Quercetin: a silent retarder of fatty acid oxidation in breast cancer metastasis through steering of mitochondrial CPT1

Recent evidence confirmed that the maximum energy in metastatic breast cancer progression is supplied by fatty acid oxidation (FAO) governed by a rate-limiting enzyme, carnitine palmitoyltransferase 1 (CPT1). Therefore, the active limitation of FAO could be an emerging aspect to inhibit breast cancer progression. Herein, for the first time, we have introduced quercetin (QT) from a non-dietary source (Mikania micrantha Kunth) to limit the FAO in triple-negative breast cancer cells (TNBC) through an active targeting of CPT1. Molecular quantification of QT was confirmed through high-performance thin-layer chromatography (HPTLC). Computational docking analyses predicted the binding affinity of QT to CPT1. Cell-based seahorse energy efflux investigated the mitochondrial respiration rate, glycolytic function and ATP production rate. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) investigated the FAO-associated gene expression. Matrigel cell invasion and fluorescence-activated cell sorting analyses investigated anti-metastatic and apoptotic cell death induction activities, respectively. In vivo antitumor activities were checked using the female breast cancer mice (BALB/c) model. QT resulted in a significant reduction in the intracellular mitochondrial respiration and glycolytic function, limiting extensive ATP production. In turn, QT elevated the reactive oxygen species (ROS) and depleted antioxidant levels to induce anti-metastatic and cell apoptosis activities. qRT-PCR resulted in active healing of altered FAO-associated gene expression which was well predicted through the successful in silico molecular binding potentiality of QT to CPT1. Subsequently, QT has shown excellent in vivo antitumor activities through the altered lipid profile and oxidative stress-healing capabilities. All the obtained data significantly grounded the fact that QT could be a promising metabolism-targeted breast cancer therapeutic.


Introduction
Breast cancer is one of the major cancers with a high mortality rate in women across the world. Based on diagnostic evidence, more than 15-20% of the total breast cancer depicted triple-negative breast cancer (TNBC) with high recurrence and worse survival rate compared to non-TNBC [1,2]. Metastatic TNBC is a fatal breast cancer subtype with absence of three primary receptors, namely estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) [3,4]. This is directly correlated with elevated lipolysis or fatty acid oxidation (FAO) for high energy compensation during rapid proliferation, even in hypoxic conditions [5,6]. The metastatic cancer 1 3 cell undergoes metabolic rearrangements, including alterations in fatty acid transport, accumulation of lipid droplets, de novo lipogenesis and β-oxidation to achieve extensive ATP demand [7,8]. It has been hypothesized that glycolysis solely plays a significant role in cancer progression and the fact was later at stake since the discovery of direct mitochondrial relation with cancer progression [9,10]. Moreover, the mitochondrial bioenergetics and FAO association have been witnessed to play an imperative role in cancer stemness, survival, proliferation and chemoresistance [11,12]. Recently, fatty acid synthesis became one of the prime concerns in metastatic breast cancer progression [13].
The carnitine palmitoyltransferase system acts as a pivotal mediator in cancer metabolic plasticity in active association with FAO. This system is solely responsible for long-chain fatty acid (LCFA) delivery from the cytoplasm to mitochondria for fatty acid β-oxidation, and carnitine palmitoyltransferase 1 (CPT1) catalyzes the rate-limiting steps of FAO. Thereby, CPT1 and FAO targeting breast cancer have been hypothesized to be an optimistic approach toward anticancer therapeutics [14,15]. Besides, fatty acid synthase (FASN), a rate-limiting enzyme, plays a decisive role in LCFA processing in FAO. Overexpression in FASN has been considered to promote breast cancer progression, while up-regulation in acetyl-CoA carboxylase (ACC) has shown negative feedback limiting breast cancer regression [16,17]. Till date, etomoxir has been considered to be a most efficient CPT1 inhibitor that can actively limit FAO, but exhibited severe concern regarding the side effects [18,19]. Therefore, introduction of novel drugs without any toxic effects is most desirable to encounter metabolism-associated cancer progression. Interestingly, the direct involvement of different enzymes and carriers in the FAO pathway, modulating beta-oxidation in cancer cells, is of intense interest since time immemorial. Moreover, the inhibition exerted by them remains associated with antitumor activities and thereby needs quick surveillance over time.
Despite the availability of several synthetic anticancer drugs, natural drugs have gained immense attention in recent times due to their minimal side effects [20]. Natural phytochemicals from edible plants have shown remarkable possibilities toward effective anticancerous treatment with beneficial clinical advantages [21,22]. Mikania micrantha Kunth (bitter vine or mile-a-minute vine), an invasive weed of the family Asteraceae, contains various bioactive compounds such as flavonoids, phenols, and polyphenols. This globally famed antiseptic and folk medicine is applied for assessment of antibacterial activities, mostly and rarely explored in cancer therapies [23]. Quercetin (QT) [IUPAC name: 2-(3, 4-dihydroxyphenyl)-3, 5, 7-trihydroxy-4H-chrome-4-one; CAS number: 117-39-5 6151-25-3], a flavonol with antilipid oxidative property, has been widely used in cancer treatment. It performs various activities such as decreasing the FASN expression, inhibiting the proliferation of carcinoma cells and significantly improving the plasma non-enzymatic antioxidant capacity during chemotherapy [24]. It also elevates the lipid peroxidase level, reduces tumor size and the cumulative number of papilloma, and acts as a potent inhibitor of lipogenesis in prostate and breast cancer cells by inhibiting FASN activity [25,26]. Reports have evidenced that QT potentially inhibits lipid synthesis by suppression of peroxisome proliferator-activated receptor-gamma (PPARγ) and CCAAT enhancer-binding protein a (C/EBPα) and activation of AMP-activated protein kinase (AMPK) in 3T3-L1 cells [27]. Eventually, QT has been proven to modulate the activity of hepatic cholesterol and hepatic cholesterol 7α-hydroxylase, the enzyme involved in cholesterol metabolism, promoting the strategic conversion of cholesterol-to-bile acid [28]. Furthermore, the antiinflammatory effects of QT have been reported observing the increased level of oxidative stress marker-nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO1), along with the decreased level of nuclear factor kappa B (NFƙB) in mice supplemented with high fat diet [29]. Dietary QT is intriguingly reported to limit FAO in metastatic breast cancer to stimulate DNA damage and apoptosis [30]. Therefore, it will be worth exploring QT from non-dietary sources as effective anticancer therapeutics, especially, for TNBC.
In this study, we synthesized a flavonoid subtype, QT, from Mikania micrantha Kunth and evaluated its metabolism-targeted anticancer activities via checking the outcome of FAO-associated rate-limiting enzyme. QT determined the fate of total intracellular glycolysis and mitochondrial oxidation rate, promoting cell death and apoptosis. Additionally, the overall molecular action was successfully predicted through in silico molecular binding assay via 3D (three-dimensional) protein structure preparation of CPT1, followed by an extra-precision (XP) molecular docking, elucidating the plausible mechanistic binding interactions of QT with CPT1. Lastly, in vivo experimentation in female BALB/c breast cancer mice model portrayed the intracellular lipid profiling and oxidative stress management enabling antitumor activities of QT, confirming its successive metabolism-targeted anticancer activities.

Extract preparation, characterization and estimation of quercetin
The invasive plant, Mikania micrantha Kunth, was collected from IIEST Shibpur, Howrah, West Bengal, India. After washing and oven-drying at 35 °C for 30-40 min, Soxhlet extractions were performed followed by spectrometric analysis of the specific compounds (Supporting informations).

Intracellular mito and glyco stress analysis
Seahorse XFe24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) was used to check the intracellular mito and glyco stress in 24 XF well plates via the investigation of the overall oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in MDA-MB-231 cells, respectively [31]. The detailed experimentation method has been elaborated in supporting informations.

Anti-invasion and migration study
Anti-invasion and migration activities of MDA-MB-231 cells in the presence of QT were investigated using the Transwell assay kit (ECM 555, Millipore) and monolayer cell scratching assay, respectively, by the invasive and wound healing cell imaging under an inverted fluorescence microscope (Olympus, Japan) and the measurement of relative fluorescence intensities utilizing a microplate reader (Bio Rad, India) following standard protocol (Supporting information).

RNA extraction, cDNA synthesis and gene expression analysis
TRIzol methods were applied for total RNA extraction, followed by the preparation of cDNA using normal polymerase chain reaction (PCR, Bio-Rad, Hercules, CA, USA). Then the individual gene expression was achieved through comparative CT method (ΔΔCT) by qRT-PCR (Applied Biosystems, Waltham, MA,USA) after the selection of genespecific forward and reverse primer (IDT technologies, Iowa, USA (Table S1) (Supporting information).

Cell apoptosis analysis
Annexin V-FITC and PI double-staining method was applied to investigate the apoptotic cell death in MDA-MB-231 cells. The QT-treated cells were washed with chilled 1X PBS buffer before their incubation with 100 µl of annexin-binding buffer. Then the cells were allowed to mix with 5 µl of annexin V-FITC (2 µg/ml) and 5 µl of PI (0.5 mg/ml) and following a 15 min incubation at room temperature in the dark. The results were recorded via the FACS (BD Biosciences, San Jose, CA).

In silico molecular modeling and docking studies
Due to the unavailability of the complete information of 3D crystal structure of CPT1, prediction of 3D structure of CPT1 was carried out in I-TASSER (Iterative Threading Assembly Refinement) server available at https:// zhang lab. ccmb. med. umich. edu/I-TASSER/ followed by the active validation through the Ramachandran plot analysis. [32]. The Glide-XP docking execution was performed employing the 'Ligand docking' module of Schrödinger's Maestro interface following default settings to analyze the CPT1 protein-ligand QT interactions [33]. As an output parameter, docking result was allowed to generate a maximum of six docked poses per ligand (Supporting information).

Animals
The nulliparous female BALB/c mice of 6-7 weeks old with average body weight 20-30 g were used for the maintenance and experiment following NIH guidelines and institutional animal ethical committee approval (RKC/IAEC/A/03 dated 14/12/17) for laboratory animals at R.G. Kar Medical College, Kolkata, India. A detailed animal care and use has been reported earlier [31]. Moreover, a successive in vivo anticancer assessment including toxicity profiling and therapeutic dosage confirmation were achieved following standard protocols (Supporting information).

Statistical analysis
Mean ± standard error of mean (SEM) was preferred to express the experimental data. Multiple comparison tests or one-way analysis of variance (ANOVA) and post hoc Dunnett's test software (SPSS v20) were used to analyze the groups statistically. Also, unpaired two-tailed Student's t test was used to measure the level of significance in some experiments. Statistical significant level was considered with a p value of less than 0.001 and 0.05.

Results
The spectrophotometric analysis of total flavonoids in MIKA was estimated to 132.66 µg equitant to QT for each mg of MIKA. Among the flavonoids, QT was identified and quantified by HPTLC. Densitometric HPTLC was performed accordingly to ICH guidelines for instrumental precision, repeatability, specificity and accuracy. Rf values of marker standard QT was determined as 0.38 and the regression analysis via area of QT was 0.99662 (sdv = 3.29) (Fig. S1). The amount of QT present in MIKA was calculated from the calibration curves and is presented in Table S2.

Fate of the major antioxidant system and ROS
A high amount of ROS generation is almost a common phenomenon for all cancerous cells, while excessive production leads to negative feedback [34]. Herein, almost a 50% increment in ROS production was investigated with QT-15 treatment, while it was almost doubled with QT-30 compared to the untreated control (Fig. S3a). This observation indicated that QT triggered ROS-mediated anticancer possibilities. The fate of intracellular antioxidant system, mainly GSH and NADPH level, is crucial to predict the anticancer possibilities actively, as a high level of GSH/NADPH is a common phenomenon in most of the cancer cells. At QT-45, GSH and NADPH levels dropped around 65% and 60%, respectively, while at QT-30, GSH and NAPDH dropped almost 55% and 45%, respectively (Fig. S3b).

Mitochondrial respiration and total glycolysis
Intracellular mitochondrial respiration was calculated from the comparison of the total oxygen consumption rate (OCR) (Fig. 1a). In our previous report, we have shown that palmitate-BSA alone can elevate the intracellular OCR and ECAR rate in breast cancer cells [31]. In this current experiment, intracellular basal and maximal respiration rate were decreased about 23.78% and 68.69%, respectively, in the presence of QT-30 compared to the control. Also, 35.07% depletion in maximal respiration was observed in the presence of QT-30 with palmitate-BSA (1:1, v/v), but a slight increase of about 17.33% in basal respiration was also investigated (Fig. 1b, c). A significant decrement of about 62.43% and 30.92% in proton leak was found in the presence of QT-30 and QT-30 with palmitate-BSA, respectively, compared to the untreated control. Similarly, 54.33% and 42.03% reductions in spare respiratory capacity were also observed in the presence of QT-30 and QT-30 with palmitate-BSA respectively compared to the control, which in turn indicates the active depletion in intracellular ATP production (Fig. 1d,  e). Importantly, intracellular total proton leak and spare respiratory capacity together exhibit the ATP production manually and accounted for a significant reduction in ATP production rate compared to untreated control (Fig. S4).
The glycolytic functions were calculated from the comparison of the total extracellular acidification rate (ECAR) (Fig. 2a). Apart from the mitochondrial respiration alteration, significant reductions in total glycolysis of about 77.68% and 49.16% were investigated in the presence of QT-30 and QT-30 with palmitate-BSA, respectively, compared to the control. Likewise, the glycolytic capacity was also reduced around 94.86% and 54.28% in the presence of QT alone or with palmitate-BSA compared to the control, respectively (Fig. 2b, c). This significant depletion in glycolytic function has further enhanced the QT-triggered energy-mediated anticancer possibilities. The molecular mechanism behind these phenomena was further hypothesized by comparing the relative protein expression profiling related to cancer cell metabolism. In most of the cancer cells, CPT1 expressions are always high due to the continuation of excessive energy supply via fatty acid β-oxidation. Interestingly, in the presence of QT-30, almost a twofold and 2.5-fold decrement has also been observed in CPT1A and FASN expression, respectively, whereas a remarkable threefold increment was evidenced in ACCα (Fig. 2d). From this observation, it can be concluded that QT-triggered increment in ACCα directly interferes in fatty acid metabolism via downregulation of both CPT1A and FASN.

Invasion and migration assay
An active invasion and migration abilities are the key phenomena in metastatic cancer progression. A dose-dependent QT-triggered wound-healing ability or cell migration was confirmed through scratch assay analysis. After 24 h of QT-15 and QT-30 treatment, the wound closure percentages were found about 28% and 38%, respectively, whereas it was about 11% and 21% after 48 h of treatment, Bar reports mean ± s.e.m; one of two experiments completed with or without human subject samples is shown. *p < 0.05, **p < 0.001 1 3 respectively (Fig. 3a, b). In untreated control, the wound closure percentage was about 78% after 24 h and 100% after 48 h. From Transwell invasion assay, almost 39% reduction in cell invasion was observed in the presence of QT-15, whereas QT-30 resulted in about 63% reduction in cell invasion compared to the untreated control (Fig. 3c, d). Also, in previous studies, scientists have shown a significant anti-migration and anti-proliferation effect of QT from dietary sources at a concentration range of 0.01-100 µM [35]. However, in comparison to those reports, our study possesses the significance of anticancer activity of QT from a non-dietary source. Moreover, MMP-2 level in QT-15-and QT-30-treated cells was found to be about 54 pg/ml and 43 pg/ml, respectively, whereas it was about 69 pg/ml in the untreated control. Similarly, MMP-9 level was investigated about 71 pg/ml in the untreated control, whereas it was about 57 pg/ml and 46 pg/ml in QT-15-and QT-30-treated cells, respectively.

Cell apoptosis analysis
A dose-dependent apoptotic cell death was observed in the presence of QT. In untreated control cells, the percentage of apoptotic cells was very low at about 0.1, whereas the live cell percentage was very high with 99.94 as expected. In QT-15-treated cells, the total cell apoptosis and live cell percentage were 15.6 and 34.9, respectively, whereas it was 36.6 and 13.4 in QT-30-treated cells (Fig. S5a, b). Therefore, QT has successfully enhanced the live to apoptotic cell transition in breast cancer cells effectively to affirm its anticancer abilities profoundly.

In silico molecular binding interaction analyses of QT to modeled CPT1 protein
I-TASSER-based structural prediction of CPT1 protein revealed that above 90% of residues in the "core" regions Changes in the expression of FAO-associated major rate-limiting enzymes, i.e., FASN, ACCα and CPT1A were represented in the presence of QT-30 with or without palmitate-BSA along with the untreated control. Bar reports mean ± s.e.m; one of two experiments completed with or without human subject samples is shown. **p < 0.001 possessed acceptable stereo-chemical quality of the modeled CPT1 protein as illustrated in the Ramachandran plot analysis (Fig. 4a). Moreover, major numbers of amino acid residues were found in the most favored regions of the plot, which was validated through the high-quality modeled protein.
Moreover, the comprehensive docking analysis demonstrated that QT formed several numbers of H-bond interactions with amino acid residues Trp236, Tyr241, Ser252, Tyr589 and Thr602 of CPT1 (Fig. 4b). Precisely, several active functional hydroxyl (-OH) groups in QT participated to create H-bond interactions with side-chain residues of CPT1 and the bond distances were measured between 1.80 and 2.83 Å. In addition, a number of hydrophobic interactions have also been observed with residues Trp236, Ile240, Tyr589, Ala591 and Val715 of CPT1. In addition, the binding mode for QT inside the hollow catalytic tunnel of CPT1 has been generated and is depicted in Fig. 4c. The surface view orientation of the represented binding mode of QT with CPT1 strongly suggests that QT can hold its position tightly inside the hollow catalytic tunnel of CPT1 at the COOH terminal. Moreover, the observed multiple H-bond and hydrophobic interactions has also suggested the binding selectivity between QT and CPT1 protein and such intermolecular interactions can influence the exhibition of some degree of biological activity, considering the all-important roles of H-bond and hydrophobic interactions in stabilizing the protein-ligand systems.

In vivo anticancer activity analysis
From acute oral toxicity profiling, the dose with 2000 mg/kg oral administration of QT was selected as non-toxic as well as a safe dose for other experiments (Table S3).
An excellent antitumor activity was observed in the presence of QT (Fig. 5a-f). Briefly, the average body weight gain of 4T1 cells-induced mice was 2.7 g in 3 weeks, but the body weight was drastically decreased by the tumor progression. Remarkably, the body weight gain was restored dose-dependently in the presence of QT. QT-15 and QT-30 restored around 21.42% and 24.96% of body weight gain, respectively, whereas the positive inhibitor, ETO-30, restored around 33% of body weight gain compared to the untreated tumor control (Fig. 5c). Likewise, QT-15 reduced 37.01% and 31.81% of tumor volume and weight, respectively, whereas QT-30 lowered 70.27% and 61.11% of tumor volume and weight compared to tumor control. ETO-30 also lowered 72.22% and 73.19% of tumor weight and volume, respectively (Fig. 5a, d, e). In addition, a time-dependent tumor regression was also investigated. After 21 days of treatment, QT-15 and QT-30 successfully reduced 54.29% and 70.99% tumor growth compared to untreated tumor control (Fig. 5f). Similarly, ETO-30 reduced 75.42% of tumor growth. Microscopic histopathological evaluations of breast tissues have further supported these findings. In tumor control breast tissue, the lumen of the duct was filled with proliferative cells in a disorderly pattern and nuclear basophilicity was noted to be enhanced. QT-15-treated breast Fig. 4 In silico molecular docking analysis. a The Ramachandran plot shows phi-psi torsion angles for all residues of the modeled CPT1 crystal structure. Glycine residues are indicated by triangles as those are not restricted to the regions of the plot appropriate to the other side-chain types. The darkest areas in red correspond to the "core" regions representing the most favorable amino acids of CPT1 and their combinations of phi-psi values. b Intermolecular interactions profile between QT and CPT1 obtained in Glide-XP molecular docking analysis. c Close surface view representation of the Glide-XP docking-based binding mode of QT in the active site of human CPT1modeled protein tissue showed lumen of duct with less proliferative cells in an orderly pattern, and nuclear basophilicity was much less than tumor control. In QT-30-and ETO-30-treated breast tissues, the ductal epithelial cells and myoepithelial cells were noted in terminal duct-lobule units surrounded by a thick basement membrane along with improved necrotic lesion in the center of ducts (Fig. 5b).
Altered oxidative stress and lipidomic profile management have been evidenced to induce apoptosis in breast cancer cell [31,36]. The obtained data showed an excellent decrement in LPO and GSH level, whereas a significant increment in SOD level in the presence of both QT and ETO-30 compared to untreated control was observed in Fig. 6a. Total LPO in tumor control was about 2.93 nM MDA/mg protein, whereas it decreased to 2.47 and 2.04 nM MDA/mg protein in the presence of QT-15 and QT-30, respectively. Likewise, GSH in QT-15-and QT-30-treated tumor tissue was decreased to 5.63 and 4.76 nM/mg protein, respectively, whereas in tumor control, it was 6.06 nM/mg protein. Also, SOD level in the tumor control was around 5.02 U/mg protein, whereas it was increased to 6.08 and 6.85 U/mg protein in QT-15-and QT-30-treated tumor tissue, respectively. In addition, a dose-dependent decrement in total cholesterol, LDL, VLDL and triglycerides level was observed in both concentrations of QT and ETO-30treated tissue compared to untreated tumor control, whereas a sharp increase in HDL was also observed in QT-and ETOtreated tissue samples (Fig. 6b). The respective total cholesterol level in QT-15-and QT-30-treated tissue was 143.5 and 128.83 mg/dl, but in untreated tumor control, it was 158.67 mg/dl. Also, LDL and VLDL in QT-30-treated tissue was found to be about 72.33 and 16.31 mg/dl, respectively, while in QT-15-treated tissue, this respective level was witnessed at about 82.05 and 20.33 mg/dl compared to 98.04 and 25.67 mg/dl in the untreated tumor control, respectively. Total HDL in tumor control was calculated about 26.83 mg/ dl, whereas it increased to 32.17 and 40.67 mg/dl in QT-15and QT-30-treated tissue, respectively. FAO-associated triglyceride levels were also found to be decreased to 99.83 and 81 mg/dl, respectively, in QT-15-and QT-30-treated tissue compared to untreated tumor control with 123.83 mg/dl of triglycerides (Fig. 6c).Thereby, these results have indicated Bar reports mean ± s.e.m; one of two experiments completed with or without human subject samples is shown. *p < 0.05, **p < 0.001 that QT has an active control over these major oxidative stress and lipid parameter to promote antitumor activities or apoptotic cell death. Perhaps, the liver enzymes (SGOT, SGPT and ALP) and renal functions (urea and creatinine) in the serum remain unchanged compared to QT-15, QT-30, ETO-30 and mock-treated mice signifying the advantages of QT as a potent anticancer drug (Fig. S6).

Discussion
FAO is the key phenomenon to promote metabolic plasticity in metastatic cancer cells starting from the cytoplasm to mitochondria [13,37]. In accordance with the above fact, the active limitation of FAO in metastatic TNBC could be a novel anticancer approach. Surprisingly, plant-based flavonoids derivative, QT, has shown noteworthy activities over available anticancer drugs due to its non-toxic effects along with high efficacy and potentiality [38]. Moreover, QT has been judiciously envisaged to be the most promising anticancer agent, but needs further investigation for its establishment as an au courant anticancer therapeutics [39,40]. Importantly, metabolism-targeted anticancer therapies are the current need to check metastatic growth with better diagnostic outcome.
Herein, QT has shown remarkable CPT1-targeted anticancer activities with excellent control over intracellular glycolysis, mitochondrial respiration and FAO. It has successfully up-regulated ACCα to inhibit the FASN and CPT1 expression, decreasing mitochondrial β-oxidation which has been further confirmed through the significant depletion in mitochondrial respiration and overall ATP production along with the reduction in glycolytic functions, limiting the extensive energy requirements of TNBC. Moreover, mitochondrial respiration has been influenced by the changes in mitochondrial membrane potential and excessive ROS generation regulating the intracellular antioxidant system, which in turn enhanced the anti-metastatic activities of QT, followed by the induction of programmed cell death or apoptosis. This also promotes antitumor activity via the management of major in vivo oxidative stress and altered lipid profile associated with metastatic cancer progression. These activities have been further justified through the in silico molecular binding potentiality of QT to CPT1.
In conclusion, QT from non-dietary source has successfully targeted mitochondrial CPT1 and inhibited FAO in metastatic TNBC, both in vitro and in vivo, decreasing the extensive energy supply to promote successive growth inhibition and apoptosis induction. Therefore, it could be adjudged as the most promising current age fat metabolismtargeted anticancer therapeutics.