Clinical Cancer Research Bridging the Lab and the Clinic in Cancer Medicine Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pootrakul, L.
Right arrow Articles by Cote, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pootrakul, L.
Right arrow Articles by Cote, R. J.
Clinical Cancer Research Vol. 12, 5987-5993, October 15, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Expression of Stress Response Protein Grp78 Is Associated with the Development of Castration-Resistant Prostate Cancer

Llana Pootrakul1, Ram H. Datar1, Shan-Rong Shi1, Jie Cai4, Debra Hawes1, Susan G. Groshen2, Amy S. Lee3 and Richard J. Cote1,4

Authors' Affiliations: 1 Department of Pathology, University of Southern California, Keck School of Medicine/USC Norris Comprehensive Cancer Center; Departments of 2 Preventive Medicine, 3 Biochemistry, and 4 Urology, University of Southern California, Los Angeles, California

Requests for reprints: Richard J. Cote, University of Southern California/Norris Comprehensive Cancer Center, 1441 Eastlake Avenue NOR 2424, Los Angeles, CA 90033. Phone: 323-865-0212; Fax: 323-865-0077; E-mail: cote_r{at}ccnt.hsc.usc.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Background: Induction of molecular chaperone Grp78 (78-kDa glucose-regulated protein) occurs in stress conditions that often characterize tumor microenvironments. We investigated the role of Grp78 in prostate cancer progression and the development of castration resistance, where cancer cells continue to survive despite the stress of an androgen-starved environment.

Experimental Design: Immunohistochemistry was done to examine Grp78 expression in 219 prostate cancers from patients with pathologic stage T3N0M0 disease [androgen ablation naive (untreated) and androgen ablation exposed (treated)] and castration-resistant prostate cancer. Classification of tumors was based on intensity of Grp78 cytoplasmic immunoreactivity and percentage of immunoreactive tumor cells. The associations of Grp78 expression with prostate cancer recurrence (clinical and/or serum prostate-specific antigen) and survival were examined in the untreated stage T3N0M0 group. Grp78 expression was also analyzed in the androgen-dependent LNCaP and castration-resistant C42B cell lines.

Results: The percentage of tumor cells expressing Grp78 was strongly associated with castration-resistant status (P = 0.005). Increased Grp78 expression was consistently associated with greater risk of prostate cancer recurrence and worse overall survival in patients who had not undergone prior hormonal manipulation. Grp78 expression was also increased in the castration-resistant LNCaP-derived cell line C42B and in LNCaP cells grown in androgen-deprived conditions compared with LNCaP cells grown in androgen-rich media.

Conclusion: Our findings show that up-regulation of Grp78 is associated with the development of castration resistance, possibly in part by augmenting cell survival as previously suggested, and may serve as an important prognostic indicator of recurrence in a subset of patients with T3N0M0 disease.

Resistance to castration therapies persists as the predominant challenge in the treatment of advanced prostate cancer. Androgen-dependent prostate cancer is characterized by the ability of cancer cells to undergo apoptosis in response to hormone depletion. The transition to castration-resistant prostate cancer requires the survival of tumor cells in such conditions, which may be attributed to a number of molecular mechanisms resulting in the evasion of apoptosis. One potential cellular survival mechanism in castration resistance is through up-regulation of stress response pathways, which confers protection to cells when they are subject to adverse conditions.

The glucose-regulated proteins (GRP) were initially identified as such in transformed chick embryo fibroblasts growing in glucose-deprived medium (1, 2). The most well studied member of the GRP family is Grp78, a 78-kDa protein also recognized as immunoglobulin heavy-chain binding protein (BiP; ref. 3). Normal functions of Grp78, which resides in the endoplasmic reticulum (ER) lumen, include proper folding and assembly of other polypeptides leading to formation of functional proteins, retention of unassembled precursors to the ER, targeting misfolded protein for degradation, ER Ca2+ binding, and the regulation of trans-membrane ER stress inducers (4–6).

The involvement if Grp78 in enhanced cell survival is suggested by the remarkable elevation of GRP78 transcription rates under various stress conditions (2). Recently, Grp78 has been shown to directly interact with intermediates of the apoptotic pathway, blocking caspase activation, where Grp78 induction results in increased cell survival and inhibition of apoptosis (7–9). As an ER chaperone, Grp78 is a key component of the unfolded protein response, promoting cell survival under ER stress (10, 11). The inherent roles and antiapoptotic capabilities of Grp78 indicate a potential role in cancer progression. Suppression of Grp78 level by antisense in fibrosarcoma results in inhibition of tumor growth (12). Elevation of Grp78 in the microenvironment of tumors due to nutrient deprivation or hypoxia confers survival advantage to cancer cells and leads to resistance to therapeutics (13). The association between increased Grp78 and malignancy has previously been implicated in various cancer cell lines and tumors (10, 14–18). Grp78 serum reactivity has recently been identified in patient sera as a putative marker of castration resistance (14). We were interested in assessing the role of Grp78 in prostate cancer progression and the development of castration resistance.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patient population. The recruitment and studies of patients described here have been approved by local institutional review boards (HS #-006044). This study included tumor samples from 219 patients with prostate cancer, comprised of three distinct cohorts of patients. One hundred ninety-one patients were classified as pathologic stage T3N0M0 disease (19), and specimens were obtained through radical retropubic prostatectomy with bilateral pelvic lymph node dissection at the University of Southern California/Norris Comprehensive Cancer Center between 1982 and 1996. These patients were further subdivided according to treatment status. Treatment consisted of neoadjuvant androgen ablation therapy with 1 mg diethylstilbestrol two or four times per day for 3 days to 20 weeks before radical prostatectomy. The stage T3N0M0 untreated group included 164 patients who were not exposed to preoperative androgen ablation therapy. The group of 27 men comprising the stage T3N0M0 treated group had received neoadjuvant androgen ablation therapy, and these patients were considered responsive to androgen (20). Tumor samples were obtained from 28 patients with castration resistance who underwent hormone ablation via orchiectomy and systemic hormone therapy but continued to show increasing prostate-specific antigen (PSA). Between 1990 and 1992, these men underwent transurethral resection to relieve urinary obstruction at Ruhr University, Bochum, Germany. All tumor grading was in concordance with the Gleason system (21).

Patient follow-up. Evaluations of the T3N0M0 patients were done at 1, 2, and 6 months postoperatively, at 6-month intervals for 5 years following surgery, then yearly. Biopsy was used to assess clinical recurrence of prostate cancer, and metastatic disease was determined according to bone scan or alternate clinical findings. PSA recurrence was designated to patients showing serum PSA levels ≥0.4 ng/mL on two consecutive tests. Median follow-up in the untreated group of 164 patients was 12.7 years with a range of 1.6 to 20 years. Median age was 67 years, ranging from 47 to 81 years.

Immunohistochemistry. Formalin-fixed 5-µm sections were taken from paraffin-embedded prostate cancer specimens and cell lines and mounted on poly-L-lysine–coated slides. The slides were deparaffinized in xylene, washed with 100% ethanol, followed by rehydration in 95% ethanol; 3% hydrogen peroxide in absolute methanol was used to quench endogenous peroxidase. Antigen retrieval was done using citrate buffer (pH 6) and microwaving for 30 minutes (22) followed by cooling at room temperature for 20 minutes. The slides were then blocked with normal horse serum for 20 minutes and incubated for 1 hour with anti-Grp78 rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:100 dilution in PBS. Incubation with biotinylated horse anti-rabbit secondary antibody at a 1:200 dilution was followed by avidin-biotin conjugate (Vector Laboratories, Inc., Burlingame, CA). Chromogen of 0.03% diaminobenzidine was then applied, with hematoxylin counterstaining. Negative controls consisting of diluent with no antibody and positive prostate cancer controls with heterogeneous immunoreactivity were used in all experiments.

Cultured prostate cancer cells (LNCaP and C42B) were harvested, cytospun on poly-L-lysine–coated slides at 250,000 per slide, and formalin fixed. Antigen retrieval was done using citrate buffer (pH 6) and microwaving for 5 minutes (22) followed by cooling at room temperature for 15 minutes. Subsequent steps in immunohistochemistry protocol follow as described above.

Immunoreactivity assessment of clinical samples. All slides were interpreted by two pathologists (S.R.S. and D.H.), who were blinded to all outcome data. Tumor scores were categorized based on two criteria: (a) percentage of tumor cells showing cytoplasmic immunoreactivity and (b) intensity of cytoplasmic immunostaining. For assessment according to percentage of cytoplasmic reactivity, tumors were classified as showing low Grp78 expression (≤50%) or high Grp78 expression (>50%). For intensity of cytoplasmic immunoreactivity, tumors were classified as having low Grp78 expression (≥1), moderate Grp78 expression (≥2), or high Grp78 expression (≥3). Grp78 status was assigned as negative to cases with <10% Grp78 immunoreactivity or weak (≥1) staining. All other cases were assigned positive Grp78 status. Upon identification of focal areas where Grp78 expression levels were markedly intense, tumors were further categorized by percentage of cells showing intense (≥3) Grp78 immunoreactivity (<5%, low Grp78; ≥5%, high Grp78). Due to the heterogeneity of Grp78 immunoreactivity, scoring corresponds with an overall evaluation of the entire tissue section. Lymphocytes, which are highly immunoreactive with anti-Grp78, were used as internal positive controls.

Cell culture. Androgen-responsive LNCaP and androgen-resistant LNCaP-derived C42B cells were grown in RPMI 1640 (Invitrogen, Carlsbad, CA) with 50 units/mL penicillin, 50 units/mL streptomycin, and 10% FCS (Mediatech, Inc., Herndon, VA). For preparation of androgen-depleted medium, FCS and RPMI 1640 were replaced by 10% dextran/charcoal-stripped serum (Omega Scientific, Inc., CA) and phenol-free RPMI 1640 (Invitrogen), as previously described in the literature (23). All cell lines were maintained in a humidified incubator at 5% CO2 and 37°C.

Automated cellular imaging. Immunostaining and evaluation of immunostained cell lines were carried out in triplicate, where immunoreactivity was assessed using ACIS II (Clarient, Inc., Aliso Viejo, CA; refs. 24–27). The ACIS II system consists of a computer-assisted bright-field microscope (x4, x10, x20, x40, and x60 objectives) coupled to a SONY 3-chip CCD camera. This fully automated system creates a reconstructed image of an immunohistochemistry stained slide and uses wavelength-specific technology to detect color differences between objects. Immunostained slides of cytospun cell lines were scanned at x4 magnification followed by image capture, transformation to pixels, and quantification by hue (color), saturation (color purity), and luminosity (brightness). Five regions of interest at x4 magnification were manually selected for each sample slide, and brown color (3,3'-diaminobenzidine chromogen) was assessed by ACIS software, which counts pixels based on 256 levels of color intensity. Representative areas were analyzed for intensity and percentage of cells positive for brown color.

Western blot analysis. For Western blot analysis, cell lysates from LNCaP and C42B cells were prepared by lysing in 1 mL ice-cold radioimmunoprecipitation assay buffer. Equal amounts of total protein from each sample were subjected to SDS-PAGE in a 7.5% Tris-HCl gel (Bio-Rad Laboratories, Hercules, CA). Following electrophoresis, the proteins were transferred to a pure nitrocellulose membrane (Bio-Rad Laboratories). The membrane was then incubated in Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NE) followed by overnight incubation with primary rabbit polyclonal anti-Grp78 antibody (1:500 dilution; Santa Cruz Biotechnology). Signal detection was done using Alexa Fluor 680 goat anti-rabbit antibody (Molecular Probes, Eugene, OR) and subsequent scanning of the membrane by the Odyssey Infrared Imager (model 9120, Li-Cor Biosciences). All bands from Western analysis were quantified for protein expression with Odyssey Infrared Imaging Software (Li-Cor Biosciences) to assess integrated intensity (pixel volume) as a measure of absorbance. Band density was represented as the ratio of average band intensity (I) of each sample to the average band intensity of the corresponding ß-actin control band.

Statistical analysis. {chi}2 and Fisher's exact tests were used to compare differences in Grp78 expression among all groups; if Ps for the overall test were significant at the 0.05 level, then these were used to analyze pairwise comparisons. Kaplan-Meier plots and the log-rank test were used to analyze the association of Grp78 expression with time to clinical and/or serum PSA recurrence and survival in the untreated stage T3N0M0 group; the stratified log-rank test was used for multivariable analyses. Results were considered significant at P < 0.05 for two-sided analyses.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Grp78 expression in localized prostate cancer. Immunohistochemistry was employed to evaluate Grp78 protein levels in tumors from 164 stage T3N0M0 untreated and 27 stage T3N0M0 treated prostate cancer patients. In the untreated group, 120 of 164 cases (73%) showed high Grp78 expression by percentage of cytoplasmic immunoreactivity (>50% stained tumor cells), as shown in Fig. 1 and Table 1 . Of the 27 cases in the treated group, however, 18 (67%) cases showed high Grp78 percent immunoreactivity (Fig. 1; Table 1). According to intensity of Grp78 immunoreactivity, 91 of 164 (55%) untreated cases showed moderate to high expression of Grp78 (Fig. 1; Table 1). In the treated group, 14 of 27 (52%) tumors showed moderate to high Grp78 expression (Fig. 1; Table 1). For percent immunoreactivity and intensity, the differences between the untreated and treated groups did not reach statistical significance (P = 0.484 and P = 0.913).


Figure 1
View larger version (76K):
[in this window]
[in a new window]
  		Fig. 1. Grp78 expression in prostate cancer. A, tumor from untreated T3N0M0 group showing >50% of tumor cells with moderately (≥2) intense Grp78 cytoplasmic immunoreactivity. Original magnification, x200. B, tumor from treated T3N0M0 group showing intense (≥3) focal Grp78 cytoplasmic immunoreactivity. Original magnification, x200. C, inset from (B), subpopulations of prostate cancer cells show intense Grp78 cytoplasmic immunoreactivity (arrows). D, tumor from castration-resistant group showing high (≥3) intensity cytoplasmic immunoreactivity. Original magnification, x200. E, immunostaining of Grp78 in LNCaP cells grown in FCS, LNCaP cells grown in androgen-depleted medium for 6 days (6d CSS), and C42B cells. AICS II–assisted computer imaging analysis shows that C42B (84% tumor cells reactive) and LNCaP cells grown in CSS for 6 days (64.2% tumor cells reactive) showed higher Grp78 cytoplasmic immunoreactivity than cells grown in FCS (24.5% tumor cells reactive). Percentages given as mean ± SD of five representative areas. Negative control with no primary antibody (left). Original magnification, x100. Inset magnification, x200.

 

View this table:
[in this window]
[in a new window]
  		Table 1. GRP78 expression (immunoreactivity) in untreated T3N0M0, treated T3N0M0, and castration-resistant prostate cancer

 
We identified focal areas of cells showing intense Grp78 immunoreactivity (≥5%; ≥3 intensity) in both the untreated and treated stage T3N0M0 cases (Fig. 1B and C). In the untreated cases, 36% (59 of 164) versus 44% (12 of 27) of treated cases were classified as positive for this intense focal immunoreactivity.

Grp78 expression in castration-resistant prostate cancer. Of the 28 castration-resistant tumors immunostained for Grp78, 28 (100%) showed high Grp78 expression by percent cytoplasmic immunoreactivity (Fig. 1; Table 1). Compared with the untreated and treated stage T3N0M0 cases, Grp78 expression according to percentage of immunoreactive tumor cells was significantly increased in castration resistance (P = 0.005). This elevation in Grp78 expression remained significant even when comparing castration-resistant cases to the untreated and treated groups separately (P = 0.002 and P < 0.001). When Grp78 expression was examined in castration-resistant tumors by intensity of cytoplasmic immunoreactivity, 22 of 28 (79%) cases showed moderate to high expression (Fig. 1; Table 1). Compared with the stage T3N0M0 cases, the number of tumors showing moderate to high intensity Grp78 expression in the castration-resistant group was significantly greater than both the untreated group (P = 0.033) and the treated group (P = 0.053). Furthermore, when Grp78 expression was examined as a combined measure of percentage of overall immunoreactive tumor cells and intensity (Grp78 status), Grp78 expression remained significantly elevated in the castration-resistant group when compared with both the untreated group (P = 0.018) and the treated group (P = 0.037).

In vitro expression of Grp78 corroborates clinical observations. Our cell line model consisted of LNCaP-derived castration-resistant C42B cells and androgen-dependent LNCaP cells grown in medium with FCS or in androgen-deprived conditions where FCS was replaced with charcoal-stripped serum. We found that C42B cells and LNCaP cells maintained in medium with charcoal-stripped serum (androgen depleted) for 6 days showed prominent cytoplasmic Grp78 immunoreactivity compared with LNCaP cells grown with FCS, which showed faint cytoplasmic Grp78 immunostaining (Fig. 1E). Quantitation of Grp78 cytoplasmic immunoreactivity by AICS II computer imaging of five representative areas on each sample slide showed that C42B cells had a mean of 84.0% immunoreactive tumor cells; LNCaP cells grown in charcoal-stripped serum for 6 days showed a mean of 64.2% reactive tumor cells; and LNCaP cells grown in FCS were found to have an average of 24.5% tumor cells showing cytoplasmic reactivity to Grp78 antibody. The ACIS II system reported a mean of 1.2% reactive tumor cells for the negative control LNCaP FCS cells excluding primary antibody. Intensity of each sample analyzed by ACIS II was also found to be greater in C42B and 6-day hormone-starved LNCaP cells (data not shown) than in LNCaP cells grown in FCS. As shown in Fig. 2 and Table 2 , these results were corroborated by Western blot analysis of cell lysates prepared from LNCaP cells grown with FCS; LNCaP cells grown with charcoal-stripped serum for 2, 4, and 6 days; and C42B cells. Comparison of Grp78 protein levels, expressed as band intensity ratios, showed that Grp78 expression in LNCaP cells was lowest in cells grown with FCS (1.00 standardized ratio), increased upon androgen starvation for 2 and 4 days (3.63 and 2.63 ratios), even further increased upon 6 days of hormone depletion (8.37 ratio), and was highest in castration-resistant C42B cells (13.94 ratio).


Figure 2
View larger version (8K):
[in this window]
[in a new window]
  		Fig. 2. Grp78 expression in prostate cancer cells during brief and prolonged androgen starvation. Western blot analysis for Grp78 expression in LNCaP cells grown in FCS and charcoal-stripped serum (2, 4, and 6 days) and castration-resistant C42B cells. ß-Actin loading control (bottom). Numbers are the ratio of sample band intensity to ß-actin band intensity, using the lowest ratio (LNCaP FCS) as the reference point of 1.00.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Quantification of protein expression from Western blot analysis of Grp78

 
Association of Grp78 expression with prostate cancer recurrence and survival. To evaluate Grp78 as a potential marker of prostate cancer progression, we examined the association of Grp78 expression with cancer recurrence risk and overall survival in untreated stage T3N0M0 patients. Treated cases were excluded due to potential alterations in Grp78 expression as a result of exposure to hormone ablation. Untreated cases were stratified by age, PSA level, and Gleason grade. We examined the associations between Grp78 expression and prostate cancer recurrence and survival in untreated stage T3N0M0 patients (n = 164, Table 3 ). At median follow-up of 12 years in the stage T3N0M0 untreated cohort, the probability of remaining recurrence free in cases expressing low Grp78 (<5% cells with intense immunoreactivity to Grp78) was 64% versus 54% in those expressing high (≥5% cells with intense immunoreactivity) levels of Grp78 (Fig. 3 ). Stratification of Grp78 expression in the stage T3N0M0 untreated cohort, by the standard clinical variables (multivariable analyses adjusting for age, PSA measurements, and Gleason score), consistently showed that the risk of recurring or dying was greater for patients with tumors that expressed high levels of Grp78 (≥5% of tumor cells with ≥3 intensity) compared with patients with tumors that expressed low levels of Grp78, even after adjusting for these known predictors of outcome. Thus, as shown in Table 3, the relative risks, which compare patients with high Grp78 expression in tumors with those with low Grp78 expression in tumors, were not changed substantially after stratification. Although these trends did not achieve statistical significance at the 0.05 level, they are consistent across strata, for both recurrence and survival. Grp78 expression proved to be significant, however, among particular subsets of patients. Interestingly, we observed that in the untreated stage T3N0M0 patients who were below the median age of 67 (n = 80) at diagnosis, increased Grp78 immunoreactivity (≥5% cells expressing high levels of Grp78) was significantly associated with increased risk of clinical and/or PSA recurrence (Fig. 4 ; Table 3). In these cases, the probability of remaining recurrence free in cases expressing low Grp78 was 61% versus 45% in those expressing high levels of Grp78, at follow-up year 12. The median recurrence-free interval for patients with low versus high Grp78 expression was 14.5 versus 8.7 years.


View this table:
[in this window]
[in a new window]
  		Table 3. Grp78 expression and recurrence-free or overall survival of patients with untreated stage T3N0M0 tumors

 

Figure 3
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 3. Probability of recurrence-free (clinical and/or PSA) status in 164 patients with stage T3N0M0 prostate cancer, based on levels of Grp78 immunoreactivity. Untreated stage T3N0M0 patients showed greater probability of prostate cancer recurrence with higher Grp78 expression. Tick marks represent patients with no evidence of disease at last follow-up. P was obtained using the log-rank test.

 

Figure 4
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 4. Probability of recurrence-free (clinical and/or PSA) status in 80 patients with stage T3N0M0 prostate cancer stratified by median age, based on levels of Grp78 immunoreactivity. Untreated stage T3N0M0 patients were stratified by age, where patients under the cohort median age of 67 years (n = 80) showed greater probability of prostate cancer recurrence with higher Grp78 expression. Tick marks represent patients with no evidence of disease at last follow-up. P was obtained using the log-rank test.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Based on previous studies identifying the role of Grp78 in cell survival and cancer progression (10, 14, 18), we hypothesized that expression of Grp78 may be associated with prostate cancer progression and the development of castration resistance. In the current study, we examined the expression of Grp78 in three cohorts of prostate cancer patients, uniquely designed to represent successive stages in the development of castration resistance. This included men who were not exposed to hormone therapy, men who were exposed and considered responsive to hormone therapy, and men who were exposed and resistant to hormone therapy. Our results suggest that Grp78 expression is up-regulated during the transition from localized prostate cancer to metastatic castration resistance. Thus, it is possible that overexpression of Grp78 may confer resistance to apoptosis in stress conditions such as anti-androgen therapy and may be an integral component of castration-resistant growth. Clonal selection has been implicated in the development of castration resistance (28). This is corroborated by identification of focal areas of cells showing intense Grp78 immunoreactivity (≥5%, ≥3 intensity) in both the untreated and treated stage T3N0M0 cases (Fig. 1B and C) where a greater percentage of treated cases (44%) versus untreated cases (36%) were classified as positive for this intense focal immunoreactivity. We have also provided corroborating in vitro evidence, using our cell line model that mimics clinical development of castration resistance, of increased Grp78 expression in castration-resistant growth, further suggesting the feasibility of Grp78 as a novel therapeutic target for prostate cancer.

It has been postulated that Grp78 induction occurs in response to cellular stress is involved in escape from apoptosis and contributes to drug resistance (7–9, 29). Recent evidence also suggests that Grp78 may contribute to the metastatic potential of prostate cancer cells via {alpha}-2-macroglobulin–mediated signaling (30). Our findings accordingly showed significantly higher expression of Grp78 in metastatic castration resistance when compared with localized stage T3N0M0 prostate cancer. This result is also consistent with those reported by Mintz et al. (14) that show strong Grp78 epitope immunoreactivity in bone marrow metastases from castration-resistant patients versus weak immunostaining in normal prostate and higher serum immunoreactivity to Grp78 in castration resistance compared with locally advanced prostate cancer.

Interestingly, we found that Grp78 expression is relatively unchanged in androgen-responsive tumors on initial exposure to anti-androgen therapy. However, we identified focal areas of cells showing intense Grp78 immunoreactivity in both the untreated and treated stage T3N0M0 cases, where a greater percentage of treated cases versus untreated cases were classified as positive for this intense focal immunoreactivity. Craft et al. has previously established in their xenograft prostate tumor model the existence of castration-resistant cells, even before hormone ablation (28). It is possible that the focal areas of prostate tumor, which strongly express Grp78, are comprised of clones that have been conferred a survival advantage by overexpressing Grp78, which may potentially develop into the more homogeneous staining cells in castration-resistant prostate cancer that show strong Grp78 immunoreactivity. Thus, our results are suggestive of clonal selection, whereby androgen withdrawal allows for selective survival and proliferation of castration-resistant cells that existed upon initiation of therapy (31).

Using our previously established cell line model of castration resistance development (32), we showed the occurrence of increased Grp78 expression in conditions of androgen deprivation and even greater Grp78 expression in established castration-resistant cells. Furthermore, the application of quantitative methods, such as densitometry for Western blot analysis and ACIS II automated image analysis for immunohistochemistry, allowed for objective assessment of relative Grp78 levels. Given the subjective nature of immunohistochemical reactivity assessment, results from the novel ACIS II system showing increased Grp78 in castration-resistant growth compared with androgen-dependent growth provided numerical validation of our clinical findings.

In this study, we observed that increased Grp78 expression in prostate tumors is associated with greater relative risk of recurrence and worse overall survival. This pattern was still observed following stratified analyses. Furthermore, Grp78 expression may have important prognostic value in distinct subsets of patients, particularly in those for whom favorable prognosis is indicated by low PSA levels or Gleason grade, as suggested by Table 3. Our results also suggest that increased Grp78 expression may be predictive of prostate cancer recurrence among patients who are diagnosed at relatively earlier age. These and further confirmatory studies may potentially indicate more aggressive treatment in patients who show high levels of Grp78, particularly for younger patients who may be better able to undergo such therapies.

A number of other stress-induced proteins have been implicated in the development of castration resistance. Heat shock protein Hsp70, which shares 60% homology with Grp78, and other stress-response proteins, such as Hsp27 and Hsp90, seem to contribute substantially to prostate cancer progression and the development of castration resistance, where suppression of these proteins is inhibitory to castration-resistant growth (33–38). This is indicative of the potential role of Grp78 in castration resistance development and as an additional therapeutic target, which, if inhibited, may allow cells to become susceptible to environmental stresses.

A significant clinical problem in prostate cancer is that patients who are initially responsive to androgen ablation therapy often develop castration resistance, despite recent advances in cancer therapy. One of the most common molecular defects associated with castration resistance is a deregulated androgen receptor (AR) pathway, resulting in constitutively active cell proliferative machinery. A better understanding of the complex contributing molecular mechanisms underlying the development of castration resistance is necessary so that the most appropriate therapeutic targets may be identified. Feldman et al. proposed five molecular pathways through which prostate cancer cells may undergo transition from androgen-dependent to castration-resistant growth (39). Among these pathways are mechanisms that result in downstream AR activation, or those in which alternative signals are up-regulated and bypass the necessity to activate AR–regulated transcription. Although the precise role of Grp78 in the development of castration resistance is unclear, increased Grp78 expression may confer a survival advantage through a number of prospective courses, including its molecular chaperone functions in assisting proper protein folding, inhibition of the apoptotic pathway, and contribution to growth and invasive potential of prostate cancer cells via cell surface receptor functions (7, 29, 40, 41). The intrinsic functions of Grp78 indicate a potential role in the AR bypass pathway of castration resistance development, although we believe that Grp78 signaling may contribute to a multitude of molecular mechanisms resulting in castration-resistant growth. Our studies suggest that up-regulation of Grp78 stress-response protein plays a role in the progression of localized prostate cancer to castration resistance and promotes Grp78 as a potential prognostic marker and therapeutic target for prostate cancer. Based on these and previous results showing Grp78 mediation of signal transduction (42), we plan to assess the chaperone function as well as cell membrane-resident signaling activity of Grp78 during castration resistance–associated molecular changes. Additional studies are currently being done to analyze Grp78 association with key signaling molecules during the progression of castration resistance, which may lead to downstream deregulation of AR activity, such as members of the epidermal growth factor receptor family, potentially establishing Grp78 as a link between AR activation and AR bypass pathways. Analysis of these interactions can result in devising novel therapies targeted towards Grp78 and Grp78-linked molecules for rational therapeutic management of castration-resistant prostate cancer.


   Footnotes
 
Grant support: Department of Defense grants PC970406 and PC992018 (L. Pootrakul, R.H. Datar, D. Hawes, S.G. Groshen, and R.J. Cote), PC97046 and 1 R33 CA-1-3455-01 (R.H. Datar, S-R Shi, and R.J. Cote); Cancer Center Core grant P30 CA 14089 (S.G. Groshen); NIH National Cancer Center grants 5 R01 CA 027607-26 and 1 RO1 CA 111700-01A1 (A.S. Lee); Department of Pathology, University of Southern California, Keck School of Medicine (L. Pootrakul); and Department of Urology, University of Southern California/Norris Comprehensive Cancer Center (J. Cai).

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.

Received 1/19/06; revised 6/21/06; accepted 6/27/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Shiu RPC, Pouyssegur J, Pastan I. Glucose depletion for the induction of two transformation-sensitive membrane proteins in Rous sarcoma virus-transformed chick embryo fibroblasts. PNAS 1977;74:3840–4.[Abstract/Free Full Text]
  2. Lee AS. Coordinated regulation of a set of genes by glucose and calcium ionophores in mammalian cells. TIBS 1987;12:20–3.
  3. Haas IG, Wabl M. Immunoglobulin heavy chain binding protein. Nature 1983;306:387–9.[CrossRef][Medline]
  4. Little E, Ramakrishnan M, Roy B, Gazit G, Lee AS. The glucose-regulated proteins (GRP78 and GRP94): functions, gene regulation, and applications. Crit Rev in Euk Gene Exp 1994;4:1–18.
  5. Dorner AJ, Krane MG, Kaufman RJ. Reduction of Endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells. MCB 1988;8:4063–70.[Medline]
  6. Lee AS. The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 2005;35:373–81.[CrossRef][Medline]
  7. Reddy RK, Mao C, Baumeister P, Austin RC, Kaufman RJ, Lee AS. Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors: role of ATP binding site in suppression of caspase-7 activation. J Biol Chem 2003;278:20915–24.[Abstract/Free Full Text]
  8. Rao RV, Peel A, Logvinova A, et al. Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78. FEBS Letters 2002;514:122–8.[Medline]
  9. Miyake H, Hara I, Arakawa S, Kamidono S. Stress protein GRP78 prevents apoptosis induced by calcium ionophore, ionomycin, but not by glycosylation inhibitor, tunicamycin, in human prostate cancer cells. J Cell Biochem 2000;77:396–408.[CrossRef][Medline]
  10. Lee AS. The glucose-regulated proteins: stress induction and clinical applications. TIBS 2001;26:504–10.[Medline]
  11. Kaufman RJ. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes and Devel 1999;13:1211–33.[Free Full Text]
  12. Jamora C, Dennert G, Lee AS. Inhibition of tumor progression by suppression of stress protein GRP78/BiP induction in fibrosarcoma B/C10ME. PNAS 1996;93:7690–4.[Abstract/Free Full Text]
  13. Dong D, Ko B, Baumeister P, et al. Vascular targeting and antiangiogenesis agents induce drug resistance effector GRP78 within the tumor microenvironment. Cancer Res 2005;65:5785–91.[Abstract/Free Full Text]
  14. Mintz PJ, Kim J, Do K, et al. Fingerprinting the circulating repertoire of antibodies from cancer patients. Nat Biotechnol 2003;21:57–63.[CrossRef][Medline]
  15. Patierno SR, Tuscano JM, Kim KS, Landolph JR, Lee AS. Increased expression of the glucose-regulated gene encoding the Mr 78,000 glucose-regulated protein in chemically and radiation-transformed C3H 10T1/2 mouse embryo cells. Cancer Res 1987;47:6220–4.[Abstract/Free Full Text]
  16. Bini L, Magi B, Marzocchi B, et al. Protein expression profiles in human breast ductal carcinoma and histologically normal tissue. Electrophoresis 1997;18:2832–41.[CrossRef][Medline]
  17. Gazit G, Lu J, Lee AS. De-regulation of GRP stress protein expression in human breast cancer cell lines. Br Ca Res Tr 1999;54:135–46.
  18. Fernandez PM, Tabbara SO, Jacobs LK, et al. Overexpression of the glucose-regulated stress gene GRP78 in malignant but not benign human breast lesions. Br Ca Res Tr 2000;59:15–26.
  19. International Union Against Cancer. Urological tumours: Prostate. In: TNM classification of malignant tumours, 4th edition. Hermanek P and Sobin LH, editors. New York: John Wiley & Sons; 1992. p. 141–4.
  20. Fradet Y. The role of neoadjuvant androgen deprivation prior to radical prostatectomy. Urol Clin North Am 1996;23:575–85.[CrossRef][Medline]
  21. Gleason DF. Histologic grading and clinical staging of prostatic carcinoma. In: Urologic pathology: the prostate. Tannenbaum M, editor. Philadelphia: Lea & Febiger; 1977. p. 171–3.
  22. Shi SR, Cote RJ, Taylor CR. Antigen retrieval immunohistochemistry: past, present, and future. J Histochem Cytochem 1997;45:327–43.[Abstract/Free Full Text]
  23. Craft N, Shostak Y, Carey M, Sawyers CL. A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat Med 1999;5:280–5.[CrossRef][Medline]
  24. Bauer KD, de la Torre-Bueno J, Diel IJ, et al. Reliable and sensitive analysis of occult bone marrow metastases using automated cellular imaging. Clin Cancer Res 2000;6:3552–9.[Abstract/Free Full Text]
  25. Wang S, Saboorian MH, Frenkel EP, et al. Assessment of HER-2/neu status in breast cancer. Am J Clin Path 2001;116:495–503.[CrossRef][Medline]
  26. Shah RB, Mehra R, Chinnaiyan AM, et al. Androgen-independent prostate cancer is a heterogeneous group of diseases: lessons from a rapid autopsy program. Cancer Res 2004;64:9209–16.[Abstract/Free Full Text]
  27. Messersmith W, Oppenheimer D, Peralba J, et al. Assessment of epidermal growth factor receptor (EGFR) signaling in paired colorectal cancer and normal colon tissue samples using computer-aided immunohistochemical analysis. Cancer Biol Ther 2005;5:70–5.
  28. Craft N, Chhor C, Tran C, et al. Evidence for clonal outgrowth of androgen-independent prostate cancer cells from androgen-dependent tumors through a two-step process. Cancer Res 1999;59:5030–6.[Abstract/Free Full Text]
  29. Shen J, Hughes C, Chao C, et al. Coinduction of glucose regulated proteins and doxorubicin resistance in Chinese hamster cells. PNAS USA 1987;84:3278–82.[Abstract/Free Full Text]
  30. Misra UK, Deedwania R, Pizzo SV. Binding of activated {alpha}2-macroglobulin to its cell surface receptor GRP78 in 1-LN prostate cancer cells regulated PAK-2-dependent activation of LIMK. JBC 2005;280:26278–86.[Abstract/Free Full Text]
  31. So A, Gleave M, Hurtado-Col A, Nelson C. Mechanisms of the development of androgen independence in prostate cancer. World J Urol 2005;23:1–9.[CrossRef][Medline]
  32. Shi Y, Chatterjee SJ, Brands FH, et al. Role of coordinated molecular alterations in the development of androgen-independent prostate cancer: an in vitro model that corroborates clinical observations. Br J Urol Int 2006;97:170–8.
  33. Munro S, Pelham HRB. An Hsp70-like protein in the ER: Identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 1986;46:291–300.[CrossRef][Medline]
  34. Zhao Z, Ma Q, Xu C. Abrogation of heat-shock protein (HSP) 70 expression induced cell growth inhibition and apoptosis in human androgen-independent prostate cancer cell line PC-3m. Asian J Androl 2004;6:319–24.[Medline]
  35. Rocchi P, So A, Kojima S, et al. Heat shock protein 27 increases after androgen ablation and plays a cytoprotective role in hormone-refractory prostate cancer. Cancer Res 2004;64:6595–602.[Abstract/Free Full Text]
  36. Cornford PA, Dodson AR, Parsons KF, et al. Heat shock protein expression independently predicts clinical outcome in prostate cancer. Cancer Res 2000;60:7099–105.[Abstract/Free Full Text]
  37. Neckers L. Heat shock protein 90 inhibition by 17-allylamino-17-demethoxygeldanamycin: a novel therapeutic approach for treating hormone-refractory prostate cancer. Clin Cancer Res 2002;8:962–6.[Free Full Text]
  38. Solit DB, Zheng FF, Drobjnak M, et al. 17-Allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptor and HER-2/neu and inhibits the growth of prostate cancer xenografts. Clin Cancer Res 2002;8:986–93.[Abstract/Free Full Text]
  39. Feldman BJ, Feldman D. The development of androgen-independent prostate cancer. Nat Rev 2001;1:34–45.
  40. Li J, Lee AS. Stress induction of Grp78/BiP and its role in cancer. Curr Mol Med 2006;6:45–54.[CrossRef][Medline]
  41. Arap MA, Lahdenranta J, Mintz PJ, et al. Cell surface expression of the stress response chaperone GRP78 enables tumor targeting by circulating ligands. Cancer Cell 2004;6:275–84.[CrossRef][Medline]
  42. Misra UK, Gonzalez-Gronow M, Gawdi G, Hart JP, Johnson CE, Pizzo SV. The role of Grp78 in {alpha} 2-macroglobulin-induced signal transduction. Evidence from RNA interference that the low density lipoprotein receptor-related protein is associated with, but not necessary for, GRP 78-mediated signal transduction. J Biol Chem 2002;277:42082–7.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
Y. Tang, L. Wang, O. Goloubeva, M. Afnan Khan, B. Zhang, and A. Hussain
Divergent Effects of Castration on Prostate Cancer in TRAMP Mice: Possible Implications for Therapy
Clin. Cancer Res., May 15, 2008; 14(10): 2936 - 2943.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. Dong, M. Ni, J. Li, S. Xiong, W. Ye, J. J. Virrey, C. Mao, R. Ye, M. Wang, L. Pen, et al.
Critical Role of the Stress Chaperone GRP78/BiP in Tumor Proliferation, Survival, and Tumor Angiogenesis in Transgene-Induced Mammary Tumor Development
Cancer Res., January 15, 2008; 68(2): 498 - 505.
[Abstract] [Full Text] [PDF]

Home page
 Neuro OncolHome page
H. K. Lee, C. Xiang, S. Cazacu, S. Finniss, G. Kazimirsky, N. Lemke, N. L. Lehman, S. A. Rempel, T. Mikkelsen, and C. Brodie
GRP78 is overexpressed in glioblastomas and regulates glioma cell growth and apoptosis
Neuro-oncol, January 1, 2008; 10(3): 236 - 243.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. C. Jiang, L. H. Chen, S. Gillespie, Y. F. Wang, K. A. Kiejda, X. D. Zhang, and P. Hersey
Inhibition of MEK Sensitizes Human Melanoma Cells to Endoplasmic Reticulum Stress-Induced Apoptosis
Cancer Res., October 15, 2007; 67(20): 9750 - 9761.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. G. Jakobsen, N. Rasmussen, A.-V. Laenkholm, and H. J. Ditzel
Phage Display Derived Human Monoclonal Antibodies Isolated by Binding to the Surface of Live Primary Breast Cancer Cells Recognize GRP78
Cancer Res., October 1, 2007; 67(19): 9507 - 9517.
[Abstract] [Full Text] [PDF]

Home page
 Ann. N. Y. Acad. Sci.Home page
G. BANHEGYI, P. BAUMEISTER, A. BENEDETTI, D. DONG, Y. FU, A. S. LEE, J. LI, C. MAO, E. MARGITTAI, M. NI, et al.
Endoplasmic Reticulum Stress
Ann. N.Y. Acad. Sci., October 1, 2007; 1113(1): 58 - 71.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. S. Lee
GRP78 Induction in Cancer: Therapeutic and Prognostic Implications
Cancer Res., April 15, 2007; 67(8): 3496 - 3499.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pootrakul, L.
Right arrow Articles by Cote, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pootrakul, L.
Right arrow Articles by Cote, R. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online