Multifocal epithelial tumors and field cancerization from loss of mesenchymal CSL signaling

Abstract

It is currently unclear whether tissue changes surrounding multifocal epithelial tumors are a cause or consequence of cancer. Here, we provide evidence that loss of mesenchymal Notch/CSL signaling causes tissue alterations, including stromal atrophy and inflammation, which precede and are potent triggers for epithelial tumors. Mice carrying a mesenchymal-specific deletion of CSL/RBP-Jκ, a key Notch effector, exhibit spontaneous multifocal keratinocyte tumors that develop after dermal atrophy and inflammation. CSL-deficient dermal fibroblasts promote increased tumor cell proliferation through upregulation of c-Jun and c-Fos expression and consequently higher levels of diffusible growth factors, inflammatory cytokines, and matrix-remodeling enzymes. In human skin samples, stromal fields adjacent to multifocal premalignant actinic keratosis lesions exhibit decreased Notch/CSL signaling and associated molecular changes. Importantly, these changes in gene expression are also induced by UVA, a known environmental cause of cutaneous field cancerization and skin cancer.

Figure 1. Mesenchymal CSL/RBP-Jκ deletion results in spontaneous skin tumor formation

(A) Overview of multiple spontaneous skin tumors in a mouse with mesenchymal RBP-Jκ deletion (ColI-Cre x RBP-Jκ^loxP/loxP) at 4 months. (B) Low and high magnification histological images of spontaneous tumors and surrounding skin in 4 months old mice. Tumors at this stage exhibited features of in situ carcinomas of keratoacanthoma and/or Bowen’s disease type, with disrupted epidermal architecture, parakeratotic nuclei and foci of aberrant keratinization. Immunohistochemical analysis of tumor stromal component is shown in Fig. S1A, D. (C) Number of tumor-bearing mice with mesenchymal RBP-Jk deletion (−/−) versus littermate controls (+/+) until 18 weeks after birth or earlier, if mice had to be sacrificed due to tumor burden. Fraction of males and females was similar, with no differences in tumor formation. (D) Number of independent (physically separated) tumors in mutant mice. Shown are average tumor numbers per mouse per time point, as well as variation among individual mice. Values may be underestimated, due to coalescence of individual lesions. (E) Histology of skin tumors in mice at 6 months of age. Higher magnification analysis showed loss of epidermal architecture, pronounced cellular atypia and inflammatory infiltration (Fig. S1B). Lesions with similar characteristics were found in 7 of 7 mice that survived until this age. Tumors were classified as well to moderately differentiated SCCs (Grade I-II). (F) Immunohistochemical analysis with anti-Pankeratin antibodies showing invading epithelial islands and tumor front in a 6 months old mouse. Quantification of results is provided in Fig. S1C. (G) Summary of CGH DNA analysis of skin tumors versus unrelated normal tissue (brain) from the same mice. For each tumors are listed specific chromosomal regions that were duplicated (+), deleted (−) or, for pairs of tumors derived from the same mouse, unaffected (N). Raw CGH data and statistical significance are provided in Table S1A. Statistical permutation tests showed that the repeated identification of 4 overlapping rearrangements in 5 mice (as found, for instance, for 7qC and 8qA) is highly significant (P = 0.0005). (H) Upper : Map of mouse chromosomal 7qC region and overlapping segments of duplication (green bars) in the indicated multiple tumors. Maps of other chromosomal regions with overlapping alterations in multiple tumors are provided in Table S1A. Lower: qPCR analysis of tumor and normal DNA samples utilized for CGH was performed with primers for three sites within the 7qC region (indicated by arrows in the previous panel). Results are in arbitrary units after internal normalization for an unrelated chromosomal region (15qB3.1). Similar results were obtained after normalization for three other chromosomal regions (5qC1 and 13qB1, data not shown). Further qPCR analysis showed duplications of the 7qC and 8qA regions in 4 and 5 additional tumors, respectively, out of 6 that were analyzed, from 3 more mice (Table S1B). (I) and (J), qRT-PCR analysis of LCM samples from tumors versus flanking normal epidermis (identified by double immunofluorescence with anti-K14 and -Tenascin C antibodies, as shown in Fig. S1D) from two mice, two tumors per mouse. Expression of indicated genes is in arbitrary units utilizing ß-Actin for normalization. Analysis of other genes is in Fig. S1F. *: p<0.01 by one way ANOVA followed by Bonferroni’s test. Scale bars: 200μm for B and D; 40μm for C and E.

Figure. 2. Early skin defects in mice with mesenchymal CSL/RBP-Jκ deletion

(A) Dermal thickness (distance between dermal-epidermal junction and panniculum carnosum; double end arrows) was quantified in five RBP-Jκ mutant mice and five controls (black and white bars) at 4 days and 3 weeks of age (* p<0.01). Histological images are in Fig. S2A. (B) Verhoeff-Masson-trichrome staining and quantification of elastic fibers (x10⁵/mm²) in back skin dermis of two P4 mice per genotype, counting 3 independent fields for each section (* p<0.01). (C) Masson-trichrome staining of total collagen in P4 mouse skin (blue) with Nuclear Fast Red for counterstaining. Images are representative of four mice per genotype. Collagen I and III immunostaining is in Fig. S2B. (D) Biochemical quantification of total collagen and ratio of soluble versus insoluble fractions in 3 weeks old mice. 5 mice per genotype were analyzed. (* p<0.01). (E) In situ zymography of P4 mouse skin with MMP3 and MMP13 preferential substrates. Little signal was observed in presence of MMP inhibitor GM6001 (not shown). MMP activity in epidermis and hair follicles is likely due to diffusion, consistent with MMP3 and MMP13 mRNA levels being increased selectively in fibroblast compartment of RBP-Jκ mutant mice (Fig. S4A and data not shown). Increased MMP3 and MMP13 protein expression in skin of mutant mice was also observed by immunohistochemistry (not shown). (F) P0 mice were injected with BrdU, followed by BrdU labeling determination in interfollicular epidermis. Four mice per genotype were analyzed (* p<0.01). (G) Immunofluorescence of back skin sections of E16.5 and E18.5 embryos and P0 mice with antibodies recognizing the activated phospho-Tyr 653/654 form of FGFR1 and, to a less extent, other FGF receptors. Images are representative of 3-4 independent fields, 2 mice per genotype. (H) Immunofluorescence signal intensity in epidermal compartment of above skin samples by computer-assisted quantification (* < 0.01). (I) Immunofluorescence of back skin sections of E16.5 and E18.5 embryos and P0 mice with antibodies against Loricrin. Images are representative of 3-4 independent fields, 2 mice per genotype. (J) Epidermal compartment of E16.5 and E18.5 embryos and P0 mice was analyzed by LCM followed and RT-PCR arrays for cytokines and growth factors. Results are shown as heat map fold changes of expression of indicated genes in mutant versus control samples. (K) Immunofluorescence analysis of P6 and P10 mouse back skin, illustrating foci of leukocyte infiltration (CD45 positive) and Keratin 6 (K6) and Tenascin C expression. Parallel immunofluorescence with anti-K6 and F4/80 antibodies illustrated hyperplastic epithelium and infiltrating macrophages, respectively. (L) Quantification of focal lesion size (CD45 positive areas demarked by arrows in the previous panel) showing a time-dependent increase. Large skin sections, 5 lesions per mouse, two mice per time point, were analyzed (*<0.01). Scale bars: 20μm for A, C, G and I; 200 μm for E and K.

Figure 3. Expanding multifocal skin lesions and counteracting effects of anti-inflammatory treatment as detected by Fluorescence Diagnosis (FD)

(A) Mice at indicated times after birth were topically treated with 5-ALA, followed by fluorescent light excitation and digital image acquisition, using same exposure conditions for age-matched mutant (−/−) and control (+/+) animals. FD signal intensity (x1e8 photons/sec/cm²/sr) was calculated as in (Troy et al., 2004) (* p<0.01). (B) Intense FD positivity in mouse mutant back skin at > 4 weeks of age corresponds to multifocal areas of tumor development (areas of macroscopic pictures marked by dotted lines). (C) Mice with mesenchymal RBP-Jκ deletion were treated, starting at birth, with the COX2 inhibitor Celebrex or vehicle alone (intraperitoneal injections, 20μg per g body weight, in DMSO, twice weekly, 5 mice per group), followed by FD analysis at indicated times. (D) Quantification of FD signal intensity and body surface with high FD signal (above a threshold of 2e7 photons/sec/cm²/sr for mice up to 6 days old and 1.5e8 photons/sec/cm²/sr for older mice) for each mouse (*<0.01). For macroscopic and histological images of mice at the end of the experiment, see Fig. S3A.

Figure 4. RBP-Jκ deficient dermal fibroblasts enhance growth/tumorigenic behavior of SCC-derived and normal primary keratinocytes

(A) Weakly tumorigenic mouse (Pam212) or human (SCC13) keratinocytes were admixed with cultured (second passage) dermal fibroblasts from mice plus/minus RBP-Jκ deletion prior to injection at the dermal-epidermal junction of NMRI athymic mice. Mice were sacrificed for tumour weight determination either 1 or 8 weeks later. Weight difference between tumor pairs in each mouse and between the two groups of tumors was highly significant (* p<0.01). (B) Mice as in previous panel were BrdU labeled for 2 hours prior to sacrifice, followed by determination of BrdU labeling of keratinocyte tumor cells (identified by Keratin 14 staining) in presence of control versus RBP-Jκ −/− fibroblasts (* p<0.01). (C) Histological analysis of tumors formed by SCC13 cells admixed with dermal fibroblasts plus/minus RBP-Jκ deletion 8 weeks after injection. Shown are low and corresponding high magnification images (left and right panels, boxed areas). (D-G) Immunofluorescence analysis of the indicated proteins in tumors formed by SCC13 cells admixed with fibroblasts plus/minus RBP-Jκ deletion. Besides the epithelial-mesenchymal border, basement membrane of underlying blood vessels (BV) is also positive for Laminin staining (E). Dotted lines mark the epithelial-mesenchymal junction. Nuclei were visualized by DAPI staining. (H) Histological images of epidermal islands formed by HKCs admixed with control versus RBP-Jκ −/− fibroblasts 1 week after intra-dermal mouse injection. Note granular layer differentiation features in epidermal islands formed in presence of control but not mutant fibroblasts. (I) Mice as in previous panel were BrdU labeled for 2 hours prior to sacrifice, followed by determination of BrdU labeling of keratinocytes (identified by Keratin 14 staining) in the presence of control versus mutant fibroblasts (* p<0.01). (J-M) Immunofluorescence analysis of epidermal islands formed by HKCs together with control versus mutant fibroblasts with antibodies against indicated proteins. Scale bars: 40μm for C-F, L and M; 20μm for G, H, J and K.

Figure 5. Dermal RBP-Jκ regulates tumor growth through AP-1

(A) Differential expression of AP-1 family members in dermal fibroblasts freshly isolated from a pool of three newborn mice (P0) plus/minus RBP-Jκ deletion. qRTPCR results are in arbitrary values after normalization for β-Actin (* p<0.01). (B) Immunoblot analysis of c-Jun and c-Fos expression in freshly isolated dermal fibroblasts from P0 mice. Densitometric scanning of autoradiographs with normalization for γ-Tubulin showed, respectively, a 2 and 1.5 fold increase of c-Jun and c-Fos expression in mutant versus control cells. (C) Immunofluorescence analysis of c-Jun (red) expression in back skin of P0 mice. Dermal fibroblasts were identified by co-staining with anti-Vimentin (green) antibodies. Dotted lines mark dermal-epidermal borders. Additional immunofluorescence images with antibodies against c-Jun and phospho-c-Jun and results quantification are provided in Fig. S5A,B. (D) Expression of indicated genes in dermal fibroblasts with RBP-Jκ deletion plus/minus siRNA-mediated c-Jun knockdown, in parallel with similarly cultured control fibroblasts (black and white bars, respectively). qRT-PCR results are expressed in arbitrary units with β-Actin for normalization (* p<0.01). Similar results with a second set of siRNA against c-Jun, and siRNAs against c-Fos are shown in Fig. S5C,D. Functional consequences of knock-down of other AP1 family members are shown in Fig. S5E. (E) Immunofluorescence analysis of c-Jun expression in tumors formed 1 week after intradermal injection of SCC13 cells admixed with RBP-Jκ deficient dermal fibroblasts transfected with siRNA against c-Jun or scrambled siRNA control. Dermal fibroblasts were identified by co-staining with anti-Vimentin antibodies. Tumor-stromal border is indicated by dotted lines. Similar results were obtained by immunofluorescence analysis of the other tumors formed in presence of dermal fibroblasts plus/minus c-Jun knock-down, and are consistent with biochemical analysis of cultured cells, showing persistent knockdown effects for at least one week after siRNA transfection. (F) Histological analysis of tumors formed by SCC13 cells admixed with RBP-Jκ deficient fibroblasts plus/minus c-Jun knock-down as in the previous panel. To minimize individual animal variations, mice were injected in parallel with the two combinations of cells. Shown are low and corresponding high magnification images (left and right panels, boxed areas). In the tumor formed by SCC13 cells admixed with RBP-Jκ −/− fibroblasts with c-Jun knock-down, pronounced apoptosis and necrosis of cells in the more central areas resulted in tissue loss - and an empty space -at the time of cutting. Similar findings were obtained with other 4 tumor pairs as shown in Fig. S5F-I, and with 5 additional tumor pairs from an independent experiment (not shown). (G) Mice as in (E,F) were BrdU labeled for 2 hrs prior to sacrifice. BrdU labeling of tumor keratinocytes (identified by Keratin 14 staining) admixed with RBP-Jκ deficient fibroblasts plus/minus c-Jun knockdown (black and white bars, respectively) was determined in parallel with tumor weight (* p<0.01). (H-J) Same samples were analyzed by immunostaining for Keratin 1 (H) and Tenascin C (I) and in situ zymography with a preferential MMP13 substrate (J). Asterisks indicate the epithelial tumor compartment. Scale bars: 20μm for C, E, F (right two panels), H-J; 200μm for F (left two panels).

Figure 6. Tumor-enhancing phenotype of human dermal fibroblasts with CSL knock-down

(A) Human dermal fibroblasts (HDFs) were infected with two anti-CSL shRNA lentiviral vectors (#1 and 2; black bars) in parallel with empty vector control (white bar), followed by 6 days selection for puromycin resistance. Cells were analyzed by qRT-PCR for indicated genes, using 36β4 for normalization(*p<0.01). Analysis of an independent strain of HDFs plus/minus CSL knockdown is shown in Fig. S6A,B. (B) SCC13 keratinocytes were admixed with HDFs plus/minus CSL knockdown (by shRNA vector #1) prior to intra-dermal injection into NMRI athymic mice. Each mouse received parallel injections of keratinocytes admixed with control versus CSL knockdown fibroblasts (white and black bars, respectively). Mice were sacrificed for tumor weight determination 8 weeks later (* p<0.01). (C) Histological analysis of tumors formed by SCC13 cells admixed with HDFs plus/minus CSL knockdown. Shown are low and high magnification images. Histological analysis of other tumors is shown in Fig. S6C. (D) Immunofluorescence analysis of tumors from the above experiment with antibodies against indicated proteins. Scale bars: 100μm.

Figure 7. Down-regulation of Notch signaling in stromal fields adjacent to keratinocyte pre-malignant lesions and impact of UVA exposure

(A) Excised skin samples from 10 patients, containing, in each case, a field of normal epidermis well separated from one with actinic keratosis (AK) lesions were utilized for LCM of the AK underlying stroma versus stroma fields further away, followed by qRT-PCR analysis of indicated genes, using 36β4 for normalization. Results are expressed as heat map of fold changes of the indicated genes in AK-underlying stroma versus stroma further away. Expression of a less complete set of genes could be assessed in part of the cases (from patients #6-10), because of limited sample availability. Effectiveness of the LCM procedure was evaluated by histological analysis of each skin sample before and after stromal removal. Only upper stromal regions immediately adjacent to the epithelium were captured, which, even in the AK underlying regions, were relatively free of leukocytes (localized instead to the deeper regions). The possibility that leukocyte contamination contributes to observed differences in gene expression was further ruled out by qRT-PCR analysis, showing that Notch1 and 2 expression was, if anything, higher in purified leukocyte than fibroblast populations, with the reverse being observed for other genes like c-Jun (data not shown). (B) Triple immunofluorescence analysis with antibodies against Notch 2 (green), c-Jun (red) and Vimentin (light blue) of an excised skin sample with an AK lesion. Upper panel: low magnification image illustrating the expected pronounced expression of Notch 2 in upper epidermal layers with Notch2 down-modulation and c-Jun up-regulation in the AK lesion (yellow arrow). Lower panels: higher magnification images for assessment of Notch 2 (green) and c-Jun (red) expression in Vimentin positive (blue) dermal fibroblasts of stromal fields underlying normal epidermis, a transition zone and AK lesion as indicated (fields 1, 2 and 3, respectively). The Notch2 fluorescent signal masked the Vimentin signal and single channel image analysis was used for quantification of Notch2 versus c-Jun signal intensity in Vimentin positive cells (right panel). For this, acquisition of digital images was followed by computer-assisted determination of fluorescence intensity on an individual cell basis. Dots refer to individual measurement values (*p<0.01). Scale bars: 200μm, (upper panel); 20μm (lower panels). Analysis of additional AK-containing skin excisions is shown in Fig. S7A-C. (C) Freshly excised human skin samples placed in semi-solid culture medium and treated with indicated doses of UVA (J/cm2) were withdrawn at indicated times thereafter (hours, days). LCM of upper dermal region followed by qRT-PCR was used to assess expression of indicated genes (*p<0.01). Similar results were obtained in other independent experiments with human skin explants and with experiments with cultured human dermal fibroblasts (Fig. S7D-G and data not shown). (D) Human skin explants exposed to indicated UVA doses were processed at various times thereafter for LCM of the upper dermal region, followed by isolation of CpG-methylated DNA by a binding protein capture method. Methylation levels of the Notch2 proximal promoter region (161 ntd position rel. to the Notch2 start site) in untreated versus UVA-treated samples were assessed by qPCR, utilizing total input DNA for normalization. Similar analysis of a distal GC-rich region of the Notch2 gene (172,856 ntd position rel. to the Notch2 start site) was performed as equal loading control of partially methylated DNA that is not subject to UVA-induced modulation. Similar analysis of UVA-exposed dermal fibroblasts is shown in (Fig. S7H). (E) Same skin samples of the first five patients analyzed in (A) were utilized for LCM of AK-underlying versus distal stromal (normal skin, NS) fields, followed by isolation of CpG-methylated DNA as in the previous panel. qPCR analysis was used to assess methylation levels of the Notch2 promoter and distal GC-rich region (*p<0.01). Scale bars: 200μm, (upper panel of B); 20μm (lower four panels of B).