Disruption of Stat3 reveals a critical role in both the initiation and the promotion stages of epithelial carcinogenesis (original) (raw)
Stat3 deficiency reduces TPA-mediated epidermal hyperproliferation. During tumor promotion in mouse skin, initiated cells undergo selective clonal expansion to form precancerous lesions (i.e., papillomas) (3). Tumor promoters such as the phorbol ester TPA induce a dramatic proliferative response in mouse epidermis, which is necessary for the clonal expansion of initiated cells (3). To examine the role of Stat3 in tumor promoter–induced epidermal hyperproliferation in vivo, Stat3-deficient mice were treated topically with TPA. Following 4 topical treatments of 6.8 nmol TPA, the epidermis of control mice showed a dramatic hyperplasia (acanthosis) (Figure 1, B and E), while the epidermis of Stat3-deficient mice exhibited a significantly milder acanthosis (Figure 1, D and E). Stat3-deficient mice also showed a significantly reduced epidermal labeling index (LI) as revealed by a reduction (by ∼80%) in the number of BrdU-positive cells following treatment with TPA at a dose of 6.8 nmol (Figure 1F). These data indicate that functional Stat3 protein is necessary for TPA-induced epidermal hyperproliferation.
Response of Stat3-deficient and control mice to TPA-induced epidermal hyperproliferation. (A–D) Groups of mice (n = 3) were treated topically with 4 applications of TPA and sacrificed 24 hours after the last treatment. BrdU was injected 30 minutes prior to sacrifice. H&E staining of epidermis from (A) untreated control mice, (B) control mice treated with TPA, (C) untreated Stat3-deficient mice, and (D) Stat3-deficient mice treated with TPA. (E) Quantitation of epidermal thickness from control (white bars) and Stat3-deficient (black bars) mice treated with different doses of TPA. (F) Percentage of BrdU-positive epidermal basal cells in control (white bars) and Stat3-deficient (black bars) mice treated with different doses of TPA. Scale bar: 50 μm. **P < 0.01 by Mann-Whitney U test.
Altered cell cycle progression in epidermis of Stat3 deficient mice following TPA treatment. We next examined the time course for epidermal DNA synthesis in epidermis of Stat3-deficient mice following TPA treatment. In nontransgenic mice, the percentage of epidermal basal cells in S phase reached a peak (LI ∼70%) at about 17 hours following a single topical application of 6.8 nmol TPA (Figure 2A, solid line). In contrast, there was a delayed epidermal proliferative response in Stat3-deficient mice with a significantly reduced peak (LI ∼30%) at about 24 hours after a single TPA treatment (Figure 2A, broken line). To analyze the underlying mechanisms contributing to the altered response of Stat3-deficient mice to TPA-induced DNA synthesis, we examined the levels of several critical regulatory proteins governing G1-to-S-phase cell cycle progression. In recent experiments, we found that topical treatment with diverse skin tumor promoters, including TPA, led to rapid activation of Stat3 (10). Immunohistochemical analyses showed an intense nuclear staining for tyrosine-phosphorylated Stat3 (PYStat3) 4 hours after TPA treatment in both basal and suprabasal keratinocytes (Figure 2B, lower left panel) in nontransgenic mice. In contrast, PYStat3 was not detectable in the epidermal keratinocytes of Stat3-deficient mice and was only detected in the dermal compartment (e.g., in fibroblasts and macrophages) after TPA treatment (Figure 2B, arrows). Western blot analyses confirmed this activation of Stat3 in epidermis of nontransgenic mice treated with TPA (Figure 2C). The Stat3 and PYStat3 protein detected in the epidermal preparations from Stat3-deficient mice by Western blotting (Figure 2C) was likely due to (a) the presence of other cells that reside in the epidermis, including Langerhans cells, and melanocytes; and/or (b) contamination from the dermis.
Analysis of cell cycle regulatory proteins in Stat3-deficient mice following treatment with TPA. (A–D) Groups of mice (n = 3) were treated with a single application of 6.8 nmol TPA and sacrificed at various times thereafter, as indicated. BrdU was injected 30 minutes prior to sacrifice. (A) Percentage of BrdU-positive epidermal basal cells in control (solid line) and Stat3-deficient (broken line) mice. (B) Immunohistochemical analysis of p-Stat3 localization in skin sections from Stat3-deficient mice (–/–) and control littermates (+/+) with (lower panels) or without (upper panels) TPA treatment. Positive staining for tyrosine-phosphorylated Stat3 in the nuclei of epidermal keratinocytes is shown by brown staining. Arrows, positive signals found in dermal cells such as fibroblasts and macrophages. Scale bar: 50 μm. (C) Western blot analyses of Stat3 and tyrosine-phosphorylated Stat3 protein levels in relation to levels of cyclin D1, cyclin E, and c-Myc at different time points in control and Stat3-deficient mouse epidermis following TPA treatment. (D) Semiquantitative analysis of the mRNA levels of cyclin D1 and c-Myc without treatment and 4 hours after TPA treatment. (E) Western blot analysis of Erk1/2 and phosphorylated Erk1/2 levels at different time points following TPA treatment in epidermis of control and Stat3-deficient mice.
Following TPA treatment, there was an initial drop in the levels of c-Myc, cyclin E, and cyclin D1 proteins (at 4 hours), followed by a recovery of these proteins to control or to slightly above control levels by 10–17 hours (Figure 2C). These data in control mice are similar to those reported by Rodriguez-Puebla et al. (12) and are consistent with the finding that cyclin D1–cdk4 and cyclin E–cdk2 complexes phosphorylate retinoblastoma protein (Rb), thus relieving its repression of E2F transcription factors and leading to activation of critical genes that drive cells from G1 into S phase (13). In epidermis of Stat3-deficient mice, we first noted reduced levels of cyclin D1, cyclin E, and c-Myc relative to those in epidermis from control mice (Figure 2C). Furthermore, following TPA treatment, the recovery of cyclin D1 and cyclin E protein levels was significantly delayed. Notably, c-Myc levels remained significantly reduced throughout the time course examined (Figure 2C). Semiquantitative RT-PCR analysis showed reduced c-Myc and cyclin D1 mRNA levels in epidermis of Stat3-deficient mice and a reduced induction of these mRNAs at 4 hours following TPA treatment (Figure 2D). These data are consistent with other data suggesting that c-Myc and cyclin D1 may be transcriptional targets of Stat3 (7). The delayed recovery of cyclin E in Stat3-deficient mice is most likely a direct effect of the delay in cyclin D1 expression. This conclusion is supported by the observation that knock-in of cyclin E into cyclin D1–deficient mice can rescue its phenotype (14). Collectively, the delayed recovery of cyclin D1 and cyclin E and the persistent downregulation of c-Myc are likely responsible for the delayed entry of Stat3-deficient epidermal cells into S phase following topical TPA treatment.
Next, we examined the impact of Stat3 deficiency on the MAPK pathway with and without tumor promoter treatment. The Erk-MAPK pathway has been implicated in regulating cyclin D1 expression and driving G1-to-S-phase cell cycle progression in murine keratinocytes (15). In Stat3-deficient mice, we found that the activity of Erk-MAPK remained intact in the epidermis, as shown by Western blot analysis of the phosphorylated form of Erk1/2 (Figure 2E). Following TPA treatment, the levels of phospho-Erk1/2 were increased in both control and Stat3-deficient mice at all the time points examined. However, the activation of Erk1/2 was enhanced in the Stat3-deficient mice treated with TPA. These data indicate that the delay in the recovery of cyclin D1 levels seen in Stat3-deficient mice treated with TPA is not due to a defect in the Erk-MAPK signaling pathway. Rather, enhanced Erk1/2 signaling could not compensate for the lack of Stat3 to drive entry from G1 to S phase following TPA treatment. Collectively, these data (Figure 2) suggest that Stat3 likely plays a critical role in the commitment to cell cycle progression in murine keratinocytes following treatment with TPA.
Stat3-deficient keratinocytes are more sensitive to DMBA-induced apoptosis. In mouse epidermis, the initiation of skin tumors occurs following treatment with a carcinogen such as DMBA. DMBA is metabolized to reactive diol-epoxides that covalently bind to DNA bases and induce mutations in certain target cells (16). Initiated cells are likely to have escaped the normal mechanisms (DNA repair, apoptosis) that provide protection against carcinogen-induced DNA damage and thus are available for selective clonal expansion during tumor promotion (17). To determine the effect of Stat3 deficiency on tumor initiation, we examined the survival of keratinocytes in response to DMBA treatment. In initial experiments using cultured keratinocytes in the absence of treatment, no significant differences were observed in the viability of Stat3-deficient keratinocytes compared with keratinocytes from nontransgenic littermates. In contrast, DMBA (30 nM) induced apoptosis in a significantly higher population of Stat3-deficient keratinocytes (Figure 3B) as compared with control keratinocytes (Figure 3A). Quantitation of DMBA-induced apoptosis in cultured keratinocytes is shown in Figure 3C. Western blot analyses of wild-type (control) keratinocytes did not reveal any significant changes in Stat3 protein levels or phosphotyrosine levels at any of the DMBA doses used (Supplemental Figure 1: supplemental material available at http://www.jci.org/cgi/content/full/114/5/720/DC1). In further experiments, topical application of DMBA to Stat3-deficient mice resulted in a significantly increased number of epidermal cells undergoing apoptosis compared with that in control mice, analyzed by both caspase-3–positive staining (Figure 3, D and E) and the presence of sunburn cells in H&E-stained sections (data not shown). Examination of the caspase-3–positive cells revealed that the majority of DMBA-induced apoptotic cells were located in a specific region of the hair follicles, with a smaller number appearing in the interfollicular epidermis (Figure 3D, arrows and arrowhead, respectively). The distribution of caspase-3–positive cells in the epidermis of nontransgenic littermates following DMBA treatment was similar to that observed in Stat3-deficient mice, although the total number was lower (Figure 3E).
Response of Stat3-deficient keratinocytes to DMBA-induced apoptosis both in vitro and in vivo. (A and B) Cultures of primary keratinocytes from (A) control mice and (B) Stat3-deficient mice treated with DMBA. Scale bar: 200 μm. (C) Percentage of apoptotic cells in control (white bars) and Stat3-deficient (black bars) keratinocytes treated with DMBA. *P < 0.05 by Mann-Whitney U test. (D) Caspase-3 staining of epidermis from Stat3-deficient mice. Note that most of the caspase-3–positive cells are located in a restricted region of the hair follicles (arrows), while a smaller number are located in the interfollicular epidermis (arrowhead). IFE, interfollicular epidermis; HF, hair follicle; D, dermis. Scale bar: 100 μm. (E) Number of caspase-3–postive cells per centimeter of epidermis in control (white bars) and Stat3-deficient mice (black bars). *P < 0.05 and **P < 0.01 by Mann-Whitney U test. (F) Double staining of label-retaining cells (BrdU; red) and caspase-3–positive cells (green) in skin of Stat3-deficient mice treated with DMBA (25 nmol) and sacrificed 24 hours later. Scale bar: 20 μm. Dotted lines mark the margins of hair follicles, and “b” indicates the bulge region of hair follicle.
Evidence has accumulated suggesting that keratinocyte stem cells reside primarily in the bulge region of the hair follicle, where they are physically protected by the epidermis (18, 19). To determine whether DMBA-induced apoptotic cells in Stat3-deficient mice were primarily localized in the bulge region, keratinocyte stem cells were pulse-chase labeled in vivo using BrdU as previously described (18) and then the location of these cells and the DMBA-induced apoptotic cells was examined. Staining for BrdU (red) to trace the “label-retaining keratinocytes” showed that they were localized primarily in the bulge region of the hair follicle, as expected (Figure 3F). Furthermore, the DMBA-induced apoptotic cells (caspase-3–positive cells, shown in green) were also located in this region, usually adjacent to label-retaining cells (Figure 3F, “b”). These findings provide evidence that Stat3 may be critical for maintaining the survival of keratinocyte stem cells following DNA damage induced by DMBA at the time of tumor initiation.
Stat3-deficient mice are completely resistant to skin tumor development. To further assess the role of Stat3 in multistage skin carcinogenesis, Stat3-deficient mice and nontransgenic littermates were treated with DMBA, followed by repetitive application of TPA. After 25 weeks of TPA treatment, Stat3-deficient mice did not develop any skin papillomas (Figure 4, A–C). At this point, over 90% of the nontransgenic littermates developed papillomas, with an average of 10 papillomas per mouse. These results were confirmed in a repeat experiment using a similar number of nontransgenic and Stat3-deficient mice (data not shown). Collectively, these data indicate a critical role for functional Stat3 protein in the development of skin tumors in mouse skin, using the DMBA-TPA, initiation-promotion protocol.
Responsiveness of Stat3-deficient mice to 2-stage carcinogenesis. (A–C) Groups of mice (n = 15) were treated with 25 nmol DMBA and, starting 2 weeks later, treated with twice-weekly applications of TPA (6.8 nmol) for the duration of the experiment. (A) Percentage of mice with papillomas. (B) Average number of papillomas per mouse. Circles, control mice; triangles, Stat3-deficient mice. (C) Representative photograph of Stat3-deficient mice and tumor-bearing control mice at the end of the experiment shown in A and B.
Stat3 is required for clonal expansion of initiated keratinocytes and maintenance of tumor cell growth. Further experiments were conducted to evaluate whether abrogation of Stat3 function inhibited growth of initiated keratinocytes and tumor cells harboring an activated Ha-ras gene. Keratinocytes transduced with v-Ha-ras were used as an in vitro model for initiated keratinocytes (20, 21). Treatment of v-Ha-ras keratinocytes with a Stat3 decoy oligonucleotide for 2 days in culture led to a marked reduction (70–90%) in cell number compared with those treated with a nonfunctional mutant control oligonucleotide (Figure 5A). Initial Western blot analysis demonstrated the presence of a significant level of PYStat3 protein in v-Ha-ras keratinocytes (data not shown). Further Western blot analyses demonstrated that Stat3 decoy significantly reduced the protein level of two Stat3 downstream targets in v-Ha-ras–transduced keratinocytes, cyclin D1 (6.7-fold reduction based on densitometry) and Bcl-xL (3.8-fold reduction) (Figure 5B). The mutant Stat3 oligonucleotide had no significant effects on the levels of cyclin D1 or Bcl-xL (Figure 5B). Stat3 protein was also downregulated (2.8-fold) by Stat3 decoy treatment (Figure 5B). A previous report has shown that Stat3 can regulate its own transcription (22). The reduced levels of Stat3, cyclin D1, and Bcl-xL proteins in Stat3 decoy–treated v-Ha-ras keratinocytes is consistent with inhibition of Stat3 function. Furthermore, these results suggested that Stat3 is required for growth of initiated keratinocytes, through its effects on both cell proliferation and survival.
Effects of a Stat3 decoy oligonucleotide on growth of initiated keratinocytes in vitro and in vivo. (A) Effect of vehicle control (TE), mutant oligonucleotide control, and Stat3 decoy oligonucleotide on growth of _v-Ha-ras_–transduced keratinocytes 48 hours after treatment. Scale bar: 100 μm. (B) Western blot analysis of Stat3, Bcl-xL, and cyclin D1 levels in v-Ha-ras keratinocytes treated with vehicle control (con), mutant decoy (mut), or Stat3 decoy (decoy) for 48 hours. (C) Effect of mutant oligonucleotide control (left 2 mice) and Stat3 decoy oligonucleotide (right 3 mice) on TPA-induced papilloma formation in TG.AC mice at 6 weeks and 13 weeks after first TPA treatment. Arrows indicate papillomas developing in TG.AC mice that received the mutant oligonucleotide together with TPA. (D) Immunohistochemical stain of PYStat3 in a representative section from a skin papilloma. Scale bar: 25 μm. (E) Immunohistochemical stain of Ki67 in a representative section of a skin papilloma. Scale bar: 25 μm. (F) Effect of mutant oligonucleotide and Stat3 decoy oligonucleotide on growth of primary skin papillomas after 2 weeks of treatment. Arrows, tumors injected with mutant oligonucleotide; arrowhead, tumor injected with Stat3 decoy.
To further substantiate a role for Stat3 in clonal expansion of initiated keratinocytes in vivo, we used TG.AC mice. TG.AC mice are transgenic mice that were generated using a ζ-globin promoter linked to a v-Ha-ras gene (23). These mice are an in vivo model for initiated keratinocytes, since topical TPA treatment promotes the development of papillomas (23). As shown in Figure 5C, treatment (as described in Methods) with Stat3 decoy inhibited the early development of papillomas promoted with TPA (6 weeks, upper panels), while the mutant decoy did not. At 6 weeks, no tumors were detected in skin of decoy-treated TG.AC mice, while papillomas were already present in skin of mice treated with mutant decoy oligonucleotide (Figure 5C, arrows). At 13 weeks of observation (Figure 5C, lower panel), there was a marked inhibition of papilloma development in the decoy-treated mice compared with the mutant decoy–treated mice.
We have recently reported that Stat3 is activated (as assessed by Western blot analysis for PYStat3) in skin papillomas and squamous cell carcinomas that develop during 2-stage carcinogenesis (10). Immunohistochemical analyses of papillomas from control littermates showed an intense PYStat3 staining in the nucleus of both basal and suprabasal cells (Figure 5D), which represent the proliferative compartment for these tumors as demonstrated by intense Ki67 staining (Figure 5E). Thus, Stat3 is activated in the proliferative compartment of these tumors. The functional importance of Stat3 in maintaining growth of papilloma cells was examined by injection of Stat3 decoy into primary tumors. Papilloma cells initiated in mouse skin using DMBA have an Ha-ras mutation in codon 61 (3). As shown in Figure 5F, direct injection of Stat3 decoy into primary skin papillomas led to a significant reduction in the volume compared with that of papillomas injected with the mutant decoy. Table 1 provides a summary of data from 13 papillomas injected with Stat3 decoy and 10 papillomas injected with mutant decoy. While not all papillomas injected with Stat3 decoy responded to this treatment protocol, approximately 50% of the papillomas injected with Stat3 decoy underwent significant reduction in size (>60%) compared with those treated with the mutant decoy. Collectively, the data presented in this section demonstrate that inhibition of Stat3 function inhibits the growth of initiated keratinocytes in mouse skin.
Effects of Stat3 decoy oligonucleotide and mutant oligonucleotide control on skin tumor volume