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[Cancer Research 65, 8655-8661, October 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Haploinsufficiency of Atp2a2, Encoding the Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2 Ca2+ Pump, Predisposes Mice to Squamous Cell Tumors via a Novel Mode of Cancer Susceptibility

Vikram Prasad1, Gregory P. Boivin2, Marian L. Miller3, Lynne H. Liu1, Christopher R. Erwin4, Brad W. Warner4 and Gary E. Shull1

Departments of 1 Molecular Genetics, Biochemistry and Microbiology, 2 Pathology and Laboratory Medicine, and 3 Environmental Health, University of Cincinnati College of Medicine and 4 Division of Pediatric Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio

Requests for reprints: Gary E. Shull, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524. Phone: 513-588-0056; E-mail: gary.shull{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A null mutation in one copy of the Atp2a2 or ATP2A2 gene, encoding sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2), leads to squamous cell tumors in mice and to Darier disease in humans, a skin disorder that also involves keratinocytes. Here, we examined the time course and genetic mechanisms of tumor development in the mutant animals. Atp2a2+/– mice overexpressed keratins associated with keratinocyte hyperactivation in normal forestomachs as early as 2 months of age. By the age of 5 to 7 months, 22% of mutants had developed papillomas of the forestomach, and 89% of mutants older than 14 months had developed squamous cell papillomas and/or carcinomas, with a preponderance of the latter. Tumors occurred in regions that had keratinized epithelium and were subjected to repeated mechanical irritation. The genetic mechanism of tumorigenesis did not involve loss of heterozygosity, as tumor cells analyzed by laser capture microdissection contained the wild-type Atp2a2 allele. Furthermore, immunoblot and immunohistochemical analysis showed that tumor keratinocytes expressed the SERCA2 protein. Mutations were not observed in the ras proto-oncogenes; however, expression of wild-type ras was up-regulated, with particularly high levels of K-ras. Loss of the p53 tumor suppressor gene occurred in a single massive tumor, whereas other tumors had increased levels of p53 protein but no mutations in the p53 gene. These findings show that SERCA2 haploinsufficiency predisposes mice to tumor development via a novel mode of cancer susceptibility involving a global change in the tumorigenic potential of keratinized epithelium in Atp2a2+/– mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The endoplasmic reticulum (ER) is the principal Ca2+ storage organelle in cells, and one of the most critical determinants of Ca2+ levels in the ER of most cell types is the activity of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2; refs. 1, 2). Encoded by the Atp2a2 gene, this pump sequesters Ca2+ in the ER, and for this reason, its activity is an important regulator of normal Ca2+ homeostasis and signaling (3). Alterations in Ca2+ handling are associated with cell proliferation and differentiation (4, 5), and perturbations of Ca2+ homeostasis have been suggested to contribute to the development of cancer (6, 7). Reduced levels of SERCA2, caused by a null mutation in one allele of the ATP2A2 gene, leads to Darier disease in humans, an autosomal dominant skin disorder characterized by multiple keratotic papules (8). Although loss of one copy of the Atp2a2 allele in mice did not cause symptoms characteristic of Darier disease, it did lead to a very high incidence of squamous cell tumors (9), involving keratinocytes, the same cell type affected in humans.

Atp2a2 heterozygous mutant (Atp2a2+/–) mice developed hyperkeratinized tumors in regions of stratified squamous epithelia that included the oral mucosa, tongue, palate, esophagus, nonglandular mucosa of the stomach, skin, and genitalia. SERCA2 protein expression was reduced in the affected tissues, consistent with the hypothesis that the tumors were due to SERCA2 haploinsufficiency (9). This is an exciting possibility, as it would represent a novel mode of tumor susceptibility. However, an alternative possibility is that the tumors arise in rare cells in which the loss of Atp2a2 heterozygosity has occurred, which would be similar to mechanisms involving known tumor suppressor genes. Thapsigargin, an irreversible inhibitor of SERCA2 activity, serves as a tumor promoter in multistage mouse skin carcinogenesis (10) and has been shown to induce DNA synthesis (11) and cause growth stimulation (12) in cultured keratinocytes. It is unclear, however, whether these effects are due to partial inhibition of SERCA2, which would resemble haploinsufficiency, to complete inhibition, which would resemble loss of heterozygosity (LOH), or to some other effect that is independent of SERCA2.

The primary genetic lesions in a wide variety of tumors are gain-of-function mutations in the ras family of proto-oncogenes (13). Ras oncogene expression alone has been considered insufficient for tumor progression, with malignant conversion requiring additional lesions resulting in loss of tumor suppressor activity (14); the p53 gene is the most commonly mutated tumor suppressor gene. Thus, tumorigenesis often involves the accrual of a succession of stochastic, genetic mutations that occur over time within a specific cell, allowing that cell to progress toward malignancy (14). However, tumors induced by nongenotoxic carcinogens typically have revealed mutations in neither the H-ras nor the p53 gene (15). The generation of these tumors was associated with irritant damage and with hyperproliferation, with early effects characterized by hyperplasia and hyperkeratosis, progressing to papillomas and squamous cell carcinomas (16). These observations are consistent with the proposal that enforced cell proliferation can lead to alternative pathways of tumor development (17).

In this report, we present our analyses of the kinetics of tumor development in Atp2a2+/– mice and of possible genetic deficiencies involving the ras and p53 genes. We present evidence that Atp2a2 haploinsufficiency itself, rather than LOH, is responsible for the cancer phenotype of these mice, with enhanced tumor susceptibility occurring in keratinized epithelia. Lesions, such as hyperplasia and hyperkeratosis, occur in the early stages followed by tumor initiation and progression via nonclassic pathways involving elevated expression of wild-type H-ras, K-ras, and, surprisingly, p53.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. The generation of the Atp2a2+/– mice has been reported earlier (1). The mice were maintained on both a mixed 129/Svj and Black Swiss background and an inbred FVB/N background (back-crossed 10 generations). Genotypes were determined by PCR analysis of tail DNA as described before (1). All animals were maintained in accordance with institutional guidelines. Age-matched wild-type and mutant mice, usually siblings, were paired and observed as they aged; >90 pairs of mice were euthanized at different ages [5-7 or 8-13 and ≥14 months (when morbidity set in)] and tissues observed for any overt tumors. This allowed us (a) to study the kinetics of tumor development and (b) to determine genetic deficiencies, especially in the early tumors. At necropsy, the oral cavity and various organs were examined for tumors or abnormalities; tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with H&E. Histologic diagnosis was carried out to identify lesions, which included squamous cell papillomas and squamous cell carcinomas. Tumor tissue isolated for DNA or protein analysis was frozen in liquid nitrogen and stored at –80°C until further use.

Morphometry. Forestomach samples were fixed in 2.5% glutaraldehyde/2% paraformaldehyde, dehydrated, and embedded in Spurr's resin. Sections (2 µm) were stained with toluidine blue and used for morphometry. Avoiding areas of tangential cut, the thickness of the forestomach epidermis was measured with a line beginning at the basement membrane drawn to the lumen of the stomach (~10 measurements per animal) using SigmaScan Pro software (Jandel Scientific, Palo Alto, CA) and Excel. In the epidermis, mitotic cells and apoptotic bodies were counted and relative percentages were obtained. Data were analyzed using SAS version 8.0 (Cary, NC), and means and SEs of the means were determined by genotype, gender, and age using the General Linear Model. Results were considered significant when P < 0.05. When a variable in wild-type mice showed no significant differences among the categories of age and/or gender, these data were pooled. Apoptotic index was also determined using the Klenow FragEL DNA Fragmentation Detector kit (Calbiochem Biochemicals, La Jolla, CA).

Laser capture microdissection and PCR analysis. The protocol was adapted from the National Institute of Child Health and Human Resources LCM Research Resources. Tumors or tissue sections with hyperplasia (5 µm thickness) were stained with H&E and stored in a desiccator until microdissection. Cells were captured on CapSure Macro LCM Caps (Arcturus Bioscience, Inc., Mountain View, CA) using a PixCell II laser capture microscope (Arcturus Bioscience) at the Cincinnati Children's Hospital Laser Capture Core. Captured cells were digested in DNA extraction buffer [100 mmol/L Tris (pH 8.5), 10 mmol/L EDTA, 200 mmol/L NaCl, 0.2% SDS + 0.6 mg/mL proteinase K) for ~20 hours at 55°C. Protein and SDS were precipitated using the Puregene DNA Isolation kit (Gentra Systems, Minneapolis, MN). Genomic DNA was then isolated by isopropanol precipitation in the presence of glycogen and probed for the presence of the wild-type and mutant Atp2a2 alleles using PCR analysis as described earlier (1).

PCR amplification and sequencing analysis. Genomic DNA was isolated from tumor samples by phenol/chloroform extraction. The Ensembl Genome Browser,5 developed and maintained by the European Molecular Biology Laboratory and the Sanger Institute, was used to design primers for the various exons in the H-ras, K-ras, and p53 genes; for the p53 gene, exons 3 to 9 were sequenced with primers located in the intronic regions to permit analysis of all intron-exon boundaries. Primers were synthesized by Sigma-Genosys (The Woodlands, TX); primer sequences and PCR protocols can be obtained by contacting the authors. PCR-amplified fragments were resolved on a 2.5% agarose gel and extracted using the QIAQuick gel extraction kit (Qiagen, Valencia, CA). The University of Cincinnati DNA Core Facility using a 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) conducted sequencing of both strands, and the Basic Local Alignment Search Tool at the National Center for Biotechnology Information was used for alignments.

Immunoblot and immunohistochemical analysis. Tissues were pulverized in liquid nitrogen and suspended in homogenization buffer [10 mmol/L NaCl, 20 mmol/L PIPES (pH 7.0), 0.5% NP40, 0.05% ß-mercaptoethanol, 5 mmol/L EDTA, 50 mmol/L NaF, and a protease inhibitor cocktail (Sigma, St. Louis, MO)]. Tissue was homogenized manually using a glass homogenizer and solubilized on ice for ~2 hours. Protein concentration was estimated using the Coomassie Plus protein assay reagent (Pierce, Rockford, IL). Proteins were separated by reducing SDS-PAGE and transferred to nitrocellulose membranes. The various antibodies used were polyclonal anti-SERCA2 (antibody N1; ref. 18; provided by Jonathan Lytton, University of Calgary, Calgary, Alberta, Canada), monoclonal anti-H-ras (clone 7D7.2, Chemicon International, Temecula, CA), monoclonal anti-K-ras (clone F234, Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal anti-p53 (clone PAb 240, Novocastra Laboratories, Newcastle upon Tyne, United Kingdom), and polyclonal anti-mouse keratin 6 (K6) and keratin 10 (K10; Covance Research Products, Berkeley, CA). Horseradish peroxidase–conjugated secondary antibodies were from KPL, Inc. (Gaithersburg, MD). Chemiluminescence was developed using the LumiGlo Chemiluminescent substrate kit (KPL), and autoradiograms were developed using BioMax Films ML and MR (Kodak, Rochester, NY).

For immunohistochemistry, tumor tissue was excised, fixed in 10% buffered formalin, and embedded in paraffin. Sections (5 µm thickness) were deparaffinized, and on rehydration, antigen retrieval was carried out in 10 mmol/L citric acid (pH 3.0) at 37°C for 30 minutes. Sections were stained for SERCA2 protein using the N1 antibody in conjunction with the Cell and Tissue Staining kit (R&D Systems, Minneapolis, MN). Stained sections were dehydrated and mounted using Fluoromount G (Southern Biotechnology Associates, Inc., Birmingham, AL). Sections were analyzed using the Axioskop and Axiovision 4.0 software (Carl Zeiss, Germany).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Atp2a2+/– mice develop squamous cell tumors. We have reported previously that Atp2a2+/– mice on the mixed 129/Svj and Black Swiss background spontaneously developed squamous cell tumors on aging (9). In the current study, the cancer phenotype was found to persist even when the mice were transferred onto the FVB/N background, and there seemed to be no difference in penetrance of the phenotype for either background. As shown in Tables 1 and 2, 22% (6 of 27 mice) of 5- to 7-month-old mutant animals had papillomas in the forestomach. In addition, 11 other mutants in this group had foci of hyperplasia in the forestomach, 4 of which were associated with hyperkeratosis. Carcinomas were not observed in any of the 5- to 7-month-old mutants and no squamous cell tumors were observed in the 27 wild-type controls.


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  		Table 1. Penetrance of cancer phenotype: animals with tumors of keratinized epithelia/total number of animals

 

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  		Table 2. Spectra of squamous tumors in Atp2a2+/– animals

 
Thirty-three pairs of mice (and 11 additional unpaired mutants) were studied between 8 and 13 months of age. Of these 44 mutants, 29 animals developed squamous cell tumors, which included 21 carcinomas and 19 papillomas. The forestomach was the most affected tissue with 13 papillomas and 9 carcinomas. None of the 33 wild-type controls developed tumors of any type (Tables 1 and 2). When Atp2a2+/– mice were aged to ≥14 months, the cancer phenotype exhibited 89% penetrance (32 of 36 mice). Of the 55 mutants (from 36 pairs plus 19 unpaired mutants) studied at this age, only one 15-month-old female had no abnormalities, whereas 5 other animals developed moderate to severe hyperplasia of keratinized epithelia but no tumors. Although the most frequently affected tissue was forestomach, with 22 papillomas and 14 carcinomas, the tissue exhibiting the highest frequency of carcinomas was skin (17 of 55 mice, with 3 mice having multiple skin tumors). Interestingly, there were no papillomas in the skin, suggestive of a higher rate of tumor progression and in sharp contrast to forestomach, oral cavity, and esophagus, where papillomas persisted in mice older than 14 months of age. All of the tumors were well differentiated and had extensive keratinization (Fig. 1). Of the 36 wild-type mice aged to at least 14 months, only 2 animals developed squamous cell tumors, a papilloma on the lip and a carcinoma on the penis, at 18 and 17 months, respectively.



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  		Figure 1. Tumors in keratinized epithelia of Atp2a2+/– mice. A, squamous cell carcinoma in esophagus of 13-month-old FVB/N male. B, squamous cell papilloma in forestomach of 6-month-old FVB/N female. C, squamous cell carcinoma in palate of 14-month-old 129/Svj and Black Swiss female. D, squamous cell carcinoma in vagina of 13-month-old 129/Svj and Black Swiss female (identified as S2 in laser capture microdissection analysis in Fig. 2A). Bar, 200 µm (A) and 100 µm (B-D).

 


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  		Figure 2. Tumor cells retain the wild-type Atp2a2 allele and express SERCA2 protein. A, genomic DNA from both tumor keratinocytes (n = 12 tumors) and two foci of hyperplasia were isolated by laser capture microdissection and probed for the wild-type Atp2a2 allele by PCR analysis. PCR-amplified fragments from five samples resolved on a 2.5% agarose gel: S1, squamous cell carcinoma (penis); S2, squamous cell carcinoma (vagina); S3, squamous cell carcinoma (forestomach); S4, foci of hyperplasia (esophagus); S5, squamous cell papilloma (forestomach); H2O, water control; +/+, wild-type control; +/–, mutant control. B, immunoblot analysis of SERCA2 protein (12.5 µg/lane) in normal and tumor tissues (n = 8). Results from four tumor samples and both wild-type and mutant controls: T1, squamous cell papilloma (forestomach); T2, squamous cell carcinoma (forestomach); T3, squamous cell papilloma (tongue); T4, squamous cell papilloma (forestomach); WT1 and WT2, normal wild-type forestomachs; Het1 and Het2, normal mutant forestomachs. C, immunohistochemical analysis showing expression of SERCA2 protein in tumor keratinocytes. Sections (5 µm) were processed as described in Materials and Methods and probed for SERCA2 protein using the rabbit, polyclonal N1 antibody (18). Representative of results observed in all tumor sections analyzed and shows robust expression of SERCA2 in keratinocytes in squamous cell carcinoma from 12-month-old male FVB/N mutant. D, H&E staining of serial section from same tumor analyzed in (C). Bar, 200 µm (C and D).

 
Tumorigenesis in Atp2a2+/– mice does not involve loss of heterozygosity or loss of expression of the remaining wild-type allele. The susceptibility of keratinized epithelial cells in Atp2a2+/– mice to neoplastic transformation could result from either a cell-specific loss of wild-type Atp2a2 expression or Atp2a2 haploinsufficiency. In cases of a preexisting null mutation in one allele, the major genetic mechanism for loss of expression of the wild-type allele is LOH (19, 20). To test this hypothesis, we did laser capture microdissection, which allows selective isolation of tumor cells free of stromal cells, followed by analysis of the Atp2a2 alleles present in the tumor cells. The tumors were selected from a spectrum of affected tissues that included skin, palate, esophagus, forestomach, penis, and vagina. PCR analysis of genomic DNA from the laser-captured tumor cells revealed the presence of the wild-type allele in all of the samples (Fig. 2A). Given the number of tumors analyzed, this result rules out loss of the remaining wild-type allele as the primary genetic mechanism of tumorigenesis.

To determine whether SERCA2 protein expression from the wild-type allele may have been down-regulated via epigenetic mechanisms, immunoblot and immunohistochemical analyses were done. Tumors analyzed by immunoblotting included squamous cell papillomas and/or squamous cell carcinomas from forestomach, tongue, and palate. This experiment revealed that expression of the SERCA2 protein, when normalized to actin (Fig. 2B), tended to exceed levels seen in normal mutant tissues. Furthermore, immunohistochemical analysis of sections from squamous cell papillomas and squamous cell carcinomas revealed robust expression of the SERCA2 protein in tumor keratinocytes (Fig. 2C and D).

These results indicate that loss or inactivation of the single, wild type Atp2a2 allele was therefore not the primary genetic lesion driving tumor development. An exciting alternative would be that the single wild-type Atp2a2 allele, unable to compensate for the loss of the targeted allele, causes a tissue-level alteration in keratinized epithelia, making it susceptible to tumorigenesis.

Atp2a2+/– forestomachs with increased levels of keratin associated with keratinocyte hyperactivation. To elucidate the effects, if any, of reduced SERCA2 expression on keratinized epithelia before development of lesions, protein lysates of forestomach from 2-month-old Atp2a2+/+ and Atp2a2+/– animals were analyzed by immunoblotting to determine expression levels of keratins. This tissue, which is free of lesions at this age, was chosen because stomach was the most commonly affected organ. Keratinocytes express highly specific keratin markers depending on their state of proliferation and/or differentiation. Keratin 1 and K10 are expressed by postmitotic keratinocytes in the suprabasal layers during normal differentiation, whereas expression of alternative keratins, such as K6, is often indicative of enforced proliferation and activation, typically occurring during wound healing and inflammation. Expression of K6 was elevated 76% in histologically normal mutant forestomachs when compared with wild-type controls (Fig. 3). In contrast, there was no significant alteration in expression of K10 (Fig. 3).



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  		Figure 3. Expression of K6 and K10 in prelesional forestomachs from 2-month-old Atp2a2+/– animals. Immunoblot analysis (10 µg protein/lane) was carried out using homogenates of histologically normal forestomachs from 2-month-old animals. Note the increased expression of K6.

 
Despite the frequent association of higher K6 levels with increased proliferation, the keratinized epithelium of Atp2a2+/– forestomach did not exhibit evidence of increased proliferation. Morphometric analysis of forestomachs revealed no significant differences in epidermal thickness (Atp2a2+/+, 40.4 ± 2.7 µm; Atp2a2+/–, 36.9 ± 2.1 µm; P = 0.35) or mitotic index (percent mitosis: Atp2a2+/+, 0.57 ± 0.3; Atp2a2+/–, 0.70 ± 0.3; P = 0.78) between wild-type and mutant tissues. Similarly, there was no significant difference in the apoptotic index as determined by morphometry and an in situ end-labeling assay, similar to the TUNEL assay (data not shown). These results suggest that tissue level alterations occurring in prelesional Atp2a2+/– keratinized epithelia, as revealed by aberrant K6 expression in forestomach, are not due to alterations in keratinocyte proliferation or apoptosis but rather reflect altered differentiation.

Atp2a2+/– tumors lacked mutations in the ras and p53 genes, but ras and p53 protein levels were increased. Initiation of squamous cell tumors is typically associated with oncogene expression, with gain-of-function mutations occurring in one or more of codons 12, 13, or 61 of the ras genes. Furthermore, tumor progression is believed to require additional genetic lesions targeting tumor suppressor genes, such as p53, which may otherwise play a role in ras-mediated apoptosis or cell cycle arrest.

To determine whether these mutations were occurring, sequence analysis of H-ras, K-ras, and p53 exons was carried out to identify any such point mutations. Genomic DNA was isolated from five squamous cell papillomas and six squamous cell carcinomas from various tissues, including palate, esophagus, forestomach, and penis. For analysis of H-ras and K-ras genes, primers were designed to span exons 1 and 2, which include codons 12, 13, and 61. No mutations were found at these codons in any of the tumors. For mutational analysis of the p53 gene, primers were designed to span exons 3 to 9 because of increasing evidence that mutations can occur outside the gene segment traditionally analyzed (exons 5-8). In addition to the coding sequence, the intron-exon boundaries for each of the exons were also sequenced. No point mutations were observed in any of the samples analyzed (n = 7), and there was only a single tumor in which complete loss of the p53 gene had occurred. This tumor, observed in a 15-month-old FVB/N Atp2a2+/– female mouse, was unusual in that it was a massive squamous cell carcinoma weighing 10.7 g (>30% of total body weight), which originated in the forestomach and invaded the surrounding organs.

Protein homogenates of Atp2a2+/– forestomach tumors (five squamous cell papillomas and three squamous cell carcinomas) were examined by immunoblot analysis for expression of ras and p53 proteins. Expression of H-ras was increased 3.9-fold in the tumors, whereas no differences in expression were observed between normal wild-type and mutant forestomach tissues (Fig. 4A). The anti-H-ras antibody in all tumor samples identified a band of apparently higher molecular weight. Although the nature of this band was not investigated, the presence of two bands is presumed to be indicative of the extensive post-translational modifications, especially palmitoylation, of the H-ras protein. Similarly, K-ras protein expression exhibited a dramatic increase in the tumor samples, whereas it was below detectable levels in normal tissue (Fig. 4A). Similarly, the protein level of p53 was also significantly increased in all tumors analyzed (Fig. 4B), whereas it was below our limits of detection in normal tissue. The Atp2a2+/– tumors therefore had increased levels of wild-type H-ras, K-ras, and p53 proteins.



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  		Figure 4. Tumor cells express higher levels of wild-type ras and p53 proteins. Immunoblot analysis (10 µg protein/lane) was done using homogenates of both normal tissues and tumor tissues (n = 8 tumors). A, H-ras and K-ras proteins. B, p53 protein. WT1 and WT2, normal wild-type forestomachs; Het1 and Het2, normal mutant forestomachs; T1, squamous cell papilloma (forestomach); T2, squamous cell carcinoma (forestomach); T3, squamous cell papilloma (tongue); T4, squamous cell papilloma (forestomach); T7, squamous cell carcinoma (palate); T8, squamous cell carcinoma (forestomach).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The study of membrane transport proteins and channels in cancer has been largely restricted to their role in chemoresistance or chemosensitivity of neoplastic cells (21). The contribution of transport proteins to tumor growth and invasion is only beginning to be elucidated (22). Analyses of the Atp2a2+/– mouse, which lacks one copy of the gene encoding the ubiquitous ER Ca2+ pump, is important as it is the first animal model to establish a direct link between altered Ca2+ handling and cancer. Squamous cell tumors were observed in 22% of 5-7-month-old mutants, 61% of 8-13-month-old mutants, and 89% of mutants that were ≥14 months of age, with multiple tumors occurring in many animals. In contrast, only two squamous cell tumors were observed among 96 wild-type controls, and the incidence of spontaneous squamous cell neoplasms in the same tissues of B6C3F1 control mice, analyzed in the National Toxicology Program studies, was only 0.26% (23). The data discussed below indicate that Atp2a2 haploinsufficiency, rather than LOH, is responsible for the massive increase in tumor susceptibility of Atp2a2+/– keratinocytes.

The presence of the wild-type Atp2a2 allele in tumor keratinocytes from 12 different tumor samples, isolated by laser capture microdissection and therefore free of nontumor cells, argues strongly against the hypothesis that loss of the single remaining wild-type Atp2a2 allele is the primary genetic lesion underlying the cancer phenotype in the Atp2a2+/– mice. Furthermore, immunoblot analysis of tumor tissue showed that the intact SERCA2 protein was expressed in the tumor samples and revealed no evidence for truncation mutants, which were frequently found in human Darier disease patients (8). Although inactivating point mutations or small deletions or insertions are a formal possibility, in cases of a preexisting null mutation in one allele, loss of wild-type function most frequently occurs via loss of the wild-type allele rather than by point mutations (19, 20). In a study of human oral squamous cell carcinomas (24), no such ATP2A2 point mutations were identified in the 52 tumor samples analyzed. The same study reported, however, that SERCA2 protein levels were reduced in human oral squamous cell carcinomas apparently due to methylation of the promoter, leading to the proposal that ATP2A2 may function as a class II tumor suppressor gene (24). Although ATP2A2 down-regulation via promoter methylation may serve as an epigenetic mechanism in the development of oral squamous cell carcinomas in humans, the high levels of SERCA2 observed during immunoblot analysis and the immunohistochemical studies showing robust SERCA2 expression in tumor keratinocytes rules out such a mechanism in Atp2a2+/– mice. These findings provide strong evidence against loss of wild-type function as a mechanism of tumorigenesis in Atp2a2+/– mice. Down-regulation of SERCA2 expression in highly tumorigenic thyroid cell lines (25) and SERCA3 in colon cancer cell lines and tissue (26) have been reported; however, these were shown to be associated with dedifferentiation of transformed cells rather than neoplastic transformation per se.

These findings establish that SERCA2 haploinsufficiency, and the consequent alterations in Ca2+ homeostasis and/or signaling, which would have pleiotropic effects on cellular function (27–32), predisposes Atp2a2+/– mice to squamous cell tumors. Although morphometric analysis of histologically normal forestomachs revealed no significant differences in epidermal thickness and mitotic/apoptotic indices between wild-type and mutant tissues, aberrant expression of K6, typically associated with keratinocyte hyperactivation (33), was observed in 2-month-old prelesional mutant forestomachs. This finding strongly suggests that SERCA2 haploinsufficiency has a significant effect on keratinocyte gene expression and differentiation, causing an altered tissue environment. SERCA2 haploinsufficiency, as the mechanism of tumorigenesis in Atp2a2+/– mice, is consistent with the observation that SERCA2 haploinsufficiency is responsible for Darier disease in ATP2A2+/– humans (8). Although there have been only occasional reports of squamous cell carcinomas in Darier disease patients (34–36), the same cell type is affected in the Atp2a2+/– mice, suggesting that the underlying cellular perturbations emanating from reduced SERCA2 expression are similar. The basis for the species difference in tumor susceptibility is uncertain but not surprising given the major differences in overall cancer susceptibility between humans and mice, which may involve factors, such as telomerase activity and telomere lengths (37).

Studies of the pathogenesis of squamous cell carcinomas have indicated that the earliest stages of tumor development typically involve H-ras mutations (38). The lack of mutations in exons 1 and 2 of either the H-ras or K-ras genes indicated that the initiating event in Atp2a2+/– tumorigenesis was not the classic gain-of-function mutations affecting ras genes. Increased expression of wild-type ras, as observed in all Atp2a2+/– tumors analyzed, is a major determinant in the proliferation of head and neck squamous cell carcinoma cells and keratinocytes (39) and promotion of tumor growth and progression (40). Although it is well documented that Ca2+ cycling affects ras activation (41), the role of increased wild-type ras expression in Atp2a2+/– tumors remains uncertain. Ras is known to support both cell proliferation and differentiation/growth arrest (42). Studies involving spatially localized ras activity have suggested that it may be an important player in epidermal homeostasis with divergent effects in undifferentiated, proliferating basal epidermal cells when compared with postmitotic suprabasal epidermal cells (43).

Mutations in the ras genes have been considered insufficient for tumor progression (14), as oncogenic ras increases stability of p53 through p19ARF (44) and may cooperate directly with the tumor suppressor to induce senescence (45). Typically, loss-of-function mutations in the p53 gene permit continued tumor growth despite accumulation of the p53 protein. Furthermore, the accumulated p53 mutant proteins, in addition to lacking tumor suppression activity, may also function as oncogenic factors, driving tumor progression (46). The lack of p53 mutations in the Atp2a2+/– tumors expressing increased levels of p53 was intriguing but could account for the slow progression of these tumors; papillomas were observed in forestomachs of mice ~22 weeks old, but tumors did not cause morbidity until mice were ≥60 weeks of age. The single tumor we identified that lacked the p53 gene was the largest of at least 78 squamous cell carcinomas that we have observed in Atp2a2+/– mice. Although it was only a single occurrence, this observation supported the possibility that the increased wild-type p53 levels may have reduced the rate of Atp2a2+/– tumor growth or progression. Nevertheless, the apparent inability of wild-type p53 to halt Atp2a2+/– tumor progression could be an important molecular event linking reduced SERCA2 expression with tumor development and is currently under investigation. ER stress induced by thapsigargin, a SERCA2 inhibitor, has been shown to prevent p53-mediated apoptosis (32).

Tumors lacking mutations in both H-ras and p53 genes have been reported in rat forestomach in response to nongenotoxic carcinogens (15). The chronic effects of such carcinogens are mediated by enforced cell proliferation associated with the regeneration resulting from the toxicity of these agents (15, 16). Interestingly, tumors in the Atp2a2+/– mice also lacked mutations in the principal ras genes and were restricted to regions of repeated wounding or mechanical irritation (due, for example, to the abrasive action of the coarse mouse chow). In this regard, it is interesting that K6, which was increased in histologically normal Atp2a2+/– forestomachs, is up-regulated in response to wounding (33).

In conclusion, our data show that squamous cell tumors in the Atp2a2+/– mouse do not arise as a result of the loss of the remaining wild-type allele. LOH would have indicated that tumors in the Atp2a2+/– animals arise from rare, cell-specific events as in relatively well-understood mechanisms involving tumor suppressor genes. Rather, our findings suggest that SERCA2 haploinsufficiency, which would be expected to perturb both cellular Ca2+ homeostasis and Ca2+ signaling, alters the tissue environment in keratinized epithelia, making it uniquely permissive for tumor development.


   Acknowledgments
 
Grant support: NIH grants HL61974, DK50594, and ES06096.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Maureen Bender for expert animal husbandry.


   Footnotes
 
5 http://www.ensembl.org. Back

Received 1/ 5/05. Revised 5/31/05. Accepted 7/27/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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