Abstract 227: Mass spectrometry-based proteomics analysis of the non-small cell lung cancer secretome
Authors: Emmanuel K. Cudjoe, Tareq Saleh, David A. Gewirtz, Adam M. Hawkridge
Journal: Cancer Research (2017)

Abstract Non-small cell lung cancer (NSCLC) is the more common subtype (~80%) of lung cancer, a leading cause of cancer death worldwide. NSCLC has one of the lowest 5-year relative survival rates due to a combination of late stage diagnosis and treatment relapse. Autophagy, a stress response mechanism in which cancer cells recycle organelles, and proteins to generate the necessary nutrients and metabolic intermediates for survival, is thought to be one of the principal mechanisms of treatment resistance and relapse in cancer. Due to its relatively refractory nature to chemo/radiotherapy, our long-term goal is to identify molecular signatures and pathways of autophagy that can be targeted to improve the sensitivity of NSCLC to radiotherapy. H1299 +/-p53 cells were cultured to ~85% confluency, the media exchanged to serum-free DMEM, and treated with 6Gy of ionizing radiation. After 12 hours, secreted proteomes were collected, processed and analyzed by reverse-phase nanoLC-MS/MS (Q-Exactive, ThermoFisher). A total of 364 secreted proteins were identified, of which 163 were quantified in at least 2 replicates of one condition. 29 unique proteins were identified to be present in all samples except H1299 +p53 cells which were not treated with ionizing radiation. These proteins included HMGA1, LSM8, CAST, CAPZB, CHGB, GSS, and HSPA9 and were associated principally with protein secretion and chaperone activities. Functional enrichment analysis using FunRich revealed that the secretome was enriched for exosomal, lysosomal, cytoplasmic, and cytoskeletal proteins (&amp;gt;2 fold, p-value &amp;lt; 0.0001). Using ANOVA, 24 proteins were found to be significantly differentially secreted between IR treated and untreated +/-p53 cells. Amongst these proteins are GPI, TXNRD1, FAM3C, DSTN, CLEC11A, HSPA9, and ALDOA. Of the significant genes, only GPI and TXNRD1 showed an interaction effect i.e. the effect of p53 status on the expression of GPI and TXNRD1 in the secretome depends on IR treatment status. In the pairwise comparison between –p53 and +p53 cells treated with IR, proteins over-secreted in –p53 cells by ≥ 2 fold include LSM8, CANX, CHGB and SCG2 whereas GNB2L1 was over-expressed by &amp;gt;2 fold in +p53 cell secretomes. Calnexin, an ER chaperone protein that is associated with senescence, has been identified in previous studies as a sero-marker of lung cancer but not as a marker for distinguishing between the autophagic responses of NSCLC to IR. The novel functional and potential diagnostic value of these differentially secreted proteins as a function of p53 expression, irradiation and functional autophagy status in the context of NSCLC will be discussed. Citation Format: Emmanuel K. Cudjoe, Tareq Saleh, David A. Gewirtz, Adam M. Hawkridge. Mass spectrometry-based proteomics analysis of the non-small cell lung cancer secretome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 227. doi:10.1158/1538-7445.AM2017-227