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CYLD has been recognized as a tumor suppressor due to its dominant genetic linkage to multiple types of epidermal tumors and a range of other cancers. The molecular mechanisms governing CYLD control of skin cancer are still unclear. Here, we demonstrated that K14-driven epidermal expression of a patient relevant and catalytically deficient CYLD truncation mutant (CYLDm) sensitized mice to skin tumor development in response to DMBA/TPA-challenge. Tumors developed on transgenic mice were prone to malignant progression and lymph node metastasis, and displayed increased activation of JNK and the downstream c-Jun and c-Fos proteins. Most importantly, topical application of a pharmacological JNK inhibitor significantly reduced tumor development and abolished metastasis in the transgenic mice. Further in line with these animal data, exogenous expression of CYLDm in A431, a human squamous cell carcinoma (SCC) cell line, markedly enhanced cell growth, migration and subcutaneous tumor growth in an AP1-depdendent manner. In contrast, expression of the wild type CYLD inhibited SCC tumorigenesis and AP1 function. Most importantly, CYLDm not only increased JNK activation but also induced an upregulation of K63-ubiquitination on both c-Jun and c-Fos, leading to sustained AP1 activation. Our findings uncovered c-Jun and c-Fos as novel CYLD-targets and underscore that CYLD controls epidermal tumorigenesis through blocking the JNK/AP1 signaling pathway at multiple levels.
Human cyld (chromosome 16q12-13) encodes a deubiquitinase that primarily removes lysine-63(K-63) linked poly-ubiquitin chains from an array of target proteins, including TRAF2/6, IKKγ, Bcl3, plk1, Tak1 and lck 1–8. K63-ubiquitin is distinct from K48- ubiquitin with the former leading to protein activation and the latter targeting protein for degradation. Thus, CYLD generally acts a negative regulator to target protein function 9. CYLD was initially discovered as a tumor suppressor due to its autosomal-dominant genetic linkage to multiple types of cutaneous adnexal tumors, including Brooke-Spiegler syndrome (BSS), familial cylindromas, multiple familial tricoepithelioma (MFT) and spiradenoma 9,10. Up to 51 different truncation and missense mutations have been characterized thus far in skin tumors; all of these mutations result in the production of catalytically deficient CYLD mutants (CYLDm) 10. In addition, loss of heterozygosity (LOH) of the normal allele has been detected in about 70% of tumors carrying a CYLDm 11–14. These data underscore that the catalytic function of CYLD is important for tumor suppression. Moreover, CYLD loss-of-function has been associated with many other cancers, including melanoma and myeloma 15,16, as well as breast, colon, liver, kidney and cervical cancers 17–21. Despite the broad relevance of CYLD to cancer, the molecular mechanisms governing CYLD-effects on tumorigenesis are poorly understood.
The NF-κB pathway is a major downstream target of CYLD and is predicted to be the central regulator in driving the pathogenesis of skin cancers associated with CYLD-deficiency 2–4. In particular, Bcl3, a non-canonical NF-κB subunit and a direct downstream target of CYLD, is identified as an essential regulator for the TPA-induced hyperproliferation of cyld−-− keratinocytes 22. However, clinical trials targeting NF-κB for the cure of cylindromas have not been satisfactory 23, which could be attributed to the common issues associated with bench-to-bedside applications. On the other hand, recent studies using either mouse or human tissue models have demonstrated that NF-κB inhibits epidermal malignancy 24–26, suggesting that there could be other key regulators acting downstream of CYLD to promote malignancy.
In this study, we used transgenic mouse and human SCC models to investigate how CYLD loss-of-function leads to abnormal signal transduction and promotes tumorigenesis. We demonstrated that expression of a catalytically deficient and patient-relevant CYLD mutant (CYLDm) sensitizes the epidermis to malignancy and metastasis in a JNK/AP1-dependent manner. We also showed that CYLDm enhanced, whereas wild type CYLD inhibited, human SCC tumorigenesis both in vitro and in vivo. Moreover, we found that CYLDm not only increased JNK activity but also increased K63-ubiqutination on both c-Jun and c-Fos, and ultimately potentiated AP1 transcriptional activity. Our findings indicate that the abnormal induction of the JNK/AP1 signaling pathway underlies epidermal tumorigenesis associated with CYLD loss-of-function.
K14-CYLDm (HA-CYLD.932) expression construct was generated with the PCR-product with pcDNA.HA-CYLD as a template 2. The purified PCR product was cloned into the pENTR1A vector (Invitrogen, Carlsbad, CA) and then gateway cloned into pBskII.K14 plasmid 27, which was then linearlized with KpnI and SmaI for the generation of transgenic mice. LZRS.CYLDWT and LZRS.CYLDm were generated by using the PmeI fragment from pcDNA.HA.CYLD and the PCR-fragment encoding HA-CYLD.932. All plasmids were sequence verified at Duke DNA sequencing core facility. Retroviruses were produced in phoenix cells as described 28.
A431 and 293T cells were obtained from ATCC and cultured in 5% fetal bovine serum (FBS) in DMEM. A431 cells were confirmed to express cytokeratin 14 by immunostaining but no additional cell line authentication was performed by the authors. DNA transfection was performed with GenJet transfection reagent (SignaGen laboratories, Ijamsville, MD) followed by selection with puromycin (1μg/ml) for 3–4 days for stable expression of LacZ, CYLDWT or CYLDm. For protein analysis, cells were serum starved for 24 hours and then incubated with fresh media containing 5% FBS and 25 ng/ml EGF for 1 hour. Protein extracts were collected in RIPA or NP-lysis buffer supplemented with the cocktails of inhibitors for protease (Roche) and ubiquitin hydrolase (20 mM N-ethylmaleimide and 5μM ubiquitin aldehyde. For ubiquitin analysis, 293T cells stably expressing LacZ, CYLDWT or CYLDm were further transfected with pcDNA plasmids encoding AU1-ubiquitinWT or mutants that contained 6 lysine to arginine mutations 29, and then used for protein analysis. Dual-luciferase gene reporter analysis, cell growth and soft agar colony formation were performed as previously described 25. For scratch-wound assay, transduced A431 cells were grown to near confluence and wounded with a 10 μl pipette tip. Images were taken at 0 and 18 hours post-wounding.
Animal studies were conducted in accordance with protocols approved by Duke Animal Care and Use Committee. K14-CYLDm transgenic mice were generated in Duke transgenic facility using FVB mice. Two independent lines of transgenic mice were identified by PCR-genotyping and validated by immunoblotting with epidermal protein lysates. For the two-stage carcinogenesis, transgenic and wild type littermates (n≥20, 1–3 days old) were treated with one dose of 50 μg 7,12-dimethylbenz[α]anthracene (DMBA, Sigma, St. Louis, MO) in 50 μl acetone as previously described 30. Three weeks later, mice were shaved on the dorsal skin and treated with 2.5 μg 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma, St. Louis, MO) in 200 μl acetone biweekly for a total of 20 weeks. Tumors on each mouse were counted weekly following TPA-treatment. Those scored as SCC based on the invaginated growth pattern were confirmed by histological analysis at the end-point of growth. JNK inhibition was achieved by topical treatment with 250 μg SP600125 (LC laboratories, Woburn, MA) in 200 μl DMSO 30 minutes before each biweekly TPA- application for a total of 20 week. Of note, no apparent health problems other than tumor growth were observed on WT and transgenic mice following the topical applications of DMBA, TPA, SP600125 or control vehicle. Statistical values were obtained through t-test analysis using GraphPad InStat 3.05.
For subcutaneous tumor growth, A431 cells were transduced with retrovirus for expression of LacZ, CYLDWT or CYLDm. Cells were trypsinized 3 days post-infection and suspended in DMEM at 5 X 106/ml and then mixed with Matrigel (BD Bioscience, San Jose, CA) at 1:1 ratio. 200 ul of the cell suspension were injected subcutaneously into CB17.SCID mice as previously described 32. The tumors were measured on day 28 and 35.
For immunoprecipitation (IP), protein lysates (250 μg/sample) isolated from A431 or 293T cells were incubated with polyclonal antibodies against c-Fos, c-Jun or Ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours at 4°C followed by 2 hour incubation with protein A-agarose beads. The beads were washed three times with NP-40 lysis buffer and then eluted for immunoblotting with antibodies against CYLD, c-Fos, or c-Jun or p-c-Jun (GenScript, Piscataway, NJ). Immunohistochemistry and immunofluorescent staining were performed with paraffin and frozen tissue sections, respectively, as described 32. Mouse keratinocytes were isolated from newborns and cultured to about 80% confluence as described 31. Cells were then treated with 0 or 100 nM TPA along with or without 10 μM SP600125 for 24 hours prior to whole cell protein analysis and nuclei isolation. Nulear proteins were extracted in [25 mM Hepes pH7.9, 400 mM NaCl, 1 mM DTT, 20% glycerol, 0.2 mM EDTA in the presence of protease and phosphatase inhibitor cocktails (Roche, South San Franciso, CA)] and 1.5 ug protein of each sample were used for AP1-gel shift assay using odyssey-dye conjugated AP1-oligonucleotides and assay kit (Li-cor Biotechnology, Lincoln, NB).
To examine how CYLD affects epidermal homeostasis and tumorigenesis, we generated transgenic mice with keratin 14 promoter-driven expression of a human CYLD mutant-932 (CYLDm) which lacks the 21 amino acid residues at the C-terminal end (Supplementary Fig. S1A). CYLDm was catalytically deficient when tested with TRAF2/6 as substrates 4; and consistently, it increased IκBα-phosphorylation and NF-κB-gene reporter function (Supplementary Fig. S1B-C). Epidermal expression of CYLDm was verified by both immunoblotting and immunostaining with an antibody against CYLD or the HA-epitope in two independent lines of transgenic mice (Fig. 1A and Supplementary Fig. S1D). The transgenic mice had no obvious developmental abnormalities other than mild epidermal hyperproliferation as indicated by the increased numbers of Ki-67-positive and nucleated cells (Supplementary Fig. S1D-E).
To determine the role of CYLDm in tumor development, we subjected both WT and transgenic mice to a two-stage skin carcinogenesis protocol. Newborn mice were initiated with one topical dose of DMBA followed by TPA twice weekly for 20 weeks. Tumor incidence and multiplicity were scored in each group for 40 weeks. Transgenic mice reached a 100% tumor incidence by week 13 following TPA promotion and developed an average of 11.4 tumors per mouse by week 21. In contrast, WT mice did not reach the 100% tumor incidence until week 21 and developed an average of 6.4 tumors per mouse (Fig. 1B). In addition, 66% of the transgenic lesions, as compared to 25% of WT lesions, developed clinical features of squamous cell carcinoma (SCC) by week 32 (Fig. 1C), as judged by the invaginated growth pattern accompanied either with or without a cauliflower-like or ulcerated appearance. In corroboration with the clinical features, transgenic tumors showed histological features of malignancy, including the epidermal tissue invasion down to the dermis and the increased numbers of atypical and Ki-67-positive cells (Fig. 1D). In addition, these tumors displayed signs of epithelial-mesenchymal transformation (EMT) as indicated by the absence of E-cadherin and the presence of mesenchymal cell markers, including N-cadherin and Vimentin. In contrast, the tumors developed on WT mice retained the expression of E-cadherin and were negative of N-cadherin and Vimentin (Fig. 1D). The WT tumors generally maintained an epithelial cell morphology although some of them were hyperproliferative and locally invasive. These data indicate that CYLDm not only sensitizes mouse skin to tumor development but also promotes malignant conversion.
The aggressive nature of the transgenic tumors prompted us to perform whole body necropsy following the collection of primary tumors. Surprisingly, over 50% of the transgenic mice, as compared to none of the WT siblings, had tumors in the lymph nodes located primarily at the axillary and inguinal regions (Fig. 2A). The lymph node tumors displayed a mixture of keratinized and spindle cell morphologies, expressed cytokeratin 5 (K5), an epidermal cell marker, and were highly proliferative, as indicated by the high number of Ki-67-positive cells (Fig. 2A). These results indicate that CYLDm promotes epidermal tumors to metastasize to lymph node.
Among the known downstream targets, NF-κB has been presumed as the major culprit in the tumorigenesis associated with CYLD mutation 3,4,22. However, NF-κB inhibitors showed a limited efficacy in a recent clinical trial 23, suggesting that other CYLD downstream targets might be involved in the tumorigenesis. Specifically, we examined the JNK signaling pathway whose receptor-mediated induction was subject to CYLD-inhibition 5. As predicted, primary and lymph node tumors from the transgenic mice displayed strong nuclei localization of phosphorylated JNK (p-JNK) and c-Fos, an AP1 subunit involved epidermal malignant conversion 9 (Fig. 2B). In contrast, RelA, a major NF-κB subunit that translocates from cytoplasm to nuclei upon activation 33, was primarily located in the cytoplasm of tumor cells (Fig. 2B). In addition, AP1 activity was increased in keratinocytes isolated from transgenic mice as demonstrated by immunoblotting for phosphorylated c-Jun (p-c-Jun) and AP1-gel mobility shift assay (Fig. 2C). Treatment with the JNK-specific inhibitor SP600125 reduced p-c-Jun levels, indicating that c-Jun activation is dependent on JNK function. In contrast to cyld−/− keratinocytes 22, the transgenic tumors did not show a significant increase of nuclear Bcl3 (Supplementary Fig. S2). These data indicate that JNK/AP1 but not NF-κB activation is increased in tumors expressing CYLDm and are consistent with our previous findings demonstrating a clinical relevance of NF-κB loss-of-function and JNK gain-of-function in human SCC 24,25,32,34. To determine whether JNK/AP1 is essential for the tumorigenesis enhanced by CYLDm, we challenged mice with the DMBA/TPA protocol and incorporated the topical treatment of SP600125 before each TPA-application. SP600125 significantly reduced tumor multiplicity and incidence in both WT and transgenic mice (Fig. 2D). In addition, SP600125 prevented the transgenic skin tumors from progressing into sarcomatoid SCC or metastasis to lymph node or other internal organs (Fig. 2E). Of interest, the transgenic mice have normal profiles of T-lymphocytes as analyzed by flow cytometry (Supplementary Fig. S3), which is in concordance with the notion that CYLD regulates T-cell development in a cell-autonomous manner 7. Thus, the tumor-prone phenotype of the transgenicmice is unlikely caused by potential immune defects. Taken together, these findings underscore that JNK/AP1 signaling pathway underlies tumor development and metastasis caused by CYLD-deficiency.
Next, we tested CYLDm-effects on A431, a spontaneous human SCC cell line. We found that CYLDm significantly increased the rate of monolayer cell growth and 3-dimensional soft agar colony formation, whereas CYLDWT decreased them (Fig. 3A-B). AP1 inhibition by expression of DNc-Jun, a dominant negative c-Jun mutant 35, significantly reduced the number of soft agar colonies. In addition, CYLDm induced an increased rate of cell migration as assessed by a scratch-wounding assay, while CYLDWT markedly slowed cell migration (Fig. 3C). Again, AP1 inhibition by siRNA-mediated gene silencing of c-Jun and c-Fos abolished the CYLDm-effect on cell migration (Supplementary Fig. S4). Moreover, CYLDm noticeably enhanced subcutaneous tumor growth of A431 cells in immunodeficient mice. In contrast, CYLDWT abolished tumor growth in mice (Fig. 3D). These findings indicate that CYLDWT inhibits whereas CYLDm promotes tumorigenesis of human SCC in an AP1-dependent manner.
To further confirm that AP1 is subject to CYLD-regulation at a functional level, we performed luciferase gene reporter analysis. We found that CYLDm increased AP1-driven expression both in the presence and absence of EGF; whereas CYLDWT reduced AP1 activity in both conditions (Fig. 4A). These data are in line with the recent finding demonstrating that CYLD controls JNK activity at a level upstream of MKK7 5. In addition, CYLDWT markedly reduced while CYLDm significantly potentiated AP1 activity driven by exogenous c-Fos expression; conversely, gene silencing of c-Jun or c-Fos abolished AP1-induction by CYLDm (Fig. 4A). These data suggest that there could be a direct link between CYLD and AP1 subunits. To test for this link, we first examined the protein levels of c-Jun and c-Fos in response to altered CYLD function in 293T cells. In response to EGF, c-Fos and c-Jun and p-c-Jun, a product of JNK activation, were increased in cells expressing LacZ or CYLDm but not in those expressing CYLDWT (Fig. 4B). Moreover, co-immunoprecipitation (co-IP) analysis revealed that EGF increased c-Jun-c-Fos dimerization, a process necessary for c-Fos function 36; this induction was enhanced by CYLDm and reduced by CYLDWT (Fig. 4C). Surprisingly, both CYLDm and CYLDWT were also precipitated by the c-Fos-antibody (Fig. 4C). The interactions between c-Jun, c-Fos and CYLD were also observed in A431 cells as demonstrated by the bidirectional co-IP analyses with three different antibodies (Supplementary Fig. S5A). These findings suggest that CYLD have an enzyme-substrate relationship with c- Jun and c-Fos. We tested this idea by IP for c-Jun and c-Fos and immunoblotting for ubiquitin and vice versa, and found that the levels of ubiquitinated c-Fos and c-Jun were increased by EGF and further augmented by CYLDm but diminished by CYLDWT; the same result was obtained by IP for ubiquitin and immunoblotting for c-Fos (Fig. 5A). Since the IP-ubiquitination analyses were performed under stringent buffer conditions that disrupted protein-protein interactions, the observed change of ubiquitination is unlikely a result of protein complex formation but rather is directly linked to c-Jun and c-Fos.
To further determine whether K63-ubiquitin is present on c-Fos and c-Jun, we coexpressed CYLDm or CYLDWT with AU1-tagged ubiquitinWT, ubiquitin-K63 or ubiquitin-K48 in 293T cells, and then performed IP for c-Jun or c-Fos and immunoblotting for AU1. In cells expressing either ubiquitinWT or ubiquitin-K63, the levels of ubiquitinated c-Jun and c-Fos were increased by EGF and were further enhanced by CYLDm but reduced by CYLDWT. In contrast, the status of c-Jun and c-Fos ubiquitination was minimally responsive to CYLD in cells expressing ubiquitin-K48 (Fig. 5B). Thus, CYLD is directly involved in regulating K63- but not K48-ubiqitination of c-Jun and c-Fos. In agreement with this finding, CYLDm did not induce protein degradation, instead resulted in an elevation of c-Fos and c-Jun in response to cell starvation and subsequent treatment with EGF (Supplementary Fig. 5B). Taken together, our findings identified c-Fos and c-Jun as the novel downstream targets of CYLD and the dominant regulators in epidermal malignancy associated with CYLDm-expression.
Cyld displays a dominant genetic linkage to multiple types of cutaneous adnexal tumors that often develop in bulky clusters in the head, neck, trunk and pubic areas 10,37. Although predominantly benign 10, these tumors are painful and disfiguring, can undergo malignant transformation with metastasis over time, and eventually lead to mortality 38–41. Thus, the malignant features of the tumors developed on K14-CYLDm transgenic mice are in line with the clinical manifestations seen in patients. Our transgenic tumor models allowed us to define JNK/AP1 signaling cascade as a key regulator in CYLDm-driven epidermal malignancy.
Cyld loss-of-function is not only relevant to cutaneous adnexal tumors but also to many other cancers, including SCC 22,42. It is worth noting that cyld−-− mice are sensitive to chemically induced carcinogenesis, but the tumors developed on these mice are not more malignant than those of WT mice 22. We predict that the differential tumor growth phenotypes observed in cyld−-− and CYLDm-transgenic mice could be explained by multiple possibilities. First, CYLDm might have dominant negative effects such that the N-terminus of CYLD possesses oncogenic functions that are independent of the C-terminal catalytic function. Such a scenario is in line with the fact that every patient-relevant cyld mutation characterized so far produces a catalytically deficient CYLD mutant 10. Second, CYLD is required for endothelia cell migration 43; thus, its absence in endothelial cells of cyld−-− mice might result in an impairment of angiogenesis, and consequently affect tumor progression. In contrast, expression of CYLDm is limited to epidermal cells in the transgenic mice. Third, the differences in mice genetic backgrounds might also contribute to the differential sensitivity to carcinogenesis, which can be addressed by cross-breeding of the transgenic and knockout mice in future studies.
K14-CYLDm transgenic mice did not develop spontaneous skin tumors, indicating that other genetic or environmental challenges are required to promote tumorigenesis. Because cutaneous adnexal tumors are frequently located on the exposed areas, UV irradiation has been considered as the major cause of tumor initiation. However, recent studies have demonstrated that the pubic area is also susceptible to cylindromatosis, a phenomenon that has been previously underreported. This datum suggests that hormonal factors might be involved in tumor induction in patients 37. Future efforts are necessary to determine how UV, hormonal factors and LOH of the WT cyld allele contribute to CYLDm-driven epidermal malignancy.
Bcl3 is a direct substrate of CYLD; and upon activation via K63-ubiquitination, it forms hetero-dimers with p50/p52 to induce expression of cyclin D1. Thus, Bcl3 is recognized as an important regulator in skin carcinogenesis of cyld−-− mice 22. Interestingly, despite the inhibitory role of CYLD on NF-κB, neither Bcl3 nor RelA displayed increased induction in the CYLDm-transgenic tumors. It is possible that this is due to the negative cross-talk from JNK/AP1 as described in our previous studies 34. These findings implicate that NF-κB is unlikely the sole key regulator in the malignant tumor phenotype developed on transgenic mice. To this end, we found that JNK and its downstream c-Jun and c-Fos proteins were highly activated in both primary and metastatic tumors from the transgenic mice. Additionally, CYLDm increased the basal levels of c-Jun and c-Fos, and sustained their activation status in response to EGF-treatment. Moreover, both CYLDWT and CYLDm interacted with c-Jun and c-Fos, but with opposite effects; the latter increased c-Fos/c-Jun K63-ubiquitination and potentiated their transcriptional activity. Presumably, K63-ubiquitination excludes the degradation-targeting K48-ubiqutination, and thereby increases c-Jun/c-Fos protein stability. Findings to date indicate that signals transmitted through membrane receptors are subjected to CYLD-regulation at multiple levels. Specifically, CYLD not only suppresses IKK/NF-κB and MKKK7/JNK/AP1 signaling through TRAF/TRADD 1–5, but also directly regulates IKKγ, Bcl3 and c-Jun/c-Fos ubiquitination 3,22. In contrast to the canonical NF-κB pathway which suppresses epidermal growth and neoplasia 24,26,44, Bcl3 and the JNK/AP1 signaling cascades support epidermal growth and tumorigenesis 22,25,32. Taken together, our data established an important and broad role for CYLD in malignant and metastatic tumor development and identified c-Jun and c-Fos as novel CYLD-downstream regulators. These findings provide mechanistic insights to therapeutic targeting of the JNK/AP1 pathway for cancers associated with CYLD loss-of-function.
We thank CM Counter, RP Hall, T Lechler (Duke University) and A Oro (Stanford University) for their great comments and/or critical reading of the manuscript. We also thank R Bernard (Netherland Cancer Institute) and G Mosialos (Alexander Fleming) for providing the CYLD expression constructs, K-L Kuan (University of California, San Diego) for the Ubiquitin, and M Kato (University of Tsukuba) for the c-Fos expression constructs, as well as T Hensely and M Benson (Duke University) for their assistance in DNA-cloning.
GRANT SUPPORT: This work was supported by R03AR051470 and K01AR051470 from NIH/NIAMS and research grants from dermatology and skin cancer foundations to JY Zhang, as well as a NIH F32-training grant to P Miliani de Marval.