Inducible cutaneous inflammation reveals a protumorigenic role for keratinocyte CXCR2 in skin carcinogenesis

Abstract

Transgenic mice that overexpress PKCalpha in the epidermis (K5-PKCalpha mice) exhibit acute CXCR2-mediated intraepidermal neutrophilic inflammation and a strong epidermal hyperplasia in response to application of 12-O-tetradecanoylphorbol-13-acetate (TPA). We now show that hyperplasia is independent of infiltrating neutrophils. Furthermore, when K5-PKCalpha mice were initiated with 7,12-dimethylbenz(a)anthracene (DMBA) and promoted with a low dose of TPA, 58% of K5-PKCalpha mice developed skin papillomas that progressed to carcinoma, whereas wild-type mice did not develop tumors. We confirmed that CXCR2 is expressed by keratinocytes and showed that transformation by oncogenic ras (a hallmark of DMBA initiation) or TPA exposure induced all CXCR2 ligands. Ras induction of CXCR2 ligands was mediated by autocrine activation of epidermal growth factor receptor and nuclear factor-kappaB, and potentiated by PKCalpha. Oncogenic ras also induced CXCR2 ligands in keratinocytes genetically ablated for CXCR2. However, ras transformed CXCR2 null keratinocytes formed only small skin tumors in orthotopic skin grafts to CXCR2 intact hosts, whereas transformed wild-type keratinocytes produced large tumors. In vitro, CXCR2 was essential for CXCR2 ligand-stimulated migration of ras-transformed keratinocytes and for ligand activation of the extracellular signal-regulated kinase (ERK) and Akt pathways. Both migration and activation of ERK and Akt were restored by CXCR2 reconstitution of CXCR2 null keratinocytes. Thus, activation of CXCR2 on ras-transformed keratinocytes has both promigratory and protumorigenic functions. The up-regulation of CXCR2 ligands after initiation by oncogenic ras and promotion with TPA in the mouse skin model provides a mechanism to stimulate migration by both autocrine and paracrine pathways and contribute to tumor development.

Figure 1

Two stage skin carcinogenesis in K5-PKCα mice and their wildtype littermates (A) and persistence of epidermal hyperplasia in neutrophil depleted K5-PKCα mice after a single topical treatment with TPA (B–D). A, Mice were initiated by treating with 100μg (390 nmoles) DMBA/0.2ml acetone at zero time and promoted by treating with 1μg (1.6 nmoles)/0.2 ml acetone twice a week from weeks 1–20. Tumors were counted every week. The percent with papilloma is plotted versus time for each group. Tumor multiplicity of 4.6 tumors/tumor bearing mice is not shown. B–D,18 hours prior to TPA application, K5-PKCα mice were injected IP with 100μg anti Ly-6G antibody (C) or isotype control (B) Arrows point to neutrophilic microabscesses present in IgG control group only, right panels represent higher magnification of the epidermis. Skin biopsies were collected three days after TPA treatment and H&E stained. D, Basal BrdU-labeled as well as unlabeled basal nuclei were counted in randomly selected regions. Between 400 and 700 cells were counted for each skin section. Data are reported as mean ± SE. Each group contains five mice and data are representative of two independent experiments.

Figure 2

Oncogenic H-ras upregulates CXCR2 ligands in primary keratinocytes through EGFR, NF-κB and PKCα. A, real-time PCR analysis of KC/CXCL1, MIP-2/CXCL2/3, GCP-2/CXCL6 and CXCR2 mRNA expression in control and v-ras^Ha-transduced keratinocytes at indicated days after transduction. Bars represent mean value of triplicate determinations. B, MIP-2 secreted into culture supernatant collected from control or v-ras transduced K5-PKCα or WT keratinocytes infected with A-CMV (control) or degradation-resistant IκBα (IκBαSR) adenovirus to block NF-κB activity. Bars represent the mean ± SEM of triplicate determinations. Results are representative of three independent experiments. *P < 0.01 compared with respective non-ras transduced control; #, P < 0.05 compared with v-ras^Ha WT keratinocytes A-CMV transduced. C, Culture supernatants from EGFR WT or KO primary keratinocytes were collected after control or v-ras^Ha transduction. KC and MIP-2 concentrations were determined by ELISA. Bars represent the mean ± SEM of triplicate determinations.

Figure 3

Targeted disruption of CXCR2 impairs growth of v-ras^Ha-induced squamous tumors. A, 2 × 10⁶ CXCR2 WT or KO primary v-ras^Ha-transduced keratinocytes combined with CXCR2 WT fibroblasts (5 × 10⁶) were grafted onto nude mice as a reconstituted skin and average tumor volume was calculated as described in methods. Data are reported as mean ± SE. *, P < 0.05 compared with CXCR2 WT for the same time-point. Each group contains 10–12 mice and results are representative of two independent experiments. Only mice bearing tumors were included in this figure. Insert shows H-ras expression in an aliquot of v-ras keratinocyte used for grafting. B, BrdU-labeled nuclei were counted in 5–7 randomly selected regions. Data are reported as mean ± SE. *, P < 0.05 compared with CXCR2 WT. Each group contains 7–9 tumors and graphed data are representative of two independent experiments. C, Apoptotic cells were identified using the Apoptag kit, which stains nuclei containing nicked DNA. Positive nuclei were counted in 5–7 randomly selected regions. Data are reported as mean ± SE. Each group contains 7–9 tumors and graphed data are representative of two independent experiments. D, immunostaining for CD31 antigen outlining blood vessels within tumors originating from CXCR2 WT (WT) or CXCR2 KO (KO) v-ras^Ha-transduced keratinocytes. Bottom graph, microvessels density based on CD31⁺ cells. The average number of positive cells in at least 5 fields per tumor originating from CXCR2 WT (WT) or CXCR2 KO (KO) v-ras^Ha-transduced keratinocytes is shown. Each group contains 7–9 tumors and graphed data are representative of two independent experiments. Data are reported as mean ± SE.

Figure 4

CXCR2 mediates migration of keratinocytes. A, Five days after v-ras^Ha transduction, migration of CXCR2 WT and KO keratinocytes was tested in Transwell assays as described in methods. MIP-2 or TGFα (10 ng/ml) diluted in low –serum containing media (0) was used as stimulus in the bottom chamber while cells were seeded in the top chamber in low –serum containing media. The CXCR2 antagonist was added to the cell suspension prior to the seeding in the top well. Fold increase (chemotactic index) of keratinocyte migration in response to treatment was compared to basal migration in the absence of stimulus. Bars represent the mean ± SEM of two triplicate determinations. *, P < 0.001; #, P < 0.0001 compared with untreated control, § P < 0.001 compared with untreated respective control. B, Delayed epidermal regeneration in K5-PKCα mice deficient for CXCR2 after a single topical application of TPA. Skin biopsies were collected at 2, 3 and 5 days after a single TPA application on K5-PKCα/CXCR2 WT and K5-PKCα/CXCR2 KO and H&E stained. Bar, 50μm. C, Immunohistochemistry for filaggrin and loricin was performed on similar biopsies as in B collected at day 3 and 5.

Figure 5

Reconstitution of CXCR2 KO keratinocytes with CXCR2 restores chemotaxis and signaling after MIP-2 treatment. On the fourth day after v-ras transduction, CXCR2 null primary keratinocytes were transduced with an empty adenovirus (Control Ad) or CXCR2 expressing adenovirus (CXCR2 Ad) for an additional 24 hours and assayed for their ability to migrate in response to MIP-2 using the Transwell assays (A) as described in figure 4 or (B) stimulated with 200ng/ml MIP-2 or 10ng/ml TGFα for 5 minutes. Western blots were performed for total and phospho-ERK 1/2 (p-ERK1/2) and total and phospho-Akt (p-Akt) as described under “materials and methods”. Relative levels of phospho-Akt and phospho-ERK are indicated in each figure normalized to the PBS treated cells. *, P < 0.05 compared with untreated control.