Molekulare Grundlage der Selektion von Adenomatöse Polyposis Coli Genmutationen im kolorektalen Karzinom

• Antragsteller: Jean Schneikert, Ph.D.
• Fachliche Zuordnung: Hämatologie, Onkologie
• Förderung: Förderung von 2009 bis 2013
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 128047551

• Erstellungsjahr: 2013

Zusammenfassung der Projektergebnisse

Adenomatous Polyposis Coli (APC) and axin/axin2 are components of the destruction complex that initiates the phosphorylation-dependent degradation of ®-catenin. ®-catenin is a transcription factor, the main effector of the wnt signaling pathway that stimulates the proliferation of the few stem cells located at the bottom of the crypts of the colonic epithelium. The accumulation of ®-catenin in stem cells is tightly regulated by the destruction complex. The mechanistic steps occurring on APC are still not completely understood. APC contains beta-catenin binding sites called 15 and 20 amino acid repeats (15R and 20R), the latter being intermingled with the SAMP repeats that are Axin/Axin2 binding sites. The 15R bind to beta-catenin and are important to target beta-catenin for degradation. The 20R are heterogeneous. Both 20R1 and 20R3 bind to beta-catenin with different affinities, but the 20R2 cannot bind to beta-catenin. APC has drawn a particular attention, because truncating mutations have been found in approximately 80% of colorectal tumors. The truncations occur almost always after the first 15R, suggesting that it plays an important role in tumor development. We have shown that colon cancer cell lines express also the paralog APC-like (APCL or APC2). APC and APCL display similar topological organizations, but APCL lacks the 15R region. RNA interference revealed that APCL controls the level and/or the activity of ®-catenin, but it is less efficient and binds less well to ®-catenin than APC, thereby providing one explanation as to why sequence alterations of APCL have never been reported in colon cancer so far. A further comparison indicated that APCL down-regulates the ®-catenin level despite the lack of the 15R region known to be important in APC. To understand this discrepancy, we performed immunoprecipitation experiments that revealed that phosphorylated ®-catenin displays a preference for binding to the 15 amino acid repeats (15R) rather than the first 20 amino acid repeat of APC. This suggested that the 15R region constitutes a gate connecting the steps of ®-catenin phosphorylation and subsequent ubiquitination/degradation. Using RNA interference and domain swapping experiments, we showed that APCL benefits from the 15R of truncated APC to target ®-catenin for degradation, in a process likely involving heterodimerization of the two partners. Our data suggest that the functional complementation of APCL by APC constitutes a substantial facet of tumor development, because the truncating mutations of APC in colorectal tumors from familial adenomatous polyposis patients are almost always selected for the retention of at least one 15R. We found that Axin interacts with the second twenty amino acid repeat (20R2) of APC and APCL. Our results suggest that beta-catenin phosphorylation depends on Axin bound to the 20R2 of either APC or APCL. Our results indicate that Axin stimulates the displacement of APC, but not APCL, from the cytoskeleton and its incorporation into bright cytoplasmic dots that others recognize as the beta-catenin destruction complex. The SAMP repeats of APC are known to interact with the N-terminal RGS domain of Axin. Our data suggest that the SAMP repeats of APC stimulate Axin oligomerisation by releasing internal inhibitory constraints imposed by the RGS domain of Axin. This occurs independently of Axin interacting with the 20R2. Our study provides evidence that Axin bound to the second twenty amino acid repeat (20R2) of APC fulfils a different role than Axin bound to the SAMP repeats of APC. Considering our data together with those from others, we discuss a working model whereby phosphorylation of beta-catenin would occur with Axin bound to the 20R2 of APC or APCL, whereas degradation of phospho-beta-catenin with oligomerisation of Axin bound to the SAMP repeats of APC.

Projektbezogene Publikationen (Auswahl)

• Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation. EMBO J. 2011 Apr 20;30(8):1433-43.
  Tanneberger, Kristina; Pfister, Astrid S; Brauburger, Katharina; Schneikert, Jean; Hadjihannas, Michel V; Kriz, Vitezslav; Schulte, Gunnar; Bryja, Vitezslav & Behrens, Jürgen
  
• APC mutations in colorectal tumours from FAP patients are selected for CtBP- mediated oligomerization of truncated APC. Hum Mol Genet. 2011 Sep 15;20(18):3554-64
  Schneikert, Jean; Brauburger, Katharina & Behrens, Jürgen
  
• A common role for various human truncated adenomatous polyposis coli isoforms in the control of beta-catenin activity and cell proliferation. PLoS One. 2012;7(4):e34479
  Vijaya Chandra, Shree Harsha; Wacker, Ingrid; Appelt, Uwe Kurt; Behrens, Jürgen & Schneikert, Jean
  
• Functional Comparison of Human Adenomatous Polyposis Coli (APC) and APC-Like in Targeting Beta-Catenin for Degradation. PloS ONE, 2013, 8: e68072
  Schneikert, Jean; Vijaya Chandra, Shree Harsha; Ruppert, Jan Gustav; Ray, Suparna; Wenzel, Eva Maria & Behrens, Jürgen
  
• Different Roles for the Axin Interactions with the SAMP versus the Second Twenty Amino Acid Repeat of Adenomatous Polyposis Coli. PloS ONE, 2014, 9: e94413.
  Schneikert, Jean; Ruppert, Jan Gustav; Behrens, Jürgen & Wenzel, Eva Maria