Ewa Surmacz

Different Effects on Mitogenesis and Transformation of a Mutation at Tyrosine 1251 of the Insulin-like Growth Factor I Receptor

The wild type insulin-like growth factor I (IGF-I) receptor has both mitogenic and transforming activities. We have examined the effect of point mutations at tyrosine residues 1250 and 1251 on these two properties of the receptor. For this purpose, we stably transfected plasmids expressing mutant and wild type receptors into R cells, which are 3T3-like cells, derived from mouse embryos with a targeted disruption of the IGF-I receptor genes, and therefore devoid of endogenous IGF-I receptors. A tyrosine to phenylalanine mutation of either the 1250 or 1251 residue, or both, has no effect on the ability of the receptor to transmit a mitogenic signal. However, the tyrosine 1251 mutant receptor and the double mutant have lost the ability to transform R cells (colony formation in soft agar), even when the receptors are expressed at very high levels, while the Y1250F mutant is fully transforming. These experiments show that the 1251 tyrosine residue is required for the transforming activity of the IGF-I receptor.

Type I insulin-like growth factor receptor function in breast cancer.

Experimental evidence suggests an important role of the type I IGF receptor (IGF-IR) in breast cancer development. Breast tumors and breast cancer cell lines express the IGF-IR. IGF-IR levels are higher in cancer cells than in normal breast tissue or in benign mammary tumors. The ligands of the IGF-IR are potent mitogens promoting monolayer and anchorage-independent growth of breast cancer cells. Interference with IGF-IR activation, expression, or signaling inhibits growth and induces apoptosis in breast cancer cells. In addition, recent studies established the involvement of the IGF-IR in the regulation of breast cancer cell motility and adhesion. We have demonstrated that in MCF-7 cells, overexpression of the IGF-IR promotes E-cadherin-dependent cell aggregation, which is associated with enhanced cell proliferation and prolonged survival in three-dimensional culture.The expression or function of the IGF-IR in breast cancer cells is modulated by different humoral factors, such as estrogen, progesterone, IGF-II, and interleukin-1. The IGF-IR and the estrogen receptor (ER) are usually co-expressed and the two signaling systems are engaged in a complex functional cross-talk controlling cell proliferation.Despite the convincing experimental evidence, the role of the IGF-IR in breast cancer etiology, especially in metastatic progression, is still not clear. The view emerging from cellular and animal studies is that abnormally high levels of IGF-IRs may contribute to the increase of tumor mass and/or aid tumor recurrence, by promoting proliferation, cell survival, and cell-cell interactions. However, in breast cancer, except for the well established correlation with ER status, the associations of the IGF-IR with other prognostic parameters are still insufficiently documented.

Differential roles of IRS-1 and SHC signaling pathways in breast cancer cells

Several polypeptide growth factors stimulate breast cancer growth and may be involved in tumor progression. However, the relative importance of diverse growth factor signaling pathways in the development and maintenance of the neoplastic phenotype is largely unknown. The activation of such growth factor receptors as the insulin‐like growth factor I receptor (IGF‐I R), erbB‐type receptors (erbB Rs) and FGF receptors (FGF Rs) controls the phenotype of a model breast cancer cell line MCF‐7. To evaluate the function of 2 post‐receptor signaling molecules, insulin receptor substrate‐1 (IRS‐1) (a major substrate of the IGF‐IR) and SHC (a common substrate of tyrosine kinase receptors), we developed several MCF‐7‐derived cell clones in which the synthesis of either IRS‐1 or SHC was blocked by antisense RNA. In MCF‐7 cells, down‐regulation of IRS‐1 by 80–85% strongly suppressed anchorage‐dependent and ‐independent growth and induced apoptotic cell death under growth factor‐ and estrogen‐reduced conditions. The reduction of SHC levels by approximately 50% resulted in the inhibition of monolayer and anchorage‐independent growth but did not decrease cell survival. Importantly, cell aggregation and the ability of cells to survive on the extracellular matrix were inhibited in MCF‐7/anti‐SHC clones, but not in MCF‐7/anti‐IRS‐1 clones. Cell motility toward IGF was not attenuated in any of the tested cell lines, but motility toward EGF was decreased in MCF‐7/anti‐SHC clones. Our results suggest that in MCF‐7 cells: 1) both IRS‐1 and SHC are implicated in the control of monolayer and anchorage‐independent growth; 2) IRS‐1 is critical to support cell survival; 3) SHC is involved in EGF‐dependent motility; and 4) normal levels of SHC, but not IRS‐1, are necessary for the formation and maintenance of cell‐cell interactions. Int. J. Cancer 72:828–834, 1997.

Insulin receptor substrate 1 is a target for the pure antiestrogen ICI 182,780 in breast cancer cells.

The pure antiestrogen ICI 182,780 inhibits insulin‐like growth factor (IGF)‐dependent proliferation in hormone‐responsive breast cancer cells. However, the interactions of ICI 182,780 with IGF‐I receptor (IGF‐IR) intracellular signaling have not been characterized. Here, we studied the effects of ICI 182,780 on IGF‐IR signal transduction in MCF‐7 breast cancer cells and in MCF‐7‐derived clones overexpressing either the IGF‐IR or its 2 major substrates, insulin receptor substrate 1 (IRS‐1) or src/collagen homology proteins (SHC). ICI 182,780 blocked the basal and IGF‐I‐induced growth in all studied cells in a dose‐dependent manner; however, the clones with the greatest IRS‐1 overexpression were clearly least sensitive to the drug. Pursuing ICI 182,780 interaction with IRS‐1, we found that the antiestrogen reduced IRS‐1 expression and tyrosine phosphorylation in several cell lines in the presence or absence of IGF‐I. Moreover, in IRS‐1‐overexpressing cells, ICI 182,780 decreased IRS‐1/p85 and IRS‐1/GRB2 binding. The effects of ICI 182,780 on IGF‐IR protein expression were not significant; however, the drug suppressed IGF‐I‐induced (but not basal) IGF‐IR tyrosine phosphorylation. The expression and tyrosine phosphorylation of SHC as well as SHC/GRB binding were not influenced by ICI 182,780. In summary, downregulation of IRS‐1 may represent one of the mechanisms by which ICI 182,780 inhibits the growth of breast cancer cells. Thus, overexpression of IRS‐1 in breast tumors could contribute to the development of antiestrogen resistance. Int. J. Cancer 81:299–304, 1999.

Inhibition of Cell Cycle Progression by Antisense Oligodeoxynucleotidesa

We have used the antisense strategy to study the role of certain genes in cell cycle progression. In particular, we used antisense oligodeoxynucleotides to study: (1) the role of the IGF-1 receptor in the control of cell proliferation; and (2) the sequence of gene expression during the cell cycle. Our results can be summarized as follows: (1) the activation of the IGF-1 receptor by its ligand, IGF-1, is an obligatory step in the proliferation of fibroblasts and hemopoietic cells; and (2) the expression of DNA synthesis genes, such as PCNA, DNA polymerase alpha, and cdc2, is dependent on the expression of previous genes. A tentative temporal order is: c-myc > c-myb > IGF-1 receptor > DNA synthesis genes.

The role of the IGF1 receptor in the regulation of cdc2 mRNA levels in fibroblasts.

The levels of cdc2 mRNA increase when quiescent cells are stimulated by growth factors. In BALB/c 3T3, both platelet-derived growth factor and insulin-like growth factor 1 (IGF-1) are required to increase cdc2 mRNA levels. In p6 cells, which constitutively overexpress the IGF-1 receptor, IGF-1 is sufficient. The importance of the IGF-1/IGF-1 receptor interaction in regulating the levels of cdc2 mRNA was further confirmed by showing that an antisense oligodeoxynucleotide to the IGF-1 receptor RNA inhibited the IGF-1-mediated increase.