Suppressing STAT5 signaling affects osteosarcoma growth and stemness

Abstract Osteosarcoma (OS) is the most common primary bone tumor that primarily affects children and adolescents. Studies suggested that dysregulation JAK/STAT signaling promotes the development of OS. Cells treated with pimozide, a STAT5 inhibitor suppressed proliferation and colony formation and i...

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Main Authors: Dharmalingam Subramaniam, Pablo Angulo, Sivapriya Ponnurangam, Prasad Dandawate, Prabhu Ramamoorthy, Pugazhendhi Srinivasan, Tomoo Iwakuma, Scott J. Weir, Katherine Chastain, Shrikant Anant
Format: Article
Language:English
Published: Nature Publishing Group 2020-02-01
Series:Cell Death and Disease
Online Access:https://doi.org/10.1038/s41419-020-2335-1
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author Dharmalingam Subramaniam
Pablo Angulo
Sivapriya Ponnurangam
Prasad Dandawate
Prabhu Ramamoorthy
Pugazhendhi Srinivasan
Tomoo Iwakuma
Scott J. Weir
Katherine Chastain
Shrikant Anant
author_facet Dharmalingam Subramaniam
Pablo Angulo
Sivapriya Ponnurangam
Prasad Dandawate
Prabhu Ramamoorthy
Pugazhendhi Srinivasan
Tomoo Iwakuma
Scott J. Weir
Katherine Chastain
Shrikant Anant
author_sort Dharmalingam Subramaniam
collection DOAJ
description Abstract Osteosarcoma (OS) is the most common primary bone tumor that primarily affects children and adolescents. Studies suggested that dysregulation JAK/STAT signaling promotes the development of OS. Cells treated with pimozide, a STAT5 inhibitor suppressed proliferation and colony formation and induced sub G0/G1 cell cycle arrest and apoptosis. There was a reduction in cyclin D1 and CDK2 expression and Rb phosphorylation, and activation of Caspase-3 and PARP cleavage. In addition, pimozide suppressed the formation of 3-dimensional osteospheres and growth of the cells in the Tumor in a Dish lung organoid system. Furthermore, there was a reduction in expression of cancer stem cell marker proteins DCLK1, CD44, CD133, Oct-4, and ABCG2. More importantly, it was the short form of DCLK1 that was upregulated in osteospheres, which was suppressed in response to pimozide. We further confirmed by flow cytometry a reduction in DCLK1+ cells. Moreover, pimozide inhibits the phosphorylation of STAT5, STAT3, and ERK in OS cells. Molecular docking studies suggest that pimozide interacts with STAT5A and STAT5B with binding energies of −8.4 and −6.4 Kcal/mol, respectively. Binding was confirmed by cellular thermal shift assay. To further understand the role of STAT5, we knocked down the two isoforms using specific siRNAs. While knockdown of the proteins did not affect the cells, knockdown of STAT5B reduced pimozide-induced necrosis and further enhanced late apoptosis. To determine the effect of pimozide on tumor growth in vivo, we administered pimozide intraperitoneally at a dose of 10 mg/kg BW every day for 21 days in mice carrying KHOS/NP tumor xenografts. Pimozide treatment significantly suppressed xenograft growth. Western blot and immunohistochemistry analyses also demonstrated significant inhibition of stem cell marker proteins. Together, these data suggest that pimozide treatment suppresses OS growth by targeting both proliferating cells and stem cells at least in part by inhibiting the STAT5 signaling pathway.
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spelling doaj.art-5d310a6efbe6464b9de53da6369710ad2022-12-21T23:02:42ZengNature Publishing GroupCell Death and Disease2041-48892020-02-0111211510.1038/s41419-020-2335-1Suppressing STAT5 signaling affects osteosarcoma growth and stemnessDharmalingam Subramaniam0Pablo Angulo1Sivapriya Ponnurangam2Prasad Dandawate3Prabhu Ramamoorthy4Pugazhendhi Srinivasan5Tomoo Iwakuma6Scott J. Weir7Katherine Chastain8Shrikant Anant9Department of Cancer Biology, The University of Kansas Medical CenterDivision of Hematology and Oncology, Children’s Mercy HospitalDepartment of Cancer Biology, The University of Kansas Medical CenterDepartment of Cancer Biology, The University of Kansas Medical CenterDepartment of Cancer Biology, The University of Kansas Medical CenterDepartment of Cancer Biology, The University of Kansas Medical CenterDepartment of Cancer Biology, The University of Kansas Medical CenterDepartment of Cancer Biology, The University of Kansas Medical CenterDivision of Hematology and Oncology, Children’s Mercy HospitalDepartment of Cancer Biology, The University of Kansas Medical CenterAbstract Osteosarcoma (OS) is the most common primary bone tumor that primarily affects children and adolescents. Studies suggested that dysregulation JAK/STAT signaling promotes the development of OS. Cells treated with pimozide, a STAT5 inhibitor suppressed proliferation and colony formation and induced sub G0/G1 cell cycle arrest and apoptosis. There was a reduction in cyclin D1 and CDK2 expression and Rb phosphorylation, and activation of Caspase-3 and PARP cleavage. In addition, pimozide suppressed the formation of 3-dimensional osteospheres and growth of the cells in the Tumor in a Dish lung organoid system. Furthermore, there was a reduction in expression of cancer stem cell marker proteins DCLK1, CD44, CD133, Oct-4, and ABCG2. More importantly, it was the short form of DCLK1 that was upregulated in osteospheres, which was suppressed in response to pimozide. We further confirmed by flow cytometry a reduction in DCLK1+ cells. Moreover, pimozide inhibits the phosphorylation of STAT5, STAT3, and ERK in OS cells. Molecular docking studies suggest that pimozide interacts with STAT5A and STAT5B with binding energies of −8.4 and −6.4 Kcal/mol, respectively. Binding was confirmed by cellular thermal shift assay. To further understand the role of STAT5, we knocked down the two isoforms using specific siRNAs. While knockdown of the proteins did not affect the cells, knockdown of STAT5B reduced pimozide-induced necrosis and further enhanced late apoptosis. To determine the effect of pimozide on tumor growth in vivo, we administered pimozide intraperitoneally at a dose of 10 mg/kg BW every day for 21 days in mice carrying KHOS/NP tumor xenografts. Pimozide treatment significantly suppressed xenograft growth. Western blot and immunohistochemistry analyses also demonstrated significant inhibition of stem cell marker proteins. Together, these data suggest that pimozide treatment suppresses OS growth by targeting both proliferating cells and stem cells at least in part by inhibiting the STAT5 signaling pathway.https://doi.org/10.1038/s41419-020-2335-1
spellingShingle Dharmalingam Subramaniam
Pablo Angulo
Sivapriya Ponnurangam
Prasad Dandawate
Prabhu Ramamoorthy
Pugazhendhi Srinivasan
Tomoo Iwakuma
Scott J. Weir
Katherine Chastain
Shrikant Anant
Suppressing STAT5 signaling affects osteosarcoma growth and stemness
Cell Death and Disease
title Suppressing STAT5 signaling affects osteosarcoma growth and stemness
title_full Suppressing STAT5 signaling affects osteosarcoma growth and stemness
title_fullStr Suppressing STAT5 signaling affects osteosarcoma growth and stemness
title_full_unstemmed Suppressing STAT5 signaling affects osteosarcoma growth and stemness
title_short Suppressing STAT5 signaling affects osteosarcoma growth and stemness
title_sort suppressing stat5 signaling affects osteosarcoma growth and stemness
url https://doi.org/10.1038/s41419-020-2335-1
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