Sage Journals: Discover world-class research

Abstract

At present, chemotherapy is the most effective strategy for treating triple-negative breast cancer (TNBC), but its efficacy was limited by the development of chemo-resistance. The exact mechanism of chemoresistance still remains unclear. This study aims to examine whether 6-phosphogluconate dehydrogenase (6PGD), a key enzyme in the oxidative pentose phosphate pathway (PPP), could promote the resistance of TNBC cells to epirubicin. A TNBC epirubicin-resistant cell line was developed by increasing concentration and the effectiveness was tested. The expression and knockdown efficiency of 6PGD were further validated by performing quantitative real-time PCR (qPCR) and Western blot. The effects of 6PGD on parental and drug-resistant TNBC cell lines were verified based on proliferation and apoptosis experiments. Finally, nicotinamide adenine dinucleotide phosphate (NADPH) and lactate quantitative experiments were performed to examine the mechanism of 6PGD in promoting drug resistance. Epirubicin-resistant cancer cells exhibited a higher level of 6PGD in contrast to epirubicin-sensitive cells. In addition, 6PGD inhibited by genetic and pharmacological approaches significantly suppressed the growth and survival of both epirubicin-sensitive and epirubicin-resisteant TNBC cells. It should be noted that 6PGD inhibition sensitized epirubicin-resistant TNBC cells to epirubicin treatment. Moreover, it was also found that the levels of NADPH and lactate increased in epirubicin-resistant TNBC cells but decreased in response to 6PGD inhibition. The present results indicated that 6PGD inhibition disrupted metabolic reprogramming in epirubicin-resistant TNBC cells. Our work demonstrated that 6PGD inhibition reversed the resistance of TNBC cells to epirubicin, providing an alternative therapeutic choice to tackle the challenge of epirubicin resistance in TNBC treatment.

Keywords

6PGD chemotherapy resistance epirubicin metabolic reprograming triple-negative breast cancer (TNBC)

Introduction

Triple-negative breast cancer (TNBC) is considered as a type of breast cancer (BC) with the highest heterogeneity due to its strong aggressiveness and a lack of specific therapeutic targets such as estrogen receptors, progesterone receptors, and HER2 receptors.^1–3 TNBC shows a higher rate of recurrence and distant metastases as well as a poorer overall survival (OS) in comparison to other types of BC.^4,5 At present, neoadjuvant chemotherapy (NAC) is the predominant therapeutic method for treating TNBC, with taxane and anthracycline-based regimens being used the most frequently.^6,7 Although NAC has been shown to be effective in treating some patients with TNBC, the pathological complete response (pCR) is only 33.6% and resistance develops in approximately half of patients, resulting in a low OS rate.^8–10

Chemotherapy resistance has contributed greatly to treatment failure, especially in cases of metastatic diseases, the failure rate of which might reach 90%.¹¹ From the current guidelines, preferred recommendation for neoadjuvant treatment is a combination of dose-dense or the use of doxorubicin (or epirubicin) and cyclophosphamide every 3 weeks, followed by paclitaxel with or without carboplatin.^12,13 In recent years, numerous studies have demonstrated that the chemoresistance of TNBC is multidimensional and is dependent on the intricate interplay of the drug efflux, tumor microenvironment, bulk tumor cells, cancer stem cells, and even metabolic abnormalities.^11,14 However, the underlying mechanism resulting in TNBC chemotherapy resistance is highly complex and has not been fully understood.

Recently, 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in the oxidative pentose phosphate pathway (PPP), has been found to be responsible for decarboxylating the attenuation of 6PG to ribulose 5-phosphate, which could cause the production of nicotinamide adenine dinucleotide phosphate (NADPH).¹⁵ 6PGD is overexpressed in several different cancer types,¹⁶ including in renal cell carcinoma¹⁷ and anaplastic thyroid cancer,¹⁸ and plays significant roles. 6PGD not only promotes the proliferation and metastasis potential of cancer cells but also contributes to chemotherapy resistance and radiation resistance. It frequently exhibits aberrant activation in various cancers and offers a metabolic advantage by promoting glycolysis and anabolic biosynthesis to support cancer cell progression.¹⁹ Notably, upregulated expression of 6PGD has been found in BC patients, and lower enzyme activity is present in patients with a free period of more than 3 years than in those who relapsed within 3 years after receiving initial treatment.²⁰ Moreover, it is reported that 6PGD expression contributes to chemotherapy resistance in a variety of cancers and that its inhibition sensitizes tumor cells toward cytotoxic drug treatment.^17,18 Cancers with 6PGD inhibition had slowed growth, H₂O₂-mediated cell death, and increased chemosensitivity.^21–23 However, the mechanism underlying these functions of 6PGD remains unknown,²⁴ and whether 6PGD inhibition plays an important role in reversing TNBC chemoresistance is also unclear. Here, we explored how 6PGD affected the ability of TNBC cells to survive after exposure to chemotherapeutic agents of epirubicin, hoping to develop a different treatment for TNBC.

Materials and Methods

Cell Culture and Establishment of Epirubicin-Resistant Breast Cancer Cells

The human TNBC cell lines MDA-MB-231 (bio-69126) and BT-549 (bio-68460) were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA) or collaborators and incubated in Gibco RPMI 1640 with 10% FBS and 1% penicillin-streptomycin, according to normally applied methods. The chemoresistant cell line was cultured from the parental cells by being exposed to progressively increasing epirubicin doses (FN5235, Pharmorubicin, China) from 20 nM to 320 nM for more than 60 days. The concentration of epirubicin increased by twofold every 2 weeks. Next, the chemoresistant cells were kept in a culture medium containing 320 nM epirubicin.

Proliferation and Apoptosis Measurement

Cell proliferation activity was evaluated by using the Cell Counting Kit-8 (CCK-8, C0037, Beyotime, Haimen, China) according to the manufacturer's protocol. Briefly, the 96-well plates were filled with 100 µL of medium containing 2000 cells per well. CCK-8 solution was added to each well after the prescribed durations of culturing. Each well was then incubated at 37 °C for 2 hours (h) and the optical density of each well was detected at 450 nm using a microplate reader (Infinite 200 PRO, TECAN, Männedorf, Switzerland). The assessment of cell apoptosis was performed by conducting flow cytometry (FC) analysis. After digesting and centrifuging, the cells were washed twice using ice-cold PBS and incubated with Annexin V-FITC (#237499-000, Invitrogen, US) and PI (#233904-000, Invitrogen, US) in the dark for 15 minutes (min). Flowjo was utilized to analyze apoptosis results following the instructions.

Quantitative Real-Time RT-PCR Analysis

TRIzol (Life Technologies Inc., Carlsbad, CA, USA) was applied to extract genomic RNA from cell lines in accordance with the manufacturer's instructions. With the aid of a NanoDropTM 2000 (Thermo Fisher Scientific), spectrophotometry was used to quantify the concentration of the samples. GoScriptTM reverse transcriptase (Promega Inc., Madison, WI, USA) was performed for reverse transcription of RNA under the same reaction conditions as previously reported.²⁵ Maxima SYBR Green/ROX qPCR Master Mix (MBI Fermentas, Germany) was used for real-time PCR (qRT-PCR) (HT7500 System, Applied Biosystems, Foster, USA). To calculate the relative expression, the 2^–△Ct algorithm was applied. GAPDH was amplified as RNA integrity controls (6PGDF：5’-ATTCGGAAGGCACTCTACG, 6PGDR: 5’-TGAGAGTCCAGCCAAACTC, GAPDHF: 5’-GTGATGGGATTTCCATTGAT, GAPDHR: 5’-GTGATGGGATTTCCATTGAT).

Protein Extraction and Western Blot

The cells were washed twice with ice-cold PBS and collected, pellets of cells were resuspended in an appropriate volume of lysis buffer. Then the cells were incubated for 5 min at 37 °C and quickly aspirated several times with a 1 mL syringe to make them fully lysed. NanoDropTM 2000 (Thermo Fisher Scientific) was applied to determine the protein concentrations. Western blot was carried out as previously described after the total proteins had been extracted.²⁶ The antibodies of 6PGD (1：1000, ab210702, Abcam, UK), GAPDH (1：1000, AT-5009, Beyotime, Haimen, China), HRP-conjugated goat anti-rabbit (1：5000, #21336496, Biosharp, China) or goat anti-mouse (1：5000, #21336156, Biosharp, China) were used in this experiment.

Evaluation of the Enzyme Activity of 6PGD

According to the protocol described, the 6PGD Activity Colorimetric Assay Kit (MAK015,

Sigma–Aldrich, USA) was applied to measure the enzyme activity. In this assay, NADPH produced during the conversion from 6PGD to 6-phosphogluconate converted a colorless probe into a colored product visible at the absorbance of 450 nm.

SiRNA Transfection

The cells were washed twice using ice-cold PBS, 6PGD siRNA (Guangzhou Ribobio, Guangzhou, China) was transfected into MDA-MB-231 and BT-549 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) at a dose of 10 nM. Existing medium was replaced with a fresh one after 6 h and incubated at 37 °C with 5% CO₂. RT-qPCR and Western blot were performed to validate the transfection efficacy after cell culture for 48 h.

Measuring the Levels of Lactate and NADPH

The Lactate Assay Fluorometric Kit (K607-100, BioVision, US) was utilized to measure the synthesis of cellular lactate, while NADPH colorimetric quantification kits (ab186031, Abcam, UK) were applied to assess NADPH's level. All tests were conducted precisely in accordance with the manufacturer's instructions.

Statistical Analyses

All the experiments were repeated at least three times. Data were presented as the means ± SD. The data were analyzed using one-way analysis of variance and two-tailed unpaired Student's t tests. GraphPad (Version 9.3.1, GraphPad Prism Software, USA) for the statistical analysis. P-value <0.05 was defined as statistically significant. Differences were considered significant at *P < .05, **P < .01 and ***P < .001, ****P < .0001.

Results

Establishment of Epirubicin-Resistant Human TNBC Cells via Gradually Increasing Epirubicin Concentration

To confirm whether 6PGD played a role in the response of human TNBC cells to epirubicin, we first constructed epirubicin-resistant cell lines of TNBC cells, namely MDA-MB-231-r (231r) and BT-549-r (549r). As shown in Figure 1a, the parental cells were cultured with epirubicin at doses that gradually increased from 20 nM to 320 nM for more than 60 days before producing the epirubicin-resistant cell lines. Furthermore, the parental cells and epirubicin-resistant cells were treated with 0.5 μM epirubicin for 72 h, and then cell proliferation and apoptosis were measured. We discovered from the results of CCK8 and FC of Annexin that the potential of epirubicin to induce apoptosis and inhibit proliferation was significantly diminished in epirubicin-resistant cells when compared to the parental cells (Figure 1b and 1c). This indicated that the cells developed effective resistance to epirubicin during prolonged treatment. Importantly, we observed a markedly upregulated expression level of 6PGD in resistant cell lines when compared to parental cell lines of TNBC cells (Figure 1d).

Figure 1.

Comparison of proliferation and apoptosis between the parental cells and epirubicin-resistant cells. (a) Establishing the TNBC cell lines MDA-MB-231 and BT-549 resistant to epirubicin. (b) Proliferation of both cell lines after treatment with 0.5 µM epirubicin for 72 h. (c) Apoptosis of both cell lines after treatment with 0.5 µM epirubicin for 72 h. (d) Expression level of 6PGD in parental and resistant cell lines. All experiments were repeated independently at least three times, * represents statistically significant (*p < .05; **p < .01; ***p < .001; ****p < .0001).

Inhibiting 6PGD Effectively Inhibited the Growth and Survival of Epirubicin-Resistant Breast Cancer Cells

To investigate the effect of 6PGD in both parental and epirubicin-resistant cell lines, we examined the growth and survival of 6PGD-inhibited cells. A pharmacological inhibitor of 6PGD, physcion,²⁷ significantly reduced 6PGD enzyme activity (Figure 2a), declined cell growth (Figure 2b) and suppressed survival (Figure 2c) of both epirubicin-resistant cells and the parental cells in a dose-dependent manner. In line with the results obtained from pharmacological inhibition, we examined the genetic approach via depleting 6PGD from cells. A significant reduction of 6PGD mRNA (Figure 2d) and protein level (Figure 2e) in epirubicin-resistant cell lines transfected with 6PGD siRNA was detected. Moreover, we found that knockdown of 6PGD significantly reduced cell proliferation (Figure 2f) and increased apoptosis (Figure 2g) in a group of parental cell lines. These findings demonstrated that 6PGD inhibition efficiently targeted TNBC cells with a lower sensitivity to epirubicin.

Figure 2.

6PGD inhibition effectively influenced tumor progression. (a) Relative activity of 6PGD in various concentrations of its inhibitor (Physcion, µM). (b) Proliferation of two kinds of cell lines after physcion inhibition of 6PGD. (c) Apoptosis of two kinds of cell lines, in which 6PGD was inhibited by physcion. (d) and (e) The transfection efficacy of 6PGD knockdown cells (231r and 549r cells) was evaluated by RT-qPCR and Western blot. (f) and (g) Proliferation and apoptosis of cells in which 6PGD was effectively knocked down by siRNA. All the experiments were independently repeated at least three times, * represents statistically significant (*p < .05; **p < .01; ***p < .001; ****p < .0001).

6PGD Inhibition Significantly Sensitized the Epirubicin-Resistant Cell Lines to Epirubicin

To study whether the 6PGD inhibition sensitized epirubicin-resistant cells to epirubicin, we analyzed the synergy of 6PGD inhibition with epirubicin in the parental cell lines and the epirubicin-resistant cell lines. It has been found that 6PGD inhibition by 20 µM physcion alone suppressed the cell growth of both the parental cell lines and the epirubicin-resistant cell lines, with much better efficacy in the epirubicin-resistant cell lines. Epirubicin alone reduced the proliferation of tumor cells more strongly in the parental cell lines than in the epirubicin-resistant cell lines, which again confirmed a successful establishment of epirubicin-resistant cell lines. Interestingly, 6PGD inhibition in combination with epirubicin showed a better effect on suppressing the proliferation than treatment with epirubicin or physcion alone in TNBC-resistant cell lines (Figure 3a). This demonstrated that inhibition of 6PGD enhanced the sensitivity of epirubicin-resistant cells to epirubicin stimulation and further inhibited the proliferation ability of the tumor cells. Meanwhile, using epirubicin alone caused more apoptosis in the parental cell lines than in the resistant cell lines, whereas 6PGD inhibition by 20 µM physcion alone increased apoptosis in TNBC-resistant cells than in parental cells. More importantly, the combination of 6PGD inhibition and epirubicin significantly increased apoptosis efficacy in epirubicin-resistant cell lines (Figure 3b). These findings clearly showed that inhibiting 6PGD together with epirubicin notably increased the sensitivity of epirubicin-resistant cells to epirubicin.

Figure 3.

Combination 6PGD inhibition with epirubicin significantly influenced growth and apoptosis of TNBC cells. (a) Proliferation of the parental cell lines and the resistant cell lines in the kinds of treatments. (b) Apoptosis of the parental cell lines and the resistant cell lines in the kinds of treatments. con: control; E+: epirubicin (0.5 µM) treated; P+: physcion (20 µM) treated; E + P: combination treated of epirubicin (0.5 µM) with physcion (20 µM); 231p: MDA-MB-231 parental cells; 549p: BT-549 parental cells; 231r: MDA-MB-231 eripubicin-resistant cells; 549r: BT-549 eripubicin-resistant cells. All the experiments were independently repeated at least three times, * represents statistically significant (*p < .05; **p < .01; ***p < .001; ****p < .0001).

6PGD Inhibition Disrupted Metabolic Reprogramming in Epirubicin-Resistant TNBC Cells

In PPP, 6PGD is a crucial enzyme that transforms 6PG into ribulose-5-phosphate using NADP+ as a cofactor, which is the major source of NADPH in cells. Previous studies have shown that the knockdown of 6PGD in lung cancer H1975 cells could attenuate cell proliferation and tumor size in xenograft mice. However, suppression of 6PGD in these cells does not cause defects in the oxidative PPP or affect the intracellular levels of NADPH and lactate.²⁸ To explore the underlying mechanisms of 6PGD inhibition in epirubicin-resistant TNBC cells, we first compared the levels of NADPH and lactate in the parental cells and the epirubicin-resistant cells. It could be found that both levels of NADPH and lactate increased in epirubicin-resistant cells than in parental cells, which was consistent with more 6PGD in the resistant cell lines (Figure 4a and 4b). We then investigated the levels of NADPH after 6PGD inhibition. The results demonstrated that 6PGD inhibition lowered the level of NADPH in epirubicin-resistant cells (Figure 4c), suggesting that 6PGD inhibition disrupts NADPH homeostasis. In addition, 6PGD inhibition significantly reduced lactate levels (Figure 4d), indicating that 6PGD is involved in metabolic reprogramming in epirubicin-resistant TNBC cells.

Figure 4.

6PGD inhibition decreased NADPH and lactate in metabolism reprogramming. (a) NADPH levels in parental and resistant cells were compared. (b) Lactate levels in parental and resistant cells were compared. (c) 6PGD inhibition by physcion decreased the level of NADPH in epirubicin-resistant cells. (d) 6PGD inhibition by physcion decreased the level of lactate in epirubicin-resistant cells. All experiments were independently repeated at least three times, * represents statistically significant (*p < .05; **p < .01; ***p < .001; ****p < .0001).

Discussion

The current majority of TNBC systemic treatments are still standard chemotherapy, and many patients would develop cytotoxic drug resistance.^8,29 Once drug resistance occurs, patients’ prognosis could be significantly poor.^9,10 Molecular testing can identify a variety of biomarkers to predict TNBC patients’ resistance to general chemotherapy or to particular chemotherapeutic medications. Circular RNAs (CircRNAs) and miR-449 family have been regarded as predictive biomarkers of anthracycline resistance. Previous research³⁰ examined the processes and roles of circUBE2D2 (has_circ_0005728) in TNBC development and chemoresistance. The miR-449 family³¹ mediates doxorubicin resistance in TNBC cells by governing cell cycle factors. Epirubicin, which was licensed in 1999 for the adjuvant treatment of BC,³² is an epimer of doxorubicin and therefore has a similar therapeutic profile.³³ However, increasing use of epirubicin in TNBC treatment in recent years has greatly promoted the prevalence of epirubicin resistance. Nevertheless, there is still a lack of study in this area. Investigating the effects and possible causes of epirubicin resistance in the chemotherapeutic treatment of TNBC is crucial. Meanwhile, 6PGD expression has been found to be upregulated in a number of malignancies, involving cervical, thyroid, colon, hepatocellular carcinoma and BCs in comparison to normal tissues.^21,34–36 Previous findings showed that 6PGD inhibition sensitizes hepatocellular carcinoma to chemotherapy.³⁷ Notably, our study confirmed that 6PGD expression gradually elevated during the development of drug resistance of TNBC cells. Inhibition of 6PGD inhibited the growth of both chemotherapy-sensitive cells and drug-resistant cells and promoted their apoptosis. As we expected, the combination of 6PGD inhibition with epirubicin achieved a greater efficacy on the resistant cells than using epirubicin alone, which corresponded to the results of recent studies that 6PGD inhibition reversed cisplatin resistance in lung and ovarian cancers²² and supported conventional treatment for leukemia.³⁸

Moreover, a majority of studies agreed that altered cellular metabolism is a key feature of cancers.³⁹ In turn, increased aerobic glycolysis, PPP, and fatty acid biosynthesis processes of cellular metabolism further promote tumor progression and drug resistance.^40–42 As in the Warburg effect, cancer cells tend to metabolize glucose to lactate even under aerobic conditions, thereby appearing to differ from nonmalignant cells in the way they metabolize, which points to new therapeutic opportunities.⁴³ As in the PPP, 6PGD has been proven to meet several biosynthetic and energetic requirements of cancer cells. Due to its overexpression in some tumors, 6PGD has been identified as a possible therapeutic target for cancer therapy in recent years.⁴⁴ In our study, we found a significant increase in lactate and NADPH content in drug-resistant cell lines, and that inhibition of 6PGD reversed such a process. This demonstrated that 6PGD was indispensable in the process of metabolic reprogramming toward epirubicin in TNBC cells, suggesting that upregulated 6PGD was required for NADPH level in epirubicin-resistant cells. These results supported the important roles of 6PGD in the metabolism of BC cells as well as in the metabolic reprogramming process of TNBC-resistant cell lines. 6PGD could be a potential option for reversing chemotherapy resistance. Our study might provide a novel strategy of treatment for TNBC patients who are resistant to chemotherapy and improve patients’ OS. The limitation of our study was that we were not able to reveal more deeply the mechanism of targeting 6PGD in reversing chemoresistance in TNBC cells. However, to the best of our knowledge, the present research showed its significance in revealing the anticancer potential of reversing epirubicin resistance in TNBC cells via 6PGD inhibition. The correlation between 6PGD expression level and prognosis in TNBC-resistant patients deserves further study, and the safety of 6PGD inhibitors in normal cells, animals, and clinical practice should be validated so as to provide a safe and effective treatment for TNBC patients.

Conclusions

In summary, this study investigated the important role of 6PGD in promoting TNBC progression and attenuating chemotherapy response efficacy of chemotherapy-resistant cells. Inhibition of 6PGD and epirubicin exerted synergistic effects on resistant cells, effectively increasing the sensitivity of resistant cells to chemotherapeutic agents through metabolic remodeling. Therefore, 6PGD might be a potential and important metabolic target in clinical applications such as reversing chemotherapy resistance in TNBC and tumor therapies.

Footnotes

Abbreviations

Acknowledgments

The authors thank the staff of the Chongqing Key Laboratory of Molecular Oncology and Epigenetics for their help in technical assistance.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Qiao Cheng

References

Britt

Cuzick

Phillips

. Key steps for effective breast cancer prevention. Nat Rev Cancer. 2020;20(8):417‐436. http://doi.org/10.1038/s41568-020-0266-x

Yin

Jiang-Jie Duan

Xiu-Wu Bian

, et al. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Res. 2020;22(1):61. http://doi.org/10.1186/s13058-020-01296-5

Wolff

Hammond ME

Hicks DG

, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013;31(31):3997‐4013. http://doi.org/10.1200/jco.2013.50.9984

Ferrari

Cristian Scatena

Matteo Ghilli

, et al. Molecular mechanisms, biomarkers and emerging therapies for chemotherapy resistant TNBC. Int J Mol Sci. 2022;23(3):1665. http://doi.org/10.3390/ijms23031665

Dent

Trudeau M

Pritchard KI

, et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007;13(15 Pt 1):4429‐4434. http://doi.org/10.1158/1078-0432.Ccr-06-3045

Edwardson

Narendrula R

Chewchuk S

, et al. Role of drug metabolism in the cytotoxicity and clinical efficacy of anthracyclines. Curr Drug Metab. 2015;16(6):412‐426. http://doi.org/10.2174/1389200216888150915112039

Lebert

Lester R

Powell E

, et al. Advances in the systemic treatment of triple-negative breast cancer. Curr Oncol. 2018;25(Suppl 1):S142-S150. http://doi.org/10.3747/co.25.3954

Kim

Gao R

Sei E

, et al. Chemoresistance evolution in triple-negative breast cancer delineated by single-cell sequencing. Cell. 2018;173(4):879‐893.e13. http://doi.org/10.1016/j.cell.2018.03.041

Foulkes

Smith

Reis-Filho

. Triple-negative breast cancer. N Engl J Med. 2010;363(20):1938‐1948. http://doi.org/10.1056/NEJMra1001389

10.

Liedtke

Mazouni C

Hess KR

, et al. Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer. J Clin Oncol. 2008;26(8):1275‐1281. http://doi.org/10.1200/jco.2007.14.4147

11.

Longley

Johnston

. Molecular mechanisms of drug resistance. J Pathol. 2005;205(2):275‐292. http://doi.org/10.1002/path.1706

12.

NCCN Guidelines 2021. [web] accessed on 15 January 2022; https://www.nccn.org/login?ReturnURL=https://www.nccn.org/professionals/physician_gls/pdf/breast.pdf.

13.

ESMO Guidelines. accessed on 15 January 2022; https://www.esmo.org/guidelines/guidelines-by-topic/breast-cancer.

14.

Nedeljković

Damjanović

. Mechanisms of chemotherapy resistance in triple-negative breast cancer-how we can rise to the challenge. Cells. 2019;8(9):957. http://doi.org/10.3390/cells8090957

15.

Lin

Elf S

Shan C

, et al. 6-Phosphogluconate dehydrogenase links oxidative PPP, lipogenesis and tumour growth by inhibiting LKB1-AMPK signalling. Nat Cell Biol. 2015;17(11):1484‐1496. http://doi.org/10.1038/ncb3255

16.

Sarfraz

Rasul A

Hussain G

, et al. 6-Phosphogluconate dehydrogenase fuels multiple aspects of cancer cells: from cancer initiation to metastasis and chemoresistance. Biofactors. 2020;46(4):550‐562. http://doi.org/10.1002/biof.1624

17.

Cao

Sun X

Zhang X

, et al. 6PGD upregulation is associated with chemo- and immuno-resistance of renal cell carcinoma via AMPK signaling-dependent NADPH-mediated metabolic reprograming. Am J Med Sci. 2020;360(3):279‐286. http://doi.org/10.1016/j.amjms.2020.06.014

18.

Cheng

. Inhibiting 6-phosphogluconate dehydrogenase reverses doxorubicin resistance in anaplastic thyroid cancer via inhibiting NADPH-dependent metabolic reprogramming. Biochem Biophys Res Commun. 2018;498(4):912‐917. http://doi.org/10.1016/j.bbrc.2018.03.079

19.

Shan

Elf S

Ji Q

, et al. Lysine acetylation activates 6-phosphogluconate dehydrogenase to promote tumor growth. Mol Cell. 2014;55(4):552‐565. http://doi.org/10.1016/j.molcel.2014.06.020

20.

Smith

King RJ

Meggitt BF

, et al. Enzyme activity, acidic nuclear proteins, and prognosis in human breast cancer. Br Med J. 1970;2(5711):698‐701. http://doi.org/10.1136/bmj.2.5711.698

21.

Guo

Xiang Z

Zhang Y

, et al. Inhibiting 6-phosphogluconate dehydrogenase enhances chemotherapy efficacy in cervical cancer via AMPK-independent inhibition of RhoA and Rac1. Clin Transl Oncol. 2019;21(4):404‐411. http://doi.org/10.1007/s12094-018-1937-x

22.

Zheng

Feng Q

Liu J

, et al. Inhibition of 6-phosphogluconate dehydrogenase reverses cisplatin resistance in ovarian and lung cancer. Front Pharmacol. 2017;8:421. http://doi.org/10.3389/fphar.2017.00421

23.

Elf

Lin R

Xia S

, et al. Targeting 6-phosphogluconate dehydrogenase in the oxidative PPP sensitizes leukemia cells to antimalarial agent dihydroartemisinin. Oncogene. 2017;36(2):254‐262. http://doi.org/10.1038/onc.2016.196

24.

Liu

Li W

Tao B

, et al. Tyrosine phosphorylation activates 6-phosphogluconate dehydrogenase and promotes tumor growth and radiation resistance. Nat Commun. 2019 l;10(1):991. http://doi.org/10.1038/s41467-019-08921-8

25.

Feng

Wu M

Li S

, et al. The epigenetically downregulated factor CYGB suppresses breast cancer through inhibition of glucose metabolism. J Exp Clin Cancer Res. 2018;37(1):313. http://doi.org/10.1186/s13046-018-0979-9

26.

Huang J

Zeng B

, et al. PSMD2 regulates breast cancer cell proliferation and cell cycle progression by modulating p21 and p27 proteasomal degradation. Cancer Lett. 2018;430:109‐122. http://doi.org/10.1016/j.canlet.2018.05.018

27.

Ericsson M

Rabasha B

, et al. 6-Phosphogluconate dehydrogenase links cytosolic carbohydrate metabolism to protein secretion via modulation of glutathione levels. Cell Chem Biol. 2019;26(9):1306‐1314.e5. http://doi.org/10.1016/j.chembiol.2019.05.006

28.

Sukhatme

Chan

. Glycolytic cancer cells lacking 6-phosphogluconate dehydrogenase metabolize glucose to induce senescence. FEBS Lett. 2012;586(16):2389‐2395. http://doi.org/10.1016/j.febslet.2012.05.052

29.

Almendro

Cheng Y

Randles A

, et al. Inference of tumor evolution during chemotherapy by computational modeling and in situ analysis of genetic and phenotypic cellular diversity. Cell Rep. 2014;6(3):514‐527. http://doi.org/10.1016/j.celrep.2013.12.041

30.

Dou

Ren X

Han M

, et al. CircUBE2D2 (hsa_circ_0005728) promotes cell proliferation, metastasis and chemoresistance in triple-negative breast cancer by regulating miR-512-3p/CDCA3 axis. Cancer Cell Int. 2020;20:454. http://doi.org/10.1186/s12935-020-01547-7

31.

Tormo

Ballester S

Adam-Artigues A

, et al. The miRNA-449 family mediates doxorubicin resistance in triple-negative breast cancer by regulating cell cycle factors. Sci Rep. 2019;9(1):5316. http://doi.org/10.1038/s41598-019-41472-y

32.

Khasraw

Bell

Dang

. Epirubicin: Is it like doxorubicin in breast cancer? A clinical review. Breast. 2012;21(2):142‐149. http://doi.org/10.1016/j.breast.2011.12.012

33.

Findlay

Tonkin K

Crump M

, et al. A dose escalation trial of adjuvant cyclophosphamide and epirubicin in combination with 5-fluorouracil using G-CSF support for premenopausal women with breast cancer involving four or more positive nodes. Ann Oncol. 2007;18(10):1646‐1651. http://doi.org/10.1093/annonc/mdm277

34.

Yang

Peng

Huang

. Inhibiting 6-phosphogluconate dehydrogenase selectively targets breast cancer through AMPK activation. Clin Transl Oncol. 2018;20(9):1145‐1152. http://doi.org/10.1007/s12094-018-1833-4

35.

Giusti

Lacconi P

Ciregia F

, et al. Fine-needle aspiration of thyroid nodules: proteomic analysis to identify cancer biomarkers. J Proteome Res. 2008;7(9):4079‐4088. http://doi.org/10.1021/pr8000404

36.

Nordenberg

Aviram R

Beery E

, et al. Inhibition of 6-phosphogluconate dehydrogenase by glucose 1,6-diphosphate in human normal and malignant colon extracts. Cancer Lett. 1984;23(2):193‐199. http://doi.org/10.1016/0304-3835(84)90154-x

37.

Chen

Wu D

Bao L

, et al. 6PGD inhibition sensitizes hepatocellular carcinoma to chemotherapy via AMPK activation and metabolic reprogramming. Biomed Pharmacother. 2019;111:1353‐1358. http://doi.org/10.1016/j.biopha.2019.01.028

38.

Bhanot

Weisberg E

Reddy MM

, et al. Acute myeloid leukemia cells require 6-phosphogluconate dehydrogenase for cell growth and NADPH-dependent metabolic reprogramming. Oncotarget. 2017;8(40):67639‐67650. http://doi.org/10.18632/oncotarget.18797

39.

Gillis

Hinneh JA

Ryan NK

, et al. A feedback loop between the androgen receptor and 6-phosphogluoconate dehydrogenase (6PGD) drives prostate cancer growth. Elife. 2021;10. http://doi.org/10.7554/eLife.62592

40.

Vander Heiden

DeBerardinis

. Understanding the intersections between metabolism and cancer biology. Cell. 2017;168(4):657‐669. http://doi.org/10.1016/j.cell.2016.12.039

41.

Hoy

Nagarajan

Butler

. Tumour fatty acid metabolism in the context of therapy resistance and obesity. Nat Rev Cancer. 2021;21(12):753‐766. http://doi.org/10.1038/s41568-021-00388-4

42.

Currie

Schulze A

Zechner R

, et al. Cellular fatty acid metabolism and cancer. Cell Metab. 2013;18(2):153‐161. http://doi.org/10.1016/j.cmet.2013.05.017

43.

Icard

Shulman S

Farhat D

, et al.

How the Warburg effect supports aggressiveness and drug resistance of cancer cells?

Drug Resist Updat. 2018;38:1‐11. http://doi.org/10.1016/j.drup.2018.03.001

44.

Zara