TY - JOUR
T1 - Twist1-induced epithelial dissemination requires PRKD1 signaling
AU - Georgess, Dan
AU - Padmanaban, Veena
AU - Sirka, Orit Katarina
AU - Coutinho, Kester
AU - Choi, Alex
AU - Frid, Gabriela
AU - Neumann, Neil M.
AU - Inoue, Takanari
AU - Ewald, Andrew J.
N1 - Funding Information:
We would like to thank Dr. Timothy J. Mitchison (Harvard University) for thoughtful discussion on the role of microtubules in Prkd activation, Dr. Isaac S. Chan (JHU) for sharing clinical expertise on taxane therapeutics, Dr. Jin Zhu (JHU) for expert advice on qPCR, and Mr. Matthew Perrone and Dr. Joel S. Bader (JHU) for thoughtful discussion of the data and manuscript. D. Georgess was supported by a Postdoctoral Fellowship Grant from the Susan G. Komen Foundation (PDF15332336) and a research grant from the LAU SAS School Research and Development Council (SRDC-F-2018-145). A.J. Ewald received support for this project through grants from the Breast Cancer Research Foundation/Pink Agenda (BCRF-18-048), the Metastatic Breast Cancer Network, the Twisted Pink Foundation, Hope Scarves, and the NIH/NCI (U01CA217846, U54CA2101732, 3P30CA006973). K. Coutinho and V. Padmanaban were supported by the Isaac Morris Hay and Lucille Elizabeth Hay Fellowship. N.M. Neumann was supported by the NIH/NIGMS (3T32GM007309) and T. Inoue was supported by the NIH/NIGMS (GM123130).
Publisher Copyright:
© 2019 American Association for Cancer Research.
PY - 2020/1/15
Y1 - 2020/1/15
N2 - Dissemination is an essential early step in metastasis but its molecular basis remains incompletely understood. To define the essential targetable effectors of this process, we developed a 3D mammary epithelial culture model, in which dissemination is induced by overexpression of the transcription factor Twist1. Transcriptomic analysis and ChIP-PCR together demonstrated that protein kinase D1 (Prkd1) is a direct transcriptional target of Twist1 and is not expressed in the normal mammary epithelium. Pharmacologic and genetic inhibition of Prkd1 in the Twist1-induced dissemination model demonstrated that Prkd1 was required for cells to initiate extracellular matrix (ECM)-directed protrusions, release from the epithelium, and migrate through the ECM. Antibody-based protein profiling revealed that Prkd1 induced broad phosphorylation changes, including an inactivating phosphorylation of β-catenin and two microtubule depolymerizing phosphorylations of Tau, potentially explaining the release of cell-cell contacts and persistent activation of Prkd1. In patients with breast cancer, TWIST1 and PRKD1 expression correlated with metastatic recurrence, particularly in basal breast cancer. Prkd1 knockdown was sufficient to block dissemination of both murine and human mammary tumor organoids. Finally, Prkd1 knockdown in vivo blocked primary tumor invasion and distant metastasis in a mouse model of basal breast cancer. Collectively, these data identify Prkd1 as a novel and targetable signaling node downstream of Twist1 that is required for epithelial invasion and dissemination. Significance: Twist1 is a known regulator of metastatic cell behaviors but not directly targetable. This study provides a molecular explanation for how Twist1-induced dissemination works and demonstrates that it can be targeted.
AB - Dissemination is an essential early step in metastasis but its molecular basis remains incompletely understood. To define the essential targetable effectors of this process, we developed a 3D mammary epithelial culture model, in which dissemination is induced by overexpression of the transcription factor Twist1. Transcriptomic analysis and ChIP-PCR together demonstrated that protein kinase D1 (Prkd1) is a direct transcriptional target of Twist1 and is not expressed in the normal mammary epithelium. Pharmacologic and genetic inhibition of Prkd1 in the Twist1-induced dissemination model demonstrated that Prkd1 was required for cells to initiate extracellular matrix (ECM)-directed protrusions, release from the epithelium, and migrate through the ECM. Antibody-based protein profiling revealed that Prkd1 induced broad phosphorylation changes, including an inactivating phosphorylation of β-catenin and two microtubule depolymerizing phosphorylations of Tau, potentially explaining the release of cell-cell contacts and persistent activation of Prkd1. In patients with breast cancer, TWIST1 and PRKD1 expression correlated with metastatic recurrence, particularly in basal breast cancer. Prkd1 knockdown was sufficient to block dissemination of both murine and human mammary tumor organoids. Finally, Prkd1 knockdown in vivo blocked primary tumor invasion and distant metastasis in a mouse model of basal breast cancer. Collectively, these data identify Prkd1 as a novel and targetable signaling node downstream of Twist1 that is required for epithelial invasion and dissemination. Significance: Twist1 is a known regulator of metastatic cell behaviors but not directly targetable. This study provides a molecular explanation for how Twist1-induced dissemination works and demonstrates that it can be targeted.
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U2 - 10.1158/0008-5472.CAN-18-3241
DO - 10.1158/0008-5472.CAN-18-3241
M3 - Article
C2 - 31676574
AN - SCOPUS:85077917735
SN - 0008-5472
VL - 80
SP - 204
EP - 218
JO - Cancer Research
JF - Cancer Research
IS - 2
ER -