Am J Cancer Res 2015;5(12):3516-3524 www.ajcr.us /ISSN:2156-6976/ajcr0005402

Review Article TOX : a novel target for human cancer gene therapy

Xin Yu1*, Zheng Li2*

1Department of Dermatology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100042, China; 2Department of Orthopaedic Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. *Equal contributors. Received December 31, 2014; Accepted June 26, 2015; Epub November 15, 2015; Published December 1, 2015

Abstract: Thymocyte selection-associated high mobility group box factor (TOX) is a member of an evolutionarily con- served DNA-binding protein family and is expressed in several immune-relevant cell subsets. TOX encodes a nuclear protein of the high-mobility group box superfamily. It contains a DNA-binding domain, which allows it to regulate tran- scription by modifying local structure and modulating the formation of multi-protein complexes. Previous studies have shown that TOX play important roles in immune system. More recently, several studies have described TOX expression is frequently upregulated in diverse types of human tumors and the overregulation often associates with tumor progression. Moreover, TOXis involved in the control of cell apoptosis, growth, metastasis, DNA repair and so on. In this review, we provide an overview of current knowledge concerning the role of TOX in tumor development and progression biology function. To our knowledge, this is the first review about the role of thisnew oncogene in tumor development and progression.

Keywords: TOX, oncogene, cancer, proliferation, apoptosis

Introduction and leukemia. In addition, TOX family members were also involved in non-tumor diseases, such TOX (Thymocyte selection-associated HMG as pulmonary tuberculosis and HIV infection. In bOX) represent a novel gene family and some cases, TOX acted as an oncogene, con- encode a novel nuclear DNA binding protein trolling cancer cells physiology via promoting belonging to a large superfamily of HMG (high oncogenic signaling. Among them, expressions mobility group)-box family. TOX proteins consist and roles of TOX1 and TOX3 in malignancies a small subfamily of proteins, including TOX1, were largely studied while studies on TOX2 and TOX2, TOX3, and TOX4. Although they shared TOX4 are relatively limited. TOX members were similar structures, different member of TOX also proved to be potential diagnostic or prog- family plays different biological roles. TOX1 was nostic markers in some malignancies, such as originally identified by microarray as a thymic and cutaneous lymphomas. transcript that was highly upregulated in However, the roles and molecular mechanisms CD4+CD8+ double positive (DP) thymocytes by of TOX in malignancies remain unspecified. Wilkinson B et al. TOX1 is proved to be a crucial regulator in immune system differentiation. In this review, we summarize the current infor- However, the biological roles of the other mem- mation on TOX, focusing on their biological roles bers of TOX family remain largely unspecified. and their involvement in human malignancies. TOX3 is predominantly expressed in brain neu- rons and breast while TOX4 is recently proved The structure of TOX genes and proteins to be a DNA interacting protein with unknown biological role. TOX gene family members are located on four different human . TOX1, originally Recently, emerging evidence has shown that identified as KIAA0808, is located at chromo- TOX genes are aberrantly expressed or mutat- some 8q12.1 in human and on 4 ed in various diseases, especially in several dif- A1 in mice. TOX2 is located on 20q13.12 with ferent kinds of malignancies, such as lung can- 12 exons [1, 2]. TOX3, also known as TNRC9 cer, breast cancer, gastric cancer, lymphomas (trinucleotide-repeat-containing 9), is located TOX gene as target for human cancer gene therapy

TOX proteins include a small sub- family of proteins, which include TOX1, TOX2, TOX3, and TOX4 [8]. The TOX subfamily members are highly conserved among verte- brates [2]. Similar to other HMG- box proteins, the sequences of the TOX family members contain an HMG-box DNA binding domain, which comprises three helices folding into an L-shaped structure. The DNA binding domains between different TOX family members are nearly iden- tical. In addition, the N-terminal domain is fairly conserved while the C-terminal domain is family- member specific. Similar to other HMG proteins, TOX seem to operate by bending DNA, there- by altering chromatin structure and modifying the accessibility of transcription factors to DNA [9]. However, the genomic bind- Figure 1. TOX expression in T cell development in the thymus during positive selection. Downregulation of CD4 and CD8 receptors in DP cells ing sites of TOX remain unspeci- yield a double dull (DD) phenotype. Re-expression of CD4 in DD fied. Further studies are needed results in CD4+CD8lo cells. CD4+CD8lo are precursors of both CD4+CD8- to understand how this DNA (CD4SP) and CD4-CD8+ (CD8SP) thymocytes. CD8SP can derive either + lo binding factor is targeted to spe- from CD4 CD8 or directly from DD cells. TOX is induced to unregulated cific regions of DNA. when DP cells were selected to DD cells. TOX were significantly up-regulat- ed at the CD4+CD8lo stage. After thymocytes differentiated into mature SP cells, TOX returned to baseline levels. Expression patterns and biologi- cal roles of TOX on 16q12 and consists of seven exons. TOX3 TOX1 is proved to be a crucial regulator in was first identified in brain in a screen for tran- immune system differentiation while the bio- scripts containing trinucleotide (CAG) repeat logical roles of the other members of TOX family expansions in 1997 [3]. TOX3 protects neuro- remain largely unspecified. In this section, we ns from death by inducing Ca2+-dependent will discuss the expression patterns and bio- transcription from different cytoprotective pro- logical roles of TOX family, focusing on the moters in neurons [4, 5]. TOX3 is also a co-fac- expressions and roles of TOX1 in immune sys- tor of CREB and CBP. TOX4 is located on tem differentiation and maturation. 14q11.2 with 9 exons. TOX4, also known as TOX1 gene is most abundantly expressed in the migration-inducing protein 7 or epidermal thymus. It is also highly expressed in the liver Langerhans cell protein LCP1, interact with a and brain. However, TOX1 is poorly expressed phosphatase complex involved in cell cycle pro- or even absent in other tissues, including heart, gression from mitosis into interphase [6]. In kidney, lung, muscle, skin, intestine, spleen addition, similar to other HMG box proteins, stomach and testis [1]. TOX1 is expressed in TOX4 protein is proved to be involved in the pro- many subsets of immune cells, suggesting its cess of DNA repair damaged by platinum anti- significant roles in immune system, including cancer drugs [6]. development of CD4 T cells and NK cells, as well as lymph node organogenesis [10-12]. TOX proteins belong to a large superfamily of HMG (high mobility group)-box family and con- TOX1 is transiently upregulated in thymus dur- tain a DNA-binding domain (the HMG-box) [7]. ing β-selection and positive selection of devel-

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Table 1. Clinical relevances of TOX Cancer Expression or gene abnormality Function in the clinic References Breast cancer Downregulated and hypermethylated TOX hypermethylation improves diagnosis; [21, 33, 35] TOX3 and LOC643714 predict adverse outcome. Lung cancer Downregulated and hypermethylated Not mentioned [21] Cutaneous lymphoma Upregulated Molecular marker for diagnosis of mycosis fungoides; [36-41] High TOX mRNA levels correlated with increased risks of disease progression and disease-specific mortality. Gastric cancer Not investigated TOX3 associated with favorable prognosis [42] Leukemia Downregulated and deleted Not mentioned [43, 44] Central neural lymphoma Downregulated and deleted Not mentioned [47] oping thymocytes [7]. Accumulating evidence ogenesis [16]. TOX1 is broadly induced and has proved that TOX1 plays a critical role in the reciprocally controls classical NK and lymphoid generation of all T lineage subsets, including tissue-inducer (LTi) cells [17]. TOX1-/- mice also CD4 and CD8 thymocytes. In the absence of lack identified fetal and adult LTi cells, explain- TOX1 (TKO), the development of CD4 T cells ing the cause of the absence of Peyer’s patches was blocked, suggesting its important role in and lymph node [15]. CD4 T cell lineage development [10]. TOX-/- (TKO) mice blocked all CD4 T lineage cells, While TOX1 is proved to be a crucial regulator in including not only production of conventional immune system differentiation, little is known CD4 thymocytes, but also development of NKT about the expressions and roles of other TOX and FOXP3+ regulatory T cells as well. However, family members. The role of TOX2 in humans CD8SP T cells developed in TKO mice. These remains unclear. The rat ortholog of TOX2 with results emphasize that the CD4+ SP transitional 100% HMG-box domain homology (GCX-1) is stage of development is not necessary for all primarily expressed and functions in the hypo- CD8 T cell development [10, 13]. In addition, thalamic-pituitary-gonadal axis of reproduction transgenic mice that express TOX1 (TOX- [1]. A recent study has shown that TOX2 is high- transgenic [Tg]) in the majority of thymocytes ly expressed in mature NK cells and is upregu- showed an increased CD8 SP thymocytes and lated during the differentiation of NK cells from decreased CD4 SP thymocytes [10]. There human umbilical cord blood (UCB)-derived might exist another pathway for CD8 but not CD34+ cells [2]. Furthermore, silence of TOX2 CD4 T cell development (Figure 1). expression inhibited the early differentiation of NK cells and overexpression of TOX2 promoted TOX1 was also highly expressed during in vitro UCB CD34+ cells to differentiate into mature NK NK differentiation and down-regulation of TOX1 cells [2]. TOX3 is predominantly expressed in led to a decreased population of natural killer brain neurons. It is also highly expressed in (NK) cells, suggesting that TOX1 plays a critical breast. In addition, breast cancer expresses role in human NK cell development [14]. higher levels of TOX3 than the normal breast Furthermore, Tox-/- hematopoietic stem cells tissue [4, 18]. The biological role of TOX4 can differentiate into NK cell precursors, but remains to be elucidated. Its N-terminus the NK cell precursors do not differentiate fur- domain contains a strong transcription activa- ther. Thus TOX1 is considered to play a critical tor and it was recently proved to be a platinat- role in immature NK cells [15]. In addition, TOX1 ed-DNA interacting protein [6, 19]. was proved to play key roles in differentiation of cultured CD34+ human cord blood precursor TOX and cancer cells into NK cells [14]. Increasing evidence has demonstrated that In addition, TKO mice lacked lymph nodes and TOX gene family members are aberrantly had a significant decrease in the frequency and expressed or mutated in various human diseas- size of Peyer’s patches, suggesting its role in es, especially in many kinds of malignancies. In lymph node organogenesis. LTi cells were this section, we will discuss the current findings proved to be the central hematopoietic system- of TOX in various human diseases, focusing on derived orchestrators of lymphoid tissue organ- their roles in malignancies (Table 1).

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TOX and breast cancer of TOX3 in breast cancer [34]. In addition, knockdown of TOX3 expression increased cel- It is now well known that aberrant methylation lular proliferation in breast cancer [35]. of hundreds of genes is prevalent during cancer development [20]. Gene silencing through aber- TOX and lung cancer rant promoter CpG island hypermethylation is In a genome-wide comparison of DNA methyla- the most frequent epigenetic abnormality tion between normal and tumor cells, the pro- observed in various malignancies. In a genome- moter CpG islands of TOX1 were also methylat- wide comparison of DNA methylation, the pro- ed in 20% lung cancers cell lines, whereas the moter CpG islands of TOX1 are hypermethyl- distant normal lung tissue from lung cancer ated in 43% of breast tumors while it was patients were unmethylated. In addition, in unmethylated and in the distant normal breast methylated lung cancer, the TOX1 transcripts tissue. In addition, in vitro studies proved that expression was silenced [21]. It was considered TOX1 was completely or partially methylated in that TOX1 was silenced through CpG hyper- three out of four breast cancer cell lines. The methylation in lung cancer, which provide a expression of TOX1 in the methylated cell lines possible mechanism for the development lung is downregulated compared to the unmethyl- cancer. TOX2 were methylated in 28% lung ated cell line, indicating that TOX1 is epigeneti- (n=190) tumors while it was unmethylated in cally silenced by promoter hypermethylation normal cells. Expression of TOX2 transcripts [21]. In conclusion, TOX1 was hypermethylated was significantly decreased in methylated lung in breast cancers but not in the adjacent nor- cancer cells [21]. mal tissue, suggesting it might be a potential novel tumor biomarker. Another study proved TOX1 and cutaneous lymphoma that TOX2 was unmethylated in normal cells but it was methylated in 23% breast (n=80) Primary cutaneous lymphomas (PCLs) are a tumors. Furthermore, expression of two novel heterogeneous group of neoplasias that are TOX2 transcripts was silenced in methylated characterized clonal proliferations of neoplas- breast cancer cells [21]. tic T or B lymphocyte in the skin with no evi- dence of extracutaneous disease at the time of Recent genome-wide association studies diagnosis [36, 37]. PCLs can be classified into (GWAS) have identified several single-nucleo- to cutaneous T-cell lymphomas (CTCL) and tide polymorphisms (SNPs) related with breast cutaneous B-cell lymphomas (CBCL). In CTCL, cancer risk [22]. TOX3 gene is located on there are indolent subtypes such as mycosis 16q12, a region commonly lost in breast can- fungoides (MF) and lymphomatoid papulosis, cers and recently proved as the risk of breast whereas other CTCL subtypes have a more cancer [23]. Several polymorphisms existed in pejorative prognosis such as Sézary syndrome the TOX3 gene, including the SNP rs3803662, (SS) and CD30-lymphomas. Recently, Youwen rs12443621, and rs805154. Among them, Zhou et al. proved that TOX1 was highly SNP rs3803662 was proved to have a strong expressed in early MF skin biopsies in immuno- association of breast cancer [24-29]. No asso- histochemistry and immunofluorescence [38]. ciation was found between the rs12443621 or The authors further found that thicker MF rs3803662 alleles and breast cancer risk [24, lesions such as plaques and tumors expressed 25, 28-31]. However, the role of the germline higher TOX1 levels than thinner patches, sug- polymorphisms in the TOX3 remains unspeci- gesting its association with MF progression. fied [29]. Although it was previously reported in High TOX1 mRNA levels correlated with one study that TOX3 expression was not associ- increased risks of disease prognosis [39]. In ated with prognosis of breast cancer patients addition, SohshiMorimura et al. demonstrated [32]. Another study showed that the expression higher levels of TOX1 mRNA in SS using immu- levels of TOX3 and/or LOC643714 associated nohistochemistry [40]. Furthermore, TOX1 is a with the progression of breast cancer, depend- direct target of microRNA-223, which could ing on the subtype and developmental stage of reduce cell growth and clonogenic potential the tumor [33]. TOX3 might act as a tumor sup- of MF [41]. In conclusion, TOX1 was highly pressor gene since the risk allele rs3803662 is expressed in some CTCLs, such as MF and SS, significantly associated with lower expression suggesting its role in the pathogenesis of CTCL.

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TOX3 and gastric cancer power for MF, demonstrated by an area under the curve (AUC) value of 0.87. For MFdiagnosis, In a recent study, the effect of TOX3 rs3803662 TOX1 mRNA levels showed sensitivity of 90.3% on survival of gastric cancer patients was 75.0% specificity of 75.0%, when cutoff was set investigated. TOX3 rs3803662 was proved to at 2.99. In addition, high TOX1 mRNA levels cor- be associated with a significantly favorable related with increased risks of disease progres- effect among diffuse-type gastric cancer sion and disease-specific mortality [39]. In patients. Thus it was concluded that TOX3 diagnosing breast cancer, the combination of rs3803662 might play an important role in the EGFR5 or TOX1 hypermethylation showed a prognostic outcome and treatment of gastric sensitivity of 92% and specificity of 92% and cancer [42]. accuracy of 93%. The combination of DPYS or TOX1 hypermethylation showed a sensitivity of TOX1 and leukemia 88%, specificity of 96% and accuracy of 91% [48]. Expression levels of TOX3 and/or Most childhood acute lymphoblastic leukemia LOC643714 were proved to affect the progno- (ALL) can be cured, but the prognosis is dismal sis of breast cancer. Tumors with the risk allele in patients who relapse after treatment. had shorter overall survival (OS) and high TOX3 Genome-wide DNA copy number analyses on and/or LOC643714 correlated with positive 61 pediatric patients with relapsed ALL identi- lymph nodes in breast cancer [33]. The TOX3 fied copy number alterations of TOX1 (8q12.1) rs3803662 CT/TT genotype associated with at relapse [43]. In contrast to acute lympho- better survival among diffuse-type gastric can- blastic leukemia in children, adult cases are cer patients, serving as an independent prog- associated with a very poor prognosis. nostic marker [42]. Recurrent deletions of TOX1 were also seen in relapse samples of adult ALL, suggested that Mechanisms of TOX deregulation in cancers TOX might be associated with relapsing ALL [44]. As mentioned above, TOX was frequently dereg- ulated in a variety of human malignances. TOX1 and primary central nervous system lym- Deregulation of can be caused phomas by two mechanisms: one is genetic alteration, namely gene mutation or loss of heterozygosity Primary central nervous system lymphoma (LOH), and the other is epigenetic event, such (PCNSL) is a very aggressive rare brain tumor as CpG island promoter hypermethylation. In characterized by accumulation of malignant this section, we will discuss the upstream regu- cells. The prognosis of PCNSL is typically worse lations of deregulation TOX gene expression in without treatment and the incidence of PCNSL diseases. is increasing [45, 46]. In patients with newly diagnosed PCNSL, the molecular characteriza- One possible mechanism is that TOX1 itself is tion of TOX1 showed biallelic deletions in copy mutated and subsequently resulted in its aber- number abnormalities (CNA) [47]. rant expression. Evidence to support this notion is that recurrent deletions of TOX1 was seen in TOX as prognostic markers relapse samples of adult ALL and childhood ALL [43, 44]. In addition, in patients with newly Given that deregulated expressions of TOX diagnosed PCNSL, TOX1 showed biallelic dele- were identified many cancers, it provides an tions in copy number abnormalities (CNA) [47]. attractive approach for cancer management. In a mutation screen study for TOX3, four muta- First of all, TOX can be used as biomarkers for tions were identified (three missense, one in- cancer diagnosis and prognosis. By monitoring frame deletion of 30 base pairs) in six primary TOX status in an individual tumor, the risk of tumors out of 133 breast tumors [49]. cancer development and progression can be predicted, as well as the prognosis of the can- The other upstream regulation might due to the cer. For example, early MF diagnosis is a major epigenetic change in TOX genes. Epigenetic challenge in the clinical practice. TOX1 was change refers to a stable change in gene highly expressed in early MF skin biopsies and expression that can be inherited through sub- TOX1 mRNA levels had good discriminatory sequent cell divisions, without a change in DNA

3520 Am J Cancer Res 2015;5(12):3516-3524 TOX gene as target for human cancer gene therapy sequence. Epigenetic regulation plays an Disclosure of conflict of interest important role during carcinogenesis and tumor development. Epigenetic changes include DNA None. methylation and histone modifications [50, 51]. DNA methylation results in transcriptional Address correspondence to: Zheng Li, Department repression, which occurs mainly in CpG islands of Orthopaedic Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical of the promoter region [52, 53]. The promoter Sciences and Peking Union Medical College, Beijing, CpG islands of TOX1 were hypermethylated in China. Tel: 86-010-69152812; Fax: 86-010- both lung cancers and breast cancer, which 69152812; E-mail: [email protected] silenced TOX1 transcripts expression [21]. Thus TOX1 was supposed to be silenced through References CpG hypermethylation in cancers [54-56]. However, the methylation status in other can- [1] Kajitani T, Mizutani T, Yamada K, Yazawa T, cers remains unknown. Further studies are Sekiguchi T, Yoshino M, Kawata H and needed to elucidate the possible relationship Miyamoto K. Cloning and characterization of between the expression of TOX1 and promo- granulosa cell high-mobility group (HMG)-box ter hypermethylation in other malignancies. protein-1, a novel HMG-box transcriptional MicroRNAs (miRs) are endogenous 18-25 regulator strongly expressed in rat ovarian nucleotide non-coding RNAs that target specific granulosa cells. Endocrinology 2004; 145: mRNA for translational repression or degrada- 2307-2318. [2] Vong QP, Leung WH, Houston J, Li Y, Rooney B, tion [57, 58]. MiRs can regulate various cellular Holladay M, Oostendorp RA and Leung W. functions, including cell survival, proliferation, TOX2 regulates human natural killer cell devel- migration, invasion and metastasis and expres- opment by controlling T-BET expression. Blood sion levels of miRs is often deregulated in can- 2014; 124: 3905-13. cer. TOX1 is a direct target of microRNA-223, [3] Margolis RL, Abraham MR, Gatchell SB, Li SH, which could reduce cell growth and clonoge Kidwai AS, Breschel TS, Stine OC, Callahan C, nic potential of MF [41]. The study provides a McInnis MG and Ross CA. cDNAs with long novel sight involving a miR-TOX1 axis to un- CAG trinucleotide repeats from human brain. derstand the mechanism of TOX deregu- Hum Genet 1997; 100: 114-122. lation. [4] Dittmer S, Kovacs Z, Yuan SH, Siszler G, Kogl M, Summer H, Geerts A, Golz S, Shioda T and Conclusions and future perspectives Methner A. TOX3 is a neuronal survival factor that induces transcription depending on the TOX was initially identified through its associa- presence of CITED1 or phosphorylated CREB in the transcriptionally active complex. J Cell tion with T lymphocyte differentiation. TOX pro- Sci 2011; 124: 252-260. teins contain a small subfamily of proteins, [5] Yuan SH, Qiu Z and Ghosh A. TOX3 regulates including TOX1, TOX2, TOX3, and TOX4. calcium-dependent transcription in neurons. Different member of TOX family plays different Proc Natl Acad Sci U S A 2009; 106: 2909- biological and pathological roles. It is known 2914. that TOX is a DNA binding protein; however the [6] Bounaix Morand du Puch C, Barbier E, Kraut A, genomic binding site of TOX is unknown. Future Coute Y, Fuchs J, Buhot A, Livache T, Seve M, investigations are needed to identify genomic Favier A, Douki T, Gasparutto D, Sauvaigo S binding sites of TOX, to understand how it is tar- and Breton J. TOX4 and its binding partners geted to specific regions of DNA. In addition, recognize DNA adducts generated by platinum anticancer drugs. Arch Biochem Biophys 2011; although emerging evidence has shown that 507: 296-303. TOX was deregulated in many tumors, the func- [7] Wilkinson B, Chen JY, Han P, Rufner KM, tional roles of TOX in tumors remain unspeci- Goularte OD and Kaye J. TOX: an HMG box pro- fied, which also requires future investigations. tein implicated in the regulation of thymocyte selection. Nat Immunol 2002; 3: 272-280. Acknowledgements [8] O’Flaherty E and Kaye J. TOX defines a -con served subfamily of HMG-box proteins. BMC This work was supported by grants from the Genomics 2003; 4: 13. National Natural Science Foundation of China [9] Reeves R. Nuclear functions of the HMG pro- (NSFC) (Grant Number: 81401847). teins. Biochim Biophys Acta 2010; 1799: 3-14.

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