Uncovering the role of the Reptin/Pontin multiprotein complex in Non-Small Cell Lung Cancer (NSCLC)
Final Report Abstract
In this project, we conducted for the first time a comprehensive examination of the Reptin/Pontin complex in non-small cell lung cancer (NSCLC). Using SILAC-based mass spectrometry for quantitative proteomics, we meticulously analysed the entire interactome surrounding these two AAA+-ATPases. This investigation led to the discovery of several novel functional partners, such as the RNA binding motif protein 14 (RBM14), which play crucial roles in both NSCLC adenocarcinoma (AC) and squamous cell carcinoma (SCC) cells. Our analysis of data from 1145 NSCLC patients revealed that elevated levels of RBM14 RNA expression were significantly linked to shorter progression-free and overall survival. While we observed mutual protein stabilization between Reptin and Pontin, RBM14 knockout did not affect the expression levels of these two partner proteins. However, the absence of RBM14 notably reduced their oncogenic function, hindering clonogenic growth, proliferation, cell cycle progression, and survival of human NSCLC cells. Furthermore, comparative proteomic analysis of NSCLC cells with or without RBM14 knockout unveiled nine functional clusters of highest significance, including processes like secretion/transport of granules and vesicles, and several other mechanisms which are known to play crucial roles in tumors growth and metastasis. These pathways correlated significantly with those identified in the functional Reptin/Pontin interactome. Moreover, while searching for controls for our NSCLC project, we discovered high expression of Reptin in various human Ewing sarcoma (ES) cell lines as well as in the majority of 100 primary ES tumors samples. Consequently, we extended our investigation to assess the role of Reptin in human ES. Knockdown of Reptin in human ES cells significantly inhibited growth ex vivo and eliminated engraftment in a murine xenograft model. Additionally, coimmunoprecipitation analyses revealed that Reptin is recruited by the key oncofusion EWSR1-FLI1, suggesting a potential novel oncogenic mechanism specific to ES. Proteomic analysis of human ES cells with Reptin knockout versus controls revealed several crucial pathways that are either up- or downregulated in ES cells by Reptin. The investigation into the fundamental mechanisms underlying ES promotion by the functional Reptin/Pontin interactome and its clinical significance for ES patients is currently ongoing.
