UNC119 is a binding partner of tumor suppressor Ras-association domain family 6 and induces apoptosis and cell cycle arrest by MDM2 and p53.

Ras-association domain family 6 (RASSF6) is a tumor suppressor that interacts with MDM2 and stabilizes p53. Caenorhabditis elegans unc-119 encodes a protein that is required for normal development of the nervous system. Humans have 2 unc-119 homologues, UNC119 and UNC119B. We have identified UNC119 as a RASSF6-interacting protein. UNC119 promotes the interaction between RASSF6 and MDM2 and stabilizes p53. Thus, UNC119 induces apoptosis by RASSF6 and p53. UNC119 depletion impairs DNA repair after DNA damage and results in polyploid cell generation. These findings support that UNC119 is a regulator of the RASSF6-MDM2-p53 axis and functions as a tumor suppressor.

INTRODUCTION

The human genome has 10 genes encoding proteins designated as Ras‐association (RA) domain family (RASSF) proteins.1, 2, 3, 4, 5 RASSF1 to RASSF6 have a RA domain near the C‐terminal region and a coiled‐coiled motif in the C‐terminal region. This coiled‐coil domain is called the Salvador/RASSF/Hippo (SARAH) domain. RASSF7 to RASSF10 have a RA domain in the N‐terminal region and lack the SARAH domain.6, 7 RASSF1 to RASSF6 are collectively called C‐RASSF, whereas RASSF7 to RASSF10 are named N‐RASSF. C‐RASSF proteins are regarded as tumor suppressors. RASSF1A, a major splicing variant of RASSF1, was identified in the lung tumor suppressor locus.8 RASSF1A expression is frequently suppressed by hypermethylation in the promoter region in cancers, and its low expression is associated with poor prognosis.4 Other C‐RASSF proteins including RASSF6 are also frequently downregulated in human cancers.4 The molecular mechanism underlying the tumor suppressive function of RASSF1A is well studied. RASSF1A stabilizes microtubules, regulates the G2/M checkpoint, and causes apoptosis and cell‐cycle arrest through MDM2 and p53.9, 10, 11, 12, 13 RASSF1A interacts with mammalian Ste20‐like kinases (MST1 and MST2) and activates the tumor suppressor Hippo pathway.14, 15, 16 These properties are partially, not entirely, shared by other C‐RASSF proteins. RASSF6 interacts with MDM2, stabilizes p53, and induces apoptosis and cell‐cycle arrest.17 RASSF6 forms a complex with MST1/2, but, in contrast to RASSF1A and MST2, RASSF6 and MST1/2 form a complex and inhibit each other under basal conditions.18 However, when certain stimuli, such as okadaic acid treatment, trigger dissociation of the complex, the Hippo pathway is activated and, simultaneously, RASSF6 induces apoptosis independently of the Hippo pathway. Thus, RASSF6 and the Hippo pathway cooperate with each other as tumor suppressors. Nevertheless, the mechanism by which RASSF6‐mediated apoptosis is triggered is not yet clarified. Therefore, it is important to identify molecules that interact with and regulate RASSF6.

unc‐119 was identified as 1 of the genes whose mutations cause uncoordinated movement in Caenorhabditis elegans.19 The human UNC119 gene was found as a retina‐enriched gene and named human retina gene 4 (HRG4).20 The gene is registered as UNC119 in the database of the National Center for Biotechnology Information (ID:9094). Truncation mutation of UNC119 is detected in human patients and causes retinal degeneration in transgenic mice.21 Humans have another closely related gene, UNC119B (ID:84747).22 UNC119 is frequently depicted as UNC119A in research papers. To avoid confusion, we will also use UNC119A for the gene and UNC119A for the protein in this report. UNC119A has two splicing variants, UNC119Aa and UNC119Ab, which encode different amino acids in the C‐terminal sequences.23 UNC119A is localized at a ribbon synapse in retina.20 UNC119A is similar in structure to lipid‐binding proteins, cGMP phosphodiesterase (PDEδ), and Rho GDP dissociation inhibitor (RhoGDI).24 Lipid‐modified proteins such as transducin, Lyn, Fyn, Abl1, and Abl2 interact with UNC119A.25 Another important binding partner is RIBEYE, a major component of ribbon synapse.26 UNC119B is implicated in the transport of myristoylated nephrocystin‐3.22 Through the interaction with these proteins, UNC119A and UNC119B play important roles in ribbon synapse formation, T‐cell activation, and cilia formation.

We identified UNC119A as a RASSF6‐binding partner. Endogenous UNC119A and RASSF6 are coimmunoprecipitated from human colon cancer SW480 cells and show a similar distribution in rat kidney. UNC119A promotes interactions between RASSF6 and MDM2 and regulates apoptosis and the cell cycle by p53. These findings suggest that UNC119A is implicated in the regulation of the RASSF6‐MDM2‐p53 axis.

MATERIALS AND METHODS

Cell cultures and transfection. HEK293FT, HCT116, U2OS, SW480, HeLa, H1299, and TIG3 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 10 mM Hepes‐NaOH at pH 7.4 under 5% CO2 at 37°C. DNA transfection was performed with Lipofectamine 2000 (Thermo Fisher Scientific). HCT116 p53‐/‐ cells were infected with lentivirus vector (lenti‐CRSIPR‐MDM2‐CR) and selected with puromycin. Clones were isolated and MDM2 depletion was confirmed by the immunoblotting. Other Materials and methods are described in Doc S1.

RESULTS

RASSF6 interacts with UNC119A

We obtained UNC119A as a putative RASSF6‐binding partner through yeast 2‐hybrid screening with human kidney cDNA library. To detect interaction between endogenous RASSF6 and UNC119A, we used human colon cancer SW480 cells, which express both RASSF6 and UNC119A at a high level (Figure 1A). RASSF6 was coimmunoprecipitated with UNC119A (Figure 1B). We previously characterized the localization of RASSF6 in rat kidney and showed that RASSF6 is detected in the renal glomerulus and renal tubular epithelial cells.27 We immunostained rat kidney with anti‐UNC119A antibody. UNC119A signals were detected in the renal glomerulus (Figure 1C). In the glomerulus, RASSF6 and UNC119A were partially colocalized with nephrin and synaptopodin, which are markers of the slit diaphragm. Like RASSF6, UNC119A was also detected in renal tubular epithelial cells. These findings support that UNC119A and RASSF6 are similarly distributed in the renal glomerulus and renal tubular epithelial cells.

Figure 1

Interaction between Ras‐association domain family 6 (RASSF6) and UNC119A and the localization of UNC119A in rat kidney. A, RASSF6 and UNC119A in various cell lines. Whole‐cell lysates of indicated cell lines (50 μg total protein) were immunoblotted by anti‐RASSF6 and anti‐UNC119A antibodies. RASSF6 was detected in SW480 cells only. RASSF6 knockdown abolished the signal detected by anti‐RASSF6 antibody in SW480 cells (second lane). UNC119A was detected in SW480, HCT116, HEK293FT, H1299, HeLa, and TIG3 cells. UNC119A knockdown abolished signals detected by anti‐UNC119A antibody (second lane). B, UNC119A was immunoprecipitated from SW480 cells and immunoblotted with anti‐UNC119A and anti‐RASSF6 antibodies. C, Rat kidney was immunostained with the indicated antibodies. UNC119A (green) and RASSF6 (green) were detected in the glomerulus (G) and in the renal tubules (T). Demarcated areas are shown at higher magnification in the lower row. UNC119A and RASSF6 are partially overlapped by nephrin (red) and synaptopodin (red). Bar, 20 μm

RASSF6 interacts with UNC119A, but not with UNC119B

UNC119A has two splicing variants, UNC119Aa and UNC119Ab.23 Myc‐UNC119Aa and Myc‐UNC119Ab were equally coimmunoprecipitated with FLAG‐RASSF6 from HEK293FT cells (Figure 2A). UNC119B did not bind to RASSF6 under the same condition that UNC119Ab interacted with RASSF6 (Figure 2B). We confirmed the results in the reverse immunoprecipitations. FLAG‐RASSF6 was coimmunoprecipitated with Myc‐UNC119Aa and Myc‐UNC119Ab, but not with Myc‐UNC119B (Figure 2C,D). We used UNC119Ab (hereafter named UNC119A) for the following studies. We further confirmed that GST‐UNC119A as well as GST‐MDM2 bound MBP‐RASSF6 in vitro, supporting the direct interaction between UNC119A and RASSF6 (Figure 2E). We next compared the subcellular distribution of UNC119A and RASSF6. In the subcellular fractionation, exogenously expressed UNC119A and RASSF6 were recovered in both the cytoplasmic and nuclear fractions (Figure 2F, left). Interaction between UNC119A and RASSF6 was observed in both fractions (Figure 2F, left). In contrast, MDM2, even when exogenously expressed, was mainly recovered in the nuclear fraction (Figure 2F, right). As a matter of course, the interaction between RASSF6 and MDM2 took place in the nuclear fraction. Proximity ligation assay also supported the interaction between UNC119A and RASSF6 in the cytoplasm and the nucleus (Figure 2G).

Figure 2

Interaction between Ras‐association domain family 6 (RASSF6) and UNC119A variants. HEK293FT cells were transfected with pCIneoMyc‐UNC119Aa, pCIneoMyc‐UNC119Ab, pCIneoMyc‐UNC119B, and pCIneoFHF‐RASSF6. Immunoprecipitation was conducted with anti‐DYKDDDDK (1E6) beads in (A), and (B), and with anti‐Myc antibody in (C) and (D). A,B, Myc‐UNC119Aa and Myc‐UNC119Ab were coimmunoprecipitated with FLAG‐RASSF6, but Myc‐UNC119B was not. Asterisks indicate the immunoglobulin. C,D, FLAG‐RASSF6 was coimmunoprecipitated with Myc‐UNC119Aa and Myc‐UNC119Ab (arrows in (C)), but not with Myc‐UNC119B (arrow in (D)). E, Eluted MBP‐RASSF6 was incubated with control GST, GST‐UNC119A, and GST‐MDM2 fixed on glutathione cellulose beads. MBP‐RASSF6 attached to beads was detected with anti‐MBP antibody. Lower panels show GST proteins in Coomassie Brilliant Blue staining. F, FLAG‐RASSF6, Myc‐UNC119A, and HA‐MDM2 were expressed in HEK293FT cells. Subcellular fractionation was carried out. Immunoprecipitation was conducted by use of anti‐DYKDDDDK (1E6) beads from the cytoplasmic and nuclear fractions. α‐Tubulin and anti‐poly (ADP‐ribose) polymerase (PARP) were used as cytoplasmic and nuclear markers, respectively. UNC119A was coimmunoprecipitated with RASSF6 in both the cytoplasmic and nuclear fractions, whereas MDM2 was coimmunoprecipitated in the nuclear fraction. G, Myc‐UNC119A and FLAG‐RASSF6 were expressed in HEK293FT cells. Proximity ligation assay was carried out according to the manufacturer's protocol. The image without the antibody is also shown as a control. Bars, 10 μm

UNC119A induces apoptosis depending on p53 and RASSF6 and is implicated in UV‐induced apoptosis

RASSF6 induces apoptosis and cell‐cycle arrest.17 This function of RASSF6 partly depends on p53. We raised the question of whether UNC119A induces apoptosis and, if it does, whether UNC119A‐induced apoptosis is mediated by RASSF6 and p53. UNC119A overexpression caused nuclear condensation and cytochrome‐C release in p53‐positive HCT116 cells (Figure 3A, arrowheads). Ratios of cells with nuclear condensation and cytochrome‐C release were smaller in p53‐negative HCT116 cells (Figure 3A right graphs). RASSF6 depletion attenuated UNC119A‐induced nuclear condensation and cytochrome‐C release (Figure 3B). FACS analysis indicated an increase in the sub‐G1 population in UNC119A‐expressing HCT116 cells, but the depletion of p53 or RASSF6 suppressed it (Figure 3C). UV exposure increased the sub‐G1 population from 3.88% to 59.38% (Figure 4, siCont, No treatment, and UV treatment). As previously reported, depletion of p53 or RASSF6 suppressed UV‐induced sub‐G1 population (Figure 4, siRASSF6 and sip53).17 Likewise, UNC119A depletion suppressed UV‐induced the sub‐G1 population (Figure 4A, siUNC119A#1). Another siRNA against UNC119A (siUNC119A#2) showed a similar effect (Figure S1A). The knockdown efficiencies were similar for both siRNAs (Figure 4B). These findings support that UNC119A regulates apoptosis by RASSF6 and p53.

Figure 3

UNC119A‐induced apoptosis depends on Ras‐association domain family 6 (RASSF6) and p53. A, p53‐positive‐ (p53 +/+) and p53‐negative‐ (p53 −/−) HCT116 cells were transfected with pBudGFP‐SUMO (GFP‐Cont.) or pCIneoGFP‐UNC119A. 24 h later, cells were immunostained with anti‐cytochrome‐C antibody. Nuclei were visualized with Hoechst 33342. Cytochrome‐C remained in mitochondria in HCT116 cells expressing control GFP and HCT116 p53−/− cells expressing GFP‐UNC119A. In HCT116 p53 +/+ cells, GFP‐UNC119A‐induced nuclear condensation and cytochrome‐C release (arrowheads). 50 GFP‐positive cells were observed in 3 independent experiments and cells with nuclear condensation and with cytochrome‐C release were counted. Data are shown as mean with SEM. ***P < .001. Bar, 10 μm. B,C, HCT116 cells were transfected with control or ‐targeted siRNA. 72 h later, cells were transfected with pBudGFP‐SUMO (GFP‐Cont.) or pCIneoGFP‐UNC119A. In (B), 24 h later, apoptosis was evaluated as described for Figure 3A. GFP‐UNC119A induced nuclear condensation and cytochrome‐C release in control cells (arrowheads) but not in RASSF6‐depleted cells. Data are shown as mean with SEM. **P < .01; and ***P < .001. Bar, 10 μm. In (C), 24 h later, the cells were harvested. The sub‐G1 population was evaluated with FACS. Numbers indicate the percentages of the sub‐G1 population. GFP‐UNC119A increased the sub‐G1 population, but knockdown of p53 or RASSF6 reduced it. ***P < .001

Figure 4

UNC119A is implicated in UV‐induced apoptosis. A, HCT116 cells were transfected with control, UNC119A‐ (#1), Ras‐association domain family 6 (RASSF6‐), or targeted siRNA. 72 h later, cells were exposed to 25 J/m2 ultraviolet (UV). 24 h and 48 h later, cells were harvested and the sub‐G1 population was evaluated as described for Figure 3C. Left panel shows the results at 24 h. Numbers indicate the percentages of the sub‐G1 population. ***P < .001. B, Validation of the knockdown by ‐, ‐, and ‐targeted siRNAs. HCT116 cells were harvested 72 h after transfection. qRT‐PCR was carried out by use of GAPDH as a reference. ***P < 0.001. Cell lysates (50 μg/total protein) were immunoblotted with anti‐UNC119A and anti‐p53 antibodies

UNC119A induces cell‐cycle arrest depending on RASSF6 and p53

We next tested the function of UNC119A in cell‐cycle regulation. We expressed UNC119A in p53‐positive HCT116 cells, which blocked BrdU incorporation (Figure 5A, siCont, arrowheads). Knockdown of p53 or RASSF6 recovered BrdU incorporation in UNC119A‐expressing cells (Figure 5A, sip53 and siRASSF6, arrowheads). UNC119A reduced BrdU incorporation even in HCT116 p53−/− cells, although the inhibitory effect was less remarkable (Figure S2). UV exposure induced G1/S arrest (Figure 5B, white arrow). UNC119A‐depleted cells overrode the arrest, and the cell cycle proceeded 20 hours later (Figure 5B, black arrow). Another siRNA against UNC119A (siUNC119A#2) showed a similar effect (Figure S1B).

Figure 5

UNC119A‐induced cell‐cycle arrest depends on Ras‐association domain family 6 (RASSF6) and p53 and UNC119A is implicated in UV‐induced cell‐cycle arrest. A, p53 or RASSF6 was knocked down in HCT116 cells. 72 h later, cells were transfected with pCIneoMyc‐UNC119A. 24 h later, cells were incubated in the medium containing 10 μmol/L BrdU for 1 h. BrdU was detected by use of BrdU labeling and detection kit (Sigma‐Aldrich, St Louis, MO, USA). Cells were immunostained with anti‐BrdU (green) and anti‐Myc (red) antibodies. Nuclei were visualized with Hoechst 33342. Cells expressing Myc‐UNC119A (arrowheads) did not incorporate BrdU, whereas cells without Myc‐UNC119A did (siCont). When p53 or RASSF6 was knocked down, Myc‐positive cells also incorporated BrdU (sip53 and siRASSF6, arrowheads). B‐D, HCT116 cells were transfected with control or ‐targeted (#1) siRNA. In B, 72 h later, cells were transferred to the medium without serum for synchronization. 24 h later, cells were exposed to 10 J/m2 UV and were cultured in the medium with serum. Cells were harvested at 0 h, 12 h, 16 h, and 20 h and analyzed by FACS. UV‐induced G1/S arrest (white arrow) in control cells, whereas UNC119A‐depleted cells overrode G1/S arrest (black arrow). In (C), 72 h later, cells were exposed to 10 J/m2 UV. 16 h later, cells were harvested. qRT‐PCR was carried out by use of GAPDH as a reference. UV exposure enhanced (P21), and mRNAs and UNC119A knockdown blocked the enhancement. ***P < .001. In D, HCT116 cells were treated with 10 μmol/L VP‐16. 24 h later, cell lysates were immunoblotted with indicated antibodies

UNC119A depletion suppresses p53‐dependent transcription after UV exposure

We surmised that UNC119A plays a role in UV‐induced p53‐dependent apoptosis and cell‐cycle arrest. To confirm this assumption, we quantified p53 target genes. UV exposure upregulated PUMA, CDKN1A, and BTG2 (Figure 5C). UNC119A depletion abrogated the UV‐induced enhancement of these genes. We treated HCT116 cells with 10 μmol/L VP‐16 for 24 hours and observed an increase in p21, PUMA, BAX, and BTG3 in western blotting (Figure 5D, first and third lanes). UNC119A itself was slightly enhanced by VP‐16. UNC119A silencing abolished the enhancement of these proteins (Figure 5D, third and fourth lanes).

UNC119A regulates the stability of p53 by MDM2

We previously reported that RASSF6 blocks MDM2‐mediated p53 degradation.17 We hypothesized that UNC119A regulates apoptosis and cell‐cycle progression through RASSF6‐MDM2‐p53. To test this hypothesis, we examined the effect of UNC119A on the RASSF6‐MDM2‐p53 axis. UNC119A coexpression increased p53 expression (Figure 6A, left). To evaluate endogenous p53, we used TIG3 cells, in which p53 induces senescence. Endogenous p53, BAX, and p21 were, indeed, enhanced by UNC119A in TIG3 cells (Figure 6A, right). p53 degradation by treatment with cycloheximide was facilitated by UNC119A silencing (Figure 6B). Another siRNA against UNC119A (siUNC119A#2) showed a similar effect (Figure S1C). UNC119A depletion by siUNC119A#1 or #2 attenuated UV‐induced enhancement of p53 (Figure 6C). p53 expression was remarkably enhanced by MDM2 depletion, and the additional knockdown of UNC119A did not affect p53 expression (Figure 6D). We prepared MDM2‐depleted cells (MDM KO cells) from HCT116 p53−/− cells by the CRISPR/Cas9 system and reintroduced p53 to evaluate the effect of UNC119A depletion on p53 expression. UNC119A depletion attenuated p53 expression in parent HCT116 p53−/− cells (Figure 6E, first and second lanes). p53 expression was enhanced in MDM2 KO cells (Figure 6E, third lane). UNC119A silencing did not significantly affect p53 expression in MDM2 KO cells (Figure 6E, fourth lane). Likewise, UNC119A depletion did not decrease p53 expression by treatment with Nutlin‐3, an inhibitor of MDM2 (Figure 6F). These findings support that MDM2 is implicated in the rapid degradation of p53 in the UNC119A‐negative background.

Figure 6

UNC119A stabilizes p53. A, FLAG‐p53 was expressed in HEK293FT cells with or without GFP‐UNC119A. p53 expression was enhanced by GFP‐UNC119A (left). GFP‐UNC119A was expressed in TIG3 cells (right). Endogenous p53, BAX, and p21 were detected. B, HCT116 cells were transfected with control or ‐targeted siRNA #1. 72 h later, cells were treated with 50 mg/L cycloheximide. Endogenous p53 was detected at the indicated periods of time. Signals were measured by ImageJ. The signal at time 0 was set at 1.0. C, HCT116 cells were transfected with control or ‐targeted (#1 and #2) siRNAs. 72 h later, cells were exposed to 10 J/m2 UV. Endogenous p53 was detected at the indicated periods of time. D, HCT116 cells were transfected with control, ‐targeted (#1), and/or ‐targeted siRNAs. 72 h later, cells were treated with 50 mg/L cycloheximide and, 4 h later, cells were harvested. Cell lysates were immunoblotted with indicated antibodies. E, MDM2‐depleted HCT116 p53−/− cells (MDM2 KO cells) were prepared by CRISPR/CAS9 technology. UNC119A was further knocked down in parent HCT116 p53−/− cells and MDM2 KO cells (siUNC119A #1). FLAG‐p53 was exogenously expressed in these cells. silencing did not reduce p53 expression in MDM2 KO cells. (F) HCT116 cells were transfected with control or ‐targeted (#1) siRNA. 72 h later, the cells were treated with DMSO or 10 μmol/L Nutlin‐3. 18 h later, the cells were harvested. Cell lysates were immunoblotted with the indicated antibodies

UNC119A strengthens the interaction between RASSF6 and MDM2

UNC119A enhances the stability of p53 and regulates apoptosis and cell‐cycle progression by RASSF6 and p53. RASSF6 binds MDM2 and blocks MDM2‐mediated p53 degradation.17 These findings prompted us to examine the effect of UNC119A on the interaction between RASSF6 and MDM2. HA‐MDM2 was more efficiently coimmunoprecipitated with FLAG‐RASSF6 in the presence of Myc‐UNC119A (Figure 7A, left, white arrowhead). In the reverse immunoprecipitation, GFP‐RASSF6 was more efficiently coimmunoprecipitated with FLAG‐MDM2 in the presence of Myc‐UNC119A (Figure 7A, middle, white arrowhead). In contrast to Myc‐UNC119A, Myc‐UNC119B did not enhance the interaction between HA‐MDM2 and FLAG‐RASSF6 (Figure 7A, right, white arrowhead).

Figure 7

UNC119A enhances the interaction between Ras‐association domain family 6 (RASSF6) and MDM2 and reduces p53 ubiquitination. A, HEK293FT cells were transfected with pCIneoFHF‐RASSF6 (FLAG‐RASSF6), pCIneoMyc‐UNC119Ab (Myc‐UNC119A), and pCIneoHAHA‐MDM2 (HA‐MDM2) as indicated (left). Immunoprecipitation was carried out with anti‐DYKDDDDK (1E6) beads. Immunoprecipitates were immunoblotted with the indicated antibodies. UNC119A coexpression increased the coimmunoprecipitated HA‐MDM2 (left, white arrowhead). Likewise, GFP‐RASSF6, Myc‐UNC119A, and FLAG‐MDM2 were expressed in HEK293FT cells (middle). UNC119A coexpression increased the coimmunoprecipitated GFP‐RASSF6 (middle, white arrowhead). In contrast to Myc‐UNC119A, Myc‐UNC119B did not increase the coimmunoprecipitated HA‐MDM2 (right, white arrowhead). B, Luciferase‐fused p53 (Luc‐p53) was coimmunoprecipitated with FLAG‐MDM2 in the presence or absence of Myc‐UNC119A. Luciferase activity in the immunoprecipitates was measured. Lower panels show FLAG‐MDM2 in the immunoprecipitates and Myc‐UNC119A in the input. Data are shown as mean with SEM. *P < .01; ns, not significant. C, FLAG‐His6‐p53 and HA‐UNC119A were expressed with Myc‐UNC119A and Myc‐MDM2 as indicated. Cells were harvested and FLAG‐His6‐p53 was precipitated by Ni‐NTA beads. Immunoblottings were carried out with anti‐HA antibody

Effect of UNC119A on the interaction between MDM2 and p53 and on MDM2‐dependent ubiquitination

We next examined the effect of UNC119A on the interaction between MDM2 and p53. For this purpose, we carried out the LUMIER assay by using luciferase‐fused p53 (Luc‐p53). Luc‐p53 was coimmunoprecipitated with FLAG‐MDM2 in the presence or absence of Myc‐UNC119A. Luciferase activity detected in the immunoprecipitate was not changed by Myc‐UNC119A (Figure 7B). Nevertheless, UNC119A decreased MDM2‐mediated ubiquitination of p53 (Figure 7C). This finding is consistent with the previous observation that RASSF6 does not block the interaction between MDM2 and p53, but attenuates MDM2‐mediated ubiquitination of p53.33

UNC119A depletion leads to the generation of polyploid cells

RASSF6 is important for DNA repair and prevents genomic instability.17 In the final set of experiments, we tested whether and how UNC119A is involved in DNA repair after DNA damage. HCT116 cells induced the expression of γ‐H2A.X, a hallmark of DNA damage, after exposure to VP‐16, a topoisomerase inhibitor. In control cells, γ‐H2A.X decreased in a time‐dependent way (Figure 8A). In UNC119A‐depleted cells, γ‐H2A.X was detected even under the basal condition (Figure 8A, left, siUNC119A#1, NT; right, immunoblot). Consistently, UNC119 silencing induced the phosphorylation of ATM without VP‐16 treatment (Figure 8A, right, lower immunoblot, arrow). γ‐H2A.X remained detectable even at 24 hours after VP‐16 removal in UNC119A‐depleted cells (Figure 8A, left). Another siRNA against UNC119A (siUNC119A#2) showed a similar effect on DNA repair (Figure S1D). We further cultured HCT116 cells that were exposed to VP‐16 and analyzed DNA content with FACS. UNC119A depletion generated polyploid cells after exposure to VP‐16 (Figure 8B). We also treated the cells with doxorubicin. UNC119A depletion increased polyploid cells in doxorubicin‐treated cells (Figure 8C). Finally, we examined whether UNC119A suppression causes poor prognosis in human cancers by using the PrognoScan database (http://www.prognoscan.org).29 A total of 91 datasets provided information regarding UNC119A. In 6 datasets, low expression of UNC119A was associated with shorter overall or disease‐free survival (Cox P‐value < .05) (Figure 9).

Figure 8

UNC119A depletion enhances genomic instability. A‐C, HCT116 cells were transfected with control or ‐targeted siRNA. 72 h later, cells were exposed to 50 μmol/L VP‐16 for 3 h and returned to the medium without VP‐16. In (A), cells were fixed and immunostained with anti‐γ‐H2A.X antibody at the indicated time points. Nuclei were visualized with Hoechst 33342. Cell lysates after 24 h were immunoblotted with anti‐γ‐H2A.X and anti‐phospho‐ATM antibodies. γ‐H2A.X and phosphorylated ATM were detected in UNC119A‐depleted cells without VP‐16 treatment (an arrow). In (B), cells were harvested at 96 h. DNA content was analyzed by FACS as described for Figure 3C. In (C), cells were treated with 0.1 μmol/L doxorubicin for 24 h after siRNA transfection and were cultured for 72 h in the absence of doxorubicin

Figure 9

Kaplan‐Meier plots of cancer patients with high and low expression of UNC119A. High and low UNC119A expression groups were determined according to the criteria of PrognoScan database. Kaplan‐Meier plots of cancer patients are shown for GSE2658 (multiple myeloma), GSE7849 (breast cancer), GSE16131‐GPL96 (follicular lymphoma), GSE30929 (liposarcoma), jacob‐00182‐UM (lung cancer), and MGH‐glioma (glioma). Horizontal axis and vertical axis indicate time and disease‐free survival, respectively. Cox P‐values are shown

DISCUSSION

We carried out yeast 2‐hybrid screening by use of RASSF6 as bait and obtained UNC119A. UNC119A was coimmunoprecipitated with RASSF6 from human colon cancer SW480 cells (Figure 1). The in vitro binding assay supported the direct interaction between RASSF6 and UNC119A (Figure 2). Although we could not demonstrate colocalization of endogenous RASSF6 and UNC119A by double immunostaining, RASSF6 and UNC119A showed similar distributions in rat kidney (Figure 1) and the proximity ligation assay also supported colocalization of RASSF6 and UNC119A in cells (Figure 2). UNC119A binds RASSF6, whereas UNC119B does not (Figure 2). A previous study showed that UNC119B, but not UNC119A, regulates the transport of the myristoylated ciliopathy protein nephrocystin‐3.22 Furthermore, UNC119A is reported to be localized to the centrosome, whereas UNC119B is not. This means that UNC119A and UNC119B are distinct in protein interaction and localization. UNC119A has 2 variants, UNC119Aa and UNC119Ab. UNC119Aa and UNC119Ab have 240 and 220 amino acids, respectively. The N‐terminal 204 amino acids are shared by UNC119Aa and UNC119Ab. UNC119B is composed of 251 amino acids. The N‐terminal 59 amino acids of UNC119A diverge from the N‐terminal 67 amino acids of UNC119B, whereas the middle portions of UNC119A and UNC119B are well conserved. Therefore, RASSF6 is likely to bind to the N‐terminal region of UNC119A.

Overexpression of the full‐length UNC119A causes apoptosis and cell‐cycle arrest in p53‐positive HCT116 cells, whereas UNC119A depletion attenuates UV‐induced apoptosis and cancels UV‐induced cell cycle arrest (Figures 3,4, and 5). These properties are reminiscent of the regulation of apoptosis and cell‐cycle progression by RASSF6.17 The underlying mechanism of the tumor‐suppressive role of RASSF6 is not yet fully understood, but one of the important mechanisms is the inhibition of MDM2‐mediated degradation of p53. UNC119A‐mediated apoptosis and cell‐cycle arrest also depend on p53. Mechanistically, UNC119A enhances the interaction of RASSF6 and MDM2, reduces p53 ubiquitination, and enhances p53 protein expression (Figures 6 and 7). These findings suggest that UNC119A is implicated in the regulation of the RASSF6‐MDM2‐p53 axis. However, UNC119A also induces apoptosis and inhibits BrdU incorporation in HCT116 p53−/− cells, suggesting that UNC119A can, to some extent, regulate apoptosis and cell‐cycle arrest by a certain mechanism, which is independent of p53. We also observed that exogenously expressed UNC119A is detected and interacts with RASSF6 not only in the cytoplasm but also in the nucleus. As RASSF6 interacts with MDM2 mainly in the nucleus, it is intriguing to study how the subcellular distribution of UNC119A is regulated.

UNC119A was originally reported as a human retina‐enriched gene product but is ubiquitously expressed in various tissues. In the retina, UNC119A binds transducin and RIBEYE, and plays critical roles in ribbon synapse formation.25, 26 In hematopoietic cells, UNC119A interacts with tyrosine kinases—Fyn, Lck, and Lyn—to regulate T‐cell activation, eosinophil survival, and the production of cytokines.30, 31, 32 In lung fibroblasts, UNC119A promotes myofibroblast differentiation through Fyn.33 In HeLa cells, UNC119A is localized at centrosomes and the midbody, and controls cytokinesis via Fyn.32 Thus, UNC119A plays versatile roles in many tissues depending on which lipid‐modified protein(s) it interacts with. It is an intriguing question as to which lipid‐modified protein is involved in the regulation of the RASSF6‐MDM2‐p53 axis by UNC119A.

RASSF6 suppression by DNA hypermethylation is frequently observed in human cancers and is associated with poor clinical prognosis.34 Stabilization of p53 is an important mechanism by which RASSF6 exerts its tumor‐suppressive function. In the present study, we show that UNC119A depletion impairs DNA repair after DNA damage induced by VP‐16 and doxorubicin (Figure 8B,C). These findings suggest that UNC119A shows a tumor‐suppressive role through p53 in cancer cells. Consistently, low expression of UNC119A is associated with poor prognosis in cancer patients (Figure 9). Among 91 datasets that provided information regarding UNC119A, 6 datasets showed correlation of the low expression of UNC119A with shorter survival. This ratio is apparently not high, but we need to consider the possibility that UNC119A cannot completely suppress tumor without intact RASSF6 and p53. Interestingly, high expression of UNC119A is associated with a poor prognosis in hepatocellular carcinoma.35 In 2 datasets of the PrognoScan database (GSE9893, breast cancer; and GSE11595, esophageal cancer), high UNC119A expression is associated with poor prognosis. As UNC119A activates SRC kinases, in cancers with dysregulation of RASSF6 or p53, it is possible that UNC119A rather promotes tumors. To confirm the importance of UNC119A as a tumor suppressor in human cancers, further studies are awaited.

CONFLICTS OF INTEREST

Authors declare no conflicts of interest for this article.

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