World J Urol (2013) 31:359–364 DOI 10.1007/s00345-012-0977-z
ORIGINAL ARTICLE
Association of legumain expression pattern with prostate cancer invasiveness and aggressiveness Yoshio Ohno • Jun Nakashima • Miki Izumi Makoto Ohori • Takeshi Hashimoto • Masaaki Tachibana
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Received: 2 September 2012 / Accepted: 16 October 2012 / Published online: 3 November 2012 Ó Springer-Verlag Berlin Heidelberg 2012
Abstract Objectives To investigate the clinical implication of legumain, an asparaginyl endopeptidase that is highly expressed in several types of cancer, expression in prostate cancer. Methods Legumain expression in prostate cancer cell lines was determined by real-time reverse transcriptase PCR and Western blot. Furthermore, legumain expression in 88 prostatectomy specimens was evaluated by immunohistochemistry. The association between legumain expression and clinicopathological factors was analyzed. Results Legumain expression was confirmed at the mRNA and protein levels in all the cells. Although all the cancer tissues were positive for legumain, 2 staining patterns were observed in the cytoplasm: diffuse cytoplasmic and vesicular positivity. The rates of Gleason score C8, extracapsular extension, and perineural invasion in the group with vesicular staining were significantly higher than those in the diffuse cytoplasmic group (p \ 0.05). The maximum size of the tumor with vesicular staining was significantly greater than that of the tumor with diffuse cytoplasmic staining (p = 0.0302). The 5-year biochemical recurrence-free rate in the patients with vesicular legumain staining was 53.2 %; this rate was significantly lower than that (78.8 %) in the patients with diffuse cytoplasmic staining (p = 0.0269). Y. Ohno (&) J. Nakashima M. Ohori T. Hashimoto M. Tachibana Department of Urology, Tokyo Medical University, 6-7-1, Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan e-mail:
[email protected] M. Izumi Department of Diagnostic Pathology, Tokyo Medical University, Tokyo, Japan
Conclusions Tumors that showed a vesicular staining pattern of legumain had the potential of being highly invasive and aggressive in patients with prostate cancer who were treated with radical prostatectomy. This suggests that legumain might contribute to the invasiveness and aggressiveness of prostate cancer. Keywords Legumain Prostate cancer Biochemical recurrence Prognostic factor
Introduction Prostate cancer is one of the most common cancers and the second leading cause of cancer death of men in the United States [1]. Most patients have early-stage cancer and can be cured by radical prostatectomy or radiation therapy. Nonetheless, a substantial fraction of patients with clinically localized prostate cancer will eventually experience disease recurrence with metastasis [2]. Although androgen deprivation therapy is the treatment of choice in patients with metastatic disease, prostate cancer will eventually progress to a castration-resistant condition. Decetaxelbased chemotherapy is currently the standard choice for castration-resistant prostate cancer but shows limited efficacy. Recently, several new agents such as cabazitaxel and abiraterone acetate have been approved in the United States and Europe [3, 4]. However, the clinical efficacy of these agents is still modest. Thus, it is necessary to investigate the mechanisms of prostate cancer progression and acquisition of androgen independence for developing new treatment strategies for advanced or recurrent prostate cancer. Recently, we identified legumain as a candidate protein involved in prostate cancer progression, using serum
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proteomic analysis (unpublished data). Legumain, an asparaginyl endopeptidase, belongs to the C13 family of cysteine proteases, which include caspases and separases [5]. Under physiological conditions, legumain is present in acidic endosomes/lysosomes and is involved in intracellular protein degradation [6]. Overexpression of legumain is associated with breast and colorectal cancer progression [7–9]. As for prostate cancer, the implication of legumain expression remains unclear though Liu et al. [9] showed high positivity of legumain in prostate cancer specimens by immunohistochemical analysis. In this study, we first evaluated legumain expression in 4 prostate cancer cell lines: androgen-dependent LNCaP cells, and androgen-independent PC-3, DU-145, and C4-2 cells [10, 11]. Second, we investigated the clinical implication of legumain expression in prostate cancer via immunohistochemical analyses of radical prostatectomy specimens.
Materials and methods Cell lines Four human prostate cancer cell lines (PC-3, DU-145, LNCaP, and C4-2) were maintained in RPMI-1640 medium containing 10 % fetal bovine serum and grown at 37 °C in a humidified atmosphere. PC-3 and DU-145 cells are reported to cause osteolytic bone metastases in animals, and C4-2 cells cause osteoblastic bone metastases [10, 11]. Real-time reverse transcriptase polymerase chain reaction Cells at 70–80 % confluence were harvested, and total RNA was extracted using Isogen (Nippon Gene, Toyama, Japan). One microgram of total RNA was subjected to reverse transcription using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Real-time PCR amplifications were then carried out using the Fast SYBR Green Master Mix (Applied Biosystems) and StepOnePlus System (Applied Biosystems). The total PCR volume was 20 lL and contained 20 ng of cDNA. The sequences of the primers used are as follows: legumain forward primer, 50 -GATGAACCACCTGCCGGATAA-30 ; legumain reverse primer, 50 -CATCATAGTAACAGGCG TAGGACGA-30 ; b-actin forward primer, 50 -ATTGCCGA CAGGATGCAGA-30 ; b-actin reverse primer, 50 -GAGTAC TTGCGCTCAGGAGGA-30 . Data were analyzed according to the comparative Ct method and were normalized to b-actin expression in each sample.
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Western blot analysis Twenty micrograms of total protein lysates were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membrane was probed with a goat anti-human legumain antibody (2009, R&D Systems, Minneapolis, MN, USA). The membrane was then incubated with anti-goat horseradish peroxidase-conjugated IgG secondary antibody (dilution at 1:1200; Amersham Pharmacia, Piscataway, NJ, USA). Immunoreactive proteins were detected with an enhanced chemiluminescence detection kit (Amersham Pharmacia) and LumiVision HS (Taitec Co., Tokyo, Japan). Subsequently, the membranes were stripped using the Restore Western Blot Stripping Buffer (Pierce, Rockford, IL, USA) and reprobed with a mouse anti-human b-actin monoclonal antibody (dilution at 1:2000; SigmaAldrich, St Louis, MO, USA), followed by an anti-mouse secondary horseradish peroxidase-conjugated IgG antibody (dilution at 1:2000; Amersham Pharmacia). The legumain protein level was expressed as a relative ratio (RR) with the following formula, using the signal intensities (SIs) of legumain and b-actin. RR ¼ SIlegumain =SIbactin Immunohistochemical analysis of the surgical specimens Surgical specimens were obtained from 88 patients who underwent radical prostatectomy between September 2003 and December 2005 at our institute. None of the patients received androgen ablation or radiation therapy before surgery. The median age was 70 years (range, 50–76 years). The mean preoperative serum prostate-specific antigen (PSA) level was 12.3 ± 14.4 ng/mL. Tumor stage was classified according to the 2002 TNM staging system [12]: 44 patients had clinical T1c tumor; 28, clinical T2 tumor; and 6, clinical T3 tumor. The median follow-up period was 77 months (range, 12–99 months). Twentyeight patients experienced a biochemical recurrence after a median time of 10 months (range, 2–44 months). Archival formalin-fixed paraffin embedded tissues of a representative area of the surgical specimens, which included the highest Gleason grade cancer, were sectioned into 4-mm-thick slices and mounted on poly-L-lysinecoated slides. The slides were incubated with a mouse antihuman legumain monoclonal antibody (dilution at 1:100; R&D Systems). Bound antibodies were detected with the avidin–biotin complex peroxidase method (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, USA) and visualized with diaminobenzidine. Legumain expression was scored as positive if more than 10 % of the cells were clearly stained in each slide under microscopic observation.
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Statistical analysis All the statistical analyses were performed using the JMP Ver. 9.0 (SAS Institute Inc., Cary, NC, USA). Differences in the legumain mRNA and protein expression levels in the different cell lines were determined by ANOVA. The correlation between legumain expression and clinicopathological factors was assessed using the chi-square test, Fisher’s exact test, and Mann–Whitney U test. Biochemical recurrence was defined as 2 consecutive serum PSA values C0.2 ng/mL [13]. The date of recurrence was the date of the first PSA value C0.2 ng/mL. The biochemical recurrence-free rate was estimated using the Kaplan–Meier method, and the difference between the curves was tested using the log-rank test. p \ 0.05 was considered statistically significant.
Results
361
A 0 LNCaP
PC3
DU145
C4-2
-1 -2
ΔCt
-3 -4 -5 -6 -7
B
β-actin
legumain
Legumain expression in the prostate cancer cell lines Legumain expression was confirmed in all the prostate cancer cell lines at the mRNA and protein levels. The realtime PCR analysis showed that legumain expression level was highest in the DU-145 cells, followed by the PC3, C4-2, and LNCaP cells (Fig. 1).
Fig. 1 Legumain expression at the mRNA and protein levels. a Legumain expression was confirmed in all the cell lines by realtime PCR. Expression level was highest in the DU-145 cells. b The Western blot showed similar legumain expression levels in all the cell lines
Immunohistochemistry in tissue samples In the normal prostate gland, the legumain staining had a vesicular pattern and most vesicles were located in the luminal side of the prostate glands (Fig. 2a). The immunohistochemical staining showed that all the tumor samples were positive for legumain at various degrees. However, we identified 2 staining patterns in the cytoplasm: diffuse cytoplasmic and vesicular staining (Fig. 2b, c). In addition, vesicles were scattered in the cytoplasm and were sometimes located in the basal side of the cytoplasm (Fig. 2c). Nuclear or membrane-associated positivity was not observed. Legumain expression pattern, clinicopathological factors, and prognosis The 2 staining patterns were sometimes observed in combination with the tumor cells, a similar finding to that previously reported in breast cancer. Therefore, the tumor cases were divided into 2 groups based on the predominant staining pattern. The diffuse cytoplasmic pattern predominated in 49 tumors (59 %), and the vesicular pattern predominated in 39 tumors (41 %). The association between the clinicopathological variables and legumain staining pattern is summarized in
Table 1. The rates of pathological T3 (59.0 %), Gleason score C8 (55.6 %), extracapsular extension (52.7 %), and perineural invasion (58.3 %) in the vesicular pattern group were significantly higher than those in the diffuse cytoplasmic pattern group (21.5, 11.5, 36.5, and 36.5 %, respectively; p \ 0.05). The maximum size of the tumor in the samples with a vesicular staining pattern was significantly greater than that in those with diffuse cytoplasmic pattern (23.3 mm vs. 18.8 mm; p = 0.0302). The 5-year biochemical recurrence-free rate in the patients with a vesicular staining tumor was 51.7 %, which is significantly lower than that observed in the patients with the diffuse cytoplasmic staining tumors (79.4 %; Fig. 3, p = 0.021).
Discussion Legumain, an asparaginyl endopeptidase, is expressed in several human tissues such as the kidney, placenta, liver, spleen, testis, and bone marrow plasma [9, 14]. Legumain plays an important role in antigen presentation, regulation of osteoclast formation, and extracellular matrix remodeling [14–17]. An earlier study by Liu et al. [9] demonstrated that legumain was expressed in several types of cancers
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Fig. 2 Legumain expression by immunohistochemistry. a Legumain expression in a normal prostate gland. Vesicular staining was observed, and most vesicles were located in the luminal side of the prostate glands. b Prostate cancer tissue. The tumor cells exhibited
diffuse positive staining in the cytoplasm. c Prostate cancer tissue. The tumor cells exhibited vesicular positivity in the cytoplasm. Vesicles were scattered in the cytoplasm and were sometimes located in the basal side of the cytoplasm (arrow)
such as colon, breast, and prostate cancers. In human colorectal cancers, legumain expression was associated with poorer differentiation/mucinous carcinoma and patients with tumors that showed lower legumain expression levels had better prognoses [7]. Gawenda et al. [8] reported the association of legumain staining pattern by immunohistochemistry with prognosis in patients with breast cancer. Wang et al. [18] reported that a higher legumain expression level was associated with poor outcome in patients with ovarian cancer. In this study, we demonstrated that legumain is expressed in prostate cancer cell lines and surgically resected prostate cancer tissues. Although it has been previously reported that legumain was highly expressed in human prostate cancer specimens, the clinical implication of its expression remained unclear. In this study, we demonstrated that a vesicular staining pattern of legumain was significantly associated with advanced tumor stage, high Gleason score, perineural invasion, and larger tumor. Furthermore, these patients had lower biochemical recurrence-free rates. To our knowledge, this is the first study that investigated the clinical implication of legumain expression in prostate cancer.
So far, the biological roles of legumain in cancer development and progression have not been fully elucidated. Legumain is a stress-response gene, and its expression was elevated in cells under serum starvation or during in vivo growth. The legumain-positive membranous vesicles were often observed at the invadopodia of tumor cells. It is thought that tumor cells with high legumain expression levels exhibited increased invasion capacity through increased extracellular matrix degradation by activation of progeratinase A. Furthermore, legumain is reported to enhance the invasive and metastatic potential of mouse colon cancer models [9]. Based on its high expression in many human tumors and its unique enzymatic properties, legumain represents a potential target for cancer therapeutics. Several prodrugs of anticancer agents such as doxorubicin [19], etoposide [20], and auristatin [21] are activated by legumain and demonstrated cytotoxicity against several cancer cell lines. On the other hand, a research group developed a legumain-based vaccine strategy that induced the production of cytotoxic T lymphocytes specific to tumor-associated macrophages and suppressed breast cancer growth and angiogenesis [22, 23].
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Table 1 Association between clinicopathological variables and legumain staining pattern
Staining pattern Cytoplasmic
p value Vesicular
No. of patients
49 (%)
39 (%)
Median age (range)
67
68
0.4081
PSA level at diagnosis
12.6
12.1
0.8847
B2
33
15
0.0163
3
16
23
B6
17
4
7
21
15
C8
11
20
17.8
23.3
0.0179 0.0315
Pathological T stage
Gleason score
Maximum diameter of cancer (mm) Extracapsular extension Neg.
33
17
Pos.
16
22
0.0048
Lymphovascular invasion Neg.
38
26
Pos.
11
13
0.3361
Biochemical recurrence-free rate
Perineural invasion Neg.
33
15
Pos.
16
24
Diffuse cytoplasmic (n= 49)
Vesicular (n= 39)
Log-rank: P = 0.021
Followup (months) Fig. 3 Biochemical recurrence-free rate. The 5-year biochemical recurrence-free rate in the patients with tumors showing legumain vesicular staining was significantly lower than that in the patients with diffuse cytoplasmic staining (p = 0.021)
However, further studies are needed to develop applications of these approaches to prostate cancer treatment. Prostate cancer has a heterogeneous natural history, ranging from an indolent to an aggressive course. For localized prostate cancer, radical prostatectomy or radiotherapy is the standard treatment. However, up to 40 % of patients have been reported to experience postoperative recurrence, and some patients eventually die of prostate cancer [2]. Thus, it is important to identify patients at
0.0097
increased risk of disease progression and cancer death. There are many established indicators of aggressiveness of prostate cancer including Gleason grade, stage, and various biomarkers [24–27]. In the present study, we demonstrated that a vesicular staining pattern of legumain was associated with lower biochemical recurrence rate. It might be possible to use the legumain expression pattern as an additional prognostic marker in patients with prostate cancer. Further studies including a larger patient cohort study with a longer follow-up are required to validate the prognostic significance of legumain expression patterns in prostate cancer. In addition, although we confirmed legumain expression in all 4 prostate cancer cell lines, only marginal differences in its expression were observed among the cell lines. Additional functional analyses, such as the effects of androgen stimulation on legumain expression and the association of legumain expression levels with the invasive potential of prostate cancer cells, are also necessary to clarify the pathophysiological roles of legumain in prostate cancer progression.
Conclusion Prostate tumors that showed a vesicular staining pattern of legumain are highly invasive and aggressive in patients treated with radical prostatectomy. This suggests that
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legumain might contribute to the invasiveness and aggressiveness of prostate cancer. Acknowledgments This study was partially supported by the Strategic Research-Based Support Project for private universities, with matching funds from the Ministry of Education, Culture, Sports, and Science, Japan. Conflict of interest No author has any financial disclosures or conflict of interest to declare.
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