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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. Reference
  9. Supporting Information

Mitochondria are key organelles for ATP production and apoptosis. Therefore, impairment of mitochondria can modulate or accelerate cancer progression. p32, originally identified as a pre-mRNA splicing factor SF2/ASF-associated protein, is localized predominantly in the mitochondrial matrix and involved in mitochondria respiration. Recently, p32 was implicated in apoptosis and resultantly cancer progression. However, little is known about the expression and function of p32 in human tumors including prostate cancer. Here, we investigated the expression of p32 in 148 prostate carcinoma tissues by immunohistochemistry and found a positive correlation of p32 expression to clinicopathological parameters including follow-up data. p32 is highly expressed in prostate tumor samples and its expression is significantly associated with the Gleason score, pathological stage and relapse. For localized cancers, high p32 is a strong and independent predictor of clinical recurrence in multivariate analysis (= 0.01). In addition, p32 is overexpressed in the prostate cancer cell lines examined. The selective knockdown of p32 by RNA interference inhibits the growth of prostate cancer cell lines but not of a non-cancerous cell line. The p32 RNA interference decreases cyclin D1, increases p21 expression and causes a G1/S cell cycle arrest in prostate cancer cells. These data suggest that p32 is critical for prostate cancer cell proliferation and may be a novel marker of clinical progression in prostate cancer. (Cancer Sci 2011; 102: 639–647)

p32 (C1QBP/gC1qR/HABP1) was first isolated from a membrane preparation of Raji cells historically and was originally copurified with the pre-mRNA splicing factor SF2/ASF in human HeLa cell.(1–3) p32 protein is a doughnut-shaped trimer(4) that is primarily localized in the mitochondria,(1,5–7) but has also been reported to be present at the cell surface(6,8) and in the nucleus(5,9,10) Human p32 has been reported to interact with a variety of molecules including human immunodeficiency virus Tat, complement 1q (C1q) and hyaluronic acids.(11) These results suggest that p32 may be a multifunctional chaperone protein.(12) Recently, it was reported that p32 was implicated in mediating the cellular apoptotic response.(13) ARF interacts with p32 and the interaction is critical in order for ARF to localize to the mitochondria and induce apoptosis.(14,15) It has also been proposed that p32 is a link to autophagy.(16,17) However, the role of p32 in mammalian cancer cells is unclear.

Previously, we identified a Saccharomyces cerevisiae homologue of the human p32 gene, referred to as mam33, which was localized in the mitochondrial matrix. Disruption of the mam33 gene caused growth retardation and impairment of mitochondrial ATP synthesis. The growth impairment was restored by the introduction of human p32 cDNA, indicating that mam33 is a functional yeast counterpart of human p32. Taken together, both human p32 and yeast mam33 reside in the mitochondrial matrix and play an important role in maintaining mitochondrial oxidative phosphorylation.(1)

Prostate cancer is the second commonest cause of male cancer death in the developed world and is the most frequent from of malignancy diagnosed.(18,19) Various lifestyles and nutritional factors, particularly high-energy consumption, were reported in relation to a risk of prostate cancer.(18,20) In patients with locally advanced disease or metastatic cancer, androgen ablation remains the mainstay of treatment. However, the effect of this treatment is only transient, with most patients developing hormone-refractory disease within 2–3 years. A better understanding of the mechanism involved in regulating tumor growth and the identification of novel growth factors implicated in disease progression would be useful to develop new therapeutic approaches. Currently, there are few markers clinically available for accurate prediction of the prognosis of prostate cancer patients besides prostate-specific antigen (PSA) and Gleason score.(21)

The metabolic phenotype is characterized by a shift from oxidative phosphorylation (OXPHOS) towards aerobic glycolysis as the main source of ATP production, a phenomenon first described by Warburg.(22) It is now recognized that the Warburg effect represents a prominent metabolic characteristic of malignant cells. Although Warburg has speculated that mitochondrial respiration is decreased in cancer cells, mitochondrial activity is essential for cancer cell survival. The mitochondria also participates in many essential and fundamental metabolic pathways including the synthesis of pyrimidine through dihydroorotate dehydrogenase (DHODH) in addition to respiration.(23)

The cell cycle phases are coordinated by the expression and/or activation of regulatory proteins, including cyclins (e.g. cyclins D, E and A), cyclin-dependent kinases (CDK) and cyclin-dependent kinase inhibitors (CDKIs).(24) CDK4 and CDK6 form complexes with one of several D-type cyclins and function early in the G1 phase. CDK2 complexed with cyclins A and E is essential for DNA replication and G1/S transition, respectively.(25) Five membrane-anchored protein complexes (complex I–V) are the main machineries for mitochondrial oxidative phosphorylation, and defects in this process have been implicated in the progression of ageing and in diverse age-related disorders such as cancer and degenerative diseases. Thus, it has been broadly accepted that there is a close relationship between respiratory control and apoptosis/cell cycle progression.

Considering that p32 is a key molecule of oxidative phosphorylation and apoptosis in mitochondria, it might be involved in cancer progression. In fact, the expression levels of p32 were significantly increased in multiple human cancer tissues compared with normal tissues. In the current study, we investigated the expression of p32 in prostate carcinomas of patients by immunohistochemistry and found a strong correlation of the expression to clinicopathological parameters including follow-up data. We also investigated the expression of p32 in human cancer cell lines and examined the effects of its small interference RNA (siRNA)-mediated knockdown on cell proliferation and the cell cycle. Here we propose that mitochondrial matrix protein p32 may be a novel progressive marker in prostate cancer.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. Reference
  9. Supporting Information

Patients, tissue and clinicopathological data.  The subjects were 148 patients (age, 47–78 years; mean, 65.6 years), who received radical prostatectomy with no chemotherapy or hormonal therapy before surgery at the Kyushu University Hospital, Fukuoka, Japan, between 1997 and 2006 and had enough carcinoma area for evaluation of immunohistochemistry. All patients underwent surgery for clinically localized prostate cancer as determined by preoperative PSA concentration, digital rectal examination and prostate needle biopsy. Detailed clinicopathological findings of the patients are summarized in Supporting Information Table S1. All prostatectomy specimens were completely reviewed to establish stage and grade of the respective prostate cancers.(21) Four patients received adjuvant hormonal therapy after surgery. Sixteen patients had <2 years of follow up. Clinical follow-up data were available for 128 patients. Their serum PSA was monitored; a PSA level >4 ng/mL was defined as PSA recurrence. Prostate-specific antigen recurrence was found in 20% (26/128) of patients. The median follow-up time of patients still relapse-free at the end of analysis was 49.5 months.(19)

Antibody.  Anti-TFAM were raised in our laboratory.(26) Anti-p32 mouse monoclonal antibody (clone1) was produced in the Japan Clinical Laboratories Incorporation (Kyoto, Japan). Anti-β-actin was purchased from Sigma–Aldrich (Steinheim, Germany). Anti-cyclinD1, p15INK4B, p21Waf1/cip1, p27kip1, CDK4 and CDK6 were purchased from Cell Signaling (Danvers, MA, USA) and anti-B23 was purchased from Invitrogen (San Diego, CA, USA).

Immunohistochemistry.  Immunohistochemistry was performed using the streptavidin–biotin–peroxidase method with a Histofine streptavidin–biotin–peroxidase kit (Nichirei, Tokyo, Japan). The primary antibody used in the present study was anti-p32 antibody (mouse monoclonal IgG1, 13 μg/mL). The specificity of p32 antibodies was tested using pre-absorption with recombinant p32 antigens with optimally diluted antiserum overnight at 4°C (Supporting Information Fig. S1). Sections, 4-μm thick, from 10% formalin-fixed, paraffin-embedded material were deparaffinized in xylene and dehydrated through ethanol. Their endogenous peroxidase activity was then blocked by methanol containing 0.3% hydrogen peroxidase for 30 min. After the peroxidase activity blocking, the sections were immunized to 10% rabbit serum in phosphate-buffered saline for 10 min; the sections were incubated at room temperature for 2 h with anti-p32. Those sections were then incubated with the second antibody for 20 min at room temperature. The reaction products were visualized by 3,3′-diaminobenzidine tetrahydrocholoride (DAB) as a chromogen. Finally, the sections were counterstained with hematoxylin.

Immunohistochemical analysis.  To assess p32 expression, we evaluated normal prostatic epithelium and carcinoma cells. In addition, an Allred score, which is a semi-quantitative system that takes into consideration the proportion of positive cells (scored on a scale of 0–5) and staining intensity (scored on a scale of 0–3), was determined for each case. The proportion and intensity were then summed to produce total scores of 0 or 2 through 8. A score of 0 was regarded as negative while weak was 2, moderate was 3–6 and strong was 7–8, as previously described.(19) Three people including a pathologist analyzed the expression independently and we used an average score of the intensity levels.

Statistical analysis.  The statistical analyses were performed using JMP version 7.0.1. (SAS Institute Inc., Cary, NC, USA). We used the Chi-squared test for statistical analysis of the correlations between immunohistochemical p32 expression and clinicopathological parameters. Disease-free survival was taken as the period between surgery and the date of the last follow up or PSA recurrence by disease (n = 128). Survival curves were calculated by the Kaplan–Meier method, and the significance was analyzed by log-rank test. The Cox proportional hazards model was used for multivariate survival analysis, in which we estimated the hazard ratio. Two-sided < 0.05 was considered statistically significant.

Cell culture.  The cells used were the human prostate cancer cell lines LNCaP, PC3, Du145, 22Rv1 and primary prostate epithelial cells, RWPE-1. All cell lines were obtained from the American Type Culture Collection (ATCC). PC3, Du145, 22Rvl and LNCaP cells were grown in RPMI-1640 medium (Invitrogen) containing 5% fetal bovine serum (FBS). Normal epithelial RWPE-1 cells were grown in keratinocyte-SFM medium (Invitrogen) containing 5 ng/mL epidermal growth factor and 50 μg/mL bovine pituitary extract. All cells were cultured in a humidified incubator with 5% CO2 and 95% air at 37°C. Charcoal-stripped FBS was used for assays of proliferation and p32 expression on the prostate cancer cell line.

Immunoblot analysis.  Cells were lysed with Lysis buffer (50 mM Tris–HCl, pH7.5, 1 mM EDTA, 150 mM NaCl and 0.5% NP-40) and subjected to immunoblot analysis as described. Proteins were separated by SDS–PAGE and immunoblotted with the indicated specific antibodies. The signals were visualized with horseradish peroxidase-labelled anti-rabbit immunoglobulin G and an ECL reagent (GE Healthcare, Uppsala, Sweden). The chemiluminescence was recorded and quantified with a chilled charge-coupled device camera, LAS1000plus (Fuji Photo Film, Tokyo, Japan). Cell fractionation and intracellular localization of p32 were performed as previously described.(27)

Immunofluorescent imaging of prostate cancer cells.  Human PC3, LNCaP RWPE-1 cells were incubated in the presence of 100 nM MitoTracker Red for 20 min. The cells were fixed, permealized and stained with 250-fold diluted anti-p32 antibody as previously described.(27)

Knockdown analysis using siRNA.  The 25 bp double-stranded RNA targeting p32 was generated (Stealth Select RNAi; Invitrogen): 5′-AUAAUGACAGUCCAACACAAGGGCC-3′ and 5′-GGCCCUUGUGUUGGACUGUCAUUAU-3′. The siRNA transfection was performed according to the manufacturer’s instructions (Invitrogen). The cells were seeded in six-well plates and assayed for the indicated time of western blotting and FACS analysis.

Cell proliferation assay.  To determine cell proliferation, RWPE-1 and PC3 cells transfected with control- or p32-siRNA were seeded in 24-well plates at a density of 2 × 104 cells per well. After 24 h, one set of cells was trypsinized, resuspended in PBS and counted by a cell counter (Beckman Coulter Inc., Brea, CA, USA). The cells were counted in a similar way every 24 h, up to 120 h.

Cell cycle analysis.  The cell cycle of RWPE-1 and PC3 cells were analyzed by flow cytometry with FACS caliber (Becton Dickinson, Franklin Lakes, NJ, USA). Cells were trypsinized and collected by centrifugation, washed and re-suspended in 0.1% BSA + PBS, and fixed in 70% ethanol at a density of 1 × 106 cells/mL. After the addition of 1000 units RNase A and 15 min incubation at room temperature, the cells were stained with 10 μL propidium iodide for 1 h. The cell cycle distribution was analyzed using the FlowJo software.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. Reference
  9. Supporting Information

Overexpression of p32 protein in prostate tumor tissues.  Normal prostate tissue in the vicinity of prostate carcinoma was evaluated by immunohistochemistry for all 148 cases. p32 expression was mainly localized to the cytoplasm in epithelial cells and strong staining was observed in the cytoplasm of basal cells in normal glands (Fig. 1A). Luminal epithelial cells of normal prostate glands showed an homogenous moderate positivity. In tumor cells, p32 expression was strong and observed in the cytoplasm, but not in the plasma membrane (Fig. 1B–E). Increased intensity of homogeneous p32 expression was noted throughout the epithelium in high-grade prostate cancer. p32 expression of high-grade carcinoma was very strong, especially in poorly differentiated adenocarcinoma (Fig. 1D). Expression of p32 in normal prostatic epithelial cells was weak compared with that in the adjacent high-grade carcinoma cells (Fig. 1F). A total of 23/148 patients’ carcinoma tissue showed strong expression of p32 also in the nucleus (Fig. 1G, arrows). Nuclear p32 expression was much less than the cytoplasm. Strong p32 expression was observed in mucinus adenocarcinoma in two samples (Fig. 1H).

image

Figure 1.  Immunohistochemistry examination of the expression of p32. (A) A benign gland tissue. Weak p32 staining was evident in the cytoplasm of luminal epithelial cells of normal prostate glands, whereas the majority of basal epithelial cells revealed moderate or strong p32 positivity (arrows). (B) Weak p32 staining and (C) moderate p32 staining in a low-grade malignant tumor. (D) Strong p32 staining in a high-grade prostate cancer. (E) Moderate p32 expression in the cytoplasm with high magnification. (F) Normal prostate glands and adjacent high grade prostate carcinoma. (G) High-grade prostate carcinoma with nuclear staining of p32 (arrows). (H) Mucinus adenocarcinoma. Scale bar, 50 μm.

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Correlations of p32 expression with clinicopathological factors and survival.  We have summarized the correlations between p32 expression and the clinicopathological parameters in Table 1. p32 expression was significantly increased in higher Gleason score compared with lower Gleason score cancer (< 0.001) and p32 expression was significantly increased in pT3 or more advanced cancer compared with pT2 cancer (= 0.01). We also observed that p32 expression was increased in extraprostatic extension (EPE) positive compared with EPE negative (Supporting Information Table S2). A statistically strong significance was also observed between p32 expression and PSA relapse (< 0.001). However, there was no significant association between p32 expression and patient’s age, preoperative PSA, surgical margin or invasion (Table 1).

Table 1.   Relationship between the expression of p32 and various clinicopathological characteristics
Variablep32 expression
n (%)Weak∼ModStrongP-value
  1. †Statistically significant (Chi-squared test). PSA, prostate-specific antigen.

Age (years)
 <7099 (66.9)57 (38.5)42 (28.4)0.45
 ≥7049 (33.1)25 (16.9)24 (16.2)
Gleason score
 ≤639 (26.4)31 (21.0)8 (5.4)<0.001†
 797 (65.5)49 (33.1)48 (32.4)
 ≥812 (8.1)2 (1.4)10 (6.8)
Preoperative PSA (ng/mL)
 <1089 (62.2)50 (35.0)39 (27.3)0.88
 ≥1054 (37.8)31 (21.7)23 (16.1)
Pathological stage
 pT2101 (68.2)63 (42.6)38 (25.7)0.01†
 pT3∼T447 (31.8)19 (12.8)28 (18.9)
Surgical margin
 Negative81 (54.7)49 (33.1)32 (21.6)0.17
 Positive67 (45.3)33 (22.3)34 (23.0)
Invasion
 Negative136 (91.9)77 (52.0)59 (39.9)0.31
 Positive12 (8.1)5 (3.4)7 (4.7)
Relapse
 (−)115 (81.6)72 (51.1)43 (30.5)<0.001†
 (+)26 (18.4)5 (3.6)21 (14.9)

We observed strong expression in the nuclei of 23/148 patients’ carcinoma tissue (Fig. 1G). We investigated the correlations between p32 nuclear expression and the clinicopathological parameters in Table 2. A statistically strong significance was observed between p32 nuclear expression and p32 cytoplasmic staining (< 0.01). p32 nuclear expression was significantly increased in higher Gleason score (= 0.03), pathological stage (= 0.02) and preoperative PSA (= 0.01) (Table 2).

Table 2.   Relationship between the nuclear expression of p32 and various clinicopathological characteristics
Variablep32 expression of nucleus
n (%)+P-value
  1. †Statistically significant (Chi-squared test). PSA, prostate-specific antigen.

Age (years)
 <7099 (66.9)84 (56.8)15 (10.1)0.85
 ≥7049 (33.1)41 (27.7)8 (5.4) 
p32 expression
 Weak∼moderate82 (55.4)80 (54.1)2 (1.4)<0.01†
 Strong66 (44.6)45 (30.4)21 (14.2)
Preoperative PSA (ng/mL)
 <10.089 (62.2)80 (55.9)9 (6.3)0.01†
 ≥10.054 (37.8)40 (28.0)14 (9.8)
Gleason score
 ≤639 (26.4)38 (25.7)1 (0.7)0.03†
 797 (65.5)77 (52.3)20 (13.5)
 ≥812 (8.1)10 (6.8)2 (1.4)
Pathological stage
 pT2101 (68.2)90 (60.8)11 (7.4)0.02†
 pT3-447 (31.8)35 (23.7)12 (8.1)

Multivariate survival analysis of localized prostate carcinomas.  The PSA recurrence-free survival rate was used as an end-point. Kaplan–Meier survival curve analyses of 102 prostate cancer tumors with clinical follow-up information showed that patients with high p32 expression in their tumors had a significantly reduced PSA recurrence-free survival rate compared with patients who had low p32 expression (log-rank test, = 0.0001) (Fig. 2).

image

Figure 2.  Disease-free survival curves of patients in high and low p32 expression groups. Disease-free survival was analyzed using the Kaplan–Meier method for high and low p32 expression in localized prostate cancer with clinical recurrence. P-values were obtained by a log-rank test (= 0.0001).

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Next, we performed the Cox regression multivariate analysis with parameters including age, high p32 expression, Gleason score, high PSA and local metastasis. Table 3 summarizes the results of PSA recurrence-free survival analyzed by univariate and multivariate analysis. The Cox proportional hazards model revealed that strong p32 expression (4.10: hazard ratio [HR], 95% confidence interval [CI], 1.39–12.07; = 0.01) and high PSA level (4.56: HR, 95% CI, 1.86–11.20; = 0.001) were independent prognostic factors. The higher Gleason score (≥8) (3.22: HR, 95% CI, 0.87–11.89; = 0.08) was not a significantly independent prognostic factor. Thus, high p32 expression consistently showed a strong and independent prognostic effect on clinical recurrence.

Table 3.   PSA recurrence-free survival in Cox regression analysis
 PSA recurrence-free survival univariate analysisP-valuePSA recurrence-free survival multivariate analysisP-value
Hazard ratio95% CIHazard ratio95% CI
  1. †Statistically significant. CI, confidence interval; PSA, prostate-specific antigen.

Age (years)
 <701 0.661 0.58
 ≥701.190.54–2.620.790.34–1.82
p32 expression
 Weak∼moderate1 0.001†1 0.01†
 Strong5.432.04–14.414.101.39–12.07
Preoperative PSA (ng/mL)
 <10.01 <0.01†1 0.001†
 ≥10.04.581.99–10.554.561.86–11.20
Gleason score
 ≤61  1  
 71.500.49–4.580.460.880.27–2.830.83
 ≥87.752.32–25.840.001†3.220.87–11.890.08
Pathological stage
 pT21 0.002†1 0.60
 pT3-43.451.593–7.4731.270.51–3.17
Surgical margin
 Negative1 0.02†1 0.51
 Positive2.481.10–5.591.350.54–3.40

p32 expression of human prostate cancer cell line.  To find the molecular mechanism of p32 in prostate tumor progression, we first investigated the expression of p32 in human prostate cancer cell lines and noncancerous cell line RWPE-1. High p32 expression was seen in all prostate cancer cell lines examined (PC3, 22Rv1, LNCaP and Du145) compared with the noncancerous cell line RWPE-1 (Fig. 3A). There was no difference in the β-actin and TFAM (mitochondrial DNA nucleoid protein) expressions among the cell lines (Fig. 3A).

image

Figure 3.  Expression of p32 in prostate cancer cell lines. (A) Western blot analysis of p32 expression in prostate cancer and noncancerous cell lines. β-actin and TFAM were used as the internal control. (B) Immunocytochemistry of p32 in prostate cancer cells. Mitochondria and p32 were visualized with a mitochondria-staining dye, MitoTracker Red (middle panels), and anti-p32 antibodies (left panel), respectively. The right panels are merged. Scale bar, 20 μm. (C) Subcellular localization of p32 in prostate cancer cells. The prostate cell lysates (WC) were separated into nuclear extract (NE) and cytosolic (Cyto) fractions. The indicated proteins were detected by immunoblotting. B23 and TFAM were used as markers for nuclei and cytosolic, respectively. (D) Downregulation of p32 by androgen deprivation. Cells were treated with charcoal-treated FBS medium for 6 days. The p32 protein was analyzed by western blot.

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p32 localized in mitochondria.  Because we observed strong expression of p32 in the nuclei of carcinoma tissue, we investigated the localization of p32 in prostate cancer cell lines. In the immunocytochemistry, p32 was granularly stained with antibodies in the PC3, LNCaP and RWPE-1 cells (Fig. 3B). The p32 staining was completely co-localized with the mitochondria visualized with a MitoTracker Red dye, suggesting that the expressed p32 is exclusively localized at mitochondria. Androgen deprivation did not lead nuclear translocation of p32 in these cells (data not shown). We then examined the intracellular localization of p32 in the prostate cancer cell line by western blotting. The cells were separated into nuclear extract and cytosolic fractions. B23 and TFAM were used as markers for the nuclei and cytosolic fraction, respectively. In all prostate cell lines, p32, as well as TFAM, was found mostly in the cytosolic fraction (Fig. 3C). These results suggest that p32 is localized at mitochondria but not at nuclear in prostate cancer cell lines.

Androgen receptor (AR) signaling plays a critical role in prostate cancer. We investigated p32 expression on prostate cancer cell lines after androgen deprivation. We observed reduced expression of p32 in androgen-sensitive LNCaP cells, but not insensitive PC3 and Du145 cells (Fig. 3D and Supporting Information Fig. S2). These results suggest that p32 expression might be regulated by AR signaling.

Involvement of p32 in prostate cancer cell proliferation.  To examine the role of p32 in prostate cancer cell proliferation, the cell growth curve was determined by counting cells after siRNA treatment. As shown in Figure 4, a significant inhibitory effect on PC3 cell proliferation was observed in p32 siRNA-treated cells compared with the control cells that were not transfected with siRNA or cells treated with control siRNA. In contrast, siRNA-p32 had no inhibitory effect on RWPE-1 cell proliferation. The expression of p32 protein was significantly reduced in RWPE-1, PC3 and LNCaP cells by the p32 siRNA compared with the control siRNA treatments (Fig. 5A). β-actin was not affected by the p32 knockdown for 72 h in RWPE-1, PC3 and LNCaP cells (Fig. 5A). These results suggest that knockdown of p32 specifically inhibits growth of prostate cancer cells PC3 but not non-cancerous prostate epithelial cells.

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Figure 4.  RNA interference-mediated depletion of p32 inhibits PC3 cell proliferation. Cell proliferation monitored in cell lines PC3 (A) and RWPE-1 (B). The diamond, square, triangle and cross represent cells treated with nothing, 40 μM control siRNA, 20 μM p32 siRNA and 40 μM p32 siRNA, respectively. The cell number was counted at 24, 48, 72, 96 and 120 h after siRNA transfection.

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image

Figure 5.  Analysis of cell cycle in p32 knockdown. (A) siRNA against p32 (p32 siRNA) in PC3, LNCaP and RWPE-1. Cells were transfected with p32 or control siRNA. After subsequent culture for 72 h, the p32 protein levels were analysed by western blot. Likewise, the expressions of specific cell cycle regulators such as CDK4, CDK6, cyclinD1 and p21Waf1 were analyzed. (B) At 72 h after siRNA transfection, cells were fixed and stained with propidium iodide. Then the DNA content was measured by flow cytometry. Three separate experiments were carried out, all of which exhibited similar trends. The results of one representative experiment are shown. Cell cycle phase distributions or the number (%) of cells present at the G1, S or G2/M phases in each experimental condition were determined.

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Cell cycle arrest by p32 siRNA mediated knockdown.  To gain insight into the molecular roles of p32, we tested whether p32 siRNA could affect cell cycle progression by using flow cytometry. p32 knockdown-induced growth suppression was associated with cell cycle arrest in PC3 cells. As shown in Figure 5B, compared with the control cells, the percentage of cells in the G1 phase (29.8%) decreased whereas the percentage in the S phase (63.3%) increased significantly after the p32-siRNA treatment in PC3 cells. On the other hand, statistically significant change was not observed in the noncancerous RWPE-1 cells. PC3 cells with downregulated p32 expression left out the S phase more slowly than the noncancerous cells. These results show that p32 depletion inhibits PC3 cell proliferation due to the blocking of progression from the G1 to S phases.

Downregulation of cyclin D1 and induction of p21 in p32 siRNA-mediated knockdown.  To confirm the molecular mechanism of the G1/S phase arrest after p32 siRNA treatment, we examined specific cell cycle regulators for the G1/S phase transition: CDK4, CDK6, cyclinD1, p27Kip1 and p21Waf1. Cells treated with or without p32 siRNA for 72 h were subjected to western blot analysis. Expression of cyclin D1 was significantly (>60%) reduced or downregulated (compared with controls), while p21Waf1 expression was upregulated in p32 siRNA-treated PC3 and LNCaP cells (Fig. 5A). In noncancerous RWPE-1 cells, expression of p21Waf1 and CDK6 was observed in p32-depleted cells, while cyclin D1 expression was not changed. In the present study, we report, for the first time, that p32 depletion resulted in significant accumulation of p21, a decrease in cyclin D1 levels, a significant inhibition of cyclinD-CDK2 activity and cell cycle arrest.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. Reference
  9. Supporting Information

In this experiment, we have shown that p32 is important for cancer progression based on the following evidence: (i) p32 is highly expressed in prostate tumor samples and associated with the Gleason score, PSA and pathological stage; (ii) high p32 expression is adversely related to survival; (iii) p32 is overexpressed in several prostate cancer cell lines and knocking down p32 inhibits the growth of prostate cancer cells but not noncancerous cells; and (iv) p32-mediated knockdown in PC3 induces G1/S phase arrest through increased p21Waf1 and decreased cyclin D1 expression. The increased expression of p32 in human prostate cancer suggests that mitochondrial p32 is a key molecule of prostate tumorigenesis and p32 expression might be a potential molecular marker for the diagnosis of prostate cancer.

The current study is the first to demonstrate the relation between clinicopathological factors and patient survival with the expression of p32 in patients with prostate cancer. Chen et al.(28) reported that p32 might be an independent predictive factor for breast cancer prognosis and upregulation of p32 might play an important role in the metastasis of breast cancer. A possible tumor suppressor role had been previously introduced,(29,30) but the overexpression of p32 had been reported in several cancer tissue samples including breast cancer.(28,31) Ghosh et al.(29) have reported that p32 accumulates in inflammatory subsquamous tissue during tumor initiation and is overexpressed with progression in papillomatic and acanthotic tissues. These observations suggest that high expression of p32 might play an important role in the tumorigenesis of several cancers. It was reported that p32 overexpression was common in breast cancers but rare in some other malignant tissues, such as prostate cancer.(11,31) However, in the present study, p32 overexpression was observed in Japanese prostate cancer patients who were very strictly defined and sorted. Reasons for the discrepancy are not clear at present. Our in vitro studies also support that overexpression of p32 accelerates prostate tumor progression. It might be possible because of different race or different environments, such as lifestyle and nutritional factors.

The p32 knockdown cells exhibited reduced synthesis of the mitochondrial DNA-encoded OXPHOS polypeptides and were less tumorigenic in vivo.(32) We also observed that knockdown of p32 in MEF cells showed decreased expression of the mitochondrial-encoded component of complex IV (COX I and COX II). These observations suggest that p32 was involved in mitochondrial translation and depletion of p32 impaired the mitochondrial respiratory activity (manuscript in preparation).

In mammalian cells there are two main ways to generate energy in the form of adenosine triphosphate from glucose, oxidative phosphorylation and glycolysis. Oxidative phosphorylation occurs in mitochondria with carbon dioxide and water as end products, whereas glycolysis from glucose to lactic acid takes place in cytoplasm. In carcinogenesis, glucose will be metabolized into lactic acid instead of carbon dioxide and water, even in the presence of oxygen, which is known as the Warburg effect.(22,33–35) Recently, Fogal et al.(32) reported that knocking down p32 expression in human cancer cells strongly shifted their metabolism from OXPHOS to glycolysis. They suggested that tumor cells used p32 to regulate the balance between OXPHOS and glycolysis.

p32 is involved in mitochondrial respiration through the expression of mitochondrial-encoded polypeptide.(32) Knockdown of p32 inhibits mitochondrial respiration, and this inhibition causes a collapse of the proton gradient across the mitochondrial inner membrane, thereby collapsing the mitochondrial membrane potential (ΔΨm).(36) This inhibition can produce reactive oxygen species (ROS).(36) Excessive production of ROS gives rise to the activation of events that lead to death and survival as signaling molecules.(37) Han et al.(38) reported that antimycin A, an electron transport chain inhibitor in mitochondria, can produce ROS in cells and ROS might have roles in cell cycle progression via regulating cell cycle-related proteins. Therefore, it is possible that ROS levels in PC3 cells can affect the cell cycle-related proteins, resulting in G1/S phase arrest of the cell cycle. In this experiment, depletion of p32 might induce cell cycle arrest through the ROS or oxidative stress because p32 is important for the maintenance of OXPHOS.

p32 was detected in various cellular compartments other than mitochondria, such as Golgi, nucleus, cytosol and the cell surface in different cell types.(1,7,9,10,17) This evolutionarily conserved protein seems not to be expressed equally throughout the cell because it shows different localization in different cell types under different physiological conditions. It is possible to be involved in many pathological conditions, especially in cancers. We observed nuclear localization of p32 in 23 samples. Strong statistical significance was observed between p32 nuclear expression and strong p32 cytoplasmic staining (< 0.01). p32 nuclear expression was significantly increased in a higher Gleason score, pathological stage and preoperative PSA (Table 2), suggesting that nuclear function of p32 might be involved in tumor progression. p32 may shed light on previous reports that it interacts with nuclear proteins like alternate mRNA splicing factor SF2,(1) lamin B receptor,(39) suggesting that nuclear p32 might be involved in the regulation of alternative splicing.

Fogal et al.(32) also reported that p32 knockdown breast cancer cell clones (MDA-MB-435) in nude mice was monitored. The author demonstrated that p32 knockdown cells produced smaller tumors than the controls, suggesting that p32 is involved in tumorgenesis in vivo. This result suggests that p32 is an important factor for cell growth of cancer cells in vivo.

In the present study, we are the first to identify that p32 is essential for cell proliferation in prostate cancer, suggesting that p32 may be a novel marker of clinical progression in prostate cancer. Its unique localization in tumors and its tumor cell-specific suppression of proliferation may make p32 a useful target in the diagnosis and therapy for prostate tumors and possibly for other tumors.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. Reference
  9. Supporting Information

The authors would like to acknowledge the technical expertise of the Support Center for Education and Research, Kyushu University. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Technology, Sports, and Culture of Japan (#22249018 and #21590337).

Reference

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. Reference
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. Reference
  9. Supporting Information

Table S1. Clinicopathological parameters in 148 patients with prostate cancer.

Table S2. Relationship between the expression of p32 and Gleason score, EPE.

Fig. S1. Immunohistochemical staining of prostate cancer with purified monoclonal p32 antibodies result in cytoplasm reactivity.

Fig. S2. Growth retardation after androgen deprivation in LNCaP cell (*P < 0.05 vs androgen withdrawal).

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