Study of the melanocytic differentiation applied to vitiligo and melanoma: from the patient to the molecular mechanisms

Background and Previous activities

The pigmentation of the skin is mostly due to the melanin pigments that are produced by melanocytes. Pigmentary disorders account for up to 20% of all dermatological consultations in some populations. They are now well demonstrated to induce a marked alteration of the quality of life of affected individuals. Most pigmentary disorders are still poorly understood and lack of effective therapeutic approaches. Pigmentation is a tightly regulated process under the control of more than 170 genes (http://www.espcr.org/micemut/). Many players are involved in this process and interact with melanocytes. Clinical observations along with patient samples are mandatory to better understand these complex mechanisms. Melanoma is the malignant transformation of melanocytes. At metastatic stage, the mortality of melanoma is one of the highest of all cancers. Despite some recent advances, a majority of patients will not respond to current therapies or will develop resistances. The genetic heterogeneity of melanomas and of patients emphasize the limitation of studying melanoma on cell lines.

Project Research

A- MELANOMA

1- Role of the non-canonical NF-kB pathway in melanoma

Enhancer of Zeste homologue 2 (EZH2) is a key oncogene. We identified the non-canonical-NF-kB pathway as a key regulator of EZH2 expression in melanoma. We showed a striking correlation between NF-kB2 and EZH2 expression in human melanoma metastases. We demonstrated that the inhibition of the non-canonical NF-kB pathway by targeting NF-kB2/p52 or the upstream kinase NIK restores the senescence program in melanoma cells through the decrease of EZH2. We showed in mouse models that the inhibition of the non-canonical NF-kB pathway restores senescence and induces a dramatic reduction in tumor growth compared to controls, thus providing potential drug targets for the re-induction of senescence in melanoma and other cancers where EZH2 is overexpressed (Oncogene. 2015 Sep 14. doi:10.1038/onc.2015.331). NIK inhibitors have been developed in in collaboration with Dr. Benhida’s Team and provide very encouraging results in vitro but also in mouse models of melanoma but also of several other malignancies. Three patents have been filled to protect these results. These compounds are actually further developed with the creation of the startup YUKIN therapeutics with the objective to test the lead compound in clinical trials for selected cancers.

2-Identification of new anti-melanoma compounds

Using our previous results and structure/activity relationship studies in collaboration with Dr. Benhida’s Team, we developed and selected candidates (Thiazole Benzensulfonamides) exhibiting a strong death-promoting effects in melanoma cells with HA15 as the lead compound of this series. Interestingly, HA15 induces death of all melanoma cells independently of mutational status and melanoma cells freshly isolated from patients sensitive or resistant to BRAF inhibitors. HA15 exhibited also a strong efficacy in xenograft mouse models performed with melanoma cells sensitive and resistant to BRAF inhibitors without any sign of toxicity. We next performed pan-genomic, proteomic and biochemical studies to decipher the signaling pathway, the mechanism of action and the target of the best candidates. We identified BIP, an endoplasmic reticulum protein, as the specific target of our compound. We demonstrated clearly that the interaction between our compound and BIP increases Endoplasmic Reticulum Stress and leads to melanoma cell death by concomitant induction autophagy and apoptosis mechanisms. Overexpression of target BIP in various cancers is described, it is thus not surprising that this molecule was also found to be active against other liquid and solid tumors. Taken together, our data suggest that our molecule has an important impact on inhibition of melanoma growth by targeting ER stress, and may therefore be developed for treatment of patients with melanoma in particular and other cancers in general. This work has been published in Cancer Cell journal and 2 international patents have been deposited.

 3-Anti-melanoma effects of biguanides

Metformin pertain to the biguanide class and is the most widely used antidiabetics due to its major clinical advantage of not inducing hypoglycemia and being well tolerated. Metformin acts on the AMPK/mTOR pathway and reduces cell proliferation. Numerous studies have show that metformin can potentially be used as an efficient anticancer drug in various cancers. These findings are strengthened by retrospective epidemiological studies that pointed out a decrease in cancer risk in diabetic patients treated with metformin. This theme is common to several teams in C3M since team 7 and 4 have demonstrated that metformin inhibited viability of prostate cancer and leukemia cells, respectively. In our team, we show that metformin exerts anti-proliferative effects on melanoma cells, without affecting normal melanocyte viability. We showed that metformin induced the following sequence of events in melanoma cells: cell cycle arrest (24h), autophagy (72h) and robust apoptosis (96h). The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers (Patent: PCT/EP2O1 1/002268 and Tomic et al., Cell Death and Disease, 2011). In a second part, we investigated the effect of metformin on melanoma invasion and metastasis development. Using different in vitro approaches, we found that metformin inhibits cell invasion without affecting cell migration and independently of anti-proliferation action (Patent EP N° BIO 12336 and Cerezo et al., MCT, 2013). Considering our preclinical results, we set up an open phase I / II clinical trial in collaboration with the Dermatology department of Nice CHU on patients with metastatic melanoma in therapeutic failure to evaluate the efficacy of metformin in human. Unfortunately, very few patients have responded to this treatment (Montaudie et al., PCMR, 2017). As a result, we started a medicinal chemistry program aimed at developing new metformin-derived molecules with improved potency and suitable pharmacological profile to obtain a better response in patients. After an initial screening, an extensive structure-activity-based hit-to-lead optimization led us to identify a new potential candidate called CRO15. This molecule exerts anti-proliferative effects on the melanoma cells tested, including cells isolated from patients and cells resistant to BRAF inhibitors. In addition, CRO15 was able to decrease the cell viability of other cancer types. This compound acts by two separate different mechanisms. The first is AMPK pathway activation induced by mitochondrial disorder. The second mechanism involves inhibition of MELK kinase activity, which induces proliferation arrest and activation of DNA damage repair pathways by p53 and REDD1 activation. All of these mechanisms activate autophagic and apoptotic processes to induce melanoma cell death. CRO15 also exhibits strong activity in xenograft and allograft mice models with melanoma cells that are either sensitive or resistant to BRAF inhibitors (One international Patent 12336 and Manuscript in preparation).

4- Implication of E2F1 transcription factor in melanoma

E2F transcription factor family is known to regulate the expression of genes involved in cell cycle. Aberrant expression of E2F1 transcription factor has been found in high-grade tumors associated with poor prognosis including metastatic melanoma. Although E2F1 overexpression is found in many cancers, its role remains controversial. We showed that E2F1 is overexpressed in several melanoma cell lines and in melanoma cells freshly isolated from patients. We also found that E2F1 inhibition by RNA interference or a pharmacological inhibitor, leads to cell death through apoptosis, senescence characterized by a typical morphology, and biochemical changes associated with cell cycle arrest in G2/M. These mechanisms were shown to be dependant of p53 and p27 pathways resulting in generation of ROS and DNA damage. Moreover, we showed that p53 mutated cells are resistant to the effects induced by E2F1 inhibition. These data reinforce our hypothesis concerning the involvement of p53 in E2F1 effects, suggesting a potential role of E2F1 in the treatment of melanoma (Rouaud et al., CDDis, 2018).

B-PIGMENTATION

1-Role of the microvascularization on skin pigmentation

Histological studies have clearly shown a significant increase in vascularization within melasma lesions compared to that in the surrounding healthy skin, although the significance of this increased vascularization in melasma remained poorly understood. We therefore examined the role of dermal microvascular endothelial cells in regulating skin pigmentation. We first used high magnification digital epiluminescence dermatoscopy, laser confocal microscopy and histological examination on benign vascular proliferations in patients. Then we further studied the biological mechanisms involved using melanocytes or epidermal reconstructed epidermis cultured with microvascular endothelial cells. We showed that benign vascular lesions of the skin have a restricted but significant hyperpigmentation compared to the surrounding skin. Microvascular endothelial cells release endothelin 1 which induces the activation of endothelin receptor B at the surface of melanocytes. Downstream the MAPK ERK1/2 and p38 pathways are activated and lead to the phosphorylation of MITF and the increased expression of tyrosinase and DCT to finally increase pigmentation. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared to perilesional skin. Finally, culture of reconstructed skins with microvascular endothelial cells lead to an increased skin pigmentation that can be prevented by inhibiting the EDRNB activation.

Taken together these results demonstrated the role for underlying microvascularization in skin pigmentation (J Invest Dermatol. 2015;135:3096-104). These findings open up a new field of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma.

2-Vitiligo

We performed transcriptome analysis on lesional, perilesional, and non-depigmented skin from vitiligo patients and on matched skin from healthy subjects. We found a significant increase in CXCL10 in non-depigmented and perilesional vitiligo skin compared to levels in healthy control skin; however, neither CXCL10 nor other immune factors were deregulated in depigmented vitiligo skin. Interestingly, the WNT pathway, which is involved in melanocyte differentiation, was altered specifically in vitiligo skin. We demonstrated that oxidative stress decreases WNT expression/activation in keratinocytes and melanocytes. We developed an ex vivo skin model and confirmed the decrease activation of the WNT pathway in human skin subjected to oxidative stress. Finally, using pharmacological agents that activate the WNT pathway, we treated ex vivo depigmented skin from vitiligo patients and successfully induced differentiation of resident stem cells into pre-melanocytes. These results shed light on the previously unrecognized role of decreased WNT activation in the prevention of melanocyte differentiation in depigmented vitiligo skin. Furthermore, these results support further clinical exploration of WNT agonists to repigment vitiligo lesions (J Invest Dermatol. 2015;135:3105-14). These results are protected by two patents. Topical formulations of WNT agonists or GSK3b inhibitors will be further developed to enhance melanocyte differentiation and to repigment vitiligo lesions.

T-cells play a crucial role in progression of vitiligo however initial steps in triggering melanocyte death remain unknown. Using blood and skin samples from vitiligo patients and healthy volunteers, we demonstrated increased presence of type-1 innate lymphoid cells (NK and ILC1)-producing interferon gamma (IFNg) in the blood and in non-lesional skin of vitiligo patients. Melanocytes of vitiligo patients have strong basal expression of chemokine-receptor-3 (CXCR3) isoform B which is directly regulated by IFNg. CXCR3B activation by CXCL10 at the surface of melanocytes induces their initial apoptosis. The remaining melanocytes, activated by the IFNg production, express co-stimulatory markers which trigger T-cell proliferation and subsequent anti-melanocytic immunity. Inhibiting the CXCR3B activation prevents this initial apoptosis and the further activation of T cells. Our results emphasize the key role of CXCR3B in the initial apoptosis of melanocytes and identify CXCR3B as a new target to prevent and to treat vitiligo by acting at the very early steps in their destruction (results submitted).

3-Visible light in pigmentation

Acquired facultative pigmentation also called tan is achieved after sun exposure. The impact of ultraviolet (UV) B and A is now well demonstrated. More recently, irradiation of the skin of healthy volunteers with visible light showed a potent and long lasting hyperpigmentation. However, data on other cell types previously shown that the effect of the different wavelengths of visible light can produce different and sometimes opposite biologic effects. Thus we studied in healthy volunteers’ skin the impact of blue-violet (415nm) and red (630nm) light. We demonstrated that blue light induces a potent and long lasting hyperpigmentation using ‘physiological’ irradiances that are delivered by following 1h30 to 3h of sun exposure during summer period while the red light does not induce any pigmentation. Histological study showed a significant increase of sunburn cells and p53 positive cells after UVB but not after blue light exposure. The conclusions are that the shorter wavelengths of the visible spectrum are responsible for the visible-induced pigmentation and that the processes involved are different from those involved in UV-induced pigmentation (Pigment Cell Melanoma Res. 2014;27:822-6). Furthermore, we have demonstrated in a clinical trial that sunscreen having protection against UVA/UVB and against the shorter wavelengths of visible light provides statistically significant improved protection against melasma relapses compared to the same sunscreen with UVA/UVB protection but devoid of protection against visible light (J Am Acad Dermatol. 2015;72:189-90). This sunscreen is now commercialized. More recently, we dissected the pathway involved in visible light-induced hyperpigmentation. We showed that melanocytes sense the blue light with a specific senor called OPSIN3. We demonstrated all the downstream pathway and explained why dark-skinned individuals are more prone to develop hyperpigmentation after blue light exposure. We also showed that after irradiation, tyrosinase complexes are formed leading to a stabilization of tyrosinase activity, thus explaining why visible light induces such a long-lasting hyperpigmentation (J Invest Dermatol. 2018 Jan;138(1):171-178).  Further works on the impact of visible light on the skin are ongoing.

Team Publications