Obesity and triple‐negative‐breast‐cancer: Is apelin a new key target?

Abstract Epidemiological studies have shown that obese subjects have an increased risk of developing triple‐negative breast cancer (TNBC) and an overall reduced survival. However, the relation between obesity and TNBC remains difficult to understand. We hypothesize that apelin, an adipokine whose levels are increased in obesity, could be a major factor contributing to both tumour growth and metastatization in TNBC obese patients. We observed that development of obesity under high‐fat diet in TNBC tumour‐bearing mice significantly increased tumour growth. By showing no effect of high‐fat diet in obesity‐resistant mice, we demonstrated the necessity to develop obesity‐related disorders to increase tumour growth. Apelin mRNA expression was also increased in the subcutaneous adipose tissue and tumours of obese mice. We further highlighted that the reproduction of obesity‐related levels of apelin in lean mice led to an increased TNBC growth and brain metastases formation. Finally, injections of the apelinergic antagonist F13A to obese mice significantly reduced TNBC growth, suggesting that apelinergic system interference could be an interesting therapeutic strategy in the context of obesity and TNBC.


| INTRODUC TI ON
Breast cancer (BC) is the most prevalent type of cancer and the primary cause of cancer-related death among women with an estimated 2.1 million diagnosed cases and 627.000 deaths in 2018. 1 This highly heterogeneous disease comprises multiple subsets of BC characterized by different expression patterns of key receptors and protumoral proteins. 2,3 Therefore, the different classes of BC vary in terms of prognosis, with the most severe form being the triple-negative breast cancer (TNBC) that accounts for 10%-20% of all BC. TNBC is characterized by the lack of oestrogen receptor (ER) and progesterone receptor (PR) expression as well as the absence of human epidermal growth factor receptor 2 (HER-2) up-regulation, making them incompatible with current targeted therapies. [4][5][6] Hence, TNBC patients have higher recurrence rate, increased susceptibility to form brain or lung metastases and have a decreased overall survival. 7,8 Several risk factors for BC have been identified and include family history of breast cancer, 9 ethnicity, 10,11 oestrogen exposure 12,13 and contraceptive use. 14 Moreover, it has been recently stated that obesity is another factor increasing the risk of developing BC. [15][16][17] This relation is most described for obese post-menopausal women suffering from ER-positive BC as obesity increases aromatase activity in the adipose tissue after the menopause, leading to local oestrogen production. 18 High concentration of oestrogen can either induce DNA damages that increase the risk of developing cancer, 19,20 or promote the proliferation of existing ER-positive BC cells. Regarding the relation between obesity and TNBC, the situation is more complex and still poorly understood. Several epidemiological studies have shown that obese subjects have an increased risk of developing TNBC, [21][22][23] larger tumours 24 and shorter disease-free and overall survival. 25 As global obesity rates are continuously increasing and that TNBC cannot benefit from current targeted therapies, there is a need to better understand the implications of obesity on TNBC development and progression. Several obesity-related disorders have already been studied for their potential role in cancer progression such as hyperinsulinaemia, high circulating levels of IGF-1, inflammation or alteration of adipokine secretion. 17,26 Among the latter, the obesity-related increased levels of leptin have been shown to contribute to tumour progression by stimulating cancer cell proliferation, survival and invasion. [27][28][29] However, owing to leptin physiological functions, the use of leptin receptor antagonist LPrA2 in vivo increased significantly the bodyweight of obese mice. 30 Apelin is another adipokine whose levels are increased with obesity. 31 This adipokine is first expressed as a precursor form of 77 amino acids called preproapelin. 32 Upon sequential cleavages, this precursor form will generate different mature isoforms as apelin-36, apelin-17, apelin-13 or a pyroglutaminated form of apelin 13 (pyr apelin-13) that is more stable and therefore the major circulating form of the adipokine. 33 The apelin receptor (Aplnr) is a class A G protein-coupled receptor that is expressed in many tissues. 34 The apelin-Aplrn interaction is involved in several physiological functions as suggested by the wide expression pattern of both ligand and receptor. Apelin functions comprise blood vessels formation, energy metabolism, fluid homeostasis and blood pressure regulation. [35][36][37] Recently, several correlation studies [38][39][40] and one study of artificial apelin overexpression in cancer cells 41 have highlighted a potential implication of apelin in tumour progression. Strikingly, no study has analysed the impact of the obesity-related increased levels of apelin on tumour growth. Given that obesity is considered as a risk factor for developing TNBC and that the mechanisms are poorly understood, we explored the hypothesis that obesity-increased levels of apelin could be a major factor contributing to either tumour growth or metastatization, thereby explaining the poor prognosis in TNBC patients.

| Animal studies
Animal studies were undertaken in accordance with the Belgian law concerning the protection and welfare of animals and were ap-

| Mice
All mice were purchased from Janvier Labs (Saint Berthevin, France) and housed in a controlled environment (

| Treatment of apelinergic antagonist
Apelinergic antagonist F13A was purchased from Bachem. Daily intraperitoneal injection of either F13A (0.5 µg/g) or PBS started on the fifth day of TNBC growth until the end of the experiment.

| TNBC cell lines
The TNBC 4T1 cell line was acquired from the American Type Culture Collection (ATTC); these cells are derived from Balb/c mice.

| Cell proliferation
Cell proliferation was assayed with a 5-bromo-2′-deoxyuridine (BrdU)-ELISA-based kit (Roche, Basel, Switzerland) following the provider's instructions. Cells were seeded in complete medium for 24 hours prior to medium replacement by a medium depleted of FBS for 24 hours.  For E0771 TNBC tumours, we considered as successfully grown TNBC any tumour above 100 mm 3 at the end of experiment.

| Tissue sampling
At the end of TNBC growth, mice were anaesthetized with isoflurane, and blood was sampled by cardiac puncture. After exsanguination, mice were killed by cervical dislocation. Subcutaneous adipose tissue is immediately immersed in liquid nitrogen. TNBC tumours were collected, any necrotic area was removed, and tumours were cut into halves: one half was stored in liquid nitrogen for mRNA and protein analysis, and the other was prepared for histological analysis. Frozen tumour pieces for mRNA and protein analysis were ground with a pestle in liquid nitrogen to obtain homogeneous tissue powder.

| RNA preparation and real-time qPCR analysis
Total RNA was prepared from tissues using TriPure Isolation Reagent

| Immunohistochemistry
Tumours were fixed in 4% paraformaldehyde for 24 hours at room temperature before processing for paraffin embedding. For each tumour, three 5-µm sections spaced 500 µm apart were submitted to antigen retrieval using citrate buffer. Sections were then incubated in BSA 5% in TBS/Triton 0.05% to block non-specific binding, then overnight at 4°C with primary antibodies for Pecam1 (Cell Signaling Technology).

| TNBC metastases formation assay
A suspension of 2 x 10 5 4T1 Luc-GFP cells in HBSS was injected into the tail vein of Balb/c nude mice under isoflurane anaesthesia.
Bodyweight evolution was followed twice a week. Mice were killed by cervical dislocation after 12 days. Ten minutes before killing, 0.15 mg/g of K + d-luciferin (Perkin Elmer) was injected intraperitoneally. During killing, lungs were inflated with 1 mL of a 15 mg/mL solution of K + dluciferin. Lungs and brain were collected and bathed separately in the K + d-luciferin solution for 5 minutes. Bioluminescent signals were acquired during 10 minutes with a Xenogen IVIS 50 bioluminescence imaging system (Perking Elmer), and signal quantification was performed with Living Image software (Perkin Elmer).

| Statistical analysis
Unpaired t test, one-way ANOVA followed by Turkey's multiple comparison test, two-way ANOVA followed by Bonferroni's multiple comparison test and Fisher's exact test were performed via GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA), with P ≤ .05 considered significant. Results are represented as mean + SEM.

| Association between obesity and TNBC growth in vivo
To investigate the potential role played by apelin on tumour growth, we combined different models and pharmacological approaches.
First, we tested the association between obesity and TNBC growth in vivo. Obesity was induced by feeding C57BL/6 mice with a highfat diet (HFD) for 5 weeks ( Figure 1A). Compared with mice receiving a normal diet (ND), mice fed a HFD had a significant increase of bodyweight gain and fat mass accumulation (by about 200%) after 5 weeks ( Figure 1B,C). Next, we injected E0771 TNBC cells derived from C57BL/6 mice into the mammary fat pad and monitored tumour growth. We observed that development of obesity significantly increased TNBC growth, leading to tumours having twice the volumes of the ND group after 17 days ( Figure 1D). In order to discriminate the impact of obesity-related disorders on TNBC growth from the impact of the HFD itself, we tested the growth of 4T1 TNBC cells derived from Balb/cJRj mice, which are described as an obesity-resistant model upon HFD feeding ( Figure 1E). After 5 weeks of either ND or HFD, Balb/cJRj mice showed no difference of bodyweight gain or fat mass accumulation ( Figure 1F,G), confirming resistance to obesity development. We followed 4T1 TNBC growth after orthotopic injection in Balb/cJRj mice and found that HFD alone had no impact on TNBC growth ( Figure 1H), thereby showing the necessity to develop obesity and increase fat depots to favour tumour growth. To further explore whether apelin could be associated with these phenotypes, we measured apelin mRNA expression. We found that besides an increased TNBC growth, apelin mRNA expression increased by 120% in the subcutaneous adipose tissue of obese mice ( Figure 1I).
Interestingly, TNBC that is growing in obese mice also displays increased tumoral apelin expression by approximately 80% compared with TNBC harvested from mice fed a ND ( Figure 1I). By contrast, the obesity-resistant mice showed no difference in apelin expression in the adipose tissue and had a tendency of only ±35% increase tumoral apelin ( Figure 1J). Moreover, increased apelin expression in E0771 TNBC correlates with bigger TNBC volumes ( Figure 1K), suggesting that increased apelin expression might be one of the mechanisms involved in the increase TNBC growth of obese mice.

| Obesity relevant infusion of apelin favours TNBC growth
To further investigate the potential implication of apelin in the association between obesity and TNBC growth, we reproduced the increase of apelin observed during obesity by chronically administering apelin using osmotic mini pumps. In order to discriminate the influence of apelin on tumour growth from obesity-related protumoral mechanisms, we performed this experiment in ND-fed obesity-resistant Balb/cJRj mice injected with 4T1 TNBC (Figure 2A). Mice infused with apelin for 17 days had no difference in bodyweight gain compared with PBS-infused mice ( Figure 2B). Interestingly, in two independent experiments, the infusion of apelin facilitated 4T1 TNBC growth in lean mice, leading to significantly bigger tumour sizes ( Figure 2C) than in vehicle-treated animals. In order to understand the mechanisms F I G U R E 1 Obesity development favours TNBC growth. A, C57BL6 obesity sensitive model. B, C, Bodyweight gain (B) and total fat mass (C) in C57BL6 models after 5 wk of diet. D, E0771 TNBC growth in ND/HFD-fed C57BL6 mice. E, Balb/cJRj obesity-resistant model. F, G, Bodyweight gain (F) and total fat mass (G) in Balb/CJRj mice after 5 wks of diet. H, 4T1 TNBC growth in ND/HFD-fed Balb/cJRj mice. I, Expression of apelin in subcutaneous adipose tissue (SAT) of C57BL6 mice and E0771 TNBC tumours. J, Expression of apelin in SAT of Balb/ cJRj mice and 4T1 TNBC tumours. K, Correlation between E0771 tumour apelin expression and E0771 tumour volume. Data are presented as mean ± SEM. Number of mice per group for (A-D, I tumour, K): ND: 9, HFD: 5-6, for (E-H, J tumour): ND: 5-6, HFD: 5-6. Number of subcutaneous adipose tissue per group for I: ND: 8, HFD: 3, for J: ND: 6, HFD: 5. Data were analysed using Student's t test for B, C; F, G; I-L. Data were analysed using two-way ANOVA followed by Bonferroni post hoc test for D, H. Data were analysed using Pearson's correlation coefficient for K. *P < 0.05; **P < 0.01; ***P < 0.001 involved in the increased 4T1 TNBC growth upon apelin infusion, we tested a potential effect of apelin treatments on in vitro proliferation of 4T1 TNBC cells. Western blot analysis revealed low basal expression of Aplnr in 4T1 TNBC cells ( Figure 2D). Despite this, treatment of 4T1 cells with apelin significantly increased mRNA expression of the proliferation marker Mki67 that has been previously correlated with poor prognosis ( 44 ; Figure 2E). However, the increased Mki67 expression did not favour proliferation of 4T1 cells as such in our experimental conditions ( Figure 2F). This suggests that Aplnr expression in 4T1 TNBC cells might be too low to directly affect cell proliferation. Ex vivo analysis of infused tumours revealed that apelin infusion significantly increased tumour apelin mRNA expression ( Figure 2G). However, apelin infusion had a limited impact on the expression of different key genes involved in cancer cells proliferation, survival and energy metabolism ( Figure 2G). These results further supported the hypothesis of a lack of direct Aplnr activation in 4T1 TNBC cells. Therefore, we hypothesized that apelin protumoral effects might come from a modulation of the tumour microenvironment. Hence, we performed a tyrosine kinase activity assay (PamGene) on tumours samples to analyse the signalling pathways that were impacted by apelin. Globally, apelin had a subtle but clear impact on tyrosine phosphorylation profile ( Figure 2H,I). We found that apelin infusion increases tyrosine phosphorylation of the key angiogenic marker vascular endothelial growth factor receptor 1 (VEGFR1) (Figure 2I), thereby suggesting an increased tumour neoangiogenesis. This hypothesis was further confirmed by the significant increase of angiopoietin 1 and Pecam1 mRNA expression ( Figure 2J) and by histological analysis of Pecam1 staining in apelin-infused tumours ( Figure 2K,L). Data were analysed using one-way ANOVA followed by Bonferroni post hoc test for (B, C, E). Data for (D) were analysed with two-way ANOVA followed by Bonferroni post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001

| Obesity relevant infusion of apelin favours TNBC brain metastases
We next tested whether obesity-related levels of apelin could also influence TNBC metastasization in addition to an increase in primary TNBC tumour growth. In order to detect metastases efficiently, we injected 4T1 TNBC cells harbouring a fluorescent Luc-GFP transgene into the tail veins of ND-fed Balb/c nude mice with or without infusion of apelin. Breast cancer cells are known to form metastases in diverse distant tissue like the lungs and brains. Indeed, after a period of 12 days of metastasization, the lungs of both PBS and apelin-infused mice were metastatic ( Figure 3A). However, mice infused with apelin tended to have more highly metastatic areas ( Figure 3B). Interestingly, 70% of apelin-infused mice had metastases in brain whereas most of the brains of the PBS-infused group were metastases-free ( Figure 3C,D). This set of data highlights the significant prometastatic effect of apelin.

| Apelin as a therapeutic target for TNBC growth in obese mice
Having established that the administration of apelin to lean mice, thereby mimicking the obesity situation, led to an increased TNBC growth and brain metastases formation, we next wondered whether the apelinergic system could be a realistic therapeutic target for TNBC in obese conditions. To test this, we administrated a commonly used apelinergic antagonist, F13A, during TNBC growth in lean or obese C57BL6 mice ( Figure 4A). Similar to the first experiment, we fed C57BL6 mice either a ND or HFD for 5 weeks in order to trigger development of obesity ( Figure 4B). After 5 weeks of diet, we injected E0771 TNBC cells in the mammary fat pad of lean and obese mice. After 5 days of tumour growth, we started daily intraperitoneal injections of either PBS or F13A. We conducted two independent experiments with these parameters. First, we observed that F13A injections did not affect the obese phenotype as the two HFD-fed groups had the same bodyweight at the end of the experiment ( Figure 4C). Second, we confirmed that obesity development with HFD favoured TNBC growth compared with the ND group ( Figure 4D). Interestingly, we found that F13A injections in obese mice significantly reduced TNBC growth compared with PBSinjected obese mice ( Figure 4D) leading to significantly lower final tumour volume ( Figure 4E). These results suggest that interfering with the apelinergic system in obesity could be an interesting therapeutic strategy ( Figure 4F).

| D ISCUSS I ON
During the past few years, several studies have shown a correlation between tumour growth and plasma levels or tumoral expression levels of apelin. 38,39,40 . Moreover, the artificial modulation of apelin expression in cancer cell lines by knockout ( 45 ) or induced overexpression 41 has suggested a direct implication of apelin on cancer progression. Although informative, these experiments remain descriptive and artificial because they are focusing only on apelin expression in cancer cells. Hence, the implication of the host circulating apelin on tumour progression remains largely unstudied.
The main condition known to modulate circulating apelin levels is the development of obesity that increases apelin expression in adipose tissue. 31 In this study, we tested the impact of obesity and HFD alone on TNBC growth. Only the development of obesity led to an increased tumour growth, suggesting that a diet rich in lipids is not sufficient to promote TNBC progression and that obesity conditions are required. One contributing factor to tumour growth in obesity could be the modulation of apelin expression as it is increased in obese mice showing large TNBC tumours but unaffected in HFD-fed mice showing normal TNBC tumours. We confirmed a causal link between increased apelin expression in obese mice and larger TNBC tumours with two different approaches. First, by accurately mimicking apelin levels as observed in obesity condition, we showed that apelin is sufficient to promote TNBC growth in lean mice and to increase tumour angiogenesis. This effect is independent of obesity development as apelin infusion did not affect the bodyweight. Next, we showed that antagonizing the Aplnr in obese mice limits TNBC growth. This reduction of TNBC growth occurs without the necessity of targeting obesity confirming a key implication of apelin protumoral effects in the context of obesity.
It has been estimated that roughly 70% of breast cancer deaths are caused by metastases ( 46 ). In our experiments, the reproduction of obesity-related levels of apelin in lean mice led to a marked increase of brain TNBC metastase production. A trend to form more highly metastatic area in the lungs was also observed. As TNBC cells in mice form brain metastases only after lung metastases, it might suggest that the tumoral spread was already too advanced in our experimental design to correctly assess secondary tumour formation in the lungs. Interestingly, we did not observe brain metastases in the control group, indicating that apelin facilitated the seeding of circulating tumours cells into the brain. This would imply that obese patients suffering from TNBC have a potential additional risk of being more prone to develop secondary tumours in the brain. A correlation between high circulating apelin and tumour grade or overall bad prognosis has previously been described. 38 On the other hand, another study suggested that tumour apelin expression was more accurately correlated with increased tumour progression. 39 In our work, we showed that in obese mice or in mice in which apelin was infused to attain obesity-like levels, TNBC growth was facilitated, but also that tumour apelin expression was increased.
Different mechanisms are possibly involved. First, high circulating apelin may not affect tumour growth directly but rather increase tumour apelin expression. In this scenario, tumour apelin would be the main driver of TNBC progression and circulating apelin would only have an indirect effect on TNBC growth by increasing tumour apelin expression. Second, high circulating apelin could promote tumour growth but also increase tumour apelin expression directly, generating a positive feedback loop that drives TNBC progression. In this case, both the circulating and the subsequent local apelin expression would favour TNBC growth in a direct manner. Third, high circulating apelin could promote tumour growth without affecting tumour apelin expression. The increased tumour apelin expression could be a characteristic of highly proliferative cancer cells in more advanced tumours rather than a direct consequence of high circulating apelin.
In this scenario, high circulating apelin levels promote TNBC growth and the subsequent fast-growing tumours would increase their apelin expression in order to promote angiogenesis and support their growth. A fourth mechanism, quite similar to the previous, could be that as high circulating apelin promote tumour neoangiogenesis, the apelin expression measured in the tumour could originate from proliferating endothelial cells rather than cancer cells.
In this study, we deliberately dissociated increased apelin levels caused by obesity from additional disorders associated with obesity and found that the increased TNBC growth is slightly smaller than the effect observed in obese mice. An explanation probably lies in the fact that obese mice have obesity-linked disorders that can further promote tumour growth, such as increase in hyperinsulinaemia or high leptin expression. 17,26 This hypothesis is supported by the fact that the apelinergic antagonist F13A in obese mice did not completely abolish the impact of obesity on TNBC growth. Another possibility could be that the apelin infusion used in this study cannot account for all effects on the apelinergic system in obesity conditions. Indeed, a second ligand of Aplnr has been recently discovered, a peptide called ELABELA (Toddler/Apela) ( 50 ). To our knowledge, there is not measurement of ELABELA levels in obese subjects or mice. Taking into account, the obesity-related levels of ELABELA and apelin for an infusion in lean mice could reproduce even more accurately the activation of the apelinergic system in obesity. Such an infusion could have an even greater impact on TNBC growth than the already significant increase described in this study.
Finally, apelin is also an adipokine that has recently been proposed as a key target to treat type 2 diabetes 37 or sarcopenia ( 51 ) in human beings. Indeed, apelin has gained attention for its ability to increase insulin sensitivity and its beneficial role on glucose homeostasis. In line with these effects, apelin is now considered as a potential therapeutic agent for diabetic patients. However, many patients suffering from type 2 diabetes have a BMI corresponding to obesity. Hence, extreme caution would be required to select patients who could benefit from apelin as a therapeutic agent because our results strongly suggest that either obesity-related levels of apelin as well as apelin infusion is able to increase TNBC growth and seeding to peripheric tissues, like the brain. Therefore, the balance risk-benefit effects of using apelin as a therapeutic target warrant careful assessment.
In this study, we showed for the first time that obesity-increased apelin expression favours breast cancer progression. We suggest that apelin is a critical driver of obesity-induced TNBC progression.
We found that blocking the Aplnr during obesity reduces tumour growth likely by affecting tumour microenvironment thereby highlighting that apelinergic system interference could be an interesting therapeutic strategy in the context of obesity and TNBC.

PD Cani is Senior Research Associate and BF Jordan is Research
Director, of the Belgian National Fund for Scientific Research (FNRS). was supported by the European Regional Development Fund and the Walloon Region, Belgium. F Gourgue is a FRIA grant holder. We thank Prof. Castan-Laurell and Prof. Knauf for their scientific discussions. We also thank Michele de Beukelaer from the 2IP imaging platform of UCLouvain and Rose-Marie Goebbels from the medical oncology unit at Saint-Luc Hospital for technical assistance.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.