Metformin Treatment for the Prevention and/or Treatment of Breast/Mammary Tumorigenesis.

There is increasing interest in metformin's effects on the development, treatment and/or progression of breast cancer. This emerges from observational studies that diabetic women treated with metformin in comparison to other antidiabetic compounds had lower breast cancer incidence and/or mortality rates. The mechanism of action is considered to be activation of hepatic AMPK resulting in reduced gluconeogenesis. Calorie restriction, which consistently reduces mammary tumorigenesis in rodents, is also thought to act through this pathway leading to the hypothesis that metformin's anticancer effects are mediated in a similar fashion. Here we review the literature evaluating metformin's anticancer effects in relation to breast/mammary tumorigenesis. We include clinical observations, as well as studies utilizing rodent models and mammary cell lines. In addition to the anticancer effect of metformin mediated through the AMPK pathway, additional mechanisms of action that directly target tissues have been identified including effects on stem cells, apoptosis, STAT3 and HER2.

Epidermal Fatty Acid binding protein promotes skin inflammation induced by high-fat diet.

Defining specific cellular and molecular mechanisms in most obesity-related diseases remains an important challenge. Here we report a serendipitous finding that consumption of a high-fat diet (HFD) greatly increased the occurrence of skin lesions in C57BL/6 mice. We demonstrated that HFD induced the accumulation of a specific type of CD11c(+) macrophages in skin preceding detectable lesions. These cells primed skin to induce IL-1ß and IL-18 signaling, which further promoted the cytokines IFN-γ- and IL-17-mediated skin inflammation. Mechanistically, epidermal fatty acid binding protein (E-FABP) was significantly upregulated in skin of obese mice, which coupled lipid droplet formation and NLRP3 inflammasome activation. Deficiency of E-FABP in obese mice decreased recruitment of CD11c(+) macrophages in skin tissues, reduced production of IL-1ß and IL-18, and consequently dampened activation of effector T cells. Furthermore, E-FABP-deficient mice are completely resistant to HFD-induced skin lesions. Collectively, E-FABP represents a molecular sensor triggering HFD-induced skin inflammation.

Combination of intermittent calorie restriction and eicosapentaenoic acid for inhibition of mammary tumors.

There are a number of dietary interventions capable of inhibiting mammary tumorigenesis; however, the effectiveness of dietary combinations is largely unexplored. Here, we combined 2 interventions previously shown individually to inhibit mammary tumor development. The first was the use of the omega-3 fatty acid, eicosapentaenoic acid (EPA), and the second was the implementation of calorie restriction. MMTV-Her2/neu mice were used as a model for human breast cancers, which overexpress Her2/neu. Six groups of mice were enrolled. Half were fed a control (Con) diet with 10.1% fat calories from soy oil, whereas the other half consumed a diet with 72% fat calories from EPA. Within each diet, mice were further divided into ad libitum (AL), chronic calorie-restricted (CCR), or intermittent calorie-restricted (ICR) groups. Mammary tumor incidence was lowest in ICR-EPA (15%) and highest in AL-Con mice (87%), whereas AL-EPA, CCR-Con, CCR-EPA, and ICR-Con groups had mammary tumor incidence rates of 63%, 47%, 40%, and 59%, respectively. Survival was effected similarly by the interventions. Consumption of EPA dramatically reduced serum leptin (P < 0.02) and increased serum adiponectin in the AL-EPA mice compared with AL-Con mice (P < 0.001). Both CCR and ICR decreased serum leptin and insulin-like growth factor I (IGF-I) compared with AL mice but not compared with each other. These results illustrate that mammary tumor inhibition is significantly increased when ICR and EPA are combined as compared with either intervention alone. This response may be related to alterations in the balance of serum growth factors and adipokines.

The balance between leptin and adiponectin in the control of carcinogenesis - focus on mammary tumorigenesis.

A number of studies indicate that a growing list of cancers may be influenced by obesity. In obese individuals these cancers can be more frequent and more aggressive resulting in reduced survival. One of the most prominent and well characterized cancers in this regard is breast cancer. Obesity plays a complex role in breast cancer and is associated with increased inflammation, angiogenesis and alterations in serum levels of potential growth factors such as insulin, adiponectin, leptin and estrogen. Reduced levels of serum adiponectin have been reported in breast cancer patients compared to healthy controls, particularly in postmenopausal women and the level of adiponectin has been shown to be inversely associated with insulin resistance. The role of serum leptin levels in breast cancer appears to be more complex. Some studies have shown leptin to be increased in women with breast cancer but other studies have found leptin to be decreased or unchanged. This may be due to a number of confounding issues. We and others propose that it may be the levels of adiponectin and leptin as well as the balance of adiponectin and leptin that are the critical factors in breast and other obesity related cancer tumorigenesis. This review will focus on the current understanding of the interplay between obesity and the functions of leptin and adiponectin. It will then examine what is known about their potential roles in cancer particularly as pertains to breast cancer and how the ratio of adiponectin to leptin may play a role in tumorigenesis.

Growth and Progression of TRAMP Prostate Tumors in Relationship to Diet and Obesity.

To clarify effects of diet and body weight on prostate cancer development, three studies were undertaken using the TRAMP mouse model of this disease. In the first experiment, obesity was induced by injection of gold thioglucose (GTG). Age of prostate tumor detection (~33 wk) and death (~43 wk) was not significantly different among the groups. In the second study, TRAMP-C2 cells were injected into syngeneic C57BL6 mice and tumor progression was evaluated in mice fed either high-fat or low-fat diets. The high fat fed mice had larger tumors than did the low-fat fed mice. In the third study, tumor development was followed in TRAMP mice fed a high fat diet from 6 weeks of age. There were no significant effects of body weight status or diet on tumor development among the groups. When the tumors were examined for the neuroendocrine marker synaptophysin, there was no correlation with either body weight or diet. However, there was a significant correlation of the expression of synaptophysin with earlier age to tumor detection and death. In summary, TRAMP-C2 cells grew faster when the mice were fed a high-fat diet. Further synaptophysin may be a marker of poor prognosis independent of weight and diet.

The manner in which calories are restricted impacts mammary tumor cancer prevention.

Although treatments for breast cancer have improved and long-term survival after diagnosis is now common, prevention of the disease is the ultimate goal. Weight loss or weight maintenance is one approach that has been recommended to reduce the risk of breast cancer, particularly for peri/postmenopausal women. This approach is supported by decades of data indicating that calorie restriction prevents spontaneous and chemically induced mammary tumor development in rodents. In most cases, calorie restriction was implemented by a consistent daily reduction of calories, i.e. chronic calorie restriction (CCR). There have also been several studies where periods of reduced caloric intake were followed by periods of refeeding, i.e. intermittent calorie restriction (ICR), resulting in the prevention of spontaneous mammary tumorigenesis. In most of the early studies, there were no direct comparisons of CCR to ICR. One study using moderate calorie restriction in a chemically induced breast cancer rat model found a slight increase in mammary tumor incidence compared with ad libitum fed and CCR rats. However, recently, it has been demonstrated in several transgenic mouse models of breast cancer that ICR consistently provided a greater degree of protection than CCR. This review will provide a detailed comparison of ICR and CCR for breast cancer prevention. It will also examine potential mechanisms of action that may include periods of reduced IGF-I and leptin as well as an increase in the adiponectin:leptin ratio. Application of this approach to at-risk women may provide an approach to lower the risk of breast cancer in overweight/obese women.

Obesity and breast cancer: status of leptin and adiponectin in pathological processes.

It is well recognized that obesity increases the risk of various cancers, including breast malignancies in postmenopausal women. Furthermore, obesity may adversely affect tumor progression, metastasis, and overall prognosis in both pre- and postmenopausal women with breast cancer. However, the precise mechanism(s) through which obesity acts is/are still elusive and this relationship has been the subject of much investigation and speculation. Recently, adipose tissue and its associated cytokine-like proteins, adipokines, particularly leptin and adiponectin, have been investigated as mediators for the association of obesity with breast cancer. Higher circulating levels of leptin found in obese subjects could be a growth-enhancing factor as supported by in vitro and preclinical studies, whereas low adiponectin levels in obese women may be permissive for leptin's growth-promoting effects. These speculations are supported by in vitro studies which indicate that leptin promotes human breast cancer cell proliferation while adiponectin exhibits anti-proliferative actions. Further, estrogen and its receptors have a definite impact on the response of human breast cancer cell lines to leptin and adiponectin. More in-depth studies are needed to provide additional and precise links between the in vivo development of breast cancer and the balance of adiponectin and leptin.

Punicic acid is an omega-5 fatty acid capable of inhibiting breast cancer proliferation.

Pomegranate extracts have been used as anticancer agents and they contain a large number of potentially bioactive substances. Punicic acid is an omega-5 long chain polyunsaturated fatty acid found in Punica granatum (pomegranate) seed oil. A number of long chain fatty acids have been reported to have cancer preventive actions. Here we investigated the potential ability of punicic acid to affect growth of both an estrogen insensitive breast cancer cell line (MDA-MB-231) and an estrogen sensitive cell line developed from the MDA-MB-231 cells (MDA-ERalpha7). Proliferation was inhibited 92 and 96% for MDA-MB-231 and MDA-ERalpha7 cells, respectively compared to untreated cells by 40 microM punicic acid. Furthermore, punicic acid induced apoptosis in the MDA-MB-231 and MDA-ERalpha7 cells by 86 and 91%, respectively compared to untreated control cells and disrupted cellular mitochondrial membrane potential. We also investigated whether lipid oxidation was required for the function of punicic acid by adding 20 microM of the antioxidant tocotrienol to the assays. This resulted in reversal of the effects of punicic acid on proliferation inhibition, apoptosis and disruption of the mitochondrial membrane potential. Finally, we evaluated the role of PKC signaling in the anti-cancer effects of punicic acid by performing proliferation assays in the presence of the PKC inhibitor bisindolymaleimide I. Proliferation inhibition by punicic acid was partially blocked in both the MDA-MB-231 and MDA-ERalpha7 cells. These results suggest that punicic acid has breast cancer inhibitor properties that are dependent on lipid peroxidation and the PKC pathway.

Eleostearic Acid inhibits breast cancer proliferation by means of an oxidation-dependent mechanism.

Eleostearic acid (alpha-ESA) is a conjugated linolenic acid that makes up approximately 60% of Momordica charantia (bitter melon) seed oil. Prior work found that water extract from bitter melon was able to inhibit breast cancer. Here, we investigated effects of alpha-ESA on both estrogen receptor (ER)-negative MDA-MB-231 (MDA-wt) and ER-positive MDA-ERalpha7 human breast cancer cells. We found that alpha-ESA inhibited proliferation of both MDA-wt and MDA-ERalpha7 cells, whereas conjugated linoleic acid had comparatively weak antiproliferative activity at 20 to 80 micromol/L concentrations. We also found that alpha-ESA (40 micromol/L) treatment led to apoptosis in the range of 70% to 90% for both cell lines, whereas conjugated linoleic acid (40 micromol/L) resulted in only 5% to 10% apoptosis, similar to results for control untreated cells. Addition of alpha-ESA also caused loss of mitochondrial membrane potential and translocation of apoptosis-inducing factor as well as endonuclease G from the mitochondria to the nucleus. Additionally, alpha-ESA caused a G(2)-M block in the cell cycle. We also investigated the potential for lipid peroxidation to play a role in the inhibitory action of alpha-ESA. We found that when the breast cancer cells were treated with alpha-ESA in the presence of the antioxidant alpha-tocotrienol (20 micromol/L), the growth inhibition and apoptosis effects of alpha-ESA were lost. An AMP-activated protein kinase inhibitor (Dorsomorphin) was also able to partially abrogate the effects of alpha-ESA, whereas a caspase inhibitor (BOC-D-FMK) did not. These results illustrate that alpha-ESA can block breast cancer cell proliferation and induce apoptosis through a mechanism that may be oxidation dependent.

Targeting the adiponectin:leptin ratio for postmenopausal breast cancer prevention.

Obesity is a risk factor for postmenopausal breast cancer. Elevated estrogen levels are thought to be a growth factor associated with this relationship. However, there is increasing evidence that factors produced directly in adipose tissue, adipokines, specifically adiponectin and leptin, impact breast cancer development. Serum adiponectin levels are reduced in women diagnosed with breast cancer and in vitro studies using human breast cancer cell lines have shown antiproliferative action of adiponectin. In contrast, elevated serum leptin levels were associated with breast cancer in some studies. In mice which lack the leptin receptor or are leptin deficient oncogene-induced mammary tumors were not detected while leptin enhanced proliferation of breast cancer cell lines, particularly those that express estrogen receptors. Of particular interest, one recent study reported that the adiponectin:leptin ratio was reduced in women with breast cancer. Here we speculate that the ratio of these adipokines may be more important in breast cancer than their absolute concentrations. Additionally, we propose strategies to alter this ratio and thus provide protection against the development of breast cancer.

Effects of adiponectin and leptin co-treatment on human breast cancer cell growth.

Obesity is a risk factor for postmenopausal breast cancer (BC), but the specific mechanisms for this relationship are not well understood. Studies on adipocyte-derived adiponectin and leptin reveal opposing effects on BC cell proliferation in vitro, suggesting they may play a role in BC pathogenesis. In the current study we examined effects on proliferation of five BC cell lines treated with varying adiponectin:leptin (A/L) ratios. A decrease in proliferation was noted for MCF-7 and T47-D cells with increasing ratios (2-500), while an increase was seen in similarly treated MDA-MB-231 and MDA-MB-361 cells. For SK-BR-3 cells, an increase was seen at a ratio of 8. We identified differential effects on some pro-mitogenic, survival and apoptosis-related proteins in MCF-7 and T47-D cells treated at an A/L ratio of 100. Specifically, the AKT and MAPK pathways were not activated in MCF-7 cells, but AKT activation occured within 30 min and MAPK activation was sustained at 48 h in T47-D cells. p53 and Bax were elevated in MCF-7, but were below basal in T47-D cells at 30 min. While co-treatment enhanced apoptosis in MCF-7, similar treatment had the opposite effect in T47-D cells. There were no differences in cell cycle distribution between treated or untreated MCF-7 or T47-D, although T47-D cells had a slightly higher proportion in the G1/G0 phase after co-treatment. The effects of A/L ratio on mediating proliferation may have some specificity since the cell lines exhibited different responses. This may explain previous inconsistencies for the relationship of serum leptin with BC. More studies are needed to better understand the complex interactions that exist between these two adipokines.

Minireview: Obesity and breast cancer: the estrogen connection.

There is now substantial evidence that overweight and/or obesity and/or weight gain are risk factors for the development of postmenopausal breast cancer. In addition, obesity and/or elevated body mass index at breast cancer diagnosis has a negative impact on prognosis for both premenopausal and postmenopausal women. Therefore, understanding the mechanism of how obesity affects the mammary tumorigenesis process is an important health issue. Elevated serum estrogen levels as well as enhanced local production of estrogen have been considered primary mediators of how increased body weight promotes breast cancer development in postmenopausal women. Here, we provide an overview of estrogen's relationship with both obesity and breast cancer as separate entities. Human and relevant preclinical studies are cited. In addition, other growth factors that may be involved in this relationship are considered.

PDLIM4, an actin binding protein, suppresses prostate cancer cell growth.

We investigated the molecular function of PDLIM4 in prostate cancer cells. PDLIM4 mRNA and protein-expression levels were reduced in LNCaP, LAPC4, DU145, CWR22, and PC3 prostate cancer cells. The re-expression of PDLIM4 in prostate cancer cells has significantly reduced the cell growth and clonogenicity with G1 phase of cell-cycle arrest. We have shown the direct interaction of PDLIM4 with F-actin. Restoration of PDLIM4 expression resulted in reduction of tumor growth in xenografts. These results suggest that PDLIM4 may function as a tumor suppressor, involved in the control of cell proliferation by associating with actin in prostate cancer cells.

Intermittent calorie restriction delays prostate tumor detection and increases survival time in TRAMP mice.

Prostate cancer is the most frequently diagnosed cancer in men. Whereas chronic calorie restriction (CCR) delays prostate tumorigenesis in some rodent models, the impact of intermittent caloric restriction (ICR) has not been determined. Here, transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were used to compare how ICR and CCR affected prostate cancer development. TRAMP mice were assigned to ad libitum (AL), ICR (2 wk 50% AL consumption followed by 2 wk pair feeding to AL consumption), and CCR (25% AL consumption) groups at 7 wk of age and followed until disease burden necessitated euthanasia or mice reached terminal endpoints (48 or 50 wk of age). Body weights fluctuated in response to calorie intake (P < 0.0001). ICR mice were older at tumor detection than AL (P = 0.0066) and CCR (P = 0.0416) mice. There was no difference for age of tumor detection between AL and CCR mice (P = 0.3960). Similar results were found for survival. Serum leptin, adiponectin, insulin, and IGF-I were all significantly different among the groups. These results indicate that the way in which calories are restricted impacts both time to tumor detection and survival in TRAMP mice, with ICR providing greater protective effect compared to CCR.

Cross-sectional analysis of intermittent versus chronic caloric restriction in the TRAMP mouse.

BACKGROUND: Previously we found that intermittent calorie restriction (ICR) delayed the age of prostate tumor detection and death in TRAMP mice in comparison to chronic calorie restricted (CCR) and ad libitum fed (AL) TRAMP mice. METHODS: In the present study the same protocol was used in a cross-sectional experiment whereby mice were either ad libitum fed, intermittently calorie restricted at 50% of the consumption of AL mice for 2 weeks followed by 2 weeks of refeeding matched to AL intake or were pair-fed to the ICR. Both ICR and CCR protocols resulted in a 25% reduction in caloric intake. Mice were enrolled in the study at 7 weeks of age to be euthanized at designated time points in cycles 3, 6, and 9 with mice euthanized at the end of restriction and refeeding. RESULTS: At the youngest time point in cycle 3 ICR impacted body weight, fat pad weights and serum factors the most. Additionally, the incidence of detectable prostate cancer pathology was reduced for ICR mice compared to AL and CCR mice. However, by cycle 5 when the mice were 28-30 weeks of age all mice except one ICR mouse had pathologically confirmed prostate cancer. Furthermore, at the two older time points many of the mice assigned to the study did not survive to reach their designated endpoints. CONCLUSIONS: Overall these findings are consistent with other studies indicating protective effects of various interventions on the development of prostate cancer in young TRAMP mice.

Role of the adiponectin leptin ratio in prostate cancer.

We hypothesize that adiponectin and leptin may be capable of mediating some of the effects that body weight has on prostate cancer and that a mouse model may be effective to examine this hypothesis. We found that tumors from the TRAMP prostate cancer model expressed adiponectin and leptin receptors. TRAMP-C2 prostate cancer cell proliferation was reduced by adiponectin. Leptin was able to block the ability of adiponectin to reduce cell proliferation through altered signaling of the ERK pathway. Overall, this work suggests that adiponectin, leptin, and their receptors may play an important role in prostate cancer.

Prevention of mammary tumorigenesis by intermittent caloric restriction: does caloric intake during refeeding modulate the response?

Chronic caloric restriction (CCR) prevents mammary tumorigenesis in rodents, but a protective effect for intermittent caloric restriction (ICR) is less well documented. We recently reported that ICR reduced mammary tumor (MT) incidence of mouse mammary tumor virus-transforming growth factor (MMTV-TGF)-alpha mice to a greater extent than did CCR. Here, we repeated this protocol and obtained serum and tissue samples. Ad libitum (AL) MMTV-TGF-alpha mice were fed AIN-93M diet. Beginning at 10 weeks of age, ICR mice received isocaloric AIN-93M-mod diet (2-fold increases in protein, fat, vitamins, and minerals) at 50% of ad libitum for 3 weeks followed by 3 weeks refeeding with AIN-93M diet. CCR mice were pair-fed AIN-93M:AIN-93M-mod (2:1) matching intakes for restriction/refeeding cycles. Mice were sacrificed for MT size, at 79 (end of 12th restriction) or at 80 (1 week after 12th refeeding) weeks of age. AL and ICR-80 mice had heavier body weights than ICR-79 and CCR mice (P < 0.0001). Cumulative food intakes of ICR and CCR mice were reduced 12% and 15% versus AL mice (P < 0.0001). However, ICR mice consumed significantly (P < 0.0001) more food than did AL mice during refeeding. MT incidence was 84%, 13%, and 27% for AL, ICR, and CCR mice, respectively. MT weight (P < 0.0011) and number (P < 0.01) were higher for AL mice compared with ICR and CCR mice. AL and ICR-80 mice had similar serum IGF-I levels, but only AL values were higher than those of ICR-79 and CCR mice (P < 0.0017). ICR mice had more MT DNA breaks compared with AL and CCR mice, suggesting enhanced apoptosis (P < 0.02). AL mice had higher mammary fat pad ObR and ObRb leptin receptor mRNA expression than did ICR and CCR mice (P < 0.001), but there was no effect on MTs. These results confirm that ICR prevents development of MTs to a greater extent than does CCR, although "overeating" during refeeding may compromise this protection.

Proteomics shows Hsp70 does not bind peptide sequences indiscriminately in vivo.

Heat shock protein 70 (Hsp70) binds peptide and has several functions that include protein folding, protein trafficking, and involvement with immune function. However, endogenous Hsp70-binding peptides had not previously been identified. Therefore, we eluted and identified several hundred endogenously bound peptides from Hsp70 using liquid chromatography ion trap mass spectrophotometry (LC-ITMS). Our work shows that the peptides are capable of binding Hsp70 as previously described. They are generally 8-26 amino acids in length and correspond to specific regions of many proteins. Through computationally assisted analysis of peptides eluted from Hsp70 we determined variable amino acid sequences, including a 5 amino acid core sequence that Hsp70 favorably binds. We also developed a computer algorithm that predicts Hsp70 binding within proteins. This work helps to define what peptides are bound by Hsp70 in vivo and suggests that Hsp70 facilitates peptide selection by aiding a funneling mechanism that is flexible but allows only a limited number of peptides to be processed.