The decline and fall of the IGF-I receptor.

This review examines the effect of targeting the insulin-like growth factor 1 receptor (IGF-IR) in human cancers. The results are disappointing. The causes for the failure are discussed, as well as the possible use of the IGF-IR as a secondary target.

Growth of v-src-transformed cells in serum-free medium through the induction of growth factors.

The v-src oncogene is one of only two oncogenes capable of transforming mouse embryo fibroblasts (MEFs) lacking the IGF-IR gene (R-cells). R-/v-src cells grow robustly in the absence of serum, suggesting the hypothesis that they may produce one or more growth factors that would sustain their ability to proliferate in serum-free condition. Using proteomic approaches on serum-free conditioned media derived from v-src-transformed cells, we have identified two growth promoting factors: ostepontin and proliferin. Subsequent experiments have indicated that osteopontin plays a prevalent role in promoting growth of v-src-transformed cells in serum-deprived condition.

Nutrient restriction and radiation therapy for cancer treatment: when less is more.

Calorie restriction (CR), or a diet modification aiming to reduce the total intake of calories by 20%-40%, has been shown to increase longevity across multiple species. Recently, there has been growing interest in investigating the potential role of CR as a treatment intervention for age-related diseases, such as cancer, because an increasing body of literature has demonstrated a metabolic component to both carcinogenesis and tumor progression. In fact, many of the molecular pathways that are altered with CR are also known to be altered in cancer. Therefore, manipulation of these pathways using CR can render cancer cells, and most notably breast cancer cells, more susceptible to standard cytotoxic treatment with radiation and chemotherapy. In this review article we demonstrate the laboratory and clinical evidence that exists for CR and show compelling evidence through the molecular pathways CR induces about how it may be used as a treatment in tandem with radiation therapy to improve our rates of disease control.

Regulation of microRNA-145 by growth arrest and differentiation.

MicroRNA145 (miR145), a tumor suppressor miR, has been reported to inhibit growth of human cancer cells, to induce differentiation and to cause apoptosis, all conditions that result in growth arrest. In order to clarify the functional effects of miR145, we have investigated its expression in diverse conditions and different cell lines. Our results show that miR145 levels definitely increase in differentiating cells and also in growth-arrested cells, even in the absence of differentiation. Increased expression during differentiation sometimes occurs as a late event, suggesting that miR145 could be required either early or late during the differentiation process.

Expression of micro-RNA-145 is regulated by a highly conserved genomic sequence 3' to the pre-miR.

Micro-RNA-145 (miR145), a tumor suppressor miR, dramatically inhibits growth of cancer cells in culture and plays a significant role in human stem cells differentiation. We have isolated a human genomic sequence of 864 bp comprising the pre-miR and its flanking sequences. The cloned miR145 genomic sequence expresses a mature miR145 in transfected cells. We show here that flanking sequences on either side of the pre-miR sequence can modulate its expression levels. Surprisingly, a highly conserved sequence 3' to the pre-miR plays a crucial role in miR145 expression.

microRNA, cell cycle, and human breast cancer.

The discovery of microRNAs as a novel class of gene expression regulators has led to a new strategy for disease diagnostics and therapeutics. Cell cycle, cell proliferation, and tumorigenesis are all regulated by microRNAs. Several general principles linking microRNAs and cancer have been recently reviewed; therefore, the current review focuses specifically on the perspective of microRNAs in control of cell cycle, stem cells, and heterotypic signaling, as well as the role of these processes in breast cancer. Altered abundance of cell cycle regulation proteins and aberrant expression of microRNAs frequently coexist in human breast cancers. Altered microRNA expression in breast cancer cell lines is associated with altered cell cycle progression and cell proliferation. Indeed, recent studies have demonstrated a causal role for microRNA in governing breast tumor suppression or collaborative oncogenesis. This review summarizes the current understanding of the role for microRNA in regulating the cell cycle and summarizes the evidence for aberrant microRNA expression in breast cancer. The new evidence for microRNA regulation by annotated genes and the involvement of microRNA in breast cancer metastasis are discussed, as is the potential for microRNA to improve breast cancer diagnosis and therapy.

Mutual interaction and reciprocal down-regulation between c-met and insulin receptor substrate-1.

The insulin receptor substrate-1 (IRS-1) and c-met, the receptor for the hepatocyte growth factor (HGF) co-immuno-precipitate from lysates treated with the respective antibodies. The interaction between IRS-1 and c-met requires a tyrosyl phosphorylated IRS-1 and results in reciprocal down-regulation. IRS-1 inhibits cell motility, while the activated c-met promotes it. These and other results suggest an explanation for reports in the literature indicating that c-met levels are high and IRS-1 levels are low in human cancer metastases.

The microRNA miR-92 increases proliferation of myeloid cells and by targeting p63 modulates the abundance of its isoforms.

MicroRNAs (miRs) are 21- to 23-nucleotide RNA molecules that regulate the stability or translational efficiency of target messenger RNAs of proteins involved in cell growth and apoptosis. miR-92 is part of the mir-17-92 cluster, which comprises members with an effect on cell proliferation. However, the role of miR-92 is unknown, and its targets have not been identified. Here, we describe a mechanism through which miR-92 contributes to regulate cell proliferation. Using a miR-92 synthetic double-strand oligonucleotide, we demonstrate that miR-92 increases 32D myeloid cell proliferation and 5-bromo-2-deoxyuridine (BrdU) incorporation and inhibits cell death. The effect is miR-92 specific since the miR-92 antagomir inhibits cell proliferation. Moreover, we show that miR-92 acts by modulating p63-isoform abundance through down-regulatation of endogenous DeltaNp63beta. Using luciferase reporters containing p63 3'UTR fragments with wild-type or mutant miR-92 complementary sites, we demonstrate that the wild-type 3'UTR is a direct target of miR-92. Finally, we observed that a miR-92-resistant DeltaNp63beta isoform (without 3'UTR) inhibits cell proliferation and parallels the effect of the antagomir. We conclude that one of the molecular mechanisms through which miR-92 increases cell proliferation is by negative regulation of an isoform of the cell-cycle regulator p63.

The insulin receptor substrate-1: a biomarker for cancer?

This review focuses on IRS-1 and the evidence of its role in cell transformation. The literature strongly suggests that IRS-1 should be considered a biomaker for cancers susceptible to IGF-IR targeting. In addition, I would like to propose that IRS-1 may have a more general role in cancer, and could be considered as a protein having the opposite effect of tumor suppressors, a sort of anti-p53 molecule.

Mechanism of growth inhibition by MicroRNA 145: the role of the IGF-I receptor signaling pathway.

MicroRNA 145 (miR145) has been proposed as a tumor suppressor. It was previously shown that miR145 targets the 3' UTR of the insulin receptor substrate-1 (IRS-1) and dramatically inhibits the growth of colon cancer cells. miR145 also targets the type 1 insulin-like growth factor receptor (IGF-IR). We show here that an IRS-1 lacking its 3' UTR is no longer down-regulated by miR145 and rescues colon cancer cells from miR145-induced inhibition of growth. An IGF-IR resistant to miR145 (again by elimination of its 3' UTR) is not down-regulated by miR145 but fails to rescue colon cancer cells from growth inhibition. These and other results, taken together, indicate that down-regulation of IRS-1 plays a significant role in the tumor suppressor activity of miR145.

Customizing the targeting of IGF-1 receptor.

The type 1 IGF receptor (IGF-IR) is activated by two ligands, IGF-1 and IGF-2, and by insulin at supraphysiological concentrations. It plays a significant role in the growth of normal and abnormal cells, and antibodies against the IGF-IR are now in clinical trials. Targeting of the IGF-IR in cancer cells (by antibodies or other means) can be improved by the appropriate selection of responsive tumors. This review focuses on the optimization of IGF-IR targeting in human cancer.

Insulin receptor substrate-1 regulates the transformed phenotype of BT-20 human mammary cancer cells.

Although originating from a human breast cancer, BT-20 cells do not form colonies in soft agar. BT-20 cells do not express insulin receptor substrate-1 (IRS-1), which is known to promote both normal and abnormal growth and to inhibit differentiation. Stable expression of IRS-1 confers to BT-20 cells the ability to form colonies in soft agar. BT-20 cells form tumors in xenografts in mice, but the size of tumors is twice as large when the cells express IRS-1. The increased transformed phenotype is characterized by occupancy of the rDNA and cyclin D1 promoters by IRS-1 and the activation of the cyclin D1, c-myc, and rDNA promoters. In addition, the retinoblastoma protein, which is detectable in the rDNA promoter of quiescent BT-20/IRS-1 cells, is replaced by IRS-1 after insulin-like growth factor-I stimulation. Our results indicate that in BT-20 human mammary cancer cells, expression of IRS-1 activates promoters involved in cell growth and cell proliferation, resulting in a more transformed phenotype. Targeting of IRS-1 could be effective in inhibiting the proliferation of mammary cancer cells.

The role of insulin receptor substrate-1 in transformation by v-src.

The insulin receptor substrate-1 (IRS-1), a docking protein for both the insulin (InR) and the insulin-like growth factor-1 (IGF-IR) receptors, sends a mitogenic, anti-differentiation and transforming signal. We now show that down-regulation of IRS-1 in cells transformed by v-src reverses the transformed phenotype (growth in serum-free medium and colony formation in soft agar). IRS-1 translocates to nuclei and is found in the cyclin D1 and rDNA promoters. Stat3, which is activated by src, requires both IRS-1 and src for promoter occupancy. IRS-1 (by itself or in combination with src) also markedly increases transcription from these two promoters. We also show that IRS-1 binds to src via its two PI3-K binding tyrosine residues, and that these two residues are required for transformation of mammary cancer cells expressing v-src. Taken together, these results indicate a significant role of IRS-1 in the activation of cell cycle progression genes and transformation of cells by v-src.

Regulation of insulin receptor substrate-1 expression levels by caveolin-1.

The insulin receptor substrate-1 (IRS-1), a docking protein of the type 1 insulin-like growth factor receptor (IGF-IR) plays a significant role in cell proliferation and differentiation. The expression of IRS-1 is down-regulated in mouse embryo fibroblasts (MEFs) with a deletion of caveolin-1 (cav1) genes (KO cells). Levels of IRS-1 mRNA are not affected. Re-introduction of cav1 into KO cells rescues IRS-1 expression. Stabilization of protein levels is reciprocal and a strict correlation between IRS-1 and cav1 levels was confirmed in five cell lines, and in mouse tissues. IRS-1 binds through its phosphotyrosine binding (PTB) domain to tyrosine 14 (Y14) of cav1, the residue phosphorylated by IGF-1 stimulation and by v-src. The down-regulation of IRS-1 in cav-/- cells occurs via the proteasome pathway. These results indicate a novel mechanism for the regulation of IRS-1 expression levels, an important finding in view of IRS-1 role in cell proliferation and transformation.

A mechanism for cell size regulation by the insulin and insulin-like growth factor-I receptors.

Deletion of the type 1 insulin-like growth factor receptor (IGF-IR) or of the insulin receptor substrate-1 (IRS-1) genes in animals causes a 50% reduction in body size at birth. Decrease in body size is due to both a decreased number of cells and a decreased cell size. Deletion of the insulin receptor (InR) genes results in mice that are normal in size at birth. We have used 32D-derived myeloid cells to study the effect of IGF-IR and InR signaling on cell size. 32D cells expressing the IGF-IR and IRS-1 are almost twice as large as 32D cells expressing the InR and IRS-1. A mechanism for the difference in size is provided by the levels of the upstream binding factor 1 (UBF1), a nucleolar protein that participates in the regulation of RNA polymerase I activity and rRNA synthesis and therefore cell size. When shifted to the respective ligands, UBF1 levels decrease in cells expressing the InR and IRS-1, whereas they remain stable in cells expressing the IGF-IR and IRS-1. The expression of the IGF-IR and IRS-1 is crucial to the stability of UBF1.

Role of pescadillo and upstream binding factor in the proliferation and differentiation of murine myeloid cells.

Pescadillo (PES1) and the upstream binding factor (UBF1) play a role in ribosome biogenesis, which regulates cell size, an important component of cell proliferation. We have investigated the effects of PES1 and UBF1 on the growth and differentiation of cell lines derived from 32D cells, an interleukin-3 (IL-3)-dependent murine myeloid cell line. Parental 32D cells and 32D IGF-IR cells (expressing increased levels of the type 1 insulin-like growth factor I [IGF-I] receptor [IGF-IR]) do not express insulin receptor substrate 1 (IRS-1) or IRS-2. 32D IGF-IR cells differentiate when the cells are shifted from IL-3 to IGF-I. Ectopic expression of IRS-1 inhibits differentiation and transforms 32D IGF-IR cells into a tumor-forming cell line. We found that PES1 and UBF1 increased cell size and/or altered the cell cycle distribution of 32D-derived cells but failed to make them IL-3 independent. PES1 and UBF1 also failed to inhibit the differentiation program initiated by the activation of the IGF-IR, which is blocked by IRS-1. 32D IGF-IR cells expressing PES1 or UBF1 differentiate into granulocytes like their parental cells. In contrast, PES1 and UBF1 can transform mouse embryo fibroblasts that have high levels of endogenous IRS-1 and are not prone to differentiation. Our results provide a model for one of the theories of myeloid leukemia, in which both a stimulus of proliferation and a block of differentiation are required for leukemia development.

Role of pescadillo in the transformation and immortalization of mammalian cells.

The murine and human homologs of the zebrafish pescadillo protein (Pes1 and PES1, respectively) play important roles in ribosome biogenesis and DNA replication. We investigated the effect of Pes1 on the growth of mouse embryo (3T3-like) fibroblasts and conditionally immortalized human fibroblasts expressing the SV40 T antigen (AR5 cells). Increased expression of Pes1 causes transformation of mouse and human fibroblasts in culture (colony formation in soft agar). Although Pes1 can replace the SV40 T antigen in inducing colony formation in soft agar, it cannot substitute the T antigen in the immortalization of human fibroblasts, indicating that it distinguishes between the two functions. As the biological effects of Pes1 are similar to those of the insulin receptor substrate-1 (IRS-1), we investigated the interactions of Pes1 with IRS-1 itself and with the SV40 T antigen. The Pes1 protein (which localizes to the nuclei and nucleoli of cells) interacts with both IRS-1 and the SV40 T antigen, and markedly decreases the interaction of T antigen with p53. Taken together, these results suggest mechanisms for the ability of Pes1 to transform cells, and its failure to immortalize them.

Insulin-like growth factor I receptor signaling and nuclear translocation of insulin receptor substrates 1 and 2.

The insulin receptor substrate 1 (IRS-1) can translocate to the nuclei and nucleoli of several types of cells. Nuclear translocation can be induced by an activated insulin-like growth factor 1 receptor (IGF-IR), and by certain oncogenes, such as the Simian virus 40 T antigen and v-src. We have asked whether IRS-2 could also translocate to the nuclei. In addition, we have studied the effects of functional mutations in the IGF-IR on nuclear translocation of IRS proteins. IRS-2 translocates to the nuclei of mouse embryo fibroblasts expressing the IGF-IR, but, at variance with IRS-1, does not translocate in cells expressing the Simian virus 40 T antigen. Mutations in the tyrosine kinase domain of the IGF-IR abrogate translocation of the IRS proteins. Other mutations in the IGF-IR, which do not interfere with its mitogenicity but inhibit its transforming capacity, result in a decrease in translocation, especially to the nucleoli. Nuclear IRS-1 and IRS-2 interact with the upstream binding factor, which is a key regulator of RNA polymerase I activity and, therefore, rRNA synthesis. In 32D cells, wild-type, but not mutant, IRS-1 causes a significant activation of the ribosomal DNA promoter. The interaction of nuclear IRS proteins with upstream binding factor 1 constitutes the first direct link of these proteins with the ribosomal DNA transcription machinery.

Deletion of the pleckstrin and phosphotyrosine binding domains of insulin receptor substrate-2 does not impair its ability to regulate cell proliferation in myeloid cells.

32D IGF-I receptor (IR) cells are IL-3-dependent myeloid cells that can be induced to differentiate into granulocytes by IGF-I. Like the parental 32D cells, 32D IGF-IR cells do not express the insulin receptor substrate (IRS)-1 or IRS-2. We investigated the effect of ectopic expression of IRS-2 in 32D IGF-IR cells. Expression in these cells of a wild-type IRS-2 inhibits IGF-I-induced differentiation, and the cells grow indefinitely in the absence of IL-3. We also investigated the effect of a mutant IRS-2 lacking both the pleckstrin (PH) and the phosphotyrosine-binding (PTB) domains, which are known to bind to the IR. The partial differentialPHPTB IRS-2 is fully as capable as the wild-type IRS-2 (and wild-type IRS-1) to stimulate the growth and inhibit the differentiation of 32D IGF-IR cells. In contrast, an IRS-1 protein lacking the same PH and PTB domains is completely inactive in blocking differentiation and stimulating IL-3-independent growth of 32D IGF-IR cells. The partial differentialPHPTB IRS-2 protein is dependent for its effect on an activated IGF-IR, is cytoplasmic, binds to the beta-subunit of the IGF-IR, and requires for its action the presence of phosphatidylinositol 3-kinase binding sequences. These experiments show that the PH and PTB domains of IRS-2 (but not IRS-1) are dispensable for the IGF-I/IRS-2-mediated growth of 32D myeloid cells. Our results also indicate that IRS-2 (either wild type or partial differentialPHPTB) is capable of inhibiting the differentiation of 32D cells.