Content uploaded by J. Silvio Gutkind
Author content
All content in this area was uploaded by J. Silvio Gutkind
Content may be subject to copyright.
2006;66:168-174. Cancer Res
Silvia Montaner, Akrit Sodhi, Amanda K. Ramsdell, et al.
of Kaposi's Sarcoma
Coupled Receptor as a Therapeutic Target for the Treatment
−Associated Herpesvirus G Protein−The Kaposi's Sarcoma
Updated version
http://cancerres.aacrjournals.org/content/66/1/168
Access the most recent version of this article at:
Cited Articles
http://cancerres.aacrjournals.org/content/66/1/168.full.html#ref-list-1
This article cites by 18 articles, 11 of which you can access for free at:
Citing articles
http://cancerres.aacrjournals.org/content/66/1/168.full.html#related-urls
This article has been cited by 9 HighWire-hosted articles. Access the articles at:
E-mail alerts
related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
.pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
.permissions@aacr.orgDepartment at
To request permission to re-use all or part of this article, contact the AACR Publications
Research.
on June 9, 2013. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
The Kaposi’s Sarcoma–Associated Herpesvirus G Protein–Coupled
Receptor as a Therapeutic Target for the Treatment of
Kaposi’s Sarcoma
Silvia Montaner,
1,2
Akrit Sodhi,
3,4
Amanda K. Ramsdell,
3
Daniel Martin,
3
Jiadi Hu,
1
Earl T. Sawai,
4
and J. Silvio Gutkind
3
1
Department of Diagnostic Sciences and Pathology;
2
Greenebaum Cancer Center, University of Maryland, Baltimore, Maryland;
3
Oral and
Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland; and
4
Department of Comp arative Patholog y, University of California at Davis, Davis, California
Abstract
The Kaposi’s sarcoma–associated herpesvirus (KSHV) encodes
a G protein–coupled receptor (vGPCR) that has been implicat-
ed in the initiation of Kaposi’s sarcoma, identifying vGPCR as
an attractive target for preventing Kaposi’s sarcoma. However,
as only a fraction of cells in advanced Kaposi’s sarcoma lesions
express vGPCR, it is unclear whether this unique viral oncogene
contributes to Kaposi’s sarcoma progression. We therefore set
out to determine whether the few cells that express vGPCR in
established tumors represent an appropr iat e therapeutic
target for the treatment of patients with preexisting Kaposi’s
sarcoma. To this end, we generated endothelial cell lines stably
expressing vGPCR or key KSHV latently expressed proteins
(vCyclin, vFlip, and LANA1). The endothelial cell line expressing
vGPCR was rendered sensitive to treatment with the nucleoside
analogue ganciclovir by using a bicistronic construct coex-
pressing the herpes simplex virus 1 thymidine kinase. S.c.
injection into nude mice with mixed-cell populations formed
tumors that approximate the ratio of vGPCR-expressing and
KSHV lat ent gene-expressing cells. These mice were then
treated with ganciclovir to specifically target only the vGPCR-
expressing cells. Surprisingly, despite the expression of KSHV
latent genes in the vast majority of tumor cells, specifically
targeting only the few vGPCR-expressing cells in established
tumors resulted in tumor regression. Moreover, we observed an
increase in apoptosis of latent gene-expressing cells after the
pharmacologic deletion of the vGPCR-expressing cells. These
findings indicate that vGPCR may play a key role in Kaposi’s
sarcoma progression and provide experimental justification
for developing molecular-based therapies specifically targeting
vGPCR and its effectors for the treatment of Kaposi’s sarcoma
patients. (Cancer Res 2006; 66(1): 168-74)
Introduction
The Kaposi’s sarcoma–associated herpesvirus (KSHV or human
herpesvirus-8), the etiologic agent for Kaposi’s sarcoma (1), encodes
an arsenal of putative oncogenes that harbor transforming potential
in vitro (2). Several of these candidate oncogenes are latent genes,
expressed in the majority of Kaposi’s sarcoma tumor or spindle cells,
and are therefore thought to play an important role in Kaposi’s
sarcomagenesis. However, accumulating evidence suggests that the
expression of latent proteins may not be sufficient to initiate Kaposi’s
sarcoma. Conversely, expression of the KSHV-encoded viral G
protein–coupled receptor (vGPCR) as a transgene or by endothelial-
specific retroviral infection is sufficient to induce Kaposi’s sarcoma–
like tumors in mice (3–5), implicating this viral oncogene in the
initiation of Kaposi’s sarcoma (6, 7). This suggests that therapies
targeting this receptor or its downstream effectors (8) may be an
effective approach to prevent the formation of new Kaposi’s sarcoma
lesions in KSHV-infected patients. However, immunohistochemical
examination of biopsies from patients with established Kaposi’s
sarcoma lesions has revealed that expression of vGPCR is detected in
only a fraction of tumor cells (9), raising whether vGPCR would be an
appropriate therapeutic target in patients with preexisting Kaposi’s
sarcoma. Indeed, as the majority of Kaposi’s sarcoma tumor cells
primarily express latent genes, these viral gene products would be
expected to represent more suitable targets for the treatment of
Kaposi’s sarcoma (10). Unfortunately, due the lack of suitable animal
models to study Kaposi’s sarcoma promotion in vivo, it has been
difficult to assess the relative contribution of, and complex interplay
among, these genes to Kaposi’s sarcomagenesis.
We show here that coinjection of endothelial cells expressing
latent genes with a few endothelial cells stably expressing vGPCR—
at a ratio that approximates the proportion of vGPCR-expressing
and latent gene-expressing tumor cells found in human Kaposi’s
sarcoma—synergistically enhances latent gene-driven tumorigenic-
ity. Indeed, immunohistochemical analysis of tumors formed using
this system revealed that the majority of cells expressed the latent
genes, whereas vGPCR is expressed in only rare tumor cells,
analogous to human Kaposi’s sarcoma. Surpris ingly, however,
pharmacologic deletion of these rare vGPCR-expressing cells is
sufficient to cause tumor regression. We observed an increase in
apoptosis of tumor cells as a consequence of the pharmacologic
deletion of the vGPCR-expressing cells. Furthermore, although
expression of KSHV latent genes was still detected in few surviving
cells, these residual cells lost their tumorigenic potential in the
absence of the paracrine secretions from vGPCR -expressing cells.
Thus, using a Kaposi’s sarcoma model system that can recapitulate
the complex interplay between lytic and latent infected cells, we
provide evidence for the feasibility of specifically targeting vGPCR-
expressing cells as a therapeutic approach for the treatment of
patients with Kaposi’s sarcoma.
Materials and Methods
Expression plasmids and reagents. The expression plasmids for
vGPCR, vGPCR (R143A), vCyclin, vFlip, vCyclin/vFlip, Kaposin, vBcl2, vIRF1,
vIL6, and enhanced green flu orescent protein have been described
Requests for reprints: J. Silvio Gutkind, Oral and Pharyngeal Cancer Branch,
National Institute of Dental and Craniofacial Research, NIH, 30 Convent Drive,
Building 30, Room 211, Bethesda, MD 20892-4330. Phone: 301-496-6259; Fax: 301-402-
0823; E-mail: sg39v@nih.gov or Silvia Montaner; E-mail: SMontaner@umaryland.edu.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-1026
Cancer Res 2006; 66: (1). January 1, 2006
168
www.aacrjournals.org
Research Article
Research.
on June 9, 2013. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
previously (3, 11). The expression plasmid for LANA1 was generously
provided by Dr. C. Boshoff (University College of London, London, United
Kingdom) and subsequently subcloned into the pCEFL euk ar yotic
expression vector. The expression plasmid for K1 was generously provided
by Dr. J. Jung (Harvard University, Cambridge, MA) and subsequently
subcloned into the pCEFL eukaryotic expression vector. The bicistronic
construct vGPCR-herpes simplex virus 1 thymidine kinase (HSV1-TK) was
obtained by first inserting vGPCR and then HSV1-TK into pCEFL internal
ribosome entry site (12). Ganciclovir was purchased from EMD Biosciences
(San Diego, CA). For in vitro studies, this compound was reconstituted in
PBS as a 50 mmol/L stock solution, which was further diluted to the
working concentration (0-1,000 nmol/L) in culture medium.
Cell lines and transfections. SV40 large T-antigen immortalized murine
endothelial cells (SVEC) were grown in DMEM supplemented with 10% fetal
bovine serum and 1% penicillin/streptomycin. Transfection was done using
Fugene reagent (Roche Applied Science, Indianapolis, IN) according to the
manufacturer’s protocol. Stable SVEC cell lines were obtained by stable
transfection of the corresponding pCEFL-derived plasmids as described
previously (3).
ELISA. Conditioned medium from EC-vGPCR, EC-R143A, or SVEC
parental cell lines was prepared as described previously (13). Assays for
cytokine secretion were done by Pierce Biotechnology (Rockford, IL).
Assessment of [
3
H]thymidine incorporation. Assessment of cell
proliferation by uptake of [
3
H]thymidine (ICN Pharmaceuticals, Inc., Costa
Mesa, CA) was essentially as described previously (13).
Establishment and treatment of tumor allografts in athymic nu/nu
mice. All animal studies were carried out using the appropriate NIH
animal care and user protocol. SVECs (10
6
cells) stably expressing vGPCR
were used to induce allografts in 6-week-old athymic (nu/nu) nude
females mice as described (3). For drug treatment, tumor-bearing
animals were randomly grouped (control, n = 10; test, n = 10) and
treated with ganciclovir (50 mg/kg/d) or an equal volume of diluent
(PBS). Treatment schedule was a single injection per animal given i.p.
for 4 consecutive days. For analysis, tumor weight was determined
as described previously (3), whereby tumor volume (LW
2
/ 2, where
L and W represent the longest length and shortest width of the tumor,
respectively) was converted to weight. Results of animal experiments
were expressed as mean F SE. At the end of the study period, animals
were euthanized for tissue retrieval, which was fixed (4% paraformalde-
hyde overnight before processing for paraffin embedding) for immuno-
histochemical analysis.
Immunohistochemistry. Tissues were fixed in 4% paraformaldehyde-1
PBS for 36 hours, transferred to 70% ethanol/PBS, and embedded in
paraffin. Immunohistochemical analysis of tissues has been described
previously (3).
Results
KSHV vGPCR potently renders expressing endothelial cells
tumorigenic. To establish a mouse model in which we could
study the contribution of candidate KSHV oncogenes to Kaposi’s
sarcomagenesis, we took advantage of the availability of non-
tumorigenic SVECs. When injected s.c. into nude mice, 10
6
SVECs are unable to form tumors up to 6 months after injection
(data not shown) but are rendered tumorigenic by stable
introduction of an oncogene, enabling rapid in vivo screening
for tumorigenic proteins. We generated SVEC lines stably
expressing key KSHV latent genes vCyclin (EC-vCyclin), vFlip
(EC-vFlip), LANA1 (EC-LANA1), and Kaposin (EC-Kaposin) or
lytic genes K1 (EC-K1), vBcl2 (EC-vBcl2), IRF1 (EC-IRF1), vIL6
(EC-vIL6), or vGPCR (EC-vGPCR). We first confirmed the
expression of these genes by Western blot analysis (data not
shown) before examining their tumorigenic potential in vivo.
Despite that many of these genes may harbor transforming
capability in vitro (2), only endothelial cells expressing the lytic
gene, vGPCR (EC-vGPCR), consistently formed tumors when
injected s.c. into nude mice (Fig. 1A). To assess whether latent
genes could cooperate in endothelial cell transformation, we also
prepared endothelial cell lines stably coexpressing vCyclin and
vFlip (EC-vCyclin/vFlip) or vCyclin, vFlip, and LANA1 (EC-
vCyclin/vFlip/LANA1). As shown in Fig. 1B, EC-vCyclin/vFlip
cells were only weakly tumorigenic. Surprisingly, addition of a
third KSHV latent gene, LANA1 (EC-vCyclin/vFlip/LANA1), failed
to further enhance the tumorigenic potential of expressing
endothelial cells (Fig. 1B), suggesting that cooperation among
latent genes may not be sufficient either to explain the
tumorigenic potential of KSHV infected cells. This is in striking
contrast to the potent tumorigenic potential of endothelial cells
expressing the KSH V lytic gene, vGPCR (Fig. 1A and B).
Collectively, prior studies (3–7) and these results suggest that
vGPCR may be one of the most potent oncogene encoded by
KSHV.
Figure 1. vGPCR is sufficient to render expressing endothelial cells oncogenic.
A, weight of tumors formed from s.c. injection of nude mice with 10
6
SVECs
stably expressing vCyclin (EC-vCyclin), vFlip (EC-vFlip), LANA1 (EC-LANA1),
Kaposin (EC-Kaposin), K1 (EC-K1), vBcl2 (EC-vBcl2), IRF1 (EC-IRF1), vIL6
(EC-vIL6), or vGPCR (EC-vGPCR). SVEC parental cells were used as a control.
B, weight of tumors formed from s.c. injection of nude mice with endothelial cell
lines stably expressing vCyclin and vFlip (EC-vCyclin/vFlip), vCyclin, vFlip, and
LANA1 (EC-vCyclin/vFlip/LANA1), vGPCR (EC-vGPCR), or the inactive vGPCR
R143A mutant (EC-R143A). SVEC parental cells were used as a control.
Tumor weight was estimated as described in Materials and Methods.
KSHV vGPCR as Therapeutic Target for Kaposi’s Sarcoma
www.aacrjournals.org
169
Cancer Res 2006; 66: (1). January 1, 2006
Research.
on June 9, 2013. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
Figure 2. vGPCR promotes the tumorigenic potential of a combination of KSHV latent genes in a mouse allograft model for Kaposi’s sarcoma. A, secretion of
key Kaposi’s sarcoma cytokines, IL-6, murine IL-8/Gro-a homologue (KC), SDF-1, and IL-2 by endothelial cells expressing vGPCR (EC-vGPCR) or its inactive mutant,
R143A (EC-R143A). Columns, mean fold induction with respect to secretion by control (SVEC) cells; bars, SD. B, proliferation of EC-vCyclin/vFlip treated with
conditioned medium of cultured endothelial cells expressing vGPCR (EC-vGPCR) or its inactive mutant, R143A (EC-R143A), determined by the incorporation of
[
3
H]thymidine. Columns, mean fold induction with respect to results obtained using conditioned medium from control (SVEC) cells; bars, SD. C, schematic
representation of the Kaposi’s sarcoma allograft model in which mixed-cell populations of lytic (vGPCR) and latent gene-expressing endothelial are coinjected into nude
mice in a ratio that approximates their expression pattern in human Kaposi’s sarcoma. D, weight of tumors formed from coinjection with mixed-cell populations of these
cell lines. E, immunohistochemical analysis of tumors formed using this model system for Kaposi’s sarcoma showing rare tumor cells expressing vGPCR (left),
with the majority of cells expressing latent genes (right ).
Figure 3. vGPCR-expressing endothelial
cells are rendered sensitive to ganciclovir
treatment by coexpressing HSV1-TK.
A, schematic representation of an
endothelial cell line stably expressing a
bicistronic construct encoding both vGPCR
and HSV1-TK (EC-vGPCR/TK), rendering
this cell line sensitive to treatment with the
nucleoside analogue, ganciclovir (GAN ).
B, increasing doses (0.1-1,000 Amol/L)
of ganciclovir inhibits cell proliferation
of EC-vGPCR/TK but not EC-vGPCR
as determined by incorporation of
[
3
H]thymidine. C, extended treatment of
EC-vGPCR/TK with 10 Amol/L ganciclovir
induced cell death in f100% of treated
cells within 4 days. Cells treated with
vehicle control are indicated. EC-vGPCR
was completely insensitive to this dose of
ganciclovir, reaching confluence within
3 days.
Cancer Research
Cancer Res 2006; 66: (1). January 1, 2006
170
www.aacrjournals.org
Research.
on June 9, 2013. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
A mouse allograft model for Kaposi’s sarcoma. vGPCR is a
constitutively active GPCR closely related to the mammalian
cytokine receptor, CXCR2. Prior work suggests that the potent
oncogenic potential of vGPCR may, in part, be facilitated by the
paracrine secretions of vGPCR-expressing cells (7, 14–16). Indeed,
conditioned medium from EC-vGPCR cells showed elevated levels
of key Kaposi’s sarcoma cytokines, such as interleukin (IL)-6,
murine IL-8 (KC), and stromal cell–derived factor-1 (SDF-1;
Fig. 2A). To determine if these paracrine secretions could
contribute to the proliferation of latently infected endothelial cells,
we treated EC-vCyclin/vFlip cells with supernatants obtained from
cultured EC -vGPCR. Surprisingly, conditioned medium from
vGPCR-expressing cells promoted the proliferation of endothelial
cells expressing KSHV latent genes (Fig. 2B). These results
suggested that the paracrine secretions from vGPCR-expressing
cells may also promote the tumorigenic potential of latently
infected spindle cells.
To study the complex interplay among lytic and latent gene-
expressing endothelial cells in Kaposi’s sarcomagenesis in vivo,
we s.c. injected mixed-cell populations of EC-vGPCR along with
EC-vCyclin/vFlip into nude mice (Fig. 2C) in a ratio that
approximates the proportion of vGPCR-expressing and latent
gene-expressing tumor cells found in human Kaposi’s sarcoma
(3). Coinjection of EC-vCyclin/vFlip (10
6
cells) with a few
endothelial cells stably expressing vGPCR (EC-vGPCR; 10
5
cells)
synergistically enhanced EC-vCyclin/vFlip tumorigenicity (Fig. 2D).
Coexpression of a third latent gene, LANA1, failed to further
promote tumor growth (data not shown). Immunohistochemical
analysis of tumors formed using this allograft model for Kaposi’s
sarcoma revealed that vGPCR -expressing cells promote the
tumoral growth of EC-vCyclin/vFlip, as only few tumor cells
expressed vGPCR , whereas the majority of cells expressed
these latent genes (Fig. 2E), similar to human Kaposi’s sarcoma
lesions (9).
vGPCR-expressing endothelial cells can be rendered sensi-
tive to ganciclovir treatment by coexpressing HSV1-TK. These
results implicate vGPCR in Kaposi’s sarcoma tumor development
through the secretion of key Kaposi’s sarcoma cytokines and
growth factors, raising whether specifically targeting the vGPCR-
expressing cells, the reby quenchi ng the secretion of these
paracrine growth factors, could be an effective approach for
treating established Kaposi’s sarcoma lesions. To address this
question, we set out to establish a vGPCR-expressing cell line
that could be selectively and specifically targeted in vivo. To this
end, we took advantage of the fact that the HSV1-TK renders
expressing cells exquisitely sensitive to treatment with acyclic
guanidine analogues (e.g., ganciclovir; ref. 17). We employed
a bicistronic construct encoding both vGPCR and HSV1-TK
(Fig. 3A) to ensure that all cells expressing vGPCR coexpressed
HSV1-TK and were therefore rendered sensitive to ganciclovir
treatment. We first confirmed coexpression of both genes in
transiently transfected cells (data not shown) before generating
an endothelial cell line stably expressing this bicistronic
construct (EC-vGP CR/TK). We next tested the effects of
increasing doses of ganciclovir on the EC-vGPCR/TK cell line.
Proliferation of EC-vGPCR/TK, as determined by incorporation
of [
3
H]thymidine, was similar to that of EC-vGPCR during the
same time interval (data not shown), suggesting that coex-
pression of the HSV1-TK in the absence of ganciclovir did not
affect the proliferative potential of EC-vGPCR/TK. Conversely,
proliferation of EC-vGPCR/TK in the presence of ganciclovir was
dramatically reduced (Fig. 3B), with an IC
50
of f1 Amol/L
compared with f1,000 Amol/L for EC-vGPCR. Extended
treatment of EC-vGPCR/TK with 10 Amol/L ganciclovir induced
cell death in f100% of treated cells within 4 days (Fig. 3C);
terminal deoxynucleotidyl transferase–mediated dUTP nick end
labeling (TUNEL) assays revealed that cell death was not
through apoptosis (data not shown). Conversely, EC-vGPCR cells
Figure 4. vGPCR/TK-expressing endothelial cells are sensitive to ganciclovir
treatment in vivo . A, established tumors formed from s.c. injection of nude mice
with EC-vGPCR or EC-vGPCR/TK were treated with PBS (cont ) or a tolerable
dose of ganciclovir (50 mg/kg) for 4 consecutive days when they reached f50
mg. Tumor weight was calculated (LW
2
/ 2, where L and W represent the longest
length and shortest width of the tumor, respectively) and recorded biweekly.
B, immunohistochemical analysis of EC-vGPCR or EC-vGPCR/TK tumors
treated with PBS (control) or ganciclovir revealing a complete loss of all
vGPCR-expressing cells in EC-vGPCR/TK tumors 24 hours after completion
of the 4-day treatment cycle.
KSHV vGPCR as Therapeutic Target for Kaposi’s Sarcoma
www.aacrjournals.org
171
Cancer Res 2006; 66: (1). January 1, 2006
Research.
on June 9, 2013. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
were completely insensitive to this dose of ganciclovir and
continued to proliferate until reaching confluence (Fig. 3C).
Collectively, these results suggest that the EC-vGPCR/TK cell line
is specifically and exquisitely sensitive to cell death in the
presence of ganciclovir.
vGPCR/TK-expressing endothelial cells are sensitiv e to
ganciclovir treatment in vivo. We next set out to determine if
EC-vGPCR/TK was sensitive to ganciclovir treatment in vivo.To
this end, we established tumor allografts by injecting EC-vGPCR/
TK s.c. into nude mice. Tumors formed from EC-vGPCR/TK grew
rapidly and were similar in size to those formed from EC-vGP CR
during the same time interval (Fig. 4A), suggesting that coex-
pression of the HSV1-TK in the absence of ganciclovir did not
affect the ability of vGPCR-expressing cells to induce tumors.
However, treatment of animals with established EC-vGPCR/TK
tumors (100 mg) with a tolerable dose of ganciclovir (50 mg/kg/d;
ref. 18) for 4 consecutive days completely abolished tumor growth
and induced tumor regression (Fig. 4A), with only residual scar
tissue remaining up to 2 months after treatment (data not shown).
Conversely, EC-vGPCR formed tumors in the presence or absence
of ganciclovir, confirming the specificity of the targeted cells
in vivo. Immunohistochemical analysis of tumors formed from
EC-vGPCR/TK and treated for 4 consecutive days with ganciclovir
revealed a complete loss of all vGPCR-expressing cells within 1 day
of the completion of the treatment cycle (Fig. 4B). Conversely,
vGPCR-expressing cells were readily detected in tumors formed
from EC-vGPCR and treated with ganciclovir (Fig. 4B). Collectively,
these results show that coexpression of HSV1-TK in vGPCR/TK-
expressing cells renders these cells sensitive to treatment with
ganciclovir in vivo.
Selectively targeting only rare vGPCR-expressing tumor
cells in mixed-cell tumors composed primarily of cells
expressing latent KSHV genes induces tumor regression. We
next set out to determine if targeting the few vGPCR-expressing
cells in a tumor formed from mixed-cell populations of
EC-vGPCR/TK with EC-vCyclin/vFlip could affect the growth of
tumors formed primarily from cells expressing the KSHV latent
genes, vCyclin and vFlip. To this end , we coinjected 10
6
EC-vCyclin/vFlip cells with a smaller number (10
5
) of endothelial
cells stably expressing vGPCR and HSV1-TK (EC-vGPCR/TK;
Fig. 5A). Similar to the EC-vGPCR cell line, EC-vGPCR/TK
synergistically enhanced EC-vCyclin/vFlip tumorigenicity (Fig. 5B).
Figure 5. Selectively targeting only rare vGPCR-expressing tumor cells is sufficient to induce tumor regression. A, schematic representation of experiment in
which tumors formed from the s.c. injection of 10
5
EC-vGPCR/TK cells with 10
6
EC-vCyclin/vFlip cells are treated with ganciclovir to specifically target only the
vGPCR-expressing cells. B, established tumors arising from mixed-cell populations of 10
5
EC-vGPCR/TK cells with 10
6
EC-vCyclin/vFlip cells treated with PBS
(control) or ganciclovir (50 mg/kg) for 4 consecutive days when they reached f50 mg. Tumor weight was calculated (LW
2
/ 2, where L and W represent the longest
length and shortest width of the tumor, respectively) and recorded biweekly. C, size of tumors arising from mixed-cell populations of 10
5
EC-vGPCR or 10
5
EC-vGPCR/TK cells with 10
6
EC-vCyclin/vFlip 1 week after treatment with ganciclovir (1 wk Post-Tx ). Columns, mean fold induction with respect to tumor size before
treatment; bars, SD. D, representative TUNEL analysis showing an increase in apoptotic cells (*) within tumors generated by the indicated mixed-cell populations
1 week after treatment with ganciclovir. Original magnification,
20. E, immunohistochemical staining showing expression of KSHV latent genes within these tumors
1 week after treatment with ganciclovir. Original magnification,
40.
Cancer Research
Cancer Res 2006; 66: (1). January 1, 2006
172
www.aacrjournals.org
Research.
on June 9, 2013. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
Surprisingly, although only few of the tumor cells expressed
vGPCR, treatment of animals with established tumors with
ganciclovir (50 mg/kg) for 4 consecutive days induced tumor
regression (Fig. 5B) and sustained inhibition of tumor growth for
up to 4 weeks following treatment (data not shown). In contrast,
the small tumors formed from EC-vCyclin/vFlip alone were
unaffected by treatment with ganciclovir (data not shown).
Furthermore, the administration of ganciclovir did not prevent
the rapid growth of tumors arising from EC-vCyclin/vFlip
cells mixed with EC-vGPCR, which served as a specificity control
(Fig. 5C). Immunohistochemical analysis of tumors 1 week after
treatment revealed an increase in apoptosis in the remaining
tumor cells in tumors that included EC-vGPCR/TK cells (Fig. 5D).
To further investigate the effect of specifically targeting only the
rare vGPCR-expressing cells on tumor cells expressing KSHV
latent genes, we examined tumors 1 week after treatment of
animals with 4 consecutive days of ganciclovir, after which
EC-vGPCR/TK cells could no longer be detected (Fig. 3 B; data not
shown). Surprisingly, immunohistochemical staining indicated
that although ganciclovir treatment halted tumor growth a
reduced number of cells expressing KSHV latent genes could be
detected (Fig. 5E), suggesting that the remaining cells expressing
KSHV latent genes are not sufficient for tumor growth in the
absence of the paracrine secretions from vGPCR -expressing cells.
These results suggest that established Kaposi’s sarcoma tumors
may be dependent on the presence of the rare vGPCR-expressing
cells for tumor growth.
Discussion
Despite over a century since its initial description, Kaposi’s
sarcoma remains a poorly understood disease. The recent discovery
of KSHV as the viral etiologic agent of Kaposi’s sarcoma has exposed
many potential therapeutic targets. Among all candidate oncogenes
encoded by KSHV, only vGPCR has been thus far shown to induce
Kaposi’s sarcoma–like lesions in several independent transgenic
animal models (3–5). Indeed, emerging evidence suggests that
dysregulated expression of this potent oncogene in nonlytic cells
may represent an early event initiating Kaposi’s sarcomagenesis (6).
Nonetheless, as expression of vGPCR is associated with only a
subpopulation of spindle cells in Kaposi’s sarcoma animal models
and in human Kaposi’s sarcoma, it is possible that the expression of
this receptor may create an environ ment permissive for the
subsequent tumor development driven by other KSHV survival
(latent) genes, after which receptor expression is no longer
necessary. This ‘‘hit-and-run’’ mechanism would imply that although
vGPCR could be an attractive therapeutic target in preventing the
initiation of Kaposi’s sarcoma it might not be an appropriate target
for the treatment of established Kaposi’s sarcoma lesions.
Surprisingly, however, using a Kaposi’s sarcoma allograft
mouse model and a novel approach to specifically eliminate
pharmacologically all vGPCR -expressing c ells, we obtained
evidence here that the paracrine secretions from the few
vGPCR-expressing cells in established Kaposi’s sarcoma lesions
may still be required to promote growth of established Kaposi’s
sarcoma lesions. Of note, the role of vGPCR-expressing cells in
paracrine-driven tumorigenesis is not without precedent. A
similar function has been attributed previously to Reed-Sternberg
cells in Hodgkin’s lymphoma. Thus, tumor cells expressing
vGPCR, although rare, might ser ve as vulnerable targets in
established Kaposi’s sarcoma lesions. Indeed, as cellular GPCRs
are the target of f60% of all current pharmaceutical drugs,
vGPCR and its downstream effectors (11–16, 19, 20) represent
attractive candidates for the development of novel therapies for
Kaposi’s sarcoma.
Of note, despite evidence of an increased rate of apoptosis in
mixed-cell tumors in which vGPCR-expressing cells have been
pharmacologically removed, few tumor cells still persisted after
ganciclovir treatment, which expressed vCyclin and vFlip. These
cells may have been protected from apoptosis by the prosurvival
effect of these KSHV latent genes. Nonetheless, these remaining
cells did not regrow tumors even after prolonged observation
(2 months). Collectively, these results, along with prior work,
suggest a model in which vGPCR plays a key role in the early
events of Kaposi’s sarcomagenesis by triggering endothelial cell
transformation and promoting the subsequent tumoral growth of
cells expressing KSHV latent genes. Ultimately, a collaborative
approach to Kaposi’s sarcoma treatment in which antiviral and
antiangiogenic therapies are combined with gene product-
targeted therapies directed against key KSHV latent and lytic
genes may ultimately prove to be the most effective therapeutic
strategy for the treatment of patients suffering from this still
enigmatic and disabling disease.
Acknowledgments
Received 3/25/2005; revised 9/5/2005; accepted 10/18/2005.
Grant support: In part by the Intramural Research Program of the National
Institutes of Health, NIDCR.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
KSHV vGPCR as Therapeutic Target for Kaposi’s Sarcoma
www.aacrjournals.org
173
Cancer Res 2006; 66: (1). January 1, 2006
References
1. Chang Y, C esarman E, Pessin MS, et al. Identificati on
of herpesvirus-like DNA sequences in AIDS-associated
Kaposi’s sarcoma. Science 1994;266:1865–9.
2. Boshoff C, Chang Y. Kaposi’s sarcoma-associated
herpesvirus: a new DNA tumor virus. Annu Rev Med
2001;52:453–70.
3. Montaner S, Sodhi A, Molinolo A, et al. Endothelial
infection with KSHV genes in vivo reveals that vGPCR
initiates Kaposi’s sarcomagenesis and can promote the
tumorigenic potential of viral latent genes. Cancer Cell
2003;3:23–36.
4. Yang TY, Chen SC, Leach MW, et al. Transgenic
expressi on of the chemokine receptor encoded by
human herpesvirus 8 induces an angioproliferative
disease resembling Kaposi’s sarcoma. J Exp Med 2000;
191:445–54.
5. Guo HG, Sadowska M, Reid W, Tschachler E, Hayward
G, Reitz M. Kaposi’s sarcoma-like tumors in a human
herpesvirus 8 ORF74 transgenic mouse. J Virol 2004;77:
2631–9.
6. Sodhi A, Montaner S, Gutkind JS. Does dysregulated
expression of a deregulated viral GPCR trigger Kaposi’s
sarcomagenesis? FASEB J 2004;18:422–7.
7. Cesarman E, Mesri EA, Gershengorn MC. Viral G
protein-coupled receptor and Kaposi’s sarcoma: a
model of paracrine neoplasia? J Exp Med 2000;191:
417–22.
8. Sodhi A, Montaner S, Gutkind JS. Viral hijacking of G-
protein-coupled-receptor signalling networks. Nat Rev
Mol Cell Biol 2004;5:998–1012.
9. Chiou CJ, Poole LJ, Kim PS, et al. Patterns of gene
expression and a transactivation function exhibited by
the vGCR (ORF74) ch emokine receptor protein of
Kaposi’s sarcoma-associated herpesvirus. J Virol 2002;
76:3421–39.
10. Staudt MR, Dittmer DP. Viral latent proteins as
targets for Kaposi’s sarcoma and Kaposi’s sarcoma-
associated herpesvirus (KSHV/HHV-8) induced lym-
phoma. Curr Drug Targets Infect Disord 2003;3:
129–35.
11. Sodhi A, Montaner S, Patel V, et al. Akt plays a central
role in sarcomagenesis induced by Kaposi’s sarcoma
herpesvirus-encoded G protein-coupled receptor. Proc
Natl Acad Sci U S A 1998;101:4821–6.
12. Montaner S, Sodhi A, Servitja JM, et al. The small
GTPase Rac1 links the Kaposi sarcoma -associated
Research.
on June 9, 2013. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
Cancer Research
Cancer Res 2006; 66: (1). January 1, 2006
174
www.aacrjournals.org
herpesvirus vGPCR to cytokine secretion and paracrine
neoplasia. Blood 2004;104:2903–11.
13. Montaner S, Sodhi A, Pece S, Mesri EA, Gutkind JS.
The Kaposi’s sarcoma-associated herpesvirus G protein-
coupled receptor promotes endothelial cell survival
through the activation of Akt/protein kinase B. Cancer
Res 2001;61:2641–8.
14. Pati S, Cavrois M, Guo HG, et al. Activation of NF-nB
by the human herpesvirus 8 chemokine receptor ORF74:
evidence for a paracrine model of Kaposi’s sarcoma
pathogenesis. J Virol 2001;75:8660–73.
15. Bais C, Santomasso B, Coso O, et al. G-protein-
coupled receptor of Kaposi’s sarcoma-associated her-
pesvirus is a viral oncogene and angiogenesis activator.
Nature 1998;391:86–9.
16. Sodhi A, Montaner S, Patel V, et al. The Kaposi’s
sarcoma-associated herpes virus G protein-coupled
receptor up-regulates vascular endothelial growth
factor expression and secretion through mitogen-
activated protein kinase and p38 pathways acting on
hypoxia-inducible factor 1a. Cancer Res 2000;60:
4873–80.
17. Fillat C, Carrio M, Cascante A, Sangro B. Suicide
gene therapy mediated by the herpes simplex vir us
thymidine kinase gene/ganciclovir system: fifteen years
of application. Curr Gene Ther 2003;3:13–26.
18. Hemminki A, Zinn KR, Liu B, et al. In vivo
molecular chemotherapy and noninvasive imaging with
an infectivity-enhanced adenovirus. J Natl Cancer Inst
2002;94:741–9.
19. Bais C, Van Geelen A, Eroles P, et al. Kaposi’s
sarcoma associated herpesvirus G protein-coupled
receptor immortalizes human endothelial cells by
activation of the VEGF receptor-2/KDR. Cancer Cell
2003;3:131–43.
20. Couty JP, Geras-Raaka E, Weksler BB, Gershengorn
MC. Kaposi’s sarcoma-associated herpesvirus G protein-
coupled receptor signals through multiple pathways in
endothelial cells. J Biol Chem 2001;276:33805–11.
Research.
on June 9, 2013. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from
