The Expression of SPARC in Human
Urothelial Cells (UROtsa) Exposed
to or Malignantly Transformed by
Cadmium or Arsenite
Jennifer Larson
Doctoral Candidate
University of North Dakota
October 5, 2011
Outline
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Background Info
Purpose
Methods
Results
Conclusions
Future directions
Bladder Cancer
• Transitional cell carcinoma of the bladder is the 9th most common cancer worldwide and the
4th in U.S (Bischoff & Clark, 2010)
• Highest cost per patient of all cancers (Sullivan et al., 2010; Jacobs et al., 2010)
• Bladder cancer was the first cancer in which industrial carcinogens were found to play the
major role in disease causation
 Link between exposure to aromatic amines and development of bladder cancer in factory
workers (Rehn, 1895)
• Cd⁺² and As⁺³ are human carcinogens (IARC, 1993; IARC, 1980) and linked to the development of
bladder cancer (Steinmaus et al., 1994 & 2000)
• Association between cigarette smoking and bladder cancer
 2-4 times increased risk (Clavel et al., 1989; Morrison et al., 1984; Silverman et al., 1992)
UROtsa Cell Line
• Derived from the urothelium lining the ureter of a 12 year-old female.
• Immortalized with SV40 large T-antigen (Perzoldt et al., 1995)
• Cd⁺² and As⁺³ are able to cause the malignant transformation of UROtsa
cells (Sens et al., 1994 & 2000)
 Generated 7 different Cd+2 & 6 different As+3 cell lines.
UROtsa Parent
UROtsa Cd⁺²
UROtsa As⁺³
Sens et al. Toxicological Sciences (2004)
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UROtsa parent cells are nontumorigenic:
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Do not grow in soft agar
No not form tumors in nude
mice
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Transformed cell lines are
tumorigenic:
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Grow in soft agar
Form tumors in nude mice
SPARC
• Secreted Protein, Acidic and Rich in Cysteine
 Also known as: Osteonectin & BM-40
• Belongs to the Matricellular group of proteins
 Secreted macromolecules that interact with cell-surface receptors, ECM, and/or
growth factors and proteases, but do NOT have structural roles
 Known to bind to structural matrix proteins
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Collagen and Vitronectin
 Found in tissues undergoing repair/remodeling (Salonen et al. 1990)
 Upregulated during embryological development (Sage et al., 1989)
 Other matricellular proteins include:
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Thrombosponbin 1 & 2
Tenascin C & X
Osteopontin
CTGF
Testican 1, 2, & 3 (SPARC-related protein)
Hevin (SPARC-related protein)
SPARC
• 3 General functions of SPARC:
 1. De-adhesion:
• Exogenous SPARC inhibits cell spreading and cell attachment (Sage et al. 1989)
• Disassemble focal adhesions (Murphy-Ullrich et al. 1995, Murphy-Ullrich 2001)
• Reorganizes actin stress fibers to periphery of cell (Murphy-Ullrich et al. 1995)
• May play a role in cell migration
 2. Anti-proliferation:
• Cell cycle inhibitor (Funk & Sage 1991, 1993; Sage et al. 1995)
• Promoted prostate cancer cell migration (De et al. 2003) and stimulated fibroblasts during myocardial
infraction (Wu et al. 2006)
• May play a role in cell migration
 3. Regulation of ECM & growth factors:
• Binds to many ECM components (Brekken & Sage 2000)
• Known activator of MMP-2 (Tremble et al. 1993)
• Regulates PDGF, VEGF, & TGF-β1 expression (Raines et al, 1992, Kuppricon et al. 1998, Abe et al. 2004, Hasselaar & Sage 1992,
Lane & Sage 1994)
• After exogenous treatment, capable of inducing the expression of SPARC
• May play a role in the progression of metastatic tumors
Purpose
• To determine the role SPARC plays in the
formation and progression of bladder cancer.
SPARC Expression in UROtsa Cell Lines &
Normal Bladder Tissue
Larson et al 2010
SPARC Expression in Normal and
Cancerous Bladder Tissue
Normal Bladder Tissue
High Grade Carcinoma
Larson et al 2010
SPARC Expression after Treatment
with MS-275 & 5-AZC
• Possible role of epigentic
modification?
• 24, 48, and 72h treatment
 48h shown
• MS-275 is a histone deacetylase
inhibitor
 Prevents acetyl groups from being
removed from histones
• 5-Aza-2’-deoxycytidine (5-AZC) is
a methylation inhibitor
 Inhibits the methylation of
histones
• Conclusion: No change in SPARC
expression
Larson et al 2010
SPARC Expression after Drug
Combination Treatment
• Treatment with both MS-275 & 5-AZC for 72hrs
• Conclusion: No change in SPARC expression
Larson et al 2010
SPARC Expression after Treatment
with As⁺³ & Cd⁺²
Larson et al 2010
SPARC Expression during Transformation
of UROtsa Cell Lines
Summary: SPARC Expression
• Moderate SPARC expression in Normal Tissue
• Low or undetectable SPARC expression in Cancerous/Tumor Tissue
• SPARC expression does not appear to be regulated epigenetically in our
system
• SPARC mRNA and protein expression decreases with increasing
concentrations of As3+ and Cd2+
SPARC Transfection
• 4 cell lines were chosen for stable transfection of
SPARC: As#3, As#6, Cd#1, Cd#4
• All cell lines were characterized:
 mRNA
 Protein
 Secretion of SPARC protein
 Growth
 Migration
SPARC Transfection
• 4 cell lines were
chosen for stable
transfection of SPARC:
As#3, As#6, Cd#1,
Cd#4
SPARC Expression within Cultured
Media
• All cells were grown in Serum Free growth media
 Media was harvested, centrifuged, and filtered
 Protein was precipitated from growth media
 Loaded equal total protein per lane
• SPARC transfected cell lines had
similar staining
 SPARC expression does not
appear to be matrix incorporated
Growth: MTT
Cell Line
Doubling Time
Parent
33.2 ± 0.8 h
As#3
33.3 ± 1.4 h
As#3-SPARC
27.4 ± 1.0 h*
As#6
21.6 ± 1.6 h
As#6-SPARC
25.6 ± 0.5 h
Cd#1
27.8 ± 0.6 h
Cd#1-SPARC
23.8 ± 0.6 h*
Cd#4
20.7 ± 1.1 h
Cd#4-SPARC
22.0 ± 0.6 h
Cao et al 2010; Somji et al 2010
Migration
• Wound/Scratch assay:
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Cells were grown to confluence
Conditions: Untx or treated with Mitomycin C (MMC) for 2h
Scratch performed with 200ul pipette tip
Cells were rinsed with PBS 2x’s
Pictures taken at 0h and 24h
MTT performed to determine cell viability
• Determine best concentration of MCC to prevent cell
death
• Performed in duplicate
Migration
• Wound/Scratch assay:
Migration
• Transwell Migration assay:
• Top chamber = 2.4 x 105 cells/ml in serum free
media
• Bottom chamber = 1.5% fetal calf serum
• 8h incubation for migration
• Count total number of cells on insert
• Remove cells from top of insert and count
remaining
• Total of 20 fields per insert, performed in
duplicate
Migration
• Transwell Migration assay:
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Conclusions
• Expression of SPARC is not epigenetically
regulated in our system
• SPARC expression decreased with treatment
with Cd2+ & As3+
• SPARC tranfections:
 Secrete SPARC into media
 2 transfected cell lines had decreased migration
Future Directions
• Assess the ability of SPARC transfected cell lines to form
tumors in nude mice
 Tumor size
 Subcutaneous vs. intraperitoneal tumor formation
 Expression of SPARC in tumor
• Assess the structural differences of SPARC between UROtsa
Parent and transfected cell lines
 Glycosylation
 Post-translational modifications
• Characterization of the promoter elements that turn off
SPARC expression with treatment of Cd2+ & As3+
Acknowledgements
• Dr Jane Dunlevy
 Holly Hewitt
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Dr Don Sens
Dr Mary Ann Sens
Dr Seema Somji
Dr Scott Garrett
Dr XuDong Zhou
Questions?
SPARC
• First identified as the most abundant non-collagenous component of
bone (Termine et al.1981)
• Highly conserved among different species
 Human gene has 92% homology with mice
• Predicted molecular mass of 32,511 Daltons
 Secreted form of the protein migrates at 43kD on SDS-PAGE
• Due to addition of carbohydrates (Sage et al. 1984)
Bradshaw & Sage 2001
SPARC Expression in Mouse
Heterotransplants
Larson et al 2010
SPARC Expression in Cancerous Tissue
• Tumor Promoter:
 Increased expression of
SPARC
• Brain: Glioblastomas,
Astrocytomas, Meningioma
• Breast: Invasive ductal
carcinoma
• Lung: NSCLC, squamous cell
carcinoma, adenocarcinoma
• Pancreas: Pancreatic ductal
adenocarcinoma
• Skin: Melanoma
• Tumor Suppressor:
 Decreased expression of
SPARC
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Brain: Neuroblastoma
Ovary: carcinoma
Lung: NSCLC & SCLC
Pancreas: PDAC
Skin: Melanoma
Uterus: Cervical &
endometrial carcinoma
Epigenetic Regulation of SPARC
• Ovarian cancer:
 Treatment with the demethylating agent 5-aza-2'-deoxycytidine (5AZC) rescued SPARC mRNA and protein expression in 9 ovarian
cancer cell lines
 1 cell line showed a significant increase in SPARC mRNA after TSA
treatment, histone deacetylase inhibitor (Socha et al. 2009)
• Colon cancer:
 Induction of SPARC was observed in 5 of the 7 cell lines after 5-AZC
treatment (Yang et al. 2007)
• Non-small cell lung cancer (NSCLC):
 SPARC expression was restored after 5-AZC treatment in all 11
NSCLC cell lines (Suzuki et al. 2005)
• Pancreatic adenocarcinoma:
 SPARC mRNA expression was restored in seven 7 of 8 cell lines after
5-AZC treatment
 SPARC expression was not restored 1 cell line after 5-AZC treatment
did not restore the SPARC expression.
 Treatment with an HDAC inhibitor TSA or with a combination of 5-AZC
and TSA did not induce SPARC expression (Sato et al. 2003)