A New TGF-ß Mimetic, XEP™-716 Miniprotein™, Exhibiting Regenerative Properties Objectivized by Instrumental Evaluation.

INTRODUCTION: Skin aging, which results from intrinsic and extrinsic factors, is characterized by a rough, uneven and wrinkled appearance of the skin at the macroscopic level. At the microscopic level, aging shows lowered keratinocyte turnover, flattened dermal-epidermal junction and reduced collagen fiber density; however, use of skin biopsies to evaluate characteristic properties of these microscopic changes is too limiting for panelists and rarely used. The development of non-invasive techniques is an opportunity to be considered for such evaluations. Our objective was to demonstrate the rejuvenating effects of XEP™-716 Miniprotein™ on skin, a miniprotein having TGF-ß beta-like properties, in vitro on normal human fibroblasts and at the clinical level. METHODS: In vitro, the skin rejuvenation properties of XEP™-716 Miniprotein™ were studied by quantification of well-known dermal components such as collagen type I, hyaluronic acid and elastin. At the clinical level, we used a non-invasive technique, the confocal laser scanning microscopy (CLSM) system, which enabled non-invasive morphological characterization of skin structures (stratum corneum thickness, viable epidermis, full epidermis, dermal-epidermal junction, papillae, dermal collagen density) and high-frequency ultrasonography to quantify the dermal density and thickness, which are useful parameters for quantifying rejuvenating effects on skin. Lastly, a cutometer was used to assess the skin's biomechanical properties, mainly firmness and elasticity. This monocentric double-blind, split-face, randomized, placebo-controlled clinical trial compared the active ingredient XEP™-716 Miniprotein™ in a vehicle on one hemiface versus vehicle alone on the other (placebo) and enrolled panelists aged 40 to 60 years old. All measurements were carried out on the malar area before and after 28 and 56 days of twice daily application of a cosmetic cream formulation containing either 2.5% or 5% XEP™-716 Miniprotein™. The skin rejuvenating properties were demonstrated by studying dermo-epidermal junction (DEJ) flattening reduction using the measure of two parameters by CLSM: the DEJ length and number of edged papillae. Dermis rejuvenation was assessed by measuring the collagen fiber perimeters (CLSM), dermal density and dermal thickness (ultrasonography). RESULTS: The in vitro results confirmed the ability of XEP™-716 Miniprotein™ to stimulate the key extracellular macromolecules, namely collagen type I, hyaluronic acid and elastin, at a level comparable to that induced by TGF beta growth factor. The clinical data showed that after 28 and 56 days of topical XEP™-716 Miniprotein™ application, there was a statistically significant increase of DEJ length, number of edged papillae and collagen fiber perimeters. At the same time point, the B-scan images of facial skin showed a statistically significant increase of dermal density and thickness. These results reveal that the DEJ became more undulated and tightly attached to the dermis, while the papillary dermis was densified, both traits being typical characteristic of younger skin. Rejuvenation was also confirmed by an improvement of skin firmness and elasticity. CONCLUSION: The in vitro and clinical results presented in this article show that XEP™-716 Miniprotein™ is a potent ingredient to rejuvenate the DEJ and dermis of mature skin.

Role of Sialyl-O-Acetyltransferase CASD1 on GD2 Ganglioside O-Acetylation in Breast Cancer Cells.

The O-acetylated form of GD2, almost exclusively expressed in cancerous tissues, is considered to be a promising therapeutic target for neuroectoderm-derived tumors, especially for breast cancer. Our recent data have shown that 9-O-acetylated GD2 (9-OAcGD2) is the major O-acetylated ganglioside species in breast cancer cells. In 2015, Baumann et al. proposed that Cas 1 domain containing 1 (CASD1), which is the only known human sialyl-O-acetyltransferase, plays a role in GD3 O-acetylation. However, the mechanisms of ganglioside O-acetylation remain poorly understood. The aim of this study was to determine the involvement of CASD1 in GD2 O-acetylation in breast cancer. The role of CASD1 in OAcGD2 synthesis was first demonstrated using wild type CHO and CHOΔCasd1 cells as cellular models. Overexpression using plasmid transfection and siRNA strategies was used to modulate CASD1 expression in SUM159PT breast cancer cell line. Our results showed that OAcGD2 expression was reduced in SUM159PT that was transiently depleted for CASD1 expression. Additionally, OAcGD2 expression was increased in SUM159PT cells transiently overexpressing CASD1. The modulation of CASD1 expression using transient transfection strategies provided interesting insights into the role of CASD1 in OAcGD2 and OAcGD3 biosynthesis, and it highlights the importance of further studies on O-acetylation mechanisms.

Profiling of O-acetylated Gangliosides Expressed in Neuroectoderm Derived Cells.

The expression and biological functions of oncofetal markers GD2 and GD3 were extensively studied in neuroectoderm-derived cancers in order to characterize their potential as therapeutic targets. Using immunological approaches, we previously identified GD3, GD2, and OAcGD2 expression in breast cancer (BC) cell lines. However, antibodies specific for O-acetylated gangliosides are not exempt of limitations, as they only provide information on the expression of a limited set of O-acetylated ganglioside species. Consequently, the aim of the present study was to use structural approaches in order to apprehend ganglioside diversity in melanoma, neuroblastoma, and breast cancer cells, focusing on O-acetylated species that are usually lost under alkaline conditions and require specific analytical procedures. We used purification and extraction methods that preserve the O-acetyl modification for the analysis of native gangliosides by MALDI-TOF. We identified the expression of GM1, GM2, GM3, GD2, GD3, GT2, and GT3 in SK-Mel28 (melanoma), LAN-1 (neuroblastoma), Hs 578T, SUM 159PT, MDA-MB-231, MCF-7 (BC), and BC cell lines over-expressing GD3 synthase. Among O-acetylated gangliosides, we characterized the expression of OAcGM1, OAcGD3, OAcGD2, OAcGT2, and OAcGT3. Furthermore, the experimental procedure allowed us to clearly identify the position of the sialic acid residue that carries the O-acetyl group on b- and c-series gangliosides by MS/MS fragmentation. These results show that ganglioside O-acetylation occurs on both inner and terminal sialic acid residue in a cell type-dependent manner, suggesting different O-acetylation pathways for gangliosides. They also highlight the limitation of immuno-detection for the complete identification of O-acetylated ganglioside profiles in cancer cells.

Identification of 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) as main O-acetylated sialic acid species of GD2 in breast cancer cells.

Mainly restricted to the nervous system in healthy adults, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. Interestingly, O-acetylated forms of GD2, not expressed in human peripheral nerve fibers, are highly expressed in GD2+ tumor cells. Very little information is known regarding the expression of O-acetylated disialogangliosides in breast cancer (BC) cell lines. Here, we analyzed the expression of GD2, GD3 and their O-acetylated forms O-acetyl-GD2 (OAcGD2) and O-acetyl-GD3 (OAcGD3) in BC cells. We used Hs 578T and SUM159PT cell lines, as well as cell clones over-expressing GD3 synthase derived from MDA-MB-231 and MCF-7. Using flow cytometry and immunocytochemistry/confocal microscopy, we report that BC cells express b-series gangliosides GD3 and GD2, as well as significant amounts of OAcGD2. However, OAcGD3 expression was not detected in these cells. O-acetylation of gangliosides isolated from BC cells was examined by LC-MS analysis of sialic acid DMB-derivatives. We report that the main acetylated form of sialic acid expressed in BC gangliosides is 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). These results highlight a close interrelationship between Neu5,9Ac2 and OAcGD2 expression, and suggest that OAcGD2 is synthetized from GD2 and not from OAcGD3 in BC cells.

Gene expression profile predicts outcome after anthracycline-based adjuvant chemotherapy in early breast cancer.

Prognosis of early beast cancer is heterogeneous. Today, no histoclinical or biological factor predictive for clinical outcome after adjuvant anthracycline-based chemotherapy (CT) has been validated and introduced in routine use. Using DNA microarrays, we searched for a gene expression signature associated with metastatic relapse after adjuvant anthracycline-based CT without taxane. We profiled a multicentric series of 595 breast cancers including 498 treated with such adjuvant CT. The identification of the prognostic signature was done using a metagene-based supervised approach in a learning set of 323 patients. The signature was then tested on an independent validation set comprising 175 similarly treated patients, 128 of them from the PACS01 prospective clinical trial. We identified a 3-metagene predictor of metastatic relapse in the learning set, and confirmed its independent prognostic impact in the validation set. In multivariate analysis, the predictor outperformed the individual current prognostic factors, as well as the Nottingham Prognostic Index-based classifier, both in the learning and the validation sets, and added independent prognostic information. Among the patients treated with adjuvant anthracycline-based CT, with a median follow-up of 68 months, the 5-year metastasis-free survival was 82% in the "good-prognosis" group and 56% in the "poor-prognosis" group. Our predictor refines the prediction of metastasis-free survival after adjuvant anthracycline-based CT and might help tailoring adjuvant CT regimens.