Reprogramming Of Mature Astrocytes By Abrogation Of Rb Tumor Suppression Leads To Astrocytoma Initiation In Gemms

Astrocytomas are the most common malignant brain tumors which maintain a poor survival rate despite decades of research effort. Understanding the initiation process and identifying astrocytoma cell(s) of origin have been of immense interest. Genetically engineered mouse models (GEMMs) are ideal for studying the initiation process compared to evolved human tumor cells. Our lab has developed an adult inducible GEMM that alters key human glioblastoma (Grade IV) pathways. Inactivation of Rb tumor suppression (TS) by expressing N-terminal 121aa of large T antigen (T121) under GFAP promoter is sufficient to initiate grade II astrocytoma. Addition of mutant KrasG12D leads to grade III and further PTEN deletion to glioblastomas. KrasG12D activation and PTEN deletion alone or in combination failed to initiate astrocytomas, suggesting that Rb TS inactivation is essential for its initiation. Studies from several cancers have identified tumor-initiating cells, a subset of tumor cells with stem cell-like properties responsible for generating the entire tumor mass. Their origin remains a mystery. We hypothesize that Rb TS inactivation reprograms astrocytes into stem/progenitor-like state generating potential cell(s) of origin for astrocytoma. Immunostaining of progenitor and proliferation markers (e.g. nestin and Ki-67) showed their co-expression in the cortex with T121, suggesting a possible reprogramming of mature astrocytes. Moreover, astrocyte differentiation marker S100beta was downregulated in T121 cells. To rule out the possibility of migration resulting in the observed cortical progenitor cells, focal induction of T121 in cortex using lenti-cre injection was performed. We found expression of Ki-67 and nestin and reduced expression of S100beta, suggesting that the reprogramming was independent of the germinal zones in the brain. Furthermore, cortical T121 expressing astrocytes were able to generate neurospheres but astrocytes from wildtype cortex failed to do so. These cells also showed self-renewability and multilineage ability, emphasizing their dedifferentiated status upon Rb-TS inactivation. We were able to achieve in-vitro reprogramming of mature astrocytes upon T121 expression suggesting that the reprogramming does not require brain microenvironment. Sphere assays using cortical cells from mice where individual Rb family member was inactivated suggests that loss of individual Rb family members alone is insufficient in reprogramming the mature astrocytes. With the Rb pathway known to be altered in more than 75% of glioblastomas, our results are a step in the direction of unraveling the process of tumor initiation for this deadly disease. Citation Format: Amit S. Adhikari, Teresa Sullivan, Yurong Song, Xiuyun Lu, Norene O9Sullivan, Terry Van Dyke. Reprogramming of mature astrocytes by abrogation of Rb tumor suppression leads to astrocytoma initiation in GEMMs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5014. doi:10.1158/1538-7445.AM2013-5014