Yu Lei, Teoh Shao Thing, Ensink Elliot, Ogrodzinski Martin P, Yang Che, Vazquez Ana I, Lunt Sophia Y
1Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI USA.
2Department of Physiology, Michigan State University, East Lansing, MI USA.
Cancer Metab. 2019 Dec 30;7:13. doi: 10.1186/s40170-019-0205-z. eCollection 2019.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with limited treatment options. Pyruvate kinase, especially the M2 isoform (PKM2), is highly expressed in PDAC cells, but its role in pancreatic cancer remains controversial. To investigate the role of pyruvate kinase in pancreatic cancer, we knocked down PKM2 individually as well as both PKM1 and PKM2 concurrently (PKM1/2) in cell lines derived from a pancreatic mouse model.
We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine metabolic profiles of wildtype and PKM1/2 knockdown PDAC cells. We further used stable isotope-labeled metabolic precursors and LC-MS/MS to determine metabolic pathways upregulated in PKM1/2 knockdown cells. We then targeted metabolic pathways upregulated in PKM1/2 knockdown cells using CRISPR/Cas9 gene editing technology.
PDAC cells are able to proliferate and continue to produce pyruvate despite PKM1/2 knockdown. The serine biosynthesis pathway partially contributed to pyruvate production during PKM1/2 knockdown: knockout of phosphoglycerate dehydrogenase in this pathway decreased pyruvate production from glucose. In addition, cysteine catabolism generated ~ 20% of intracellular pyruvate in PDAC cells. Other potential sources of pyruvate include the sialic acid pathway and catabolism of glutamine, serine, tryptophan, and threonine. However, these sources did not provide significant levels of pyruvate in PKM1/2 knockdown cells.
PKM1/2 knockdown does not impact the proliferation of pancreatic cancer cells. The serine biosynthesis pathway supports conversion of glucose to pyruvate during pyruvate kinase knockdown. However, direct conversion of serine to pyruvate was not observed during PKM1/2 knockdown. Investigating several alternative sources of pyruvate identified cysteine catabolism for pyruvate production during PKM1/2 knockdown. Surprisingly, we find that a large percentage of intracellular pyruvate comes from cysteine. Our results highlight the ability of PDAC cells to adaptively rewire their metabolic pathways during knockdown of a key metabolic enzyme.
胰腺导管腺癌（PDAC）是一种侵袭性癌症，治疗选择有限。丙酮酸激酶，尤其是M2亚型（PKM2），在PDAC细胞中高表达，但其在胰腺癌中的作用仍存在争议。为了研究丙酮酸激酶在胰腺癌中的作用，我们在源自胰腺小鼠模型的细胞系中分别敲低PKM2以及同时敲低PKM1和PKM2（PKM1/2）。
我们使用液相色谱串联质谱（LC-MS/MS）来确定野生型和PKM1/2敲低的PDAC细胞的代谢谱。我们进一步使用稳定同位素标记的代谢前体和LC-MS/MS来确定在PKM1/2敲低的细胞中上调的代谢途径。然后，我们使用CRISPR/Cas9基因编辑技术靶向PKM1/2敲低的细胞中上调的代谢途径。
尽管PKM1/2被敲低，PDAC细胞仍能够增殖并继续产生丙酮酸。丝氨酸生物合成途径在PKM1/2敲低期间部分促进了丙酮酸的产生：该途径中磷酸甘油酸脱氢酶的敲除降低了葡萄糖产生丙酮酸的量。此外，半胱氨酸分解代谢在PDAC细胞中产生了约20%的细胞内丙酮酸。丙酮酸的其他潜在来源包括唾液酸途径以及谷氨酰胺、丝氨酸、色氨酸和苏氨酸的分解代谢。然而，这些来源在PKM1/2敲低的细胞中并未提供大量的丙酮酸。
PKM1/2敲低不影响胰腺癌细胞的增殖。在丙酮酸激酶敲低期间，丝氨酸生物合成途径支持葡萄糖向丙酮酸的转化。然而，在PKM1/2敲低期间未观察到丝氨酸直接转化为丙酮酸。对几种丙酮酸替代来源的研究确定了PKM1/2敲低期间半胱氨酸分解代谢用于丙酮酸的产生。令人惊讶的是，我们发现很大一部分细胞内丙酮酸来自半胱氨酸。我们的结果突出了PDAC细胞在关键代谢酶敲低期间适应性重新连接其代谢途径的能力。
