Basal Cell Carcinoma in Gorlin’s Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

International audienceBasal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings suggest that defects in dermo/epidermal interactions could contribute to BCC susceptibility in NBCCS patients

Culture supernatants from NBCCS fibroblasts promote SA-β-Gal activity in WT but not in transformed (E6-E7) or tumoral (SCC13) keratinocytes.

<p>(A) SA-β-Gal activity in WT1, WT1 E6-E7, and SCC13 keratinocytes after treatment with either non conditioned medium (NCM), culture supernatant conditioned by WT (WT1), or NBCCS (NBCCS6 and NBCCS10) fibroblasts. (B) SA-β-Gal positive cells were blind counted by three independent investigators. Bars represent the confidence intervals (α = 0.05). Number of SA-β-Gal positive cells was increased in WT keratinocytes treated with NBCCS supernatants. No increase of SA-β-Gal positive cells was detected in both WT1 E6-E7 and SCC13 keratinocytes.</p

Abrogation of P53 in NBCCS keratinocytes confers invasive properties in the presence of NBCCS fibroblasts.

<p>(A) Western blot analysis of P53 showed a strong attenuation in WT (WT1), NBCCS6 and NBCCS10 keratinocytes transduced with a retrovirus expressing the HPV16 E6 and E7 oncoproteins. (B) Representative images of H&E coloration of paraffin-embedded sections of organotypic invasion assays composed of either WT E6-E7 or NBCCS E6-E7 keratinocytes overlaying a dermis equivalent comprising either WT or NBCCS fibroblasts. Bar: 200 μm. (C) Invasion index of keratinocytes from assays as in (B). Values represent the average of 3 total sections from two independent organotypic cultures. Bars represent the confidence intervals, α = 0.05.</p

NBCCS fibroblasts stimulate <i>PATCHED1</i> transcription in WT keratinocytes.

<p>(A), <i>PATCHED1</i> and <i>GAPDH</i> mRNAs amounts expressed by WT keratinocytes cultured with either WT or NBCCS fibroblasts (NBCCS6 or NBCCS10) were determined after semi-quantitative PCR and agarose gel electrophoresis (upper panel). Relative <i>PATCHED1</i> mRNA levels were increased in WT keratinocytes (WT1) treated with culture media derived from NBCCS fibroblasts (NBCCS6 and NBCCS10) (lower panel). (B), Activity of the <i>PATCHED1</i> promoter in WT keratinocytes cultured in media conditioned from either WT or NBCCS fibroblasts (NBCCS6 or NBCCS10). Note the substantial increase of the relative transcriptional activity of the <i>PATCHED1</i> promoter in WT keratinocytes treated with culture media conditioned by NBCCS fibroblasts. Experiments were performed two times in triplicates, p<0.09.</p

NBCCS fibroblasts alter morphology and differentiation of WT epidermis.

<p>WT keratinocytes were seeded onto dermis equivalents comprising skin fibroblasts isolated from either a WT (WT1) donor or from two independent NBCCS patients (NBCCS6 and NBCCS10). H&E staining reveals the absence of cornified layers (CL) and slight epidermal thinning in NBCCS OSC. Note increased accumulation of Laminin B1 and β1 Integrin in the basal layer (BL) and the para basal layers of epidermis. Star (*) points dermo epidermal cleft between the dermal and the epidermal compartments. Also note delayed expression of Keratin 10 (K10) and absence of expression of Loricrin (Lor) in NBCCS OSC; PI, nuclei counter staining using propidium iodide. Dash lines delineate the dermo epidermal junction. Bar: 110 μm</p

Neutralizing SHH in organotypic skin cultures attenuates alterations of WT epidermis in the presence of NBCCS fibroblasts.

<p>OSC comprising either WT (WT1) or NBCCS fibroblasts (NBCCS6 or NBCCS10) were developed in the presence of either the 5E1 anti-SHH blocking mAb (5E1 mAb) or an isotype-matched (anti-Myc 9E10) control antibody (Control mAb). OSC sections were stained with either H&E, or immunolabeled with the anti-β1 Integrin mAb. Note that developing OSC in the presence of the 5E1 antibody improved epidermal atrophy (as pointed out by black arrows) and β1 Integrin delocalization otherwise observed in the presence of NBCCS fibroblasts. Bar: 110 μm</p

SHH secretion depends on the presence of both fibroblasts and keratinocytes and is further increased in the presence of NBCCS fibroblasts.

<p>(A), Western blot analysis of N-SHH secreted from either WT (WT1) or NBCCS (NBCCS6 or NBCCS10) fibroblasts. Secretion of N-SHH was not detectable in culture supernatants of WT and NBCCS fibroblasts. (B), Western blot analysis of N-SHH secreted from co-cultures of WT keratinocytes with WT (WT1) or NBCCS fibroblasts (NBCCS6 or NBCCS10). N-SHH became slightly detectable in the presence of WT fibroblasts (WT1). N-SHH amount was substantially increased in the presence of NBCCS fibroblasts (NBCCS6, NBCC10). Lower panel: quantitation of N-SHH in culture supernatant obtained under the circumstances described for the upper panel. Note that detection of SHH is limited to the N-SHH processed protein (22 kDa, arrow).</p

NBCCS fibroblasts alter proliferation of WT keratinocytes in organotypic skin cultures.

<p>(A) Rates of cycling keratinocyte (Ki67, green/yellow nuclei), and P53-positive keratinocytes (P53, brown nuclei) were blind counted by 3 independent investigators on sections of OSCs, as indicated. Bar: 170 μm. (B) Ki67- (left panel) and P53- (right panel) positive keratinocytes were counted in the basal layer of 10 different fields spread along 3 independent sections. Error bars indicate the mean ± SD of the counted fields. (C), Western blot analysis shows stabilization of P53 in WT keratinocytes (WT1) treated for 24 h in culture supernatants conditioned by NBCCS (NBCCS6 and NBCCS10) fibroblasts (left panel). Right panel shows levels of P53 relative to GAPDH and expressed as fold induction relative to the WT fibroblasts strain. Error bars indicate the mean ± SD of triplicate experiments.</p