BackgroundPancreatic 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 KrasG12D/-; p53-/- pancreatic mouse model.MethodsWe 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.ResultsPDAC 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.ConclusionPKM1/2 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.

中文翻译：

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半胱氨酸分解代谢和丝氨酸生物合成途径支持在胰腺癌细胞中丙酮酸激酶敲低过程中产生丙酮酸

背景胰腺导管腺癌（PDAC）是一种侵袭性癌症，治疗选择有限。丙酮酸激酶，尤其是 M2 异构体 (PKM2)，在 PDAC 细胞中高度表达，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 KrasG12D/- 的细胞系中单独敲除 PKM2 以及同时敲除 PKM1 和 PKM2 (PKM1/2)；p53-/- 胰腺小鼠模型。方法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 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 敲低细胞中上调的代谢途径。然后，我们使用 CRISPR/Cas9 基因编辑技术针对 PKM1/2 敲低细胞中上调的代谢途径。结果尽管 PKM1/2 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 敲低期间部分促成了丙酮酸的产生：在该途径中敲除磷酸甘油酸脱氢酶会降低葡萄糖产生的丙酮酸。此外，半胱氨酸分解代谢在 PDAC 细胞中产生约 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和谷氨酰胺、丝氨酸、色氨酸和苏氨酸的分解代谢。然而，这些来源并没有在 PKM1/2 敲低细胞中提供显着水平的丙酮酸。结论 PKM1/2 敲低不影响胰腺癌细胞的增殖。丝氨酸生物合成途径支持在丙酮酸激酶敲低期间将葡萄糖转化为丙酮酸。然而，在 PKM1/2 敲低期间未观察到丝氨酸直接转化为丙酮酸。研究丙酮酸的几种替代来源，确定了在 PKM1/2 敲低期间产生丙酮酸的半胱氨酸分解代谢。令人惊讶的是，我们发现大部分细胞内丙酮酸来自半胱氨酸。我们的研究结果突出了 PDAC 细胞在关键代谢酶的敲低过程中自适应地重新连接其代谢途径的能力。研究丙酮酸的几种替代来源，确定了在 PKM1/2 敲低期间产生丙酮酸的半胱氨酸分解代谢。令人惊讶的是，我们发现大部分细胞内丙酮酸来自半胱氨酸。我们的研究结果突出了 PDAC 细胞在关键代谢酶的敲低过程中自适应地重新连接其代谢途径的能力。研究丙酮酸的几种替代来源，确定了在 PKM1/2 敲低期间产生丙酮酸的半胱氨酸分解代谢。令人惊讶的是，我们发现大部分细胞内丙酮酸来自半胱氨酸。我们的研究结果突出了 PDAC 细胞在关键代谢酶的敲低过程中自适应地重新连接其代谢途径的能力。