Introduction

Prostate cancer is a complex disease that affects millions of men worldwide, mainly in areas with a high human development index. Patients with localized disease at low to intermediate risk of recurrence generally have a favorable outcome with an overall survival of 99% over 10 years if the disease is detected and treated at an early stage. Key genetic alterations include fusions of TMPRSS2 with ETS family genes, amplification of the MYC oncogene, deletion and/or mutation of PTEN and TP53 and, in advanced disease, amplification and/or mutation of the androgen receptor (AR) [1].

Insulin-like growth factor 1 (IGF-1) is a key mediator in human physiology and pathophysiology, including cancer. The IGF-1 receptor type I (IGF-1R) mediates the effects of IGF-1 by activating two important intracellular signaling cascades: the phosphatidylinositol-3-kinase/AKT-kinase (PI3K/AKT) pathway and the Raf/AKT kinase pathway. Mitogen-activated protein kinase (Raf/MAPK) [2,3]. IGF-1R-dependent signaling regulates a wide range of cellular responses, including cell proliferation. IGF-1 can act as both a capacity growth factor, stimulating the “G0 to G1 transition” of quiescent/dormant cells, and a progression growth factor, stimulating the “G1 to G2 transition” of somatic cells in the cell cycle [4,5].

By alternative splicing of exons 5 and 6, human igf1 produces three transcripts, namely IGF-1Ea, IGF-1Eb and IGF1Ec [2]. Since they contain exons 3 and 4, all can produce mature IGF-1. However, translation of these transcripts produces different peptides of the E structure, namely Ea, Eb and Ec. It is, therefore, possible that the preferential expression of IGF-1Ec detected in various experimental settings after tissue injury supports the need of the injured tissue for an adjuvant of IGF-1, the Ec-related bioactivity [6,7]. Indeed, several studies have confirmed that synthetic human Ec peptide (hEc) and murine E peptide (mE) [product of the E domain of the mouse IGF-1Eb transcript of igf1] possess mitogenic, angiogenic, and migratory growth factor activity in vitro [8,9].

The role of IGF-1 as a potent growth and survival factor in human cancer has long been established. The IGF-1 gene produces multiple heterogeneous transcripts, which result in the mature form of IGF-1 [10,11]. Recent studies have indicated the preferential expression of the IGF-1Ec isoform in prostate cancer [6], whereas exogenous administration of a 24 amino acid synthetic peptide of the COO-terminal part of the Ec isoform (parts of exons 5 and 6 of IGF-1 gene), has been associated with a statistically significant increase in proliferation in PC-3 and LNCaP prostate cancer cells [6]. It has been suggested that the effects of this synthetic PEc are mediated by extracellular signal-regulated kinase (ERK) but not Akt, promoting prostate cancer cell growth in vitro [6].

Unlike IGF-1, the effects of PEc are not mediated through the IGF-1 receptor (IGF-1R), the insulin receptor (IR), or any of the hybrid receptors (IGF-1R/IR) [6,12]. Mechanistically, synthetic PEc has been suggested to be involved in mouse mesenchymal cell migration and invasion [11,13-15]. Despite this evidence, some studies show conflicting results regarding the action of synthetic PEc on muscle cells [12,15]. Therefore, the role of Ec in cancer biology remains to be elucidated.

The Ec Peptide of the IGF1Ec Isoform in Relation to Prostate Cancer

IGF-1Ec expression levels were examined in randomly selected prostate cancer sections. In a work carried out in our country, seventy-eight patients with prostate cancer were examined and the mean immunohistochemical expression of IGF-1Ec was found to be significantly lower in patients with prostate cancer stage ≤ IIb (AJCC) compared to the tissues of stage III and IV patients (p<0.004). Mean IGF1Ec expression was significantly lower in stage IIa-IIb prostate tumors compared to stage III (mean IGF1Ec expression was 101.3 vs. 144.7, p = 0.005). These results are in agreement with those previously described [6] and suggest that IGF-1Ec expression is related to prostate cancer stage. Overexpression of Ec peptide leads to increased proliferation in PC-3 cells.

Thus, the wild-type prostate cancer cell line PC-3 (wtPC-3) was selected, which expresses only a small amount of IGF-1Ec compared to LNCaP prostate cancer cells and to human prostate epithelial cells (HPrEc under tissue culture [6]. PEc-overexpressing PC-3 cells (PC-3PEc) were generated and compared with control wild-type PC-3 (mPC-3) cells. mPC-3 cells were compared with PC -3 wild type in terms of cell proliferation and ERK1/2 phosphorylation and showed no statistically significant differences.

PEc Induces PC-3 Multiplication through an Autocrine/ Parecrine Mode of Action

To determine whether PEc in PC-3PEc cells becomes extracellular, PEc levels were examined in the media of PC-3PEc cells and mPC-3 cells by multiple reaction (MRM) analysis. PC3PEc cells were found to secrete a significant amount of PEc in contrast to mPC-3 cells [6].

Relationship of the Ec Peptide with the Phenomenon of Epithelial-Meseghymal Transition

PC-3PEc cells grown in vitro exhibited a fibroblast-like morphology under light microscopy compared to mPC-3 epithelial cells. Recent evidence shows that the IGF-1 pathway induces the EMT effect through the IGF-1 receptor (IGF-1R) by regulating the zinc ring enhancer-binding protein 1 (ZEB1) in prostate carcinoma cells, which is stimulated by ERK1/2 phosphorylation [16-18]. ZEB1 represses E-cadherin and is involved in further chromatin condensation and gene silencing [19-21]. Loss of E-cantherin is associated with increased tumor migration and invasion both in vitro and in vivo. The decrease in E-cantherin occurs gradually and the increase in vimentin starts at 48 hours, synchronously, with a peak effect within 72 hours [22]. Treatment of PC-3PEc cells with the anti-PEc antibody (administered in medium) results in reversal of the mesenchymal phenotype (increased E-cadherin expression and decreased vimentin expression) at 24 h. This finding reinforces the idea of an autocrine/paracrine mode of action of PEc. In addition PC-3PEc cells showed a more than fivefold increase in ZEB1 expression level compared to mPC-3 cells. On the other hand, it has also been suggested that, in cancer, EMT can also be promoted by the direct effect of cdc6 (cell division cycle 6), a protein involved in the replication licensing mechanism, at the E-cadherin locus, as a molecular switch [22].

In PC-3 IGF-1R KD cells it has been found that, although exogenous administration of IGF-1 did not affect ZEB1 expression, exogenous administration of PEc increased ZEB1 expression. These data suggest that PEc induces EMT in PC-3 cells through a different receptor molecule than IGF-1R. Furthermore, IGF1R silencing in PC-3PEc cells did not affect their mesenchymal phenotype (morphology and E-cadherin and vimentin patterns). The effect of PEc on the migration ability of mPC-3 prostate cancer cells was also examined by migration assays, where PC-3PEc cells and mPC-3 cells were examined under the influence of exogenous synthetic PEc. It was observed that PC-3PEc cells showed an increased migration rate compared to mPC-3 cells similar to that of mPC-3 cells under the influence of exogenous synthetic PEc. It was also observed that in both cases (PC3PEc cells and mPC3 cells after PEc administration) anti-IGF-1Ec antibody led to a decrease in their migration rate [6].

Association of Ec Peptide with Prostate Cancer Metastasis

Subcutaneous injection of mPC-3 cells into SCID mice is mainly associated with lymph node metastases [23]. Approximately 80% of men are diagnosed with organ-confined disease, 15% with local metastases, and 5% with distant metastases [24]. Men diagnosed with advanced stage disease (distant metastases) have a poor overall survival of only 30% at 5 years [24]. Early detection of local disease may also be instrumental in efforts to increase the life expectancy of prostate cancer patients, while also preventing the occurrence of metastatic disease [25]. Furthermore, tailoring treatment to men who are likely to benefit from immediate definitive treatment and those who are not remains a key clinical challenge [26].

Discussion

Prostate cancer is the most common cancer in older men. Cells within the prostate often form tumors, most often in mid- and late life. The adult human prostate can be divided into a central, transitional, and peripheral zone, and also contains fibromuscular and periurethral regions. In young adult men, the distal zone constitutes >70% of prostatic glandular tissue and contributes the most to normal prostate function. It is also the most common site of origin for neoplasms in the aged prostate, as nearly 80% of prostate tumors arise from this region [27,28].

There are no curative treatments for metastatic prostate cancer. The most common treatment for recurrent disease is surgical or medical castration. However, more commonly, patients progress and develop castration-resistant, metastatic growth [2931]. Our results suggest that PEc is a key molecule in tumor growth and survival and is also involved in the Epithelial-Mesenchymal Transition (EMT) phenomenon of prostate cancer cells leading to metastasis. In a mouse study, endogenous PEc overproduction in PC-3 cells was associated with increased ERK1/2-driven proliferation (similar to exogenous PEc administration) and larger tumor formation in SCID mice compared to those are generated by mPC-3 cells. Furthermore, the fact that PEc becomes extracellular and its biological effects are inhibited by anti-IGF-1Ec antibody suggests an autocrine/paracrine mode of action. The importance of PEc in tumor growth and progression is also indicated by inoculation of PC-3 IGF-1Ec KD cells, where only two of 10 (20%) mice developed palpable tumors ten weeks after subcutaneous injection. Our data also show that PEc expression is associated with the EMT phenomenon. Administration of exogenous PEc to mPC-3 cells, as well as overexpression and secretion of PEc from PC-3PEc cells, induces vimentin expression and suppresses E-cantherin expression. These in vitro results were consistent in mPC-3 and PC-3PEc-induced tumors in SCID mice. Furthermore, although IGF-1 induces EMT in a manner involving ERK1/2 activation and ZEB1 expression via IGF-1R, PEc appears to induce the same effect, but using an IGF-1R-independent mechanism. Further in vivo evidence for the involvement of PEc in the metastatic process of prostate cancer came from the orthotopic injection of immortalized HPrEcPEc cells into SCID mice, where four of seven mice developed metastases to nearby tissues [32]. These results are in agreement with those obtained from immunohistochemical analysis of tumors from prostate cancer patients, where the IGF1Ec isoform was found to be positively associated with prostate cancer stage and grade. These observations provide new insights into prostate cancer biology, whereby peptide E (PEc) resulting from proteolytic cleavage of the IGF-1Ec isoform induces prostate cancer progression, metastasis, and repair. Accordingly, Ec peptide may be an attractive candidate target for the treatment of prostate cancer without affecting the actions of mature IGF-1.

Conflict of Interest

All authors declare no conflict of interest.

References

1. Hjelmborg JB, Scheike T, Holst K, Skytthe A, Penney KL, et al. (2014) The Heritability of Prostate Cancer in the Nordic Twin Study of Cancer. Cancer Epidemiol Biomark Prev 23: 2303-2310.
2. Philippou A, Halapas A, Maridaki M, Koutsilieris M (2007) Type I insulinlike growth factor receptor signaling in skeletal muscle regeneration and hypertrophy. J Musculoskelet Neuronal Interact 7: 208-218.
3. Philippou A, Maridaki M, Pneumaticos S, Koutsilieris M (2014) The complexity of the IGF1 gene splicing, posttranslational modification and bioactivity. Mol Med 20: 202-214.
4. Ma QL, Yang TL, Yin JY, Peng ZY, Yu M, et al. (2009) Role of insulinlike growth factor-1 (IGF-1) in regulating cell cycle progression. Biochem Biophys Res Commun 389: 150-155.
5. Mairet-Coello G, Tury A, DiCicco-Bloom E (2009) Insulin-like growth factor-1 promotes G(1)/S cell cycle progression through bidirectional regulation of cyclins and cyclin-dependent kinase inhibitors via the phosphatidylinositol 3-kinase/Akt pathway in developing rat cerebral cortex. J Neurosci 29: 775-788.
6. Armakolas A, Philippou A, Panteleakou Z, Nezos A, Sourla A, et al. (2010) Preferential expression of IGF-1Ec (MGF) transcript in cancerous tissues of human prostate: evidence for a novel and autonomous growth factor activity of MGF E peptide in human prostate cancer cells. Prostate 70: 1233-1242.
7. Kuo YH, Chen TT (2002) Novel activities of pro-IGF-I E peptides: regulation of morphological differentiation and anchorage-independent growth in human neuroblastoma cells. Exp Cell Res 280: 75-89.
8. Alagaratnam S, Loizidou M, Yang SY, Fuller B, Ramesh B (2020) Increased expression of IGF-1Ec with increasing colonic polyp dysplasia and colorectal cancer. J Cancer Res Clin Oncol 146: 28612870.
9. Wang G, Zhao D, Spring DJ, DePinho RA (2018) Genetics and biology of prostate cancer. Genes Dev 32: 1105-1140.
10. Dai Z, Wu F, Yeung EW, Li Y (2010) IGF-IEc expression, regulation and biological function in different tissues. Growth Horm IGF Res 20: 275-281.
11. Matheny RW Jr, Nindl BC, Adamo ML (2010) Minireview: Mechanogrowth factor: a putative product of IGF-I gene expression involved in tissue repair and regeneration. Endocrinology 151: 865-875.
12. Brisson BK, Barton ER (2012) Insulin-like growth factor-I E-peptide activity is dependent on the IGF-I receptor. PLoS One 7: e45588.
13. Collins JM, Goldspink PH, Russell B (2010) Migration and proliferation of human mesenchymal stem cells is stimulated by different regions of the mechano-growth factor prohormone. J Mol Cell Cardiol 49: 10421045.
14. Cui H, Yi Q, Feng J, Yang L, Tang L (2014) Mechano growth factor E peptide regulates migration and differentiation of bone marrow mesenchymal stem cells. J Mol Endocrinol 52: 111-120.
15. Fornaro M, Hinken AC, Needle S, Hu E, Trendelenburg AU, et al. (2014) Mechano-growth factor peptide, the COOH terminus of unprocessed insulin-like growth factor 1, has no apparent effect on myoblasts or primary muscle stem cells. Am J Physiol Endocrinol Metab 306: E150-E156.
16. Graham TR, Zhau HE, Odero-Marah VA, Osunkoya AO, Kimbro KS, et al. (2008) Insulin-like growth factor-I-dependent up-regulation of ZEB1 drives epithelial-to-mesenchymal transition in human prostate cancer cells. Cancer Res 68: 2479-2488.
17. Zhau HE, Odero-Marah V, Lue HW, Nomura T, Wang R, et al. (2008) Epithelial to mesenchymal transition (EMT) in human prostate cancer: lessons learned from ARCaP model. Clin Exp Metastasis 25: 601-610.
18. Peinado H, Olmeda D, Cano A (2007) Snail, ZEB and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev 7: 415-428.
19. Ohira T, Gemmill RM, Ferguson K, Kusy S, Roche J, et al. (2003) WNT7a induces E-cadherin in lung cancer cells. Proc Natl Acad Sci U S A 100: 10429-10434.
20. Spaderna S, Schmalhofer O, Hlubek F, Berx G, Eger A, et al. (2006) A transient, EMT-linked loss of basement membranes indicates metastasis and poor survival in colorectal cancer. Gastroenterology 131: 830-840.
21. Grooteclaes ML, Frisch SM (2000) Evidence for a function of CtBP in epithelial gene regulation and anoikis. Oncogene 19: 3823-3828.
22. Sideridou M, Zakopoulou R, Evangelou K, Liontos M, Kotsinas A, et al. (2011) Cdc6 expression represses E-cadherin transcription and activates adjacent replication origins. J Cell Biol 195: 1123-1140.
23. Bastidel C, Bagnis C, Mannoni P, Hassoun J, Bladoul F (2002) A Nod Scid mouse model to study human prostate cancer. Prostate Cancer Prostatic Dis 5: 311-315.
24. Siegel RL, Miller KD, Jemal A (2018) Cancer statistics. CA Cancer J Clin 68: 7-30.
25. Sammon JD, Abdollah F, D’Amico A, Gettman M, Haese A, et al. (2015) Predicting life expectancy in men diagnosed with prostate cancer. Eur Urol 68: 756-765.
26. Papageorgiou E, Philippou A, Armakolas A, Christopoulos PF, Dimakakos A, et al. (2016) The human Ec peptide: the active core of a progression growth factor with species-specific mode of action. Hormones (Athens) 15: 423-434.
27. Timms BG (2008) Prostate development: a historical perspective. Differentiation 76: 565-577.
28. Zlotta AR, Egawa S, Pushkar D, Govorov A, Kimura T, et al. (2013) Prevalence of prostate cancer on autopsy: cross-sectional study on unscreened Caucasian and Asian men. J Natl Cancer Inst 105: 10501058.
29. Nelson WG, De Marzo AM, Isaacs WB (2003) Prostate cancer. N Engl J Med 349: 366-381.
30. Strope AS, Andriole LG (2010) Prostate cancer screening: current status and future perspectives. Nat Rev Urol 9: 487-493.
31. Guillonneau B (2012) Prostate cancer: new treatments for localized tumours-what matters first? Nat Rev Urol 9: 414-415.
32. Milingos DS, Philippou A, Armakolas A, Papageorgiou E, Sourla A, et al. (2011) Insulin like growth factor-1Ec (MGF) expression in eutopic and ectopic endometrium: characterization of the MGF E-peptide actions in vitro. Mol Med 17: 21-28.