Self-inflicted DNA double-strand breaks sustain tumorigenicity and stemness of cancer cells

Abstract

DNA double-strand breaks (DSBs) are traditionally associated with cancer through their abilities to cause chromosomal instabilities or gene mutations. Here we report a new class of self-inflicted DNA DSBs that can drive tumor growth irrespective of their effects on genomic stability. We discover a mechanism through which cancer cells cause DSBs in their own genome spontaneously independent of reactive oxygen species or replication stress. In this mechanism, low-level cytochrome c leakage from the mitochondria leads to sublethal activation of apoptotic caspases and nucleases, which causes DNA DSBs. In response to these spontaneous DNA DSBs, ATM, a key factor involved in DNA damage response, is constitutively activated. Activated ATM leads to activation of transcription factors NF-κB and STAT3, known drivers of tumor growth. Moreover, self-inflicted DNA DSB formation and ATM activation are important in sustaining the stemness of patient-derived glioma cells. In human tumor tissues, elevated levels of activated ATM correlate with poor patient survival. Self-inflicted DNA DSBs therefore are functionally important for maintaining the malignancy of cancer cells.

Spontaneous DNA damage propels tumorigenicity

“…Thus, knockout of executioner caspases, ENDOG or CAD, reduced the level of phosphorylated ATM (pATM) and STAT3 (pSTAT3), and abrogated NF-κB activation. In addition, cells deficient for ATM did not constitutively activate the NF-κB/STAT3 axis, and displayed reduced soft-agar colony-forming ability and in vivo tumor growth [10]. In these experimental settings, knockout of ATR abolished the clonogenic and tumorigenic potential of ATM −/− cancer cells, confirming the functional interrelationship between these DDR kinases that has previously been reported [6].…”

“…They demonstrated that the subpopulation of cancer cells with elevated levels of spDSBs (spDSBs high ) displayed higher clonogenicity in soft-agar assay and grew more efficiently when xenografted in nude mice than the spDSBs low fraction. In line with this evidence, strategies aimed at minimizing spDSBs (e.g., by knocking out effector caspases or knocking down pro-apoptotic BCL2 proteins) and boosting spDSBs (e.g., by irradiating spDSBs low cells with low doses of x-rays) dwindled and enhanced the in vitro clonogenicity and in vivo tumorigenicity of malignant cells, respectively [10].…”

“…By employing CRISPR/Cas9 technology on these patient-derived glioma cells, Liu et al demonstrated that ATM knockout decreased the CSC fraction. This was accompanied by the downregulation of STAT3 and resulted in diminished sphere-growing ability, clonogenicity and in vivo tumor growth [10]. In these experimental settings, the forced expression of constitutively active STAT3 rescued the stemness and tumorigenic potential of patient-derived glioma cells.…”

“…In a recent paper published by Cell Research, the group of Chuan-Yuan Li reports an alternative, intriguing point of view about the source of endogenously induced DSBs and their contribution to cancer evolution [10]. When performing a comparative analysis of a panel of malignant, immortalized/untransformed and primary cell lines previously synchronized in the G 1 phase, these authors found high levels of endogenously induced DSBs exclusively in tumor cells.…”

“…These lesions, which were dubbed as "self-inflicted" or spontaneous DSBs (spDSBs), originated in viable cancer cells independently of ROS and RS (as they occurred before S phase entry) through the order of events involving: (1) the partial permeabilization of the outer mitochondrial membrane, (2) the release of cytochrome c1 (CYC1) from mitochondria, (3) sublethal activation of executioner caspases (caspase 3 (CASP3), CASP6 and CASP7), and (4) cleavage of chromatin DNA by the apoptotic nucleases endonuclease G (ENDOG) and DNA fragmentation factor β (DFFB/CAD) ( Figure 1). This circuitry was elegantly unveiled by CRISPR/Cas9 gene editing-mediated knockout and epistatic experiments revealing limited nuclease activation in CASP3 −/− , CASP3 −/− CASP6 −/− and CASP3 −/− CASP6 −/− CASP7 −/− cancer cells, as well as a significant decrease of spDSBs in tumor cells deficient for executioner caspases, ENDOG or CAD [10]. This mechanism is reminiscent to that observed upon exogenous perturbations [11], even though it occurred in unperturbed conditions.…”