Promising new antitumor agents usually appear as our understanding of oncogenesis advances. Combination chemotherapy of DAC and chemotherapeutic agents have been investigated since 2004, the results suggesting that DAC could increase the cytotoxicity of chemotherapeutic agents against lung cancer cells and melanoma cells
in vitro[
17,
18]. In the previous study, we also reported the synergistic growth suppression of DAC with PTX in RCC [
13]. DAC is a demethylation agent, which was shown to suppress the proliferation of malignant tumors by reactivating the expression of specific methylated genes or causing genome-wide demethylation [
19,
20]. However, another study suggested that DAC-induced antineoplastic activity was dependent on DNA damage [
21]. Whether DAC acts on tumors primarily through its effect on DNA methylation or through synergistic cytotoxicity with PTX remains unknown.
In this study, we investigated the gene transcriptional alteration by the cDNA microarray and revealed possible molecular mechanism and pathways implicated in the synergy of DAC and PTX against RCC cells. Several key regulatory genes were identified and may play critical roles in the synergy of these two agents. These include lymphoid enhancer-binding factor 1 (LEF1), transforming growth factor β-induced (TGFBI), C-X-C motif ligand 5 (CXCL5) and myelocytomatosis viral related oncogene (c-myc). LEF1 was initially identified as a pre-B and T lymphoid-specific gene encoding a DNA-binding protein of high mobility group proteins [
22,
23]. In addition, LEF1 is a member of the lymphoid-enhancing factor/T-cell factor (LEF/TCF) family of transcription factors, which acts through the Wnt/β-catenin signaling pathway to regulate gene expression and coordinate many cellular processes in normal development and carcinogenesis [
24‐
26]. A study showed that the proliferation and invasion of the melanoma cell is regulated by LEF1/TCF activity [
27]. Upon Wnt stimulation, LEF1 could combine with β-catenin and activate Wnt-responsive target genes [
28]. Our previous study has confirmed that LEF1 can enhance the proliferation of RCC cells and that suppressing the expression of LEF1/β-catenin complex plays an important role in the synergistic mechanism of DAC and PTX against RCC cells [
29]. TGFBI is a target of TGF-β and secreted into the extracellular space, where it binds to fibronectin and collagen as well as integrins to stimulate adhesion, migration, spreading, and proliferation in renal proximal tubular epithelial cells [
30,
31]. Studies showed that TGFBI was induced by TGF-β in the lung adenocarcinoma cell line and overexpression of TGFBI was associated with some malignancies, such as RCC and hemangioblastoma [
32,
33]. Our previous study has demonstrated that TGFBI-promoted metastasis of RCC cells depends on inactivation of the von Hippel-Lindau (VHL) tumor suppressor and that TGFBI could be a therapeutic target against RCC in the future [
34]. CXCL5, a member of the CXC chemokine family, has been shown to be involved in angiogenesis, tumor growth, and metastasis. CXCL5 is upregulated significantly in sporadic endometrioid endometrial adenocarcinomas compared to normal endometrium [
35]. CXCL5 overexpression was also associated with late stage gastric cancer and high N stage, suggesting CXCL5 is involved in the progression of gastric cancer, especially in lymph node metastasis [
36]. However, whether CXCL5 could stimulate phenotypic responses in renal epithelial cells with malignant progression remains unknown. c-myc is a multifunctional, nuclear phosphoprotein that plays multiple roles in eukaryotic cells including cell progression, differentiation, apoptosis and neoplasia [
37]. It interferes with the regulators of G1/S transition, as well as other regulators of cell growth and metabolism, inducing several translation factors and adhesion molecules [
38]. Alterations of the c-myc genomic region are usually observed in prostate cancer [
39‐
41] and bladder cancer [
42], however, genomic alterations of c-myc are mostly subordinate for conventional RCC with the exception of papillary renal cancer [
43‐
45].
At present, the signaling pathways underlying the synergy of DAC and PTX against RCC have not been investigated. In this study, we found that some cell cycle-related pathways were involved in the synergy of both agents by IPA, such as cyclins, DNA replication and cell cycle/mitotic regulation; these results are consistent to our previous study. Moreover, we also confirmed that the synergy of DAC and PTX may be mediated by inhibiting the PI3K/Akt pathway. PI3K could enhance production of PIP3 and trigger a signaling cascade, which results in the activation of Akt [
46]. Because LEF1/β-catenin is a target of the PI3K/Akt pathway, we speculated that synergistic suppression of LEF1/β-catenin expression by DAC and PTX is dependent on the inactivation of the PI3K/Akt pathway.