{'fraction_non_alphanumeric': 0.04553668232743043, 'fraction_numerical': 0.027827972533429706, 'mean_word_length': 4.865197117422636, 'pattern_counts': {'":': 0, '<': 5, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
CENP‐50 is required for papilloma development in the two‐stage skin carcinogenesis model

Abstract CENP‐50/U is a component of the CENP‐O complex (CENP‐O/P/Q/R/U) and localizes to the centromere throughout the cell cycle. Aberrant expression of CENP‐50/U has been reported in many types of cancers. However, as Cenp‐50/U‐deficient mice die during early embryogenesis, its functions remain poorly understood in vivo. To investigate the role of Cenp‐50/U in skin carcinogenesis, we generated Cenp‐50/U conditional knockout (K14CreER‐Cenp‐50/U fl/fl) mice and subjected them to the 7,12‐dimethylbenz(a)anthracene (DMBA)/terephthalic acid (TPA) chemical carcinogenesis protocol. As a result, early‐stage papillomas decreased in Cenp‐50/U‐deficient mice. In contrast, Cenp‐50/U‐deficient mice demonstrated almost the same carcinoma incidence as control mice. Furthermore, mRNA expression analysis using DMBA/TPA‐induced papillomas and carcinomas revealed that Cenp‐50/U expression levels in papillomas were significantly higher than in carcinomas. These results suggest that Cenp‐50/U functions mainly in early papilloma development and it has little effect on malignant conversion.

As the centromere is essential for chromosome segregation during cell division, disruption of most centromere proteins in mice can cause embryonic lethality, accompanied by enlarged nuclei containing an increased number of nucleoli, nuclear bridging, chromosome condensation and spindle fiber bundling. For example, Cenp-c null mutation results in embryonic lethality at 3.5 d, with a chromosome missegregation phenotype and mitotic arrest. 20,21 Cenp-a null mice fail to survive beyond 6.5 d post-conception, whereas heterozygous knockout mice are healthy and fertile. 22 Therefore, it is difficult to generate centromere protein-deficient mice. As this lethality is caused by a chromosome segregation defect, cultured cells with knockout of centromere proteins should be lethal. However, we demonstrated that chicken DT40 cells with knockout of each CENP-O complex protein were viable, 16,17 (Table S1), suggesting that cells with knockout of the CENP-O complex proteins were good models to study CIN. Next, we attempted to generate centromere protein-deficient cells and demonstrated that Cenp-r knockout mice were viable, 23 (Table S1). Conversely, Cenp-50/U-deficient mouse ES cells died after they exhibited abnormal mitotic behavior, unlike the chicken DT40 cells. 24 In addition, Cenp-50/U-deficient mice died during early embryogenesis (at approximately E7.5), 24 (Table S1).
Although these results were unexpected, the phenotype of cells with knockout of CENP-O complex proteins was weak compared with cells with other centromere proteins knocked out. Therefore, in this study, we generated Cenp-50/U conditional knockout mice and characterized their function related to carcinogenesis.
We have demonstrated previously the essential role of Cenp-r, which forms a complex with other Cenp-O proteins (Cenp-O/P/Q/ R/U), in the two-stage skin carcinogenesis model. Cenp-r functions as a tumor suppressor in early papilloma development, but as an oncogene at the late papilloma stage and in malignant conversion. 23 Based on this study, we hypothesized that other Cenp-O complex proteins function in skin carcinogenesis. We therefore generated K14(Keratin 14)Cre ER -Cenp-50/U fl/fl mice to investigate the function of Cenp-50/U in vivo, especially in the process of carcinogenesis.
It has been shown previously that the pathology of the two-stage chemically induced skin carcinogenesis mouse model is almost identical to the development of human skin cancers, and therefore it is an ideal model to study skin cancer initiation and growth. 25,26 In the first step of this chemically induced carcinogenesis protocol, mice were treated with a low dose of the mutagen 7,12-dimethylbenz(a) anthracene (DMBA) to start tumor development. This first chemical treatment step led to "tumor initiation." In the second step, mice were treated continuously with TPA to stimulate epidermal tumor proliferation. This second chemical treatment step influenced "tumor promotion." During tumor promotion, benign tumors, known as papillomas, are thought to develop from additional mutations caused by TPA chemical treatment. After prolonged treatment (~20 wk), some papillomas will progress into carcinogenic tumors such as squamous cell carcinomas (SCC). The roles of numerous genes and cell-signaling pathways in skin tumor development could be explored with this two-stage skin carcinogenesis model using genetically engineered mouse models. [27][28][29][30][31][32][33] In this study, we demonstrated the function of Cenp-50/U in the epidermis and its role in two-stage skin carcinogenesis. We clarified the effects of Cenp-50/U deficiency on mouse normal epidermis and its oncogenic role in papillomagenesis.

| Mice
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The protocol was approved by the Committee on the Ethics of Animal Experiments of Chiba Cancer Center (Permit Number: [13][14][15][16][17][18]. All efforts were made to minimize suffering. C57BL/6 strain mice were used as recipients for targeted ES cells and as the background strain in this study. The generation of mice carrying the floxed allele of the Cenp-50/U gene (Cenp-50/U fl/fl ) is described elsewhere. 24 Cenp-50/U fl/fl mice were mated to K14-Cre ER transgenic mice. 38 K14-Cre ER transgenic mice were obtained from the Jackson Laboratory (JaX 005107). To disrupt Cenp-50/U, K14Cre ER -Cenp-50/ U fl/fl and Cenp-50/U fl/fl mice at 8 wk old were injected subcutaneously 5 times with 1.0 mg/mL of tamoxifen (Sigma-Aldrich) that had been prepared following the manufacturer's instructions by completely dissolving tamoxifen into 1 mL of 100% ethanol at 50°C, adding 9 mL of sunflower oil and mixing this thoroughly by vortexing.

| RT-PCR and quantitative real-time RT-PCR
Total RNA was isolated from normal skins, papillomas, carcinomas and metastatic carcinomas of mice after 40 wk of DMBA/ TPA treatment using TRIzol (Invitrogen) in accordance with the manufacturer's protocol. cDNA was generated with the iScript™ Select cDNA Synthesis Kit (Bio-Rad) using 100 ng of DNasepretreated total RNA. RT-PCR was performed using the Prime Taq DNA Polymerase Kit (Genet Bio). The products were subjected to agarose gel electrophoresis. Gapdh was used as a control. qRT-PCR was performed using GeneAce SYBR ® qPCR Mix α Low ROX following the manufacturer's protocol (NIPPON GENE). mRNA expression levels were measured using the Applied Biosystems ® 7500 system (Life Technologies) and normalized to the levels of
The endogenous peroxidase activity in the specimens was blocked

| Statistical analysis
At least 3 replicates were performed for all experiments. The significance of differences was calculated using unpaired one-and two-tailed Student t tests or two-way ANOVA. A P-value < .05 was considered significant and a value < .01 was highly significant.
These analyses were performed using GraphPad Prism software (GraphPad). Cenp-r expression levels in carcinomas were significantly higher than in papillomas ( Figure 1B). In contrast, Cenp-50/U expression levels in carcinomas were significantly lower than in papillomas ( Figure 1A). In other words, Cenp-50/U and Cenp-r exhibited opposite expression profiles in skin carcinogenesis.

| mRNA expression profiles of the Cenp-O complex are altered in skin tumors
Our previous study revealed that Cenp-r functions as a tumor suppressor-type gene in papilloma development and as an oncogene-type gene during the progression stage. 23 These results suggested that Cenp-50/U plays a opposing role to Cenp-r in skin carcinogenesis.

| Generation of Cenp-50/U conditional knockout mice
A previous study demonstrated that Cenp-50/U knockout mice die during early embryogenesis (approximately E7.5). 24 To investigate the function of Cenp-50/U in vivo, we generated mice harboring conditional alleles of Cenp-50/U, in which exons 4-6 of the Cenp-50/U gene are flanked by loxP sites (Figure 2A). First, we isolated mice with Cenp-50/U fl/+ alleles and finally generated Cenp-50/U fl/fl alleles by intercrossing ( Figure 2B). Next, we crossed the Cenp-50/ U fl/fl conditional knockout mice with mice carrying a K14Cre ER allele, 34 which is specifically expressed in the epidermis, to generate

| Cenp-50/U deficiency affects cell proliferation in normal skins
As we successfully generated epidermis-specific Cenp-50/U knockout mice, we investigated the function of Cenp-50/U in the epidermis using these mice. First, we performed histologi-

| Cenp-50/U deficiency partially inhibits papillomagenesis
Our previous study demonstrated the essential function of Cenp-r in the two-stage skin carcinogenesis model. 23 In this study, our expression analysis suggested that Cenp-50/U functions might be opposite to those of Cenp-r in skin carcinogenesis (Figure 1 Table S2).
In addition, we classified these papillomas into 3 categories based on size (<2, 2-6 and >6 mm). K14Cre ER -Cenp-50/U fl/fl mice demonstrated a significant decrease only in small papillomas < 2 mm in diameter between 10 and 14 wk after initiation ( Figure 4B-D, Table S2). These results suggested that Cenp-50/U mainly functions in early papilloma development, but has a weak effect on papilloma growth.

| Cenp-50/U deficiency increases apoptotic cells in papillomas
Skin carcinogenesis experiments revealed that Cenp-50/U functions in early papilloma development. Therefore, we investigated cell proliferation and apoptosis in papillomas of K14Cre ER -Cenp-50/ U fl/fl and Cenp-50/U fl/fl mice. First, we performed histological analysis using early-stage papillomas (<6 mm) from K14Cre ER -Cenp-50/ U fl/fl and Cenp-50/U fl/fl mice. As HE staining revealed no significant morphological changes between these mice ( Figure S1C,D), we carried out immunohistochemical analysis with the cell proliferation marker Ki67 and TUNEL staining to investigate apoptotic cells in papillomas. Papillomas of K14Cre ER -Cenp-50/U fl/fl mice had a significantly higher number of TUNEL-positive cells than those of Cenp-50/ U fl/fl mice ( Figure 5C,D,F). Conversely, no significant difference between K14Cre ER -Cenp-50/U fl/fl and Cenp-50/U fl/fl mice was observed by immunohistochemical analysis with the cell proliferation marker Ki67 ( Figure 5A,B,E). Therefore, Cenp-50/U deficiency promotes cell death by inducing apoptotic cells rather than controlling cell proliferation in papillomas. These finding are consistent with previous reports, which stated that CENP-50/U-deficient DT40 cells undergo apoptosis. 17 Therefore, Cenp-50/U regulates abnormal mitosis both in mice and cell lines.

| Cenp-50/U is required for early papilloma development, but has little effect on malignant conversion
To investigate the effects of Cenp-50/U on malignant conversion, we monitored carcinoma development up to 34 wk after initiation.
The carcinoma incidence was almost the same in K14Cre ER -Cenp-50/  Figure 6A). In addition, malignant conversion ratio per papilloma was almost the same in K14Cre ER -Cenp-50/U fl/fl (13.4%) and Cenp-50/U fl/fl (16.2%) mice at 34 wk after initiation ( Figure 6B). We then performed histological analysis using carcinomas from K14Cre ER -Cenp-50/U fl/fl and Cenp-50/U fl/fl mice. HE staining revealed no significant morphological changes between these mice (Fig. S1E,F). These results suggested that Cenp-50/U has almost no effect on malignant conversion.
In this study, we found a decrease in early-stage papillomas in Cenp-50/U-deficient mice due to the induction of cell death, but there was little effect on malignant conversion. Taken together, Cenp-50/U mainly functions in papilloma development, and does not play a role in papilloma growth or malignant conversion (Figure 7).

| D ISCUSS I ON
In this study, we specifically knocked out Cenp-50/U in the epidermis, and clarified its function in two-stage skin carcinogenesis.
Cenp-50/U deficiency promoted cell death in normal mouse skins.
In addition, our skin carcinogenesis experiments revealed that Cenp-50/U deficiency downregulates early papillomagenesis by inducing cell death.
Centromere proteins play a fundamental role in accurate chromosome segregation during mitosis and meiosis. Chromosome segregation errors cause genetic diseases, including some cancers.
In addition, several centromere proteins play an important role in embryonic development. 4,[20][21][22]35 Therefore, it was difficult to generate centromere protein-deficient mice and perform functional analysis in vivo. As Cenp-50/U-deficient mice also died during early embryogenesis (approximately E7.5), 24 its functions were poorly understood. Therefore, we generated Cenp-50/U conditional knockout mice and analyzed its function in vivo.
Cenp-50/U is a centromere protein, but unlike other centromere Recent studies have reported aberrant expression of CENP-50/U in many types of cancers, including breast cancer, prostate cancer, colorectal carcinoma, ovarian cancer, glioblastoma, erythroleukemia, and lymphoma. 36,37 These might be related to variety of CENP-50/U effects in various cell types and also suggested that CENP-50/U is a molecular marker for cancer therapy. We demonstrated that Cenp-50/U is highly expressed in papillomas and is involved in the early papillomagenesis stage, and this positive correlation suggested that Cenp-50/U can be a molecular marker in carcinogenesis.

ACK N OWLED G M ENTS
This study was supported by JSPS KAKENHI Grant Number 23790435, 25221106 and 15H05972.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.