Abstract
Mutations in the KRAS proto-oncogene are present in 50% of all colorectal cancers and are increasingly associated with chemotherapeutic resistance to frontline biologic drugs. Accumulating evidence indicates key roles for overactive KRAS mutations in the metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis in cancer cells. Here, we sought to exploit the more negative membrane potential of cancer cell mitochondria as an untapped avenue for interfering with energy metabolism in KRAS variant-containing and KRAS WT colorectal cancer cells. Mitochondrial function, intracellular ATP levels, cellular uptake, energy sensor signaling, and functional effects on cancer cell proliferation were assayed. 3-Carboxyl proxyl nitroxide (Mito-CP) and Mito-Metformin, two mitochondria-targeted compounds, depleted intracellular ATP levels and persistently inhibited ATP-linked oxygen consumption in both KRAS WT and KRAS variant-containing colon cancer cells and had only limited effects on nontransformed intestinal epithelial cells. These anti-proliferative effects reflected the activation of AMP-activated protein kinase (AMPK) and the phosphorylation-mediated suppression of the mTOR target ribosomal protein S6 kinase B1 (RPS6KB1 or p70S6K). Moreover, Mito-CP and Mito-Metformin released Unc-51-like autophagy-activating kinase 1 (ULK1) from mTOR-mediated inhibition, affected mitochondrial morphology, and decreased mitochondrial membrane potential, all indicators of mitophagy. Pharmacological inhibition of the AMPK signaling cascade mitigated the anti-proliferative effects of Mito-CP and Mito-Metformin. This is the first demonstration that drugs selectively targeting mitochondria induce mitophagy in cancer cells. Targeting bioenergetic metabolism with mitochondria-targeted drugs to stimulate mitophagy provides an attractive approach for therapeutic intervention in KRAS WT and overactive mutant-expressing colon cancer.
Highlights
HCT116 cells expressing constitutively active KRAS were somewhat more resistant to mitochondria-targeted drug (MTD) treatment with proliferation decreasing by 80% with 1 M Mito-CP or 50 M Mito-Met10 (Fig. 1, A and C)
Together with the reduction in p-ULK and the structural mitochondrial abnormalities noted in MTD-treated cells, these data support the idea that Mito-CP, and to a lesser extent Mito-Met10, stimulate mitophagy, contributing to the proliferation inhibitory effects of these compounds in WT and oncogenic KRAS colon cancer cells
We demonstrate here that disruption of complex I and mitochondrial bioenergetics potently arrested the proliferation capacity of colon cancer epithelial cells
Summary
Mutations in the KRAS proto-oncogene are present in 50% of all colorectal cancers and are increasingly associated with chemotherapeutic resistance to frontline biologic drugs. 3-Carboxyl proxyl nitroxide (Mito-CP) and Mito-Metformin, two mitochondria-targeted compounds, depleted intracellular ATP levels and persistently inhibited ATP-linked oxygen consumption in both KRAS WT and KRAS variant– containing colon cancer cells and had only limited effects on nontransformed intestinal epithelial cells These anti-proliferative effects reflected the activation of AMP-activated protein kinase (AMPK) and the phosphorylation-mediated suppression of the mTOR target ribosomal protein S6 kinase B1 (RPS6KB1 or p70S6K). Pharmacological inhibition of the AMPK signaling cascade mitigated the anti-proliferative effects of Mito-CP and Mito-Metformin This is the first demonstration that drugs selectively targeting mitochondria induce mitophagy in cancer cells. Metformin is posited to inhibit the mitochondrial electron transport complex I and indirectly activates the AMP-activated protein kinase (AMPK) signaling cascade, leading to suppressed colon carcinoma proliferation and reduced polyp formation [27, 28] These results encouraged us to determine whether Metformin conjugated to TPPϩ (Mito-Met10) might impact colon cancer cell dynamics. This study is the first to demonstrate the molecular mechanisms by which compounds engineered to localize within the mitochondria limit colon cancer proliferation and progression
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