Colorectal cancer (CRC) is among the most frequent cancer entities worldwide. Multiple factors are causally associated with CRC development, such as genetic and epigenetic alterations, inflammatory bowel disease, lifestyle and dietary factors. During malignant transformation, the cellular energy metabolism is reprogrammed in order to promote cancer cell growth and proliferation. In this review, we first describe the main alterations of the energy metabolism found in CRC, revealing the critical impact of oncogenic signaling and driver mutations in key metabolic enzymes. Then, the central role of mitochondria and the tricarboxylic acid (TCA) cycle in this process is highlighted, also considering the metabolic crosstalk between tumor and stromal cells in the tumor microenvironment. The identified cancer-specific metabolic transformations provided new therapeutic targets for the development of small molecule inhibitors. Promising agents are in clinical trials and are directed against enzymes of the TCA cycle, including isocitrate dehydrogenase, pyruvate dehydrogenase kinase, pyruvate dehydrogenase complex (PDC) and α-ketoglutarate dehydrogenase (KGDH). Finally, we focus on the α-lipoic acid derivative CPI-613, an inhibitor of both PDC and KGDH, and delineate its anti-tumor effects for targeted therapy.

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs.

DNA double-strand breaks (DSBs) are critical DNA lesions, which threaten genome stability and cell survival. DSBs are directly induced by ionizing radiation (IR) and radiomimetic agents, including the cytolethal distending toxin (CDT). This bacterial genotoxin harbors a unique DNase-I-like endonuclease activity. Here we studied the role of DSBs induced by CDT and IR as a trigger of autophagy, which is a cellular degradation process involved in cell homeostasis, genome protection and cancer. The regulatory mechanisms of DSB-induced autophagy were analyzed, focusing on the ATM-p53-mediated DNA damage response and AKT signaling in colorectal cancer cells. We show that treatment of cells with CDT or IR increased the levels of the autophagy marker LC3B-II. Consistently, an enhanced formation of autophagosomes and a decrease of the autophagy substrate p62 were observed. Both CDT and IR concomitantly suppressed mTOR signaling and stimulated the autophagic flux. DSBs were demonstrated as the primary trigger of autophagy using a DNase I-defective CDT mutant, which neither induced DSBs nor autophagy. Genetic abrogation of p53 and inhibition of ATM signaling impaired the autophagic flux as revealed by LC3B-II accumulation and reduced formation of autophagic vesicles. Blocking of DSB-induced apoptotic cell death by the pan-caspase inhibitor Z-VAD stimulated autophagy. In line with this, pharmacological inhibition of autophagy increased cell death, while ATG5 knockdown did not affect cell death after DSB induction. Interestingly, both IR and CDT caused AKT activation, which repressed DSB-triggered autophagy independent of the cellular DNA-PK status. Further knockdown and pharmacological inhibitor experiments provided evidence that the negative autophagy regulation was largely attributable to AKT2. Finally, we show that upregulation of CDT-induced autophagy upon AKT inhibition resulted in lower apoptosis and increased cell viability. Collectively, the findings demonstrate that DSBs trigger pro-survival autophagy in an ATM- and p53-dependent manner, which is curtailed by AKT2 signaling.

Lipoic acid (LA) is a redox-active disulphide compound, which functions as a pivotal co-factor for mitochondrial oxidative decarboxylation. LA and chemical derivatives were shown to target mitochondria in cancer cells with altered energy metabolism, thereby inducing cell death. In this study, the impact of LA on the tumor suppressor protein p53 was analyzed in various colorectal cancer (CRC) cell lines, with a focus on the mechanisms driving p53 degradation. First, LA was demonstrated to trigger the depletion of both wildtype and mutant p53 protein in all CRC cells tested without influencing its gene expression and preceded LA-triggered cytotoxicity. Depletion of p53 coincided with a moderate, LA-dependent ROS production, but was not rescued by antioxidant treatment. LA induced the autophagy receptor p62 and differentially modulated autophagosome formation in CRC cells. However, p53 degradation was not mediated via autophagy as shown by chemical inhibition and genetic abrogation of autophagy. LA treatment also stabilized and activated the transcription factor Nrf2 in CRC cells, which was however dispensable for p53 degradation. Mechanistically, p53 was found to be readily ubiquitinylated and degraded by the proteasomal machinery following LA treatment, which did not involve the E3 ubiquitin ligase MDM2. Intriguingly, the combination of LA and anticancer drugs (doxorubicin, 5-fluorouracil) attenuated p53-mediated stabilization of p21 and resulted in synergistic killing in CRC cells in a p53-dependant manner.