Defects in the Fanconi Anemia Pathway in Head and Neck Cancer Cells Stimulate Tumor Cell Invasion through DNA-PK and Rac1 Signaling

Abstract

Purpose Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and FA gene mutations and transcriptional repression are common. Invasive tumor behaviour is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, in order to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells. Experimental Design Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classical hallmarks of FA following exposure to DNA crosslinkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-PK and Rac1 inhibitors. Results We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-dependent protein kinase (DNA-PK) and downstream Rac1 GTPase activity. Conclusions These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes.

Drug Sensitivity Prediction Models Reveal a Link between DNA Repair Defects and Poor Prognosis in HNSCC

“…have reported that knockdown of FA genes in HNSCC cell lines results in increased invasion through activation of the NHEJ repair pathway and via DNA-PK and Rac1 signaling (56). Our DNA-PK inhibition data and differential pathway expression analyses support the engagement of these migration-promoting mechan-isms in DNA repair-deficient HNSCC cell lines and provides a mechanistic link to a higher propensity to metastasize.…”

“…DNA-PK activation-mediated migration is also in line with an increased engagement of NHEJ as indicated by the PARP inhibitor resistance and connects repair defects to the reports from others that show a role for these proteins in migration, invasion, and metastasis. Consistent with the notion that loss of FA/HR DNA repair may induce metastatic behavior, knockdown of BRCA1 and FANCD2 was reported to cause dedifferentiation and induce EMT in human mammary epithelial cells in vitro (56). Cells that have undergone EMT downregulate apoptosis (57) and show increased resistance to DNA damage (58).…”

DNA‐PkCS expression in oropharyngeal squamous cell carcinoma: Correlations with human papillomavirus status and recurrence after transoral robotic surgery

“…Although DNA‐Pk CS is perhaps best known for its role in nonhomologous end‐joining mediated DNA damage repair, it has also been implicated as a transcriptional regulator affecting cellular processes, such as migration, invasion, and metastasis . Even in head and neck cancer, a recent report suggests that loss of function of Fanconi anemia proteins promotes tumor invasion through the activity of DNA‐Pk CS . In this study, we show evidence that DNA‐Pk CS expression may be a marker for recurrence within HPV‐positive oropharyngeal SCCs, even when controlling for clinical characteristics and adjuvant therapy.…”

“…15 Even in head and neck cancer, a recent report suggests that loss of function of Fanconi anemia proteins promotes tumor invasion through the activity of DNA-Pk CS . 32 In this study, we show evidence that DNA-Pk CS expression may be a marker for recurrence within HPV-positive oropharyngeal SCCs, even when controlling for clinical characteristics and adjuvant therapy. We also observed a possible association between DNA-Pk CS and the number of involved lymph nodes, which has previously been described as a predictor of recurrence and survival after surgical resection in HNSCCs.…”

The Role of the Core Non-Homologous End Joining Factors in Carcinogenesis and Cancer

“…It is believed that the FA pathway blocks NHEJ from engaging one-sided DSBs at replications forks by preventing the inappropriate engagement of the DNA-PK complex with damaged replication forks to divert DSB repair toward the HR pathway. Loss of the FA pathway in head and neck squamous cell carcinoma (HNSCC) cells promotes cellular invasion and this response requires DNA-PKcs activity [ 35 ]. Finally, recent evidence has emerged that a single catastrophic event, termed chromothripsis, that results in multiple DSBs of a chromosome are randomly rejoined by NHEJ in a chaotic genomic structure ( Figure 2 C) [ 36 ].…”