Suppressive Roles of Calreticulin in Prostate Cancer Growth and Metastasis
2009; Elsevier BV; Volume: 175; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2009.080417
ISSN1525-2191
AutoresMahesh Alur, Minh M. Nguyen, Scott Eggener, Feng Jiang, Soheil S. Dadras, Jeffrey A. Stern, Simon Kimm, Kim Roehl, James M. Kozlowski, Michael Pins, Marek Michalak, Rajiv Dhir, Zhou Wang,
Tópico(s)Peptidase Inhibition and Analysis
ResumoCalreticulin is an essential, multifunctional Ca2+-binding protein that participates in the regulation of intracellular Ca2+ homeostasis, cell adhesion, and chaperoning. Calreticulin is abundantly expressed and regulated by androgens in prostate epithelial cells. Given the importance of both calreticulin in multiple essential cellular activities and androgens in prostate cancer, we investigated the possibility of a role for calreticulin in prostate cancer progression. Immunohistochemistry revealed the down-regulation of calreticulin in a subset of human prostate cancer specimens. Prostate cancer cells overexpressing exogenous calreticulin produced fewer colonies in both monolayer culture and soft agar. Furthermore, calreticulin overexpression also inhibited tumor growth in the orthotopic PC3 xenograft tumor model and macroscopic lung metastasis in the rat Dunning AT3.1 prostate tumor model. To address the potential mechanism of calreticulin suppression of prostate cancer, we generated calreticulin mutants with different functional domains deleted. The calreticulin mutants containing the P-domain, which binds to other endoplasmic reticulum chaperone proteins, were sufficient for the suppression of PC3 growth in colony formation assays. Overall, our data support the hypothesis that calreticulin inhibits growth and/or metastasis of prostate cancer cells and that this suppression requires the P-domain. Calreticulin is an essential, multifunctional Ca2+-binding protein that participates in the regulation of intracellular Ca2+ homeostasis, cell adhesion, and chaperoning. Calreticulin is abundantly expressed and regulated by androgens in prostate epithelial cells. Given the importance of both calreticulin in multiple essential cellular activities and androgens in prostate cancer, we investigated the possibility of a role for calreticulin in prostate cancer progression. Immunohistochemistry revealed the down-regulation of calreticulin in a subset of human prostate cancer specimens. Prostate cancer cells overexpressing exogenous calreticulin produced fewer colonies in both monolayer culture and soft agar. Furthermore, calreticulin overexpression also inhibited tumor growth in the orthotopic PC3 xenograft tumor model and macroscopic lung metastasis in the rat Dunning AT3.1 prostate tumor model. To address the potential mechanism of calreticulin suppression of prostate cancer, we generated calreticulin mutants with different functional domains deleted. The calreticulin mutants containing the P-domain, which binds to other endoplasmic reticulum chaperone proteins, were sufficient for the suppression of PC3 growth in colony formation assays. Overall, our data support the hypothesis that calreticulin inhibits growth and/or metastasis of prostate cancer cells and that this suppression requires the P-domain. Prostate cancer was the most frequently diagnosed non-skin cancer and the third leading cause of cancer death among American men in 2008.1Jemal A Siegel R Ward E Hao Y Xu J Murray T Thun MJ Cancer statistics, 2008.CA Cancer J Clin. 2008; 58: 71-96Crossref PubMed Scopus (10310) Google Scholar Androgens are intimately associated with prostate cancer progression and as such, androgen ablation remains the standard therapy for patients with metastatic prostate cancer.2Kozlowski J Grayhack J Gillenwater J Grayhack J Howards S Duckett J Carcinoma of the prostate. Mosby Year Book, Chicago1991: 1277Google Scholar However, hormone therapy is not curative and the vast majority of treated patients eventually experience disease progression. Elucidating the mechanism of androgen influence on prostate cancer is important as it may facilitate the development of more effective therapies and methods of disease prevention. Androgen action is mediated through the androgen receptor, which controls the expression of androgen-responsive genes.3Zhou Z Wong C Sar M Wilson E The androgen receptor:an overview. [Review].Rec Prog Hormone Res. 1994; 49: 249-274PubMed Google Scholar As androgen-responsive genes likely play important roles in prostate cancer progression, the characterization of their expression patterns and functions should provide insight into the roles of androgen in disease development. Calreticulin is one of the androgen-responsive genes in the prostate.4Wang Z Tufts R Haleem R Cai X Genes regulated by androgen in the rat ventral prostate.Proc Natl Acad Sci USA. 1997; 94: 12999-13004Crossref PubMed Scopus (94) Google Scholar, 5Zhu N Pewitt EB Cai X Cohn EB Lang S Chen R Wang Z Calreticulin: an intracellular Ca++-binding protein abundantly expressed and regulated by androgen in prostatic epithelial cells.Endocrinology. 1998; 139: 4337-4344Crossref PubMed Scopus (40) Google Scholar Androgen ablation by castration rapidly down-regulates calreticulin at both the mRNA and protein levels by more than tenfold. In contrast, androgen replacement rapidly restores the expression of calreticulin in the regrowth of the castrated prostate. Northern blot analysis of the tissue-specificity of calreticulin expression in the rat model shows that the most abundant levels occur in the prostate, as compared with the liver, kidney, brain, heart, muscle, and seminal vesicles. In situ hybridization and immunohistochemistry studies demonstrate that prostatic epithelial cells specifically express calreticulin.5Zhu N Pewitt EB Cai X Cohn EB Lang S Chen R Wang Z Calreticulin: an intracellular Ca++-binding protein abundantly expressed and regulated by androgen in prostatic epithelial cells.Endocrinology. 1998; 139: 4337-4344Crossref PubMed Scopus (40) Google Scholar, 6Zhu N Wang Z Calreticulin expression is associated with androgen regulation of the sensitivity to calcium ionophore-induced apoptosis in LNCaP prostate cancer cells.Cancer Res. 1999; 59: 1896-1902PubMed Google Scholar The expression profile and androgen-responsiveness of calreticulin in the prostate indicate that calreticulin may play a key role in androgen action in prostate epithelial cells. Calreticulin performs a variety of functions within the cell. This evolutionarily conserved protein localizes to the endoplasmic reticulum (ER),7Michalak M Milner R Burns K Opas M Calreticulin. 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The N-domain (residues 1 to 180) is thought to bind heavy metals (Zn2+) and interact with other ER chaperones, nuclear receptors, and nucleic acids.29Michalak M Corbett EF Mesaeli N Nakamura K Opas M Calreticulin: one protein, one gene, many functions.Biochem J. 1999; 344: 281-292Crossref PubMed Scopus (702) Google Scholar The P-domain (residues 181 to 290) contains a proline-rich region that forms an extended arm structure and interacts with other chaperones in the lumen of the ER. Lastly, the C-domain (residues 291 to 400) is a highly acidic region that binds Ca2+ and is involved in Ca2+ storage.30Nakamura K Zuppini A Arnaudeau S Lynch J Ahsan I Krause R Papp S De Smedt H Parys JB Muller-Esterl W Lew DP Krause KH Demaurex N Opas M Michalak M Functional specialization of calreticulin domains.J Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (243) Google Scholar Although the role of calreticulin in normal cellular functions and during embryogenesis is well-established, its role in human carcinogenesis remains poorly understood.31Gelebart P Opas M Michalak M Calreticulin, a Ca2+-binding chaperone of the endoplasmic reticulum.Int J Biochem Cell Biol. 2005; 37: 260-266Crossref PubMed Scopus (222) Google Scholar In the current study, we demonstrate the down-regulation of calreticulin protein in a subset of human prostate cancer specimens. Furthermore, we show that calreticulin overexpression in prostate cancer cells inhibits prostate tumor growth and metastasis and that its growth inhibitory role requires the P-domain. Overall, our present study provides evidence, for the first time, that calreticulin is capable of suppressing prostate cancer progression. Immunohistochemistry was performed as described previously.32Xiao W Zhang Q Jiang F Pins M Kozlowski JM Wang Z Suppression of prostate tumor growth by U19, a novel testosterone-regulated apoptosis inducer.Cancer Res. 2003; 63: 4698-4704PubMed Google Scholar We used 21 paraffin-embedded archival blocks obtained from human prostatectomy specimens. In 11 of the specimens was found high grade prostate intraepithelial neoplasia; in 10 specimens, cancer with Gleason grade 3 + 3; and in 3 specimens, cancer with Gleason grade 4 + 4. All of the specimens also contained benign glandular epithelial cells. For the immunohistochemistry, we used unstained sections, 4-μm thick. The immunostaining was performed using a polyclonal anti-calreticulin antibody as described previously.5Zhu N Pewitt EB Cai X Cohn EB Lang S Chen R Wang Z Calreticulin: an intracellular Ca++-binding protein abundantly expressed and regulated by androgen in prostatic epithelial cells.Endocrinology. 1998; 139: 4337-4344Crossref PubMed Scopus (40) Google Scholar Sections immunostained in the absence of primary antibody were included as controls for nonspecific staining. The slides were scored under a ×10 microscopic field by two pathologists (S.S.D. and M.P.) and the results were recorded as either no down-regulation (dash), mild down-regulation (arrow), or significant down-regulation (two arrows) of the calreticulin level in cancer cells relative to the benign glandular cells in the same slide. We also evaluated calreticulin expression in prostate cancer tissue microarrays. Tissue microarray blocks were prepared by transferring paraffin-block tumor cylindrical cores from 'donor' blocks to one 'recipient' block.33Dhir R Tissue microarrays: an overview.Methods Mol Biol. 2008; 441: 91-103Crossref PubMed Google Scholar Each tissue core was characterized by a genitourinary pathologist (M.P.). Immunostaining of the tissue cores was performed with anti-calreticulin antibodies. Two observers (S.E.E. and S.K.) then independently isolated the area of characterized tissues within the tissue core and scored the intensity of immunostaining using a subjective score of 0 (no staining) to 3 (intense staining). If the scoring discrepancy between the two observers was 1.5 or greater, the tissue core was discarded from analysis. Average immunostaining score for each type of prostatic histology was then determined. Specimens in tissue microarrays were grouped according to their pathology and histological Gleason grade. Statistical analysis was performed using SPSS statistical software (SPSS Inc, Chicago, IL). The analysis of calreticulin expression levels using the immunostaining score among individual histological groups was performed by the Games-Howell post hoc test and overall evaluation using an analysis of variance test. Evaluation of immunostaining scores greater or less than 1 was done using the Pearson χ2 test. Values of P < 0.05 were considered statistically significant. The vectors pcDNA3.1 (Hygro) and pcDNA3.1 (Neo) were obtained from Invitrogen (Carlsbad, CA). Through PCR, HindIII and XhoI sites were added to the 5′ and 3′ ends, respectively, of rat calreticulin (rtCrt), using the following primers: 5′RtCrt-HindIII, 5′-GGGAAGCTTATGCTCCTTTCGGTGCCGCTCC-3′ and RtCrtREV-XhoI, 5′-GGGCTCGAGGCCAGTGGCATCCTCCTCATCTTC-3′. The resultant PCR product was cloned into a pcDNA3.1 (N) vector containing the hemaglutinnin (HA) epitope, followed by the KDEL sequence that is found at the carboxyl-terminus of calreticulin. Domain mutant constructs were created similarly. All sequences were confirmed either by the Northwestern University Sequencing Core Facility (Chicago, IL) or Macrogen (Seoul, S. Korea). To clone green fluorescent protein (GFP)-tagged calreticulin expression vectors, GFP was PCR-amplified from pEGFP-C1 (Invitrogen, Carlsbad, CA), using the following primers: GFPCRTFOR.2, 5′-GATATCCTCGAGATGGTGAGCAAGGGCGAGGAG-3′ and GFPREV.1, 5′-GGGTCTAGACTACAGCTCGTCCTTGGCCTGCTTGTACAGCTCGTCCATGCCGAG-3′. We then replaced the HA tag with the GFP cDNA in the expression constructs. The human androgen receptor-negative prostate cancer line PC3 and the human androgen receptor-positive androgen-sensitive prostate cancer lines LNCaP were acquired from ATCC (Manassas, VA). The rat Dunning AT3.1 tumor cell line was kindly provided by Dr. Allen Gao (University of California, Davis, CA). PC3 and LNCaP cells were maintained in RPMI media supplemented with 10% fetal bovine serum, penicillin/streptomycin, and glutamine (complete media without drug) (Mediatech, Inc., Manassas, VA). AT3.1 cells were grown in RPMI-complete media that contained 250 nmol/L dexamethasone. (VWR, West Chester, PA). In colony formation assays, PC3 cells, at approximately 50% confluence in 6 well plates, were transfected in duplicate using Fugene 6.0 (Roche, Indianapolis, IN). The following day, the duplicate transfectants were combined and then diluted 1:50 and 1:100 in media containing either hygromycin (150 μg/ml) (VWR, West Chester, PA) or G418 (500 μg/ml) (Gemini Bio-Products, West Sacramento, CA). After 2 weeks in the drug-containing media, the stable clones were counted. Similarly, LNCaP cells, at approximately 60% to 70% confluence, were transfected in 6-well plates using Lipfectamine 2000 (Invitrogen, Carlsbad, CA). After 2 days, cells were resuspended in media containing either hygromycin (150 μg/ml) or G418 (500 μg/ml) and then cultured for approximately 3 weeks, at which time the colonies were counted. To control for transfection efficiency in colony formation assay, cells cultured in 6-well plates were transfected with 2.0 μg of pcDNA3 empty vector, pcDNA-Calreticulin, or pcDNA-HA-Calreticulin expression vectors in the presence of 0.25 μg of the pEGFP-C1 plasmid via Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in OPTI-MEM medium (Invitrogen, Carlsbad, CA) and after 4 hours the medium was replaced with RPMI 1640 medium. Twenty hours after transfection, cells were lysed in modified radioimmunoprecipitation buffer [50 mmol/L Tris (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA (pH 8.0), 1 mmol/L NaF, 2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, and protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO)]. GFP and HA-Calreticulin expression were determined by Western analysis using mouse anti-GFP polyclonal (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and mouse anti-HA monoclonal (Covance, Denver, PA) antibodies as described previously.34Saporita AJ Ai J Wang Z The Hsp90 inhibitor, 17-AAG, prevents the ligand-independent nuclear localization of androgen receptor in refractory prostate cancer cells.Prostate. 2007; 67: 509-520Crossref PubMed Scopus (73) Google Scholar Stable transfection was done similarly as above. When colonies were approximately 3 mm in diameter, they were isolated either by pipette tip or through the use of a cloning ring. Western blots were performed to determine relative levels of calreticulin, as described previously.6Zhu N Wang Z Calreticulin expression is associated with androgen regulation of the sensitivity to calcium ionophore-induced apoptosis in LNCaP prostate cancer cells.Cancer Res. 1999; 59: 1896-1902PubMed Google Scholar PC3 cells were plated on coverslips in 6-well plates. At approximately 30% to 50% confluence, cells were transfected with the GFP-tagged constructs as described above. After 1 to 2 days, the coverslips were fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 1% Triton X-100 (VWR, West Chester, PA) for 5 minutes, and then stained with calnexin (Cnx) antibody (Stressgen, Victoria, BC, Canada). Following extensive washing, coverslips were incubated in tetramethylrhodamine B isothiocyanate-conjugated anti-mouse secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 hour. Cells were observed and imaged using a Nikon TE2000U microscope attached to a digital camera. Images were captured and edited using Metamorph software 6.0 (Downingtown, PA) and Adobe Photoshop 7.0 (San Jose, CA). Soft agar assays were performed as described previously.35Kaighn ME Narayan KS Ohnuki Y Lechner JF Jones LW Establishment and characterization of a human prostatic carcinoma cell line (PC-3).Invest Urol. 1979; 17: 16-23PubMed Google Scholar Briefly, 2 ml of bottom agar in complete media (0.6%) was poured into 6-well plates. After solidification and drying, 1 ml of top agar (0.4%) containing 30,000 stably-transfected cells was applied to the bottom layer. After cooling, plates were placed in a 37°C CO2 incubator for 15 days and then the number of colonies was determined in five random fields per well in two different wells. Athymic nude mice were obtained from Harlan Sprague Dawley (Indianapolis, IN) and maintained in the Northwestern Animal Care Facility, according to approved animal care and use protocols. Orthotopic injections were performed similarly as described previously.36Rembrink K Romijn JC van der Kwast TH Rubben H Schroder FH Orthotopic implantation of human prostate cancer cell lines: a clinically relevant animal model for metastatic prostate cancer.Prostate. 1997; 31: 168-174Crossref PubMed Scopus (83) Google Scholar, 37Stephenson RA Dinney CP Gohji K Ordonez NG Killion JJ Fidler IJ Metastatic model for human prostate cancer using orthotopic implantation in nude mice.J Natl Cancer Inst. 1992; 84: 951-957Crossref PubMed Scopus (284) Google Scholar Briefly, PC3 cells (1 × 106 cells in 20 μl) overexpressing calreticulin were injected orthotopically into the prostate of anesthetized nude mice using a 30-gauge needle along with a Hamilton syringe. Proper injection was demonstrated by the production of a bubble within the prostate capsule. The mice were sutured using Ethicon sutures (Johnson & Johnson, Somerville, NJ) and stapled. Mice were euthanized approximately 30 days after the injection of tumor cells. The urogenital organs (bladder, prostate, and seminal vesicles) were excised and weighed immediately. This experiment was performed as described previously.38Shevrin DH Gorny KI Kukreja SC Patterns of metastasis by the human prostate cancer cell line PC-3 in athymic nude mice.Prostate. 1989; 15: 187-194Crossref PubMed Scopus (51) Google Scholar, 39Eggener SE Stern JA Jain PM Oram S Ai J Cai X Roehl KA Wang Z Enhancement of intermittent androgen ablation by "off-cycle" maintenance with finasteride in LNCaP prostate cancer xenograft model.Prostate. 2006; 66: 495-502Crossref PubMed Scopus (32) Google Scholar Briefly, 1 × 105 AT3.1 cells in 500 μl were injected subcutaneously into the right flank of 4- to 6-week-old male nude mice (Charles River Laboratories, Frederick, MD). Ten mice were used for each cell line. After 20 to 25 days, when the tumor burden became excessive, the mice were euthanized. Primary tumors at the site of injection were excised and weighed. The lungs were also removed, fixed in Bouin's solution and the number of macrometastases recorded. To investigate a possible role for calreticulin in prostate cancer progression, we performed immunohistochemical analysis of human prostate cancer specimens and demonstrated a decrease in calreticulin expression, as compared with benign tissue adjacent to the tumor (Figure 1A). After surveying benign tissue, high grade prostatic intraepithelial neoplasia lesions and tumors with a Gleason score of 6 or 8, we also found that the frequency and magnitude of calreticulin down-regulation appear to increase with Gleason score (Figure 1B). To further evaluate calreticulin expression in prostate cancer progression, we conducted immunohistochemical analysis on prostate tissue microarrays containing a total of 341 tissue cores, of which 277 (81.2%) met the criteria that the scoring discrepancy between the two observers was less than 1.5 and were further analyzed (Figure 1C). The average immunostaining score for benign prostate tissue (1.21) was higher than that of intermediate (Gleason 7: 0.91, P = 0.007) and high grade prostate cancers (Gleason 8 or 9: 0.77, P = 0.006). Low grade prostate cancer stained more intensely for calreticulin when compared with high grade disease (1.10 vs. 0.77, P = 0.048). Calreticulin expression in nodal metastases, while significantly different from benign tissue (0.85 vs. 1.21, P = 0.023), was most similar to intermediate grade prostate cancer. We then extended our analysis to evaluate if down-regulation of calreticulin expression was more common in prostate cancer than in benign tissue. We defined the immunostaining score of 1.0 or less as calreticulin down-regulation, and a score greater than 1.0 as normal or increased calreticulin expression. Calreticulin down-regulation was more common in cancerous versus benign tissue (Figure 1D). Benign tissue exhibited an immunostaining score of 1.0 or less in 45% of the cores compared with Gleason 5 or 6 (49%), Gleason 7 (63%), and Gleason 8 or 9 (77%) cancers. The incidence of down-regulation also increased along with histological aggressiveness (Pearson χ2, P = 0.004), as Gleason 8 or 9 cores had immunostaining scores of 1.0 or less much more frequently than Gleason 5 or 6 cores (77% vs 49%). While nodal metastases were the most frequent tissue type to demonstrate calreticulin down-regulation, the low number of tissue cores (n = 17) prevented any meaningful analysis. The ability to form colonies in vitro represents one characteristic of the transformed phenotype in prostate cancer.40Baron V De Gregorio G Krones-Herzig A Virolle T Calogero A Urcis R Mercola D Inhibition of Egr-1 expression reverses transformation of prostate cancer cells in vitro and in vivo.Oncogene. 2003; 22: 4194-4204Crossref PubMed Scopus (100) Google Scholar To investigate whether calreticulin expression modulates in vitro growth, we performed monolayer colony formation assays using PC3 and LNCaP cells stably transfected with either calreticulin-expression or control vectors. Experiments were done with both HA-tagged and untagged exogenous calreticulin. In PC3 cells, the untagged and HA-tagged calreticulin constructs both caused a twofold decrease in colony formation as compared with the empty control vectors (P < 0.05) (Figure 2, A and B). In LNCaP cells, calreticulin expression caused an even greater fold inhibition of colony formation (P < 0.05) (Figure 2, C and D). The reason for the decrease in colony formation was not due to a decrease in the transfection efficiency of the cultures that received the calreticulin construct, because the calreticulin constructs used in our experiments did not affect the co-transfection efficiency of a GFP expression vector (data not shown). These observations suggest a growth inhibitory role for calreticulin. To further investigate the growth inhibitory activity of calreticulin in prostate cancer cells, we tested the effect of calreticulin overexpression on prostate cancer cell growth in soft agar. Figure 3, B and C show the results from two representative PC3 clones that stably overexpress HA-tagged calreticulin, CrtHA.6 and CrtHA.10. Both clones expressed approximately twofold more calreticulin than either the parental PC3 cells alone or cells transfected with the control vector, as determined by Western blot (Figure 3A). When placed in soft agar in multiple independent experiments, CrtHA.6 and CrtHA.10 exhibited statistically-significant reductions in anchorage-independent growth as compared with control cells (P < 0.05, Figure 3C). As shown in Figure 3B, the diameter of the largest CrtHA.6 and CrtHA.10 colonies was approximately half that of the largest parental or the control vector-transfected PC3 colonies, which is also consistent with a growth suppressive function of calreticulin. Transfection with the control vector had no effect on growth. Anchorage-independent growth, considered an indication of cellular transformation, often corresponds with tumor growth in nude mice. Therefore, our result in soft agar assay suggests that calreticulin may possess tumor suppressive activity and so its down-regulation in prostate cancer specimens may contribute to malignant growth in vivo. To test whether calreticulin has tumor suppressive function in vivo, we examined the effect of calreticulin overexpression in a PC3 orthotopic xenograft tumor model in nude mice. We had initially isolated a group of six PC3 clones that overexpress calreticulin. Based on
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