Ras is frequently activated in cutaneous squamous cell carcinoma, a prevalent form of skin cancer. However, the pathways that contribute to Ras-induced transformation have not been entirely elucidated. We have previously demonstrated that in transgenic mice, overexpression of the Ras activator RasGRP1 promotes the formation of spontaneous skin tumors and enhances malignant progression in the multistage carcinogenesis skin model that relies on the oncogenic activation of H-Ras. Utilizing a RasGRP1 knockout mouse model (RasGRP1 KO), we now show that lack of RasGRP1 reduced the susceptibility to skin tumorigenesis. The dependency on RasGRP1 was associated with a diminished response to the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Specifically, we found impairment of epidermal hyperplasia induced by TPA through keratinocyte proliferation. Using a keratinocyte cell line that carries a ras oncogenic mutation, we also demonstrated that RasGRP1 could further activate Ras in response to TPA. Thus, we propose that RasGRP1 upregulates signaling from Ras and contributes to epidermal tumorigenesis by increasing the total dosage of active Ras.
Cutaneous squamous cell carcinoma (SCC) is a prevalent form of non-melanoma skin cancer that results from the transformation of epidermal keratinocytes (1,2). Although the oncogenic events that drive this malignancy remain to be fully characterized, Ras is frequently activated in human SCC (3,4). Mouse models that recapitulate several aspects of the genesis and progression of cutaneous SCC, like the multistage skin carcinogenesis model, have corroborated the pathogenic role of Ras and continue to serve in the investigation of pathways that control Ras oncogenic effects in the skin (5,6). Ras comprises a family of small GTPases that cycle between inactive guanosine diphosphate-bound (RasGDP) and active guanosine triphosphate (GTP)-bound (RasGTP) states (7,8). It is in its GTP-bound state that Ras can interact and activate downstream effector molecules such as Raf and phosphatidylinositol 3-kinase to regulate various cellular functions. Certain somatic mutations render Ras proteins constitutively active, mainly due to their inability to catalyze GTP hydrolysis and to respond to GTPase-activating proteins (9,10). This constitutive activation participates in cellular transformation in many tissues, including the epidermis. However, high levels of active RasGTP could also be achieved by biochemical activation of wild-type (Wt) Ras proteins from upstream activators. Potential candidates for aberrant biochemical Ras activation in the skin include epidermal growth factor receptor overexpression (11–13) as well as increased secretion of growth factors that could act in an autocrine/paracrine manner to stimulate Ras (14,15).
Ras activation requires the function of guanine nucleotide exchange factors (GEFs) that catalyze the guanosine diphosphate–GTP exchange (16). Although GEF mutations are uncommon in cancer (17), GEFs are nevertheless an important component in the upstream Ras pathway and could represent a potential molecular target from the therapeutic standpoint. We have shown previously that epidermal keratinocytes express RasGRP1 (18), a GEF activated in response to diacylglycerol and its phorbol ester analogs like the skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) (19,20). Our studies have revealed that RasGRP1 overexpression augments Ras activation in primary keratinocytes in response to TPA in a protein kinase C (PKC)-independent manner (18). Furthermore, a transgenic mouse model for RasGRP1 overexpression in the skin (K5.RasGRP1) develops spontaneous cutaneous SCC associated with wounding (21,22) and displays higher susceptibility to tumor progression in response to multistage carcinogenesis (23), suggesting that RasGRP1 plays a role in skin tumorigenesis. Because those studies involved the use of ectopic overexpression of RasGRP1, the question remained as to what extent the observations reflected a pathophysiological role of RasGRP1 in the epidermis. To address this issue, we recently utilized keratinocytes isolated from a RasGRP1 knockout mouse model (RasGRP1 KO) and found that RasGRP1 is critical in the activation of Ras by TPA (24). Encouraged by those observations, we decided to examine the response of the RasGRP1 KO mice to multistage carcinogenesis. Here we show that the skin carcinogenesis response was significantly reduced in the RasGRP1 KO mice. The lack of RasGRP1 also impaired the response of the mice to TPA-induced hyperplasia due to diminished keratinocyte proliferation with no increase in apoptosis. Additionally, we show that in a keratinocyte cell line carrying a Ras oncogenic mutation, RasGRP1 could further activate Ras in response to TPA. Taken together, the findings demonstrate an important role for RasGRP1 in skin tumorigenesis and support the idea that RasGRP1 participates in keratinocyte transformation by contributing to the increase in the total active Ras dosage.
Materials and methods
All animal studies were done according to Institutional Animal Care and Use Committee guidelines at the University of Hawaii Animal Facility. The RasGRP1 KO mice were generated as described previously (25) and backcrossed for >10 generations to the FVB/N background. For the multistage carcinogenesis experiment, 6- to 8-week-old mice were utilized. Eighteen RasGRP1 KO and 17 Wt mice were topically treated with 26 μg of 7,12-dimethylbenz(a)anthracene (DMBA) in 200 μl of acetone on the shaved dorsal skin. Two weeks later, tumor promotion was initiated by topical administration of 2 μg TPA in 200 μl acetone, twice a week for 20 weeks. Mice were monitored weekly, and tumors formed were measured with a caliper at least once a week; lesions of at least 1mm were counted as tumors. After the TPA treatment ended, mice were followed for an additional 8 weeks and then euthanized. Tumors were collected for histology and DNA analysis.
For evaluation of TPA-induced epidermal hyperplasia, five 6- to 8-week-old mice of Wt, RasGRP1 KO or transgenic K5.RasGRP1 background were treated with two topical applications of 2 μg TPA in 200 μl acetone administered 48h apart on the shaved dorsal skin. Mice were also treated with acetone alone as vehicle control. The treated skin was collected 24h after the last TPA application and processed for histology. The epidermal thickness was determined by microscopic analysis of hematoxylin and eosin-stained skin specimens using MetaMorph (Molecular Device Corporation). The grid of a hemacytometer was used for calibration. Samples were measured at six different locations before calculating the average thickness of each specimen in micrometers.
Analysis of H-ras mutations
We determined the presence of H-ras mutations in codon 61 in the tumors as described before (23), using a mutation-specific PCR assay developed by Nelson et al. (26). Briefly, DNA was extracted from a minimum of two 10 μm sections of paraffin-embedded tumors using the QIAamp DNA Micro kit (Qiagen) according to the manufacturer’s instructions. One hundred nanograms of DNA were used for the PCR reaction with the following primers: upstream ras primer, 5′-CTA AGC CTG TTG TTT TGC AGG AC-3′; downstream mutant ras primer, 5′-CAT GGC ACT ATA CTC TTC TA-3′. This primer combination produced a 110 bp band. Wt H-ras was also amplified as a control (upstream ras primer, 5′-CTA AGC CTG TT G TTT TGC AGG AC-3′; downstream Wt ras primer: 5′-CAT GGC ACT ATA CTC TTC TT-3′), which also generated a 110 bp PCR product.
Histopathology and immunohistochemistry
Tumors and skin from hyperplasia protocols were fixed in 4% paraformaldehyde for 24h and maintained in 70% ethanol until paraffin-embedded. Hematoxylin and eosin-stained slides were used for descriptive histopathology and hyperplasia measurements. For immunohistochemical analysis of Ki-67, deparaffinized sections were subjected to heat-induced epitope retrieval using citrate buffer. After blocking, tissues were incubated with a 1:500 dilution of a rabbit polyclonal anti-Ki-67 antibody (EMD Millipore) overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibody for 1 h. 3,3′-Diaminobenzidine was used as a substrate. Tissues were counterstained with hematoxylin and mounted. All the immunohistochemistry analysis was done using the same samples used to measure the hyperplastic response of the three groups to TPA. We utilized three most hyperplastic tissues from each group for Ki-67 staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Ki-67-positive cells were counted using the cell counter function of Image J. The procedures were performed at the University of Hawaii Cancer Center Pathology Shared Resource, and the Microscopy & Imaging Shared Resource.
The presence of apoptotic cells in skin specimens was evaluated by using a modified TUNEL assay kit designed for detection of apoptosis in dermal tissue sections (DermaTACS; Trevigen). Briefly, slides were treated with Proteinase K (1 μg in 50 μl of DNase-free H2O) and the activity of endogenous peroxidase was quenched using 3% H2O2 in methanol. Then, DNA fragmentation was measured by labeling the samples with terminal deoxynucleotidyl transferase and the thymidine analog bromodeoxyuridine. Incorporated bromodeoxyuridine was detected using a biotinylated anti-bromodeoxyuridine antibody followed by streptavidin-horseradish peroxidase and incubation in TACS Blue Label™ according to the manufacturer’s instructions. Positive controls (DNA endonuclease-treated specimens to generate fragmentation of DNA) and negative controls (omission of terminal deoxynucleotidyl transferase in the assay) were run for each assay.
Cell culture and western blots
SP-1 cells (papilloma-derived keratinocytes generated by chemical-induced carcinogenesis in SENCAR mice) were obtained from Dr S.Yuspa (National Cancer Institute, Bethesda, MD), and were cultured as described previously (27). Briefly, cells were plated in dishes coated with Coating Matrix solution (Invitrogen) in Eagle’s minimum essential medium containing 1.2mM CaCl2, antibiotics, antimycotics and 8% fetal bovine serum. After 24h, cells were washed with Dulbecco’s phosphate-buffered saline and cultured in 154 medium (Invitrogen) supplemented with 50 μM CaCl2, antibiotics, antimycotics, 2% calcium-free fetal bovine serum and a human epidermal growth factor supplement containing epidermal growth factor, pituitary extract, insulin, transferrin and hydrocortisone (Invitrogen). For transduction of the SP-1 cells with RasGRP1, recombinant adenoviral vectors encoding rat RasGRP1 were used as described before (18), along with adenoviral vectors for expression of bacterial β-galactosidase (LacZ) that served as control for the adenoviral infection. To evaluate levels of GTP-loaded Ras (RasGTP), we utilized the glutathione S-transferase-tagged Ras-binding domain of Raf-1 (GST-Raf) as a probe in a Ras affinity precipitation or pulldown assay. SP-1 cells were serum-starved overnight and then treated with TPA or vehicle (dimethyl sulfoxide) for 15 min. Each 60 mm culture dish was harvested on ice with 500 μl of lysis buffer containing 50mM Tris pH 8 at working temperature, 10mM MgCl2, 0.5M NaCl, 2% Igepal and protease inhibitors (Mini Complete with EDTA; Roche Applied Science). The lysates were vortexed and incubated on ice for 5min followed by centrifugation at 13 000 r.p.m. for 5min at 4°C. Half of the supernatant was then incubated with GST-Raf bound to colored sepharose beads (Cytoskeleton) for 1h with rotation in the cold. The affinity complexes were washed twice with washing buffer containing 25mM Tris pH 7.5, 30mM MgCl2 and 40mM NaCl and then resuspended in 60 μl of 2× Laemmli buffer and boiled. Thirty microliters of each pulldown sample were resolved on 15% acrylamide gels along with 30 μl of the original lysate that served as measurement of total Ras input in the assay. The proteins were blotted onto nitrocellulose membranes, and immunostaining was done using the pan anti-Ras clone RAS10 antibody (EMD Calbiochem). The following antibodies were used for analysis of RasGRP1 and extracellular signal-regulated kinase levels from total lysates containing 50 μg of protein: mouse monoclonal anti-RasGRP (199; Santa Cruz Biotechnology), rabbit polyclonal anti-p44/42 MAPK (Erk1/2) (9102; Cell Signaling Technology) and rabbit polyclonal phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101; Cell Signaling Technology).
Ablation of RasGRP1 impairs skin tumor formation
To evaluate the role of RasGRP1 in Ras-dependent skin tumorigenesis, we examined the response of a KO mouse model for RasGRP1 (RasGRP1 KO) in the multistage carcinogenesis protocol. As shown in Figure 1A and B, the RasGRP1 KO mice were markedly resistant to tumorigenesis, with only 3 out of 18 mice developing tumors compared with 13 out of 17 for Wt mice. The few tumors produced in the mutant group were also smaller compared with the size of the Wt tumors (Figure 1C). Histological analysis of the lesions revealed mainly microinvasive, well-differentiated cutaneous SCC in both groups (Figure 1D and E). However, no extensively invasive SCCs were observed in the RasGRP1 KO group, in contrast to a few that developed within the Wt mice. Genotyping of tumor DNA confirmed the presence of the KO allele in the tumors from the mutant mice, ruling out a somatic event that would have allowed RasGRP1 KO keratinocytes to express some functional form of RasGRP1 (Figure 1F). Taken together, the data suggest that while RasGRP1 is not indispensable, as some tumors formed in the RasGRP1 KO mice, it significantly contributes to tumorigenesis in the multistage carcinogenesis model.
Tumors generated in the RasGRP1 KO mice show the H-ras codon 61 mutation
Development of tumors in the multistage carcinogenesis protocol using DMBA is dependent on oncogenic Ras activation, mainly as a result of H-ras mutations at codon 61 (6). Using a PCR approach that discriminates between Wt and codon 61 mutant alleles for H-ras, we confirmed the presence of oncogenic H-ras in the RasGRP1 KO-generated tumors as well as in a sample of Wt tumors that was screened for comparison (Figure 2A). This result suggests that DMBA is able to induce H-ras mutations in the RasGRP1 KO mouse skin; however, it does not exclude the possibility that the mutation rate might differ from that produced in the Wt mice, resulting in a lower number of initiated cells that could be promoted by TPA. One common cause for a reduced mutation rate in the multistage carcinogenesis model is an increased susceptibility to cell death in keratinocytes that had been exposed to DMBA (28). To test this possibility, we examined the response of the epidermis to DMBA exposure and assessed apoptosis by the TUNEL assay. As shown in Figure 2B, there was no significant DNA fragmentation detected in RasGRP1 KO mouse skin 24 h after DMBA treatment, ruling out increased apoptosis as a mechanism for the reduced response to tumorigenesis of the mutant mice. Whereas the results above do not preclude a defect in initiation, the fact that some tumors still form in the RasGRP1 KO mice underscore a more important role of RasGRP1 during the TPA-mediated tumor promotion stage.
The hyperplastic effects of TPA in the skin are impaired in the RasGRP1 KO mice
To address the possibility that the RasGRP1 KO mice were refractory to skin carcinogenesis due to a reduced sensitivity to the effects of TPA, we examined the consequences of RasGRP1 ablation on the ability of TPA to induce acute epidermal hyperplasia. Relative to the littermate Wt controls, the skin of the RasGRP1 KO mice failed to thicken in response to TPA exposure (Figure 3), which was consistent with a reduction in proliferation as estimated by Ki-67 staining (Figure 4A). Notably, epidermal thickness increased >3-fold in transgenic mice overexpressing RasGRP1 in the skin and was accompanied by increased Ki-67 staining of the epidermis (Figures 3 and 4A). Since none of the groups sustained significant apoptosis as a result of the TPA treatment (Figure 4B), the results suggest that RasGRP1 influences the proliferative effects of TPA on the epidermis and, in this way, participates in tumor promotion.
TPA can increase Ras activation via RasGRP1 in the context of an oncogenic Ras mutant
Our previous studies demonstrated that RasGRP1 is essential for Ras activation by TPA in keratinocytes (29). Furthermore, the results above suggest an important role for RasGRP1 in the effects of TPA in the skin during tumor promotion. However, given that tumors produced by DMBA/TPA carry H-ras mutations, we questioned the contribution of RasGRP1 when a Ras oncoprotein was already present in the initiated cells. Using a keratinocyte cell line derived from DMBA/TPA-induced papillomas (SP-1) that express RasGRP1 endogenously, we set out to test the hypothesis that even in the presence of an active Ras mutant, RasGRP1 could further mediate the increase in the total active Ras dosage in the cells. We observed that SP-1 keratinocytes transduced to overexpress RasGRP1 displayed elevated levels of extracellular signal-regulated kinase phosphorylation compared with cells transduced with an irrelevant protein (Figure 5A), suggesting stimulation of Ras cascades. When RasGTP levels were measured, TPA treatment resulted in a clear surge in Ras activation in the RasGRP1-overexpressing cells compared with the mock (LacZ) controls (Figure 5B). We detected an increase in activity of Ras proteins of at least two gel mobilities, suggesting activation of other family members in addition to H-Ras. Thus, RasGRP1 can contribute to Ras activation in the presence of oncogenic Ras in keratinocytes.
The contribution of RasGRP1 to tumorigenesis in cutaneous SCC has remained inconclusive. Here, we found that RasGRP1 influences epidermal carcinogenesis leading to cutaneous SCC. In the absence of RasGRP1, carcinogenesis is significantly reduced in response to the classic combination DMBA/TPA. On the other hand, as we have shown before using the transgenic K5.RasGRP1 mice, RasGRP1 overexpression produces bigger and more aggressive tumors (23). Moreover, we show that exogenous expression of RasGRP1 increases total Ras dose (likely affecting more than one Ras isoform). Thus, the results of our two models combined clearly support an important role for RasGRP1 in Ras-driven skin cancer.
Ras hyperactivation in keratinocytes is a critical driving force in transformation and tumorigenesis. Models of cutaneous SCC like the multistage model used in this study, heavily rely on induction of H-ras oncogenic mutations that constitutively activate H-Ras (30). Given that most oncogenic Ras forms show an impairment in the intrinsic as well as the GTPase-activating protein-induced GTPase activity (9,10), which turns them constitutively active, they are expected to be less dependent on GEF proteins like RasGRP1. Therefore, although RasGRP1 contribution to transformation may be associated to the initial and/or recurrent activation of oncogenic H-Ras, other possibilities are more likely to explain its role in tumorigenesis. We propose that RasGRP1 is required to stimulate Wt Ras isoforms (H-ras, K-ras and/or N-ras), which combined with the activity of a Ras oncoprotein (H-ras codon 61) provide the high RasGTP levels necessary for tumorigenesis. In fact, a number of studies support the contribution of high intensity Ras signals to epidermal transformation, which could be achieved by increase in mutant ras alleles in mouse skin tumors (31,32) but potentially also by elevated epidermal growth factor receptor ligands that could activate Wt Ras (14,15,33). In our model, it is plausible that TPA stimulation of RasGRP1 results in the additional Ras activation—above that provided by the Ras oncoprotein—required for transformation, and our results using the H-ras mutant SP-1 cells suggest that Ras can be further activated by TPA stimulation of RasGRP1 in initiated keratinocytes. An alternative, although not mutually exclusive, explanation is based on the concept of complementary rather than redundant roles of the different Ras isoforms. If activation of different isoforms leads to stimulation of non-redundant downstream pathways, then an H-Ras oncoprotein may not be sufficient—even at high levels of activity—to fulfill the whole tumorigenesis program. In this regard, we have recently demonstrated that the lack of RasGRP1 impairs JNK2 activation by TPA in keratinocytes (24). As JNK2 is critical in DMBA/TPA-induced skin tumorigenesis (34), a decrease in TPA-induced JNK2 activation could alternatively explain the diminished response of the RasGRP1 KO mice to tumorigenesis.
The tumor-promoting effects of TPA have been associated in part with its ability to induce epidermal hyperplasia (6,35), which results from its direct proliferative effects on keratinocytes and an inflammatory reaction that combined allow the expansion of initiated cells. Several molecular pathways mediate the action of TPA in the skin, in particular, PKC and activation of growth factor signaling cascades in epidermal and non-epidermal tissues (6,33,36,37). Based on our present findings, we can also add the contribution of RasGRP1 to TPA-induced hyperplasia and thus, to tumor promotion. Yet, our previous work with the transgenic RasGRP1 mice did not detect differences in hyperplasia compared with Wt mice (23). This apparent discrepancy could be the result of the different phorbol ester regimens used in both studies; in fact, a small decrease in TPA dose administered over a 2-day period (present study) has now revealed a RasGPR1 dosage effect in hyperplasia, which we did not see at a slightly higher, single dose of TPA (23). Discrepancies associated with the TPA regimen were also observed in carcinogenesis studies involving a transgenic mouse model for PKCα, where the effects of PKCα overexpression in tumorigenesis could be appreciated only at a very low TPA dose (6,34,35). Although the mechanism to explain these differences remains elusive, it may result from the interplay of the multiple phorbol ester targets in different cell types, contributing to the pleiotropic effects of TPA.
RasGRP1 plays a critical role in thymocyte differentiation and T-cell receptor signaling (25); consequently, the RasGRP1 KO mice possess defects in T-cell number and activation, specifically a decrease in CD4+ and CD8+ T cells, a defect in activation of TCRγδ and a reduction in TCRαβ T cells (25,38,39). Given the role of the immune system in cancer (10,40), the immunodeficiency of the RasGRP1-mutant mice could have influenced the response to the multistage carcinogenesis protocol. A number of studies using multistage carcinogenesis on immunocompromised mice have revealed that CD4+ and CD8+ single-positive T cells have opposite effects in tumorigenesis and their contributions depend on the background strain and the TPA dose used during tumor promotion. For example, CD8+ T cells antagonize while CD4+ T cells favor tumorigenesis in the C3H/HeN mouse strain promoted with high doses of TPA (40 nmol = 25 μg) (41). On the other hand, CD8+ αβ+ T cells appear to have a protumorigenic role in FVB mice under a high dose DMBA/TPA regime (42,43). Furthermore, the type of TCR also influences carcinogenesis, and while TCRγδ+ T cells generally oppose tumorigenesis, the contribution of the TCRαβ+ T cells varies depending on whether they are CD4+ or CD8+ T cells (44–46). Girardi et al. (45) have shown that TCRδ−/− mice in FVB background have an increased tumor susceptibility to multistage carcinogenesis at a low dose of TPA (5 nmol = 3 μg), whereas TCRβ−/− mice do not differ from the response of the Wt mice unless the tumor-promoting regime is increased to 40 nmol (25 μg), when they show resistance to tumor formation. Considering the strain of mice used in our studies (FVB) and the low dose of TPA for tumor promotion (2 μg), we speculate that the RasGRP1 KO mice should have displayed a higher tumorigenic response to DMBA/TPA if based solely on the T-cell deficiencies; instead, we observed a reduced response. This strongly suggests the importance of a cell autonomous action of RasGRP1 in the keratinocytes. In an initial attempt to discern the contribution of the epidermal RasGRP1 in skin carcinogenesis, we crossed heterozygous K5.RasGRP1 transgenic mice onto the KO RasGRP1 background. Interestingly, the double-mutant K5.RasGRP1+/Tg; RasGRP1−/− mice developed spontaneous papillomas and cutaneous SCCs in a similar fashion as the transgenic K5.RasGRP1 animals (21) (Supplementary Figure 1, available at Carcinogenesis Online), a phenomenon that we have previously demonstrated to be associated with skin wounding (22). Although TCRγδ T cells have been deemed required for wound-induced skin tumors (47), the deficiency in activation of this cell population due to the RasGRP1 KO background did not appear to affect tumor response in the RasGRP1 double-mutant mice. Therefore, although RasGRP1 may contribute to the response to carcinogens and tumor-promoting signals through immune-mediated mechanisms, the findings on the spontaneous tumors in the RasGRP1 double-mutant mice provide support to the importance of the cell autonomous effects of RasGRP1 in the skin.
In summary, our studies demonstrate the contribution of RasGRP1 to epidermal carcinogenesis through its function as a Ras activator. Given the prevalence of Ras activation in cutaneous SCC, defining relevant upstream and downstream Ras pathways in keratinocyte transformation can contribute to the development of novel therapeutic agents to treat this malignancy. The identification of RasGRP1 as a tumor promoter and progression target in mouse skin tumorigenesis provides the rationale for further studies of this Ras GEF in the development of human cutaneous SCC, and of its potential as a therapeutic target.
National Institutes of Health (R01 CA096841 to J.W.R. and J.J.). Procedures performed at the University of Hawaii Microscopy & Imaging Shared Resource and the Pathology Shared Resource were funded in part by National Institutes of Health grant (P30 CA071789), as a Cancer Center Support Grant.
guanine nucleotide exchange factor
protein kinase C
squamous cell carcinoma
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
The authors dedicate the publication to the memory of Dr P.S.L. who passed away during revision of the manuscript. Her research will be continued by her lab under the guidance of Drs J.W.R. and J.J both of whom contributed to revision and submission of this manuscript. Any inquiries or requests for reagents should be addressed to Dr J.W.R.
Conflict of Interest Statement: None declared.