Structural and Functional Characterization of the RAI2 Protein
Final Report Abstract
The purpose of this study was to reveal the poorly characterized molecular function of the RAI2 protein and to explore whether it could represent a novel drug-able target molecule. The search for novel interacting partners revealed that RAI2 shows affinity to PARylated proteins that are known to be involved in nuclear organization and the DNA damage response. Although the exact meaning of the newly identified molecular interactions for the maintenance of chromosomal integrity remains to be determined in future studies, we could already show that RAI2 depleted cells possess increased PARP1 activation and thus reduced DNA repair capacity as indicated by the reduced NAD/NADH ratios after short-term peroxide treatment. Based on our results we propose that RAI2 acts in the presence of genotoxic stress as a recruitment factor for scaffolding proteins of the DNA damage response, which contributes to an efficient DNA repair especially for repairing single-strand breaks. Among the identified interacting proteins CtBP1 showed by far the highest affinity to RAI2. Consequently, the molecular interaction between RAI2 and CtBP’s were studied in more detail at the molecular level. We have established that truncated RAI2 proteins (RAI2303-465 and RAI2303-362) binds CtBP’s in the low micromolar range and unexpectedly induces the formation of high molecular weight complexes when both the motifs are intact. Based on these data we engaged into a number of different structural biology approaches. By using X- ray crystallography, we determined the high-resolution structure of tetrameric CtBP2 in the presence of a short binding fragment of RAI2, showing a hetero-octameric CtBP24RAI22 complex. By using NMR- spectroscopy (in collaboration with O. Ohlenschläger, Jena), we determined the structure of the C-terminal part of RAI2, demonstrating that the two CtBP1/2 binding sites are in a flexible orientation with respect to each other. Finally, using SAXS and electron microscopy we found that wt-RAI2 with two intact CtBP binding sites leads to polymerization. A present negative stain EM map reveals a polymer with a thickness of 9 nm, matching the overall dimensions of a tetrameric CtBP, as observed by X-ray crystallography. Our present analysis suggests a stacking interaction of CtBP tetramers. How these CtBP tetramers are “glued” by RAI2 is still under investigation. We are also in the process of evaluating whether these polymers are suitable for high-resolution cryo electron microscopy. Based on these structural data, we reasoned that RAI2 with two CtBP binding sites could become a mediator for RAI2-induced polymerization. Interestingly, we concordantly observe RAI2-mediated CtBP polymerization in vivo through the formation of nuclear speckles in breast cancer cells. Abrogation of RAI2-CtBP interaction leads to their disappearance showing the importance of the tandem motif found in RAI2 on the cell biological level. Also, we could show that high RAI2 levels are inhibiting the co-repressor activity of CtBPs as shown by increased transactivation of the proximal promotor of the CDKN1A gene. We are planning to verify whether our observations on nuclear speckles in tumor cells and RAI2/CtBP polymerization are showing the same of similar features under different conditions (in vitro, in cells) and at different resolution scales, by using electron tomography (together with Julia Mahamid, EMBL Heidelberg). In conclusion, we demonstrate that RAI2 by using the synergistic effect of its two CtBP-binding motifs induces the polymeric assembly of CtBPs and thus disabling their corepressor function. Interestingly, our observations could become a blueprint for RAI2- related viral proteins with a double CtBP binding motif. A similar polymerization pattern as observed for RAI2/CtBP has indeed also been observed for Epstein Barr Virus Nuclear Antigen 3A (EBNA3A) protein from the Epstein-Barr virus, which has been shown to have tumor-promoting effects. Taking these data together, we are presently preparing a proof-of-principle study to exploit RAI2-induced CtBP polymerization, and hence inactivation as a transcriptional co-regulator, for potential drug discovery. We further performed analysis with breast cancer cell lines and with clinical specimens from published breast cancer cohorts. We found out that RAI2 depletion in breast cancer cell lines leads to impaired mitotic fidelity and induces chromosomal instability. Additionally, we were able to show that RAI2- depletion is sensitizing KPL-1 and MCF-7 breast cancer cells for the treatment with Topoisomerase I and Aurora-A inhibitors, indicating a synthetic lethality in relation to RAI2 status and again provides evidence for a functional role of RAI2 in the DNA damage response. By analyzing gene expression profiles of published breast cancer cohorts, we concordantly showed that RAI2 is among the top genes (rank = 36 out of 12961) whose expression anti-correlates with aneuploidy in primary breast tumours. Taken together, herein we have provided evidence that RAI2 is important for the maintenance of genomic integrity of breast cancer cells. The identification of synthetic lethality associated with low RAI2 expression in combination with drugs such as Aurora-A and Topoisomerase I inhibitors could be perhaps exploited in clinical setting, which needs obviously to be proven in further experimental settings. On the molecular level we found out that the core region of the RAI2 protein is valid to induce oligomerization of CtBP factors which is prone to inhibit their biological activity as transcriptional corepressors. Based on these findings, we propose that ligands comprising two CtBP binding motifs may have an enhanced effect on functional deactivation of CtBP caused by polymerization.
Publications
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Expression of Epithelial Mesenchymal Transition and Cancer Stem Cell Markers in Circulating Tumor Cells. In: Advances in Experimental Medicine & Biology, Isolation and Molecular Characterization of Circulating Tumor Cells. ISBN 978-3-319-55947-6, pp 205- 228
Werner S, Stenzl A, Pantel K, Todenhöfer T
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Tracing the Seeds in the Soil. Clin Chem. 2017 Jul 7
Werner S, Pantel K
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AACR Annual Meeting 2018. Chicago, USA. Deciphering the interactome of the metastasis-suppressor protein RAI2
Werner S. et al.
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AACR Annual Meeting 2019. Atlanta, USA. Blood-borne tumor cell dissemination in cancer patients (extended abstract)
Pantel K & Werner S.
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Epithelial cell adhesion molecule (EpCAM) as key marker of circulating tumor cells in cancer patients. Cell Stress. 2019 May 21
Keller L, Werner S, Pantel K
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Epithelial keratins: Biology and implications as diagnostic markers for liquid biopsies. Molecular Aspects of Medicine, 2019 Sep. 25
Stefan Werner, Laura Keller, Klaus Pantel
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Liquid biopsy and minimal residual disease - latest advances and implications for cure. Nat Rev Clin Oncol 2019;16: 409-24
Pantel K, Alix-Panabieres C
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Unravelling tumour heterogeneity by single-cell profiling of circulating tumour cells. Nat Rev Cancer. 2019 Aug 27
Keller L, Pantel K
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Pathophysiology of Tumor Cell Release into the Circulation and characterization of CTC. In: Tumor Liquid Biopsies. Springer Print ISBN 978-3-030-26438-3
Todenhöfer T, Stenzl A, Pantel K, Werner S
