A moderate elevation of circulating levels of IGF-I does not alter ErbB2 induced mammary tumorigenesis

Abstract

Background: 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.

Methods: 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 mm(3). 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.

Results: 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.

Conclusion: 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.

Figure 1

Liver specific transgene overexpression of IGF-I results in increased levels of circulating IGF-I and increased body weight. A.) Overexpression of liver IGF-I (black bar) resulted in a significant (p < 0.01) increase in female serum levels of IGF-I measured at 6 weeks of age compared to mice without the TTR-IGF-I transgene (white bar). B.) Females expressing the IGF-I transgene exhibited significantly (p < 0.05) larger body weights at 7, 10-12 weeks of age due to increased circulating IGF-I compared to wt/ErbB2 only controls. No transgene = wt + ErbB2 only females; IGF-I Transgene = TTR-IGF-I transgenic + TTR-IGF-I-ErbB2 bigenic females. N for each group is represented within their respective bars. N = 30 animals per group for weight. ** p < 0.01; * p < 0.05.

Figure 2

Increased circulating levels of IGF-I had no effect on pubertal mammary gland growth. Representative whole mounts of wild type (wt) and IGF-I transgenic (TTR-IGF-I) mammary glands at 4 wks (top 2 panels), 6 wks (middle 2 panels), and 9 wks of age (bottom 2 panels). N = 20 per group at 4 wks of age; N = 4 for WT and N = 7 for TG at 6 wks of age; N = 7 for WT and N = 8 for TG at 9 wks of age. WT = wild type and TG = TTR-IGF-I.

Figure 3

Increased levels of circulating IGF-I does not initiate mammary tumorigenesis. A.) Kaplan-Meier tumor curve illustrating the percent of animals without mammary gland tumors vs. the day tumors were first palpated. Increased IGF-I had no effect on ErbB2 initiated mammary tumorigenesis (p = 0.59). Additionally, the moderate increase in circulating IGF-I did not induced mammary tumors in females expressing only the IGF-I transgene (purple) compared to controls (maroon line). B.) Increased circulating levels on IGF-I resulted in significantly higher (p < 0.05) IGF-I protein levels in tumors from bigenic females compared to tumors in ErbB2 transgenics (right panel). Middle panel shows IGF-I protein levels measured in contralateral normal mammary glands from these same bigenic and ErbB2 transgenic females. Left panel shows a significant increase (p < 0.05) in serum IGF-I in these same bigenic females compared to ErbB2 transgenics. Bars indicate the mean (±SEM) serum or protein levels of IGF-I assayed by ELISA. N = indicated with bars; MG = Mammary Gland; ErbB2 = ErbB2 transgenic only; Bigenic = TTR-IGF-I-ErbB2; MTTF (mean time to tumor formation) = weeks/days/months; * = p < 0.05.

Figure 4

Increased circulating IGF-I does not alter growth in ErbB2-induced mammary gland turmors. Graphs plotting individual tumor growth curves for ErbB2 (left) and bigenic (right) females. Statistical analysis revealed that increased circulating levels of IGF-I (right) had no significant (p = 0.23) affect on mammary tumor growth compared to ErbB2 only induced mammary tumors. N = 16 for ErbB2 group and 15 for TTR-IGF-I-ErbB2 bigenic group.

Figure 5

Increased levels of circulating IGF-I have no effect on tumor type or mammary gland signaling. A.) Representative H&E staining of mammary tumors promoted by MMTV-ErbB2 (top) or TTR-IGF-I-ErbB2 bigenic (bigenic-bottom). Both groups had similar tumor phenotypes which were predominantly adenocarcinomas. B.) Representative immunoblot of phosphorylated (p) and total (T) AKT and Erk ½ signaling in normal mammary glands (M) and tumors (T). *** = p < 0.001; Magnification: 40 ×. C = representative wild type females; I = representative TTR-IGF-I transgenic females