Mapping the global interactome of the ARF family reveals spatial organization in cellular signaling pathways.

The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ∼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.

The Deubiquitylase Otub1 Regulates the Chemotactic Response of Splenic B Cells by Modulating the Stability of the γ-Subunit Gng2.

The ubiquitin proteasome system performs the covalent attachment of lysine 48-linked polyubiquitin chains to substrate proteins, thereby targeting them for degradation, while deubiquitylating enzymes (DUBs) reverse this process. This posttranslational modification regulates key features both of innate and adaptative immunity, including antigen presentation, protein homeostasis and signal transduction. Here we show that loss of one of the most highly expressed DUBs, Otub1, results in changes in murine splenic B cell subsets, leading to a significant increase in marginal zone and transitional B cells and a concomitant decrease in follicular B cells. We demonstrate that Otub1 interacts with the γ-subunit of the heterotrimeric G protein, Gng2, and modulates its ubiquitylation status, thereby controlling Gng2 stability. Proximal mapping of Gng2 revealed an enrichment in partners associated with chemokine signaling, actin cytoskeleton and cell migration. In line with these findings, we show that Otub1-deficient B cells exhibit greater Ca2+ mobilization, F-actin polymerization and chemotactic responsiveness to Cxcl12, Cxcl13 and S1P in vitro, which manifests in vivo as altered localization of B cells within the spleen. Together, our data establishes Otub1 as a novel regulator of G-protein coupled receptor signaling in B cells, regulating their differentiation and positioning in the spleen.

Mapping the global interactome of the ARF family reveals spatial organization in cellular signaling pathways.

The ADP-ribosylation factors (ARFs) and ARF-like (ARLs) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we utilized proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ~3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.

Second TURB, restaging TURB or repeat TURB in primary T1 non-muscle invasive bladder cancer: impact on prognosis?

PURPOSE: A re-transurethral resection of the bladder (re-TURB) is a well-established approach in managing non-muscle invasive bladder cancer (NMIBC) for various reasons: repeat-TURB is recommended for a macroscopically incomplete initial resection, restaging-TURB is required if the first resection was macroscopically complete but contained no detrusor muscle (DM) and second-TURB is advised for all completely resected T1-tumors with DM in the resection specimen. This study assessed the long-term outcomes after repeat-, second-, and restaging-TURB in T1-NMIBC patients. METHODS: Individual patient data with tumor characteristics of 1660 primary T1-patients (muscle-invasion at re-TURB omitted) diagnosed from 1990 to 2018 in 17 hospitals were analyzed. Time to recurrence, progression, death due to bladder cancer (BC), and all causes (OS) were visualized with cumulative incidence functions and analyzed by log-rank tests and multivariable Cox-regression models stratified by institution. RESULTS: Median follow-up was 45.3 (IQR 22.7-81.1) months. There were no differences in time to recurrence, progression, or OS between patients undergoing restaging (135 patients), second (644 patients), or repeat-TURB (84 patients), nor between patients who did or who did not undergo second or restaging-TURB. However, patients who underwent repeat-TURB had a shorter time to BC death compared to those who had second- or restaging-TURB (multivariable HR 3.58, P = 0.004). CONCLUSION: Prognosis did not significantly differ between patients who underwent restaging- or second-TURB. However, a worse prognosis in terms of death due to bladder cancer was found in patients who underwent repeat-TURB compared to second-TURB and restaging-TURB, highlighting the importance of separately evaluating different indications for re-TURB.

CdGAP is a talin-binding protein and a target of TGF-ß signaling that promotes HER2-positive breast cancer growth and metastasis.

Epithelial-to-mesenchymal transition (EMT) plays a crucial role in metastasis, which is the leading cause of death in breast cancer patients. Here, we show that Cdc42 GTPase-activating protein (CdGAP) promotes tumor formation and metastasis to lungs in the HER2-positive (HER2+) murine breast cancer model. CdGAP facilitates intravasation, extravasation, and growth at metastatic sites. CdGAP depletion in HER2+ murine primary tumors mediates crosstalk with a Dlc1-RhoA pathway and is associated with a transforming growth factor ß (TGF-ß)-induced EMT transcriptional signature. CdGAP is positively regulated by TGF-ß signaling during EMT and interacts with the adaptor talin to modulate focal adhesion dynamics and integrin activation. Moreover, HER2+ breast cancer patients with high CdGAP mRNA expression combined with a high TGF-ß-EMT signature are more likely to present lymph node invasion. Our results suggest CdGAP as a candidate therapeutic target for HER2+ metastatic breast cancer by inhibiting TGF-ß and integrin/talin signaling pathways.

FIRRM cooperates with FIGNL1 to promote RAD51 disassembly during DNA repair.

Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator of recombination and mitosis (FIRRM) as a sensitizer of the ICL-inducing agent mafosfamide. Mechanistically, we showed that FIRRM, like its interactor Fidgetin like 1 (FIGNL1), contributes to the resolution of RAD51 foci at ICL-induced DSBs. While the stability of FIGNL1 and FIRRM is interdependent, expression of a mutant of FIRRM (∆WCF), which stabilizes the protein in the absence of FIGNL1, allows the resolution of RAD51 foci and cell survival, suggesting that FIRRM has FIGNL1-independent function during DNA repair. In line with this model, FIRRM binds preferentially single-stranded DNA in vitro, raising the possibility that it directly contributes to RAD51 disassembly by interacting with DNA. Together, our findings establish FIRRM as a promoting factor of ICL repair.

Clinical Assessment of Judgment in Adults and the Elderly: Development and Validation of the Three Domains of Judgment Test-Clinical Version (3DJT-CV).

(1) Background: This article discusses the first two phases of development and validation of the Three Domains of Judgment Test (3DJT). This computer-based tool, co-constructed with users and capable of being administered remotely, aims to assess the three main domains of judgment (practical, moral, and social) and learn from the psychometric weaknesses of tests currently used in clinical practice. (2) Method: First, we presented the 3DJT to experts in cognition, who evaluated the tool as a whole as well as the content validity, relevance, and acceptability of 72 scenarios. Second, an improved version was administered to 70 subjects without cognitive impairment to select scenarios with the best psychometric properties in order to build a future clinically short version of the test. (3) Results: Fifty-six scenarios were retained following expert evaluation. Results support the idea that the improved version has good internal consistency, and the concurrent validity primer shows that 3DJT is a good measure of judgment. Furthermore, the improved version was found to have a significant number of scenarios with good psychometric properties to prepare a clinical version of the test. (4) Conclusion: The 3DJT is an interesting alternative tool for assessing judgment. However, more studies are needed for its implementation in a clinical context.

Coordination of non-professional efferocytosis and actomyosin contractility during epithelial tissue morphogenesis.

In developing embryos, specific cell populations are often removed to remodel tissue architecture for organogenesis. During urinary tract development, an epithelial duct called the common nephric duct (CND) gets shortened and eventually eliminated to remodel the entry point of the ureter into the bladder. Here we show that non-professional efferocytosis (the process in which epithelial cells engulf apoptotic bodies) is the main mechanism that contributes to CND shortening. Combining biological metrics and computational modeling, we show that efferocytosis with actomyosin contractility are essential factors that drive the CND shortening without compromising the ureter-bladder structural connection. The disruption of either apoptosis, non-professional efferocytosis, or actomyosin results in contractile tension reduction and deficient CND shortening. Actomyosin activity helps to maintain tissue architecture while non-professional efferocytosis removes cellular volume. Together our results demonstrate that non-professional efferocytosis with actomyosin contractility are important morphogenetic factors controlling CND morphogenesis.

Prognosis of Primary Papillary Ta Grade 3 Bladder Cancer in the Non-muscle-invasive Spectrum.

BACKGROUND: Ta grade 3 (G3) non-muscle-invasive bladder cancer (NMIBC) is a relatively rare diagnosis with an ambiguous character owing to the presence of an aggressive G3 component together with the lower malignant potential of the Ta component. The European Association of Urology (EAU) NMIBC guidelines recently changed the risk stratification for Ta G3 from high risk to intermediate, high, or very high risk. However, prognostic studies on Ta G3 carcinomas are limited and inconclusive. OBJECTIVE: To evaluate the prognostic value of categorizing Ta G3 compared to Ta G2 and T1 G3 carcinomas. DESIGN, SETTING, AND PARTICIPANTS: Individual patient data for 5170 primary Ta-T1 bladder tumors from 17 hospitals were analyzed. Transurethral resection of the tumor was performed between 1990 and 2018. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Time to recurrence and time to progression were analyzed using cumulative incidence functions, log-rank tests, and multivariable Cox-regression models with interaction terms stratified by institution. RESULTS AND LIMITATIONS: Ta G3 represented 7.5% (387/5170) of Ta-T1 carcinomas of which 42% were classified as intermediate risk. Time to recurrence did not differ between Ta G3 and Ta G2 (p = 0.9) or T1 G3 (p = 0.4). Progression at 5 yr occurred for 3.6% (95% confidence interval [CI] 2.7-4.8%) of Ta G2, 13% (95% CI 9.3-17%) of Ta G3, and 20% (95% CI 17-23%) of T1 G3 carcinomas. Time to progression for Ta G3 was shorter than for Ta G2 (p < 0.001) and longer than for T1 G3 (p = 0.002). Patients with Ta G3 NMIBC with concomitant carcinoma in situ (CIS) had worse prognosis and a similar time to progression as for patients with T1 G3 NMIBC with CIS (p = 0.5). Multivariable analyses for recurrence and progression showed similar results. CONCLUSIONS: The prognosis of Ta G3 tumors in terms of progression appears to be in between that of Ta G2 and T1 G3. However, patients with Ta G3 NMIBC with concomitant CIS have worse prognosis that is comparable to that of T1 G3 with CIS. Our results support the recent EAU NMIBC guideline changes for more refined risk stratification of Ta G3 tumors because many of these patients have better prognosis than previously thought. PATIENT SUMMARY: We used data from 17 centers in Europe and Canada to assess the prognosis for patients with stage Ta grade 3 (G3) non-muscle-invasive bladder cancer (NMIBC). Time to cancer progression for Ta G3 cancer differed from both Ta G2 and T1 G3 tumors. Our results support the recent change in the European Association of Urology guidelines for more refined risk stratification of Ta G3 NMIBC because many patients with this tumor have better prognosis than previously thought.

Structural biology of DOCK-family guanine nucleotide exchange factors.

DOCK proteins are a family of multi-domain guanine nucleotide exchange factors (GEFs) that activate the RHO GTPases CDC42 and RAC1, thereby regulating several RHO GTPase-dependent cellular processes. DOCK proteins are characterized by the catalytic DHR2 domain (DOCKDHR2 ), and a phosphatidylinositol(3,4,5)P3 -binding DHR1 domain (DOCKDHR1 ) that targets DOCK proteins to plasma membranes. DOCK-family GEFs are divided into four subfamilies (A to D) differing in their specificities for CDC42 and RAC1, and the composition of accessory signalling domains. Additionally, the DOCK-A and DOCK-B subfamilies are constitutively associated with ELMO proteins that auto-inhibit DOCK GEF activity. We review structural studies that have provided mechanistic insights into DOCK-protein functions. These studies revealed how a conserved nucleotide sensor in DOCKDHR2 catalyses nucleotide exchange, the basis for how different DOCK proteins activate specifically CDC42 and RAC1, and sometimes both, and how up-stream regulators relieve the ELMO-mediated auto-inhibition. We conclude by presenting a model for full-length DOCK9 of the DOCK-D subfamily. The involvement of DOCK GEFs in a range of diseases highlights the importance of gaining structural insights into these proteins to better understand and specifically target them.

Biasing the conformation of ELMO2 reveals that myoblast fusion can be exploited to improve muscle regeneration.

Myoblast fusion is fundamental for the development of multinucleated myofibers. Evolutionarily conserved proteins required for myoblast fusion include RAC1 and its activator DOCK1. In the current study we analyzed the contribution of the DOCK1-interacting ELMO scaffold proteins to myoblast fusion. When Elmo1-/- mice underwent muscle-specific Elmo2 genetic ablation, they exhibited severe myoblast fusion defects. A mutation in the Elmo2 gene that reduced signaling resulted in a decrease in myoblast fusion. Conversely, a mutation in Elmo2 coding for a protein with an open conformation increased myoblast fusion during development and in muscle regeneration. Finally, we showed that the dystrophic features of the Dysferlin-null mice, a model of limb-girdle muscular dystrophy type 2B, were reversed when expressing ELMO2 in an open conformation. These data provide direct evidence that the myoblast fusion process could be exploited for regenerative purposes and improve the outcome of muscle diseases.

Systematic proximal mapping of the classical RAD51 paralogs unravel functionally and clinically relevant interactors for genome stability.

Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC.

Breast Cancer Metastatic Dormancy and Relapse: An Enigma of Microenvironment(s).

Multiple factors act in concert to define the fate of disseminated tumor cells (DTC) to enter dormancy or develop overt metastases. Here, we review these factors in the context of three stages of the metastatic cascade that impact DTCs. First, cells can be programmed within the primary tumor microenvironment to promote or inhibit dissemination, and the primary tumor can condition a premetastatic niche. Then, cancer cells from the primary tumor spread through hematogenous and lymphatic routes, and the primary tumor sends cues systematically to regulate the fate of DTCs. Finally, DTCs home to their metastatic site, where they are influenced by various organ-specific aspects of the new microenvironment. We discuss these factors in the context of breast cancer, where about one-third of patients develop metastatic relapse. Finally, we discuss how the standard-of-care options for breast cancer might affect the fate of DTCs.

T1G1 Bladder Cancer: Prognosis for this Rare Pathological Diagnosis Within the Non-muscle-invasive Bladder Cancer Spectrum.

BACKGROUND: The pathological existence and clinical consequence of stage T1 grade 1 (T1G1) bladder cancer are the subject of debate. Even though the diagnosis of T1G1 is controversial, several reports have consistently found a prevalence of 2-6% G1 in their T1 series. However, it remains unclear if T1G1 carcinomas have added value as a separate category to predict prognosis within the non-muscle-invasive bladder cancer (NMIBC) spectrum. OBJECTIVE: To evaluate the prognostic value of T1G1 carcinomas compared to TaG1 and T1G2 carcinomas within the NMIBC spectrum. DESIGN, SETTING, AND PARTICIPANTS: Individual patient data for 5170 primary Ta and T1 bladder tumors from 17 hospitals in Europe and Canada were analyzed. Transurethral resection (TUR) was performed between 1990 and 2018. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Time to recurrence and progression were analyzed using cumulative incidence functions, log-rank tests, and multivariable Cox regression models stratified by institution. RESULTS AND LIMITATIONS: T1G1 represented 1.9% (99/5170) of all carcinomas and 5.3% (99/1859) of T1 carcinomas. According to primary TUR dates, the proportion of T1G1 varied between 0.9% and 3.5% per year, with similar percentages in the early and later calendar years. We found no difference in time to recurrence between T1G1 and TaG1 (p = 0.91) or between T1G1 and T1G2 (p = 0.30). Time to progression significantly differed between TaG1 and T1G1 (p < 0.001) but not between T1G1 and T1G2 (p = 0.30). Multivariable analyses for recurrence and progression showed similar results. CONCLUSIONS: The relative prevalence of T1G1 diagnosis was low and remained constant over the past three decades. Time to recurrence of T1G1 NMIBC was comparable to that for other stage/grade NMIBC combinations. Time to progression of T1G1 NMIBC was comparable to that for T1G2 but not for TaG1, suggesting that treatment and surveillance of T1G1 carcinomas should be more like the approaches for T1G2 NMIBC in accordance with the intermediate and/or high risk categories of the European Association of Urology NMIBC guidelines. PATIENT SUMMARY: Although rare, stage T1 grade 1 (T1G1) bladder cancer is still diagnosed in daily clinical practice. Using individual patient data from 17 centers in Europe and Canada, we found that time to progression of T1G1 cancer was comparable to that for T1G2 but not TaG1 cancer. Therefore, our results suggest that primary T1G1 bladder cancers should be managed with more aggressive treatment and more frequent follow-up than for low-risk bladder cancer.

Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entry.

At mitotic entry, reorganization of the actomyosin cortex prompts cells to round-up. Proteins of the ezrin, radixin, and moesin family (ERM) play essential roles in this process by linking actomyosin forces to the plasma membrane. Yet, the cell-cycle signal that activates ERMs at mitotic entry is unknown. By screening a compound library using newly developed biosensors, we discovered that drugs that disassemble microtubules promote ERM activation. We further demonstrated that disassembly of interphase microtubules at mitotic entry directs ERM activation and metaphase cell rounding through GEF-H1, a Rho-GEF inhibited by microtubule binding, RhoA, and its kinase effector SLK. We finally demonstrated that GEF-H1 and Ect2, another Rho-GEF previously identified to control actomyosin forces, act together to drive activation of ERMs and cell rounding in metaphase. In summary, we report microtubule disassembly as a cell-cycle signal that controls a signaling network ensuring that actomyosin forces are efficiently integrated at the plasma membrane to promote cell rounding at mitotic entry.

AXL Receptor Tyrosine Kinase as a Promising Therapeutic Target Directing Multiple Aspects of Cancer Progression and Metastasis.

The receptor tyrosine kinase AXL is emerging as a key player in tumor progression and metastasis and its expression correlates with poor survival in a plethora of cancers. While studies have shown the benefits of AXL inhibition for the treatment of metastatic cancers, additional roles for AXL in cancer progression are still being explored. This review discusses recent advances in understanding AXL's functions in different tumor compartments including cancer, vascular, and immune cells. AXL is required at multiple steps of the metastatic cascade where its activation in cancer cells leads to EMT, invasion, survival, proliferation and therapy resistance. AXL activation in cancer cells and various stromal cells also results in tumor microenvironment deregulation, leading to modulation of angiogenesis, fibrosis, immune response and hypoxia. A better understanding of AXL's role in these processes could lead to new therapeutic approaches that would benefit patients suffering from metastatic diseases.

Defining the interactomes of proteins involved in cytoskeletal dynamics using high-throughput proximity-dependent biotinylation in cellulo.

Proximity-dependent biotinylation (BioID) screens are excellent tools to capture in cellulo interactomes for a large variety of baits, including transient and weak affinity interactions, as well as localization-specific proximity components, which are much harder to detect with conventional approaches. Here, we describe the major starting steps and a detailed protocol on how to perform BioID in mammalian cells. We also describe the mass spectrometry procedure and the bioinformatics pipeline for the data analysis. For complete details on the use and execution of this profile, please refer to Bagci et al. (2020).