The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 2 February 2013
Loss of SPARC in bladder cancer enhances
carcinogenesis and progression
Neveen Said,1,2 Henry F. Frierson,3 Marta Sanchez-Carbayo,4
Rolf A. Brekken,5 and Dan Theodorescu2,6,7
1Department of Radiation Oncology, 2Department of Urology, and 3Department of Pathology, University of Virginia, Charlottesville, Virginia, USA.
4Spanish National Cancer Institute (CNIO), Madrid, Spain. 5Departments of Surgery and Pharmacology, University of Texas Southwestern Medical Center,
Dallas, Texas, USA. 6Departments of Surgery and Pharmacology, University of Colorado, Aurora, Colorado, USA.
7University of Colorado Comprehensive Cancer Center, Aurora, Colorado, USA.
Secreted protein acidic and rich in cysteine (SPARC) has been implicated in multiple aspects of human cancer.
However, its role in bladder carcinogenesis and metastasis are unclear,with some studies suggesting it may
be a promoter and others arguing the opposite. Using a chemical carcinogenesis model in Sparc-deficient
mice and their wild-type littermates, we found that loss of SPARC accelerated the development of urothelial
preneoplasia (atypia and dysplasia), neoplasia, and metastasis and was associated with decreased survival.
SPARC reduced carcinogen-induced inflammation and accumulation of reactive oxygen species as well as uro-
thelial cell proliferation. Loss of SPARC was associated with an inflammatory phenotype of tumor-associated
macrophages and fibroblasts, with concomitant increased activation of urothelial and stromal NF-κB and
AP1 in vivo and in vitro. Syngeneic spontaneous and experimental metastasis models revealed that tumor-
and stroma-derived SPARC reduced tumor growth and metastasis through inhibition of cancer-associated
inflammation and lung colonization. In human bladder tumor tissues, the frequency and intensity of SPARC
expression were inversely correlated with disease-specific survival. These results indicate that SPARC is pro-
duced by benign and malignant compartments of bladder carcinomas where it functions to suppress bladder
carcinogenesis, progression, and metastasis.
Bladder cancer is caused primarily by tobacco use and exposure
to industrial chemicals (1) and will affect an estimated 73,510
patients and lead to 14,880 deaths in 2012 (2). N-nitrosodibu-
tylamine (BBN) was first identified as a bladder carcinogen in
rodents (3) and is detected in tobacco smoke and environmen-
tal and infectious metabolites (3). Carcinogenesis is a multistep
process consisting of initiation, promotion, and progression
and is governed by cumulative genetic and epigenetic alterations
and microenvironmental cues (4), with inflammation playing
an important role as is a tumor promoter via proinflammatory
cytokines/chemokines and ROS (5, 6).
Secreted protein acidic and rich in cysteine (SPARC, osteonec-
tin, BM-40) is regulated in tissues undergoing remodeling, during
normal development, during tissue repair, and in cancer (reviewed
in refs. 7–10). Interest in SPARC has grown in recent years because
of its apparent role in regulating tumor growth via its produc-
tion by both cancer and or stromal cells and based on its interac-
tions with biologically active cytokines/chemokines in the tumor
milieu (summarized in refs. 7–10). However, some of the litera-
ture appears contradictory regarding how SPARC regulates tumor
growth, and many questions remain. For example, increased
expression of SPARC is associated with an aggressive tumor phe-
notype in melanomas and gliomas (reviewed in refs. 7–10), while
other studies have reported SPARC as a tumor suppressor whose
expression is frequently lost in cancerous compartment due to
promoter methylation (8–10). Use of SPARC knockout (Sparc–/–)
mice revealed that SPARC suppresses syngeneic and oncogene-
driven tumors (9, 11–14) through regulation of matrix deposition
and through antiinflammatory, antiangiogenic, antiprolifera-
tive, and proapoptotic effects, while SPARC has been found to be
upregulated in the stroma (9, 15). SPARC exerts autocrine and
paracrine inhibition of cancer cell proliferation through cell-cycle
arrest (8, 9, 13, 16, 17) and suppression of survival signaling (7, 11,
12, 18, 19), cancer cell adhesion, and invasion (8, 11, 12, 17, 18).
The role of SPARC in urinary bladder physiology and cancer
is also unclear and conflicting. SPARC is expressed in normal
murine and human urothelia and suburothelial stroma (20) and
in primary cultures of human urothelial cells (21–23). SPARC has
been shown to exert an antiadhesive and antiproliferative effect
on human and murine urothelial cells (21–23). A few studies have
reported expression of SPARC in urothelial tumors, with con-
flicting results between gene expression profiling and immuno-
localization of SPARC protein in tumor tissues. In one study,
gene expression profiling of invasive bladder tumors revealed
that higher expression of SPARC inversely correlated with prog-
nosis and patient survival (24). Another study (25) showed via
immunostaining that SPARC protein was exclusively localized to
the desmoplastic stroma within and around the SPARC-negative
invasive carcinoma. Additionally, a recent study (26) reported
that carcinogenic heavy metals downregulated SPARC expres-
sion during malignant transformation of immortalized UROtsa
cells. Immunostaining of xenografts of the transformed cells
exhibited strong immunoreactivity for the SPARC protein in the
host (murine) stromal component surrounding SPARC-nega-
tive tumor cells. Furthermore, in bladder cancer cell lines, gene
expression profiling of poorly tumorigenic T24 cells and its iso-
genic metastatic variant T24T revealed an approximately 7.2-fold
decrease in SPARC mRNA expression in the invasive T24T (27).
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2013;123(2):751–766. doi:10.1172/JCI64782.
752 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 2 February 2013
Taken together, the current literature does not provide a uni-
fying picture of the role of SPARC in cancer, nor does it assign
the relative contributions of tumor- and stroma-derived SPARC.
Herein we address these issues by using urothelial cancer as a
model and find that SPARC is produced by benign and malig-
nant compartments of bladder carcinomas, where it functions
to suppress bladder carcinogenesis, progression, and metastasis.
SPARC expression in human bladder cancer is associated with stage
and outcome. To determine the relevance of SPARC expression
to human disease, we evaluated a human bladder cancer tis-
sue microarray (TMA) (28, 29) for SPARC protein expression
(Figure 1, A and B) and found that in non–muscle invasive
(NMI) disease, SPARC was expressed in the cancerous urothe-
lium and adjacent stroma. In contrast, in muscle invasive (MI)
disease, the expression of SPARC in the cancerous tissue was
decreased and staining was mainly observed in the tumor-asso-
ciated stroma. We sought to distinguish the differential com-
partmentalization of SPARC in human tumors with respect to
the frequency of SPARC expression (number of cells express-
ing the protein) as well as the intensity of expression in can-
cer cells. SPARC staining was exclusively cytoplasmic in tumor
and stromal cells in all the examined cores. The specificity of
SPARC immunostaining was confirmed as described in Meth-
ods. We used 2 scoring systems, as described in Methods, and
determined SPARC tumor cell expression as a function of dis-
ease outcome. We found that both the frequency (Figure 1C)
and the intensity (Figure 1D) of SPARC staining were positively
correlated with disease-specific survival (DSS). However, there
was no relationship between the intensity and/or frequency of
stromal SPARC expression and DSS.
Loss of SPARC accelerates BBN-induced bladder carcinogenesis and metas-
tasis. To determine the relevance of SPARC in the pathobiology of
bladder cancer, we used Sparc–/– mice and their wild-type counter-
parts (Sparc+/+) in a carcinogen-induced model of bladder cancer,
as described in Methods (3), that shares molecular similarities to
human disease (30). It is noteworthy to mention that there were
no developmental, functional, or histologic differences observed
between normal Sparc–/– and Sparc+/+ bladders. In both genotypes,
the sequence of bladder abnormalities after carcinogen exposure
progressed from inflammation with urothelial atypia and dysplasia
(IAD) and carcinoma in situ (CIS) to invasive carcinoma (Figure 2, A
and B). Sparc–/– mice exhibited accelerated urothelial pathology in all
cohorts (Figure 2B). Sparc–/– mice exhibited significantly decreased
survival compared with their Sparc+/+ littermates, with a median sur-
vival of 42 and 20 weeks for Sparc+/+ and Sparc–/–, respectively, and
a hazard ratio of 0.0173 and 95% CI of the ratio 0.004 to 0.07 (Fig-
ure 2C). Mortality of both genotypes was primarily due to obstruc-
tive uropathy. Loss of SPARC was also associated with pathological
changes in the renal urothelium similar to those in the bladder (Fig-
ure 2D). Only mice with invasive primary bladder cancers developed
metastases mainly to the para-aortic LNs and lungs (Figure 2E),
with Sparc–/– mice exhibiting a higher incidence of metastases. Mac-
roscopic and microscopic examination of lung lesions with metasta-
ses revealed that Sparc–/– lungs exhibited greater number and size of
metastatic nodules (Figure 2, F and G). In addition, the expression
of SPARC protein in bladders exhibited distinctive expression as the
Expression of SPARC in human bladder cancer tumors.
(A and B) IHC staining of SPARC in NMI (n = 92) and MI
(n = 102) tumors showing expression of SPARC in cancer-
ous and stromal cells with an overall decrease in tumor
cells in MI disease. Original magnification, ×100; ×200
(inset). (C) Kaplan-Meier curve showing disease-specific
survival in high- and low-scoring tumors. Tumors were
scored by counting the number of cancer cells express-
ing the protein or the intensity of SPARC expression (D)
as determined by SPARC staining in cancer cells (see
Methods) and DSS. Results of log-rank test shown.
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 2 February 2013
Loss of SPARC accelerates bladder carcinogenesis and progression. (A) Representative H&E-stained sections of bladder pathology.
Original magnification, ×200. (B) Mice were assigned to 4 cohorts as a function of BBN exposure, and the distribution of the ensuing
bladder pathology in each cohort is represented in bar graphs showing the percentage of animals (P < 0.05, χ2 test). (C) Kaplan-Meier
curves showing significantly decreased survival in Sparc–/– mice as compared with Sparc+/+ counterparts. (D) The incidence of upper tract
urothelial tumors in the 15- to 25-week and greater than 25-week cohorts. *P < 0.05; Fisher exact test. (E) The incidence of metastasis in
Sparc+/+ and Sparc–/– mice in 15- to 25-week and greater than 25-week cohorts *P < 0.05; Fisher exact test. (F) Incidence and number of
visible lung metastases in the 15- to 25-week and greater than 25-week cohorts. *P < 0.05; χ2 test; **P < 0.05, Student’s t test. (G) Repre-
sentative H&E-stained lung sections showing larger size of lung metastases in Sparc–/– lungs. Original magnification, ×100. Only animals
with invasive primary tumors were considered for analyses in D–G.
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 2 February 2013
parisons were considered significant at P < 0.05. Statistical analysis of the
CES of SPARC, phospho–p65–NF-kB, and phospho–c-Jun in mouse tissue
was performed using Kruskal-Wallis 1-way ANOVA followed by Dunn’s
and Bonferroni’s multiple comparison post-hoc analyses (GraphPad Prism
5.0). Associations of SPARC expression in TMAs with DSS were evaluated
using the log-rank test. DSS time was equivalent to months between trans-
urethral resection or cystectomy and death of disease. Survival plotted
using Kaplan-Meier methodology (SPSS) (28, 29)
Study approval. All animal experiments were performed after approval of
protocol and in compliance with guidelines of the Animal Care and Use
Committee of the University of Virginia. Bladder cancer TMAs were con-
structed at the Spanish National Cancer Institute (28, 29). These arrays
included primary urothelial cell carcinomas of the bladder belonging to
patients recruited under Institutional Review Board–approved protocols;
patients gave informed consent in studies referenced (28, 29).
This study was supported by NIH grant CA143971 (to D. Theo-
dorescu), the Paul Mellon Urologic Cancer Institute, University
of Virginia (to N. Said), and CA118240 (to R.A. Brekken). The
authors wish to thank Sharon Birdsall, John Sanders, Melissa Bev-
ard, and Jeremy Gatesman for technical help with experiments.
Received for publication May 14, 2012, and accepted in revised
form November 8, 2012.
Address correspondence to: Dan Theodorescu, Departments of
Surgery and Pharmacology and Comprehensive Cancer Center,
University of Colorado, Aurora, Colorado 80045, USA. Phone:
303.724.7135; Fax: 303.724.3162; E-mail: dan.theodorescu@
Immunostaining of mouse tumor tissues was performed with Vecta-
stain ABC ELITE or a MOMA Kit (Vector Laboratories, Inc.) according
to manufacturer’s instructions and counterstained with hematoxylin.
Antigen retrieval was achieved as described above. We used monoclonal
and polyclonal antibodies against the following: phospho–p65–NF-κB,
phospho–c-Jun (Cell Signaling), Ki67 (Dako Cytomation), mac1 and CD31
(eBioscience), and goat anti-mouse SPARC (R&D Systems Inc.). Scoring of
the expression of phospho–p65–NF-κB, and phospho–c-Jun in the cyto-
plasm and nuclei of urothelial (normal and cancerous) and stromal cells
was carried out using a composite expression score (CES) combining the
frequency and intensity of staining. The frequency of positive staining
was determined by the number of positive cells per 100 cells counted in 6
independent fields/bladder section. Frequency score was set based on the
percentage of positive cells: 10% (score 0); +, 11%–40% (+), 41%–70% (++),
and more than 70% (+++). The intensity of staining intensity score was as
described above. For each section, a CES was generated by transforming
the frequency and intensity scores into numeric values. This scoring sys-
tem allows compensation for tumor heterogeneity as well as the sequential
phosphorylation and nuclear translocation of these transcription factors.
In all immunostaining experiments, negative controls were performed
omitting the primary antibodies. In SPARC immunostaining, the specific-
ity of the antibodies was validated by including recombinant human and
murine SPARC (R&D Systems Inc.) during staining to competitively inhibit
the reaction of the antibodies with tissue SPARC (Supplemental Figure 12).
Statistics. The relationships among tumor histology, genotype, and age
were assessed by applying the χ2 test. All other comparisons between
Sparc−/− and Sparc+/+ groups were performed with 2-tailed Student’s t test.
The changes of the levels of cytokines/chemokines, growth factors, and
inflammatory mediators as a function of tumor progression in either
Sparc−/− or Sparc+/+ tumors were analyzed by 1-way ANOVA. Analyses were
carried out with GraphPad Prism 5.0 software and Microsoft Excel. Com-
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