DNA damaging agents cause a variety of lesions, of which DNA double-strand breaks (DSBs) are the most genotoxic. Unbiased approaches aimed at investigating the relationship between the number of DSBs and outcome of the DNA damage response have been challenging due to the random nature in which damage is induced by classical DNA damaging agents. Here, we describe a CRISPR/Cas9-based system that permits us to efficiently introduce DSBs at defined sites in the genome. Using this system, we show that a guide RNA targeting only a single site in the human genome can trigger a checkpoint response that is potent enough to delay cell cycle progression. Abrogation of this checkpoint leads to DNA breaks in mitosis which gives rise to aneuploid progeny.

The glucocorticoid receptor (GR) regulates gene expression, governing aspects of homeostasis, but is also involved in cancer. Pharmacological GR activation is frequently used to alleviate therapy-related side-effects. While prior studies have shown GR activation might also have anti-proliferative action on tumours, the underpinnings of glucocorticoid action and its direct effectors in non-lymphoid solid cancers remain elusive. Here, we study the mechanisms of glucocorticoid response, focusing on lung cancer. We show that GR activation induces reversible cancer cell dormancy characterised by anticancer drug tolerance, and activation of growth factor survival signalling accompanied by vulnerability to inhibitors. GR-induced dormancy is dependent on a single GR-target gene, CDKN1C, regulated through chromatin looping of a GR-occupied upstream distal enhancer in a SWI/SNF-dependent fashion. These insights illustrate the importance of GR signalling in non-lymphoid solid cancer biology, particularly in lung cancer, and warrant caution for use of glucocorticoids in treatment of anticancer therapy related side-effects.

Microtubules are cellular targets for a variety of anticancer therapies because of their critical function in mitosis. Taxol belongs to a class of microtubule targeting agents that suppresses microtubule dynamics and interferes with the functioning of the mitotic spindle, thereby effectively blocking cell cycle progression of rapidly proliferating tumor cells. Despite its antitumor activity, drug resistance remains a common obstacle in improving its overall clinical efficacy. Previous studies have shown that the expression of a specific β-tubulin isotype, βIII-tubulin/TUBB3, is dysregulated in drug-refractory tumors. However, whether enhanced TUBB3 expression is directly involved in promoting taxol resistance remains a subject of debate. Here, we have used several approaches to assess the functional relation of TUBB3 overexpression and taxol resistance. First, we generated a number of taxol-resistant cell lines, to find that TUBB3 expression was elevated in a resistant cell line (RPE-20) derived from untransformed retinal pigment epithelial (RPE) cells, but the abundance of TUBB3 remained unchanged in four other cell lines after taxol treatment. However, although RPE-20 cells displayed enhanced TUBB3 levels, we find that simultaneous up-regulation of the P-glycoprotein (P-gP) drug-efflux pump is responsible for the resistance to taxol. Indeed, we could show that TUBB3 levels were dynamically regulated upon taxol exposure and withdrawal, unrelated to the resistance phenotype. Next, we generated cell lines in which we could induce robust overexpression of TUBB3 from its endogenous locus employing the CRISPRa system. We demonstrate that solely enhancing TUBB3 expression results in a very minor decrease in the sensitivity to taxol. This was further substantiated by selective depletion of TUBB3 in a series of breast cancer cell lines expressing high levels of TUBB3. We find that TUBB3 depletion had a minimal effect on the sensitivity to taxol in one of these cell lines, but had no effect in all of the others. Based on these findings we propose that TUBB3 overexpression can only marginally affect the sensitivity to taxol in cultured cell lines.