The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. We have previously shown that vitamin D and vitamin D analogs such as EB 1089 can enhance the response to radiation in breast cancer through the promotion of a cytotoxic form of autophagy. In A549 and H460 non-small cell lung cancer (NSCLC) cells, 1,25-D3 (the hormonally active form of vitamin D) and EB 1089 prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack VDR/vitamin D receptor or functional TP53, respectively, 1,25-D3 failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post-irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when TP53 was induced in otherwise tp53-null H1299 NSCLC cells. Sensitization was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis, or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D3 or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by VDR, TP53, and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D3 and EB 1089 in NSCLC.
Keywords: ACTB, actin, β; AMPK, AMP activated protein kinase; ANXA5, annexin A5; ATG5, autophagy related 5; AVO, acidic vesicular organelles; BECN1, Beclin 1, autophagy-related; Baf, bafilomycin A1; FACS, fluorescence activating cell sorting; GFP, green fluorescent protein; H2AFX/H2AX, H2A histone family, member X; GLB, galactosidase; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; NSCLC; NSCLC, non-small cell lung cancer; PI, propidium iodide; SQSTM1, sequestosome 1; TP53, tumor protein p53; VDR, vitamin D (1, 25-dihydroxyvitamin D3) receptor; WT, wild-type; LC3 and LC3-II acronyms have been used to indicate the LC3B and LC3BII isoforms respectively; autophagy; cytoprotective; cytostatic; radiation; β.