Background

Breast cancer is the most commonly diagnosed cancer among women and its metastases results in poor survival rates in patients. The ability to alter metabolism is a key attribute cancer cells use to survive within different metastatic microenvironments and cause organ failure. We hypothesized that evaluation of metabolic alterations within tumor cells could provide a better understanding of cancer metastasis. Therefore, to investigate underlying metabolic alterations during metastases, we utilized human MDA-MB-231 and mouse 4T1 models that closely mimic human breast cancer metastasis.

Methods

The glycolysis and glutamine pathway-related changes were examined in bone metastatic cells by XF-24 extracellular flux analyzer and western blotting. The expression levels of genes related to metabolism were examined by PCR arrays.

Results

The MDA-MB-231 cells isolated after bone metastases showed reduced glucose uptake and glycolysis compared to parental cells, suggesting that these cells could alter metabolic requirements for survival. To understand these metabolic changes, we investigated glutamine, a common and naturally occurring non-essential amino acid. Interestingly, in reduced glucose conditions both cell lines showed dependence on glutamine for cell survival, and with glutamine withdrawal significantly increasing apoptotic cell death. Glutamine was also critical for normal cell proliferation even in the presence of high glucose concentrations. To further understand this metabolic switch in metastatic cells, we examined the genes related to metabolism and identified a more than seven-fold downregulation of protein kinase C zeta (PKC-ζ) expression levels in bone-derived MDA-MB-231 cells compared to the parental population. The PKC-ζ levels were also significantly reduced in metastatic 4T1 cells compared to non-metastatic MT1A2 cells. Since PKC-ζ deficiency promotes glutamine utilization via the serine biosynthesis pathway, we examined glutamine metabolism. The ectopic expression of PKC-ζ inhibited glutamine conversion to glutamate, while mutant PKC-ζ reversed this effect. Furthermore, the gene expression levels of enzymes involved in serine biosynthesis, phosphoserine phosphatase (PSPH), phosphoserine aminotransferase (PSAT1), and phosphoglycerate dehydrogenase (PHGDH) showed upregulation following glucose deprivation with PKC-ζ deficiency. The PHGDH upregulation was inhibited by ectopically expressing wild type but not mutated PKC-ζ in glucose-deprived conditions.

Conclusions

Our results support the upregulation of serine biosynthesis pathway genes and downregulation of PKC-ζ as potential metabolic alterations for bone metastatic breast cancer cells.

中文翻译：

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骨转移性乳腺癌细胞显示谷氨酰胺代谢增强，PKC-ζ下调

背景

乳腺癌是女性中最常被诊断出的癌症，其转移导致患者生存率低下。改变新陈代谢的能力是癌细胞用于在不同的转移微环境中生存并导致器官衰竭的关键属性。我们假设评估肿瘤细胞内的代谢变化可以更好地了解癌症转移。因此，为了研究转移过程中潜在的代谢改变，我们利用了人类MDA-MB-231和小鼠4T1模型来紧密模拟人类乳腺癌的转移。

方法

通过XF-24细胞外通量分析仪和western印迹检查骨转移细胞中糖酵解和谷氨酰胺途径相关的变化。通过PCR阵列检查与代谢相关的基因的表达水平。

结果

与亲代细胞相比，骨转移后分离出的MDA-MB-231细胞显示葡萄糖吸收和糖酵解降低，这表明这些细胞可以改变生存的代谢要求。为了了解这些代谢变化，我们研究了谷氨酰胺，一种常见且自然发生的非必需氨基酸。有趣的是，在降低的葡萄糖条件下，两种细胞系都显示出对谷氨酰胺的依赖，以维持细胞存活，而谷氨酰胺的撤离则明显增加了凋亡细胞的死亡。即使存在高葡萄糖浓度，谷氨酰胺对于正常细胞增殖也至关重要。为了进一步了解转移细胞中的这种代谢转换，我们检查了与代谢相关的基因，并确定了蛋白激酶C zeta（PKC-ζ）的下调超过7倍。与亲本群体相比，骨来源的MDA-MB-231细胞中的表达水平）。与非转移性MT1A2细胞相比，转移性4T1细胞中的PKC-ζ水平也显着降低。由于PKC-ζ缺乏会通过丝氨酸生物合成途径促进谷氨酰胺利用，因此我们研究了谷氨酰胺代谢。PKC-ζ的异位表达抑制了谷氨酰胺向谷氨酸的转化，而突变体PKC-ζ逆转了这一作用。此外，参与PKC-ζ缺乏的葡萄糖剥夺后，参与丝氨酸生物合成的酶，磷酸丝氨酸磷酸酶（PSPH），磷酸丝氨酸氨基转移酶（PSAT1）和磷酸甘油酸脱氢酶（PHGDH）的基因表达水平显示出上调。该PHGDH在葡萄糖缺乏的条件下，异位表达野生型可抑制上调，但PKC-ζ不会突变。

结论

我们的结果支持丝氨酸生物合成途径基因的上调和PKC-ζ的下调作为骨转移性乳腺癌细胞的潜在代谢改变。