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. Author manuscript; available in PMC: 2014 Jun 27.
Published in final edited form as: Mol Cell. 2013 Jun 27;50(6):908–918. doi: 10.1016/j.molcel.2013.05.017

FANCD2 Activates Transcription of TAp63 and Suppresses Tumorigenesis

Eunmi Park 1, Hyungjin Kim 1, Jung Min Kim 1, Benjamin Primack 1, Sofia Vidal-Cardenas 1, Ye Xu 1, Brendan D Price 1, Alea A Mills 2, Alan D D’Andrea 1,3,*
PMCID: PMC3897475  NIHMSID: NIHMS483037  PMID: 23806336

SUMMARY

Fanconi Anemia (FA) is a rare genetic disorder characterized by an increased susceptibility to squamous cell cancers. Fifteen FA genes are known, and the encoded proteins cooperate in a common DNA repair pathway. A critical step is the monoubiquitination of the FANCD2 protein, and cells from most FA patients are deficient in this step. How monoubiquitinated FANCD2 suppresses squamous cell cancers is unknown. Here we show that Fancd2-deficient mice are prone to Ras oncogene-driven skin carcinogenesis, while Usp1-deficient mice, expressing elevated cellular levels of Fancd2-Ub, are resistant to skin tumors. Moreover, Fancd2-Ub activates the transcription of the tumor suppressor TAp63, thereby promoting cellular senescence and blocking skin tumorigenesis. For FA patients, the reduction of FANCD2-Ub and TAp63 protein levels may account for their susceptibility to squamous cell neoplasia. Taken together, Usp1 inhibition may be a useful strategy for upregulating TAp63 and preventing or treating squamous cell cancers in the general non-FA population.

Keywords: Fanconi Anemia, Deubiquitinating Enzymes, Senescence, Ras, USP1

INTRODUCTION

Squamous cell carcinoma (SCC) of the skin is one of the most common forms of cancer in humans, and its rising incidence may reflect increased sun exposure and a diminished ozone layer (Alam and Ratner, 2001). One of the most common molecular abnormalities observed in SCC is the overexpression of the p53 family member, p63 (Hu et al., 2002). Like p53, p63 is a transcriptional regulator, and it is expressed as two primary isoforms, the tumor suppressive TAp63 isoform (Koster and Roop, 2004) and the oncogenic ΔNp63 isoform (Rocco and Ellisen, 2006; Rocco et al., 2006). Moreover, expression of p63 is required for normal skin development (Mills et al., 1999) (Yang et al., 1999). The TAp63 isoform is primarily expressed in the dermis, and ΔNp63 is primarily expressed in the epidermis (Koster and Roop, 2004). Acquired mutations in the p63 gene derived from human squamous cell tumors are predicted to disrupt the expression of the tumor suppressive TAp63 isoform, suggesting that this isoform plays a critical role in suppressing the progression of SCC in the general (non-FA) population (Stransky et al., 2011).

Fanconi Anemia (FA) is a rare autosomal recessive or X-linked recessive disease characterized by multiple congenital abnormalities, bone marrow failure, and an increased susceptibility to squamous cell cancers, including skin, oral, and esophageal carcinomas (Alter et al., 2003). Primary cells isolated from FA patients exhibit hypersensitivity to DNA interstrand crosslinking agents, such as mitomycin C and diepoxybutane, resulting from an underlying defect in DNA repair (Auerbach, 2009). The fifteen known FA proteins cooperate in a common DNA repair pathway (reviewed in (Kee and D’Andrea, 2010). Eight of the FA proteins (A, B, C, E, F, G, L, M) are assembled into a nuclear protein complex, the FA core complex, which binds to DNA, primarily at stalled replication forks containing an interstrand crosslink (ICL). The FA core complex is a multisubunit E3 ubiquitin ligase which subsequently monoubiquitinates two additional FA proteins (FANCD2 and FANCI) (Garcia-Higuera et al., 2001) (Smogorzewska et al., 2007). The monoubiquitinated FANCD2 protein (FANCD2-Ub) then functions as a landing pad, recruiting an additional downstream FA protein, FANCP/SLX4 (Yamamoto et al., 2011) as well as the FAN1 nuclease (O’Donnell and Durocher, 2010). This regulated recruitment of endonucleases is required for the excision of the DNA crosslink. Downstream FA proteins (D1, N, O, and J) are further required for additional downstream repair events. Whether monoubiquitinated FANCD2 protein, the critical intermediate in the FA pathway, has additional cellular functions beyond DNA repair is unknown.

Recent studies have suggested that monoubiquitinated FANCD2 (FANCD2-Ub) may have an additional role as a tumor suppressor. Fancd2-deficient mice have an increase in spontaneous epithelial cancers (Houghtaling et al., 2003), and the incidence of these cancers is further increased in the setting of p53 deficiency (Houghtaling et al., 2005). Moreover, FANCD2-Ub is expressed at high levels in tissues which are prone to malignant transformation, such as the squamous cell epithelia of the head and neck region and the uterine cervix, suggesting that the protein may function as a tumor suppressor in these tissues (Holzel et al., 2003). Whether FANCD2-Ub suppresses squamous cell cancers by regulating the level of other known suppressor proteins, such as the TAp63 protein, is unknown.

In the current study, we demonstrate that FANCD2-Ub and its binding partner FANCP/SLX4 directly suppress squamous cell cancer development by transcriptionally upregulating the cellular level of TAp63. Fancd2-deficient mice have an increased incidence of Ras oncogene-induced squamous cell cancers, compared to normal sibling control mice. Conversely, mice deficient in Usp1, the protease which deubiquitinates Fancd2 (Nijman et al., 2005) (Kim et al., 2009), have elevated cellular levels of Fancd2-Ub and are resistant to Ras-induced squamous cell cancers. Fancd2-Ub promotes normal Ras-induced cellular senescence, and absence of Fancd2-Ub results in increased cellular transformation. Moreover, Fancd2-Ub binds to the promoter of the TAp63 gene and promotes its transcription. Taken together, our results indicate that the FA pathway suppresses squamous cell tumor development in a TAp63-dependent mechanism, accounting, at least in part, for the specific predisposition of FA patients to this class of cancer.

RESULTS

Fancd2-deficient mice exhibit increased squamous cell tumorigenesis

Previous studies have indicated that Fancd2 (-/-) mice have elevated levels of spontaneous epithelial carcinomas (Houghtaling et al., 2003). We initially tested whether topical treatment with DMBA (dimethylbenz(a)anthracene), followed by a 28 week course of TPA (12-O-tetradecanoylphorbol-13-acetate) (Park et al., 2007) (Figure 1A), would further enhance squamous cell carcinogenesis in a strain of Fancd2-deficient mice developed in our laboratory (Parmar et al., 2010). As expected, Fancd2 (+/-) mice exhibited an increased incidence and number of tumors following DMBA/TPA exposure (Figure 1B). All of the Fancd2 (+/-) mice developed tumors by 21 weeks of DMBA/TPA treatment. These tumors did not exhibit loss of heterozygosity at the Fancd2 locus (data not shown). In contrast, only 60% of the sibling control Fancd2 (+/+) mice developed tumors by 28 weeks of DMBA/TPA treatment. Fancd2 (+/-) mice developed more papillomas than Fancd2 (+/+) mice (mean 4.5 vs 2.3 tumors per mouse, P<0.01) (Figure 1C). Moreover, the papillomas from the Fancd2 (+/-) mice had a six times higher rate of progression to malignant carcinoma than the papillomas from the Fancd2 (+/+) mice (Figure S1), which rarely developed invasive cancers (data not shown). Skin papillomas were excised from Fancd2 (+/+) and Fancd2 (+/-) mice exposed to the DMBA/TPA treatment and further analyzed. As expected, activating Ras mutations resulting from the exposure to carcinogen were found in tumors derived from the treated mice, regardless of genotype. Specifically, the H-RasV61 mutation was detected in most tumors by PCR. H-Ras mutations were not detected in the skin surrounding the tumors.

Figure 1. Fancd2-deficient mice exhibit increased squamous cell tumorigenesis.

Figure 1

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(A) Schematic diagram shows a DMBA/TPA two-stage tumor induction procedure. (B) Tumor incidence in Fancd2 (+/+) and Fancd2 (+/-) mice treated with DMBA/TPA. Increased skin tumor formation in Fancd2 (+/-) mice versus wild-type Fancd2 (+/+) mice (P<0.01). For each group, n = 18 mice (See also Figure S1, lane 1-4). (C) The distribution of total number of tumors per mouse in Fancd2 (+/-) mice versus Fancd2 (+/+) mice 28 weeks after TPA treatment (P<0.01). (D) Loss of Fancd2 enhances skin proliferation after 4 weeks of TPA treatment. TPA-induced epithelial cell proliferation was evaluated in each indicated group (four mice per group). The photomicrograph shows skin from a representative mouse at 100X magnification. Arrows indicate the epidermis. Scale bar =100 μm. (E) In vivo senescence detected by β-gal staining. Skin from mice of the indicated genotype was stained. The Fancd2 (+/+) skin shows enhanced blue senescent staining in the base of the hair follicles (lower magnification) and in the interfollicular epidermis (arrows, higher magnification). Scale bar=25μm. The blue senescent staining was reduced in the skin from the Fancd2 (-/-) mouse.

FANCD2 monoubiquitination is required for Ras oncogene induced senescence

We next examined the effect of TPA application alone on the epidermis of Fancd2-deficient mice (Figure 1D). Interestingly, Fancd2 (-/-) and Fancd2 (+/-) mice exhibited increased epidermal thickening following TPA exposure, compared to sibling control wild-type mice, consistent with the role of Fancd2 as a suppressor of cell proliferation. Furthermore, we applied TPA to the skin of Fancg-deficient mice, which also have a cellular defect in the synthesis of monoubiquitinated Fancd2. Like Fancd2-deficient mice, the Fancg (-/-) mice also exhibited enhanced epidermal thickening following TPA treatment (Figure S2A).

This increase in skin thickening suggested that Fancd2 (-/-) cells may have a defect in Ras oncogene-induced senescence (OIS), a normal cellular process which limits cell proliferation after oncogene activation (Serrano et al., 1997) (Keyes et al., 2005). To test this hypothesis, we examined the level of senescence-associated (SA)-β galactosidase staining, a measure of in vivo senescence (Lin et al., 2010), in skin extracted from the TPA exposed animals (Figure 1E). Consistent with the reduced proliferation observed in TPA treated Fancd2 (+/+) epithelial cells, we detected a higher accumulation of SA-β galactosidase staining in this tissue, compared to Fancd2 (+/-) epithelium.

To further quantify cellular senescence, we measured OIS in Fancd2 (-/-) primary murine embryo fibroblasts (MEFs) (Figure 2). Fancd2 (-/-) MEFs showed decreased OIS (Figure 2A), as measured by RAS oncogene-induced SA-β galactosidase staining, and exhibited an increased number of transformed foci in soft agar (Figure 2B). Interestingly, complementation with the cDNA encoding the wild-type human FANCD2, but not with the cDNA encoding the FANCD2-K561R mutant protein, which fails to undergo monoubiquitination (Garcia-Higuera et al., 2001) (Figure 2C), restored OIS and suppressed cell proliferation. Consistent with these results, primary bone marrow stromal cells derived from a Fancd2 (-/-) mouse were also defective in Fancd2-monoubiquitination and RAS-induced senescence (data not shown). Thus, monoubiquitination of FANCD2 is critical for oncogene-induced senescence and its loss may account for the increased TPA-inducible epithelial thickening in Fancd2 (-/-) and Fancg (-/-) mice.

Figure 2. FANCD2 monoubiquitination is required for Ras oncogene-induced senescence and for TAp63 induction after DNA damage.

Figure 2

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(A-C)Primary Fancd2 (-/-) MEFs (passage 1) were infected with either control retrovirus (EV) or a retrovirus carrying wild type FANCD2 (WT) or mutant FANCD2 (K561R). After puromycin selection, cells were infected with H-RasV12 or control virus. Data shown are mean ± standard errors of three independent experiments, at passage 2 of the MEFs. Each experiment was performed in triplicate plates for three sibling MEFs of each genotype. (A) Cells were stained for SA-β-gal expression after 2 weeks from infection. (B) Cell proliferation was assayed by crystal violet staining. (C) Immunoblot confirming expression of FANCD2 in retrovirus-infected cells. The FANCD2 (K561R) protein is not monoubiquitinated. (D) TAp63 mRNA was measured by Real-Time PCR in primary Fancd2 (+/+) and Fancd2 (-/-) MEFs after MMC treatment (500 nM). Data shown are mean ± standard errors of triplicate plates for three sibling MEFs of each genotype. Data shown are representative of three independent experiments. (E) Immunoblot showing expression of Fancd2 and TAp63 protein in primary MEFs infected with retrovirus. *, P<0.001; **, P<0.005. See also Figure S2.

The failure of Fancd2-deficient MEFs to undergo OIS suggested that these cells may also be deficient in the expression or activation of a tumor suppressor protein. The TAp63 protein is a tumor suppressor known to be required for Ras oncogene-induced senescence (Guo et al., 2009). The Fancd2-deficient MEFs had reduced expression of DNA damage-inducible TAp63 mRNA (Figure 2D and Figure S2B) and protein (Figure 2E, lanes 3 and 4), compared to Fancd2 (+/+) MEFs, suggesting a possible molecular mechanism for the loss of OIS in the Fancd2-deficient cells. Specifically, the reduced expression of TAp63 in Fancd2-deficient cells may result in reduced expression of TAp63-inducible genes. Such genes encode proteins known to mediate DNA repair and to suppress cell growth (Lin et al, 2009). Like Fancd2-deficient MEFs, human FANCD2-deficient keratinocytes (HaCat cells) also exhibited decreased OIS (Figure S2 C, D), suggesting that Fancd2-Ub may play a broader role in activating senescence, limiting cell proliferation, and suppressing tumorigenesis.

Usp1-deficient mice are resistant to Ras-driven skin carcinogenesis

We reasoned that mice with elevated levels of Fancd2-Ub may therefore have elevated cellular senescence and may be resistant to DMBA/TPA induced tumors. The protease Usp1 normally deubiquitinates Fancd2-Ub (Nijman et al., 2005) (Cohn et al., 2007), and Usp1 (-/-) mice have elevated cellular levels of Fancd2-Ub (Kim et al., 2009). Usp1 (-/-) mice are born at sub-Mendelian frequency and exhibit small size, infertility, bone marrow defects, and cellular hypersensitivity to DNA interstrand crosslinking agents. Further analysis revealed that Usp1 (-/-) mice exhibit abnormal ectodermal phenotypes such as malformed eyes (data not shown) and a thin epidermal layer (Figure 3A), compared to Usp1 (+/+) sibling control mice. In contrast to Fancd2-deficient mice, treatment of the skin of Usp1 (-/-) mice with TPA resulted in decreased epidermal cellularity and thickening (Figure 3B), compared to wild-type sibling control mice.

Figure 3. Usp1-deficient mice are resistant to Ras-driven skin carcinogenesis.

Figure 3

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(A) Histology of epidermis and dermis of Usp1 (+/+) versus Usp1 (-/-) newborn mice. The epidermis is indicated with a bracket. (B) Loss of Usp1 decreases skin proliferation after TPA treatment. Usp1 (+/+), Usp1 (+/-) and Usp1 (-/-) mice were exposed to topical TPA for four weeks (four mice per group). Skin thickness was examined by microscopy at 100x magnification. Scale bar =100μm. (C) Delayed tumor formation in Usp1 (-/-) and Usp1 (+/-) mice following DMBA/TPA treatment (P<0.05). Tumor multiplicity was not statistically different between Usp1 (+/+) and Usp1 (+/-) mice. These data are representative of two independent experiments.

Furthermore, Usp1 (-/-) and Usp1 (+/-) mice were resistant to DMBA/TPA-induced tumorigenesis, compared to wild-type sibling controls (Figure 3C) and developed skin tumors later than the wild-type animals (P<0.05). Tumor multiplicity was not statistically significantly different between Usp1 (+/+) and Usp1 (+/-) mice. The delayed tumorigenesis of the Usp1 (+/-) mice, compared to Usp1 (+/+) mice, was not a result of decreased H-RasV61 mutations or tumor initiation (Figure S1A), but instead was a result of delayed malignant progression (Kemp et al., 1993), presumably due to increased Fancd2-Ub mediated senescence. Interestingly, the Usp1(-/-) mice did not develop detectable papillomas.

The papillomas excised from the Usp1(+/-) mice exhibited a benign, differentiated phenotype (data not shown). In contrast, the papillomas excised from Fancd2 (+/+) mice ranged in histology from benign to malignant (Figure S1B, C). In general, the benign, more differentiated papillomas expressed high levels of Fancd2-Ub and low levels of Usp1, while the less differentiated papillomas or skin carcinomas expressed low levels of Fancd2-Ub and high levels of Usp1. The papillomas excised from the Fancd2 (+/-) mice more often displayed the less differentiated cellular phenotype (Figure S1D). Taken together, these results suggest that low levels of Usp1 and the corresponding high levels of Fancd2-Ub may promote OIS, thereby providing a barrier to oncogene-induced tumorigenesis, as previously described (Bartkova et al., 2006).

Enhanced Ras oncogene-induced senescence due to Usp1 depletion requires TAp63 and FANCD2-Ub

The p63 isoforms are abundantly expressed in human keratinocytes, and p63 (-/-) mice exhibit a severe defect in skin development and differentiation (Mills et al., 1999; Yang et al., 1999), similar to the ectodermal phenotype of the Usp1 (-/-) mice. Since TAp63 suppresses epithelial cancers by promoting cellular senescence (Guo et al., 2009), we reasoned that the Usp1 (+/-) mice may exhibit elevated TAp63 expression. The severe ectodermal phenotypes of the Usp1 (-/-) newborn mice further suggested a possible defect in p63 expression or function. Therefore, we examined the skin of Usp1 (-/-) newborn mice for defects in the expression of the various p63 isoforms (Figure 4). Indeed, skin extracted from Usp1 (-/-) newborn mice exhibited increased TAp63 expression (Figure 4A). Other epithelial tissues from the newborn Usp1 (-/-) mice, including mammary tissue, ovarian tissue, and cultured keratinocytes also exhibited increased TAp63 expression (data not shown). TAp63 was identified by its immunoreactivity with an anti-p63 antibody and by its characteristic gel migration which contrasts with other p63 isoforms (Figure S3). The level of p21 was also elevated in the skin from Usp1 (-/-) mice. The p53 level, in contrast, was comparable to the level in skin extracted from a wild-type control mouse (Figure 4A). Elevated TAp63 protein resulted, at least in part, from an increase in the TAp63 mRNA transcript level, as observed in non-damaged Usp1 (-/-) MEFs (Figure 4B). An increase in TAp63 mRNA expression was also observed in wild-type MEFs following DNA damage (Figure S5E).

Figure 4. Enhanced Ras oncogene-induced senescence by Usp1 depletion requires TAp63 and FANCD2-Ub.

Figure 4

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(A) Skin was obtained from a Usp1 (+/+) or Usp1 (-/-) newborn mouse, and tissue lysates were analyzed by immunoblotting with the indicated antibodies (See Figure S3). (B) TAp63 mRNA expression was measured by Real-Time PCR and found to be elevated in Usp1 (-/-) primary MEFs, as indicated. *, P<0.001. Data shown are mean ± standard errors (S. E.) of triplicate plates for three sibling MEFs of each genotype. Data shown are representative of three independent experiments. (C) Usp1 (+/+), Usp1 (+/-) or Usp1 (-/-) primary MEFs (passage 1) were infected with a retrovirus expressing H-RasV12 or vector only. H-RasV12 -expressing Usp1 (-/-) and Usp1 (+/-) MEFs exhibited significantly increased senescence (i.e., SA-ß-galactosidase staining) after 2 weeks. Each experiment was performed in triplicate plates for three sibling MEFs of each genotype. Data shown are mean ± standard errors of three independent experiments, at passage 2 of the MEFs. (D-F) TAp63 (+/+) or TAp63 (-/-) MEFs (passage 2) were infected with lentivirus expressing murine Usp1 shRNA or vector only. After puromycin selection, cells were infected with H-RAS V12 retrovirus or vector only. (D) The quantification of SA-β-gal staining is shown. Data shown are mean ± standard errors of triplicate plates for three sibling MEFs of each genotype, representatively of the three independent experiments. (E) Primary MEFs were stained for SA-β-gal expression 2 weeks after retrovirus infection, and the cells were photographed at 100x magnification. (F) The efficiency of Usp1 depletion was shown by immunoblotting. (G-H) Fancd2 (+/+) and Fancd2 (-/-) primary MEFs (passage 2) were infected with a lentivirus expressing Usp1 shRNA or vector only. After puromycin selection, cells were infected with H-RasV12 or control retrovirus. (G) Cell lysates were collected 2 days after retrovirus infection and immunoblotted with the indicated antibodies. (H) Indicated cells were stained for SA-β-gal expression 10 days after retrovirus infection. Data shown are mean ± standard errors of triplicate plates for three sibling MEFs of each genotype, representatively of the three independent experiments. *, P<0.001 (paired t-Test); **, P<0.005. See also Figure S3 and S4.

We reasoned that the elevated TAp63 levels may result in elevated oncogene-induced cellular senescence, accounting for the decreased tumorigenesis of Usp1-deficient mice. We therefore tested MEFs derived from Usp1 (+/+), Usp1 (+/-), and Usp1 (-/-) mice for RAS oncogene-induced senescence (OIS) (Figure 4C, Figure S4). Infection with a retrovirus encoding oncogenic H-RASV12 strongly induced senescence in Usp1 (-/-) MEFs, as shown by elevated SA-β-galactosidase staining. Moreover, Usp1 depletion induced senescence in wild-type MEFs (Figure 4D). To further test the requirement of TAp63 in OIS promoted by Usp1 depletion, we depleted Usp1 in TAp63 (-/-) MEFs using shRNA and examined OIS (Figure 4 D-F) (Guo et al., 2009). Usp1 depletion failed to induce senescence in the absence of TAp63, whereas Usp1 depletion in wild-type MEFs enhanced cellular senescence. Taken together, these results indicate that the increased OIS, resulting from Usp1 depletion and Fancd2-Ub elevation, requires TAp63 activity.

In wild-type MEFs, Usp1 depletion increased the levels of Fancd2-Ub, TAp63, and p21 (Figure 4G, lanes 1, 2) and enhanced OIS (Figure 4H). In contrast, Usp1 depletion failed to induce TAp63 or p21 expression levels and only slightly induced senescence in Fancd2 (-/-) MEFs. Taken together, these results further indicate that elevated Fancd2-Ub is required for TAp63 induction and for Ras-induced senescence.

Monoubiquitinated FANCD2 promotes transcription of TAp63 after DNA damage

Although FANCD2-Ub has well known functions as a DNA repair protein, little is known regarding its possible function as a transcription factor. We reasoned that FANCD2-Ub may play a direct role in transcriptionally activating TAp63 expression. Consequently, we performed chromatin immunoprecipitation (ChIP) assays to determine whether FANCD2-Ub could directly bind to the promoter region of TAp63 (Figure 5). The promoter regions of the p63 gene have previously been analyzed (Buckley et al., 2011) (Waltermann et al., 2003). We initially divided the promoter of TAp63 into six regions (A1-A6), shown schematically in Figure 5A. The nucleotide sequence of the promoter is shown in Figure S5A. FANCD2 binds specifically to the A3 region of the TAp63 promoter in a DNA damage-inducible manner (Figure 5B). The same pattern of inducible FANCD2-Ub binding was obtained following cellular exposure to other DNA damaging agents (MMC, IR) (Figure S5H).

Figure 5. Monoubiquitinated FANCD2 promotes transcription of TAp63.

Figure 5

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(A) Schematic diagram of primer sets for the TAp63 promoter. (A1: +99 bp to -20 bp of TAp63 promoter, A2: -21 to -320, A3: -321 to -801, A4: -802 to -980, A5: -981 to -1220, A6: -1221 to -1601). (B) 293T cells were damaged with UVC (30 J/m2) or left untreated. After 3 hours, cells were cross-linked with formaldehyde, pellets were harvested, and cross-linked chromatin was sonicated, followed by immunoprecipitation with an anti-FANCD2 antibody. Genomic DNA was purified from the immunoprecipitates and subjected to Real-Time PCR using the indicated primer sets (A1 to A6). ChIP data were calculated as FANCD2 IP/Input (%). IgG (negative control) had no signal. For B-H, the ChIP experiments shown were performed three times. Data are mean ± standard errors of triplicate plates. Data shown are representative of three independent experime nts. (C) Binding of FANCD2-Ub to the TAp63 promoter detected by ChIP assay with an anti-FANCD2 antibody, followed by PCR using the A3 primer set for the promoter region of TAp63. PD20 cells are FANCD2-deficient human fibroblasts. PD20+ V, PD20+D2WT, and PD20+D2K561R cells were treated with UVC (30 J/m2), cross-linked with formaldehyde after 3 hours, and harvested pellets. Cells were used in ChIP assays, using an anti-FANCD2 antibody, followed by Real-Time PCR with the indicated primer set. ChIP data are represented as FANCD2 IP/Input (%). (D) 293T cells were transfected with siRNA against USP1 and were cross-linked with formaldehyde 48 hours after transfection. ChIP assay was performed with the anti-FANCD2 antibody. (E) FA-A cells (FANCA-deficient GM6914 fibroblasts) transduced with empty vector (V) or corrected with the cDNA encoding FANCA were treated with UVC (30 J/m2) or left untreated, followed by formaldehyde fixation. Cells were analyzed by ChIP with anti-FANCD2 antibody and subjected to Real-Time PCR using the indicated primer sets. (F)ChIP assay was performed with an anti-FANCP/SLX4 antibody in 293T cells. (G)ChIP assay was performed with an anti-FANCD2 antibody in 293T cells, as indicated. (H)ChIP assay was performed with an anti-FANCP/SLX4 antibody in PD20 (FANCD2-deficient) cells. (I) Schematic representation of the TAp63 promoter and luciferase reporter gene constructs. (J) PD20 (FANCD2-deficient) cells were transiently co-transfected with luciferease reporter constructs bearing various lengths of the human TAp63 promoter region (100 ng) and Renilla luciferease reporter plasmid. TAp63 luciferase activity of UVC treated (30 J/m2) cells was normalized to untreated cells and represented as fold induction from individual duplicate experiments. Data shown are mean ± standard errors of three independent experiments. *, P<0.001 (t-Test); **, P<0.005. See also Figure S5, Table S1 and Table S2.

We next determined whether monoubiquitination of FANCD2 is required for promoter binding. Interestingly, the wild-type FANCD2 protein, but not the unubiquitinated FANCD2-K561R mutant protein, interacted with the A3 sequence of the TAp63 promoter following DNA damage (Figure 5C), demonstrating that FANCD2 monobubiquitination is critical for promoter binding. USP1 depletion, which also causes an increase in FANCD2-Ub, even in the absence of DNA damage, increased promoter binding of FANCD2-Ub (Figure 5D). We also examined FANCA-deficient cells (FA-A cells) which have a defect in the FA core ubiquitin E3 ligase complex (Kee and D’Andrea, 2010) and are therefore unable to monoubiquitinate FANCD2. These cells also exhibited decreased FANCD2-Ub binding to the A3 promoter sequence (Figure 5E), further indicating that FANCD2 monoubiquitination is required for its targeting to the TAp63 promoter.

FANCP/SLX4 was recently identified as another downstream protein in the FA pathway (Crossan et al., 2011) (Stoepker et al., 2011) (Kim et al., 2011). FANCP/SLX4 has structural features consistent with other direct transcriptional activators, including a SAP domain and a BTB domain (Fekairi et al., 2009; Kim et al., 2013; Svendsen et al., 2009). FANCP/SLX4 also has two ubiquitin binding domains (UBZ4), and it interacts with the ubiquitin moiety of FANCD2-Ub (Yamamoto et al., 2011). We next tested whether FANCP/SLX4 can also bind to the A3 region of the TAp63 gene (Figure 5 F, G, H). Interestingly, FANCP/SLX4 interacted with the A3 region following DNA damage with UV light (Figure 5F) or following MMC treatment (data not shown). Knockdown of SLX4 using siRNA reduced the UV-inducible binding of FANCD2-Ub to the A3 region, suggesting that FANCD2-Ub and FANCP/SLX4 cooperate in the transcriptional activation of TAp63 (Figure 5G). Consistent with this hypothesis, Slx4 (-/-) MEFs have reduced levels of TAp63 mRNA and protein (data not shown). Moreover, FANCD2-Ub was not required for the FANCP/SLX4 interaction with the A3 sequence (Figure 5H), suggesting that FANCP/SLX4 binds to the A3 promoter region independently and functions, at least in part, to recruit FANCD2-Ub to this site. In contrast, another downstream FA protein, FANCD1/BRCA2, was not required for damage-inducible localization of FANCD2-Ub to the A3 site (data not shown). Taken together, these data suggest that a complex of FANCD2-Ub and FANCP/SLX4 may cooperate in the transcriptional activation of the TAp63 tumor suppressor gene and perhaps other genes.

In order to identify other genes which are transcriptionally regulated by FANCD2-Ub, we next performed a genome-wide ChIP-Seq analysis (Figure S5B-G). Human 293T cells were treated with or without UV radiation, and an anti-FANCD2 antibody was used to immunoprecipitate chromatin fragments. DNA damage activated a 163-fold increase in the number of annotated peaks of FANCD2 protein binding. We hypothesized that some of these FANCD2-Ub binding sites were non-specific sites of DNA damage while other sites were bonafide sites of transcriptional activation. The genome-wide distribution of inferred DNA binding sites is shown in Figure S5B. To determine whether the DNA damage-induced binding sequences map to genes with distinct functional annotations, we used the USCS genome browser. Interestingly, FANCD2-Ub-induced binding sequences were highly associated with genes that are essential for epithelial development and morphogenesis (Figure S5C). Representative genes in this epithelium-related ontology, which exhibited FANCD2-Ub binding after DNA damage, were further identified (Table S2). Examination of the ChIP-seq tracks indicated specific genes which have DNA damage-inducible FANCD2-Ub binding (Figure S5D-G). The sequence preferences for FANCD2-Ub are shown in Table S1. To confirm that these binding sites participate in the transcriptional activation of the genes, we performed a quantitative PCR (qPCR) validation of the targets (Figure S5E-G). Indeed, DNA damage activated the Fancd2-Ub dependent transcription of the TAp63, ΔNp63, and Brca2 mRNAs. The level of induction varied, depending, at least in part, on the kind of genotoxic stress used. For instance, while IR and UV yielded relatively strong induction of these transcripts, MMC was a weaker stimulant. IR and UV may be more potent inducers since they are delivered during a short time interval. In contrast, MMC is delivered overnight and must be taken up by cells before it crosslinks the DNA. This delay may account for its reduced transcriptional induction. Damage by IR, MMC, or UV failed to upregulate transcription of these genes in Fancd2-deficient MEFs. The FANCD2-Ub mediated upregulation of both p63 isoforms is surprising, since TAp63 and ΔNp63 appear to exhibit antagonist functions (Koster and Roop, 2004).

The ChIP results demonstrate that FANCD2-Ub can bind to relevant cis-acting regulatory sequences of the TAp63 gene, leading to enhanced TAp63 expression and enhanced cellular senescence. To further determine the functional role of FANCD2-Ub in transcription, these regulatory regions were used to construct luciferase reporters (Figure 5I schematic). The constructs were transfected into FA cells and corrected FA cells. FANCD2-Ub, but not the K561R (unubiquitinated) mutant form of FANCD2, enhanced the damage-inducible transcription of the TAp63 promoter (enhancer) constructs (Figure 5J). Taken together, these results demonstrate that monoubiquitinated FANCD2 localizes to the promoter of TAp63 and contributes to its DNA damage-inducible transcription.

Usp1 knockdown promotes senescence and reduces tumor growth in vivo

These results suggest that Usp1 has a growth-promoting activity while its substrate, Fancd2-Ub, promotes senescence and opposes cell growth by inducing TAp63 mRNA and protein expression. Indeed, recent studies have indicated that Usp1 acts as an oncogene, and its overexpression can transform 3T3 fibroblasts (Williams et al.). To further test this hypothesis, we depleted USP1 in a RAS-driven human lung cancer cell line, A549, and tested the resulting clones in a mouse xenograft model (Figure 6). As predicted, A549 cells expressing USP1 shRNA showed an increase in the level of FANCD2-Ub (Figure 6A). The reduction of USP1 expression by shRNA, and the increased FANCD2-Ub expression, inhibited the colony formation of A549 cells in soft agar (Figure 6B) and inhibited the tumor xenograft growth in nude mice (Figure 6 C, D). Consistent with these results, doxycycline induction of the shRNA to FANCD2 in A549 cells resulted in increased tumor growth in a xenograft model (Figure 6 E, F). Taken together, these data further indicate that depletion of USP1 results in increased cellular levels of FANCD2-Ub, causing increased senescence and decreased epithelial tumor growth in vivo.

Figure 6. Usp1 knockdown promotes senescence and reduces tumor growth in vivo.

Figure 6

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(A-B) The USP1 shRNA or a control vector were stably expressed in the Ras driven human lung adenocarcinoma epithelial cell line, A549. (A) Cell lysates were immunoblotted with the indicated antibodies. (B) A549 cells were plated in soft agar and transformed foci were counted at 100x magnification 2 weeks later. Data shown are mean ± standard errors of three independent experiments in cell lines. Each experiment was performed triplicated plates. **, P<0.005, t-Test. (C-D)Comparison of tumorigenicity of USP1 shRNA or control shRNA transfected A549 cells. A total of 2 × 106 A549 cells were injected as xenografts into nude mice. Data shown are mean ± standard errors from obtained all tumors. (C) Every 3 days, tumor volume (mm) was measured for the indicated time period. The data represent the average tumor size of 10 mice in each group (P < 0.05). (D) Quantification of tumor volume of the xenografts at day32. (E-F) A549 cells expressing an inducible shRNA targeting FANCD2 or control vector, following doxycycline treatment. (E) Immunoblotting showing expression of FANCD2 in A549 cells grown in doxycycline for 3 days. (F) Growth of A549 cells with shRNA targeting FANCD2 tumor xenografts. In each group, 10 mice were given doxycycline (200 μg/ml) by drinking water when the tumor reached 200 mm3. RTV is relative tumor volume based on 200 mm3 for individual tumors. Data shown are mean ± standard errors from obtained all tumors.

Depletion of Usp1 decreases epidermal proliferation in Fancd2 (+/-) mice

We next examined the effect of knocking down Usp1 on epidermal proliferation in a Fancd2-deficient genetic background (Figure 7). For these studies, we used a double heterozygous mouse strain, Fancd2 (+/-)/ Usp1 (+/-), which was previously described (Kim et al., 2009). Topical TPA treatment increased epidermal proliferation in Fancd2 (+/-) mice. Interestingly, this hyperproliferation was reduced in Fancd2 (+/-)/ Usp1 (+/-) double heterozygous mice (Figure 7A, B). The increased proliferation in the Fancd2 (+/-) mice correlated with increased Ki67 staining (Figure 7A) and decreased senescence (data not shown). Usp1 heterozygosity resulted in elevated Fancd2-Ub levels in the Fancd2 (+/-) heterozygote background (Figure 7C, lane 3), correlating with the reduced epidermal proliferation, and consistent with the function of Fancd2-Ub as a tumor suppressor. These data further indicate that a decrease in Usp1 expression causes an increase in Fancd2-Ub levels, and that this increase may account for the decreased epidermal cell proliferation in the double heterozygote animals.

Figure 7. Usp1 depletion decreases epidermal proliferation in Fancd2 (+/-) mice.

Figure 7

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(A) Mice from the indicated genotypes were exposed topically to TPA or acetone alone for four weeks. Mice were sacrificed, and skin histology was evaluated by microscopy. Arrows indicate the sites of epidermal proliferation. Scale bar=100 μm. Ki67 staining was performed to evaluate cellular proliferation. (B) Bar graphs showing skin thickness of the three mouse cohorts after TPA exposure (n = 3 female mice per cohort). **, P<0.005 (t-Test). Data shown are mean ± standard errors of triplicate plates for three sibling MEFs of each genotype. Data shown are representative of three independent experiments. (C) TPA- treated skin was collected, and tissue lysates were immunoblotted with the indicated antibodies. The double heterozygote has increased Fancd2-Ub expression (lane 3), consistent with the decreased epithelial cell proliferation observed. (D) Proposed role of Fancd2 monoubiquitination in tumor suppression.

DISCUSSION

Our results indicate that FANCD2-Ub, a critical downstream product of the FA pathway, acts as a tumor suppressor in squamous epithelium. FANCD2-Ub accumulates in epithelial cells in response to DNA damage or oncogenic stress, or following USP1 knockdown, resulting in enhanced cellular senescence (Figure 7D). In addition to promoting DNA repair via the canonical FA pathway (Kee and D’Andrea, 2010), FANCD2-Ub, along with its binding partner FANCP/SLX4, is also a transcriptional regulator of TAp63. TAp63 may in turn activate cellular senescence (Guo et al., 2009) by transcriptionally upregulating other tumor suppressor proteins, such as RAD51 and BRCA2 (Lin et al., 2009). Indeed, Fancd2 (-/-) and TAp63 (-/-) MEFs have reduced levels of Brca2 mRNA and protein, compared to wild-type MEFs (data not shown). Taken together, our results suggest that the squamous cell-specific tumorigenesis in FA patients results from loss of this tumor suppressor pathway. Somatic disruption of the pathway may also account for the development of SCC in the general (non-FA) population.

Recent studies suggest that FANCD2-Ub has transcriptional activity. For example, FANCD2-Ub appears to bind to a concensus sequence for NF-κB on the TNFα promoter and to repress transcription of the TNFα gene (Matsushita et al., 2011). Although FANCD2-Ub binds to a region of the TAp63 promoter (Region A3), which also contains two NF-κB binding motifs (Figure S5A), our ChIP-Seq study did not reveal a statistically significant sequence preference of FANCD2-Ub for these motif (Table S1). Other recent studies indicate that FANCD2-Ub forms a protein complex with BRG1 and localizes to the promoters of anti-oxidant genes (Du et al., 2012). Taken together, our study further supports a direct role of FANCD2-Ub in transcription of the TAp63 gene and provides a mechanism for the tumor suppressor activity of the FA pathway.

Fancd2-Ub appears to exert its anti-tumor activity through both cell autonomous and cell extrinsic mechanisms. First, Fancd2-Ub functions as a tumor suppressor in a cell autonomous manner. Indeed, Fancd2-Ub is a DNA damage response (DDR) protein which directly promotes TAp63-mediated cellular senescence and inhibits the growth of tumor xenografts (Figure 6). In this regard, the Fancd2-Ub tumor suppressor activity is similar to the pro-senescence tumor suppressor activity observed for other DDR proteins, such as ATM (Bartkova et al., 2006) and CHK2 (Di Micco et al., 2006). The decrease in epidermal tumors observed in the DMBA/TPA treated Usp1 (+/-) mice likely results directly from the tumor suppressor activity of Fancd2-Ub generated by the FA pathway in H-Ras mutant epidermal cells. Second, Fancd2-Ub may also suppress squamous cell tumorigensis indirectly, through a cell extrinsic mechanism. For instance, for Usp1 (-/-) newborn mice, the Fancd2-Ub-mediated senescence of dermal fibroblasts may account for their thin overlying epidermal layer. The thin epidermis of the Usp1-deficient mice contains fewer total epidermal target cells, thus providing another mechanism for the decreased incidence of DMBA/TPA induced tumors. Other functions of FANCD2-Ub in the underlying stromal cells, such as its ability to limit vascularization, may further suppress tumor growth of the overlying epithelium.

Our study specifically examined the mechanism by which Usp1 disruption and FANCD2-Ub elevation can suppress the development of H-Ras-driven tumors of the epidermis. It will be interesting to determine whether Usp1 knockout mice are also resistant to other Ras driven tumors, such as K-Ras driven lung cancers or pancreatic cancers. Usp1 knockout has recently been shown to confer an anti-cancer activity by promoting the differentiation of mesenchymal cells (Williams et al., 2011), suggesting that Usp1-deficient mice may be resistant to mesenchymal tumors, such as sarcomas and leukemias. Thus, FANCD2-Ub may have a more general anti-tumor function in other tissues, although the suppression of SCCs appears to be especially pronounced, as demonstrated by the specific increased incidence of this tumor class in FA patients.

Our model (Figure 7D) predicts that somatic disruption of FA genes or of TAp63 may contribute to squamous cell carcinogenesis in the general (non-FA) population. A mutational screen of head and neck cancers has recently shown that some SCCs have mutations in the p63 gene. These mutations are predicted to result in the loss of the TAp63 isoform and the upregulation of the oncogenic ΔNp63 isoform (Stransky et al., 2011). Somatic mutations in FA genes were also identified in sporadic SCCs (Romick-Rosendale et al., 2013).

TAp63 also plays a critical role in maintaining the female germline (Suh et al., 2006). Recent studies have shown that TAp63 is the predominant p63 isoform in primordial follicle oocytes, and it regulates DNA damage inducible apoptosis of oocytes in vivo (Kerr et al., 2012). Fancd2-deficient mice (Houghtaling et al., 2003) and other FA pathway deficient mice (Parmar et al., 2009) also have dysplastic ovaries and reduced fertility, and exhibit an increased incidence of ovarian cancer (Bakker et al., 2012; Wong et al., 2003). Taken together, these findings further support a model in which activation of TAp63 expression by the FA pathway is required, not only for suppression of SCC, but also for normal ovarian development.

Finally, our results suggest a possible therapeutic strategy for human SCCs by inducing tumor cell senescence. Small molecule inhibitors of Usp1 may enhance FANCD2-Ub levels and TAp63 levels and perhaps promote cellular senescence. USP1 inhibitors may therefore exert both chemosensitization and tumor cell senescence activities.

METHODS

Animals and DMBA/TPA-induced skin carcinogenesis

Fancd2-, Fancg, and Usp1-deficient mice were maintained on a C57BL/6 background and genotyped as previously described (Kim et al., 2009) (Parmar et al., 2010). All animal studies were carried out under an Animal Care and Use Committee-approved protocol. For mouse skin carcinogenesis, the back skin of each female mouse (6-8 weeks) was shaved 1 day before topical treatment, with 7,12-dimethylbenz(a)anthracene (DMBA; D3254, Sigma-Aldrich, Inc, St. Louis, MO), 100 μg in 200 μL of acetone. One week later, mice were topically treated with 2.5 μg of 12-O-tetradecanoylphorbol-13-actate (TPA; p-1680, LC Laboratories, Woburn, MA) in 200 μL of acetone, 5 times a week for 28 weeks. Onset, number and size of tumors were monitored once a week. Paraffin-embedded sections of tumor tissue and skin were prepared and fixed with 4% paraformaldehyde, and H&E staining was done in our Histology and Tissue Core.

Cell proliferation assay

Primary MEFs were seeded in 6-well plates (103 cells per well) in triplicate. After 2 weeks, cells were fixed with methanol and stained with 0.05 % crystal violets. For in vivo cell proliferation assays, mice were topically treated with TPA (2.5 μg) for four weeks. Skin samples were fixed with formalin and paraffin-embedded sections of skin were stained with H&E and Ki67.

SA-β-Galactosidase Staining

Staining for SA-β-galactosidase activity in cultured cells was carried out using a staining kit (Cell signaling catalog, #9860). Briefly, cells were seeded in 6-well plates (104 cells per well) in triplicate. After 10 days, the cells were fixed with β-Gal Fixative and stained with complete β-Gal Stain Solution followed by addition of β-Gal Holding Solution. For in vivo senescence staining, frozen sections of skin were prepared with optimal cutting temperature (O.C.T.) compound and followed by staining. The sections were counterstained with Eosin.

Supplementary Material

01

HIGHLIGHTS.

  • Monoubiquitinated FANCD2 is a tumor suppressor protein

  • Fancd2-deficient mice have an increased incidence of DMBA/TPA-induced skin cancers

  • Usp1-deficient mice have elevated Fancd2-Ub and are resistant to skin cancers

  • FANCD2-Ub transcriptionally increases TAp63 and promotes cellular senescence

Acknowledgments

We thank Youngeun Choi for help in real-time PCR analysis and Jason T. Tsichlis for technical assistance. We thank Markus Grompe, KJ Patel, Alec Kimmelman, David Pellman, Dipanjan Chowdhury, Geoffrey Shapiro, Kalindi Parmar, David Kozono, Younghoon Kee, Min Huang and other members of the D’Andrea Laboratory for helpful discussions. E.P. is supported by postdoctoral fellowships from the Susan G. Komen Foundation (KG101186) and the ASCO-Gianni Bonadonna Fellowship. This study was also supported by NIH grants R01DK43889, R01HL52725, and P01CA092584 to A.D.D.

Footnotes

AUTHOR CONTRIBUTIONS

E.P and A.D.D conceived and designed the study. E.P. performed all experiments and analyzed the data. J.M.K. was involved in the establishment of mouse models and animal experiments. H.K. contributed to the xenograft study and B.P. provided technical assistance during animal experiments. Y.X. and B.P. contributed to the elucidation of the transcriptional activity of the FANCD2-Ub protein. E.P., J.M.K., H.K., A.M, and A.D.D. participated in discussions and interpretations of the experiments. E.P., J.M.K., and A.D.D. wrote the manuscript.

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests

Accession Numbers

Chi-seq data have been deposited in the GEO database and the NCBI Sequence Read Archive database under accession numbers GSE46902.

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References

  1. Alam M, Ratner D. Cutaneous squamous-cell carcinoma. N Engl J Med. 2001;344:975–983. doi: 10.1056/NEJM200103293441306. [DOI] [PubMed] [Google Scholar]
  2. Alter BP, Greene MH, Velazquez I, Rosenberg PS. Cancer in Fanconi anemia. Blood. 2003;101:2072. doi: 10.1182/blood-2002-11-3597. [DOI] [PubMed] [Google Scholar]
  3. Auerbach AD. Fanconi anemia and its diagnosis. Mutat Res. 2009;668:4–10. doi: 10.1016/j.mrfmmm.2009.01.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bakker ST, van de Vrugt HJ, Visser JA, Delzenne-Goette E, van der Wal A, Berns MA, van de Ven M, Oostra AB, de Vries S, Kramer P, et al. Fancf-deficient mice are prone to develop ovarian tumours. J Pathol. 2012;226:28–39. doi: 10.1002/path.2992. [DOI] [PubMed] [Google Scholar]
  5. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, Vassiliou LV, Kolettas E, Niforou K, Zoumpourlis VC, et al. Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature. 2006;444:633–637. doi: 10.1038/nature05268. [DOI] [PubMed] [Google Scholar]
  6. Buckley NE, Conlon SJ, Jirstrom K, Kay EW, Crawford NT, O’Grady A, Sheehan K, Mc Dade SS, Wang CW, McCance DJ, et al. The DeltaNp63 proteins are key allies of BRCA1 in the prevention of basal-like breast cancer. Cancer Res. 2011;71:1933–1944. doi: 10.1158/0008-5472.CAN-10-2717. [DOI] [PubMed] [Google Scholar]
  7. Chen J, Dexheimer TS, Ai Y, Liang Q, Villamil MA, Inglese J, Maloney DJ, Jadhav A, Simeonov A, Zhuang Z. Selective and Cell-Active Inhibitors of the USP1/ UAF1 Deubiquitinase Complex Reverse Cisplatin Resistance in Non-small Cell Lung Cancer Cells. Chem Biol. 2011;18:1390–1400. doi: 10.1016/j.chembiol.2011.08.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohn MA, Kowal P, Yang K, Haas W, Huang TT, Gygi SP, D’Andrea AD. A UAF1-containing multisubunit protein complex regulates the Fanconi anemia pathway. Mol Cell. 2007;28:786–797. doi: 10.1016/j.molcel.2007.09.031. [DOI] [PubMed] [Google Scholar]
  9. Crossan GP, van der Weyden L, Rosado IV, Langevin F, Gaillard PH, McIntyre RE, Gallagher F, Kettunen MI, Lewis DY, Brindle K, et al. Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia. Nat Genet. 2011;43:147–152. doi: 10.1038/ng.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, Luise C, Schurra C, Garre M, Nuciforo PG, Bensimon A, et al. Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature. 2006;444:638–642. doi: 10.1038/nature05327. [DOI] [PubMed] [Google Scholar]
  11. Du W, Rani R, Sipple J, Schick J, Myers KC, Mehta P, Andreassen PR, Davies SM, Pang Q. The FA pathway counteracts oxidative stress through selective protection of antioxidant defense gene promoters. Blood. 2012 doi: 10.1182/blood-2011-09-381970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fekairi S, Scaglione S, Chahwan C, Taylor ER, Tissier A, Coulon S, Dong MQ, Ruse C, Yates JR, 3rd, Russell P, et al. Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases. Cell. 2009;138:78–89. doi: 10.1016/j.cell.2009.06.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garcia-Higuera I, Taniguchi T, Ganesan S, Meyn MS, Timmers C, Hejna J, Grompe M, D’Andrea AD. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol Cell. 2001;7:249–262. doi: 10.1016/s1097-2765(01)00173-3. [DOI] [PubMed] [Google Scholar]
  14. Guo X, Keyes WM, Papazoglu C, Zuber J, Li W, Lowe SW, Vogel H, Mills AA. TAp63 induces senescence and suppresses tumorigenesis in vivo. Nat Cell Biol. 2009;11:1451–1457. doi: 10.1038/ncb1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Holzel M, van Diest PJ, Bier P, Wallisch M, Hoatlin ME, Joenje H, de Winter JP. FANCD2 protein is expressed in proliferating cells of human tissues that are cancer-prone in Fanconi anaemia. J Pathol. 2003;201:198–203. doi: 10.1002/path.1450. [DOI] [PubMed] [Google Scholar]
  16. Hoskins EE, Morris TA, Higginbotham JM, Spardy N, Cha E, Kelly P, Williams DA, Wikenheiser-Brokamp KA, Duensing S, Wells SI. Fanconi anemia deficiency stimulates HPV-associated hyperplastic growth in organotypic epithelial raft culture. Oncogene. 2009;28:674–685. doi: 10.1038/onc.2008.416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Houghtaling S, Granville L, Akkari Y, Torimaru Y, Olson S, Finegold M, Grompe M. Heterozygosity for p53 (Trp53+/-) accelerates epithelial tumor formation in fanconi anemia complementation group D2 (Fancd2) knockout mice. Cancer Res. 2005;65:85–91. [PubMed] [Google Scholar]
  18. Houghtaling S, Timmers C, Noll M, Finegold MJ, Jones SN, Meyn MS, Grompe M. Epithelial cancer in Fanconi anemia complementation group D2 (Fancd2) knockout mice. Genes Dev. 2003;17:2021–2035. doi: 10.1101/gad.1103403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hu H, Xia SH, Li AD, Xu X, Cai Y, Han YL, Wei F, Chen BS, Huang XP, Han YS, et al. Elevated expression of p63 protein in human esophageal squamous cell carcinomas. Int J Cancer. 2002;102:580–583. doi: 10.1002/ijc.10739. [DOI] [PubMed] [Google Scholar]
  20. Kee Y, D’Andrea AD. Expanded roles of the Fanconi anemia pathway in preserving genomic stability. Genes Dev. 2010;24:1680–1694. doi: 10.1101/gad.1955310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kemp CJ, Donehower LA, Bradley A, Balmain A. Reduction of p53 gene dosage does not increase initiation or promotion but enhances malignant progression of chemically induced skin tumors. Cell. 1993;74:813–822. doi: 10.1016/0092-8674(93)90461-x. [DOI] [PubMed] [Google Scholar]
  22. Kerr JB, Hutt KJ, Michalak EM, Cook M, Vandenberg CJ, Liew SH, Bouillet P, Mills A, Scott CL, Findlay JK, Strasser A. DNA damage-induced primordial follicle oocyte apoptosis and loss of fertility require TAp63-mediated induction of Puma and Noxa. Mol Cell. 2012;48:343–352. doi: 10.1016/j.molcel.2012.08.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Keyes WM, Wu Y, Vogel H, Guo X, Lowe SW, Mills AA. p63 deficiency activates a program of cellular senescence and leads to accelerated aging. Genes Dev. 2005;19:1986–1999. doi: 10.1101/gad.342305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kim JM, Parmar K, Huang M, Weinstock DM, Ruit CA, Kutok JL, D’Andrea AD. Inactivation of murine Usp1 results in genomic instability and a Fanconi anemia phenotype. Dev Cell. 2009;16:314–320. doi: 10.1016/j.devcel.2009.01.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kim Y, Lach FP, Desetty R, Hanenberg H, Auerbach AD, Smogorzewska A. Mutations of the SLX4 gene in Fanconi anemia. Nat Genet. 2011;43:142–146. doi: 10.1038/ng.750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kim Y, Spitz GS, Veturi U, Lach FP, Auerbach AD, Smogorzewska A. Regulation of multiple DNA repair pathways by the Fanconi anemia protein SLX4. Blood. 2013;121:54–63. doi: 10.1182/blood-2012-07-441212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Koster MI, Kim S, Mills AA, DeMayo FJ, Roop DR. p63 is the molecular switch for initiation of an epithelial stratification program. Genes Dev. 2004;18:126–131. doi: 10.1101/gad.1165104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Koster MI, Roop DR. Transgenic mouse models provide new insights into the role of p63 in epidermal development. Cell Cycle. 2004;3:411–413. [PubMed] [Google Scholar]
  29. Lin HK, Chen Z, Wang G, Nardella C, Lee SW, Chan CH, Yang WL, Wang J, Egia A, Nakayama KI, et al. Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence. Nature. 2010;464:374–379. doi: 10.1038/nature08815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lin YL, Sengupta S, Gurdziel K, Bell GW, Jacks T, Flores ER. p63 and p73 transcriptionally regulate genes involved in DNA repair. PLoS Genet. 2009;5:e1000680. doi: 10.1371/journal.pgen.1000680. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  31. Matsushita N, Endo Y, Sato K, Kurumizaka H, Yamashita T, Takata M, Yanagi S. Direct inhibition of TNF-alpha promoter activity by Fanconi anemia protein FANCD2. PLoS One. 2011;6:e23324. doi: 10.1371/journal.pone.0023324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature. 1999;398:708–713. doi: 10.1038/19531. [DOI] [PubMed] [Google Scholar]
  33. Nijman SM, Huang TT, Dirac AM, Brummelkamp TR, Kerkhoven RM, D’Andrea AD, Bernards R. The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway. Mol Cell. 2005;17:331–339. doi: 10.1016/j.molcel.2005.01.008. [DOI] [PubMed] [Google Scholar]
  34. O’Donnell L, Durocher D. DNA repair has a new FAN1 club. Mol Cell. 2010;39:167–169. doi: 10.1016/j.molcel.2010.07.010. [DOI] [PubMed] [Google Scholar]
  35. Park E, Zhu F, Liu B, Xia X, Shen J, Bustos T, Fischer SM, Hu Y. Reduction in IkappaB kinase alpha expression promotes the development of skin papillomas and carcinomas. Cancer Res. 2007;67:9158–9168. doi: 10.1158/0008-5472.CAN-07-0590. [DOI] [PubMed] [Google Scholar]
  36. Park JW, Pitot HC, Strati K, Spardy N, Duensing S, Grompe M, Lambert PF. Deficiencies in the Fanconi anemia DNA damage response pathway increase sensitivity to HPV-associated head and neck cancer. Cancer Res. 2010;70:9959–9968. doi: 10.1158/0008-5472.CAN-10-1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Parmar K, D’Andrea A, Niedernhofer LJ. Mouse models of Fanconi anemia. Mutat Res. 2009;668:133–140. doi: 10.1016/j.mrfmmm.2009.03.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Parmar K, Kim J, Sykes SM, Shimamura A, Stuckert P, Zhu K, Hamilton A, Deloach MK, Kutok JL, Akashi K, et al. Hematopoietic stem cell defects in mice with deficiency of Fancd2 or Usp1. Stem Cells. 2010;28:1186–1195. doi: 10.1002/stem.437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rocco JW, Ellisen LW. p63 and p73: life and death in squamous cell carcinoma. Cell Cycle. 2006;5:936–940. doi: 10.4161/cc.5.9.2716. [DOI] [PubMed] [Google Scholar]
  40. Rocco JW, Leong CO, Kuperwasser N, DeYoung MP, Ellisen LW. p63 mediates survival in squamous cell carcinoma by suppression of p73-dependent apoptosis. Cancer Cell. 2006;9:45–56. doi: 10.1016/j.ccr.2005.12.013. [DOI] [PubMed] [Google Scholar]
  41. Romick-Rosendale LE, Lui VW, Grandis JR, Wells SI. The Fanconi anemia pathway: Repairing the link between DNA damage and squamous cell carcinoma. Mutation research. 2013 doi: 10.1016/j.mrfmmm.2013.01.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell. 1997;88:593–602. doi: 10.1016/s0092-8674(00)81902-9. [DOI] [PubMed] [Google Scholar]
  43. Smogorzewska A, Matsuoka S, Vinciguerra P, McDonald ER, 3rd, Hurov KE, Luo J, Ballif BA, Gygi SP, Hofmann K, D’Andrea AD, Elledge SJ. Identification of the FANCI Protein, a Monoubiquitinated FANCD2 Paralog Required for DNA Repair. Cell. 2007;129:289–301. doi: 10.1016/j.cell.2007.03.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stoepker C, Hain K, Schuster B, Hilhorst-Hofstee Y, Rooimans MA, Steltenpool J, Oostra AB, Eirich K, Korthof ET, Nieuwint AW, et al. SLX4, a coordinator of structure-specific endonucleases, is mutated in a new Fanconi anemia subtype. Nat Genet. 2011;43:138–141. doi: 10.1038/ng.751. [DOI] [PubMed] [Google Scholar]
  45. Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, Kryukov GV, Lawrence MS, Sougnez C, McKenna A, et al. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011;333:1157–1160. doi: 10.1126/science.1208130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Suh EK, Yang A, Kettenbach A, Bamberger C, Michaelis AH, Zhu Z, Elvin JA, Bronson RT, Crum CP, McKeon F. p63 protects the female germ line during meiotic arrest. Nature. 2006;444:624–628. doi: 10.1038/nature05337. [DOI] [PubMed] [Google Scholar]
  47. Svendsen JM, Smogorzewska A, Sowa ME, O’Connell BC, Gygi SP, Elledge SJ, Harper JW. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair. Cell. 2009;138:63–77. doi: 10.1016/j.cell.2009.06.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Waltermann A, Kartasheva NN, Dobbelstein M. Differential regulation of p63 and p73 expression. Oncogene. 2003;22:5686–5693. doi: 10.1038/sj.onc.1206859. [DOI] [PubMed] [Google Scholar]
  49. Williams SA, Maecker HL, French DM, Liu J, Gregg A, Silverstein LB, Cao TC, Carano RA, Dixit VM. USP1 deubiquitinates ID proteins to preserve a mesenchymal stem cell program in osteosarcoma. Cell. 2011;146:918–930. doi: 10.1016/j.cell.2011.07.040. [DOI] [PubMed] [Google Scholar]
  50. Wong JC, Alon N, McKerlie C, Huang JR, Meyn MS, Buchwald M. Targeted disruption of exons 1 to 6 of the Fanconi Anemia group A gene leads to growth retardation, strain-specific microphthalmia, meiotic defects and primordial germ cell hypoplasia. Hum Mol Genet. 2003;12:2063–2076. doi: 10.1093/hmg/ddg219. [DOI] [PubMed] [Google Scholar]
  51. Yamamoto KN, Kobayashi S, Tsuda M, Kurumizaka H, Takata M, Kono K, Jiricny J, Takeda S, Hirota K. Involvement of SLX4 in interstrand cross-link repair is regulated by the Fanconi anemia pathway. Proc Natl Acad Sci U S A. 2011;108:6492–6496. doi: 10.1073/pnas.1018487108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, McKeon F. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature. 1999;398:714–718. doi: 10.1038/19539. [DOI] [PubMed] [Google Scholar]

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