This study used a syngeneic model of murine primary and metastatic mammary cancer to evaluate and compare the effects of nutrient availability, dietary energy restriction, and metabolic therapeutic intervention with metformin. The 66cl4 cells are a subpopulation of a spontaneously occurring mammary tumor from a Balb/c mouse and are known to metastasize to the lung [27
]. The aggressive nature of this tumor model suggests highly activated growth factor signaling as evidence by high proliferation rates in vitro, MAPK activation signals and lack of estrogen dependence. These cells do not express the hormone receptors for estrogen or progesterone or the Her-2 receptor and appear to represent a murine model of triple negative breast cancer. Triple negative breast cancer is more frequent in younger women as well as women who are obese. In addition, women diagnosed with triple negative breast cancer have especially poor prognosis and rapid occurrence of metastases [35
]. It is especially important to develop adjuvant interventions for women with this aggressive subtype of breast cancer.
From the perspective of primary tumor expansion, DER was the most effective at suppressing tumor growth and metastatic disease in this model. The application of metformin resulted in moderate, yet significant, effects on the primary tumor phenotype. Most prominently, increased apoptosis and reduced proliferation was observed. This data are in agreement with the in vitro observations that metformin treatment reduced proliferation, protein synthesis, and MAPK signaling in the 66cl4 cells and is consistent with findings reported in the human triple negative breast cancer cells, MDA-MB-231, when treated with metformin [37
]. Primary tumor expansion was promoted by increased dietary energy availability and the effect attenuated by the addition of metformin, which is consistent with the report by Algire et al. that utilized a HED and metformin in the Lewis Lung Carcinoma tumor model [16
]. These two studies confirm the efficacy of metformin to repress the growth of aggressive primary tumors in the presence of excess dietary energy in two independent tumor models. When metastatic disease was evaluated, only the application of DER resulted in a significant repression of lung lesions. Even though metformin effectively repressed primary tumor growth when used with HED, it was unable to repress the number of metastatic lung lesions in conjunction with either diet. Importantly, the HED group developed an increased number of metastatic lesions as well as larger lesions when compared to the standard diet group. Even though metformin was able to induce autophagy as demonstrated by increased cell density and LC3b staining of the HED group, metformin therapy was not sufficient to reduce the disease burden in the lungs of these animals.
It has been proposed that growth factor pathways can be critical in supporting breast cancer growth, especially in relation to obesity and diabetes. In addition, the efficacy of DER against tumor progression has been attributed to reduced IGF-1 levels and downstream signaling [38
]. The use of DER most efficiently repressed IGF-1 levels as well as blood glucose and insulin levels, while the use of metformin repressed blood glucose and IGF-1 levels only when given with the HED, as has been reported in other studies [19
]. These data demonstrate that, while metformin can repress glucose and IGF-1 levels in the presence of excess dietary energy, metformin is not as effective under a balanced energy status and falls short of DER in this regard.
The effect of increased dietary energy in the form of free sugar and therapeutic modifiers such as metformin on the metastatic process is a more complex process to model and interpret. A suppression of distal metastasis was observed with DER compared to control diet, but surprisingly, metformin did not suppress metastasis in conjunction with the HED diet despite the fact that metformin was effective at controlling both IGF-1 and insulin levels. The implications of these results may be significant and imply that the contribution of glucose and IGF-1 in secondary metastasis is offset by other pro-metastatic events as suggested by other recent reports [40
]. Not surprisingly, the HED regimen dramatically increases both leptin and resistin levels well above the standard diet. The increased levels of both adipokines were not suppressed by metformin treatment. These data suggest that clinical measures other than glucose, IGF-1, and insulin may be useful when considering the effectiveness of metformin or other metabolic therapeutics when used as adjuvant breast cancer therapies.
As recent reports indicate, there is a growing interest in circulating adipokines as proinflammatory and tumorigenic factors for primary and metastatic cancer [42
]. These data suggest that the effects of metformin on primary tumors are distinct from the events required for secondary metastasis to the lung, especially in this aggressive breast cancer model. It would be expected that HED may be promoting distal tissue priming to promote tumor cell homing, which is strongly supported by the evidence of significant infiltration of myeloid cells within the lung parenchyma with the HED regimen. It should be emphasized that leptin and resistin levels are only two adipokines that may be involved in this process, but any number of proinflammatory agents and growth factors may be involved, including cytokines such as TNF-α, IL-6, IL-8, or SDF-1α. Additional work is needed to investigate the role for each of these adipokines and local mediators to determine their contribution to the metastatic process.
Since epidemiological studies suggest that up to 50% of our population over the age of 50 exhibit metabolic syndrome [45
], representing a pre-diabetic condition related to high caloric intake and a sedentary lifestyle, the relationship of metabolic imbalance and the initiation and/or progression of breast cancer has become critically relevant. The data presented here imply that primary tumor growth and secondary tumor cell metastasis is supported by high energy consumption. Second, where potential novel therapies might be employed to suppress metastatic tumor growth, the identification of adipokines and local mediators that promote tissue priming may be critical in targeting tumor cell inoculation and/or survival in metastatic sites.