Author Manuscript Published OnlineFirst on September 20, 2018; DOI: 10.1158/1541-7786.MCR-18-0332 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Targeting TAZ-driven Human Breast Cancer by Inhibiting a SKP2-p27 Signaling Axis He Shen 1, Nuo Yang 2, Alexander Truskinovsky 3, Yanmin Chen 1, Ashley L. Mussell 1, Norma J. Nowak4, Lester Kobzik5, Costa Frangou 5* and Jianmin Zhang 1* 1. Department of Cancer Genetics & Genomics, Roswell Park Cancer Institute, Buffalo, NY 14263 2. Department of Anesthesiology, Jacobs School of Medicine & Biomedical Sciences, University at Buffalo, The State University of New York, NY 14214 3. Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY 14263 4. Department of Biochemistry, Jacobs School of Medicine & Biomedical Sciences, University at Buffalo, The State University of New York, NY 14214 5. Harvard TH Chan School of Public Health, Molecular and Integrative Physiological Sciences, 665 Huntington Avenue, Boston, MA 02115 Running Title: TAZ-induced BLBC Tumor Maintenance Through SKP2-p27 Key words: TAZ, SKP2, breast cancer, cell cycle, oncogene dependence, tumor maintenance. 1 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 20, 2018; DOI: 10.1158/1541-7786.MCR-18-0332 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Additional information: Financial support: National Cancer Institute (NCI) R01 CA207504 and the American Cancer Society Research Scholar Grant RSG-14-214-01-TBE (to J.Z.). Correspondence: * Dr. Costa Frangou, Harvard TH Chan School of Public Health, Molecular and Integrative Physiological Sciences, 665 Huntington Avenue, Boston, MA 02115 [email protected] * Dr. Jianmin Zhang, Department of Cancer Genetics & Genomics, Roswell Park Cancer Institute, Buffalo, NY 14263 [email protected] The authors declare no potential conflicts of interest. Word count: 5935 Number of figures: 6 2 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 20, 2018; DOI: 10.1158/1541-7786.MCR-18-0332 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Deregulated expression of the transcriptional co-activator with PDZ-binding motif (WWTR1/TAZ) is a common feature of basal-like breast cancer (BLBC). Yet, how oncogenic TAZ regulates cell cycle progression and proliferation in breast cancer remains poorly understood, and whether TAZ is required for tumor maintenance has not been established. Here, using an integrative oncogenomic approach, TAZ-dependent cellular programs essential for tumor growth and progression were identified. Significantly, TAZ-driven tumor cells required sustained TAZ expression, given that its withdrawal impaired both genesis and maintenance of solid tumors. Moreover, temporal inhibition of TAZ diminished the metastatic burden in established macroscopic pulmonary metastases. Mechanistic investigation revealed that TAZ controls distinct gene profiles that determine cancer cell fate through cell cycle networks, including a specific, causal role for S-phase kinase-associated protein 2 (SKP2) in mediating the neoplastic state. Together, this study elucidates the molecular events that underpin the role of TAZ in BLBC and link to SKP2, a convergent communication node for multiple cancer signaling pathways, as a key downstream effector molecule. Implications: Understanding the molecular role of TAZ and its link to SKP2, a signaling convergent point and key regulator in BLBC, represents an important step toward the identification of novel therapeutic targets for TAZ-dependent breast cancer. 3 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 20, 2018; DOI: 10.1158/1541-7786.MCR-18-0332 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Breast cancer (BC) is a multifaceted disease with distinct histopathologic features, genetic and genomic variability, and prognostic outcomes. Breast tumor heterogeneity is dynamic and evolves unpredictably during disease progression, creating considerable variability within primary tumors and metastases (1). However, BC can be classified into at least four major molecular subtypes based on gene expression programmes: luminal A, luminal B, HER2-enriched, and basal-like (2). These subtypes exhibit important differences in risk factors, response to treatment, likelihood of disease progression, and propensity to metastasize to multiple organs, including bones, lungs, and liver (3). Basal-like BCs (BLBCs) are especially associated with an aggressive clinical history, early recurrence, distant metastasis, and reduced survival rates in patients. BLBCs lack oestrogen (ER), progesterone (PR) and HER2 expression (4). These characteristics make BLBCs challenging to treat, as they do not respond to endocrine treatments or targeted therapies, and treatment options are restricted to conventional chemotherapy. Despite extensive efforts to characterize the BLBC subtype, the oncogenic drivers of BLBC remain elusive (5). Several genomic and genetic studies have identified multiple candidate oncogenic alterations in BLBC (6). These include genes associated with signal transduction, angiogenesis, the cell cycle and proliferation, cell survival, DNA replication and recombination, motility and invasion (7). For example, PTEN loss, PI3K/AKT pathway activation, and TP53 mutations are frequently observed in BLBCs (8, 9). But widespread chromosomal instability is typical in BLBC (10), making it difficult to distinguish genes driving cancer development from those playing a bystander role. 4 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 20, 2018; DOI: 10.1158/1541-7786.MCR-18-0332 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. The blockade of transcription factor interactions can be especially attractive in targeting cellular pathways that promote oncogenic transformation and typically involve many signaling proteins that ultimately converge on a much smaller set of oncogenic transcription factors. Recently, we and others reported that protein expression of the Hippo transducer transcriptional co-activator with PDZ-binding motif (TAZ) is associated with a decreased survival rate and shorter time to relapse in BLBC patients (11, 12). The oncogenic activity of TAZ involves the regulation of diverse signal transduction pathways that direct such processes as proliferation, migration, and resistance to apoptosis, albeit through poorly characterized gene expression programmes (13). Ectopic expression of TAZ in human cell and transgenic murine tissues results in their oncogenic transformation (11, 14). However, whether oncogenic TAZ is required for the maintenance of BLBC has not been established. To test whether TAZ is necessary for maintaining as well as developing a cancer, we generated a transplant-based model of BLBC in which constitutively active TAZ expression is controlled in a doxycycline (dox)-dependent manner. We exploited this ability to precisely control the timing of TAZ expression during tumorigenesis and metastasis, which allowed us to examine the consequence of sustained TAZ activity on the maintenance of the transformed state in a relevant experimental setting. Using this system, we found that TAZ is not only a driver of BLBC progression and a novel prognostic factor but is also required for the maintenance of tumors and already established metastases. Importantly, this finding credentials TAZ as a legitimate BC therapeutic target. Furthermore, through iterative rounds of hypothesis generation and testing, we demonstrated that TAZ drives an SKP2-regulated cell cycle programme 5 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 20, 2018; DOI: 10.1158/1541-7786.MCR-18-0332 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. involving genes linked to tumorigenic potential and poor prognosis in BC. As a result, our work provides biological insight and proof-of-concept evidence for targeting TAZ- regulated genes that support tumour maintenance to identify novel treatments for BC. 6 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 20, 2018; DOI: 10.1158/1541-7786.MCR-18-0332 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and methods Cell culture: MCF10A, HMEC, T47D, BT474, MDA-MB-453, MDA-MB-468, ZR751, BT549, CAL120, CAL51, MDA-MB-231, MDA-MB-361, MDA-MB-435, SUM159, HCC38, HCC1143, HCC1937 cell culture was performed as previously described (12). MDA-MB-231, T47D, MDA-MB453, BT-474, HCC38 HCC1143 and HCC1937 cells were purchased from American Type Culture Collection (ATCC, VA); MCF10A and HMEC were obtained from Dr. Joan Brugge, Harvard Medical School; CAL51, CAL120 and SUM159 were obtained