DNA damage is well recognized as a critical factor in cancer development and progression. DNA lesions create an abnormal nucleotide or nucleotide fragment, causing a break in one or both chains of the DNA strand. When DNA damage occurs, the possibility of generated mutations increases. Genomic instability is one of the most important factors that lead to cancer development. DNA repair pathways perform the essential role of correcting the DNA lesions that occur from DNA damaging agents or carcinogens, thus maintaining genomic stability. Inefficient DNA repair is a critical driving force behind cancer establishment, progression and evolution. A thorough understanding of DNA repair mechanisms in cancer will allow for better therapeutic intervention. In this review we will discuss the relationship between DNA damage/repair mechanisms and cancer, and how we can target these pathways.

Internal tandem duplication of FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (dsbs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational downregulation of p22phox, a small membrane-bound subunit of NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD have a higher protein level of p22phox and p22phox-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22phox and NOX proteins knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H2O2)-specific dye, Peroxy Orange 1 (PO1) and nuclear H2O2, as measured with Nuclear Peroxy Emerald 1 (NucPE1). These reductions in the level of H2O2 following the NOX knockdowns, were accompanied by a decrease in the number of DNA double strand breaks (dsbs). We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA dsbs than their wild type counterparts. We also observed that NOX4 and p22phox localize to the nuclear membrane in MV4-11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22phox mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability.

Surgery induced inflammation is a potent promoter of tumour recurrence and metastasis in colorectal cancer. The recently discovered family of Nox enzymes represent a major source of endogenous reactive oxygen species (ROS) and are now heavily implicated in tumour cell metastasis. Interestingly, Nox enzymes can be ‘purposefully’ activated by inflammatory cytokines and growth factors which are present in abundance in the peri-operative window. As colon cancer cells express Nox enzymes and Toll-like receptor 4 (TLR-4), we hypothesised that LPS may potentiate the ability of colon cancer cells to metastasise via Nox enzyme mediated redox signalling. In support of this hypothesis, this paper demonstrates that LPS induces a significant, transient increase of endogenous ROS in SW480, SW620 and CT-26 colon cancer cells. This increase in LPS-induced ROS activity is completely abrogated by a Nox inhibitor, diphenyleneiodonium (DPI), Nox1 siRNA and an NF-κB inhibitor, Dihydrochloride. A significant increase in Nox1 and Nox2 protein expression occurs following LPS treatment. Inhibition of NF-κB also attenuates the increase of Nox1 and Nox2 protein expression. The sub-cellular location of LPS-induced ROS generation lies mainly in the endoplasmic reticulum. LPS activates the PI3K/Akt pathway via Nox generated ROS and this signal is inhibited by DPI. This LPS activated Nox mechanism facilitates a significant increase in SW480 colon cancer cell adhesion to collagen I, which is inhibited by DPI, Nox1 siRNA and a PI3K inhibitor. Altogether, these data suggest that the LPS-Nox1 redox signalling axis plays a crucial role in facilitation of colon cancer cell adhesion, thus increasing the metastatic potential of colon cancer cells. Nox1 may represent a valuable target in which to prevent colon cancer metastasis.

The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H₂O₂-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H₂O₂ after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H₂O₂ in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H₂O₂ production in AML cells is mediated by p22phox and is critical for STAT5 signalling.

Neuroblastoma is the most frequent extracranial childhood tumour but effective treatment with current immunotherapies is challenging due to its immunosuppressive microenvironment. Efforts to date have focused on using immunotherapy to increase tumour immunogenicity and enhance anticancer immune responses, including anti-GD2 antibodies; immune checkpoint inhibitors; drugs which enhance macrophage and natural killer T (NKT) cell function; modulation of the cyclic GMP-AMP synthase-stimulator of interferon genes pathway; and engineering neuroblastoma-targeting chimeric-antigen receptor-T cells. Some of these strategies have strong preclinical foundation and are being tested clinically, although none have demonstrated notable success in treating paediatric neuroblastoma to date. Recently, approaches to overcome heterogeneity of neuroblastoma tumours and treatment resistance are being explored. These include rational combination strategies with the aim of achieving synergy, such as dual targeting of GD2 and tumour-associated macrophages or natural killer cells; GD2 and the B7-H3 immune checkpoint; GD2 and enhancer of zeste-2 methyltransferase inhibitors. Such combination strategies provide opportunities to overcome primary resistance to and maximize the benefits of immunotherapy in neuroblastoma.