Our data show that cells from the growing margin of keloid scars have a higher production of collagen I and III compared with other lesional sites. Additionally, temporal extension of cell passage affects collagen production. Clinically these findings may influence selection and interpretation of extended cell passage and provide future direction for lesional site-specific therapy in keloid scars.

IntroductionThe cytochrome P450 (CYP) enzymes are a class of heme-containing enzymes involved in phase I metabolism of a large number of xenobiotics. The CYP family member CYP2E1 metabolises many xenobiotics and pro-carcinogens, it is not just expressed in the liver but also in many other tissues such as the kidney, the lung, the brain, the gastrointestinal tract and the breast tissue. It is induced in several pathological conditions including cancer, obesity, and type II diabetes implying that this enzyme is implicated in other biological processes beyond its role in phase I metabolism. Despite the detailed description of the role of CYP2E1 in the liver, its functions in other tissues have not been extensively studied. In this study, we investigated the functional significance of CYP2E1 in breast carcinogenesis.MethodsCellular levels of reactive oxygen species (ROS) were measured by H2DCFDA (2 2.9.2 2′,7′-dichlorodihydrofluorescein diacetate) staining and autophagy was assessed by tracing the cellular levels of autophagy markers using western blot assays. The endoplasmic reticulum stress and the unfolded protein response (UPR) were detected by luciferase assays reflecting the splicing of mRNA encoding the X-box binding protein 1 (XBP1) transcription factor and cell migration was evaluated using the scratch wound assay. Gene expression was recorded with standard transcription assays including luciferase reporter and chromatin immunoprecipitation.ResultsEctopic expression of CYP2E1 induced ROS generation, affected autophagy, stimulated endoplasmic reticulum stress and inhibited migration in breast cancer cells with different metastatic potential and p53 status. Furthermore, evidence is presented indicating that CYP2E1 gene expression is under the transcriptional control of the p53 tumor suppressor.ConclusionsThese results support the notion that CYP2E1 exerts an important role in mammary carcinogenesis, provide a potential link between ethanol metabolism and breast cancer and suggest that progression, and metastasis, of advanced stages of breast cancer can be modulated by induction of CYP2E1 activity.

Mutations in leucine-rich-repeats and immunoglobulin-like-domains 2 (LRIG2) or in heparanase 2 (HPSE2) cause urofacial syndrome, a devastating autosomal recessive disease of functional bladder outlet obstruction. It has been speculated that urofacial syndrome has a neural basis, but it is unknown whether defects in urinary bladder innervation are present. We hypothesized that urofacial syndrome features a peripheral neuropathy of the bladder. Mice with homozygous targeted Lrig2 mutations had urinary defects resembling those found in urofacial syndrome. There was no anatomical blockage of the outflow tract, consistent with a functional bladder outlet obstruction. Transcriptome analysis revealed differential expression of 12 known transcripts in addition to Lrig2 , including 8 with established roles in neurobiology. Mice with homozygous mutations in either Lrig2 or Hpse2 had increased nerve density within the body of the urinary bladder and decreased nerve density around the urinary outflow tract. In a sample of 155 children with chronic kidney disease and urinary symptoms, we discovered novel homozygous missense LRIG2 variants that were predicted to be pathogenic in 2 individuals with non-syndromic bladder outlet obstruction. These observations provide evidence that a peripheral neuropathy is central to the pathobiology of functional bladder outlet obstruction in urofacial syndrome, and emphasize the importance of LRIG2 and heparanase 2 for nerve patterning in the urinary tract.

Dupuytren's disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH) - Xiaflex® - is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex® against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex® exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex® did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex® significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex® enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex® on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.

Aim:The aim of the study is to investigate the impact of the cytochrome P450 2E1, which is the most efficient CYP450 family member in generating reactive oxygen species (ROS), on cellular energy metabolism of breast cancer cells and therefore the effects of CYP2E1 on breast carcinogenesis. Methods: The estrogen receptor positive MCF-7 and the triple negative MDA-MB-231 breast cancer cells were used as experimental system to estimate ROS generation in these cells overexpressing CYP2E1 and treated with the glycolytic inhibitors 3-bromopyruvate or 2-deoxyglucose in the presence or absence of the CYP2E1 inhibitor chlormethiazole. Adenosine triphosphate (ATP) assay was used to measure ATP production and lactate assay to quantify the efflux of lactic acid in breast cancer cells treated with the CYP2E1 inhibitor chlormethiazole, the mitochondrial membrane potential and cell viability assays were employed to assess the pathway of cellular energy production and cellular death respectively after treatment of MCF-7 and MDA-MB-231 with the CYP2E1 activator acetaminophen or the CYP2E1 inhibitor chlormethiazole. Results:The results indicated increased ROS generation in breast cancer cells overexpressing CYP2E1. ROS generation was differentially regulated in breast cancer cells upon treatment with the CYP2E1 inhibitor chlormethiazole. Chlormethiazole treated MCF-7 cells exhibited reduced lactate efflux implying that CYP2E1 directly or indirectly regulates the glycolytic rate in these cells. Furthermore the mitochondrial membrane potential of both MCF-7 and MDA-MB-231 cells was differentially affected by the CYP2E1 activator acetaminophen versus the CYP2E1 inhibitor chlormethiazole providing additional support for the involvement of CYP2E1 in energy metabolic pathways in breast cancer. Conclusion: Results presented in this study provide evidence to suggest that CYP2E1 regulates cellular energy metabolism of breast cancer cells in a manner dependent on cell type and potentially on the clinical staging of the disease therefore CYP2E1 is a possible breast cancer biomarker.

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Female breast cancer (BC) became the most commonly diagnosed cancer globally in 2020. One of the hallmarks of BC is both intratumor and intertumor heterogeneity. Efforts have been made to categorize this heterogeneity based on the presence or absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2/ERBB2). Receptor expression patterns are used to stratify BC and the subsequent treatments. Much of the current knowledge of breast cancer comes from in vitro and in vivo studies using well established cell lines, some of which have been cultured since the 1950s. Given that serial passaging over many years has been reported to alter the mutational profile of cell lines and therefore their relevance to clinical decisions on the primary tumour, new near-tumor cell models need to be generated. In this study, tumour biopsies or ascitic fluids were obtained from a local cancer research biobank, as well as directly from metastatic BC patients across the globe. Using mechanical dissociation and cell separation techniques we isolated tumor cells and grew them in proprietary media. The resultant models were subjected to whole exome and RNA sequencing. In addition, the models were tested in a 3-day cell death assay against a library of more than 50 FDA-approved drugs. The models and four BC cell lines (HCC1937, ZR-75-1, MDA-MB-436 and HCC1428) were also tested in a longer term 7-day assay against a panel of four poly(ADP-ribose) polymerase (PARP) inhibitors. A total of 10 patient-derived cell (PDC) models were successfully established from biopsies or ascitic fluids taken from patients with primary (1/10) or metastatic (9/10) BC. Models were established from patients with ER+ BC, HER2+ BC and triple negative BC (TNBC) within an average of 23 days from sample arrival. All models displayed a greater sensitivity to the non-targeted drugs in the library compared with the targeted drugs. Mutations in breast cancer associated 1 (BRCA1) and BRCA2 genes that were not present in BC cell lines were identified in a subset of the PDCs. All models showed some sensitivity to PARP inhibitors, and most were exquisitely sensitive to talazoparib with IC50s of less than 100 nM. Determining cell death responses in near-tumour PDCs should thus be adopted to enable more informed clinical decisions for treatment pathways and in parallel will be promising tools for oncology drug discovery. Citation Format: Immaculate Nalubowa, Subir Singh, Yuen Ngan Fan, Rachel Howard-Jones, Albert Bezman, Dominic I. James, Geoff Muckle, Gareth J. Griffiths. Differential sensitivity to poly(ADP-ribose) polymerase inhibitors in patient-derived cell models of breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P119.