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Angiogenin (ANG) is a 14-kDa multifunctional proangiogenic secreted protein whose expression level correlates with the aggressiveness of several tumors. We observed increased ANG expression and secretion in endothelial cells during de novo infection with Kaposi's sarcoma-associated herpesvirus (KSHV), in cells expressing only latency-associated nuclear antigen 1 (LANA-1) protein, and in KSHV latently infected primary effusion lymphoma (PEL) BCBL-1 and BC-3 cells. Inhibition of phospholipase Cγ (PLCγ) mediated ANG's nuclear translocation by neomycin, an aminoglycoside antibiotic (not G418-neomicin), resulted in reduced KSHV latent gene expression, increased lytic gene expression, and increased cell death of KSHV+ PEL and endothelial cells. ANG detection in significant levels in KS and PEL lesions highlights its importance in KSHV pathogenesis. To assess the in vivo antitumor activity of neomycin and neamine (a nontoxic derivative of neomycin), BCBL-1 cells were injected intraperitoneally into NOD/SCID mice. We observed significant extended survival of mice treated with neomycin or neamine. Markers of lymphoma establishment, such as increases in animal body weight, spleen size, tumor cell spleen infiltration, and ascites volume, were observed in nontreated animals and were significantly diminished by neomycin or neamine treatments. A significant decrease in LANA-1 expression, an increase in lytic gene expression, and an increase in cleaved caspase-3 were also observed in neomycin- or neamine-treated animal ascitic cells. These studies demonstrated that ANG played an essential role in KSHV latency maintenance and BCBL-1 cell survival in vivo, and targeting ANG function by neomycin/neamine to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV-associated malignancies.
Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a γ2 human herpesvirus which is etiologically associated with the pathogenesis of Kaposi's sarcoma (KS), an angioproliferative tumor of endothelial origin. KSHV is also associated with two B-cell-proliferative neoplasms: body cavity-based lymphoma (BCBL) or primary effusion B-cell lymphoma (PEL) and multicentric Castleman's disease (MCD) (1–3). PEL is a rare aggressive form of non-Hodgkin's lymphoma that occurs most frequently in AIDS patients. This B-cell monoclonal malignancy is observed in various body cavities, such as the pleura, pericardium, and peritoneum (2, 4). Occasionally, PEL can be present as a solid mass in lymph nodes and other organs (5, 6). PEL is associated with a poor prognosis and resistance to conventional chemotherapy, with a survival time of 2 to 6 months (7). Histologically, PEL cells are large B cells having the appearance of anaplastic or immunoblastic cells (8). They express CD45, CD30, and immunoglobulin genes but lack B-cell differentiation antigens (8). Among the PEL B-cell lines isolated from patients, BC-1, HBL-6, and JSC carry both KSHV and Epstein-Barr virus (EBV) genomes, whereas BCBL-1 and BC-3 carry only the KSHV genome (9). Available treatment strategies to control HHV-8 infection-associated malignancies are limited and of low efficacy. Hence, there is a vital requisite for designing therapies that target viral infection and tumor formation.
Similar to that of other members of the herpesvirus family, the KSHV life cycle can be divided into latent and lytic cycles. In PEL cells, 50 to 150 copies of the viral genome are maintained as nuclear episomes (10). During the latent phase, no new viral particles are produced, and the cells express KSHV latency-associated genes, such as open reading frame (ORF) 73 (latency-associated nuclear antigen 1 [LANA-1]), ORF 72 (vCyclin), ORF 71 (vFlip), K12 (kaposin), ORF 10.5 (LANA-2), 12 viral microRNAs, and occasionally the viral interleukin 6 (vIL-6) gene. The oncogenesis of PEL is predominantly mediated by latent KSHV genes. In PEL cells, proteins expressed from the latent genes are responsible for the maintenance of the episomal KSHV genome, inhibition of tumor suppressor p53, cell cycle regulation, inhibition of apoptosis, host gene regulation, stabilization of cytokine expression, antiapoptosis, antiautophagy, immune evasion, and proliferation (11–18). In addition, KSHV latency-associated microRNAs are also involved in cell survival (19, 20), and recently miR-K12-11 has been shown to promote B-cell expansion in vivo (21).
Only about 1 to 3% of PEL cells spontaneously enter the lytic cycle, induced by the KSHV lytic switch replication and transcription activator (RTA) (ORF 50) protein. However, about 10% to 25% of cells enter the lytic phase after chemical treatment, such as phorbol esters or histone deacetylase inhibitors (sodium butyrate). The lytic nonstructural genes mediate several functions, such as immune evasion, inhibition of apoptosis, host gene modulation, host protein expression shutoff, and modulation of signal transduction (9). In contrast to PEL pathogenesis, both the latent and lytic cycles of KSHV, along with the infection-induced angiogenic inflammatory network, are involved in KS pathogenesis (9).
Angiogenin (ANG), a 14-kDa multifunctional protein, was first isolated as an angiogenic-secreted protein produced by HT-29 human colon adenocarcinoma (22, 23). The level of expression of ANG correlates with the aggressiveness of several tumors, such as urothelial carcinoma and tumors of the pancreas, gastric system, colon, ovary, endometrium, cervix, and breast (24–31). ANG is a multifunctional protein with different functions dependent on its localization. In addition to being a secreted protein, ANG has also been detected at the plasma membrane, in the cytoplasm, in the nucleus, and in the nucleolus of cells. Secreted ANG has been shown to interact with actin on the cell membrane and is involved in the migration of endothelial cells by promoting the production of plasmin from plasminogen (32, 33). ANG activates several signaling pathways, including phospholipase Cγ (PLCγ), phospholipase A2 (PLA2), protein kinase B (PKB/AKT), and extracellular signal-related kinase 1/2 (ERK1/2) (34–36). ANG is also called RNase 5, as it contains 35% identity with the human pancreatic RNase 1 and is involved in the generation of 18S and 28S rRNA (37).
The nuclear translocation of ANG is necessary for its angiogenic potential, as both the inhibition and mutation of the nuclear localization sequence inhibits angiogenic activity (38, 39). In the nucleolus, ANG binds to CT repeats of rRNA promoters and promotes their transcription (40). Several studies have elucidated the role of nuclear ANG in cancer cell proliferation and angiogenesis (38, 41–43). Treatment of cancer cells with the aminoglycoside antibiotic neomycin (distinct from neomycin G418) mediated antiproliferative and antiangiogenic effects, which was shown to be due to the inhibition of ANG nuclear translocation (44). Investigation regarding the mechanism by which neomycin inhibits ANG nuclear translocation revealed that the PLCγ-inhibiting activity of neomycin was involved (44). Neomycin inhibited PLCγ by binding to phosphatidylinositol 4,5-bisphosphate (PIP2) (45). The inhibition of ANG nuclear translocation was also observed with U73122, a PLCγ inhibitor. Other members of the aminoglycoside antibiotic family, such as streptomycin, kanamycin, gentamicin, paromomycin, and amikacin, did not inhibit ANG nuclear translocation and consequently were unable to inhibit ANG-induced proliferation or angiogenesis (44). In particular, paromomycin is structurally very similar to neomycin, as the difference between these two drugs is a positive-charged amino group (present in neomycin) replacing a neutral hydroxyl (present in paromomycin). However, it has been shown that paromomycin does not inhibit ANG nuclear translocation and ANG-induced proliferation (44). ANG nuclear translocation was also unaffected by inhibitors of tyrosine kinases, phosphotyrosine phosphatase, and protein kinase C (44). In normal cells, though neomycin inhibits the nuclear translocation of ANG by inhibiting PLCγ activation, it did not affect the viability of the cells, and even a concentration of 1 mM is nontoxic (46).
We have previously reported a novel role of ANG in the biology of KSHV. ANG expression and secretion was increased upon de novo KSHV infection of human dermal microvascular endothelial cells (HMVEC-d) and was elevated in long-term KSHV-infected endothelial cells (telomerase-immortalized human umbilical vein endothelial long-term-infected cells [TIVE-LTC]) (47). Expression of KSHV latency protein LANA-1 and lytic protein viral G protein-coupled receptor (vGPCR) induced ANG gene expression and ANG protein secretion. In addition, we have shown that ANG expression and secretion was increased in PEL cells (BCBL-1 and BC-3), which was not observed however in EBV+ lymphoma and lymphoblastoid cells (46). Our studies suggested that ANG plays important roles in KSHV pathogenesis through its antiapoptotic, cell proliferation, migration, and angiogenic properties (46, 47). We have also shown that ANG addition induced KSHV ORF 73 (LANA-1) gene expression (46). Inhibition of its nuclear translocation with neomycin reduced latent ORF 73 gene expression and increased the lytic ORF 50 gene both during de novo infection and in latently infected TIVE-LTC and PEL cells. The role of ANG was confirmed, as silencing ANG with short hairpin RNA (shRNA) had a similar effect on viral gene expression as that of neomycin treatment. A greater quantity of infectious KSHV was detected in the supernatants of neomycin-treated BCBL-1 cells than 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells (46, 48). This suggested a role for ANG in the regulation of KSHV latent and lytic cycles (in vitro model, see Fig. 2A).
In addition, we observed that ANG is crucial for the antiapoptotic effect of KSHV observed after serum starvation of endothelial cells (47). Whereas KSHV infection protected endothelial cells from apoptosis, blocking nuclear translocation of ANG with neomycin allowed apoptosis to proceed. We also observed a role for ANG in KSHV oncogenesis of PEL cells, as nuclear ANG was essential for BCBL-1 cell survival in vitro (46). Indeed, treatment with neomycin significantly decreased the viability of KSHV-positive lymphoma cells (BCBL-1, BJAB-KSHV, BC-3, and JSC-1 cells) as well as latently infected endothelial TIVE-LTC cells but had no effect on EBV-positive cells (LCL or Raji) or KSHV- and EBV-negative cells (BJAB, Akata, Ramos, and Loukes) (46). Similarly, knocking down ANG with shRNA decreased PEL cell viability, thus confirming the role of ANG in PEL cell survival (46) (in vitro model, see Fig. 2A). Treatment of normal endothelial cells with ANG also induced PLCγ and AKT phosphorylation, while treatment with neomycin and ANG silencing inhibited PLCγ and AKT phosphorylation (46). Our studies demonstrated that blockage of PLCγ activation by neomycin mediated the inhibition of latent gene expression, and the conventional PLCγ inhibitor U73122 showed similar results. Collectively, these studies suggested that KSHV has evolved to exploit ANG for its advantage via the PLCγ pathway for maintaining its latency (in vitro model, see Fig. 2A).
Correlation of ANG's expression level with the aggressiveness of several tumors and inhibition of progression and metastasis of human cancer cells by anti-ANG monoclonal antibodies in athymic mice suggested that actively proliferating cancer cells could be inducing ANG for inhibiting apoptotic pathways (24–31, 49, 50). However, how ANG regulates cell survival and apoptosis was not known. We have recently demonstrated that ANG interacts with p53 and colocalizes in the nucleus of KSHV-negative cancer cells (51). Silencing endogenous ANG induced p53 promoter activation and p53 target gene expression, downregulated the expression of the antiapoptotic Bcl-2 gene, and increased p53-mediated cell death. In contrast, ANG expression blocked proapoptotic Bax and p21 expression, induced Bcl-2, and blocked cell death. ANG also coimmunoprecipitated (co-IPed) with Mdm2, a p53 regulator protein. ANG expression inhibited p53 phosphorylation, increased p53-Mdm2 interaction, and increased p53 ubiquitination. These studies demonstrated that ANG inhibits p53 functions to promote antiapoptosis and cell survival of cancer cells and suggested that targeting ANG could be an effective therapy for several cancers.
In the context of KSHV-infected cells, we observed that LANA-1 and ANG colocalized and co-IPed in de novo-infected endothelial cells and in latently infected PEL BCBL-1 and BC-3 cells (48). LANA-1 and ANG interaction occurred in the absence of the KSHV genome and other viral proteins. ANG coeluted with LANA-1, p53, and Mdm2, while LANA-1, p53, and Mdm2 also co-IPed with ANG. LANA-1, ANG, and p53 colocalized in KSHV-infected cells. Silencing ANG or inhibiting its nuclear translocation resulted in decreased nuclear LANA-1 and ANG levels, decreased interactions between ANG–LANA-1, ANG-p53, and LANA-1–p53, the induction of p53, p21, and Bax proteins, the increased cytoplasmic localization of p53, the downregulation of Bcl-2, the increased cleavage of caspase-3, and the apoptosis of cells. Together, these studies suggested that the antiapoptosis observed in KSHV-infected cells and the suppression of p53 functions are mediated in part by ANG, and KSHV has probably evolved to utilize ANG's multiple functions for the maintenance of its latency and cell survival. These studies also suggested that targeting ANG to induce the apoptosis of cells latently infected with KSHV is a potential therapeutic strategy against KSHV infection and associated malignancies.
In the present study, we tested the in vivo antitumor activity of the ANG nuclear translocation inhibitor neomycin as well as neamine, a derivative of neomycin known to have fewer adverse side effects (41–43). Our studies show that in vivo treatment of BCBL-1 cell-injected NOD/SCID mice with neomycin and neamine significantly prolongs their survival by inhibiting tumor establishment. At the time of initial tumor detection, the weight, ascites development, and BCBL-1 infiltration in the animals' spleens were reduced in neomycin-and neamine-treated animals compared to those of phosphate-buffered saline (PBS)-treated mice. At the cellular level, we observed a decrease of KSHV latent gene expression and an increase of lytic gene expression in BCBL-1-injected and treated animals. In addition, we observed increased BCBL-1 cell apoptosis in neomycin- and neamine-treated mice. These findings suggest that neomycin and neamine could be used as potential therapeutic candidates for the treatment of KSHV-associated PEL.
Neomycin, paromomycin, and CD19 antibody (for immunofluorescence assay [IFA], 1:100 dilution) were from Sigma-Aldrich, St. Louis, MO. Neamine was a generous gift from G. F. Hu, Sackler School of Graduate Biomedical Sciences, Tufts University, Massachusetts. ANG antibody (for IFA, 1:100 dilution) was from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. Total caspase-3 and cleaved caspase-3 antibodies (for Western blotting [WB], 1:1,000 dilution; for IFA, 1:100 dilution) were from Cell Signaling Technology, Danvers, MA. Human CD19 antibody (for WB, 1:1,000 dilution) was from GeneTex, Irvine, CA. Rabbit polyclonal gB (UK-218) (for IFA, 1:100 dilution), rabbit polyclonal LANA-1 (for WB, 1:1,000 dilution; for IFA, 1:80 dilution), and mouse monoclonal LANA-1 (for IFA, 1:50 dilution) antibodies were generated in our laboratory (52). Horseradish peroxidase-linked antibodies (for WB, 1:5,000 dilution) were from KPL Inc., Gaithersburg, MD. Alexa 488 (for IFA, 1:500 dilution) and Alexa 594 (for IFA, 1:1,000 dilution) secondary antibodies and DAPI (4′,6-diamidino-2-phenylindole) were from Molecular Probes, Invitrogen, Grand Island, NY.
BCBL-1 cells were propagated and maintained as per procedures described previously (53–55). BCBL-1 cells were routinely tested for mycoplasma by the Lonza MycoAlert kit (LT37-618) (Lonza, New Jersey) as per the manufacturer's instructions and were found to be negative. NOD.CB17-Prkdcscid/J (NOD/SCID) mice (Jackson Laboratory, Bar Harbor, ME) were kept at the Biological Resource Facility at Rosalind Franklin University of Medicine and Sciences, North Chicago, IL. NOD/SCID mice were housed in microisolator cages. All animal experiments were approved by the Institutional Animal Care and Use Committee of Rosalind Franklin University of Medicine and Sciences (IACUC protocol no. 10-06). Mice were weighed as a criterion for ascites growth and tumorigenesis. Animals were monitored and euthanized when signs of distress were clearly visible, according to our protocol. For the engraftment of BCBL-1 cells, BCBL-1 cells were injected intraperitoneally (i.p.) into NOD/SCID mice at 107 cells per mouse.
Comparison of survival curves was done using the log rank test (56).
The assay was performed in a 48-well-plate format. The base agar matrix layer was prepared as per the manufacturer's protocol (cell transformation assay soft agar with cell recovery, catalog no. CBA-135; Cell Biolabs, California). BCBL-1 cells, resuspended at 5 × 105 cells/ml, were added to the agar matrix layer. After solidification, medium containing 200 μM neomycin was added on top of the cell/agar matrix layer. Six days later, the colonies were viewed under a Nikon eclipse TE2000-5 microscope using the Nikon MetaMorph digital imaging system. Quantification of anchorage-independent growth was performed as per the manufacturer's guidelines, using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)-based assay. Briefly, the cell-containing matrix was solubilized, MTT solution was added, and the absorbance was read at 570 nm in a Synergy HT microplate reader (BioTek Instruments) after the addition of detergent solution.
The tissue samples were excised and fixed in 4% paraformaldehyde (PFA) for 7 days and kept in 20% sucrose in PBS. The samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) at the Northwestern University Mouse Histology and Genotyping Core, Chicago, IL.
Sections of skin biopsy samples from healthy subjects or KS patients as well as sections from healthy lung or PEL solid lung lesions were obtained from the AIDS and Cancer Specimen Resource (ACSR). The sections were deparaffinized and hydrated with water before antigen retrieval using Dako target retriever solution in a steamer for 20 min. Slides were cooled, rinsed, blocked using 1% bovine serum albumin (BSA) in 0.025% Triton X-100–PBS for 30 min, and used for staining of ANG alone, double-staining with anti-ANG and mouse monoclonal anti-CD19 antibodies, or double-staining with anti-ANG and mouse monoclonal anti-LANA-1 antibodies. Sections were washed and incubated with a 1:200 dilution of Alexa 488-coupled anti-rabbit antibody or Alexa 594-coupled anti-mouse antibody (Molecular Probes) for 1 h at room temperature. Nuclei were visualized using DAPI, and stained cells were viewed with the appropriate filters under a fluorescence microscope (Nikon 80i) with a 20× objective and the Nikon MetaMorph digital imaging system.
The ascites fluids recovered from the different animals were centrifuged. Cell pellets were washed in PBS, fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 for 10 min, blocked with Image-iTFX signal enhancer (Invitrogen) for 20 min, and incubated for 2.5 h with the primary antibodies indicated in the respective figures. After three washes, the cells were incubated for 1.5 h with the secondary anti-rabbit antibodies. Nuclei were visualized using DAPI (Molecular Probes, Invitrogen), and stained cells were viewed with the appropriate filters under a fluorescence microscope with a 20× objective.
Cells were harvested in RIPA lysis buffer (125 mM NaCl, 0.01 M sodium phosphate [pH 7.2], 0.1% SDS, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, and 50 mM sodium fluoride) with protease inhibitor and phosphatase inhibitor cocktails (Sigma). Cellular debris was removed by centrifugation at 13,000 × g for 5 min at 4°C, and equal amounts of protein samples were resolved by 10% SDS-PAGE and subjected to Western blotting with the antibodies as indicated in each figure. To confirm equal protein loading, blots were also probed with antibodies against human tubulin or actin. Secondary antibodies conjugated to horseradish peroxidase were used for detection. Immunoreactive bands were visualized by enhanced chemiluminescence.
Total RNA was extracted by using TRIzol reagent (Invitrogen), quantified by densitometric analysis at 260 nm, and analyzed by real-time reverse transcription (RT)-PCR using primers to ORF 73 (57). PCR was performed using an ABI Prism 7500 real-time PCR system utilizing TaqMan EZ RT-PCR core reagents (Applied Biosystems).
In our previous studies, we have shown that de novo KSHV infection of HMVEC-d cells resulted in increased secretion of ANG (47, 58). In addition, we have shown that ANG expression and secretion were increased in KSHV-associated B-lymphoma cell lines (46). To determine whether ANG is expressed in KSHV-associated tumors, we analyzed skin sections from healthy subjects and KS-positive patients with anti-ANG and anti-LANA-1 antibodies in immunofluorescence assays (IFA) (Fig. 1A). In contrast to healthy tissues, intense ANG staining colocalizing with LANA-1 staining was observed in KS lesions (Fig. 1A, compare top and bottom panels). Similarly, we analyzed the expression of ANG in tissues from healthy lung and lung with solid PEL lesions (Fig. 1B). We observed a striking increase in ANG expression in PEL lesions. ANG staining in PEL lesions was specific to the B-cell lymphoma, as it colocalized with the B-cell marker, CD19 (Fig. 1B). In addition, we performed a costaining with ANG and LANA-1 antibodies in the solid PEL lesions of lungs (Fig. 1C). We observed increased ANG staining in the areas of cells expressing LANA-1. These results suggested that the expression pattern of ANG is consistent with the presence of latent KSHV in the lesions. Taken together, increased detection of ANG in KSHV-associated malignancies highlighted the importance of ANG in KSHV pathogenesis.
We have previously shown that ANG localized predominantly in the nuclei and nucleoli of KSHV-infected cells (47). In addition, blocking ANG nuclear translocation by neomycin treatment decreased the survival of latently infected endothelial cells and BCBL-1 cells (46). The results of our extensive previous in vitro studies are summarized in Fig. 2A. A characteristic of tumor development is the ability of the cells to proliferate independently of anchorage, and the oncogenic capacity of BCBL-1 cells to form colonies on soft agar has been previously shown (59, 60). Hence, we examined the growth of BCBL-1 cells in soft agar in the absence or presence of neomycin (Fig. 2). We chose a 200 μM concentration of neomycin, as it has previously been used and showed no toxicity on normal endothelial, KSHV-negative TIVE, BJAB, Akata, or EBV+ cells, whereas it reduced survival of KSHV+ cells. We observed loose, disaggregated BCBL-1 cell colonies in soft agar (Fig. 2B, left). The morphology of these colonies is similar to that of the colonies observed with the BCP-1 cell line (61). However, in the presence of 200 μM neomycin, the quantity and the size of the colonies formed in soft agar were reduced (Fig. 2B, right). As manual counting of colonies was less quantitative and does not reflect colony size, we used the assay developed by Cell Biolabs to quantify the anchorage-independent growth. Following the manufacturer's protocol, the semisolid medium was solubilized, and the anchorage-independent growth was quantified by an MTT solution. We observed a significant decrease in BCBL-1 cell viability after growth in soft agar in neomycin treatment conditions, with roughly 65% decrease in MTT assay (Fig. 2C). These results suggested that nuclear translocation of ANG plays an important role for the survival and tumorigenic properties of BCBL-1 cells.
Transfer of KSHV-infected PEL cells to immunodeficient mice leads to tumor engraftment without any spread of KSHV infection to murine tissues (61, 62). After intraperitoneal (i.p.) injection of 107 BCBL-1 cells into NOD/SCID mice, we observed tumor development starting at day 28, and all animals developed tumors with a mean survival time of 44 days (Fig. 3A). To determine the in vivo effect of inhibiting the nuclear transport of ANG by neomycin, we injected the drug after BCBL-1 cell injection. Mice were injected with 107 cells followed by the injection of 10 mg of neomycin/kg of body weight every 2 days for 1 week and once a week thereafter. We observed a significant delay (P < 0.004) in tumor development in the neomycin-treated mice (Fig. 3B). The mean survival time was improved from 56 days in nontreated animals to 96 days in neomycin-treated mice. The effect of blocking ANG was confirmed using neamine, a derivative of neomycin known to have fewer adverse side effects (41–43). We observed an even greater delay in tumor development in the neamine-treated mice (Fig. 3C). The mean survival time was increased from 56 days in nontreated animals to 118 days in neamine-treated mice (P < 0.0015). To determine that these effects were specific to blocking the nuclear localization of ANG, we used paromomycin as a negative control. Paromomycin, an analogue of neomycin, does not affect the nuclear transport of angiogenin. When mice were injected with paromomycin, BCBL-1 tumor development was not significantly inhibited. Indeed, the survival of paromomycin-treated mice was comparable to PBS-injected animals, with a mean survival time of 60 and 56 days, respectively (Fig. 3D). Altogether, these results suggested that agents that block ANG nuclear translocation in BCBL-1 cells in vitro are also effective in vivo, resulting in protection from BCBL cell tumor development with increased survival time of mice, and neamine had a greater protective effect than neomycin.
To determine the effect of ANG inhibitors early during tumor development, all mice were injected i.p. with 107 BCBL-1 cells followed by the injection of the corresponding drugs (10 mg/kg) every 2 days for 1 week and once a week thereafter. Seven weeks after the injection of tumor cells, all the animals were euthanized at the same time. At this time, we observed some abdominal distention in the PBS-treated animals but none in the neomycin- or neamine-treated animals (Fig. 4Aa and b). Abdominal distention is a well-established sign of ascites development. In addition, the PBS-treated animals were significantly heavier than the animals treated with neomycin and neamine (Fig. 4Ac). Whereas the average weight of an NOD/SCID mouse at 7 weeks was 20 g, the weight of BCBL-1-injected mice treated with PBS was around 29 g. However, the body weight of the mice injected with BCBL-1 cells and treated with neomycin was significantly reduced to 24 g, and the weight of neamine-treated animals was comparable to the average weight of NOD/SCID mice at the same age (20 g) (Fig. 4Ac). An increase in body weight is a second sign indicating tumor formation.
To confirm that the abdominal distension and gain of weight were due to tumor formation, we extracted the ascites cells from these mice for further analysis (Fig. 4B). Animals not injected with BCBL-1 cells did not show any ascites formation (data not shown). However, all of the mice injected with BCBL-1 cells and treated with PBS developed ascites (5/5). In contrast, ascites formation was observed in 3 of the 5 neomycin-treated mice and only in 1 mouse of the 5 neamine-treated mice (Fig. 4B). In addition, we collected the ascites and measured the volume produced in each mouse. We collected an average of 1.5 ml of ascites from PBS-treated animals, and the volumes of ascites from neomycin- and neamine-treated animals were reduced to an average of 0.35 and 0.05 ml, respectively (Fig. 4B). The presence and quantity of ascites correlated with the increased weight observed in Fig. 4Ac, confirming that the weight gain observed in Fig. 4A was due to tumor establishment. These data demonstrated a significant delay in tumor formation in neomycin- and neamine-treated animals and indicated that neamine treatment was more potent in inhibiting BCBL-1 tumor formation.
We observed that mice injected i.p. with BCBL-1 cells presented significantly enlarged spleens compared to those of normal NOD/SCID mice (data not shown). We next evaluated the effect of neomycin and neamine on the spleens of BCBL-1 cell-injected mice euthanized 7 weeks postinjection. We observed significantly smaller spleens in neomycin- and neamine-treated mice than those from PBS-treated animals. Representative pictures of the spleens are shown in Fig. 5Aa. The spleens from uninjected animals weighed around 0.05 g, whereas BCBL-1-injected and PBS-treated mice weighed approximately 0.2 g. Interestingly, the spleens were significantly smaller in neomycin- and neamine-treated animals, with an average weight of 0.1 g and 0.05 g, respectively (Fig. 5Ab).
To determine the cause of the enlarged spleens, we performed histologic analysis using H&E staining of the spleen sections (Fig. 5Ba). In BCBL-1-injected mice treated with PBS, we observed the presence of infiltrating cells (Fig. 5Ba, top; enlarged in the top right). These infiltrated cells are large and have the appearance of anaplastic cells. This morphology is similar to the morphology of PEL cells (8). However, the numbers of infiltrating cells were significantly reduced in neomycin- and neamine-treated animals (Fig. 5Ba, middle and bottom, respectively). We observed an average of 15, 6, and 4 infiltrating cells per field in PBS-, neomycin-, and neamine-treated animals, respectively (Fig. 5Bb). The number of infiltrating cells is proportional to the weight of the spleens, suggesting that these cells are responsible for spleen enlargement.
To confirm that enlargement of the spleens was due to BCBL-1 cell infiltrations, we quantified the expression of the KSHV latency ORF 73 gene from the spleen RNA. In mice injected with BCBL-1 cells and treated with PBS, we observed significantly more ORF 73 expression than in mice injected with BCBL-1 cells and treated with neomycin or neamine (Fig. 5C). The ORF 73 expression is proportional to the weight of the spleen and to the number of infiltrating cells observed in the histologic analysis, indicating that enlargement of the spleens is likely due to BCBL-1 cell infiltration. Altogether, these results demonstrated that neomycin and neamine treatment decreased BCBL-1 cell dissemination into the spleens of NOD/SCID mice.
Our earlier in vitro studies have shown that the decrease of BCBL-1 viability after neomycin treatment was due partially to a decrease in KSHV latency gene expression, and ANG plays a role in the maintenance of KSHV latency (46). Because we observed a decrease of BCBL-1 oncogenesis in vivo, we analyzed the recovered ascites cells for the expression of the latency protein LANA-1. In Western blot analysis of ascites cells, we observed a reduction in LANA-1 expression (bands at 220, 130, and 110 kDa) in cells isolated from animals treated with neomycin or neamine compared with that of the cells isolated from PBS-treated animals (Fig. 6Aa). We observed about 39% and 52% reduction of LANA-1 expression in the cells from neomycin- and neamine-treated animals, respectively. Actin was used as a loading control. In addition, we performed a Western blot analysis using an antibody against the human B-cell marker CD19. We did not observe significant changes in CD19, indicating that the decrease in LANA-1 is not due to an increase in mouse cells collected with the ascites. To confirm the decrease in LANA-1 expression, ascites cells were analyzed by IFA with anti-LANA-1 antibodies (Fig. 6Ab). We observed a decrease in the expected nuclear punctate LANA-1 staining in the ascites cells from neomycin- and neamine-treated animals. We quantified the level of LANA-1 in the IFA experiment by counting the number of LANA-1 puncta per cell (Fig. 6Ac). Whereas 30 puncta were observed in the ascites cells from PBS-treated animals, only 17 and 7 puncta were observed in the neomycin and neamine-treated animals, respectively (43% and 77% reduction, respectively).
In vitro treatment of BCBL-1 cells with neomycin increased lytic gene expression with an increase in the early lytic ORF 50 mRNA levels after 3 days of neomycin treatment (46). In addition, the early and late lytic proteins, ORF 59 and K8.1A proteins, respectively, were also increased after 3 days of neomycin treatment (46). To determine if the reduction of the observed latent gene expression in NOD/SCID mice was associated with a concomitant in vivo increase in the KSHV lytic cycle, the ascites cells from the different mice were stained with anti-KSHV envelope glycoprotein gB antibodies (Fig. 6Ba). In PBS-treated animals, 3% of the ascites were expressing gB, which is consistent with the estimated 3 to 5% of BCBL-1 cells that undergo spontaneous lytic reactivation. In contrast, about 37% and 22% of the ascites cells were positive for gB staining in neomycin- and neamine-treated mice, respectively (12- and 7-fold increases, respectively) (Fig. 6Bb). Taken together, these results indicated that in vivo treatment of BCBL-1-injected NOD/SCID mice with neomycin and neamine results in a decrease of the latent gene expression, with a concomitant increase in KSHV lytic gene expression.
In vitro neomycin treatment of BCBL-1 cells resulted in reduced viability (46). Our studies have demonstrated an antiapoptotic role for ANG. It is well established that the expression of KSHV latency proteins, such as vFlip and LANA-1, are essential for BCBL-1 cell survival. To further elucidate the consequence of neomycin/neamine treatment (blocking ANG nuclear translocation) and the decrease of viral latency protein expression on ascites cell apoptosis, we examined the activation of caspase-3, a crucial executioner of apoptosis. Like all caspases, caspase-3 activation requires its proteolytic cleavage. The induction of apoptosis in the ascites cells was measured by Western blotting using an antibody specific for the cleaved form of caspase-3 (Fig. 7Aa). Whereas cleaved caspase-3 was absent (mice 1 and 2) or low (mice 3 and 4) in the ascites recovered from PBS-treated animals, we observed the presence of active caspase-3 in all the ascites recovered from neomycin- and neamine-treated mice (mice 5 to 8). We quantified the Western blot and estimated a 3.3- and 2.9-fold increase in caspase-3 activation in neomycin- and neamine-treated mice, respectively (Fig. 7Ab). Actin and a total procaspase-3 Western blot were used as the loading control. This result was confirmed by an IFA experiment, wherein cleaved caspase-3 staining was increased in ascites cells from neomycin- and neamine-treated animals compared with the staining in cells from PBS-treated animals (Fig. 7Ba). The percentage of cells stained with cleaved caspase-3 antibody was quantified, and we observed 34% of the ascites cells stained by cleaved caspase-3 isolated from PBS-treated animals (Fig. 7Bb). However, apoptosis was increased to 93% and 97% of the ascites cells isolated from neomycin- and neamine-treated animals, respectively (Fig. 7Bb). Taken together, these results indicated that the delay of BCBL-1-induced tumorigenesis observed in neomycin- and neamine-treated animals was collectively due to a reduction of KSHV latency, an increase in the lytic cycle, and a concomitant increase in apoptosis of BCBL-1 cells.
We observed in the present study a higher expression of ANG in Kaposi's sarcoma lesions than with healthy skin as well as an increase of ANG expression in lung PEL compared with that in healthy lungs (Fig. 1). We have also previously shown that human B-cell lines isolated from PEL expressed higher levels of ANG than EBV+ lymphoma and lymphoblastoid cells, and we demonstrated in vitro that ANG was a determinant factor in PEL cell proliferation and survival (46, 48). Indeed, blocking ANG nuclear translocation with neomycin treatment significantly decreased the viability of KSHV+ lymphoma cells as well as latently infected endothelial cells but had no effect on EBV+ cells or KSHV− and EBV− cells (46, 48). Our present studies extended these observations and demonstrate reduction in the in vitro growth of BCBL-1 cells in soft agar by blocking ANG nuclear translocation (Fig. 2). Finally, the studies here demonstrate for the first time that blocking ANG nuclear translocation significantly decreased the pathology of BCBL-1-induced tumors in NOD/SCID mice. In neomycin- and neamine-treated animals, tumor establishment was reduced, and the lifespan of the animals was significantly increased (Fig. 8 A and B). Analysis of ascites cells from treated mice demonstrated that neomycin and neamine disrupted KSHV latency, induced the induction of the viral lytic cycle, and increased apoptosis in these cells (Fig. 8C), validating our finding that ANG plays a critical role in the maintenance of KSHV latency (46, 48).
Our previous in vitro studies demonstrated that silencing ANG or inhibition of its nuclear translocation with neomycin inhibited latent ORF 73 gene expression and increased the lytic switch ORF 50 gene both during de novo infection and in latently infected cells (46, 48). Interestingly, ANG treatment activated PLCγ and AKT, whereas neomycin inhibited the activation of both proteins. In addition, the PLCγ inhibitor U73122 induced KSHV reactivation, similar to neomycin, suggesting that KSHV has evolved to exploit ANG for its advantage via the PLCγ pathway for maintaining its latency (46, 48). The therapeutic effect of neomycin and neamine could be due to a direct effect on ANG nuclear translocation and ANG cellular function but also to a cumulative effect on viral gene expression. For better understanding, we have summarized the potential implications of the multiple roles that ANG could play in KSHV biology and KSHV-associated malignancies below.
The observation that neomycin and neamine treatment resulted in an increase in apoptosis of the in vivo-injected KSHV+ BCBL-1 cells (Fig. 7) likely reflects the in vivo inhibition of ANG nuclear translocation by these drugs. ANG has been shown to prevent apoptosis induced by serum withdrawal in human endothelial and mouse carcinoma cells (47, 63). A potential antiapoptotic mechanism of ANG during serum withdrawal was the inhibition of the nuclear translocation of apoptosis-inducing factor (AIF), thereby preventing AIF-induced chromatin condensation and DNA fragmentation (64). Another antiapoptotic mechanism of ANG is the upregulation of antiapoptotic genes and downregulation of proapoptotic genes (63). These effects were dependent on Bcl-2 and NF-κB (63). Interestingly, we have shown that ANG is upregulated during KSHV infection through an NF-κB-dependent pathway (47, 58). At 8 and 24 h postinfection of endothelial cells, ANG-mediated mRNA levels were significantly reduced with the NF-κB inhibitor Bay11-7082. NF-κB is a well-established antiapoptotic protein and is constitutively active in PEL (65). Similar to our results, blocking the NF-κB pathway with Bay11-7082 has been shown to prevent or delay PEL tumor growth in NOD/SCID mice and prolong their disease-free survival (66). The therapeutic potential of blocking the NF-κB pathway has been confirmed by blocking the proteosome with Bortezomib, using the new NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ), or using the biscoclaurine alkaloid cepharanthine (67–71). In all these studies, blocking the NF-κB pathway induced the apoptosis of PEL. We postulate that the observed effect of neomycin and neamine could be due to blocking an antiapoptotic regulatory loop between NF-κB and ANG.
We have also shown that ANG activated the AKT pathway and neomycin treatment decreased AKT activation in BCBL-1 cells (46, 48). Interestingly, the inhibition of AKT with miltefosine and perifosine, two alkylphospholipids, inhibited PEL cell growth, induced apoptosis in vitro, and delayed PEL tumor progression in vivo (72, 73). Altogether, these studies indicated that ANG could also be protecting the PEL cells from apoptosis in part through the regulation of crucial antiapoptotic pathways, such as NF-κB and AKT.
To better understand the role of ANG in KSHV biology, we previously performed a proteomic analysis of ANG-interacting proteins. We observed that 28 cellular proteins, with diverse functions, interacted with both ANG and LANA-1 (74). We further analyzed the interaction between ANG and annexin A2. We observed that silencing annexin A2 by small interfering RNA (siRNA) resulted in significant cell death of KSHV+ BCBL-1 cells but had no effect on KSHV− B cell lines such as Ramos or BJAB. In addition, silencing annexin A2 impaired cell cycle progression specifically in BCBL-1 cells by decreasing some cell cycle-associated proteins (74). These results indicate a role for ANG in cell cycle and apoptosis regulation through its interaction with annexin A2.
Furthermore, we demonstrated that ANG decreased p53-mediated cell death (51). The expression of ANG correlated with p53 levels in several cancer cell lines, and we observed a colocalization between ANG and p53 in human colon carcinoma. The silencing of ANG induced p53 target gene expression and increased p53-mediated cell death, whereas its overexpression had the opposite effect (51). In a recent study, we also confirmed that ANG participated in the antiapoptosis state of PEL cells by the suppression of p53. Suppressing ANG nuclear translocation activated p53 and increased the expression of its target genes, such as the p53, p21, and Bax genes, in KSHV+ BCBL-1 cells but not in KSHV− BJAB cells, leading to selective cell death (48).
In addition to a direct role for ANG in oncogenesis, ANG could regulate cell viability through the regulation of KSHV gene expression. We observed that blocking ANG nuclear translocation induced a decrease in KSHV latent gene expression and an increase in lytic gene expression (Fig. 6). As several latency proteins have antiapoptotic roles, a decrease of these proteins would likely be associated with an increase in apoptosis. For example, it has been shown that LANA-1 interacts with and inhibits p53, whereas vFlip inhibits apoptosis through the activation of the transcription factor NF-κB (12, 15, 75–78). KSHV microRNAs have also been shown to contribute to the inhibition of apoptosis in infected cells. For example, miR-K12-1, K12-3, and K12-4-3p regulate caspase-3 expression (79). More recently, KSHV microRNAs were shown to target numerous proapoptotic factors (80, 81). ANG could be protecting PEL cells from apoptosis through multiple pathways, including upregulation of the latency gene cluster, and the observed apoptosis of KSHV+ cells by blocking ANG's nuclear translocation could be due to the cumulative effects of reduction in latent gene expression and consequent reduction in antiapoptotic functions of viral gene products as well as ANG.
As we have seen in our study, targeting ANG, by the use of blocking antibodies or downregulation of ANG by siRNA or inhibitory drugs, has been proposed as an anticancer therapy in other cancer models.
The role of ANG in tumor formation has been evaluated using RNA interference (RNAi) technology to downregulate ANG expression, targeting ANG independently of its localization. ANG siRNA decreased the cell proliferation and colony formation of human lung adenocarcinoma A549 and PC-3 human prostate cancer in vitro, and it significantly inhibited A549 and PC-3 tumor formation in mouse models (82, 83). In addition, downregulation of ANG has also been shown to prevent AKT-driven prostate intraepithelial neoplasia in murine prostate-restricted AKT transgenic mice (84).
The use of siRNA as a therapeutic is challenging, as all the cancerous cells need to be targeted. Therefore, several pharmacologic approaches have been proposed to block the effect of ANG on oncogenesis. Mutagenesis analyses have shown that reducing the ribonucleotic activity of ANG also reduced its angiogenic properties (85–90). N65828, an inhibitor of ANG ribonucleotic activity, inhibited PC-3 prostate tumor cell oncogenesis as well as a model of AKT-induced prostate intraepithelial neoplasia in vivo (84, 91). Neomycin has been previously shown to inhibit ANG nuclear translocation and consequently to reduce ANG-induced cell proliferation and angiogenesis (44). In vivo, neomycin inhibited lung adenocarcinoma development, human prostate cancer PC-3 cell tumor growth in athymic mice, and the development of AKT-driven prostate intraepithelial neoplasia in murine prostate-restricted AKT transgenic mice (82–84). The use of neomycin as a chemotherapeutic agent was unfortunately accompanied with nephrotoxicity and ototoxicity. Interestingly, neamine, another member of the aminoglycoside antibiotic family and a derivative of neomycin, has been shown to present reduced toxicity compared to that of neomycin but retain the effects on ANG nuclear translocation and ANG-induced angiogenesis and cell proliferation (38, 41–43). For example, neamine inhibited the proliferation, migration, and invasion of the H7402 human hepatoma cell line in vitro (92). In vivo, neomycin and neamine decreased both the tumor weight and the formation of neovessels after injection of athymic mice with HT-29 human colon carcinoma and MDA-MB-435 breast cancer cells or A431 human epidermoid carcinoma cells (43).
The role of ANG in tumor formation has also been evaluated using neutralizing antibodies, specifically targeting the functions dependent on the secreted form of ANG (33, 50, 93). In vitro, an anti-ANG polyclonal antibody inhibited ANG-induced endothelial cell invasiveness (33). The mouse monoclonal anti-ANG antibody MAb 26-2F inhibited the ribonucleotic, angiogenic, and mitogenic activities of ANG and decreased in a dose-dependent manner the establishment of human colon adenocarcinoma after injection of HT-29 cells in athymic mice (49, 50). As the use of murine antibodies in human patients is problematic, a chimeric mouse/human antibody based on the structure of MAb 26-2F has been developed, and it inhibited the formation of human breast cancer xenografts after injection of MDA-MB-435 and MCF-7 cells in athymic mice (93). The use of anti-ANG antibodies as a PEL therapeutic agent is beyond the scope of the present study and will be evaluated in the future.
Our earlier in vitro studies demonstrated that blocking nuclear transport of angiogenin disrupted KSHV latency, resulting in apoptosis and cell death in KSHV+ PEL and endothelial cells. Our present in vivo studies extended our in vitro observations and demonstrate that neomycin and neamine inhibit the oncogenesis of PEL cells. Currently available clinically validated treatments for PEL include cytotoxic chemotherapy agents and mTOR inhibitors (94). Since no targeted agents have been added to the clinical practice even after 20 years of KSHV discovery, ANG's specific associations with KSHV biology and latency, but not with EBV, coupled with the relatively low adverse side effects of neamine, suggest that it could be considered an attractive therapeutic candidate for PEL treatment.
This study was supported in part by Public Health Service grants AI 097540 to V.B., AI 091767 and CA 075911 to B.C., and RFUMS–H.M. Bligh Cancer Research Fund to B.C.
We thank Robert Marr and Keith Philibert for critically reading the manuscript.
Published ahead of print 28 August 2013