t10,c12-CLA stimulates mammary tumorigenesis independently of the time of its administration
Previously, we found that continuous feeding of a diet supplemented with 0.5% t10,c12-CLA from weaning, or from 10 weeks of age, markedly accelerated mammary tumor development and lung metastasis in ErbB2 transgenic mice (12
). In the current study, we asked whether short-term administration of this CLA isomer might be deleterious. We focused on two 4-week time periods: (i) post-puberty, from 10 to 14 weeks of age, and (ii) immediately prior to the time when palpable mammary tumors were expected, from 21 to 25 weeks of age. At the former time, although all the cells can be considered ‘initiated’ since ErbB2 is overexpressed, preneoplastic lesions are not readily detected in the mammary gland. In contrast, although palpable tumors are not seen at 21 weeks of age, preneoplastic and subclinical neoplastic lesions are expected to be present. For comparison purposes, two additional groups of mice were fed the t10,c12-CLA-supplemented diet continuously from 10 or 21 weeks of age. The latter group also allowed us to examine the effect of delayed t10,c12-CLA feeding on tumorigenesis. Finally, an additional control group was fed a diet supplemented with 0.5% c9,t11-CLA from 10 weeks of age.
Consistent with our previous studies, continuous supplementation with t10,c12-CLA from 10 weeks of age markedly stimulated mammary tumorigenesis in ErbB2 transgenic mice, with latency, the time when 50% of the mice in each group had a palpable tumor, decreased from 40.6 weeks in the control to 26.6 weeks in the CLA group ( and , P < 0.001). Overall survival time, calculated based on the time each mouse had to be killed because of tumor size, was also significantly decreased in the CLA group (). In contrast to the effects with t10,c12-CLA, c9,t11-CLA did not have any significant effect on latency or survival ().
Effect of time of administration of CLA on mammary tumorigenesis and survival
Short-term administration of t10,c12-CLA appeared to temporarily accelerate mammary tumor development. To quantify this, we compared weekly differences in the percent of tumor-bearing mice between the control and short-term supplementation groups beginning ~10 weeks (determined empirically) after the end of the supplementation. The group fed the t10,c12-CLA diet between 10 and 14 weeks of age had an average of 14.3% (±1.6) more tumor-bearing animals each week in the 15-week time period between 25 and 40 weeks of age (). Similarly, in the group fed t10,c12-CLA between 21 and 25 weeks of age, there were 14.2% (±1.4) more tumor-bearing animals each week in the 10-week time period between 36 and 46 weeks of age (). However, for each of the short-term supplementation groups, overall survival and survival time after detection of a palpable tumor did not differ from control ().
Initiation of continuous t10,c12-CLA supplementation starting just prior to expected tumor development (21 weeks of age) accelerated mammary tumor development compared with control ( and ), although the decreased latency just failed to achieve statistical significance (P
0.068), probably because the progression timeline was so far advanced. Importantly, CLA was considerably more deleterious when started at this later age, since both overall survival of the mice as well as time of survival after detection of a palpable tumor were significantly shortened compared with the control group (, P
< 0.01). The latter measurement suggests that tumor growth rate was considerably enhanced by this t10,c12-CLA supplementation protocol.
t10,c12-CLA stimulates the growth of established ErbB2-overexpressing mammary tumors
The stimulation of tumorigenesis in the older mice, as well as their decreased survival time, suggested that in addition to enhancing growth of preneoplastic/subclinical neoplastic lesions, t10,c12-CLA supplementation might also stimulate the growth of established mammary tumors. Since women with breast cancer might take a CLA supplement, we addressed this question by randomizing tumor-bearing mice into two groups and feeding them control or 0.5% t10,c12-CLA-supplemented diets for 4 weeks. As seen in , growth of established tumors was significantly increased at the 3- and 4-week time points. Even more significantly, however, during the treatment period, 9 out of 11 mice in the t10,c12-CLA group developed a new palpable tumor; by comparison, only one out of nine mice in the control group developed a new tumor (, inset).
Long-term but not short-term administration of t10,c12-CLA increases the size of the kidney, liver, heart and spleen
We reported previously that when ErbB2 transgenic mice were fed the t10,c12-CLA-supplemented diet from weaning, the weights of the liver, heart and spleen were significantly increased compared with mice fed the control or c9,t11-CLA-supplemented diets (12
). In the current study, we asked whether delaying the start of the t10,c12-CLA supplementation to 10 or 21 weeks of age, or short-term supplementation for only 4 weeks, would have an adverse effect on organ size. As seen in supplementary Table I
(available at Carcinogenesis
Online), continuous feeding of the t10,c12-CLA diet from 10 or 21 weeks of age not only resulted in a doubling of the relative size (organ weight per 100 g body wt) of the heart, liver and spleen but also increased the relative kidney size by 40%. These changes were not seen in mice fed the t10,c12-CLA-supplemented diet for only 4 weeks. No changes in organ sizes were observed in the mice fed the diet supplemented with 0.5% c9,t11-CLA (supplementary Table I
is available at Carcinogenesis
Short-term administration of t10,c12-CLA to ErbB2 transgenic mice induces morphological changes in the mammary gland
Long-term supplementation with 0.5% t10,c12-CLA but not with c9,t11-CLA induced a dramatic lobular hyperplasia of the mammary epithelium, which was associated with the increased mammary tumorigenesis in this group (12
). Since a short, 4 week, supplementation with t10,c12-CLA resulted in a temporary acceleration of mammary tumor development in the ErbB2 transgenic mice, we asked whether 4 weeks were sufficient to induce morphological changes in the mammary gland and, if so, whether these changes were maintained once the mice were switched back to the control diet. shows that the mammary glands of mice fed the diet containing 0.5% t10,c12-CLA for 4 weeks were more developed, with significantly increased branching, ductal budding and lobular development when compared with mice fed the control diet or the diet supplemented with 0.5% c9,t11-CLA. Concurrently, the length of the epithelium from the lymph node to the edge of the fat pad was significantly decreased (data not shown). These t10,c12-CLA-induced morphological changes were independent of estrous cycle stage at the time of killing. From the H&E sections, it can be seen that in the mice fed t10,c12-CLA, there was significant loss of the white adipose tissue in the mammary gland, and almost complete loss of the brown adipose tissue (). Additionally, there was a marked infiltration of polymorphonuclear leukocytes (PMNs) into the stroma surrounding the mammary epithelium (, vi, arrow), and mammary lymph node size was increased by 35% (, right panel). These differences were not seen in mice fed c9,t11-CLA.
Since one or more of these changes could be associated with the accelerated tumor development, we asked whether they persisted in mice given the t10,c12-CLA-supplemented diet for 4 weeks and then switched back to the control diet for the duration of the tumorigenesis study. As seen in , although prolonged continuous t10,c12-CLA administration stimulated an extensive and statistically significant lobular and ductal hyperplasia, mammary development scores in the mice fed the t10,c12-CLA diet for only 4 weeks, before being returned to the control diet, were not significantly different from those in the mice fed the control diet. This would suggest that the morphological changes were reversible. In contrast, mammary lymph node size, which was significantly increased by long-term administration of t10,c12-CLA, remained elevated in mice fed this isomer for 4 weeks before being returned to the control diet (). No effect of c9,t11-CLA was noted on lymph node size.
Mammary glands in the groups fed t10,c12-CLA continuously from 10 or 21 weeks of age were very small compared with the control and c9,t11-CLA groups, and had lost all of the brown adipose tissue and the majority of the white adipose tissue (data not shown). PMN infiltration into the ductal stroma was marked (data not shown). When the t10,c12-CLA diet was fed for only 4 weeks before returning the mice to the control diet, the mammary glands were noticeably smaller than those in the control group, although larger than those fed t10,c12-CLA continuously. Return to the control diet resulted in repopulation with white adipose tissue, although very little brown adipose tissue; PMN infiltration in the mammary ductal stroma of these mice was variable.
CLA alters proliferation and apoptosis of ErbB2-overexpressing mammary epithelium in a time-dependent manner
We recently reported that a short, 10 day, dietary supplementation with t10,c12-CLA starting at 10 weeks of age significantly stimulated proliferation of ErbB2-overexpressing mammary epithelium, suggesting that this may be a major factor contributing to the accelerated mammary tumor development (12
). In the current experiments, we asked whether this stimulatory effect on the mammary epithelium was maintained with prolonged feeding of the t10,c12-CLA isomer. Two independent studies with ErbB2 transgenic mice were performed. In the first, mice were fed control or 0.5% CLA-supplemented diets from 10 to 14 weeks of age and then killed. In the second, supplementation was initiated at 10 weeks of age, and continued for the lifetime of the mice (defined as the time when a mouse had to be killed because of tumor size). Proliferation was evaluated by Ki67 immunohistochemistry.
As seen in (left panel), the Ki67 labeling index was modestly increased in the ductal and lobular epithelium of mice fed the t10,c12-CLA-supplemented diet for 4 weeks; however, this was not statistically significant, unlike the situation with 10 days of feeding [, (12
)]. The third epithelial structure examined was the terminal end bud (TEB). In young animals, this is the bulbous structure at the end of the growing ducts that consists of multiple cell layers. In the older, transgenic ErbB2 mice in these studies, however, preneoplastic and small neoplastic lesions also have multiple cell layers when examined by cross-section, so the structures referred to as TEBs/lesions in the two older groups are most probably lesions, rather than TEBs that have generally disappeared by this age. In the lesions of mice fed the control diet for 4 weeks, proliferation was quite high and was not further increased by CLA. In contrast, there was a marked increase in the labeling index in TEBs of mice fed the diet for only 10 days.
In contrast to the mice fed CLA diets for 10 days or 4 weeks, the Ki67 labeling index in all epithelial structures was slightly but significantly decreased by lifetime supplementation of the diet with t10,c12-CLA. An even greater decrease was observed in the ducts and lesions of mice supplemented with c9,t11-CLA (). Although we cannot perform a statistical analysis because the experiments were performed independently, it is noteworthy that the proliferative index in the control mice markedly increased with age. This is not unexpected given the overexpression of ErbB2 in the epithelium, and it could explain the apparent altered CLA responsiveness in the older animals.
Apoptosis in the mammary epithelium was examined concurrently by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling immunohistochemistry. Overall, the apoptotic index (, right panel) was much lower than the proliferative index. However, t10,c12-CLA stimulated apoptosis in the mammary epithelium of mice fed this isomer for 10 days or 4 weeks. Interestingly, this effect was lost after long-term administration (‘lifetime’), when a significant decrease in apoptosis was observed in each of the mammary epithelial structures from the t10,c12-CLA group. In comparison, c9,t11-CLA stimulated apoptosis in the mammary epithelium after 4 weeks supplementation, and had no effect or modestly decreased apoptosis when supplemented for the lifetime of the mice.
t10,c12-CLA stimulates survival and proliferative signaling
To gain leads as to how t10,c12-CLA might be affecting tumorigenesis, two major signaling pathways were examined in mammary tumors from mice fed control or CLA-supplemented diets. As seen in , phosphorylation of PI3K and Akt was increased 12.0- and 9.7-fold, respectively, in tumors from mice fed the t10,c12-CLA-supplemented diet. Although total PI3K and Akt each decreased by 50%, the increased phosphorylation of these two proteins far exceeded the decrease in total protein, suggesting activation of survival signaling in these tumors. Additionally, phosphorylation of MEK and extracellular signal-regulated kinase (ERK) was increased 3.2- and 3.7-fold in the tumors from mice fed the t10,c12-CLA-supplemented diet, concurrent with a 56% decrease in total MEK and a 1.1-fold increase in total ERK, suggesting an increase in proliferative pathways. No significant differences were observed in tumors from mice fed c9,t11-CLA. We tested the hypothesis that this stimulatory effect was mediated by an increased signaling through ErbB2. Since ErbB2 is activated by phosphorylation, western blot analysis was used to examine the phosphorylation status of ErbB2. No significant difference in phospho-Tyr877 () or phospho-Tyr1221/1222 (data not shown) was observed among the three groups. Moreover, total ErbB2 was not changed. We also used an antibody against total phospho-tyrosine, but did not find any significant change at ~185 kDa, the molecular weight of ErbB2 (). We next asked whether t10,c12-CLA activated the IGF-IR, since both the Akt and ERK pathways are downstream targets of this receptor (23
). Indeed this was the case, and shows that phosphorylation of IGF-IR and/or insulin receptor (the phospho-antibody detects both) was significantly increased 7-fold in mice fed t10,c12-CLA. Total IGF-IR was not significantly changed by dietary CLA.
Fig. 4. t10,c12-CLA activates the PI3K/Akt and MEK/ERK pathways. ErbB2 transgenic mice were fed control or 0.5% CLA-supplemented diets continuously from 24 or 70 days of age, and protein expression evaluated in mammary tumors. (A) Phospho-PI3K p85 (Tyr458)/p55 (more ...)
Fig. 5. t10,c12-CLA activates IGF-IR/IR, but not ErbB2. ErbB2 transgenic mice were fed control or 0.5% CLA-supplemented diets continuously from 24 days or 70 days of age and protein expression evaluated in mammary tumors. (A) Phospho-IGF-IRβ (Tyr1135/1136)/insulin (more ...)