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Cited by 168Signaling Pathways in DiseaseRetinoids in leukemia and cellular processesCancer-related Molecular Pathways

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Arsenic targets Pin1 and cooperates with retinoic acid to inhibit cancer-driving pathways and tumor-initiating cells

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Kozono, Shingo, Lin, Yu-Min, Seo, Hyuk-Soo, Pinch, Benika, Lian, Xiaolan, Qiu, Chenxi, Herbert, Megan K., Chen, Chun-Hau, Tan, Li, Gao, Ziang Jeff, Massefski, Walter, Doctor, Zainab M., Jackson, Brian P., Chen, Yuanzhong, Dhe-Paganon, Sirano, Lu, Kun Ping, Zhou, Xiao Zhen·2018·Nature Communications
DOI 10.1038/s41467-018-05402-2PMC PMC6085299
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Abstract

Arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) combination safely cures fatal acute promyelocytic leukemia, but their mechanisms of action and efficacy are not fully understood. ATRA inhibits leukemia, breast, and liver cancer by targeting isomerase Pin1, a master regulator of oncogenic signaling networks. Here we show that ATO targets Pin1 and cooperates with ATRA to exert potent anticancer activity. ATO inhibits and degrades Pin1, and suppresses its oncogenic function by noncovalent binding to Pin1’s active site. ATRA increases cellular ATO uptake through upregulating aquaporin-9. ATO and ATRA, at clinically safe doses, cooperatively ablate Pin1 to block numerous cancer-driving pathways and inhibit the growth of triple-negative breast cancer cells and tumor-initiating cells in cell and animal models including patient-derived orthotopic xenografts, like Pin1 knockout, which is substantiated by comprehensive protein and microRNA analyses. Thus, synergistic targeting of Pin1 by ATO and ATRA offers an attractive approach to combating breast and other cancers.

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Figures

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
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 testCC BY · PubMed Central
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)
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)CC BY · PubMed Central
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
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 miceCC BY · PubMed Central
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
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 testCC BY · PubMed Central
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
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 miceCC BY · PubMed Central
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
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 controlsCC BY · PubMed Central
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)
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)CC BY · PubMed Central
Fig. 8 A model for the cooperation of ATO and ATRA in targeting Pin1 to block multiple oncogenic pathways and eliminate cancer stem cells, two major sources of cancer drug resistance
Fig. 8 A model for the cooperation of ATO and ATRA in targeting Pin1 to block multiple oncogenic pathways and eliminate cancer stem cells, two major sources of cancer drug resistanceCC BY · PubMed Central

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Authors
Kozono, S., Lin, Y.-M., Seo, H.-S., Pinch, B., Lian, X., Qiu, C., Herbert, M. K., Chen, C.-H., Tan, L., Gao, Z. J., Massefski, W., Doctor, Z. M., Jackson, B. P., Chen, Y., Dhe-Paganon, S., Lu, K. P., & Zhou, X. Z.
Affiliations
  • Beth Israel Deaconess Medical Center (US)
  • Dana-Farber Cancer Institute (US)
  • Harvard University (US)
  • Fujian Medical University (CN)
  • Broad Institute (US)
  • Dartmouth College (US)
Corresponding author
  • Lu, Kun Ping
  • Zhou, Xiao Zhen
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-08-09
Volume
9
Issue
1
Pages
3069-3069
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-018-05402-2
PMC
PMC6085299
OpenAlex
W2886147882
Cited by
168
References
82
Citations by year
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FWCI
6.49
Type
journal article
Language
en
Open access
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  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
U.S. Department of Health & Human Services | National Institutes of Health (R01CA167677, R01CA205153)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Signaling Pathways in Disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinoids in leukemia and cellular processes
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
Keywords
arsenic trioxide, all-trans retinoic acid, Pin1, isomerase, drug synergy, AQP9, triple-negative breast cancer, cancer stem cells, acute promyelocytic leukemia, protein degradation, microRNA, patient-derived xenografts
MeSH terms
NIMA-Interacting Peptidylprolyl Isomerase, Arsenic Trioxide, Animals, Antineoplastic Agents, Female, Fibroblasts, Humans, Mice, Inbred BALB C, Neoplasm Transplantation, Neoplasms, Magnetic Resonance Spectroscopy, Tretinoin, Signal Transduction, Leukemia, Promyelocytic, Acute, Gene Expression Regulation, Neoplastic, Mice, Knockout, Gene Expression Profiling, Proteomics, Cell Proliferation, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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