Uev1A upregulates CT45A expression in a Ubc13-dependent manner
We performed a microarray analysis by comparing UEV1A-overexpressed and vector control MDA-MB-231 breast cancer cells, which revealed 47 genes upregulated by more than fivefold in UEV1A-overexpressed MDA-MB-231 cells (Supplementary Table S2). Interestingly, 16 out of 47 belong to cancer/testis antigens (CTAs), among which CT45A family members are most highly elevated in UEV1A-overexpressed MDA-MB-231 cells (Figure S1A). CTAs are tumor-associated and testis-derived specific immunogenic antigens closely associated with spontaneous immune responses in cancer patients [32, 33]. They are not expressed in nearly all normal tissues except testis after birth, but are highly expressed in various types of cancers [34-39]. Within CTAs, the CT45A gene family comprises 10 genes designated as CT45A1 to CT45A10, which are distinct but highly conserved, as their amino-acid sequences exhibit more than 98% identity  (Figure S1B). To independently examine the role of Uev1A in upregulating CT45A expression and its biological implications, UEV1A was cloned into a pcDNA4.0/TO/HA(+) vector and then trasfected into MDA-MB-231-TR and MCF7 cells to construct stable cell lines as previously reported . The level of UEV1A ectopic expression was monitored by western blot against the HA-tag after 10 μg/ml doxycyline (Dox) treatment (Figure S2A,B). Then CT45A expression was measured by qRT-PCR and found to be signifcantly upregulated in UEV1A-overexpressed MDA-MB-231-TR (Figure 1A) and MCF7 (Figure 1B) cells. It has been reported that UEV1A is upregulated in MDA-MB-231 and MCF7 cells by 2.8- and 4-fold, respectively . To ask whether this moderate overexpression of UEV1A contributes to CT45A upregulation in breast cancer cells, we suppressed the endogenous UEV1A expression in MDA-MB-231 and MCF7 cells using shRNAs delivered by lentiviral particles as previously reported . It was found that two independent shUEV1A constructs, shUEV1A-1 and shUEV1A-2, reduced UEV1A expression in MDA-MB-231 cells by 43% and 60% (Figure S3A), and in MCF7 cells by 71% and 85% (Figure S3B), respectively, compared to contral shRNA-treated cells. Meanwhile, the CT45A transcript levels were also reduced (Figure 1C, D). To further ask whether Uev1A upregulates CT45A expression in a Ubc13-dependent manner, we constructed stable MDA-MB-231-TR and MCF7 cell lines expressing Dox-inducible Uev1A-F38E mutant protein (Figure S2A,B), which is known to abolish physical interaction between Mms2/Uev1A and Ubc13 [7, 17, 19]. As expected, UEV1A-F38E failed to upregulate CT45A mRNA levels in both MDA-MB-231-TR (Figure 1E) and MCF7 (Figure 1F) cells. These observations collectively indicate that Uev1A upregulates CT45A expression in a Ubc13-dependent manner in breast cancer cells.
Uev1A positively regulates CT45A downstream gene expression in breast cancer cells
CT45A has been reported to act as a proto-oncogene through upregulating tumorigenic and metastatic genes . We first measured the transcript level of several CT45A previously-reported  downstream genes thought to be involved in tumoregenesis, EMT, stemness and metastasis after CT45A ectopic expression. The expression of some tumoregenesis-associated genes, including those encoding RAS exchange factor (RASGEF1A), melanoma antigen family member (MAGED4B), homeobox B6 (HOXB6 and HOXD13) was indeed significantly higher in CT45A-overexpressed MDA-MB-231 (Figure 2A) and MCF7 (Figure 2B) cells than their respective control cells. Expression of several EMT, stemness and metastasis related genes, including TWIST1, KIT, aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), CXCR4 and/or SULF2 were also upregulated in CT45A-overexpressed MDA-MB-231 (Figure 2C) and MCF7 (Figure 2D) cells. Since UEV1A can upregulate CT45A expression, we asked whether UEV1A could also upregulate the expression of CT45A downstream genes in breast cancer cells. Indeed, the majority of CT45A downstream genes, including HOXB6, HOXD13, RASGEF1A, MAGED4B, ALDH1A1, TWIST1, KIT, CXCR4 and SULF2, were upregulated in UEV1A-overexpressed MDA-MB-231 (Figure 2E, G) and MCF7 (Figure 2F, H) cells. Taken together, we conclude that UEV1A positively regulates CT45A downstream gene expression in breast cancer cells.
CT45A is a critical regulator for Uev1A-induced breast cancer cell migration
To ask whether an elevated CT45A level alone is indeed sufficient to promote breast cancer development and metastasis, CT45A was cloned into plasmid pcDNA4.0/TO/HA(+), transiently transfected into MDA-MB-231 and MCF7 cells and the level of CT45A ectopic expression after 200 μg/ml zeocin treatment was monitored by western blot against an HA-tag antibody (Figures 3A and 4A). The effects of CT45A ectopic expression on MDA-MB-231 (Figure 3) and MCF7 (Figure 4) cells were then assessed. The transwell without matrigel experiments show that overexpression of CT45A nearly tripled the MDA-MB-231 cell mobility compared with vector-transfected cells (Figure 3B,C). Similarly, after induction the migration of MCF7 CT45A transfectants was more than 2.3-fold higher than the control cells (Figure 4B,C), indicating that CT45A regulates breast cancer cell migration in vitro.
To ask whether Uev1A is a critical regulator for CT45A-induced migration, we successfully depleted CT45A by approximately 50% using siRNA in MDA-MB-231 (Figure S3C) and MCF7 (Figure S3D) cells. Similar effects were also observed in UEV1A-overexpressed MDA-MB-231 (Figure 3D) and MCF7 (Figure 4D) cells. The above treatment does not affect the expression of UEV1A (Figures 3E and 4E), but the moderate CT45A depletion in UEV1A-overexpressed cells markedly reduced cell migration as determined by a transwell assay without matrigel (Figures 3F,G and 4F,G). The above findings allow us to conclude that CT45A is a critical regulator for Uev1A-induced migration in breast cancer cells, as partial depletion of CT45A can reverse cell migration in UEV1A-overexpressed breast cancer cells.
CT45A promotes metastasis in other type of cancer cells
To ask whether UEV1A overexpression also increases CT45A expression in other cancer cells, we created stable UEV1A-transfected HCT116 colorectal cancer cell lines as previously described (Figure 5A) , in which CT45A was moderately upregulated upon UEV1A ectopic expression, and this upregulation was not observed in the UEV1A-F38E-expressed HCT116 cells (Figure 5B). To ask whether this moderate overexpression of UEV1A contributes to CT45A upregulation in colorectal cancer cells, we suppressed the endogenous UEV1A expression in HCT116 cells by using shRNAs delivered by lentiviral particles as previously reported . It was found that two independent shUEV1A constructs, shUEV1A-1 and shUEV1A-2, reduced UEV1A expression in HCT116 cells by 55% and 65%, respectively (Figure S4A), compared to control shRNA-treated cells. Meanwhile, the CT45A transcript levels were also reduced (Figure 5C). To ask whether ectopic expression of CT45A could promote metastasis in other types of cancer cells, HCT116 cells were transiently transfected with pcDNA4.0/TO/HA-CT45A and the CT45A expression was monitored by western blot analysis against HA-tagged CT45A (Figure 5D). The CT45A ectopic expression resulted in concommitant increase in HCT116 cell migration by sevenfold (Figure 5E,F), indicating that CT45A could also promote tumorigenesis and metastasis in other types of cancer cells. To further ask whether CT45A is a critical regulator for UEV1A-induced migration, we depleted CT45A by using siRNA in UEV1A-overexpressed HCT116 cells. As shown in Figure S4B, CT45A was depleted by 44%. The above treatment does not affect the expression of UEV1A (Figure S4C), but the moderate CT45A depletion in UEV1A-overexpressed HCT116 cells markedly reduced cell migration as determined by a transwell assay without matrigel (Figure 5G,H). The above findings indicate that Uev1A induces colorectal cancer cell migration through upregulating CT45A genes.
Depletion of CT45A can reverse EMT in UEV1A-overexpressed breast cancer cells
Epithelial-mesenchymal transition (EMT) is closely associated with cancer progression, cancer cell metastasis and drug resistance [42, 43]. Cells undergoing EMT display increased expression of mesenchymal genes including N-cadherin, fibronectin and vimentin, and decreased expression of epithelial genes including E-cadherin, occulin and ZO-1 . It was reported that overexpression of CT45A could induce breast cancer EMT, and thus foster cancer metastasis by upregulating EMT master gene TWIST1 . To further investigate the potential molecular mechanisms by which CT45A regulates breast cancer cell migration, we monitored alterations of EMT markers, including N-cadherin and vimentin, two well-characterized mesenchymal markers, and E-cadherin, a well-known epithelial marker [44, 45]. Consistent with breast cancer cell migration, increased mRNA levels of N-cadherin and vimentin and decreased E-cadherin were found upon CT45A overexpression in MDA-MB-231 (Figure 6A) and MCF7 (Figure 6B) cells. We also assessed the effect of CT45A on cellular N-cadherin and E-cadherin at protein levels. Firstly, we monitored cellular N-cadherin and E-cadherin levels in MDA-MB-231 and MCF7 cells and found that MDA-MB-231 and MCF7 cells produced detectable N-cadherin and E-cadherin, respectively (Figure 6C). Interestingly, ectopic expression of CT45A increased N-cadherin in MDA-MB-231 cells and decreased E-cadherin in MCF7 cells (Figure 6C,E,F), suggesting that cell migration stimulated by ectopic CT45A expression is likely due to the enhanced EMT in breast cancer cells. To address whether Uev1A is a critical upstream regulator of CT45A-induced EMT, we depleted CT45A by using siRNA in UEV1A-overexpressed MDA-MB-231 and MCF7 breast cancer cells (Figures 3D and 4D), which significantly increased E-cadherin protein levels in UEV1A-overexpressed MCF7 cells and decreased N-cadherin protein levels in UEV1A-overexpressed MDA-MB-231 cells (Figure 6D,G,H). Collectively, these results support a notion that Uev1A can serve as an important regulator for CT45A-induced EMT in breast cancer cells.
Uev1A regulates CT45A expression through the AKT signaling pathway
Since Uev1A has been reported to be associated with NF-κB activation [19-21] and AKT activation , we wish to investigate molecular mechanisms by which Uev1A regulates CT45A expression. To ask whether Uev1A regulates CT45A expression through the NF-κB pathway, MDA-MB-231 and HCT116 cells transiently overexpressing UEV1A were treated with the NF-κB pathway inhibitor Bay11-7082  and its efficacy was measured by the nuclear P65 level (Figure S5A,C). The CT45A transcript level was not significantly reduced in UEV1A-ovexpressed MDA-MB-231 (Figure S5B) and HCT116 cells (Figure S5D) by treatment with Bay11-7082, indicating that Uev1A upregulation of CT45A expression is independent of the NF-κB pathway. To ask whether Uev1A regulates CT45A expression through the AKT signaling pathway in breast cancer cells, phosphorylation levels of both AKT-Thr308 and AKT-Ser473 in MDA-MB-231 and MCF7 cells transiently overexpressing UEV1A were first monitored by western blot and found to be increased (Figure 7A). In contrast, overexpression of UEV1A-F38E failed to induce AKT phosphorylation at both residues (Figure 7A), indicating that the effects of Uev1A on AKT is dependent on its interaction with Ubc13. These observations allow us to conclude that excessive Uev1A promotes the Uev1A-Ubc13 complex formation, which activates the AKT signaling pathway. To further address whether Uev1A promotes CT45A expression through the AKT signaling pathway, we examined effects of PI3K/AKT pathway inhibitor LY294002  on MDA-MB-231 and MCF7 cells with ectopic UEV1A expression. As seen in Figure 7B, the AKT-Ser473 phosphorylation level was markedly decreased after LY294002 treatment in UEV1A-overexpressed MDA-MB-231 and MCF7 cells compared to those without the inhibitor treatment. We then examined CT45A expression and found that, compared to cells without LY294002 treatment, the CT45A transcript level was significantly reduced in UEV1A-overexpressed MDA-MB-231 (Figure 7C) and MCF7 (Figure 7D) cells after 10 μM LY294002 treatment. After 20 μM LY294002 treatment, the CT45A transcript further decreased to levels below the vector control cells without the inhibitor treatment (Figure 7C,D). It was previously reported that insulin-like growth factor (IGF-1) is an important activator of the PI3K/AKT signaling pathway [48, 49]. To further investigate whether CT45A is indeed a direct downstream gene of the AKT signaling pathway, we treated MDA-MB-231 (Figure 7E) and MCF7 (Figure 7F) cells with IGF-1, and found that the AKT-Ser473 phosphorylation level was dramatically increased after IGF-1 treatment compared to untreated cells. Under the above experimental conditions, the CT45A mRNA levels were significantly increased in MDA-MB-231 (Figure 7G) and MCF7 (Figure 7H) cells after IGF-1 treatment. Collectively, we conclude that Uev1A-Ubc13 regulates CT45A expression through the AKT signaling pathway in breast cancer cells.