[Cancer Research 60, 4320-4323, August 15, 2000]
© 2000 American Association for Cancer Research
Inactivation of Retinoblastoma Protein in Uveal Melanoma by Phosphorylation of Sites in the COOH-Terminal Region1
Milam A. Brantley, Jr. and
J. William Harbour2
Center for Ocular Oncology, Department of Ophthalmology and Visual Sciences [M. A. B., J. W. H.], and Division of Molecular Oncology [J. W. H.], Washington University, St. Louis, Missouri 63110
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ABSTRACT
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Uveal melanoma is the most common malignancy of the eye, but little is
known about its underlying genetic defects. Melanomas of uveal origin,
unlike those of the skin, are rarely familial and have not been linked
consistently to mutations in tumor suppressor genes. Here, we
investigated the Rb pathway in uveal melanoma. Most tumors displayed
strong immunostaining for Rb and p16, suggesting that they were not
mutationally inactivated. However, Rb was frequently phosphorylated at
serine-807 and serine-811, and cyclin D1 was expressed in many of the
tumors. Mutation of these serine residues prevented cyclin D-dependent
phosphorylation from inactivating Rb in cultured cells. We conclude
that Rb is frequently inactivated in uveal melanoma by phosphorylation
of residues in the COOH-terminal region that regulate its activity, and
one mechanism for this phosphorylation is overexpression of cyclin D.
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Introduction
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Uveal melanoma is the most common cancer of the eye and leads to
metastatic death in up to 53% of patients (1)
. Unlike
cutaneous melanoma, little is known about the underlying genetic
changes in uveal melanoma.
Rb3
is the prototype tumor suppressor protein and is a major target for
mutations in cancer (2)
. Rb is inactivated in most tumors,
either by mutation of the Rb gene or by
hyperphosphorylation of the protein as a result of mutations elsewhere
in the Rb pathway (e.g., loss of p16, overexpression of
cyclin D or cdk4; Ref. 2
). Mutations in the Rb pathway,
particularly those that affect p16 and cdk4, play an important role in
cutaneous melanoma (3)
, but there is little evidence that
these mutations are prevalent in uveal melanoma. Germ-line mutations in
p16 are extremely rare in uveal melanoma patients, even among those
with a family history of melanoma (4, 5, 6)
. Loss of
heterozygosity at the chromosome 9p21 locus has been reported in up to
32% of uveal melanomas, but mutation of the p16 gene is
rarely observed (7
, 8)
. Likewise, germ-line or tumor
mutations in cdk4 are rare in uveal melanoma (5
, 9)
. The
status of Rb itself has not been investigated adequately in this tumor.
In the present study, we have used immunohistochemical analysis in 32
tumor specimens and transcriptional assays in cultured cells to examine
the Rb pathway in uveal melanoma. By immunostaining, both Rb and p16
were expressed in the vast majority of tumors. Cyclin D was also
expressed in most melanoma cells, and immunostaining with a phospho-Rb
antibody revealed that two specific serine residues in the
COOH-terminal region of Rb were frequently phosphorylated in these
tumors. In transcriptional repression assays, these serine residues
were required for cyclin D-mediated inactivation of Rb. Thus, our
results suggest that the tumor suppressor activity of Rb is frequently
inhibited in uveal melanoma by phosphorylation of specific residues in
the COOH-terminal region of Rb, and that one mechanism for this
phosphorylation is overexpression of cyclin D.
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Materials and Methods
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Immunohistochemistry.
Immunohistochemistry was performed using the streptavidin-biotin method
with the Vector ABC Elite kit (Vector Laboratories, Inc., Burlingame,
CA). Specimens consisted of paraffin-embedded sections of 32 enucleated
globes containing melanomas involving the choroid and ciliary body.
Four-µm sections were obtained, deparaffinized, rehydrated with
ethanol, and treated with 0.3% hydrogen peroxide and methanol to
inhibit endogenous peroxidase activity. Heat-induced antigen retrieval
was performed using microwave treatment in citrate buffer (Rb and p16
antibodies) or EDTA (cyclin D1 antibody) for 15 min. Primary antibodies
were applied at 4°C overnight. Antibodies for Rb (C-15, 1:50
dilution; and IF-8, 1:40 dilution) and p16 (F-12; 1:75 dilution) were
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The
antibody for phospho-Rb-serine 807/811 (1:25 dilution) was obtained
from New England Biolabs, Inc. (Beverly, MA). The antibody for cyclin
D1 (NCL-CYCLIN D1-GM, 1:40 dilution) was obtained from Novocastra
Laboratories Ltd. (Newcastle Upon Tyne, United Kingdom).
Positive controls included normal choroidal melanocytes (Rb and p16
antibodies), a mantle cell lymphoma (cyclin D1 antibody), and p16-null
U20S osteosarcoma cells that constitutively hyperphosphorylate Rb
(phospho-Rb antibody). Negative controls included Rb-null C33A cervical
carcinoma cells (Rb antibody), U2OS cells (p16 antibody), and normal
choroidal melanocytes (phospho-Rb and cyclin D1 antibodies). The
secondary antibody alone was used as an additional negative control for
all antibodies. The percentage of positive cells was estimated by
counting at least 200 cells in at least eight x40 fields for each
specimen. In most cases, at least two sections from each tumor were
analyzed for each antibody.
Transcription Assays and Plasmid Constructs.
For CAT assays, 0.2 µg of the reporter plasmid pSVEC-G (Gal4 sites
upstream of the SV40 enhancer and the E1b TATA box driving the
CAT gene) along with 0.5 µg of the indicated expression
vectors was transfected into Rb-null C33A cells in a total of 10 µg
of DNA by the calcium phosphate method as described previously
(10)
. A phosphorimager was used to quantify CAT activity.
Expression plasmids included the following Gal4-tagged Rb proteins: G-A
(domain A, amino acids 379–602), G-B (domain B, amino acids 620–792),
and RbC (the COOH-terminal region, amino acids 767–928), as described
previously (10)
. RbC
2 was created by subcloning an
SspI/EcoRI (amino acids 767–928) fragment from
PSM.2S (which contains serine-to-alanine substitutions at serine-807
and serine-811; Ref. 11
) into the Gal4 DNA binding domain
expression vector pM2. Rb was functionally reconstituted in these
assays by coexpressing domains A and B and the COOH-terminal region on
separate proteins, as described previously (10)
. Cyclin D
was expressed as an RC.CMV vector.
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Results
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Immunohistochemistry.
Using two separate antibodies that detect hypo- and hyperphosphorylated
forms of Rb, 94% of tumors had strong nuclear staining (
20%
positive cells) with a mean of 64% positive cells/tumor (Table 1
; Fig. 1 A
). Most normal choroidal melanocytes had positive nuclear
staining. Immunostaining for p16 revealed strong nuclear expression
(20% positive cells) in all cases, with a mean of 76% positive
cells/tumor (Fig. 1B
). Most normal choroidal melanocytes
also had positive nuclear staining. For cyclin D1, all tumors contained
cells with positive nuclear staining (mean, 18% positive cells; range,
1–60%; Fig. 1C
). Strong staining (20% positive cells)
was observed in 41% of tumors; 59% contained 5% positive cells.
Normal choroidal melanocytes were negative, suggesting that detection
of cyclin D1 in melanoma cells reflected abnormally high expression.
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Fig. 1. A, immunohistochemical staining of Rb in a
representative uveal melanoma. Most melanoma cells had nuclear staining
for Rb. x40. B, immunohistochemical staining of p16 in
a representative uveal melanoma. Most melanoma cells had nuclear
staining for p16. x40. C, immunohistochemical staining
of cyclin D1 in a representative uveal melanoma. x40.
D, immunohistochemical staining for Rb phosphorylated on
serine-807/811 in a representative uveal melanoma. The fraction of
positive cells was similar to the fraction of cycling cells reported
previously for uveal melanomas (12)
. Positively staining
mitotic figures were observed frequently (arrow).
x40.
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Using an antibody that specifically detects Rb that is phosphorylated
at serine-807 and serine-811 ("phospho-Rb"), normal choroidal
melanocytes were negative, but all tumors contained malignant cells
with intense nuclear staining (Fig. 1D
). The percentage of
positive cells ranged from 0.1 to 5% of cells/tumor, consistent with
the fraction of cycling cells in uveal melanomas stained for Ki-67
(12)
. Virtually all mitotic figures were positive and
represented 
4% of all positive cells (Fig. 1D
), further
supporting the idea that phospho-Rb is expressed in cycling cells.
There was a trend for increased cyclin D1 and phospho-Rb staining among
melanomas of lower (spindle and mixed) histological grades (Table 1)
.
Thus, although Rb does not appear to be mutated in most uveal
melanomas, it is frequently phosphorylated on serine-807 and
serine-811, and this phosphorylation may functionally inactivate Rb.
Overexpression of cyclin D may be a common mechanism for maintaining Rb
in a phosphorylated state in these tumors.
Transcription Assays.
To determine the functional consequence of phosphorylating Rb on
serine-807/811, we transfected Rb into an Rb-null cell line and
measured active transcriptional repression, an activity that is
required for Rb to arrest cells in G1 phase
(13)
. Transfection of Rb repressed the activity of a CAT
reporter by 85% (Fig. 2
, Lane 2). Coexpression of cyclin D efficiently blocked this
Rb repressor activity, presumably by activating endogenous kinases to
phosphorylate Rb (Fig. 2
, Lane 3). However, when serine-807
and serine-811 in Rb were converted to alanine, cyclin D was unable to
block Rb repressor activity (Fig. 2
, Lane 4).
Phosphorylation of Rb was reduced in vitro when these serine
residues were mutated (data not shown). Taken together, these results
suggest that serine-807 and serine-811 are genuine targets for cyclin
D-dependent phosphorylation, and that phosphorylation of these sites
can inhibit Rb repressor activity.
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Fig. 2. The phosphoacceptor sites serine-807 and serine-811 in the
COOH-terminal region of Rb are required for cyclin D-mediated
inhibition of Rb transcriptional repressor activity. To assay for
active repression, Rb was fused to the DNA binding domain of Gal4 and
coexpressed in Rb-null C33a cells, along with the pSVEC-G reporter
containing Gal4 binding sites upstream of the SV40 enhancer. CAT
activity from the reporter was measured with a phosphorimager.
Rb, constructs containing wild-type Rb sequence (amino
acids 379–928); Rb2, serine-807 and
serine-811 have been converted to alanine to prevent phosphorylation of
these sites. Cyclin D was coexpressed where indicated. Transfection of
0.5 µg of each expression vector (or vector control) and 0.2 µg of
reporter was performed using the calcium phosphate method. Note that
cyclin D blocks most of the transcriptional repression by Rb, but this
effect is lost with mutation of serine-807/811.
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Discussion
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Rb inhibits proliferation by arresting cells in the
G1 phase of the cell cycle (2)
. For
cell division to occur, Rb is hyperphosphorylated and inactivated by
cdks that interact with their cyclin partners to form active kinase
complexes (2)
. cdks are in turn restrained by inhibitors
such as p16, which blocks cdk4/6 and allows hypophosphorylated Rb to
accumulate (14)
. The result of these interactions is a
tightly regulated pathway that allows cell division only under
appropriate physiological circumstances. In most cancers, this "Rb
pathway" is disrupted such that Rb is inactivated, either by mutation
of the Rb gene or by functional inactivation of Rb by
hyperphosphorylation of the protein (2)
. In this study, we
wished to determine the status of the Rb pathway in uveal melanoma.
We found that Rb is expressed strongly in most of the uveal melanomas,
suggesting that the Rb gene is not commonly mutated in this
cancer. However, we also found that serine-807 and serine-811 of Rb are
often phosphorylated in these tumors, and this phosphorylation may
block the tumor suppressor activity of Rb. Mutation of serine-807/811
prevented inhibition of Rb repressor activity by cyclin D-dependent
phosphorylation. Furthermore, these sites have been shown to regulate
Rb binding to the proto-oncogene c-abl (11)
,
and this binding is important for tumor suppression by Rb
(15)
. We showed previously that phosphorylation of two
other sites in the COOH-terminal region (threonine-821/826) blocks
active repression by Rb through induction of an intramolecular
interaction that displaces histone deacetylases from the pocket
(10)
. It is interesting that serine-807/811 can
independently regulate active repression by Rb, possibly by inducing a
similar intramolecular interaction. Taken together, our findings
support the idea that Rb is functionally inactivated in uveal melanomas
by phosphorylation of these (and potentially other) cdk phosphoacceptor
sites.
One mechanism for inappropriately phosphorylating Rb is by mutation of
p16 (2)
. However, we found no evidence for p16
inactivation in uveal melanoma. In one recent report, loss of
heterozygosity at the p16 locus was observed in 24% of
uveal melanomas, half of which had a homozygous deletion that included
this locus (8)
. However, no mutations within the
p16 gene were found, and no other evidence was presented
that p16 was specifically targeted by these genetic rearrangements.
Thus, most available evidence suggests that p16 is not a frequent
target of inactivating mutations in uveal melanoma.
Another mechanism for hyperphosphorylating Rb is by overexpression of
cyclin D (2)
. We found positive immunostaining for cyclin
D in most tumors, whereas normal choroidal melanocytes were negative.
Overexpression of cyclin D has been observed in a number of cancers as
a result of amplification, translocation, or other rearrangement of the
gene, and these mutations presumably contribute to tumorigenesis by
activating endogenous cdk4/6 to phosphorylate Rb (2)
. In
support of this possibility, we show that overexpression of cyclin D in
cultured cells blocks active transcriptional repression by Rb, which is
required for Rb to arrest cells in G1 (10
, 13)
. Others have further shown that overexpression of cyclin D
can overcome Rb-mediated tumor suppression in vivo
(16)
. Therefore, the tumor suppressor function of Rb
appears to be inhibited in uveal melanomas by phosphorylation of
specific cdk phosphoacceptor sites, and this phosphorylation may be
attributable to, at least in some cases, overexpression of cyclin D.
Because some of the tumors were only weakly positive for cyclin D1,
other proteins in the Rb pathway (e.g., cyclin D2, cyclin
D3, cyclin E, or cdk4/6) may also be deregulated in some tumors.
Overexpression of cyclin D may also serve to deregulate the Rb pathway
by another recently described mechanism (17)
. Cyclin D,
when complexed with cdk4/6, can sequester p21 and p27 so that they are
unavailable to inhibit cdk2 (Ref. 17
; Fig. 3
). Cyclin D also competes directly with p16 for binding to cdk4/6
(18
, 19) , as demonstrated in cultured uveal melanoma cells
where p16 protein levels were normal, but p16-cdk4 complexes were not
found as in normal choroidal melanocytes (20)
. Thus,
cyclin D can activate cdk4/6 to phosphorylate Rb, and it can interfere
directly and indirectly with several cdk inhibitors, resulting in the
downstream activation of cdk2 and circumvention of the Rb checkpoint
(Fig. 3
). This study provides new insights into abnormalities of the Rb
pathway in uveal melanoma, and it suggests that the molecular
pathophysiology of this form of melanoma may be distinct from its
cutaneous counterpart.
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Fig. 3. Overexpression of cyclin D may disrupt the Rb pathway at
several points: (a) it competes with p16 for interaction
with cdk4/6, which then becomes activated to phosphorylate Rb
(top); (b) cyclin D-cdk4/6 complexes
sequester the cdk inhibitors p21 and p27 so that they are unavailable
to block cdk2 (bottom). Cyclin E-cdk2 can then act
downstream of Rb to initiate cell cycle progression into S phase. See
text for details.
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Acknowledgments
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We thank Belinda McMahan in the Immunomorphology Core Laboratory
for performing immunohistochemistry and Dr. Morton Smith (University of
Wisconsin) for assistance in obtaining tumor specimens.
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FOOTNOTES
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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.
1 These studies were supported by grants (to
J. W. H.) from the NIH and Research to Prevent Blindness, Inc. 
2 To whom requests for reprints should be
addressed, at Ophthalmology and Molecular Oncology, Washington
University School of Medicine, Box 8069, 660 South Euclid Avenue, St.
Louis, MO 63110. Phone: (314) 747-1738; Fax: (314) 747-5073; E-mail: harbour{at}vision.wustl.edu
3 The abbreviations used are: Rb, retinoblastoma
protein; cdk, cyclin-dependent kinase; CAT, chloramphenicol
acetyltransferase.
Received 4/27/00.
Accepted 6/29/00.
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Top
ABSTRACT
Introduction
