Mammary Tumors Growing in the Absence of Growth Hormone Are More Sensitive to Doxorubicin Than Wild-Type Tumors

Abstract

Previously, we reported that N-methyl-N-nitrosourea (MNU)-induced mammary tumors could be established in mutant spontaneous dwarf rats (SDRs), which lack endogenous growth hormone (GH) by supplementing with exogenous GH, and almost all such tumors regressed upon GH withdrawal. When the highly inbred SDR line was outcrossed to wild-type (WT) Sprague-Dawley rats, MNU-induced mammary tumors could still be established in resulting outbred SDRs by supplementing with exogenous GH. However, unlike tumors in inbred SDRs, 65% of mammary tumors established in outbred SDRs continued growth after GH withdrawal. We further tested whether these tumors were more sensitive to doxorubicin than their WT counterparts. To accomplish this, MNU-induced mammary tumors were established in WT rats and in SDRs supplemented with exogenous GH. Once mammary tumors reached 1 cm3 in size, exogenous GH was withdrawn from SDRs, and the subset that harbored tumors that continued or resumed growth in the absence of GH were selected for doxorubicin treatment. Doxorubicin was then administered in 6 injections over 2 weeks at 2.5 mg/kg or 1.25 mg/kg for both the WT and SDR groups. The SDR mammary tumors that had been growing in the absence of GH regressed at both doxorubicin doses while WT tumors continued to grow robustly. The regression of SDR mammary tumors treated with 1.25 mg/kg doxorubicin was accompanied by reduced proliferation and dramatically higher apoptosis relative to the WT mammary tumors treated with 1.25 mg/kg doxorubicin. These data suggest that downregulating GH signaling may decrease the doxorubicin dose necessary to effectively treat breast cancer.

Keywords: IGF-1; MNU; breast cancer; doxorubicin; growth hormone; mammary tumor.

Figure 1.

Growth curves of MNU-induced mammary tumors in SDRs treated with supplemental GH from first appearance of tumor up to doxorubicin treatment start. When GH was halted, (indicated by the dashed line), some tumors continued to grow or resumed growth after an initial regression [SDRs #1, 2, 3, 4 (L5), 5 (L2), 9, 10, and 11 (L2)], new tumors emerged [SDRs #5 (R2, M5), 7 (R2, M5), and 8 (R2)], and some tumors regressed [SDRs #4 (R5), 6, 7(L3), 8 (L5), 9 (R2-1), and 11 (R2)].

Figure 2.

Blood was collected for analysis of serum IGF-I from WT rats at time of sacrifice (WT), SDRs treated with bovine GH (SDR + GH), and SDRs treated with bovine GH after GH withdrawal but before doxorubicin treatment (SDR − GH). Asterisks indicate significant difference in mean (**P < 0.01, ***P < 0.001; analysis of variance).

Figure 3.

Tumor volumes in experimental rats were modeled based on observable tumor volumes. (A) An outline of the experimental design used for this study. (B) Model-estimated tumor growth curves, along with approximate 95% CIs, are shown for both WT (n = 7 tumors) and SDR (n = 11 tumors) type rats treated with 1.25 mg/kg doxorubicin. (C) Model-estimated tumor growth curves, along with approximate 95% CIs, are shown for both WT (n = 6 tumors) and SDR (n = 4 tumors) type rats treated with 2.5 mg/kg doxorubicin. At both doses, SDR tumors had an overall decreasing tumor trajectory while the WT tumors grew over the observed time period, this difference in slopes was found to be statistically significant (P < 0.0001).

Figure 4.

H&E staining of the mammary tumors in WT (A) and SDRs (B and C) treated with 1.25 mg/kg doxorubicin showed evidence of hemosiderin deposits (golden-brown stain) in SDRs indicated by arrows in B and C. (D) Blinded semiquantitative hemosiderin stain scoring indicates a complete lack of hemosiderin in WT rats (n = 3) with extensive evidence in SDRs (n = 8; ***P < 0.001; Student’s t-test). (E) Simple linear regression analysis shows a positive correlation between hemosiderin score and tumor volume decrease over the course of treatment in SDRs treated with 1.25 mg/kg doxorubicin. Scale bars in A and B = 100 µm. Scale bar in C = 20 µm.

Figure 5.

Ki67 immunohistochemistry staining is a marker of proliferative cells. (A and B) Representative Ki67-stained images of mammary tumors treated with 1.25 mg/kg doxorubicin in WT (A) and SDR (B) are shown. Scale bar = 50µm. (C) Blinded cell counting shows a decrease in Ki67-positive cells in mammary tumors found in SDRs (n = 6) relative to WT (n = 3; *P < 0.05; Student’s t-test).

Figure 6.

Immunofluorescent TUNEL staining is a marker of apoptotic cells. DAPI stain (A), TUNEL stain (B), and merged image (C) are shown for mammary tumors from WT rats treated with 1.25 mg/kg doxorubicin. Representative TUNEL stain (D) is also shown for mammary tumors treated with 1.25 mg/kg doxorubicin in SDRs. Scale bar = 50µm. (E) Blinded cell stain analysis showed an increase in TUNEL-positive cells for mammary tumors from SDRs (n = 6) treated with 1.25 mg/kg doxorubicin compared to WT (n = 3) treated with 1.25 mg/kg doxorubicin (*P < 0.05; Student’s t-test).