Arsenic Suppresses Cell Survival via Pirh2-mediated Proteasomal Degradation of ΔNp63 Protein*

Background: ΔNp63 exhibits an oncogenic potential and is often overexpressed in squamous cell carcinomas. Results: Arsenic degrades ΔNp63 protein at least in part via Pirh2-mediated proteolysis, and inhibition of ΔNp63 expression facilitates tumor cells to arsenic-induced death. Conclusion: Arsenic trioxide induces Pirh2 to target ΔNp63 for degradation. Significance: Targeting ΔNp63 may be explored to manage tumors overexpressing ΔNp63. Transcription factor p63, a member of the p53 family, shares a high degree of sequence similarity with p53. Because of transcription from two distinct promoters, the p63 gene encodes two isoforms, TAp63 and ΔNp63. Although TAp63 acts as a tumor suppressor, ΔNp63 functions as an oncogene and is often overexpressed in squamous cell carcinomas. Thus, therapeutic agents targeting ΔNp63 might be used to manage tumors that overexpress ΔNp63. Here we found that arsenic trioxide, a frontline agent for acute promyelocytic leukemia, inhibits ΔNp63 but not TAp63 expression in time- and dose-dependent manners. In addition, we found that arsenic trioxide decreases the stability of ΔNp63 protein via a proteasome-dependent pathway but has little effect on the level of ΔNp63 transcript. Furthermore, we found that arsenic trioxide activates the Pirh2 promoter and consequently induces Pirh2 expression. Consistent with this, we found that knockdown of Pirh2 inhibits, whereas ectopic expression of Pirh2 enhances, arsenic-induced degradation of ΔNp63 protein. Importantly, we found that knockdown of ΔNp63 sensitizes, whereas ectopic expression of ΔNp63 inhibits, growth suppression induced by arsenic. Together, these data suggest that arsenic degrades ΔNp63 protein at least in part via Pirh2-dependent proteolysis and that inhibition of ΔNp63 expression facilitates tumor cells to arsenic-induced death.

The transcription factor p63, a member of the p53 family, shares a high degree of sequence similarity with p53, particularly in the central DNA-binding domain (1). Because of transcription from two distinct promoters, the p63 gene yields two types of transcripts, TAp63 and ⌬Np63 (2,3). Both TAp63 and ⌬Np63 transcripts consist of at least five variants because of alternative splicing at the 3Ј terminus (4). TAp63 contains an activation domain similar to the first activation domain in p53 and, thus, has a strong transcriptional activity. Like p53, overexpression of TAp63 is capable of inducing cell cycle arrest and apoptosis (5,6). In contrast, ⌬Np63 loses such an activation domain but gains 14 unique residues at the N terminus. These 14 residues, together with the adjacent proline-rich region, constitute an activation domain for ⌬Np63 (7,8). Thus, ⌬Np63 also possesses a transcriptional activity.
Although p53 functions as a classical tumor suppressor, the role for p63 in tumorigenesis is still uncertain. A study showed that p63 ϩ/Ϫ mice are predisposed to develop spontaneous tumors (9), suggesting that the p63 gene acts as a tumor suppressor. Consistently, TAp63 is found to induce senescence and suppress tumorigenesis in TAp63 conditional knockout mice (10). On the other hand, many studies have highlighted the oncogenic properties of ⌬Np63. ⌬Np63 is frequently found to be amplified and overexpressed in squamous cell carcinomas (11,12). ⌬Np63␣ overexpression promotes cell proliferation in vitro and tumor growth in vivo (13,14). In addition, ⌬Np63␣ represses apoptosis-related genes and, thereby, contributes to chemoresistance of hepatocellular carcinoma (15). In line with this, knockdown of ⌬Np63␣ induces apoptosis and sensitizes cells to DNA damage (16). Clinically, high levels of ⌬Np63 expression in tumors are associated with an aggressive phenotype and chemoresistance (17,18).
The role of ⌬Np63 in tumorigenesis might be partially due to its transcriptional activity. We found previously that GPX2 and BMP7, two direct targets of ⌬Np63, inhibit oxidative stressinduced apoptosis in a p53-dependent manner and are required for survival of tumor cells (19,20). Other studies also found that ⌬Np63 regulates genes involved in cell cycle progression and cell survival (2,21). Interestingly, ⌬Np63 was found to regulate the splicing pattern of CD44, which may affect the adhesion and metastasis of cancer cells (14). The oncogenic potential of ⌬Np63 might be also due to its dominant-negative activities to suppress p53-or TAp63-mediated transactivation (2,7,15,23). In addition, ⌬Np63␣ is found to exhibit a survival function in squamous epithelial malignancy by repressing TAp73-dependent pro-apoptotic activity (24). However, the unique transcriptional and dominant-negative abilities in ⌬Np63 may be explored for a new therapeutic approach modulating ⌬Np63 expression to manage tumors that overexpress ⌬Np63 but harbor TAp63, TAp73, and/or wild-type p53.
Arsenic is a metalloid with a substantial efficacy in treating patients with acute promyelocytic leukemia, myeloma, and myelodysplastic syndromes (25). Evidences showed that arsenic functions as an anticancer agent at least in part via targeting proteins for degradation (26 -31). Recently, we found that arsenic targets mutant p53 for degradation, which mediates arsenic-induced growth suppression in solid tumor cells (32). The structural and functional similarity between ⌬Np63 and mutant p53 prompts us to examine whether arsenic has an effect on ⌬Np63 expression. Indeed, we found that arsenic induces ⌬Np63 degradation via the proteasome-dependent pathway. Our finding suggests that targeting ⌬Np63 with arsenic may be explored further to manage tumors that are carrying a high level of ⌬Np63.

EXPERIMENTAL PROCEDURES
Cell Culture-Human keratinocyte cell line HaCaT, human cervical carcinoma cell line ME-180, and human pancreatic cancer cell line MIA PaCa-2 were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone). The human mammary epithelial cell line MCF10A was cultured in DMEM/F12 supplemented with 5% donor horse serum, 20 ng/ml of EGF, 10 g/ml of insulin, 0.5 g/ml of hydrocortisone, and 100 ng/ml of cholera toxin.
Antibodies-Mouse anti-p63(4A4) was purchased from Santa Cruz Biotechnology, Inc. Rabbit anti-Pirh2 was purchased from Bethyl Laboratories, Inc. Rabbit anti-actin and mouse anti-FLAG were purchased from Sigma.
Protein Half-life Assay-ME-180 cells were left untreated or pretreated with 10 M arsenic trioxide for 1 h and then incubated with 50 g/ml of cycloheximide to inhibit de novo protein synthesis for various times. The relative level of ⌬Np63␣ protein was quantified by densitometry and normalized by the level of actin protein, which was then plotted versus time (h) to calculate the half-life of ⌬Np63␣.
Proteasome Inhibition Assay-Cells were seeded for 24 h, left untreated or pretreated with proteasome inhibitor MG132 (5 M) for 4 h, and then treated with arsenic trioxide for various times.
Luciferase Assay-The dual luciferase assay was done in triplicate according to the instructions of the manufacturer (Promega). Briefly, 0.5 g of a luciferase reporter and 3 ng of pRL-SV40-Renilla luciferase reporter (Promega) were cotransfected into HaCaT cells by using the Expressfect transfection reagent (Denville). The fold increase in relative luciferase activity is a product of the luciferase activity induced by arsenic treatment divided by that induced by mock treatment.
Cell Survival Assay-To determine whether ectopically expressed ⌬Np63␣ makes tumor cells resistant to arsenic trioxide treatment, ME-180 cells were transiently transfected with pcDNA3 or pcDNA3-myc-⌬Np63␣ for 1 day and then left untreated or treated with 7.5 M arsenic trioxide for 2 days. The survival cells were collected and counted.
To determine whether knockdown of ⌬Np63 sensitizes tumor cells to arsenic trioxide treatment, ME-180 cells were transfected with scrambled siRNA or siRNA against p63 for 1 day and then left untreated or treated with 7.5 M arsenic trioxide for 2 days. The survival cells were collected and counted.
Statistics-All experiments were performed in triplicates. Two-group comparisons were analyzed by two-sided Student's t test. p values were calculated, and p Ͻ 0.05 was considered significant.

Arsenic Trioxide Inhibits ⌬Np63 Expression in Time-and
Dose-dependent Manners-Arsenic trioxide is a therapeutic agent for acute promyelocytic leukemia, which potentially targets proteins for degradation (26 -31). We found previously that arsenic-induced degradation of mutant p53 leads to growth suppression (32). ⌬Np63, a member of p53 family, shares the high degree of sequence similarity to mutant p53 (1) and confers a proliferative and chemoresistant advantage to tumor cells (16,18). Thus, we rationalized that targeting ⌬Np63 with arsenic may be explored as a therapeutic strategy to tumors that carry a high level of ⌬Np63.
Studies showed that although TAp63 is expressed at a very low level, ⌬Np63, especially ⌬Np63␣, is highly expressed in MCF10A mammary epithelial cells, HaCaT keratinocyte cells, and ME-180 cervical carcinoma cells (34 -36). Thus, the effect of arsenic on ⌬Np63 expression was examined in these cells. We found that upon treatment with 10 M arsenic trioxide, the ⌬Np63, a Novel Target of Arsenic level of ⌬Np63␣ protein was promptly decreased in ME-180 cells, reaching maximum reduction within 12-24 h (Fig. 1A, top  panel). The level of actin protein was used as a loading control (Fig. 1A, bottom panel). Similar results were found in HaCaT cells (Fig. 1B) and MCF10A cells (C).
To determine the dose-response relationship between arsenic and ⌬Np63␣ expression, ME-180 cells were treated with various doses of arsenic trioxide for 12 h. We found that the level of ⌬Np63␣ protein in ME-180 cells was decreased markedly by arsenic trioxide at a concentration of as low as 2.5 M (Fig. 1D). This concentration is close to that for degradation of mutant p53 in cultured cells (32) and the plasma peak values of 1.5-3.4 M in acute promyelocytic leukemia patients treated with arsenic trioxide (37). As arsenic concentration was increased to 5 M, the level of ⌬Np63␣ was decreased further in ME-180 cells (Fig. 1D). However, the arsenic-induced decrease of ⌬Np63␣ was not further enhanced at concentrations of more than 5 M (Fig. 1D). A similar result was seen in HaCaT cells (Fig. 1E) and MCF10A cells (F).
Because TAp63 isoforms function as tumor suppressors by inducing senescence (10) and preventing invasiveness and metastasis (38), it is vitally important to determine how arsenic alters TAp63 expression in tumor cells. For this purpose, MIA PaCa-2 cells, which carry a high level of TAp63␣, were left untreated or treated with arsenic trioxide as indicated (Fig. 1, G  and H). Interestingly, we found that arsenic trioxide had little, if any, effect on the level of TAp63␣ protein in MIA PaCa-2 cells (Fig. 1, G and H).
Arsenic Trioxide Decreases the Stability of ⌬Np63 Protein but Has Little Effect on the Level of ⌬Np63 Transcript-To examine whether arsenic-induced reduction of ⌬Np63 is through a posttranslational mechanism, ME-180 cells were treated with 50 g/ml of cycloheximide for various times in the absence or presence of arsenic trioxide. The relative level of ⌬Np63␣ protein was quantified by densitometry and normalized by level of actin protein, which was then plotted versus time (h) to calculate the half-life of ⌬Np63␣. We found that the half-life for ⌬Np63␣ protein was decreased from about 2 h in the control cells to about 1 h in cells treated with arsenic trioxide (Fig. 2, A  and B).
Next, to test whether ⌬Np63 transcription is suppressed by arsenic trioxide, RT-PCR analysis was performed to measure the level of ⌬Np63 transcript in ME-180, HaCaT, and MCF10A cells, which were mock-treated or treated with 10 M arsenic trioxide as indicated. The actin transcript was measured as a control. We found that the level of ⌬Np63 mRNA was not obviously altered by arsenic trioxide in ME-180 (Fig. 2C), HaCaT (D), and MCF10A (E) cells. These results suggest that arsenic trioxide does not inhibit ⌬Np63 transcription but rather decreases the stability of ⌬Np63 protein.
Arsenic Trioxide Degrades ⌬Np63 Protein via the Proteasome-dependent Pathway-Because the stability of ⌬Np63 protein was decreased by arsenic, we further explored whether ⌬Np63 is degraded via the proteasome-dependent pathway. To test this, ME-180 cells were left untreated or treated with 4 M MG132, an inhibitor of 26 S proteasome, in the absence or presence of arsenic trioxide. We found that arsenic-induced ⌬Np63 degradation was almost abolished by MG132 (Fig. 3A). Similarly, we found that arsenic-induced degradation of ⌬Np63 protein was inhibited by MG132 in HaCaT cells (Fig. 3B). Taken together, these results suggest that arsenic-induced ⌬Np63 degradation is at least in part via the proteasome-dependent pathway.
Here, multiple E3 ligases, known to degrade p63 or other p53 family members, were screened in arsenic-treated cells with a Western blot assay. We found that upon treatment with arsenic trioxide, the level of Pirh2 protein was promptly increased in HaCaT cells (Fig. 4A) and ME-180 cells (B). To further examine whether arsenic increased the level of Pirh2 mRNA, RT-PCR analysis was performed with HaCaT cells, which were left untreated or treated with 5 M arsenic trioxide for 6 or 12 h. We found that the level of Pirh2 mRNA in HaCaT cells was obviously increased by arsenic (Fig. 4C).
Next, to explore how arsenic trioxide transactivates the Pirh2 gene, several luciferase reporters containing various regions of the Pirh2 promoter were generated (Fig. 4D). Each of these reporters was cotransfected into HaCaT cells with pRL-SV40-Renilla. We found that arsenic was able to increase the luciferase activities of all the Pirh2 promoter reporters (Fig. 4E). Interestingly, the effect of arsenic trioxide on the luciferase activity reached the maximum in Pirh2-500 promoter reporter and then slightly decreased in longer promoter reporters. Thus, it is most likely that the response element to arsenic treatment is located in the proximal region of the Pirh2 promoter. Together, these results suggest that arsenic trioxide can transactivate the expression of Pirh2 gene.
Knockdown of Pirh2 Inhibits whereas Ectopic Expression of Pirh2 Promotes Arsenic-induced Degradation of ⌬Np63 Protein-To further examine whether Pirh2 mediates arsenic-induced degradation of ⌬Np63 protein, ME-180 and HaCaT cells were transiently transfected with scrambled siRNA or siRNA against Pirh2 for 3 days and then mock-treated or treated with arsenic. We showed that the level of Pirh2 was significantly decreased by Pirh2 but not scrambled siRNA (Fig. 5, A and B). Importantly, we found that Pirh2 knockdown obviously increased ⌬Np63 expression regardless of arsenic treatment (Fig. 5, A and  B, compare lanes 1 and 3 with lanes 2 and 4, respectively). We also noted that arsenic treatment increased Pirh2 expression concomitantly with a decreased expression of ⌬Np63 (Fig. 5, A  and B, compare lane 1 with lane 3).
Next, we examined whether ectopic expression of Pirh2 enhances arsenic-induced degradation of ⌬Np63 protein. Consistently, we found that upon ectopic expression of Pirh2 in ME-180 and HaCaT cells, the level of ⌬Np63 protein was obviously decreased under normal and arsenic-treated conditions (Fig. 5, C and D, compare lanes 1 and 3 with lanes 2 and 4,  respectively). Together, these data suggest that arsenic-induced degradation of ⌬Np63 protein is at least in part mediated by Pirh2 E3 ligase.
Knockdown of ⌬Np63 Sensitizes whereas Ectopic Expression of ⌬Np63 Desensitizes Tumor Cells to Arsenic Treatment-Because ⌬Np63 is implicated in promoting cell survival and conferring tumor cells resistant to DNA damage (16), we tested whether ⌬Np63 knockdown affects cell survival in arsenictreated cells. To this end, ME-180 cells were transfected with scrambled siRNA or siRNA against p63. We found that the level of ⌬Np63␣ was significantly decreased by p63 but not scrambled siRNA (Fig. 6A, compare lane 1 with lane 2). We also found that when combined with arsenic treatment, the level of ⌬Np63␣ was further decreased in ME-180 cells with p63 knockdown (Fig. 6A, compare lane 4 with lanes 1-3). In addition, we found that short-term knockdown of ⌬Np63 alone had little effect on cell survival in ME-180 cells (Fig. 6B). However, we found that upon arsenic treatment, ⌬Np63 knockdown significantly reduced the number of survival cells by 42.2% as compared with that in control cells (Fig. 6B).
Next, we tested whether ectopic expression of ⌬Np63 is capable of making tumor cells resistant to arsenic treatment. For this purpose, ME-180 cells were transiently transfected with an empty vector or a vector expressing ⌬Np63␣ for 24 h and then treated with or without arsenic trioxide for 48 h. We found that the level of ⌬Np63␣ was increased significantly in ME-180 cells transfected with a vector expressing ⌬Np63␣, FIGURE 2. Arsenic trioxide decreases the stability of ⌬Np63␣ protein but has little effect on the level of ⌬Np63 transcript. A, the half-life of ⌬Np63␣ protein was shortened by arsenic trioxide in ME-180 cells. Western blot analyses were prepared with extracts from ME-180 cells that were treated with cycloheximide (50 g/ml) (CHX) in the absence or presence of 10 M arsenic trioxide for 0 -8 h and then probed with antibodies against ⌬Np63 and actin, respectively. B, the relative levels of ⌬Np63 protein measured in A were normalized by levels of actin protein and then plotted versus time. C-E, the level of ⌬Np63 transcript is not obviously altered by arsenic trioxide. RT-PCR analysis was performed with total RNAs isolated from ME-180 (C), HaCaT (D), and MCF10A (E) cells untreated or treated with 10 M arsenic trioxide for 6 or 9 h. Actin mRNA was amplified as a loading control. ⌬Np63, a Novel Target of Arsenic regardless of arsenic treatment (Fig. 7A, compare lanes 1 and 3  with lanes 2 and 4, respectively). As expected, we found that arsenic also decreased the ectopically expressed ⌬Np63␣ (Fig.  7A, compare lane 2 with lane 4). Furthermore, we found that upon arsenic treatment, ectopic expression of ⌬Np63 significantly increased the number of survival cells by 1.6-fold of that in cells transfected with an empty vector. We would like to mention that short-term overexpression of ⌬Np63 alone had little effect on cell survival in ME-180 cells (Fig. 7B). Together, these results suggest that ⌬Np63 plays a role in arsenic-induced inhibition on cell survival and that knockdown of ⌬Np63 sensitizes tumor cells to arsenic trioxide.

DISCUSSION
⌬Np63 is often highly expressed in squamous cell carcinomas (11,12,44). In addition to transactivation of pro-survival genes, ⌬Np63 protein is dominant-negative over TAp63, TAp73, and wild-type p53 (2,7,15,23,24). Thus, the imbalance of ⌬Np63 with TA isoforms of the p53 family members and wild-type p53 may lead to tumorigenesis via altered expression  HaCaT (B) cells that were transfected with scrambled siRNA or siRNA against Pirh2 for 3 days and then treated with 5 M arsenic trioxide for 9 h. The blots were then probed with antibodies against Pirh2, ⌬Np63, and actin, respectively. C and D. Western blot analyses were prepared with extracts from ME-180 (C) and HaCaT (D) cells transfected with pcDNA3 or pcDNA3-2ϫFLAG-Pirh2 for 2 days and then treated with 5 M arsenic trioxide for 9 h. The blots were then probed with antibodies against the FLAG tag, ⌬Np63, and actin, respectively.  of genes related to cell cycle arrest, apoptosis, and inhibition of metastasis. Here we showed that arsenic trioxide, a frontline therapeutic agent for acute promyelocytic leukemia, inhibits ⌬Np63 but not TAp63 expression in time-and dose-dependent manners. In addition, we found that arsenic trioxide destabilizes ⌬Np63 protein via the proteasome-dependent pathway. Importantly, we found that knockdown of ⌬Np63 sensitizes whereas ectopic expression of ⌬Np63 inhibits growth suppression induced by arsenic trioxide. Thus, identification of arsenic trioxide as an effective agent for ⌬Np63 but not TAp63 degradation provides a promising approach to promptly decrease the level of ⌬Np63 in tumor cells. This finding is of vital importance and potentially developed as a therapeutic strategy to tumors highly expressing ⌬Np63, given that high ⌬Np63 expression confers proliferative and chemoresistant advantage to tumor cells (16 -18).
In an effort to probe into the mechanisms by which arsenic promotes ⌬Np63 degradation, we found that arsenic potently up-regulates the level of Pirh2 E3 ligase. Knockdown of Pirh2 inhibits whereas ectopic expression of Pirh2 promotes arsenicinduced degradation of ⌬Np63 protein. Pirh2, a p53-induced RING finger E3 ubiquitin ligase, is involved in the negative regulation of both TAp63 and ⌬Np63 expression through physical interaction and ubiquitin-mediated and proteasome-dependent proteolysis (43). Interestingly, we found that the protein level of ⌬Np63 but not TAp63 is decreased by arsenic. This result is similar to the selective decrease of mutant p53 protein, but not wild-type p53 protein, in arsenic-treated cells (32). This may be due to arsenic-activated signal pathways related to reactive oxygen species (45,46). Consistent with the postulation, reactive oxygen species generated from hypoxia and reoxygenation increase the expression of p63 protein in human lymphocytes (47), a group of cells in which TAp63 is dominantly expressed (48).
Previously, Pirh2 was regarded as an oncogene because of overexpression in tumor tissues (49,50) and targeting several tumor suppressors for proteasome-dependent degradation, such as p53 (51), TAp73 (52), and p27 (53). However, Pirh2 has an opposing function by suppressing tumorigenesis. A recent study showed that Pirh2 expression is reduced in various human cancers and that lower levels of Pirh2 expression correlate with decreased survival of patients with lung, breast, or ovarian cancer (22). Furthermore, Pirh2 negatively regulates the expression of c-Myc oncoprotein in vivo (22). Here, we showed that arsenic treatment induces Pirh2, which then targets ⌬Np63 for proteasome-dependent degradation. Thus, Pirh2 may function as a tumor suppressor. Although the Pirh2 promoter was found to be activated in response to treatment of arsenic trioxide, we are still challenged with unresolved questions, including the identity of the transcriptional factor recruited by arsenic to induce Pirh2 expression. Thus, future studies are warranted to solve this issue.