Abstract
Context:
Prostate cancer metastases preferentially target bone, and the calcium-sensing receptor (CaSR) may play a role in promoting this metastatic progression.
Objective:
We evaluated the association of prostate tumor CaSR expression with lethal prostate cancer.
Design:
A validated CaSR immunohistochemistry assay was performed on tumor tissue microarrays. Vitamin D receptor (VDR) expression and phosphatase and tensin homolog tumor status were previously assessed in a subset of cases by immunohistochemistry. Cox proportional hazards models adjusting for age and body mass index at diagnosis, Gleason grade, and pathological tumor node metastasis stage were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association of CaSR expression with lethal prostate cancer.
Setting:
The investigation was conducted in the Health Professionals Follow-up Study and Physicians' Health Study.
Participants:
We studied 1241 incident prostate cancer cases diagnosed between 1983 and 2009.
Main Outcome:
Participants were followed up or cancer-specific mortality or development of metastatic disease.
Results:
On average, men were followed up 13.6 years, during which there were 83 lethal events. High CaSR expression was associated with lethal prostate cancer independent of clinical and pathological variables (HR 2.0; 95% CI 1.2–3.3). Additionally, there was evidence of effect modification by VDR expression; CaSR was associated with lethal progression among men with low tumor VDR expression (HR 3.2; 95% CI 1.4–7.3) but not in cases with high tumor VDR expression (HR 0.8; 95% CI 0.2–3.0).
Conclusions:
Tumor CaSR expression is associated with an increased risk of lethal prostate cancer, particularly in tumors with low VDR expression. These results support further investigating the mechanism linking CaSR with metastases.
“We studied the role of calcium sensing receptor (CaSR) in lethal prostate cancer in two large prospective cohorts. High CaSR expression was linked with a 2-fold higher risk of lethal prostate cancer.”
The calcium-sensing receptor (CaSR) is a transmembrane receptor that plays a key role in calcium homeostasis through the regulation of the PTH. Although it was initially described in bone, parathyroid, and kidney tissues, CaSR is widely expressed in tissues that do not play a role in maintaining calcium homeostasis, including prostate tissue (1–3). Moreover, there is compelling experimental evidence that suggests that CaSR plays an important role in prostate cancer progression. Prostate cancer most commonly metastasizes to bone, especially to areas of high bone turnover. Bone remodeling results in the release of extracellular calcium and growth factors, such as TGFβ, IGF-1, and IGF-2, which may activate the CaSR and promote metastatic progression. In prostate cancer cell lines, the CaSR mediates the effects of extracellular calcium to stabilize cyclin D, promote phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling, and increase metastatic potential (4). Our group has previously found in the Health Professionals Follow-up Study (HPFS) that common genetic variation in CaSR were associated with both higher and lower risk for lethal prostate cancer; in addition, there was evidence that the associations between some of the genetic variants with lethal prostate cancer may be modified by circulating vitamin D (5).
Prostate tumor cells that express high levels of CaSR may have a higher propensity to metastasize. However, the role of tumor CaSR expression in lethal prostate cancer has not been studied. We conducted a large pathoepidemiology investigation among prostate cancer patients in the HPFS and the Physicians' Health Study (PHS) to address whether high CaSR tumor expression is associated with lethal prostate cancer (defined as prostate cancer death or metastases to bone or other organ). Moreover, we investigated whether the association of CaSR with lethal prostate cancer progression may be modified by dietary calcium intake and by predicted circulating 25-hydroxyvitamin D (25[OH]D). Furthermore, we sought to examine whether the association of CaSR with lethal prostate cancer may differ according to prostate cancer molecular subtypes defined by vitamin D receptor (VDR) expression and phosphatase and tensin homolog (PTEN) tumor status.
Materials and Methods
Study population
We included 1241 men diagnosed with prostate cancer and treated by radical prostatectomy who were participants in the PHS (6, 7) or HPFS (8). The men were diagnosed with cancer between 1983 and 2009 and had available archival prostate tumor materials for evaluation. The PHS included two randomized trials investigating aspirin and vitamin supplements in the prevention of cardiovascular disease and cancer among a total of 29 071 male physicians, including 22 071 men aged 40–84 years starting in 1982 and an additional 7000 men aged 50 years or older starting in 1999, with all subjects receiving annual follow-up questionnaires. The HPFS is an ongoing cohort of 51 529 male health professionals followed up with biennial questionnaires since 1986. Incident prostate cancers (International Classification of Diseases, ninth revision, 185) were self-reported and confirmed through medical record and pathology report review. Details of the prostate cancer tumor cohorts within these two studies are available elsewhere (9).
Clinical and follow-up data
We abstracted data on tumor stage, prostate-specific antigen (PSA) level at diagnosis, and treatments from medical records and pathology reports. Standardized histopathological review by study pathologists of hematoxylin and eosin slides of each case provided uniform Gleason grading (10). Prostate cancer patients were followed up with written questionnaires to collect detailed information regarding treatments and clinical progression. For prostate cancer cases in the HPFS, treating physicians were contacted to collect clinical course information and to confirm development of metastases. For prostate cancer cases in the PHS, self-reported prostate cancer metastases have been validated to be reported with high accuracy. Cause of death was determined by medical record and death certificate review. Follow-up for mortality in the cohorts is greater than 98%.
Calcium intake and predicted circulating 25(OH)D
Calcium intake and predicted vitamin D levels were available for men in the HPFS cohort who answered validated food frequency questionnaires in 1986 and every 4 years thereafter. The questionnaire contained a list of 131 food and beverage items and included an open-ended section (11). Energy-adjusted cumulative average calcium intake was calculated until the date closest to but before diagnosis. Predicted circulating 25(OH)D levels at the date closest to, but before, diagnosis was calculated based on geographic region, skin pigmentation, dietary intake, supplement intake, body mass index (BMI), and leisure-time physical activity (a surrogate of exposure to solar UV-B) (12–14). In the HPFS, predicted circulating 25(OH)D is modestly correlated with measured circulating 25(OH)D (r2 = 0.30), and predicted circulating 25(OH)D was significantly associated with lower total cancer incidence and lower cancer mortality (13, 14).
Immunohistochemical assessment of protein expression
Immunohistochemistry (IHC) was performed using tumor tissue available from a biorepository of archival radical prostatectomy tissue. Hematoxylin and eosin slides were reviewed by our study pathologists to confirm prostate cancer and to identify tumor areas for tissue microarray (TMA) construction. Fourteen TMAs were constructed by sampling at least three 0.6-mm cores of tumor per case from the dominant nodule or nodule with the highest Gleason pattern. Five-micrometer sections of each TMA were deparaffinized in xylene, followed by a graded alcohol rehydration. The methods describing the IHC staining and scoring protocols for VDR, PTEN, Ki67, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL), CD34, pS6, and stathmin have been previously described (15–19) and are available in the Supplemental Methods. Below we describe the IHC staining and scoring protocol for CaSR.
Calcium-sensing receptor
For CaSR IHC, antigen retrieval was performed in EDTA/Tween 20 solution with pressure cooker (BioCare) according to the manufacturer's protocol. Rabbit anti-CaSR polyclonal antibody (Abcam; catalog number ab137408) was applied at 1:300 for 1 hour. Detection of the primary CaSR antibody was carried out using horseradish peroxidase-based EnVision kit (Dako). Sections were subsequently counterstained with hematoxylin, and the sections were dehydrated in a graded series of alcohol prior to coverslip application. CaSR intensity was scored manually (authors N.T. and S.F.) using a semiquantitative four-level scale: 0 (no staining by any tumor cells), 1 (faint staining), 2 (moderately intense staining), and 3 (intense staining). CaSR staining was predominantly membranous/cytoplasmic, but occasionally nuclear staining was observed. However, the scoring was only in reference to the membranous/cytoplasmic stain. The staining was almost invariably homogenous and uniform in distribution, and for this reason, only CaSR intensity was evaluated (Supplemental Figure 1). For the occasional cases in which staining was noted to be heterogeneous in distribution, the overall staining pattern was averaged (eg, if intensity grades 1 and 3 were present in approximately equal distribution, an average grade of 2 was given. Ten percent of cores were rereviewed by a study pathologist, and there was no major categorical discrepancy between the grades. In the rare instances of discordance in the CaSR grading, disagreement tended to be between grades 2 and 3 and, to a lesser extent, between grades 0 and 1.
Statistical methods
Based on the expertise of the study pathologists, tumor CaSR expression was dichotomized (<2 vs ≥2) to compare clinical and dietary characteristics and prognostic outcomes. We used Cox proportional hazards models to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between CaSR expression and the time to the development of lethal prostate cancer. Person-time was calculated from the date of cancer diagnosis to the earliest of the following time points: development of lethal prostate cancer, death from other causes, or end of follow-up (March 2011 for the PHS and March 2015 for the HPFS). The minimally adjusted models were adjusted for age at diagnosis (years, continuous), and the fully adjusted models were adjusted for age at diagnosis (years, continuous), BMI at diagnosis (kilograms per square meter, continuous), Gleason grade (≤6, 3+4, 4+3, 8–10, ordinal), pathological tumor stage (T2 N0/Nx M0/Mx, T3 N0/Nx M0/Mx, and T4 N1 M1, ordinal), and study cohort. If pathological tumor node metastasis (TNM) data were missing (n = 36), then we assigned the most common pathological TNM stage (T2 N0/Nx M0/Mx). As a sensitivity analysis, we tested alternative categorization of CaSR expression (0, <1, <2, ≥2). We additionally tested the association of CaSR with lethal prostate cancer stratified by pathological tumor stage (T2, N0/Nx, M0/Mx vs T3/T4 or N1 or M1) and Gleason score (2–7 vs 8–10). To investigate whether the association between CaSR and lethal prostate cancer may be modified by tumor VDR expression or PTEN loss, we cross-classified CaSR with VDR and PTEN and tested the association of the cross-classified terms with lethal progression. We also tested the significance of multiplicative interaction terms between CaSR and VDR and PTEN using the Wald test. Based on previous findings of the association between VDR expression and lethal progression (16), we dichotomized VDR expression according to the lower two-thirds of expression.
Analyses were conducted using SAS version 9.3 (SAS institute Inc), and all statistical tests were two sided with values of P < .05 considered statistically significant.
Written informed consent was obtained from each subject. This research project was approved by the Institutional Review Boards at Partners Healthcare and the Harvard T. H. Chan School of Public Health.
Results
The prostatectomy cohort included 1241 incident prostate cancer cases diagnosed from 1983 to 2009. Cases in the HPFS on average had a slightly higher BMI at diagnosis, PSA at diagnosis, and tended to have a higher Gleason grade compared with cases in the PHS (Supplemental Table 1). In both cohorts the combined mean age at diagnosis was 65.6 years, the median PSA at diagnosis was 6.5 ng/mL, and most cases were diagnosed in the PSA era (Table 1). Higher CaSR expression was not significantly associated with clinical characteristics, BMI, predicted circulating 25(OH)D, or calcium intake (Table 2), although the mean BMI at diagnosis was slightly higher (P = .11) and there was a higher proportion of people in the most extreme category of calcium intake (>1500 mg/d; Ptrend = .05). CaSR expression tended to be higher in tumor tissue compared with normal appearing tissue (P = .19, data not shown).
Table 1.
Selected Clinical Characteristics Among Men With Prostate Cancer Between 1982 and 2009 With Measured CaSR in the HPFS and the PHS
| Characteristic | n | |
|---|---|---|
| Follow-up time, y, median (q1, q3) | 1241 | 13.3 (9.7, 17.6) |
| Age at diagnosis, y, mean (SD) | 1241 | 65.6 (5.8) |
| BMI at diagnosis, kg/m2, mean (SD) | 1241 | 25.8 (3.4) |
| PSA at diagnosis, median (q1, q3)a | 1142 | 6.5 (4.8, 10.0) |
| Gleason grade, n (%) | 1241 | |
| <6 | 237 (19) | |
| 3 + 4 | 449 (36) | |
| 4 + 3 | 298 (24) | |
| 8+ | 257 (21) | |
| Pathological TNM, n, %a | 1205 | |
| T2 N0/Nx | 879 (73) | |
| T3 N0/Nx | 293 (24) | |
| T4 N1/M1 | 33 (3) | |
| Year of diagnosis, n, % | 1241 | |
| 1982–1989, pre-PSA era | 58 (5) | |
| 1990–1993, peri-PSA era | 269 (22) | |
| 1994 or later, PSA era | 914 (74) |
Abbreviation: q, quartile.
PSA at diagnosis data are missing for 99 cases; pathological TNM data are missing for 36 cases.
Table 2.
Association of CaSR Expression With Clinical Characteristics, Predicted 25(OH)D, and Calcium Intake in Men Diagnosed With Prostate Cancer Between 1983 and 2009 in the HPFS and the PHS
| CaSR 0 to <2 | CaSR ≥2 | P Valuea | |
|---|---|---|---|
| BMI at diagnosis, mean (SD) | 25.8 (3.4) | 26.3 (3.1) | .11 |
| Gleason grade, n, % | |||
| <7 | 209 (19) | 28 (19) | .55 |
| 3 + 4 | 400 (37) | 49 (33) | |
| 4 + 3 | 258 (24) | 40 (27) | |
| ≥8 | 225 (21) | 32 (21) | |
| Pathological TNM, n, % | |||
| T2 N0/Nx | 770 (73) | 109 (73) | .78 |
| T3 N0/Nx | 259 (25) | 34 (23) | |
| T4/N1/M1 | 27 (3) | 6 (4) | |
| Predicted 25(OH)D, n, %b | |||
| Quartile 1 | 180 (24) | 38 (28) | .40 |
| Quartile 2 | 181 (25) | 36 (26) | |
| Quartile 3 | 190 (26) | 31 (22) | |
| Quartile 4 | 184 (25) | 33 (24) | |
| Calcium intake, mg/d, n, %b | |||
| ≤750 | 248 (34) | 43 (31) | .05 |
| 751–1000 | 241 (33) | 42 (31) | |
| 1001–1250 | 124 (17) | 23 (17) | |
| 1251–1500 | 60 (8) | 9 (7) | |
| >1500 | 49 (7) | 20 (15) |
Cochran-Armitage trend test used for Gleason grade, pathological TNM, predicted 25(OH)D, and calcium intake; a t test was used for BMI at diagnosis
Predicted 25(OH)D and calcium intake were available only in the HPFS.
During a mean follow-up of 13.6 years (range 0.08–27.2 y), there were 83 lethal events. Of the 83 lethal events, 68 were fatal prostate cancer cases, 11 were cases of bone metastases, and four were organ metastases. We evaluated and satisfied the proportional hazards assumption by testing the significance of the interaction between CaSR status and follow-up time in a model adjusting for age and BMI at diagnosis, Gleason grade, pathological TNM, and study cohort (Wald test P = .48). In the age-adjusted model, high CaSR expression was associated with a higher risk for lethal progression (HR 2.2; 95% CI 1.3–3.6). This result persisted after adjusting for BMI at diagnosis, Gleason grade, pathological TNM, and cohort (HR 2.1; 95% CI 1.2–3.5; Table 3). The findings were similar for the alternate categorization of CaSR expression (0, <1, <2, ≥2; data not shown). The association between CaSR and lethal progression was not statistically different by tumor stage or grade at diagnosis; advanced tumors (defined as T3-T4 or N1 or M1; HR 2.8; 95% CI 1.5–5.2) compared with nonadvanced tumors (defined as T2, N0-Nx, M0-Mx; HR 1.6; 95% CI 0.6–4.3; Pinteraction = .37), and Gleason seven or fewer tumors (HR 3.0; 95% CI 1.4–6.3) compared with Gleason 8–10 tumors (HR 1.6; 95% CI 0.8–3.3; Pinteraction = .20; Supplemental Table 2).
Table 3.
Association of CaSR Expression With Lethal Prostate Cancer Among Men Diagnosed With Prostate Cancer Between 1983 and 2009 in the HPFS and the PHS
| CaSR 0 to <2 | CaSR ≥2 | |
|---|---|---|
| Lethal events, n | 63 | 20 |
| Total, n | 1092 | 149 |
| Person-time, y | 14 729 | 2157 |
| Model 1, HR (95% CI)a | 1.0 (reference) | 2.2 (1.3–3.6) |
| Model 2, HR (95% CI)b | 1.0 (reference) | 2.2 (1.4–3.7) |
| Model 3, HR (95% CI)c | 1.0 (reference) | 2.0 (1.2–3.3) |
| Model 4, HR (95% CI)d | 1.0 (reference) | 2.1 (1.2–3.5) |
Time to event analysis using Cox proportional hazards model adjusted for age at diagnosis.
Model 1 + BMI at diagnosis and Gleason grade.
Model 2 + pathological TNM.
Model 3 + cohort.
Table 4 presents the association of CaSR with selected molecular tumor characteristics. Higher CaSR expression was associated with measures of angiogenesis including smaller (P = .02) and more irregular blood vessels (P ≤ .0001). CaSR expression was significantly associated with higher expression of the PI3K/AKT/mTOR pathway markers pS6 (P = .01) and stathmin (P = .005) but was not associated with PTEN loss. There was a suggestive trend for a positive correlation between CaSR expression and higher VDR expression (P = .07). No association was found with the proliferation marker, Ki67, or the apoptosis marker, TUNEL. Of note, adjusting CaSR for angiogenesis attenuated the association between CaSR and lethal prostate cancer (HR 1.1; 95% CI 0.5–2.3). Adjusting CaSR for tumor expression of PTEN, pS6, stathmin, or VDR did not appreciably alter the association between CaSR and lethal progression (data not shown).
Table 4.
Association of CaSR Expression With Selected Tumor Biomarkers Among Men Diagnosed With Prostate Cancer Between 1983 and 2009 in the HPFS and the PHSa
| Characteristic | n | CaSR 0 to <2 | CaSR ≥2 | P Valueb |
|---|---|---|---|---|
| Proliferation indexc | 762 | 0.1 (0, 0.5) | 0.1 (0, 0.4) | .30 |
| Apoptosis indexd | 650 | 0.5 (0, 2.0) | 0.5 (0, 3.0) | .46 |
| VDR high, n, %e | 729 | 214 (33) | 37 (43) | .07 |
| PTEN loss, n, %f | 977 | 138 (16) | 19 (19) | .43 |
| pS6g | 739 | 0.12 (0.05, 0.28) | 0.17 (0.07, 0.38) | .01 |
| Stathminh | 713 | 0.016 (0.007, 0.04) | 0.022 (0.01, 0.05) | .005 |
| Angiogenesis | ||||
| Vessel areai | 388 | 486 (373, 671) | 405 (317, 574) | .02 |
| Vessel irregularityj | 388 | 3.9 (3.2, 4.6) | 4.8 (4.0, 5.3) | <.0001 |
All values are median (quartile 1, quartile 3) unless otherwise noted.
P values are based on the Wilcoxon-Mann-Whitney test for Ki67, TUNEL, pS6, stathmin, vessel area, and vessel irregularity and the χ2 test for PTEN and VDR.
Proliferation index is Ki67%-positive nuclear staining.
Apoptosis index is TUNEL percentage-positive nuclear staining.
VDR expression was dichotomized according to the lower two-thirds of expression.
A tissue core was considered to have PTEN protein loss if the intensity of cytoplasmic and nuclear staining was markedly decreased or entirely negative across greater than 10% of tumor cells compared with the surrounding benign glands and/or stroma.
pS6 is phospho-S6 ribosomal protein, expressed as mean area score.
Stathmin is expressed as mean area score.
Vessel area is the area comprised by microvessels (in square micrometers); smaller vessels are more angiogenic.
Vessel irregularity is calculated as the perimeter2/4π × area, with a value of 1.0 indicating a perfect circle and values greater than 1.0 indicating increasing irregularity.
The association of CaSR expression and progression to lethal cancer stratified by VDR expression and PTEN loss is presented in Table 5. There was suggestive evidence for effect modification by VDR status. Compared with the reference group (men with lower CaSR expression and higher VDR expression), men with higher CaSR expression and lower VDR expression had a higher risk for lethal progression (HR 4.5; 95% CI 2.0–10.0; Pinteraction = .16). Findings were similar after adjusting for Gleason grade and pathological tumor stage (HR 3.2; 95% CI 1.4–7.3; Pinteraction = .07). We also observed suggestive evidence for effect modification by tumor PTEN status. Compared with the reference group (men with lower CaSR expression and intact PTEN), men with higher CaSR expression and PTEN loss had the highest risk for lethal progression (HR 7.8; 95% CI 3.6–17.1; Pinteraction = .13). However, this association was markedly attenuated in the fully adjusted model (HR 3.1; 95%CI 1.4–7.1; Pinteraction = .56). The association of CaSR expression with lethal progression was not modified by calcium intake (Pinteraction = .52) or predicted circulating 25(OH)D (Pinteraction = .89; data not shown).
Table 5.
Association of CaSR Expression With Lethal Prostate Cancer Stratified by VDR Expression and PTEN Tumor Status Among Men Diagnosed With Prostate Cancer Between 1983 and 2009 in the HPFS and the PHS
| Na | Lethalb | Person-Years | Model 1c | Pinteraction Valued | Model 2e | Pinteraction Valued | |
|---|---|---|---|---|---|---|---|
| CaSR/VDR expression | |||||||
| Low CaSR/high VDRf | 214 | 12 | 3114 | Reference | .16 | Reference | .07 |
| Low CaSR/low VDRf | 429 | 32 | 6260 | 1.3 (0.7–2.6) | 1.0 (0.5–2.0) | ||
| High CaSR/high VDR | 37 | 3 | 641 | 1.2 (0.3–4.3) | 0.8 (0.2–3.0) | ||
| High CaSR/low VDR | 49 | 12 | 711 | 4.5 (2.0–10.0) | 3.2 (1.4–7.3) | ||
| CaSR/PTEN status | |||||||
| Low CaSR/PTEN intact | 649 | 29 | 8788 | Reference | .13 | Reference | .56 |
| Low CaSR/PTEN loss | 227 | 21 | 3030 | 2.1 (1.2–3.7) | 1.4 (0.8–2.4) | ||
| High CaSR/PTEN intact | 76 | 5 | 1080 | 1.4 (0.5–3.6) | 1.6 (0.6–4.1) | ||
| High CaSR/PTEN loss | 25 | 8 | 333 | 7.8 (3.6–17.1) | 3.1 (1.4–7.1) | ||
Number of total observations.
Number of lethal events.
Adjusted for age at diagnosis.
P value of VDR and CaSR or PTEN and CaSR multiplicative interaction is based on the Wald test.
Adjusted for age at diagnosis, BMI at diagnosis, Gleason grade, and pathological TNM.
High VDR is the upper one-third of expression; low VDR is the lower two-thirds of expression.
Discussion
To our knowledge, this is the first report to characterize CaSR expression in the prostate and to relate CaSR expression to lethal prostate cancer. In this large pathoepidemiology investigation, higher CaSR tumor expression was associated with an approximately 2-fold higher risk for lethal progression that was independent of Gleason grade and pathological stage. Higher CaSR expression was significantly associated with lethal progression among cases with lower tumor VDR expression but not among cases with high tumor VDR expression. These results are in line with our previous findings linking common genetic variation in the CaSR gene with lethal prostate cancer and collectively suggest that CaSR may play a role in promoting lethal prostate cancer. These findings support further investigation to better understand the mechanisms linking CaSR with lethal prostate cancer progression and further investigation of the potential of tumor CaSR expression to be used in conjunction with other tumor markers to improve prognostication.
The mechanism by which the CaSR may influence prostate cancer progression is not clear; however, growing experimental evidence implicates the bone remodeling environment in promoting the spread and growth of prostate tumor cells (20, 21). Bone remodeling releases calcium and other growth factors into circulation that may activate the CaSR and promote disease metastasis. In the skeletal metastatic prostate tumor cell lines, PC-3 and C4-2B, extracellular calcium activated the CaSR, leading to the up-regulation of the PI3K/AKT pathway, increased proliferation, and metastatic progression. These effects were not observed in the epithelial-derived LNCaP prostate tumor cells (4). Similarly, in bone metastasizing renal cell carcinoma and breast cancer cell lines, extracellular calcium treatment promoted the up-regulation of angiogenic growth factors (22) and the PI3K/AKT pathway and down-regulated PTEN (22, 23). Yano et al (3) reported CaSR activation in PC-3 prostate tumor cells by extracellular calcium induced production of the PTH-related protein, which may in turn promote bone resorption and further calcium release, potentially creating a cycle that is favorable for inducing bony metastases (21). Notably, these previous investigations of the role of CaSR in prostate cancer were limited to prostate cancer cell lines derived from bone metastasis and lymph nodes, whereas our investigation was concerned with the role of CaSR in primary prostate tumors. We evaluated the association between CaSR expression and molecular markers of disease aggressiveness. In line with previous reports, we found high CaSR expression to be significantly associated with higher expression of stathmin, a downstream target of PI3K/AKT signaling, and pS6, a marker of mTOR signaling. However, CaSR expression was not associated with cell proliferation or apoptosis, as characterized by Ki67 and TUNEL, respectively. We also observed a significant association between high CaSR and smaller irregularly shaped blood vessels. Adjusting for angiogenesis resulted in CaSR no longer being associated with lethal prostate cancer, suggesting that the effect of CaSR signaling may in part be mediated through promoting vascular growth. Taken together, these findings suggest that higher tumor CaSR expression may support prostate cancer progression, in part, through increased PI3k/AKT/mTOR signaling and by promoting vascular growth.
Prior to our investigation, there were no reports that related CaSR tumor expression with prostate cancer progression and only a few reports for other cancers. In a smaller study from Li et al (24), on a TMA of 148 primary breast tumors, protein expression of CaSR was reported to be down-regulated in neoplastic tissue compared with normal-appearing tissue, and lower CaSR expression was significantly associated with an increased risk of lethal breast cancer progression. The findings by Li et al contrast with our own findings and with prior experimental evidence that suggest CaSR signaling promotes metastatic growth of breast cancer to bone (1, 22, 25). However, in the context of colorectal cancer and parathyroid tumors, higher CaSR expression has been observed to be protective (26). In colorectal cancer, activation of CaSR promotes E-cadherin expression and down-regulates the Wnt/β-catenin pathway (27, 28).
There is strong biological plausibility that vitamin D signaling may modify the association of CaSR with prostate cancer progression. Vitamin D plays an important role in calcium metabolism and bone remodeling (29). The CaSR gene contains two promoters, both of which contain a vitamin D response element (30), providing a mechanism by which vitamin D regulates CaSR. Also, higher circulating 25(OH)D (31) and tumor VDR (16) expression have been associated with a lower risk of lethal prostate cancer. We observed evidence that signaling through the VDR may modify the role of CaSR in disease progression. High CaSR was associated with lethal progression in tumors with low VDR expression but not in tumors with high VDR expression. This finding should be interpreted cautiously and replicated in other studies, but it suggests that higher vitamin D signaling may inhibit CaSR tumor promoting signaling in the prostate.
There is growing evidence that tumor PTEN (32, 33) status may distinguish distinct molecular subtypes of prostate cancer. Moreover, as mentioned above, experimental research suggests that CaSR signaling promotes PI3K/AKT/mTOR signaling (22, 23), which is negatively regulated by PTEN (34, 35). We therefore evaluated the association between CaSR and PTEN loss and tested PTEN tumor status as a potential effect modifier of CaSR on lethal prostate cancer. CaSR expression was not associated with PTEN status, but we found suggestive evidence that tumor PTEN status may modify the association of CaSR with lethal progression. High CaSR was associated with lethal progression in tumors with PTEN loss but not in tumors with intact PTEN. After adjustment for Gleason grade and tumor stage, the interaction between CaSR and PTEN was markedly attenuated, but high CaSR remained significantly associated with lethal progression only in tumors with PTEN loss.
We also investigated whether calcium intake and predicted 25(OH)D may modify the association of CaSR with lethal progression. The association between calcium intake and prostate cancer is not clear. A meta-analysis of 12 studies and a recent analysis in the HPFS suggest that high calcium intake is associated with a higher risk of prostate cancer (36, 37). In the HPFS we previously found a significant inverse association between circulating 25(OH)D and lethal prostate cancer (31); however, a pooled analysis of five cohorts (including HPFS) did not find evidence for a significant association (38). In this study, we observed suggestive evidence that higher CaSR expression is associated with higher calcium intake, suggesting an intriguing possible mechanism through which calcium intake may influence prostate cancer risk. We did not find evidence that calcium intake modified the association between CaSR and lethal progression. Furthermore, we did not find an association between CaSR expression and predicted 25(OH)D, nor did predicted circulating 25(OH)D modify the association between CaSR and lethal progression.
This study is part of one of the largest prostate tumor tissue resources, with a prospective follow-up of up to 27 years for validated prostate cancer outcomes. Our study pathologists reviewed and provided uniform Gleason grades for all cases, reducing measurement error. We used cause-specific death and distant metastasis as the outcome, which are the most clinically relevant end points for prostate cancer. Despite our large sample size, this study is limited by the relatively small number of lethal events (n = 83) and would benefit from replication in other cohorts, particularly replication in large biopsy cohorts. We were limited to the use of estimated circulating vitamin D. Also, our study was limited to a subset of the cases in HPFS and PHS who received a radical prostatectomy, so the generalizability of our findings may be limited.
In conclusion, higher CaSR expression was associated with an approximately 2-fold higher risk of lethal progression that was independent of Gleason grade and tumor stage. The association between CaSR expression and lethal progression was most apparent in tumors with lower VDR expression. Collectively with the strong biological plausibility and evidence from experimental research, our findings suggest that tumor CaSR signaling may play an important role in metastatic progression of prostate cancer. These findings support further investigation to better understand the mechanisms linking CaSR with lethal prostate cancer progression and the potential of tumor CaSR expression as a prognostic biomarker.
Acknowledgments
We are grateful to the participants and staff of the Physicians' Health Study and Health Professionals Follow-up Study for their valuable contributions. In addition we thank the following state cancer registries for their help: Alabama, Arizona, Arkansas, California, Colorado, Connecticut, Delaware, Florida, Georgia, Idaho, Illinois, Indiana, Iowa, Kentucky, Louisiana, Maine, Maryland, Massachusetts, Michigan, Nebraska, New Hampshire, New Jersey, New York, North Carolina, North Dakota, Ohio, Oklahoma, Oregon, Pennsylvania, Rhode Island, South Carolina, Tennessee, Texas, Virginia, Washington, and Wyoming. We also thank Meir J. Stampfer for his guidance on this project. We acknowledge Channing Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School. The authors assume full responsibility for analyses and interpretation of these data.
This work was supported by National Institute of Health Grants R01CA136578, R01CA179129, UM1 CA167552, P50 CA090381, and T32 CA09001 (to T.U.A.); the Prostate Cancer Foundation Young Investigators Awards (to L.A.M. and K.M.W.); the American Cancer Society-Ellison Foundation Postdoctoral Fellowship PF-14-140-01-CCE (to T.U.A.); a Department of Defense Prostate Cancer Research Program Fellowship (to I.M.S.). The Physicians' Health Study is supported by Grants CA-34944 and CA-40360, Grant CA-097193 from the National Cancer Institute, and Grants HL-26490 and HL-34595 from the National Heart, Lung, and Blood Institute (Bethesda, Maryland).
Disclosure Summary: The authors have nothing to disclose.
Footnotes
- BMI
- body mass index
- CaSR
- calcium-sensing receptor
- CI
- confidence interval
- HPFS
- Health Professionals Follow-up Study
- HR
- hazard ratio
- IHC
- immunohistochemistry
- mTOR
- mammalian target of rapamycin
- 25(OH)D
- 25-hydroxyvitamin D
- PHS
- Physicians' Health Study
- PI3K
- phosphatidylinositol 3-kinase
- PSA
- prostate-specific antigen
- PTEN
- phosphatase and tensin homolog
- TMA
- tissue microarray
- TNM
- tumor node metastasis
- TUNEL
- terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling
- VDR
- vitamin D receptor.
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