Regulation of cancer stem cell properties by SIX1, a member of the PAX-SIX-EYA-DACH network

CHAPTER ONE

Regulation of cancer stem cell properties by SIX1, a member of the PAX-SIX-EYA-DACH network Tami J. Kingsburya,b,c,*, MinJung Kima,b,d, Curt I. Civina,b,c,d

a Center for Stem Cell Biology & Regenerative Medicine, University of Maryland School of Medicine, Baltimore, MD, United States b Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, MD, United States c Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, United States d Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, United States *Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Cancer stem cells (CSCs) 3. SIX1 and CSCs 4. Sustained proliferative signaling 5. Evading growth suppressors 6. Enabling replicative immortality 7. Resisting cell death 8. Genome instability and mutation 9. Deregulating cellular energetics 10. Inducing angiogenesis 11. Regulation of SIX1 expression 12. SIX1 clinical significance 13. EYA 14. DACH 15. Concluding remarks Acknowledgments Conflict of interest References

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Abstract The PAX-SIX-EYA-DACH network (PSEDN) is a central developmental transcriptional regulatory network from Drosophila to humans. The PSEDN is comprised of four conserved protein families; including paired box (PAX), sine oculis (SIX), eyes absent (EYA), and dachshund (DACH). Aberrant expression of PSEDN members, particularly SIX1, has been observed in multiple human cancers, where SIX1 expression correlates

Advances in Cancer Research, Volume 141 ISSN 0065-230X https://doi.org/10.1016/bs.acr.2018.12.001

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2019 Elsevier Inc. All rights reserved.

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with increased aggressiveness and poor prognosis. In conjunction with its transcriptional activator EYA, the SIX1 transcription factor increases cancer stem cell (CSC) numbers and induces epithelial-mesenchymal transition (EMT). SIX1 promotes multiple hallmarks and enabling characteristics of cancer via regulation of cell proliferation, senescence, apoptosis, genome stability, and energy metabolism. SIX1 also influences the tumor microenvironment, enhancing recruitment of tumor-associated macrophages and stimulating angiogenesis, to promote tumor development and progression. EYA proteins are multifunctional, possessing a transcriptional activation domain and tyrosine phosphatase activity, that each contributes to cancer stem cell properties. DACH proteins function as tumor suppressors in solid cancers, opposing the actions of SIX-EYA and reducing CSC prevalence. Multiple mechanisms can lead to increased SIX1 expression, including loss of SIX1-targeting tumor suppressor microRNAs (miRs), whose expression correlates inversely with SIX1 expression in cancer patient samples. In this review, we discuss the major mechanisms by which SIX1 confers CSC and EMT features and other important cancer cell characteristics. The roles of EYA and DACH in CSCs and cancer progression are briefly highlighted. Finally, we summarize the clinical significance of SIX1 in cancer to emphasize the potential therapeutic benefits of effective strategies to disrupt PSEDN protein interactions and functions.

1. Introduction Transcription factors function as master regulators of developmental programs, determining stem-progenitor self-renewal and multipotency, cell survival, proliferation, specification, maturation, and function (Goode et al., 2016; Niwa, 2018). Aberrant structure/function or altered expression of transcription factors, notably disruption of a lineage-specific differentiation transcription factor or constitutive expression of a developmentallyrestricted transcription factor, can cause dysregulated cellular characteristics that include cancer stem cell phenotypes and hallmarks of cancers (Bradner, Hnisz, & Young, 2017; Rosenbauer & Tenen, 2007). Over the past decade, re-expression of the developmental homeobox transcription factor SIX1 and its transcriptional activators belonging to the EYA protein family have emerged as frequent features in the transcriptomes and proteomes of solid cancers (Blevins, Towers, Patrick, Zhao, & Ford, 2015; Kong et al., 2016; Liu et al., 2016; Wu et al., 2015). SIX1 and EYA belong to the PAX-SIX-EYA-DACH Network (PSEDN), which is comprised of four protein families—paired box proteins (PAX), sine oculis homeobox proteins (SIX), eyes absent homologs (EYA), and dachshund homologs (DACH)—that are all highly conserved across taxa. The PSEDN was first described in Drosophila as the retinal determination gene network (RDGN), based on the requirement of an intact fly

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PSEDN for eye formation and the ability of enforced expression of PSEDN members to induce ectopic eye formation (Pappu & Mardon, 2004). PSEDN members orchestrate the development of multiple organs in mammals (Christensen, Patrick, McCoy, & Ford, 2008; Wu et al., 2015). SIX proteins regulate the genesis, survival and proliferation of stem-progenitor cells (Li et al., 2003; Wu et al., 2015). Consistent with these roles, Six1 knockout mice exhibit reduced or absent kidneys, profound muscle developmental defects, rib cage deformations and loss of inner ear structures (Li et al., 2003). SIX1 expression is largely absent from normal adult mammalian tissues, except muscle satellite cells, where SIX1 regulates satellite cell myoblast myogenic commitment and differentiation via satellite cell niche occupancy (Le Grand et al., 2012). Mutations in PSEDN members have also been implicated in human disorders (Christensen et al., 2008). Dominant mutations of SIX or EYA cause branchio-oto-renal (BOR) syndrome (hypoplastic or absent kidneys with resultant renal insufficiency, malformation or absence of the middle ear with resultant hearing loss, and branchial arch anomalies including clefts, fistulae, and cysts) (Kochhar et al., 2008; Musharraf et al., 2014; Ruf et al., 2004; Sanggaard et al., 2007). Genetic analysis of fly PSEDN has revealed complex positive and negative interactions between PSEDN members that fine-tune development (Davis & Rebay, 2018; Kumar, 2009; Liu et al., 2016). In mammalian systems, distinct physical and functional interactions among the PAX, SIX, EYA and DACH proteins coordinate to shape the cellular transcriptome (Liu et al., 2016). SIX proteins are defined by their conserved SIX domain (SD) and homeobox DNA-binding domain (HD) (Fig. 1A). Only some SIX family members appear to possess an intrinsic transactivation domain (TAD) (Brodbeck, Besenbeck, & Englert, 2004; Ohto et al., 1999; Ruf et al., 2004). EYA proteins are multifunctional, possessing a N-terminal proline-serine-threonine rich TAD (Ohto et al., 1999; Xu, Cheng, Epstein, & Maas, 1997) and a conserved C-terminal EYA domain (ED) that mediates interaction with SIX proteins (Patrick et al., 2013) and harbors tyrosine phosphatase activity (Fig. 1B) ( Jemc & Rebay, 2007; Li et al., 2003; Rayapureddi et al., 2003; Tootle et al., 2003). EYA proteins directly bind to and co-activate SIX protein complexes to activate expression of myriad cellular genes. In contrast, DACH1, either independently or in conjunction with SIX1, can inhibit gene expression via recruitment of repressor complexes (Ikeda, Watanabe, Ohto, & Kawakami, 2002; Li et al., 2003). EYA3 tyrosine phosphatase activity can switch SIX-DACH from an inhibitory to a stimulatory transcriptional complex (Li et al., 2003). DACH proteins bind DNA via their conserved

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A SIX1

SD

SIX2

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98%

SIX3

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61%

SIX4

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SIX5

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69%

SIX6

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HD

284 TAD 291 (73%) 332 (45%) TAD TAD

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538 (63%)

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573 (50%)

EYA4

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639 (74%)

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C DACH1

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DD2

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706 599 (52%)

Fig. 1 Human SIX, EYA, and DACH family members. Overview of protein family members with amino acid length and the percent identity to (A) SIX1, (B) EYA1, and (C) DACH1 of full length protein and conserved domains indicated. (A) SIX family. Human SIX proteins possess two conserved domains: the SIX-specific protein-protein interaction domain (SD, yellow) and the SIX-type DNA binding homeodomain (HD, green). In addition, SIX2, SIX4, and SIX5 possess the transactivation domain (TAD, blue) at the C-terminus. (B) EYA family. Human EYA proteins possess one conserved domain: the EYA protein-protein interaction domain, which possesses protein tyrosine phosphatase activity (ED, blue). (C) DACH family. Human DACH proteins possess two conserved domains: the Dachshund domain 1 (DD1, red) that binds DNA and has homology to SKI/SNO/DAC co-repressors and the Dachshund domain 2 (DD2, purple) that mediates EYA interaction in Drosophila.

Dachshund domain 1 (DD1 or box-N), which has homology to the SKI/ SNO family of co-repressor proteins (Fig. 1C). The conserved DD2 domain (box-C) mediates binding of Drosophila Dachshund to Eyes absent (EYA) (Chen, Amoui, Zhang, & Mardon, 1997). Mammalian DACH and EYA proteins, however, do not appear to directly bind each other, but instead interact indirectly via CREB binding protein (Ikeda et al., 2002). Consistent with their most common functions in normal cellular physiology, SIX1 and EYA proteins are frequently upregulated in solid cancers with oncogenic effects, while DACH proteins are often downregulated and function primarily as tumor suppressors (Fig. 2). In contrast, expression of SIX, EYA or DACH

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Fig. 2 SIX1-EYA1-DACH1 transcriptional regulatory network in solid cancers. SIX1 and EYA1 form a transcriptional complex that stimulates expression of genes that promote CSC phenotypic and functional properties, enhancing tumor formation and metastasis. DACH1, either alone or in combination with additional transcription factors, including SIX1, represses expression of genes necessary for the acquisition of CSC properties and cancer progression.

can exert oncogenic effects in hematopoietic cells (Lee, Kim, Hwang, et al., 2012; Lee, Kim, Kim, et al., 2012; Wang et al., 2011), highlighting importance of cell context in shaping transcriptional output and cell responses. Humans possess 9 PAX, 6 SIX, 4 EYA and 2 DACH family members, making comprehensive overview of the entire PSEDN beyond the scope of a single review. In this chapter of a volume on cancer stem cells (CSCs), we focus primarily on SIX1, which has emerged as a crucial factor in diverse cancers. High SIX1 expression in solid cancers confers CSC and epithelial-mesenchymal transition (EMT) phenotypes and generally correlates with poor prognosis. We begin with a brief discussion of CSCs and an overview of SIX1’s ability to promote features of CSCs. We then describe mechanisms by which SIX1 can confer multiple hallmarks and enabling characteristics of cancer (Hanahan & Weinberg, 2011), followed by a brief synopsis of mechanisms by which SIX1 expression is up-regulated in cancers. Next, we provide examples of SIX1 associations with human cancers and clinical outcomes, noting exceptions to general family rules observed for SIX3 and EYA4 in multiple cancers. Finally, we briefly highlight the roles of EYA and DACH proteins in regulating CSCs, cancer development and progression.

2. Cancer stem cells (CSCs) The heterogeneity of cancers, both from patient-to-patient within a cancer type (inter-tumor heterogeneity) and from cell-to-cell within a single patient’s cancer (intra-tumor heterogeneity), has long been noted

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(Dagogo-Jack & Shaw, 2018; Meacham & Morrison, 2013; Prasetyanti & Medema, 2017). By analogy to the cellular hierarchies of normal organsystems, the CSC model predicts that relatively rare CSCs possessing the cardinal stem cell properties of extensive self-renewal and multipotent differentiation potential establish the inherent cellular heterogeneity of the cancer. Thus, in the CSC model, only a CSC can grow immortally and generate all of the heterogeneous cell types in the given cancer (Batlle & Clevers, 2017; Clevers, 2011). A CSC can be functionally defined by its capacity to regenerate a cancer after transplant to an experimental animal. To emphasize this experimental definition, an operationally defined CSC is often referred to as a tumor-initiating cell (TIC). The frequency of TICs can vary widely among cancer types and cases. Within a particular cancer type, higher frequencies of CSCs correlate with increased adverse patient outcomes (Rasheed, Kowalski, Smith, & Matsui, 2011; Visvader & Lindeman, 2012). Self-renewal and stemness gene signatures also correlate with poor prognosis (Visvader & Lindeman, 2012). TIC numbers underestimate CSC frequencies due to cross-species growth factor or niche incompatibility and immune recognition (Meacham & Morrison, 2013) and can vary dramatically depending on xenograft conditions (Quintana et al., 2008). Alternatively, CSCs have been identified by expression of specific cell markers, based on their presumed tissue of origin, such as CD34+ CD38 cells in acute myeloid leukemias (AMLs) (Bonnet & Dick, 1997; Lapidot et al., 1994). The challenges of phenotypic CSC identification are discussed in chapter “Assays for functionally defined normal and malignant mammary stem cells” by Aalam et al. in this volume. While CSCs were initially proposed to comprise a fixed subset of quiescent cancer cells uniquely hardwired with extensive self-renewal capacity and ability to regenerate the cancer (Batlle & Clevers, 2017; Clevers, 2011), it is now recognized that cell plasticity enables differentiated cancer cells to de-differentiate and acquire stem cell properties (Batlle & Clevers, 2017; Chaffer et al., 2013; Gupta et al., 2011). Studies in mice have shown the capacity of the microenvironment to confer CSC properties, converting cells lacking CSC capacities into cells that meet the CSC definition (de Sousa e Melo et al., 2017; Korkaya et al., 2012; Schwitalla et al., 2013; Shimokawa et al., 2017; Vermeulen et al., 2010). Factors that enhance EMT also increase CSC numbers (Mani et al., 2008; Morel et al., 2008). It seems likely that dynamic regulation of cell characteristics occurs within cancers, orchestrated by both the cancer and its environment. Observations on cancer cell plasticity predict that strategies designed to selectively eliminate CSCs

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may fail due to reemergence of new CSCs (Batlle & Clevers, 2017; de Sousa e Melo et al., 2017). Inclusion of methods to target factors and networks, such as SIX-EYA, that support the acquisition of CSC properties and the hallmarks required for cancer expansion and progression may be more successful.

3. SIX1 and CSCs SIX family members promote progenitor cell survival, proliferation and migration during development (Christensen et al., 2008; Yajima et al., 2014), functions that can readily be coopted to promote cancer development and progression. SIX1 increases CSC numbers in vitro and in vivo: SIX1 increased phenotypic and functional CSCs in breast (Iwanaga et al., 2012), colorectal (Xu, Zhang, Pena, Pirisi, & Creek, 2017) and esophageal cancer (Nishimura et al., 2017) and phenotypic CSCs in pancreatic cancers (Lerbs et al., 2017). SIX1 also blocked the differentiation of human papilloma virus (HPV) 16-immortalized human keratinocytes used to model aspects of cervical cancer in vitro (Xu et al., 2014). The increase in CSC numbers mediated by SIX1 involves multiple mechanisms, including stimulation of mitogenactivated protein kinase (MAPK) and diversion of transforming growth factor beta (TGFβ) cell signaling pathways (Iwanaga et al., 2012; Nishimura et al., 2017). SIX1 has been most extensively studied in the context of breast cancer. Experimental overexpression of SIX1 in the non-malignant MCF12A mammary cell line was sufficient to transform cells, enabling the generation of aggressive cancers in immunodeficient mice (Coletta et al., 2008). SIX1 overexpression in the MCF7 luminal mammary carcinoma cell line conferred a human breast CSC gene signature, with fivefold increased numbers of phenotypic (CD24lowCD44hi) CSCs, twofold increased secondary tumorspheres (an index of self-renewal), and fourfold increased functional TICs as quantitated by serial dilution mouse xenograft experiments (Iwanaga et al., 2012). These findings demonstrate that SIX1 can drive acquisition of CSC properties as well as development of breast cancers. Further support for the oncogenic role of SIX1 was obtained using a genetic mouse model. Expression of SIX1 in mouse mammary epithelium resulted in precocious secretory differentiation and hyperplasia throughout the mammary gland and increased numbers of phenotypic (CD29hiCD24+) and functional (mammosphere formation) mammary epithelial stem cells

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(McCoy et al., 2009). SIX1 overexpression resulted in mammary tumors in up to 40% of mice. The tumors exhibited diverse histology and Wnt signaling activation, consistent with derivation from stem-progenitor cells. Most SIX1-induced tumors exhibited molecular markers of EMT and progressed to aggressive malignant neoplasms. During development, EMT is essential for proper body patterning and morphogenesis (Lim & Thiery, 2012; Thiery, Acloque, Huang, & Nieto, 2009). SIX1 and SIX4 are required for EMT during limb myogenesis (Grifone et al., 2005). SIX1 overexpression promotes EMT across a wide spectrum of cancer cell types, including those derived from breast (Iwanaga et al., 2012; McCoy et al., 2009; Micalizzi et al., 2009; Xu et al., 2014), colon (Ono et al., 2012), cervix (Sun et al., 2016), esophagus (Nishimura et al., 2017), stomach (Xie et al., 2018), pancreas (Lerbs et al., 2017), and muscle (Yu, Davicioni, Triche, & Merlino, 2006). EMT not only drives cancer metastasis, but also increases CSC numbers, as assessed by both marker expression and TICs (Mani et al., 2008; Morel et al., 2008; Sato, Semba, Saya, & Arima, 2016). SIX1 expression in luminal breast cancer cells stimulated EMT and TICs via activation of both TGFβ and MEK/ERK signaling (Iwanaga et al., 2012; McCoy et al., 2009). In patient samples, SIX1 expression correlated with increased phospho-ERK (Iwanaga et al., 2012), indicating that SIX1 can regulate ERK signaling in breast cancers. TGFβ signaling possesses dual opposing roles during carcinogenesis, switching between cancer-suppressive and cancer-promoting functions depending on cell context (Ikushima & Miyazono, 2010; Micalizzi, Farabaugh, & Ford, 2010). SIX1 can orchestrate the switch in TGFβ signaling output to drive EMT via upregulation of transforming growth factor beta receptor 1 (TGFβR1) (Micalizzi, Wang, Farabaugh, Schiemann, & Ford, 2010). SIX1 also stimulates expression of the miR-106b-25 cluster, which overcomes TGFβ growth suppressive properties by targeting cyclin dependent kinase inhibitor 1A (p21) and BCL2-like protein 11 (BIM) (Smith et al., 2012). The miR-106b-25 cluster additionally targets the inhibitory SMAD family member 7 (SMAD7), leading to increased expression of TGFβR1 and activation of downstream signaling. Enforced expression of the miR-106b-25 cluster was sufficient to induce EMT and TICs. In HPV-immortalized keratinocytes, SIX1 expression induced EMT via activation of TGFβ signaling and MAPK signaling with increased phospho-ERK (Xu, Zhang, et al., 2017). TGFβ signaling was also required for SIX1mediated EMT in cervical cancer cells (Sun et al., 2016).

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4. Sustained proliferative signaling Sustained proliferation is a key hallmark of cancer cells. According to the classic CSC model, CSCs are predicted to be rare and quiescent. In contrast, however, CSC frequencies and proliferative status vary dramatically, with CSCs being abundant and proliferative in some cancers, including melanomas, colorectal carcinomas (CRCs) and certain AML cases (Batlle & Clevers, 2017; Visvader & Lindeman, 2012). Multiple mechanisms can lead to sustained cancer cell proliferation, including autocrine proliferative signaling mediated by cancer-derived growth factors, modulation of the cancer microenvironment leading to release of growth factors from cancer-associated stromal cells or altered signaling pathways that either enhance the sensitivity of cancer cells to limiting concentrations of growth factors or enhance downstream proliferative signaling responses (Hanahan & Weinberg, 2011). Gain- and loss-of-function analyses have demonstrated that SIX1 stimulates cancer cell proliferation by multiple mechanisms, including controlling expression of cell cycle regulators or activity of cell signaling pathways and bypassing checkpoints that modulate cell proliferation (Blevins et al., 2015; Wu et al., 2015). Experimental overexpression of SIX1 in immortalized MCF12A cells, which have low endogenous SIX1 levels, increased cyclin A1 levels via direct binding of SIX1 to the cyclin A1 promoter and consequent stimulated gene expression (Coletta et al., 2004). Cyclin A1 binds to and activates cyclin-dependent kinase 2 (CDK2) and CDK1, to promote the G1/S transition, progression through S phase and the G2/M transition (Muller-Tidow et al., 2004; Yang et al., 1999). SIX1 overexpression in MCF7 cells accelerated cell cycle progression and resulted in larger tumors following transplant into immunodeficient mice (Coletta et al., 2004). Conversely, SIX1 downregulation in MCF7 cells reduced cyclin A levels, cell proliferation and tumor size (Coletta et al., 2004). SIX1 knockdown in the 21PT mammary carcinoma cell line also reduced cell cycle progression and proliferation. The ability of SIX1 to stimulate cell proliferation was dependent upon cyclin A1 in both MCF7 and 21PT cells (Coletta et al., 2004). SIX1 similarly stimulated cyclin A expression and cell proliferation in ovarian cancer cells (Behbakht et al., 2007). In cervical cancer, HPV E7 oncoprotein stimulated SIX1 expression (Liu, Zhang, et al., 2014). SIX1 overexpression in the HPV-negative C33a cervical cancer cell line increased expression of genes encoding factors

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required for DNA replication, including DNA polymerases and minichromosome maintenance complex components. SIX1 enhanced DNA synthesis, accelerated the G1/S cell cycle progression and increased cervical cancer cell proliferation in vitro and in vivo, as assessed in mouse ectopic and orthotopic cervical cancer xenograft models (Liu, Zhang, et al., 2014). SIX1 knockdown in the HPV-positive Caski cervical cancer cell line reduced DNA synthesis, blocked the G1/S transition and inhibited cell proliferation and xenograft tumor formation (Liu, Zhang, et al., 2014). SIX1 overexpression also stimulated cell proliferation of PANC-1 and MIA PaCa-2 pancreas cancer cell lines, whereas SIX1 knockdown reduced cell proliferation (Li, Tian, Lv, et al., 2013). In mouse xenograft assays, transplant of SIX1-overexpressing PANC-1 cells generated tumors that were nearly sixfold larger than controls. SIX1 directly bound to the CCND1 promoter to stimulate cyclin D1 expression. Cyclin D was necessary for SIX1mediated growth advantage in PANC-1 cells, and cyclin D expression rescued the cell proliferation deficit observed in SIX1-knockdown cells (Li, Tian, Lv, et al., 2013). Thus, SIX1 regulates pancreatic cancer cell line proliferation at least in part via regulation of cyclin D expression. SIX1 also stimulated cyclin D1 expression in endometrial (Xin, Li, & Yang, 2016) and gastric cancer cells (Xie et al., 2018) and cyclin E in endometrial (Xin et al., 2016) and esophageal (He, Li, Tang, & Li, 2017) cancer cells. shRNA-mediated knockdown of SIX1 in the human hepatocellular cancer (HCC) cell line MHCC97L reduced cell proliferation by delaying the G2/M transition (Ng et al., 2010). Subcutaneous xenograft tumors derived from SIX1-knockdown cells were fivefold smaller compared to controls, indicating reduced tumor growth due to SIX1 knockdown. Furthermore, SIX1 mediated the increased growth of HCC cells observed in response to histone deacetylase 5 (HDAC5) expression (Feng et al., 2014). SIX1 overexpression has also been shown to increase cell proliferation via activation of cell signaling pathways. In SAOS-2 and U2OS osteosarcoma cell lines, SIX1 reduced phosphatase and tensin homolog (PTEN) expression, thereby activating AKT signaling via suppression of PTEN inhibition (Yu, Zhang, Li, & Yu, 2018). SIX1-overexpressing SAOS-2 and U2OS cells generated larger tumors in mice compared to controls, and the increase could be blocked by PTEN co-expression. SIX1 overexpression in HEC1B endometrial adenocarcinoma cells, which have low endogenous SIX1 levels, increased cell growth and colony formation, whereas SIX1 knockdown in Ishikawa endometrial adenocarcinoma cells (which have high endogenous SIX1) reduced these phenotypes (Xin et al., 2016).

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Pharmacologic inhibition of either ERK or AKT signaling blocked SIX1 stimulation of cell proliferation, demonstrating that these pathways were required for SIX1-mediated effects on endometrial cancer cell growth (Xin et al., 2016). In sonic hedgehog (SHH)-dependent medulloblastoma in mouse models, SIX1 stimulated growth via activation of SHH signaling (Eisner et al., 2015). SIX1 additionally enhances cell proliferation via stimulation of growth factors and modulation of the cancer microenvironment. SIX1 overexpression increased glioblastoma multiforme (GBM) cell proliferation in vitro by stimulating expression of connective tissue growth factor (CTGF) (Tian et al., 2015). In contrast, overexpression of SIX1 in human or mouse CRC cell lines did not significantly alter cell proliferation in vitro, but stimulated proliferation in vivo via recruitment of tumor-associated macrophages and angiogenesis (Xu, Zhang, et al., 2017). Additionally, SIX1 overexpression in the MC38 CRC cell line increased the number of mice that developed cancers following injection into the cecum from 25% to 75%, the average tumor size by threefold, the percentage of proliferative (Ki67+) cells within tumors and the number of cells expressing CRC stem cell markers CD44, CD166 and aldehyde dehydrogenase 1 (ALDH1) (Xu, Zhang, et al., 2017). Since no differences in caspase 3 staining were observed between SIX1 and control tumors, SIX1 likely increased tumor size by stimulating in vivo cell proliferation rather than blocking cell death. The ability of SIX1 to increase cell proliferation in vivo, but not in vitro, suggests that SIX1 coordinates with the cancer microenvironment to orchestrate increased cell proliferation. shRNA-mediated knockdown of SIX1 in the human hepatocellular cancer (HCC) cell line MHCC97L reduced cell proliferation by delaying the G2/M transition (Ng et al., 2010). Subcutaneous xenograft tumors derived from SIX1-knockdown cells were fivefold smaller compared to controls, indicating reduced tumor growth due to SIX1 knockdown. SIX1 also mediated the increased growth of HCC cells in response to histone deacetylase 5 (HDAC5) (Feng et al., 2014).

5. Evading growth suppressors The tumor suppressor p53 guards against unrestrained cell proliferation and regulates stem cell self-renewal, proliferation and differentiation ( Jain & Barton, 2018). TP53 is disrupted in over 50% of human cancers,

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and its function is compromised in still more cancers (Soussi & Wiman, 2007). Multiple lines of evidence indicate that SIX1 can disable TP53 (p53) restraint of cell proliferation. Modest overexpression of SIX1 in MCF7 cells (to levels lower than may be present in cancers) reduced levels of nuclear p53 and expression of downstream target genes, such as p21, in the presence and absence of DNA damage (Towers et al., 2015). In contrast, SIX1 knockdown in HEK293T or HCT-116 cells increased p53 levels and expression of p53 downstream target genes. Breast cancers induced by enforced SIX1 expression in mouse mammary tissue shared a gene expression signature with genetic engineered mouse models of p53-deficient cancers (Towers et al., 2015). SIX1 did not alter p53 transcript levels, but rather inhibited p53 expression post-transcriptionally via upregulation of the known oncogenic miR, miR-27a-3p, which can directly target and inhibit p53 expression (Towers et al., 2015). SIX1 also repressed expression of ribosomal protein L26 (RPL26), which competes with miR-27a-3p for p53 mRNA binding, enabling more effective targeting of p53. Analysis of human cancers revealed that SIX1 expression correlated with reduced RPL26 across cancers, suggesting that SIX1 regulates p53 expression in patients (Towers et al., 2015). Expression of p53 also limits stem cell expansion via inhibiting selfrenewal, symmetric cell divisions and de-differentiation of somatic cells into more primitive stem-progenitor cells primed for transformation and cancer development (Bonizzi, Cicalese, Insinga, & Pelicci, 2012; Tschaharganeh et al., 2014). These p53 functions are consistent with increased stem cells numbers and tumorigenesis observed following enforced SIX1 expression in mouse mammary tissue (McCoy et al., 2009), suggesting that reduced p53 may contribute to multiple aspects of SIX1 function in cancer. Retinoblastoma protein (pRB) limits cell proliferation by repressing expression of E2F transcription factor target genes required for the G1/S transition (Dyson, 2016). Expression of endogenous SIX1 is cell cycleregulated, becoming detectable near the G1/S boundary and increasing until mitosis. SIX1 is then phosphorylated by casein kinase 2 (CK2), leading to loss of DNA binding and degradation by the anaphase promoting complex (APC) (Ford et al., 2000; Sun et al., 2013). Consistent with its expression pattern, SIX1 is a direct target of E2F transcription factor 1 (E2F1) (Young, Nagarajan, & Longmore, 2003). Observations that manipulating SIX1 expression regulates cancer cell proliferation, suggest that SIX1 is a critical E2F1 target for determining entry into S phase and that SIX1 overexpression may enable cancer cells to bypass pRb growth-inhibitory signaling.

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TGFβ signaling exerts an antiproliferative function that can suppress cancer cell proliferation and survival (Siegel & Massague, 2003). Inactivating mutations and loss of heterozygosity in transforming growth factor beta receptor 2 (TGFBR2) and SMAD4 are common in multiple cancers, enabling cancer cells to bypass the tumor suppressor function of TGFβ signaling (Ikushima & Miyazono, 2010). As discussed above, SIX1 can co-opt TGFβ signaling to stimulate EMT in multiple types of cancer. By diverting TGFβ signaling to EMT, SIX1 enables cancer cells to evade TGFβ growth suppression and progress to a more invasive and aggressive cancer.

6. Enabling replicative immortality Highly proliferative CSCs must also acquire characteristics consistent with replicative immortality. Pre-neoplastic senescence serves as a barrier tumor progression. Senescent cells are metabolically active, but remain irreversibly arrested in G1 (Kuilman, Michaloglou, Mooi, & Peeper, 2010; Lee & Lee, 2014). In non-malignant human fibroblast IMR90 cells, SIX1 was downregulated by multiple senescence inducing stimuli, including RAS oncogene expression and DNA damage (Adrados et al., 2016). SIX1 was not downregulated in quiescent IMR90 cells, demonstrating that loss of SIX1 was associated with senescence and not loss of proliferation. Furthermore, SIX1 depletion was sufficient to trigger senescence. Loss of SIX1 increased expression of cyclin dependent kinase inhibitor 2A (p16), a key mediator of senescence cell cycle arrest, and blocking p16 blunted the induction of senescent phenotypes (Adrados et al., 2016). Enforced SIX1 expression also inhibited the ability of RAS to induce p16 expression and partially reverted RAS-mediated senescent arrest. SIX1-mediated repression of p16 provides a mechanism for cancer cells to escape senescence and enhance cancer formation in CSC functional assays.

7. Resisting cell death Cancer cells establish multiple mechanisms to avoid cell intrinsic and extrinsic apoptosis, including removal of key sensors, such as p53, increased levels of pro-survival signals and anti-apoptotic effectors, as well as reduced levels of pro-apoptotic factors (Hanahan & Weinberg, 2011). Successful metastasis of solid cancers requires the ability to overcome anoikis, a form of apoptosis triggered when cells lose attachment to an extracellular matrix (ECM) or neighboring cells (Kim, Koo, Sung, Yun, & Kim, 2012).

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CSCs have been proposed to underlie metastasis, therapy resistance and disease recurrence (Peitzsch, Tyutyunnykova, Pantel, & Dubrovska, 2017). CSCs can possess multiple intrinsic properties that enhance their resistance to therapy, including increased reactive oxygen species (ROS) scavenging capacity, high levels of multidrug resistance efflux pumps or increased pro-survival pathways (Cojoc, Mabert, Muders, & Dubrovska, 2015; Nassar & Blanpain, 2016). Extrinsic factors provided by the niche also contribute to therapy resistance by providing paracrine factors or a hypoxic environment that promotes cell survival. During normal development, SIX family members promote stemprogenitor cell survival. Six1 knockout mice exhibit striking phenotypes, including failure of renal organogenesis, associated not only with reduced proliferation, but also increased apoptosis (Li et al., 2003; Xu et al., 2003). Several lines of evidence have demonstrated that SIX1 can inhibit mitochondrial apoptosis, the main cell death pathway activated by hypoxia, chemotherapeutic drugs and ionizing radiation. Experimental overexpression of SIX1 in SAOS2 and U2OS osteosarcoma cell lines increased cell growth and reduced cellular levels of cleaved caspase-3 and caspase-7, whereas siRNA knockdown of SIX1 reduced cell growth and increased levels of cleaved caspase-3 and -7 (Yu et al., 2018). SIX1 knockdown via siRNA in the SGC-7901 gastric cancer cell line reduced colony formation without overt alterations in cell cycle distribution (Du et al., 2017). Instead, SIX1 knockdown increased the number of apoptotic cells via reducing B-cell lymphoma 2 protein (BCL2) expression and mitochondrial membrane potential while increasing caspase-7 expression and activation. Consistent with the ability of SIX1 to oppose mitochondria-mediated apoptosis, SIX1 knockdown resulted in a 10-fold increase in the sensitivity of SGC-7901 cells to 5-fluorouracil (Du et al., 2017). SIX1 increased BCL2 expression and radioresistance in TE-1 esophageal squamous cell carcinoma (ESCC) cells via an AKT signaling-dependent mechanism (He et al., 2017). SIX1 also blocked mitochondria-mediated apoptosis in ovarian cancer cells (OCC) by stimulating mitochondria fission and increasing dynaminrelated protein-1 (DRP1) signaling, without the loss of mitochondrial membrane potential (Yang et al., 2017). Conversely, SIX1 depletion increased mitochondrial fusion and reduced DRP1 signaling. SIX1 expression protected cells from cisplatin- and paclitaxel-induced apoptosis, consistent with previous observations that mitochondria fission in the absence of reduced membrane potential increased resistance to cellular stress (Yang et al., 2017). Regulation of mitochondrial morphology and function therefore likely

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also enhances the ability of SIX1 to promote cancer development and resistance to antineoplastic chemotherapy. SIX1 can also increase resistance to paclitaxel in breast cancer cells and HCC cells. Across 6 breast cancer cell lines with varying SIX1 levels, higher SIX1 levels correlated with increased resistance (Li, Tian, Hu, et al., 2013). Experimental overexpression of SIX1 in cells with low endogenous SIX1 levels increased paclitaxel resistance by two to threefold, whereas SIX1 knockdown in cells with high SIX1 levels reduced paclitaxel resistance (Li, Tian, Hu, et al., 2013). SIX1 expression reduced levels of cleaved caspase-3 and poly [ADP-ribose] polymerase 1 (PARP1) observed following paclitaxel treatment. In the HepG2 HCC cell line, paclitaxel stimulated SIX1 expression, suggesting that SIX1 may (in part) mediate responses to cellular stress induced by paclitaxel. Indeed, SIX1 knockdown increased cell ROS levels and paclitaxel-induced apoptosis (Li, Zhao, Geng, Huo, & Zhang, 2018). Separately, SIX1 overexpression increased and SIX1 knockdown decreased OCC resistance to tumor necrosis factor-related apoptosis by inducing ligand (TRAIL)-mediated apoptosis (Behbakht et al., 2007). As described above, SIX1 overexpression reduces expression of p53 and its target genes, which include mouse double minute 2 homolog (MDM2), an E3 ligase that targets p53 for degradation. Paradoxically, SIX1 overexpression made MCF7 cells 20-fold more resistant to Nutlin-3, an MDM2-targeted agent, by enabling cells to survive MDM2 knockdown by reducing PARP cleavage (Towers et al., 2015). Examination of public data on 451 human cancer lines across cancer types, revealed an 10-fold increase in SIX1 expression in cell lines resistant to Nutlin-3, suggesting that SIX1 can lead to resistance to therapies targeting the p53-MDM2 interaction (Towers et al., 2015).

8. Genome instability and mutation Genomic instability is common in cancers, enabling the accumulation of mutations that enhance cancer progression. Given the capacity of CSCs to drive cancer development and progression, acquisition of pro-cancer mutations in CSCs are strategically poised to generate aggressive cancers with increased resistance to therapy due to increased CSC numbers, tumorigenicity and metastasis. SIX1 overexpression in the MCF7 breast cancer cell line attenuated the damage-induced G2 checkpoint, providing a mechanism by which cells can accumulate unrepaired lesions (Ford, Kabingu, Bump, Mutter, & Pardee, 1998). SIX1-overexpressing cells initially exhibited cell

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cycle profiles similar to controls, but following irradiation, were unable to maintain G2 arrest (Ford et al., 1998). Overexpression of SIX1 in the nonmalignant MCF12A mammary epithelial cell line increased genomic instability, as measured by the occurrence of double-stranded DNA breaks using comet assays, and chromosomal aberrations and anomalies observed in spectral karyotypes (Coletta et al., 2008). High expression of cyclin A1 and cyclin D1, established SIX1 target genes, can reduce DNA repair efficiency and stimulate DNA double strand breaks and chromosome instability, respectively (Casimiro & Pestell, 2012; Muller-Tidow et al., 2004; Shimura et al., 2013).

9. Deregulating cellular energetics The metabolic landscape of cancer cells differs dramatically from normal cells. Central to this altered metabolism is the Warburg effect, a switch to aerobic glycolysis fueled by increased glucose uptake, although it is now recognized that aerobic and anaerobic metabolism are not mutually exclusive in cancer cells (Wolpaw & Dang, 2018). Aerobic glycolysis occurs in response to rapid high demand for ATP, and cancer cells co-opt this mechanism for their advantage. The exact reason(s) for the Warburg effect is not clear, but several hypotheses, including rapid requirement for ATP, high biosynthetic demand, and direct signaling via ROS and chromatin modifications have recently been reviewed by Liberti and Locasale (Liberti & Locasale, 2016). CSCs exhibit metabolic plasticity, utilizing glycolysis and/or oxidative phosphorylation for energy production (De Francesco, Sotgia, & Lisanti, 2018; Snyder, Reed-Newman, Arnold, Thomas, & Anant, 2018). CSCs also derive energy from fatty acid oxidation and exhibit increased utilization of metabolites such as glutamine and glutamate. Metabolic flexibility enables CSC to adapt to local their microenvironment and contributes to therapy resistance. The concept of “metabostemness” derives from our emerging understanding of the role cellular metabolism plays in regulating self-renewal and pluripotency (Menendez & Alarcon, 2014). Recently, SIX1 was shown to regulate the Warburg Effect at the level of transcription (Li, Liang, et al., 2018). Stable SIX1 knockdown in the ZR75-1 breast cancer cell line decreased, whereas overexpression increased expression of a panel of glycolysis-related genes, including glucose transporter 1 (GLUT1), phosphoglycerate kinase 1 (PGK1) and lactate dehydrogenase A (LDHA), by stimulating hypoxia-inducible factor 1-alpha

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(HIF-1α) expression (Li, Liang, et al., 2018). SIX1 also stimulated glycolysis-related genes, independently of HIF-1α. In this case, direct SIX1 binding to the promoters and recruitment of histone acetyltransferases lysine acetyltransferase 7 (HBO1) and amplified in breast cancer 1 (AIB1) mediated transcriptional activation of the glycolytic genes. Li and colleagues further showed that SIX1 was targeted by miR-548a-3p and implicated miR-548a-3p/SIX1 axis as determinant of glycolytic gene expression and corresponding phenotypes, including glucose uptake, pyruvate levels, lactate production and ATP levels in multiple cell types (Li, Liang, et al., 2018). Importantly, aerobic glycolysis was necessary for SIX1 stimulation of cell proliferation in vitro and tumor growth in vivo. In patient samples, SIX1 expression correlated with LDHA and PGK1, and HIF-1α levels, but inversely correlated with miR-548a-3p levels. Furthermore, miR-548a-3p levels are reduced in breast cancer and negatively correlated with tumor size (Li, Liang, et al., 2018). Although most studies report increased levels of SIX1, an oncogenic SIX1 (SIX1Q177R) mutation present in 10% of pediatric Wilms tumors which modulates SIX1 DNA interactions (Walz et al., 2015; Wegert et al., 2015), results in a gain-offunction phenotype with further enhanced stimulation of glycolytic genes (Li, Liang, et al., 2018).

10. Inducing angiogenesis Development of tumor-associated neovasculature enables the acquisition of nutrients and removal of cellular waste products necessary to support tumor growth. CSCs can reside in a perivascular niche, in which endothelial cells secrete factors to maintain CSC properties (Calabrese et al., 2007). CSCs can be an important source of angiogenic factors, as demonstrated for glioma CSCs that secrete VEGF (Bao et al., 2006; Li et al., 2009). SIX1 regulates the expression of genes, such as vascular endothelial growth factor (VEGF), lysyl oxidase (LOX) and matrix metalloproteinases (MMP9), involved in promoting tumor stroma remodeling needed to support tumor cancer growth and escape during metastasis (He et al., 2017; Liu, Li, et al., 2014; Wang et al., 2012; Xie et al., 2018; Xu, Zhang, et al., 2017). Analysis of tumors derived from SIX1-expressing MC38 CRC tumors revealed increased numbers of vessels within the tumor mass, compared to controls (Xu, Zhang, et al., 2017). CD31 and alpha-smooth muscle actin (α-SMA) staining confirmed the presence of endothelial cells and pericytes in the vessels, consistent with

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SIX1-mediated stimulation of tumor angiogenesis. SIX1 overexpression in MC38 adenocarcinoma cells recruited tumor-associated macrophages (TAM) by increasing expression of macrophage-specific colony-stimulating factor-1 (CSF-1), chemokine C–C motif ligand 2/5 (CCL2/5) and VEGF (Xu, Zhang, et al., 2017). VEGF is a potent angiogenic factor that induces endothelial cell proliferation and migration to promote tumor angiogenesis. In papillomas, VEGF has also been shown to expand CSC numbers by promoting CSC symmetric self-renewing cell division (Beck et al., 2011). Similarly, VEGF signaling increased HCC CSC numbers and self-renewal capacity, consistent with observations that increased VEGF is associated with early recurrence in HCC patients (Liu, Hao, Ouyang, Zheng, & Chen, 2017). In CRC, infiltrating immune cells and cytokines are important for tumor initiation, growth, progression and metastasis. In the MC38 subcutaneous CRC model, no differences were observed in white blood cell (WBC) counts or circulating pro-inflammatory cytokines (IL-1β, IL-6, TNFα) between mice with control vs SIX1 overexpressing cancers (Xu, Zhang, et al., 2017). Comparison of infiltrating cells, however, revealed a threefold increase in macrophages and mild decreased in neutrophils in SIX1 vs control cancers. Staining revealed co-localization with VEGF+ cells, suggesting that SIX1 overexpressing cancers recruited TAM, which in turn increased angiogenesis. Cancers overexpressing SIX1 exhibited elevated levels of chemokine (C-X-C motif ) ligand 1 (CXCL1) mRNA, but reduced CSF-2 and CSF-3 levels, consistent with increased macrophage and reduced neutrophil recruitment. Increased HIF-1α was also observed in response to SIX1 expression. In human CRC cells, SIX1 similarly stimulated CCK2 and CCL5, which recruit macrophages.

11. Regulation of SIX1 expression SIX1 can be (re-)expressed in malignancies via transcriptional and post-transcriptional mechanisms. In breast cancer, amplification of the SIX1 gene has been reported (Reichenberger, Coletta, Schulte, VarellaGarcia, & Ford, 2005). In leukemias, where SIX1 function has not been well described, SIX1 and EYA1 are overexpressed in mixed-lineage leukemia (MLL)-rearranged leukemias and have been shown to be transcriptional targets of MLL-ENL fusion proteins in mouse AML models, implicating SIX/EYA function in leukemic transformation involving MLL (KMT2A)

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gene rearrangements (Wang et al., 2011). SIX1 was also stimulated following inducible expression of the MLL-AF9 oncogenic fusion protein in mouse granulocyte-macrophage progenitors (Stavropoulou et al., 2016). Hypoxia and HIF-1α can increase SIX1 expression, which reciprocally stimulates HIF-1α expression under both normoxic and hypoxic conditions, thereby creating a positive feedback regulatory loop (Xu, Zhang, et al., 2017). Multiple tumor suppressor miRs target SIX1 to exert anti-tumorigenic effects (Table 1). MiR-548 inhibits aerobic glycolysis by targeting SIX1 in breast cancer (Li, Liang, et al., 2018). MiR-204 targets SIX1 in non-small cell lung cancer (NSCLC), and SIX1 expression, tumor size and presence of metastases negatively correlated with miR-204 expression (Xia et al., 2014). In HCC, miR-204-5p was downregulated compared to non-tumor Table 1 miRNAs that target SIX, EYA, and DACH. Family Gene miR Cancer

References

SIX

miR-23b-3p Osteosarcoma

Liu et al. (2018)

miR-30a

Prostate cancer

Zhu, Li, Li, and Jiang (2017)

miR-30b

Colorectal cancer

Zhao, Xu, Qin, Gao, and Gao (2014)

miR-185

Breast cancer, ovarian cancer, hepatocellular carcinoma, rhabdomyosarcoma, Wilms tumor

Imam et al. (2010)

miR-188

Oral squamous cell carcinoma

Wang and Liu (2016)

miR-204

Non-small cell lung cancer

Xia et al. (2014)

SIX1

SIX2

miR-204-5p Hepatocellular carcinoma Breast cancer (EMT)

Chu et al. (2018) Zeng et al. (2016)

miR-362

Cervical cancer

Shi and Zhang (2017)

miR-488

Ovarian cancer

Yang et al. (2017)

miR-548-3p Breast cancer

Li, Liang, et al. (2018)

miR-185

Zhu et al. (2016)

Hepatocellular carcinoma

Continued

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Table 1 miRNAs that target SIX, EYA, and DACH.—cont’d Family Gene miR Cancer

References

EYA

EYA1

miR-562

Sporadic Wilms tumor

Drake et al. (2009)

EYA2

miR-30a

Lung adenocarcinoma Breast cancer

Yuan et al. (2016) Fu et al. (2014)

miR-338-3p Breast cancer (pulmonary Liang et al. (2017) metastasis)

EYA3

miR-375

Cervical carcinoma

Bierkens et al. (2013)

miR-708

Ewing sarcoma

Robin et al. (2012)

Pancreatic ductal adenocarcinoma

Zhang et al. (2014)

miR-217

Breast cancer

Zhang, Yuan, Cui, Xiao, and Jiang (2015)

miR-552

Colorectal cancer

Cao et al. (2017)

DACH DACH1 miR-194

controls and low miR-204 levels predicted poor patient outcomes (Chu et al., 2018). Ectopic re-expression of miR-204-5p reduced HCC cell line proliferation and clonogenicity with SIX1 being a direct target. MiR-2045p also suppressed EMT in breast cancer cell lines by inhibiting SIX1 expression (Zeng et al., 2016). Conversely, SIX1 inhibited miR-204 expression, suggesting that balance of SIX1/miR-204 regulates breast cancer migration and invasiveness (Zeng et al., 2016). An inverse relationship between SIX1 and miR-185 levels was observed in ovarian cancer, Wilms tumor and breast cancer cell lines (Imam et al., 2010). MiR-185 exerted a tumor suppressive function, reducing tumor growth and cell migration and resistance to TRAIL-mediated apoptosis, all features of SIX1, which miR-185 was shown to directly target. In ovarian cancer, SIX1 levels negatively correlated with levels of the tumor suppressor miR-488 which was shown directly SIX1 (Yang et al., 2017). MiR-30 suppresses breast and prostate cancer cell proliferation as well as migration and invasiveness of colorectal, prostate and breast cancer cells via targeting SIX1 and EYA2 (Fu et al., 2014; Zhao et al., 2014; Zhu et al., 2017). MiR-362 targeted SIX1 to block cell proliferation, migration and invasion of cervical cancer cell lines (Shi & Zhang, 2017). In cervical cancer, miR-362 was downregulated and inversely correlated with SIX1 (Shi & Zhang, 2017), suggesting an important role for the miR-362/SIX1 axis in cervical cancer progression.

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12. SIX1 clinical significance SIX1 expression is associated with increased aggressiveness and poor patient outcomes in multiple cancers, consistent with its ability to increase CSC numbers and induce EMT. High SIX1 levels were prevalent in prostate cancer (Zeng et al., 2015), gastric adenocaricnomas (Du et al., 2017; Lv et al., 2014), cervical cancer (Zheng et al., 2010), colorectal cancer (Xu, Zhang, et al., 2017), gliomas (Zhang & Xu, 2017), ovarian cancer (Qamar et al., 2012), hepatocellular carcinoma (Kong et al., 2014), pancreatic ductal adenocarcinoma (Lerbs et al., 2017), esophageal squamous cell carcinoma (Nishimura et al., 2017), and osteosarcoma (Chao, Liu, & Zhao, 2017) and correlated with increased metastasis and reduced diseasefree and overall survival, frequently with independent prognostic value. SIX1 has been most extensively studied in breast cancer, where it is expressed in 50% of primary tumors and 90% of metastatic lesions (Reichenberger et al., 2005). In contrast, SIX1 is absent or expressed at very low levels in normal mammary tissue. High SIX1 expression was associated with reduced disease-free and overall survival (Micalizzi et al., 2009). The effect of SIX1 is particularly striking in luminal breast cancer, particularly the B subtype, which is comprised of highly aggressive and tamoxifen-refractory breast cancers (Iwanaga et al., 2012). Analysis of publically available data from 3555 patients across 20 published gene expression omnibus (GEO) datasets confirmed that SIX1 overexpression was associated with shorter time to relapse and reduced overall survival, especially in luminal breast cancer patients (Xu et al., 2016). High SIX2 expression was also associated with shorter time to relapse and metastasis (Fig. 3). In contrast, high SIX3 expression was found to be a potentially protective factor for overall survival and relapse-free survival for patients with basal-like breast cancer (Fig. 3). SIX1 has also been implicated in lung cancer. SIX1 mRNA was increased 15-fold in 71% of human immunodeficiency virus (HIV)-associated lung cancer samples (Zheng, Wang, et al., 2018). In phyllodes tumor, a biphasic tumor comprised of stromal and epithelial components, SIX1 protein expression in the cytoplasm of stromal cells was associated with increased tumor size, hypercellularity, increased stromal mitotic activity, stromal outgrowth and shorter time to recurrence (Tan, Thike, Bay, & Tan, 2014). SIX1 upregulation was also implicated in progression of lung adenocarcinoma invasiveness (Mimae et al., 2012). In contrast, SIX3 expression in adenocarcinoma

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Fig. 3 The schematic diagram showing the correlation between patient prognosis and the expression levels of SIX, EYA, and DACH in solid cancers. Individual PSEDN members exert either promote or antagonize cancer aggressiveness and recurrence. Altered expression of PSEDN members frequently predicts patient outcomes. High expression of most, but not all, SIX and EYA family members is associated with poor clinical outcomes, including reduced overall survival. In contrast, high expression of DACH1, SIX3 or EYA4, which function as tumor suppressors in solid cancers, correlated with more favorable patient outcomes, including prolonged survival.

patients (and adenocarcinoma patients with bronchioalveolar carcinoma features) correlated with improved overall survival and progression-free survival, consistent with a protective effect. Enforced SIX3 expression in NSCLC cell lines H322 and H1299 (in which endogenous SIX3 was repressed by promoter hypermethylation) reduced cell proliferation, colony formation and migration in vitro (Mo et al., 2013), suggesting that SIX3 function antagonizes key cancer-promoting cell phenotypes. These findings reiterate the need to understand the SIX3 function in positive cancer outcomes. It will be interesting to know whether the inhibitory action of SIX3 reflects its previously reported inability to translocate EYA1 into the nucleus (Ohto et al., 1999). In gliomas, which account for 80% of all malignant brain tumors, SIX1 and its cofactor EYA1 were identified as part of a set of 8 “core” genes highly (>10-fold) upregulated in A2B5+ glioblastoma progenitor cells (Auvergne et al., 2013). SIX1 protein was observed in 49% of glioma tissues samples, with increasing prevalence in high-grade vs low-grade samples (Zhang & Xu, 2017). High SIX1 expression levels correlated with shorter patient survival times across malignancy grades, and multivariate analysis indicated that SIX1 level was an independent prognostic factor (Zhang & Xu, 2017). Again, SIX3 exhibited tumor suppressive functions, reducing astrocytoma cell proliferation and invasiveness in vitro as well as tumor growth in vivo. SIX3 post-translationally increased p53 expression via repressing expression of aurora kinase A (AURKA) and aurora kinase B (AURKB),

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which can directly phosphorylate p53 and enhance its degradation via MDM2-mediated ubiquitination (Yu et al., 2017). SIX3 overexpression downregulated E2F1, triggering G1/S cell cycle arrest (Yu et al., 2017). Thus, SIX1 and SIX3 have antagonistic roles in cell cycle regulation. SIX3 overexpression also reduced the number of cells exhibiting extra centrosomes and misaligned metaphase chromosomes, suggesting that SIX3 reduces chromosome mis-segregation and aneuploidy (Yu et al., 2017). High SIX3 also reduced the sensitivity of cells to aurora kinase inhibitors, potentially providing a biomarker for clinical trials. SIX1 has also been implicated in hematologic malignancies. Increased levels of SIX1 were associated with advanced stage B cell lymphomas and failure to achieve remission in childhood acute lymphoblastic leukemias (ALL) (Micalizzi et al., 2009). SIX1 is also overexpressed in Hodgkins lymphoma, a B cell malignancy, where evidence suggests that SIX1 exerts positive and negative regulation on downstream transcription factors to coordinately block differentiation of T-cell and B-cell lineages (Nagel, Meyer, Kaufmann, Drexler, & MacLeod, 2015). SIX1 and its co-factor EYA1 are among a set of only 12 MLL-ENL bound genes upregulated in mixed lineage leukemia mouse models and overexpressed in human leukemias (Wang et al., 2011), implicating EYA1 and SIX1 in mixed lineage leukemia rearrangements (MLLr) acute leukemia development.

13. EYA EYA proteins are direct SIX-binding partners and function as SIX transcriptional co-activators. EYA proteins also possess an EYA domain (ED), which has homology to the haloacid dehydrogenase (HAD) superfamily and has tyrosine phosphatase activity (Fig. 1) (Tootle et al., 2003). Thus, EYA proteins are multifunctional and contribute to CSC biology and cancer progression via transcriptional and non-transcriptional mechanisms (Blevins et al., 2015; Zhou, Zhang, Vartuli, Ford, & Zhao, 2018). EYA1 overexpression promotes breast cancer cell growth via increased cell proliferation and reduced apoptosis (Wu et al., 2013), whereas EYA1 knockdown sensitizes breast cancer cells to irradiation (Sun, Kaneko, Li, & Li, 2015). EYA proteins promote SIX1 oncogenic function, as demonstrated by the requirement for SIX1-EYA interaction to promote breast cancer metastasis (Patrick et al., 2013). EYA2 knockdown in MCF7 cells blocked ability of SIX1 overexpression to induce TGFβ signaling, EMT and CSC

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phenotypes (Farabaugh, Micalizzi, Jedlicka, Zhao, & Ford, 2012), stimulating efforts to develop therapeutic strategies based on targeting the SIX1-EYA protein-protein interaction. Beyond breast cancer, gain- and loss-of-function studies have demonstrated that EYA2 stimulates glioma cell proliferation, cell cycle progression and cyclin D expression (Wen, Liang, Pan, & Wang, 2017). EYA2 also increases cell invasiveness via upregulating MMP9 expression in a SIX1dependent manner. Knockdown of EYA2 in A549 NSCLC cells reduced cell proliferation, migration and invasiveness and increased apoptosis (Gao et al., 2015). EYA3 is highly expressed in Ewing sarcoma and its knockdown increased the number of apoptotic cells, reduced efficiency of DNA repair and sensitized cells to DNA damaging chemotherapeutic agents (Robin et al., 2012). The EWS-FLI1 fusion protein, responsible for Ewings sarcoma, increases EYA3 expression via repressing expression of miR-708, which directly targets EYA3 (Robin et al., 2012). EYA phosphatase activity contributes to cancer development and progression via multiple mechanisms. EYA1 phosphatase activity is required to build a transcriptional complex that activates CCND1 transcription to drive breast cancer cell proliferation (Wu et al., 2013). EYA2 dephosphorylation of estrogen receptor beta (ERβ) inhibits its transcriptional activity thereby blocking the antiproliferative and anti-tumorigenic activity of ERβ (Yuan et al., 2014). Knockdown of EYA3 in MDA-MB-231 cells reduced metastasis, which could be rescued by wild type but not tyrosine phosphatase mutant EYA3. EYA-mediated dephosphorylation of H2AX Y142 is necessary for the DNA damage response (DDR) and cell survival under hypoxic conditions (Cook et al., 2009). Enhancement of DNA repair may provide an additional mechanism by which EYA proteins stimulate therapy resistance. Interestingly, EYA phosphatase activity can also contribute to cancer progression in non-tumor cells. Using conditional EYA3 knockout mice, Wang et al. recently showed that EYA3 expressed in endothelial cells is necessary for tumor vascularization (Wang et al., 2018). The ability of EYA3 to stimulate angiogenesis was dependent upon tyrosine phosphatase activity, consistent with previous data demonstrating EYA phosphatase inhibition reduced endothelial cell motility and tubulogenesis in vitro (Tadjuidje et al., 2012). The requirement for EYA phosphatase in promoting cancer phenotypes and CSC resistance to chemotherapy has stimulated efforts to identify small molecule inhibitors of EYA phosphatase activity for anti-cancer therapeutic approaches (Krueger et al., 2013, 2014).

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EYA proteins also promote dephosphorylation of MYC on threonine 58, thereby increasing MYC stability and oncogenic capacity (Cook et al., 2009; Krishnan et al., 2009). Initially, EYA proteins were proposed to possess threonine phosphatase activity (Li et al., 2017; Okabe, Sano, & Nagata, 2009; Sano & Nagata, 2011). More recently, it was shown that EYA3 lacks intrinsic threonine phosphatase activity, but instead orchestrates MYC dephosphorylation via interaction with protein phosphatase 2 (PP2A), a serine/threonine protein phosphatase with well-known links to cancer. EYA3 interaction with PP2A leads to c-MYC dephosphorylation and stabilization, stimulating tumor progression (Zhang et al., 2018). In immune competent mouse models of triple-negative breast cancer, EYA3 supported tumor growth via suppression of the anti-cancer immune response (Vartuli et al., 2018). EYA3 expression reduced, and knockdown increased, the number of infiltrating CD8+ T cells. The effect of EYA3 was mediated by MYC-dependent upregulation of programmed death-ligand 1 (PD-L1), and EYA3 levels correlated with PD-L1 in human breast tumors (Vartuli et al., 2018). Cytoplasmic function of EYA3 phosphatase has also been shown to regulate cancer cell phenotypes. Targeting EYA3 to the nucleus using a C-terminal nuclear localization signal reduced the ability of EYA3 to stimulate cell motility (Pandey et al., 2010). Overexpression of EYA3 harboring a point mutation that disrupts tyrosine phosphatase activity in MCF7 cells blocked the ability of EYAs to increase motility and invasiveness of breast cancer cells in vitro. In contrast, increased cell proliferation was not dependent upon tyrosine phosphatase activity. EYA3 phosphatase activity supported cell motility by reducing cellular actin stress fibers via modulation of RAC, CDC42 and RHO-GTP (Pandey et al., 2010). Full understanding of EYA proteins, including their cytoplasmic functions in normal and malignant development, will require identification of additional EYA phosphatase targets. Enforced expression of EYA1 transformed primary mouse hematopoietic stem-progenitor cells (HSPCs), as assessed by in vitro colony serial replating assays (Wang et al., 2011). Co-expression of EYA1 and SIX1 resulted in more robust colony formation, suggesting SIX1 enhanced the capacity of EYA1. These observations may reflect a requirement for SIXEYA interaction, in cells where EYA, but not SIX1, is expressed in limiting amounts. EYA2 is a target of promyelocytic leukemia zinc finger (PLZF), and EYA2 immortalized mouse HSPCs in serial replating assays and conferred a leukemia stem cell gene program signature (Ono, Masuya, Ishii, Katayama, & Nosaka, 2017). Interaction of EYA2 with SIX, but

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not the tyrosine phosphatase activity of EYA2, was required for EYA2 immortalization of mouse HSPCs. EYA2 knockdown also reduced the clonogenicity of HSPCs transformed with PLZF-retinoic acid receptor alpha (RARA) fusion protein responsible for acute promyelocytic leukemia (APL), consistent with EYA2 being required for PLZF-RARA induced self-renewal (Ono et al., 2017). The function of SIX and EYA factors in normal and malignant hematopoiesis remain to be defined. In contrast to other EYA family members, EYA4 functions as a tumor suppressor in multiple cancers, including colon (Kim et al., 2015), liver (Mo et al., 2018), esophagus (Luo et al., 2018), lung (Wilson et al., 2014), mouth (Towle, Truong, & Garnis, 2016), and peripheral nerves (Miller et al., 2010). EYA4 is downregulated in nearly 80% of esophageal squamous cell carcinomas (ESCC) (Luo et al., 2018). EYA4 knockdown in ESCC cell lines increased EMT phenotypes, whereas EYA4 overexpression reduced cell migration and invasion capacity. EYA4 suppressed EMT via inhibition of p-AKT/GSK3β/slug signaling. Conversely, TGFβ signaling, which induces EMT in ESCC cell lines, inhibited EYA4 expression (Luo et al., 2018). A notable exception to EYA4 serving a tumor suppressive role was observed in glioma, where elevated EYA4 predicted reduced patient survival. Consistent with an oncogenic role, EYA4 overexpression in U87MG malignant glioma cells increased cell proliferation via SIX1-dependent repression of cyclin-dependent kinase inhibitor 1B (CDKN1B), the gene encoding cell cycle inhibitor CDKN1B (p27KIP1) (Li, Qiu, Qiu, & Tian, 2018).

14. DACH DACH family members are often downregulated in cancers, suggesting that they function as tumor suppressors. Reduced DACH1 expression is observed in brain (Wang et al., 2018), breast (Wu et al., 2006; Xu et al., 2017), liver (Liu et al., 2015), prostate (Wu et al., 2009) and uterine (Nan et al., 2009) cancers, where it correlates with poor clinical outcomes and increased metastasis. Breast cancer patients retaining higher DACH1 expression had prolonged disease-free and overall survival (Xu, Yu, et al., 2017). In breast cancer cells, DACH1 opposed SIX1 action, via transcriptional repression of common targets, including CCND1 and IL8 (interleukin 8) to inhibit cell proliferation and migration in vitro, as well as tumor growth and metastases in vivo (Popov et al., 2010; Wu et al., 2008, 2006, 2007, 2003). Expression of DACH1 reduced the frequency of phenotypic

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(CD44hi/CD24low) CSCs and functional TICs in breast cancers in vitro and in vivo. DACH1 repressed by mechanisms that include direct repression of NANOG, SOX2 and LIN28 expression (Wu et al., 2011; Xu, Yu, et al., 2017). Conversely, DACH1 knockdown increased functional CSCs. DACH1 also binds to and inhibits the activity of Y-box binding protein 1 (YB-1), an oncogenic factor that mediates transcriptional and post-transcriptional control of tumor cell proliferation and EMT (Wu, Chen, et al., 2014). Additionally, DACH1 inhibits YB-1-mediated cap independent translation of key EMT inducers, including Snail, by depleting the cytoplasmic pool of YB-1 via nuclear accumulation. DACH1 also restrained prostate cancer via repression of genes required for contact-independent growth (cyclin A1, A2 and E1) and cell migration (IL8, IL6) (Chen et al., 2015). In CRC, DACH1 is targeted by oncogenic miR552, to promote expression of CCND1 and c-MYC (Cao et al., 2017). DACH1 expression reduced HCC proliferation, tumorigenesis and increased cell death via increasing p53 expression (Cheng et al., 2018). DACH1 inhibits cell growth and migration in part via inhibition of Wnt/β-catenin signaling (Liu et al., 2015; Wang, Zou, et al., 2018). Homozygous deletion of 13q21, which contains DACH1, has been observed in gliomas, and enforced DACH1 expression inhibits glioblastoma cell line growth in vitro and tumorigenicity in vivo (Watanabe et al., 2011). DACH1 inhibited glioma CSC numbers by direct repression of fibroblast growth factor 2 (FGF2) gene expression (Watanabe et al., 2011). The regulation of cytokines by DACH1 has recently been reviewed (Zheng, Liu, Yi, Qin, & Wu, 2018). Ectopic expression of DACH1 in gastric and hepatocellular cancer cells, which lack endogenous DACH1 due to DACH1 promoter hypermethylation, inhibited cancer phenotypes and increased sensitivity to chemotherapeutic agents (Yan et al., 2014; Zhu et al., 2013). DACH1 suppressed esophageal cancer growth via activating TGFβ signaling (Wu, Herman, et al., 2014). In contrast, DACH1 inhibited TGFβ signaling in breast and pancreatic cancer cells, blocking the induction of TGFβ-induced apoptosis and TGFβ-induced Smad- and activator protein 1 (AP-1)-mediated changes in gene expression (Wu et al., 2003). Thus, as is well-established for TGFβ signaling (Ikushima & Miyazono, 2010), DACH1 may offer multiple opposing roles in tumor development and cancer progression. In contrast to its well-established role as a tumor suppressor in multiple solid cancers, DACH1 appears to function as an oncogene in hematologic

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malignancies. DACH1 expression is increased in myeloid leukemias, and is directly upregulated by the MLL-AF9 oncogenic fusion protein that drives aggressive myelo-monoblastic AML (Lee, Kim, Hwang, et al., 2012). Consistent with this phenotype, DACH1 increased the self-renewal capacity of primary mouse bone marrow progenitor cells as assessed by in vitro serial replating assays (Lee, Kim, Hwang, et al., 2012). Expression of DACH1 in myeloid progenitors increased cyclin D and CDKN1C (p57KIP2) expression, whereas DACH1 knockdown in HL-60 promyelocytic leukemia cells reduced cyclinD/CDK4/6 levels and blocked cell cycle progression (Lee, Kim, Kim, et al., 2012). Furthermore, in AML cells, DACH1 binds to and activate homeobox protein HOXA9 (Lee, Kim, Hwang, et al., 2012), involved in stem cell expansion and leukemias (Collins & Hess, 2016). The pro-leukemic roles of DACH1 are consistent with its previously proposed function in hematopoietic stem cell (HSC) self-renewal based on findings that DACH1 mRNA is selectively expressed in murine long-term HSCs as opposed to short term HSCs or multipotent progenitors (Forsberg et al., 2005). The divergent roles of DACH1 in distinct cancers likely reflect a unique role of the PSEDN in specific cell lineages and highlight the importance of cell context-specific molecular interactions in cancer development and progression.

15. Concluding remarks An increasing body of evidence points to a pivotal role for PSEDN members in regulation of cancer stem cell genesis and the emergence of cancer hallmarks and enabling characteristics. Compilation of available data suggests that the PSEDN functions as a central hub in cancer biology, with the output of SIX-EYA vs DACH determining cancer development, progression and aggressiveness. More detailed understanding of the mechanisms regulating SIX-EYA-DACH expression, sub-cellular localization and functional interactions is needed to fully understand the multiple mechanisms by which this conserved transcriptional network regulates fundamental cellular processes that can drive CSC phenotypes and functional capacity. In particular, efforts to determine the mechanisms by which certain SIX and EYA family members exert oncogenic vs tumor suppressive functions may provide insight into specific protein interactions to target for future therapeutic strategies.

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Acknowledgments Support for our research has been provided by the Maryland Stem Cell Center Research Fund (Discovery Award to T.J.K.), the Mark Foundation for Cancer Research (Momentum Award to T.J.K.), and the American Cancer Society (Institutional Research Grant subaward to M.J.K.).

Conflict of interest The authors declare that they have no competing financial interests.

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