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. 2015 Jan 6;21(1):167-79.
doi: 10.2119/molmed.2014.00222.

Oncogenic Role of the Ec Peptide of the IGF-1Ec Isoform in Prostate Cancer

Athanasios Armakolas  1 , Maria Kaparelou  1 , Andreas Dimakakos  1 , Efstathia Papageorgiou  1 , Nikolaos Armakolas  2 , Athanasios Antonopoulos  2 , Constantina Petraki  3 , Maria Lekarakou  3 , Pavlos Lelovas  4 , Martha Stathaki  1 , Constantinos Psarros  1 , Ismene Donta  5 , Panos S Galanos  6 , Paul Msaouel  1 , Vassilis G Gorgoulis  6   7   8   9 , Michael Koutsilieris  1
Affiliations

Oncogenic Role of the Ec Peptide of the IGF-1Ec Isoform in Prostate Cancer

VSports手机版 - Athanasios Armakolas et al. Mol Med. .

Abstract

IGF-1 is one of the key molecules in cancer biology; however, little is known about the role of the preferential expression of the premature IGF-1 isoforms in prostate cancer. We have examined the role of the cleaved COO- terminal peptide (PEc) of the third IGF-1 isoform, IGF-1Ec, in prostate cancer. Our evidence suggests that endogenously produced PEc induces cellular proliferation in the human prostate cancer cells (PC-3) in vitro and in vivo, by activating the ERK1/2 pathway in an autocrine/paracrine manner. PEc overexpressing cells and tumors presented evidence of epithelial to mesenchymal transition, whereas the orthotopic injection of PEc-overexpressing, normal prostate epithelium cells (HPrEC) in SCID mice was associated with increased metastatic rate. In humans, the IGF-1Ec expression was detected in prostate cancer biopsies, where its expression correlates with tumor stage. Our data describes the action of PEc in prostate cancer biology and defines its potential role in tumor growth, progression and metastasis. VSports手机版.

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Figure 1
Figure 1
PEc is associated with the induction of proliferation in PC-3 cells. Immunohistochemical analysis of IGF-1Ec expression in human prostate biopsies: (A) benign columnar prostate epithelium and prostate cancer (arrows), 100×; (B) benign columnar prostate epithelium and prostate cancer (arrows), 200×; (C) prostate adenocarcinoma and high grade prostatic intraepithelial neoplasia [PIN] 200×; (D) prostate adenocarcinoma and PIN, 400×. Actions of PEc in prostate cancer. (E) Detection of IGF-1Ec expression levels by qRT-PCR in mPC-3 cells (white column) and in PC-3 cells overexpressing the PEc (PC-3PEc) cells (black column). An at least six-fold increase of IGF-1Ec mRNA expression was detected in the PC-3PEc cells compared with the mock PC-3 (mPC-3) cells using GAPDH as a reference gene. (Student t test, p < 0.05, n = 6. Error bars refer to SD). (F) Immunofluorescence analysis of the PEc expression in mPC-3 and the PC-3PEc cells, using a polyclonal rabbit anti-human IGF-1Ec antibody. (G) Cell proliferation analysis using MTT assay. Exogenous PEc administration (17 nmol/L) induced the growth rate of wtPC-3 cells at 24 and 48 h. Increased cellular proliferation also was observed in PC-3PEc cell lines compared with mPC-3 cells, as measured at 24 and 48 h. (Student t test, p < 0.0001, n = 5. Error bars refer to SD). (H) Cell cycle analysis of mPC-3 and PC-3PEc cells using flow cytometry. PC-3PEc cells presented significantly lower distribution in the G1/G0 phase and higher distribution in the S phase as compared with mPC-3 cells (Student t test, p < 0.001, n = 3). RN2: range in gating; FL5: fluorescent light detector 5. (I) Western analysis of ERK1 and ERK2 (ERK1/2) and Akt phosphorylation in mPC-3 and PC-3PEc cells. Exogenous administration of synthetic PEc (17 nmol/L) induced ERK1/2 but not Akt phosphorylation in mPC-3 cells (0, 15, 30 and 60 min). PC-3PEc possessed constantly constitutive ERK1/2 activation without Akt being affected. (J) Exogenous IGF-1 (17 nmol/L) induced Akt and ERK1/2 phosphorylation in mPC-3 cells at 15, 30 and 60 min (Western blot).
Figure 2
Figure 2
The PEc effects are generated in an autocrine/paracrine manner. (A) Multiple Reaction Monitoring (MRM) protein analysis for the detection of the PEc-specific trypric products, (YQPPSTNK and GSTFEER) in the media of the mPC-3 and PC-3PEc. Sample 1: Tryptic products obtained from the media of mPC-3 cells. Sample 2: tryptic products of media obtained from PC-3PEc cells. Sample 3: tryptic products of synthetic PEc peptide (control). Significant amounts of both PEc tryptic products were detected into the PC-3PEc media. (B) Western analysis of ERK1/2 phosphorylation in PC-3PEc cells and in mPC-3 cells after the administration of exogenous synthetic PEc, incubated with increasing concentrations of rabbit anti-human PEc (anti-PEc) antibody (1:1,000; 1:500 and 1:50), for 1 h. Anti-IGF-1Ec antibody significantly decreased ERK1/2 phosphorylation in both cell types in a fashion that was directly proportional to the antibody concentration added in the culture media. In addition, nonimmunized rabbit serum did not affect the ERK1/2 phosphorylation in either system. In both cases, GAPDH was used as a control.
Figure 3
Figure 3
PEc overexpression induces EMT in PC-3 cells. (A) Analysis of the morphology of mPC-3 and PC-3PEc cells by light microscopy. PC-3PEc cells have a more spindle-like appearance compared with mPC-3 cells cultured under identical conditions. (B) PC-3PEc cells presented a significant decrease of E-cadherin and a significant increase of vimentin expression compared with mPC-3 cells as assessed by qRT-PCR (Student t test, P < 0.001 for both cases, n = 3). (C) Immunofluorescence (IF) analysis of E-cadherin and vimentin expression in mPC-3 cells and in PC-3PEc cells. E-cadherin expression was suppressed and vimentin was increased in PC-3PEc cells. (D) The treatment of PC-3PEc cells with the anti-human IGF-1Ec antibody (anti-PEc, 1/50) reversed their E-cadherin and vimentin expression profile toward the mPC-3 phenotype (IF analysis). Nonimmunized rabbit serum did not affect E-cadherin and vimentin expression in PC-3PEc cells. (E) Western blot analysis of the Snail, Twist, ZEB1 and cdc6 proteins in mPC-3 and in PC-3PEc cells. These proteins’ expression was induced in PC-3PEc cells. GAPDH and β-actin were used as controls. (F) ZEB1 silencing was associated with an increase in the E-cadherin expression and a decrease in vimentin expression in PC-3PEc cells at 24 h after induction of the silencing, as shown by immunofluorescence. The silencing of the IGF-1R did not affect the E-cadherin and vimentin expression in PC-3PEc cell lines, suggesting that the IGF-1R is not involved in the PEc effects on E-cadherin and vimentin. (G) Analysis of the ZEB1 expression by qRT-PCR in wtPC-3 cells and PC-3 cells after silencing the IGF-1R (PC-3 IGF-1R KD), after the exogenous administration of IGF-1 and PEc (17 nmol/L in both cases). Exogenous IGF-1 induced the ZEB1 expression in wtPC-3 cells 24 h after its administration, but it did not affect the ZEB1 expression in PC-3 IGF-1R KD cells. Exogenous PEc had similar effects on ZEB1 expression that maintained in the PC-3 IGF-1R KD cells. Positive control: human mesenchymal cells (HMC).
Figure 4
Figure 4
Association of PEc with increased PC-3 cell mobility. (A–F) Wound healing assay, PEc effect on PC-3 mobility. Evaluation of the mPC-3 and PC-3PEc cells migration capacity at various time points (0, 8, 16 and 24 h). (A) PC-3PEc cells, (B) mPC-3 cells, (C) PC-3 cells after the exogenous administration of 17 ng/mL synthetic PEc, (D) PC-3PEc cells after the administration of the anti-PEc antibody in the media (1/50), (E) mPC-3 cells after the administration of synthetic PEc and of the anti-PEc antibody (1/50), (F) mPC-3 cells after the administration of synthetic PEc and of nonimmunized rabbit serum (1/50). PC-3PEc cells presented elevated migration capacity compared with mPC-3 cells. The administration of exogenous PEc stimulated the migration capacity in wtPC-3 cells. Treatment of PC-3PEc cells with the anti-PEc antibody decreased their migration capacity. Similarly the administration of the anti-PEc antibody to mPC-3 cells suppressed the stimulation of their migration capacity caused by the exogenous synthetic PEc. The migration pattern of PC-3PEc cells (single cells) was suggestive of mesenchymal nature in contrast to the migration pattern of wtPC-3 cells (sheet-like) indicating their epithelial nature.
Figure 5
Figure 5
In vivo effects of the PC-3PEc cells. (A) SCID mice injected with PC-3PEc cells produced larger tumors, in size and in weight, than those obtained from mPC-3 cells at the same time points. (Student t test, p < 0.001, n = 15. Error bars refer to SD) Arrows: point of the primary tumors. Arrowheads: points of tumors developed distal from the injection sites (35 d post-injection). (B) Immunohistochemi-cal analysis of mPC-3 induced and PC-3PEc induced tumors. PC-3PEc tumors documented with elevated expression of PEc compared with the wtPC-3 tumors. PC-3PEc tumors also contained an increased Ki-67 expression, which is a proliferation marker, and vimentin expression, while they had a decrease in E-cadherin expression as compared with mPC-3 tumors. In addition, PC-3PEc tumors presented decreased CKHMW levels and elevated chromogranin and synaptophysin levels compared with mPC-3 tumors, suggesting a possible neuroendocrine differentiation. Furthermore, we detected a relatively increased p53 expression in PC-3PEc tumors compared with the mPC-3 tumors. (C) Analysis of tumorigenesis in SCID mice injected with PC-3 cells after the silencing of the IGF-1Ec expression (PC-3 IGF-1Ec KD). These tumors presented slower tumor growth, compared with mPC-3 tumors and low tumorigenicity rate (20%, 2 out of 10). PC-3 IGF-1Ec KD tumors presented a similar expression pattern of prostate cancer markers examined in mPC-3 tumors, as observed by IHC.
Figure 6
Figure 6
Association of PEc with metastases. (A) qRT-PCR analysis indicated a significant increase of PEc expression in HPrEC-PEc cells compared with mHPrEC cells, (reference gene: GAPDH) (Student t test, P < 0.001, n = 3). (B) Similar results were observed by immunofluorescence analysis. (C) The growth rate was significantly induced as assessed by MTT assay, in HPrEC-PEc cells and in mHPrEc cells after the exogenous administration of PEc (17 nmol/L) at 24 and 48 h, as compared with the untreated immortalized mHPrEC cells (p < 0.001 in both cases, Student t test, n = 3. Error bars refers to SD). (D) PEc administration induced ERK1/2 phosphorylation in immortalized mHPrEC cells, while the immortalized HPrEC-PEc cells possessed constantly activated ERK1/2 (Western blot). (E) Kaplan Meier survival analysis of orthotopically injected SCID mice with mHPrEC or HPrEC-PEc cells. The mice injected with HPrEC-PEc cells (seven) presented a statistically significant decrease in survival at 12 wks compared with the mice injected with immortalized HPrEC cells (nine) (P < 0.002) (Cum. Survival: cumulative survival). (F,G,H) Orthotopic injections of HPrEC-PEc cells in SCID mice were associated with metastases in proximal tissues (seminal cord, colon, peritoneum).
Figure 7
Figure 7
Mode of action of PEc. PEc is secreted from prostate cancer cells acting in an autocrine and/or paracrine manner, and stimulates prostate cancer cell growth by activating ERK1/2 via an IGF-1R-, IR- and IGF-1R/IR-independent mechanism and induces EMT via ZEB1 expression, inducing prostate cancer metastatic ability.
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