DAPK3 Suppresses Acini Morphogenesis and Is Required for Mouse Development Brandon A

Published OnlineFirst October 10, 2014; DOI: 10.1158/1541-7786.MCR-14-0333 Oncogenes and Tumor Suppressors Molecular Cancer Research DAPK3 Suppresses Acini Morphogenesis and Is Required for Mouse Development Brandon A. Kocher1, Lynn S. White1, and David Piwnica-Worms1,2 Abstract Death-associated protein kinase (DAPK3) is a serine/threonine augmented acinar size in DAPK3-depleted structures indicating kinase involved in various signaling pathways important to tissue an epistatic interaction. Using a validated gene trap strategy to homeostasis and mammalian biology. Considered to be a puta- generate a constitutive DAPK3-knockout mouse, it was demon- tive tumor suppressor, the molecular mechanism by which strated that DAPK3 is vital for early mouse development. The DAPK3 exerts its suppressive function is not fully understood Dapk3 promoter exhibits spatiotemporal activity in developing and the field lacks an appropriate mouse model. To address these mice and is actively expressed in normal breast epithelia of adult gaps, an in vitro three-dimensional tumorigenesis model was used mice. Importantly, reduction of DAPK3 expression correlates with and a constitutive DAPK3-knockout mouse was generated. In the the development of ductal carcinoma in situ (DCIS) and more 3D morphogenesis model, loss of DAPK3 through lentiviral- aggressive breast cancer as observed in the Oncomine database of mediated knockdown enlarged acinar size by accelerated acini clinical breast cancer specimens. proliferation and apoptosis while maintaining acini polarity. Depletion of DAPK3 enhanced growth factor–dependent mTOR Implications: Novel cellular and mouse modeling studies of activation and, furthermore, enlarged DAPK3 acini structures DAPK3 shed light on its tumor-suppressive mechanisms and were uniquely sensitive to low doses of rapamycin. Simultaneous provide direct evidence that DAPK3 has relevance in early devel- knockdown of RAPTOR, a key mTORC1 component, reversed the opment. Mol Cancer Res; 13(2); 358–67. Ó2014 AACR. Introduction Similar to other family members, DAPK3 is considered to be a tumor suppressor. Overexpression of DAPK3 in mammalian cells Death-associated protein kinase 3 (DAPK3 or ZIPK) is a mem- results in cell death and cell-cycle inhibition, whereas kinase- ber of the DAPK serine/threonine protein kinase family and is inactivating mutations along with recurrent deleterious somatic known to regulate smooth muscle contraction, cell–cell adhesion, mutations are observed in lung and breast cancers, respectively cytoskeleton dynamics, inflammation, as well as cardiovascular (2–4). Knockdown of DAPK3 increases proliferation of various functions and is thought to serve as a tumor suppressor through cell lines (2). Clinically, reduced DAPK3 mRNA correlates with regulation of caspase-dependent and -independent apoptosis, increased tumor invasion, metastasis, and overall survival in proliferation, and autophagy (1). The DAPK family contains 4 patients with gastric carcinoma (5). Abrogation of DAPK3 mRNA additional members, including DAPK1 (herein referred to as expression was shown to significantly decrease cisplatin sensiti- DAPK), DAPK2 (also DAPK-related protein 1), DRAK-1 and vity in various lung cancer cell lines and may impact overall DRAK-2 (DAPK-related apoptosis-inducing protein kinase-1 and survival of patients with non–small cell lung cancer treated -2), which all share homology within their kinase domain. DAPK3 with platinum-based therapy (6). DAPK3 is also considered contains an N-terminal kinase domain that shares 80% amino a potentially novel breast cancer gene as recurrent DAPK3 acid homology with the prototypical DAPK and differs from other alterations were observed in both BRCA1 mutant and non-mutant family members by the presence of a C-terminal leucine zipper breast cancers (3). In addition, human DAPK3 regulates a variety motif and absence of calmodulin-regulated (CaM) and death of signaling pathways commonly deregulated in cancer. For domains. example, DAPK3 negatively regulates the canonical Wnt/b-cate- nin (CTNNB1) pathway by disrupting the interaction between Nemo-like kinase and T-cell factor 4 in colon cancer cell lines (7). – 1Molecular Imaging Center, Mallinckrodt Institute of Radiology, It also regulates androgen receptor mediated transcription via Washington University School of Medicine, St. Louis, Missouri. ubiquitination and degradation of androgen receptor in various 2Department of Cancer Systems Imaging, University of Texas MD cancer cell lines (8). DAPK3 also interacts with and/or phosphor- Anderson Cancer Center, Houston, Texas. ylates various cancer-associated proteins in vitro, including ATF4, Note: Supplementary data for this article are available at Molecular Cancer AATF, DAXX, PRKC, and STAT3 (4, 9–12). Research Online (http://mcr.aacrjournals.org/). Despite these in vitro and clinical observations, the full phys- Corresponding Author: David Piwnica-Worms, Department of Cancer Systems iologic significance of DAPK3 is not well understood. In addition, Imaging, The University of Texas MD Anderson Cancer Center, 1400 Pressler compared with the prototypical DAPK family member DAPK, Street, Unit 1479, FCT16.5098, Houston, TX 77030. Phone: 713-745-0850; Fax: relatively little is known about the functional tumor-suppressive 713-745-7540; E-mail: [email protected] mechanisms regulated by DAPK3. These limitations are poten- doi: 10.1158/1541-7786.MCR-14-0333 tially exacerbated by the lack of a knockout mouse model as well Ó2014 American Association for Cancer Research. as inadequate cell culture models that cannot recapitulate the 358 Mol Cancer Res; 13(2) February 2015 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst October 10, 2014; DOI: 10.1158/1541-7786.MCR-14-0333 DAPK3 Tumor Suppressor Acts through mTORC1 physiologic context of tissue development or carcinogenesis. incubated for 8 hours with filtered viral supernatants after which Three-dimensional (3D) in vitro tumor systems provide the ability fresh media were added. The following day the media were to functionally investigate the contribution of tumor suppressors replaced with a second round of viral supernatant after which and oncogenes to the complex development and architecture of fresh media were added for 24 hours. Stable cells were then tumor spheroids (13). Given the use of 3D tumor systems and the selected using 1 mg/mL puromycin for 2 passages. Stable cells clinically observed mutations of DAPK3 in breast cancer (3), we were then allowed to recover from selection for one passage before chose to further explore the functional significance of DAPK3 in a use in 2D and 3D assays. For 2D assays, stable cells were plated at MCF10A 3D morphogenesis model. When grown on an extra- an equal density such that they would be about 75% confluent at cellular enriched matrix (Matrigel), the immortalized MCF10A initiation of the experiment. PLKO.1-hygro shRNA constructs epithelial cell line forms hollow spheroids that undergo a regu- against RICTOR and RAPTOR (RPTOR) were generated by sub- lated and coordinated series of biochemical and phenotypic cloning hygromycin in place of puromycin within the pLKO.1- events (14). This model has been used to investigate the contri- constructs and similarly selected using 500 mg/mL hygromycin. bution of loss-of-function (LOF) alterations to acini development For double knockdown, MCF10A were simultaneously infected and early events in tumor formation. with both appropriate shRNA-puromycin (shRNAP) and shRNA- Herein, we describe the functional significance of DAPK3 in hygromycin (shRNAH) virus and subsequently selected with both MCF10A acini morphogenesis and characterize the early lethality 1 mg/mL puromycin and 500 mg/mL hygromycin for one passage observed in a DAPK3-knockout mouse. We identified that DAPK3 followed by one passage in normal media. negatively regulates MCF10A morphogenesis through an mTORC1/S6 (RPS6) pathway that is independent of enhanced Immunofluorescence and confocal microscopy AKT and ERK signaling. Our studies also shed light on an unex- MCF10A acini were grown in 8-well chamber slides and at the pected role for DAPK3 in mouse development as well as a spatially indicated time points were fixed with 4% paraformaldehyde distinct expression pattern with potential relevancy to human (PFA) in PBS for 30 minutes at room temperature. Slides were development and breast cancer. We also identified that DAPK3 is washed 3 times (15 minutes each wash) at room temperature in downregulated in aggressive breast cancer relative to less aggres- 100 mmol/L glycine in PBS and subsequently permeabilized with sive and normal patient samples. 0.05% Tween-20 in PBS, pH 7.4 for 20 minutes. Fixed acini were blocked in IF buffer (0.2% Triton X-100, 0.1% BSA-radioimmu- Materials and Methods noassay grade from Sigma-Aldrich, 0.05% Tween-20 in PBS, pH 7.4) plus 10% normal goat serum for 1.5 hours at room temper- Cell culture and reagents ature and then blocked in secondary block containing IF buffer MCF10A and 293T cells were obtained directly from the ATCC 0 plus 10% goat serum and 20 mg/mL goat anti-mouse IgG F(ab )2 in 2011 and not further tested. MCF10A cells were cultured as fragment-specific (Jackson ImmunoResearch, Cat. 115-006-006) previously described (14) and 293T were cultured in DMEM in a humidified chamber for 30 minutes. Fixed acini were then supplemented with 10% FBS and L-glutamine. 3D morphogenesis stained with 1:100 primary

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