- Email: [email protected]
Inactivation of myopodin expression associated with prostate cancer relapse
ADULT UROLOGY
INACTIVATION OF MYOPODIN EXPRESSION ASSOCIATED WITH PROSTATE CANCER RELAPSE YAN PING YU, GEORGE C. TSENG,
AND
JIAN-HUA LUO
ABSTRACT Objectives. Myopodin has recently been characterized as a tumor suppressor gene whose encoded product inhibits prostate cancer growth and metastasis both in vitro and in animal models. However, the clinical evidence of tumor suppression in humans is still lacking. In this study, we conducted a large-scale analysis of myopodin expression in prostate cancer samples to examine the relationship between myopodin expression and prostate cancer grade and stage and the probability of clinical relapse. Methods. Immunostaining using anti-myopodin antibodies was performed on 746 formalin-fixed paraffinembedded tissue samples to evaluate the level of myopodin expression in normal and malignant prostatic tissue. Results. The expression of myopodin was semiquantitatively determined by immunostaining. The myopodin expression levels were examined in relation to prostate cancer grade and stage, preoperative prostatespecific antigen level, surgical margin involvement, tumor volume, and clinical relapse. The mean myopodin expression score (range 0 to 3) was 2.1 and 0.88 for benign prostatic tissue and prostate cancer, respectively. Minimal variation in myopodin expression was observed among the various grades, stages, tumor volumes, and preoperative prostate-specific antigen levels of prostate cancer. However, samples with positive surgical margins were associated with lower myopodin expression. Complete inactivation of myopodin expression correlated with a greater than 86% rate of clinical relapse. Conclusions. Our results suggest that myopodin is an important predictor of prostate cancer metastasis, independent of Gleason score, preoperative prostate-specific antigen level, and tumor stage. UROLOGY 68: 578–582, 2006. © 2006 Elsevier Inc.
P
rostate cancer is the second most commonly diagnosed malignancy in American men. At the present rate of diagnosis, 1 in 6 men will be diagnosed with the disease during his lifetime.1 Although approximately 30,000 men die of this disease annually,2 much more commonly, prostate cancer is not clinically aggressive. Against this backdrop of a very common malignancy, it is still not clear what key molecular events are responsible for the progression of prostate cancer to the lethal form of the disease. Myopodin was initially identified as a gene that is frequently deleted in advanced-stage prostate can-
This work was supported by grants from the National Cancer Institute (1UO1CA88110-01 and R01 CA098249) and John Rangos Foundation for Enhancement of Research in Pathology. From the Departments of Pathology and Biostatistics, University of Pittsburgh, Pittsburgh, Pennsylvania Reprint requests: Jian-Hua Luo, M.D., Ph.D., Department of Pathology, University of Pittsburgh, Scaife Hall S-760, 3550 Terrace Street, Pittsburgh, PA 15261. E-mail: [email protected] Submitted: December 15, 2005, accepted (with revisions): March 13, 2006 © 2006 ELSEVIER INC. 578
ALL RIGHTS RESERVED
cer.3 The protein encoded by myopodin shares some homology with “synaptopodin,” whose expression is closely related to terminal differentiation of neurons and podocytes.4,5 Recent studies have suggested that the myopodin protein may contain tumor suppressor activity for urothelial carcinoma and prostate cancer.6,7 In this study, we performed a large-scale myopodin expression analysis in prostate cancer tissue and investigated whether inactivation of myopodin expression is associated with prostate cancer relapse. Our results have indicated that expression inactivation of myopodin is independent of tumor grade and stage and that complete inactivation of myopodin expression is associated with a high rate of metastasis. MATERIAL AND METHODS SAMPLE PREPARATION Seven tissue array slides and 40 thin sections of formalinfixed paraffin-embedded tissue were used. The tissue arrays of prostatic tissues were constructed from a total of 746 formalin-fixed paraffin-embedded tissue blocks using a 1-mm diameter array donor needle into seven receiver paraffin blocks by 0090-4295/06/$32.00 doi:10.1016/j.urology.2006.03.027
FIGURE 1. Downregulation of myopodin protein expression in prostate cancer. Representative images of immunostaining of myopodin shown. Myopodin expression scores of normal liver (negative control, A), normal prostate (B), and prostate cancer (C and D) indicated.
a semiautomatic tissue arrayer (Chemicon, San Diego, Calif). Each block contained 50 to 160 tissue cores. Thin tissue sections (3 m) were collected and used for immunostaining assays. The institutional review board approved the procedure of archived tissue collection, sample coding, collection of clinical follow-up data, and experimental protocols.
GENERATION OF ANTIBODIES AGAINST MYOPODIN The procedure of generating anti-myopodin has been previously described.7 In brief, myopodin antisera were raised from rabbits against regions of myopodin nonhomologous to synaptopodin in the C-terminus (myoC, KMGKKKGKKPLNALDVMKHQ). These antisera were peptide affinity purified using the Aminolink Kit (Pierce, Rockford, Ill). The purified antisera were tested for specificity for myopodin in Western blotting with protein extracts from cells known to overexpress myopodin (I4) or cells not expressing myopodin (LNCaP, 293 cells) as described by Jing et al.7 The antisera were then titrated for antibody avidity. Normal liver samples not expressing myopodin were used as negative controls in immunostaining (Fig. 1A).
IMMUNOHISTOCHEMISTRY STAINING AND TISSUE ARRAY ANALYSIS
Sections of the tissue arrays 3 m thick were collected and mounted onto glass slides. The sections were deparaffinized in
UROLOGY 68 (3), 2006
xylene and ethanol. Endogenous peroxidase was inactivated by treatment with 3% hydrogen peroxide. Antigen retrieval was achieved by placing slides in 10 mM citric acid (pH 5.0) at 90°C in a 2100 Retriever (PickCell Laboratories, Amsterdam, The Netherlands) for 20 minutes. After slowly cooling to room temperature, the tissue sections were incubated with antimyopodin antibodies at 1:100 dilution at room temperature for 30 minutes. The sections were then incubated with biotinylated secondary IgG for 30 minutes at room temperature. Labeling was amplified by allowing the secondary antibodies to bind a compound reagent (avidin-biotinylated horseradish peroxidase complex, Vector Laboratories, Burlingame, Calif) for 30 minutes. The slides were exposed to 3,3= diaminobenzidine solution to visualize immunostaining (Vector Laboratories). Cells within tissue samples were counterstained with hematoxylin.
SEMIQUANTITATIVE ANALYSIS AND DATA PROCESSING Immunostaining was analyzed under a light microscope. Immunostaining specificity was verified by comparing labeled tissue with tissue incubated without a primary antibody. The expression of myopodin was graded as 0 if no cells within the tumor stained, 0.5⫾ if focally positive cells for myopodin were evident, 1⫹ if most of the cells were weakly positive, 2⫹ if staining was moderately intensive, and 3⫹ if staining was strongly positive. The overall score for each sample repre579
sented a consensus of scores by three observers who were unaware of the patients’ clinical outcomes.
RESULTS We constructed a prostatic tissue microarray containing seven slides and a total of 746 tissue samples. Of these array slides, 590 samples, including 334 tumor and 256 normal prostate specimens, had an adequate amount of tissue for analysis. The normal prostate samples included tissue from 40 organ donors and 216 benign prostatic specimens adjacent to prostate cancer tissue. Patient age ranged from 49 to 75 years. A combined Gleason score of 6 and 7 was predominant, accounting for 38% and 31% of the samples, respectively. Most (69.2%) of the prostate cancer samples were organ confined at surgery (Stage T2). Of the analyzable tumor samples, clinical follow-up information for a postoperative period of at least 5 years was available for 231 cases. To investigate whether expression of myopodin is inactivated in prostate cancer, immunostaining was compared among four classifications: (a) prostate cancer, (b) all benign prostatic tissue (from organ donors and morphologically benign prostatic tissue adjacent to malignant tissue [AT]), (c) benign tissue from organ donors only, and (d) AT samples only. As shown in Figure 1, normal acinar and basal cells of prostate glands were readily stained by anti-myopodin antibodies. Myopodin was primarily localized to the nucleus. However, the cytoplasmic expression of myopodin was notably greater in cancer cells than in normal cells (Fig. 1B,C). The average score for overall myopodin expression in normal prostatic tissue was 2.1, and the average score for prostate cancer was 0.88, representing a nearly 2.4-fold decrease (P ⬍0.0001) in the cancer cells relative to the normal cells. The average score for organ donor-derived noncancerous tissue was 2.3 and that for AT tissue was 2.0. This reduced myopodin expression in the AT samples relative to tissue from cancer-free donors was small but significant (log-rank test, P ⬍0.0001), implying that a subtle downregulation of myopodin may occur in tissue adjacent to cancer (Fig. 2A). When the prostate cancer samples were stratified by Gleason score, minimal variation was found in the averaged myopodin expression scores among the samples (range 0.875 to 0.885; Fig. 2B). Similarly, the averaged myopodin expression score in samples representing various pathologic stages at radical prostatectomy were strikingly similar (range 0.88 to 0.94; Fig. 2C). These analyses suggest that although inactivation of myopodin expression is associated with the presence of cancer, it may be independent of cancer cell differentiation and cancer stage. Similar analyses were also per580
formed to evaluate the expression of myopodin with preoperative prostate-specific antigen level (Fig. 2D), positive surgical margins (Fig. 2E), and tumor volume (Fig. 2F). Even though a slight increase was found in the preoperative prostate-specific antigen level with lower myopodin scores, the Wilcoxon rank-sum test suggested that such a difference was not statistically significant (P ⫽ 0.6 to 0.7). Similar conclusions were also drawn for the association between myopodin expression and tumor volume (P ⫽ 0.06 to 0.3). Prostate cancer samples with positive surgical margins were associated with a slight, but statistically significant, decrease in myopodin expression (0.75 versus 0.99, P ⫽ 0.018). To investigate whether myopodin expression is related to tumor behavior, we divided the cancer samples into five groups according to the myopodin expression score and determined whether the expression score correlated with the likelihood of clinical relapse within 5 years of surgical prostate resection. As shown in Figure 2G, only limited variation in the clinical relapse rate was observed among patients whose tissues had a score of 0.5⫾ to 2⫹; the relapse rates in these cases ranged from 28% to 34%. However, complete inactivation of myopodin produced a markedly elevated recurrence rate of 85.7% (30 of 35). Kaplan-Meier analysis (Fig. 3) indicated that the clinical outcome was quite poor for patients with samples that received a 0 score in myopodin expression compared with patients with all other scores (14.3% versus 83.4% versus 14.3%, P ⬍0.0001). This striking difference implies that myopodin may be a useful marker for predicting prostate cancer metastasis. COMMENT The myopodin gene was localized to a minimal common deletion region of chromosome 4q25 in prostate cancer cells by the differential subtraction chain approach.3 In addition, polymerase chain reaction survey studies have revealed deletion mutations affecting the myopodin gene in some aggressive prostate cancers.3 Additional study indicated that deletion of myopodin, either partial or complete, was associated with high rates of metastasis and clinical prostate cancer relapse.3 Hot spots of deletion were identified within the synaptopodin homologous region of myopodin.3 The absence of a myopodin deletion, however, was associated with a low rate of invasion and clinical relapse, regardless of the Gleason tumor grade.3,7 In vitro and in vivo tumor suppression analysis has indicated that myopodin expression in invasive prostate cancer cell lines (PC3 and LNCaP) results in decreased invasiveness and suppression of cell proliferation.7 UROLOGY 68 (3), 2006
FIGURE 2. Inactivation of myopodin expression was independent of tumor grade and stage, but complete inactivation correlated with clinical relapse. (A) Myopodin expression scores in prostate cancer (PC), all benign prostatic tissues, organ-donor prostatic tissues only, and AT only. (B) Myopodin expression was independent of Gleason tumor grade. (C) Myopodin expression was independent of pathologic tumor stage. (D) Myopodin expression was independent of preoperative prostate-specific antigen. (E) Myopodin expression inactivation correlated mildly with positive surgical margins. (F) Myopodin expression was independent of tumor volume. (G) Myopodin expression inactivation correlated with clinical relapse within 5 years of surgical resection.
plicating myopodin dysfunction in tumor growth and metastasis, and further indicate that complete inactivation of myopodin expression correlates highly with prostate cancer metastasis and relapse. CONCLUSIONS
FIGURE 3. Kaplan-Meier analysis of clinical relapse of prostate cancer for samples with and without myopodin expression. Metastasis was determined by physical and chemical evidence.
Overall, myopodin appears to function as a tumor growth and metastasis suppressor. The present findings complement these studies, imUROLOGY 68 (3), 2006
Myopodin inactivation in prostate cancer cells has important clinical implications. First, as a tumor suppressor gene implicated in metastasis, inactivation of myopodin may have a negative impact on a patient’s clinical outcome. As a result, additional precautions in the treatment and monitoring plan for patients with complete inactivation of myopodin expression in their tumor tissue should be considered. This is clinically relevant because inactivation of myopodin expression is largely independent of tumor grade and clinical stage. Assays on myopodin expression may supplement Gleason grading as an added objective diagnostic 581
to be considered in prostate cancer prognosis prediction. In addition to its potential as a diagnostic, myopodin may ultimately provide a potential target for clinical intervention. Because restoration of wild-type myopodin expression in tumor cells significantly modifies the aggressive behavior of these tumors, gene-targeted therapy to enhance the activity of myopodin might be useful in the treatment or even prevention of metastasis. REFERENCES 1. Isaacs JT: Molecular markers for prostate cancer metastasis: developing diagnostic methods for predicting the aggressiveness of prostate cancer. Am J Pathol 150: 1511–1521, 1997. 2. Jemal A, Murray T, Ward E, et al: Cancer statistics, 2005. CA Cancer J Clin 55: 10 –30, 2005.
582
3. Lin F, Yu YP, Woods J, et al: Myopodin, a synaptopodin homologue, is frequently deleted in invasive prostate cancers. Am J Pathol 159: 1603–1612, 2001. 4. Mundel P, Reiser J, Zuniga Mejia Borja A, et al: Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236: 248 –258, 1997. 5. Nagata M, Nakayama K, Terada Y, et al: Cell cycle regulation and differentiation in the human podocyte lineage. Am J Pathol 153: 1511–1520, 1998. 6. Sanchez-Carbayo M, Schwarz K, Charytonowicz E, et al: Tumor suppressor role for myopodin in bladder cancer: loss of nuclear expression of myopodin is cell-cycle dependent and predicts clinical outcome. Oncogene 22: 5298 –5305, 2003. 7. Jing L, Liu L, Yu YP, et al: Expression of myopodin induces suppression of tumor growth and metastasis. Am J Pathol 164: 1799 –1806, 2004.
UROLOGY 68 (3), 2006
INACTIVATION OF MYOPODIN EXPRESSION ASSOCIATED WITH PROSTATE CANCER RELAPSE YAN PING YU, GEORGE C. TSENG,
AND
JIAN-HUA LUO
ABSTRACT Objectives. Myopodin has recently been characterized as a tumor suppressor gene whose encoded product inhibits prostate cancer growth and metastasis both in vitro and in animal models. However, the clinical evidence of tumor suppression in humans is still lacking. In this study, we conducted a large-scale analysis of myopodin expression in prostate cancer samples to examine the relationship between myopodin expression and prostate cancer grade and stage and the probability of clinical relapse. Methods. Immunostaining using anti-myopodin antibodies was performed on 746 formalin-fixed paraffinembedded tissue samples to evaluate the level of myopodin expression in normal and malignant prostatic tissue. Results. The expression of myopodin was semiquantitatively determined by immunostaining. The myopodin expression levels were examined in relation to prostate cancer grade and stage, preoperative prostatespecific antigen level, surgical margin involvement, tumor volume, and clinical relapse. The mean myopodin expression score (range 0 to 3) was 2.1 and 0.88 for benign prostatic tissue and prostate cancer, respectively. Minimal variation in myopodin expression was observed among the various grades, stages, tumor volumes, and preoperative prostate-specific antigen levels of prostate cancer. However, samples with positive surgical margins were associated with lower myopodin expression. Complete inactivation of myopodin expression correlated with a greater than 86% rate of clinical relapse. Conclusions. Our results suggest that myopodin is an important predictor of prostate cancer metastasis, independent of Gleason score, preoperative prostate-specific antigen level, and tumor stage. UROLOGY 68: 578–582, 2006. © 2006 Elsevier Inc.
P
rostate cancer is the second most commonly diagnosed malignancy in American men. At the present rate of diagnosis, 1 in 6 men will be diagnosed with the disease during his lifetime.1 Although approximately 30,000 men die of this disease annually,2 much more commonly, prostate cancer is not clinically aggressive. Against this backdrop of a very common malignancy, it is still not clear what key molecular events are responsible for the progression of prostate cancer to the lethal form of the disease. Myopodin was initially identified as a gene that is frequently deleted in advanced-stage prostate can-
This work was supported by grants from the National Cancer Institute (1UO1CA88110-01 and R01 CA098249) and John Rangos Foundation for Enhancement of Research in Pathology. From the Departments of Pathology and Biostatistics, University of Pittsburgh, Pittsburgh, Pennsylvania Reprint requests: Jian-Hua Luo, M.D., Ph.D., Department of Pathology, University of Pittsburgh, Scaife Hall S-760, 3550 Terrace Street, Pittsburgh, PA 15261. E-mail: [email protected] Submitted: December 15, 2005, accepted (with revisions): March 13, 2006 © 2006 ELSEVIER INC. 578
ALL RIGHTS RESERVED
cer.3 The protein encoded by myopodin shares some homology with “synaptopodin,” whose expression is closely related to terminal differentiation of neurons and podocytes.4,5 Recent studies have suggested that the myopodin protein may contain tumor suppressor activity for urothelial carcinoma and prostate cancer.6,7 In this study, we performed a large-scale myopodin expression analysis in prostate cancer tissue and investigated whether inactivation of myopodin expression is associated with prostate cancer relapse. Our results have indicated that expression inactivation of myopodin is independent of tumor grade and stage and that complete inactivation of myopodin expression is associated with a high rate of metastasis. MATERIAL AND METHODS SAMPLE PREPARATION Seven tissue array slides and 40 thin sections of formalinfixed paraffin-embedded tissue were used. The tissue arrays of prostatic tissues were constructed from a total of 746 formalin-fixed paraffin-embedded tissue blocks using a 1-mm diameter array donor needle into seven receiver paraffin blocks by 0090-4295/06/$32.00 doi:10.1016/j.urology.2006.03.027
FIGURE 1. Downregulation of myopodin protein expression in prostate cancer. Representative images of immunostaining of myopodin shown. Myopodin expression scores of normal liver (negative control, A), normal prostate (B), and prostate cancer (C and D) indicated.
a semiautomatic tissue arrayer (Chemicon, San Diego, Calif). Each block contained 50 to 160 tissue cores. Thin tissue sections (3 m) were collected and used for immunostaining assays. The institutional review board approved the procedure of archived tissue collection, sample coding, collection of clinical follow-up data, and experimental protocols.
GENERATION OF ANTIBODIES AGAINST MYOPODIN The procedure of generating anti-myopodin has been previously described.7 In brief, myopodin antisera were raised from rabbits against regions of myopodin nonhomologous to synaptopodin in the C-terminus (myoC, KMGKKKGKKPLNALDVMKHQ). These antisera were peptide affinity purified using the Aminolink Kit (Pierce, Rockford, Ill). The purified antisera were tested for specificity for myopodin in Western blotting with protein extracts from cells known to overexpress myopodin (I4) or cells not expressing myopodin (LNCaP, 293 cells) as described by Jing et al.7 The antisera were then titrated for antibody avidity. Normal liver samples not expressing myopodin were used as negative controls in immunostaining (Fig. 1A).
IMMUNOHISTOCHEMISTRY STAINING AND TISSUE ARRAY ANALYSIS
Sections of the tissue arrays 3 m thick were collected and mounted onto glass slides. The sections were deparaffinized in
UROLOGY 68 (3), 2006
xylene and ethanol. Endogenous peroxidase was inactivated by treatment with 3% hydrogen peroxide. Antigen retrieval was achieved by placing slides in 10 mM citric acid (pH 5.0) at 90°C in a 2100 Retriever (PickCell Laboratories, Amsterdam, The Netherlands) for 20 minutes. After slowly cooling to room temperature, the tissue sections were incubated with antimyopodin antibodies at 1:100 dilution at room temperature for 30 minutes. The sections were then incubated with biotinylated secondary IgG for 30 minutes at room temperature. Labeling was amplified by allowing the secondary antibodies to bind a compound reagent (avidin-biotinylated horseradish peroxidase complex, Vector Laboratories, Burlingame, Calif) for 30 minutes. The slides were exposed to 3,3= diaminobenzidine solution to visualize immunostaining (Vector Laboratories). Cells within tissue samples were counterstained with hematoxylin.
SEMIQUANTITATIVE ANALYSIS AND DATA PROCESSING Immunostaining was analyzed under a light microscope. Immunostaining specificity was verified by comparing labeled tissue with tissue incubated without a primary antibody. The expression of myopodin was graded as 0 if no cells within the tumor stained, 0.5⫾ if focally positive cells for myopodin were evident, 1⫹ if most of the cells were weakly positive, 2⫹ if staining was moderately intensive, and 3⫹ if staining was strongly positive. The overall score for each sample repre579
sented a consensus of scores by three observers who were unaware of the patients’ clinical outcomes.
RESULTS We constructed a prostatic tissue microarray containing seven slides and a total of 746 tissue samples. Of these array slides, 590 samples, including 334 tumor and 256 normal prostate specimens, had an adequate amount of tissue for analysis. The normal prostate samples included tissue from 40 organ donors and 216 benign prostatic specimens adjacent to prostate cancer tissue. Patient age ranged from 49 to 75 years. A combined Gleason score of 6 and 7 was predominant, accounting for 38% and 31% of the samples, respectively. Most (69.2%) of the prostate cancer samples were organ confined at surgery (Stage T2). Of the analyzable tumor samples, clinical follow-up information for a postoperative period of at least 5 years was available for 231 cases. To investigate whether expression of myopodin is inactivated in prostate cancer, immunostaining was compared among four classifications: (a) prostate cancer, (b) all benign prostatic tissue (from organ donors and morphologically benign prostatic tissue adjacent to malignant tissue [AT]), (c) benign tissue from organ donors only, and (d) AT samples only. As shown in Figure 1, normal acinar and basal cells of prostate glands were readily stained by anti-myopodin antibodies. Myopodin was primarily localized to the nucleus. However, the cytoplasmic expression of myopodin was notably greater in cancer cells than in normal cells (Fig. 1B,C). The average score for overall myopodin expression in normal prostatic tissue was 2.1, and the average score for prostate cancer was 0.88, representing a nearly 2.4-fold decrease (P ⬍0.0001) in the cancer cells relative to the normal cells. The average score for organ donor-derived noncancerous tissue was 2.3 and that for AT tissue was 2.0. This reduced myopodin expression in the AT samples relative to tissue from cancer-free donors was small but significant (log-rank test, P ⬍0.0001), implying that a subtle downregulation of myopodin may occur in tissue adjacent to cancer (Fig. 2A). When the prostate cancer samples were stratified by Gleason score, minimal variation was found in the averaged myopodin expression scores among the samples (range 0.875 to 0.885; Fig. 2B). Similarly, the averaged myopodin expression score in samples representing various pathologic stages at radical prostatectomy were strikingly similar (range 0.88 to 0.94; Fig. 2C). These analyses suggest that although inactivation of myopodin expression is associated with the presence of cancer, it may be independent of cancer cell differentiation and cancer stage. Similar analyses were also per580
formed to evaluate the expression of myopodin with preoperative prostate-specific antigen level (Fig. 2D), positive surgical margins (Fig. 2E), and tumor volume (Fig. 2F). Even though a slight increase was found in the preoperative prostate-specific antigen level with lower myopodin scores, the Wilcoxon rank-sum test suggested that such a difference was not statistically significant (P ⫽ 0.6 to 0.7). Similar conclusions were also drawn for the association between myopodin expression and tumor volume (P ⫽ 0.06 to 0.3). Prostate cancer samples with positive surgical margins were associated with a slight, but statistically significant, decrease in myopodin expression (0.75 versus 0.99, P ⫽ 0.018). To investigate whether myopodin expression is related to tumor behavior, we divided the cancer samples into five groups according to the myopodin expression score and determined whether the expression score correlated with the likelihood of clinical relapse within 5 years of surgical prostate resection. As shown in Figure 2G, only limited variation in the clinical relapse rate was observed among patients whose tissues had a score of 0.5⫾ to 2⫹; the relapse rates in these cases ranged from 28% to 34%. However, complete inactivation of myopodin produced a markedly elevated recurrence rate of 85.7% (30 of 35). Kaplan-Meier analysis (Fig. 3) indicated that the clinical outcome was quite poor for patients with samples that received a 0 score in myopodin expression compared with patients with all other scores (14.3% versus 83.4% versus 14.3%, P ⬍0.0001). This striking difference implies that myopodin may be a useful marker for predicting prostate cancer metastasis. COMMENT The myopodin gene was localized to a minimal common deletion region of chromosome 4q25 in prostate cancer cells by the differential subtraction chain approach.3 In addition, polymerase chain reaction survey studies have revealed deletion mutations affecting the myopodin gene in some aggressive prostate cancers.3 Additional study indicated that deletion of myopodin, either partial or complete, was associated with high rates of metastasis and clinical prostate cancer relapse.3 Hot spots of deletion were identified within the synaptopodin homologous region of myopodin.3 The absence of a myopodin deletion, however, was associated with a low rate of invasion and clinical relapse, regardless of the Gleason tumor grade.3,7 In vitro and in vivo tumor suppression analysis has indicated that myopodin expression in invasive prostate cancer cell lines (PC3 and LNCaP) results in decreased invasiveness and suppression of cell proliferation.7 UROLOGY 68 (3), 2006
FIGURE 2. Inactivation of myopodin expression was independent of tumor grade and stage, but complete inactivation correlated with clinical relapse. (A) Myopodin expression scores in prostate cancer (PC), all benign prostatic tissues, organ-donor prostatic tissues only, and AT only. (B) Myopodin expression was independent of Gleason tumor grade. (C) Myopodin expression was independent of pathologic tumor stage. (D) Myopodin expression was independent of preoperative prostate-specific antigen. (E) Myopodin expression inactivation correlated mildly with positive surgical margins. (F) Myopodin expression was independent of tumor volume. (G) Myopodin expression inactivation correlated with clinical relapse within 5 years of surgical resection.
plicating myopodin dysfunction in tumor growth and metastasis, and further indicate that complete inactivation of myopodin expression correlates highly with prostate cancer metastasis and relapse. CONCLUSIONS
FIGURE 3. Kaplan-Meier analysis of clinical relapse of prostate cancer for samples with and without myopodin expression. Metastasis was determined by physical and chemical evidence.
Overall, myopodin appears to function as a tumor growth and metastasis suppressor. The present findings complement these studies, imUROLOGY 68 (3), 2006
Myopodin inactivation in prostate cancer cells has important clinical implications. First, as a tumor suppressor gene implicated in metastasis, inactivation of myopodin may have a negative impact on a patient’s clinical outcome. As a result, additional precautions in the treatment and monitoring plan for patients with complete inactivation of myopodin expression in their tumor tissue should be considered. This is clinically relevant because inactivation of myopodin expression is largely independent of tumor grade and clinical stage. Assays on myopodin expression may supplement Gleason grading as an added objective diagnostic 581
to be considered in prostate cancer prognosis prediction. In addition to its potential as a diagnostic, myopodin may ultimately provide a potential target for clinical intervention. Because restoration of wild-type myopodin expression in tumor cells significantly modifies the aggressive behavior of these tumors, gene-targeted therapy to enhance the activity of myopodin might be useful in the treatment or even prevention of metastasis. REFERENCES 1. Isaacs JT: Molecular markers for prostate cancer metastasis: developing diagnostic methods for predicting the aggressiveness of prostate cancer. Am J Pathol 150: 1511–1521, 1997. 2. Jemal A, Murray T, Ward E, et al: Cancer statistics, 2005. CA Cancer J Clin 55: 10 –30, 2005.
582
3. Lin F, Yu YP, Woods J, et al: Myopodin, a synaptopodin homologue, is frequently deleted in invasive prostate cancers. Am J Pathol 159: 1603–1612, 2001. 4. Mundel P, Reiser J, Zuniga Mejia Borja A, et al: Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236: 248 –258, 1997. 5. Nagata M, Nakayama K, Terada Y, et al: Cell cycle regulation and differentiation in the human podocyte lineage. Am J Pathol 153: 1511–1520, 1998. 6. Sanchez-Carbayo M, Schwarz K, Charytonowicz E, et al: Tumor suppressor role for myopodin in bladder cancer: loss of nuclear expression of myopodin is cell-cycle dependent and predicts clinical outcome. Oncogene 22: 5298 –5305, 2003. 7. Jing L, Liu L, Yu YP, et al: Expression of myopodin induces suppression of tumor growth and metastasis. Am J Pathol 164: 1799 –1806, 2004.
UROLOGY 68 (3), 2006