Original Research CDK5RAP3 is a co-factor for the oncogenic transcription factor STAT3 Susana P. Egusquiaguirre; Suhu Liu; Isidora Toi 1 ; Kevin Jiang; Sarah R. Walker; Maria Nicolais; Tzuen Yih Saw; Michael Xiang; Katarina Bartel; Erik A. Nelson; David A. Frank Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215; Departments of Medicine, Brigham and Womens Hospital and Harvard Medical School, Boston, MA 02115, United States Abstract The transcription factor STAT3 regulates genes governing critical cellular processes such as proliferation, survival, and selfrenewal. While STAT3 transcriptional function is activated rapidly and transiently in response to physiologic signals, through a variety of mechanisms it can become constitutively activated in the pathogenesis of cancer. This leads to chronic expression of genes that underlie malignant cellular behavior. However, STAT3 is known to interact with other proteins, which may modulate its function. Understanding these interactions can provide insights into novel aspects of STAT3 function and may also suggest strategies to ther- apeutically target the large number of cancers driven by constitutively activated STAT3. To identify critical modulators of STAT3 transcriptional function, we performed an RNAinterference based screen in a cellbased system that allows quantitative measurement of STAT3 activity. From this approach, we identified CDK5 kinase regulatorysubunit associated protein 3 (CDK5RAP3) as an enhancer of STAT3dependent gene expression. We found that STAT3 transcriptional function is modulated by CDK5RAP3 in cancer cells, and silencing CDK5RAP3 reduces STAT3mediated tumorigenic phenotypes including clonogenesis and migration. Mechanistically, CDK5RAP3 binds to STAT3regulated genomic loci, in a STAT3dependent manner. In primary human breast can- cers, the expression of CDK5RAP3 expression was associated with STAT3 gene expression signatures as well as the expression of individual STAT3 target genes. These findings reveal a novel aspect of STAT3 transcriptional function and potentially provide both a biomarker of enhanced STAT3dependent gene expression as well as a unique mechanism to therapeutically target STAT3. Neoplastic (2019) xx xxx–xxx Introduction The transcription factor STAT3 is activated constitutively in a large fraction of human cancers, including approximately 70% of breast cancers [1,2]. When activated by IL6 and other cytokines, STAT3 becomes phos- phorylated on a unique carboxyterminal tyrosine residue, which leads to the formation of an active dimer that translocates from the cytoplasm to the nucleus, and binds to nine base pair sequences in the regulatory regions of target genes (TTCN 3 GAA). Under normal, physiologic condi- tions, STAT3 is activated for a period of 30 to 90 minutes, after which it is rapidly dephosphorylated and shuttles out of the nucleus. This tight reg- ulation of STAT3 function reflects the fact that target genes of STAT3 control critical cellular processes such as proliferation, survival, and selfrenewal. When activated inappropriately in a cancer cell, either through activation of kinases, loss of negative regulatory factors, or a combination, it leads to continuous expression of genes that drive the malignant behav- ior of a cell [3,4]. Abundant evidence suggests that the activation of STAT3 is not a bystander event in oncogenesis, but it directly drives cancer pathogenesis [2,5,6]. Since loss of STAT3 function in normal cells can be tolerated [7], inhibiting STAT3 function is a promising strategy for targeted cancer Received 2 May 2019; received in revised form 13 October 2019; accepted 14 October 2019 2019 The Authors. Published by Elsevier Inc. on behalf of Neoplasia Press, Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). https://doi.org/10.1016/j.neo.2019.10.002 Corresponding author at: Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, United States. e-mail address: david_frank@dfci.harvard.edu (D.A. Frank). 1 I.T. also holds an appointment in the Department of Biochemistry, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia. Volume 22 Number 1 January 2020 pp. 47–59 47