The present invention is in the domain of pharmacy and more specifically in skin cancer area and particular for Basal Cell Carcinoma (BCC). The present invention provides a cellular model targeting the Sonic Hedgehog/Patched (SHH/PTCH) pathway dysregulation or inappropriately activated as well as screening method using this cellular model to screen pharmacological compounds able to treat or prevent BCC lesions.
The Hedgehog pathway is normally active during embryonic development and plays a central role in cell differentiation and proliferation.
Inappropriate activation or dysregulation of the Hedgehog pathway is believed to play a critical role in the proliferation and survival of certain cancer cells, including in basal cell carcinoma and medulloblastoma.
Known pathway-activating mutations include those that impair the ability of PTCH, a transporter-like Hh receptor, to restrain Smoothened (SMO) activation of transcriptional targets via the GLI family of latent transcription factors.
Binding of Hh ligand to PTCH is functionally equivalent to genetic loss of PTCH, in that pathway activation by either requires activity of SMO, a seven transmembrane protein that binds to and is inactivated by the pathway antagonist, cyclopamine.
The implication of PATCHED pathway activation in several cancer conditions, most notably in BCCs, has motivated much effort to set up experimental systems to assess the inhibitory activity of small molecules.
The existing systems to measure activation or inhibition of the activated SHH/PTCH pathway, are based on cell lines from human or mouse origin. Theses cells can schematically be classified in two categories destined to measure i-) endogenous cellular events after treatment; these events include triggering of a differentiation process and modulation of gene expression, notably of those genes known as transcriptional targets of pathway activation; ii-) cell lines engineered to report pathway activation/inhibition after transient or permanent introduction of reporter constructs made of responsive DNA driving a reporter gene. Cell lines developped so far are:
Human normal primary keratinocytes and fibroblasts in reconstructed skin where expression of GLI1 and GLI2 mRNA have been measured to demonstrate inhibtion by the small Robotnikinin molecule of SHH/PTCH pathway activation by SHH (Stanton et al., 2009)
Healthy human primary keratinocytes from patients with nevoid bas3al cell carcinoma or Gorlin syndrome have been isolated to mimic the somatic loss of one PATCHED allele in sporadic BCC epidermal cells (Brellier et al., 2008a).
However, the above described cell lines have some disadvantages. Most of cell lines are not stable in the sense that after several passages the expression of inserted genes decreases strongly or is shut down. Either those cell lines are not sufficiently robust to be efficient and sensitive to be used in a drug screening as a model. None of the cited prior art provides a system to allow a simple detection of activation in human skin cells, including keratinocytes and fibroblasts.
Thus, there is a need for developing a human cell line easy to produce and to use, efficient, being a relevant model and sensitive for the screening or assessment of molecules libraries.
The inventors have developed a new cell line providing strong advantages. Indeed, the present invention provides an immortalized cell line of human skin fibroblasts which responds to pathway stimulation without the need to introduce an exogenous reporter cassette using genetic engineering, thus that cell line is characterized in that reporter gene is endogenous with its own elements of response to pathway activation. The reporter capacity of this human cell line is stable overtime, particularly even after a high rate from passages in tissue culture.
In preferred embodiment of invention, the immortalized reporter fibroblast cell line according to the invention, express the PTCH protein and/or other members of the pathway that are necessary to convey responses to agonists and antagonists of the said pathway as shown in
The invention provides also a process for obtaining immortalized human fibroblasts as describe above, comprising the following steps:
The invention provides also a drug screening method, wherein said immortalized fibroblast cell line as described above is used to screen. In a preferred embodiment of the invention, the drug screening method comprises the following steps:
The present invention also provides an In vitro method for the screening of candidate compounds for the preventive and/or curative treatment of cutaneous cancer and preferentially basal cell carcinoma.
The invention regards also to a drug obtainable with the drug screening method as described above.
Basal Cell Carcinomas (BCC) of the skin is the commonest human cancer. BCCs derive from epidermal keratinocytes. The great majority of BCCs occurs on photo-exposed skin due to ultraviolet induced mutagenesis. The steadily rising incidence of BCCs in the last decades is attributed to increasing enthusiasm for recreational sun exposure. Although BCC rarely metastasize, they can result in local destruction and invasion of underlying tissues and consequently, life threatening complications. BCC are usually treated by local surgical excision, topical chemotherapy, photodynamic therapy, but, according to tumor size, location and frequency, there may be considerable aestetic sequelae. Thus, drawbacks of current BCC treatments strongly support the need for pharmacological innovations that should specifically target the SONIC in so far as inappropriate and constitutive activation of this pathway is associated with the vast majority of BCC (see below). In several cancers including BCCs, It has been suggested that accumulation of mRNA corresponding to target genes of the pathway depends on interactions of tumor cells with fibroblasts of the microenvironment. Furthermore, molecules that target the SHH pathway could also be of interest in the treatment of other/non-BCC cancer conditions where the influence of stromal microenvironment (e. g. melanoma, pancreas, oeasophagus, liver, prostate, lung, muscle, colon) is though to play a role in inappropriate activation of the SHH/PTCH pathway (for revue see, (Scales and de Sauvage, 2009)).
The SHH/PTCH signaling pathway is essential during embryogenesis and development where it controls cell fate by modulating proliferation and differentiation. Animal models, notably the fruit fly drosophila melanogaster, have shown that at specific stages of development, some cells produce and emit a signal, the Hedgehog molecule (HH), wich, in turn, is received by target cells. In vertebrates, the family of Hedgehog molecules is composed of Sonic Hedgehog, SHH, Desert Hedgehog, DHH, and Indian Hedgehog, IHH. Target cells (of these ligands) express PATCHED (PTCH), a putative twelve pass transmembrane protein acting as the receptor of HH molecules. When HH molecules are not expressed and/or not secreted at the vicinity of target cells, PTCH acts as a repressor of the pathway by inhibiting another transmembrane protein called SMOTHENED (SMO). SMO is a putative seven pass transmembrane protein apparented to G-protein coupled receptors. The inhibition of SMO by PTCH is relieved in the presence of HH molecules bound to PTCH. De-repression of SMO leads to activation of transcription factors of the GLI family (named GLI 1, 2 and 3) that activate (GLI1 and 2) or repress (GLI3), the transcription of their target genes. Interestingly, PTCH1 is a transcriptional target of GLI1 and GLI2 factors (Scales and de Sauvage, 2009).
The importance of the SHH/PTCH pathway is illustrated by severe diseases due to mutations affecting its integrity at different levels. Notably, in the human, heterozygous mutations in the PTCH1 gene are responsible for the dominantly inherited genetic syndrome called nevoid basal cell carcinoma syndrome (NBCCS or Gorlin syndrome). NBCCS patients are highly prone to BCCs that generally (about 50% cases) present with a loss of heterozygosity in the PTCH1 locus. In Gorlin patients, more than 50% BCCs also bear mutation in the tumor suppressor gene TP53, suggesting some cooperation of the P53 and the SHH/PTCH pathways toward development of BCCs. Very interestingly, the two PTCH1 alleles are also lost in most sporadic (general population) BCCs; in the latter case, again, the two TP53 alleles are found mutated in 10 to 50% sporadic BCCs. 20-30% sporadic BCCs are mutated in both TP53 and PTCH1. In both NBCSS and sporadic BCCs, inactivation of PTCH results in constitutive activation of the pathway with accumulation GLI1 and GLI2 mRNAs (Dahmane et al., 1997; Unden et al., 1996). In contrast to sporadic BCCs that almost exclusively develop in photo exposed skin area, about 40% NBCCS BCCs develop in non exposed skin. In addition, our previous observations have indicated that primary NBCCS primary fibroblasts isolated from healthy skin expressed a transcriptome resembling that characterized in fibroblasts associated to sporadic carcinomas (CAF) (Valin et al., 2009; Valin and Magnaldo, 2008). Together, these observations strongly support the idea of a strong contribution of dermal fibroblasts in carcinoma development in NBCCS patients.
The implication of the SHH/PTCH pathway activation in several cancer conditions, most notably in BCCs, has motivated much effort to set up experimental systems to assess the inhibitory activity of small molecules.
The existing systems of activity measure are based on cell lines from human or mouse origin. Theses cells can schematically be classified in two categories destined to measure i-) endogenous cellular events after treatment; these events include triggering of a differentiation process and modulation of gene expression, notably of those genes known as transcriptional targets of pathway activation; ii-) cell lines engineered to report pathway activation/inhibition after transient or permanent introduction of reporter constructs made of responsive DNA driving a reporter gene. Cell lines developed so far in the prior art reveals that none of those system allows simple detection of activation in human epidermal keratinocytes.
To provide a simple detection system, the inventors have worked to develop a human cell line in the respect of the following specifications (i.e. what we need for easy, efficient, relevant, sensitive assessment of molecules libraries):
In the specific case of NBCCS cells, other advantages of abrogating P53 stem from our and others reports indicating that : i-) P53 stabilisation after a single UVB irradiation is higher and prolonged in NBCCS compared to control cells (Brellier et al., 2008b); ii-) there is a mutual inhibition of Gli1 and P53 (Stecca and Ruiz i Altaba, 2009); iii-) 20-30% BCCs bear mutations in both TP53 and PATCHED suggesting that attenuation of P53 could enhance sensitivity to PTCH pathway activators.
Thus, it is reasoned that abrogation or attenuation of the P53 pathway using E6-E7 oncogenic proteins of Human Papilloma Virus 16 (HPV16) would favor activation of the SHH/PTCH pathway in human skin cells, including fibroblasts. Preferably, it was decided to use human primary fibroblasts from healthy skin (non photo exposed non tumoral) from individuals having NBCCS or Gorlin syndrome, after transformation with the E6-E7 oncogenic proteins (NBCCS 6 E6/E7 fibroblasts).
Thus, the present invention provides an immortalized cell line of human fibroblasts from healthy individuals having NBCCS or Gorlin syndrome expressing an enhanced and stable response toward PTCH pathway activation overtime, particularly even after a high rate from passages in tissue culture.
It meant by stable expression of the endogenous reporter gene overtime that after high number of passages the level expression is the same as the initial level without recombination or lost of chromosomal material, as this can be observed in cell lines that express an exogenous reporter gene (i.e. Luciferase) under the control of GLI binding sequence in direct repeat (×8) tandem configuration (Sasaki et al., 1997).
In a preferred embodiment of the invention, the immortalized fibroblasts from healthy individuals having NBCCS or Gorlin syndrome cell line according to the invention express the PTCH protein and/or other members of the pathway that are necessary to convey responses to agonists and antagonists of the said pathway as shown in
In addition, this cell line is produced in standard culture medium (DMEM based containing non sophisticated, ready to use goodies). In addition the cell line stands temporary serum starvation (0.5% serum) which allows avoiding interference with the activity response. Thus, response of cells to pathway activation in a quasi defined medium providing the advantage of growing cells in medium which does not interfere with activity response. The invention provides thus a robust model with expected or calibrated response which avoids any interfering factors.
The invention provides also a drug screening method, wherein said immortalized fibroblast cell line as described above is used to screen. The invention relates to an in vitro screening method of the PTCH pathway inhibitors for treating skin cancer and preferably BCC, comprising determining the capacity of said drug to inhibit or down regulate expression or biological activity of the PTCH pathway.
In a preferred embodiment of the invention, the drug screening method comprises the following steps:
The following figures illustrate the invention:
The examples which follow illustrate the invention without limiting the scope thereof.
Human primary fibroblasts (named CTRL or NBCCS) were isolated from a healthy non photo-exposed skin biopsy of either a control patient or a patient with caracteristic NBCCS after informed consent (Otto et al., 1999; Valin et al., 2009)
The CTRL and NBCCS6 cell lines were then immortalized by retroviral transduction with pLE6/E7SN resulting in CTRL and NBCCS-E6-E7 (
G418 is an aminoglycoside antibiotic similar in structure to gentamicin B1, produced by Micromonospora rhodorangea. G418 blocks polypeptide synthesis by inhibiting the elongation step in both prokaryotic and eukaryotic cells. Resistance to G418 is conferred by the Neomycin resistance gene (neo) from Tn5 encoding an aminoglycoside 3′-phosphotransferase, APH 3′ II.
Selection in mammalian cells is usually achieved in three to seven days with concentrations ranging from 200 to 1000 μg/ml (Arnaudeau-Bégard et al., 2003).
Real Time Quantitative PCR
Total RNA was extracted with RNeasy® Mini kit according to manufacturer's instructions (Qiagen, Hilden, Germany). Reverse transcription was performed on 1 μg total RNA with Superscript II Reverse Transcriptase (Roche Applied Science, Basel, Switzerland) by using random primers. Q-PCR was carried out using the 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA).
Effective transformation of the CTRL and NBCCS6 cell line was assessed by analysing their growth properties and attenuation of expression of the P53 tumor suppressor protein.
As shown in
RT-Q-PCR was performed from total cDNAs prepared from the indicated cells. CTRL, human primary dermal fibroblasts before or after CTRL E6/E7 immortalization by pLE6E7 retroviral transduction. NBCC6, primary fibroblast from Nevoid Basal Cell Carcinoma Syndrome patient # 6 before or after (NBCCS6 E6/7) immortalization by pLE6E7 retroviral transduction.
To evaluate small Molecules Modulators of the SHH/PTCH pathway, the Purmorphamine (SMO agonist) was used. Purmorphamine (SMO agonist) was diluted in DMSO at stock concentrations of 50 mM and 10 mM, respectively. To avoid side effects and toxicity, the final concentration of DMSO was fixed to 0.1% DMSO.
As shown in
It should be noted that NBCCS 6 E6/E7 exhibit a 7× times higher response than CTRL cells in the same experimental conditions.
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Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/EP2012/068777 | 9/24/2012 | WO | 00 | 3/24/2014 |
Number | Date | Country | |
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61538586 | Sep 2011 | US |