WCRC-25: A novel luminal Invasive Lobular Carcinoma cell line model.

Breast cancer is categorized by the molecular and histologic presentation of the tumor, with the major histologic subtypes being No Special Type (NST) and Invasive Lobular Carcinoma (ILC). ILC are characterized by growth in a single file discohesive manner with stromal infiltration attributed to their hallmark pathognomonic loss of E-cadherin ( CDH1 ). Few ILC cell line models are available to researchers. Here we report the successful establishment and characterization of a novel ILC cell line, WCRC-25, from a metastatic pleural effusion from a postmenopausal Caucasian woman with metastatic ILC. WCRC-25 is an ER-negative luminal epithelial ILC cell line with both luminal and Her2-like features. It exhibits anchorage independent growth and haptotactic migration towards Collagen I. Sequencing revealed a CDH1 Q706* truncating mutation, together with mutations in FOXA1, CTCF, BRCA2 and TP53 , which were also seen in a series of metastatic lesions from the patient. Copy number analyses revealed amplification and deletion of genes frequently altered in ILC while optical genome mapping revealed novel structural rearrangements. RNA-seq analysis comparing the primary tumor, metastases and the cell line revealed signatures for cell cycle progression and receptor tyrosine kinase signaling. To assess targetability, we treated WCRC-25 with AZD5363 and Alpelisib confirming WCRC-25 as susceptible to PI3K/AKT signaling inhibition as predicted by our RNA sequencing analysis. In conclusion, we report WCRC-25 as a novel ILC cell line with promise as a valuable research tool to advance our understanding of ILC and its therapeutic vulnerabilities. Financial support: The work was in part supported by a Susan G Komen Leadership Grant to SO (SAC160073) and NCI R01 CA252378 (SO/AVL). AVL and SO are Komen Scholars, Hillman Foundation Fellows and supported by BCRF. This project used the UPMC Hillman Cancer Center and Tissue and Research Pathology/Pitt Biospecimen Core shared resource which is supported in part by award P30CA047904. This research was also supported in part by the University of Pittsburgh Center for Research Computing, RRID:SCR_022735, through the resources provided. Specifically, this work used the HTC cluster, which is supported by NIH award number S10OD028483. Finally, partial support was provided by the Magee-Womens Research Institute and Foundation, The Shear Family Foundation, and The Metastatic Breast Cancer Network.

The role of tumor microenvironment and exosomes in dormancy and relapse.

Recent advancements in the field of cancer have established that the process of metastasis is organ-specific with tumor cell dissemination occurring in the very early stages of disease. Pre-metastatic niches are actively remodeled and transformed by both primary tumor specific factors and by influences from the extracellular matrix.Although improvements in cancer therapies have significantly improved outcomes in patients with early stage disease, the risk of recurrence and relapse leading to mortality remains high. Recent studies have emerged highlighting the influence of dormant tumor cells and exosomes as key players in cancer relapse. In this review we discuss the critical mediators of tumor progression and their link to cancer dormancy, while also exploring possible therapeutics for targeting relapse.

Peroxiredoxin-1 Tyr194 phosphorylation regulates LOX-dependent extracellular matrix remodelling in breast cancer.

BACKGROUND: Peroxiredoxin 1 (PRDX1) belongs to an abundant family of peroxidases whose role in cancer is still unresolved. While mouse knockout studies demonstrate a tumour suppressive role for PRDX1, in cancer cell xenografts, results denote PRDX1 as a drug target. Probably, this phenotypic discrepancy stems from distinct roles of PRDX1 in certain cell types or stages of tumour progression. METHODS: We demonstrate an important cell-autonomous function for PRDX1 utilising a syngeneic mouse model (BALB/c) and mammary fibroblasts (MFs) obtained from it. RESULTS: Loss of PRDX1 in vivo promotes collagen remodelling known to promote breast cancer progression. PRDX1 inactivation in MFs occurs via SRC-induced phosphorylation of PRDX1 TYR194 and not through the expected direct oxidation of CYS52 in PRDX1 by ROS. TYR194-phosphorylated PRDX1 fails to bind to lysyl oxidases (LOX) and leads to the accumulation of extracellular LOX proteins which supports enhanced collagen remodelling associated with breast cancer progression. CONCLUSIONS: This study reveals a cell type-specific tumour suppressive role for PRDX1 that is supported by survival analyses, depending on PRDX1 protein levels in breast cancer cohorts.

The peroxidase PRDX1 inhibits the activated phenotype in mammary fibroblasts through regulating c-Jun N-terminal kinases.

BACKGROUND: Reactive oxygen species (ROS), including hydrogen peroxide, drive differentiation of normal fibroblasts into activated fibroblasts, which can generate high amounts of hydrogen peroxide themselves, thereby increasing oxidative stress in the microenvironment. This way, activated fibroblasts can transition into cancer-associated fibroblasts (CAFs). METHODS: Mammary fibroblasts from either female 8 weeks old PRDX1 knockout and wildtype mice or Balb/c mice were studied for characteristic protein expression using immunofluorescence and immunoblotting. Cancer-associated fibroblasts was examined by transwell migration and invasion assays. The binding of PRDX1 to JNK1 was assessed by co-immuneprecipitation and JNK regulation of CAF phenotypes was examined using the JNK inhibitor SP600125. Extracellular hydrogen peroxide levels were measured by chemiluminescence via the reaction between hypochlorite and luminol. Statistical analyses were done using Students t-test. RESULTS: We show here PRDX1 activity as an essential switch in regulating the activated phenotype as loss of PRDX1 results in the development of a CAF-like phenotype in mammary fibroblasts. We also show that PRDX1 regulates JNK kinase signaling thereby inhibiting CAF-like markers and CAF invasion. Inhibition of JNK activity reduced these behaviors. CONCLUSIONS: These data suggest that PRDX1 repressed the activated phenotype of fibroblasts in part through JNK inhibition which may present a novel therapeutic option for CAF-enriched cancers such as breast cancer.

Signals Getting Crossed in the Entanglement of Redox and Phosphorylation Pathways: Phosphorylation of Peroxiredoxin Proteins Sparks Cell Signaling.

Reactive oxygen and nitrogen species have cell signaling properties and are involved in a multitude of processes beyond redox homeostasis. The peroxiredoxin (Prdx) proteins are highly sensitive intracellular peroxidases that can coordinate cell signaling via direct reactive species scavenging or by acting as a redox sensor that enables control of binding partner activity. Oxidation of the peroxidatic cysteine residue of Prdx proteins are the classical post-translational modification that has been recognized to modulate downstream signaling cascades, but increasing evidence supports that dynamic changes to phosphorylation of Prdx proteins is also an important determinant in redox signaling. Phosphorylation of Prdx proteins affects three-dimensional structure and function to coordinate cell proliferation, wound healing, cell fate and lipid signaling. The advent of large proteomic datasets has shown that there are many opportunities to understand further how phosphorylation of Prdx proteins fit into intracellular signaling cascades in normal or malignant cells and that more research is necessary. This review summarizes the Prdx family of proteins and details how post-translational modification by kinases and phosphatases controls intracellular signaling.

Influence of extruded lentil containing high chromium nutritional yeast on the main physiological factors associated with metabolic syndrome in rodent models.

Insulin resistance, obesity and dyslipidemia are the main physiological factors associated with metabolic syndrome. The objectives of this study were to understand the effects of diets containing extruded lentil fortified with high chromium nutritional yeast (YCr) or chromium picolinate on glucose tolerance, clearance and fasting blood glucose concentrations in Normal and Obese (Ob/Ob) mice and to determine the effects of the diets on the mice plasma lipid profiles. Diets A, B and C contained YCr in different doses and concentrations, as follows: Diet A = 15.7 g and 16 ppm, B = 157.1 g and 16 ppm, and C = 299.3 g and 27 ppm, respectively. Diet D contained chromium picolinate at a dose and concentration of 15.7 g and 16 ppm, respectively. Intraperitoneal glucose tolerance tests and intraperitoneal insulin tolerance tests were conducted at 4-weeks and 8-weeks post diet initiation, in addition to, plasma lipoprotein profiles and organ weights. Normal mice showed only slight variability with respect to the studied biological parameters compared to the Ob/Ob mice group. Results indicated that following 4-weeks of diet supplementation, Ob/Ob mice fed diets A, C and D had significantly (p < 0.05) lower fasting blood glucose (FBG) than Ob/Ob mice fed Diet B. However, after 8-weeks Ob/Ob mice fed Diet C, containing YCr, had a significantly (p < 0.05) lower FBG than mice supplemented with Diet D, containing chromium picolinate. Therefore, based on these findings, it was concluded that YCr at the highest concentration and dose was more effective than chromium picolinate. These results indicate that ready-to-eat snacks and breakfast cereal type products supplemented with chromium in the form of YCr could be used as vehicles for the amelioration of main physiological factors associated with metabolic syndrome.

The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

Senescent cells withdraw from the cell cycle and do not proliferate. The prevalence of senescent compared to normally functioning parenchymal cells increases with age, impairing tissue and organ homeostasis. A contentious principle governing this process has been the redox theory of aging. We linked matricellular protein thrombospondin 1 (TSP1) and its receptor CD47 to the activation of NADPH oxidase 1 (Nox1), but not of the other closely related Nox isoforms, and associated oxidative stress, and to senescence in human cells and aged tissue. In human endothelial cells, TSP1 promoted senescence and attenuated cell cycle progression and proliferation. At the molecular level, TSP1 increased Nox1-dependent generation of reactive oxygen species (ROS), leading to the increased abundance of the transcription factor p53. p53 mediated a DNA damage response that led to senescence through Rb and p21cip, both of which inhibit cell cycle progression. Nox1 inhibition blocked the ability of TSP1 to increase p53 nuclear localization and p21cip abundance and its ability to promote senescence. Mice lacking TSP1 showed decreases in ROS production, p21cip expression, p53 activity, and aging-induced senescence. Conversely, lung tissue from aging humans displayed increases in the abundance of vascular TSP1, Nox1, p53, and p21cip Finally, genetic ablation or pharmacological blockade of Nox1 in human endothelial cells attenuated TSP1-mediated ROS generation, restored cell cycle progression, and protected against senescence. Together, our results provide insights into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence and suggest potential targets for controlling the aging process at the molecular level.