The role of insulin-like growth factor-II in cancer growth and progression evidenced by the use of ribozymes and prostate cancer progression models

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Abstract

Towards understanding the IGF system during cancer growth and progression, progressive prostate cancer models, such as SV40 large T antigen immortalized human prostate epithelial cells (P69, M2182, M2205, and M12) and LNCaP sublines (C4, C4-2, and C4-2B4), were used. IGF-II mRNA levels progressively increase as prostate cancer cells become more tumorigenic and metastatic, suggesting that IGF-II contributes in part to prostate cancer progression. The role of IGF-II in cancer cell growth was evaluated in LNCaP, PC3, and M12 prostate cancer cell lines and MCF-7 breast cancer cell line by ribozyme/antisense strategies which were previously shown to suppress endogenous IGF-II expression and cell growth in PC-3 cells [Xu et al., Endocrinol 140 (1999) 2134]. Retroviral mediated transient expression of IGF-II-specific ribozyme (RZ) caused extensive cell death. In stably cloned cell lines, both RZ and mutant ribozyme (MRZ) inhibited cancer cell growth, suggesting that antisense effects of MRZ may be sufficient for cell growth inhibition. These results confirm an important role of IGF-II in cancer cell growth and progression, and support further development of gene therapy targeting IGF-II.

Introduction

An increased number of studies have addressed the insulin-like growth factor (IGF) system in various cancers since recent population studies provided strong circumstantial evidence that serum IGF-I levels influence prostate cancer risk in 1998 [1], [2]. Previously, we and others suggested an important role of the IGF system, in particular, IGF-II in prostate cancer growth and progression [3], [4], [5]. Both IGF-I and -II exert mitogenic actions via the IGF-I receptor. IGF-II is a 7.5 kDa single-chain polypeptide, which is processed from its precursor [6]. It has been reported that an incompletely processed 15 kDa IGF-II is expressed more abundantly than the 7.5-kDa form in many cancers [7], [8], [9]. We showed that the 15-kDa IGF-II has a mitogenic potency greater than that of the 7.5 kDa [10]. In an examination of tumor tissues, we reported that IGF-II was expressed by more than 50% of prostate, breast, and bladder tumors, and in 100% of paraganglioma. Among IGF-II positive tumors, greater expression of the 15 kDa IGF-II relative to the 7.5 kDa IGF-II form was confirmed [11].

Ribozymes are RNA enzymes that specifically cleave their respective target RNAs, thereby inhibiting the expression of specific gene products. Ribozymes have been utilized to functionally destroy mRNAs as well as for the repair of mutant RNAs [12]. Hammerhead-type ribozymes work in cis (intramolecularly) in nature, but separation of cis-acting ribozymes into two RNA fragments can convert them into trans-acting RNAs capable of site-specific cleavage of substrate RNAs. We have demonstrated that suppression of IGF-II expression by IGF-II specific hammerhead-type ribozymes in prostate cancer cells, PC-3, leads to cell growth inhibition [13]. This observation together with IGF-II up-regulation in prostate cancer in situ is consistent with the hypothesis that the 15-kDa form of IGF-II expressed in cancerous cells contributes to prostate cancer cell growth in vivo.

Progressive prostate cancer models, SV40 large T antigen immortalized human prostate epithelial cells (P69, M2182, M2205, and M12) and LNCaP sublines (C4, C4-2, and C4-2B), would provide unique experimental systems for studying the sequence of genetic changes involved in the emergence of the metastatic phenotype during human prostate cancer progression [14], [15], [16], [17]. Using these models, we now show that IGF-II mRNA levels progressively increase as prostate cancer cells become more tumorigenic and metastatic. To investigate further the role of IGF-II in cancer cell growth, we utilized the ribozyme strategy to analyze other established cancer cell lines including LNCaP prostate cancer and MCF-7 breast cancer cells, which also express IGF-II mRNA. We demonstrate that intracellular expression of the IGF-II-specific ribozyme inhibited cell growth in all four cancer cell lines tested, which included LNCaP, PC-3, and M12 prostate cancer cell lines as well as MCF-7 breast cancer cell line. A catalytically inactive mutant ribozyme inhibited cancer cell growth as well, suggesting that antisense effects of the mutant ribozyme may be sufficient for cancer cell growth inhibition.

Section snippets

Cells and culture conditions

M2182, M2205, and M12 cell lines are tumorigenic sublines obtained by sequential passage in male athymic mice from human prostate epithelial cells immortalized by transfection with the SV40T antigen gene [14], [15]. In brief, rare tumors that developed in 2 of 18 mice injected subcutaneously with the parental line after a 6-month latent period, were cultured in vitro and subsequently re-injected subcutaneously into athymic mice. Lineage I cells were derived from a tumor arising in a mouse

IGF-II mRNA levels in lineage-derived prostate cancer cell lines

To correlate IGF-II up-regulation with the sequence of genetic changes during human prostate cancer progression, we first quantitated IGF-II mRNA levels in prostate cell lines, P69, M2182, M2205, and M12, which were established from SV40 large T antigen immortalized human prostate epithelial cells [14], [15]. Fig. 1A summarizes the results of quantitative competitive reverse transcription-polymerase chain reaction (QC RT-PCR). IGF-II mRNA was not detectable in parental P69 cells, but was

Discussion

This paper describes two aspects concerning the role of IGF-II in cancer. First, potential roles of IGF-II in cancer progression were addressed. Second, the role of IGF-II in cancer cell growth was evaluated by ribozyme/antisense strategies in various cancer cells expressing IGF-II.

Although we and other researchers suggested that IGF-II up-regulation might contribute to prostate cancer progression, it has not been possible to determine the role of IGF-II in cancer progression in vivo. In this

Acknowledgements

Mineo Rossana is a recipient of a FIRC (Fondazione Italiana per la Ricerca sul Cancro) fellowship and supported by MURST 98 Progetto Biomedicina V8. This work was supported by the California Breast Cancer Research Program (3CB-0186), the US Army Prostate Cancer Research Program (DAMD17-98-1-8579 and 17-00-1-0049), NIH grants (CA65767, CA85859, and DK52683), and a research grant from Tokai University.

References (32)

  • W.H. Daughaday et al.

    Measurement of derivative of proinsulin-like growth factor-II in serum by a radioimmunoassay directed against the E-domain in normal subjects and patients with nonislet cell tumor hypoglycemia

    J. Clin. Endocrinol. Metab.

    (1992)
  • T. Enjoh et al.

    Characterization of new monoclonal antibodies to human Insulin-like growth factor-II and their application in Western immunoblot analysis

    J. Clin. Endocrinol. Metab.

    (1993)
  • J.F. Perdue et al.

    Binding specificities and transducing function of the different molecular weight forms of insulin-like growth factor-II (IGF-II) on IGF-I receptors

    Endocrinology

    (1991)
  • S.L. Li et al.

    Expression of insulin-like growth factor (IGF)-II in human prostate, breast, bladder, and paraganglioma tumors

    Cell Tissue Res.

    (1998)
  • Z.D. Xu et al.

    Hammerhead ribozyme-mediated cleavage of the human insulin-like growth factor-II RNA in vitro and in prostate cancer cells

    Endocrinology

    (1999)
  • V.L. Bae et al.

    Tumorigenicity of SV40T antigen immortalized human prostate epithelial cells: Association with decreased epidermal growth factor receptor (EGFR) expression

    Int. J. Cancer

    (1994)
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