Rossana Mineo

Insulin/Insulin-like Growth Factor I Hybrid Receptors Have Different Biological Characteristics Depending on the Insulin Receptor Isoform Involved

The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R− cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-RsA) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-RsA, insulin activated the IGF-I half-receptor β-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-RsBbound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-RA-containing cells than in Hybrid-RB-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.

Insulin receptor activation by IGF-II in breast cancers: evidence for a new autocrine/paracrine mechanism

IGF-II, produced by breast cancer epithelial and stromal cells, enhances tumor growth by activating the IGF-I receptor (IGF-I-R) via autocrine and paracrine mechanisms. Previously we found that the insulin receptor (IR), which is related to the IGF-I-R, is overexpressed in breast cancer cells. Herein, we find that, in breast cancer the IR is activated by IGF-II. In eight human breast cancer cell lines studied there was high affinity IGF-II binding to the IR, with subsequent IR activation. In these lines, IGF-II had a potency up to 63% that of insulin. In contrast, in non malignant human breast cells, IGF-II was less than 1% potent as insulin. Via activation of the IR tyrosine kinase IGF-II stimulated breast cancer cell growth. Moreover, IGF-II also activated the IR in breast cancer tissue specimens; IGF-II was 10 – 100% as potent as insulin. The IR occurs in two isoforms generated by alternative splicing of exon 11; these isoforms are IR-A (Ex11−) and IR-B (Ex11+). IR-A was predominantly expressed in breast cancer cells and specimens and the potency of IGF-II was correlated to the expression of this isoform (P<0.0001). These data indicate, therefore, that the IR-A, which binds IGF-II with high affinity, is predominantly expressed in breast cancer cells and represents a new autocrine/paracrine loop involved in tumor biology.

Androgens Up-regulate the Insulin-like Growth Factor-I Receptor in Prostate Cancer Cells

In this study, we show that androgens up-regulate insulin-like growth factor-I receptor (IGF-IR) expression and sensitize prostate cancer cells to the biological effects of IGF-I. Both dihydrotestosterone and the synthetic androgen R1881 induced an approximately 6-fold increase in IGF-IR expression in androgen receptor (AR)-positive prostate cancer cells LNCaP. In accordance with IGF-IR up-regulation, treatment with the nonmetabolizable androgen R1881 sensitized LNCaP cells to the mitogenic and motogenic effects of IGF-I, whereas an IGF-IR blocking antibody effectively inhibited these effects. By contrast, these androgens did not affect IGF-IR expression in AR-negative prostate cancer cells PC-3. Reintroduction of AR into PC-3 cells by stable transfection restored the androgen effect on IGF-IR up-regulation. R1881-induced IGF-IR up-regulation was partially inhibited by the AR antagonist Casodex (bicalutamide). Two other AR antagonists, cyproterone acetate and OH-flutamide, were much less effective. Androgen-induced IGF-IR up-regulation was not dependent on AR genomic activity, because two AR mutants, AR-C619Y and AR-C574R, devoid of DNA binding activity and transcriptional activity were still able to elicit IGF-IR up-regulation in HEK293 kidney cells in response to androgens. Moreover, androgen-induced IGF-IR up-regulation involves the activation of the Src-extracellular signal-regulated kinase pathway, because it was inhibited by both the Src inhibitor PP2 and the MEK-1 inhibitor PD98059. The present observations strongly suggest that AR activation may stimulate prostate cancer progression through the altered IGF-IR expression and IGF action. Anti-androgen therapy may be only partially effective, or almost ineffective, in blocking important biological effects of androgens, such as activation of the IGF system.

In IGF-I receptor-deficient leiomyosarcoma cells autocrine IGF-II induces cell invasion and protection from apoptosis via the insulin receptor isoform A

One of the two isoforms of the human insulin receptor (isoform A or IR-A) binds IGF-II with high affinity and is predominantly expressed in fetal tissues and malignant cells. We evaluated the biological relevance of IR-A in human myosarcoma cells. Six myosarcoma cell lines were studied. All produced high amounts of IGF-II and five of them predominantly expressed IR-A. SKUT-1 leiomyosarcoma cells, that do not express the IGF-IR, were identified as a suitable model to study the effects of IR-A in the absence of the interference of IGF-IR. In these cells, which express high levels of IR with an IR-A relative abundance of ≈95%, IGF-II elicits biological effects exclusively via IR-A activation and IGF-I is almost ineffective. Blockade of autocrine IGF-II reduced unstimulated cell viability and migration. Although both insulin and IGF-II activate IR-A, these two ligands showed a different ability to activate different intracellular signaling pathways and to elicit different biological effects. Insulin was more potent than IGF-II in activating the PI3-K/Akt pathway and in protecting cells from apoptosis. In contrast, IGF-II was more potent than insulin in activating the Shc/ERK pathway and in stimulating cell migration. These data indicate that IGF-II sensitive IR-A is the predominant IR isoform in a variety of myosarcoma cells. In SKUT-1 leiomyoma cells this fetal IR isoform may vicariate the IGF-IR for cell response to both insulin and IGF-II. Acting on the same IR-A receptor IGF-II is more potent than insulin in stimulating cancer cell migration.

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

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.