Transcript Sarco/Endoplasmic Reticulum Calcium

Sarco/Endoplasmic Reticulum
Calcium-ATPase 2 Expression as a
Tumor Marker in Colorectal Cancer
Fu-Yen Chung, MS, Shiu-Ru Lin, PhD, Chien-Yu Lu, MD, ChingSheng Yeh, PhD, Fang-Ming Chen, MD, Jan-Sing Hsieh, MD, PhD,
Tsung-Jen Huang, MD, PhD, and Jaw-Yuan Wang, MD, PhD
From Kaoshiung Medical University and Chung-Ho Memorial Hospital
American Journal of Surgical Pathology‧Volume 30, No. 8, Aug. 2006
Presented by TMUH Int. Hsin-Chih Huang
Introduction
• Cytoplasmic calcium acts as second
messenger, controlling many different
aspects of cellular physiology.
• The endoplasmic reticulum (ER) is the
principal calcium storage organelle in cells,
and one of the most critical determinant of
calcium in the ER of most cell types is the
activity of sarco/endoplasmic reticulum
calcium-ATPase 2 (SERCA 2).
Introduction
• SERCA isoforms are encode by SERCA1,
2, and 3.
• The ATP2A2 gene localized on
chromosome 12q23-q24.1 expressing the
SERCA2 of the calcium pump.
Introduction
• The activity of SERCA2 is an important
regulator of normal calcium homeostasis
and signaling.
Introduction
• Lytton and MacLennan cloned human
cDNAs coding for 2 alternatively spliced
products of the cardiac calcium-ATPase
gene, SERCA2a and SERCA2b:
– SERCA2a is located primarily in the heart and
slow-twitch skeletal muscle
– SERCA2b is present in smooth muscle and
non-muscle tissue
Introduction
• Alterations in calcium-dependent signaling are
involved in cell proliferation and differentiation,
apoptosis, and disruption of calcium
homeostasis, which have been suggested to
contribute to cancer development.
• Cell exposure to the SERCA blockade
thapsigargin results in the inhibition of cell
proliferation, suggesting a potential role in
controlling cell growth.
Introduction
• Because SERCA2 gene alterations were
found to be closely related to colorectal
carcinogenesis from the authors’ results
using cDNA microarray, the authors
therefore hypothesize that the SERCA2
may have a great clinical potential in terms
of diagnosis and monitoring CRC patients.
(Wang JY, Yeh CS, Tzou WS, et al. Analysis of progressively
overexpressed genes in tumorigenesis of human colorectal
cancers using cDNA microarray. Oncology reports, 2005;
14:65-72)
Introduction
• The authors analyzed the SERCA2 mRNA
expression between cancerous tissues
and the corresponding noncancerous
tissues in CRC patients.
Materials and Methods
• Patients and samples collection
• Total RNA isolation and first strand cDNA
synthesis
• Multiplex RT-PCR
• Immunohistochemistry
• Statistical analysis
Patients and Samples Collection
• Tissues were collected from 50 patients:
– Sporadic CRC
– Surgical resection
– Department of Surgery, Kaohsiung Medical
University Hospital
– Between July 2000 and January 2001
– Written-informed consent
Patients and Samples Collection
• 28 men and 22 women
• Mean age 64.4 ± 1.3 y
Patients and Samples Collection
• The criteria of the American Joint
Commission on Cancer defined clinical
stages and pathologic features:
– Stage I – 4 patients
– Stage II – 16 patients
– Stage III – 24 patients
– Stage IV – 6 patients
Patients and Samples Collection
• All of the patients were regularly followedup at 3-month intervals until Sep. 2005
(range 2 to 62 mo, median 34 mo)
• Those lost to follow-up or dead from other
causes than CRC were regarded as
censored data for the analysis of survival
rates
Patients and Samples Collection
• All of the samples were:
– collected immediately after surgical resection
– frozen instantly in liquid nitrogen
– stored at – 70 °C until analyzed
Total RNA Isolation and First
Strand cDNA Synthesis
• Total RNA was extracted from fresh tissue
samples of the CRC patients using a
QIAamp RNA Blood Mini Kit (QIAGEN Inc,
Valencia, CA).
Total RNA Isolation and First
Strand cDNA Synthesis
• RNA concentration was determined
spectrophotometrically on the basis of
absorbance at 260nm.
Total RNA Isolation and First
Strand cDNA Synthesis
• The first strand cDNA was synthesized from total
RNA by using a reverse transcriptasepolymerase chain reaction (RT-PCR) kit
(Promega Corp, Madison, WI).
• The reaction mixture with RNA were incubated
at 42°C for 2 hours, heated to 95°C for 5 minutes,
and then stored at 4°C until the analysis.
Multiplex RT-PCR
• PCR was performed by using β-actin primers as
internal control to correct the differences in total
RNA amounts.
• Sequences of the PCR primers for SERCA2 and
β-actin were designed according a PCR primer
selection program on the basis of primer 3 at
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
(Accessed on January 25, 2005).
Multiplex RT-PCR
• The amplification cycles were 20 seconds
at 95°C, 20 seconds at 60°C, and 30
seconds at 74°C in a programmable
thermal cycler (Primus 25, MWGBIOTECH AG, Ebersberg, Germany).
• The cycle was repeated 35 times.
Multiplex RT-PCR
• The PCR products were analyzed in 3%
agarose gel.
• The signals on the ultraviolet transilluminator
were scanned with a computing laser
densitometer (Alpha Inotech, San-Leandro,
CA) to calculate the relative mRNA density
ratio.
Immunohistochemistry
• Four-micrometer paraffin sections were immuno stained
as described previously.
• Five sections of each tumor tissue were stained per case.
• Antigen retrieval was done using microwave heating for
10 minutes in 10mM citrate buffer (pH 6.0).
• Each section was dewaxed in xylene and rehydrated
with alcohol.
• The slides were then incubated in 3%hydrogen peroxide
for 5 minutes, to block endogenous peroxidase activity.
Immunohistochemistry
• After washing in Tris-buffered saline (pH
7.6), the slides were incubated with diluted
primary mouse monoclonal antibodies
against SERCA2 (1:300; Calbiochem,
Darmstadt, Germany).
Immunohistochemistry
• The primary antibodies were detected using the
DAKO LSAB2 System, and horseradish
peroxidase (DAKO Corp, Carpinteria, CA) with
biotinylated antimouse IgG as secondary
antibody.
• The reaction was developed with streptavidinhorseradish peroxidase and diaminobenzidine
chromogen, and the sections were subsequently
counterstained with light hematoxylin.
Immunohistochemistry
• To examine the possibility of false positive
results, the authors used a nonimmune
antiserum instead of the primary antibody
as negative control.
Immunohistochemistry
• The percentage of positively stained tumor
cells was evaluated for each tumor section.
• The tumors were considered as positive
immunreactive if greater than 5% of the
neoplastic cells showed distinct cytoplasm
and/or plasma membrane staining.
• When 5% or less than 5% were stained,
the results were considered negative.
Immunochemistry
• Semiquantitative scores were used for
SERCA2 stains according to the
percentage of positively stained cells
– + indicates <25%
– ++, 25% to 50%
– +++, 50% to 75%
– ++++, >75%
Immunohistochemistry
• Cancer tissues that expressed scores of
++, +++, or ++++ were regarded as the
high expression group, whereas those with
scores of + or negative staining were
regarded as the low expression group.
Statistical Analysis
• All of the data were analyzed using the
Statistical Package for the Social Sciences
Ver. 11.5 software (SPSS Inc. Chicago, IL)
• Results were expressed as mean ± SE.
Statistical Analysis
• The χ2 test and the student t test were used to
compare the clinico-pathologic parameters
between the SERCA2 positive and negative
groups.
• The overall survival rates were calculated by the
Kaplan-Meier method and the differences in
survival rates were analyzed by log rank test.
• Multivariate analysis of independent prognostic
factors was determined using the Cox
proportional hazard model.
Statistical Analysis
• A probability of P < 0.05 was considered to
be statistically significant.
Results
• SERCA2 mRNA expression
• Immunohistochemistry
• Survival rates and multivariate analysis
SERCA2 mRNA Expression
• 45 of the 50 patients (90%) showed a
higher expression level of SERCA2 mRNA
in the cancerous tissues than in the
corresponding noncancerous tissues by
RT-PCR
• Mean SERCA2 mRNA expression level
– in cancerous tissues was 0.63 ± 0.10
– In the corresponding noncancerous tissue
was 0.31 ± 0.06
– Significantly higher, P = 0.001
SERCA2 mRNA Expression
• Overexpressed SERCA2 mRNA was identified in
cancerous when compared with noncancerous tissues.
–
–
–
–
T indicates cancerous tissues
N, noncancerous tissues
M, marker
β-actin is an internal control
SERCA2 mRNA Expression
Immunohistochemistry
• SERCA2 antigen was detected in 39 (78%)
by immunohistochemical staining.
• SERCA2 was predominantly in expressed
in the cancer cells.
• All of the positive cases showed brownish
cytoplasmic staining but no nuclear
staining.
Immunohistochemistry
• In the high SERCA2 expression group:
– The incidence of serosal invasion (27/34, 79.4%)
was significantly higher (P = 0.012) than in the low
expression group (7/16, 43.8%).
– The incidence of lymph node metastasis (24/34,
70.6%) was considerably higher (P = 0.009) than in
the low expression group (5/16, 31.3%)
– The incidence of advanced stage cancer (according
to Tumor Node Metastasis classification) (25/34,
73.5%) was significantly higher (P = 0.004) than in the
low expression group (5/16, 31.3%)
Survival Rates and Multivariate Analysis
• The overall survival rate for the high SERCA2
expression group was significantly lower than that
of the low SERCA2 expression group.
Survival Rates and Multivariate Analysis
• Table 2 showed that the independent prognostic factors
identified by multivariate analysis were tumor stage
(P=0.015) and SERCA2 expression (P=0.018).
Survival Rates and Multivariate Analysis
• SERCA2 antigen staining was further
identified to be a significant and powerful
prognostic indicator in patients with CRC.
Discussion
• The result of the current study indicated
that in CRC patients, SERCA2 mRNA is
more frequently overexpressed in cancer
tissues than in noncancerous tissue
Discussion
• Calcium mobilization from the ER into the
cytosol is suggested to be a key component of
several signaling network controlling tumor cell
growth, differentiation, or apoptosis.
• Refilling of calcium into the ER from the cytosol
by active ATP-driven ion transport is ensured by
SERCA enzymes.
• SERCA enzymes that accumulate calcium in the
ER, and the subsequently anomalous calcium
homeostasis may play an important role in
neoplastic transformation.
Discussion
• The intracellular calcium homeostasis becomes
progressively anomalous during colon
carcinogenesis, as reflected by deficient
SERCA3 expression.
• SERCA3 expression is barely detectable in
moderately differentiated tumors, and is
undetectable in poorly differentiated
adenocarcinoma of the colon.
• SERCA3 constitutes a vital process in the
development of CRC, and is a new and
potentially useful marker for the study of the
state of differentiation of CRC.
Discussion
• In contrast to SERCA3 analysis from other
previous studies, the SERCA2 expression
levels in the current work are not
significantly related to cell differentiation
by histological examinations.
Discussion
• The inactivation of the SERCA2 gene is a
frequent and early event during carcinogenesis
in some cancers and that loss of its expression
may be regulated partly by an epigenetic
mechanism via promoter methylation.
• The inactivation of SERCA2 gene result in
impaired uptake of cytosolic calcium into the ER
and consequent disruption of calcium signaling,
causing Darier disease, an autosomal-dominant
skin disorder characterized by loss of cellular
adhesion, proliferation, and abnormal
keratinization.
Discussion
• SERCA2 is up-regulated in CRC, which
may result in the disturbance of calcium
homeostasis through increasing the ER
calcium uptake.
Discussion
• High SERCA2 protein expression is
considerably correlated to serosal invasion,
lymph node metastasis, advanced tumor
stage, and poor actual overall survival.
• SERCA enzyme-dependent intracellular
signaling pathways may ensure this
metastatic process.
• The induction of SERCA2 protein
expression may participate, at least in part,
in the progression of CRC.
Discussion
• The insulin growth factor-induced prostate
cancer cell proliferation through increasing
calcium concentration within the ER, and
tumor necrosis factor-α reduced cell
proliferation and induced apoptosis by
decreasing calcium concentration within
the ER.
Discussion
• The characteristics of the increased
refilling of the ER calcium store and its
consequent influence on store-operated
calcium influx in one possible explanation.
• Treatment of head and neck squamous
cell carcinoma using the calcium influx
inhibitor would inhibit cell growth and
invasion in vivo.
Discussion
• Overall, the authors’ results indicated that:
– SERCA2 mRNA was overexpressed in CRC tissue
– High SERCA2 expression level were correlated with:
• serosal invasion
• lymph node metastasis
• advanced tumor stage
• poor prognosis
– The potential role for SERCA2 expression level as a
new prognostic biomarker for CRC
Thanks for Your Attention!!
Presented by TMUH Int. Hsin-Chih Huang