NSCLC Mutated Isoforms of CCDC6 Affect the Intracellular Distribution of the Wild Type Protein Promoting Cisplatinum Resistance and PARP Inhibitors Sensitivity in Lung Cancer Cells

Abstract

CCDC6 is implicated in cell cycle checkpoints and DNA damage repair by homologous recombination (HR). In NSCLC, CCDC6 is barely expressed in about 30% of patients and CCDC6 gene rearrangements with RET and ROS kinases are detected in about 1% of patients. Recently, CCDC6 point-mutations naming E227K, S351Y, N394Y, and T462A have been identified in primary NSCLC.In this work, we analyze the effects exerted by the CCDC6 mutated isoforms on lung cancer cells. By pull-down experiments and immunofluorescence, we evaluated the biochemical and morphological effects of CCDC6 lung-mutants on the CCDC6 wild type protein. By using two HR-reporter assays, we analyzed the effect of CCDC6 lung-mutants in perturbing CCDC6 physiology in the HR process. Finally, by cell-titer assay, we evaluated the response to the treatment with different drugs in lung cancer cells expressing CCDC6 mutants. This work shows that the CCDC6 mutated and truncated isoforms, identified so far in NSCLC, affected the intracellular distribution of the wild type protein and impaired the CCDC6 function in the HR process, ultimately inducing cisplatinum resistance and PARP-inhibitors sensitivity in lung cancer cells. The identification of selected molecular alterations involving CCDC6 gene product might define predictive biomarkers for personalized treatment in NSCLC.Cancers 2020, 12, 44 2 of 18 exposed to DNA damage also decreases the levels of the G2-checkpoint phospho-protein Chk1 and shortens the G2 phase, allowing a premature entrance into the mitosis [9,10].Cancer cells carrying HR DNA repair molecular alteration, like BRCA1/2 defects, are selective target of the PARP inhibition, resulting in a synthetic lethal phenotype. Indeed, cancer cells defective for CCDC6 show an impaired HR process and switch to the non-homologous end joining process (NHEJ) to repair their DNA. As the NHEJ process is prone to errors, the cancer cells defective for CCDC6 show tolerance to the DNA damage induced by several mutagens, including cis-platinum. Furthermore, the switch to the NHEJ repair process that involves the poly (ADP-ribose) polymerase enzymes (PARP1/2) makes the CCDC6 defective cells sensitive to PARP inhibition because of a synthetic lethal effect [11].In several in vitro cancer cell systems CCDC6 protein levels have been correlated with cancer cell tolerance to DNA damage, resistance to cisplatinum and sensitivity to poly (ADP-ribose) polymerase inhibitors (PARPi) [4,[11][12][13][14][15][16].To date, besides the first CCDC6 fusion with the tyrosine kinase RET identified in papillary thyroid cancer [17,18], additional CCDC6 fusions have been reported with PDGFRb [19,20], PTEN [21], FGFR2 [22,23] ANK3 [24], and UBE2D1 [25] and with other genes in different tumor types [26].Following the fusions, the recombinant oncogenes always include, at least in part, the CCDC6 coiled-coil region that is able to induce protein dimers [27,28]. Interestingly, in the case of CCDC6/RET, the first 101 amminoacid (aa) of CCDC6 involved in the fusion can form heterodimers with wild...

The tumour suppressor CCDC6 is involved in ROS tolerance and neoplastic transformation by evading ferroptosis

“…inhibitor drugs specifically depended on the CCDC6 levels in the analysed testicular cells, as already reported in other cell systems [41,42]. The cisplatin sensitivity was poorly modulated by the CCDC6 rescue in the murine testicular cells (Fig.…”

Coiled‑coil domain‑containing 68 promotes non‑small cell lung cancer cell proliferation <em>in&nbsp;vitro</em>

“…Previous studies have explored the role of CCDC proteins in lung cancer; for example, low levels of CCDC19 are associated with an unfavorable outcome in patients with lung squamous cell carcinoma ( 26 ) and with NSCLC pathogenesis ( 27 ). CCDC6 may represent a predictive biomarker of resistance to conventional single-mode therapy and yield insight on tumor sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors in NSCLC ( 28 ). CCDC106 is highly expressed in NSCLC tissues and cell lines, and its expression levels are associated with an advanced TNM stage, lymph node metastasis and poor overall survival in patients with NSCLC ( 29 ).…”

Toll-Like Receptor 7 Mediates Inflammation Resolution and Inhibition of Angiogenesis in Non-Small Cell Lung Cancer

“…A549, NCI-H1975 (H1975 throughout the text), and HOP62 lung cancer cells were grown as previously described [ 60 , 61 , 62 ]. Human primary lung microvascular endothelial cells (HPLMECs) and human umbilical vein endothelial cells (HUVECs) from Cell Biologics were grown in human endothelial cell medium with the addition of VEGF, heparin, EGF, FGF, hydrocortisone, L-glutamine, antibiotic–antimycotic Solution, and FBS according to the manufacturer’s instructions (Cell Biologics, Chicago, IL, USA).…”