SMAD7-Expression und Radiotoxizität: Klärung von Mechanismen für die beobachtete Korrelation in Fibroblasten mit der klinischen Toxizität und Entwicklung eines Zellmodells zur Testung pharmakologischer Interventionen
Zusammenfassung der Projektergebnisse
This project was focused on the impact of SMAD7 on acute side effects of radiochemotherapy as applied in the neoadjuvant setting for rectal cancer patients. Based on previous research in our facility we hypothesized that expression and genetic variation of SMAD7 might contribute to acute clinical toxicity. To explore this hypothesis in a broader manner several attempts as defined by sub-projects were undertaken. First, as we disposed on a patient set with documented clinical toxicity and matched patient-derived fibroblasts we set up for a comprehensive investigation of expression patterns in fibroblasts upon in vitro simulation of radiochemotherapy in relation to acute treatment-related side effects of the pertinent patients. Finally, a list of promising candidates was revealed in relation to acute toxicities of the respective patients. Specifically, the differential expression (irradiated versus non-irradiated fibroblasts) of four transcripts exhibited associations with the clinical toxicity data of p<0.001 upon multiple testing adjustment. These results are pending publication. Specific elements in the SMAD7 promoter are of crucial relevance for transcription as reported in literature. We could confirm that these elements are likewise relevant under radiation conditions. Based on data ascertained prior to start of this project it was suggested that inherited genetic variants at the SMAD7 locus might significantly modulate the risk of acute toxicity of the considered neoadjuvant radiochemotherapy. These preliminary data were considered as training set. Within this funded research proposal we sought to confirm the former associations in a test set comprising a clinical cohort of similar sample size. Though, allelic frequencies of the considered SMAD7 variants were almost identical between training and test set the genotype-toxicity associations could not be confirmed for any of the variants. Thus, we conclude that common inherited genetic variation in SMAD7 does not have a relevant impact on acute toxicities in this neoadjuvant treatment setting. We aimed to establish an ambitious cell model for real-time analysis of SMAD7 expression in living cells. This bio-engineered system should then serve as a tool to test pharmacological interventions for enhancing SMAD7 expression with the perspective to alleviate acute treatment-related toxicities. This project part was particularly labor intensive. Upon having overcome technical obstacles we could obtain a cell model with correct tagging of the endogenous SMAD7 locus. Our data indicate that this model could be used to monitor SMAD7 expression following radiation and to test potential interventions. However, a limitation is that the used CRISPR/Cas9-modified HEK293T cells turned out to be irresponsive to TGFβ1 treatment usually serving as a positive control for TGFβ pathway stimulation and induction of SMAD7 transcription. We did not expect this initially as the parent HEK293 cell line is TGFβ1-responsive. Using HEK293, SMAD7 expression was induced by high concentrations of aspirin in a dose-dependent manner. Finally, we conclude from this project as the most important findings: (1) Based on comprehensive transcriptome analyses, new candidates with potential relation to acute clinical toxicity were identified. (2) Common inherited genetic variability at the SMAD7 locus does not have a significant impact on the acute side effects of clinical treatment. (3) Using CRISPR/Cas9 technology, endogenous tagging of SMAD7 is feasible and its expression, which was demonstrated to be induced by radiation, can be monitored with this system.
Projektbezogene Publikationen (Auswahl)
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“Molekular-genetische Prädiktoren der Toxizität einer neoadjuvanten Radiochemotherapie am Beispiel des Rektumkarzinoms“; MD thesis 2019
Mergler, Caroline Patricia Nadine
