Ephrin and Eph-receptor growth factor signaling in non small cell lung cancer : identification of biomarkers and therapeutic targets

Abstract: Non-small cell lung cancer (NSCLC) is the main subtype of lung cancer (LC) and unfortunately it responds very poorly to conventional chemo- and radiotherapy (RT). Moreover, NSCLC is often diagnosed at a stage where metastases are found and only for a limited number of NSCLC tumors targeted therapies can be used as their oncogenic drivers remains elusive. Thus there is a need of finding novel targets in NSCLC and this thesis focus around these topics. In Paper I the aim was to find novel RT targets in NSCLC by global genomic profiling. It was previously shown that NSCLC cells could be sensitized to RT by addition of the staurosporine analogue PKC 412. By global gene expression analyses on this NSCLC system we identified the Eph growth factor receptor ligand Ephrin B3 as a putative RT target as it was downregulated in the combined RT and PKC 412 treated NSCLC cells. Indeed, we demonstrated that Ephrin B3 ablation of NSCLC cells in combination with RT increased cellular senescence, mitotic catastrophe and apoptosis, inhibited the cell survival kinases Akt, MAPKERK, p38MAPK and decreased RT-induced G2-arrest. Thus we in Paper I identified Ephrin B3 as a driver of RT resistance. In Paper II the aim was to investigate how Ephrin B3 influences the proliferative “signalome” of NSCLC cells. The phosphoproteome of NSCLC cells with or without Ephrin B3 expression was analyzed using a peptid-based approach in which SCX and TiO2-based fractionation was used prior to identification by mass spectrometry and Ingenuity pathway analyses. Among the differentially phosphorylated proteins one candidate was the erythropoietin-producing hepatocellular receptor tyrosine kinase class A2 (EphA2), previously shown to control tumor cell signaling. We demonstrated that when Ephrin B3 expression was blocked in NSCLC cells EphA2 lost its phosphorylation on Ser897, a site previously reported to control migration in other tumor types. We also found that inhibition of Ephrin B3 expression suppressed Akt1 Ser129 phosphorylation which was reported to control EphA2 at Ser897. Thus our findings supported a hypothetical mechanism in which NSCLC cell survival signaling was mediated by an Ephrin B3 and EphA2 signaling circuit. In Paper III the purpose was to analyze how Ephrin B3 and its putative Ephs mediates their effects on migration and invasion of NSCLC of different histology in vitro as well as to reveal as to what extent these signaling components may be operative in NSCLC in vivo. Our study identified a novel function of Ephrin B3 where it similar to EphA2 controlled proliferation, migration and invasion of NSCLC cells in vitro. We showed for the first time that Ephrin B3 binds EphA2, EphA4, EphA5 and EphA3 indicating a master function of signaling of Ephrin B3 in NSCLC. Moreover, as EphA2 Ser897 and Akt Ser129 both were found in complex with Ephrin B3 in NSCLC cells and given that we observed p38MAPK and Src kinase in such complex our data further add onto how EphA2 may drive NSCLC proliferation and migration. In analyses of NSCLC clinical specimen Ephrin B3 was concomitantly expressed with EphA2 and its known ligand Ephrin A1 but did not correlate to poor survival. Several growth factor receptors, including EphA5, have been shown to control DNA damage response (DDR) signaling and hence to constitute RT sensitizing targets. In Paper IV we analyzed if EphA2, EphA4 and Ephrin B3 similarly influenced DDR components and hence could be used combat RT resistance. Our results showed that a combination of RT and ablation of EphA2, EphA4 or Ephrin B3 reduced proliferation and colony forming potential. We also described a novel interaction of EphA2, EphA4 and Ephrin B3 with the DDR proteins pATM (S1981), pDNA-PKcs (S2056) and γH2AX (S139) suggesting that this Ephrin and corresponding Ephs may directly intervene with DDR. Thus this thesis suggests that Ephrin B3 and its associated Ephs may be used as novel therapeutic targets in NSCLC alone or in combination with RT enabling further progress on precision cancer medicine.