protein expression of CAV1 (Fig. 4d–f; Fig. S4), but also repressed vIL-6-promoted cell proliferation, invasion, and angiogenesis (Fig. 4g–j). These data collectively indicate that vIL-6 downregulates CAV1 by increasing DNMT1 to promote the hypermethylation of CAV1 promoter.

Fig. 4. vIL-6 inhibits CAV1 to promote cell proliferation, invasion, and angiogenesis by upregulating DNMT1 to enhance the hypermethylation of CAV1 promoter.

Fig. 4

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a Western blotting analysis of DNMT1 in HUVECs transduced with lentiviral vIL-6 (vIL-6) or its control vector lentiviral pHAGE (pHAGE) for 48 h. b Western blotting analysis of DNMT1 in HUVECs treated with PBS (PBS) or infected by KSHV wild-type virus (KSHV_WT) (MOI of 3) and a mutant virus with a deletion of KSHV ORF-K2 (K2_Mut) (MOI of 3) for 48 h. c ChIP assay of the interaction between DNMT1 and the CAV1 promoter. Immunoprecipitation was done in HUVECs with anti-DNMT1 antibody. d Lentiviral vIL-6- or its control pHAGE-infected HUVECs were transduced with a mixture of shRNAs targeting DNMT1 (shDNMT1), and then were subjected to methylation-specific PCR assay to detect the DNA methylation status of CAV1 promoter. e qPCR analysis of CAV1 mRNA expression in HUVECs treated as in (d). f Western blotting analysis of DNMT1 and CAV1 in HUVECs treated as in (d). g Plate colony formation assay of HUVECs treated as in (d). The number of colonies was counted at 2 weeks post seeding. h, i Transwell migration and matrigel invasion analyses of HUVECs treated as in (d). The migrated and invaded HUVECs were collected at 6 and 12 h post seeding. j Lentiviral vIL-6- or its control pHAGE-infected endothelial cell line were transduced with a mixture of shRNAs targeting DNMT1 (shDNMT1), and then were subjected to CAM assay. Data were shown as mean±SD. *P <0.05, **P <0.01, and ***P <0.001, Student’s t test.

vIL-6 downregulates CAV1 by increasing acetylated STAT3, which interacts with DNMT1 and promotes DNMT1 binding to the CAV1 promoter

It has been reported that STAT3 is a major inducer of DNMT1 transcription [27], and that vIL-6 induced upregulation of DNMT1 is dependent on STAT3 activation [26]. We hypothesized that phosphorylated STAT3 might lead to greater DNMT1 expression. To test this hypothesis, we knocked-down STAT3 by using a mixture of shRNAs targeting STAT3 [28], and found that knockdown of STAT3 not only reduced DNMT1 protein, but also increased the expression level of CAV1 protein in vIL-6-transduced HUVECs (Fig. 5a). In line with a previous study [27], the ChIP assay supported STAT3 binding to the enhancer of DNMT1 gene in endothelial cells (Fig. 5b), and showed that STAT3 binding to the enhancer of DNMT1 was reduced in cells infected with the K2_mut virus- compared with KSHV-infected cells (Fig. S5). A previous study showed that K685-acetylated STAT3 interacts with DNMT1 and promotes the binding of DNMT1 to promoters of tumor-suppressor genes [29]. Thus, we asked whether acetylated STAT3 was associated with DNMT1-induced CAV1 promoter methylation in vIL-6-transduced HUVECs to cause lower CAV1 expression. Western blotting showed that acetylated STAT3 was increased in both vIL-6-transduced cells and KSHV-infected cells (Fig. 5c, d), and the level of acetylated STAT3 was lower in STAT3-silenced cells, as expected (Fig. 5a). Co-immunoprecipitation (Co-IP) analysis showed that acetylated STAT3 interacted with DNMT1. However, mutation of STAT3 at Lys685 reduced the interaction of STAT3 with DNMT1, resulting in an increase of CAV1 protein (Fig. 5e). We also reconfirmed the interaction between DNMT1 and acetylated STAT3 in KSHV-infected HUVECs (Fig. 5f). The ChIP assay further confirmed that STAT3 bound to the CAV1 promoter, and this binding was disrupted when acetylation of STAT3 was blocked (Fig. 5g). Together, these results suggest that vIL-6 downregulates CAV1 by increasing acetylated STAT3, which interacts with DNMT1 and promotes the binding of DNMT1 to the CAV1 promoter.

Fig. 5. vIL-6 downregulates CAV1 by increasing acetylated STAT3 which interacts with DNMT1 and promotes the binding of DNMT1 to CAV1 promoter.

Fig. 5

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a Western blotting analysis of phosphorylated STAT3 (p-STAT3), acetylated STAT3 (Ly685) (Ac-STAT3), STAT3, DNMT1, and CAV1 in vIL-6-expressing HUVECs transduced with a mixture of shRNAs targeting STAT3 (shSTAT3). b ChIP assay of the interaction between STAT3 and the enhancer of DNMT1 gene. Immunoprecipitation was done in HUVECs with anti-STAT3 antibody. c Western blotting analysis of acetylated STAT3 (Lys685) (Ac-STAT3), DNMT1, and CAV1 in HUVECs transduced with lentiviral vIL-6 (vIL-6) or its control vector lentiviral pHAGE (pHAGE) for 48 h. d Western blotting analysis of acetylated STAT3 (Lys685) (Ac-STAT3), DNMT1, and CAV1 in HUVECs treated with PBS (PBS) or infected by KSHV wild-type virus (KSHV_WT) (MOI of 3) for 48 h. e Co-immunoprecipitation analyses of the interaction between acetylated STAT3 and DNMT1. Immunoprecipitation was done with Flag antibody in HUVECs transduced with STAT3 wild type (STAT3-Flag) or STAT3 mutation at Lys685 (STAT3-K685R-Flag). f Co-immunoprecipitation analyses of the interaction between acetylated STAT3 and DNMT1. Immunoprecipitation was performed with anti-DNMT1 antibody in HUVECs treated with PBS (PBS) or infected by KSHV wild-type virus (KSHV_WT) (MOI of 3) for 48 h. g ChIP assay of the interaction between acetylated STAT3 and the CAV1 promoter. Immunoprecipitation was performed with Flag antibody in HUVECs treated as in (e). Data were shown as mean ± SD. ***P <0.001, Student’s t test.

vIL-6 promotes cell migration and invasion by downregulating CAV1 to activate AKT signaling

We and others have previously shown that vIL-6 activates AKT signaling by increasing levels of AKT phosphorylation [9, 10, 30]. Further, downregulation of CAV1 increases AKT activity [31]. These studies suggested that CAV1 may regulate the AKT pathway in vIL-6-expressing cells. Western blotting showed that, as expected, overexpression of CAV1 inhibited vIL-6-induced AKT activation (Fig. 6a), and knockdown of STAT3 also decreased vIL-6-induced AKT phosphorylation (Fig. 6b). Our previous studies have shown that inhibition of AKT does not affect vIL-6 induction of angiogenesis [9, 10]. To examine whether the AKT pathway plays a role in other vIL-6-induced oncogenic phenotypes, we performed migration and invasion assays. Using a mixture of shRNAs targeting AKT [32], we found that knockdown of AKT did not affect CAV1 expression, but decreased vIL-6-induced cell migration and invasion (Fig. 6c–e). Similar results were also observed in vIL-6-transduced HUVECs treated with MK2206 2HCl, a chemical inhibitor of AKT (Fig. 6f–h). Taken together, these results suggest that vIL-6 promotes cell migration and invasion by downregulating CAV1 to activate the AKT signaling.

Fig. 6. vIL-6 promotes cell migration and invasion by activating AKT pathway.

Fig. 6

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a Lentiviral vIL-6- or its control pHAGE-infected HUVECs were transduced with lentivirus expressing CAV1, and then immunoblotted with antibodies against phosphorylated AKT (p-AKT), CAV1, and AKT. Tubulin was used as a loading control. b Lentiviral vIL-6- or its control pHAGE-infected HUVECs were transduced with a mixture of shRNAs targeting STAT3 (shSTAT3), and then immunoblotted with antibodies against phosphorylated STAT3 (p-STAT3), STAT3, phosphorylated AKT (p-AKT), AKT, and CAV1. Tubulin and GAPDH were used as loading controls. c Lentiviral vIL-6- or its control pHAGE-infected HUVECs were transduced with a mixture of shRNAs targeting AKT (shAKT), and then immunoblotted with antibodies against phosphorylated AKT (p-AKT), AKT, and CAV1. GAPDH was used as a loading control. d, e Transwell migration and matrigel invasion analyses of HUVECs treated as in (c). The migrated and invaded HUVECs were counted at 6 and 12 h post seeding. f Lentiviral vIL-6- or its control pHAGE-infected HUVECs were treated with the AKT inhibitor, MK2206 2HC1 (5 μM) for 48 h, and then were immunoblotted with antibodies against phosphorylated AKT (p-AKT), AKT, and CAV1. GAPDH was used as a loading control. g, h Transwell migration and matrigel invasion analyses of HUVECs treated as in (f). The migrated and invaded HUVECs were calculated at 6 and 12 h post seeding. Data were shown as mean ± SD. *P <0.05, **P <0.01, and ***P < 0.001, Student’s t test.

vIL-6-regulated CAV1/AKT pathway mediates KSHV-induced cellular transformation

KSHV efficiently infected and transformed primary MM cells [22], and ectopic expression of vIL-6 alone transformed NIH3T3 cells [7]. We wondered whether deletion of K2 gene from the KSHV genome influenced growth transformation induced by KSHV. Western blotting showed that DNMT1, phosphorylated STAT3, acetylated STAT3, and phosphorylated AKT were increased in MM cells infected by KSHV wild-type (KSHV_WT) virus (KMM) but reversed in those infected by the K2_mut virus (Fig. 7a). Complementation with vIL-6 in K2_mut-infected MM was sufficient to reverse these effects (Fig. 7a). Importantly, deletion of vIL-6 abolished colony formation in soft agar and lowered KSHV-induced angiogenesis, while complementation with vIL-6 in K2_mut-infected MM recovered these phenotypes (Fig. 7b–d).

Fig. 7. vIL-6-regulated CAV1/AKT pathway mediates KSHV-induced cellular transformation.

Fig. 7

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a MM cells infected by KSHV wild-type virus (KSHV_WT) (MOI of 3) and a mutant virus with a deletion of KSHV ORF-K2 (K2_Mut) (MOI of 3) for 6 h were followed by transduction with lentiviral vIL-6. Forty-eight hours later, cells were collected for western blotting analysis of DNMT1, phosphorylated STAT3 (p-STAT3), acetylated STAT3 (Ac-STAT3) (Lys685), CAV1, phosphorylated AKT (p-AKT), and vIL-6. GAPDH and tubulin were used as loading controls. b Soft agar assay of MM and KMM cells transduced with lentiviral vIL-6 for 48 h. The representative images were captured at 2 weeks post seeding. Magnification, ×100. Scar bars, 40 μm. c Quantification of the results in (b). d CAM assay of cells treated as in (a). e Western blotting analysis of CAV1 in MM and KMM cells transduced with lentiviral CAV1 for 48 h. f Soft agar assay of cells treated in (e). The representative images were captured at 2 weeks post seeding. Magnification, ×100. Scar bars, 40 μm. g Quantification of the results in (f). h CAM assay of MM cells treated as in (e). i Western blotting analysis of phosphorylated AKT (p-AKT) and AKT in MM and KMM cells treated with the AKT inhibitor, MK2206 2HCl (5 μM) for 48 h. j Soft agar assay of cells treated in (i). The representative images were captured at 2 weeks post seeding. Magnification, ×100. Scar bars, 40 μm. k Quantification of the results in (j). l A schematic working model of the mechanism of how vIL-6 facilitates angiogenesis and tumorigenesis. Data were shown as mean±SD. *P < 0.05, **P < 0.01, and ***P <0.001, Student’s t test. n.s. not significant.

To examine the role of CAV1 in KSHV-induced growth transformation, we overexpressed CAV1 in KMM cells (Fig. 7e). Soft agar and CAM assays showed that overexpression of CAV1 in KMM cells impaired KSHV-induced cellular transformation and angiogenesis compared with control vector-transduced KMM cells (Fig. 7f–h). We then determined the role of AKT signaling in KSHV-induced growth transformation and angiogenesis. Treatment of KMM cells with an inhibitor of AKT, MK2206 2HCl, decreased phosphorylated AKT and suppressed the efficiency of growth transformation induced by KSHV (Fig. 7i–k), but failed to affect KSHV-induced angiogenesis compared with KMM cells treated with DMSO (Fig. S6). These results collectively suggest that vIL-6-regulated CAV1/AKT pathway mediates KSHV-induced growth transformation.

Discussion

CAV1 is an important constituent of the caveolae structure and acts as a scaffolding protein to interact with signaling molecules to modulate signal transduction, including mitogen-activated protein kinases, protein kinase A, and Src tyrosine kinases [33]. Increasing evidences indicate that the role of CAV1 in cancer biology could be either a tumor promoter or suppressor depending on the context. However, the exact mechanisms mediating these effects are still unclear [34]. When CAV1 is present in endothelial cells, it appears to inhibit vascular development and angiogenesis [35]. Consistently, in this study, we observed that CAV1 was downregulated in vIL-6-transduced endothelial cells, KSHV-infected endothelial cells, and KS tumor cells, which manifest endothelial cell markers. Overexpression of CAV1 inhibited endothelial cell invasion and angiogenesis induced by vIL-6 and KSHV. It has been reported that CAV1 was reduced in transformed NIH3T3 cells, and upregulation of CAV1 abolished the ability of colony formation in soft agar of transformed NIH3T3 cells [36]. As a further support of the inhibitory effect of CAV1 on cellular transformation, we observed that CAV1 was downregulated in KSHV-infected and -transformed MM (KMM) cells, and overexpression of CAV1 impaired efficiency of colony formation in soft agar of KSHV-transformed cells. Therefore, we concluded that CAV1 acted as an inhibitory factor influencing the development and pathogenesis of KS.

As for downstream signaling of CAV1, it interacts with PTEN via its CAV1-binding sequence to inhibit AKT activity [31]. In fact, KSHV-infected cells exhibited an aberrant activation of AKT [37]. To date, at least five of KSHV-encoded products have been shown to activate AKT signaling. They are vGPCR, ORF45, vIL-6, ORF-K1, and miR-K3 [32, 37]. We and others have previously demonstrated that vIL-6 activates AKT pathway to promote numerous oncogenic phenotypes [9, 10, 30, 38]. Here, we showed that overexpression of CAV1 inhibited vIL-6-induced AKT activation, implying that vIL-6 downregulates CAV1 to activate AKT signaling. Further, we found that inhibition of AKT signaling not only impaired vIL-6-induced cell migration and invasion in HUVECs, but also decreased KSHV-induced cellular transformation. However, inhibition of AKT activity failed to reduce angiogenesis induced by vIL-6 and KSHV, which was consistent with our previous studies [9, 10]. Therefore, our data revealed that downregulation of CAV1 activates the AKT pathway to promote tumorigenesis and cell invasion induced by vIL-6 and KSHV. Meanwhile, our results did not eliminate the possibility that CAV1 may regulate other pathway(s) or targets to enhance angiogenesis induced by vIL-6 and KSHV.

STAT3 is constitutively activated in many types of cancer and hereby regulates important cellular activities including cell differentiation, proliferation, invasion, and angiogenesis [39]. It is well known that phosphorylation of STAT3 is essential for its activation of target genes, working as a transcription factor [40]. KSHV infection induced hyper-activation of STAT3 [41]. Numerous mechanisms have been shown to mediate STAT3 activation in KSHV-infected cells [28, 42–45]. vIL-6 triggered STAT3 phosphorylation to support PEL cell proliferation and viability [46]. In this study, we revealed that phosphorylated STAT3 induced by vIL-6 binds to the enhancer of DNMT1 gene to promote its transcription and expression in endothelial cells, which is consistent with previous studies [26, 27]. Besides phosphorylation, acetylation of STAT3 at Lys685 is another type of posttranslational modification. Acetylation of STAT3 is critical for the formation of stable dimers, which is important for its transcriptional activity [47]. By interacting with DNMT1, acetylated STAT3 causes hypermethylation and transcriptional repression of some tumor-suppressor genes [29]. Here, we found that along with STAT3 phosphorylation, vIL-6 also induced STAT3 acetylation (Lys685). Acetylated STAT3 interacted with phosphorylated STAT3-induced DNMT1 to form the complex, which bound to CAV1 promoter and increased the methylation of CAV1 promoter, resulting in epigenetic silencing of CAV1. Our data elucidate a novel mechanism of vIL-6 activation of the STAT3 pathway and strengthen the role of STAT3 activation in the development and progression of KS. Therefore, targeting STAT3 by blocking both phosphorylation and acetylation of STAT3 is an attractive therapeutic strategy in KSHV-related malignancies. In fact, inhibition of STAT3 with an inhibitor is sufficient to block KSHV-induced cellular transformation [42].

It is well known that, as a homolog of human IL-6, vIL-6 could directly bind to the gp130 subunit of IL-6 receptor to active the JAK/STAT pathway, inducing STAT3 phosphorylation and activation [48, 49]. In this study, we found that, besides STAT3 phosphorylation, vIL-6 could also induce STAT3 acetylation (Lys685), however, the underlying mechanism remains unclear. Histone acetyltransferase p300 could directly acetylate STAT3 at lysine 685 [50]. Recently, several histone deacetylases have been shown to recognize acetylated STAT3 at Lys685 and catalyze the removal of acetyl group, such as SIRT1–3 and HDAC6 [51], and STAT3 phosphorylation at Tyr705 could protect the acetyl group on Lys685 from deacetylation by these enzymes. Therefore, we speculate that vIL-6 might induce STAT3 acetylation through inhibition of the expression of deacetylase enzymes, increasing the expression of acetyl-transferases, or regulating the interaction between STAT3 and these deacetylase enzymes/acetyltransferases. These potential underlying mechanisms need to be further explored.

KSHV could efficiently infect, immortalize and transform MM cells. KSHV-infected and -transformed MM cells (KMM) efficiently induce tumors in nude mice with virological and pathological features resembling KS [22]. Based on this robust model, KSHV with a deletion of miRNAs failed to cause cellular transformation of primary cells, indicating an essential role of KSHV miRNAs in tumorigenesis [52]. Deletion of vFLIP and vCyclin also abolished and reduced KSHV-induced cellular transformation [53, 54]. In the current study, we demonstrated that deletion of vIL-6 totally abolished KSHV-induced growth transformation, and significantly attenuated KSHV-induced angiogenesis, while complementation with vIL-6 in K2_mut-infected MM recovered these phenotypes. These data suggest that besides viral miRNAs, vFLIP and vCyclin, vIL-6 also acts as an essential viral determinant for KSHV-induced cellular transformation and tumorigenesis.

In summary, we revealed that vIL-6 upregulated phosphorylated STAT3, which binds to the enhancer of DNMT1 gene to promote its transcription. Meanwhile, vIL-6 also increased STAT3 acetylation, and acetylated STAT3 interacted with phosphorylated STAT3-induced DNMT1 to form a complex, which bound to CAV1 promoter and promoted the methylation of CAV1 promoter resulting in epigenetic silencing of CAV1. Epigenetic silencing of CAV1 facilitated vIL-6-induced tumorigenesis and invasion by activating AKT signaling. On the other hand, reduced CAV1 promoted vIL-6-induced angiogenesis through an unknown mechanism (Fig. 7l). Our findings uncover a novel mechanism of vIL-6 induction of tumorigenesis, angiogenesis, and cell invasion, and demonstrate a significant role of vIL-6, and its regulatory proteins, and pathways in the pathogenesis of KSHV-related malignancies.

Materials and methods

Cells and tissues

Rat primary embryonic MM cells and KSHV-transformed MM cells (KMM) were maintained as previously described [22]. Primary human umbilical vein endothelial cells (HUVECs) were cultured in complete EBM-2 culture media (LONZA, Allendale, NJ, USA) as previously described [32]. HEK293T and a human umbilical vein endothelial cell line (catalog #CRL-2922™; ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium containing 10% bovine calf serum (BCS, HyClone) in the absence of antibiotics. All cells were cultured at 37 °C in a humidified, 5% CO2 atmosphere. Cell lines used in this study were examined by mycoplasma contamination test using Myco-Blue Mycoplasma Detector (D103–01/02, Vazyme Biotech Co., Ltd, Nanjing, China).

Reagents, plasmids, and lentiviral infection

Both 5-aza (Decitabine) and MK2206 2HCl were purchased from Selleck Chemicals (Shanghai, China). The skeleton plasmid of lentiviral vectors Flag-vIL-6, His-vIL-6, Flag-STAT3, and Flag-STAT3-K685R was pHAGE-CMV-MCS-IzsGreen (pHAGE for short). pHAGE contains ZsGreen fluorescent coding sequence, which can display fluorescence similar to that produced by green fluorescent proteins in cells. All the shRNAs were constructed into the modified pCDH (named as mpCDH), a new lentiviral plasmid generated in our previous study [32]. HEK293T cells were cotransfected with lentiviral plasmids, packaging plasmid psPAX2, and pseudotyping pMD2.G using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) as previously described [10, 55, 56].

Construction and identification of KSHV vIL-6 mutant

The deletion mutagenesis of KSHV vIL-6 coding sequence was modified using a two-step ‘scarless’ homologous recombination procedure that has been previously described [20, 21]. We used GS1783 Escherichia coli (a gift from Greg Smith, Northwestern University) harboring BAC16, a bacterial artificial chromosome (BAC) clone of the KSHV genome [21, 57]. Briefly, PCR amplification was used to generate a linear DNA fragment containing a kanamycin resistance expression cassette, an I-SceI restriction enzyme site, and flanking sequences derived from the upstream or downstream region of KSHV vIL-6 coding sequence, each of which includes a roughly 40-bp copy of a duplication. The fragment was then electroporated into GS1783 cells harboring BAC16 and transiently expressing gam, bet, and exo. These three proteins are required for homologous recombination of linear DNA fragment with a target sequence and can be expressed in a temperature-inducible manner from the lambda red operon engineered within the endogenous GS1783 chromosome. Integration of the Kanr/I-SceI cassette was verified by PCR and restriction enzyme digestion of the purified BAC16 DNA. The GS1783 strain is also equipped with an arabinose-inducible gene encoding the I-SceI enzyme. Upon treatment with 1% l-arabinose, the integrated Kanr/I-SceI cassette is cleaved, resulting in a transiently linearized BAC16. A second red-mediated recombination between the duplicated sequences results in recircularization of the BAC DNA and “scarless” loss of the Kanr/I-SceI cassette. Kanamycin-sensitive clones were screened via replica plating. BAC DNA was purified from chloramphenicol-resistant colonies using an alkaline lysis procedure followed by isopropanol precipitation. Purified BAC DNA was digested with KpnI, CpoI, or SbfI and separated on a 1% agarose gel using pulse-field gel electrophoresis (CHEF-DR II, Biorad) with the following conditions: 6 V/cm for 15 h; initial and final switch times of 1 and 5 s, respectively; and 14 °C. To further verify recombinant BACmids, modified regions were PCR amplified and sequenced. The primers were 5′-AAC TGT GAC ATA TTT TTA GAG GAC TCG GA-3′ and 5′-TAG AAT CAA TGT GGT TCT AAG TCG CAC G-3′.

Generation of KSHV_WT and K2_mutant recombinant virus

iSLK-BAC16 cells and iSLK-BAC16ΔK2 cells were used for the generation of KSHV_WT virus and vIL-6_mutant virus, respectively, which were induced by sodium butyrate and doxycycline as previously described [57].

Cell proliferation assay, plate colony formation assay, transwell migration, and Matrigel invasion

Cell proliferation assay was performed using Cell Count Kit-8 (Dojindo Molecular Technologies, Tokyo, Japan) according to the manufacturer’s instructions as previously described [56]. Plate colony formation assay, transwell migration, and matrigel invasion were performed as previously described [32, 55]. For transwell migration and matrigel invasion assays, transwell chambers (8 μm) from Merck Millipore (Darmstadt, Germany) were adopted. HUVECs (1 × 105) were seeded into chambers with or without coated matrigel. After 6 or 12 h incubation, the chambers were harvested, fixed, and stained. The migrated cells were photographed and calculated by counting stained cells in a double-blinded manner by two observers.

Wound-healing assay

Cells (~80% confluence) were plated in six-well plates and scratched with a pipette tip (200 μl). After 24 h, the migrated cells were photographed using an inverted light microscopy (Olympus, Japan) and were counted as already described [32].

Chicken chorioallantoic membranes (CAMs) assay

Nine-day-old embryos were used for CAM assay. A total of 5 × 106 cells mixed with matrigel (BD Biosciences) were vertically injected into eggs through pierced opening in the upper surface. Six chicken embryos were used for each treatment. Embryos were randomly divided. Four days later, CAMs were harvested and captured under stereomicroscope as previously described [56]. The number of branching points was quantified using the software ImageJ (NIH, http://rsb.info.nih.gov/ij/) [58]. The angiogenesis index was calculated as the mean number of branch points from the experimental conditions minus the mean number of branch points from gel alone controls. No statistical method was used to predetermine sample sizes.

RT-qPCR analysis

Total RNA was prepared using TRIzol (Life Technologies, Grand Island, NY, USA). cDNA was made by HiScript Q RT SuperMix (Vazyme Biotech Co., Ltd, Nanjing, China). For RT-qPCR, AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China) and Applied Biosystems (ABI, Foster City, CA, USA) were adopted. Primers for GAPDH utilized as an internal normalization control were 5′-GAA GGT GAA GGT CGG AGT C-3′ and 5′-GAA GAT GGT GAT GGG ATT TCC-3′. Primers specific for CAV1 gene were 5′-GAG CTG AGC GAG AAG CAA GT-3′ and 5′-TCC CTT CTG GTT CTG CAA TC-3′. Primers specific for vIL-6 were 5′-GTA TTC TAG AGC CCG CTG CTA-3′ and 5′-TTA AAT CCT ATT AAC CCG CAG T-3′. Primers specific for LANA were 5′-GAA GTG GAT TAC CCT GTT GTT AGC-3′ and 5′-TTG GAT CTC GTC TTC CAT CC-3′.

Western blotting analysis

Western blotting analysis was performed as described [32]. Antibodies used were anti-CAV1 rabbit antibody, anti-Flag rabbit antibody, anti-p-STAT3 rabbit antibody, anti-Acetyl-Stat3 (Lys685) rabbit antibody, anti-STAT3 mouse antibody, anti-p-AKT rabbit antibody, and anti-AKT rabbit antibody from Cell Signaling Technologies (Beijing, China), anti-α-Tubulin mouse antibody, and anti-GAPDH mouse antibody, from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-DNMT1 mouse antibody was from Abcam (Cambridge, MA, USA). Anti-His mouse antibody was obtained from Beyotime Institute of Biotechnology (Nantong, Jiangsu, China). Anti-vIL-6 rabbit monoclonal antibody was kindly provided by Dr Robert Yarchoan from Center for Cancer Research, National Cancer Institute (Bethesda, Maryland, USA).

Immunohistochemistry (IHC)

For IHC analysis, the following antibodies were used: anti-KSHV LANA (Advanced Biotechnologies Inc., Columbia, MD, USA), anti-CAV1 (Cell Signaling Technologies, Beijing, China), and anti-rabbit immunoglobulin G (IgG) (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China). Secondary antibodies used in this study were horseradish peroxidase (HRP)-labeled goat anti-rat or anti-rabbit as appropriate (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China). DAB (3,3′-diaminobenzidine) Peroxidase (HRP) Substrate Kit (Vector Laboratories, Inc., Burlingame, USA) was used to visualize staining.

DNA methylation analysis

DNA methylation of the CpG Island of the CAV1 promoter region was examined using methylation-specific PCR (MS-PCR). Bisulfite conversion kit (Tiangen Biotech, Beijing, China) and methylation-specific PCR kit (Tiangen Biotech, Beijing, China) were adopted according to the manufacturer’s instructions. Designed MS-PCR primers for methylated (M) were 5′-AAC GTT TTT ATT CGT TTT TTG TTC-3′ and 5′-CGC CAA AAA TTT ATT CTA CTC G-3′, for unmethylated (U) were 5′-AAT GTT TTT ATT TGT TTT TTG TTT GT-3′ and 5′-CCC ACC AAA AAT TTA TTC TAC TCA C-3′.

Chromatin immunoprecipitation (ChIP) assay

ChIP analysis was performed as previously described [59]. EZ-Magna ChIP™ A/G ChIP Kit (Merck, Darmstadt, Germany) was used in accordance with the manufacturer’ s instructions. Cells (107) were cross-linked by 1% formaldehyde and harvested to suffer sonication. The obtained DNA fragments were precipitated with anti-DNMT1 (Abcam, Cambridge, MA, USA), anti-STAT3 and antiphospho-STAT3 (Tyr705) (Cell Signaling Technologies, Beijing, China), anti-RNA polymerase II (Merck, Darmstadt, Germany), or IgG (Merck, Darmstadt, Germany). Purified DNA was used in RT-qPCR using specific primers. The sequences of CAV1 promoter primers were 5′-AGC ACC CCA GCG CGG GAG-3′ and 5′-GCG GTG GCT GGG AGG GAG-3′. The primers for DNMT1 enhancer were used as previously described [27].

Co-Immunoprecipitation

Co-IPs were performed as previously described [28]. Immunoprecipitated proteins were subjected to western blotting analysis using specific antibodies. IPKine™ HRP Goat Anti-Mouse or Anti-Rabbit IgG LCS (Abbkine Scientific Co., Ltd, Wuhan, China) were used as the secondary antibodies for Co-IP, which could avoid the detection of the heavy chains of IgG by specifically reacting with kappa light chains on IgG.

Soft agar colony formation assay

First, place 2 ml of the bottom layer (0.6% agarose gel, BD Biosciences) into each well of a 6-well culture plate, and then added 1 ml of the cell-containing layer (0.3% agarose gel mixed with 2 × 104 cells). After 2 weeks, photographed and counted the number of colonies in each well using a light microscope. For data analysis, the colony sizes of 20 μm or larger were scored for calculation of the percentage of soft agar colony.

Statistical analysis

Sample sizes for relevant experiments were determined by power analyses conducted during experiment planning. Data are expressed as mean ± SD. Differences were analyzed by the Student t test. Statistical significance was set at P < 0.05. Each experiment was repeated at least three times independently, unless otherwise stated.

Supplementary Material

2

Acknowledgements

We thank Drs Robert Yarchoan and Victoria Wang at the Center for Cancer Research, National Cancer Institute for providing the purified monoclonal rabbit anti-vIL-6 antibody and information related to the detection of vIL-6 in KSHV-infected HUVECs by western blotting. We also thank Drs Yuan Chang and Patrick Moore from University of Pittsburgh for providing reagent. We are also grateful to members from Dr Lu laboratory for helpful discussion.

Funding This work was supported by grants from National Natural Science Foundation of China (81730062, 81761128003, 31800148, and 81503368), Natural Science Foundation of Jiangsu Province (BK20180681), Nanjing Medical University (KY101RC1710), a grant from NIH (R01CA213275), and grants (CA200422, AI073099, AI116585, AI129496, AI140718, AI140705, DE023926, DE027888, DE028521 and Fletcher Jones Foundation).

Footnotes

Supplementary information The online version of this article (https://doi.org/10.1038/s41388-020-1317-1) contains supplementary material, which is available to authorized users.

Conflict of interest The authors declare that they have no conflict of interest.

Compliance with ethical standards

Ethical approval The clinical section of the research was reviewed and ethically approved by the Institutional Ethics Committee of the First Affiliated Hospital of Nanjing Medical University.

Informed consent Written informed consent was obtained from all participants, and all samples were anonymized. All participants were adults.

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