Differentiation-related epigenomic changes define clinically distinct keratinocyte cancer subclasses.

Keratinocyte cancers (KC) are the most prevalent malignancies in fair-skinned populations, posing a significant medical and economic burden to health systems. KC originate in the epidermis and mainly comprise basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). Here, we combined single-cell multi-omics, transcriptomics, and methylomics to investigate the epigenomic dynamics during epidermal differentiation. We identified ~3,800 differentially accessible regions between undifferentiated and differentiated keratinocytes, corresponding to regulatory regions associated with key transcription factors. DNA methylation at these regions defined AK/cSCC subtypes with epidermal stem cell- or keratinocyte-like features. Using cell-type deconvolution tools and integration of bulk and single-cell methylomes, we demonstrate that these subclasses are consistent with distinct cells-of-origin. Further characterization of the phenotypic traits of the subclasses and the study of additional unstratified KC entities uncovered distinct clinical features for the subclasses, linking invasive and metastatic KC cases with undifferentiated cells-of-origin. Our study provides a thorough characterization of the epigenomic dynamics underlying human keratinocyte differentiation and uncovers novel links between KC cells-of-origin and their prognosis.

Identification of eusynstyelamide B as a potent cell cycle inhibitor following the generation and screening of an ascidian-derived extract library using a real time cell analyzer.

Ascidians are marine invertebrates that have been a source of numerous cytotoxic compounds. Of the first six marine-derived drugs that made anticancer clinical trials, three originated from ascidian specimens. In order to identify new anti-neoplastic compounds, an ascidian extract library (143 samples) was generated and screened in MDA-MB-231 breast cancer cells using a real-time cell analyzer (RTCA). This resulted in 143 time-dependent cell response profiles (TCRP), which are read-outs of changes to the growth rate, morphology, and adhesive characteristics of the cell culture. Twenty-one extracts affected the TCRP of MDA-MB-231 cells and were further investigated regarding toxicity and specificity, as well as their effects on cell morphology and cell cycle. The results of these studies were used to prioritize extracts for bioassay-guided fractionation, which led to the isolation of the previously identified marine natural product, eusynstyelamide B (1). This bis-indole alkaloid was shown to display an IC50 of 5 µM in MDA-MB-231 cells. Moreover, 1 caused a strong cell cycle arrest in G2/M and induced apoptosis after 72 h treatment, making this molecule an attractive candidate for further mechanism of action studies.

Pteridine-, thymidine-, choline- and imidazole-derived alkaloids from the Australian ascidian, Leptoclinides durus.

Four new acylated pteridine alkaloids, duramidines A-D, two new acylated thymidine alkaloids, leptoclinidines A and B, two new 1-acylglyceryl-3-(O-carboxyhydroxymethylcholine) alkaloids, durabetaines A and B, three new 1,3-dimethyl-5-methylsulfanylimidazole alkaloids, leptoclinidamines D-F, and the known alkaloids leptoclinidamines B and C and 6-bromo-1H-indolo-3-yl-oxoacetic acid methyl ester were isolated from the Australian ascidian Leptoclinides durus. The duramidines are the first pteridine alkaloids, possessing a three carbon side chain esterified at C-1' with a 4-hydroxy-2'-methoxycinnamic acid, and are either hydroxylated or sulfated at C-2'. The leptoclinidines are the first 3'-indole-3-carboxylic acid ester derivatives of thymidine to be reported in the literature. The durabetaines are the first glyceryl-3-(O-carboxyhydroxymethylcholine) alkaloids to be reported from an animal source and are also the only known derivatives from this class to be acylated with aromatic carboxylic acids. MS and NMR data analysis established the structures of the new compounds. All compounds were shown to be inactive when tested for cytotoxic activity against prostate (LNCaP) and breast (MDA-MB-231) cancer cell lines and antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus.

Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells.

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.

Leptin antagonism inhibits prostate cancer xenograft growth and progression.

Hyperleptinaemia is a well-established therapeutic side effect of drugs inhibiting the androgen axis in prostate cancer (PCa), including main stay androgen deprivation therapy (ADT) and androgen targeted therapies (ATT). Given significant crossover between the adipokine hormone signalling of leptin and multiple cancer-promoting hallmark pathways, including growth, proliferation, migration, angiogenesis, metabolism and inflammation, targeting the leptin axis is therapeutically appealing, especially in advanced PCa where current therapies fail to be curative. In this study, we uncover leptin as a novel universal target in PCa and are the first to highlight increased intratumoural leptin and leptin receptor (LEPR) expression in PCa cells and patients' tumours exposed to androgen deprivation, as is observed in patients' tumours of metastatic and castrate resistant (CRPC) PCa. We also reveal the world-first preclinical evidence that demonstrates marked efficacy of targeted leptin-signalling blockade, using Allo-aca, a potent, specific, and safe LEPR peptide antagonist. Allo-aca-suppressed tumour growth and delayed progression to CRPC in mice bearing LNCaP xenografts, with reduced tumour vascularity and altered pathways of apoptosis, transcription/translation, and energetics in tumours determined as potential mechanisms underpinning anti-tumour efficacy. We highlight LEPR blockade in combination with androgen axis inhibition represents a promising new therapeutic strategy vital in advanced PCa treatment.

The ascidian natural product eusynstyelamide B is a novel topoisomerase II poison that induces DNA damage and growth arrest in prostate and breast cancer cells.

As part of an anti-cancer natural product drug discovery program, we recently identified eusynstyelamide B (EB), which displayed cytotoxicity against MDA-MB-231 breast cancer cells (IC50 = 5 µM) and induced apoptosis. Here, we investigated the mechanism of action of EB in cancer cell lines of the prostate (LNCaP) and breast (MDA-MB-231). EB inhibited cell growth (IC50 = 5 µM) and induced a G2 cell cycle arrest, as shown by a significant increase in the G2/M cell population in the absence of elevated levels of the mitotic marker phospho-histone H3. In contrast to MDA-MB-231 cells, EB did not induce cell death in LNCaP cells when treated for up to 10 days. Transcript profiling and Ingenuity Pathway Analysis suggested that EB activated DNA damage pathways in LNCaP cells. Consistent with this, CHK2 phosphorylation was increased, p21CIP1/WAF1 was up-regulated and CDC2 expression strongly reduced by EB. Importantly, EB caused DNA double-strand breaks, yet did not directly interact with DNA. Analysis of topoisomerase II-mediated decatenation discovered that EB is a novel topoisomerase II poison.

Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior.

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells' characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-L-lysine, poly-L-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-L-lysine and poly-L-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.