Temozolomide Down-Regulates P-glycoprotein In Glioblastoma Stem Cells By Interfering With The Wnt3a/GSK3/beta-catenin Pathway

C Riganti1,2, IC Salaroglio1, V Caldera3, I Campia1, J Kopecka1, M Mellai3, L Annovazzi3, A Bosia1,2, D Ghigo1,2, D Schiffer3. 1Department of Oncology, University of Turin, via Santena 5/bis, 10126, Turin, Italy, 2Center for Experimental Research and Medical Studies (Ce.R.M.S.), University of Turin, via Santena 5/bis, 10126, Turin, Italy, 3Neuro-bio-oncology Center, Policlinico di Monza Foundation, Via Pietro Micca 29, 13100, Vercelli, Italy

Glioblastoma multiforme stem cells display a highly chemoresistant phenotype. The role of the ATP-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp/ABCB1), multidrug resistance related proteins (MRPs/ABCCs), breast cancer resistance protein (BCRP/ABCG2), as inducers of the chemoresistant phenotype is poorly known. Also the effects of ABC transporters on the efficacy of temozolomide (TMZ), the first-line chemotherapeutic drug for glioblastoma, are controversial [1].

To clarify these issues, we analyzed human glioblastoma cells U87-MG and primary glioblastoma samples, each grown as stem cells (neurospheres, NS) and non stem cells (adherent cells, AC), and phenotypically characterized for stemness/differentiation markers [2].

In both U87-MG and primary glioblastoma cells, TMZ (50, 100, 200 μM, for 72 and 120 h) was cytotoxic in a dose- and time-dependent way in AC, not in NS. The same results were obtained with doxorubicin, vinblastine, etoposide, methotrexate, cisplatin, mitoxantrone. Surprisingly, the pre-treatment of NS with a non cytotoxic dose of TMZ (50 μM for 48 h), followed by a non-cytotoxic dose of doxorubicin (5 μM), vinblastine (20 nM), etoposide (10 μM) for 24 h induced cytotoxicity in NS (p < 0.02). Since doxorubicin, vinblastine and etoposide are all substrates of Pgp, we investigated whether there was a correlation between the expression of Pgp and the activity of the typical stemness pathways of glioblastoma cells. NS had significantly higher expression levels (p < 0.05) of Notch 1/2/3/4, Wnt3a, Sonic Hedgehog, Pgp, MRP1, BCRP than the corresponding AC. Of note, TMZ specifically decreased Wnt3a and Pgp in NS; time-dependence experiments showed that the decrease of Wnt3a transcription preceded the decrease of Pgp, suggesting that Wnt3a may control Pgp in glioblastoma stem cells. The stable overexpression of Wnt3a in primary glioblastoma cells was indeed sufficient to transform AC cells into NS and to simultaneously increase proliferation and Pgp expression. On the contrary primary glioblastoma NS silenced for Wnt3a lost the ability to form neurospheres and reduced at the same time the proliferation rate and the Pgp levels.

The promoter of Wnt3a gene is rich of CpG islands and was strongly unmethylated in primary glioblastoma NS cells. Acting as a DNA-alkylating agent, TMZ induced the methylation of Wnt3a promoter, decreased the autocrine synthesis of Wnt3a, disrupted the glycogen syntase-3 kinase/beta-catenin axis and prevented the nuclear translocation of beta-catenin, which is a transcriptional activator of Pgp. As a consequence, TMZ reduced the amount of Pgp protein in NS.

We suggest that low dose TMZ may chemosensitize the stem cells population of glioblastoma to substrates of Pgp (like doxorubicin, vinblastine and etoposide) by down-regulating the Wnt3a-dependent signalling.

References

[1] Kestel L, Pfirrmann M, Robel K, Illmer T, Kramer M, Dill C, Ehninger G, Schackert G, Krex D (2009). A MDR1 (ABCB1) gene single nucleotide polymorphism predicts outcome of temozolomide treatment in GBM patients. Annal Oncol 20: 175-181.

[2] Caldera V, Mellai M, Annovazzi L, Piazzi A, Lanotte M, Cassoni P, Schiffer D (2011). Antigenic and Genotypic Similarity between Primary GBMs and Their Derived Neurospheres. J Oncol 2011: 314962.