Plasma Membrane Calcium ATPase Expression in Human Colon Multistep Carcinogenesis

AUTHOR QUERY SHEET Author(s):

Jan H. R¨uschoff, Timo Brandenburger, Emanuel E. Strehler, Adelaida G. Filoteo, Ernst Heinm¨oller, Gerhard Aum¨uller, and Beate Wilhelm

Article title:

Plasma Membrane Calcium ATPase Expression in Human Colon Multistep Carcinogenesis 657817 1) Query sheet 2) Article proofs

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Contrib. No.

Given name(s)

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Jan H.

R¨uschoff

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Timo

Brandenburger

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Emanuel E.

Strehler

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Adelaida G.

Filoteo

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Ernst

Heinm¨oller

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Gerhard

Aum¨uller

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Beate

Wilhelm

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AQ2. Au: Please check and clarify whether the provide fourth affiliation is OK. AQ3. Au: Please check and confirm whether the city and country name of the companies appearing in the sentence ”Total RNA (2.5 μg) . . . by 15 min at 70◦ C” is OK. AQ4. Au: A declaration of interest statement reporting no conflict of interest has been inserted. Please confirm whether the statement is correct.

Cancer Investigation, 00:1–7, 2012 ISSN: 0735-7907 print / 1532-4192 online C Informa Healthcare USA, Inc. Copyright  DOI: 10.3109/07357907.2012.657817

ORIGINAL ARTICLE

Plasma Membrane Calcium ATPase Expression in Human Colon Multistep Carcinogenesis 5

1 4 ¨ ¨ Jan H. Ruschoff, Timo Brandenburger,2 Emanuel E. Strehler,3 Adelaida G. Filoteo,3 Ernst Heinmoller, 1 1 ¨ Gerhard Aumuller, andBeate Wilhelm

Department of Anatomy and Cell Biology, Philipps-University, Marburg, Germany,1 Department of Anesthesiology, University Hospital D¨usseldorf, D¨usseldorf, Germany,2 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, USA,3 Pathology Nordhessen, Kassel, Germany4

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Expression of PMCA has been reported to be affected in neuroblastoma (11), breast (12–14), hepatocellular (15), oral (16), and colon cancer (17–19). These studies were mainly done in cancer cell lines. The investigation of colon cancer cell lines revealed an up-regulated PMCA4 expression when cells were induced to differentiate, while PMCA1 levels remained constant (17, 18). On the other hand, recent microarray analyses suggested a down-regulation of PMCA4 mRNA in colon cancers (19). To the best of our knowledge, this is the first study investigating the protein expression of the PMCA and specifically of PMCA1 and PMCA4 in a large series of clinical patient samples. 84 formalin fixed paraffin embedded (FFPE) colorectal cancer samples of different tumor stages and grades, as well as precursor lesions and lymph node metastases were analyzed in situ both at the protein and mRNA levels. The results reveal a distinct down-regulation of the PMCA4 protein level in late-stage colorectal cancer while also suggesting that posttranscriptional control mechanisms are involved.

The expression of the plasma membrane Ca2+ ATPase (PMCA) was analyzed in a series of 84 formalin-fixed and paraffin embedded colon samples including normal mucosa (n = 32), adenoma (n = 19), adenocarcinoma (n = 27), and lymph node metastasis (n = 6) using (i) immunohistochemistry, (ii) mRNA in situ hybridization, and (iii) quantitative reverse-transcriptase PCR. A marked reduction of PMCA4 protein was observed in high-grade adenoma, colon cancer as well as lymph node metastasis, pointing to its potential role in the progression of cancer. However, PMCA4 RNA transcripts were unchanged or even increased in colon carcinomas, suggesting posttranscriptional regulation of PMCA4 during carcinogenesis. Keywords: Colorectal cancer; PMCA; Signal transduction

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Calcium is involved in a wide range of cellular regulatory processes (1). Apoptosis (2), cell differentiation (3), cell cycle control (4), proliferation, and hence tumorigenesis are (5) influenced by changes in the level of Ca2+ . To maintain normal cell and tissue function, a fine-tuned regulation of the calcium level is needed that is achieved by a variety of different ion channels, pumps, and exchangers (6). One of the Ca2+ -transporting enzymes belonging to this regulatory system is the plasma membrane Ca2+ -ATPase (PMCA), which is an ATP-driven active pump that transports Ca2+ across the plasma membrane out of the cytosol. In mammals, PMCA is encoded by four different genes, resulting in the four isoforms PMCA1–4. In addition, alternative RNA splicing generates more than 30 splice variants of the four PMCAs (7–9). The distribution of the isoforms and splice variants differs in various tissues and organs. While PMCA 1 and 4 are expressed in virtually all cells, PMCA 2 and 3 are restricted to a limited number of organ tissues (10). Recent studies have shown an altered expression of various Ca2+ regulatory pumps and channels in different cancers.

MATERIALS AND METHODS Tissue samples Formalin fixed paraffin embedded (FFPE) samples and tissue microarrays (TMAs) of normal colon mucosa (n = 32), colon adenoma (n = 19; 12 low grade, 7 high grade), adenocarcinoma (n = 27), and lymph node metastasis (n = 6) were obtained from the archives of the Institute of Pathology Nordhessen, Germany (Table 1). Histological sections were prepared from each tissue block and the different types of epithelium (normal vs. neoplastic), the location (in situ vs. invasive) including the degree of dysplasia in adenoma, and the grade of differentiation in carcinoma were determined by an experienced pathologist and marked on H&E stained tissue sections. Tumor typing was performed according to the World Health International

Both authors contributed equally. Correspondence to: Beate Wilhelm, Department of Anatomy and Cell Biology, Philipps-University, Robert-Koch-Str. 8, 35337, Marburg, Germany. email: [email protected]

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Table 1. Sample specifications IHC FFPE TMA ISH FFPE qRT-PCR FFPE

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Histological Classification (WHO) and tumor staging was determined according to UICC (20, 21). Immunohistochemistry and immunoscoring For immunohistochemical staining, 5 μm thick sections from all 84 FFPE specimens were used. After deparaffinization and hydration, slides were treated with 0.3% H2 O2 for 45 min and washed in PBS for 5 min. Sections were steamed for 20 min in EDTA-buffer (pH 8.4) before incubation with pan-specific antibody against PMCA (5F10, monoclonal, Dianova, Hamburg) or isoform-specific antibody against PMCA1 (polyclonal, Dianova, Hamburg, Germany) or PMCA4 (JA9, monoclonal, Dianova, Hamburg) for 1 hr at a dilution of 1:1,000 in PBS at room temperature. After washing in PBS, the primary antibodies were detected with biotinylated secondary antibody (1:200, in PBS, Zytomed Systems, Berlin, Germany) and avidin-biotin enzyme reagent (1:200, in PBS, Zytomed Systems, Berlin, Germany), followed by color development with 3,3’ diaminobenzidine (DAB). Finally, the sections were counterstained with hematoxylin. For expression analysis of the PMCAs, a semiquantitative scoring method was established as derived from Hirsch et al (22). Accordingly, staining was scored by intensity (0–3; negative, weak, moderate, or strong) and the percentage of tumor cells fitting to these groups (area%). To compensate for staining variability between the different antibodies and dayto-day variation the maximum possible staining intensity was defined for each antibody and case by setting the corresponding maximum staining intensity of internal reference structures to 100 in each tissue section. Reference structures used were vascular endothelium for PMCA4 and pan-PMCA and granulocytes for PMCA1. The final PMCA expression level within the tumor cells was then calculated as% of the maximum staining of the normal reference structures by [Sum tumor (intensity level (I) I-1 × area%) + (I-2 × area%) + (I3 × area%) divided by I-max × 100 of Normal crypt] × 100 (see Table 1 and Figure 2). Reverse transcription PCR Total RNA from human brain tissue was bought from Ambion (Austin, USA). Total RNA (2.5 μg) was reversetranscribed to single-stranded cDNA by using 1 μl of M-MLV RNAse H(–) reverse transcriptase (200,000 U/ml, Promega, Madison, WI, USA), 2 μl of oligo dT15 (Promega, Madison, WI, USA), and 0.5 mM dNTPs (Roche Diagnostics, New Delhi, India) in a volume of 40 μl for 50 min at 42◦ C, fol-

lowed by 15 min at 70◦ C. The PCR-master mixture consisted of 5 μl of 10× PCR buffer, 2 mM MgCl2 , 2 U Taq-polymerase (Platinum Taq, Invitrogen), 1 μl of 10 mM dNTPs, 50 pmol of each forward and reverse primer, and 1 μl of cDNA in a final volume of 50 μl. Amplification was performed on an MJ Research PTC-200 Peltier Thermal cycler (Biozym, Hessisch Oldendorf, Germany). The primer sequences for PMCA4 were as follows: forward primer 5 -TCG TCT GTC CCA TCT ATG AGG TG-3 , reverse primer 5 -TTT TAT ACA TTC CAT CTC TAC CGC AAC-3 . The conditions for PMCA4 amplification were as follows: 98◦ C for 2 min, 35 cycles of denaturation at 94◦ C for 30 s, annealing at 61◦ C for 30 s, and elongation at 72◦ C for 30 s, the last elongation phase was extended to 10 min. PCR product was electrophoresed on a 1.5% agarose gel and eluted using the Qiaex Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. R II The PCR product was subsequently cloned in a pCR vector using the TA Cloning Kit Dual Promotor (Invitrogen, de Schelp, Netherlands) and transformed into E.coli strain inv-alpha F’. Plasmid preparation was performed using the Plasmid-Kit from Qiagen (Hilden, Germany). The presence of cloned insert was checked by digestion with EcoRI (Amersham, Freiburg, Germany) and gel electrophoretic analysis. Furthermore, the insert was sequenced by MWG (Ebersberg, Germany) and the sequence received was aligned using the BLAST program at NCBI (www.NCBI.nlm.nih.gov/). Labeling of riboprobes by in vitro transcription In vitro transcription and labeling of riboprobes for PMCA4 was performed using the RNA-labelling kit (Roche diagnostics, Mannheim, Germany) as previously described (23). In situ hybridization The 5 μm sections from 7 normal colon mucosa samples, 4 low-grade adenomas, 4 high-grade adenomas, 7 colon cancers, and 2 lymph node metastases were deparaffinized and hydrated. In situ hybridization was performed as previously described in detail (24). Briefly, sections were treated with 0.1 M HCl for 12 min and incubated with proteinase K (5 μg/ml and 10 μg/ml) for 30 min at 37◦ C to increase probe accessibility. After incubation with 4% paraformaldehyde in PBS (pH 7.4) for 5 min, sections were rinsed twice in PBS. Thereafter, sections were acetylated and prehybridized (50% deionized formamide, 50 mM Tris-HCl-buffer, pH 7.5 containing 25 mM EDTA, 20 mM NaCl, 250 μg/ml t-RNA, and 2.5× Denhardt’s solution) for 2 hr at 48◦ C. Hybridization was performed overnight at 48◦ C in hybridization buffer Cancer Investigation

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consisting of prehybridization buffer plus 10% dextran sulphate and DIG-labeled probe (10 ng/μl). Both the antisense and sense probe of the 406 bp PMCA4-specific fragment were used. Unbound probe was removed by sequential washing. The hybridized probe was localized after overnight incubation using an antiDIG alkaline phosphatase-conjugated Fab-fragment (diluted 1:500, Roche Diagnostics, Mannheim, Germany). Bound alkaline phosphatase was visualized by an enzyme-catalyzed color reaction utilizing 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT; Promega, Madison, WI, USA) as chromogenic substrates.

Real-time quantitative PCR Quantitative mRNA expression analysis of PMCA4 was done using 7 normal mucosa specimens, 4 adenomas (low grade), and 7 carcinomas. Each tissue type was extracted by macrodissection of the corresponding area marked on the H&E stained slide. Total RNA was isolated from the FFPE tissue using the RNeasy FFPE Kit (Qiagen, Germantown, USA) following the manufacturer’s instructions. The concentration of RNA was determined after resuspending in RNAse- and DNAse-free water measuring the absorbance spectrophotometrically at 260 nm. The RNA (200 μg) was then reverse transcribed using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche diagnostics, Mannheim, Germany) following the manufacturer’s instructions. Primers for qRT-PCR were designed with ProbeFinder version 2.43 (Roche diagnostics, Mannheim, Germany) and had the following sequences: PMCA4 forward primer 5 -TGT CAT CTT TAT CCT TGT CTT TGC-3 , reverse primer 5 -TGG CTG GGT GGT GAA TGTA -3 ; housekeeping gene hB2M forward primer 5 -CAT TCC TGA AGC TGA CAG CAT TC-3 , reverse primer 5 -CAG AAA GAG AGA GTA GCG CGA G3 . The 5 μl of transcribed cDNA was amplified using the R 480 SYBR Green I Master (Roche diagnostics, LightCycler Mannheim, Germany) and 30 μM of each primer for PMCA4 and hB2M. The PCR program contained an initial denaturation at 95◦ C for 10 min, 40 cycles consisting of denaturation at 95◦ C for 10 s, annealing at 60◦ C for 10 s, and extension at 72◦ C for 30 s. Fluorescence curves were analyzed and crossing thresholds (CP) were determined for each sample. Target PMCA4 mRNA levels were normalized to endogenous control hB2M mRNA levels (CT). This CT was then expressed as relative to the control to calculate CT. Using the formula 2−CT fold changes were calculated. All samples were run in duplicates.

RESULTS Localization and quantification of PMCA (1–4), PMCA1, and PMCA4 expression in colon carcinomas Immunohistochemistry (IHC) in normal colon mucosa enterocytes and goblet cells displayed a marked staining for pan PMCA, PMCA1, and PMCA4 in the basolateral plasma membrane, which was similar in the enterocytes of lowgrade adenomas where goblet cell numbers are reduced. In high-grade adenomas, however, where strong staining of pan PMCA and PMCA1 could still be demonstrated, PMCA4 expression was markedly reduced or even completely lost. The same alteration could be observed in invasively growing adenocarcinomas where tumor cells have penetrated the lamina muscularis mucosae: there was strong pan PMCA and PMCA1 staining but no or only weak PMCA4 labeling (Figure 1). Based on the semiquantitative IHC scoring method, no statistically significant difference of pan PMCA and PMCA1 staining could be observed with respect to the various stages of colon adenoma and carcinoma. In contrast, a significant change of expression in the different stages of colon carcinogenesis was noticed for PMCA4. While normal mucosa and low-grade adenomas reached high staining scores, highgrade adenomas, carcinomas, and metastases revealed IHC scores at a significantly lower level (p < .01) (Figure 2). Analysis of PMCA4 transcripts in colon carcinomas In situ hybridization analyses were performed on tissue sections comprising 7 normal colon mucosas, 8 adenomas (4 low grade, 4 high grade), 7 adenocarcinomas, and 2 lymph node metastases in order to evaluate the mRNA expression of PMCA4. In normal colon mucosa strong perinuclear labeling was detected in the enterocytes and goblet cells. In contrast to the immunohistochemical analysis, however, no reduction of PMCA4 mRNA could be observed in the high-grade adenoma or in carcinoma and metastatic specimen (Figure 3). In order to quantify mRNA levels of the PMCA4 gene and to confirm our in situ hybridization data, qRT-PCR was performed. RNA was extracted from each section of normal mucosa, low-grade adenoma, and carcinoma. The PMCA4 gene expression level of carcinoma (n = 7) and low-grade adenoma (n = 4) was matched with that from corresponding normal mucosa (n = 7). There was a trend toward an increased PMCA4 mRNA expression level in carcinomas as compared to normal mucosa and low-grade adenoma samples (Figure 4). However, expression changes did not reach a level of statistical significance.

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Statistical analysis Statistical analysis was performed for the immunoscoring method using SPSS software 17.0. The Wilcoxon test was used to assess the significance of intergroup differences. For qPCR the relative expression software tools (REST) was used. Data are presented as mean ± S.D. as specified in the figures. The cut-off point for significance was set to p < .05. C 2012 Informa Healthcare USA, Inc. Copyright 

PMCA1 and PMCA4 isoforms are expressed in normal colon mucosa (10). In adenocarcinoma cell lines PMCA1b has been shown as the dominant isoform, whereas PMCA4b is expressed at a suppressed level (18). The aim of the present study was to characterize the expression and protein localization of the PMCAs, and specifically of PMCA4 during colon multistep carcinogenesis (25) using primary human tissue

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Figure 1. Immunohistochemical detection of all PMCAs combined (PMCA 1–4) (panels A, D, G, J), and of PMCA1 (panels B, E, H, K), and PMCA4 (panels C, F, I, L) in human colon FPPE-tissue sections. The tissue sections showed all relevant stages of the adenoma-carcinoma sequence consisting of normal colon mucosa (panels A–C), low-grade adenoma (panels D-F), high-grade adenoma (panels G-I), and colon carcinoma (panels J-L). In normal mucosa staining for pan PMCA, PMCA1, and PMCA4 is localized in the enterocytes and goblet cells. In the transition to low-grade adenoma, goblet cells disappear. Pan PMCA and PMCA1 labeling was detected in all adenoma and carcinoma stages, while PMCA4 was detected in low-grade adenoma but showed clearly reduced intensity of staining in the adenoma high grade (panel I) and carcinoma (panel L) stages. The figure displays one representative section of each carcinoma stage. Insert: higher magnification of PMCA4 staining; n for PMCA staining: 32 (N), 11 (AdLG), 7 (AdHG), 27 (Ca); n for PMCA1: 32 (N), 12 (AdLG), 7 (AdHG), 27 (Ca); n for PMCA4: 31 (N), 12 (AdLG), 7 (AdHG), 26 (Ca), Bar = 20 μm. Cancer Investigation

PMCA Expression in Colon Cancer 

Figure 2. PMCA immunohistochemical staining scores were calculated as relative values to the reference staining of tunica intima cells and granulocytes in each section. The staining scores reveal a significantly lower level of PMCA4 protein expression in the tissue of highgrade adenomas (AdHG), carcinomas (Ca), and metastasis (Meta) in relation to normal mucosa (N), while in low-grade adenoma (AdLG) no difference of PMCA4 protein to normal mucosa was obvious. The staining scores for pan PMCA (1–4) and PMCA1 show no differences between normal mucosa, adenoma, cancer, and metastasis. The bars represent means ± S.D. ∗∗ p < .01; n for pan PMCA: 32 (N), 11 (AdLG), 7 (AdHG), 27 (Ca), 6 (LN Meta); n for PMCA1: 32 (N), 12 (AdLG), 7 (AdHG), 27 (Ca), 6 (LN Meta); n for PMCA4: 31 (N), 12 (AdLG), 7 (AdHG), 26 (Ca), 6 (LN Meta).

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samples rather than immortalized cell lines. In order to localize and to quantify PMCA protein expression, a series of 32 normal colon mucosas, 19 colon adenomas (12 low grade, 7 high grade), 27 colon adenocarcinomas, and 6 lymph node metastases were analyzed by using immunohistochemistry on tissue sections. Levels of PMCA4 mRNA were additionally determined both by in situ hybridization and quantitative RT-PCR, in a selected number of cases. The results from immunohistochemistry on colon tissue indicate for the first time that PMCA4 protein is markedly decreased in de-differentiated high-grade adenoma and colon carcinoma, whereas PMCA1 protein levels appear to be unchanged in the same lesions. These observations match with the results in various colon cancer cell lines where induction of differentiation, e.g., by sodium-butyrate or postconfluence, was shown to be associated with a significant up-regulation of PMCA4 protein and a less pronounced induction of PMCA1 protein (17, 18). These cell culture results are thus in agreement with PMCA4 down-regulation at the protein level in less differentiated colon cancer tissues. In contrast, our results on PMCA4 mRNA expression differ from the findings in colon cancer cell lines (17, 18) and in a recent study on the expression of PMCA4 in colon tissues (19). While our in situ hybridization and qRT-PCR data show that PMCA4 RNA remains unchanged or may even be up-regulated during the process of carcinogenesis, the other studies reported a reduced PMCA4 transcript level in colon C 2012 Informa Healthcare USA, Inc. Copyright 

cancer tissues and cell lines (17–19). With regard to the cell culture experiments it should be kept in mind, however, that although colon carcinoma cell lines are useful models, they do not truly reflect the native cells embedded in a tissue microenvironment. Concerning the discrepancy between our study and the earlier work on colon tissues it is not clear as to how the tumor areas were dissected in that study (19); raising the possibility that differences in the methodology of tissue dissection could explain the different results. In the present study well-characterized, adequate tissue samples (normal, adenomas, and carcinomas) were dissected with high accuracy in collaboration with a pathologist, and results were validated by two different methods, i.e., in situ hybridization and qRT-PCR. In contrast, Aung and coworkers (19) used a chip technology on purchased mRNA without well-defined tumor dissection. Our data strongly suggest that posttranscriptional regulatory mechanisms (e.g., at the level of protein translation or stability) lead to a reduction of PMCA4 protein expression in colon cancer. Whether or not microRNAs play a role in this process has not been investigated so far (26). Cytosolic calcium controls a wide range of cell functions including cell proliferation (5). Among the main regulators of calcium homeostasis are different calcium pumps and channels. Beside the PMCA, which transports Ca2+ to the extracellular milieu, it has also been shown that expression of the endoplasmic reticulum Ca2+ transporting ATPase SERCA3 is lost or markedly decreased in malignant colon cancer cells (27). Furthermore, SERCA3 protein was found to be decreased in well-differentiated adenocarcinoma, and immunohistochemically undetectable in poorly differentiated adenocarcinoma (28). In contrast, SERCA2 mRNA and protein expression increased during colon carcinogenesis (29). Changes in colon cancer were also documented for Ca2+ channels. Some channels like TRPP8 (30), Cav 1.2 (31), and Cav1.1 (32) were up-regulated while other channels like Cav 3.1 and Cav 3.3 (33) were down-regulated. These changes suggest that multiple components of the cellular calcium handling system undergo remodeling during carcinogenesis, likely resulting in enhanced cell survival, resistance to apoptosis, and maintenance of proliferative capacity. What are the functional consequences of reduced PMCA4 protein expression for carcinogenesis of the colon? Reduced PMCA4 protein expression could obviously result in altered Ca2+ homeostasis characterized by impaired handling of transient Ca2+ spikes and/or by an increased intracellular Ca2+ level. It has been shown that down-regulation of PMCA4 by small interfering RNA does not induce cell death in colon cancer cell lines, whereas overexpression of PMCA4 reduced proliferation in these cells (19). Therefore, it may be speculated that the diminished levels of PMCA4 in high-grade colon adenoma and adenocarcinoma promote cell proliferation. A similar effect has been demonstrated for other Ca2+ transporters in carcinogenesis. Blocking Ttype Ca2+ channels caused decreased cytosolic Ca2+ levels in human glioma and murine neuroblastoma cell lines resulting in reduced cell proliferation while overexpression of the same channel increased cell proliferation (34). T-Type Ca2+ channels were also important for cell proliferation in colon,

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Figure 3. In situ hybridization to detect PMCA4 transcripts in FFPE human colon tissue sections. High expression of PMCA4 RNA was shown in all different stages including normal mucosa (panel A), low-grade adenoma (panel B), high-grade adenoma (panel C), and colon carcinoma (panel D). N = 7 (N), 4 (AdLG), 4 (AdHG), 7 (Ca), 2 (LN Meta), Bar = 20 μm.

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gastric, and prostate cancer cell lines (35). Diminished PMCA4 protein expression in colon adenocarcinoma may also affect the sensitivity of the tumor cells to apoptosisinducing stimuli. Previous studies have shown that PMCA4b interacts with the proapoptotic tumor suppressor Rasassociated factor 1 RASSF1A and that this interaction inhibits the activity of the ERK-pathway (36). Reduced PMCA4

Figure 4. Real time RT-PCR analysis of PMCA4 mRNA expression relative to normal mucosa (N) and normalized to hB2M. The bars represent the mean ± S.D. for n = 7 (N), 4 (Ad LG), 7 (Ca). PMCA4 transcript levels were higher in carcinoma compared to normal and low-grade adenoma. However, differences were not significant.

protein expression at the plasma membrane may thus render colon cancer cells more resistant to apoptotic stimuli via down-regulation of the pro-apoptotic RASSF1A-ERK signaling pathway. This, together with the proliferative effect due to altered Ca2+ handling may promote cancer progression in colon carcinoma. Further studies will have to elucidate the mechanism, including posttranscriptional regulatory events, responsible for the down-regulation of PMCA4 protein expression in multistep colon carcinogenesis. At a practical level, loss of PMCA4 as detected by immunohistochemistry may be useful as a marker of colon cancer disease progression and invasiveness.

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We thank Mrs. C. Keppler and Mr. M. Dreher (Institute of Anatomy, University of Marburg) for their expertise in tissue sectioning and in situ hybridization. Immunohistochemical support by Ms. Hampacher (Pathologie Nordhessen) is gratefully acknowledged.

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DECLARATION OF INTERESTS The authors report no declaration of interest. Cancer Investigation

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