C/EBPα expression is downregulated in human nonmelanoma skin cancers and inactivation of C/EBPα confers susceptibility to UVB-induced skin squamous cell carcinomas.

Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G(1) checkpoint, and diminished or ablated expression of C/EBPα results in G(1) checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB.

Ablation of TAK1 upregulates reactive oxygen species and selectively kills tumor cells.

TAK1 kinase activates multiple transcription factors and regulates the level of reactive oxygen species (ROS). We have previously reported that ablation of TAK1 in keratinocytes causes hypersensitivity to ROS-induced cell apoptosis. It is known that some tumor cells produce ROS at higher levels compared with normal cells. We used inducible epidermal-specific TAK1 knockout mice and examined whether ablation of TAK1 in preexisting skin tumors could cause an increase in ROS and result in tumor cell death. Deletion of tak1 gene in skin tumors caused the accumulation of ROS and increased apoptosis, and skin tumors totally regressed within 5 to 10 days. Normal skin did not exhibit any significant abnormality on tak1 gene deletion. Thus, TAK1 kinase could be a new and effective molecular target for ROS-based tumor killing.

C/EBPalpha and C/EBPbeta are required for Sebocyte differentiation and stratified squamous differentiation in adult mouse skin.

C/EBPalpha and C/EBPbeta are bZIP transcription factors that are highly expressed in the interfollicular epidermis and sebaceous glands of skin and yet germ line deletion of either family member alone has only mild or no effect on keratinocyte biology and their role in sebocyte biology has never been examined. To address possible functional redundancies and reveal functional roles of C/EBPalpha and C/EBPbeta in postnatal skin, mouse models were developed in which either family member could be acutely ablated alone or together in the epidermis and sebaceous glands of adult mice. Acute removal of either C/EBPalpha or C/EBPbeta alone in adult mouse skin revealed modest to no discernable changes in epidermis or sebaceous glands. In contrast, co-ablation of C/EBPalpha and C/EBPbeta in postnatal epidermis resulted in disruption of stratified squamous differentiation characterized by hyperproliferation of basal and suprabasal keratinocytes and a defective basal to spinous keratinocyte transition involving an expanded basal compartment and a diminished and delayed spinous compartment. Acute co-ablation of C/EBPalpha and C/EBPbeta in sebaceous glands resulted in severe morphological defects, and sebocyte differentiation was blocked as determined by lack of sebum production and reduced expression of stearoyl-CoA desaturase (SCD3) and melanocortin 5 receptor (MC5R), two markers of terminal sebocyte differentiation. Specialized sebocytes of Meibomian glands and preputial glands were also affected. Our results indicate that in adult mouse skin, C/EBPalpha and C/EBPbeta are critically involved in regulating sebocyte differentiation and epidermal homeostasis involving the basal to spinous keratinocyte transition and basal cell cycle withdrawal.

Genetic ablation of CCAAT/enhancer binding protein alpha in epidermis reveals its role in suppression of epithelial tumorigenesis.

CCAAT/enhancer binding protein alpha (C/EBPalpha) is a basic leucine zipper transcription factor that inhibits cell cycle progression and regulates differentiation in various cell types. C/EBPalpha is inactivated by mutation in acute myeloid leukemia (AML) and is considered a human tumor suppressor in AML. Although C/EBPalpha mutations have not been observed in malignancies other than AML, greatly diminished expression of C/EBPalpha occurs in numerous human epithelial cancers including lung, liver, endometrial, skin, and breast, suggesting a possible tumor suppressor function. However, direct evidence for C/EBPalpha as an epithelial tumor suppressor is lacking due to the absence of C/EBPalpha mutations in epithelial tumors and the lethal effect of C/EBPalpha deletion in mouse model systems. To examine the function of C/EBPalpha in epithelial tumor development, an epidermal-specific C/EBPalpha knockout mouse was generated. The epidermal-specific C/EBPalpha knockout mice survived and displayed no detectable abnormalities in epidermal keratinocyte proliferation, differentiation, or apoptosis, showing that C/EBPalpha is dispensable for normal epidermal homeostasis. In spite of this, the epidermal-specific C/EBPalpha knockout mice were highly susceptible to skin tumor development involving oncogenic Ras. These mice displayed decreased tumor latency and striking increases in tumor incidence, multiplicity, growth rate, and the rate of malignant progression. Mice hemizygous for C/EBPalpha displayed an intermediate-enhanced tumor phenotype. Our results suggest that decreased expression of C/EBPalpha contributes to deregulation of tumor cell proliferation. C/EBPalpha had been proposed to block cell cycle progression through inhibition of E2F activity. We observed that C/EBPalpha blocked Ras-induced and epidermal growth factor-induced E2F activity in keratinocytes and also blocked Ras-induced cell transformation and cell cycle progression. Our study shows that C/EBPalpha is dispensable for epidermal homeostasis and provides genetic evidence that C/EBPalpha is a suppressor of epithelial tumorigenesis.

Diminished expression of C/EBPalpha in skin carcinomas is linked to oncogenic Ras and reexpression of C/EBPalpha in carcinoma cells inhibits proliferation.

The basic leucine zipper transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is involved in mitotic growth arrest and has been implicated as a human tumor suppressor in acute myeloid leukemia. We have previously shown that C/EBPalpha is abundantly expressed in mouse epidermal keratinocytes. In the current study, the expression of C/EBPalpha was evaluated in seven mouse skin squamous cell carcinoma (SCC) cell lines that contain oncogenic Ha-Ras. C/EBPalpha mRNA and protein levels were greatly diminished in all seven SCC cell lines compared with normal primary keratinocytes, whereas C/EBPbeta levels were not dramatically changed. Reexpression of C/EBPalpha in these SCC cell lines resulted in the inhibition in SCC cell proliferation. To determine whether the decrease in C/EBPalpha expression observed in the SCC cell lines also occurred in the carcinoma itself, immunohistochemical staining for C/EBPalpha in mouse skin SCCs was conducted. All 14 SCCs evaluated displayed negligible C/EBPalpha protein expression and normal C/EBPbeta levels compared with the epidermis and all 14 carcinomas contained mutant Ras. To determine whether oncogenic Ras is involved in the down-regulation of C/EBPalpha, BALB/MK2 keratinocytes were infected with a retrovirus containing Ras12V, and C/EBPalpha protein, mRNA and DNA binding levels were determined. Keratinocytes infected with the retrovirus containing oncogenic Ras12V displayed greatly diminished C/EBPalpha protein, mRNA and DNA binding levels. In addition, BALB/MK2 cells containing endogenous mutant Ras displayed diminished C/EBPalpha expression and the ectopic expression of a dominant-negative RasN17 partially restored C/EBPalpha levels in these cells. These results indicate that oncogenic Ras negatively regulates C/EBPalpha expression and the loss of C/EBPalpha expression may contribute to the development of skin SCCs.

Cell cycle-dependent phosphorylation of C/EBPbeta mediates oncogenic cooperativity between C/EBPbeta and H-RasV12.

CCAAT/enhancer binding protein beta (C/EBPbeta) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling. C/EBPbeta is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with RasV12 to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBPbeta in fibroblasts and report a novel proline-directed phosphoacceptor site at Ser64 within the transactivation domain. Ser64 phosphorylation was induced by activated Ras and Raf but was not blocked by chemical inhibitors of MEK1/2, phosphatidylinositol 3-kinase, JNK, or p38 mitogen-activated protein kinases. Ser64 was efficiently phosphorylated in vitro by the cyclin-dependent kinases Cdk2 and Cdc2. Thr189, previously identified as an ERK1/2 phosphorylation site that regulates C/EBPbeta activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G0) cells but was strongly increased in mid-G1 cells and in cells arrested in S or M phase. In addition, phosphorylation on both sites was blocked by treating cells with the Cdk inhibitor roscovitine. In contrast to wild-type C/EBPbeta, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited RasV12-induced focus formation. Our findings support a role for C/EBPbeta as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPbeta on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.

CCAAT/enhancer binding protein-beta is a mediator of keratinocyte survival and skin tumorigenesis involving oncogenic Ras signaling.

The basic leucine zipper transcription factor CCAAT/enhancer binding protein-beta (C/EBPbeta) is expressed in many cell types, including keratinocytes. C/EBPbeta activity can be increased by phosphorylation through pathways stimulated by oncogenic Ras, although the biological implications of Ras-C/EBPbeta signaling are not currently understood. We report here that C/EBPbeta-nullizygous mice are completely refractory to skin tumor development induced by a variety of carcinogens and carcinogenesis protocols, including 7,12-dimethylbenz[a]anthracene-initiation/12-O-tetradecanoylphorbol 13-acetate promotion, that produce tumors containing oncogenic Ras mutations. No significant differences in TPA-induced epidermal keratinocyte proliferation were observed in C/EBPbeta-null versus wild-type mice. However, apoptosis was significantly elevated (17-fold) in the epidermal keratinocytes of 7,12-dimethylbenz[a]anthracene-treated C/EBPbeta-null mice compared with wild-type mice. In v-Ha-ras transgenic mice, C/EBPbeta deficiency also led to greatly reduced skin tumor multiplicity and size, providing additional evidence for a tumorigenesis pathway linking Ras and C/EBPbeta. Oncogenic Ras potently stimulated C/EBPbeta to activate a C/EBP-responsive promoter-reporter in keratinocytes and mutating an ERK1/2 phosphorylation site (T188) in C/EBPbeta abolished this Ras effect. Finally, we observed that C/EBPbeta participates in oncogenic Ras-induced transformation of NIH 3T3 cells. These findings indicate that C/EBPbeta has a critical role in Ras-mediated tumorigenesis and cell survival and implicate C/EBPbeta as a target for tumor inhibition.