Activation of Wnt/β-catenin signaling plays a central role in the development and progression of colorectal cancer (CRC). The Wnt-transcription factor, TCF7L2, is overexpressed in primary rectal cancers that are resistant to chemoradiotherapy (CRT) and TCF7L2 mediates resistance to CRT. However, it is unclear whether the resistance is mediated by a TCF7L2 inherent mechanism or Wnt/β-catenin signaling in general. Here, inhibition of β-catenin by siRNAs or a small molecule inhibitor (XAV-939) resulted in sensitization of CRC cells to CRT. To investigate the potential role of Wnt/β-catenin signaling in controlling therapeutic responsiveness, non-tumorigenic RPE-1 cells were stimulated with Wnt-3a, a physiological ligand of Frizzled-receptors, which increased resistance to CRT. This effect could be recapitulated by overexpression of a degradation-resistant mutant of β-catenin (S33Y), also boosting resistance of RPE-1 cells to CRT, which was, conversely, abrogated by siRNA-mediated silencing of β-catenin. Consistent with these findings, higher expression levels of active β-catenin were observed as well as increased TCF/LEF reporter activity in SW1463 cells that evolved radiation resistance due to repeated radiation treatment. Global gene expression profiling identified several altered pathways, including PPAR signaling and other metabolic pathways, associated with cellular response to radiation. In summary, aberrant activation of Wnt/β-catenin signaling not only regulates the development and progression of colorectal cancer, but also mediates resistance of rectal cancers to chemoradiotherapy. Implications Targeting Wnt/β-catenin signaling or one of the downstream pathways represents a promising strategy to increase response to CRT.

Background: Preoperative chemoradiotherapy represents the standard treatment for patients with rectal cancer. However, the clinical response of individual tumors to multimodal treatment is not uniform, and ranges from complete response to complete resistance. Therefore, the identification of novel therapeutic targets whose modification could be harnessed to sensitize a priori resistant tumors is exceedingly important. In this context, we have previously demonstrated that the Wnt transcription factor TCF4 was over-expressed in rectal cancers that were resistant to chemoradiotherapy. The aim of this study was to explore the functional relevance of TCF4 and Wnt/β-catenin signaling for mediating treatment resistance. Methods: Colorectal cancer cell lines SW837, SW480, and HT-29 were transfected with shRNAs targeting TCF4. After establishing stable single-cell clone (SCC) populations, selected clones were irradiated at 0, 1, 2, 4, 6 and 8 Gy of X-rays. To assess the putative Wnt/β-catenin dependency, β-catenin was inhibited in cancer cell lines using siRNAs, the tankyrase inhibitors XAV939 / JW55 and the porcupine inhibitor LGK 974. Furthermore Wnt/β-catenin signaling activity was stimulated in normal retinal pigment epithelial (RPE) cells through incubation with Wnt3a. Results: RNAi-mediated silencing of TCF4 led to a significant radiosensitization in SW837 and SW480, two cell lines with a high basal TCF4-activity, whereas no effect was observed in HT-29 a cell line with low activity. Inhibition of β-catenin similarly caused a pronounced sensitization of CRC cells to clinically relevant doses of X-rays. Finally, exogenous stimulation of canonical Wnt signaling with Wnt3a resulted in a significantly decreased sensitivity of RPE cells to radiotherapy, i.e. induced radioresistance. Conclusion: We have uncovered a novel role of Wnt/β-catenin signaling in mediating resistance to chemoradiotherapy. Moreover, these data suggest that targeting key components of this pathway may represent a potential therapeutic strategy to increase the proportion of patients that respond to chemoradiotherapy, with considerable clinical implications. Citation Format: Georg Emons, Melanie Spitzner, Sebastian Reineke, Janneke Möller, Emil Kendziorra, Tim Beissbarth, Margret Rave-Fraenk, Jochen Gaedcke, Thomas Ried, Michael Ghadimi, Marian Grade. Wnt/ß-catenin signaling mediates resistance of colorectal cancer cells to chemoradiotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3946. doi:10.1158/1538-7445.AM2014-3946

Background: Preoperative chemoradiotherapy represents the standard treatment for patients with rectal cancer. However, the clinical response of individual tumors to multimodal treatment is not uniform, and ranges from complete response to complete resistance. Therefore, the identification of novel therapeutic targets whose modification could be harnessed to sensitize a priori resistant tumors is exceedingly important. Previously, we demonstrated that the Wnt transcription factor TCF7L2 (also known as TCF4) was overexpressed in primary rectal cancers that were resistant to chemoradiotherapy (CRT), and that TCF7L2 functionally mediates resistance of CRC cells to clinically relevant doses of ionizing radiation (IR). Methods: Using siRNAs we silenced CTNNB1 (β-catenin), another key-component of canonical Wnt-signaling, in colorectal cancer cell lines LS1034, SW480, and SW837. To asses influence on CRT, cells were exposed to 0, 1, 2, 4, 6 and 8 Gy of X-rays and 5-FU. Wnt- signaling was stimulated in retinal pigment epithelial cells (RPE) either by adding Wnt-3A, or overexpressing non degradable β-catenin (S33Y-mutated) and analyzed changes in CRT. Finally we repetitively irradiated SW1463 (68Gy) to establish an isogenic radio-resistant cell line and examined changes in protein expression. Results: Silencing of CTNNB1 resulted in (chemo-) radiation-sensitization of all three CRC-cell lines. To further investigate the potential role of Wnt/β-catenin signaling in controlling therapeutic responsiveness, non-tumorigenic RPE cells were stimulated with Wnt-3A, which significantly increased resistance to CRT. This effect could be recapitulated by overexpression of β-catenin (S33Y-mutated), resulting in a significantly increased resistance to CRT. The effect could be rescued by siRNA mediated knockdown of β-catenin. Consistent with these findings, we observed higher expression levels of active (unphosphorylated) β-catenin as well as increased TCF reporter activity in SW1463 cells that were rendered radiation-resistant due to repeated IR treatment. Conclusion: Together, these findings strongly support the interpretation that Wnt/β-catenin signaling plays a central role in mediating resistance of CRC cells to CRT. Hence, pathway inhibition may represent a promising strategy to increase therapeutic responsiveness to CRT, which represents the standard treatment for locally advanced rectal cancers. This would have considerable clinical implications. Citation Format: Georg Emons, Melanie Spitzner, Sebastian Reineke, Frank Kramer, Margret Rave-Fraenk, Jochen Gaedcke, Michael Ghadimi, Thomas Ried, Marian Grade. A novel role for Wnt/β-catenin signaling in mediating resistance of colorectal cancer to chemoradiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1653.

aktivem β-Catenin mit siβ-Catenin in der Zelllinie LS1034 führt somit zu einer signifikanten Radiotherapiesensibilisierung. Behandlung mit XAV939Die siRNA-Versuche bestätigten die Relevanz des Wnt/β-Catenin-Signalwegs für die Radiotherapieresistenz. Allerdings ist der klinische Einsatz von siRNAs bisher noch schwierig und problematisch. Daher war das nächste Ziel, die Ergebnisse mithilfe des kleinmolekularen Inhibitors XAV939 zu überprüfen, da dieser potentiell klinisch einsetzbar ist. Der Tankyraseinhibitor XAV939 führt, wie in Abbildung 5 zu sehen, durch Abbauhemmung zu einer Stabilisierung von Axin und durch Zunahme des proteasomalen Abbaus zu einer verstärkten Degradation von β-Catenin (Huang et al. 2009). Somit wird der Wnt/β-Catenin-Signalweg durch XAV939 inhibiert. Behandlung von SW480 mit XAV939Bei den Versuchen mit XAV939 wurde ebenfalls mit der Zelllinie SW480 begonnen. Zunächst erfolgte eine Zeitpunkts-und Dosisfindung (siehe Kapitel 2.2.5.1). In Abbildung 16 ist zu erkennen, dass zum Zeitpunkt 6 h weder Proteinlevel von Axin2 noch von aktivem β-Catenin zu detektieren sind. Nach 24 h Inkubation mit XAV939 ist eine Zunahme der Proteinlevel von Axin2 und eine leichte Abnahme der Proteinlevel von aktivem β-Catenin im Vergleich zur Kontrolle zu erkennen.Abbildung 16: Zeitreihe SW480 mit XAV939. Immunfärbung von Aktin, Axin2 und aktivem β-Catenin aus Zelllysaten nach 6 -48 h Inkubation mit XAV939. Nach 48 h ist eine deutliche Stabilisierung von Axin2 und ein Abnahme von aktivem β-Catenin im Vergleich zur DMSO-Kontrolle zu sehen. Nach 48 h erfolgt eine deutliche Stabilisierung von Axin2 bei gleichzeitiger deutlicher Abnahme von aktivem β-Catenin. Somit ist die erwartete Wirkung des XAV939 bei der Zelllinie SW480 nach 48 h Inkubation mit XAV939 zu erkennen. Als nächstes erfolgte eine Optimierung der XAV939-Konzentration. Dazu wurde die bereits etablierte Inkubationszeit von 48 h verwendet und die XAV939-Konzentration von 0,25 bis 20 µM variiert. Als

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