The sine oculis (SIX) homeobox family of transcription factors play important developmental roles in a wide range of species from fruit flies to humans. The founding member, sine oculis (so), was first identified in Drosophila melanogaster where it was discovered to be required for compound eye formation (Cheyette et al., 1994; Serikaku and O’Tousa, 1994). Subsequent research in fruit flies identified two additional SIX genes, optix, and DSix4 (reviewed in Kawakami et al., 2000). All three transcription factors were found to share a conserved N-terminal SIX domain adjacent to a homeodomain (HD), which function as protein-protein and DNA binding domains, respectively (Figure 1; Kawakami et al., 2000). Gene duplication during evolution expanded the SIX family of genes and created three subfamilies in vertebrates, which are composed of the so subfamily (Six1 and Six2), the optix subfamily (Six3 and Six6), and the DSix4 subfamily (Six4 and Six5). The vertebrate orthologs contain the same SIX and HD domains as the ancestral Drosophila proteins with significant amino acid sequence identity between the functional domains of family members. For example, the mouse SIX domain amino acid identity ranges from 63 to 93% while sequence identity in the HD ranges from 59 to 98% between family members, with Six3, Six4, and Six5 showing the most divergence. For a more complete review of SIX family protein structure and sequence comparisons see Kawakami et al. (2000) and Kumar (2009).

In vertebrates, SIX genes play critical roles in tissue formation and organogenesis, such as for the head, ear, retina, nose, brain, skeletal muscle, and kidney (Oliver et al., 1995; Ohto et al., 1998; Jean et al., 1999; Kobayashi et al., 2001; Laclef et al., 2003; Lagutin et al., 2003; Li et al., 2003; Xu et al., 2003; Self et al., 2006). In these tissues, the SIX family of transcription factors function as regulators of progenitor cell maintenance and differentiation. They can act as transcriptional activators or repressors depending on interactions with other highly conserved regulators including Paired-box (Pax), Eyes absent (Eya), Dachshund (Dach), and Groucho (Grg) proteins (see reviews Kawakami et al., 2000; Kumar, 2009). Correlating with their important functions during embryogenesis, several congenital defects in humans are associated with mutations in SIX genes. In cancer, SIX genes have been found to be ectopically- or over-expressed and experimental interrogation suggests SIX proteins can drive disease pathogenesis. This review will focus on recent developments on SIX gene function and regulation in order to provide insight into congenital diseases along with how SIX genes become exploited in the context of cancer formation. Common themes emerge when comparing roles of SIX genes in developmental and cancer biology which relate to progenitor cell maintenance, cell behavior, and regulation; generating new questions and opportunities for research in each field.

Members of the SIX family of transcription factors are widely expressed in the developing vertebrate embryo where they play important regulatory roles in tissue and organ formation. Unsurprisingly, several human congenital conditions have been associated with haploinsufficiency or hypomorphic mutations in SIX genes (Figure 1 and Table 1). Many human SIX gene mutations were located within the SIX domain or homeodomain, which were suggestive of impaired protein-protein interactions or DNA binding. However, mutations have been detected outside of these domains and their functional significance remains unclear (Figure 1). Mutations in human SIX1 and SIX2 have been associated with multiple congenital disorders such as branchio-oto-renal syndrome (BOR), renal dysplasia, hearing loss, and frontonasal dysplasia syndrome (Ruf et al., 2004; Kochhar et al., 2008; Weber et al., 2008; Mosrati et al., 2011; Guan et al., 2016; Hufnagel et al., 2016). BOR is an autosomal dominant disorder that is characterized by a triad of clinical symptoms including branchial arch defects, hearing loss, and renal abnormalities (Melnick et al., 1976). The most commonly identified mutations in BOR are in the SIX1 binding partner EYA1 (Abdelhak et al., 1997), however, mutations have been identified in SIX1 that disrupt DNA binding or impact the ability to form a functional transcriptional complex with EYA1 (Ruf et al., 2004; Patrick et al., 2009). Independent of BOR, dominantly inherited hearing loss has been associated with mutations in both SIX1 and SIX2 (Mosrati et al., 2011; Guan et al., 2016). Additionally, SIX2 mutations were detected in patients with renal hypodysplasia (Weber et al., 2008) and more recently in frontonasal dysplasia syndrome (Hufnagel et al., 2016).

The involvement of SIX1 and SIX2 in human syndromes is supported by functional studies in animal models. Aspects of BOR are evident in mouse knock-out models as Six1^–/– null mice fail to develop a thymus and kidney, in addition to having significant defects in structures of the inner ear and nose (Zheng et al., 2003; Xu et al., 2003; Ruf et al., 2004; Ozaki et al., 2004; Zou et al., 2006). Further analysis has shown Six1 to be required for otic vesicle and cochlea development as well as differentiation of hair cells in the ear (Ozaki et al., 2004; Zhang et al., 2017). Identified human BOR SIX1 mutations expressed in Xenopus embryos disrupted otic vesicle formation and ear morphology, further connecting SIX1 function to BOR phenotypes (Shah et al., 2020). Six2 manipulation in animals models has also been informative in its role during development. For example, Six2 knock-out mice develop renal hypoplasia, where the metaneprhic kidney forms but is significantly smaller than normal (Self et al., 2006; Kobayashi et al., 2008). Mouse Six2 mutants also display features associated with frontonasal dysplasia. Two independent Six2 animal models, a genetic knockout and a mutant from an X-ray irradiation screen (Brachyrrhine), exhibited defects of the cranial base and cleft palate (Self et al., 2006; Fogelgren et al., 2008; He et al., 2010; Sweat et al., 2020). These phenotypes were similar to a dominantly inherited form of frontonasal dysplasia syndrome linked to a chromosomal deletion of SIX2 at 2p21 (Hufnagel et al., 2016). Six1 has also been linked to craniofacial development as disruption of Six1 function leads to micrognathia and defects of the nose, mandible, and midface structures (Ozaki et al., 2004; Guo et al., 2011; Tavares et al., 2017). Furthermore, both so homologs have been shown to participate together in craniofacial development as Six1/Six2 double knockout animals have significantly more severe phenotypes than single mutants, including agenesis of the frontal and parietal bones of the skull (Liu et al., 2019a).

SIX3 and SIX6 of the optix subfamily have been associated with specific congenital defects of the developing forebrain in humans. Mutations in SIX3 result in holoprosencephaly (HPE), a heterogeneous collection of forebrain malformations, with incomplete penetrance due to SIX3 haploinsufficiency or hypomorphic function (Table 1; Wallis et al., 1999; Lacbawan et al., 2009; Hehr et al., 2010; Stokes et al., 2018). Mutations in additional genes are also known to cause HPE including Sonic Hedgehog (SHH) and other members of this signaling pathway (Dubourg et al., 2007). Mutations in SIX3 and SHH have both been implicated in schizencephaly, a syndrome which clinically overlaps with HPE (Hehr et al., 2010). SIX6 has been connected with various eye malformations including anophthalmia and microphthalmia (Gallardo et al., 1999, 2004), primary open angle glaucoma (POAG) (Iglesias et al., 2014; Carnes et al., 2014; Mohanty et al., 2018), and optic disk anomalies and macular atrophy (Yariz et al., 2015). Similar to other family members, mutations such as point mutations and allele deletions in SIX6 were thought to result in haploinsufficiency or hypomorphic function (Figure 1 and Table 1).

In the mammalian embryo, Six3 and Six6 are expressed in derivatives of the anterior neural plate including the hypothalamus, pituitary gland, olfactory placodes, and regions of the developing eye (Oliver et al., 1995; Jean et al., 1999). Functional studies have shown a requirement for Six3 in forebrain development, for example, Six3 knock-out mice do not form telencephalic or optic vesicles (Lagutin et al., 2003). Shh, which has been shown to be involved in forebrain formation and HPE presentations (Shimamura and Rubenstein, 1997; Fuccillo et al., 2004), is directly regulated by Six3 (Jeong et al., 2008; Geng et al., 2008). The Six3-Shh interaction in the anterior neural plate is required to repress Wnt signaling, allowing for proper dorsoventral patterning of the telencephalon (Geng et al., 2008; Jeong et al., 2008; Liu et al., 2010; Carlin et al., 2012). Manipulation of Six3 expression in animal models has also provided insight into the incomplete penetrance observed in familial HPE. Knock-in of human mutations or the creation of variable expressing hypomorphs in mice has modeled both semilobar and alobar HPE with variable penetrance (Geng et al., 2008, 2016). The ultimate result of haploinsufficiency or hypomorphic alleles of SIX3 is reduced SHH signaling and defective forebrain formation. Compared to Six3, Six6 was found to be more restricted during forebrain development with expression in the forming hypothalamus, pituitary, and retina (Jean et al., 1999). Disruption of Six6 function in animal models has led to defects in the visual system ranging from small eye phenotypes in zebrafish to hypoplasia of the neural retina in mice (Li et al., 2002; Iglesias et al., 2014; Carnes et al., 2014; Teotia et al., 2017; Diacou et al., 2018). Conditional knockout of Six3 or Six6 have provided additional insights into the roles of the transcription factors in eye development. Neural retina maintenance and differentiation is dependent upon the function of both Six3 and Six6 (Zhu et al., 2002; Liu et al., 2006, 2010; Manavathi et al., 2007; Samuel et al., 2016; Takata et al., 2017; Liu and Cvekl, 2017) and both genes are required to repress Wnt signaling during eye development (Diacou et al., 2018). Coordinated and overlapping roles for the two transcription factors has also been implicated in the hypothalamus and pituitary gland where they may regulate the neurons that express gonadotropin-releasing hormone and differentiation of the receptive pituitary gonadotropes (Larder et al., 2011; Xie et al., 2015).

Of the DSix4 subfamily members, Six4 and Six5, only SIX5 has thus far been linked to congenital disease in humans (Table 1). Along with SIX1, mutations in SIX5 have been identified in patients with BOR that may impact DNA binding (Figure 1 and Table 1; Hoskins et al., 2007). However, more recent investigations have questioned the causative impact of SIX5 mutations in BOR where either no mutations were detected or mutations in other genes had been identified in addition to mutations in SIX5 (Krug et al., 2011; Wang et al., 2012; Song et al., 2013). Renal malformations are part of the complex BOR phenotype and a screen of 749 patients with congenital anomalies of the kidney and urinary tract (CAKUT) did detect one family with a mutation in SIX5 (Hwang et al., 2014). Further research is needed to more concretely connect SIX5 mutations as a causative factor in BOR and associated renal malformations. Another disease connected to SIX5 expression is Myotonic dystrophy (DM1). DM1 is dominantly inherited and presents with myotonia, muscle wasting, cardiac conduction defects, fertility defects, and cataracts (Harper, 1975). The underlying genetic cause of DM1 is a CTG trinucleotide repeat expansion in the 3′ UTR of the DMPK gene on chromosome 19 (Brook et al., 1992; Fu et al., 1992; Mahadevan et al., 1992). The repeat expansion appears to disrupt the expression of neighboring genes, including SIX5, which has reduced expression in DM1 (Klesert et al., 1997; Thornton et al., 1997).

Investigation into Six5 function in animal models has begun to tease out its role in multigenic DM1. Knock-out mice for Six5, both heterozygous and homozygous animals, develop cataracts with variable penetrance replicating observed DM1 phenotypes (Klesert et al., 2000; Sarkar et al., 2000). Additional studies have shown a requirement for Six5 in spermatogonia viability and spermatozoa differentiation in male mice (Sarkar et al., 2004). Furthermore, cardiac conduction defects have been observed in heterozygous mutant mice (Wakimoto et al., 2002). One of the hallmark features of DM1 is progressive muscle wasting and hypotonia. Thorough interrogation of Six5 function in mouse models does not support a direct role for the transcription factor in muscle phenotypes of DM1 despite expression in the developing myotome and skeletal muscle (Murakami et al., 1998; Klesert et al., 2000; Personius et al., 2005; Matynia et al., 2010). Although, triple and quadruple gene knockout of loci suspected in DM1, including Six5, does result in the array of multisystem defects present in myotonic dystrophy (Yin et al., 2020). Therefore, it appears the independent role of SIX5 in DM1 is limited to the eye, spermatogonia, and cardiac conduction system.

The lack of SIX4 mutations identified in human congenital disease is not surprising based upon studies in animal models. For example, Six4 knock-out mice were found to be viable with no obvious developmental or progressive defects (Ozaki et al., 2001). However, Six4 has been found to cooperate with other SIX family members in several developmental processes. Both Six5 and Six4 were identified to function together during vertebrate body wall development where loss of both genes resulted in omphalocele (Takahashi et al., 2018). Further mouse compound knock-out studies have uncovered Six4 functional cooperation with Six1 during myogenesis (Grifone et al., 2005; Relaix et al., 2013; Wurmser et al., 2020), gonadogenesis (Fujimoto et al., 2013); thymus development (Zou et al., 2006), neurogenesis (Konishi et al., 2006; Chen et al., 2009), and kidney development (Kobayashi et al., 2007; Xu and Xu, 2015). Based upon these studies, it appears Six4 function is compensated by other family members but still plays an important supportive role during embryonic development.

The embryonic phenotypes associated with SIX gene manipulation in animal models or from human mutation greatly correlate with the developmental expression profile of each family member (Table 1; reviewed in Kawakami et al., 2000). However, associations with adult gene expression and disease have not been as clear. Adult expression has been detected in skeletal muscle and satellite progenitor cells for Six1, Six2, Six4, and Six5 (reviewed in Maire et al., 2020). Six1 has been detected in the adult thymus as well as the salivary gland, trachea, and at low levels in the mouse mammary gland (Ford et al., 1998; Coletta et al., 2004; Guo et al., 2011). Six1 expression has also been demonstrated to be induced in differentiated mouse immune cells following infection (Liu et al., 2019b). SIX2 and SIX3 gene expression has been detected in adult pancreatic β-cells (Arda et al., 2016; Bevacqua et al., 2021) while SIX3 and SIX6 were found to be expressed in the adult pituitary (Aijaz et al., 2005; Xie et al., 2015). SIX5 was detected in the epithelium of the Fallopian tube and cervix but not in the ovary or glandular epithelium (Winchester et al., 2000). Most other adult tissues appear to be negative or express very low levels of SIX genes including the lymph nodes, lung, and kidney (Ford et al., 1998; Kobayashi et al., 2008; Guo et al., 2011). The lack of significant expression of SIX transcription factors in adult tissue is of greater consequence in the context of cancer, where SIX genes become ectopically or re-expressed to drive tumorigenesis.

The features of the SIX family of transcription factors that make them critical in the development of specific tissues and organ systems also makes them potentially deleterious when ectopically expressed in adult tissues. Promotion of cell proliferation or migration by SIX proteins ectopically expressed in adult tissues can and often contribute to the formation, survival, and metastasis of a variety of tumor types (Table 2).

The so subfamily (SIX1 and SIX2), particularly SIX1, have been frequently implicated in the promotion, invasion, and survival of a variety of cancers (Blevins et al., 2015). SIX1 alone has been shown to be overexpressed in many forms of cancer such as breast (Ford et al., 1998; Coletta et al., 2004; Iwanaga et al., 2012), ovarian (Behbakht et al., 2007), cervical (Sun et al., 2016), Wilms tumor (Wegert et al., 2015), osteosarcoma (Hua et al., 2014; Chao et al., 2017), rhabdomyosarcoma (Yu et al., 2004), and several others (Table 2). Increased levels of SIX1 gene or protein expression was often found to be strongly correlated with poor prognosis regardless of tumor type (Blevins et al., 2015). SIX2, like SIX1, overexpression was detected in breast cancer (Wang et al., 2014; Oliphant et al., 2019) and appeared to promote increased survival, self-renewal, and metastasis of tumor cells (Table 2). All of these characteristics contribute to poor prognosis and decreased patient survival. SIX2 has been detected in other cancers such as hepatocellular carcinoma (HCC) (Zhu et al., 2016; Li et al., 2018; Wan et al., 2019), non-small cell lung cancer (Hou et al., 2019) and colorectal cancer (Wu et al., 2017). As one might predict, increased levels of SIX2 are highly correlated with cancers involving the kidney. SIX2 overexpression has been identified in the pediatric cancer Wilms tumor (Murphy et al., 2012; Pierce et al., 2014; Walz et al., 2015; Wegert et al., 2015) as well as in renal cell carcinoma (Senanayake et al., 2013; Cheng et al., 2019) and nephroblastoma (Senanayake et al., 2013). Specific point mutations in SIX1 and SIX2 have been detected in Wilms tumor cells where they potentially increase transcriptional activity (mutations denoted in red of Figure 1; Wegert et al., 2015).

Recently, SIX6 has been found to be associated with T-cell acute lymphoblastic leukemia (T-ALL) (Laukkanen et al., 2020), though the researchers of the study concluded that SIX6 most likely belonged to a larger regulatory gene network and increased levels of SIX6 alone were not sufficient to induce development of T-ALL. The study did conclude, however, that higher levels of SIX6 was associated with inferior treatment response and poor prognosis (Laukkanen et al., 2020). Increased SIX6 levels have also been associated with poor overall survival in non-small cell lung carcinoma and breast cancer (Liu et al., 2016; Xu H. X. et al., 2016). In stark contrast to other SIX transcription factors, increased levels of SIX3 appear to play a tumor suppressive role rather than an oncogenic one. Higher levels of SIX3 were associated with decreased tumor proliferation and metastasis, leading to better survival outcomes and/or prognosis in breast cancer (Zheng et al., 2018), astrocytoma (Yu et al., 2017), glioblastoma (Zhang B. et al., 2017), and lung adenocarcinoma (Mo et al., 2013).

SIX4 expression has been detected in non-small cell lung (Tang et al., 2019), breast (Sun et al., 2020), colorectal (Li et al., 2017; Sun et al., 2019), and hepatocellular cancers (He et al., 2020). Over- or ectopic expression of SIX4 promoted metastasis by inducing epithelial-to-mesenchymal transition and angiogenesis (Sun et al., 2019). Furthermore, significant correlations have been identified between expression levels of SIX4, tumor cell metastasis, and poor patient prognosis (Li et al., 2017; Tang et al., 2019; Sun et al., 2019, 2020; He et al., 2020). To date, few studies have analyzed SIX5 function in cancer (Table 2). In one report, SIX5 was detected in normal ovarian epithelium, as well as in malignant ovarian and borderline tumors suggesting that SIX5 could be used as a marker for epithelial differentiation in ovarian tissue rather than a specific marker for cancer (Winchester et al., 2000). A recent meta-analysis showed that high SIX5 expression levels correlated with poor overall survival in lung squamous cell carcinoma (Liu et al., 2016).

Several common functions and modes of regulation have been identified for SIX genes, not just amongst family members, but also between roles during embryonic and cancer development (Table 3).

The fields of developmental biology and cancer research together have provided great insight into the important functions of the SIX gene family in vertebrates. However, many questions remain including how the gene family is regulated, the regulation of activation or repression complexes, and whether SIX proteins are viable therapeutic targets. Efforts have been made to identify transcriptional regulators and characterize the promoters of SIX genes. For example, complex ChIP-seq analysis identified regulatory regions of Six2 in kidney progenitor cells and how Six2 may function in complex transcriptional regulatory networks (Park et al., 2012; O’Brien et al., 2018). The functional importance of the putative Six2 regulatory sequences and how they function in the balance between kidney progenitor cell maintenance and differentiation remains to be determined. It is also unclear whether any of the identified transcription factor binding sites upstream of Six2 or other SIX family members become re-engaged in cancer. In addition to the specific proteins that regulate SIX family enhancers and promoters, it is of interest to further identify upstream signal transduction pathways that can induce SIX gene expression. Evidence has shown a role for Notch, Wnt, and TGF-β pathways to not only be targets of SIX proteins but also to feedback onto SIX genes (Table 3). Identification of upstream pathways may be helpful in the context of congenital disease where clinical manifestations are commonly the result of SIX gene haploinsufficiency and compensation by wild-type alleles could reduce disease severity. Upstream activation pathways of SIX expression would also be of interest in cancer where they could provide new opportunities to reduce SIX induced tumorogenesis. Another tumorogenic target to reduce SIX function would be miRNAs, which have been found to be reduced in several SIX family associated cancers (Table 4). MicroRNAs appear to be an intriguing class of therapeutic targets and reintroduction into cancer cells may allow for specific downregulation of SIX expression in cancer (Rupaimoole and Slack, 2017).

A second area to further investigate is the regulation of activator vs. repressor complexes involving SIX proteins and other conserved factors such as Eya and Dach proteins. During development, these three protein families are commonly co-expressed to maintain a balance between progenitor cell proliferation and differentiation. Does the presence of Eya proteins always promote transcriptional activation even in the presence of repressors such as Dach (Li et al., 2003) or is there more complex regulation of additional SIX binding factors involved. Efforts have begun to better elucidate how SIX factors coordinate with other transcription factors to regulate target genes (O’Brien et al., 2018; Ogawa et al., 2019; Xu et al., 2021). The mechanisms regulating SIX transcriptional activation or repression would provide great insights for both developmental and cancer biology.

Traditionally, transcription factors have been seen as poor drug targets despite the central role they can play in disease such as cancer (Bushweller, 2019). Past difficulties have included targeting protein-DNA or protein-protein interactions due to the charge and flat shape of binding surfaces (Arkin et al., 2014). However, great progress has been made in better understanding protein structure, identifying the residues required for protein interactions, and the regulation of protein function through post-translational modifications. All of these areas are potential targets to control SIX transcription factors in the context of cancer (Bushweller, 2019). One approach has already been demonstrated in a breast cancer model where a small molecule inhibited the interaction between SIX1 and EYA2, reducing downstream TGF-β signaling and EMT leading to reduced metastasis in mouse xenografts (Zhou et al., 2020). Further exploration should identify additional novel regulators of SIX protein function and in conjunction with other therapeutic modalities, such as epigenetic modifiers, could prove effective strategies to combat SIX induced tumorogensis. Such therapeutic insights will only arise from the continual integration of developmental and cancer biology research into the function of the SIX family of transcription factors.

LM, LF, and TC conceived and developed the manuscript outline, and wrote and edited the manuscript. LM, LF, BB, and TC researched the literature and developed the tables. LM and TC created the figure. All the authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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