Tumor Architecture and Notch Signaling Modulate Drug Response in Basal Cell Carcinoma

Abstract

Hedgehog (Hh) pathway inhibitors such as vismodegib are highly effective for treating basal cell carcinoma (BCC); however, residual tumor cells frequently persist and regenerate the primary tumor upon drug discontinuation. Here, we show that BCCs are organized into two molecularly and functionally distinct compartments. Whereas interior Hh⁺/Notch⁺ suprabasal cells undergo apoptosis in response to vismodegib, peripheral Hh⁺⁺⁺/Notch^- basal cells survive throughout treatment. Inhibiting Notch specifically promotes tumor persistence without causing drug resistance, while activating Notch is sufficient to regress already established lesions. Altogether, these findings suggest that the three-dimensional architecture of BCCs establishes a natural hierarchy of drug response in the tumor and that this hierarchy can be overcome, for better or worse, by modulating Notch.

Keywords: Hedgehog; Notch; basal cell carcinoma; skin cancer; vismodegib.

Figure 1. Vismo regresses tumors in a mouse model of BCC

A. Nodular human BCC (left) and mouse Gli1;Ptch1 tumor (right). Magnified views of palisaded basal cells (asterisks) are depicted in the lower panels. B. Schematic for vismo treatment. Unt., untreated. TAM, tamoxifen. C. Tumor regression in response to varying doses of daily vismo for 1 week. Veh., vehicle control. D. Tumor regression in response to 1 or 2 weeks of daily vismo at a dose of 50 mg/kg animal weight. E. Histology of untreated or treated Gli1;Ptch1 tumors. F. Vismo completely inhibits proliferation (green). G. Quantitation for F. H. Vismo causes tumor apoptosis, as assessed by cleaved Caspase 3 (cCasp3) staining (green). I. Quantitation for H, where apoptosis is expressed as a percentage of total K5⁺ tumor cells. J. Recurrent Gli1;Ptch1 tumor, treated for 1 week with vismo, then left untreated for an additional 1 week. Red, co-staining for K5. Error bars, SE. Scale bars, 100 µm. See also Figure S1.

Figure 2. Gli1;Ptch1 tumors are comprised of 2 compartments

A. p53 (green) is detected in Gli1;Ptch1;p53^flox/+ tumors (left), but not in tumors that deleted the allele (right). B. Loss of p53 does not affect tumor response to vismo. Unt., untreated. C. Histology of untreated (left) or regressed tumor (right) after 1 week of vismo. D. Ratio of suprabasal:basal cells in untreated and treated tumors. E. Quantitation of tumor apoptosis, as in Figure 1I, divided by tumor compartment. F. Basal tumor cells express β4 integrin (green). G. Polarized basal cells display apical primary cilia, as marked by acetylated α Tubulin (AcTub, green), whereas suprabasal cells exhibit random ciliary orientation. The right panel is magnified from the boxed area. H. Basal tumor cells exhibit a modest increase in proliferation (green). I. Quantitation for H. Red, co-staining for K5. Error bars, SE. *, p < 0.05; **, p < 0.01; n.s., not significant. Scale bars, 100 µm.

Figure 3. Tumor basal and suprabasal cells are molecularly distinct

A. Gli1;SmoM2 tumor, 25 weeks after induction, with fluorescent tumor cells (green) and β4 integrin at the periphery (red). B. Validation of flow sorting of Gli1;SmoM2 tumor and non-tumor populations by qPCR for the indicated control genes. A6, α6 integrin. C. Basal Gli1;SmoM2 tumor cells display increased Hh target gene expression. D. Suprabasal Gli1;SmoM2 tumor cells display elevated Hey2 and HeyL. E. Basal Gli1;SmoM2 tumors exhibit increased Jag1 and Jag2. F. Single molecule in situ staining (black/brown dots) for Gli1 in different tumor models, as indicated. Gli1 is modestly enriched in basal tumor cells, lost upon vismo treatment, and absent in tumors that formed in a Gli1 null background (right). G. Single molecule in situ staining for Ptch2, which is also increased in basal tumor cells, lost upon vismo treatment, and largely absent in tumors that developed in a Ptch2 null background (right). Residual staining is likely due to abortive transcripts from the Ptch2-disrupted locus. H. In situ staining for HeyL, which is enriched in suprabasal cells. I. IHC for Gli1 (green) in Gli1;Ptch1 tumors that had developed in an otherwise wild-type or Gli1 null background. Staining is enriched at the tumor periphery (asterisk). Lower panels are single-channel views of the above images. J. Validation of Gli1 IHC (green) in touch dome epithelia from wild-type or Gli1 null mice. Merkel cells are identified by K8 (red). K. IHC for NICD (green) and Jag1 (red) in Gli1;Ptch1 tumors. Right panels are single-channel views of the area indicated by the asterisk. Error bars, SE. Scale bars, 100 µm. See also Figure S2–3.

Figure 4. Suppressing downstream Notch promotes tumor persistence

A. Histology of Gli1;Ptch1 (left) and Gli1;Ptch1;dnMAML (right) tumors. B. Quantitation of tumor cell density (left) and number per field (right). C. Histology showing that dnMAML expression (right panels) partially protects tumors against vismo. D. Quantitation for C. Unt., untreated. E. Tumors expressing dnMAML (dotted lines) exhibit less apoptosis, both overall (black lines), as well as in the basal (green) and suprabasal (red) compartments, relative to control tumors (solid lines). F. Histology of Gli1;Ptch1 (left) and Gli1;Ptch1;Rbpj (right) tumors. G. Quantitation of tumor cell density (left) and number per field (right). H. Histology showing that deletion of Rbpj (right panels) partially protects tumors against vismo. I. Quantitation for H. J. Tumors which delete Rbpj (dotted lines) exhibit less apoptosis, both overall (black lines), as well as in the basal (green) and suprabasal (red) compartments, relative to control tumors (solid lines). Error bars, SE. *, p < 0.05; **, p < 0.01. Scale bars, 100 µm.

Figure 5. Deleting Notch1 promotes tumor persistence, while NICD overexpression regresses established tumors

A. Histology of Gli1;Ptch1 (left) and Gli1;Ptch1;N1 (right) tumors. B. Quantitation of tumor cell density (left) and number (right). C. Histology showing that Notch1 deletion (right panels) protects tumors against vismo. D. Quantitation for C. Unt., untreated. E. Top, tumors that delete Notch1 are less apoptotic (green) after 1 day of vismo. Bottom, quantitation of overall apoptosis levels (black), or subdivided by basal (green) and suprabasal (red) compartments. F. Histology showing that tumors harboring a Notch1 activated allele (Gli1;Ptch1;NICD) (middle) resemble control tumors (left). Right, only rare cells expressed GFP (green, arrows) in Gli1;Ptch1;NICD tumors, indicating incomplete recombination. G. Strategy for tamoxifen (TAM) “boosting” to induce latent expression of NICD/GFP (green) in established tumors. H. Histology of Gli1;Ptch1;NICD tumors (right) and control tumors (left) after 2 weeks of tamoxifen boosting. I. Quantitation of tumor area after boosting. J. TAM boosting induces apoptosis in Gli1;Ptch1;NICD tumors (dotted lines), with more subtle effects in control tumors (solid lines). Overall tumor apoptosis rates are shown (black), and subdivided between basal (green) and suprabasal (red) compartments. Red, co-staining for K5. Error bars, SE. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bars, 100 µm. See also Figure S4.

Figure 6. Hh signaling opposes Notch activation

A. Hh pathway activity, assessed by Ptch2, is unaffected in Gli1;Ptch1 tumors when Notch is suppressed, as indicated. B. Gli1;SmoM2 tumors display low level Hh pathway activation, as assessed by Ptch2 (top left). Gli1;Ptch1 tumors display intermediate Hh (top middle), while tumors expressing Keratin 5 promoter-driven Gli2 (K5;Gli2) display high Hh activity (top right). Lower panels, the abundance of NICD⁺ cells (green) is inversely associated with Hh activity in these tumors. C. Quantitation of NICD⁺ cells in different models, subdivided by basal (green) and suprabasal (red) compartments. D. Gli1;Ptch1 tumors treated with vismo display increased NICD⁺ cells. E. Quantitation of NICD⁺ cells in the tumor basal (green) and suprabasal (red) compartments, in untreated (Unt.) and vismo-treated tumors. Red, co-staining for K5. Error bars, SE. *, p < 0.05; **, p < 0.01; n.s., not significant. Scale bars, 100 µm. See also Figure S5.

Figure 7. Human BCCs exhibit compartment-specific marker expression

A. Single molecule in situ staining for PTCH2 (brown, left panels) or IHC for Ki67 (green, right panels) in untreated human nodular BCCs. Top row shows tumors with basally enriched staining, whereas bottom row shows tumors with diffuse staining. Red, IHC for K5. B. Top row, some tumors contain concentrated areas of NICD⁺ suprabasal cells (green), whereas other regions are sparse for NICD⁺ cells (bottom row). Top right, abundant NICD⁺ cells (asterisk) in regions extending from a tumor mass. C. Tumor containing NICD⁺ nodule directly adjacent to NICD⁻ nodule. Right box is a magnified view of the area denoted by the asterisk. D. Table summarizing findings. As all BCCs were comprised of multiple discrete tumor nodules, an estimate of the percent of total tumor periphery displaying basally enriched Hh or Ki67, or the overall percent of tumor cells displaying suprabasal NICD, is shown. As an example for Hh or Ki67, an estimate of “80%” indicates that 80% of the total tumor periphery displayed basal enrichment for a given marker, whereas the remaining 20% displayed diffuse staining. Asterisk, nodular tumors admixed with infiltrative regions. E. Resemblance of BCC tumor architecture (left) with architecture of normal skin (right). In both cases, basement membrane contact likely confers basal cell polarity and modulates Notch signaling. Suprabasal cells that do not contact the basement membrane typically activate Notch. In BCC, vismo treatment suppresses Hh signaling and increases the proportion of Notch-activated suprabasal cells, some of which undergo apoptosis to cause tumor shrinkage. Scale bars, 100 µm. See also Figure S6.