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Short Communications |
From the Department of Microbiology and Immunology,*
Kimmel Cancer Institute, and the Department of Pathology, Anatomy, andCell Biology,
Thomas Jefferson University,Philadelphia, Pennsylvania; the Department of MedicalOncology,
Fox Chase Cancer Center,Philadelphia, Pennsylvania; the Arthur and Sonia Labatt Brain TumorResearch Centre,¶
The Hospital for Sick Children,Toronto, Ontario, Canada; and the First Department of InternalMedicine||
and the Departments ofNeurosurgery
and Tumor Genetics andBiology,**
Kumamoto University School ofMedicine, Kumamoto, Japan
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Abstract |
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The extracellular domain of a number of membrane proteins can be
proteolytically cleaved on the extracytoplasmic side.14
The proteolytic cleavage of membrane proteins has recently emerged as a
key mechanism underlying their functional regulation.15
We
have previously demonstrated a proteolysis-based model as one mechanism
involved in the regulation of CD44 function.16-18
Our
studies showed that CD44 is proteolytically cleaved at the ectodomain
through membrane-associated metalloproteases in various cancer cell
lines to produce a membrane-bound cleavage product of 
25
kd.16
This CD44 ectodomain cleavage was found to play a
critical role in CD44-mediated tumor cell migration by regulating the
dynamic interaction between CD44 and the extracellular
matrix.16,19
Transfection studies on NIH3T3 fibroblasts
have shown that overexpression of the oncogenic mutant of Ha-Ras can
result in enhancement of CD44 cleavage accompanied by the promotion of
CD44-mediated cell migration.18
We have also reported that
CD44 cleavage is regulated by multiple signaling pathways, for example,
the activation of protein kinase C, the influx of extracellular
Ca2+, and members of the Rho family of small
GTPases.17
Based on these observations, CD44 cleavage
seems to be a key event for the regulation of CD44 function in human
cancers.
Although the literature on CD44 in human tumors has grown in recent
years, no previous study has evaluated clinical human tumor specimens
for proteolytic cleavage of CD44. We have generated an antibody (Ab)
against the intracytoplasmic domain of CD44 (anti-CD44cyto Ab) that can
detect the 25-kd cleavage product (Figure 1A)
.16
In this study, we
have used this Ab to examine whether CD44 cleavage occurs in several
types of human tumor tissues. We demonstrate here for the first time
that the CD44 cleavage product is frequently detected in brain, breast,
lung, colon, and ovarian tumors and the cleavage event may contribute
to the oncogenesis of human tumors.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Human tumor tissues and corresponding uninvolved normal tissues were obtained under Internal Review Board (IRB)-approved protocols and the diagnoses confirmed by standard histological analysis. All specimens were snap-frozen and stored at -70°C until required. The normal human breast protein was purchased from Clontech (Palo Alto, CA). U251MG cells and brain tumor-derived cell lines were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Normal human astrocytes were purchased from Clonetics (San Diego, CA) and cultured in specialized media (BioWhittaker, Rockland, ME). Carbobenzoxyl-leucinyl-eucinyl-leucinal (MG132) was purchased from Sigma (St. Louis, MO). The hydroxamate-based metalloprotease inhibitor, BB94 (batimastat), was kindly provided by William G. Stetler-Stevenson (National Institutes of Health, Bethesda, MD).
Western Blot Analysis
Tissue specimens and cells were lysed with phosphate-buffered saline (PBS)/TDS buffer (10 mmol/L Na2HPO4, 150 mmol/L NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.2% NaH3, 0.004% NaF, 1 mmol/L NaVO4, 25 mmol/L ß-glycerophosphate, 100 µg/ml phenylmethyl sulfonyl fluoride, and 1 µg/ml each aprotinin and leupeptin) and centrifuged at 15,000 x g to remove insoluble material. Protein concentrations were determined using the BioRad DC protein assay (BioRad, Richmond, CA). Equal amounts of protein were electrophoresed on 4 to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose filters. The filters were blocked in PBS containing 10% skim milk for 1 hour and then incubated with either a 1:300 dilution of anti-CD44ecto monoclonal antibody (mAb) (F10-44-2; Novocastra, Newcastle, UK), a 1:750 dilution of anti-CD44cyto Ab or a 1:2500 dilution of anti-actin mAb (ICN, Costa Mesa, CA) in PBS containing 0.03% Tween 20 for 50 minutes. The filters were washed three times for 5 minutes each with PBS containing 0.3% Tween 20 and then incubated for 40 minutes with horseradish peroxidase-conjugated anti-mouse IgG Ab (Amersham, Arlington Heights, IL) for anti-CD44ecto mAb and anti-actin mAb, and with horseradish peroxidase-conjugated anti-rabbit IgG Ab (Amersham) for anti-CD44cyto Ab. Secondary Abs were detected using an enhanced chemiluminescence system (Amersham).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Initially, the expression of CD44 protein in normal human brain
and astrocytic tumors was analyzed by Western blotting using
anti-CD44ecto Ab or anti-CD44cyto Ab, which are directed against an
epitope in the CD44 ectodomain and CD44 cytoplasmic domain,
respectively (Figure 1A)
. The highly invasive human glioma cell line,
U251MG cells, served as a positive control because these cells have
previously shown evidence of high CD44 cleavage
activity.16,17
The full-length standard CD44 (CD44s)
protein (85 kd) lacking all variant exons was detected with both
anti-CD44ecto Ab and anti-CD44cyto Ab in all samples examined (Figure 1B)
. Consistent with our previous demonstrations,16,17
the
25-kd CD44 cleavage product was detected in U251MG with
anti-CD44cyto Ab, but not with the anti-CD44ecto Ab (Figure 1, A and B)
. Notably, four glioma specimens (specimens 1728 and 1380, both
glioblastomas; specimen 0952, a gliosarcoma; and specimen 1289, an
anaplastic oligodendroglioma in Figure 1B
) exhibited the 25-kd CD44
cleavage product as well as U251MG cells, whereas the CD44 cleavage
product was not observed in normal brain specimens (Figure 1B
, right).
The anti-CD44ecto Ab failed to detect this fragment (Figure 1B
, left),
indicating that it is derived from cleavage of the ectodomain. Western
blots with Abs against actin, Gab1, a 120-kd cytoplasmic protein, or
the epidermal growth factor receptor, a 180-kd transmembrane protein
revealed no proteolytic fragments (data not shown). This indicated that
the fragments observed with the anti-CD44cyto Ab are not the result of
protein degradation.
The CD44 ectodomain cleavage has been previously shown to be
mediated through metalloprotease activity.16,19
To
verify that the metalloprotease-mediated CD44 cleavage occurs in these
human gliomas, we established cell lines derived from the glioma tumor
specimens 1728 and 2005 and analyzed them by Western blotting using
anti-CD44cyto Ab. The 2005 cell line was derived from a glioblastoma
tumor in which the CD44 cleavage product was not detected (Figure 1B)
and this cell line did not show the CD44 cleavage product in culture
(Figure 1C)
. In contrast, the 1728 cell line was derived from a
recurrent glioblastoma tumor containing the CD44 cleavage product
(Figure 1B)
and the cell line also displayed the 25-kd CD44
cleavage product (Figure 1C)
. Normal human astrocytes did not
demonstrate the CD44 cleavage product (Figure 1C)
. Consistent with
earlier observations,16
the production of CD44 cleavage
products in both the 1728 and U251MG cells was markedly reduced in the
presence of BB94 (a synthetic hydroxamate metalloprotease inhibitor)
(Figure 1C)
, indicating that the proteolytic cleavage of CD44 in these
tumor cells was attributable to metalloprotease activity. Taken
together, these results demonstrate that Western blot analysis using
anti-CD44cyto Ab allows us to reliably assess the occurrence of CD44
cleavage in human tumor tissues and this method was chosen for ensuing
analyses.
CD44 cleavage was evaluated in 7 normal brain tissue specimens and 72
astrocytic tumors comprised of 17 low-grade [World Health Organization
(WHO) grades I and II] and 55 high-grade (WHO grades III and IV)
tumors. Representative results of Western blot analysis are shown in
Figure 1, B and D
. Full-length CD44s (85 kd) was expressed in all of
these tissue specimens, and gliomas generally exhibited higher
expression of full-length CD44s than normal brain (Figure 1, B and D)
.
The CD44 cleavage product was detected in 59% (10 of 17) low-grade
tumors, 58% (32 of 55) high-grade tumors, and in none of the seven
normal brain tissues (Table 1)
. These
results indicate that CD44 cleavage is a tumor-specific phenomenon,
although there was no significant association between CD44 cleavage and
pathological grade in glioma. Recurrent high-grade tumors were more
likely to be positive (6 of 7, 86%) for CD44 cleavage compared to
primary high-grade tumor (26 of 48, 54%), but this did not reach
statistical significance in this sample size.
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CD44 cleavage was next evaluated in primary human carcinomas of
the breast, lung, colon, and ovary. We examined 42 breast carcinomas
(all invasive ductal carcinomas) by Western blotting using
anti-CD44cyto Ab. All of these samples, including the normal breast
tissue specimens, showed expression of full-length CD44s protein. The
expression level of CD44 cleavage product was much higher in 28 of 42
tumors than in a normal breast specimen
obtained from a patient with no evidence
of tumor (Figure 2A
and Table 2
). For 15
tumors, normal tissue adjacent to the tumor was available and
representative blots of these paired specimens are shown in Figure 2B
.
There was enhanced expression of the CD44 cleavage product as compared
to the normal tissue (308 and 262 in Figure 2B
). CD44 variant isoform
(CD44v) expression has been described in multiple human tumors
including breast carcinomas.1,7,20-24
The
expression of the higher molecular weight CD44v protein was detected in
11 of 42 (26%) breast carcinomas (Figure 2, A and B
, and Table 2
).
Interestingly, all 11 of the samples expressing CD44v protein also
exhibited the CD44 cleavage product.
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. The enhanced CD44 cleavage was observed in 45% (5 of 11)
non-small cell lung carcinomas (Table 2)
, including three of eight
adenocarcinomas, one of two squamous cell carcinomas, and one of one
adenosquamous cell carcinoma. We found expression of CD44v protein in
27% (3 of 11) non-small cell lung carcinomas (Table 2)
. Two of the
three non-small cell lung carcinomas expressing CD44v protein also
exhibited an abundance of CD44 cleavage product (Table 2)
.
We found a very high incidence of CD44 cleavage in colon carcinomas as
compared to their matched normal tissues. The CD44 cleavage product was
detected in 90% (9 of 10) colon carcinomas, whereas no reactivity to
this product was found in 9 of the 10 paired normal colon specimens
(Figure 3A
and Table 2
). One normal colon
specimen displayed a detectable amount of CD44 cleavage product;
however, the amount was much lower than the matched tumor specimen
(data not shown). Among the panel of 10 colon carcinomas tested, 7
cases were found to express CD44v protein, and all of the 7 tumors also
co-expressed the CD44 cleavage product (Table 2)
.
|
. We found CD44 cleavage product in 3
of 12 (25%) ovarian carcinomas but not in any of three normal ovaries
tested (Figure 3B
and Table 2
). Expression of CD44v was detected in
three tumors and CD44 cleavage product was observed in two of these
three tumors (Table 2)
.
Overall, we found enhanced expression of the CD44 cleavage product in
60% (45 of 75) of these tumors, whereas CD44v expression was observed
in 32% (24 of 75) (Table 2)
. Interestingly, the percentage of CD44
cleavage was significantly higher in tumors expressing CD44v than in
tumors expressing only CD44s protein (92% versus 45%,
P < 0.001) (Table 3)
. We
also obtained data on histological grade of these tumors. There seemed
to be no direct correlation between the presence of the CD44 cleavage
product and the tumor grade (Table 4)
.27,28
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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. Because the alternative splicing
of CD44 occurs in the ectodomain, CD44 variant exon-specific Abs also
suffered from this limitation. We have developed an Ab raised to the
COOH-terminal region of the CD44 cytoplasmic domain and demonstrated
that it could detect CD44 cleavage products in various cancer cell
lines.16
In the present study, we demonstrate for the
first time that the ectodomain cleavage product of CD44 is easily and
clearly detectable by Western blot in multiple human tumor tissues
using anti-CD44cyto Ab.
We have previously shown that CD44 ectodomain cleavage can be induced
in tumor cell lines by several stimuli including
Ca2+ influx, Ras oncogene expression, and TPA
treatment of cells, and that this cleavage can be inhibited by
metalloprotease inhibitors.16-18
Our findings have been
supported by more recent reports demonstrating that membrane type 1
matrix metalloprotease (MT1-MMP) cleaves the CD44 ectodomain and
promotes cancer cell migration.19
Our present study
demonstrates that this metalloprotease-mediated CD44 cleavage is
reproduced in cell lines derived from tumor specimens examined. This
fact, and the fact that degradation of other proteins was not observed
in the primary tumor samples examined indicate that the 25-kd CD44
fragment is indeed generated endogenously in these tumors and is not
the result of protein degradation.
The CD44 cleavage product was never observed in normal brain and ovary tissue specimens. We observed detectable amounts of CD44 cleavage product on long exposures of normal breast, lung, and colon specimens, although the amount was much lower than that observed in tumor specimens. There are several potential explanations for this observation. The importance of continuous cross talk between tumor cells and surrounding nontumor cells during tumor progression has been described.29,30 Regarding the regulation of metalloprotease production in the surrounding cells of tumors, tumor cell-derived factors that increase the expression of several metalloproteases in fibroblasts have been reported.31 Furthermore, a recent report has shown a significantly higher incidence of metalloprotease expression in normal colon tissues of patients with liver metastasis than in those without metastasis.32 Thus, malignant tumor tissues may alter the expression profile of metalloproteases in their adjacent normal tissues and induce CD44 cleavage. Alternatively, because metalloproteases have been implicated in a variety of normal cellular processes,33,34 CD44 cleavage may occur at a low level in these normal tissues as part of a normal physiological process that is amplified in tumors. Finally, we cannot completely exclude the possibility that microscopically undetected tumor cells may infiltrate into the adjacent normal tissues.
In our analysis of brain tumors, we found the abundant expression of CD44s protein, yet no detectable CD44v protein was observed in any of these tumor tissues. These results are consistent with earlier studies showing that CD44s protein is the most dominant form of CD44 expressed in brain tumors.35-38 On the other hand, CD44 cleavage product was clearly detected in 58% (32 of 55) of high-grade gliomas and 59% (10 of 17) of low-grade gliomas, suggesting its potential as a target for new diagnostic and therapeutic approaches. We and others have previously reported that CD44 cleavage promotes tumor cell migration in vitro.16,18,19 However, our present finding that CD44 cleavage was also detected in low-grade (I and II) tumors as well as high-grade (III and IV) gliomas suggest that CD44 cleavage may not necessarily contribute to tumor invasiveness in vivo, although WHO grading of gliomas does not depend on invasiveness. On the other hand, this discrepancy may also be attributable to the complicated mechanisms underlying tumor invasion in vivo, a highly regulated process requiring the well-orchestrated interplay of various components (organ-specific microenvironment, growth factor signaling, adhesion molecule, proteolytic events, and so forth).
In contrast to brain tumors, 26% of breast carcinomas, 27% of lung carcinomas, 70% of colon carcinomas, and 25% of ovarian carcinoma expressed CD44v protein. With the exception of colon carcinomas, the incidence of CD44v protein expression seems to be lower than what was reported in earlier studies, perhaps because of a difference in methodology.20-22,39 These studies mainly used reverse transcriptase-polymerase chain reaction to detect expression of the variant forms or Abs specific for the splice variants. In the present study, we demonstrate that CD44 cleavage is more prevalent than CD44v expression in these tumors. Notably, tumors expressing CD44v protein show a significantly higher incidence of the enhanced CD44 cleavage than those expressing only CD44s protein. Because both CD44s and CD44v would generate the same size CD44 cleavage product16 and all CD44v-expressing tumors co-express the CD44s protein, we cannot conclude the exact origin (CD44s or CD44v) of the CD44 cleavage product observed in these tumors at this point.
The enhanced CD44 proteolytic cleavage was found irrespective of the grade of malignancy in the tumor samples examined, hence CD44 cleavage may define a very early event in tumorigenesis. More recently, we have demonstrated the functional link between CD44 cleavage and consequent signal transduction within cells.40 It is possible that the CD44 cleavage might impact on a signaling pathway to promote tumor development. The intracellular domain of CD44 has also been shown to bind to merlin and this can send a growth inhibitory signal to cells when the extracellular domain of CD44 is bound to its ligand.41 The CD44 cleavage would result in loss of the extracellular domain of CD44 and may abolish this inhibitory signal resulting in unregulated cell proliferation. Considering these potential roles for CD44 cleavage, its presence in multiple human tumor types and its high frequency of occurrence indicate that further work is warranted to establish its biological and clinical significance.
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Acknowledgements |
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Footnotes |
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Supported by grants from the National Institutes of Health (grants CA69495 and CA51093 to A. J. W.); and a grant for Cancer Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (to H. S.).
I. O. and H. T. contributed equally to this work.
Accepted for publication November 9, 2001.
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References |
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