Abstract 2048: PTEN deficient lung cancer cells are sensitive to the combination of olaparib with cisplatin through suppressing DNA damage signaling.
Authors: Minami Daisuke , Takigawa Nagio , Takeda Hiromasa , Takata Minoru , Ochi Nobuaki , Ichihara Eiki , Hisamoto Akiko , Hotta Katsuyuki , Tanimoto Mitsune , Kiura Katsuyuki 
Published: August 2014

Abstract
PARP inhibition in combination with DNA-damaging agents is one of the most promising new therapeutic approaches to cancer. PTEN is a tumor-suppressor gene deactivating PI3K downstream of EGFR signaling. In H1650 cells harboring EGFR deletion mutation in exon 19 with PTEN loss, we reported that synergistic effects of a PARP inhibitor (olaparib) and a DNA-damaging agent (cisplatin) were observed in vitro and in vivo (#4686, AACR 2012).
We further clarified how PTEN loss affected DNA damage signaling. H1299 and A549 are lung cancer cells harboring wild type PTEN. Antagonistic effects of olaparib with cisplatin were shown in H1299 and A549 cells according to combination index (CI). Sensitivity to a PARP inhibitor was reported to be associated with a defect in Mre11 expression. H1650 cells exhibited lower levels of Mre11 compared with PTEN-transfected H1650 cells (designated H1650PTEN+ cells). To address how lower expression levels of Mre11 affects the synergism between cisplatin and olaparib, Mre11 expression vector was transfected into H1650 cells (designated H1650Mre11+ cells). CIs were 0.23, 0.20, 0.57 and 0.29 when concentration ratios of cisplatin and olaparib were designed to be molar ratios of 1:1, 1:2, 1:3 and 1:5, respectively, in H1650 cells, while CIs were 0.76, 0.91, 0.97 and 0.77 in H1650Mre11+ cells. Although these CIs in H1650Mre11+ were somewhat elevated compared with those observed in original H1650 cells, these results indicated that lower levels of Mre11 alone could not be the sole reason for the synergism. On the other hand, PTEN has other nuclear functions, including transcriptional regulation of the RAD51 gene, whose product is essential for homologous recombination (HR) repair of DNA breaks. RPA is displaced from single stranded DNA by RAD51 to initiate HR. There were no significant differences of RAD51 and RPA levels by Western blotting. Meanwhile, PTEN deficiency resulted in significant reduction in RAD51 and RPA focus formation after drug-exposure or irradiation compared to H1650PTEN+ cells although γH2AX was similarly increased in both cells. As RPA binds to single stranded DNA, the RPA focus could be a marker for end resection at the double stranded DNA ends. Thus, inactivation of PTEN might lead to suppression of DNA damage signaling, leading to the lower levels of end resection and, hence, less RPA focus formation. As shown above, reduced levels of Mre11 alone could not provide a sufficient explanation for this, though Mre11 is involved in the molecular mechanisms of the end resection.
The combination of cisplatin with olaparib in PTEN deficient lung tumors might be pursued in clinical trials although further investigations should be required to clarify whether and to what extent the molecular events including Mre11, RAD51 and RPA are responsible for the synergic effect.
Citation Format: Daisuke Minami, Nagio Takigawa, Hiromasa Takeda, Minoru Takata, Nobuaki Ochi, Eiki Ichihara, Akiko Hisamoto, Katsuyuki Hotta, Mitsune Tanimoto, Katsuyuki Kiura. PTEN deficient lung cancer cells are sensitive to the combination of olaparib with cisplatin through suppressing DNA damage signaling. [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 2048. doi:10.1158/1538-7445.AM2013-2048