Heterozygous mutations in the tumor suppressor gene PATCHED provoke basal cell carcinoma-like features in human organotypic skin cultures

Abstract

Basal cell carcinoma of the skin is the most common type of cancer in humans. The majority of these tumors displays aberrant activation of the SONIC HEDGEHOG (SHH)/PATCHED pathway, triggered by mutations in the PATCHED tumor suppressor gene, which encodes a transmembrane receptor of SHH. In this study, we took advantage of the natural genotype (PATCHED þ /À ) of healthy keratinocytes expanded from patients with the nevoid basal cell carcinoma or Gorlin syndrome to mimic heterozygous somatic mutations thought to occur in the PATCHED gene early upon basal cell carcinoma development in the general population. PATCHED þ /À epidermis developed on a dermal equivalent containing wild-type (WT) PATCHED þ / þ fibroblasts exhibited striking invasiveness and hyperproliferation, as well as marked differentiation impairment. Deciphering the phenotype of PATCHED þ /À keratinocytes revealed slight increases of the transcriptional activators GLI1 and GLI2-the latter known to provoke basal cell carcinoma-like tumors when overexpressed in transgenic mice. PATCHED þ /À keratinocytes also showed a substantial increase of the cell cycle regulator cyclin D1. These data show for the first time the physiological impact of constitutive heterozygous PATCHED mutations in primary human keratinocytes and strongly argue for a yet elusive mechanism of haploinsufficiency leading to cancer proneness.

PTCH1+/− Dermal Fibroblasts Isolated from Healthy Skin of Gorlin Syndrome Patients Exhibit Features of Carcinoma Associated Fibroblasts

“…COL11A1 mRNA was also found increased in NBCCS fibroblasts, an observation reminiscent of its over-expression by stromal fibroblasts in human colorectal tumors [62]. In contrast lower amounts of mRNAs of the basement membrane components COL3A1 , COL7A1 and LAMA2 [63] (Table 2) could be responsible for loosening basement membrane and, hence, facilitate local aggressiveness of pre-tumoral ( PTCH1 +/− keratinocytes) or tumoral epidermal cells [22].…”

“…Altogether, these results strongly suggest the involvement of dermal fibroblasts in BCC predisposition in NBCCS patients including in non photo-exposed skin areas. In addition, NBCCS keratinocytes from the same NBCCS patients exhibit spontaneous invasive properties in dermal equivalents populated with control fibroblasts [22]. More investigations must now be carried out in the long term to determine the extent to which NBCCS fibroblasts may affect the fate of epidermal keratinocytes.…”

“…These data suggest that solar UVs are far from being the only etiologic factor of BCC in NBCCS patients. Other mechanisms such as altered dermo-epidermal interactions could contribute to development of NBCCS BCC in non photo-exposed skin [22]. More and more evidence accumulates indicating the role of the stroma in tumoral development in general [23], and in the particular case of BCC as well.…”

Activation of the hedgehog-signaling pathway in human cancer and the clinical implications

“…Mice heterozygous for a Ptch1 null mutation show the essential features in basal cell nevus syndrome patients, such as tumor development such as (medulloblastomas, rhabdomyosarcomas and BCCs) and developmental defects (such as spina bifida occulta) (Goodrich et al, 1997;Hahn et al, 1998;Aszterbaum et al, 1999a). In human organotypic skin cultures, keratinocytes with heterozygous mutation of PTCH1 show high invasiveness, hyperproliferation and Hedgehog signaling in human cancer L Yang et al marked differentiation impairment in comparison with wild-type keratinocytes, arguing for PTCH1 haploinsufficiency in cancer development (Brellier et al, 2008). BCC, the most common human cancer consistently has abnormalities of the hedgehog pathway with PTCH1 mutations in 50% human sporadic BCCs, suggesting alteration of additional genes in the hedgehog pathway in this type of skin cancer.…”

Proteomic Analysis ofPTCH1+/− Fibroblast Lysate and Conditioned Culture Media Isolated from the Skin of Healthy Subjects and Nevoid Basal Cell Carcinoma Syndrome Patients

“…However, in contrast to sporadic BCCs, all BCCs found in GS are observed in both sun-protected and sun-exposed areas. Moreover, there is scientific evidence for the distinct pathogenesis and clinical behaviour of those cutaneous neoplasms in the diverse [4], as well as for the increased expression of matrix metalloproteinase-3 in cultured fibroblasts and BCCs in GS [5]. …”