Yardenone 2 destabilizes HIF-1α at the protein level, reducing its ability to translocate into the nucleus

We initially evaluated the presence of HIF-1α by immunoblot in the various cells, including P69 normal epithelial cells, DU145 and PC3 prostate cancer cells and 786-O kidney cancer cells. The 786-O cells served as a negative control due to their exclusive expression of HIF-2. We exposed the cells to 1% O2 hypoxia (Hx) for 24- and 48-h and used immunoblotting to assess the presence of HIF-1α. Our results confirmed the absence HIF-1α of in the 786-O cells (Fig. 2A). We conducted an initial screening for the destabilization of HIF-1α under hypoxic conditions using Sodwanone A, B, C, E/M, G and Yardenone 2 (Fig. 1). At 10 µM, no significant effects were observed, while 30 µM was excessively toxic, resulting in a 45% decrease in cell viability in P69 cells, an 89% decrease in DU145 cells, and a 48% decrease in PC3 cells (unpublished data). We thus selected 20 µM as the optimal concentration. Sod. A effectively destabilized HIF-1α in P69 and PC3 cells but remained toxic to DU145 cells (Fig. 2B). Interestingly, Yard. 2 exclusively destabilized HIF-1α in PC3 cells (Fig. 2C). Subsequently, we focused our investigation on Sod. A and Yard. 2. To investigate the impact of these compounds on HIF-1α localization for dimer formation and subsequent gene activation, we performed immunofluorescence analyses. In PC3 cells exposed to 48 h of hypoxia, Sod. A reduced HIF-1α nuclear localization by 20.3%, while Yard. 2 significantly decreased nuclear HIF-1α, reduced to 12.8% (Fig. 2D).

Fig. 2

Effect of Sodwanones and Yardenones on HIF-1α stabilization. A P69, DU145, PC3, and 786-O cells were subjected to hypoxia 1% O2 (Hx 1%) for 24 and 48h. Cell lysates were analyzed by immunoblotting for HIF-1α. Tubulin was used as a loading control. B P69, DU145, and PC3 cells were treated with Sodwanone A (Sod. A) at 20 μM for 48h in hypoxia (Hx 1%). Cell lysates were analyzed by immunoblotting for HIF-1α. Tubulin was used as a loading control. C, P69, DU145, and PC3 cells were treated with Yardenone 2 (Yard. 2) at 20 μM for 48h in hypoxia (Hx 1%). Cell lysates were analyzed by immunoblotting for HIF-1α. Tubulin was used as a loading control. D, Immunofluorescence labeling and merge images showing the nuclear localization of HIF-1α (in green) and DAPI (in blue) in PC3 cells treated with Sod. A and Yard. 2 at 20 μM for 48h in hypoxia (Hx 1%)

We also investigated a possible effect at the level of transcriptional regulation (Suppl. Figure 1). While the mRNA expression level of HIF-1α was decreased under hypoxia in conditions where the HIF-1α protein is strongly stabilized, we did not observe any effect of Yard. 2 on the mRNA expression level of HIF-1α in PC3 cells. This indicates that the destabilization of HIF-1α by Yard.2 occurs at the protein level, rather than at the transcriptional level.

These findings confirm the targeted action of both Sod. A and Yard. 2. Sod. A exhibits significant toxicity in hypoxic DU145 cells, while Yard. 2 selectively affects PC3 cells, which are known to be among the most aggressive PCa cell lines.

Yardenone 2 selectively reduces the proliferation of PC3 cells only under hypoxic conditions

We then examined the effect of Sod. A (20 µM) or Yard.2 (20 µM) on the proliferation and viability of P69, DU145, PC3 and 786-O cells. In normoxia (Nx), 20 µM Sod. A significantly decrease proliferation at 48 h in both P69 and 786-O cells and reduced cell viability (Fig. 3A and B). In contrast, Sod.A had no effect on the proliferation or viability of DU145 and PC3 cells. Similarly, Yard. 2 did not impact any of the cell lines tested (Fig. 3A and B).

Fig. 3

Impact of the Sodwanone A (Sod. A) and Yardenone 2 (Yard. 2) on cell proliferation and viability. A and B P69, DU145, PC3, and 786-O cells were seeded at the same density and incubated in normoxia (Nx) for 48 h in the absence (Ctl) or presence of 20 µM of Sod. A, and Yard. 2. Cell proliferation (A) and cell viability (B) were measured using an ADAM cell counter. C and D P69, DU145, PC3, and 786-O cells were seeded at the same density and incubated in hypoxia (Hx 1%) for 48 h in the absence (Ctl) or presence of 20 µM of Sod. A, and Yard. 2. Cell proliferation (C) and cell viability (D) were measured using an ADAM cell counter. (E and F) PC3 cells were seeded at the same density and incubated in hypoxia (Hx 1%) for 24, 48, 72, and 96 h in the absence (Ctl) or presence of 20 µM of Yard. 2. Cell proliferation (E) and cell viability (F) were measured using an ADAM cell counter. G, Clonogenic assay of P69, DU145, PC3, and 786-O cells. Cell lines were seeded at the same density and incubated in Hx 1% O2 (Hx 1%) for 7 days (7d) in the absence (Ctl) or presence of Yard. 2 at 20 µM. H Top, Three-dimensional structures obtained from confocal image series using IMARIS software; scale bars = 200 µm. MSK-PC3 organoids have been treated for 15 days in the absence or presence of Yard. 2 (40 µM) every 3 days. Bottom, Quantification of cell area (pixels) at day 15. The 2-way ANOVA is representative of at least five different organoids. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0005. Data represent the mean ± SD of experiments performed at least three times

We validated the effects of the drug responses under hypoxic conditions. Sod. A displayed a significant negative impact on all cell lines tested, including 786-O cells that do not express HIF-1 (Fig. 3C). Viability was reduced in DU145, PC3 and 786-O cells (Fig. 3D). In contrast, Yard. 2 specifically decreased proliferation in PC3 cells without causing cell death (Fig. 3C and D). Consequently, we chose to focus exclusively on Yard. 2, which appears to be more selective under hypoxic conditions and with respect to HIF-1. Over an extended period of up to 96 h, Yard. 2 significantly inhibits proliferation under hypoxic conditions, without affecting PC3 cell viability (Fig. 3E and F). Following a 48-h incubation with 20 µM of Yard. 2 and negative controls, cell cycle stages were analyzed using FACS (Suppl. Figure 2). The results demonstrated a significant accumulation of cells in the G2/M phase (3.5 ± 0.78% for Yard. 2). However, Yard. 2 did not influence the cell cycle in 786-O cells, suggesting that this effect could be dependent on HIF-1. These findings indicate that Yard. 2 may slightly inhibit proliferation by arresting the cell cycle at the G2/M phase. Clonogenic assays, commonly used in cancer research to assess the ability of cancer cells to initiate tumors, revealed that Yard. 2 significantly reduces clonogenicity without inducing cell death over a 7-day period, as evidenced by the lack of colony growth in PC3 cells (Fig. 3G). To further validate Yard. 2's effectiveness in a 3D model, we utilized patient-derived organoid lines, specifically MSK-PCa3, from Chen’s laboratory, which mimic the molecular diversity of adenocarcinoma prostate cancer. These organoids were cultured for 15 days with Yard. 2 being added every 3 days (Fig. 3H). In the presence of Yard. 2, we observed a significant decrease in the size of the organoids, along with notable changes in their morphology and conformation. Together, these results strongly suggest that, Yard. 2 may serve as a specific HIF-1 inhibitor, acting at the protein level with a cytostatic effect. In contrast, Sod. A has unfavorable effects in both normoxia or hypoxia, regardless of HIF-1 status or whether the cells are tumoral or normal.

Yardenone 2 specifically acts on HIF-1α target genes

To validate the effects of Yard. 2 on HIF-1 and gene expressions, we performed an RNA-seq experiment using PC3 cells in both normoxia and hypoxia, in the absence or presence of Yard. 2 for 48 h.

The analysis identified 4,223 differentially expressed genes between Hx and Nx, with a significance cut-off of p-values < 0.05. Furthermore, in hypoxic cells treated with Yard. 2, 6063 genes exhibited differential expression compared to Nx, with the same p-value threshold. Notably, the expression of 2893 genes was significantly altered in PC3 cells upon Yard. 2 treatments (Fig. 4A). Analysis of the top molecular functions between hypoxia and normoxia revealed significant changes in pathways, including NADH regeneration, glycolytic process, and notably, an emphasis on the response to hypoxia and low oxygen levels (Fig. 4B). Interestingly, the addition of Yard. 2 under hypoxic conditions compared to normoxic conditions resulted in the elimination of the hypoxic response, highlighting the potential effectiveness of Yard. 2 in hypoxia (Hx) (Fig. 4C). We then examined the modulation of HIF-1 target genes to determine whether Yard. 2 had a specific impact in Hx. Significant differences were observed in the expression of HIF-1 target genes, including Ldha, Eno1, Pgk1, Ca9, Bnip3, Tpi1 and Egln1 (Fig. 4D and E). In contrast, no significant changes were found in the expression of HIF-2 target genes, such as Hes4, Cdt1 and Pparg (Suppl. Figure 3A and B), suggesting a selective effect of Yard. 2 on HIF-1 target genes. Additionally, we identified 11 distinct clusters of genes with varying expression patterns when comparing hypoxia to normoxia (Suppl. Figure 3C). Principal component analysis (PCA) further confirmed the difference between Hx and Nx, indicating homogeneous samples, particularly in Nx, and distinct separation between the two groups (Suppl. Figure 3D). Analysis of cellular components revealed enrichment in mitochondria and supramolecular fibers (Suppl. Figure 3E). When PC3 cells were treated with Yard. 2, we identified 11 clusters of differentially expressed gene with distinct functions (Suppl. Figure 3F). The PCA showed samples homogeneity in both groups, with a clear separation between them (Fig. 4F). In PC3 cells treated with Yard. 2, an enrichment of cellular components such as microtubule, cytoskeleton, focal adhesion, lysosome, secretory vesicle was observed (Fig. 4G). The molecular functions characterized were consistent with the observed cellular components, including microtubule binding, tubulin binding, cytoskeletal protein binding, ATP binding and protein dimerization activity (Fig. 4H).

Fig. 4

Yard. 2 modulates HIF-1 target genes and acts as a microtubule inhibitor. PC3 cells were treated in normoxia (Nx) and hypoxia (Hx 1%) in the absence or presence of Yard. 2 at 20μM for 48h. A Venn diagram showing the differential distribution of the genes detected between hypoxia and normoxia (in blue) and between Hx + Yard. 2 and Nx (in red). B Gene set enrichment list of RNA-Seq data comparing hypoxia (Hx) and normoxia (Nx) using “Cellular functions”. C Gene set enrichment list of RNA-Seq data comparing hypoxia + Yard. 2 (Hx + Yard. 2) and normoxia (Nx) using “Cellular functions”. D and E Heatmap (D) and the representative bar diagram (E) of some specific HIF-1 target genes in normoxia (Nx) compared to hypoxia (Hx) or hypoxia treated with Yard. 2. F PCA plot of hypoxia (Hx) and hypoxia + Yard.2 (Hx + Yard. 2) samples. Ellipses and shapes show clustering of the samples. G gene set enrichment map of RNA-Seq data comparing hypoxia + Yard. 2 (Hx + Yard. 2) and hypoxia (Hx) using “Cellular components”. H Gene set enrichment list of RNA-Seq data comparing hypoxia + Yard. 2 (Hx + Yard. 2) and hypoxia (Hx) using “Molecular functions”

These findings provide strong evidence of the modulation of HIF-1 target genes in the presence of Yard. 2, highlighting its influence on various processes occurring during hypoxic culture conditions. Moreover, Yard.2 appears to have a significant impact on microtubule-related processes.

Docetaxel and Yard. 2 exhibit similarities and distinctions in their actions on microtubules in hypoxia

To gain deeper insights into the mode of action of Yard. 2 on microtubules, we used Docetaxel (DTX) as a positive control. DTX selectively binds to microtubules, which are essential for cell division, thereby disrupting cell division. This disruption prevents the proper separation of chromosomes during cell division, resulting in the arrest of cancer cell growth. In the context of prostate cancer, DTX is commonly used as first-line chemotherapy for patients with castration-resistant prostate cancer (CRPC) [21, 22].

Consequently, we investigated the impact of varying concentrations of DTX (10, 30, and 100 nM) on the proliferation and viability of PC3 cells. Notably, even at the lowest concentration of 10 nM, a noticeable reduction in the proliferation was observed, consistent with the effects observed at 30 and 100 nM (Fig. 5A). Moreover, all concentrations led to a decrease in the viability of normoxic PC3 cells following 72 h of treatment (Fig. 5B). Subsequently, we examined the effects of DTX under hypoxic conditions. We observed a trend of notable negative impact at 24 and48h, while at 72 h, DTX demonstrated a significantly strong negative effect across all tested concentrations (Fig. 5C). Interestingly, under hypoxic conditions, viability was no longer affected (Fig. 5D). After a 48-h incubation with 30 nM of DTX and negative controls, cell cycle stages were analyzed using FACS (Fig. 5E). The results revealed a significant and strong accumulation of cells in the G2/M phase, with 69.13 ± 0.72% for DTX compared to Yard. 2 (Suppl. Figure 2). These findings suggest that inhibition of proliferation due to DTX is likely triggered by cell cycle arrest at the G2/M phase. We then examined the responses to DTX on HIF-1α stabilization, considering prior findings by several groups such as Escuin et al. [23], Carbonaro et al. [24] and Li et al. [25]. These studies reported the destabilization of HIF and an augmentation in cell death following DTX treatment. Although 10 nM was ineffective at 48 h post-treatment, 30 nM notably destabilized HIF-1α to an extent of 60.6 ± 0.4% (Fig. 5F), a more significant effect than observed in the presence of Yard. 2 (Fig. 2C).

Fig. 5

Docetaxel (DTX) and Yard. 2 exhibit similar but distinct actions. A and B PC3 cells were incubated in normoxia (Nx) for 24, 48h, and 72h in the absence (Ctl) or presence of 10, 30 or 100nM of Docetaxel (DTX). Cell proliferation (A) and cell viability (B) were measured using an ADAM cell counter. C and D PC3 cells were incubated in hypoxia (Hx 1%) for 24, 48h, and 72h in the absence (Ctl) or presence of 10, 30 or 100nM of Docetaxel (DTX). Cell proliferation (C) and cell viability (D) were measured using an ADAM cell counter. A–D * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0005. Data represent the mean ± SD of experiments performed at least three times. E Cell cycle effect of PC3 cells treated with 30nM of DTX. F PC3 cells were treated with 10 or 30nM of Docetaxel (DTX) and subjected to hypoxia 1% O2 (Hx 1%) for 48h. Cell lysates were analyzed by immunoblotting for HIF-1α. β-Actin was used as a loading control. G PC3 cells were treated in hypoxia (Hx 1%) in the presence of Yard. 2 at 20 μM and in the presence of Docetaxel (DTX) at 30nM for 48h. Venn diagram showing the differential distribution of the genes detected between Hx + Yard. 2 and Hx (in blue) and between Hx + DTX and Hx (in red). Gene set enrichment list of RNA-Seq data using “Cellular functions”

To assess the impact of DTX on gene expression patterns, we performed an RNA-seq experiment using PC3 cells in hypoxia with 30 nM of DTX for 48 h. We investigated the modulation of HIF-1 genes target genes to determine any specific impact of DTX. Despite the destabilization of HIF-1α by DTX at 30 nM, we observed either significant up-regulation or no noticeable changes in the expression levels of HIF-1 target genes, including LDHA, ENO1, PGK1, CA9, BNIP3, TPI1 and EGLN1 (Suppl. Figure 4A and B). Moreover, no significant differences were detected in the expression of HIF-2 target genes, such as HES4, CDT1 and PPARG (Suppl. Figure 4C and D). Further analysis revealed that 329 genes exhibited common expression patterns when treated with Yard. 2 or DTX, with a significance cut-off of p-values < 0.05 (Fig. 5G). These genes were specifically associated with cell cycle, mitotic nuclear division, spindle organization and strongly linked to microtubules. Additionally, 1,855 genes displayed differential expression in response to DTX compared to Yard. 2, also with p-values < 0.05, primarily with mitochondrial functions. Notably, the expression of 1,455 genes exhibited significant changes upon treatment with Yard. 2 in PC3 cells compared to DTX, predominantly associated with microtubules, thereby underlying the role of Yard. 2.

These findings suggest that both Yard. 2 and DTX have comparable effects on microtubules while manifesting distinct specificities. Intriguingly, although both compounds affect HIF-1α stabilization, Yard. 2 influences HIF-1 target genes, while DTX does not, pointing to potential differences in their kinetic mechanisms of action.

Invalidation of HIF-1α affect slightly the microtubule structure

Prolonged mitotic arrest induced by DTX triggers mitotic catastrophe. During this process, cells experience aberrant mitotic events, such as chromosome misalignment, chromosome breakage, and multipolar spindle formation. These abnormalities result in the activation of cell death pathways, leading to apoptosis or necrosis. Consequently, we investigated whether Yard. 2 could provoke similar effects. Immunofluorescence analysis revealed evidence of mitotic catastrophe induced by DTX, even in hypoxia, as indicated by condensed tubulin and the entrapment of nuclei (Fig. 6A). In contrast, treatment with Yard. 2 led to a slight condensation of tubulin, with no instances of multiple nuclei per cell. Given that the level of destabilization of HIF-1α was lower with Yard. 2 compared to DTX, we chose to silence HIF-1α using a doxycycline-inducible shHIF-1α. Following a 24-h treatment, HIF-1α was destabilized to 70% (Fig. 6B) decreasing the mRNA expression levels of HIF-1α, CA9 and GLUT1 by 86%, 80% and 54%, respectively (Fig. 6C). Given that the level of HIF-1α destabilization was comparable to that induced by Docetaxel, we further investigated whether this effect was associated with mitotic catastrophe. Following a 24-h treatment, tubulin condensation was observed, similar to the effects seen with Yard. 2. However, no evidence of mitotic catastrophe was noted (Fig. 6D).

Fig. 6

Yard. 2 directly targets HIF-1α, subsequently inhibiting the induction of genes that later impact tubulin. A Immunofluorescence labeling and merged images with HIF-1α (in green), α-tubulin (in red), and DAPI (in blue) for PC3 cells treated with 30 nM of Docetaxel (DTX) or 20 µM of Yard. 2 and incubated for 48 h in Hx. The yellow arrow indicates condensed tubulin, while the green arrow signifies mitotic catastrophe. B PC3 cells were stably transfected with the inducible (doxycycline) pLKO-TetOn-Puromycin encoding shRNA to HIF-1α. PC3 expressing shRNA HIF-1α were subjected to hypoxia (Hx1%) in the absence (−) or presence ( +) of doxycycline. Cell lysates were analyzed by immunoblotting for HIF-1α. β-Actin was used as a loading control. C Graphic representation of HIF-1α, Ca9 and Glut1 mRNA expression in PC3 cells incubated in hypoxia (Hx—1% O2) %) in the absence (−) or presence ( +) of doxycycline for 48 h. The one-way ANOVA is representative of two independent experiments. D Immunofluorescence labeling and merged images with HIF-1α (in green), α-tubulin (in red), and DAPI (in blue) for PC3 expressing shRNA HIF-1α in the absence (−) or in the presence ( +) of doxycycline and incubated for 48 h in Hx. E Graph illustrating the comparison of the modes of action of Yard. 2 versus Docetaxel

These results suggest that the direct action on HIF-1α via Yard. 2 or shHIF-1α is the initial event triggering a minor effect on microtubules, whereas DTX treatment likely targets microtubules first, leading to destabilization of HIF-1α (Fig. 6E).