Loss of TGFbeta signaling destabilizes homeostasis and promotes squamous cell carcinomas in stratified epithelia

Abstract

Although TGFbeta is a potent inhibitor of proliferation, epithelia lacking the essential receptor (TbetaRII) for TGFbeta signaling display normal tissue homeostasis. By studying asymptomatic TbetaRII-deficient stratified epithelia, we show that tissue homeostasis is maintained by balancing hyperproliferation with elevated apoptosis. Moreover, rectal and genital epithelia, which are naturally proliferative, develop spontaneous squamous cell carcinomas with age when TbetaRII is absent. This progression is associated with a reduction in apoptosis and can be accelerated in phenotypically normal epidermis by oncogenic mutations in Ras. We show that TbetaRII deficiency leads to enhanced keratinocyte motility and integrin-FAK-Src signaling. Together, these mechanisms provide a molecular framework to account for many of the characteristics of TbetaRII-deficient invasive SQCCs.

Figure 1. Adult TβRII cKO Mice Develop Spontaneous Anogenital SQCCs

(A) Efficient targeting and loss of TβRII mRNA was assessed by real-time PCR using primer pairs corresponding to the floxed exon 4 of TβRII. MK, primary keratinocytes. Mean ± SD. (B) Anal and vaginal tumors were visible in 6-month-old cKO, but not WT mice. (C) Kaplan-Meier curves depicting the probability of tumor-free survival with age. M, male; F, female. Note that >89% of cKO mice developed anogenital tumors within 7 months, while WT and heterozygous (Het) animals were tumor-free. (D) Hematoxylin- and eosinstained longitudinal sections of the distal colon, rectal, and anal/perianal tissues revealed classical SQCC pathology in 7-month-old cKO, but not in WT mice. SQCCs displayed anastomosing trabeculae and nests of keratinized squamous cells organized around central cavities, and were associated with moderate, multifocal, and chronic lymphoplasmacytic inflammation around the tumor masses.

Figure 2. Diminished TGFβ Signaling in Human Genital SQCCs

Male (B) and female (D) human genital SQCCs were scored according to their grade of severity (I-lowest to III-highest) and analyzed for TβRII, phosphorylated (activated) Smad2, TGFβ1 and/or K14 by immunohistochemistry (IHC). Examples of IHC staining from a male genital SQCC grade I (A) and from a female SQCC grade II (C) show reduced or no anti-TβRII, in contrast to perilesional, matched control skins. TβRII loss correlated with a lack of p-Smad2 staining in 10 out of 10 SQCC samples. TGFβ1 and K14 served as internal quality controls and were detected in TβRII-deficient human samples. Scoring: (+), positive staining; (−), negative staining; (low), reduced expression. Epi, epidermis; Der, dermis; Str, stroma. Lines encircle SQCCs in the top panels.

Figure 3. Tissue Homeostasis Is Impaired in the Anal Canal of Older TβRII cKO Mice

(A) The anal canal epithelium is a transitional epithelium bordering stratified squamous epithelium of anal skin and simple epithelium of large intestine. Immunofluorescence microscopy of frozen tissue sections revealed that this epithelium naturally expresses many markers of a hyperproliferative state, including K17, K6, β6-integrin (β6), and tenascin C (TnC). β4-integrin (β4) or K5 label the basal-like cells of the anal canal. Epithelial-stromal border is denoted by white lines. DAPI (blue) labels nuclear chromatin. Str, stroma. (B) (Ba) Inflammatory Macrophages (Mac1) are prevalent in the stroma underlying the anal epithelium. (Bb) Mac1-positive cells are much more prevalent in the anal region (Anal) than in backskin epidermis (Epi) in both WT and cKO mice. (C–F) TβRII null anal epithelium exhibits sustained hyperproliferation but maintains homeostasis through enhanced apoptosis, which is lost in spontaneous tumors. (Ca–E) Proliferation was assayed by BrdU incorporation and correlates with pronounced Ras/p-MAPK staining. (Fa) Apoptosis was assayed by immunostaining for activated caspase 3 (Ac-casp3) (arrows). (Cb, Db, and Fb) Quantification of proliferation and apoptosis in WT and cKO anal canal and adjacent anal epidermis at 7 weeks when the cKO anal canal is histologically normal, and 7 months when it appears hyperplastic. Accelerated proliferation is balanced by increased apoptosis when homeostasis is maintained, while alleviated apoptosis correlated with tumor formation. Graphs represent the mean (±SD) of 3 different WT and cKO mice. *p < 0.05.

Figure 4. TGFβ and Ras-MAPK Signaling Also Cooperate to Control Tissue Homeostasis in the Epidermis

(A) Proliferation and apoptosis rates are increased in the stem cell compartment (basal layer) of TβRII null epidermis (Epi) (α6+CD34+), but not in the stem cell compartment (bulge) of the hair follicle (α6+CD34+). Analyses were performed on adult mice (7 wk). Graphs represent the mean (±SD) of 3 different WT and cKO mice. *p < 0.05. (B) Schematic of tumor susceptibility assay where primary keratinocytes (MKs) from WT or cKO backskins were isolated, infected with a retrovirus expressing constitutively active (oncogenic) Ha-Ras, and grafted together with WT dermal fibroblasts (DF) onto the backs of Nude mice. HF, Hair follicle. (C) At 23 d, papillomas formed from Ha-Ras MKs, while invasive SQCCs formed from TβRII null/Ha-Ras MKs. Note necrosis in center of large tumor. (D) Hematoxylin- and eosin-stained tissue sections revealed papilloma (Pap) pathology in Ha-Ras graft and SQCC pathology in TβRII null/Ha-Ras graft. (E) Ultrastructural analysis indicated a compromised basal lamina underlying Ha-Ras transformed, TβRII null SQCCs (right frame). Scarcity of hemidesmosomes (Hd) and perturbed basal lamina (BL) is typical of invasive SQCCs. The basal lamina appeared uncompromised at the boundary between Epi and dermis (Der) in adjacent nude skin (left frame). (F) TβRII null/Ha-Ras-induced SQCCs exhibited sustained hyperproliferation without enhanced apoptosis. Graphs represent the mean (±SD) of 3 different WT and cKO mice. *p < 0.05. (G) TβRII null MKs fail to activate Smad2 and remain resistant to a G1 growth arrest when stimulated with TGFβs as judged by growth curves and cell cycle profiles. (H and I) MKs were cultured in low Ca²⁺ medium in the presence or absence of 5 ng/ml TGFβ1, added at day 2. Growth curves (H) indicate the average value of 3 independent experiments performed in triplicate (±SD). (Note: by this assay and Elisas, active TGFβs are low/absent in epidermal culture medium.) (I) Cell cycle analyses with propidium iodide staining. (J) FACS analysis for the presence of the apoptotic marker annexin-V revealed that Ha-Ras and TGFβ signaling cooperate to induce apoptosis in WT MKs, while apoptosis rates remain low and unaltered in KO MKs under these conditions. TNFα (100 ng/ml) and cycloheximide (5 μg/ml) induced apoptosis in WT MKs served as a positive control. Bar graphs indicate mean value (±SD) on duplicate experiments. (K) Keratin 17 (K17), Tenascin C (Tnc), and β6-integrin are expressed in both Ha-Ras papillomas and TβRII null/Ha-Ras SQCCs. β4-integrin marks the basal cell layer and Macrophages (Mac1) are recruited to the tumor stroma. Note similarities between the expression patterns in Ha-Ras papillomas (here) and WT anogenital epithelia (Figure 3).

Figure 5. Invasive Metastatic Skin Tumors Correlate with Increased Integrin, FAK, Src, and MAPK Activities

(A) DMBA treatment of mice revealed a marked increase in DMBA-induced tumor formation in the absence of TβRII. (B) Lung metastasis from TβRII-cKO mouse topically treated with DMBA to induce backskin SQCCs. Note typical keratinized SQCC histology as revealed by hematoxylin and eosin staining and K14 immunohistochemistry. (C–F) Anti-FAK and p-FAK immunohistochemistry on sections from WT anal canal (C), cKO pretumor and tumor stage of anogenital skin (D and E), and cKO-Ras backskin SQCC (F). Str, stroma; Epi, epidermis; Der, dermis. (G–I) Immunoblots of protein lysates from MKs grown in the presence or absence of serum, ±4 min serum stimulation where indicated. Note: Src activity was assessed by immunoprecipitation/immunoblot analysis using Src and pY417-Src antibodies. (J–M) Immunofluorescence with antibodies against β1- and active β1-integrin (Ac-β1) performed on tissue sections as indicated. (N) Quantitative FACS analysis of KO (red) versus WT (blue) cultured MKs with antibodies against the cell surface integrins indicated. Gray lines depict secondary antibody-only control.

Figure 6. Wound Closure Is Accelerated in cKO versus WT Animals

(A) Shown are representative examples at 0, 3, and 7 days after wounding. (B and C) Immunohistochemistry reveals activated FAK and MAPK at the epidermal wound edge by day 3 after wounding. Note hyperthickening of wounded cKO skin. Epi, epidermis; Der, dermis. (D and E) Quantification of proliferation and apoptosis inside and outside the wound area. Bar graphs depict mean ± SD. *p < 0.05. (F) WT and cKO skin explant outgrowth in serum-containing media indicate accelerated epidermal outgrowth from cKO skin explants, a difference that becomes even more pronounced upon addition of active TGFβ (5 ng/ml) to the culture medium on day 2. Graph indicates the mean value of three independent experiments (±SD). *p < 0.03. Phase contrast images show extent of outgrowth at 4 days in the absence of active TGFβ .

Figure 7. Elevated Cell Motility in the Absence of TbRII

(A and B) Elevated cell motility in the absence of TβRII. Transwell migration assays were carried out on Boyden chambers ± coating with Matrigel ECM on the top chamber and in the absence and presence of fibroblast-conditioned medium, used as stimulus in the bottom chamber. Bar graphs depict means of 3 independent experiments ± SD. *p < 0.03. **p < 0.004. Note that KO MKs showed elevated migration and invasion over WT MKs, and Ha-Ras transformation enhanced these effects. (C and D) Rescue experiment to show that the enhanced migratory ability of KO MKs is specifically attributable to TβRII loss. The CMV promoter was used to drive TβRII cDNA, which restored cell velocities to WT levels (C). Immunoblot shows that re-expression of TβRII in KO MKs restores TGFβ2 responsiveness (D). (E) TGFβ1 treatment has only a slight effect on the velocity of WT MKs, which are still significantly less motile than KO MKs. (F and G) Enhanced MAPK (Erk) and Src/FAK activities promote cell motility in KO MKs. (F) Cell velocities were measured ± 50 μM of the MEK1 inhibitor PD98059 and ± 5 μM of the Src inhibitor PP2, which also inhibits FAK activity. (C, E, and F) Thirty cells were tracked by phase-contrast microscopy with an acquisition rate of 1 frame/min over 12 hr. Bar graphs indicate average velocity ± SEM. *p < 0.05. **p < 0.0001. (G) Scatter plots depict the migration tracks of 5 representative cells for each condition.