Priority Report p73 and IGF1R Regulate Emergence of Aggressive Cancer Stem–like Features via miR-885-5p Control Claudia Meier, Philip Hardtstock, Sophie Joost, Vijay Alla, and Brigitte M. P€ utzer Abstract Cancer stem–like cells (CSC) have been proposed to promote cancer progression by initiating tumor growth at distant sites, suggesting that stem-like cell features can support metastatic efficiency. Here, we demonstrate that oncogenic DNp73, a dominant-negative variant of the tumor-suppressor p73, con- fers cancer cells with enhanced stem-like properties. DNp73 overexpression in noninvasive melanoma and lung cancer cells increased anchorage-independent growth and elevated the expression of the pluripotency factors CD133, Nanog, and Oct4. Conversely, DNp73 depletion in metastatic cells down- regulated stemness genes, attenuated sphere formation and reduced the tumor-initiating capability of spheroids in tumor xenograft models. Mechanistic investigations indicated that DNp73 acted by attenuating expression of miR-885-5p, a direct regulator of the IGF1 receptor (IGF1R) responsible for stemness marker expression. Modulating this pathway was sufficient to enhance chemosensitivity, overcoming DNp73- mediated drug resistance. Clinically, we established a correla- tion between low p73 function and high IGF1R/CD133/ Nanog/Oct4 levels in melanoma specimens that associated with reduced patient survival. Our work shows how DNp73 promotes cancer stem–like features and provides a mechanistic rationale to target the DNp73–IGF1R cascade as a therapeutic strategy to eradicate CSC. Cancer Res; 76(2); 197–205. Ó2015 AACR. Introduction N-terminal truncated variants of the tumor-suppressor TP73, collectively termed DNp73, are of pivotal importance in the modulation of gene expression to confer a malignant phenotype and drive tumor progression. Lacking the TA domain by differ- ential splicing events at the 5 0 -end or usage of an alternative intrinsic promoter, DNp73 isoforms are unable to directly trans- activate target gene expression. DNp73 thereby counteracts tumor-suppressive properties of wild-type p53/TAp73 through forming inactive heteromeric complexes with them or by occu- pying p53-responsive elements on gene promoters. High-expres- sion levels of DNp73 are observed especially in late-stage tumors and metastases, where they significantly correlate with poor patient outcome (1). Numerous studies have provided substan- tial evidence that DNp73 bestows chemoresistance by decreasing intracellular drug accumulation through upregulation of the ABC transporters A2, A5, and B1 (2, 3). Moreover, we recently dem- onstrated that the DNp73 variant DEx2/3p73 causes dramatic increases in cancer malignancy via promoting the transition from an epithelial to a highly motile mesenchymal (EMT) phenotype, leading to increased invasiveness and metastases formation in xenograft mouse models (4). DNp73-mediated acquisition of mesenchymal properties involves interference with wild-type TAp73-dependent transactivation of LIMA1/EPLIN that consec- utively fosters activation of the IGF1R-AKT-Slug signaling and downregulation of E-cadherin. Regardless of the function in DNp73-induced EMT and cancer aggressiveness, IGF1R signaling was implicated in several biologic processes essential for malignancy, including cell growth, DNA repair, and survival. Emerging evidence also support an involve- ment of the IGF1-receptor in differentiation events. IGF1R expres- sion levels are enriched in human embryonic and hematopoietic stem cell populations. Regulated by mutant D40p53, IGF1R maintains pluripotency by blocking differentiation of embryonic stem cells into somatic cells (5). A direct link between activated IGF1R signaling and the gain of stem cell– and EMT-associated properties was recently discovered in lung cancer stem cells, demonstrating a profound role for fibulin 3-suppressed IGF1R/ PI3K/AKT/GSK3b signaling in the regulation of mesenchymal and stem cell markers (6). Cancer stem–like cells (CSC) are a rare subpopulation of undifferentiated cells within a tumor that are proposed to be the cancer-initiating cells responsible for tumor- igenesis, tumor relapse, and metastasis. In particular, CSCs exhibit characteristics of tumor cells that have undergone EMT, and contribute to DNA damage protection. In consistence with the activation of IGF1R by DNp73 and the role of IGF1R signaling in mediating EMT, chemoresistance, and stemness, we hypothesize an involvement of DNp73 in the acquisition of stem cell features to promote therapeutic resistance and cancer cell propagation. Materials and Methods Cell culture and antisense oligonucleotide transfection The human melanoma cell lines SK-Mel-28, -29, -103, 147 (obtained from M. Soengas at the Department of Dermatology, University of Michigan, Comprehensive Cancer Center, Ann Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical Center, Rostock, Germany. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Brigitte M. P€ utzer, Rostock University Medical Center, Schillingallee 69, 18055 Rostock, Germany. Phone: 49-381-494-5066; Fax: 49- 381-494-5062; E-mail: brigitte.puetzer@med.uni-rostock.de doi: 10.1158/0008-5472.CAN-15-1228 Ó2015 American Association for Cancer Research. Cancer Research www.aacrjournals.org 197 on June 7, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst November 10, 2015; DOI: 10.1158/0008-5472.CAN-15-1228