Walnut Phenolic Extracts Induced Metabolic Reprogramming in Colon Cancer Stem Cells by Reinforcing the Mitochondrial Function: From RNA‐seq to Mitochondrial Stress Test

Previously we reported that walnut phenolic extracts (WPE) can reduce the stemness of colon cancer stem cells (CSCs). Since cancer cells commonly exhibit metabolic alterations, such as the Warburg effect, and CSCs also demonstrate impaired mitochondrial functions, we determined the metabolic influence of WPE on colon CSCs in the ramification of mitochondrial functions. From the HCT116 colon cancer cell line, CD133 + CD44 + cells were sorted by FACS and treated with or without 20μg/mL WPE. To investigate the mechanism behind the effect of walnuts on colon CSCs, RNA‐sequencing (RNA‐seq) analysis was conducted using an Illumina Hiseq 2500 platform and differentially expressed genes (DEGs) were categorized into functional clusters based on Gene Ontology functional enrichment analysis. ClueGo and CluePedia plug‐ins in Cytoscape was used to construct inter‐connected functional networks including the relative expression patterns of the DEGs within the integrated metabolic and mitochondrial cluster. RT‐PCR method validated the RNA‐seq analysis data. To evaluate the bioenergetic profile of the WPE‐treated CD133 + CD44 + HCT116 cells cellular oxygen consumption rate (OCR), an indicator of mitochondrial respiration, and extracellular acidification rate (ECAR), an indicator of glycolysis, were determined using XFp Analyzer (Seahorse Bioscience, North Billerica, MA), which can measure mitochondrial function and glycolytic activity through mitochondrial stress test. For the stress test 1 μmole/L oligomycin A, 0.4 μmole/L carbonyl cyanide‐4 phenylhydrazone, and the mixture of 1μmole/L rotenone and 1μmole/L antimycin A were added to the media sequentially to assess the global change in mitochondrial respiration. To confirm the data glucose uptake was analyzed using Colorimetric Glucose Uptake Kit (Abcam, Cambridge, UK) and lactate production was measured by L‐lactate Assay Kit (Abcam). RNA‐seq analysis found 1983 DEGs and up or down‐regulated DEGs were classified into two major functional clusters, the metabolic cluster (73 genes) and the mitochondria cluster (112 genes). WPE treatment shifted the bioenergetic profile of CD133 + CD44 + HCT116 cells towards the more glycolysis pathway (ΔECAR=36.98 mpH/min/ptn, p=0.003). Mitochondrial stimulation demonstrated an increase in mitochondrial respiration potential (ΔOCR=111.5 pmol/min/ptn, p‐value=0.0006). We also confirmed that both glucose uptake (Δ=0.39 pmol/ul, p=0.002) and lactate production (Δ=0.08 nmol/ul, p=0.005) were elevated after WPE treatment. In the present colon cancer stem cell model WPE treatment elicited a broad range of transcriptional modulation associated with mitochondria and metabolic pathways. Enhanced glycolysis illustrated that WPE treatment may drive a Warburg effect‐like phenotypic metabolic shift. A high bioenergetic signature and increased mitochondrial capacity indicate the restoration and re‐maturation of mitochondrial function by WPE treatment, which may cause the diminished stemness in WPE‐treated colon CSCs. Support or Funding Information California Walnut Commission