EGF-SNX3-EGFR axis drives tumor progression and metastasis in triple-negative breast cancers

Epidermal growth factor receptor (EGFR) has critical roles in epithelial cell physiology. Over-expression and over-activation of EGFR have been implicated in diverse cancers, including triple-negative breast cancers (TNBCs), prompting anti-EGFR therapies. Therefore, developing potent therapies and addressing the inevitable drug resistance mechanisms necessitates deciphering of EGFR related networks. Here, we describe Sorting Nexin 3 (SNX3), a member of the recycling retromer complex, as a critical player in the epidermal growth factor (EGF) stimulated EGFR network in TNBCs. We show that SNX3 is an immediate and sustained target of EGF stimulation initially at the protein level and later at the transcriptional level, causing increased SNX3 abundance. Using a proximity labeling approach, we observed increased interaction of SNX3 and EGFR upon EGF stimulation. We also detected colocalization of SNX3 with early endosomes and endocytosed EGF. Moreover, we show that EGFR protein levels are sensitive to SNX3 loss. Transient RNAi models of SNX3 downregulation have a temporary reduction in EGFR levels. In contrast, long-term silencing forces cells to recover and overexpress EGFR mRNA and protein, resulting in increased proliferation, colony formation, migration, invasion in TNBC cells, and increased tumor growth and metastasis in syngeneic models. Consistent with these results, low SNX3 and high EGFR mRNA levels correlate with poor relapse-free survival in breast cancer patients. Overall, our results suggest that SNX3 is a critical player in the EGFR network in TNBCs with implications for other cancers dependent on EGFR activity.Chemical Immunolog

GYNOCARE Update: Modern Strategies to Improve Diagnosis and Treatment of Rare Gynecologic Tumors—Current Challenges and Future Directions

More than 50% of all gynecologic tumors can be classified as rare (defined as an incidence of ≤6 per 100,000 women) and usually have a poor prognosis owing to delayed diagnosis and treatment. In contrast to almost all other common solid tumors, the treatment of rare gynecologic tumors (RGT) is often based on expert opinion, retrospective studies, or extrapolation from other tumor sites with similar histology, leading to difficulty in developing guidelines for clinical practice. Currently, gynecologic cancer research, due to distinct scientific and technological challenges, is lagging behind. Moreover, the overall efforts for addressing these challenges are fragmented across different European countries and indeed, worldwide. The GYNOCARE, COST Action CA18117 (European Network for Gynecological Rare Cancer Research) programme aims to address these challenges through the creation of a unique network between key stakeholders covering distinct domains from concept to cure: basic research on RGT, biobanking, bridging with industry, and setting up the legal and regulatory requirements for international innovative clinical trials. On this basis, members of this COST Action, (Working Group 1, “Basic and Translational Research on Rare Gynecological Cancer”) have decided to focus their future efforts on the development of new approaches to improve the diagnosis and treatment of RGT. Here, we provide a brief overview of the current state-of-the-art and describe the goals of this COST Action and its future challenges with the aim to stimulate discussion and promote synergy across scientists engaged in the fight against this rare cancer worldwide

ALCAM is indirectly modulated by miR-125b in MCF7 cells

MicroRNA (miRNA) deregulation is associated with various cancers. Among an expanding list of cancer-related miRNAs, deregulation of miR-125b has been well documented in many cancers including breast. Based on current knowledge, miR-125b is considered to be a tumor suppressor in breast cancers. While important messenger RNA (mRNA) targets have been defined for miR-125b, here, we aimed to further investigate direct/indirect consequences of miR-125b expression in breast cancer cells by using a transcriptome approach. Upon miR-125b expression, a total of 138 cancer-related genes were found to be differentially expressed in breast cancer cells. While only a few of these were predicted to be direct mRNA targets, majority of the gene expression changes were potentially downstream and indirect effects of miR-125b expression. Among these, activated leukocyte antigen molecule (ALCAM) mRNA and protein levels were found to be highly significantly increased upon miR-125b expression. Given the tumor suppressor role of miR-125b in our model system, upon silencing of ALCAM expression, cell proliferation rate re-increased in miR-125b-expressing cells. While ALCAM's possible context-dependent roles are not clear in breast cancer, a diverse expression pattern of ALCAM mRNA was detected in a panel of breast cancer patient samples. Differentially expressed/regulated cancer-related genes upon miR-125b expression along with the significant increase of ALCAM are of future interest to understand how deregulated expression of miR-125b may have a tumor suppressor role in breast and other cancers

USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7

The endosomal system is a highly dynamic multifunctional organelle, whose complexity is regulated in part by reversible ubiquitylation. Despite the wide-ranging influence of ubiquitin in endosomal processes, relatively few enzymes utilizing ubiquitin have been described to control endosome integrity and function. Here we reveal the deubiquitylating enzyme (DUB) ubiquitin-specific protease 32 (USP32) as a powerful player in this context. Loss of USP32 inhibits late endosome (LE) transport and recycling of LE cargos, resulting in dispersion and swelling of the late compartment. Using SILAC-based ubiquitome profiling we identify the small GTPase Rab7—the logistical centerpiece of LE biology—as a substrate of USP32. Mechanistic studies reveal that LE transport effector RILP prefers ubiquitylation-deficient Rab7, while retromer-mediated LE recycling benefits from an intact cycle of Rab7 ubiquitylation. Collectively, our observations suggest that reversible ubiquitylation helps switch Rab7 between its various functions, thereby maintaining global spatiotemporal order in the endosomal system

USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7

The endosomal system is a highly dynamic multifunctional organelle, whose complexity is regulated in part by reversible ubiquitylation. Despite the wide-ranging influence of ubiquitin in endosomal processes, relatively few enzymes utilizing ubiquitin have been described to control endosome integrity and function. Here we reveal the deubiquitylating enzyme (DUB) ubiquitin-specific protease 32 (USP32) as a powerful player in this context. Loss of USP32 inhibits late endosome (LE) transport and recycling of LE cargos, resulting in dispersion and swelling of the late compartment. Using SILAC-based ubiquitome profiling we identify the small GTPase Rab7—the logistical centerpiece of LE biology—as a substrate of USP32. Mechanistic studies reveal that LE transport effector RILP prefers ubiquitylation-deficient Rab7, while retromer-mediated LE recycling benefits from an intact cycle of Rab7 ubiquitylation. Collectively, our observations suggest that reversible ubiquitylation helps switch Rab7 between its various functions, thereby maintaining global spatiotemporal order in the endosomal system

Computational Prediction of Functional MicroRNA–mRNA Interactions

Proteins have a strong influence on the phenotype and their aberrant expression leads to diseases. MicroRNAs (miRNAs) are short RNA sequences which posttranscriptionally regulate protein expression. This regulation is driven by miRNAs acting as recognition sequences for their target mRNAs within a larger regulatory machinery. A miRNA can have many target mRNAs and an mRNA can be targeted by many miRNAs which makes it difficult to experimentally discover all miRNA–mRNA interactions. Therefore, computational methods have been developed for miRNA detection and miRNA target prediction. An abundance of available computational tools makes selection difficult. Additionally, interactions are not currently the focus of investigation although they more accurately define the regulation than pre-miRNA detection or target prediction could perform alone. We define an interaction including the miRNA source and the mRNA target. We present computational methods allowing the investigation of these interactions as well as how they can be used to extend regulatory pathways. Finally, we present a list of points that should be taken into account when investigating miRNA–mRNA interactions. In the future, this may lead to better understanding of functional interactions which may pave the way for disease marker discovery and design of miRNA-based drugs