Both the relative rarity of cancer stem-like cells in tumors and the absence of reproducible specific markers for glioma stem cell limit identification and isolation of this important cellular subpopulation. In this study we used the efflux of Hoechst dye, which allows identification of the side population phenotype, to isolate both tumor endothelial cells and tumor-initiating cells with cancer stem-like cells characteristic from mouse and human glioblastomas.
One of the main molecular changes accompanying progression of gliomas to high grade which increased stem cell character and enhances resistance to chemotherapy is the loss of PTEN and consequent elevation of Akt pathways activity (Hu et al., 2005
). Increased activity of the Akt pathway is associated with many forms of resistance and is found in a population of radiation resistant cancer stem-like cells in medulloblastomas where Akt inhibition appears to sensitize the cell population for radiation-induced apoptosis (Hambardzumyan et al., 2008b
). Our data provide further connections between the Akt pathway, stem-like character, and therapeutic resistance, suggesting that in addition to the induction of radiation resistance in medulloblastomas, activation of this pathway also enhances the ability of glioma cancer stem-like cells to expel drugs.
Although temozolomide is currently the first-line standard of care for the treatment of glioma, temozolomide increases the aggressiveness of surviving glioma cells by several mechanisms. We report in this study that temozolomide increases the number of SP cells, especially from PTEN-deleted gliomas. If temozolomide were a substrate for ABCG2, the data would suggest a selection for cells that efflux the drug. However, our data and other (Chua et al., 2008
) indicate it is not an ABCG2 substrate, implying that the increase in the SP phenotype might result from the enrichment of cells with stem-like properties. Therefore, in the process of increasing the number of cells in tumors with stem-like properties, temozolomide may render surviving cells even more resistant to subsequent treatment with drugs that are substrates for ABCG2. Ubiquitous distribution of temozolomide was reported into all human tissues including the brain, suggesting that the BBB efflux pumps do not contribute to temozolomide resistance (Ostermann et al., 2004
). Interestingly, we found that SP cells overexpress MGMT as compared to MP cells. The MGMT enzyme is responsible for the removal of alkyl groups induced by alkylating agents, suggesting that the resistance to temozolomide observed in glioma cancer stem-like cells might be related in part to its alkylating activity. A general trend towards high expression of several DNA repair genes, including MGMT, has been demonstrated in heamatopoeitic stem cells (Casorelli et al., 2007
), contributing to the higher drug resistance of cancer stem-like cells.
The SP cells from PTEN-deleted neurospheres were substantially more tumorigenic than the non-SP cells, as shown by a significant decrease in tumor latency. Moreover, temozolomide further enriched for highly tumorigenic cells, suggesting that temozolomide treatment could favor tumor recurrence. Because the SP phenotype in glioma cancer stem-like cells is mainly mediated by ABCG2, as shown by the almost complete abolition of the SP phenotype when blocking of its function, we subsequently studied the oncogenic potential of ABCG2. Although ABCG2 expression appears not to be oncogenic itself, selecting for the Hoechst dye exclusion phenotype, conferred by this transporter, highly enriches for tumor-promoting cells. Cancer stem-like cells simultaneously present impaired oncogenes/tumor suppressors expression and drug resistance abnormalities, two distinct properties that can be mutually exclusive. ABCG2 main function is to efflux compounds to protect the cells from cytotoxic agents, a drug-resistance characteristic of cancer stem-like cells, which defines the SP phenotype.
One focus of the current study was to elucidate the mechanisms regulating ABCG2 function. We noticed that although PI3K activity and temozolomide treatment changed the activity of ABCG2 in neurospheres, the expression levels of the mRNA and protein were unaffected. In mouse and human, inhibiting the PI3K/Akt pathway, but not mTOR, strongly decreased the activity of the transporter by modulating its localization at the cell membrane. These data suggest that therapeutic blockade of either PI3K or Akt might reduce ABCG2 function in cancer stem-like cells in vivo and improve chemotherapy efficacy for drugs that are substrates of this transporter.
In summary, our studies support a role for ABCG2 as a molecular determinant of the side population phenotype in glioma stem-like cells, a phenotype characterized by both tumorigenic and chemoresistant properties. Furthermore, we report that loss of the tumor suppressor PTEN, an important genomic alteration associated with glioma progression, greatly increases the SP and further enhances the MDR phenotype upon the standard treatment with temozolomide.