Genetic manipulation of the IGF-I axis to regulate mammary gland development and function.

Insulin-like growth factor I (IGF-I) is known to regulate mammary gland development. This regulation occurs through effects on both cell cycle progression and apoptosis. Our laboratory has studied the IGF-I-dependent regulation of these processes by using transgenic and knockout mouse models that exhibit alterations in the IGF-I axis. Our studies of transgenic mice that overexpress IGF-I during pregnancy and lactation have demonstrated that this growth factor slows the apoptotic loss of mammary epithelial cells during the declining phase of lactation but has minimal effects during early lactation on milk composition or lactational capacity. In contrast, our analysis of early developmental processes in mammary tissue from mice carrying a targeted mutation in the IGF-I receptor gene suggests that IGF-dependent stimulation of cell cycle progression is more important to early mammary gland development than potential anti-apoptotic effects. With both models, the effects of perturbing the IGF-I axis are dependent on the physiological state of the animal. The diminished ductal development that occurs in response to loss of the IGF-I receptor is dramatically restored during pregnancy, whereas the ability of overexpressed IGF-I to protect mammary cells from apoptosis does not occur if the mammary gland is induced to undergo forced involution. Data from our laboratory on the expression of IGF-signaling molecules in the mammary gland suggest that this effect of physiological context may be related to the expression of members of the insulin receptor substrate family.

Targeted disruption of the IGF-I receptor gene decreases cellular proliferation in mammary terminal end buds.

IGF-I mediates mammary ductal development through stimulation of terminal end bud (TEB) development; however, no published data exist on the mechanism through which this occurs. The mechanism of IGF-I action on the TEB was studied by determining the requirement for the IGF-I receptor (IGF-IR) in IGF-I-dependent ductal development. We hypothesized that loss of the IGF-IR would disrupt mammary ductal development through a combination of decreased proliferation or increased apoptosis. Because IGF-IR null mice die at birth, embryonic mammary gland transplantation was used to study the effects of a disrupted IGF-IR gene. Analyses of grafts after 4 or 8 wk of development demonstrated a limited growth potential of the null mammary epithelium in virgin hosts. Bromodeoxyuridine labeling and terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick-end labeling showed that cell proliferation was significantly decreased in null TEBs, but apoptosis was not. In addition, both the size and number of TEBs were reduced in null outgrowths. In pregnant hosts, null ductal growth was stimulated beyond the level seen in virgin hosts. These findings directly establish a proliferation-dependent role for the IGF-IR in the cells of the TEB. Additionally, this study indicates that pregnancy-dependent compensatory mechanisms can stimulate mammary development in the absence of an IGF-IR.

IGF and insulin action in the mammary gland: lessons from transgenic and knockout models.

Transgenic and knockout mice have become valuable experimental systems with which to study specific molecular events within the mammary gland of an intact animal. These models have provided a wealth of information about the effects of a number of oncogenes and growth factors. This review focuses on results obtained from the application of transgenic and knockout models to determine the roles of insulin and insulin-like growth factors (IGF) in the regulation of mammary gland development, lactation and tumorigenesis. Transgenic models which overexpress IGF-I or -II display specific alterations in mammary gland development and an increased incidence of mammary tumors. Analysis of mammary gland development in knockout mice which are deficient in IGF-I or the IGF-I receptor supports the conclusion that the IGF system is important for normal mammary gland development. This review discusses these observations in detail and attempts to fit them into a larger picture of IGF and insulin action in the mammary gland.

Cooperative interaction between mutant p53 and des(1-3)IGF-I accelerates mammary tumorigenesis.

Mammary tumorigenesis was analysed in transgenic mice which overexpress des(1-3)hIGF-I (WAP-DES) and/or a mutant form of p53 (p53172R-H). Nonlactating, multiparous WAP-DES mice exhibited hyperplastic lesions termed mammary interepithelial neoplasia (MIN) which constitutively expressed WAP-DES. By 23 months of age, 53% of the WAP-DES mice developed mammary adenocarcinomas. A 75% reduction in both apoptosis and proliferation was observed in the normal mammary glands of WAP-DES mice. Mammary tumor incidence in WAP-DES/p53 bitransgenic mice was similar to that of WAP-DES and 2 - 3-fold greater than that of nontransgenic and p53172R-H females. Tumor latency, however, was reduced by 8 months in bitransgenic mice as compared to mice of the other three genotypes. Aneuploidy was frequently observed in tumors from bitransgenic and p53172R-H mice, but not from mice expressing only the WAP-DES transgene. Expression of IGFBP3 was elevated in tumors from WAP-DES, but not bitransgenic mice, indicating an alteration in the p53/IGF-I axis. These studies indicate that overexpression of des(1-3)hIGF-I increases the frequency of MIN and stochastic mammary tumors and that the appearance of tumors displaying genomic instability is accelerated by mutant p53172R-H. Oncogene (2000) 19, 889 - 898.

Interactions of apoptosis, proliferation and host age in the regression of the mouse mammary preneoplasia, TM3, carrying an unusual mutation in p53.

We have developed an in vivo model system of mouse mammary preneoplasias in order to examine the cell and molecular changes that occur during tumorigenesis. Most of these preneoplasias are characterized by an alveolar hyperplasia morphologically similar to that present in normal pregnant mammary gland, but have tumor forming capabilities ranging from very low to high. One of these hyperplasias, the TM3 HOG (transformed mammary hyperplastic outgrowth), forms tumors infrequently and has the unusual characteristic of spontaneous regression. We have observed that 7-8 months post-transplantation into the cleared mammary fat pad of a BALB/c mouse, the TM3 hyperplasia will begin to regress, leaving only a sparse ductal tree with remnant alveolar structures by 10-12 months post-transplantation. We have sought to elucidate the mechanism of this regression by determining the apoptotic and proliferative rates of the alveolar cells during TM3 HOG development. Studies show that apoptotic rates in the TM3 HOG are consistently high (4-7%) at all times after transplantation. This apoptotic rate is higher than the rates found in other preneoplasias in our system and approach the rates observed in the normal involuting gland. An unusual p53 mutation, a serine insertion at codon 233, may be causally related to the high spontaneous apoptotic frequencies as well as elevated inducible apoptotic frequencies in TM3. In addition, a sudden decrease ( approximately 63%) in proliferation occurs around 8 months post-transplantation. Furthermore, transplantation experiments indicate that the ability of the 8-month-old host and/or mammary gland to support growth of preneoplastic mammary tissues is markedly diminished compared with 3- or 6-month-old hosts. The results presented here suggest that the persistent high apoptotic rates, concomitant with decreased proliferation rates, may be responsible for TM3's regression and implicate a unique mutant p53 as a causal factor. Additionally, the results suggest that host determinants can interact with specific molecular changes in the preneoplastic cells to influence growth and progression of the preneoplastic populations.