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[Cancer Research 65, 861-867, February 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Diminished Expression of C/EBP{alpha} in Skin Carcinomas Is Linked to Oncogenic Ras and Reexpression of C/EBP{alpha} in Carcinoma Cells Inhibits Proliferation

Minsub Shim, Kristina L. Powers, Sarah J. Ewing, Songyun Zhu and Robert C. Smart

Cell Signaling and Cancer Group, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina

Requests for reprints: Robert C. Smart, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633. Phone: 919-515-7245; Fax: 919-515-7169; E-mail: rcsmart{at}unity.ncsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The basic leucine zipper transcription factor, CCAAT/enhancer binding protein {alpha} (C/EBP{alpha}) 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/EBP{alpha} is abundantly expressed in mouse epidermal keratinocytes. In the current study, the expression of C/EBP{alpha} was evaluated in seven mouse skin squamous cell carcinoma (SCC) cell lines that contain oncogenic Ha-Ras. C/EBP{alpha} mRNA and protein levels were greatly diminished in all seven SCC cell lines compared with normal primary keratinocytes, whereas C/EBPß levels were not dramatically changed. Reexpression of C/EBP{alpha} in these SCC cell lines resulted in the inhibition in SCC cell proliferation. To determine whether the decrease in C/EBP{alpha} expression observed in the SCC cell lines also occurred in the carcinoma itself, immunohistochemical staining for C/EBP{alpha} in mouse skin SCCs was conducted. All 14 SCCs evaluated displayed negligible C/EBP{alpha} protein expression and normal C/EBPß 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/EBP{alpha}, BALB/MK2 keratinocytes were infected with a retrovirus containing Ras12V, and C/EBP{alpha} protein, mRNA and DNA binding levels were determined. Keratinocytes infected with the retrovirus containing oncogenic Ras12V displayed greatly diminished C/EBP{alpha} protein, mRNA and DNA binding levels. In addition, BALB/MK2 cells containing endogenous mutant Ras displayed diminished C/EBP{alpha} expression and the ectopic expression of a dominant-negative RasN17 partially restored C/EBP{alpha} levels in these cells. These results indicate that oncogenic Ras negatively regulates C/EBP{alpha} expression and the loss of C/EBP{alpha} expression may contribute to the development of skin SCCs.

Key Words: C/EBP{alpha} • Ras • keratinocytes • skin


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CCAAT/enhancer binding proteins (C/EBP) are members of the basic leucine zipper class of transcription factors that contain a basic DNA binding domain and a leucine zipper domain involved in homodimerization or heterodimerization (1, 2). There are six members of the C/EBP family (C/EBP{alpha}, C/EBPß, C/EBP{gamma}, C/EBP{delta}, C/EBP{varepsilon}, and C/EBP{zeta}; refs. 3–5). C/EBP{alpha} plays an important role in metabolism as well as in the regulation of cell proliferation and differentiation in a variety of cell types (5). C/EBP{alpha} protein can interact with several cell cycle regulatory proteins, and it has been proposed that such interactions are responsible for inhibiting cell proliferation. C/EBP{alpha} can regulate p21 expression as well as to directly interact and enhance the ability of p21 to inhibit cdk2 (6, 7). In addition, C/EBP{alpha} has been shown to inhibit cell growth through its interaction with Rb family proteins (8, 9). More recent studies suggest that C/EBP{alpha} can block cell proliferation independent of its transcriptional activity by forming a complex with cdk2 and cdk4 thereby blocking cyclin-cdk interactions and cell cycle progression (10). C/EBP{alpha} can also directly repress E2F function through its physical association with E2F and this repression is necessary for growth arrest and adipocyte and granulocyte differentiation (11, 12). Most recently, the antimitotic activity of C/EBP{alpha} was shown to require a SWI/SWF complex; however, events downstream of this interaction important in the regulation of cell proliferation are currently unknown (13).

The antiproliferative effects of forced C/EBP{alpha} expression have been shown in a variety of nontransformed cells (14, 15) as well as transformed cells, including SaOS2 osteosarcoma cells which lack functional Rb and p53, (16), SV40 transformed fibroblasts (16), HepG2 hepatocarcinoma cells (17), and lung cancer cell lines (18). Thus, C/EBP{alpha} seems to have antimitotic activity in a variety of tumor cell types, and Rb and p53 seem to be dispensable for its antimitotic activity (16). The loss of C/EBP{alpha} expression or function in certain cancers is emerging as an important event in the development of certain cancers. For example, C/EBP{alpha} is inactivated by mutation (19) or through its association with oncoprotein AML-1-ETO (20, 21) in human acute myeloid leukemia. The inactivation of C/EBP{alpha} is thought to result in differentiation block of the granulocytic blasts and has implicated C/EBP{alpha} as a putative tumor suppressor gene in acute myeloid leukemia. C/EBP{alpha} expression is greatly reduced in human hepatocellular carcinomas (22), lung cancer cell lines, and lung cancers (18), particularly adenocarcinoma and poorly differentiated lung cancers. Collectively, these studies support the notion that loss of C/EBP{alpha} expression may be permissive for tumor cell proliferation.

C/EBP{alpha} and C/EBPß are abundantly expressed in mouse and human epidermal keratinocytes (23–25). In mouse keratinocytes, C/EBPß is involved in the regulation of the early stages of squamous differentiation (26). Recently, C/EBPß has been shown to play a critical role in Ras-mediated mouse skin tumorigenesis and keratinocyte survival (27). Unlike C/EBPß, C/EBP{alpha} does not cooperate with Ras to induce transformation of NIH 3T3 cells (27). However, the forced expression of C/EBP{alpha} in keratinocytes inhibits their growth (26) and preliminary data from our laboratory on a limited number of squamous cell carcinoma (SCC) suggested that both C/EBP{alpha} and C/EBPß levels are reduced in mouse SCC (23). In the current study, we have examined C/EBP{alpha} and C/EBPß levels in seven mouse skin SCC cell lines and 14 SCCs that contain oncogenic Ras. Our results indicate that C/EBP{alpha} but not C/EBPß protein levels are greatly diminished in SCC cell lines and SCCs. We observed that oncogenic Ras negatively regulates C/EBP{alpha} levels and reexpression of C/EBP{alpha} in oncogenic Ras containing SCC cell lines blocks proliferation. Our results suggest the loss of C/EBP{alpha} expression contributes to the deregulation of cell proliferation in SCCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Cell Culture. Primary keratinocytes were isolated from newborn CD-1 mice (<3 days old) by overnight trypsin flotation at 4°C and keratinocytes were cultured in low-calcium medium [Ca2+-free EMEM supplemented with 4% Chelex-treated fetal bovine serum, 10 ng/mL of human epidermal growth factor, 100 units/mL of penicillin, 100 µg/mL of streptomycin, 250 ng/mL of amphotericin B (Fungizone), with added calcium chloride to a final concentration of 0.05 mmol/L]. Mouse SCC cell lines were isolated from skin SCCs in CD-1 mice that were induced by 7,12-dimethylbenz(a)anthracene (DMBA) initiation followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or mirex promotion. Following trypsin treatment of the SCCs, SCC cells were plated at low density in low-calcium medium without epidermal growth factor. Individual colonies were ring cloned and expanded (28). All five SCC cell lines (MT2.5, MT2.6, MT2 3r3, T6, and M9.6) contain oncogenic H-Ras mutations in the 61st codon. FVBN-217 and TGAC-43 mouse skin SCC cell lines were a kind gift from Dr. Ron Cannon (National Institute of Environmental Health Sciences, Research Triangle) and were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, 100 µg/mL streptomycin per mL, and 250 ng/mL of amphotericin B (Fungizone). BALB/MK2 and BALB/MK2-DMBA keratinocytes (a gift from Dr. B. Weissman, University of North Carolina, Chapel Hill, NC) were cultured in low-calcium medium (Ca2+-free EMEM supplemented with 8 % Chelex-treated fetal bovine serum, with (BALB/MK2) or without (BALB/MK2-DMBA) 4 ng of human epidermal growth factor per mL, and 0.05 mmol/L calcium chloride).

Immunohistochemistry for C/EBP{alpha} and C/EBPß. Paraffin-embedded tissue sections of normal skin, SCC and keratoacanthomas derived from DMBA/TPA promotion were subjected to an antigen retrieval protocol [95°C for 30 minutes in 10 mmol/L citrate buffer (pH 6.0)] followed by incubation with anti-C/EBP{alpha} antibody (1:250; sc-61, Santa Cruz Biotechnology) or anti-C/EBPß antibody (1:250; sc-150, Santa Cruz Biotechnology). A biotinylated goat anti-rabbit IgG was used as the secondary antibody. Detection was made by avidin-biotin complex kit (Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine as the chromagen (BioGenex, San Ramon, CA). Sections were counterstained with hematoxylin. No C/EBP{alpha} or C/EBPß staining was observed when the primary antibody was omitted and the control rabbit serum was applied.

Preparation of Cell Lysates. Nuclear extracts were prepared as previously described by Schreiber et al. (29). For the preparation of whole cell lysates, cells were washed with cold PBS, harvested by scraping, and collected by brief centrifugation. Cells were lysed in lysis buffer [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride, 100 µg/mL aprotinin, 100 µg/mL leupeptin, 1 mmol/L sodium orthovanadate, and 0.6 % NP40] by sonication and then one-tenth volume of 5 mol/L NaCl was added. Lysates were vortexed, incubated for 15 minutes on ice and centrifuged (14,000 x g, 10 minutes, 4°C). Supernatants were stored at –80°C until use. Protein concentration was determined using the Bio-Rad protein assay reagent.

Western Blot Analysis. Equal amounts of protein were precipitated by adding equal volume of 20% trichloroacetic acid and washed with acetone (–20°C). Protein samples were solubilized and boiled in SDS sample buffer for 2 minutes and then separated by SDS-PAGE. The separated proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA). Following incubation in blocking buffer (PBS with 1% bovine serum albumin, 5% nonfat dry milk, and 0.1% Tween 20) for 1 hour at room temperature, the membranes were probed for 2 hours at room temperature with rabbit polyclonal IgG raised against C/EBP{alpha} (1:2,000; sc-61, Santa Cruz Biotechnology) or C/EBPß (1:2,000; sc-150, Santa Cruz Biotechnology). The membranes were washed and probed with a horseradish peroxidase–linked secondary antibody (1:2,500) for 1 hour at room temperature. Detection was made with an enhanced chemiluminescence reagent followed by exposure of membrane to film. All membranes were stained to confirm equal loading.

Northern Blot Analysis. Total RNA was isolated using Promega's SV total RNA isolation kit. C/EBP{alpha} cDNA was labeled with [{alpha}-32P] dCTP by using Ready-To-Go labeling beads (Amersham, Piscataway, NJ). RNA was electrophoresed on agarose gel containing formaldehyde and transferred to zeta-probe GT membrane (Bio-Rad, Hercules, CA) and UV cross-linked. Blots were incubated at 65°C in hybridization buffer [0.25 mol/L Na2HPO4 (pH 7.2) and 7 % SDS] and sequentially washed with washing buffer 1 [20 mmol/L Na2HPO4 (pH 7.2) and 5 % SDS] and washing buffer 2 (20 mmol/L Na2HPO4 and 1% SDS) at room temperature. Films were exposed to membranes at –80°C and developed.

Transfection of SCC Cell Lines. SCC cell lines were transfected when they reached 30% to 40% confluence in 60-mm-diameter dishes with 2 µg of DNA (pcDNA3 or pcDNA3-C/EBP{alpha}) and 16 µg of LipofectAMINE (Invitrogen, Carlsbad, CA). Transfection was done in serum-free EMEM (containing 0.05 mmol/L calcium) at 37°C and 5% CO2 for 4 hours, after which the cells were refed with low-calcium medium. Forty-eight hours later, the cultures were split (1:3) and replated in the above medium containing 300 µg/mL G418 and this selection medium was changed every other day. On days 3, 5, and 7 after G418 selection, the total number of colonies in 30 random grid squares was counted and then converted to colonies per dish. The number of cells per colony was scored directly from 30 randomly chosen colonies.

Virus Infection of BALB/MK2 Cells. {phi}NX cells were cultured with DMEM medium plus 10% fetal bovine serum and transfected with pBabe-puro control vector or pBabe-puro-Ras (12V) vector (20 µg/dish) by calcium precipitation method for 6 hours. The cells were then maintained in EMEM medium containing 8% chelaxed fetal bovine serum, 4 ng/mL EGF and 0.05 mmol/L calcium chloride for overnight. Virus containing medium was collected from {phi}NX cell cultures thrice, 4 hours apart, filtered through a 0.45-µm filter, and overlaid onto BALB/MK2 cells with 5 µg/mL polybrene. Cultures were maintained with viral containing medium overnight and 24 hours later cells were shifted to a selection medium containing 2 µg/mL puromycin for 4 days before the cells were processed for nuclear extract preparation.

Electrophoretic Mobility Shift Assay. Nuclear protein (4 µg per sample) in 10 µl nuclear extract buffer (29) was incubated at room temperature for 30 minutes with 10 µL of master binding mix with 32P-labeled C/EBP probe (Santa Cruz Biotechnology, SC-2525). Samples were loaded onto 4% polyacrylamide gel and subjected to electrophoresis in 0.025x Tris-borate EDTA buffer at 200 V for 3 to 4 hours. The gel was transferred to Whatman paper, dried in a 80°C gel dryer for 1 hour, and exposed to film for appropriate time to check the DNA binding pattern.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
C/EBP{alpha} Protein and mRNA Levels Are Greatly Diminished in SCC Cell Lines. We have previously shown that C/EBP{alpha} and C/EBPß are abundantly expressed in mouse keratinocytes (23). In the current study, we examined C/EBP{alpha} protein levels in seven mouse SCC cell lines. SCC cell lines (MT23r3, MT2.5, MT2.6, T6, FVBN-217, and M9.6) were isolated from various mouse skin SCCs which were generated by in vivo treatment with DMBA and then promoted with TPA and/or mirex. All of these SCC cell lines contain oncogenic H-Ras A182 T mutations in at least one allele of the 61st codon as determined by allele-specific hybridization (28). SCC cell line TgAC-43 was isolated from a skin carcinoma that developed on transgenic Tg.AC mouse skin. TgAC-43 expresses high levels of the v-H-Ras transgene. As shown in Fig. 1A, the C/EBP{alpha} protein levels were greatly diminished in all seven SCC cell lines compared with normal primary mouse keratinocytes. The average decrease in C/EBP{alpha} protein levels was 90% (Fig 1A). In contrast to C/EBP{alpha} protein levels, C/EBPß protein levels were not dramatically changed (25% decrease on average) compared with normal primary keratinocytes (Fig 1A and B). These results show that C/EBP{alpha} protein levels are greatly diminished or nearly undetectable in all seven SCC cell lines containing oncogenic Ras compared with normal primary keratinocytes and that C/EBP{alpha} and C/EBPß are differentially regulated in SCC cells. We also compared the levels of C/EBP{alpha} in BALB/MK2 keratinocytes, an immortalized but not tumorigenic cell line to the levels in the SCC cell lines. Compared with BALB/MK2 keratinocytes, C/EBP{alpha} protein was decreased in all seven SCC by an average of 67% (range, 32-89% decrease).



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  		Figure 1. C/EBP{alpha} protein and mRNA levels are greatly diminished in SCC cell lines. A, nuclear extracts were prepared from normal mouse keratinocytes and SCC cell lines and equal amounts of protein (20 µg) were subjected to Western blot analysis using rabbit polyclonal anti-C/EBP{alpha} or anti-C/EBPß antibody. C/EBP{alpha} protein standard is histidine tagged and runs slightly slower than nontagged C/EBP{alpha}. B, densitometric analysis of results from A normalized to C/EBP{alpha} or C/EBPß in normal keratinocytes. C, total cellular RNA was isolated from normal keratinocytes and SCC cell lines and Northern blot analysis was conducted. The membrane was reprobed with 7S RNA cDNA to verify the equal loading of RNA. D, densitometric analysis of results from C normalized to 7S RNA.

 
C/EBP{alpha} can be regulated at the transcriptional level by numerous transcription factors; however, C/EBP{alpha} protein is also degraded via a ubiquitin-dependent proteasomal pathway (30). To determine whether the decrease in C/EBP{alpha} protein level in the SCC cells is associated with decreased C/EBP{alpha} mRNA expression, we examined C/EBP{alpha} mRNA levels in the SCC cell lines. As shown in Fig. 1C, C/EBP{alpha} mRNA levels in all seven SCC cell lines were also greatly diminished compared with normal primary keratinocytes. The average decrease in C/EBP{alpha} mRNA was 90% (Fig 1D). In general, the decreased level of C/EBP{alpha} mRNA expression was proportional to the decreased level of C/EBP{alpha} protein in each SCC cell line.

Reexpression C/EBP{alpha} Induces Growth Arrest in SCC Cell Lines. We have previously reported that forced expression of C/EBP{alpha} induces growth arrest and morphologic changes in BALB/MK2 keratinocytes (26). In order to examine the effect of C/EBP{alpha} on the growth of SCC cell lines, we transfected MT2.5 and MT3r3 SCC cell lines with pcDNA3-C/EBP{alpha}. Transfected SCC cells were selected in the presence of G418 and the number of colonies per dish and the number of cells per colony were counted during the selection period. As shown in Fig. 2A, B, and C, the number of colonies in C/EBP{alpha} transfected SCC cell lines was greatly reduced (>75%) compared with pcDNA3-transfected SCC cells. The number of cells per colony was also decreased (>90%) in the C/EBP{alpha} transfected cells compared with pcDNA3 transfected cells. Moreover, during the selection period the number of cells/colony in C/EBP{alpha} transfected SCC cells increased very slowly compared with the empty vector transfected cells. The above experiment was also conducted with MT2.6, T6, FVBN-217, M9.6, and TgAC43 SCC cell lines and similar results to those reported above for MT2.5 and MT3r3 were obtained (data not shown). These results indicate that C/EBP{alpha} inhibits the proliferation of SCC cells containing oncogenic Ras suggesting that antimitotic activity of C/EBP{alpha} is dominant over the proliferative effects of oncogenic Ras.



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  		Figure 2. Reexpression of C/EBP{alpha} induces growth arrest in SCC cell lines. A, MT2.5 SCC cells were transfected with pcDNA3 or pcDNA3-C/EBP{alpha} and subsequently subcultured in the presence of 300 µg/mL G418. The number of colonies per dish (left) and the number of cells per colony (right) were determined at days 3, 5, and 7 of G418 selection. Columns, mean of a representative experiment done in triplicate; bars, ±SD. B, MT2 3r3 SCC cells were transfected with pcDNA3 or pcDNA3-C/EBP{alpha} and subsequently subcultured in the presence of 300 µg/mL G418. The number of colonies per dish (left) and the number of cells per colony (right) were determined at days 3, 5, and 7 of G418 selection. Columns, mean of a representative experiment done in triplicate; bars, ±SD. C, MT2 3r3 SCC cells were transfected with pcDNA3 or pcDNA3-C/EBP{alpha} and subcultured in the presence of 300 µg/mL G418. On days 5 and 7 of G418 selection, the cells were stained with crystal violet and photographs of colonies were taken.

 
C/EBP{alpha} Expression Is Down-regulated in SCCs. To confirm that the decrease in C/EBP{alpha} expression observed in the SCC cell lines also occurs in the primary cancer lesion itself, immunohistochemical staining for C/EBP{alpha} in mouse skin SCCs was conducted. We examined 14 SCCs and two keratoacanthomas that were generated by DMBA/TPA initiation-promotion protocol. All 14 SCC and both keratoacanthomas contained oncogenic H-Ras with mutations in the 61st codon as determined by single-strand conformational polymorphism analysis of exon 2 followed by XbaI restriction analysis (31). As shown in Fig. 3A, in epidermis isolated from mice in which SCCs were induced with DMBA/TPA treatment, there is nuclear and cytoplasmic C/EBP{alpha} staining in the suprabasal layer of epidermis where post-mitotic keratinocytes undergo differentiation as well as perinuclear C/EBP{alpha} staining in basal layer. Based on immunostaining staining of C/EBP{alpha} conditional knockout epidermis the observed cytoplasmic staining in Fig. 3A is mostly nonspecific (data not shown). In contrast to the epidermis, all SCCs (14 of 14) showed little to no C/EBP{alpha} immunostaining as did the two keratoacanthomas (data not shown). A representative micrograph of a SCC is shown in Fig. 3B with intact epidermis (inset) from above the tumor on the same section included for comparison of epidermal C/EBP{alpha} immunostaining intensity. A micrograph of the SCC using higher magnification is shown in Fig. 3C. These data confirm the decrease in C/EBP{alpha} levels observed in the SCC cell lines also occurs in the primary SCC tumor itself. C/EBPß immunostaining in the DMBA/TPA–treated epidermis occurs primarily in the nuclei of the suprabasal cells (Fig. 3D). The observed cytoplasmic staining is nonspecific based on immunohistochemical staining of C/EBPß knockout epidermis. Representative micrographs of C/EBPß immunostaining in a SCC are shown in Fig. 3E and F. In contrast to C/EBP{alpha}, CEBPß was abundantly expressed in 14 of 14 SCCs. These immunostaining results in SCCs are in accord with the SCC cell line Western analysis results and clearly show C/EBP{alpha} and C/EBPß protein levels are differentially regulated in the SCCs.



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  		Figure 3. C/EBP{alpha} expression is down-regulated in SCCs. A, C/EBP{alpha} immunostaining of uninvolved epidermis of mice with SCCs induced by DMBA/TPA. B, C/EBP{alpha} immunostaining of SCC with epidermis (inset) from above the tumor (lower magnification). C, C/EBP{alpha} immunostaining of SCC. D, C/EBPß immunostaining of uninvolved epidermis of mice with SCCs induced by DMBA/TPA. E, C/EBPß immunostaining of SCC with epidermis (inset) from above the tumor (lower magnification). F, C/EBPß immunostaining of SCC.

 
Decreased C/EBP{alpha} Expression Is Associated with Oncogenic Ras. Our results in the SCC cell lines as well as in SCCs suggest an association between decreased C/EBP{alpha} expression and the presence of oncogenic Ras. This association between oncogenic Ras containing SCC cell lines and decreased C/EBP{alpha} is apparent whether oncogenic Ras results from the mutated endogenous Ras or from the expression of transgenic v-Ha-Ras. Because papillomas are the precusor tumor type of SCCs and since mutation of Ras is an early initiating event in DMBA-initiated/TPA-promoted mouse skin tumorigenesis, we examined by Western analysis the levels of C/EBP{alpha} in DMBA-initiated/TPA-promoted papillomas. We observed that the C/EBP{alpha} levels were decreased by ~70% in 4 of 4 pooled papilloma samples compared with whole skin or epidermis control and immunohistochemical staining for C/EBP{alpha} also revealed a substantial decrease compared with uninvolved epidermis (data not shown). To determine whether oncogenic Ras is involved in the down regulation of C/EBP{alpha}, BALB/MK2 keratinocytes were infected with pBABEpuro retrovirus or pBABEpuro containing Ras12V and then C/EBP{alpha} protein, mRNA and DNA binding levels were determined. As shown in Fig 4A, C/EBP{alpha} protein levels were decreased >90% in cells infected with pBABEpuro containing oncogenic Ras12V compared with cells infected with empty retrovirus (Fig 4A). Oncogenic Ras12V expressing cells displayed a ~50% decrease in C/EBP{alpha} mRNA as determined by reverse transcription-PCR. As shown in Fig 4B, keratinocytes infected with pBABEpuro-Ras12V displayed decreased C/EBP{alpha} DNA binding as determined by electrophoretic mobility shift assay analysis. To provide additional evidence that Ras is involved with the down-regulation of C/EBP{alpha} expression as well as to avoid the possible influence of supraphysiologic levels of Ras12V that may occur with the pBABEpuroRas12V retroviral infection we examined C/EBP{alpha} levels in BALB/MK2-DMBA cells that contain endogenous H-Ras A182->T mutation in the 61st codon which was induced by DMBA treatment (32). C/EBP{alpha} expression was greatly diminished (Fig. 4C) in the mutant Ras containing BALB/MK2-DMBA cells compared with BALB/MK cells. Moreover, the expression of a dominant negative RasN17 in BALB/MK2-DMBA cells (Fig. 4D) resulted in an increase in C/EBP{alpha} protein levels. Collectively, these results indicate that oncogenic Ras contributes the down-regulation of C/EBP{alpha} expression.



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  		Figure 4. Decreased C/EBP{alpha} expression is associated with oncogenic Ras. A, C/EBP{alpha} protein levels in whole cell extracts prepared from BALB/MK2 cells infected pBabe-puro or pBabe-puro-RasV12. B, EMSA analysis of nuclear extracts prepared from BALB/MK2 cells infected pBabe-puro or pBabe-puro-RasV12. C, C/EBP{alpha} rotein levels in BALB/MK2 keratinocytes and BALB/MK2-H-Ras keratinocytes which contain endogenous H-Ras mutation. D, C/EBP{alpha} protein levels in cells transfected with RasN17. NSB, nonspecific binding.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study, we have examined fourteen mouse skin SCCs and seven SCC cell lines all of which contain mutant oncogenic Ras and found that C/EBP{alpha} levels, but not C/EBPß levels, were greatly diminished in all carcinomas and SCC cell lines. These results clearly establish that loss of CEBP{alpha} expression in Ha-ras containing SCC is a frequent event and that C/EBP{alpha} and C/EBPß are differentially regulated in SCCs. This divergence in expression of the two highly related basic leucine zipper transcription factors in SCCs may reflect their emerging roles in tumorigenesis. For example, C/EBPß has a critical role in Ras-mediated mouse skin tumorigenesis and keratinocyte survival (27). C/EBPß can transform mammary epithelial cells (33) and cooperate with Ras to transform NIH 3T3 fibroblasts (27). In addition, increased expression of C/EBPß is associated with increased invasiveness of human colorectal cancer (34) and the progression of ovarian cancer (35). More recently it has been shown that cyclin D1 can interact with C/EBPß to alter gene expression and it seems that C/EBPß is important in the unique patterns of altered gene expression observed in the human cancers that overexpress cyclin D1 (36). Collectively, these studies suggest that the retention of C/EBPß expression within the tumor cells contributes to tumor cell proliferation/survival. In direct contrast, the loss of C/EBP{alpha} expression seems to be permissive for tumor growth. C/EBP{alpha} is antimitotic in numerous cell types and C/EBP{alpha} levels are decreased in human hepatocellular carcinomas (22), lung cancer, and lung cancer cell lines (18) as well as a variety of transformed cell lines (16, 17). Moreover, C/EBP{alpha} function is abrogated by mutation or through its association with oncoprotein AML-1-ETO in human acute myeloid leukemia implicating C/EBP{alpha} as a putative human tumor suppressor (19–21). Collectively, these studies support the idea that loss of C/EBP{alpha} expression may be permissive for tumor cell growth. Our findings that C/EBP{alpha} levels are greatly diminished in skin SCC cell lines and SCCs are in accord with a tumor suppressor-like function for C/EBP{alpha}.

Ras proteins function as key regulators of signaling pathways that control cell proliferation/differentiation. Ras signaling is altered in the majority of human cancers due to the mutational activation of Ras or alterations in upstream or downstream signaling pathways (37). Oncogenic Ras is known to alter the expression of genes that stimulate cell proliferation, such as cyclin D1 which participates with CDK4/6 in propelling the cell through the G1-S transition of the cell cycle. C/EBP{alpha} is thought to negatively regulate the cell cycle by binding to CDK4 and CDK2 and preventing complex formation with cyclin D and E, respectively (10). C/EBP{alpha} can also inhibit E2F transcription activity and stimulate the activity of the CDK inhibitor p21. In the current study, we show that Ras is a potent negative regulator of C/EBP{alpha} mRNA levels, protein levels, and DNA binding activity. Thus, Ras signaling seems to couple the up-regulation of genes that positively regulate proliferation (e.g., cyclin D1) with the down-regulation of genes that negatively regulate cell proliferation (e.g., C/EBP{alpha}). Importantly, we found that reexpression of C/EBP{alpha} in mutant Ras containing SCC cell lines can block cell proliferation even though these cells contain oncogenic Ras. Our results suggest the Ras-induced down regulation of C/EBP{alpha} expression is a permissive event for cell proliferation. Whereas we have shown that C/EBP{alpha} inhibits proliferation of SCC cell lines, we do not know the mechanism by which C/EBP{alpha} induces growth arrest in SCC cell lines. As described in the Introduction and above, C/EBP{alpha} negatively regulates cell proliferation by multiple mechanisms (6–13) and additional studies are required to determine which of mechanism(s) are operative in keratinocytes. Recent studies from our laboratory have demonstrated that C/EBP{alpha} is a DNA damage–inducible p53 regulated protein that has a role in the G1 checkpoint (38).

In skin, oncogenic Ras is a potent stimulator of epidermal keratinocyte proliferation and an inhibitor of stratified squamous differentiation (39). C/EBP{alpha} is involved in mitotic growth arrest and differentiation of numerous cell types. If C/EBP{alpha} has both these activities in keratinocytes then the down-regulation of C/EBP{alpha} by Ras would not only be permissive for cell proliferation but also block squamous differentiation, perhaps contributing to clonal expansion of oncogenic Ras containing cells. C/EBP{alpha} likely contributes to squamous differentiation as it can regulate the expression of involucrin, a marker of squamous differentiation (40). We have observed that in BALB/MK2 cells that overexpress C/EBP{alpha}, involucrin levels are increased.1 Whereas oncogenic Ras is frequently mutated in a variety of human epithelial cancers and in chemical carcinogen-induced mouse skin SCC, it is not frequently mutated in human skin tumors. However, oncogenic Ras is frequently mutated in certain human cancers, including pancreas (90%), colorectal adenocarcinoma (45%), lung adenocarcinoma (35%), and liver (30%; ref. 37). It is noteworthy that reports of decreased C/EBP{alpha} expression in human cancers occurred in hepatocellular carcinoma (22) and lung cancer, with lung adenocarcinomas demonstrating the most significant and frequent decreases (18). Thus, there may be a relationship between human tumors that exhibit frequent mutations in Ras and those that have been reported to exhibit diminished C/EBP{alpha} expression.

The regulation of C/EBP{alpha} can occur at the transcriptional and post-transcriptional level (30). Our results show that Ras produces decreased mRNA levels suggesting that Ras produces a decrease in the transcription of C/EBP{alpha}. The C/EBP{alpha} promoter has been shown to be subject to autoregulation by C/EBP{alpha} (41) and negative regulation by c-myc (42, 43). The exact mechanism through which Ras negatively regulates C/EBP{alpha} levels in keratinocytes remains to be determined but could involve c-myc. In terms of post-translational modification, transfected C/EBP{alpha} has been shown to be phosphorylated on serine 248 in a Ras dependent manner involving PKC in 293T kidney cells (44). This modification enhanced transcription activity of C/EBP{alpha}. We have also observed that transfected Ras stimulates transcription activity of transfected C/EBP{alpha} in Balb/MK2 keratinocytes. However, this regulation may not be relevant in nontransfected keratinocytes as endogenous C/EBP{alpha} expression is down regulated by Ras. Recently, PKC{delta} and PKC{eta} have been reported to positively regulate C/EBP{alpha} expression through a mechamism involving p38 (40). Future studies in our lab will address the signaling mechanism(s) through which Ras down-regulates C/EBP{alpha} expression.

In summary, our results suggest that the loss of C/EBP{alpha} expression contributes to the altered growth characteristics of skin SCCs. Importantly, we have observed that oncogenic Ras down-regulates the expression of C/EBP{alpha}. Whereas additional studies are required to determine how Ras negatively regulates C/EBP{alpha} expression, our studies describe a novel link between these two important proteins. Our results contribute to the emerging evidence that C/EBP{alpha} may have a tumor suppressor-like function in numerous types of cancer and identify a novel mechanism for negative regulation by Ras.


   Acknowledgments
 
Grant support: National Cancer Institute grant CA 46637 and National Institute of Environmental Health Sciences Training grant ES7046.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Sharon Meyer for the isolation of the SCC cell lines and Dr. Peter Johnson for the pBabe C/EBP{alpha} constructs.


   Footnotes
 
1 M. Shim and R. C. Smart, unpublished results. Back

Received 4/29/04. Revised 9/ 1/04. Accepted 11/17/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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