Abstract 4257: Overexpression of Gut-enriched Krüppel-like Factor (KLF-4) reduces the tumor-initiating potential of primary colon cancer derived spheres
Authors: Hoffmann Christopher M. , Ball Claudia R. , Dieter Sebastian M. , Nowrouzi Ali , Schmidt Manfred , Koch Moritz , Weitz Jürgen , von Kalle Christof , Glimm Hanno 
Published: February 2011

Compelling evidence accumulated over the last years indicates that a small subfraction of all cells within tumors, so called tumor initiating cells (TIC), drive tumor formation as well as progression to metastases in human colon cancer. Tumorsphere cultures of primary human colon cancers allow enriching TICs that form tumors in xenotransplanted mice independent of the expression of cell surface markers. Even though self renewal and differentiation of tumor initiating cells can be specifically monitored in spheroid cultures combined with xenotransplantation the mechanisms that govern colon cancer TIC biology are not fully understood. KLF-4 is highly expressed in differentiated colonic epithelial cells but not in their precursors and it is upregulated various colon cancer cell lines when they are cultured under conditions that drive their differentiation. We now analyzed whether KLF-4 modulates the biology of TIC in human colon cancer.

To address this question, primary human colon cancer cells were cultured in single cell suspension under serum-free conditions in the presence of 20ng/µL EGF and 10ng/µL FGF. After 4-6 weeks in culture, tumorsphere formation was detectable in 20% of all samples. Tumorspheres were dissociated and infected with lentiviral vectors encoding either KLF-4 or GFP (MOI 1-50). KLF-4 expression decreased the sphere forming potential 50-fold : 10 spheres were generated per 1000 seeded KLF-4+ cells after 10 days in culture as compared to 500 spheres per 1000 GFP+ or non-transduced cells. To analyze if KLF4 expression leads to apoptosis of colon cancer sphere forming cells propidium-iodide/annexin staining were performed. KLF-4 expression significantly increased the proportion of apoptotic cells (38% in KLF-4 transduced cells as compared to 12% in control cells). Tumor-forming potential of Klf4 expressing tumor sphere cells was determined by xenotransplantation under the kidney capsule of immunodeficient NOD/SCID-IL2RG−/− mice 16h after transduction. Mice were sacrificed on day 45 after transplantation and tumors were weighted. In all mice, tumor-growth and metastasis formation to the liver were detectable. However, the absolute tumor weight was reduced by about 70% in mice transplanted with KLF4 transduced cells as compared to tumors in control mice. No expression of KLF-4 was detectable in control tumors or KLF4 transduced tumors by RT-PCR, indicating that only non-transduced or non-expressing cells present in the transduction cultures contributed to tumor formation.

In summary, we provide evidence that human KLF-4 induces apoptosis in human colon cancer tumor-initiating cells and that its expression suppresses tumor-formation. Therapeutic strategies leading to an upregulation of KLF-4 may eradicate tumor initiating cells in colon cancer.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4257.