While LET was shown to reduce proliferation of MCF-7 cells in the micromolar () and PAL even in the nanomolar range (), our metabolomics results suggest no major effect of the individual agents on the proliferation of cancer cells. Typically, the stimulation of pathways related to glucose uptake and glycolysis, as well as lipid-, protein-, and nucleotide synthesis, is an indication of cellular proliferation (). However, given the very low dose of LET (10 nM) and the short incubation time of 48 hr compared with other studies (; our unpublished data), this was not unexpected. In the single treatments, no effect on amino acid metabolism and only a minor response on nucleotide metabolism were observed for both drugs. However, PAL alone reduced several intermediates of the central carbon metabolism (glycerate 3-phosphate, ribose, 6-phosphogluconic acid, lactic acid, malic acid), while for others (glucose 6-phosphate, ribulose 1,5-diphosphate, 5-phosphoribosyl diphosphate) a slight increase was seen in the LET group. Only a few fatty acids were upregulated by LET, with a generally more potent effect of PAL ( Table 1 ).

To gain a deeper understanding of the drugs’ effect on cellular metabolism, breast cancer cells were dosed with individual agents and a combination of PAL + LET ( Figure 1 ). Meta-analyses were used for initial data evaluation to compare the control group, cells dosed with the single agents, and cells receiving the combined dose (). As illustrated in Figure 2 A, the individual drugs have exclusive effects on cellular metabolism with only nine statistically significant common metabolic features changed in both treatments. System-wide, exposure to PAL at a concentration of 200 nM over a period of 48 hr led to a significant alteration of metabolic features (514) after filtering by p value and fold change (see the Star Methods ). Incubation with 10 nM LET resulted in a similar number of dysregulated features (459). These distinct effects of the agents are also reflected by the metabolomics cloud plot ( Figure S1 ) and the pathway cloud plots shown in Figure S2 , which report predicted modified metabolic pathways based on the recently implemented systems biology functionality within XCMS Online (). This tool allows for the rapid prediction of dysregulated pathways without time-consuming metabolite identification. While caution is warranted when interpreting these results, many of the key metabolites involved in the predicted pathways, including several molecules associated with central carbon and nucleotide metabolism, have been manually verified in a second step ( Table 1 ). The drug concentrations were chosen to mimic plasma levels in patients undergoing therapy and based on previous reports (and our unpublished data).

Values are fold changes when comparing dosed groups to control (dosed/control). Unpaired two-tailed t test with 0.05 set as p value for statistical significance. Trend is indicated for the combined treatment with + (1.0- to 1.5-fold), ++ (1.5- to 2.0-fold), +++ (>2.0-fold), − (1.0- to 0.75-fold), −− (0.75- to 0.5-fold), and −−− (<0.5-fold). n.s., not significant.

(B–D) Total ion chromatograms (TIC) of the hydrophilic interaction liquid chromatography- mass spectrometry measurements were automatically corrected for retention time shifts and improved the quality of the mass spectrometric data underlying (A), (C), and (D). The multigroup metabolomics cloud plot shown in (C) and the boxplots (D) highlight specific changes of the cancer cells’ metabolome following drug dosing. Boxplots (D) were extracted from (C) for six selected metabolites (1–6) whose abundances changed significantly in the combined dosing group. Palbociclib, letrozole, and the combination of both were compared with the solvent control (means ± SEM; n = 4; *p < 0.05, **p < 0.01).

(A) Venn diagram of meta-analyses between the individual and the combined dosing. Combined exposure to PAL and LET resulted in approximately twice as many altered metabolic features (1,013) than in the single treatments (PAL 514 and LET 459) after 48 hr incubation (fold change >1.5, p value <0.05).

(B) The untargeted metabolomics workflow was applied to the individual and the combined treatment of cancer drugs to study their synergism at the metabolomics level. In addition, the effect of the two xenoestrogens on the combined drug dosing was investigated. LC-QTOF-MS, liquid chromatography-quadrupole time-of-flight-mass spectrometry.

(A) The selective CDK4/6 inhibitor palbociclib and the aromatase inhibitor letrozole are a first-line treatment for ER-positive and HER-negative advanced breast cancer. The isoflavone genistein and the mycotoxin zearalenone are dietary xenoestrogens exhibiting affinity towards estrogen receptors.

Interestingly and in contrast to the combined effects of PAL and the ER antagonist FULV (our unpublished data), no metabolic signatures of cell death were observed in the study at hand. Most notable, nucleotide metabolism was overall upregulated after 2 days of PAL + LET exposure, while PAL + FULV resulted in some metabolites with dramatically decreased abundances. Thus, FULV and LET appear to have different effects on cellular metabolism when combined with PAL, and it remains to be determined whether the metabolic consequences of these drug treatments are responsible for the improved clinical outcome seen in patients on combination therapies ().

To investigate if the combined PAL + LET treatment affects the mTOR pathway, we set out to determine the phosphorylation state of the mTOR downstream targets p70-S6-kinase and ribosomal protein S6. PAL + LET reduced phosphorylation of the proteins in the two tested ER-positive cell lines MCF-7 and T47D, indicating an inhibitory effect of the combined treatment on activation of S6 kinase by mTOR ( Figure 3 D).

(D) Impact of xenoestrogens and/or combined PAL + LET treatment on phosphorylation state of mTOR downstream targets. Cells were exposed to the indicated compounds for 48 hr. Western blot analysis of phospho-p70-S6-kinase (P-S6K), total S6K, phospho-S6 (P-S6), and total ribosomal protein S6. The images are representative of three independent experiments.

(C) The effect of GEN and ZEN on the PAL + LET treatment was examined in cell proliferation assays. The human breast cancer cell lines MCF-7 and T47D were exposed to indicated drugs and/or xenoestrogens for 72 hr and viable cells were quantified using the resazurin assay. Shown are the percentages of individual samples relative to the control sample (DMSO only), which was set to 100% (means ± SD; n = 4; *p < 0.05, **p < 0.01, ***p < 0.001). ns, not significant.

(B) Non-metric multidimensional scaling visualizes the similarity level of individual samples in a dataset and also suggests the distinct effect of the xenoestrogens on the combined PAL + LET dosing samples.

(A) PCA score plots: the first (PC1) and second (PC2) principal components explain 32% and 20% of the variance, respectively, for the experiment containing GEN, with very similar variances for ZEN (33% and 17%).

The lower abundance of amino acids suggests that the mammalian target of rapamycin (mTOR) pathway, which is a negative target of AMP-activated protein kinase, is attenuated (). When activated by high amino acid levels, this pathway typically promotes anabolic reactions with multiple roles in cell differentiation and a stimulation of glycolysis, which was not apparent in our data. Since basically all amino acids were less abundant in the combined dosing group it appears reasonable to speculate that a master regulator, such as mTOR, is involved in the observed molecular events. mTOR acts as a downstream effector for many oncogenic pathways and deregulation of mTOR signaling is a hallmark of many human cancers ().

The meta-analyses performed on XCMS Online illustrated the overall highly pronounced impact of the combination treatment compared with individual exposures by way of a Venn diagram, together with a metabolomics cloud plot and extracted box-and-whisker plots ( Figure 2 ). The combination of PAL and LET resulted in 1,013 altered metabolic features in the breast cancer cells. Many of these metabolic features, however, may result from the same molecule due to the formation of adducts or in-source fragment ions (). From this dataset more than 100 significantly dysregulated metabolites (fold change >1.5, p < 0.05) were derived and 58 metabolites were putatively identified. Many of those were matched to key molecular building blocks with a prominent role in cellular metabolism. Table 1 reports on the relative changes upon single and combined treatments for major metabolites associated with central carbon, nucleotide, amino acid, and fatty acid metabolism. In line with the meta-analysis ( Figure 2 A), the effects on cellular metabolism following combined treatment are enhanced and draw a distinct picture: on the one hand, all significantly altered amino acids and central carbon metabolites (except ribose-5-phosphate) were clearly less abundant after PAL + LET dosing. On the other hand, fatty acids as well as nucleobases, nucleosides, and nucleotides (except UMP) showed the opposite behavior, with generally higher abundances ( Table 1 and Figure 2 ). This behavior is corroborated by the results of the metabolic pathway prediction ( Figure S2 ). We speculate that the increased levels of nucleic acid precursors might be caused by the PAL-mediated inhibition of CDK4/6 and the resulting cell-cycle arrest. This mode of action would prevent the utilization of precursors in the S phase where high concentrations are required for DNA replication ().

Role of Xenoestrogens during Combined Agent Dosing

Pairwise and multigroup analyses within XCMS Online were utilized to evaluate the effect of the phytoestrogen GEN and the mycoestrogen ZEN on the combined PAL + LET treatment. Principal component analysis (PCA) and non-metric multidimensional scaling of the obtained data allowed for an unbiased assessment and visualization of their impact on a global scale in MCF-7 cells ( Figures 3 A and 3B). Using these non-supervised, multivariate analyses, it became apparent that the investigated xenoestrogens counteracted the metabolic effects of the combined PAL + LET treatment group. This behavior was confirmed for specific metabolites, which seemed to be particularly interesting players in the combined drug action ( Table 1 ). Most of the highly dysregulated features observed in the univariate data were also found in the PCA model. The antagonistic effect of xenoestrogens on the combined drug treatment was also confirmed in cell-proliferation assays using the ER-positive cell lines MCF-7 and T47D. Proliferation of both cell lines was markedly reduced after exposure to PAL + LET, compared with untreated cells. Strikingly, co-administration of PAL + LET with either of the two xenoestrogens GEN or ZEN restored cell proliferation to levels that were comparable with those of untreated cells ( Figure 3 C). This functional assay together with our metabolomics data provides evidence that dietary xenoestrogens have the potential to circumvent the anti-oncogenic effects of the PAL + LET combination therapy.

Goodson et al., 2011 Goodson 3rd, W.H.

Luciani M.G.

Sayeed S.A.

Jaffee I.M.

Moore 2nd, D.H.

Dairkee S.H. Activation of the mTOR pathway by low levels of xenoestrogens in breast epithelial cells from high-risk women. Agathocleous and Harris, 2013 Agathocleous M.

Harris W.A. Metabolism in physiological cell proliferation and differentiation. Figure 4 Relative Abundances of Selected Key Metabolites Identified during Combined PAL + LET Dosing Were Significantly Altered by the Dietary Xenoestrogens Zearalenone and Genistein Show full caption The effect of the two food estrogens (100 nM ZEN or 1 μM GEN) added to the combined treatment was compared with the control (C), the single agents (PAL, P; LET, L), and the combined treatment (n = 4). The impact of the estrogens on the combination is highlighted by the blue shading. The y axis represents relative metabolite abundances (a.u.). PL, PAL + LET; PLZ, zearalenone; PLG, genistein. It has been reported in breast epithelial cells that the xenoestrogen bisphenol A induces activation of the mTOR pathway and leads to marked resistance to the mTOR-inhibitor rapamycin (). To investigate if the observed effects of GEN and ZEN on the combined PAL + LET treatment also affect the mTOR pathway, the phosphorylation state of the mTOR downstream targets p70-S6-kinase and ribosomal protein S6 was determined after xenoestrogen co-exposure as well. Our results show that the inhibitory effect of the combined drug treatment is antagonized by the tested xenoestrogens GEN and ZEN ( Figure 3 D), which is in line with the obtained metabolite data. mTOR activity is negatively regulated by low intracellular amino acid levels (). In MCF-7 cells PAL + LET significantly reduced the abundance of numerous metabolites, notably many amino acids. However, addition of GEN or ZEN to the drug combination counteracted this inhibitory effect of PAL + LET and led to cellular amino acid levels that were comparable with those of untreated cells in many cases ( Figure 4 ).

Sullivan and Vander Heiden, 2017 Sullivan M.R.

Vander Heiden M.G. When cancer needs what's non-essential. Brasili and Filho, 2017 Brasili E.

Filho V.C. Metabolomics of cancer cell cultures to assess the effects of dietary phytochemicals. Hilakivi-Clarke et al., 1999 Hilakivi-Clarke L.

Onojafe I.

Raygada M.

Cho E.

Skaar T.

Russo I.

Clarke R. Prepubertal exposure to zearalenone or genistein reduces mammary tumorigenesis. Adlercreutz, 2002 Adlercreutz H. Phytoestrogens and breast cancer. Blei et al., 2015 Blei T.

Soukup S.T.

Schmalbach K.

Pudenz M.

Möller F.J.

Egert B.

Wörtz N.

Kurrat A.

Müller D.

Vollmer G.

et al. Dose-dependent effects of isoflavone exposure during early lifetime on the rat mammary gland: studies on estrogen sensitivity, isoflavone metabolism, and DNA methylation. Zhang et al., 2017 Zhang F.F.

Haslam D.E.

Terry M.B.

Knight J.A.

Andrulis I.L.

Daly M.B.

Buys S.S.

John E.M. Dietary isoflavone intake and all-cause mortality in breast cancer survivors: the Breast Cancer Family Registry. Nechuta et al. (2012) Nechuta S.J.

Caan B.J.

Chen W.Y.

Lu W.

Chen Z.

Kwan M.L.

Flatt S.W.

Zheng Y.

Zheng W.

Pierce J.P.

et al. Soy food intake after diagnosis of breast cancer and survival: an in-depth analysis of combined evidence from cohort studies of US and Chinese women. Breast cancer is a multifaceted disease with dietary factors and hormones being key elements in its development. Diet has long been acknowledged for its role in contributing to cancer risk; however, the molecular foundations of this phenomenon remain poorly understood (). Bioactive food constituents and contaminants have been associated with both beneficial and potentially adverse effects on cancer susceptibility, progression, and outcome in general. In particular phytochemicals such as polyphenols have been used for the treatment of cancer; however, despite recent advances empowered by new metabolomic technology, their effect on cancer metabolism has not yet been fully clarified (). This is especially true for the impact of specific compounds during chemotherapy. There is considerable epidemiological evidence from Asian countries that high-soy diets (i.e., rich in isoflavones such as GEN) lower the incidence of breast cancer (). Typically, Asian women consume substantially more soy food than women in Western-style populations, resulting in high phytoestrogen early-life exposures. Thus, it was suggested that the time point in life when exposure begins seems to be a critical parameter for a potential protective effect (). A recent multiethnic US study associated higher dietary isoflavone intake with lower mortality in women with HR-negative tumors and those not treated with hormone therapy, while no negative impact on all-cause mortality was found for patients receiving (self-reported) hormone therapy ().described an association between higher consumption of soy-based foodstuff following a breast cancer diagnosis and improved treatment outcomes and reduced recurrence rates in more than 9,500 survivors from China and the United States. Interestingly, isoflavone intake was inversely associated with recurrence among both groups despite the large differences in dietary soy consumption by country.

Rice and Whitehead, 2006 Rice S.

Whitehead S.A. Phytoestrogens and breast cancer –promoters or protectors?. Rice and Whitehead, 2006 Rice S.

Whitehead S.A. Phytoestrogens and breast cancer –promoters or protectors?. Rice and Whitehead, 2006 Rice S.

Whitehead S.A. Phytoestrogens and breast cancer –promoters or protectors?. Takemura et al., 2007 Takemura H.

Shim J.-Y.

Sayama K.

Tsubura A.

Zhu B.T.

Shimoi K. Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro. Contradictory to these epidemiological surveys, GEN as well as other phytoestrogens stimulated the growth of ER+ cell lines in vitro (). This paradox might be caused by the stronger affinity of phytoestrogens toward the ERβ than the ERα form of the ER (). The xenoestrogens GEN and ZEN triggered a similar response on a significant share of metabolites in the treated cells in this study ( Figure S3 ) even though GEN has a higher affinity toward the ERβ than the ERα form of the receptor () and ZEN interacts with both in a comparable manner (). Activation of ERα is associated with growth promotion in breast tumors, while the role of ERβ is less well understood. In general, affinity of phytoestrogens to the ERs is much lower than that of the principal endogenous estrogen 17β-estradiol.

Jäger et al. (2011) Jäger W.

Gruber A.

Giessrigl B.

Krupitza G.

Szekeres T.

Sonntag D. Metabolomic analysis of resveratrol-induced effects in the human breast cancer cell lines MCF-7 and MDA-MB-231. Ju et al. (2008) Ju Y.H.

Doerge D.R.

Woodling K.A.

Hartman J.A.

Kwak J.

Helferich W.G. Dietary genistein negates the inhibitory effect of letrozole on the growth of aromatase-expressing estrogen-dependent human breast cancer cells (MCF-7Ca) in vivo. Ju et al., 2008 Ju Y.H.

Doerge D.R.

Woodling K.A.

Hartman J.A.

Kwak J.

Helferich W.G. Dietary genistein negates the inhibitory effect of letrozole on the growth of aromatase-expressing estrogen-dependent human breast cancer cells (MCF-7Ca) in vivo. Soukup et al., 2016 Soukup S.T.

Helppi J.

Müller D.R.

Zierau O.

Watzl B.

Vollmer G.

Diel P.

Bub A.

Kulling S.E. Phase II metabolism of the soy isoflavones genistein and daidzein in humans, rats and mice: a cross-species and sex comparison. Verkasalo et al., 2007 Verkasalo P.K.

Appleby P.N.

Allen N.E.

Davey G.

Adlercreutz H.

Key T.J. Soya intake and plasma concentrations of daidzein and genistein: validity of dietary assessment among eighty British women (Oxford arm of the European Prospective Investigation into Cancer and Nutrition). Warth et al., 2013 Warth B.

Sulyok M.

Berthiller F.

Schuhmacher R.

Krska R. New insights into the human metabolism of the Fusarium mycotoxins deoxynivalenol and zearalenone. Other groups have previously described changes in metabolite concentrations in breast cancer cells or xenograft models upon incubation with phytohormones. For example,found increased levels of amino acids and arachidonic acid in two breast cancer cell lines upon high resveratrol doses.showed that dietary GEN can reverse the inhibitory effect of LET on tumor growth and adversely impact breast cancer therapy. The authors even concluded that caution is warranted for consumption of dietary GEN by postmenopausal women with estrogen-dependent breast cancer taking LET treatment (). Given the results obtained in this in vitro study it seems not unlikely that xenoestrogens such as the phytoestrogen GEN and the mycoestrogen ZEN may also influence the described PAL + LET combination therapy through their bioactivity in an in vivo scenario. As we aimed to unravel the effects on metabolism at baseline xenoestrogen concentrations realistic in a Western-style society (), it is important to note that the described alterations of metabolism, cellular proliferation, and mTOR signaling were caused by rather low concentrations. It is possible that individuals consuming high-soy (GEN) or high-cereal (ZEN) diets may exceed these concentrations in their plasma.