A549 cells are not responsive to HH stimulation
We previously showed that Oxy186 and Oxy210 suppressed A549 cell proliferation, possibly by inhibiting GLI activity in the HH pathway [11, 14]. However, the transcriptional activity of a GLI-responsive luciferase reporter transiently introduced into A549 cells was not induced by treatment with conditioned media containing N-terminal SHH (SHH CM) (Fig. 1A, left panel). The same reporter can be activated in mouse embryonic fibroblasts (MEFs) by the same SHH CM (Fig. 1A, right panel), indicating that A549 cells are not responsive to HH stimulation. Consistent with the lack of HH signaling responsiveness, Oxy186, Oxy210 and two established HH/SMO inhibitors, SANT-1 and cyclopamine [16], did not inhibit HH signaling in A549 cells as they did in MEFs (Fig. 1A). Having found that A549 cells lack a HH signaling response, we repeated the previous cell proliferation assays [11, 14] and indeed found that the SMO inhibitor cyclopamine was unable to suppress A549 cell growth, but both Oxy186 and Oxy210 strongly suppressed cell growth (Fig. 1B). Since the presence of primary cilia is indispensable for HH signaling in mammalian cells [17], we examined the formation of primary cilia in over-confluent A549 cells and MEFs. Consistent with the lack of HH signaling responsiveness, primary cilia were only sparsely detected in A549 cells compared to MEFs (Fig. 1C).
Oxy186 and Oxy210 suppress A549 cell proliferation through non-canonical GLI, and WNT/β-catenin pathways.
Besides the canonical, ligand-induced HH signaling, SMO-independent, non-canonical ways to activate GLI have been reported in many cancers [18]. A549 cells were previously reported to harbor high GLI activity despite their lack of responsiveness to SMO inhibitors [19]. Consistent with these findings, at the highest concentration tested (40 µM), the GLI inhibitor GANT61 [20] was able to suppress the basal activity of the GLI-luciferase reporter in A549 cells in the absence of HH ligand stimulation (Fig. 2A). Oxy186 and Oxy210 were also able to suppress the GLI-luciferase reporter to the same extent as GANT61 (Fig. 2A). Furthermore, GANT61 inhibited A549 cell growth in a dose-dependent manner (Fig. 2B), indicating that A549 cells are subject to growth inhibition of clamping down GLI activities, which is in line with our previous report [11, 14]. Unexpectedly, FH535, an inhibitor of WNT/β-catenin signaling [21], also strongly suppressed A549 cell growth (Fig. 2B), and both Oxy186 and Oxy210 could further inhibit A549 cell growth even in the presence of the GLI inhibitor GANT61 or the WNT/β-catenin inhibitor FH535 (Fig. 2C). These results suggest that these two oxysterols probably act on both the GLI and WNT/β-catenin pathways. Since Oxy210 was also previously reported as an inhibitor of TGF-β signaling [11], we tested the effect of SB431542, a well-known inhibitor of TGF-β type I receptors [22], on A549 cell proliferation. The results clearly showed that SB431542 does not suppress A549 cell growth (Fig. 2B), indicating that the mechanism whereby Oxy210 suppresses cell growth in A549 cells is not through inhibition of TGF-β signaling.
Oxy186 and Oxy210 inhibit WNT/β-catenin signaling.
To test if Oxy186 and Oxy210 possess WNT pathway inhibitory activities, we performed the WNT-dependent TOPflash reporter assay in A549 cells and found that WNT3A conditioned medium (WNT CM) induced a robust increase in luciferase activity, which was inhibited by FH535 as expected (Fig. 3A). Both Oxy186 and Oxy210 significantly inhibited the luciferase reporter activity, but Oxy189, another oxysterol derivative previously used as a negative control, did not (Fig. 3A), suggesting that the WNT inhibitory activity is specific to Oxy186 and Oxy210. Moreover, Oxy186 and Oxy210 were able to inhibit WNT3A-induced TOPflash activity even in a cell line in which both Gli1 and Gli2 genes were knocked out (Fig. 3B), indicating that the inhibitory role of Oxy186 and Oxy210 on WNT signaling is independent of their ability to inhibit GLI signaling.
To corroborate results from the luciferase reporter assay, we examined the expression levels of several WNT target genes in A549 cells, namely AXIN2, LEF1, TCF7 and TNFRSF19, by real-time quantitative PCR (RT-qPCR). As shown in Fig. 3C, WNT CM induced the expression of all four genes, and both Oxy186 and Oxy210 inhibited this effect.
During WNT/β-catenin signaling, binding of extracellular WNT ligands to the membrane-bound FZD and LRP5/6 receptors results in the signaling transduction through DVL [7], which in cooperation with casein kinase 1ε (CSNK1E) promotes the accumulation of CTNNB1 to elicit transcriptional responses [23, 24]. To determine at which step of this signal transduction cascade Oxy186 and Oxy210 exert their inhibition, we tested the ability of these two compounds to modulate the induction of TOPflash activity by overexpression of LRP6, a combination of DVL2 and CSNK1E, or CTNNB1 in A549 cells. In this setting, Oxy186 and Oxy210, but not the negative control oxysterol Oxy189, inhibited the TOPflash activity induced by LRP6; however, Oxy186 and Oxy210 were not able to inhibit that induced by DVL2 and CSNK1E or by CTNNB1 (Fig. 3D), suggesting that Oxy186 and Oxy210 likely work at the receptor level.
Differential inhibition of xenograft tumor growth by Oxy186 and Oxy210
To examine the efficacy of the oxysterols in inhibiting tumor growth in vivo, we tested Oxy186 and Oxy210 in a subcutaneous xenograft tumor model generated from A549 cells in nude mice. After the tumor size reached an average of ~ 100 mm3, the compounds were administered daily via oral gavage at a dose of 50 mg/kg (Fig. 4A). This treatment regimen was well tolerated, as mice in all 3 randomized experimental groups maintained similar body weight throughout the 18 days of treatments (Fig. 4B, left panel), during which we were able to complete data collection on all experimental mice. From the relative tumor volume data (Fig. 4B, right panel), we found that Oxy186 started to inhibit tumor growth at day 8, and significantly reduced the relative tumor volume from day 11 to day 18 of treatment, compared to that of the vehicle control. Surprisingly, Oxy210 did not show such inhibitory effect, and its effect on tumor growth was indistinguishable from that of the vehicle control (Fig. 4B, right panel). The absolute tumor volumes were also significantly reduced in the Oxy186 treatment group starting at day 8 (Fig. 4C).
Basal cell carcinoma pathway is enriched in differentially expressed genes from Oxy186-treated xenografts
To determine which molecular pathways are targeted by Oxy186 to exert its tumor-suppressing activity, we extracted RNA from control, Oxy186-treated and Oxy210-treated xenografts and performed RNA-seq analyses. Consistent with the lack of inhibitory effect in tumor growth, Oxy210-treated samples did not separate from the control group on the principal component analysis plot (Fig. 5A). Out of the seven Oxy186-treated samples, four were clearly distinct from the rest of samples while three were mixed with the control group, indicating that these samples had different responses to the Oxy186 treatment (Fig. 5A). Using these four samples for further analysis, we found that more than 1700 genes were significantly downregulated while only 142 genes were significantly upregulated by Oxy186 treatment (Fig. 5B). From those differentially expressed genes, KEGG [25] pathway enrichment analysis identified 9 pathways that were significantly enriched (Padj < 0.05), including the basal cell carcinoma pathway (Fig. 5C).
Basal cell carcinoma is a common type of skin cancer derived from basal cells of the epidermis. Susceptible gene pathways known to affect basal cell carcinoma include the HH and WNT pathways [26] (Fig. 6A). Based on RNAseq data, we identified several genes in the HH pathway that were downregulated in Oxy186-treated samples compared to the control xenografts (Fig. 6A). We further confirmed downregulation of GLI1, GLI2 and PTCH2 in Oxy186-treated xenografts using RT-qPCR (Fig. 6B), but expression of some other HH target genes (e.g. SHH, PTCH1, HHIP) were not significantly affected by Oxy186 treatment (Fig. 6A, data not shown).
RT-qPCR analysis also showed a decreasing trend of GLI2 and PTCH2 expression in Oxy210-treated samples; however, no statistically significance could be assigned comparing to those of the control group (Fig. 6B). Because Oxy210 was found previously to inhibit TGF-β pathway [11], we measured mRNA levels of several known TGF-β target genes, namely JUNB, ATF3 and SMURF2, using Oxy210-treated samples. Similar to the levels of GLI2 and PTCH2 expression in Oxy210-treated samples, these three genes were also downregulated in the Oxy210-treated xenografts, but the difference comparing to those of the control group did not reach statistical significance (Additional file 1: Fig. S1).
RNA-seq analyses of xenograft tumors identifies the WNT pathway as a target of Oxy186
Using Gene Set Enrichment Analysis (GSEA) [27], we further identified 22 positively enriched and 49 negatively enriched gene sets in our dataset of Oxy186-treated xenografts compared to the oncogenic signature gene sets (C6) from the Molecular Signatures Database (MsigDB) [28]. Among the negatively enriched gene sets, several were related to the WNT pathway, including the WNT up-regulated gene set [29], the MYC up-regulated gene set [30], and the CCND1 up-regulated gene set [31] (Fig. 7A). MYC and CCND1 are well known WNT-induced genes that promote cell proliferation and tumor growth [32]. We confirmed that expression of MYC and CCND1 was indeed significantly downregulated in Oxy186-treated, and only slightly downregulated in Oxy210-treated xenografts (Fig. 7B). We then chose three or four genes from each gene set to validate their gene expression by RT-qPCR, and found that all of these genes were significantly downregulated in Oxy186-treated xenografts compared to their expression in control xenografts (Fig. 7C-E).
WNT/β-catenin signaling not only promote cell proliferation but also induce epithelial-mesenchymal transition (EMT) by inducing the expression of transcription factors, such as SNAIL and ZEB1 [33, 34]. These two EMT-inducing transcription factors had been demonstrated to promote the invasive ability and bone metastasis of small cell lung cancer cells [35, 36]. We found that expression of these two genes as well as expression of VIM and TJP1, which encode mesenchymal marker Vimentin and ZO-1, respectively, were also significantly downregulated in Oxy186-treated Xenografts (Fig. 7F). While Oxy210 only had a slightly effect on these gene expression in xenografts (Fig. 7F). Taken together, these results indicate that Oxy186 targets the GLI and WNT pathways in xenografts.