Human Molecular Genetics Advance Access originally published online on August 18, 2008
Human Molecular Genetics 2008 17(22):3596-3600; doi:10.1093/hmg/ddn252
© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Expression of p21waf1/Cip1 in stromal fibroblasts of primary breast tumors
George Trimis1,
Ioulia Chatzistamou2,
Katerina Politi3,
Hippokratis Kiaris1,* and
Athanasios G. Papavassiliou1
1 Department of Biological Chemistry
2 Department of Histology and Embryology
3 Department of Cytopathology, Aretaieion Hospital, University of Athens Medical School, 75 M. Asias Str., 115 27 Athens, Greece
* To whom correspondence should be addressed. Tel: +30 2107462695; Fax: +30 2107462695; Email: hkiaris{at}med.uoa.gr
Received August 11, 2008; Accepted August 15, 2008
 |
ABSTRACT
|
|---|
During carcinogenesis, stromal fibroblasts undergo certain changes
in concert with their neoplastic neighbors, an interaction that
progressively leads to a cancer-associated state. However, despite
the increasing appreciation of the importance of stromal/tumor
interactions in the progression of cancer, little is known about
the factors responsible for regulating the crosstalk between
stromal fibroblasts and neoplastic cells. Here we show that
the stage of the disease in primary human breast lesions affects
p21 expression in the fibroblasts. In stromal fibroblasts of
benign fibroadenomas, p21 exhibits a periductal pattern of staining,
which is abolished in malignant adenocarcinomas in which p21
immunopositivity exhibits a mosaic pattern that eventually is
abolished in more aggressive types of the disease. In order
to address the role of fibroblasts’ p21 in tumorigenesis,
we have reconstituted MCF7 human breast cancers in mice, with
fibroblasts differing in the p21 status. These experiments showed
that p21 deficiency in stromal fibroblasts accelerates tumor
growth through cell non-autonomous mechanism(s). In addition,
even a transient, siRNA-mediated p21 suppression in fibroblasts
sufficiently stimulates MCF7 and MDA-MB-231 growth
in vivo.
We propose that p21 regulation is intimately linked with the
ability of stromal cells to affect tumor growth.
|
INTRODUCTION
|
|---|
Fibroblasts represent the major cellular component of cancer-associated
stroma. Although, however, their role in accelerating cancer
growth and in certain instances even to cause malignant conversion
has been demonstrated, the molecular factors regulating this
process remain largely unknown (
1–
4). Recently, by performing
a series of tumor reconstituition experiments, involving the
co-inoculation of breast cancer cells with fibroblasts differing
in their p53 status, we showed that p53 mutations in stromal
fibroblasts exert a positive effect on cancer growth (
5). This
finding was subsequently confirmed by others in a transgenic
mouse model of prostate cancer involving the assessment of primary
malignant lesions (
6). Noteworthy, p53 mutations represent a
detectable genetic lesion in the stroma of primary breast and
probably other cancers, suggesting that at least in certain
cases the transition of stromal fibroblasts into a cancer-associated
state may be associated with the acquisition of p53 deficiency
(
7–
9).
Although, however, the accumulation of certain genetic lesions by the stromal fibroblasts provides an attractive molecular mechanism that may account for the transition of the fibroblasts into a cancer-associated state, it can be challenged by the fact that its operation should involve the clonal selection of the mutant fibroblasts. The latter, although formally possible, appears unlikely during the early stages of the disease at which a rapid and more global response should be evoked in the stroma. Therefore, it is conceivable that other mechanisms, operating at the level of the (de-)regulation of gene expression, might be involved in this process. In the present study, we tested the hypothesis that differential expression of p21waf1/Cip1 (p21) in stromal fibroblasts is involved in tumorigenesis by non-autonomous mechanisms. Our hypothesis was based on two lines of evidence: (i) In part, the effects of p53 in cell cycle are mediated by p21 and thus, an overlap between the consequences of these two cell cycle regulators is anticipated; and (ii) p21 is only rarely—if ever—mutated in primary human tumors; however, it is subjected to complex, yet unclear, regulation during carcinogenesis (10–12).
|
MATERIALS AND METHODS
|
|---|
Cells, mice and xenograft development
Human mammary epithelial adenocarcinoma cells MCF7 and MDA-MB-231
were originally obtained from American Type Culture Collection
(VA, USA) and maintained in DMEM containing 10% FBS and antibiotics/antimycotics.
SCID and p21-deficient mice (
13) were originally obtained by
Jackson laboratories (ME, USA) and subsequently maintained in
our laboratory. Care of animals was in accord with institutional
guidelines. For xenograft development, 1.5
x 10
6 MCF7 cells
and 0.5
x 10
6 MEFs per mouse were re-suspended in 0.2 ml of
mixture at 1:1 ratio of serum-free DMEM and ice-cold Matrigel
(BD Bioscience) and then injected s.c. into SCID female virgin
mice,
6 weeks old. Subsequently, animals were observed at least
twice per week for tumor development. Then, either the time
period until the onset of palpable tumors was scored or upon
tumor onset, tumor diameter was assessed by using a microcaliper.
Animals were sacrificed when tumor exceeded 10% of body weight
or when mice showed signs of distress.
SiRNA and western blot analysis
Suppression of p21 in the fibroblasts was achieved by transfecting wild-type MEFs with siRNA specific for mouse p21 (clone ID 160142, Ambion) according to the manufacturer's instruction. Cells were lysed 48 h after transfection by using RIPA reagent and total protein was subjected to western blot analysis. Antibodies for p21 and actin were obtained from Sigma.
Primary human breast tumors, histology and immunohistochemistry
For histological analyses, xenografts were fixed in 10% formalin, paraffin embedded and stained with hematoxylin/eosin for light microscopy. Immunohistochemistry for Ki-67 was performed with a rabbit polyclonal antibody obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) by using the Kwik-DAB kit (ThermoShandon, Pittsburgh, PA, USA), according to the manufacturer's instructions. Before observation, a weak counter stain with hematoxylin was performed. Primary human breast tumors, including six specimens from fibroblastic disease, 20 fibroadenomas (FAs), five ductal carcinomas in situ (DCIS) and 32 invasive ductal adenocarcinomas (DCs), were selected from the archives of Aretaieion University Hospital on the basis of being rich in stromal fibroblasts. Then, 0.5 µ sections were subjected to immunohistochemical analysis for p21, p53 and vimentin using antibodies obtained from Sigma followed by the application of the Kwik-DAB kit. Images shown were obtained by Pro-image Analysis Software (Media Cybernetics, Inc., MD, USA).
Statistical analysis
Statistical analysis of the animal experiment results was performed by using Student's t-test, whereas the results of human tumor analysis have been analyzed by the 
2 test.
|
RESULTS
|
|---|
Initially, we have attempted to assess the growth of human cancer
cells in mice suffering from severed combined immunodeficiency
(SCID) that differed in the status of p21. Unfortunately, double
mutant SCID/p21-null mice exhibited increased mortality prior
to the age of 8 weeks old, preventing us from performing this
experiment (data not shown). Therefore, we have concentrated
on evaluating the role of p21 directly in fibroblasts. To address
the role of p21 of stromal fibroblasts in tumor growth, we carried
out tumor reconstitution experiments in which we evaluated the
behavior of cancer cells that prior to their inoculation in
mice were mixed with embryonic fibroblasts that differed in
the status of p21. For tumor inoculation, we have used SCID
mice. We chose this particular strain of immuno-incompetent
mice because these animals can be used for tumor transplantation
studies, have a normal reproductive cycle and can support the
growth of MCF7 cells in addition to other hormone-dependent
cells (
5,
13). Furthermore, we have selected MCF7 cells because
alone they are hardly tumorigenic in SCID mice, whereas their
tumorigenicity is dramatically increased when the MCF7 inoculates
include fibroblasts, reflecting perhaps a sensitivity to stromal
factors (
5 and our unpublished observations). The experimental
strategy we adopted is shown in Figure
1A. Reconstitution
of mammary adenocarcinoma in SCID mice by fibroblasts lacking
p21 and MCF7 human breast cancer cells resulted in tumors that
were growing faster than those derived by inoculating MCF7 cells
and wild-type fibroblasts (Fig.
1B, see legend and methods
for more details).
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 1. Growth of tumors reconstituted by MCF7 cells and fibroblasts differing in the p21 status in SCID mice. MCF7 cells and MEFs isolated from p21-null and wild-type animals at E13.5 were inoculated subcutaneously, adjacent to the mammary fat pads, in SCID female virgin mice 6 weeks old. The experimental strategy of tumor reconstitution is shown in (A). (B) Tumor growth rates of one representative of three independent experiments performed with different preparations of fibroblasts. Each of these experiments involved four to six mice per group and produced similar results. For the experiment shown, n = 5. Vertical lines indicate the standard deviation. *P < 0.01; **P < 0.001.
|
|
Consistently with their increased growth rate, immunohistochemical
analysis of the tumors using Ki67, a marker for proliferating
cells (
14), indicated that MCF7 xenografts reconstituted with
p21-null fibroblasts contained more actively dividing cells
than those reconstituted with wild-type p21 stromal fibroblasts
(Fig.
1B, insets). Similar results were obtained when p21
was knocked-down by siRNA at 15 n
M in wt fibroblasts and tumors
were reconstituted with MDA-MB-231 breast cancer cells in addition
to the MCF7 cells (Fig.
2).
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 2. Transient, siRNA-mediated p21 suppression in stromal fibroblasts effectively stimulates tumor growth. (A) Western blot analysis showing the reduced expression of p21 in fibroblasts following siRNA transfection. Injection of these fibroblasts 48 h post-transfection with MDA-MB-231 (B) or MCF7 (C) human breast cancer cells stimulated tumor growth in vivo.
|
|
In order to confirm the biological relevance of this paracrine
modulation of tumor growth by fibroblasts' p21 in our experimental
system, we assessed the expression of stromal p21 in a set of
63 primary human breast tumors, including six specimens from
fibroblastic disease, 20 FAs, five DCIS and 32 invasive DCs,
which were selected on the basis of being rich in stromal cells.
Our results show that although in the fibroblastic disease specimens
p21 immunopositivity was minimal in the stromal fibroblasts
(one of six), in FAs it increased to 60% (12 of 20,
P < 0.001))
and subsequently decreased progressively in grade II (55%,
P < 0.001) and grade III (17%) adenocarcinomas (
P < 0.005)
(Figs
3 and
4 and Table
1). Besides, however, these
differences in the overall staining intensity and the percentage
of positive cells between the benign and the invasive breast
lesions tested, an apparent difference that became readily detectable
was related to the localization of the p21 immunopositive cells.
In the FAs, immunopositivity was localized predominantly in
the stromal fibroblasts adjacent to the epithelial cells, whereas
in the invasive cancers it was random and independent of the
relative position of the fibroblasts to the epithelial cells
(
P < 0.001). In FAs, only in one out of 12 positive specimens
positivity was diffuse and mosaic when compared with the 10
of 11 grade II and all two grade III adenocarcinomas analyzed
(Table
1). Furthermore, positivity for p21 in stromal fibroblasts
correlated with p21 expression in the epithelium (
P < 0.001)
(Fig.
5).
View larger version (166K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 4. Representative microphotographs showing p21 immunoreactivity in FAs and invasive ductal carcinomas. Immunoreactivity was more intense in the FAs (A and C) when compared with adenocarcinomas (B and D). Black arrowheads indicate positively stained fibroblasts. The yellow dotted line in the FAs indicates the periductal localization of p21 immunoreactivity (10x). (E)Higher magnification (40x) of (A and B) and (B and D) (F). Fibroblasts positive for p21 are indicated by red, whereas those negative for p21 are indicated by green arrows. Blue arrows show lymphocytes that are immunopositive for p21. Inserts in (B) show Ki-67 immunostaining in MCF7 tumors reconstituted with wt or p21-KO fibroblasts.
|
|
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 5. Cumulative results of the staining showing the correlation between p21 and p53 expression in the epithelium (A) and the stroma (B) and the correlation of p21 (C) and p53 (D) expression between stroma and epithelium.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1. p21waf1/Cip1 immunopositivity in stromal fibroblasts in primary human FAs and invasive breast carcinomas
|
|
A subset of the specimens, 16 FAs, 14 grade II tumors and seven
grade III tumors, was stained for p53. Consistent with previous
findings, p53 immunopositiviy was detectable in the stroma of
both benign and malignant breast tumors (Fig.
3). Immunopositivity
was increased in the epithelium in more advanced breast cancers
when compared with the FAs. p53 immunopositivity in the stroma
was not associated with the type of the disease (Fig.
6).
However, p53 and p21 immunopositivity were slightly associated
in the epithelium (
P < 0.01) but not in the stroma of the
specimens (Fig.
5). Finally, for both p21 (
P < 0.001)
and to a lesser extend p53 (
P < 0.05), immunopositivity correlated
in the stromal and the epithelial component of the same specimens
(Fig.
5).
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 6. Cumulative results of p21 (A and B) and p53 (C and D) expression in the stroma (A and C) and the epithelium (B and D) of benign and malignant breast tumors. FA, fibroadenomas; DCIS, ductal carcinoma in situ; DC, invasive ductal adenocarcinoma grade II (II) and grade III (III); L, localized; D, diffuse pattern of staining.
|
|
Selected specimens were also stained for vimentin, which represents
a widely accepted fibroblast marker, in order to identify fibroblasts
in the stromal tissue of the tumors (Fig.
3).
|
DISCUSSION
|
|---|
Certain genetic lesions, exemplified by the inactivation of
p53 tumor suppressor, account for a subset of molecular alterations
seen in stromal fibroblasts during carcinogenesis (
7,
9). These
mutations have been shown capable of affecting both the latency
of tumorigenesis and the efficacy of anticancer therapy (
15).
However, considering that the activation of stromal fibroblasts
occurs early in the development of the disease, the clonal selection
of fibroblasts carrying such genetic lesions poses some considerations
related to the extent of the contribution of such mechanism.
The latter is due to the fact that such mechanism must be operational
early in carcinogenesis, even before malignant conversion and
clonal growth of the cancer cells occur. That p21 operates—though
not exclusively—downstream of p53 in combination with
the observation that it is not targeted by mutations but is
subjected to complex regulation during carcinogenesis, raises
the possibility to play an important role in the control of
the response of stromal fibroblasts in neoplastic growth. In
the present study, we confirmed this hypothesis, providing evidence
that p21 in fibroblasts modulates the profile of tumorigenesis
by a non-autonomous mechanism in a xenograft model of the disease.
The fact that even a transient, siRNA-mediated suppression of
p21 sufficiently stimulates tumor growth suggests that the role
of stromal fibroblasts’ p21 is particularly important
in the very early stages of xenograft growth. Furthermore, we
have provided evidence that the onset of a benign neoplastic
lesion, such as the FA but for reasons unclear to us not from
the fibroblastic disease, triggers the induction of p21 expression
adjacent to the epithelial cells. This expression pattern of
p21 is abolished, however, when lesions become malignant, as
indicated by the progressive elevation of diffuse staining in
grade II adenocarcinomas and its final abolishment in grade
III tumors. Taking together this observation on the p21 expression
in stromal fibroblasts in primary breast lesions, with the negative
role of fibroblasts’ p21 in tumorigenesis, our results
indicate that during very early stages of the disease when the
tumor is still benign, a state exemplified by the FAs, stromal
p21 is induced, rendering fibroblasts protective against neoplastic
growth. This is also consistent with the observation that fibroblasts
distal from the neoplastic lesion in FAs as well as those located
closely to the malignant epithelial cells in invasive carcinomas
both express low levels of p21, despite that they possess distinct—and
in fact opposite—activities with respect to their role
in carcinogenesis. How p21 in stromal fibroblasts affects tumor
growth by paracrine mechanisms remains obscure; however, it
is unlikely to contribute directly to the CAF-like phenotype
as smooth muscle actin expression, a landmark of CAFs, remains
unaffected following p21 knock-down (data not shown).
Collectively, our results attribute a direct role in p21 of stromal fibroblasts in tumorigenesis. Whether these non-autonomous effects of p21 are reversible implying certain therapeutic implications or the tumor enters a state that becomes independent of stromal p21 expression remains to be seen.
|
FUNDING
|
|---|
This work was supported by the grant EPAN (05NONEU-13) from
GSRT, the KESY Oncology program 05 from the Greek Ministry of
Health and Kapodistrias 06 from ELKE, University of Athens.
|
ACKNOWLEDGEMENTS
|
|---|
We are grateful to the personnel of the Laboratory of Experimental
Surgery (Director, Prof. D. Perrea) for excellent animal housing
care.
Conflict of Interest statement. None declared.
|
REFERENCES
|
|---|
-
Elenbaas B., Weinberg R.A. Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation. Exp. Cell Res. (2001) 264:169–184.[CrossRef][Web of Science][Medline]
-
Tlsty T.D., Hein P.W. Know thy neighbor: stromal cells can contribute oncogenic signals. Curr. Opin. Genet. Dev. (2001) 11:54–59.[CrossRef][Web of Science][Medline]
-
Olumi A., Grossfeld G., Hayward S., Carroll P., Tlsty T., Cunha G. Carcinoma-associated fibroblasts direct tumor progression of initiated human prostate epithelium. Cancer Res. (1999) 59:5002–5011.[Abstract/Free Full Text]
-
Kiaris H., Chatzistamou I., Kalofoutis C., Koutselini H., Piperi C., Kalofoutis A. Tumor-stroma interactions in carcinogenesis: Basic aspects and perspectives. Mol. Cell. Biochem. (2004) 261:117–122.[CrossRef][Web of Science][Medline]
-
Kiaris H., Chatzistamou I., Trimis G., Frangou-Plemmenou M., Pafiti A., Kalofoutis A. Evidence for a non-autonomous role of p53 in carcinogenesis. Cancer Res. (2005) 65:1627–1630.[Abstract/Free Full Text]
-
Hill R., Song Y., Cardiff R.D., Van Dyke T. Selective evolution of stromal mesenchyme with p53 loss in response to epithelial tumorigenesis. Cell (2005) 123:1001–1011.[CrossRef][Web of Science][Medline]
-
Kurose K., Gilley K., Matsumoto S., Watson P.H., Zhou X.P., Eng C. Frequent somatic mutations in PTEN and TP53 are mutually exclusive in the stroma of breast carcinomas. Nat. Genet. (2002) 32:355–357.[CrossRef][Web of Science][Medline]
-
Moinfar F., Man Y.G., Arnould L., Bratthauer G.L., Ratschek M., Tavassoli F.A. Concurrent and independent genetic alterations in the stromal and epithelial cells of mammary carcinoma: implications for tumorigenesis. Cancer Res. (2000) 60:2562–2566.[Abstract/Free Full Text]
-
Patocs A., Zhang L., Xu Y., Weber F., Caldes T., Mutter G.L., Platzer P., Eng C. Breast-cancer stromal cells with TP53 mutations and nodal metastases. N. Engl. J. Med. (2007) 357:2543–2551.[Abstract/Free Full Text]
-
el-Deiry W.S., Tokino T., Velculescu V.E., Levy D.B., Parsons R., Trent J.M., Lin D., Mercer W.E., Kinzler K.W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell (1993) 75:817–825.[CrossRef][Web of Science][Medline]
-
Weiss R.H. p21Waf1/Cip1 as a therapeutic target in breast and other cancers. Cancer Cell (2003) 4:425–429.[CrossRef][Web of Science][Medline]
-
Brugarolas J., Chandrasekaran C., Gordon J.I., Beach D., Jacks T., Hannon G.J. Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature (1995) 377:552–527.[CrossRef][Web of Science][Medline]
-
Kuperwasser C., Chavarria T., Wu M., Magrane G., Gray J.W., Carey L., Richardson A., Weinberg R.A. Reconstruction of functionally normal and malignant human breast tissues in mice. Proc. Natl Acad. Sci. USA (2004) 101:4966–4971.[Abstract/Free Full Text]
-
Thor A.D., Liu S., Moore D.H. 2nd, Edgertom S.M. Comparison of mitotic index, in vitro bromodeoxyuridine labeling, and MIB-1 assays to quantitate proliferation in breast cancer. J. Clin. Oncol. (1999) 17:470–477.[Abstract/Free Full Text]
-
Lafkas D., Trimis G., Papavassiliou A.G., Kiaris H. p53 mutations in stromal fibroblasts sensitize tumors against chemotherapy. Int. J. Cancer (2008) 123:967–971.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
TOP
ABSTRACT
INTRODUCTION