Tipos de Acoso Escolar

Interestingly, the data obtained from the mass spectrometry analysis in MDA-MB-231 cells, displayed the EGFR amongst others as an interaction partner of the FGFR4. The EGFR is a key regulator of various processes in cancers, approved therapeutic target and the main component of tumor progression in the WAP-TGFα mouse mammary carcinoma model used

in our experiments. Therefore, the validation of the potential interaction between the EGFR and the FGFR4 preceded the validation of the other analyzed interaction partners.

First we aimed to show, that the FGFR4 gets co-immunoprecipitated with the EGFR in MDA- MB-231 cells overexpressing either the empty pLXSN, pLXSN-Gly388 or –Arg388 (Figure 39A). These data indicate a first hint for the interaction of these two receptors. In contrast to the mass spectrometry analysis, the Western Blot Analysis displayed an increased content of co-immunoprecipitated FGFR4 Arg388 compared to FGFR4 Gly388. As expected, the negative control displayed no co-immunoprecipitated FGFR4 as FGFR4 is barely expressed in MDA-MB-231 cells. Nevertheless, as proteins are mostly localized in clusters on the membrane, co-immunoprecipitation is no final evidence for an interaction of two receptors. Therefore, we investigated the EGFR-FGFR4 interaction upon EGF stimulation. As shown in Figure 39B, the EGFR displays increased phosphorylation in the presence of the overexpressed FGFR4. Furthermore, the EGFR in MDA-MB-231 cells overexpressing the FGFR4 Arg388 is even more activated than in the presence of the FGFR4 Gly388. Interestingly, the co-immunoprecipitated FGFR4-Arg388 is more active than the FGFR4- Gly388. Above that, phosphorylation of the FGFR4 increases over time upon EGF stimulation. These data are confirmed by the quantification of the Western Blot Analysis (Figure 39C) Furthermore, the activation of the downstream signalling protein Akt is increased in MDA-MB-231 cells overexpressing the FGFR4 Arg388 upon EGF stimulation. The activation of Erk did not differ between the different FGFR4 isotypes (data not shown). This result indicates a physiological interaction of the FGFR4 and EGFR upon EGF stimulation. Similarily, the EGFR-FGFR4 interaction is hardly seen in unstimulated cells. In summary, the FGFR4 and the EGFR are direct interaction partners. Here, FGFR4 seems to support EGFR induced signalling by receptor phosphorylation upon EGF stimulation, whereas the FGFR4 Arg388 enhances the signal.

Figure 39: Validation of the EGFR/FGFR4 interaction; A) Co-Immunoprecipitation of EGFR and FGFR4 in MDA-MB-231 cells overexpressing the empty pLXSN, pLXSN-Gly388 and –Arg388: Interaction of EGFR and FGFR4 Arg388 seems to be stronger than EGFR and FGFR4 Gly388; B) EGFR-FGFR4 interaction upon EGF-stimulation: increased phosphorylation of the EGFR and accelerated FGFR4 interaction and activation in MDA-MB-231 cells expressing the FGFR4 Arg388; C) Quantification of Western Blot Analysis of EGF stimulated MDA-MB-231 cells: MDA-MB-231 cells expressing the FGFR4 Ag388 display an accelerated EGFR and Akt activation, total EGFR and tubulin served as normalization value for quantification, respectively; the co-immunoprecipitated FGFR4 Arg388 displays a accelerated binding to the EGFR and increased activation comparetd to the co-immunoprecipitated FGFR4 Gly388

To further confirm the data obtained in MDA-MB-231 cells we investigated the signalling upon EGF and TGFα stimulation in MEFs derived from the FGFR4 Arg385 KI mice transformed with EGFR. MEFs transformed with EGFR displayed an accelerated and prolonged activation of Akt in the presence of the FGFR4 Arg385 allele upon EGF and TGFα stimulation (Figure 40A). The activation of Erk shows no difference between the different FGFR4 isotypes (data not shown). Similar to the MDA-MB-231 cells overexpressing the FGFR4 Arg388, MEFs transformed with EGFR and expressing the FGFR4 Arg385 display a significant increase in pEGFR levels compared to FGFR4 Gly385 MEFs (EGF5’-p=0.000073, EGF10’-p=0.0025, TGFα5’-p=0.07, TGFα10’-p=0.01) (Figure 40B). Above that, MEFs transformed with EGFR display an activation of the FGFR4 upon EGF and TGFα stimulation (Figure 40C). Similar to MDA-MB-231 cells, MEFs expressing the FGFR4 Arg385 allele display an increased activation of the FGFR4. These data confirm the results obtained in MDA-MB-231 cells. The FGFR4 Arg385 clearly supports the activation and following downstream signaling of the EGFR.

Figure 40: Western Blot analysis of MEFs derived from FGFR4 Gly385 or Arg385 homozygous mice transformed with EGFR upon EGF and TGFαααα stimulation; A) MEFs transformed with EGFR display an increased and prolonged activation of Akt upon EGF and TGFαααα stimulation when expressing the FGFR4 Arg385 allele; B) MEFs transformed with EGFR display an significantly increased activation of the EGFR upon EGF and TGFαααα stimulation when expressing the FGFR4 Arg385 allele (EGF5’-p=0.000073, EGF10’- p=0.0025, TGFαααα5’-p=0.07, TGFαααα10’-p=0.01); actin served as a normalization value for quantification C) In MEFs, transformed with EGFR, FGFR4 gets activated upon EGF and TGFαααα stimulation whereas the FGFR4 Arg385 displays an increased phosphorylation compared to the FGFR4 Gly385; All data are shown as mean ± SDM; all p-values were calculated using the students T-test and values ≤ 0.03 were considered statistically significant.

4.3.3

The FGFR4 Arg385 influences the migratory behavior and the