Layered Double Hydroxide Nanoparticles Loaded with Resveratrol Inhibit Glycolysis and Show Efficacy in the Treatment of Breast Cancer.

Immune therapies targeting the PD-1/PD-L1 pathway have been employed in the treatment of breast cancer, which requires aerobic glycolysis to sustain breast cancer cells growth. However, whether PD-L1 expression is regulated by glycolysis in breast cancer cells remains to be further elucidated. Here, we demonstrate that glycolytic enzyme hexokinase 2 (HK2) plays a crucial role in upregulating PD-L1 expression. Under high glucose conditions, HK2 acts as a protein kinase and phosphorylates IκBα at T291 in breast cancer cells, leading to the rapid degradation of IκBα and activation of NF-κB, which enters the nucleus and promotes PD-L1 expression. Immunohistochemistry staining of human breast cancer specimens and bioinformatics analyses reveals a positive correlation between HK2 and PD-L1 expression levels, which are inversely correlated with immune cell infiltration and survival time of breast cancer patients. These findings uncover the intrinsic and instrumental connection between aerobic glycolysis and PD-L1 expression-mediated tumor cell immune evasion and underscore the potential to target the protein kinase activity of HK2 for breast cancer treatment.

Background: The prevalence of obesity is steadily increasing worldwide, impacting the incidence of a variety of diseases, including breast cancer (BCa). Although epidemiological studies show a strong association between elevated body mass index (BMI) and a heightened risk of postmenopausal BCa, the underlying etiology remains relatively unknown. One potential mechanism by which obesity contributes to BCa progression and recurrence is through induction of obesity-induced stress (OBIS). Reactive oxygen species (ROS) are byproducts of metabolism, and excessive production or accumulation of ROS is known to contribute to early tumor initiating events through redox modulation of p53 transcriptional activity. Objectives: Recent studies indicate that p53 is a key regulator of glycolysis. As the obese state is known to promote aerobic glycolysis, the preferred mode of energy production by most transformed cells, and because approximately 40% of human breast cancers harbor p53 mutations, the aim of this study was to determine the impact of obesity on rates of glucose consumption and genotoxic stress in the context of p53 status. Results: Using the MMTV-Wnt1 transgenic mouse model of spontaneous breast cancer harboring a heterozygous deletion mutation for p53, we determined the impact of diet-induced obesity (DIO) on markers of aerobic glycolysis and oxidative stress. Interestingly, thioredoxin interacting protein (TXNIP), a regulator of glucose homeostasis, was found to be induced in the normal mammary fat pad (MFP) of obese p53-heterozygous (p53-het) mice versus p53-wild type (p53-wt) MFPs. Conversely, MCF-7 breast cancer cells grown in vitro in serum from these obese mice showed a 50% decrease in TXNIP expression in comparison to cells cultured in serum from the non-obese mice. These findings are consistent with TXNIP's putative role as a tumor suppressor gene. Further, glucose transporter 1 (GLUT-1), a basal glucose transporter required to sustain cellular respiration, was upregulated 5-fold in the MFP of obese p53-het mice compared to MFP taken from non-obese p53-het mice, confirming that obesity regulates glucose uptake in the MFP independently of p53 status. Additionally, the obese p53-het mice displayed elevated serum levels of 8-isoprostane, a biomarker of oxidative stress. Additional in vitro studies demonstrate that MCF-7 cells cultured in serum derived from obese versus non-obese C57BL/6 mice (Ob-serum and non-Ob serum) displayed heightened levels of ROS and significant modulation of HIF-1α, a ROS-inducible transcription factor known to regulate glycolysis. Ob-serum also enhanced lactate dehydrogenase activity and inhibited expression of pyruvate dehydrogenase (PDH), concurrently promoting glycolysis and bypassing oxidative phosphorylation (OXPHOS). This metabolic shift was associated with the accumulation of γ-H2AX foci, a known indicator of DNA double-strand breaks (DSBs). Finally, we observed that a combined shRNA-mediated knockdown of TXNIP and p53 in MCF-7 cells resulted in a dramatic induction of DSBs, and this was exacerbated by exposure to Ob-serum. Potential Impact: Based on these findings, we conclude that obesity may enhance the rate of metabolic reprogramming in breast cancer cells, resulting in OBIS and accumulation of ROS; thereby promoting disease progression by enhancing genotoxic stress in cells harboring a p53 mutation. Citation Format: David Antonio Cavazos, Karrie Wheatly, Stephen Hursting. Obesity induces a metabolic switch toward aerobic glycolysis in breast cancer cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A06.

Tamoxifen resistance is one of the major challenges for its medical uses in estrogen receptor (ER)-positive breast cancer. Aerobic glycolysis, an anomalous characteristic of glucose metabolism in cancer cells, has been shown to associate with the resistance to chemotherapeutic agents. It remains, however, largely unclear whether and how tamoxifen resistance contributes to aerobic glycolysis in breast cancer. Here, we report that tamoxifen resistance is associated with enhanced glycolysis in ER-positive breast cancer cells. We demonstrate that EREG, an agonist of EGFR, has an important role in enhancing glycolysis via activating EGFR signaling and its downstream glycolytic genes in tamoxifen-resistant breast cancer cells. We further show that EREG is a direct target of miR-186-3p and that downregulation of miR-186-3p by tamoxifen results in EREG upregulation in tamoxifen-resistant breast cancer cells. Importantly, systemic delivery of cholesterol-modified agomiR-186-3p to mice bearing tamoxifen-resistant breast tumors effectively attenuates both tumor growth and [18F]-fluoro-deoxyglucose ([18F]-FDG) uptake. Together, our results reveal a novel molecular mechanism of resistance to hormone therapies in which the miR-186-3p/EREG axis orchestrates tamoxifen resistance and aerobic glycolysis in ER-positive breast cancer, suggesting targeting miR-186-3p as a promising strategy for therapeutic intervention in endocrine-resistant breast tumors.

Increased dependence on aerobic glycolysis is characteristic of most cancer cells, whereas the mechanism underlying the promotion of aerobic glycolysis in metastatic breast cancer cells under ambient oxygen has not been well understood. Here, we demonstrated that aberrant expression of signal-induced proliferation-associated 1 (SIPA1) enhanced aerobic glycolysis and altered the main source of ATP production from oxidative phosphorylation to glycolysis in breast cancer cells. We revealed that SIPA1 promoted the transcription of EPAS1, which is known as the gene encoding hypoxia-inducible factor-2α (HIF-2α) and up-regulated the expression of multiple glycolysis-related genes to increase aerobic glycolysis. We also found that blocking aerobic glycolysis by either knocking down SIPA1 expression or oxamate treatment led to the suppression of tumor metastasis of breast cancer cells both in vitro and in vivo. Taken together, aberrant expression of SIPA1 resulted in the alteration of glucose metabolism from oxidative phosphorylation to aerobic glycolysis even at ambient oxygen levels, which might aggravate the malignancy of breast cancer cells. The present findings indicate a potential target for the development of therapeutics against breast cancers with dysregulated SIPA1 expression.

This study aimed to investigate the effect of SIK2 on cisplatin resistance induced by aerobic glycolysis in breast cancer cells and its potential mechanism. qRt-PCR and Western blot were used to detect SIK2 mRNA and protein levels, and cisplatin (DDP) resistant cell lines of breast cancer cells were established. Viability was measured and evaluated via CCK-8, cell invasion capability was evaluated via Transwell, and apoptosis rate was assessed via Flow cytometry. The glycolysis level was evaluated by measuring glucose consumption and lactic acid production. The protein levels of p-PI3K, p- protein kinase B (Akt) and p-mTOR were determined by western blot. SIK2 was highly expressed in breast cancer tissues and cells compared with adjacent tissues and normal human breast epithelial cells, and it had higher diagnostic value for breast cancer. Silencing SIK2 expression can inhibit proliferation and invasion of breast cancer cells and induce their apoptosis. In addition, SIK2 knockdown inhibits glycolysis, reverses the resistance of drug-resistant cells to cisplatin, and inhibits PI3K/AKT/mTOR signaling pathway. When LY294002 was used to inhibit PI3K/AKT/mTOR signaling pathway, the effect of pcDNA3.1-SIK2 on aerobic glycolysis of breast cancer cells could be reversed. SIK2 can promote cisplatin resistance caused by aerobic glycolysis of breast cancer cells through PI3K/AKT/mTOR signaling pathway, which may be a new target to improve cisplatin resistance of breast cancer cells.

Paridis Rhizoma saponins (PRS) are significant components of Rhizoma Paridis and have inhibitory effects on various tumors, such as bladder, breast, liver and colon cancer. Polyphyllin II (PPII), one of the PRS, has an unclear effect on breast cancer. The present study aimed to explore the effect and mechanism of PPII in breast cancer. A network pharmacology approach was employed to predict the core components and breast cancer‑related targets of PRS. Moreover, a xenograft tumor model was established to determine the anti‑breast cancer effect of PPII in vivo. The viability of MDA‑MB‑231 cells was determined by a Cell Counting Kit‑8 assay. Apoptosis was analyzed using annexin V/PI double staining. Additionally, Transwell and scratch assays were performed to evaluate invasion and migration. The potential mechanism was predicted by Kyoto Encyclopedia of Genes and Genomes enrichment analysis and molecular docking analysis and verified by western blot analysis. The effect of PPII on aerobic glycolysis in breast cancer cells was detected by lactic acid and pyruvate kits and Western blotting of glycolytic rate‑limiting enzymes. Network pharmacology analysis revealed 26 core targets involved in breast cancer and that PPII was the core active component of PRS. The in vivo studies showed that PPII could inhibit the growth of breast cancer in mice. In vitro experiments confirmed that PPII induced cancer cell apoptosis and inhibited invasion and migration. Furthermore, PPII was capable of suppressing the expression of key proteins in the PI3K/Akt signaling pathway, reducing the generation of aerobic glycolytic products, and diminishing the protein expression levels of hexokinase 2 and pyruvate kinase M2. The results indicated that PPII inhibited aerobic glycolysis in breast cancer cells through the PI3K/Akt signaling pathway, thereby inhibiting breast cancer growth.

Cancer deaths, including breast cancer, are caused by metastasis of the malignant tumors to distant locations. However, current methods of detection cannot distinguish pre-invasive breast cancer from noninvasive breast tumor or benign breast disease. Population-wide mammographic screenings have led to increased detection of ductal carcinoma in situ or DCIS, noninvasive, proliferative cells contained by the basement membrane of the terminal ductal lobular unit. DCIS is usually not associated with metastasis and/or cancer death. Each year, in the US alone, about 1.5 million of women who have been diagnosed with DCIS or a suspicious lump/lesion by mammography will require resection or breast biopsy after diagnosis for further pathologic analysis. However, ~80-85% of biopsies result in noninvasive breast disease or benign findings. As a result, a considerable number of patients suffer from side effects caused by breast biopsy and/or overtreatment. Therefore, there is an urgent need to find a biomarker for pre-invasive breast cancer. The ability of cancer cells to produce lactate through aerobic glycolysis (the Warburg effect) is a consistent hallmark of cancer, including breast cancer. Recent advancements in liquid chromatography-mass spectrometry (LC-MS) technology have significantly improved the sensitivity of this method compared to traditional NMR or GC-MS-based technologies, which make it feasible to detect very low concentrations of small molecules or metabolites. We have recently established a positional isotopic labeling and LC-MS-based targeted metabolomics method that can directly measure the conversion from [1-13C]glucose to [3-13C]lactate through glycolysis. Our results show that metastatic breast cancer cells exhibit a dramatically increased production of [3-13C]lactate from [1-13C]glucose even under aerobic conditions as compared to low- or noninvasive breast cancer cell lines. We found that the rate of aerobic glycolysis is closely correlated with glucose uptake and lactate production in breast cancer cells. We have also observed significantly elevated production of [3-13C]lactate in serum samples of early stage metastatic mammary tumors developed in mice. Since elevated levels of lactate are closely correlated to increased tumor aggressiveness, these results suggest that monitoring of lactate production from glycolysis by targeted metabolomics may provide a biomarker for pre-invasive breast cancer. These results will pave the way for further exploration of the elevated production of lactate as a promising biomarker for pre-invasive breast cancer and for assessment of therapeutic response in clinical trials. Citation Format: Da-Qing Yang, Margot Cleary. Measuring relative utilization of aerobic glycolysis in breast cancer cells by positional isotopic discrimination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1485. doi:10.1158/1538-7445.AM2017-1485

In the present study, we investigated the role of β-catenin on aerobic glycolysis in breast cancer cells and further evaluated its effects on triple-negative breast cancer (TNBC) cell migration and invasion. We used two metastatic TNBC cell lines, i.e., MDA-MB-468 and MDA-MB-231, and treated them with β-catenin antagonists (WNT5A and XAV939). Evaluation of glycolytic markers in WNT5A-expressing cell lines showed selective downregulation in the expression of β-catenin, platelet type-phosphofructokinase (PFKP), and lactate secretion, without affecting cell proliferation. Of the three phosphofructokinase isoform, we found that only PFKP expression was associated with decreased survival in breast cancer patients. We also found that the cause of PKFP and lactate downregulation was mediated by inhibition of β-catenin signaling, which is in accordance with the finding that β-catenin signaling positively regulates lactate production. Functionally, we showed that specific downregulation of β-catenin resulted in reduced lactate production accompanied by impaired TNBC cells migration and invasion. Finally, we demonstrated that apart from its inhibitory effect on lactate production, inhibition of β-catenin can also impair TNBC cell migration induced by exogenous lactate via downregulating monocarboxylate transporter 1 (MCT1). Overall, we demonstrated that inhibition of β-catenin-PFKP signaling axis impaired lactate production as well as its uptake from tumor microenvironment (via MCT1), thereby impairing TNBC cells' migration. Citation Format: Chandra P. Prasad, Tommy Andersson. Inhibition of β-catenin impairs triple-negative breast cancer (TNBC) cell migration and invasion by modulating aerobic glycolysis components [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B021.

Mitochondria are essential for energy metabolism in the tumor microenvironment and the survival of cancer cells. ADP-ribosylation factor-like GTPase 5b (ARL5B) has been found to be associated with mitochondrial dysfunction and breast cancer (BC) progression, but the underlying mechanism needs to be further understood. We investigated the effects of ARL5B on the apoptosis and glycolysis of breast cancer cells and its underlying mechanisms. Quantitative reverse transcription-PCR (qRT-PCR) and western blot assays were used to detect the expression of ARL5B in breast cancer tissues and cells. An ARL5B loss-of-function assay was performed to verify its role in BC development. ARL5B was upregulated in breast cancer tissues and cell lines. ARL5B knockdown induced apoptosis and activated the mitochondrial pathway in breast cancer cells. Interestingly, the inhibition of ARL5B repressed the aerobic glycolysis of breast cancer cells. The role of ARL5B in breast cancer cells was exerted by mediating the activation of viral RNA sensor MDA5-evoked signaling. Silencing ARL5B triggered MDA5 signaling by upregulating the key proteins involved in the MDA5 pathway. Importantly, MDA5 silencing reversed the effects of ARL5B knockdown on mitochondrial-mediated apoptosis and glycolysis, whereas poly (I:C), as a ligand for MDA5, further enhanced ARL5B knockdown- facilitated mitochondrial apoptosis and the inhibition of glycolysis. The knockdown of ARL5B activated MDA5 signaling and thus led to the enhanced mitochondrial- mediated apoptosis and glycolysis inhibition in breast cancer cells. Our study suggested that ARL5B might be a potential therapy target for BC.

Jolkinolide B inhibits proliferation or migration and promotes apoptosis of MCF-7 or BT-474 breast cancer cells by downregulating the PI3K-Akt pathway

Tumor cells metabolize more glucose to lactate in aerobic or hypoxic conditions than normal cells. Pyruvate kinase isoenzyme type M2 (PKM2) is crucial for tumor cell aerobic glycolysis. We established a role for let-7a-5p/Stat3/hnRNP-A1/PKM2 signaling in breast cancer cell glucose metabolism. PKM2 depletion via small interfering RNA (siRNA) inhibits cell proliferation and aerobic glycolysis in breast cancer cells. Signal transducer and activator of transcription 3 (Stat3) promotes upregulation of heterogeneous nuclear ribonucleoprotein (hnRNP)-A1 expression, hnRNP-A1 binding to pyruvate kinase isoenzyme (PKM) pre messenger RNA, and the subsequent formation of PKM2. This pathway is downregulated by the microRNA let-7a-5p, which functionally targets Stat3, whereas hnRNP-A1 blocks the biogenesis of let-7a-5p to counteract its ability to downregulate the Stat3/hnRNP-A1/PKM2 signaling pathway. The downregulation of Stat3/hnRNP-A1/PKM2 by let-7a-5p is verified using a breast cancer. These results suggest that let-7a-5p, Stat3, and hnRNP-A1 form a feedback loop, thereby regulating PKM2 expression to modulate glucose metabolism of breast cancer cells. These findings elucidate a new pathway mediating aerobic glycolysis in breast cancers and provide an attractive potential target for breast cancer therapeutic intervention.

Metabolic reprogramming is a hallmark of cancer. Here, we demonstrate that tumour-associated macrophages (TAMs) enhance the aerobic glycolysis and apoptotic resistance of breast cancer cells via the extracellular vesicle (EV) transmission of a myeloid-specific lncRNA, HIF-1α-stabilizing long noncoding RNA (HISLA). Mechanistically, HISLA blocks the interaction of PHD2 and HIF-1α to inhibit the hydroxylation and degradation of HIF-1α. Reciprocally, lactate released from glycolytic tumour cells upregulates HISLA in macrophages, constituting a feed-forward loop between TAMs and tumour cells. Blocking EV-transmitted HISLA inhibits the glycolysis and chemoresistance of breast cancer in vivo. Clinically, HISLA expression in TAMs is associated with glycolysis, poor chemotherapeutic response and shorter survival of patients with breast cancer. Our study highlights the potential of lncRNAs as signal transducers that are transmitted between immune and tumour cells via EVs to promote cancer aerobic glycolysis.

Patients suffering from breast cancer (BC) still have a poor response to treatments, even though early detection and improved therapy have contributed to a reduced mortality. Recent studies have been inspired on the association between microRNAs (miRs) and therapies of BC. The current study set out to investigate the role of miR-216b in BC, and further analyze the underlining mechanism. Firstly, hexokinase 2 (HK2) and miR-216b were characterized in BC tissues and cells by RT-qPCR and Western blot assay. In addition, the interaction between HK2 and miR-216b was analyzed using dual luciferase reporter assay. BC cells were further transfected with a series of miR-216b mimic or inhibitor, or siRNA targeting HK2, so as to analyze the regulatory mechanism of miR-216b, HK2 and mammalian target of rapamycin (mTOR) signaling pathway, and to further explore their regulation in BC cellular behaviors. The results demonstrated that HK2 was highly expressed and miR-216b was poorly expressed in BC cells and tissues. HK2 was also verified as a target of miR-216b with online databases and dual luciferase reporter assay. Functionally, miR-216b was found to be closely associated with BC progression via inactivating mTOR signaling pathway by targeting HK2. Moreover, cell viability, migration and invasion were reduced as a result of miR-216b upregulation or HK2 silencing, while autophagy, cell cycle arrest and apoptosis were induced. Taken together, our findings indicated that miR-216b down-regulates HK2 to inactivate the mTOR signaling pathway, thus inhibiting the progression of BC. Hence, this study highlighted a novel target for BC treatment.

Cancer-associated fibroblast-derived CCL5 enhanced aerobic glycolysis through upregulation of IP3R to promote breast cancer cell metastasis.

Breast cancer is the most common cancer in women and one of the leading causes of cancer death worldwide. Ferroptosis, a promising mechanism of killing cancer cells, has become a research hotspot in cancer therapy. Simvastatin (SIM), as a potential new anti-breast cancer drug, has been shown to cause ferroptosis of cancer cells and inhibit breast cancer metastasis and recurrence. The purpose of this study is to develop a novel strategy boosting ferroptotic cascade for synergistic cancer therapy. In this paper, iron base form of layered double hydroxide supported simvastatin (LDHs-SIM) was synthesized by hydrothermal co-precipitation method. The characterization of LDHs-SIM were assessed by various analytical techniques, including ultraviolet-visible (UV-vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and transmission electron microscopy (TEM). Biological activity, ferroptosis mechanism and biocompatibility were analyzed through in vivo and in vitro analysis, so as to evaluate its therapeutic effect on breast cancer. The constructed LDHs-SIM nanosystem can not only release SIM through mevalonate (MVA) pathway, inhibit the expression of glutathione peroxidase 4 (GPX4), inhibit the expression of SLC7A11 and reduce the synthesis efficiency of GSH, but also promote the accumulation of Fe2+ in cells through the release of Fe3+, and increase the intracellular ROS content. In addition, LDHs-SIM nanosystem can induce apoptosis of breast cancer cells to a certain extent, and achieve the synergistic effect of apoptosis and ferroptosis. In the present study, we demonstrated that nanoparticles of layered double hydroxides (LDHs) loaded with simvastatin were more effective than a free drug at inhibiting breast cancer cell growth, In addition, superior anticancer therapeutic effects were achieved with little systemic toxicity, indicating that LDHs-SIM could serve as a safe and high-performance platform for ferroptosis-apoptosis combined anticancer therapy.