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PDF file - 69K, Supplementary Table 1: Emerging metabolic targets and the inhibitors that have shown promise to overcome cancer cell resistance to therapeutics.

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Supplementary Figure 3 from Overcoming Trastuzumab Resistance in Breast Cancer by Targeting Dysregulated Glucose Metabolism

Supplementary Figure 1 from Overcoming Trastuzumab Resistance in Breast Cancer by Targeting Dysregulated Glucose Metabolism

ErbB2 is overexpressed in 15-20% of breast cancer, which is associated with malignancy and poor prognosis. We previously reported that ErbB2 supports malignant progression of breast cancer by upregulating lactate dehydrogenase A (LDHA), an important enzyme in glycolysis. However, whether ErbB2 promotes breast cancer progression through other glycolytic enzymes remains unclear. Hexokinase 1 (HK1) and hexokinase 2 (HK2) are the first rate-limiting enzymes of glycolysis and both of them are increased in breast cancer. Here, we aim to investigate whether ErbB2 upregulates HK1 and HK2 and the role of HK1 and HK2 in the malignant progression of ErbB2-overexpressing breast cancer. In current study, we found that the mRNA level of ErbB2 was positively correlated with that of HK1 and HK2, respectively. Moreover, ErbB2 upregulated the protein levels of HK1 and HK2 in breast cancer cells. We also found that both siHK1 and siHK2 significantly inhibited the proliferation, migration and invasion of ErbB2-overexpressing breast cancer cells. Taken together, our findings suggested that ErbB2 promoted the malignant progression of breast cancer cells by upregulating HK1 and HK2, and HK1 and HK2 might serve as promising therapeutic targets for ErbB2-overexpressing breast cancer.

Cerulenin is a fungal metabolite and a specific inhibitor of fatty acid synthase (FASN), which has shown a potential anticancer activity. 20-25% of breast cancer patients with ErbB2-overexpressing develop resistance to treatment. Therefore, it is urgent to find an effective new target for the treatment of ErbB2-overexpressing breast cancer. Our previous study found that cerulenin inhibits the glycolysis and migration of SK-BR-3 cells, but the effect of cerulenin on other malignant phenotypes of breast cancer is still unknown. Furthermore, the mechanism by which cerulenin displays its inhibitory effects is not fully understood. In this study, we systematically investigate the inhibitory effects of cerulenin on proliferation, migration, invasion and glycolysis of ErbB2-overexpressing breast cancer cells and its molecular mechanism. We found that cerulenin obviously suppresses the proliferation, migration, invasion as well as glycolysis. Through bioinformatic analyses, we found that PKM2 might be a target of cerulenin. In addition, ErbB2 and its signaling pathway upregulated PKM2 protein levels. Furthermore, we demonstrated that cerulenin downregulated the protein levels of ErbB2, PKM2 and EMT markers (MMP9, MMP2 and Snail2) in a dose- and time-dependent manner. Finally, the inhibitory of cerulenin on colony formation, migration, invasion and glycolysis, as well as protein levels of EMT markers were rescued by replenishing with PKM2. These findings illustrated that cerulenin inhibits proliferation, migration, invasion and glycolysis by targeting ErbB2/PKM2 pathway in ErbB2-overexpressing breast cancer cells.

乳腺癌是女性最常见的恶性肿瘤,随着肿瘤代谢研究的深入,脂代谢在乳腺癌发生发展中的作用越来越受到重视.内源性脂肪酸合成是肿瘤细胞脂肪酸的主要来源,也是肿瘤细胞的一个重要特征,靶向内源性脂肪酸合成治疗乳腺癌已经成为了一个研究热点.脂肪酸合成途径的相关酶包括ATP-柠檬酸裂解酶(adenosine triphosphate-citrate lyase,ACL)、乙酰辅酶A羧化酶1(acetyl-CoA carboxylase 1,ACC1)、脂肪酸合酶(fatty acid synthase,FASN)和硬脂酰辅酶A去饱和酶1(stearoyl-CoA desaturase-1,SCD1)在乳腺癌发生发展中发挥重要的作用,成为了乳腺癌治疗的新靶点.本文综述了ACL、ACC1、FASN和SCD1与乳腺癌的临床相关性及意义,在乳腺癌发生发展中的作用和分子机制及其抑制剂治疗乳腺癌的研究进展.

ErbB2 is overexpressed in approximately 25% of breast cancer cases and promotes metastatic potential. We previously reported that ErbB2 promoted glycolysis via heat shock factor 1 (HSF1)/lactate dehydrogenase A (LDHA) axis and ErbB2-mediated glycolysis was required for the growth of breast cancer cells. However, the importance of HSF1/LDHA axis-mediated glycolysis in ErbB2-enhanced metastatic potential remains to be elucidated. In this study, we investigated the effect of HSF1/LDHA axis-mediated glycolysis on migration and invasion in breast cancer cells. Firstly, we demonstrated that ErbB2-mediated migration and invasion were dependent on glycolysis in breast cancer cells. Secondly, we found that HSF1/LDHA axis played an important role in glycolysis, which contributed to ErbB2-enhanced migration and invasion. Finally, we showed that ErbB2 was positively correlated with HSF1/LDHA axis in invasive breast cancer patients via GEO analysis. Taken together, ErbB2 promoted metastatic potential of breast cancer cells via HSF1/LDHA axis-mediated glycolysis. And our findings indicated that targeting HSF1/LDHA axis may be a promising strategy to treat ErbB2-overexpressing breast cancer patients.

The incidence of breast cancer ranks at the top of female malignant tumors in China. Metastasis remains the main cause of death among breast cancer patients. The overexpression of ErbB2 is closely related to the metastasis and poor prognosis of breast cancer patients. Therefore, ErbB2 is an important clinical therapeutic target of breast cancer. However, the molecular mechanism of ErbB2 promoting breast cancer metastasis has not been studied clearly. Stearoyl-CoA desaturase 1 (SCD1) is a key enzyme in catalyzing the conversion of saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs). SCD1 is overexpressed in breast cancer, and its overexpression is an indicator of poor prognosis in breast cancer patients. However, the role of SCD1 in ErbB2-overexpressing breast cancer metastasis has not been reported. In this study, we investigated the role of SCD1 in the migration and invasion of ErbB2-overexpressing breast cancer cells and its molecular mechanism. First, we demonstrated that ErbB2 upregulates the expression of SCD1. Second, we found that SCD1 and its catalytic product oleic acid played crucial roles in migration and invasion of ErbB2-overexpressing breast cancer cells. Finally, we found that in breast cancer cells, ErbB2 upregulated SCD1 through lactate dehydrogenase A (LDHA). To sum up, upregulation of SCD1 by ErbB2 via LDHA promotes the migration and invasion of breast cancer cells.

目的 在体外研究小干扰RNA(siRNA)沉默己糖激酶2(HK2)对人乳腺癌SK-BR-3细胞增殖、迁移和侵袭的影响及其分子机制.方法 在SK-BR-3细胞中分别转染siRNA阴性、siHK2-A和siHK2-B,分别采用荧光定量PCR和Western Blot法检测HK2 mRNA和蛋白的表达水平;MTT法检测细胞的增殖;Transwell小室检测细胞的迁移和侵袭;葡萄糖检测试剂盒检测细胞对葡萄糖的摄取.结果 与阴性对照组比较,siHK2能显著降低SK-BR-3细胞的HK2 mRNA及蛋白水平,抑制细胞增殖、迁移和侵袭能力,减少细胞对葡萄糖的摄取.结论 siHK2可能通过降低葡萄糖摄取来抑制SK-BR-3细胞的增殖、迁移和侵袭.

Background: Numerous studies on acute ischemic stroke (AIS) have been conducted at low-altitude regions, and the related findings have been used to guide clinical management. However, corresponding studies at high altitude are few. This study aimed to analyse the clinical characteristics of AIS patients at high-altitude regions through a hospital-based comparative study between Tibet and Beijing.Methods: This study included the diagnoses of AIS patients from People’s Hospital of Tibet Autonomous Region (PHOTAR) and Peking University First Hospital (PUFH) between 1 January 2014 and 31 December 2017, where data including patient demographics, treatment time, onset season, risk factors, infarction location, laboratory data, image examination results, treatments, and AIS subtype were collected and compared. Continuous and categorical variables were analysed with a two-sample t-test or Wilcoxon rank sum test and chi-square test, respectively. Significant risk factors were examined with binary logistic regression analysis.Results: In total, 236 and 1021 inpatients from PHOTAR and PUFH were included, respectively. The PHOTAR patients were younger than the PUFH patients (P<0.001). Young adult stroke, erythrocytosis, and hyperhomocysteinemia were more frequent in PHOTAR patients (all P<0.001). Other vascular risk factors, including hypertension, diabetes mellitus, hyperlipidaemia, and smoking and alcohol consumption history, were less prevalent in PHOTAR patients than in PUFH patients. The rate of intravenous thrombolysis and the rate of within intravenous thrombolysis window time were also lower in PHOTAR patients (both P<0.001). The PHOTAR group also tended to have anterior circulation infarction. Erythrocytosis and hyperhomocysteinemia were independent risk factors in PHOTAR, and young adults accounted for a larger proportion of stroke cases.Conclusion: In Tibet, AIS patients were relatively younger, and anterior circulation infarctions were more common. Erythrocytosis and hyperhomocysteinemia may contribute to these differences. Here, young adult stroke also accounted for a higher proportion, and this may be associated with erythrocytosis. Our findings present the first hospital-based comparative study in Tibet and may contribute to policies for stroke prevention in this region.

Objective To investigate the heregulin-β1 (HRG-β1)-induced expression of stearoyl-CoA desaturase-1 (SCD1) and the effect of its activity of SCD1 on the proliferation, migration and invasion of breast cancer cells MCF7 and SK-BR-3. Methods MCF7 and SK-BR-3 cells were treated with control (PBS) or HRG-β1 at different concentrations and for different times. Then RT-qPCR and Western blotting were used to detect the mRNA and protein expression levels of SCD1. The wound healing assay and Transwell assay were employed to measure the migration and invasion of MCF7 and SK-BR-3 cells treated with PBS, HRG-β1, A939572 and oleic acid (OA). Results HRG-β1 increased both SCD1 mRNA and protein levels in MCF7 and SK-BR-3 cells at a time-dose dependent manner. Compared with the control group, HRG-β1 promoted cell proliferation in MCF7 and SK-BR-3 cells (P < 0.01), while proliferation rate was decreased after A939572 treatment (P < 0.01). Compared with the control group, HRG-β1 significantly increased the wound healing rates and invaded cell numbers of MCF7 and SK-BR-3 cells (P < 0.01). The wound healing rates and invaded cell numbers of both cell lines were significantly decreased after HRG-β1+A939572 treatment (P < 0.01). The addition of OA rescued the migration and invasion of both cell lines which were weakened by A939572 (P < 0.01). Conclusions HRG-β1 can up-regulate the expression of SCD1, and the protein partially mediates the effect of HRG-β1 in promoting the proliferation, migration and invasion in MCF7 and SK-BR-3 cells.

OBJECTIVE:To investigate the effect of small interfering RNA of lactate dehydrogenase A (siLDHA) on migration and invasion of epidermal growth factor receptor 2 (ErbB2) over expressing breast cancer cell line SK-BR-3, MDA-MB-453 and its molecular mechanism.METHODS:SK-BR-3 and MDA-MB-453 cells were transfected with siLDHA to interfere with the expression of LDHA. The transfection of scramble siRNA was used as negative control. The LDHA protein levels were detected by Western blot ( P<0.01). Cell migration and invasion was detected by Transwell assays. Lactate dehydrogenase (LDH) activity was measured by LDH assay kit. The glucose and lactate concentration in the culture media was determined by glucose and lactate assay kit, respectively, and then glucose uptake and lactate production by the cells were calculated.RESULTS:siLDHA downregulated LDHA protein levels in SK-BR-3 and MDA-MB-453 cells ( P<0.01). Compared with negative control group, siLDHA significantly decreased migration and invasion of SK-BR-3 and MDA-MB-453 cells ( P<0.001). siLDHA reduced LDH activity in SK-BR-3 cells, glucose uptake and lactate production in SK-BR-3 and MDA-MB-453 cells, the difference was significant ( P<0.05).CONCLUSION:Knockdown of LDHA by siRNA inhibits the migration and invasion via downregulation of glycolysis in ErbB2 over expressing breast cancer cell line.

目的 探讨糖酵解抑制剂2-脱氧葡萄糖(2-Deoxy-D-glucose,2-DG)对乳腺癌MCF7/ErbB2细胞增殖、迁移和侵袭的影响及其分子机制.方法 用不同浓度的2-DG(0,0.5,1,2,4,8,16,32 mM)处理MCF7/ErbB2细胞48h后,用MTS试剂盒测定细胞的增殖;用Transwell实验检测不同浓度2-DG(0,0.5,1,2 mM)对MCF7/ErbB2细胞的迁移和侵袭的影响;用不同浓度2-DG(0,0.5,1,2 mM)处理MCF7/ErbB2细胞48h后,用葡萄糖和乳酸检测试剂盒分别测定培养基中的葡萄糖浓度和乳酸浓度,并计算细胞对葡萄糖的摄取量和乳酸生成量;用1 mM 2-DG处理MCF7/ErbB2细胞0、4、8、12、24h后,用Western blot检测己糖激酶Ⅱ(hexokinase Ⅱ,HKII)蛋白表达水平.结果 与2-DG未处理组比较,2-DG(0.5～32 mM)能抑制MCF7/ ErbB2细胞的增殖;2-DG(0.5,1,2 mM)能显著抑制MCF7/ ErbB2细胞的迁移和侵袭(P＜0.001),同时显著降低葡萄糖摄取量和乳酸生成量(P＜0.001),而且上述抑制作用均表现出对2-DG剂量的依赖性;1 mM 2-DG显著下调MCF7/ErbB2细胞中HKII的蛋白水平.结论 2-DG通过下调HKII蛋白表达降低糖酵解水平,从而抑制乳腺癌MCF7/ ErbB2细胞的增殖、迁移和侵袭.

The aim of the present study was to investigate changes in the expression of ErbBs during epithelial-mesenchymal transition (EMT) of breast cancer cells and its association with the expression of fatty acid synthase (FASN). MCF-7-MEK5 cells were used as the experimental model, while MCF-7 cells were used as a control. Tumor cells were implanted into nude mice for in vivo analysis. Cerulenin was used as a FASN inhibitor. Reverse transcription-polymerase chain reaction and western blot analysis were used to detect expression levels of FASN and ErbB1-4. Immunohistochemistry was used to detect the expression of FASN and ErbB1-4 in 58 invasive ductal carcinomas (IDC), as well as their association with clinicopathological characteristics. The expression of FASN and ErbB1-4 in MCF-7-MEK5 cells and tumor tissues increased significantly compared with controls (P<0.001). Inhibition of FASN by cerulenin resulted in a significant decrease in expression of ErbB1, 2 and 4 (P<0.001), whereas there was no evident change in ErbB3. In IDC samples, the expression of FASN and ErbB1-4 increased considerably in lymph node metastases compared with non-lymph node metastases (P<0.05). ErbB2 expression increased in advanced clinical stages (II, III and IV) of IDC and in tumors with larger diameters (P<0.05). The expression of ErbB3 increased in ER-positive tumors (P<0.05). Additionally, a positive association between the expression of FASN and ErbB1, 2 and 4 was observed (P<0.05). FASN activates ErbB1, 2 and 4, and their dimers, which are polymerized via the microstructural domain of the cell membrane. This may initiate EMT and consequentlyincrease the invasion and migration of cancer cells. However, ErbB3 may also affect tumor progression via a FASN-independent pathway.

OBJECTIVES:To explore whether heregulin-β1 (HRG-β1) can induce glycolysis and the role of HRG-β1-induced glycolysis in the migration of human breast cancer cell line MCF7. METHODS:MCF7 cells were treated with PBS (PBS group) or HRG-β1 for 12, 24 and 48 h. Culture media were harvested for glucose uptake and lactate production assays, and cells were collected and lactate dehydrogenase A (LDHA) protein levels were detected by using Western blot. MCF7 cells were treated with PBS (PBS group), HRG-β1 or HRG-β1 plus oxamate (OX) for 24 h. Culture media were harvested for glucose uptake and lactate production assays, and cells were harvested and the protein levels of LDHA was detected by Western blot. The wound healing assay was used to detect the migration of MCF7 cells treated with PBS (PBS group), HRG-β1 or HRG-β1 plus OX for 48 h. RESULTS:MCF7 cells treated with HRG-β1 for 12, 24 and 48 h displayed higher levels of glucose uptake, lactate production and LDHA protein levels when the levels reached the peak at 24 h. The differences of glucose uptake, lactate production and LDHA protein levels between PBS group and HRG-β1 group were statistically significant ( P<0.05). Compared to HRG-β1 group, the glucose uptake of HRG-β1 plus OX treated group was not significantly different ( P>0.05), but the statistically significant decrease of lactate production and LDHA protein levels were noticed ( P<0.01 and P<0.05). When MCF7 cells were scratched for 48 h, the wound healing rate of control group, HRG-β1 group and HRG-β1 plus OX group was (49±5.09)%, (100±2.21)% and (51±4.10)% respectively. The difference of each group was statistically significant ( P<0.001). CONCLUSIONS:HRG-β1 induces glycolysis via upregualtion of LDHA and HRG-β1-induced glycolysis promotes the migration of breast cancer cells line MCF7.

Both fatty acid synthase (FASN) and ErbB2 have been shown to promote breast cancer cell migration. However, the underlying molecular mechanism remains poorly understood and there is no reported evidence that directly links glycolysis to breast cancer cell migration. In this study, we investigated the role of FASN, ErbB2-mediated glycolysis in breast cancer cell migration. First, we compared lactate dehydrogenase A (LDHA) protein levels, glycolysis and cell migration between FASN, ErbB2-overexpressing SK-BR-3 cells and FASN, ErbB2-low-expressing MCF7 cells. Then, SK-BR-3 cells were treated with cerulenin (Cer), an inhibitor of FASN, and ErbB2, LDHA protein levels, glycolysis, and cell migration were detected. Next, we transiently transfected ErbB2 plasmid into MCF7 cells and detected FASN, LDHA protein levels, glycolysis and cell migration. Heregulin-β1 (HRG-β1) is an activator of ErbB2 and 2-deoxyglucose (2-DG) and oxamate (OX) are inhibitors of glycolysis. MCF7 cells were treated with HRG-β1 alone, HRG-β1 plus 2-DG, OX or cerulenin and glycolysis, and cell migration were measured. We found that FASN, ErbB2-high-expressing SK-BR-3 cells displayed higher levels of glycolysis and migration than FASN, ErbB2-low-expressing MCF7 cells. Inhibition of FASN by cerulenin impaired glycolysis and migration in SK-BR-3 cells. Transient overexpression of ErbB2 in MCF7 cells promotes glycolysis and migration. Moreover, 2-deoxyglucose (2-DG), oxamate (OX), or cerulenin partially reverses heregulin-β1 (HRG-β1)-induced glycolysis and migration in MCF7 cells. In conclusion, this study demonstrates that FASN, ErbB2-mediated glycolysis is required for breast cancer cell migration. These novel findings indicate that targeting FASN, ErbB2-mediated glycolysis may be a new approach to reverse breast cancer cell migration.

OBJECTIVE To study the expression of fatty acid synthase (FASN) and adipocyte fatty acid-binding protein (A-FABP) in human infiltrating ductal breast cancer (IDC) tissues and hunman breast cancer cells and the relationship with the clinicopathogical characteristics. To further explore the relationship between FASN and A-FABP, and the relevance of the invasion in cancer cell. METHODS The expression of FASN and A-FABP was detected in 58 cases of human infiltrating ductal breast cancer and 12 cases of human normal breast tissues by immunohistochemistry technique, calculated positive expression percentage according to the number of positive cells percentage and the staining degree of positive sediment. The cell wound-healing assay was applied to detect the invasion of SKBR3 and MCF-7 cells. Western blot was used to detect the expression of FASN and A-FABP in MCF-7 and SKBR3 cells. RESULTS The positive rates of FASN and A-FABP were 8.3% (1/12) and 16.7% (1/6) respectively in 12 cases of normal breast tissues by immunohistochemistry. In 58 cases of IDC tissues, the positive rates of FASN and A-FABP were 72.4% (42/58) and 79.3% (46/58) respectively. The differences of the positive rates of FASN and A-FABP in normal breast and IDC tissues were statistically significant (P<0.01, P2 cm) when compared with lymph node metastasis negative group or the diameter < or =2 cm group, the differences were statistically significant (P<0.01). In IDC group, the expression of FASN correlated with A-FABP (r=0.797, P<0.001), The migration rate of SKBR3 was significantly higher than MCF-7 cell at 12, 24 h (P<0.05), FASN expression in SKBR3 was higher than that in MCF-7. CONCLUSION FASN and A-FABP might associated with the lymph node metastasis and tumor size, and there was correlation between FASN and A-FABP in human IDC tissues. FASN may associated with the invasion and metastasis in breast cancer cells.