sarco/endoplasmic reticulum calcium-atpase 2 expression as a tumor marker in colorectal cancer

Sarco/Endoplasmic Reticulum CSarco/Endoplasmic Reticulum Calcium-ATPase 2 Expression as a alcium-ATPase 2 Expression as a Tumor Marker in Colorectal CanTumor Marker in Colorectal CancercerFu-Yen Chung, MS, Shiu-Ru Lin, PhD, Chien-Yu Lu, MD, Ching-SheFu-Yen Chung, MS, Shiu-Ru Lin, PhD, Chien-Yu Lu, MD, Ching-Sheng Yeh, PhD, Fang-Ming Chen, MD, Jan-Sing Hsieh, MD, PhD, Tsunng Yeh, PhD, Fang-Ming Chen, MD, Jan-Sing Hsieh, MD, PhD, Tsung-Jen Huang, MD, PhD, and Jaw-Yuan Wang, MD, PhDg-Jen Huang, MD, PhD, and Jaw-Yuan Wang, MD, PhD

From Kaoshiung Medical University and Chung-Ho Memorial HospitalFrom Kaoshiung Medical University and Chung-Ho Memorial Hospital

American Journal of Surgical Pathology Volume 30, No. 8, Aug. 2006‧American Journal of Surgical Pathology Volume 30, No. 8, Aug. 2006‧

Presented by TMUH Int. Hsin-Chih HuangPresented by TMUH Int. Hsin-Chih Huang

IntroductionIntroduction

• 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).


• SERCA isoforms are encode by SERCA1, SERCA isoforms are encode by SERCA1, 2, and 3.2, and 3.

• The ATP2A2 gene localized on chromosoThe ATP2A2 gene localized on chromosome 12q23-q24.1 expressing the SERCA2 me 12q23-q24.1 expressing the SERCA2 of the calcium pump.of the calcium pump.


• The activity of SERCA2 is an important The activity of SERCA2 is an important regulator of normal calcium homeostasis regulator of normal calcium homeostasis and signaling.and signaling.


• Lytton and MacLennan cloned human cDNLytton and MacLennan cloned human cDNAs coding for 2 alternatively spliced produAs coding for 2 alternatively spliced products of the cardiac calcium-ATPase gene, Scts of the cardiac calcium-ATPase gene, SERCA2a and SERCA2b:ERCA2a and SERCA2b:– SERCA2a is located primarily in the heart and SERCA2a is located primarily in the heart and

slow-twitch skeletal muscleslow-twitch skeletal muscle– SERCA2b is present in smooth muscle and nSERCA2b is present in smooth muscle and n

on-muscle tissueon-muscle tissue


• Alterations in calcium-dependent signaling are inAlterations in calcium-dependent signaling are involved in cell proliferation and differentiation, apvolved in cell proliferation and differentiation, apoptosis, and optosis, and disruption of calcium homeostasis, disruption of calcium homeostasis, which have been suggested to contribute to canwhich have been suggested to contribute to cancer developmentcer development..

• Cell exposure to the SERCA blockade thapsigarCell exposure to the SERCA blockade thapsigargin results in the inhibition of cell proliferation, sugin results in the inhibition of cell proliferation, suggesting a potential role in controlling cell growth.ggesting a potential role in controlling cell growth.


• Because SERCA2 gene alterations were fBecause SERCA2 gene alterations were found to be closely related to colorectal caround to be closely related to colorectal carcinogenesis from the authors’ results using cinogenesis from the authors’ results using cDNA microarray, the authors therefore hycDNA microarray, the authors therefore hypothesize that pothesize that the SERCA2 may have a grthe SERCA2 may have a great clinical potential in terms of diagnosis eat clinical potential in terms of diagnosis and monitoring CRC patientsand monitoring CRC patients..

((Wang JY, Yeh CS, Tzou WS, et al. Analysis of progressively Wang JY, Yeh CS, Tzou WS, et al. Analysis of progressively overexpressed genes in tumorigenesis of human colorectal coverexpressed genes in tumorigenesis of human colorectal cancers using cDNA microarray. Oncology reports, 2005; 14:ancers using cDNA microarray. Oncology reports, 2005; 14:65-72)65-72)


• The authors analyzed the SERCA2 mRNA The authors analyzed the SERCA2 mRNA expression between cancerous tissues anexpression between cancerous tissues and the corresponding noncancerous tissues d the corresponding noncancerous tissues in CRC patients.in CRC patients.

Materials and MethodsMaterials and Methods

• Patients and samples collectionPatients and samples collection

• Total RNA isolation and first strand cDNA Total RNA isolation and first strand cDNA synthesissynthesis

• Multiplex RT-PCRMultiplex RT-PCR

• ImmunohistochemistryImmunohistochemistry

• Statistical analysisStatistical analysis

Patients and Samples CollectionPatients and Samples Collection

• Tissues were collected from Tissues were collected from 50 patients50 patients::– Sporadic CRCSporadic CRC– Surgical resectionSurgical resection– Department of Surgery, Kaohsiung Medical Department of Surgery, Kaohsiung Medical

University HospitalUniversity Hospital– Between July 2000 and January 2001Between July 2000 and January 2001– Written-informed consentWritten-informed consent


• 28 men and 22 women28 men and 22 women

• Mean age 64.4 Mean age 64.4 ± 1.3 y± 1.3 y


• The criteria of the American Joint CommisThe criteria of the American Joint Commission on Cancersion on Cancer defined clinical stages and defined clinical stages and pathologic features:pathologic features:– Stage I – 4 patientsStage I – 4 patients– Stage II – 16 patientsStage II – 16 patients– Stage III – 24 patientsStage III – 24 patients– Stage IV – 6 patientsStage IV – 6 patients


• All of the patients were All of the patients were regularly followed-regularly followed-up at 3-month intervalsup at 3-month intervals until Sep. 2005 until Sep. 2005 (range 2 to 62 mo, median 34 mo)(range 2 to 62 mo, median 34 mo)

• Those lost to follow-up or dead from other Those lost to follow-up or dead from other causes than CRC were regarded as causes than CRC were regarded as censored data for the analysis of survival censored data for the analysis of survival ratesrates


• All of the samples were:All of the samples were:– collected immediately after surgical resectioncollected immediately after surgical resection– frozen instantly in liquid nitrogenfrozen instantly in liquid nitrogen– stored at – 70 stored at – 70 °C until analyzed°C until analyzed

Total RNA Isolation and First StranTotal RNA Isolation and First Strand cDNA Synthesisd cDNA Synthesis

• Total RNA was extracted from fresh tissue Total RNA was extracted from fresh tissue samples of the CRC patients using a QIAasamples of the CRC patients using a QIAamp RNA Blood Mini Kit (QIAGEN Inc, Valemp RNA Blood Mini Kit (QIAGEN Inc, Valencia, CA).ncia, CA).


• RNA concentration was determined spectrRNA concentration was determined spectrophotometrically on the basis of absorbanophotometrically on the basis of absorbance at 260nm.ce at 260nm.


• The first strand cDNA was synthesized from total The first strand cDNA was synthesized from total RNA by using a reverse transcriptase-polymerasRNA by using a reverse transcriptase-polymerase chain reaction (RT-PCR) kit (Promega Corp, Me chain reaction (RT-PCR) kit (Promega Corp, Madison, WI).adison, WI).

• The reaction mixture with RNA were incubated aThe reaction mixture with RNA were incubated at 42t 42°C for 2 hours, heated to 95°C for 5 minutes, °C for 2 hours, heated to 95°C for 5 minutes, and then stored at 4°C until the analysis.and then stored at 4°C until the analysis.

Multiplex RT-PCR 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-PCRMultiplex 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, MWG-BIOTECH AG, Ebersberg, Germany).

• The cycle was repeated 35 times.

Multiplex RT-PCRMultiplex 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.

ImmunohistochemistryImmunohistochemistry

• 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.


• 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).


• 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 streptavidin-horseradish peroxidase and diaminobenzidine chromogen, and the sections were subsequently counterstained with light hematoxylin.


• To examine the possibility of false positive results, the authors used a nonimmune antiserum instead of the primary antibody as negative control.


• 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.

ImmunochemistryImmunochemistry

• Semiquantitative scores were used for SERCA2 stains according to the percentage of positively stained cells – + indicates <25%– ++, 25% to 50%– +++, 50% to 75%– ++++, >75%


• Cancer tissues that expressed scores of +Cancer tissues that expressed scores of ++, +++, or ++++ were regarded as the high +, +++, or ++++ were regarded as the high expression group, whereas those with scorexpression group, whereas those with scores of + or negative staining were regarded es of + or negative staining were regarded as the low expression group.as the low expression group.

Statistical AnalysisStatistical Analysis

• All of the data were analyzed using the StaAll of the data were analyzed using the Statistical Package for the Social Sciences Vetistical Package for the Social Sciences Ver. 11.5 software (SPSS Inc. Chicago, IL)r. 11.5 software (SPSS Inc. Chicago, IL)

• Results were expressed as mean Results were expressed as mean ± SE.± SE.


• The The χχ22 test and the student test and the student tt test were used to c test were used to compare the clinico-pathologic parameters betweompare the clinico-pathologic parameters between the SERCA2 positive and negative groups.en the SERCA2 positive and negative groups.

• The overall survival rates were calculated by the The overall survival rates were calculated by the Kaplan-Meier method and the differences in survKaplan-Meier method and the differences in survival rates were analyzed by log rank test.ival rates were analyzed by log rank test.

• Multivariate analysis of independent prognostic fMultivariate analysis of independent prognostic factors was determined using the Cox proportionactors was determined using the Cox proportional hazard model.al hazard model.


• A probability of A probability of PP < 0.05 was considered < 0.05 was considered to be statistically significant.to be statistically significant.

ResultsResults

• SERCA2 mRNA expressionSERCA2 mRNA expression

• ImmunohistochemistryImmunohistochemistry

• Survival rates and multivariate analysisSurvival rates and multivariate analysis

SERCA2 mRNA ExpressionSERCA2 mRNA Expression

• 45 of the 50 patients (90%) showed a high45 of the 50 patients (90%) showed a higher expression level of SERCA2 mRNA in ter expression level of SERCA2 mRNA in the cancerous tissueshe cancerous tissues than in the correspo than in the corresponding noncancerous tissues by RT-PCRnding noncancerous tissues by RT-PCR

• Mean SERCA2 mRNA expression levelMean SERCA2 mRNA expression level– in cancerous tissues was 0.63 in cancerous tissues was 0.63 ± 0.10± 0.10– In the corresponding noncancerous tissue waIn the corresponding noncancerous tissue wa

s 0.31 ± 0.06s 0.31 ± 0.06– Significantly higher, Significantly higher, PP = 0.001 = 0.001


• Overexpressed SERCA2 mRNA was identified in cancerOverexpressed SERCA2 mRNA was identified in cancerous ous when compared with noncancerous tissues.– T indicates cancerous tissues– N, noncancerous tissues– M, marker– β-actin is an internal control


• SERCA2 antigen was detected in 39 (78SERCA2 antigen was detected in 39 (78%) by immunohistochemical staining.%) by immunohistochemical staining.

• SERCA2 was predominantly in expressed SERCA2 was predominantly in expressed in the cancer cellsin the cancer cells..

• All of the positive cases showed All of the positive cases showed brownish brownish cytoplasmic stainingcytoplasmic staining but no nuclear stainin but no nuclear staining.g.


• In the high SERCA2 expression group:In the high SERCA2 expression group:– The incidence of The incidence of serosal invasionserosal invasion (27/34, 79.4%) (27/34, 79.4%)

was significantly higher (P = 0.012) than in the low was significantly higher (P = 0.012) than in the low expression group (7/16, 43.8%).expression group (7/16, 43.8%).

– The incidence of The incidence of lymph node metastasislymph node metastasis (24/34, (24/34, 70.6%) was considerably higher (P = 0.009) than in 70.6%) was considerably higher (P = 0.009) than in the low expression group (5/16, 31.3%)the low expression group (5/16, 31.3%)

– The incidence ofThe incidence of advanced stage cancer advanced stage cancer (according (according to Tumor Node Metastasis classification) (25/34, to Tumor Node Metastasis classification) (25/34, 73.5%) was significantly higher (P = 0.004) than in the 73.5%) was significantly higher (P = 0.004) than in the low expression group (5/16, 31.3%)low expression group (5/16, 31.3%)

Survival Rates and Multivariate AnalysisSurvival Rates and Multivariate Analysis

• The overall survival rate for the high SERCA2 The overall survival rate for the high SERCA2 expression group was significantly lowerexpression group was significantly lower than that than that of the low SERCA2 expression group.of the low SERCA2 expression group.


• Table 2 showed that the independent prognostic factors identified by multivariate analysis were tumor stage (P=0.015) and SERCA2 expression (P=0.018).


• SERCA2 antigen staining was further SERCA2 antigen staining was further identified to be a significant and powerful identified to be a significant and powerful prognostic indicator in patients with CRC.prognostic indicator in patients with CRC.

DiscussionDiscussion

• The result of the current study indicated thThe result of the current study indicated that in CRC patients, at in CRC patients, SERCA2 mRNA is morSERCA2 mRNA is more frequently overexpressed in cancer tissue frequently overexpressed in cancer tissueses than in noncancerous tissue than in noncancerous tissue


• Calcium mobilization from the ER into the cytosoCalcium mobilization from the ER into the cytosol is suggested to be a key component of several l is suggested to be a key component of several signaling network controlling tumor cell growth, dsignaling network controlling tumor cell growth, differentiation, or apoptosis.ifferentiation, or apoptosis.

• Refilling of calcium into the ER from the cytosol Refilling of calcium into the ER from the cytosol by active ATP-driven ion transport is ensured by by active ATP-driven ion transport is ensured by SERCA enzymes.SERCA enzymes.

• SERCA enzymes that accumulate calcium in the SERCA enzymes that accumulate calcium in the ER, and the subsequently anomalous calcium hER, and the subsequently anomalous calcium homeostasis may play an important role in neoplaomeostasis may play an important role in neoplastic transformationstic transformation..


• The intracellular calcium homeostasis becomes The intracellular calcium homeostasis becomes progressively anomalous during colon progressively anomalous during colon carcinogenesis, as reflected by deficient carcinogenesis, as reflected by deficient SERCA3 expression.SERCA3 expression.

• SERCA3 expression is barely detectable in SERCA3 expression is barely detectable in moderately differentiated tumors, and is moderately differentiated tumors, and is undetectable in poorly differentiated undetectable in poorly differentiated adenocarcinoma of the colon.adenocarcinoma of the colon.

• SERCA3SERCA3 constitutes a vital process in the constitutes a vital process in the development of CRC, and isdevelopment of CRC, and is a new and a new and potentially useful marker for the study of the potentially useful marker for the study of the state of differentiation of CRCstate of differentiation of CRC..


• In contrast to SERCA3 analysis from other In contrast to SERCA3 analysis from other previous studies, the SERCA2 expression previous studies, the SERCA2 expression levels in the current work are not levels in the current work are not significantly related to cell differentiation significantly related to cell differentiation by histological examinations.by histological examinations.


• The inactivation of the SERCA2 gene is a frequeThe inactivation of the SERCA2 gene is a frequent and early event during carcinogenesis in somnt and early event during carcinogenesis in some cancers and that loss of its expression may be e cancers and that loss of its expression may be regulated partly by an epigenetic mechanism via regulated partly by an epigenetic mechanism via promoter methylation.promoter methylation.

• The inactivation of SERCA2 gene result in impaiThe inactivation of SERCA2 gene result in impaired uptake of cytosolic calcium into the ER and cred uptake of cytosolic calcium into the ER and consequent disruption of calcium signaling, causionsequent disruption of calcium signaling, causing Darier disease, an autosomal-dominant skin ng Darier disease, an autosomal-dominant skin disorder characterized by loss of cellular adhesidisorder characterized by loss of cellular adhesion, proliferation, and abnormal keratinization.on, proliferation, and abnormal keratinization.


• SERCA2 is up-regulated in CRCSERCA2 is up-regulated in CRC, which , which may result in the disturbance of calcium may result in the disturbance of calcium homeostasis through increasing the ER homeostasis through increasing the ER calcium uptake.calcium uptake.


• High SERCA2 protein expression is consideHigh SERCA2 protein expression is considerably correlated to serosal invasion, lymph nrably correlated to serosal invasion, lymph node metastasis, advanced tumor stage, and ode metastasis, advanced tumor stage, and poor actual overall survival.poor actual overall survival.

• SERCA enzyme-dependent intracellular sigSERCA enzyme-dependent intracellular signaling pathways may ensure this metastatic naling pathways may ensure this metastatic process.process.

• The induction of SERCA2 protein expressioThe induction of SERCA2 protein expression may participate, at least in part, in the progn may participate, at least in part, in the progression of CRCression of CRC..


• The insulin growth factor-induced prostate The insulin growth factor-induced prostate cancer cell proliferation through increasing cancer cell proliferation through increasing calcium concentration within the ER, and tcalcium concentration within the ER, and tumor necrosis factor-umor necrosis factor-αα reduced cell prolife reduced cell proliferation and induced apoptosis by decreasinration and induced apoptosis by decreasing calcium concentration within the ER.g calcium concentration within the ER.


• The characteristics of the increased refillinThe characteristics of the increased refilling of the ER calcium store and its consequg of the ER calcium store and its consequent influence on store-operated calcium infent influence on store-operated calcium influx in one possible explanation.lux in one possible explanation.

• Treatment of head and neck squamous celTreatment of head and neck squamous cell carcinoma using the calcium influx inhibitl carcinoma using the calcium influx inhibitor would inhibit cell growth and invasion in or would inhibit cell growth and invasion in vivo.vivo.


• Overall, the authors’ results indicated that:Overall, the authors’ results indicated that:– SERCA2 mRNA was overexpressed in CRC tissueSERCA2 mRNA was overexpressed in CRC tissue– High SERCA2 expression level were correlated with:High SERCA2 expression level were correlated with:

• serosal invasionserosal invasion• lymph node metastasislymph node metastasis• advanced tumor stageadvanced tumor stage• poor prognosispoor prognosis

– The potential role for SERCA2 expression level as a neThe potential role for SERCA2 expression level as a new prognostic biomarker for CRCw prognostic biomarker for CRC

Thanks for Your Attention!!Thanks for Your Attention!!

Presented by TMUH Int. Hsin-Chih Huang

sarco/endoplasmic reticulum calcium-atpase 2 expression as a tumor marker in colorectal cancer

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