**1. Introduction**

Activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway is frequently detected in human malignancies, including gastrointestinal stromal tumors (GIST) and soft tissue sarcomas (STS). This pathway supports tumor growth and progression and is thought to be linked with resistance to the current therapeutic regimens, including certain chemotherapeutic agents, ionizing radiation (IR) and targeted-based therapies. In particular, activation of PI3K/Akt/mTOR pathway in GIST is due to the constitutive activation of c-KIT/PDGFRA autophosphorylation and involved in oncogenesis and tumor progression at various disease stages [1], and is also thought to be linked with GIST resistance

to imatinib mesylate (IM) [2,3]. Moreover, the preclinical experiments illustrated that a long-term exposure of IM- and sunitinib (SU)-resistant cell lines to SU induces epigenetic silencing of *PTEN* with a consequent overactivation of the PI3K/AKT pathway [4]. This also correlates with a clinical data illustrating that low/negative IHC-staining for PTEN was associated with aggressive disease [5], thereby suggesting that *PTEN* depletion and/or silencing is also associated with aggressive phenotype and resistance to RTK inhibition. Important, molecular and genomic changes in IM-resistant GISTs illustrated that the PI3K/Akt/mTOR pathway has a greater importance in IM-resistant GIST than other pathways downstream of c-KIT or PDGFRA (e.g., MEK/MAPK pathway), therefore illustrating a rationale for targeting the PI3K/Akt/mTOR pathway in GIST [6]. Indeed, inhibition of PI3K [7], AKT [8] and mTOR [1] has been shown promising results in vitro and in xenograft models and led to the clinical trials to examine an efficiency against IM-resistant GIST. However, some of them have not succeeded to date [9,10], whereas the other trials to examine the novel drugs targeting the elements of this pathway are currently ongoing (NCT01991379, NCT01735968 and NCT01468688).

Activation of the PI3K/AKT/mTOR pathway is also well-documented for soft tissue sarcomas (STS). In particular, for leiomyosarcoma (LMS) the most common genetic abnormalities include the loss of function mutations in *p53* and/or *PTEN* or activating mutations in the genes encoding the signaling molecules of the PI3K/AKT/mTOR pathway [11–13]. Similarly, to LMS, the PI3K/AKT/mTOR signaling pathway is often aberrantly activated in Ewing's sarcoma (ES) [14,15], rhabdomyosarcoma, a well-known pediatric sarcoma of soft tissues displaying very similar histology and therapeutic options with ES [16], osteosarcoma (OS) [17,18], thereby illustrating that the PI3K/AKT/mTOR pathway is a suitable therapeutic target for STS as well as for other human cancers.

Besides, the PI3K/AKT/mTOR pathway is considered as a perspective molecular therapeutic target for STS and GIST, accumulating evidence also illustrates the tight connection between this pathway and checkpoint responses and repair of DNA damage, induced by certain chemotherapeutic agents and/or ionizing radiation. This reflects, predominantly, the regulatory role of AKT in DNA double-stand break (DSB) repair, including non-homology end-joining (NHEJ) and homologous recombination (HR)—mediated DNA repair, which in turn also illustrates the AKT-mediated pathway, a perspective target to sensitize STS and GIST to DNA-damaging agents, including topoisomerase II (Topo II) inhibitors. Given that Topo II inhibitors are currently used for therapy of STS [19–22] and taking into account that GIST were also previously shown to be sensitive to the Topo II inhibitors [23,24], we thought to examine whether inhibition of the PI3K/AKT/mTOR pathway might enhance their sensitivity to Topo II inhibitor, doxorubicin (DOX) via targeting of the molecular pathways involved in DNA DSB repair.

In the present study we characterized the importance of AKT-pathway for HR-mediated repair of DSBs in STS and GIST in vitro and their relevance to the tumor cell sensitivity to topoisomerase II inhibitor, doxorubicin (Dox). We found that inhibition of AKT-signaling in GIST and STS cell lines results in a significantly decreased expression of Rad51 recombinase and number of residual Rad51/BRCA1 foci in Dox-treated tumor cells. This was due to the decreased stability of Rad51 as a consequence of the enhanced proteasomal degradation. Finally, as a result of the impaired homology-mediated DNA repair, we observed a substantial decrease of viability in AKT-inhibited tumor cells after Dox treatment, which was evidenced by MTS-based colorimetric assay and increased expression of apoptotic markers (cleaved forms of caspase-3 and poly-(ADP)-ribose-polymerase (PARP) and the numbers of hypodiploid cells).

Therefore, overactivation of AKT-signaling pathway in STS and GIST might serve as a prospective molecular target to enhance cytotoxic effects of DNA-topoisomerase II inhibitors inducing DNA DSBs in STS and GIST.
