Epidermal Notch1 loss promotes skin tumorigenesis by impacting the stromal microenvironment

Abstract

Notch1 is a proto-oncogene in several organs. In the skin, however, Notch1 deletion leads to tumor formation, suggesting that Notch1 is a "tumor suppressor" within this context. Here we demonstrate that, unlike classical tumor suppressors, Notch1 loss in epidermal keratinocytes promotes tumorigenesis non-cell autonomously by impairing skin-barrier integrity and creating a wound-like microenvironment in the skin. Using mice with a chimeric pattern of Notch1 deletion, we determined that Notch1-expressing keratinocytes in this microenvironment readily formed papillomas, showing that Notch1 was insufficient to suppress this tumor-promoting effect. Accordingly, loss of other Notch paralogues that impaired the skin barrier also predisposed Notch1-expressing skin to tumorigenesis, demonstrating that the tumor-promoting effect of Notch1 loss involves a crosstalk between barrier-defective epidermis and its stroma.

Figure 1

Msx2-N1CKO mice develop skin tumors spontaneously and in response to chemical carcinogens. A) Spontaneous skin tumorigenesis of Msx2-N1CKO mice and their wild-type littermates (n=20 for each group; p <0.0001, log rank test). B) A representative Msx2-N1CKO mouse with 2 papillomas (left) and 1 BCC (right) at P593 (scale bar: 500μm). C) Schematic diagram outlining the standard DMBA/TPA treatment protocol used for skin carcinogenesis studies. Two days prior to treatment with DMBA (black arrowhead), the back skin of the animals are shaved to ensure the hair follicles are in telogen (rest) phase. D) DMBA/TPA-induced skin tumorigenesis of Msx2-N1CKO and their wild-type littermates (n=10 for each group; p <0.0001, log-rank test). E) The average number of papillomas per each tumor-bearing mutant mouse observed over a 25-week follow-up period. Beginning at 20 weeks post initiation, >50% of Msx2-N1CKO mice have more than 20 papillomas (*: p <0.05, student’s t-test). These findings are confirmed in additional independent experiments.

Figure 2

Epidermal deletion of Notch1 leads primarily to skin tumor promotion. A) An example of Msx2-N1CKO and wild-type skins with high doses of TPA (18μg per mouse, arrow) twice weekly for 25 weeks (scale bar: 100μm). B) Schematic diagram detailing the treatment protocols. Six- to 10-week-old K14ERT-N1CKO mice are treated with 4-OHT for five days at the indicated points (red arrowheads) to induce Notch1 deletion. C, D) Notch1 deletion was induced by 4-OHT injections beginning 4 days after DMBA-mediated tumor initiation (DOT) induces skin carcinogenesis to a similar level as does Notch1 deletion 9 days prior to DMBA treatment (ODT). Both Notch1-deficient cohorts (C) develop skin tumors earlier and (D) in significantly higher numbers than controls (p <0.05 for all tumor counts after week 9, student’s t-test). The ODT group has significantly more tumors than the DOT group at the last two time points (*: p =0.017, **: p =0.014, student’s t-test). E, F) Msx2-N1CKO mice treated topically with one dose of DMBA (150μg per mouse) develop skin tumors in the absence of TPA (E) starting at 10 weeks after DMBA treatment, and (F) gain a few papillomas per each tumor-bearing mouse. No papilloma is formed in DMBA-treated, wild-type littermates (n=10 for each group; p <0.0001, log-rank test). These data are confirmed in additional independent experiments.

Figure 3

Notch1 loss in the epidermis causes malignant progression of skin tumors. A) Schematic diagram outlining the method used to address the role of Notch1 in tumor progression. Notch1 deletion was induced after papilloma formation in K14CreERT background. B) Treatment of mice with 4-OHT starting after the last TPA application (arrow) does not impact the total number of tumors formed in the Notch1-deleted (DTO) or control animals (n=8 for each group). C) A significant fraction (~50%) of tumors in DTO cohort progress to SCC, distinguishable on hematoxylin and eosin (H&E)-stained histological section (scale bar: 500μm). In contrast, papillomas on Notch1-expressing control skin show no sign of malignant progression. Note that global loss of Notch1 results in hair loss phenotype, and that SCC cells in Notch1-deficient skin invade through the subcutaneous muscle layer (insets). D) Malignant skin cancer in DTO mice has metastatic features shown by the presence of AE1⁺ epidermal keratinocytes in a draining lymph node of a representative DTO animal (scale bar: 50μm). E) Due to their severe skin cancers, DTO mice become moribund and demise prematurely (p<0.0001, log-rank test).

Figure 4

Notch1-deletion in epidermal keratinocytes acts as a non-cell autonomous tumor promoter. A) The chimeric pattern of Notch1 deletion in Msx2-N1CKO animals enables us to determine if the tumor-promoting effect of Notch1 loss in the skin is the consequence of cell autonomous or non-cell autonomous changes. Notch1-expressing skin patches (white) in Msx2-N1CKO animals serve as an internal control to determine if Notch1-expressing initiated cells can form skin tumors in an environment modified by neighboring, Notch1-deficient cells. Considering that in this genetic background none of the DMBA/TPA-treated wild-type littermates developed tumors (Figure 1), tumor growth in white territory of Msx2-N1CKO skin would point to a non-cell autonomous tumor-promoting signal from the environment conditioned by Notch1-deleted (blue) cells. B) X-gal staining of representative Msx2-N1CKO;Rosa26R mouse treated with DMBA/TPA shows the presence of a significant number of completely white tumors (arrows) adjacent to Notch1-deleted (blue) skin territory. Note the comparable size and number of white and blue tumors, consistent with a dominant non-cell autonomous effect downstream of Notch1 loss that results in tumor promotion in wild-type cells. C) PCR analysis of DNA isolated from 8 randomly selected Msx2-N1CKO tumors from one individual confirms that Notch1 locus is intact (i.e. Notch1 is not deleted) in >99% of tumor-forming cells in a substantial number of tumors (Δ: deleted allele; M: molecular marker), demonstrating that these tumors have arisen from Notch1-expressing initiated cells (see Figure S4 for the sensitivity of the method).

Figure 5

Notch1-deficient epidermis creates a wound-like microenvironment in the skin. A) H&E staining of Msx2-N1CKO and wild-type (Wt) skin at birth (P0), P9 and 1 year of age demonstrates the progression from relatively hypoplastic epidermis in newborn Msx2-N1CKO animals to distinct epidermal hyperplasia at 1 year. The dermal hyperplasia, however, is evident as early as P9. The bars on the left side of each picture show the average thickness of epidermis (black) and dermis (green) across three 100X microscope fields (scale bar: 50μm). B) X-gal staining of 1-year-old Msx2-N1CKO skin shows similar degree of hyperplasia (brackets) in Notch1-expressing epidermis (red) and in the Notch1-deleted territory (blue), consistent with the presence of a dominant proliferative microenvironment in Msx2-N1CKO skin. Immunohistochemical staining for the V1744 epitope (detecting activated Notch1) confirms the presence of Notch1 activity in hyperplastic epidermis adjacent to the Notch1-deficient epidermis (scale bar: 50μm). C) Leukocyte infiltration into the dermis of Msx2-N1CKO is detectable as early as P9 using CD45 pan-leukocyte marker (average number of CD45⁺ leukocytes in 6 randomly selected 200X microscope fields is tabulated; scale bar: 50μm). D) Flow cytometry (FC) and cell counting methods are used (Figure S9) to calculate the fold increase of dermal immune cells in 6- to 8-month-old Msx2-N1CKO animals relative to their wild-type controls (*: p <0.0001, student’s t-test). E, F) Treating Msx2-N1CKO;DPPI−/− (n=13), Msx2-N1CKO (n=14), DPPI−/− (n=12) and wild-type (Notch1^flox/flox; DPPI^+/−; n=12) littermates with DMBA/TPA excludes a dominant tumor-promoting role for mast cells in Notch1-deficient skin. Notch1-deficient mice are significantly more prone to DMBA/TPA-induced tumor growth than their Notch1-expressing counterparts (p <0.0001, log-rank test), and no major difference was detected between Msx2-N1CKO;DPPI−/− and Msx2-N1CKO animals with regard to (E) time to tumor onset (p =0.31, log-rank test) or (F) average number of tumors per each tumor-bearing mouse (p =0.12 at week 25 after initiation, student’s t-test). G) Double staining of skin with α-SMA and an endothelial marker (MECA-32) demonstrates the abundance of myofibroblasts in adult Notch1-deficient skin (asterisk). Note the expansion of microvasculature in Notch1-deficient skin as early as P9 (arrows). The arrowheads point to α-SMA-expressing arrector pili muscles (scale bar: 50μm). H) The number of dermal fibroblasts is significantly increased in Notch1-deficient skin. Dermal fibroblasts are counted in 6 random 200X microscope fields, and the average ratio of Msx2-N1CKO to wild-type fibroblast counts at P9 and 1 year of age is presented (*: p <0.001 compared to wild type, student’s t-test). I) Dermal fibroblasts in Notch1-deficient skin overexpress two major keratinocyte proliferative signals, SDF-1 and KGF, which are detected by qRT-PCR on total Skin mRNA samples (but not in epidermal mRNA, data not shown) from P9 Msx2-N1CKO and wild-type mice (*: p <0.001 compared to wild type, student’s t-test). J) Dermal view of dorsal skin shows expanded vascular network in 1-year-old Msx2-N1CKO skin compared to a wild-type littermate (scale bar: 1cm).

Figure 6

Stepwise removal of Notch paralogs in Msx2-N1CKO animals leads to progressively worse epidermal hyperplasia that accelerates the onset of spontaneous skin carcinogenesis. A) Skin histology of compound Notch-deficient mice at P9 demonstrates progressive epidermal hyperplasia, hyperkeratosis, and dermal hyperplasia as more Notch alleles are deleted. Note the disorganized epidermal cells with atypia in Notch pathway-deficient skin (i.e. Msx2-N1N2N3CKO and Msx2-PSDCKO) (scale bar: 50μm). B) Although Msx2-N1CKO epidermis appears relatively normal at P9, Msx2-PSDCKO skin that lacks all Notch signaling shows severely hyperplastic epidermis, which overexpresses keratin 14 in suprabasal keratinocytes (marking epidermal hyperplasia) and expresses keratin 6 (marking epidermal dysplasia). Note that Msx2-PSDCKO skin is severely hypoplastic at birth (Figure S5; (Demehri et al., 2008)); Msx2-PSDCKO skin has thus undergone an acute reactive hyperplasia in 9 days to repair its severe skin-barrier defect (scale bar: 50μm). C) Progressive reduction in Notch signal dosage reduces the life span (Demehri et al., 2008) and time to spontaneous tumor onset. Lifelong monitoring of 10 to 20 mice for each genotype reveals that severity of skin pathology (shown in “A”) correlates inversely with lifespan, time to tumor onset, and tumor penetrance (i.e., the percentage of animals with a given genotype that developed tumors) in compound Notch-deficient mice. Significant differences in average time to tumor onset between adjacent genotypes are marked by brackets. Although the shorting in life span for most genotypes is due directly to their intrinsic skin phenotype including exfoliation, bleeding, and infection, Msx2-N1N2CKO, Msx2-N1N2N3CKO and Msx2-PSDCKO animals die shortly after birth due to a lethal blood disorder (Demehri et al., 2008). Note that Msx2-N2CKO and Notch3^−/− (N3KO) mice have normal skin and behave like wild type. Abbreviations: Msx2-Cre/+; Notch1^flox/flox (Msx2-N1CKO), Msx2-Cre/+; Notch1^flox/flox; Notch2^flox/+ (Msx2-N1N2hCKO), Msx2-Cre/+; Notch1^flox/flox; Notch2^flox/+; Notch3^+/− (Msx2-N1N2hN3hCKO), Msx2-Cre/+; Notch1^flox/flox; Notch2^flox/+; Notch3^−/−(Msx2-N1N2hN3CKO), Msx2-Cre/+; Notch1^flox/flox; Notch2^flox/flox (Msx2-N1N2CKO), Msx2-Cre/+; Notch1^flox/flox; Notch2^flox/flox; Notch3^−/− (Msx2-N1N2N3CKO), Msx2-Cre/+; RBP-j^flox/flox (Msx2-RBP-jCKO), and Msx2-Cre/+; PS1^flox/flox; PS2^−/− (Msx2-PSDCKO).

Figure 7

Mice lacking Notch2 and Notch3 in the skin develop skin-barrier defect, epidermal hyperplasia and skin tumors. A) TSLP overexpression (a biomarker for postnatal skin-barrier defects (Demehri et al., 2008)) is seen in both Notch1-deficient and Notch2/3-deficient epidermis. mRNA for qRT-PCR analysis is isolated from epidermis of P9 Msx2-N1CKO, Msx2-N2N3CKO and their wild-type littermates. p21^wAF1/Cip1 expression (a direct target of Notch1), which is reduced in Notch1-deficient epidermis and thought to be essential for tumor formation in Notch1-deficient skin (Nicolas et al., 2003), is not altered in Msx2-N2N3CKO epidermis. However, TSLP mRNA level is highly elevated in both Msx2-N1CKO and Msx2-N2N3CKO relative to their wild-type controls (*: p <0.001, **: p <0.0001, student’s t-test). B) Serum TSLP levels remain elevated in adult (6 to 8 months old) Msx2-N2N3CKO animals implying the persistence of defective skin-barrier in these mice (*: p <0.01, student’s t-test). C) The presence of a mild allergic inflammation responding to skin-barrier defects in adult Msx2-N2N3CKO mice is evident based on their elevated serum IgE levels (n=4 for each group; *: p <0.01, student’s t-test). D) H&E staining of Msx2-Cre; Notch2^flox/flox; Notch3^−/− (Msx2-N2N3CKO) and wild-type skin at P0, P9 and 1 year of age shows a Msx2-N1CKO-like pattern of dermal hyperproliferation starting at P9 and subsequent epidermal hyperplasia in 1-year-old mutant skin. The bars on the left side of each picture show the average thickness of epidermis (black) and dermis (green) across three 100X microscope fields. Keratin 6 (K6) and keratin 14 (K14) immunofluorescence staining shows distinct epidermal hyperplasia with dysplastic changes in 1-year-old Msx2-N2N3CKO skin (scale bar: 50μm). E) While none of Notch2^flox/flox; Notch3^−/− (N3KO) or wild-type controls develop any skin tumor, Msx2-N2N3CKO mice develop spontaneous skin tumors overtime (n=4; p <0.0001, log rank test). F) A representative Msx2-N2N3CKO mouse (P418) is shown to demonstrate that the spontaneous tumors in Msx2-N2N3CKO skin are benign papillomas; H&E-stained Msx2-N2N3CKO papilloma is also examined using K6 and K14 immunostaining (lower panel; scale bar: 200μm). G) Treating Msx2-N2N3CKO and N3KO littermates with DMBA/TPA results in tumor formation in all Msx2-N2N3CKO mice (n=14). In contrast, only 3 out 16 DMBA/TPA-treated N3KO animals develop tumors (p <0.0001, log-rank test). H) The average number of tumors per each tumor-bearing Msx2-N2N3CKO mouse is significantly higher than that among N3KO controls over the 25-week experimental period (p <0.05 for all tumor counts after week 8, student’s t-test). I) Analysis of all tumors present at week 25 after initiation show that N3KO control mice have only developed papillomas. In contrast, the majority of skin tumors in DMBA/TPA-treated Msx2-N2N3CKO skin progress to SCC as confirmed by histological examination.