Suite 507, Armour Academic Building, Cell & Molecular Medicine, Rush University Medical Center, Chicago, IL 60612, United States.
Suite 507, Armour Academic Building, Cell & Molecular Medicine, Rush University Medical Center, Chicago, IL 60612, United States.
Gene. 2021 Apr 5;775:145419. doi: 10.1016/j.gene.2021.145419. Epub 2021 Jan 12.
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.
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.
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.
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.
乳腺癌是女性中最常见的癌症，其转移导致患者的生存率降低。改变代谢是癌细胞在不同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理解癌症转移。因此，为了研究转移过程中的潜在代谢变化，我们利用人 MDA-MB-231 和鼠 4T1 模型来模拟人类乳腺癌转移。
通过 XF-24 细胞外通量分析仪和 Western blot 检测骨转移细胞中糖酵解和谷氨酰胺途径相关变化。通过 PCR 芯片检测与代谢相关的基因表达水平。
与亲本细胞相比，从骨转移中分离出的 MDA-MB-231 细胞的葡萄糖摄取和糖酵解减少，表明这些细胞可能改变了生存所需的代谢需求。为了了解这些代谢变化，我们研究了谷氨酰胺，这是一种常见的天然非必需氨基酸。有趣的是，在低糖条件下，两种细胞系都依赖谷氨酰胺来维持细胞存活，而谷氨酰胺耗尽则显著增加细胞凋亡。即使在高葡萄糖浓度下，谷氨酰胺对正常细胞增殖也很重要。为了进一步了解转移细胞中的这种代谢转变，我们检查了与代谢相关的基因，并发现与亲本群体相比，骨源性 MDA-MB-231 细胞中蛋白激酶 C ζ (PKC-ζ) 的表达水平下调了七倍以上。与非转移性 MT1A2 细胞相比，转移性 4T1 细胞中的 PKC-ζ 水平也显著降低。由于 PKC-ζ 缺乏可通过丝氨酸生物合成途径促进谷氨酰胺利用，我们检查了谷氨酰胺代谢。PKC-ζ 的异位表达抑制了谷氨酰胺转化为谷氨酸，而突变 PKC-ζ 逆转了这种效应。此外，在 PKC-ζ 缺乏时，葡萄糖剥夺后，丝氨酸生物合成途径相关酶的基因表达水平上调，包括磷酸丝氨酸磷酸酶 (PSPH)、磷酸丝氨酸氨基转移酶 (PSAT1) 和磷酸甘油酸脱氢酶 (PHGDH)。在葡萄糖剥夺条件下，野生型但不是突变型 PKC-ζ 的异位表达抑制了 PHGDH 的上调。
我们的结果支持上调丝氨酸生物合成途径基因和下调 PKC-ζ 作为骨转移乳腺癌细胞的潜在代谢变化。
