Previous studies addressing functional aspects of nuclear factor κB (NF-κB) activation in normal and transformed keratinocytes revealed complex and seemingly contradictory roles of this transcription factor in this cell type. In normal skin, NF-κB signaling seems to inhibit squamous cell carcinoma development whereas, in squamous cell carcinoma themselves, deregulated NF-κB expression and/or signaling is frequently observed. To further investigate this paradox, we focused on NF-κB activation as it relates to the transformed phenotype of immortalized but nontumorigenic human keratinocytes (HaCaT cells). We observed that NF-κB activity contributed to survival and growth of cultured HaCaT keratinocytes as shown by use of pharmacologic NF-κB inhibitors, RNA interference, and inducible overexpression of a dominant interfering IκB construct. NF-κB activation was largely provided through interaction with extracellular matrix components because preventing cell attachment by forced suspension culture markedly reduced NFκB signaling associated with cell death (anoikis); conversely, anoikis was partially reversed by NF-κB activation induced either by tumor necrosis factor-α treatment or by overexpressing the NF-κB p65 subunit in HaCaT cells. Furthermore, overexpression of NF-κBp65 in HaCaT cells induced colony formation in soft agar and tumorigenicity in nude mice. In summary, as opposed to normal keratinocytes, immortalized HaCaT keratinocytes provide a cellular context in which deregulated NF-κB signaling supports multiple malignant traits in vitro and in vivo. (Cancer Res 2006; 66(10): 5209-15)

The transcription factor nuclear factor κB (NF-κB) was first identified as a nuclear factor in B lymphocytes, which binds to the enhancer of the immunoglobulin κ light chain. The NF-κB family contains several members including Rel A (p65), Rel B, c-Rel, p50 (NF-κB1), and p52 (NF-κB2), which exist as homodimers and heterodimers retained by ankyrin domain–containing IκBs in the cytoplasm of unstimulated cells (for review, see ref. 1). On stimulation of cells with inflammatory cytokines, IκB is phosphorylated by IκB kinases, ubiquitinated, and proteasomally degraded. Dissociation from IκB enables nuclear translocation of NF-κB dimers where they direct transcription of a host of target genes, many of which encode antiapoptotic proteins. In addition to their roles in immune and inflammatory processes, NF-κB family members have been observed to have oncogenic properties (2). Consistent with important roles of NF-κB in tumorigenesis, enhanced NF-κB activity has been observed in malignant tumors of diverse origin including carcinomas (for review, see ref. 3).

Investigations into the role of NF-κB in normal epidermis and the development of squamous cell carcinomas of the skin or mucous membranes revealed complex and seemingly contradictory roles of this transcription factor. As in other epithelial malignancies, deregulated NF-κB signaling is well documented in squamous cell carcinomas (46). Yet, when NF-κB signaling is disrupted in normal epidermis, squamous cell carcinomas develop at increased rates (7, 8). The present study was undertaken to probe the relevance of NF-κB signaling to the malignant phenotype of nontumorigenic human epidermal keratinocytes representing early stages of malignant transformation (HaCaT; ref. 9). We describe that NF-κB activation through interaction with extracellular matrix components provided an important survival mechanism to HaCaT cells that was disrupted by preventing cell attachment to extracellular matrix by forced suspension culture. Reduced NF-κB signaling and cell death in forced suspension culture could be partially reversed by tumor necrosis factor-α (TNFα)–induced NF-κB activation. Deregulated NF-κB signaling achieved by overexpressing the NF-κB p65 subunit similarly increased survival of HaCaT cells in forced suspension culture, induced colony formation in soft agar, and resulted in tumorigenicity of HaCaT cells in nude mice. These observations underscore that molecular alterations incurred during immortalization of HaCaT keratinocytes have conferred oncogenic properties to deregulated NF-κB signaling. This is in clear contrast to normal keratinocytes in which NF-κB activation inhibits proliferation and reduces the propensity of malignant transformation (7, 10, 11).

Cell lines and culture conditions. Immortalized human keratinocytes (HaCaT) and derivative cells were maintained in culture medium (W489) supplemented with 2% FCS as previously described (12). HaCaTp65 cells stably expressing the COOH-terminally hemagglutinin-tagged human NF-κB p65 subunit were selected using 1 μg/mL puromycin. Selection of antibiotic-resistant cells started 48 hours after transfection with NF-κBp65-pIRESpuro2 vector (BD BioSciences, Palo Alto, CA) and continued until resistant colonies appeared. In this vector, both the NF-κBp65 gene and the puromycin resistance gene are expressed from one bicistronic mRNA, in which the internal ribosomal entry site of the encephalomyocarditis virus permits translation of the puromycin N-acetyltransferase coding region downstream of the NF-κBp65 gene. Because all the surviving colonies expressed the transgene, there was no need for clonal selection, but rather all the puromycin-resistant clones were pooled and cultured under selective condition. Using this protocol, individual clonal variations are eliminated. Two independent transfections and selections were made generating two separate NF-κBp65-overexpressing cell lines exhibiting similar phenotypic characteristics. The IκBα superrepressor gene tagged at the COOH terminus with hemagglutinin (Upstate Biotechnology, Lake Placid, NY) was cloned into the tetracycline regulatable episomal expression vector pCEPTetP as previously described by us (13). The vector was transfected into the HaCaT-tTA1 cell line expressing the tetracycline-controlled transactivator (13) and hygromycin-resistant colonies were pooled. Unless noted otherwise, all experiments were done in serum-free, growth factor–free KGM medium formulated for keratinocyte growth (13).

RNA interference. Small interfering RNA (siRNA) targeted to down-regulate NF-κBp65 mRNA (siGENOME SMARTpool siRNA, Dharmacon, Lafayette, CO) was transfected into HaCaT cells using RNAiFECT reagent (Qiagen, Valencia, CA) according to the protocol of the manufacturer. Nonsense siRNA provided by the manufacturer was used as a negative control. Cell lysates were collected at 24, 48, and 72 hours posttransfection.

Antibodies and immunoblot analyses. Immunoblot analyses were done using standard procedures as previously described by us (13). Antibodies to NF-κBp65, β-actin, and IκB (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used at 1 μg/mL. The antibody against α-tubulin (Calbiochem, San Diego, CA) was used at 1 μg/mL. The antibody against cleaved poly(ADP-ribose) polymerase (PARP; Cell Signaling, Beverly, MA) was used at 1:1,000 dilution. Goat anti-rabbit secondary antibody (IRDye 800CW, Rockland Inc., Gilbertsville, PA) and goat anti-mouse secondary antibody (Alexa Fluor 680, Molecular Probes, Eugene, OR) for Odyssey IR imaging were used at 1:10,000 and 1:2,000 dilutions, respectively. Signal analysis was done either by film exposure or digitally by scanning membranes with the Odyssey IR imaging system (Li-Cor Biosciences, Lincoln, NE).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Subconfluent HaCaT cells were trypsinized and reseeded onto cell culture–treated 96-well plates in serum-free, growth factor–free KGM in the presence and absence of NF-κB inhibitor Bay11-7082 (5 μmol/L; Biomol, Plymouth Meeting, PA). Culture medium was exchanged to fresh growth medium (W489 containing 2% FCS) 24 hours after seeding. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were done 3 days later by incubating cultures with MTT solution (Sigma, St. Louis, MO) at 37°C for 4 hours. MTT solubilization solution (10% Triton X-100/0.1 N HCL in isopropanol) was added and the tissue culture plates were incubated for additional 12 hours at 37°C before absorbance was spectrophotometrically assessed at 570 nm (reference wavelength, 690 nm).

Caspase-3 activity assay. For the caspase-3 substrate cleavage assay, cells were processed and assayed using the EnzCheck Caspase-3 Assay Kit (Molecular Probes). Fluorescence was measured at emission and excitation settings of 485/530 nm with a Bio-Tek FL600 plate reader (Winooski, VT).

Anchorage-independent survival assay. Anchorage-independent cell survival was assessed as previously described (13). Briefly, tissue culture plates were coated with 0.9% agarose in serum-free medium. HaCaT cells were seeded on top of the agarose layer in serum-free KGM (1.5 mL/well) at 2 × 105/mL. Twenty-four hours later, 600-μL aliquots of cell solution were transferred to cell culture–treated plastic and supplemented with fresh media (W489 with 2% FCS). After 24 hours, reattached cells were fixed with 70% ethanol for 5 minutes. Cells were stained with crystal violet solution (0.2% in methanol), rinsed with water, and air-dried.

NF-κB DNA binding assay. Subconfluent HaCaT cells were trypsinized and seeded in serum-free medium (6 mL; 4 × 105 cells/mL) in 100-mm plates precoated with 0.9% agarose with or without TNFα (1 μg/mL). Cells were collected for nuclear extract preparation after various times in forced suspension culture (4, 8, and 24 hours). For comparison, nuclear extracts were also prepared immediately after trypsinization of HaCaT cells. DNA binding of NF-κB was determined using the NF-κB p50/p65 Transcription Factor Assay (Chemicon International, Temecula, CA) according to the instructions of the manufacturer. Briefly, nuclear extracts were prepared and incubated with double-stranded biotinylated oligonucleotides containing consensus NF-κB binding sequences. NF-κB/DNA complexes were captured on 96-well plates coated with streptavidin. NF-κB bound to DNA was detected colorimetrically (450/650 nm) after sequential incubation with an antibody against NF-κBp65 and horseradish peroxidase–conjugated secondary antibody.

NF-κB reporter (secreted alkaline phosphatase) assay. Cells were seeded at 7.5 × 104/mL in KGM for 1 to 2 days before cotransfection with pSEAP2-NF-κB vector (BD BioSciences) encoding a secreted form of human placental alkaline phosphatase driven by a NF-κB-responsive promoter and a β-galactosidase expression vector (14) for control purposes. NF-κB-dependent transcription was determined 48 hours after transfection using the Great EscAPe SEAP Reporter System 3, which is based on detection of secreted alkaline phosphatase in cell supernatants normalized to β-galactosidase activity using the luminescent β-gal detection kit (BD Biosciences).

Soft agar colony formation assay. Growth and survival of HaCaT cell variants was determined as previously described (15). Briefly, cells growing in monolayer culture were trypsinized and resuspended (104 cells) in 2 mL medium containing 0.4% agar and 10% FCS. Cell suspensions were added to 0.6% agar layers in six-well plates. Wells examined immediately after plating showed only single cells. Soft agar colonies of >50 cells were scored after 14 days in triplicate.

Tumorigenicity of HaCaT variants in nude mice. HaCaT, HaCaTII-4, and HaCaTp65 single-cell suspensions were injected s.c. into the right hind limbs (5 × 106 for HaCaTII-4 and 1 × 107 for other cell lines in 100 μL PBS) of 8-week-old athymic NCR NUM mice (Taconic Farms, Hudson, NY). Tumor growth was monitored every 2 days for 2 months. Tumor volume was determined by direct measurement with calipers and calculated by the formula [(smallest diameter2 × widest diameter) / 2]. Tumors that grew >500 mm3 and did not regress were considered to be established tumors. Tumors were not allowed to grow beyond 2,000 mm3 in accordance with Institutional Animal Care and Use Committee regulations.

Immunohistochemical analyses. Paraffin-embedded blocks containing the mouse tumor tissue were fixed in 10% neutral-buffered formalin and processed for H&E staining and immunohistochemical analysis. Tissue sections were deparaffinized in xylene, rehydrated in ethanol, rehydrated with water, and washed in 1% PBS. Primary antibodies (HA-Tag, Cell Signaling; NFκB, Santa Cruz Biotechnology) were applied to slides and incubated for 45 to 60 minutes. The immune complexes were visualized with the chromogenic substrate Dako Liquid DAB+ Substrate-Chromogen Solution (diaminobenzidine tetrahydrochloride, K3468, DAKO, Carpinteria, CA) for 5 minutes.

NF-κB activation contributes to survival of immortalized keratinocytes in steady-state culture conditions. NF-κB signaling has recently been shown to support survival of immortalized mammary epithelial cells in three-dimensional tissue reconstructs (16, 17). Here we assessed whether NF-κB signaling similarly contributed to the survival of immortalized human epidermal keratinocytes. To this end, we first used the NF-κB inhibitor Bay11-7082 in immortalized human keratinocytes (HaCaT). Bay11-7082 efficiently decreased transcriptional NF-κB activity in HaCaT keratinocytes (Fig. 1A). Consistent with an important role of NF-κB in HaCaT cell proliferation or survival, we observed that, within 24 hours of Bay11-7082 addition, metabolic activity of HaCaT cells was markedly reduced as determined by MTT assay (Supplementary Fig. S1). Similar results were obtained when using other inhibitors of NF-κB activity (i.e., the proteasome inhibitor MG-132 and parthenolide; Supplementary Fig. S1). Inhibition of MTT conversion was associated with extensive membrane blebbing and nuclear condensation (not shown) and with p85PARP cleavage (Fig. 1A), consistent with caspase-3 activation and apoptotic cell death. To obtain independent evidence for a role of NF-κB in HaCaT cell survival, we assessed the effects of down-regulating the expression of the NF-κB p65 subunit in HaCaT cells by way of siRNA (Fig. 1B). As expected, transfection of NF-κBp65-targeted siRNA was associated with reduced expression of the NF-κBp65 protein and increased PARP cleavage. To further test the role of NF-κB activation in HaCaT cell survival, we transfected these cells with a mutant IκBα [IκB superrepressor (IκBSR)] which contains serine-to-alanine mutations at amino acids 32 and 36. The IκBSR construct is resistant to IKK-dependent phosphorylation, ubiquitination, and proteasomal degradation, and serves to inhibit NF-κB-dependent transactivation by sequestering NF-κB in the cytoplasm (18). Attempts at stable, constitutive expression of this construct failed (not shown), necessitating the use of a previously established inducible expression system (14). On removal of tetracycline from the medium, expression of the hemagglutinin-tagged IκBSR was efficiently induced (Fig. 2A). As expected, induced expression of the dominant interfering IκB resulted in markedly reduced NF-κB activity as assessed by transcription of a reporter gene driven by a NF-κB-responsive promoter (Fig. 2B). In addition, induced expression of IκBSR resulted in enhanced apoptosis as determined by assessment of caspase-3 activity in cell extracts collected at several time points after transgene induction (Fig. 2C). Of note, even in the noninduced state, slightly enhanced levels of caspase-3-dependent substrate cleavage were observed over time, perhaps due to leakiness of the expression construct. Taken together, these results underscore that NF-κB activation is an important requisite for survival of cultured HaCaT cells.

Figure 1.

Induction of apoptosis in HaCaT keratinocytes by down-regulating NF-κB activity or expression. A, inhibition of NF-κB activity in HaCaT cells in monolayer culture treated with NF-κB inhibitor Bay11-7082 (5 μmol/L) for 24 hours as compared with controls that received vehicle only. NF-κB activity was determined using the Great EscAPe SEAP Reporter System 3 as described in Materials and Methods. Columns, mean of three independent experiments; bars, SD. P < 0.05, Student's t test. Inset, apoptosis induction in HaCaT cells by treatment with Bay11-7082. Cell extracts were prepared 24 hours after Bay11-7082 was added and caspase-3 activity assessed by detection of the caspase-3 cleavage product of PARP. For control purposes, blots were rehybridized with an antibody to α-tubulin. B, time-dependent changes in expression of NF-κBp65 and the p85PARP cleavage product in siRNA-treated cells. Densitometric representation of the immunoblot results relative to expression of β-actin or α-tubulin as indicated.

Figure 1.

Induction of apoptosis in HaCaT keratinocytes by down-regulating NF-κB activity or expression. A, inhibition of NF-κB activity in HaCaT cells in monolayer culture treated with NF-κB inhibitor Bay11-7082 (5 μmol/L) for 24 hours as compared with controls that received vehicle only. NF-κB activity was determined using the Great EscAPe SEAP Reporter System 3 as described in Materials and Methods. Columns, mean of three independent experiments; bars, SD. P < 0.05, Student's t test. Inset, apoptosis induction in HaCaT cells by treatment with Bay11-7082. Cell extracts were prepared 24 hours after Bay11-7082 was added and caspase-3 activity assessed by detection of the caspase-3 cleavage product of PARP. For control purposes, blots were rehybridized with an antibody to α-tubulin. B, time-dependent changes in expression of NF-κBp65 and the p85PARP cleavage product in siRNA-treated cells. Densitometric representation of the immunoblot results relative to expression of β-actin or α-tubulin as indicated.

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Figure 2.

Inhibition of NF-κB activity by overexpressing of the IκBα superrepressor induces apoptosis in HaCaT cells. A, expression of the IκBα superrepressor was induced in HaCaT cells by removal of tetracycline from culture media. Transgene expression was monitored by Western blot analysis of the hemagglutinin tag and IκBαSR itself as indicated. B, NF-κB-dependent promoter activity in induced and uninduced HaCaT cells determined 24 hours after transgene induction using a reporter gene assay. Columns, mean of three independent experiments; bars, SD. P < 0.01, Student's t test. C, effect of IκBα superrepressor expression on HaCaT cells survival. Apoptosis induction by IκBα superrepressor expression is evident by measuring caspase-3 activity using a fluorogenic substrate as described in Materials and Methods.

Figure 2.

Inhibition of NF-κB activity by overexpressing of the IκBα superrepressor induces apoptosis in HaCaT cells. A, expression of the IκBα superrepressor was induced in HaCaT cells by removal of tetracycline from culture media. Transgene expression was monitored by Western blot analysis of the hemagglutinin tag and IκBαSR itself as indicated. B, NF-κB-dependent promoter activity in induced and uninduced HaCaT cells determined 24 hours after transgene induction using a reporter gene assay. Columns, mean of three independent experiments; bars, SD. P < 0.01, Student's t test. C, effect of IκBα superrepressor expression on HaCaT cells survival. Apoptosis induction by IκBα superrepressor expression is evident by measuring caspase-3 activity using a fluorogenic substrate as described in Materials and Methods.

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Extracellular matrix adhesion maintains NF-κB activation in HaCaT cells. Next, we asked whether matrix adhesion itself contributed to NF-κB activity under steady-state culture conditions. This investigation was prompted by the observation that maintaining HaCaT cells in the absence of exogenous growth factors had only marginal effects on NF-κB activity (not shown). To address this question, we placed HaCaT cells in forced suspension culture as previously described (13) and assayed NF-κB activity under those conditions for up to 24 hours. Consistent with an important role of matrix adhesion in maintaining NF-κB activity in HaCaT cells, we observed a continuous decrease in NF-κB activity over the observation period (Fig. 3A).

Figure 3.

TNF-α enhances HaCaT cell survival in forced suspension culture through activation of NF-κB. A, HaCaT cells were suspended in plates precoated with 0.9% agarose in serum-free medium. NF-κB activity was evaluated in cell extracts prepared at the time points indicated by assessing DNA binding of the active form of NF-κB in nuclear extracts as described in Materials and Methods. Treatment of suspended cells with TNF-α (10 ng/mL) counteracted the gradual loss of NF-κB DNA binding activity observed in control cells. B, HaCaT cells were suspended in plates precoated with 0.9% agarose with or without TNF-α (10 ng/mL) and the NF-κB inhibitor Bay11-7082 (5 μmol/L) as indicated for 24 hours. Aliquots of cells were reseeded in fresh medium on tissue culture–treated plastic. Crystal violet staining of reattached viable cells was done 24 hours later. C, comparison of the effects of EGF (10 ng/mL) and of TNF-α (10 ng/mL) on HaCaT cell survival in forced suspension cultures.

Figure 3.

TNF-α enhances HaCaT cell survival in forced suspension culture through activation of NF-κB. A, HaCaT cells were suspended in plates precoated with 0.9% agarose in serum-free medium. NF-κB activity was evaluated in cell extracts prepared at the time points indicated by assessing DNA binding of the active form of NF-κB in nuclear extracts as described in Materials and Methods. Treatment of suspended cells with TNF-α (10 ng/mL) counteracted the gradual loss of NF-κB DNA binding activity observed in control cells. B, HaCaT cells were suspended in plates precoated with 0.9% agarose with or without TNF-α (10 ng/mL) and the NF-κB inhibitor Bay11-7082 (5 μmol/L) as indicated for 24 hours. Aliquots of cells were reseeded in fresh medium on tissue culture–treated plastic. Crystal violet staining of reattached viable cells was done 24 hours later. C, comparison of the effects of EGF (10 ng/mL) and of TNF-α (10 ng/mL) on HaCaT cell survival in forced suspension cultures.

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TNF-α treatment rescues NF-κB activity and enhances HaCaT cell survival in forced suspension culture. The results described above raised the question whether inflammatory cytokines known to stimulate NF-κB activity can supplant the requirement for extracellular matrix adhesion for NF-κB activation in the suspended state. Among such cytokines, TNF-α is one of the strongest NF-κB activators (1). We therefore determined whether TNF-α treatment counteracted the reduction of NF-κB activity incurred by HaCaT cells in forced suspension culture. Consistent with this notion, we observed attenuated reduction of NF-κB activity in suspended cells in the presence of TNF-α (Fig. 3A). This observation led us to assess the effect of TNF-α addition on survival of HaCaT cells in suspension culture. In agreement with an important role of NF-κB activity in HaCaT cell survival, rescue of NF-κB activity by TNF-α treatment was associated with enhanced survival of HaCaT cells in forced suspension culture (Fig. 3B). This was determined by assessing colony formation of HaCaT cells reseeded on cell culture–treated plastic after 24 hour of forced suspension culture. As compared with untreated controls, TNF-α treatment during suspension increased both the number and size of colonies. Specifically, the number of colonies in the presence of TNF-α was consistently increased by 40% whereas treatment with epidermal growth factor increased colony formation by 130% as described previously (Fig. 3C; ref. 13). As expected, the survival advantage conferred by TNF-α addition was drastically attenuated by addition of the NF-κB inhibitor Bay11-7082. Because TNF-α is known to induce apoptosis in certain cell systems and to avoid such proapoptotic effect of TNF-α, we initially included caspase 8 inhibitors in the experiments probing the effects of TNF-α on HaCaT cell survival in forced suspension. However, the inclusion of such inhibitors was not necessary for TNF-α to exert its survival effect in forced suspension cultures. Collectively, these results assigned an unexpected new role to TNF-α in support of anchorage-independent epithelial cell survival.

Deregulated NF-κB signaling supports anchorage-independent HaCaT cell survival. To independently test whether TNF-α-mediated HaCaT cell survival in forced suspension culture could be accomplished through up-regulating NF-κB activity, we established HaCaT cells stably expressing the NF-κB p65 subunit (Fig. 4A). As expected, these cells revealed increased constitutive NF-κB activity relative to mock-transfected cells. Furthermore, when subjected to forced suspension culture, these cells survived in markedly higher numbers when compared with controls (Fig. 4B). TNF-α treatment did not further enhance the rescue effect observed in NF-κBp65-expressing HaCaT cells, in agreement with the notion that the TNF-α effect on HaCaT cell survival is primarily due to activation of NF-κB. Finally, and as expected, Bay11-7082 treatment drastically attenuated NF-κB activity (Supplementary Fig. S3) and survival of NF-κBp65 HaCaT cells in suspension culture (Fig. 4B).

Figure 4.

Overexpression of the NF-κB p65 subunit supports HaCaT cell survival in forced suspension culture. A, NF-κB promoter activity in HaCaT cells stably transfected with NF-κBp65 (HaCaTp65) as compared with parental HaCaT cells. Columns, mean of three independent experiments; bars, SD. P < 0.01, Student's t test. Inset, expression levels of NF-κBp65 in parental HaCaT and HaCaTp65 cells. B, enhanced survival in forced suspension culture of HaCaTp65 cells. Cells reseeded after 24 hours of forced suspension culture. Note that survival of HaCaTp65 cells was not further stimulated by TNF-α treatment and was markedly inhibited by NF-κB inhibitor Bay11-7082.

Figure 4.

Overexpression of the NF-κB p65 subunit supports HaCaT cell survival in forced suspension culture. A, NF-κB promoter activity in HaCaT cells stably transfected with NF-κBp65 (HaCaTp65) as compared with parental HaCaT cells. Columns, mean of three independent experiments; bars, SD. P < 0.01, Student's t test. Inset, expression levels of NF-κBp65 in parental HaCaT and HaCaTp65 cells. B, enhanced survival in forced suspension culture of HaCaTp65 cells. Cells reseeded after 24 hours of forced suspension culture. Note that survival of HaCaTp65 cells was not further stimulated by TNF-α treatment and was markedly inhibited by NF-κB inhibitor Bay11-7082.

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Overexpression of NF-κBp65 induces soft agar colony formation by HaCaT cells. The observation that deregulated NF-κB activity supported anchorage-independent survival of HaCaT cells in liquid culture encouraged us to ascertain whether overexpression of NF-κBp65 also affected colony formation in soft agar, an important parameter of advanced malignancy in cells derived from solid tumors. This analysis revealed that, consistent with earlier results (9, 19), mock-transfected HaCaT cells did not form soft agar colonies (Fig. 5). In contrast, NF-κBp65-overexpressing HaCaT cells formed colonies, albeit to a lesser degree, than fully transformed Ha-RasV12-expressing HaCaTII-4 cells included as a positive control (19). This experiment was repeated with a second, independently derived p65-overexpressing HaCaT transfectant which also formed colonies in soft agar (data not shown).

Figure 5.

Overexpression of NF-κBp65 in HaCaT cells induces soft agar colony formation. Survival and growth of HaCaT, HaCaTII-4, HaCaTMock, and HaCaTp65 cells in soft agar was determined as described in Materials and Methods. Soft agar colonies of >50 cells were scored at 14 days in triplicate samples. Columns, mean of three independent experiments producing comparable results; bars, SD. P < 0.01, Student's t test.

Figure 5.

Overexpression of NF-κBp65 in HaCaT cells induces soft agar colony formation. Survival and growth of HaCaT, HaCaTII-4, HaCaTMock, and HaCaTp65 cells in soft agar was determined as described in Materials and Methods. Soft agar colonies of >50 cells were scored at 14 days in triplicate samples. Columns, mean of three independent experiments producing comparable results; bars, SD. P < 0.01, Student's t test.

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Overexpression of NF-κBp65 induces tumor formation of HaCaT cells in nude mice. Finally, we addressed the question whether NF-κBp65 overexpression rendered HaCaT cells tumorigenic in immunodeficient mice. HaCaT cells do not form tumors when xenotransplanted to immunodeficient nude mice (19). In marked contrast, we observed that HaCaTp65 cells formed tumors in all six mice injected with 1 × 107 cells per mouse (Fig. 6) whereas mock-transfected control HaCaT cells did not. Expression of the NF-κBp65 transgene in tumor tissues was confirmed in tumor cells in situ by immunostaining using antibodies to both the hemagglutinin tag and NF-κBp65 itself (Supplementary Fig. S2). To confirm that NF-κBp65 overexpression in HaCaT cells induces tumorigenicity in these cells, we used a second, independently derived p65-overexpressing HaCaT cell variant which also formed tumors (not shown). In these experiments, we used Ha-Ras-transformed HaCaTII-4 cells as a positive control; in contrast to HaCaT cells, HaCaTII-4 cells have previously been described to be tumorigenic in nude mice when injected at 5 × 106 per mouse (19). We confirmed these earlier findings for both HaCaT and HaCaTII-4 cells, which were nontumorigenic and tumorigenic, respectively. HaCaTII-4 cells formed tumors in three of six inoculated mice. HaCaTII-4 tumors presented as moderate to poorly differentiated squamous cell carcinomas (Fig. 6). These tumors grew in broad sheets and without significant keratin production. Numerous mitotic figures were present and cell borders were well delineated with intercellular bridges. By contrast, HaCaTp65 tumors represented well-differentiated squamous cell carcinomas with abundant eosinophilic cytoplasm, consistent with keratin production. In comparison with HaCaTII-4 tumors, only rare mitotic figures were present in HaCaTp65 tumors along the periphery of the neoplastic lesions. In addition, neutrophils and, to a lesser extent, lymphocytes were prominent within the tumor and at the tumor/stromal interface. Collectively, these data revealed that overexpression of NF-κBp65 in HaCaT cells was associated with tumor formation in vivo and with inflammatory infiltrates.

Figure 6.

In vivo tumorigenicity of HaCaT keratinocytes overexpressing NF-κBp65. Single-cell suspensions of HaCaT cell variants were injected into the flanks of immunodeficient nude mice. HaCaTp65 and HaCaTII-4 tumor volumes were determined using calipers and calculated [(smallest diameter2 × widest diameter) / 2] every 2 days for 2 month. Paraffin-embedded blocks containing HaCaTp65 and HaCaTII-4 tumors were processed for H&E staining.

Figure 6.

In vivo tumorigenicity of HaCaT keratinocytes overexpressing NF-κBp65. Single-cell suspensions of HaCaT cell variants were injected into the flanks of immunodeficient nude mice. HaCaTp65 and HaCaTII-4 tumor volumes were determined using calipers and calculated [(smallest diameter2 × widest diameter) / 2] every 2 days for 2 month. Paraffin-embedded blocks containing HaCaTp65 and HaCaTII-4 tumors were processed for H&E staining.

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This study presents multiple lines of evidence in support of oncogenic properties of NF-κB in an epidermal cell line representing an early stage of squamous cell carcinoma development. Perhaps most importantly, overexpression of the NF-κB p65 subunit in immortalized HaCaT keratinocytes induced soft agar colony formation and tumorigenicity in immunodeficient mice; these are canonical criteria for full malignant transformation of solid tumor cells. In addition, the results presented here assign a role to TNF-α-induced NF-κB signaling in survival of HaCaT cells, particularly in the anchorage-independent state. This result is consistent with the earlier observation that mice deficient in TNF-α exhibit greatly reduced rates of carcinogen-induced skin tumor formation (20). Collectively, our results support the notion that deregulated NF-κB signaling, as observed in squamous cell carcinomas, supports several aspects of the malignant phenotype in this cell type.

However, our results seem to be in stark contrast to the well-characterized role of NF-κB as a tumor suppressor in normal murine and human keratinocytes. Notably, down-regulating NF-κB activity by targeted overexpression of the IκBSR in mouse epidermis is associated with epidermal hyperproliferation and increased rates of squamous carcinoma development (8) and NF-κB exerts growth inhibitory effects on normal mouse keratinocytes (10, 21). Similarly, abrogating NF-κB activity in human skin reconstructs xenotransplanted to mice leads to hyperproliferation and epidermal neoplasia (7). In light of this previous evidence, our data support the concept that immortalized epidermal keratinocytes at early stages of malignant transformation have accrued molecular alterations that enable a “switch” of NF-κB function from being a tumor suppressor to being a tumor promoter as recently proposed by Perkins (22) and Aggarwal (3).

Whereas tumor initiation usually involves inactivation of tumor suppressor genes, later stages of tumor development are frequently driven by oncogenes. It is presently unknown which initiating events have occurred in HaCaT keratinocytes that create permissive conditions for NF-κB to promote tumorigenicity of these cells. However, it is known that key tumor suppressors are altered in HaCaT cells. This includes increased telomerase activity (23), mutational inactivation of both alleles of p53 (24), silencing of p16INK4A expression by promoter methylation (25), and defective regulation of either p21 expression or function (26, 27). Consistent with earlier reports (23, 28), we found p21Waf1/Cip1 protein not to be expressed in either HaCaT cells or HaCaTp65 cells under steady-state culture conditions.4

4

M.R.D. Quadros and U. Rodeck, unpublished results.

Thus, as p21/Waf1/Cip1 is a target gene for NF-κB in normal keratinocytes, it may contribute to growth arrest on NF-κB activation in this cell type (29) but not in HaCaT cells (23). It remains to be determined whether loss of p21/Waf1/Cip1 expression, as observed in HaCaT cells, is sufficient for the manifestation of oncogenic properties of NF-κB activation. Alternatively, loss of either p53 or p16INK4A/ADP ribosylation factor (ARF) function in HaCaT cells may have unmasked transcriptional properties of NF-κB normally suppressed by p53 and/or ARF (22, 30). Additional studies are necessary to define the functional importance of NF-κB-dependent growth constraints in epidermal keratinocytes in preventing the emergence of malignant cells in the differentiating epidermis.

To our knowledge, this is the first report of tumorigenic conversion of HaCaT keratinocytes by overexpression of a single proto-oncogene. Previous work by the Fusenig group has shown that forced expression of oncogenic Ha-RasV12 was similarly capable of inducing tumorigenicity of HaCaT cells in experimental mice (19), a result confirmed in the present study using HaCaTII-4 cells. In contrast to HaCaTII-4 tumors, HaCaTp65 tumors revealed marked infiltration with inflammatory cells. Previous studies have implicated both tumor cell-autonomous effects of NF-κB signaling (31, 32) and “field effects” by chronic inflammation (32) in NF-κB driven epithelial tumor formation. However, in the case of HaCaTp65 tumors, the inflammatory infiltrate may, at least in part, be caused by excessive keratin deposition in HaCaTp65 tumors. Regardless of the relative contribution of the inflammatory response to tumorigenicity in mice, persistent overexpression of NF-κBp65 in HaCaT cells was clearly associated with tumor cell-autonomous roles of NF-κB activation, in support of epithelial cell survival and colony formation in soft agar. Thus, it seems likely that NF-κB signaling contributes in a tumor cell-autonomous fashion to the development and progression of hepatomas (31), colorectal carcinomas (32), and, as shown here, squamous cell carcinomas.

In summary, these results establish that deregulated NF-κB signaling exerts powerful oncogenic effects in an immortalized keratinocyte line. They lend support to the notion that NF-κB activity serves opposite roles in squamous cell carcinoma development, depending on tumor progression stage. Whereas NF-κB activation seems to curb transformation of normal uninitiated keratinocytes, it exacerbates the malignant phenotype of initiated, immortalized keratinocytes such as HaCaT cells.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Grant support: U.S. NIH grants CA106633 (A.P. Dicker) and CA81008 (U. Rodeck) and Department of Defense grant DAMD-17-02-1-0216 (U. Rodeck).

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. N. Fusenig (Division of Carcinogenesis and Differentiation, German Cancer Research Center, Heidelberg, Germany) for providing HaCaT cells, and Yaping Sui, William R. Davidson, and Stephanie Lavorgna for technical assistance.

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Supplementary data