Abstract 3454: LIN28 is expressed in glioblastomas and promotes KRAS-mediated transformation of human neural stem cells

The pluripotency factor LIN28 is activated in many cancers arising outside the central nervous system, but its role in brain tumors is poorly understood. LIN28 inhibits the maturation of let-7, which in turn represses KRAS, c-myc, HMGA2 and other oncogenic factors, many of which are known to be activated in glioblastoma (GBM). Because malignant gliomas are thought to arise from normal human neural stem cells (hNSC), we hypothesized that the stem cell factor LIN28 plays a role in the development of high grade gliomas. Using immunohistochemistry, we detected LIN28 protein in a subset of gliomas, with higher expression in GBMs than in lower grade tumors. To examine if LIN28 would facilitate transformation of hNSC, we infected these cells with lentivirus encoding LIN28 together with dominant negative R248W p53 (DN-p53), constitutively active KRAS (CA-KRAS) and hTERT (hNSC-LIN28/DN-p53/CA-KRAS/hTERT), which are all commonly dysregulated in GBM. As controls, hNSC were also infected with DN-p53/CA-KRAS/hTERT, LIN28/DN-p53/hTERT, DN-p53/hTERT, or with GFP. The lentivirus transduced hNSCs continued to express the stem cell markers CD133, SOX2, Nestin and OLIG2, and retained the potential to differentiate along neuronal and glial lineages. The LIN28/DN-p53/CA-KRAS/hTERT expressing cells showed increased proliferation by BrdU assay compared to normal hNSCs. To test their tumorigenecity in vivo, hNSC-LIN28/DN-p53/CA-KRAS/hTERT were injected into the brains of immunocompromised mice. Eight weeks after injection, 66.7% (12 of 18) of the mice developed invasive brain tumors resembling GBMs which were positive for Nestin and GFAP, and negative for synaptophysin. In contrast, control injections including hNSC-DN-p53/hTERT (5 mice), hNSC-LIN28/DN-p53/hTERT (5 mice) and hNSC-GFP (10 mice) did not generate tumors over 6 months. Interestingly, hNSC-DN-p53/CA-KRAS/hTERT cultures proliferated very poorly, suggesting that both LIN28 and activated KRAS may play key roles in transformation. In support of this possibility, we found using quantitative RT-PCR and western blotting that hNSC-LIN28/DN-p53/CA-KRAS/hTERT expressed higher levels of KRAS and phospho-MAPK than hNSC-DN-p53/CA-KRAS/hTERT lacking LIN28, suggesting this stem cell factor is required to facilitate MAPK activation and complete transformation of hNSCs. To further confirm the role of LIN28 in the tumorigenicity of GBMs, a tumor-derived neurosphere line (JHH-GBM14) was infected with LIN28 (GBM14-LIN28) or GFP control (GBM14-GFP) and injected as orthotopic xenografts into immunocompromised mice. In addition to forming brain tumors larger than GBM14-GFP controls, GBM14-LIN28 tumors significantly invaded into normal tissues including the corpus callosum and the contralateral brain. Our data show that LIN28 is expressed in human GBMs and that LIN28 can facilitate tumorigenicity in orthotopic GBM mouse models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3454. doi:10.1158/1538-7445.AM2011-3454