BP-3 enhances estrogen-stimulated mammary gland proliferation in pubertal mice fed HFD
Since BP-3 showed estrogenic activity as a proliferative stimulus to MCF-7 breast cancer cells [29, 30], we sought to see if BP-3 could stimulate in vivo mammary epithelial proliferation. To that end, both pubertal and adult BALB/c mice were placed on LFD or HFD with and without BP-3 (see Materials and Methods), OVX, allowed time for recovery and clearance of endogenous hormones, and then treated with E2 or control for 5 d. While no BP-3 effects were seen in the adult mice (data not shown), the pubertal mice fed HFD plus BP-3 showed higher proliferation in response to E2 than did mice fed HFD alone (Figure 1A). No BP-3 effects were observed in the absence of E2, and no BP-3 effects were observed in mice fed LFD.
As the initial experiment involved relatively long-term treatment with BP-3 and acute exposure to E2, we examined the effects of BP-3 in an acute exposure regimen at our standard dose, as well as 0.1 and 0.01 doses, with and without co-treatment with E2 in OVX pubertal BALB/c mice fed HFD. While BP-3 by itself showed no effects at any dose (data not shown), BP-3 augmented the proliferative response to E2 in both ducts and duct ends at the standard dose and in duct ends at the 0.1 dose (Figure 1B).
BP-3 reduced tumorigenesis in mice fed LFD and promoted tumorigenesis in mice fed LFD-HFD
Having observed growth promoting effects, we examined whether BP-3 treatment would promote tumorigenesis in the Trp53-null mammary transplant model in BALB/c mice. We previously reported that HFD exposure at either puberty or adulthood promoted mammary tumorigenesis in this model [7]. Since our examination of BP-3 stimulated growth in the mammary gland only found effects at puberty with mice fed HFD, we examined the effects of BP-3 on tumorigenesis with mice fed LFD, pubertally-restricted HFD (HFD-LFD), and adulthood-restricted HFD (LFD-HFD). As in our earlier studies, most tumors were epithelial in composition (Figure 2A), while some were poorly differentiated spindle cell carcinomas (Figure 2B). While an adult-restricted HFD (LFD-HFD) increased the proportion of spindle cell tumors compared to LFD, the proportion of epithelial versus spindle cell tumors was increased by BP-3 treatment in mice fed LFD-HFD (Figure 2C).
Kaplan-Meier analysis revealed that BP-3 reduced tumorigenesis of epithelial tumors in mice fed LFD (Figure 3A). On the other hand, consistent with the increased proportion of epithelial tumors, BP-3 was promotional for epithelial tumorigenesis in mice fed LFD-HFD (Figure 3C), while reducing spindle cell tumorigenesis (Figure 3D). No significant effects were observed with Kaplan-Meier analysis for either spindle cell tumors in mice fed LFD (Figure 3B), or for both epithelial and spindle cell tumors from mice fed HFD-LFD (Figure 3E and F).
BP-3 treatment increased latency of both epithelial and spindle cell tumors in mice fed LFD (Figure 4A and B). No significant effects on latency were found for BP-3 treatment on other diets. ANOVA found significance for dietary effects in both epithelial and spindle cell tumors (Supplementary Table S2). This is consistent with mice fed LFD-HFD tending to have shorter tumor latencies than mice fed either LFD or HFD-LFD, although this trend is not significant by Mann-Whitney U test.
Tumor characteristics
Most of the epithelial tumors were ER- PR-, ranging from 89 to 100% among the dietary regimens and BP-3 treatments, and did not vary significantly by histological type, diet, or BP-3 treatment. Similarly, most spindle cell tumors were ER- PR-, ranging from 88 to 100% among the dietary regimens and BP-3 treatment (Supplementary Table S3).
Since unregulated proliferation and resistance to apoptosis are hallmarks of cancer, we measured tumor cell proliferation and apoptosis by quantifying BrdU incorporation and TUNEL, respectively. Epithelial tumors arising in mice fed LFD-HFD and HFD-LFD showed greater proliferation with BP-3 treatment (Figure 5A) and spindle cell tumors arising in mice fed LFD and LFD-HFD showed greater proliferation with BP-3 treatment (Figure 5B). Both epithelial and spindle cell tumors arising in mice fed HFD-LFD showed reduced proliferation compared to LFD-fed mice. ANOVA (Supplementary Table S2) found that overall dietary effects on proliferation of epithelial tumors were not significant, while BP-3 effects were significant and showed a significant interaction with diet. ANOVA found both significant dietary and BP-3 effects on proliferation of spindle cell tumors, but no interaction between these treatments.
BP-3 did not significantly alter apoptosis, except for spindle cell tumors arising in mice fed LFD (Figure 6). Apoptosis in spindle cell tumors from LFD + BP-3 mice was reduced by half compared to those from LFD mice (Figure 6B). We also observed a reduction of apoptosis in epithelial tumors arising in mice fed HFD-LFD (Figure 6A) and in spindle cell tumors arising in mice fed LFD-HFD compared to LFD (Figure 6B). It is noteworthy that the spindle cell tumors arising in mice fed LFD + BP-3 showed both higher proliferation and lower apoptosis, but longer latency. ANOVA (Supplementary Table S2) found a significant dietary effect on apoptosis in epithelial tumors, but no significance to the effects of diet and BP-3 in spindle cell tumors. However, the interaction between diet and BP-3 was significant for spindle cell tumors, consistent with BP-3 having a significant effect only on tumors arising in mice fed LFD.
Having observed increased proliferation with BP-3 treatment in epithelial tumors arising in mice fed LFD-HFD and HFD-LFD and in spindle cell tumors arising in mice fed LFD and LFD-HFD, we examined whether this would be reflected in the number of epithelial proliferative lesions observed in the mammary glands of 26-week old mice, prior to the appearance of palpable tumors, as well as in the proliferation of normal mammary tissue. BP-3 treatment only increased the number of lesions in mice fed HFD-LFD (Figure 7A), while proliferation was increased by BP-3 treatment in all dietary groups (Figure 7B). No association was observed between BP-3 treatment effects on normal cellular proliferation and tumorigenesis, or between BP-3 treatment effects on epithelial proliferative lesions and tumorigenesis. Mice fed HFD-LFD also showed increased lesions compared to mice fed LFD (Figure 7A). ANOVA (Supplementary Table S2) found both significant dietary and BP-3 effects on the number of lesions, although these effects show no significant interaction. This is consistent with BP-3 showing a trend toward increased lesions in all dietary groups, and the HFD-LFD group showing a significantly higher number of lesions compared to the LFD and LFD-HFD groups. ANOVA only found a significant effect for BP-3 treatment on proliferation in mammary glands of 26-week old mice.
Because we found that HFD promoted angiogenesis among epithelial tumors arising in both the Trp53-null transplantation model [7] and in the 7,12-dimethylbenz[a]anthracene (DMBA) model [5], we examined the intra-tumoral vascularization of tumors with and without BP-3 treatment using the endothelial cell marker, CD31. Epithelial tumors arising in mice fed LFD-HFD showed increased vascularization in response to BP-3 treatment (Figure 8A). Spindle cell tumors as a group showed increased vascularization compared to epithelial tumors (Figure 8A and B; Supplementary Figure S2), as previously reported [7]. ANOVA (Supplementary Table S2) found significant BP-3 effects for both epithelial and spindle cell tumors, consistent with small but statistically insignificant increases in vascularization of LFD and HFD-LFD epithelial tumors, as well as small but statistically insignificant decreases in vascularization of spindle cell tumors with BP-3 treatment.
Effects on metabolic parameters
Animal weight was followed over the time course of tumorigenesis and BP-3 exposure had no significant impact on body weight (Supplementary Figure S3). BALB/c mice were previously reported to be obesity-resistant when fed HFD [5, 6,7, 8]. In the present study, we observed considerable weight gain among mice fed LFD-HFD. The mice fed LFD-HFD and LFD-HFD + BP-3 were significantly heavier than those fed LFD and LFD + BP-3 by 11 weeks of age, with at least 20% weight gain by 16 weeks of age in (Supplementary Figure S3). While certainly not obesity-resistant in these experiments, the weight gain of mice fed LFD-HFD is not correlated with elevated non-fasting plasma glucose levels (Supplementary Figure S4A), although non-fasting plasma insulin levels were elevated in mice fed LFD-HFD (Supplementary Figure S4B). BP-3 modestly reduced glucose levels in mice fed LFD-HFD and HFD-LFD (Figure S4A), and modestly increased insulin levels in mice fed HFD-LFD (Figure S4B). ANOVA (Supplementary Table S2) found significant BP-3 effects on glucose levels, consistent with the modest reductions found across dietary groups. ANOVA (Supplementary Table S2) also found significant dietary effects on insulin levels, consistent with modest elevation of insulin across dietary groups. Kaplan-Meier analysis revealed that the weight gain in mice fed LFD-HFD did not significantly alter epithelial tumorigenicity, whether with or without BP-3 treatment (Supplementary Figure S5A and B). Segregation of spindle cell tumors between weight groups do not provide an adequate sample size for meaningful analysis. We speculate that this change in weight gain from prior experiments may be the result of housing changes in our mouse facility. Weight gain in mice has been attributed to a shift from sub-thermoneutral to thermoneutral conditions [31].