Phosphoproteomic Changes Induced by Cell-Derived Matrix and Their Effect on Tumor Cell Migration and Cytoskeleton Remodeling.

Increased fibrotic extracellular matrix (ECM) deposition promotes tumor invasion, which is the first step of the metastatic cascade. Yet, the underlying mechanisms are poorly understood as conventional studies of tumor cell migration are often performed in 2D cultures lacking the compositional and structural complexity of native ECM. Moreover, these studies frequently focus on select candidate pathways potentially overlooking other relevant changes in cell signaling. Here, we combine a cell-derived matrix (CDM) model with phosphotyrosine phosphoproteomic analysis to investigate tumor cell migration on fibrotic ECM relative to standard tissue culture plastic (TCP). Our results suggest that tumor cells cultured on CDMs migrate faster and in a more directional manner than their counterparts on TCP. These changes in migration correlate with decreased cell spreading and increased cell elongation. While the formation of phosphorylated focal adhesion kinase (pFAK)+ adhesion complexes did not vary between TCP and CDMs, time-dependent phosphoproteomic analysis identified that the SRC family kinase LYN may be differentially regulated. Pharmacological inhibition of LYN decreased tumor cell migration and cytoskeletal rearrangement on CDMs and also on TCP, suggesting that LYN regulates tumor cell migration on CDMs in combination with other mechanisms. These data highlight how the combination of physicochemically complex in vitro systems with phosphoproteomics can help identify signaling mechanisms by which the fibrotic ECM regulates tumor cell migration.

A conditional mouse expressing an activating mutation in NRF2 displays hyperplasia of the upper gastrointestinal tract and decreased white adipose tissue.

Activation of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co-complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL-Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2-activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3-dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)-positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA-seq profiling and follow-up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2-regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14-restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 transcription factor.

Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5'-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.

The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes.

MYO19 interacts with mitochondria through a C-terminal membrane association domain (MyMOMA). Specific mechanisms for localization of MYO19 to mitochondria are poorly understood. Using promiscuous biotinylation data in combination with existing affinity-capture databases, we have identified a number of putative MYO19-interacting proteins. We chose to explore the interaction between MYO19 and the mitochondrial GTPase Miro2 by expressing mchr-Miro2 in combination with GFP-tagged fragments of the MyMOMA domain and assaying for recruitment of MYO19-GFP to mitochondria. Coexpression of MYO19898-970 -GFP with mchr-Miro2 enhanced MYO19898-970 -GFP localization to mitochondria. Mislocalizing Miro2 to filopodial tips or the cytosolic face of the nuclear envelope did not recruit MYO19898-970 -GFP to either location. To address the kinetics of the Miro2/MYO19 interaction, we used FRAP analysis and permeabilization-activated reduction in fluorescence analysis. MyMOMA constructs containing a putative membrane-insertion motif but lacking the Miro2-interacting region displayed slow exchange kinetics. MYO19898-970 -GFP, which does not include the membrane-insertion motif, displayed rapid exchange kinetics, suggesting that MYO19 interacting with Miro2 has higher mobility than MYO19 inserted into the mitochondrial outer membrane. Mutation of well-conserved, charged residues within MYO19 or within the switch I and II regions of Miro2 abolished the enhancement of MYO19898-970 -GFP localization in cells ectopically expressing mchr-Miro2. Additionally, expressing mutant versions of Miro2 thought to represent particular nucleotide states indicated that the enhancement of MYO19898-970 -GFP localization is dependent on Miro2 nucleotide state. Taken together, these data suggest that membrane-inserted MYO19 is part of a larger complex, and that Miro2 plays a role in integration of actin- and microtubule-based mitochondrial activities.

The Cancer/Testes (CT) Antigen HORMAD1 promotes Homologous Recombinational DNA Repair and Radioresistance in Lung adenocarcinoma cells.

The Cancer/Testes (CT) Antigen HORMAD1 is germ cell-restricted and plays developmental roles in generation and processing of meiotic DNA Double Strand Breaks (DSB). Many tumors aberrantly overexpress HORMAD1 yet the potential impact of this CT antigen on cancer biology is unclear. We tested a potential role of HORMAD1 in genome maintenance in lung adenocarcinoma cells. We show that HORMAD1 re-distributes to nuclear foci and co-localizes with the DSB marker γH2AX in response to ionizing radiation (IR) and chemotherapeutic agents. The HORMA domain and C-term disordered oligomerization motif are necessary for localization of HORMAD1 to IR-induced foci (IRIF). HORMAD1-depleted cells are sensitive to IR and camptothecin. In reporter assays, Homologous Recombination (HR)-mediated repair of targeted ISce1-induced DSBs is attenuated in HORMAD1-depleted cells. In Non-Homologous End Joining (NHEJ) reporter assays, HORMAD1-depletion does not affect repair of ISce1-induced DSB. Early DSB signaling events (including ATM phosphorylation and formation of γH2AX, 53BP1 and NBS1 foci) are intact in HORMAD1-depleted cells. However, generation of RPA-ssDNA foci and redistribution of RAD51 to DSB are compromised in HORMAD1-depleted cells, suggesting that HORMAD1 promotes DSB resection. HORMAD1-mediated HR is a neomorphic activity that is independent of its meiotic partners (including HORMAD2 and CCDC36. Bioinformatic analysis of TCGA data show that similar to known HR pathway genes HORMAD1 is overexpressed in lung adenocarcinomas. Overexpression of HR genes is associated with specific mutational profiles (including copy number variation). Taken together, we identify HORMAD1-dependent DSB repair as a new mechanism of radioresistance and a probable determinant of mutability in lung adenocarcinoma.

Computer-Aided Design and Synthesis of 1-{4-[(3,4-Dihydroxybenzylidene)amino]phenyl}-5-oxopyrrolidine-3-carboxylic Acid as an Nrf2 Enhancer.

The design and synthesis of a novel nuclear factor erythroid 2-related factor 2 (Nrf2) enhancer is reported. Using a structure-based virtual screening approach, several commercially available compounds were identified as having high probability to interact with the Nrf2-binding pocket in the Kelch-like ECH-associated protein 1 (Keap1). Keap1 is an adaptor protein that recruits Nrf2 to a cullin-3-dependent ubiquitin ligase complex. The identified compounds were tested against rat pheochromocytoma PC-12 cells for their cytoprotective activity, and one compound (SKT359126) demonstrated an Nrf2-mediated cell-protective effect. Based on the structure of SKT359126, 23 novel derivatives were synthesized and evaluated. Of the screened derivatives, 1-{4-[(3,4-dihydroxybenzylidene)amino]phenyl}-5-oxopyrrolidine-3-carboxylic acid demonstrated better activity than the parent molecules in activating the Nrf2 transduction pathway in a dose- and time-dependent manner. This compound represents a promising starting point for the development of therapeutics for the treatment of oxidative-stress-related diseases.

5-Fluorouracil mediated anti-cancer activity in colon cancer cells is through the induction of Adenomatous Polyposis Coli: Implication of the long-patch base excision repair pathway.

Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLß and FEN1, thus blocking LP-BER via APC's DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain's interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant.

WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors.

About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAF(V600E/K)) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance.