The Candidate Tumor Suppressor Gene ZAC Is Involved in Keratinocyte Differentiation and Its Expression Is Lost in Basal Cell Carcinomas

Eugenia Basyuk,1,2 Vincent Coulon,3 Anne Le Digarcher,1 Marjorie Coisy-Quivy,2 Jean-Pierre Moles,3 Alberto Gandarillas,2,3 and Laurent Journot1

1Institut de Geńomique Fonctionnelle, CNRS-UMR5203, INSERM-U661, Universite´Montpellier 1, Universite´Montpellier 2; 2Institut de Geńe´tique Molećulaire, CNRS-UMR5535, Universite´Montpellier 2; and 3Laboratoire de Dermatologie Molećulaire, IURC, EA3754, Montpellier, France

Abstract displays transactivation or transrepression activities depending ZAC is a zinc finger transcription factor that induces on the precise arrangement of its binding sites (2), and apoptosis and cell cycle arrest in various cell lines. accumulating evidence suggests its involvement in the control The corresponding gene is maternally imprinted and of cell proliferation and differentiation. Thus, inhibition of Zac1 localized on chromosome 6q24-q25, a region harboring expression by antisense oligonucleotides enhances pituitary cell an unidentified tumor suppressor gene for a variety proliferation (3), and expression of Lot1, the rat orthologue of of solid neoplasms. ZAC expression is lost or Zac1, is lost during the spontaneous transformation of rat ovary down-regulated in some breast, ovary, and pituitary surface epithelial cells in vitro (4). Interestingly, Zac1/Lot1 is tumors and in an in vitro model of ovary epithelial negatively regulated by epidermal growth factor, suggesting cell transformation. In the present study, we examined that it might be involved in a feedback loop controlling cell ZAC expression in normal skin and found a high proliferation on mitogen activation (5). In addition to its role as expression level in basal keratinocytes and a lower, a transcription factor, Zac1 serves as a transcriptional more heterogeneous, expression in the first suprabasal coactivator or corepressor of nuclear receptors and binds to differentiating layers of epidermis. In vitro, ZAC was the nuclear receptor coactivators CBP/p300 and p160 (6). More up-regulated following induction of keratinocyte recently, it was suggested that Zac1 is a transcriptional differentiation. Conversely, ZAC expression triggered coactivator for p53 (7) and that it regulates the proapoptotic keratinocyte differentiation as indicated by induction of gene Apaf-1 (8). involucrin expression. Interestingly, we found a dramatic The human orthologue ZAC/LOT1/PLAGL1 is widely loss of ZAC expression in basal cell carcinoma, a expressed in normal tissues and the corresponding protein neoplasm characterized by a relatively undifferentiated binds DNA and displays transactivation activity (9). As its morphology. In contrast, ZAC expression was mouse counterpart, human ZAC displays an antiproliferative maintained in squamous cell carcinomas that retain activity by inducing apoptosis and cell cycle arrest in the squamous differentiated phenotype. Altogether, transfected cells. In addition, alternative splicing of human these data suggest a role for ZAC at an early stage of ZAC transcript generates two mRNA species encoding proteins keratinocyte differentiation and further support its role in with different number of zinc finger domains (5 versus 7), carcinogenesis. (Mol Cancer Res 2005;3(9):483–92) which differentially regulate apoptosis and cell cycle arrest (10). Noteworthy, ZAC maps to chromosome 6q24-q25 (9, 11), Introduction a region frequently deleted in many solid tumors and thought Zac1 is a mouse zinc finger transcription factor, which to harbor at least one tumor suppressor gene. Loss of induces apoptosis and cell cycle arrest in vitro, and abrogates heterozygosity at 6q24-q25 occurs in breast and ovary cancer, tumor formation in nude mice (1). Zac1 binds DNA and melanomas, astrocytomas, and renal cell carcinomas. The functional properties of ZAC and the chromosomal location of the corresponding gene make it a plausible candidate for the tumor suppressor gene at 6q24-q25. Received 2/22/05; revised 8/5/05; accepted 8/11/05. Grant support: Centre National de la Recherche Scientifique, La Ligue In this context, loss or down-regulation of ZAC expression is Nationale contre le Cancer, and L’Association pour la Recherche contre le observed in a large proportion of mammary (12) and ovary Cancer (A. Gandarillas and L. Journot), La Ligue Nationale contre le Cancer (11, 13) tumor-derived cell lines and tumors, in nonfunctioning fellowship (V. Coulon), and European Molecular Biology Organization fellowship (A. Gandarillas). pituitary adenomas (14), and in head and neck squamous cell The costs of publication of this article were defrayed in part by the payment of carcinoma (SCC; ref. 15). Expression profiling of human breast page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tumors (16), lung adenocarcinomas (17), and breast tumor– Requests for reprints: Laurent Journot, Institut de Ge´nomique Fonctionnelle, derived cell lines (16, 18) with microarrays confirmed down- 141, rue de la cardonille, F-34094 Montpellier Cedex 5, France. Phone: 33-467- regulation of ZAC expression. We found that ZAC expression is 142-932; Fax: 33-467-542-432. E-mail: [email protected] Copyright D 2005 American Association for Cancer Research. reinduced in breast tumor–derived cell lines by treatment with doi:10.1158/1541-7786.MCR-05-0019 the demethylating agent azacytidine, suggesting that the

methylation status of ZAC gene critically modulates its and two common types of nonmelanoma skin cancer [i.e., expression (12). This hypothesis was further supported by the basal cell carcinomas (BCC) and SCCs]. Our data suggest a demonstration of ZAC imprinting in human (19, 20) and mice role for ZAC in keratinocyte differentiation and BCC (21) and by the finding that methylation of ZAC promoter is formation. responsible for its observed monoallelic expression (22, 23). Although the above-mentioned data support a role for ZAC as a Results tumor suppressor gene, its precise mechanism of action and ZAC Expression in Normal Skin target genes are mostly unknown at present. We first examined ZAC expression in five normal human Because ZAC is potentially involved in the homeostasis of skin samples using nonradioactive in situ hybridization with mammary and ovary epithelial cells, we assessed its digoxigenin-labeled riboprobes. As a positive control for in situ expression in different epithelial tissues, including epidermis. hybridization, we used a probe for the differentiation marker Human epidermis is a stratified, constantly renewed epithelium cytokeratin K1, which prominently labeled the suprabasal in which proliferation and differentiation are compartmental- spinous layers of epidermis and, to a lesser extent, the granular ized and tightly controlled. It consists of basal, suprabasal layer but not the basal layer (Fig. 1A). As a negative control, (lower spinous, upper spinous, and granular), and cornified identical samples were hybridized with a probe for lacZ that layers. The basal layer comprises two populations of produced no signal (Fig. 1B). In situ hybridization with the keratinocytes: stem cells and transit amplifying cells that ZAC probe showed a strong and homogenous expression in initiated their differentiation program after a rapid phase of the basal layer of epidermis (Fig. 1C and D). We detected as clonal expansion (24, 25). Differentiating keratinocytes detach well a faint and more heterogeneous expression in the spinous from the basal membrane, increase in size, and transform into layers. Interestingly, in one normal skin sample, we detected a cornified envelope that sheds from the surface of the skin ZAC expression in all suprabasal keratinocytes, including the (26). Therefore, the epidermis is an excellent model to study lower and upper spinous and granular layers (data not shown), the regulation of proliferation and differentiation in vivo and indicative of some individual variations. We also observed ZAC its alteration during carcinogenesis. expression in the dermal fibroblasts (Fig. 1D) and in the In the present study, we analyzed ZAC expression during epithelial cells of the sudoriferous glands (Fig. 1E). In contrast, differentiation of human primary keratinocytes in vitro and no expression was observed in the endothelium of the blood in normal and pathologic skin samples, including psoriasis vessels (Fig. 1F).

FIGURE 1. ZAC expression in normal skin. Sections from normal skin samples were processed for in situ hybridization with probes for cytokeratin 1 (A), h-galactosidase (B), or ZAC (C-F). A-D. Epidermis. E. Sudoriferous gland. F. Blood vessel. A, B, and D-F. The field is 1.5 Â 1.2 mm. C. The field is 3 Â 2.4 mm.

Mol Cancer Res 2005;3(9). September 2005 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. ZAC Is Involved in Keratinocyte Differentiation 485

ZAC Expression Is Induced in Early Differentiating Keratinocytes In vitro To gain insights into the role of ZAC in epithelial cell biology, we analyzed ZAC expression during keratinocyte differentiation in vitro. Primary culture of human keratinocytes is widely used to study factors that regulate stem cell proliferation, the initiation of terminal differentiation and tissue assembly, and the process of neoplastic transformation. Primary keratinocytes from human foreskin were cultured under low- calcium and low-serum conditions in a specialized keratinocyte growth medium that keeps these cells as a monolayer, in an undifferentiated stage, for extended periods of time (27, 28). Terminal differentiation under these conditions can be initiated in keratinocytes, and a proportion of cells within the monolayer express the differentiation marker involucrin (29) and eventu- ally shed into the culture medium (30). When the calcium concentration is raised, cells start to form desmosomes and stratify with selective migration of involucrin-positive cells out of the basal layer (31). We detected low level of ZAC expression in the keratinocyte cultures grown under low- calcium conditions by in situ hybridization (Fig. 2B). Because addition of calcium to the culture medium increases the proportion of differentiating keratinocytes in the stratified layer, this system allows the evaluation of ZAC expression during differentiation. We did real-time reverse transcription- PCR (RT-PCR) with total RNAs derived from control keratinocytes cultured in low-calcium medium and from keratinocytes at different time points after raising calcium concentration (Fig. 2A). We observed an induction of ZAC expression in keratinocytes stimulated with calcium as early as 8 hours following calcium addition. ZAC expression levels remained high until 24 hours and then gradually declined at 48 and 72 hours. This finding was further corroborated by in situ hybridization data (Fig. 2B and C). We noted a heterogeneous distribution of ZAC signal in cells grown in the presence of 1.5 mmol/L calcium where cells in cluster expressed high levels of ZAC (Fig. 2C), whereas isolated cells displayed low ZAC expression, reminiscent of the situation in control cells (Fig. 2B). FIGURE 2. ZAC expression in primary keratinocytes is induced by calcium. Normal keratinocytes were grown in vitro in a specialized medium To further assess the regulation of ZAC expression during with low-calcium concentration. A. ZAC expression level was quantified by keratinocyte differentiation, we used another differentiation real-time RT-PCR at different time points after calcium concentration was system (i.e., anchorage-independent growth in methylcellu- raised to 2 mmol/L. Normalization of ZAC expression level was done using geometric averaging of B2M, ARP, and hypoxanthine phosphoribosyl- lose). In contrast to the calcium-induced stratification system, transferase expression levels. Points, mean of two independent experi- keratinocytes grown under these conditions exhibit a certain ments done in triplicate; bars, SE. *, P < 0.01, Student’s t test. B and C. extent of synchronization. Keratinocytes are irreversibly In situ hybridization on keratinocytes grown in low calcium (B) and 24 hours after raising calcium concentration (C). committed by 5 to 6 hours and undergo terminal differentiation by 24 hours when most of them express the differentiation marker involucrin (32). Total RNAs from adherent keratino- committed to differentiation. After 12 hours in suspension, cytes or from keratinocytes placed in suspension for different DNA synthesis in the keratinocytes is inhibited. At this time period of time were analyzed by Northern blot hybridization point, ZAC expression decreased significantly and remained with ZAC cDNA as a probe (Fig. 3A). To control for RNA low after 24 hours in suspension when the cells terminally quality and equal loading, we rehybridized the Northern blots differentiate (Fig. 3A and B). Interestingly, trypsinization of with a probe against 18S rRNA. We repeated the same the cells rapidly up-regulated ZAC expression as evidenced by experiment with an independent preparation of primary Northern blot (Fig. 3C) or real-time RT-PCR (data not shown) keratinocytes and analyzed ZAC expression levels using real- analyses. time RT-PCR (Fig. 3B). In both experiments, loss of anchorage resulted in a transient increase in ZAC expression ZAC-Induced Keratinocyte Differentiation In vitro after 3 to 6 hours in suspension. At that time, keratinocytes Because ZAC was expressed during early stages of loose their ability to proliferate and become irreversibly keratinocytes differentiation in normal skin and in primary

keratinocytes, we tested whether it was able to induce keratinocytes differentiation in vitro. We transfected HaCaT, a keratinocyte-derived cell line, with plasmids encoding ZAC splice variants, ZAC and ZACD2, which are both expressed in normal epidermis (data not shown). ZACD2 is a variant of ZAC, which lacks the two NH2-terminal zinc fingers, and differs from ZAC in its ability to control cell cycle exit and apoptosis (10). A green fluorescent protein–encoding vector was transfected as a control in our experiments. Thirty-six hours after transfection, we did immunostainings for ZAC and the differentiation marker involucrin. The number of cells positive for both involucrin and ZAC was determined and compared with the number of green fluorescent protein– and involucrin-positive cells in the control. ZAC or ZACD2 expression led to a significant increase of involucrin level in transfected cells (Fig. 4). These results suggest that ZAC expression is sufficient to promote keratinocyte differentiation in vitro.

ZAC Expression in Pathologic Skin Samples To find out whether the up-regulation of ZAC expression observed during differentiation in vitro was associated with the decrease of the proliferative potential and the initiation of terminal differentiation in vivo, we analyzed ZAC expression in hyperproliferative disorders of the skin (i.e., psoriasis and skin tumors). Psoriasis is a pathology of the skin characterized by an increased proliferation rate and abnormal differentiation that leads to thickening of the proliferative and differentiated layers and to an increase of the epidermal turnover (33). We did in situ hybridization on sections of psoriatic human skin biopsies. In all five samples examined, we observed rather extended ZAC expression with respect to normal skin. ZAC mRNA was expressed in the thickened basal and suprabasal layers of the psoriatic epidermis (Fig. 5A and C). The expression was strong and homogeneous in the basal layers and much weaker and heterogeneous in the suprabasal layers. Control hybridization with the h-galactosidase probe produced no signal (data not shown) and a probe for cytokeratin K1–labeled suprabasal layers (Fig. 5B and D). Thus, we observed increased expression of ZAC in psoriatic epidermis, consistent with the delay in the initiation of terminal differentiation that is typical for this type of skin pathology. We further examined ZAC expression in two types of skin cancer, BCCs and SCCs. Morphologically, SCC is characterized by an irregular and highly disorganized keratinocyte proliferation, forming diffuse structures (34). SCC is locally more invasive than BCC. It consists of squamous cells, which are usually more differentiated than BCC-forming cells, and it has a high ability to develop metastasis. We found a strong ZAC expression in all eight SCCs examined by in situ hybridization (Table 1; Fig. 6A and B). On one section, the epidermis FIGURE 3. ZAC expression is regulated by anchorage-independent growth. A and B. Normal keratinocytes were grown in a specialized overlaying the tumor was present and we noted that ZAC was medium containing methylcellulose for the indicated period of time. A. expressed in the basal and suprabasal layers. We also observed Total RNAs were prepared and analyzed by Northern blotting with a ZAC ZAC expression in the lymphocytes infiltrating the tumors (data probe and a 18S rRNA probe. B. ZAC expression level was quantified as in Fig. 2A but for the control genes B2M, ARP, glyceraldehyde-3- not shown). Altogether, our data suggest that ZAC is not phosphate dehydrogenase, and PP1A. Points, mean of a representative involved in SSC formation. experiment done in triplicate; bars, SE. *, P < 0.01, Student’s t test. C. Northern blot analysis of ZAC and 18S rRNA expression in keratinocytes BCC is the most common type of human cancer (35). before and after trypsin treatment. Morphologically, this tumor is characterized by tumor nodules

infiltrating dermis, with characteristic peripheral palisading. BCC nodules are composed of small nondifferentiated cells with a basal cell-like phenotype (36). Familial BCCs, called Gorlin syndrome or nevoid BCC syndrome, have loss-of- function mutations of PATCHED (PTCH), which encodes a SONIC HEDGEHOG (SHH) receptor (37, 38). We examined 13 sporadic and 5 familial BCCs by in situ hybridization and found loss of ZAC expression in all sporadic BCC (Table 1). ZAC expression was absent from all tumor nodules but weakly present in the basal and suprabasal layers of the epidermis overlaying the tumor on the same tissue section (Fig. 6C-E). In several cases, ZAC expression was not detected even in the epidermis overlaying the tumor. In the biopsies of BCC taken from patients with Gorlin syndrome, ZAC expression was completely lost in three of five cases. In the other two cases, it was strongly diminished when compared with normal skin taken from the same patients where expression was noted in the basal and suprabasal layers of epidermis (Fig. 6F). In these cases, a very weak ZAC expression was observed in the middle of the tumoral nodules but not on their periphery where most proliferating cells appear to be located (39).

ZAC Expression Is Not Regulated by the SHH Pathway Constitutive activation of the SHH pathway is sufficient to induce BCC in transgenic mice (40) and human skin (41). The observed loss or decrease of ZAC expression in all BCC examined suggested a possible down-regulation of ZAC by the activation of the SHH pathway. To test this hypothesis, we analyzed ZAC expression in primary keratinocytes grown in the presence of recombinant SHH. It was shown previously that SHH opposes calcium-induced cell cycle arrest in differentiating keratinocytes (42). Primary keratinocytes were grown to 60% to 80% confluence in low-calcium conditions before the calcium concentration was raised to 1.5 mmol/L and SHH was simultaneously added to the culture medium. Total RNAs from cells cultivated with or without SHH for 4 and 24 hours were analyzed by RT-PCR. To test for RNA integrity, each RNA sample was analyzed for actin expression. No effect on ZAC expression was observed after addition of SHH to the culture medium (Fig. 7). To verify that recombinant SHH was active in this experiment, the up-regulation of BMP-2B, a member of the transforming growth factor-h family and a known target of SHH in mammals (43), was verified and confirmed by RT-PCR (Fig. 7).

Discussion We undertook the present study to test a possible involvement of ZAC in epidermal differentiation and to evaluate its implication in the development of skin cancer. We detected ZAC expression in the basal and, to a lower level, suprabasal layers of normal epidermis. Using two models of FIGURE 4. ZAC promotes involucrin expression in HaCaT cells. HaCaT cells were transfected with green fluorescent protein (GFP), keratinocyte differentiation in vitro, we observed an early up- ZAC, or ZACD2 and stained with 4,6-diamidino-2-phenylindoleV (blue), regulation of ZAC expression following induction of differen- anti-ZAC (red), and anti-involucrin (green) antisera. Top, high-magnifi- tiation. Interestingly, in both models, ZAC induction is transient cation pictures of ZAC- and ZACD2-positive cells; bottom, involucrin- positive cells and transfected cells (i.e., green fluorescent protein – or and followed by a down-regulation. These observations are in ZAC-positive cells) were scored and the ratios of involucrin-positive good agreement with the distribution of ZAC mRNA in normal cells to transfected cells were calculated for each condition. Columns, mean of three independent experiments; bars, SD. *, P < 0.01, Student’s skin. Keratinocytes initiate differentiation within the basal layer t test. where ZAC is abundantly expressed. The differentiation process

FIGURE 5. ZAC expression in psoriasis samples. Two psoriasis samples (A-D) were analyzed by in situ hybridization for ZAC (A and C) and cytokeratin 1 (B and D) expression. A-C. The field is 1.2 Â 1.2 mm. D. The field is 2.4 Â 2.4 mm.

proceeds during the migration of these cells toward the outmost expressed in all sporadic and familial BCCs, where ZAC is lost. layers of the skin where ZAC expression gradually disappears. We thus assessed whether the SHH-PTCH-SMOH-GLI path- In addition, we showed that ZAC expression in an immortalized way modulates ZAC expression and found no regulation of keratinocyte cell line promotes keratinocyte differentiation. ZAC by recombinant SHH. Hence, ZAC loss of expression is Collectively, this set of data suggests that ZAC expression apparently not directly controlled by the aberrant activation of contributes to keratinocyte differentiation. the SHH pathway in BCC. We found a complete loss or a significant decrease in ZAC Besides PTCH, very few other tumor suppressor genes have expression in all sporadic and familial BCCs examined. This been involved in BCC formation and/or development. One observation is in agreement with the proposed tumor interesting candidate is TP53 for which a specific, UV-induced suppressor function for ZAC and with the observed loss or mutation spectrum has been reported in sporadic BCC (48, 49). down-regulation of ZAC expression in breast, ovary, and The recent demonstration that mouse Zac1 behaves as a p53 pituitary tumors (12–14). BCC is the most common type of coactivator (7) suggests that the loss of ZAC expression in neoplasm and its incidence is dramatically increasing. BCC sporadic BCC may correlate with a decrease in the p53-induced metastasis occurs only locally and sporadically in relation to response to DNA damage. Noteworthy, Zac1 cotransfection sun exposure. Its incidence is increased significantly in some was reported to potentiate p53-mediated stimulation of rare genetic disorders, such as Gorlin syndrome. Familial p21WAF1/Cip1 expression (7), which is involved in keratinocyte BCCs and about one-third of sporadic BCCs have loss-of- differentiation (50). function mutations of PATCHED (37, 38), which encodes a Our data regarding ZAC expression in vitro and in vivo receptor for the morphogen SHH. Activation of PATCHED suggest a role for ZAC in the early stages of keratinocyte negatively regulates the activity of SMOOTHENED, a seven- differentiation. Due to its functional properties (1, 9, 10), one transmembrane domain receptor controlling the activation of would predict that ZAC would be involved in the cell cycle the GLI family of transcription factors (44, 45). Moreover, arrest, which usually accompanies the induction of terminal human sporadic BCCs consistently express GLI-1 (46). In differentiation in most tissues. Indeed, in normal epidermis, the agreement with these data, overexpression of Shh (40) or GLI-1 progressive loss of ZAC expression in the suprabasal layers (47) is sufficient to induce BCC-like tumors in transgenic mice. could be correlated with an irreversible proliferation shutdown. Interestingly, ZAC and GLI-1 display opposite patterns of However, several lines of evidence indicate that ZAC is most expression. Indeed, GLI-1 expression is not detected in basal likely not directly involved in negative regulation of cell cycle keratinocytes of the normal skin, which display high expression progression in the epidermis. First, the maintaining of ZAC of ZAC in our in situ experiments. Conversely, GLI-1 is expression in the hyperproliferative psoriasis and SCC indicates

no link between ZAC expression and a proliferation block. Finally, our data also unveil a potential role for the loss of Second, epidermal differentiation is not invariably accompanied the candidate tumor suppressor gene ZAC in the development by a complete withdrawal from the cell cycle, because of BCC. Additional experiments aiming at identifying ZAC keratinocytes retain the ability to synthesize DNA (51, 52) target genes will provide insights into the process of and continue to grow in size during differentiation (26). Finally, keratinocyte differentiation that is lost in BCC. SCCs and psoriasis retain the capacity to differentiate and express ZAC, whereas BCC cells display a nondifferentiated phenotype and do not express ZAC. All these data suggest that Materials and Methods ZAC plays a role in early events of keratinocyte differentiation, Cell Culture at a step distinct from the control of the cell cycle progression. HaCaT keratinocyte cells were maintained in DMEM plus Interestingly, keratinocytes of suprabasal layers of normal and 10% fetal bovine serum and transfected using Lipofectamine psoriatic epidermis and SCC display increased cell size, 2000 (IVG, Cergy, France). Plasmids encoding ZAC and whereas BCCs are formed by small-sized cells. ZAC may thus ZACD2 were described previously (10). Monolayer cultures have a role in the cellular growth that takes place at the of primary human keratinocytes were prepared from neonatal initiation of differentiation that is altered in SCCs and lost in foreskins as described (54). Cells were maintained in serum- BCCs. free keratinocyte growth medium K-SFM (IVG). Stratifica- In addition, it was shown recently that cytokeratin 14 is a tion was induced by addition of 2 mmol/L calcium or 10% direct target of mouse Zac1 and that several other cytokeratins fetal bovine serum to the medium. Cells were harvested for are positively or negatively regulated by Zac1 (2). Consistent RNA isolation at different time points after induction. To with the putative role of its human counterpart in keratinocytes study the effect of SHH on ZAC expression, the keratino- differentiation, Zac1 was observed to repress cytokeratin 19, cytes were grown to 60% to 80% confluence before the which has been proposed as an interfollicular stem cell marker concentration of calcium in the medium was raised to 1.5 to (53), and to activate cytokeratin 14, a component of the basal 2 mmol/L. Recombinant SHH (Research Diagnostics, Inc., layer where ZAC is also present (24). Flanders, NJ) was simultaneously added at a final concentration of 20 Ag/mL. Cells were harvested for RNA isolation at different time intervals after SHH addition. To induce Table 1. ZAC expression in normal and pathologic skin terminal differentiation, the keratinocytes were trypsinized samples was assessed by in situ hybridization and visual and maintained in suspension in 1.75% methylcellulose as scoring described previously (32). n Diagnosis ZAC expression RNA Isolation and Northern Blot Analysis 308 BCC À Total RNA was isolated from keratinocyte cultures using 281 BCC À Trizol (LTI, Cergy, France). Northern blots of total RNAs (20 280 BCC À A 221 BCC À g) were hybridized with a human ZAC probe (nucleotides 684- 220 BCC À 2,800 of the human cDNA; Genbank accession no. AJ006354) 392 BCC À and a probe for 18S rRNA. Hybridization was in Church buffer 314 BCC À 394 BCC À [0.5 mol/L phosphate buffer (pH 7), 7% SDS, 2% bovine serum 466 BCC À albumin] and final washing step was at 65jCin1Â SSC and 366-2 BCC À 0.2% SDS. 436 BCC À 218bs BCC À 448 BCC À Real-time and Semiquantitative PCR Analysis 354 BCC (NBCCS) À 355 BCC (NBCCS) À cDNAs were generated from 1 Ag total RNAs treated with 357 BCC (NBCCS) +/À DNase I by using random hexamers and Moloney murine 315 BCC (NBCCS) À 318 BCC (NBCCS) +/À leukemia virus reverse transcriptase (LTI). Real-time PCR was 366-1 SCC + done using Applied Biosystems (Courtaboeuf, France) SYBR 430 SCC +++ Green PCR mix according to the manufacturer’s instructions. 432 SCC ++ 345 SCC + Housekeeping genes used to normalize ZAC expression levels 397 SCC + were selected using the geNorm process (55). The sequences of 287 SCC + the primers for real-time PCR are ZAC 5V-CCACTCACAG- 442 SCC ++ 431 SCC + GAGCTGATGAAA and 5V-GCAGCCTTCAGTTGGAAT- 467 Normal skin ++ GAA, B2M 5V-TGACTTTGTCACAGCCCAAGATA and 5V- 442n Normal skin + 455 Normal skin ++ CGGCATCTTCAAACCTCCA, ARP 5V-CTCCAAGCAGATG- 428 Normal skin ++ CAGCAGA and 5V-CCCATCAGCACCACAGCC, hypoxan- 356 Normal skin (NBCCS) +++ thine phosphoribosyltransferase 5V-CGGCTCCGTTATGGCG 408 Normal skin (scalp) + 427 Normal skin (scalp) + and 5V-GGTCATAACCTGGTTCATCATCAC, and PP1A 5V- GTCGACGGCGAGCCC, 5V-TCTTTGGGACCTTGTCTGCAA, NOTE: À,noZAC expression; +/À, very weak ZAC expression; +, ++, and +++, and glyceraldehyde-3-phosphate dehydrogenase 5V-TGTTC- increasing levels of ZAC expression. Abbreviation: NBCCS, a sample from a patient with nevoid BCC (Gorlin) GACAGTCAGCCGC and 5V-GGTGTCTGAGCGATGTGGC. syndrome. The sequences of the primers for semiquantitative PCR are

FIGURE 6. ZAC expression in skin tumors. Two SCCs (A and B), two sporadic BCCs (C and D), a BCC from a patient with Gorlin syndrome (E), and normal skin from the same patient (F) were analyzed for ZAC expression by in situ hybridization. Sections were counterstained with hematoxylin. Arrows, tumor nodules in BCC samples; arrowheads, tumor on SCC sections.

ZAC 5V-AACCGGAAAGACCACCTGAAAAACCAC and nucleotide–long RNA probe, corresponding to nucleotides 5V-GTCGCACATCCTTCCGGGTGTAGAA (amplicon 303 bp), 1,889 to 2,182 of the human cDNA (Genbank accession no. actin 5V-ACTGGCATCGTGATGGACTCC and 5V-GTTG- AJ006354). The specificity of this probe was shown GCGTACAGGTCTTTGCG (amplicon 444 bp), and BMP-2B previously (56). For detection of cytokeratin 1, we used a 5V-CACCATGATTCCTGGTAACC and 5V-TCTCCAGATG- TTCTTCGTGG (amplicon 390 bp). Semiquantitative PCR was done as follows: 2 minutes at 94jC followed by 20 to 30 cycles: 30 seconds at 94jC, 30 seconds at 55jC for BMP-2B primers, 57jC for ZAC primers, and 60jC for actin primers, and 30 seconds at 72jC. Lower numbers of cycles were used to verify linearity of the amplification process. Half of the PCR reaction was run on a 6% polyacrylamide gel and analyzed by Southern blotting.

In situ Hybridization FIGURE 7. ZAC expression is not repressed by recombinant SHH. In situ hybridization on sections of normal skin, psoriasis, Normal keratinocytes were grown in low-calcium medium. The calcium concentration was raised to 1.5 mmol/L for the indicated period of time in and skin tumors obtained after biopsy was done as described the presence (+) or absence (À) of recombinant SHH. Total RNAs were previously (56). For detection of ZAC, we used a 294- isolated and analyzed by RT-PCR for ZAC, actin, and BMP-2B expression.

1-kb probe corresponding to the coding region of cytokeratin expression of ZAC/LOT1 in squamous cell carcinomas of head and neck. Head Neck 2004;26:338 – 44. 1 cDNA, which was hydrolyzed before hybridization. 16. Perou CM, Jeffrey SS, van de Rijn M, et al. Distinctive gene expression Following ISH, sections of BCC and SCC were stained patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad with hematoxylin. Sci U S A 1999;96:9212 – 7. 17. Singhal S, Amin KM, Kruklitis R, et al. Alterations in cell cycle genes in early stage lung adenocarcinoma identified by expression profiling. Cancer Biol Immunocytochemistry Ther 2003;2:291 – 8. Cells grown on coverslips were fixed with 4% formaldehyde 18. Ross DT, Scherf U, Eisen MB, et al. Systematic variation in gene expression in PBS for 30 minutes, washed with PBS thrice, and patterns in human cancer cell lines. Nat Genet 2000;24:227 – 35. permeabilized with 0.1% Triton in PBS for 10 minutes. Double 19. Gardner RJ, Mackay DJG, Mungall AJ, et al. An imprinted locus associated labeling was done with anti-ZAC rabbit polyclonal antibodies with transient neonatal diabetes mellitus. Hum Mol Genet 2000;9:589 – 96. (diluted 1:1,000) and anti-involucrin mouse monoclonal anti- 20. Kamiya M, Judson H, Okazaki Y, et al. The cell cycle control gene ZAC/ PLAGL1 is imprinted—a strong candidate gene for transient neonatal diabetes. bodies SY5 (Interchim, Montluc¸on, France), diluted 1:100 in Hum Mol Genet 2000;9:453 – 60. PBS containing 1% bovine serum albumin. Secondary anti- 21. Piras G, El Kharroubi A, Kozlov S, et al. Zac1 (Lot1), a potential tumor bodies anti-rabbit FITC (Sigma, San Quentin Fallavier, France) suppressor gene, and the gene for e-sarcoglycan are maternally imprinted genes: identification by a substractive screen of novel uniparental fibroblast lines. Mol and anti-mouse CY3 (Sigma) were diluted 1:300 and 1:3,000, Cell Biol 2000;20:3308 – 15. respectively, in PBS with 1% bovine serum albumin. No cross- 22. Abdollahi A, Pisarcik D, Roberts D, Weinstein J, Cairns P, Hamilton TC. reactivity was observed in control experiments. The coverslips LOT1 (PLAGL1/ZAC1), the candidate tumor suppressor gene at chromosome were mounted in 4V,6-diamidino-2-phenylindole–containing 6q24-25, is epigenetically regulated in cancer. J Biol Chem 2003;278:6041 – 9. medium (56). 23. Varrault A, Bilanges B, Mackay DJG, et al. Characterization of the methylation-sensitive promoter of the imprinted ZAC gene supports its role in transient neonatal diabetes mellitus. J Biol Chem 2001;276:18653 – 6. Acknowledgments 24. Fuchs E, Byrne C. The epidermis: rising to the surface. Curr Opin Genet We thank Drs. Edouard Bertrand, Jean-Marie Blanchard, and Annie Varrault for Dev 1994;4:725 – 36. critical reading of the article and helpful comments. 25. Watt FM, Jones PH. Expression and function of the keratinocyte integrins. Dev Suppl 1993;185 – 92. 26. Watt FM, Green H. Involucrin synthesis is correlated with cell size in human References epidermal cultures. J Cell Biol 1981;90:738 – 42. 1. Spengler D, Villalba M, Hoffmann A, et al. Regulation of apoptosis and cell cycle arrest by Zac1, a novel zinc finger protein expressed in the pituitary gland 27. Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH. and the brain. EMBO J 1997;16:2814 – 25. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 1980;19:245 – 54. 2. Hoffmann A, Ciani E, Boeckardt J, Holsboer F, Journot L, Spengler D. Transcriptional activities of the zinc finger protein Zac are differentially 28. Wille JJJ, Pittelkow MR, Shipley GD, Scott RE. Integrated control of controlled by DNA binding. Mol Cell Biol 2003;23:988 – 1003. growth and differentiation of normal human prokeratinocytes cultured in serum- free medium: clonal analyses, growth kinetics, and cell cycle studies. J Cell 3. Pagotto U, Arzberger T, Ciani E, et al. Inhibition of Zac1, a new gene Physiol 1984;121:31 – 44. differentially expressed in the anterior pituitary, increases cell proliferation. Endocrinology 1999;140:987 – 96. 29. Murphy GF, Flynn TC, Rice RH, Pinkus GS. Involucrin expression in normal and neoplastic human skin: a marker for keratinocyte differentiation. J Invest 4. Abdollahi A, Godwin AK, Miller PD, et al. Identification of a gene containing Dermatol 1984;82:453 – 7. zinc-finger motifs based on lost expression in malignantly transformed rat ovarian surface epithelial cells. Cancer Res 1997;57:2029 – 34. 30. Watt FM, Green H. Stratification and terminal differentiation of cultured epidermal cells. Nature 1982;295:434 – 6. 5. Abdollahi A, Bao R, Hamilton TC. LOT1 is a growth suppressor gene down- regulated by the epidermal growth factor receptor ligands and encodes a nuclear 31. Boisseau AM, Donatien P, Surleve-Bazeille JE, et al. Production of zinc-finger protein. Oncogene 1999;18:6477 – 87. epidermal sheets in a serum free culture system: a further appraisal of the role of extracellular calcium. J Dermatol Sci 1992;3:111 – 20. 6. Huang SM, Stallcup MR. Mouse Zac1, a transcriptional coactivator and repressor for nuclear receptors. Mol Cell Biol 2000;20:1855 – 67. 32. Adams JC, Watt FM. Fibronectin inhibits the terminal differentiation of human keratinocytes. Nature 1989;340:307 – 9. 7. Huang S-M, Scho¨nthal AH, Stallcup MR. Enhancement of p53-dependent gene activation by the transcriptional coactivator Zac1. Oncogene 2001;20:2134 – 43. 33. McKay IA, Leigh IM. Altered keratinocyte growth and differentiation in psoriasis. Clin Dermatol 1995;13:105 – 14. 8. Rozenfeld-Granot G, Krishnamurthy J, Kannan K, et al. A positive feedback mechanism in the transcriptional activation of Apaf-1 by p53 and the coactivator 34. Kim J, Shin DM. Biomarkers of squamous cell carcinoma of the head and Zac-1. Oncogene 2002;21:1469 – 76. neck. Histol Histopathol 1997;12:205 – 18. 9. Varrault A, Ciani E, Apiou F, et al. hZAC encodes a zinc finger protein with 35. Katz MH. Nonmelanoma skin cancer. Md Med J 1997;46:239 – 42. antiproliferative properties and maps to a chromosomal region frequently lost in 36. Maloney ME. Histology of basal cell carcinoma. Clin Dermatol 1995;13: cancer. Proc Natl Acad Sci U S A 1998;95:8835 – 40. 545 – 9. 10. Bilanges B, Varrault A, Mazumdar A, et al. Alternative splicing of the 37. Gailani MR, Stahle-Backdahl M, Leffell DJ, et al. The role of the human imprinted candidate tumor suppressor gene ZAC regulates its antiproliferative and homologue of Drosophila patched in sporadic basal cell carcinomas. Nat Genet DNA binding activities. Oncogene 2001;20:1246 – 53. 1996;14:78 – 81. 11. Abdollahi A, Roberts D, Godwin AK, et al. Identification of a zinc-finger 38. Hahn H, Wicking C, Zaphiropoulous PG, et al. Mutations of the human gene at 6q25: a chromosomal region implicated in development of many solid homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. tumors. Oncogene 1997;14:1973 – 9. Cell 1996;85:841 – 51. 12. Bilanges B, Varrault A, Basyuk E, et al. Loss of expression of the candidate 39. Grimwood RE, Ferris CF, Mercill DB, Huff JC. Proliferating cells of human tumor suppressor gene ZAC in breast cancer cell lines and primary tumors. basal cell carcinoma are located on the periphery of tumor nodules. J Invest Oncogene 1999;18:3979 – 88. Dermatol 1986;86:191 – 4. 13. Cvetkovic D, Pisarcik D, Lee C, Hamilton TC, Abdollahi A. Altered 40. Oro AE, Higgins KM, Hu Z, Bonifas JM, Epstein EH, Scott MP. Basal cell expression and loss of heterozygosity of the LOT1 gene in ovarian cancer. carcinomas in mice overexpressing Sonic Hedgehog. Science 1997;276:817 – 21. Gynecol Oncol 2004;95:449 – 55. 41. Fan H, Oro AE, Scott MP, Khavari PA. Induction of basal cell carcinoma 14. Pagotto U, Thomas A, Theodoropoulou M, et al. The expression of the features in transgenic human skin expressing Sonic Hedgehog. Nat Med 1997;3: antiproliferative gene ZAC is lost or highly reduced in non functioning pituitary 788 – 92. adenomas. Cancer Res 2000;60:6794 – 8. 42. Fan H, Khavari PA. Sonic Hedgehog opposes epithelial cell cycle arrest. 15. Koy S, Hauses M, Appelt H, Friedrich K, Schackert HK, Eckelt U. Loss of J Cell Biol 1999;147:71 – 6.

43. Bitgood MJ, McMahon AP. Hedgehog and Bmp genes are coexpressed at 50. Dotto GP. p21WAF1/Cip1: more than a break to the cell cycle? Biochim many diverse sites of cell-cell interaction in the mouse embryo. Dev Biol 1995; Biophys Acta 2000;1471:M43 – 56. 172:126 – 38. 51. Gandarillas A, Davies D, Blanchard JM. Normal and c-Myc- 44. Hahn H, Wojnowski L, Miller G, Zimmer A. The patched signaling pathway promoted human keratinocyte differentiation both occur via a novel cell in tumorigenesis and development: lessons from animal models. J Mol Med 1999; cycle involving cellular growth and endoreplication. Oncogene 2000;19: 77:459 – 68. 3278 – 89. 45. Ruiz i Altaba A. Gli proteins and Hedgehog signaling: development and 52. Zanet J, Pibre S, Jacquet C, Ramirez A, de Alboran IM, Gandarillas cancer. Trends Genet 1999;15:418 – 25. A. Endogenous Myc controls mammalian epidermal cell size, hyper- 46. Dahmane N, Lee J, Robins P, Heller P, Ruiz i Altaba A. Activation of the proliferation, endoreplication and stem cell amplification. J Cell Sci 2005; transcription factor Gli1 and the Sonic Hedgehog signalling pathway in skin 118:1693 – 704. tumours. Nature 1997;389:876 – 81. 53. Larouche D, Hayward C, Cuffley KLG. Keratin 19 as a stem cell marker 47. Nilsson M, Unden AB, Krause D, et al. Induction of basal cell carcinomas in vivo and in vitro. Methods Mol Biol 2005;289:103 – 10. and trichoeptheliomas in mice overexpressing GLI-1. Proc Natl Acad Sci U S A 54. Rheinwald JG. Serial cultivation of normal human epidermal keratinocytes. 2000;97:3438 – 43. Methods Cell Biol 1980;21A:229 – 54. 48. Moles J-P, Moyret C, Guillot B, et al. p53 gene mutations in human epithelial 55. Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real- skin cancers. Oncogene 1993;8:583 – 8. time quantitative RT-PCR data by geometric averaging of multiple internal control 49. Ouhtit A, Nakazawa H, Armstrong BK, et al. UV-radiation-specific p53 genes. Genome Biol 2002;3:RESEARCH0034. mutation frequency in normal skin as a predictor of risk of basal cell carcinoma. 56. Basyuk E, Bertrand E, Journot L. Alkaline fixation drastically improves J Natl Cancer Inst 1998;90:523 – 31. signal of in situ hybridization. Nucleic Acids Res 2000;28:e46.

Mol Cancer Res 2005;3(9). September 2005 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. The Candidate Tumor Suppressor Gene ZAC Is Involved in Keratinocyte Differentiation and Its Expression Is Lost in Basal Cell Carcinomas

Eugenia Basyuk, Vincent Coulon, Anne Le Digarcher, et al.

Mol Cancer Res 2005;3:483-492.

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