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+/−) 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+/− epidermis developed on a dermal equivalent containing wild-type (WT) PATCHED+/+ fibroblasts exhibited striking invasiveness and hyperproliferation, as well as marked differentiation impairment. Deciphering the phenotype of PATCHED+/− 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+/− 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.

Main

In humans, the two major classes of non-melanoma skin cancers are basal (BCC) and squamous cell carcinoma, both of which arise from epidermal keratinocytes. Prevalence of BCC has incrHeased constantly over the last decades and is currently the most common malignant neoplasm in Caucasian populations (DePinho, 2000). Molecular clues to BCC development became known in 1996 with the discovery that a germinal mutation in the PATCHED gene (9q22.3) is associated with a human, dominantly inherited syndrome, called nevoid basal cell carcinoma (NBCCS) or Gorlin syndrome (Hahn et al., 1996; Johnson et al., 1996). NBCCS patients present a high predisposition to BCCs and variable developmental traits (Gorlin, 1987; Chidambaram and Dean, 1996). PATCHED encodes a transmembrane protein, acting as a receptor of the diffusible morphogen protein SONIC HEDGEHOG (SHH) (Marigo et al., 1996; Stone et al., 1996). In the absence of SHH, PATCHED constitutively represses the pathway, whereas this repression is relieved in the presence of SHH. In sporadic BCCs, mutations of PATCHED have been described at high frequency (67%) (Reifenberger et al., 2005) and loss of heterozygosity (LOH) has also frequently been found (93%) (Teh et al., 2005).

As a tumor suppressor gene, PATCHED inactivation occurs in two hits: the first hit may occur in one allele following exposure to a genotoxic stress such as ultraviolet irradiation. Subsequently, inactivation of the second allele of PATCHED by LOH is believed to confer SHH-independent growth, genetic instability and, potentially, tumor development (Johnson et al., 1996). As a consequence, in most BCCs analysed, PATCHED inactivation results in overexpression of mRNAs encoding downstream transcriptional effectors of the pathway, GLI1 and GLI2 that control cell proliferation and differentiation, as well as PATCHED itself (Gailani et al., 1996; Dahmane et al., 1997; Unden et al., 1997).

Although the etiologic role of ultraviolet irradiation is clear in sporadic BCCs, it remains much less evident for familial BCCs as they also frequently develop in non-photoexposed skin areas. Genetic manipulations have shown that one-third of Patched+/− mice develop microscopic skin tumors, in the absence of exposure to external genotoxic stress (Aszterbaum et al., 1999). Furthermore, overexpression of either Shh (Oro et al., 1997), Gli1 (Nilsson et al., 2000), or Gli2 (Grachtchouk et al., 2000) in the basal epidermal layer provokes a pseudo-neoplastic phenotype. Taken together, these observations suggest that either inactivation of one allele of PATCHED, or artificial expression of positive effectors (Gli factors) and activator (SHH) of the pathway in epidermal cells induce similar phenotypes and seem sufficient to promote cancer development in the absence of evident exogenous mutagenic challenge.

Here, we took advantage of the natural PATCHED+/− genotype of epidermal keratinocytes isolated from healthy skin of NBCCS patients to mimic the somatic loss of one PATCHED allele in sporadic BCC epidermal cells. Primary keratinocyte strains were expanded from small skin biopsies of 10 independent NBCCS patients. Growth properties and lifespans (data not shown) of NBCCS PATCHED+/− (n=10) and control PATCHED+/+ (n=4) keratinocytes could not be distinguished in two-dimensional cultures. The two NBCCS patients studied here bore premature stop codons in the PATCHED open reading frame (H652X for NBCCS 6 and L489X for NBCCS 10, see Figure 1) (Brellier et al., 2008) as most NBCCS germline (Evans et al., 2000; Boutet et al., 2003; Soufir et al., 2006) and sporadic somatic PATCHED mutations (Reifenberger et al., 2005). NBCCS 6 and 10 PATCHED+/− keratinocytes (passage 3–6) were cultured in a three-dimensional organotypic skin system comprising a wild-type (WT) dermal equivalent containing PATCHED+/+ fibroblasts.

Figure 1
figure1

Abnormal histological features, early and late differentiation in PATCHED+/− epidermis. Fibroblast and keratinocyte were isolated from non-photoexposed healthy skin of control and nevoid basal cell carcinoma syndrome (NBCCS) individuals (Rheinwald and Green, 1975). NBCCS6 is a 49-year-old female. NBCCS10 is a 47-year-old male. Detailed clinical traits are described in Brellier et al., (2008). Both patients developed numerous basal cell carcinomas (BCCs). Organotypic skin cultures were prepared at least twice in duplicates as described in detail (Bernerd et al., 2001). H652X, and L489X, are PATCHED germinal mutations detected in NBCCS6 and NBCCS10 patients, respectively (Pruvost-Balland et al., 2006), and resulting in the illustrated putative forms of the protein. (ac) HES, hematoxylin-eosin-safron staining of 5 μm paraffin sections. SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum, BL, basal layer. (df) Immunolabelings of laminin B1 (B1 lam.) using rabbit anti-laminin antibody (Novotec, Paris, France; 1/400 in phosphate-buffered saline (PBS)). (gi) Immunolabelings of beta 1 integrin (β1 Int.) using mouse monoclonal anti-beta-1 integrin antibodies (clone 4b7, Oncogene Research Products, Boston, MA, USA; 1/50 in PBS). Bar: 150 μm. (jl) Immunolabelings of 5 μm cryosections of K10 keratin (K10) using anti-keratin 10 mouse monoclonal antibody (RKSE 60, Sanbio, Uden, The Netherlands; 1/10). (mo) Immunolabelings of loricrin (Lor.) using A8.73 anti-loricrin rabbit serum (T.Magnaldo; 1/200). Bar: 50 μm.

Either PATCHED+/+ or PATCHED+/− organotypic epidermis presented a full thickness with well-organized basal, spinous and granular layers, and a stratum corneum (Figures 1a–c). However, PATCHED+/− keratinocytes exhibited a strong and spontaneous trend to form numerous epidermal invasions within the WT dermal equivalent (Figures 1b and c), which were never observed with PATCHED+/+ keratinocytes. Epidermal invasions did not clearly exhibit all the characteristics of human BCCs, notably typical pallisadic cells.

As the stepwise program of epidermal differentiation attests proper control of cutaneous homeostasis, the expression of early and late differentiation markers was examined. Laminin B1, a major component of the basement membrane, was found slightly increased in PATCHED+/− in comparison with WT PATCHED+/+ epidermis (Figures 1d–f). In control cultures, labeling of beta-1 integrin was faint and restricted to basal keratinocytes and the basement membrane zone (Figure 1g) but it was much stronger and extended to one to two suprabasal layers in PATCHED+/− epidermis (Figures 1h and i). As expected, in WT epidermis, K10 keratin was detected from the first suprabasal layer throughout the entire depth of the epithelium (Figure 1j). The major cell envelope precursor, loricrin, was detected in granular layers (Figure 1m). In PATCHED+/− epidermis, onset of expression of K10 keratin was delayed to the median part of the spinous layers (Figures 1k and l) and loricrin became barely detectable (Figures 1n and o).

The proliferative status of keratinocytes in the basal layer was measured by labeling of Ki67 (Figure 2). PATCHED+/+ and PATCHED+/− epidermis exhibited similar rates of proliferation in intact zones of the basal epidermal layer. However, the number of Ki67-positive keratinocytes was very high in epidermal invasions formed by PATCHED+/− keratinocytes within the PATCHED+/+ dermal equivalent thus contributing to increasing the global rate of proliferation (Figure 2).

Figure 2
figure2

Alterations of proliferation in PATCHED+/− epidermis. (ac) Cryosections of organotypic skins were immunolabeled using the anti-Ki67 mouse monoclonal antibody (NovoCastra, Newcastle, UK; 1/20). Ki67 positive keratinocytes were counted on ten fields of 0.5 mm length. In keratinocyte microinvasions (b and c), Ki67 labeling could be observed at very high levels (up to 100% in some sections), whereas it appeared normal in non-invasive areas. Bar: 50 μm. Bottom panel: quantification of Ki67 positive keratinocytes in the total epidermis (basal layer and intradermal invasions) of control versus NBCCS organotypic skin cultures.

In BCCs, cyclin D1 overexpression may attest to the constitutive activation of the SHH/PATCHED pathway (Adolphe et al., 2006) as it is a transcriptional target of GLI2. Amounts of mRNAs encoding GLI1 and GLI2 were increased by 1.75 and 2.3, respectively, in PATCHED+/− compared with PATCHED+/+ keratinocytes in two-dimensional cultures (Figure 3a). The amount of cyclin D1 was increased significantly (P<0.001 and P<0.0001; about 40%) in PATCHED+/− keratinocytes (Figure 3b). To determine whether the SHH/PATCHED pathway is activated in PATCHED+/− epidermal invasions, cyclin D1 was immunolabeled in sections of organotypic skins, BCC and adjacent epidermis isolated from patient NBCCS 10 (L489X) (Figure 3, Right panel). In adjacent epidermis and in control organotypic skin cultures, cyclin D1 was faint and exhibited a clear and dense nuclear intensity in solely a few basal and directly suprabasal keratinocytes (Figure 3c and e). In contrast, both the frequency of cyclin D1-positive epidermal keratinocytes and the density of nuclear labeling were increased in organotypic skins with a PATCHED+/− epidermis (Figures 3f and g) most notably in, or above, epidermal invasions (Figures 3h and i). In BCC, cyclin D1 was detected in variable fractions (10–100%) of invasive keratinocytes depending on the tumoral zone (Figure 3d).

Figure 3
figure3

(a and b) Gli1, Gli2, and cyclin D1 increases in PATCHED+/− keratinocytes. Keratinocytes were cultured in keratinocyte serum-free medium (GIBCO/BRL, Bethesda, MD, USA) supplemented with 30 μg/ml bovine pituitary extract, 0.1 ng/ml epidermal growth factor and 0.1 mM CaCl2. (a) Total RNA was extracted from PATCHED+/− and wild-type (WT) PATCHED+/+ keratinocytes using RNeasy kit (Qiagen, Hilden, Germany). Reverse transcription from 1 μg total RNA using Superscript II reverse transcriptase (Roche Applied Science, Basel, Switzerland) was performed. Q-PCR was carried out in a Taqman device (Applied Biosystems, Foster City, CA, USA), using specific primers as described in Supplementary Data 2. Results were normalized using the geNORM software (http://medgen.ugent.be/~jvdesomp/genorm/). Quantifications indicate a significant increase of GLI2 (*P<0.035) in the case of NBCCS10. Values represent the mean of at least four independent experiments, each of them performed in triplicates. (b) PATCHED+/+ and PATCHED+/− keratinocyte protein extracts (40 μg) were separated by 10% SDS–PAGE gel electrophoresis, transferred onto polyvinyl difluoride (PVDF) membrane and probed with purified mouse monoclonal anti-cyclin D1 antibody (clone DCS-6; Santa Cruz Biotechnology; Santa Cruz, CA USA; 1/1000) and then with the mouse monoclonal anti-GAPDH antibody (clone 9484, Abcam, Cambridge, UK; 1/5000) as loading control. Blots were analysed using electrochemiluminescence (ECL+) reagents (Amersham Biosciences, Uppsala, Sweden) and cyclin D1 amount relative to the GAPDH was quantified using GeneGnome software (Ozyme, St-Quentin-en-Yvelines, France). Mean of four independent experiments is illustrated. Note that there is a significant (***P<0.001; ****P<0.0001; t-test) increase in cyclin D1 amount in PATCHED+/− (H652X, L489X) compared with WT PATCHED+/+ keratinocytes. (ci) Nuclear cyclin D1 expression in PATCHED+/− keratinocytes. Paraffin sections (5 μm) of skin organotypic cultures and skin biopsies were immunolabeled with a rabbit monoclonal anti-cyclin D1 antibody (Clone SP4; Lab Vision Corp, Fremont, CA, USA; 1/25) using a Benchmark XT automated stainer and DAB detection kit. Antigen retrieval was achieved in CC2 buffer (Ventana Medical Systems, Tucson, AZ, USA;). (c) Healthy adjacent epidermis of BCC shown in (d). (e) Control organotypic (PATCHED+/+) skin culture. (f and g) PATCHED+/− organotypic skin cultures developed from NBCCS6 and NBCCS10 keratinocytes, respectively. (h and i) show higher magnification of areas delineated in (f and g). Note numerous positive nuclei for cyclin D1 labeling in the BCC (d), as well as in epidermal invasions in (h and i). Bar: 63 μm.

Cyclin D1 has been associated with cell invasiveness (Kenney and Rowitch, 2000; Mill et al., 2003). Overexpression of GLI2 in PATCHED+/− keratinocytes could be connected to delayed and reduced differentiation (Figure 1) as described previously (Regl et al., 2004). Whether the active forms of GLI2 are present at the sites of high expression of cyclin D1 could not be determined in the absence of reliable antibody. In excellent agreement with our data, conditional loss of one Patched allele in mouse epidermis resulted in the increase of nuclear cyclins B1 and D1 (Adolphe et al., 2006). Also in excellent agreement, activation of the pathway by SHH ectopic expression in human epidermal keratinocytes was shown to promote proliferation (Fan and Khavari, 1999) as well as epidermal invasions and delayed differentiation in HaCat cells (Bigelow et al., 2005).

Our attempts by laser microdissection to determine whether epidermal invasions could result from LOH at the PATCHED locus failed, presumably due to the very small size of these structures. Sequences of genomic DNA after long-term serial passages in culture showed that NBCCS6 and NBCCS10 keratinocyte strains retained both the WT and the mutated PATCHED alleles (Figure 4). In contrast, only the WT PATCHED cDNA could be detected from mRNAs of both NBCCS keratinocyte strains, suggesting either absence or rapid degradation of mutant transcripts (Figure 4). These observations argued strongly against loss or inactivation of the WT PATCHED allele in NBCCS keratinocytes even under highly proliferative culture conditions. That LOH could have occurred in epidermal invasions within the very short period of time (14 days) necessary for organotypic skin culture, seems unlikely. In addition, we could not detect any aberrant level or P53 mutation in both NBCCS strains used for organotypic skin cultures (Brellier et al., 2008). Finally, similar amounts of PATCHED mRNA were measured in control and in both NBCCS keratinocyte strains suggesting a mechanism of transcriptional compensation (Supplementary Informations 1).

Figure 4
figure4

Sequences of genomic DNA and cDNA in NBCCS patients. Keratinocytes were cultured and passed serially before confluency. After eight passages, genomic DNA and RNA were extracted. DNA was prepared using gDNA extraction kit (Promega Corporation, Madison, WI, USA) and cDNA were prepared as described in Figure 3. Genomic DNA and cDNA were amplified and sequenced using primers indicated in Supplementary Informations 2. (a, c, e and g) Sequences of genomic DNA in WT, NBCCS6 and NBCCS10 keratinocytes after long-term culturing. Note the presence of both the WT and the mutated allele in the DNA of NBCCS6 (c) and NBCCS10 (g) strains (with addition of a cytosine in position 1925 and deletion of an adenine in position 1366 of the PATCHED sequence, respectively). (b, d, f and h) Sequences of cDNA in WT, NBCCS6 and NBCCS10 keratinocytes cultured in the long term. Both NBCCS6 (d) and NBCCS10 (h) strains express the PATCHED WT allele. In contrast, note that the cDNAs bearing the respective PATCHED mutations are not detected in both NBCCS strains.

Organotypic skin cultures with PATCHED+/− exhibited some characteristics of BCCs but typical pallisadic cells of human BCCs could not be observed. These features could result from unbalanced dermo–epidermal interactions. Future investigations should contribute to preventive or therapeutic measures intended both for patients suffering from NBCCS and for individuals from the general population susceptible to BCC development.

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Acknowledgements

VB, AV, SB, have contributed equally to this work. FB and AV were recipients of PhD fellowships from the CNRS and the Ligue Nationale contre le Cancer, and MESR, respectively. SB was an IGR post-doctoral fellow. We gratefully thank Dr Françoise Bernerd, Dr Howard Green, Ms Valérie Vélasco, Virginie Marty, Dr Rune Toftgard, Dr Paule Opolon, and Marianne Brown-Luedi for their kind help. This work was supported by the CNRS, the ARC (no. 9500), the Fondation de l’Avenir, the SFD and the AFM.

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Correspondence to M-F Avril or T Magnaldo.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Brellier, F., Bergoglio, V., Valin, A. et al. Heterozygous mutations in the tumor suppressor gene PATCHED provoke basal cell carcinoma-like features in human organotypic skin cultures. Oncogene 27, 6601–6606 (2008). https://doi.org/10.1038/onc.2008.260

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Keywords

  • PATCHED
  • skin
  • keratinocyte
  • basal cell carcinoma
  • organotypic cultures
  • Gorlin syndrome

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