A moderate elevation of circulating levels of IGF-I does not alter ErbB2 induced mammary tumorigenesis
© Dearth et al; licensee BioMed Central Ltd. 2011
Received: 26 May 2011
Accepted: 25 August 2011
Published: 25 August 2011
Epidemiological evidence suggests that moderately elevated levels of circulating insulin-like growth factor-I (IGF-I) are associated with increased risk of breast cancer in women. How circulating IGF-I may promote breast cancer incidence is unknown, however, increased IGF-I signaling is linked to trastuzumab resistance in ErbB2 positive breast cancer. Few models have directly examined the effect of moderately high levels of circulating IGF-I on breast cancer initiation and progression. The purpose of this study was to assess the ability of circulating IGF-I to independently initiate mammary tumorigenesis and/or accelerate the progression of ErbB2 mediated mammary tumor growth.
We crossed heterozygous TTR-IGF-I mice with heterozygous MMTV-ErbB2 mice to generate 4 different genotypes: TTR-IGF-I/MMTV-ErbB2 (bigenic), TTR-IGF-I only, MMTV-ErbB2 only, and wild type (wt). Virgin females were palpated twice a week and harvested when tumors reached 1000 mm3. For study of normal development, blood and tissue were harvested at 4, 6 and 9 weeks of age in TTR-IGF-I and wt mice.
TTR-IGF-I and TTR-IGF-I/ErbB2 bigenic mice showed a moderate 35% increase in circulating total IGF-I compared to ErbB2 and wt control mice. Elevation of circulating IGF-I had no effect upon pubertal mammary gland development. The transgenic increase in IGF-I alone wasn't sufficient to initiate mammary tumorigenesis. Elevated circulating IGF-I had no effect upon ErbB2-induced mammary tumorigenesis or metastasis, with median time to tumor formation being 30 wks and 33 wks in TTR-IGF-I/ErbB2 bigenic and ErbB2 mice respectively (p = 0.65). Levels of IGF-I in lysates from ErbB2/TTR-IGF-I tumors compared to ErbB2 was elevated in a similar manner to the circulating IGF-I, however, there was no effect on the rate of tumor growth (p = 0.23). There were no morphological differences in tumor type (solid adenocarcinomas) between bigenic and ErbB2 mammary glands.
Using the first transgenic animal model to elevate circulating levels of IGF-I to those comparable to women at increased risk of breast cancer, we showed that moderately high levels of systemic IGF-I have no effect on pubertal mammary gland development, initiating mammary tumorigenesis or promoting ErbB2 driven mammary carcinogenesis. Our work suggests that ErbB2-induced mammary tumorigenesis is independent of the normal variation in circulating levels of IGF-I.
IGF-I has the characteristics of both a circulating hormone and a tissue growth factor. While numerous studies have focused on the autocrine and/or paracrine ability of IGF-I to regulate mammary gland development and tumorigenesis , only a few have focused on the role of circulating IGF-I . Although it is known that circulating levels of IGF-I vary considerably within the normal population, meta-analysis of several studies have shown that elevated serum IGF-I levels are associated with increased risk of breast cancer in premenopausal women . Mammographic density is strongly related to breast cancer risk  and evidence supports a positive correlation between circulating IGF-I levels and mammographic density . Supporting this, the IGF-I axis correlates with birth weight, height, and parity, all which have been show to be contributing breast cancer risk factors [6, 7]. Increased IGF-I signaling has also been linked to trastuzumab resistance in ErbB2 positive breast cancer  and blocking both the IGF-I receptor (IGF-IR) and the ErbB2 receptor (ErbB2-R) inhibits ErbB2 driven breast cancer cell growth . Conversely, antiestrogens that are effective in the treatment and prevention of breast cancer have been consistently found to lower serum IGF-I levels .
The idea that circulating IGF-I may regulate breast cancer is persuasive, especially taking into account that IGF-I, mediated by the endocrine actions of growth hormone (GH), plays a vital role in regulating the developing mammary gland . The mammary gland can proliferate in response to IGF-I both in organ culture , and by treatment of mice with an implant containing IGF-I in the mammary gland . Conversely, it has been shown that there is very limited growth of IGF-IR-null mammary epithelium . In addition, IGF-I-null mice have severely retarded mammary ductal development and branching . These and other studies support a model whereby GH acts upon mammary stroma which produces IGF-I to stimulate pubertal mammary ductal outgrowth in a paracrine manner. However, recent studies in mice with only circulating IGF-I and no local production have shown that endocrine IGF-I can also support mammary ductal growth [15, 16]. We have previously shown that circulating IGF-I, via tail vein administration, results in activation of IGF signaling in the mammary gland . Conversely, using a novel GH antagonist, pegvisomant, we showed that blocking GH action, which results in a lowering of serum IGF-I levels can block IGF-I signaling in the mammary gland and results in a delay of mammary gland development .
It has been shown that little (lit/lit) mice, which have only 10% of circulating IGF-1 levels, displayed a significant reduction of growth of human MCF-7 cell xenografts . Similarly, deletion of the IGF-I gene in the liver, resulting in an 80% reduction in circulating IGF-I, delays chemically and transgenically-induced mammary tumorigenesis and metastasis. As compelling as these studies may be in supporting the notion that reduced circulating IGF-I may limit breast cancer initiation and progression, the 10% level of circulating IGF-I doesn't mimic epidemiologic studies.
There are few mouse models that show moderate changes in circulating IGF-I that are comparable to human variation. We originally developed TTR-IGF-I transgenic mice which overexpress IGF-I mRNA exclusively in the liver and show a moderate but significant increase in circulating levels of IGF-I . Importantly the 35% increase in circulating IGF-I demonstrated in this model is comparable to the 31% increase in circulating IGF-I levels shown to increase breast cancer risk in premenopausal women . The increase in our model is significantly less than the 2.5-fold increase seen in a similar TTR-IGF-I model recently reported by Wu et al. . To determine if increased levels of IGF-I can initiate mammary tumorigenesis and/or promote ErbB2-induced tumorigenesis, we crossed TTR-IGF-I transgenic mice with the well characterized MMTV-ErbB2 transgenic mice. Increased circulating IGF-I did not alter pubertal mammary gland development or total body composition. We show that although TTR-IGF-I and bigenic mice had a 35% increase in circulating total IGF-I compared to ErbB2 transgenic and control mice, IGF-I alone was insufficient to cause mammary tumorigenesis. Furthermore, the elevation of systemic IGF-I had no effect on ErbB2-induced mammary tumorigenesis. Analysis of ErbB2-intiated tumors revealed no major effect of increased circulating IGF-I on tumor type (solid adenocarcinomas) or mammary gland signaling. This is the first study using a transgenic animal model that mimics the variation of normal circulating levels of IGF-I in epidemiological studies. Our studies show that elevated circulating IGF-I has no effect on normal mammary gland development or ErbB2-induced mammary tumorigenesis.
Animals and Experimental Design
All animals were housed under controlled conditions of temperature (23°C), lights (lights on: 0600 h; lights off: 1800 h) and ad libitum access to food (Harland Teklad Diet, Madison, WI) and tap water. The care and handling of animals used in this study followed the guidelines established by the National Institutes of Health (NIH) and all humane procedures were pre-approved by the University Laboratory Animal Care Committee (ULACC).
Homozygous TTR-IGF-I (tg/tg) transgenic females on a C57/Bl6 background were developed and characterized previously . Due to the known ability of the C57/Bl6 genetic background to inhibit oncogene-induced mammary tumorigenesis, homozygous TTR-IGF-I mice were first backcrossed to FVB/N mice for six generations to create homozygous TTR-IGF-I/FVB/N animals in order to cross these animals with the ErbB2 mouse model MMTV-c-Neu FVB/N. MMTV-c-Neu FVB/N mice (Jackson Laboratories) are based on a mammary specific overexpression of the ErbB2 receptor (a frequently amplified oncogene in human breast cancer), which results in mammary specific tumorigenesis.
Homozygous TTR-IGF-I and homozygous MMTV-c-Neu FVB/N (further referred to as MMTV-ErbB2) mice were crossed to FVB/N wild type mice to produce heterozygous transgenics. For mammary gland developmental studies heterozygous TTR-IGF-I (tg/wt) FVB females and age-matched wild type FVB/N littermates (controls) were sacrificed at 4 weeks, 6 weeks and 9 weeks of age. Blood was collected and mammary glands processed for paraffin blocks or whole mount analysis.
For the tumor studies heterozygous TTR-IGF-I (tg/wt) mice were crossed with heterozygous MMTV-ErbB2 (tg/wt) mice. The resulting offspring had one of 4 genotypes; heterozygous TTR-IGF-I (tg/wt), heterozygous MMTV-ErbB2 (wt/tg), bigenic TTR-IGF-I/MMTV-ErbB2 (tg/tg), or wild type controls (wt/wt). Female were weaned at 21 days and housed five per cage and monitored for mammary gland tumorigenesis. In all studies age-matched wild type littermates were used as controls.
Mice were palpated twice weekly to determine time to tumor formation, and once palpated, the rate of tumor growth was determined by measuring the tumor size with caliper measurements (millimeters/mm) and using the formula for an ellipsoid sphere: L × W2/2 = mm3. Mice were sacrificed when tumors reach 1000 mm3. Once tumors reached 1000 mm3, mice were injected with BrdU (100 mg/kg) for 2 hrs, sacrificed; and normal mammary glands and mammary glands with tumors were processed for paraffin blocks or frozen in liquid nitrogen.
Whole gland morphological and histological analysis
For tumor and developmental studies mammary gland whole mounts were processed as previously described by Williams and Daniel  with the following modifications. The #4 inguinal mammary glands from the right side were removed and spread flatly on the inner surface of a 50 ml tube and fixed with with 10% Formalin in PBS. The next day, tissue was placed in a cassette and fat was removed using acetone for 48 hrs. Samples were dehydrated in 100% ethanol (EtOH) for l hr, 95% EtOH for 1 hr, and stained with Carmine Alum. Mammary glands were destained as follows: H20 for 1 hr.; 70% EtOH for 1 hr.; 95% EtOH for 1 hr.; 100% EtOH 3 × for 1 hr.; and cleared in xylene 3 × for 1 hr. Finally tissues were permanently stored in glass vials filled with methylsalicylate until analyzed.
Percentage of fat pad filled was determined by measuring (mm) the length of ductal out growth of #4 inguinal normal mammary glands and dividing it by the length of the total length of the mammary gland fat pad at 4, 6 and 9 weeks of age.
Mammary gland tumors and #4 inguinal normal mammary glands were harvested, placed in cassettes and fixed in 4% paraformaldehyde in PBS overnight. The following day, paraformaldehyde was replaced by 70% EtOH and samples were embedded in paraffin. Serial sections (5 μm thick) cut from paraffin blocks were placed on Superfost Plus slides (Fisher Scientific, Fair Lawn, NJ), deparaffinized, gradually hydrated and all sections stained with Hematoxylin-Eosin (H&E) and then examined microscopically.
Frozen mammary glands were first crushed under liquid nitrogen using a metal pestle and mortar. Crushed tissue was lysed in TNESV buffer and 50 μg of tissue protein lysate was immuonblotted as described previously . We used the following antibodies at the listed concentrations: Anti-P85 1:1000 (Upstate Group, Inc., Lake Placid, NY, USA), p-AKT 1:1000 (Cell Signaling Technology, Beverly, MA, USA), p-ERK1/2 1:1000 (Cell Signaling Technology, Beverly, MA, USA), AKT 1:1000 (Cell Signaling Technology, Beverly, MA, USA), ERK1/2 1:4000 (Upstate Group, Inc., Lake Placid, NY, USA), and β-actin 1:4000 (BD Biosciences, San Jose, CA, USA).
IGF-I levels were measured in serum using rat/mouse IGF-I ELISA assay purchased from Immunodiagnostic Systems (Boldon, Tyne & Wear, UK). The assay sensitivity was 82 ng/ml. IGF-I levels measured in mammary gland tissue were measured by first crushing the tissue under liquid nitrogen using a metal pestle and mortar. Crushed tissue was then lysed in TNESV buffer and 10 μl of supernatant was assayed by a newly developed mouse IGF-I High Sensitivity (HS) ELISA assay from Immunodiagnostic Systems. The assay sensitivity was 2.8 ng/ml.
In the tumor studies, weights between animals that expressed the TTR-IGF-I transgene (TTR-IGF-I and TTR-IGF-I/MMTV-ErbB2 bigenics) and those that did not (wild type and MMTV-ErbB2 trangenics) were analyzed using ANOVA. All other analysis was done by unpaired Student's t test assuming random sampling. Probability values < 0.05 were considered to be statistically significant. The IBM PC programs INSTAT and PRISM software (GraphPad, San Diego, CA, USA) were used to calculate and graph the results. Time to tumor formation was analyzed using Kaplan-Meier survival curves  and compared using the generalized Wilcoxon test . Tumor growth rates were analyzed by computing the individual growth curve measurements from time of primary tumor appearance to attaining a size of 500 mm3. Then the growth rates for each mouse were compared using a Student's t test.
Results and Discussion
A moderate increase in levels of circulating IGF-I doesn't alter pubertal mammary gland development
Analysis of Mammary Gland Growth
Wild Type (control)
Average. % Fat Pad Filled 4 wks
Average. % Fat Pad Filled 6 wks
Average. % Fat Pad Filled 9 wks
Increased circulating IGF-I doesn't affect mammary gland tumorigenesis
Bigenic IGF-I/ErbB2 virgin female mice showed palpable mammary tumors beginning at 24 weeks of age and had a mean time to tumor formation (MTTF) of 33 weeks (Figure 3A). ErbB2 only transgenic virgin females showed a similar tumor formation with the earliest mammary tumors palpated at 24 weeks of age and a MMTF of 30 weeks (Figure 3A). There was no difference in MTTF (p = 0.59). Importantly, increased circulating levels of IGF-I had no affect on mammary tumor incidence, as no mammary tumors were detected in the TTR-IGF-I only mice (Figure 3A). While species variation in IGF-I signaling may play a role in our findings compared to epidemiological data that currently exists  a wealth of evidence confirms the common functionality of IGF-I signaling between humans and mice in both normal growth and development, and numerous human malignancies not just breast cancer .
Comparing Groups: End of study tumor summary
Number of Tumors
19(20) = 95%
19(21) = 90%
Mean Time to Tumor Formation (MTTF)
224 days 30 wks/7.5 m
233 days 33 wks/8.3 m
Average Number of Tumors per Animal
2.2 (11) = 55%
2.3 (13) = 60%
Epidemiological data suggests that increased circulating IGF-I is associated with a women's risk of developing breast cancer . On the other hand, experimental evidence that increased circulating IGF-I is able to initiate and/or regulate tumor growth has not yet been established. Using the first transgenic animal model to simulate circulating levels of IGF-I that may be comparable to levels in women susceptible to breast cancer, our data suggest that modest elevation of circulating IGF-I do not have a role in mammary tumor initiation or promotion. More so, we showed that circulating IGF-I does not alter normal pubertal mammary gland development; thus supporting the established dogma that paracrine/autocrine IGF-I regulated by GH is the preferred regulatory pathway responsible for mammary gland development .
The inability of increased circulating IGF-I to initiate mammary tumorigenesis in our model suggest that the breast cancer risk associated with higher levels of circulating IGF-I in women may, in part, be due to IGF-I being associated or modulating another risk factor for breast cancer. Thus IGF-I may enhance a women's sensitivity to oncogenic promoters like ER (estrogen receptor) or ErbB2. IGF-I has been shown to increase transcriptional activation of ER in breast cancer cell lines increasing cellular sensitivity to the actions of estrogen [35, 36]. However, estrogen has been shown to activate the IGF pathway in MCF7L xenographs independent of the level of circulating IGF-I . Furthermore, we showed that increased circulating IGF-I levels had no effect on ErbB2 promoted mammary tumorigenesis. This would suggest the local interaction between the between the ErbB2 and IGF-I pathways previously shown [9, 38] is independent of circulating IGF-I and more likely dependent upon alterations in autocrine/paracrine signaling pathways in the breast.
This work culminates to suggest that circulating IGF-I itself may not be directly altering breast cancer risk and thus may not be a suitable target for successful treatment. Further studies are required in additional models to determine if this result is common across other breast cancer subtypes.
Insulin-like growth factor-I
Hepatic IGF-I transgenic
knockout mice expressing the hepatic IGF-I transgene
Liver IGF-I Deficient
little transgenic mouse
We would like to thank Susan Durham, PhD and IDS for the generous donation of the mouse IGF-I HS ELISA assay kits. This work was supported in part by pilot project funding from the Dan L. Duncan Cancer Center at Baylor College of Medicine. RK Dearth was supported by a postdoctoral fellowship from The Susan G. Komen Breast Cancer Foundation (PDF113306): Yu-Fen Wang is supported by a DOD (BC083130) pre-doctoral award. AV Lee is a recipient of a T.T. Chao Scholar Award (Department of Medicine, Baylor College of Medicine). J Xu is supported by a NIH grant (CA112403).
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