ATO induces Pin1 degradation and inhibits cell growth

ATO, especially in combination with ATRA, effectively cures the fatal disease APL10,11,12. Since ATRA inhibits APL, AML, breast cancer, and liver cancer by targeting Pin124,40,41,42,43, we wondered whether ATO has any effects on Pin1. Using concentrations (0.1–2 µM) that have widely and safely been used in APL cells47,48,49, we surprisingly found that ATO dose-dependently downregulated Pin1 protein levels in mouse embryonic fibroblasts (MEFs) (Fig. 1a, b), human TNBC MDA-MB-231 (231) cells (Fig. 1c, d) and many other breast cancer cells (see below). ATO had no effects on Pin1 mRNA levels (Fig. 1e, f), and ATO-induced Pin1 degradation was rescued by the proteasome inhibitor MG132 (Fig. 1g, h and Supplementary Fig. 1a, b). Moreover, ATO dose-dependently reduced Pin1’s protein half-life in MEFs and 231 cells (Fig. 1i, j and Supplementary Fig. 1c, d). Thus, ATO induces proteasome-dependent Pin1 degradation.

Fig. 1 ATO induces Pin1 degradation and inhibits cell growth at clinically relevant concentrations. a–d ATO dose-dependently reduces Pin1 in MEFs (a, b) and 231 cells (c, d). Pin1 WT and KO MEFs or 231 cells, or Pin1 KO MEFs or 231 cells reconstituted with Pin1 were treated with different concentrations of ATO for 3 days, followed by Pin1 immunoblot (a, d) and quantification (b, d). e, f ATO does not affect Pin1 mRNA levels in MEFs (e) and 231 cells (f). MEFs and 231 cells were treated with different concentrations of ATO for 3 days, followed by assaying Pin1 mRNA using real-time PCR analysis. g, h ATO-induced Pin1 downregulation is rescued by proteasome inhibition in MEFs (g) and 231 cells (h). MEFs or 231 cells were treated with different concentrations of ATO in the absence or presence of MG132, followed by Pin1 immunoblot. i, j ATO dose-dependently reduces Pin1 protein stability in MEFs (i) and 231 cells (j). MEFs or 231 cells were treated with different concentrations of ATO in the absence or presence of cycloheximide, followed by Pin1 immunoblot. k, l ATO dose-dependently inhibits cell growth of Pin1 WT, but not Pin1 KO MEFs and 231 cells, which can be rescued by re-expressing Pin1. Pin1 WT and KO MEFs (k) or 231 cells (l) or Pin1 KO MEFs or 231 cells reconstituted with Pin1 were treated with different concentrations of ATO for 3 days, followed by assaying cell growth. The results are expressed as mean ± S.D. and the P values (*P < 0.05, **P < 0.01, ***P < 0.001) were determined by ANOVA test Full size image

To determine whether ATO inhibits Pin1 function in cells, we examined its effects on the growth of Pin1 KO (Pin1−/−) and wild-type (WT, Pin1+/+) MEFs, which display a differential response to Pin1 inhibition by ATRA24. ATO dose-dependently inhibited Pin1 WT MEF growth, but was less effective against Pin1 KO MEF growth, and the effect was restored by stably re-expressing Pin1 (Fig. 1k). To confirm these results, we generated Pin1 KO 231 cells using the CRISPR-Cas9 system, and verified them using DNA sequencing and protein analysis (Fig. 1c). Again, Pin1 CRISPR KO cells were more resistant to ATO, except when Pin1 levels were brought back to endogenous levels using a lentiviral vector containing an altered Kozak sequence (Fig. 1c, l)34. Thus, ATO inhibition of Pin1 contributes to its anti-proliferative effects.

ATO directly and noncovalently binds to and inhibits Pin1

It has been shown that ATO exerts its cellular effects by covalently interacting with vicinal Cys residues in its targets including PML-RARα9,16,50,51. Pin1 has two Cys residues, Cys113 and Cys57. To examine whether they are required for ATO to induce Pin1 degradation, we mutated them to Ala or Ser individually or in combination, and stably expressed the Pin1 mutants in Pin1 CRISPR KO 231 cells at levels similar to endogenous Pin1 (Fig. 2a). ATO equally degraded the single and double Pin1 Cys mutants (Fig. 2a) as WT protein (Figs. 1c, 3c), indicating that Pin1’s Cys residues are not necessary for ATO-induced Pin1 degradation.

Fig. 2 ATO directly binds to specific residues in the Pin1 active site via a previously unknown mechanism. a Cys residues in Pin1 are not required for ATO to degrade Pin1. Pin1 KO cells stably expressing Cys single or double Pin1 Ala or Ser mutants were treated with different concentrations of ATO, followed by Pin1 immunoblot. b ATO inhibits PPIase activity of Pin1, but not Cyp18 or FKBP12. Pin1, Cyp18, or FKBP12 was incubated with different concentrations of ATO, followed by chymotrypsin-coupled PPIase assay. c Biotin-ATO binds to Pin1. Pin1 was incubated with biotin-ATO, followed by isolating biotin-ATO-bound Pin1 for immunoblot (top) and plotting against ATO concentrations (bottom). d ATO binding to recombinant Pin1 can be competed by ATO. Biotin-ATO was incubated with recombinant Pin1, followed by incubation with ATO before subjecting biotin-ATO pulldown assay and Pin1 immunoblot. e ATO binding to cellular Pin1 can be competed by ATO. The 231 cells were treated with or without ATO and then subjected to biotin-ATO pulldown assay, followed by Pin1 immunoblot, along with Pin1 imputs. f, g NMR analysis of ATO-Pin1 binding. Weighted average chemical shift difference Δ of 15N-Pin1 upon addition of 5× ATO was calculated as |ΔH| + (1/5)|ΔN| in p.p.m. and plotted as a function of residue number (f). Upon addition of 2.5× or 5× ATO to Pin1, total six residues in Pin1 show significant chemical shift changes at both ATO concentrations, with two Cys that show no obvious chemical shift changes being highlighted (g). h–j The co-crystal structure of the Pin1-ATO complex. ATO was mixed and co-crystallized with 500 µM Pin1, followed by collecting diffraction data at synchrotron beamline 24ID using and integrating and scaling data sets using XDS. Identical novel trigonal electron density in shape was noted at the Pin1 active site in multiple co-crystals (h, i). The apexes of this electron density are positioned within hydrogen bonding distances (dark green) of side chains from Met130, Gln131, Thr152, Ser154, and His157 and within van der Waals distance (blue) of side chain from Leu122 in the Pin1 active site (j) Full size image

Fig. 3 Disrupting ATO binding to Pin1 impairs its ability to induce Pin1 degradation and to inhibit breast cancer tumor growth. a The co-crystal structure of ATO and Pin1 complexes suggests that the M130V, but not M130I Pin1 mutation, would prevent ATO from binding to the Pin1 active site (arrows). b Biotin-ATO binds to the M130I, but not M130V Pin1 mutant. Pin1 and its mutants were incubated with different concentrations of biotinylated ATO, followed by isolating biotin-ATO-bound Pin1 or its mutants using NeutrAvidin beads. ATO-bound Pin1 were detected by immunoblot and plotted against ATO concentrations. c–f The M130V, but not M130I, Pin1 mutation impairs ATO’s ability to induce Pin1 degradation and inhibit cell growth. Pin1 CRISPR cells stably expressing Pin1 or its M130V or M130I mutant (c) were treated with ATO, followed by assaying Pin1 levels (d, e) or cell growth (f). g–j The M130V, but not M130I Pin1, mutation impairs the ability of ATO to inhibit tumor growth in mice. Female nude mice were flank inoculated with 1 × 106 Pin1 CRISPR cells that stably re-expressed Pin1, or its M130V or M130I mutant, and 1 week later, treated with ATO (2 mg/kg, i.p., 3 times/week). Tumor sizes were weekly measured (g) and mice were sacrificed after 5 weeks to collect tumor tissues (h) and measure their weights (i), as well as their expression of Pin1 and selected Pin1 substrate (j). The results are expressed as mean ± S.D. and the P values (*P < 0.05, **P < 0.01, ***P < 0.001) were determined by ANOVA test. n = 4–6 mice Full size image

To examine whether ATO would affect Pin1 catalytic activity, we used the standard chymotrypsin-coupled peptidyl-prolyl isomerase (PPIase) assay52. ATO dose-dependently inhibited Pin1 PPIase activity (Ki = 0.116 µM) (Fig. 2b), which is phosphorylation-specific, but had minimal effects on cyclophilin (Cyp18) or FKBP12 (Fig. 2b), members of the two major non-phosphorylation-specific PPIase families, cyclophilins and FK506-binding proteins52. To examine whether ATO would directly bind to Pin1 and to determine its binding affinity, we synthesized a biotinylated arsenate compound (biotin-ATO) and performed a binding assay using recombinant Pin1. Biotin-ATO directly bound to Pin1 in a concentration-dependent manner (apparent Kd = 0.238 µM) (Fig. 2c), consistent with the PPIase results (Fig. 2b), and was dose-dependently competed by ATO (Fig. 2d). Biotin-ATO also pulled down Pin1 from 231 cells, and binding was competed by ATO (Fig. 2e). Thus, ATO directly binds and inhibits Pin1 catalytic activity with an affinity of 0.1–0.2 µM.

To understand how ATO binds and inhibits Pin1 catalytic activity, we assessed the dynamics of ATO binding to 15N-labeled Pin1 PPIase domain using nuclear magnetic resonance (NMR) spectroscopy. Upon addition of ATO, select cross-peaks in the 1H-15N HSQC spectrum of Pin1 shifted and broadened in a dose-dependent manner, indicating binding. The residues perturbed upon ATO binding were located in the Pin1 active site, with particularly significant changes observed for Leu60, Leu122, Gly123, Met130, Gln131, and His157 (Fig. 2f, g and Supplementary Fig. 2a). Notably, ATO titration did not affect the cross-peaks for Cys57 or Cys113 (Fig. 2f, g and Supplementary Fig. 2a), further supporting that Pin1 binding of ATO is not Cys-mediated.

A search in the NCBI structure database showed several dozens of arsenic–protein complexes with covalent interactions between arsenic compounds and vicinal Cys or Cys-like cofactors or functional groups in targets, as per the commonly known mechanism16. A similar covalent interaction has been proposed to mediate ATO binding to PML-RARα50,51. To explore our unexpected noncovalent binding mode of ATO to Pin1, we co-crystallized excess ATO with the Pin1 PPIase domain and refined the structure to 1.6 Å resolution with excellent statistics (Supplementary Table 1). We noted well-defined novel electron density in the prolyl binding pocket of the Pin1 active site that was trigonal in shape with significant Fo-Fc values at 4σ (Fig. 2h, i). Although anomalous signal at 1.0438 Å was weak, isomorphous Fo-Fo maps calculated from ATO-soaked and Apo data sets showed clear density for what appeared to be ATO with central arsenic density peak >6σ. The electron density was nicely situated within the Pin1 catalytic active site positioned within van der Waals or hydrogen bonding distances of Leu122, Met130, Gln131, Thr152, Ser154, and His157 (Fig. 2j and Supplementary Fig. 2b). This model of ATO binding was consistent with the degree of change in chemical shift for all backbone amides in Pin1 revealed by NMR analysis. Again, neither Cys57 nor C113 were close to the ATO-binding pocket. Thus, ATO inhibits and induces Pin1 degradation via a novel noncovalent mechanism, distinct from the previous action modes of ATO on PML-RARα and others16,50,51.

Disruption of Pin1 binding to ATO leads to ATO resistance

To demonstrate the significance of the novel interaction between Pin1 and ATO, we sought to identify a Pin1 point mutant that would disrupt ATO binding and determine the importance of direct ATO-Pin1 binding in vitro and in vivo. Since most of the ATO-binding residues are also involved in binding of Pin1 to proline residue in its substrate53, we were careful to select a mutation that would not severely impair Pin1 enzymatic activity. Indeed, point substitutions at T152 or H157 almost completely inactivated Pin1 PPIase activity (Supplementary Fig. 2c, d). We did manage to generate a pair of enzymatically active Pin1 M130 mutants, albeit with slightly lower activity than the WT protein (Supplementary Fig. 2c, d), likely caused by altered proline binding of the substrate. The Pin1-ATO co-crystal structure predicted that an M130V mutation would disrupt ATO binding, whereas an M130I mutant would bind to ATO like the WT protein (Fig. 3a). Indeed, Pin1 M130I mutant-bound biotin-ATO with a similar affinity to the WT protein, whereas Pin1 M130V mutant had a much reduced affinity for Biotin-ATO (Fig. 3b). ATO dose-dependently inhibited the PPIase activity of Pin1 M130I, but not Pin1 M130V mutant (Supplementary Fig. 2e). Thus, the M130V, but not M130I, mutation in Pin1 disrupts Pin1 binding to ATO, as predicted.

If direct binding to Pin1 is critical for ATO to target Pin1 in TNBC, we would expect expression of the Pin1 M130V mutant in Pin1 KO cells to reduce the sensitivity to ATO in vitro and in vivo. To test this possibility, we stably expressed Pin1, Pin1 M130V, and M130I mutants in Pin1 CRISPR KO 231 cells at endogenous levels (Fig. 3c), and then assayed their response to ATO. Pin1 CRISPR KO cells were used to avoid the potential effects of endogenous Pin1. As expected, cells expressing the Pin1 M130V mutant showed impaired ATO-induced Pin1 degradation and inhibition of cell growth, whereas cells expressing the Pin1 M130I mutant behaved similarly to the WT protein (Fig. 3d–f). To confirm these results, we orthotopically xenografted Pin1 CRISPR KO 231 cells expressing Pin1 or its mutants into mice, and 1 week later when tumor growth was notable, the xenografted mice was treated with ATO at 2 mg/kg 3 times/week, a standard concentration that has widely and safely been used for treating APL in mouse models and human patients47,48,49. Pin1 CRISPR KO 231 cells failed to grow any tumors in mice (Fig. 3g–i), consistent with the findings that Pin1 KO mice are highly resistant to cancer development27,28,29,30. In contrast, tumors did develop in Pin1 CRISPR KO 231 cells expressing Pin1 or its M130I or M130V mutant, although the tumors of the Pin1 mutants were slightly smaller than WT Pin1 tumors (Fig. 3g–i), consistent with their lower PPIase activity (Supplementary Fig. 2c, d). Importantly, ATO treatment effectively inhibited the growth of tumors derived from Pin1 or its M130I mutant, but not at all from the M130V mutant (Fig. 3g–i). Moreover, ATO reduced the levels of Pin1 and its substrate oncoproteins such as NF-κB/p6554, β-catenin55, and Rab2A34, and increased the levels of Pin1 substrate tumor suppressors such as Fbw756 in breast tumors derived from xenografts expressing Pin1 or M130I mutant, but not M130V mutant (Fig. 3j). Thus, ATO binding to Pin1 is essential for ATO to induce Pin1 degradation, block oncogenic pathways, and inhibit tumor growth.

ATO uptake via AQP9 regulates its ability to inhibit Pin1

To further support ATO’s potent anticancer activity via targeting Pin1, we examined the effects of ATO on cell growth using 10 different human breast cancer cell lines. Cells were treated with increasing concentrations of ATO and assessed for Pin1 levels (Fig. 4a and Supplementary Fig. 3a) and cell growth (Fig. 4b). ATO-induced Pin1 degradation was tightly and positively correlated with ATO-inhibited cell growth (Fig. 4c). However, ATO sensitivity was surprisingly variable among different cell lines. To identify the underlying mechanisms, we examined expression of AQP9, a membrane transporter that mediates cellular uptake of ATO known to correlate with ATO sensitivity in APL57,58. Indeed, AQP9 was readily detected in ATO-responsive cells, but not in ATO-resistant cells (Fig. 4f), with AQP9 expression being inversely correlated with Pin1 level and cell growth (Fig. 4d, e). Thus, ATO’s ability to inhibit breast cancer is positively correlated with Pin1 degradation and AQP9 expression.

Fig. 4 ATRA cooperates with ATO to induce Pin1 degradation and inhibit cancer cell growth by increasing cellular ATO uptake via the induction of AQP9 expression. a–c Correlation between the ability of ATO to induce Pin1 degradation and to inhibit cell growth. Ten human breast cancer cells were treated with ATO for 3 days, followed by Pin1 immunoblot (a) and counting cell numbers (b), and determining their correlation (c). d–f Correlation between AQP9 expression and the ability of ATO to degrade Pin1 and to inhibit cell growth. AQP9 expression were assayed by immunoblot (f) and their correlations with the ability of ATO to degrade Pin1 (d) and inhibit cell growth (e) were calculated using the data from a, b, and f. g–i AQP9 KD reduces ATO sensitivity in ATO-sensitive cells. Stable AQP9 KD 231 cells generated using two unrelated shRNA vectors were treated with different concentrations of ATO for 3 days, followed by assaying Pin1 levels (g) and cell growth (h), or with 1 µM ATO for different times, followed by assaying cellular ATO concentrations by ICP-Mass Spec (i). j–l AQP9 OX reverses ATO resistance in ATO-resistant cells. Stable AQP9 OX MCF7 cells were treated with different concentrations of ATO for 3 days, followed by assaying Pin1 levels (j), cell growth (k), or with 1 µM ATO for different times, followed by assaying cellular ATO concentrations by ICP-Mass Spec (l). m–p ATRA induces AQP9 protein expression, increases ATO uptake, and cooperates with ATO in inhibiting cell growth in TNBC cells. The 231 cells were treated with different concentrations of ATRA for 7 days, followed by AQP9 immunoblot (m) or with 1 µM ATO for different times before subjecting to ATO concentrations by ICP-Mass Spec (n), or with different concentrations of ATO and/or ATRA for 3 days, followed by counting cell number (o) and determining their synergy using CalcuSyn (p). q–s AQP9 KD abolishes ATRA cooperation with ATO, but does not affect ATRA sensitivity. Control or AQP9 KD 231 cells were treated with ATO and/or ATRA, followed by Pin1 immunoblot (q) and cell growth assay (r), followed by using CalcuSyn to calculate their synergy (s). The results are expressed as mean ± SD and the P values were determined by ANOVA test Full size image

To demonstrate the functional significance of AQP9 expression in determining ATO sensitivity, we stably knocked down AQP9 in two ATO-sensitive cells (Supplementary Fig. 3b) and overexpressed AQP9 in three ATO-resistant cells (Supplementary Fig. 3e). Two different AQP9 short hairpin RNA (shRNA) constructs effectively silenced AQP9 (Supplementary Fig. 3b), and also abrogated the ability of ATO to induce Pin1 degradation (Fig. 4g and Supplementary Fig. 3c) and inhibit cell growth (Fig. 4h and Supplementary Fig. 3d) in both cell lines, with shAQP9-2 being more effective. In contrast, AQP9 overexpression (Supplementary Fig. 3e) converted all three ATO-resistant cells to become ATO-sensitive cells in terms of Pin1 degradation (Fig. 4j and Supplementary Fig. 3f) and growth inhibition (Fig. 4k and Supplementary Fig. 3g). These results are further supported by measuring cellular ATO uptake using inductively coupled plasma mass spectrometry (ICP-MS). Whereas AQP9 knockdown (KD) reduced ATO uptake in ATO-sensitive cells (Fig. 4i), and AQP9 overexpression increased ATO uptake in ATO-resistant cells (Fig. 4l). Thus, ATO uptake via AQP9 regulates its ability to induce Pin1 degradation and inhibit cancer cells.

ATO and ATRA cooperately inhibit Pin1 and oncogenic pathways

To demonstrate the cooperation and translational significance of ATO and ATRA in targeting Pin1 for treating cancers, we chose TNBC as a model system because unlike APL, which is basically cured by ATO and ATRA10,11,12, TNBC has the worst prognosis of all breast cancer subtypes and no targeted therapy is available59. Furthermore, Pin1 plays an essential oncogenic role in breast cancer27,31,60,61, and chemical ablation of Pin1 by ATRA exerts antitumor activity against TNBC24. Finally, as shown in APL57,58, ATRA dose-dependently increased both AQP9 mRNA (Supplementary Fig. 4a, b) and protein expression (Fig. 4m and Supplementary Fig. 4c) in TNBC cells, likely due to activation of the AQP9 promoter activity by ATRA, as shown by promoter reporter and mutagenesis analyses (Supplementary Fig. 4d). Moreover, ATRA and ATO combination increased time-dependent ATO uptake (Fig. 4n), and cooperately ablated Pin1 in two TNBC cells (Supplementary Fig. 5a, b).

To independently confirm the cooperative effects of ATO and ATRA on Pin1 levels, we established an in-cell enzyme-linked immunosorbent assay (ELISA) to quantify Pin1 protein levels after drug treatments, which correlated well with the Pin1 levels quantified using immunoblotting (Supplementary 5c–e). Importantly, the in-cell ELISA confirmed that, while either ATO or ATRA alone dose-dependently reduced Pin1 levels, their combination displayed strong synergy (Supplementary Fig. 5f), as calculated by the CalcuSyn program with the Chou–Talalay method62. As single agents, ATO and ATRA caused dose-dependent inhibition of cell growth in two TNBC cells, but their combination displayed synergistic effects (Fig. 4o, p and Supplementary Fig. 5g, h). To confirm the potential effects of ATRA on ATO response, we treated two AQP9 KD TNBC cells with either ATO, ATRA, or their combination. AQP9 KD did not affect the ability of ATRA to induce Pin1 degradation (Fig. 4q and Supplementary Fig. 5i) or inhibit cell growth (Fig. 4r and Supplementary Fig. 5j), but did largely abrogate its ability to synergize with ATO, which prevented additional Pin1 degradation (Fig. 4q and Supplementary Fig. 5i) and cell growth inhibition (Fig. 4s and Supplementary Fig. 5k). Thus, ATO cooperates with ATRA to promote Pin1 degradation and inhibit cell growth by inducing AQP9 expression in TNBC.

Pin1 simultaneously activates and inactivates numerous oncoproteins and tumor suppressors, respectively7,25, as well as globally downregulates microRNAs in cancer cells by inhibiting their biogenesis32. We next assessed the extent to which ATO and/or ATRA affect protein levels of a selected subset of Pin1 substrate oncoproteins and tumor suppressors, whose protein stability is regulated by Pin1 in TNBC25. ATO and ATRA alone caused the dose-dependent reduction of Pin1 protein and its substrate oncoproteins, including cyclin D161, NF-κB/p6554, β-catenin55, Akt63, c-Jun64, c-Myc65, Rab2A34, and caused the dose-dependent induction of Pin1 substrate tumor suppressors such as Fbw756 and Smad2/366 in two TNBC cell lines (Fig. 5a and Supplementary Fig. 5l). Moreover, their combination displayed cooperative effects, with the phenotypes similar to those resulting from Pin1 KO using CRISPR (Fig. 5a and Supplementary Fig. 5l). Thus, ATO and ATRA cooperately ablate Pin1 to simultaneously block multiple cancer-driving pathways.

Fig. 5 ATO and ATRA cooperatively ablate Pin1 and inhibit multiple Pin1-regulated oncogenic pathways and tumor growth in TNBC in vitro and in vivo including PDOXs. a ATO and ATRA cooperatively turn off many oncoproteins and on many tumor suppressors, like Pin1 KO. The 231 and 159 cells were treated with different concentrations of ATO and/or ATRA for 72 h, followed by IB, with Pin1 KO cells as controls. b–d ATO and ATRA cooperatively induced global protein expression like Pin1 KO. The 231 cells were treated with ATO and/or ATRA or DMSO for 72 h, followed by quantitative mass spectrometry analyses, with Pin1 KO 231 cells as a control. Three thousand seven hundred and fifty-eight proteins passed the abundance filter (b), and 209 proteins were altered by >1.5-fold (c). The log 2 transformed ratio of treated versus control was used to generate the heatmap in GENE-E. The Spearman's correlation matrix for the 209 altered proteins are shown and their P values are all below 2.2e−16, except P value for ATO and Pin1 KO being 3.5e−12 (d). e ATO and ATRA globally upregulates microRNA expression like Pin1 KO. MicroRNAs of ATO-treated and/or ATRA-treated 231 cells and Pin1 KO 231 cells were profiled by NanoString. Data are presented as relative to microRNA expression of DMSO-treated (Ctrl) 231 cells or vector CRISPR 231 cells through the dot density plot. The P values were determined by Student’s t test. f–h ATO and ATRA cooperatively inhibit tumor growth in TNBC 231 orthotopic xenografts. The 231 cells were transplanted into mammary fat pads, and 1 week later, treated with ATO and/or ATRA. Tumor sizes were measured (f) and mice were sacrificed after 6 weeks to collect tumor tissues (g) and measure their weights (h). i–n ATO and ATRA cooperatively inhibit tumor growth in TNBC PDOXs. TNBC patient-derived tumors were transplanted, followed by treating mice with ATO and/or ATRA 2–3 weeks after xenograft when tumors were notable (i–k) or reached about 360 mm3 (l–n). o ATRA induces AQP9 to cooperate with ATO to downregulate Pin1 and Pin1 oncogenic substrates and upregulate Pin1 tumor-suppressive substrates in human cells and PDOXs, assayed by immunoblot. The results are expressed as mean ± SD and the P values were determined by ANOVA or Student’s t test. n = 4–5 mice Full size image

To independently confirm the cooperative ablation of Pin1 by ATO and ATRA in TNBC cells, we performed global analyses of protein and microRNA expression after treating 231 cells with ATO and/or ATRA for 3 days. Global alterations in proteins and microRNAs in mock-treated cells were compared to the positive control Pin1 CRISPR KO 231 cells using a tandem mass tag (TMT9plex)-based proteomic approach67 and an NanoString nCounter microRNA Expression Assay32, respectively. Out of the 7003 proteins quantified across all 10 samples, 3758 proteins passed the abundance filter and were reliably quantified. Among them, 209 were altered by 1.5-fold in abundance in Pin1 CRISPR 231 cells compared with the parental WT control cells. Although ATO, ATRA, and Pin1 KO had some difference in overall expression pattern, ATO and ATRA conferred similar effects at the proteomic level, but their cooperation was obvious, with their combination most closely resembling the Pin1 KO effect (Spearman's correlation coefficient 0.69, P value <2.2e−16) (Fig. 5b–d and Supplementary Fig. 6a). Similarly, although ATO, ATRA, and Pin1 KO also had some different effects on individual microRNAs (Supplementary Fig. 6b), ATO and ATRA, especially in their combination, globally upregulated microRNA expression, similar to Pin1 KO (Fig. 5e). Strikingly, many of the consistently downregulated proteins across all treatments are oncogenic, and many of the consistently upregulated proteins are tumor suppressive (Supplementary Table 2). Global upregulation of microRNAs in Pin1 KO or inhibited cancer cells is also consistent with the findings that Pin1 regulates microRNA biogenesis32,68. Thus, multiple independent analyses demonstrate that ATO and ATRA synergistically target Pin1 to inhibit its numerous cancer-related pathways.

ATO and ATRA cooperately inhibit Pin1 and tumor growth

Given the striking anticancer effects of ATO and ATRA in vitro, a critical question is whether they have any cooperative effects on Pin1 levels, Pin1-regulated oncogenic pathways, and tumor growth of TNBC in vivo. We thus orthotopically xenografted TNBC 231 cells into cleared mouse mammary fat pads and then treated them with ATO, ATRA, or their combination 1 week after xenograft when tumor growth was notable. Since regular ATRA has a half-life of only 45 min in humans46, we used 5 mg 21-day slow-releasing pellets21. For ATO, we used 2 mg/kg 3 times/week, a standard concentration that has widely and safely been used for treating APL in mouse models and human patients47,48,49. While ATRA and ATO alone inhibited tumor growth, their combination displayed cooperative activity, markedly inhibiting tumor growth (Fig. 5f–h).

To better recapitulate human TNBC tumors and their microenvironment, we established PDOX models for two different human TNBC tumors and treated them with ATO and/or ATRA at the same doses as above. Again, ATRA and ATO alone inhibited tumor growth, but their combination displayed cooperative antitumor activity in both PDOXs when the treatments were started after tumor growth was notable (Fig. 5i–k), or tumor volume reached 270 mm3 (Fig. 5l–n), or even 360 mm3 (Supplementary Fig. 6c–e). Notably, ATRA also induced AQP9 expression and cooperated with ATO to induce Pin1 degradation, destabilization of Pin1’s substrate oncoproteins, and stabilization of Pin1’s substrate tumor suppressors, in both TNBC cell orthotopic and PDOX tumors (Fig. 5o and Supplementary Fig. 6f). Thus, ATO and ATRA cooperatively ablate Pin1 to block multiple cancer-driving pathways and inhibit tumor growth in TNBC cell xenografts and PDOXs.

ATO and ATRA cooperatively inhibit Pin1 and TIC self-renewal

As an independent approach to demonstrate that ATO has anticancer activity by targeting Pin1 oncogenic function and cooperating with ATRA, we chose to study TICs/CSCs of TNBCs, which are a proposed source of tumor initiation, growth, and metastasis, but are not effectively targeted by current cancer drugs4,5. Moreover, Pin1 is highly enriched in breast TICs and drives TIC self-renewal and tumor initiation and growth33,34,35, but whether Pin1 inhibitors would effectively target TICs is not known.

To examine the effects of ATO and ATRA on TICs in TNBC, we first treated 231 and 159 cells with ATO (1 µM), ATRA (10 µM), or their combination, followed by assaying the breast TIC-enriched CD24−CD44+ or ALDH+ population using fluorescence-activated cell sorting (FACS)33,34 While ATO and ATRA both significantly reduced breast TIC-enriched population, their combination cooperatively reduced the TIC population to the levels (Fig. 6a, b) close to Pin1 CRISPR cells (Fig. 6e, f). To examine the effects of ATO and ATRA on self-renewal of breast TICs, we treated TNBC cells with ATO, ATRA, or their combination, followed by a serial mammosphere formation assay. Both TNBC 231 and 159 cells formed very fast-growing spheres that did not decrease when propagated to M4 (Fig. 6c, d), indicating that mammosphere-forming cells were self-renewing at a constant rate35. However, after treatment with ATO or ATRA, the cells formed fewer and smaller mammospheres displaying strongly impaired mammosphere formation efficiency at M2–4. Moreover, their co-treatment displayed cooperative effects, almost completely inhibiting mammosphere formation efficiency at M1 (Fig. 6c, d), similar to Pin1 CRISPR KO (Fig. 6g). Similar results were also obtained in TNBC MDA-MB-468 cells (Supplementary Fig. 7a–d). Moreover, ATO effectively inhibited mammosphere formation efficiency at M1 in Pin1 CRISPR 231 cells expressing Pin1 or the M130I mutant, but not the M130V mutant (Supplementary Fig. 7e, f), consistent with their ATO binding (Fig. 3) Thus, Pin1 binding to ATO is required for ATO to target TICs.

Fig. 6 ATO and ATRA cooperatively inhibit the population and self-renewal of TICs in TNBC. a, b ATO and ATRA cooperatively reduce the population of TICs in TNBCs. Human TNBC 231 (a) and 159 (b) cells were treated with ATO (1 µM) or ATRA (10 µM) or their combination, followed by FACS analysis of the TIC-enriched CD24−CD44+ population and ALDH+ population. c, d ATO and ATRA cooperatively inhibit the self-renewal of TICs in TNBCs. TNBC 231 (c) and 159 (d) cells were treated with ATO (1 µM) or ATRA (10 µM) or their combination, followed by serial mammosphere formation assay to measure their TIC self-renewal. Scale bar = 150 μm. e–g Pin1 KO using CRISPR reduce the population and self-renewal of TICs in TNBCs. Pin1 CRISPR KO and control 231 cells (e, g) or 159 cells (f) were subjected to FACS analysis of the TIC-enriched CD24−CD44+ population (e) and ALDH+ population (f) or serial mammosphere formation assay (g). Scale bar = 150 μm. h–j ATO and ATRA cooperatively inhibit the EMT of TNBC cells. TNBC 231 cells were treated with ATO (1 µM) or ATRA (10 µM) or their combination, followed by measuring the expression of E-cadherin (h), and slug, vimentin, and ZEB-1 (i) using real-time PCR or immunoblot (j). k, l ATO and ATRA cooperatively inhibit migration and invasion of TNBC cells. TNBC 231 cells were treated with ATO (1 µM) or ATRA (10 µM) or their combination, followed by assaying cell migration (k) and invasion (l) using Pin1 CRISPR KO cells as controls Full size image

Since the epithelial-to-mesenchymal transition (EMT) phenotype is another breast TIC property69, and is reversed by Pin1 KD or KO33, we also examined the effects of ATO and/or ATRA on EMT. ATO and ATRA, especially in combination, strongly induced the mesenchymal-to-epithelial transition (MET), as displayed by upregulation of epithelial markers, such as E-cadherin (Fig. 6h–j), and downregulation of mesenchymal markers, such as slug, vimentin, and ZEB-1 (Fig. 6i–j), as well as reduced cell migration and invasion equivalent to Pin1 KO using CRISPR (Fig. 6k, l and Supplementary Fig. 8a, b). Thus, ATO and ATRA cooperatively reduce the population, self-renewal, and EMT of TICs in TNBC, similar to Pin1 KO.

ATO and ATRA cooperatively inhibit Pin1 and TIC tumor growth

Breast TICs are notoriously resistant to cytotoxic chemotherapy drugs such as taxol4,5, commonly used to treat TNBC70. Since ablation of Pin1 by ATO and ATRA eliminates breast TICs, we expected that taxol-resistant TNBC cells would still be sensitive to ATO and ATRA co-treatment. To test this, we generated taxol-resistant 231 and 159 cells (Fig. 7a), followed by drug treatments. Compared with parental cells, taxol-resistant 231 and 159 cells had increased population of TICs (Fig. 7b), elevated levels of multiple CSC regulators (Fig. 7c), and increased migration and invasion (Supplementary Fig. 8a–d), as expected4,5. Importantly, these TIC-related phenotypes were drastically inhibited by ATO and ATRA, and particularly their combination (Fig. 7c and Supplementary Fig. 8a–d). Moreover, ATO and ATRA, especially in their combination, potently inhibited the growth of taxol-resistant cells (Fig. 7d), and also effectively inhibited self-renewal of taxol-resistant breast TICs (Fig. 7e and Supplementary Fig. 8e). Thus, ATO and ATRA combination eliminates resistance of TICs to taxol.

Fig. 7 ATO and ATRA cooperatively inhibit taxol resistance, tumor initiation, and tumor growth of TICs in TNBC. a Generation of taxol-resistant 231 and 159 cells by treating cells with an increasing concentration of taxol over time, followed by assaying cell growth after taxol treatment. b Taxol-resistant 159 cells have an increased population of TICs, as assayed by ALDH using FACS analysis. c–e ATO and ATRA cooperatively reduce multiple cancer stem cell regulators, cell growth, and self-renewal of taxol-resistant TNBC cells in vitro. Taxol-resistant TNBC 231 and 159 cells were treated with ATO (1 µM) or ATRA (10 µM) or their combination, followed by measuring selected stem cells regulators using IB (c), cell growth (d), self-renewal of TICs using serial mammosphere formation assay, followed by calculating the average area of all mammospheres formed (e). f–i ATO and ATRA cooperatively reduce tumor initiation and growth, and CSC regulators of TNBC cells in mice similar to Pin1 KO using CRISPR. TNBC cells were treated with ATO (1 µM) and ATRA (10 µM) for 3 days, followed by being injected into subcutaneous sites of nude mice in limiting dilutions and treated with ATO (2 mg/kg, i.p., 3 times/week) and ATRA (5 mg in 21-day slow release) (f, h). Pin1 CRISPR and vector control 231 cells were used in parallel as a control (g, i). Mice were sacrificed and evaluated for tumor weight (f, g), and expression of selected CSC regulators (h). Pin1 CRISPR cells were analyzed for CSC regulators by immunoblot (i). j, k ATO and ATRA cooperatively reduce TIC population and CSC regulators in PDOXs. TNBC patient-derived tumors were transplanted into cleared mouse mammary fat pads, followed by treating mice with ATO and/or ATRA or their combination for 5 weeks. Mice were sacrificed and evaluated for the TIC population by FACS (j), and selected CSC regulator expression (k) Full size image

This raises the question of whether ATO and ATRA combination would inhibit tumor initiation and growth of breast TICs in vivo. We assayed the effects of ATO and ATRA combination therapy on tumor initiation of TNBCs using a limiting dilution assay in mice, a standard approach to determine tumor initiation71. Importantly, ATO and ATRA co-treatment not only effectively reduced breast TIC frequency by ~90-fold (P < 0.0001), but also dramatically reduced tumor growth (Fig. 7f and Table 1), similar to Pin1 KO (Fig. 7g and Table 1). Moreover, ATO and ATRA co-treatment potently downregulated multiple CSC regulators in tumors (Fig. 7h), like Pin1 KO (Fig. 7i). Finally, ATO and ATRA co-treatment also cooperatively reduced breast TIC-enriched population (Fig. 7j) and multiple CSC regulators (Fig. 7k) in PDOX tumors. Thus, ATO and ATRA cooperatively ablate Pin1 to inhibit the self-renewal, drug resistance, tumor initiation, and growth of TICs in TNBC, similar to Pin1 KO.