Background
Lifestyle factors such as physical inactivity, obesity and hypercaloric nutrition have been shown to substantially impact breast cancer (BC) risk [
1‐
7]. To what extent modifiable lifestyle factors affect cancer risk in women genetically at high risk of developing BC, such as carriers of a germline mutation in the genes
BRCA1 and
BRCA2 (g
BRCA1/2) has been far less widely researched. Of note is that the penetrance of certain inherited
BRCA1 mutations has been increasing 24% to 67% as generations proceed [
8‐
10]. Amongst other reasons this increase might be attributed to an alteration of the social and lifestyle environment [
8,
11].
In light of this the LIBRE-1 trial, a prospective lifestyle-intervention randomized controlled trial, was initiated to investigate whether a structured 1-year intervention program ameliorates cardiopulmonary fitness, body mass index (BMI) and the nutritional pattern in female
gBRCA1/2 mutation carriers [
12‐
14]. Additionally, the identification of lifestyle-related biomarkers for molecular mechanisms that are involved in tumorigenesis in g
BRCA1/2 mutation carriers is important and therefore certain promising biomarkers were measured in the LIBRE-1 trial. Two of these, which are the focus of this work: neurotensin (NT) and enkephalin (ENK) have been identified as markers linking cardiovascular, cardiometabolic and cancer risk.
Neurotensin/pro-neurotensin (pro-NT)
Neurotensin (NT) is a 13-amino acid peptide, which is mainly found in the central nervous system and in the small intestine. It behaves as a neuromodulator in the brain by regulating the anorectic effect and is an important factor in nutrient metabolism, particularly through lipid ingestion and fat storage [
15‐
19]. Clinical studies indicate that increased levels of peripheral NT occur in obesity and are directly linked to features of insulin resistance in humans [
17,
19‐
21]. Within the Framingham Heart Study, NT was associated with incident major cardiovascular events, independently of traditional cardiovascular risk factors [
22]. Melander et al. conducted a large cohort study, which confirmed a contribution of NT to obesity-derived diseases such as cardiovascular and in particular BC [
21]. Recently, Li and colleagues were able to demonstrate a protection from obesity in NT-deficient mice fed with a high-fat diet [
17].
Enkephalin/pro-enkephalin (pro-ENK)
As part of the endogenous opioid peptide system, Methionine-ENK (MetENK), is deemed to be of considerable importance in tumorigenesis by averting tumor cell proliferation [
23‐
27]. Within the framework of two Swedish cohort studies, fasting plasma of 3498 non-diseased women was examined to quantify pro-ENK, the stable precursor of ENK, and predict its impact on BC risk [
28]. After a median follow-up of 14.7 years, women with pro-ENK values in the lowest quartile were found to be three to five times more susceptible to BC than women with pro-ENK values in the highest quartile [
28,
29]. Intriguingly, more recent findings indicate a favourable effect of MetENK concerning obesity and its related diseases [
30]. Cardiovascular function has also been found to be affected by this biomarker and seems to be modulated by physical activity [
31‐
35].
Within an experimental study, the effect of weekly moderate-intensity exercise training on the activation of the cardiac opioid system of a diet-induced obesity model in rats was compared [
36]. While sedentary animals fed with a high fat diet presented lower cardiac pro-ENK messenger ribonucleic acid (mRNA) levels, a level-dependent activation of the cardiac opioid system was seen in the active group [
36]. This could be relevant in the context of our study, as there is emerging evidence on an association between
BRCA1/2 deficiency and various cardiovascular disorders such as atherosclerosis and ischemic heart disease [
37]. Certainly, premature menopause as a consequence of prophylactic bilateral salpingo-oophorectomy (PBSO) and /or potential cardiotoxic effects of cancer treatment (chemotherapy/radiotherapy in case of breast cancer) may have a decisive impact. Recently, a causal part for the genes
BRCA1 and
BRCA2 in cardiovascular diseases was described [
37]. The role of these two genes as gatekeepers in cardiac function and structure can increase susceptibility to cardiac damage when their function is compromised [
38].
We, therefore, wanted to answer two specific questions: i) whether gBRCA1/2 mutation carriers differ from women of the general population, that are not indicative for hereditary breast and ovarian cancer regarding plasma concentration of NT and ENK, ii) what is the effect of a 1-year lifestyle-intervention on NT and ENK plasma levels in gBRCA1/2 mutation carriers.
Discussion
We aimed to identify lifestyle-related biomarkers for molecular mechanisms that are involved in tumorigenesis in g
BRCA1/2 mutation carriers. NT and ENK are known to be linked with cardiovascular, cardiometabolic and cancer risk, and emerging evidence suggests direct associations with lifestyle factors [
17,
19‐
21,
30,
33,
45]. We therefore aimed to investigate if NT and ENK plasma levels might be altered by a structured lifestyle-intervention program consisting of endurance training alongside the MedD in the context of g
BRCA1/2 mutation carriers, within the lifestyle-intervention trial LIBRE-1 [
12‐
14].
Initially, we wanted to know whether g
BRCA1/2 mutation carriers inherently differ from women of the general population regarding NT and ENK fasting plasma concentrations. Therefore, plasma levels of the stable precursors of NT and ENK, pro-NT and pro-ENK were analyzed in the LIBRE-1-total, as well as the OMA-reference group at SE. The median pro-NT fasting plasma concentration was higher in LIBRE-1-total participants, reaching statistical significance in the comparison of LIBRE-cancer and OMA-cancer subgroups. Higher pro-NT levels would be expected with overweight or obesity, however the median BMI was comparable throughout our studied subgroups [
17,
19,
20,
40]. The OMA-cancer subgroup was in median older than the others, however we found age not to be significantly associated with pro-NT within uni- and multivariate analyses. A large control collective from the Malmö cancer and diet (MDC) study of 1929 healthy subjects did not show a significant correlation between age and pro-NT, which points to an age-independent pro-NT activity [
16]. This might point to an intrinsic disposition in the LIBRE-cancer subgroup given that pro-NT is in turn associated with elevated BC risk [
21]. Discrepant results were obtained for pro-ENK. Participants of the LIBRE-non-cancer subgroup had lower median pro-ENK plasma concentrations compared to their OMA-non-cancer peers, which may indicate an adverse risk constellation for g
BRCA1/2 mutation carriers. Yet, compared with this, the subgroup LIBRE-cancer presented more favorable pro-ENK levels than participants of OMA-cancer, a difference that was marginally statistically significant (63 pmol/L vs. 69 pmol/L,
p = 0.045). Univariate analysis showed a significant age-dependency, yet this disappeared in the multivariate analysis. Intriguingly, recent findings indicate a potential age–dependent increase of pro-ENK [
46]. In contrast to these, the LIBRE-cancer subgroup had higher pro-ENK levels compared to the OMA-cancer subgroup, who were in median 16 years older. This might be explained by pre-existing stronger health awareness of the LIBRE-cancer subgroup, who for instance had a better cardiopulmonary fitness level compared to the LIBRE-non-cancer subgroup at SE, reflected by higher initial levels of VO2peak (mL/min/kg). A comparison with the cardiopulmonary fitness of the OMA-cancer subgroup is however not possible as CPET was not carried out in the OMA-reference study. Another influencing factor might be an endogenous compensation mechanism subsequent to cardiotoxic cancer therapy, especially when considering a possibly higher susceptibility to cardiac muscle disorder in g
BRCA1/2 mutation carriers [
34]. However, further research is required to answer this question.
We then investigated the effect of a 1-year lifestyle-intervention on pro-NT and pro-ENK plasma levels in the g
BRCA1/2 mutation carriers within the lifestyle-intervention feasibility trial LIBRE-1. We saw a statistically significant inverse correlation between pro-NT plasma levels and VO2peak changes over the 1-year intervention (− 0.435; CI − 0.653 to − 0.151;
p = 0.004), which also remained consistent in univariate and multivariate regression analyses. Based on previous reports from the LIBRE-1 trial [
11,
12], which point out the over-motivation of the LIBRE-1 participants particularly in the CG, we consider VO2peak derived from CPET to be the most objective parameter for cardiopulmonary fitness.
Pro-ENK plasma concentration was positively correlated to increasing VO2peak at the study time point SE (0.323; CI 0.061–0.544;
p = 0.017), which is in agreement with corresponding studies that postulated a relationship between physical training and plasma concentration of pro-ENK [
47,
48]. Yet, this was not reproducible in follow-ups at V1 and V2, nor for the change through the 1-year lifestyle-intervention. Particularly, participants of LIBRE-cancer, who had the highest pro-ENK levels at SE and made up the majority of our study population, barely improved their pro-ENK plasma levels. A possible explanation might be that the impact of additional exercise sessions on proportionally elevated pro-ENK levels in LIBRE-cancer are less pronounced compared to LIBRE-non-cancer, who were physically less active at SE, reflected by lower proENK concentration. Existing evidence indicates a trainable response regarding pro-ENK concentrations [
47,
48]. Kjær et al. described highest pro-ENK concentrations at 54% of the VO2peak in trained endurance athletes, however with further increasing exercise intensities the pro-ENK level decreased to resting concentrations [
48]. Compared to this, the response patterns of untrained probands resulted in an increase in pro-ENK plasma concentrations with increasing exercise intensities [
48]. The precise mechanism and explanation remains unknown, but this may be the emergence of a type of “adrenal exhaustion” due to a potential over-stimulation of the adrenal gland, from which pro-ENK is released [
47].
We also built statistical models (linear regression) for pro-NT and pro-ENK plasma concentrations taking into consideration physical, clinical as well as nutritional parameters. Pro-NT showed a consistent, yet non-significant inverse association with the ratio of Omega 6/Omega 3 FA (an objective assessment of dietary patterns) in uni-and multivariate analysis (Estimate: − 32.7 and − 37.9,
p = 0.097 and 0.080, respectively). These results are in agreement with previous findings in the literature, where NT has been identified as an important factor in nutrient metabolism particularly through lipid ingestion and fat storage [
15,
16,
18‐
20]. Increased levels of peripheral NT occur in obesity and are directly linked to features of insulin resistance in humans [
17,
19‐
21]. Li et al. were recently able to demonstrate a protection from obesity in NT-deficient mice fed with a high-fat diet [
17], our findings provide additional support that dietary intervention may contribute to a reduction in pro-NT plasma levels.
Limitations
Our study needs to be interpreted in light of its limitations. First, the study population was small, resulting in a restricted ability to draw definitive conclusions. Second, we had only one measurement of the biomarkers for the OMA-reference population. However, as a feasibility trial, LIBRE-1 aimed at generating hypotheses. Further investigation will be conducted in the framework of the larger LIBRE-2 trial with a sample size of more than 600 gBRCA1/2 mutation carriers.
Conclusion
Our study gives first indications that pro-NT, which is involved in tumorigenesis, cardiovascular- and obesity-related diseases, is inversely associated with improving cardiopulmonary fitness and nutritional parameters in gBRCA1/2 mutation carriers. Yet, the situation was discrepant for pro-ENK, which showed a positive correlation with VO2peak at SE, however, further training did not lead to a corresponding increase in pro-ENK levels, possibly due to a type of “adrenal exhaustion”.
gBRCA1/2 mutation carriers seem to have an adverse constellation of pro-NT and to a lesser extent pro-ENK, possibly due to intrinsic disposition.
Our findings support our supposition that improvements of lifestyle behavior including physical activity and dietary pattern, might constitute a strategy to reduce cancer risks, as well as mitigate cardiovascular risk in these mutation carriers.
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