Netrin-1 blockade inhibits tumour growth and EMT features in endometrial cancer.

Netrin-1 is upregulated in cancers as a protumoural mechanism1. Here we describe netrin-1 upregulation in a majority of human endometrial carcinomas (ECs) and demonstrate that netrin-1 blockade, using an anti-netrin-1 antibody (NP137), is effective in reduction of tumour progression in an EC mouse model. We next examined the efficacy of NP137, as a first-in-class single agent, in a Phase I trial comprising 14 patients with advanced EC. As best response we observed 8 stable disease (8 out of 14, 57.1%) and 1 objective response as RECIST v.1.1 (partial response, 1 out of 14 (7.1%), 51.16% reduction in target lesions at 6 weeks and up to 54.65% reduction during the following 6 months). To evaluate the NP137 mechanism of action, mouse tumour gene profiling was performed, and we observed, in addition to cell death induction, that NP137 inhibited epithelial-to-mesenchymal transition (EMT). By performing bulk RNA sequencing (RNA-seq), spatial transcriptomics and single-cell RNA-seq on paired pre- and on-treatment biopsies from patients with EC from the NP137 trial, we noted a net reduction in tumour EMT. This was associated with changes in immune infiltrate and increased interactions between cancer cells and the tumour microenvironment. Given the importance of EMT in resistance to current standards of care2, we show in the EC mouse model that a combination of NP137 with carboplatin-paclitaxel outperformed carboplatin-paclitaxel alone. Our results identify netrin-1 blockade as a clinical strategy triggering both tumour debulking and EMT inhibition, thus potentially alleviating resistance to standard treatments.

CLK1 reorganizes the splicing factor U1-70K for early spliceosomal protein assembly.

Early spliceosome assembly requires phosphorylation of U1-70K, a constituent of the U1 small nuclear ribonucleoprotein (snRNP), but it is unclear which sites are phosphorylated, and by what enzyme, and how such modification regulates function. By profiling the proteome, we found that the Cdc2-like kinase 1 (CLK1) phosphorylates Ser-226 in the C terminus of U1-70K. This releases U1-70K from subnuclear granules facilitating interaction with U1 snRNP and the serine-arginine (SR) protein SRSF1, critical steps in establishing the 5' splice site. CLK1 breaks contacts between the C terminus and the RNA recognition motif (RRM) in U1-70K releasing the RRM to bind SRSF1. This reorganization also permits stable interactions between U1-70K and several proteins associated with U1 snRNP. Nuclear induction of the SR protein kinase 1 (SRPK1) facilitates CLK1 dissociation from U1-70K, recycling the kinase for catalysis. These studies demonstrate that CLK1 plays a vital, signal-dependent role in early spliceosomal protein assembly by contouring U1-70K for protein-protein multitasking.

A conserved sequence motif bridges two protein kinases for enhanced phosphorylation and nuclear function of a splicing factor.

The assembly and activation of the spliceosome rely upon the phosphorylation of an essential family of splicing factors known as the serine-arginine (SR) proteins. Although it has been demonstrated recently that two enzyme families, the SR protein kinases (SRPKs) and the Cdc2-like kinases (CLKs), can function as a complex to efficiently phosphorylate these SR proteins in the nucleus, the molecular features involved in such a connection are unknown. In this study, we identified a group of conserved residues in the large lobe of SRPK1 that interact with the N terminus of CLK1 stabilizing the SRPK1-CLK1 complex. Mutations in this motif not only disrupt formation of the kinase-kinase complex but also impair SRPK1-dependent release of the phospho-SR protein from CLK1. The binding motif potently up-regulates CLK1-specific phosphorylation sites, enhances SR protein diffusion from nuclear speckles, and impacts the alternative splicing of several target genes. These results indicate that CLK1 binds a conserved, electronegative surface on SRPK1, thereby controlling SR protein phosphorylation levels for enhanced subnuclear trafficking and alternative splicing regulation.

Matrix Rigidity Controls Epithelial-Mesenchymal Plasticity and Tumor Metastasis via a Mechanoresponsive EPHA2/LYN Complex.

Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes via distinct mechanotransduction pathways. In breast cancer, increased ECM stiffness promotes epithelial-to-mesenchymal transition (EMT), cell invasion, and metastasis. Here, we identify a mechanosensitive EPHA2/LYN protein complex regulating EMT and metastasis in response to increasing ECM stiffness during tumor progression. High ECM stiffness leads to ligand-independent phosphorylation of ephrin receptor EPHA2, which recruits and activates the LYN kinase. LYN phosphorylates the EMT transcription factor TWIST1 to release TWIST1 from its cytoplasmic anchor G3BP2 to enter the nucleus, thus triggering EMT and invasion. Genetic and pharmacological inhibition of this pathway prevents breast tumor invasion and metastasis in vivo. In human breast cancer samples, activation of this pathway correlates with collagen fiber alignment, a marker of increasing ECM stiffness. Our findings reveal an EPHA2/LYN/TWIST1 mechanotransduction pathway that responds to mechanical signals from the tumor microenvironment to drive EMT, invasion, and metastasis.

RREB1 Integrates TGF-ß and RAS Signals to Drive EMT.

TGF-ß is long known to require Ras activation to induce EMT. In a recent issue of Nature, Massagué and colleagues (Su et al., 2020) identify RAS-responsive element binding protein 1 (RREB1) as a critical integrator of TGF-ß and Ras signals during both developmental and cancer EMT programs.

Disordered protein interactions for an ordered cellular transition: Cdc2-like kinase 1 is transported to the nucleus via its Ser-Arg protein substrate.

Serine-arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation. In the nucleus, the cdc2-like kinases (CLKs) phosphorylate SR proteins on their intrinsically disordered Arg-Ser (RS) domains, mobilizing them from storage speckles to the splicing machinery. The CLKs have disordered N termini that bind tightly to RS domains, enhancing SR protein phosphorylation. The N termini also promote nuclear localization of CLKs, but their transport mechanism is presently unknown. To explore cytoplasmic-nuclear transitions, several classical nuclear localization sequences in the N terminus of the CLK1 isoform were identified, but their mutation had no effect on subcellular localization. Rather, we found that CLK1 amplifies its presence in the nucleus by forming a stable complex with the SR protein substrate and appropriating its NLS for transport. These findings indicate that, along with their well-established roles in mRNA splicing, SR proteins use disordered protein-protein interactions to carry their kinase regulator from the cytoplasm to the nucleus.

Apical-basal polarity inhibits epithelial-mesenchymal transition and tumour metastasis by PAR-complex-mediated SNAI1 degradation.

Loss of apical-basal polarity and activation of epithelial-mesenchymal transition (EMT) both contribute to carcinoma progression and metastasis. Here, we report that apical-basal polarity inhibits EMT to suppress metastatic dissemination. Using mouse and human epithelial three-dimensional organoid cultures, we show that the PAR-atypical protein kinase C (aPKC) polarity complex inhibits EMT and invasion by promoting degradation of the SNAIL family protein SNAI1. Under intact apical-basal polarity, aPKC kinases phosphorylate S249 of SNAI1, which leads to protein degradation. Loss of apical-basal polarity prevents aPKC-mediated SNAI1 phosphorylation and stabilizes the SNAI1 protein to promote EMT and invasion. In human breast tumour xenografts, inhibition of the PAR-complex-mediated SNAI1 degradation mechanism promotes tumour invasion and metastasis. Analyses of human breast tissue samples reveal negative correlations between PAR3 and SNAI1 protein levels. Our results demonstrate that apical-basal polarity functions as a critical checkpoint of EMT to precisely control epithelial-mesenchymal plasticity during tumour metastasis.

Dynamically stiffened matrix promotes malignant transformation of mammary epithelial cells via collective mechanical signaling.

Breast cancer development is associated with increasing tissue stiffness over years. To more accurately mimic the onset of gradual matrix stiffening, which is not feasible with conventional static hydrogels, mammary epithelial cells (MECs) were cultured on methacrylated hyaluronic acid hydrogels whose stiffness can be dynamically modulated from "normal" (<150 Pascals) to "malignant" (>3,000 Pascals) via two-stage polymerization. MECs form and remain as spheroids, but begin to lose epithelial characteristics and gain mesenchymal morphology upon matrix stiffening. However, both the degree of matrix stiffening and culture time before stiffening play important roles in regulating this conversion as, in both cases, a subset of mammary spheroids remained insensitive to local matrix stiffness. This conversion depended neither on colony size nor cell density, and MECs did not exhibit "memory" of prior niche when serially cultured through cycles of compliant and stiff matrices. Instead, the transcription factor Twist1, transforming growth factor ß (TGFß), and YAP activation appeared to modulate stiffness-mediated signaling; when stiffness-mediated signals were blocked, collective MEC phenotypes were reduced in favor of single MECs migrating away from spheroids. These data indicate a more complex interplay of time-dependent stiffness signaling, spheroid structure, and soluble cues that regulates MEC plasticity than suggested by previous models.

Biomaterials to model and measure epithelial cancers.

The use of biomaterials has substantially contributed to both our understanding of tumorigenesis and our ability to identify and capture tumour cells in vitro and in vivo. Natural and synthetic biomaterials can be applied as models to recapitulate key features of the tumour microenvironment in vitro, including architectural, mechanical and biological functions. Engineered biomaterials can further mimic the spatial and temporal properties of the surrounding tumour niche to investigate the specific effects of the environment on disease progression, offering an alternative to animal models for the testing of cancer cell behaviour. Biomaterials can also be used to capture and detect cancer cells in vitro and in vivo to monitor tumour progression. In this Review, we discuss the natural and synthetic biomaterials that can be used to recreate specific features of tumour microenvironments. We examine how biomaterials can be applied to capture circulating tumour cells in blood samples for the early detection of metastasis. We highlight biomaterial-based strategies to investigate local regions adjacent to the tumour and survey potential applications of biomaterial-based devices for diagnosis and prognosis, such as the detection of cellular deformability and the non-invasive surveillance of tumour-adjacent stroma.

Molecular interactions connecting the function of the serine-arginine-rich protein SRSF1 to protein phosphatase 1.

Splicing generates many mRNA strands from a single precursor mRNA, expanding the proteome and enhancing intracellular diversity. Both initial assembly and activation of the spliceosome require an essential family of splicing factors called serine-arginine (SR) proteins. Protein phosphatase 1 (PP1) regulates the SR proteins by controlling phosphorylation of a C-terminal arginine-serine-rich (RS) domain. These modifications are vital for the subcellular localization and mRNA splicing function of the SR protein. Although PP1 has been shown to dephosphorylate the prototype SR protein splicing factor 1 (SRSF1), the molecular nature of this interaction is not understood. Here, using NMR spectroscopy, we identified two electrostatic residues in helix α2 and a hydrophobic residue in helix α1 in the RNA recognition motif 1 (RRM1) of SRSF1 that constitute a binding surface for PP1. Substitution of these residues dissociated SRSF1 from PP1 and enhanced phosphatase activity, reducing phosphorylation in the RS domain. These effects lead to shifts in alternative splicing patterns that parallel increases in SRSF1 diffusion from speckles to the nucleoplasm brought on by regiospecific decreases in RS domain phosphorylation. Overall, these findings establish a molecular and biological connection between PP1-targeted amino acids in an RRM with the phosphorylation state and mRNA-processing function of an SR protein.

Mobilization of a splicing factor through a nuclear kinase-kinase complex.

The splicing of mRNA is dependent on serine-arginine (SR) proteins that are mobilized from membrane-free, nuclear speckles to the nucleoplasm by the Cdc2-like kinases (CLKs). This movement is critical for SR protein-dependent assembly of the macromolecular spliceosome. Although CLK1 facilitates such trafficking through the phosphorylation of serine-proline dipeptides in the prototype SR protein SRSF1, an unrelated enzyme known as SR protein kinase 1 (SRPK1) performs the same function but does not efficiently modify these dipeptides in SRSF1. We now show that the ability of SRPK1 to mobilize SRSF1 from speckles to the nucleoplasm is dependent on active CLK1. Diffusion from speckles is promoted by the formation of an SRPK1-CLK1 complex that facilitates dissociation of SRSF1 from CLK1 and enhances the phosphorylation of several serine-proline dipeptides in this SR protein. Down-regulation of either kinase blocks EGF-stimulated mobilization of nuclear SRSF1. These findings establish a signaling pathway that connects SRPKs to SR protein activation through the associated CLK family of kinases.

Redirecting SR Protein Nuclear Trafficking through an Allosteric Platform.

Although phosphorylation directs serine-arginine (SR) proteins from nuclear storage speckles to the nucleoplasm for splicing function, dephosphorylation paradoxically induces similar movement, raising the question of how such chemical modifications are balanced in these essential splicing factors. In this new study, we investigated the interaction of protein phosphatase 1 (PP1) with the SR protein splicing factor (SRSF1) to understand the foundation of these opposing effects in the nucleus. We found that RNA recognition motif 1 (RRM1) in SRSF1 binds PP1 and represses its catalytic function through an allosteric mechanism. Disruption of RRM1-PP1 interactions reduces the phosphorylation status of the RS domain in vitro and in cells, redirecting SRSF1 in the nucleus. The data imply that an allosteric SR protein-phosphatase platform balances phosphorylation levels in a "goldilocks" region for the proper subnuclear storage of an SR protein splicing factor.

Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing.

Phosphorylation has been generally thought to activate the SR family of splicing factors for efficient splice-site recognition, but this idea is incompatible with an early observation that overexpression of an SR protein kinase, such as the CDC2-like kinase 1 (CLK1), weakens splice-site selection. Here, we report that CLK1 binds SR proteins but lacks the mechanism to release phosphorylated SR proteins, thus functionally inactivating the splicing factors. Interestingly, CLK1 overcomes this dilemma through a symbiotic relationship with the serine-arginine protein kinase 1 (SRPK1). We show that SRPK1 interacts with an RS-like domain in the N terminus of CLK1 to facilitate the release of phosphorylated SR proteins, which then promotes efficient splice-site recognition and subsequent spliceosome assembly. These findings reveal an unprecedented signaling mechanism by which two protein kinases fulfill separate catalytic features that are normally encoded in single kinases to institute phosphorylation control of pre-mRNA splicing in the nucleus.

Nuclear protein kinase CLK1 uses a non-traditional docking mechanism to select physiological substrates.

Phosphorylation-dependent cell communication requires enzymes that specifically recognize key proteins in a sea of similar, competing substrates. The protein kinases achieve this goal by utilizing docking grooves in the kinase domain or heterologous protein adaptors to reduce 'off pathway' targeting. We now provide evidence that the nuclear protein kinase CLK1 (cell division cycle2-like kinase 1) important for splicing regulation departs from these classic paradigms by using a novel self-association mechanism. The disordered N-terminus of CLK1 induces oligomerization, a necessary event for targeting its physiological substrates the SR protein (splicing factor containing a C-terminal RS domain) family of splicing factors. Increasing the CLK1 concentration enhances phosphorylation of the splicing regulator SRSF1 (SR protein splicing factor 1) compared with the general substrate myelin basic protein (MBP). In contrast, removal of the N-terminus or dilution of CLK1 induces monomer formation and reverses this specificity. CLK1 self-association also occurs in the nucleus, is induced by the N-terminus and is important for localization of the kinase in sub-nuclear compartments known as speckles. These findings present a new picture of substrate recognition for a protein kinase in which an intrinsically disordered domain is used to capture physiological targets with similar disordered domains in a large oligomeric complex while discriminating against non-physiological targets.

The Disintegrin and Metalloprotease ADAM12 Is Associated with TGF-ß-Induced Epithelial to Mesenchymal Transition.

The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-ß and promotes TGF-ß-dependent signaling through interaction with the type II receptor of TGF-ß. Here we explore the implication of ADAM12 in TGF-ß-mediated epithelial to mesenchymal transition (EMT), a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A), we demonstrate that TGF-ß-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-ß receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-ß-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.

TIF1γ interferes with TGFß1/SMAD4 signaling to promote poor outcome in operable breast cancer patients.

BACKGROUND: The Transforming growth factor ß (TGFß) signaling has a paradoxical role in cancer development and outcome. Besides, the prognostic significance of the TGFß1, SMAD4 in breast cancer patients is an area of many contradictions. The transcriptional intermediary factor 1γ (TIF1γ) is thought to interact with the TGFß/SMAD signaling through different mechanisms. Our study aims to define the prognostic significance of TGFß1, SMAD4 and TIF1γ expression in breast cancer patients and to detect possible interactions among those markers that might affect the outcome. METHODS: Immunohistochemistry was performed on tissue microarray (TMA) blocks prepared from samples of 248 operable breast cancer patients who presented at Centre Léon Bérard (CLB) between 1998 and 2001. The intensity and the percentage of stained tumor cells were integrated into a single score (0-6) and a cutoff was defined for high or low expression for each marker. Correlation was done between TGFß1, SMAD4 and TIF1γ expression with the clinico-pathologic parameters using Pearson's chi-square test. Kaplan-Meier method was used to estimate distant metastasis free survival (DMFS), disease free survival (DFS) and overall survival (OS) and the difference between the groups was evaluated with log-rank test. RESULTS: 223 cases were assessable for TIF1γ, 204 for TGFß1 and 173 for SMAD4. Median age at diagnosis was 55.8 years (range: 27 to 89 years). Tumors were larger than 20 mm in 49.2% and 45.2% had axillary lymph node (LN) metastasis (N1a to N3). 19.4% of the patients had SBR grade I tumors, 46.8% grade II tumors and 33.9% grade III tumors. ER was positive in 85.4%, PR in 75.5% and Her2-neu was over-expressed in 10% of the cases. Nuclear TIF1γ, cytoplasmic TGFß1, nuclear and cytoplasmic SMAD4 stainings were high in 35.9%, 30.4%, 27.7% and 52.6% respectively. TIF1γ expression was associated with younger age (p=0.006), higher SBR grade (p<0.001), more ER negativity (p=0.035), and tumors larger than 2 cm (p=0.081), while TGFß1 was not associated with any of the traditional prognostic factors. TGFß1 expression in tumor cells was a marker of poor prognosis regarding DMFS (HR=2.28; 95% CI: 1.4 to 3.8; p=0.002), DFS (HR=2.00; 95% CI: 1.25 to 3.5; p=0.005) and OS (HR=1.89; 95 % CI: 1.04 to 3.43; p=0.037). TIF1γ expression carried a tendency towards poorer DMFS (p=0.091), DFS (p=0.143) and OS (p=0.091). In the multivariate analysis TGFß1 remained an independent predictor of shorter DMFS, DFS and OS. Moreover, the prognostic significance of TGFß1 was more obvious in the TIF1γ high patient subgroup than in the patients with TIF1γ low expression. The subgroup expressing both markers had the worst DMFS (HR=3.2; 95% CI: 1.7 to 5.9; p<0.0001), DFS (HR=3.02; 95 % CI: 1.6 to 5.6; p<0.0001) and OS (HR=2.7; 95 % CI: 1.4 to 5.4; p=0.005). CONCLUSION: There is a crosstalk between the TIF1γ and the TGFß1/SMAD4 signaling that deteriorates the outcome of operable breast cancer patients and when combined together they can serve as an effective prognostic tool for those patients.

Matrix stiffness drives epithelial-mesenchymal transition and tumour metastasis through a TWIST1-G3BP2 mechanotransduction pathway.

Matrix stiffness potently regulates cellular behaviour in various biological contexts. In breast tumours, the presence of dense clusters of collagen fibrils indicates increased matrix stiffness and correlates with poor survival. It is unclear how mechanical inputs are transduced into transcriptional outputs to drive tumour progression. Here we report that TWIST1 is an essential mechanomediator that promotes epithelial-mesenchymal transition (EMT) in response to increasing matrix stiffness. High matrix stiffness promotes nuclear translocation of TWIST1 by releasing TWIST1 from its cytoplasmic binding partner G3BP2. Loss of G3BP2 leads to constitutive TWIST1 nuclear localization and synergizes with increasing matrix stiffness to induce EMT and promote tumour invasion and metastasis. In human breast tumours, collagen fibre alignment, a marker of increasing matrix stiffness, and reduced expression of G3BP2 together predict poor survival. Our findings reveal a TWIST1-G3BP2 mechanotransduction pathway that responds to biomechanical signals from the tumour microenvironment to drive EMT, invasion and metastasis.