Integrin inactivators: balancing cellular functions in vitro and in vivo.

Integrins mediate cell-matrix and cell-cell interactions and integrate extracellular cues to the cytoskeleton and cellular signalling pathways. Integrin function on the cell surface is regulated by their activity switching such that intracellular proteins interacting with the integrin cytoplasmic domains increase or decrease integrin-ligand binding affinity. It is widely accepted that integrin activation by specific proteins is essential for cell adhesion and integrin linkage to the actin cytoskeleton. However, there is also increasing evidence that integrin-inactivating proteins are crucial for appropriate integrin function in vitro and in vivo and that the regulation of integrin-ligand interactions is a fine-tuned balancing act between inactivation and activation.

SHARPIN S146 phosphorylation mediates ARP2/3 interaction, cancer cell invasion and metastasis.

SHARPIN is involved in several cellular processes and promotes cancer progression. However, how the choice between different functions of SHARPIN is post-translationally regulated is unclear. Here, we characterized SHARPIN phosphorylation by mass spectrometry and in vitro kinase assay. Focusing on S131 and S146, we demonstrate that they have a role in SHARPIN-ARP2/3 complex interaction, but play no role in integrin inhibition or LUBAC activation. Consistent with its novel role in ARP2/3 regulation, S146 phosphorylation of SHARPIN promoted lamellipodia formation. We also demonstrate that SHARPIN S146 phosphorylation-mediated ARP2/3 interaction is sensitive to inhibition of ERK1/2 or reactivation of protein phosphatase 2A (PP2A). Notably, CRISPR/Cas9-mediated knockout of SHARPIN abrogated three-dimensional (3D) invasion of several cancer cell lines. The 3D invasion of cancer cells was rescued by overexpression of the wild-type SHARPIN, but not by SHARPIN S146A mutant. Finally, we demonstrate that inhibition of phosphorylation at S146 significantly reduces in vivo metastasis in a zebrafish model. Collectively, these results map SHARPIN phosphorylation sites and identify S146 as a novel phosphorylation switch defining ARP2/3 interaction and cancer cell invasion. This article has an associated First Person interview with the first author of the paper.

Hepsin regulates TGFß signaling via fibronectin proteolysis.

Transforming growth factor-beta (TGFß) is a multifunctional cytokine with a well-established role in mammary gland development and both oncogenic and tumor-suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFß activity by acting as a storage compartment of latent-TGFß, but how TGFß is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFß signaling through the release of latent-TGFß from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFß signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent-TGFß1, while overexpression of hepsin in mammary tumors increased TGFß signaling. Cell-free and cell-based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent-TGFß and, importantly, that the ability of hepsin to activate TGFß signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFß pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.

The Sharpin interactome reveals a role for Sharpin in lamellipodium formation via the Arp2/3 complex.

Sharpin, a multifunctional adaptor protein, regulates several signalling pathways. For example, Sharpin enhances signal-induced NF-κB signalling as part of the linear ubiquitin assembly complex (LUBAC) and inhibits integrins, the T cell receptor, caspase 1 and PTEN. However, despite recent insights into Sharpin and LUBAC function, a systematic approach to identify the signalling pathways regulated by Sharpin has not been reported. Here, we present the first 'Sharpin interactome', which identifies a large number of novel potential Sharpin interactors in addition to several known ones. These data suggest that Sharpin and LUBAC might regulate a larger number of biological processes than previously identified, such as endosomal trafficking, RNA processing, metabolism and cytoskeleton regulation. Importantly, using the Sharpin interactome, we have identified a novel role for Sharpin in lamellipodium formation. We demonstrate that Sharpin interacts with Arp2/3, a protein complex that catalyses actin filament branching. We have identified the Arp2/3-binding site in Sharpin and demonstrate using a specific Arp2/3-binding deficient mutant that the Sharpin-Arp2/3 interaction promotes lamellipodium formation in a LUBAC-independent fashion.This article has an associated First Person interview with the first author of the paper.

Negative regulators of integrin activity.

Integrins are heterodimeric transmembrane adhesion receptors composed of α- and ß-subunits. They are ubiquitously expressed and have key roles in a number of important biological processes, such as development, maintenance of tissue homeostasis and immunological responses. The activity of integrins, which indicates their affinity towards their ligands, is tightly regulated such that signals inside the cell cruicially regulate the switching between active and inactive states. An impaired ability to activate integrins is associated with many human diseases, including bleeding disorders and immune deficiencies, whereas inappropriate integrin activation has been linked to inflammatory disorders and cancer. In recent years, the molecular details of integrin 'inside-out' activation have been actively investigated. Binding of cytoplasmic proteins, such as talins and kindlins, to the cytoplasmic tail of ß-integrins is widely accepted as being the crucial step in integrin activation. By contrast, much less is known with regard to the counteracting mechanism involved in switching integrins into an inactive conformation. In this Commentary, we aim to discuss the known mechanisms of integrin inactivation and the molecules involved.

Hepsin promotes breast tumor growth signaling via the TGFß-EGFR axis.

Hepsin, a type II transmembrane serine protease, is commonly overexpressed in prostate and breast cancer. The hepsin protein is stabilized by the Ras-MAPK pathway, and, downstream, this protease regulates the degradation of extracellular matrix components and activates growth factor pathways, such as the hepatocyte growth factor (HGF) and transforming growth factor beta (TGFß) pathway. However, how exactly active hepsin promotes cell proliferation machinery to sustain tumor growth is not fully understood. Here, we show that genetic deletion of the gene encoding hepsin (Hpn) in a WAP-Myc model of aggressive MYC-driven breast cancer inhibits tumor growth in the primary syngrafted sites and the growth of disseminated tumors in the lungs. The suppression of tumor growth upon loss of hepsin was accompanied by downregulation of TGFß and EGFR signaling together with a reduction in epidermal growth factor receptor (EGFR) protein levels. We further demonstrate in 3D cultures of patient-derived breast cancer explants that both basal TGFß signaling and EGFR protein expression are inhibited by neutralizing antibodies or small-molecule inhibitors of hepsin. The study demonstrates a role for hepsin as a regulator of cell proliferation and tumor growth through TGFß and EGFR pathways, warranting consideration of hepsin as a potential indirect upstream target for therapeutic inhibition of TGFß and EGFR pathways in cancer.

Respiratory complex I regulates dendritic cell maturation in explant model of human tumor immune microenvironment.

BACKGROUND: Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. METHODS: Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity. RESULTS: We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity. CONCLUSIONS: Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.

Novel pyrimidine-2,4-diamine derivative suppresses the cell viability and spindle assembly checkpoint activity by targeting Aurora kinases.

Mitosis represents a clinically important determination point in the life cycle of proliferating cells. One potential drug target within the mitotic machinery is the spindle assembly checkpoint (SAC), an evolutionarily conserved signaling pathway that monitors the connections between microtubules (MTs) and chromosomes. Mistakes in SAC signaling may lead to cell division errors that can trigger elimination of cancer cells at M phase or soon after exit from mitosis. In this study, we describe the cellular effects of a novel pyrimidine-2,4-diamine derivative that we discovered to inhibit the activity of SAC. The compound caused rapid escape from the mitotic arrest induced by lack of interkinetochore tension but not by lack of MT-kinetochore attachments. In cycling cells, the compound disrupted the architecture of mitotic spindle that triggered a transient M-phase arrest that was rapidly followed by a forced mitotic exit. The premature termination of M phase was found to be a consequence of precocious inactivation of SAC caused by a direct inhibitory effect of the compound on Aurora B kinase in vitro and in cells. The compound also targets Aurora A kinase and tubulin in vitro and in cells, which can explain the observed spindle anomalies. The reduced activity of Aurora B kinase resulted in polyploidy and suppression of cancer cell viability. Our data suggest that this new pharmacophore possesses interesting anticancer properties that could be exploited in development of mitosis-targeting therapies.

Loss of p38gamma MAPK induces pleiotropic mitotic defects and massive cell death.

The p38 mitogen-activated protein kinase (p38 MAPK) family, which is comprised of four protein isoforms, p38α, p38ß, p38γ and p38δ, forms one of the key MAPK pathways. The p38 MAPKs are implicated in many cellular processes including inflammation, differentiation, cell growth, cell cycle and cell death. The function of p38 MAPKs in mitotic entry has been well established, but their role in mitotic progression has remained controversial. We identify p38γ MAPK as a modulator of mitotic progression and mitotic cell death. In HeLa cells, loss of p38γ results in multipolar spindle formation and chromosome misalignment, which induce a transient M phase arrest. The majority of p38γ-depleted cells die at mitotic arrest or soon after abnormal exit from M-phase. We show that p38 MAPKs are activated at the kinetochores and spindle poles throughout mitosis by kinase(s) that are stably bound to these structures. Finally, p38γ is required for the normal kinetochore localization of polo-like kinase 1 (Plk1), and this contributes to the activity of the p38 MAPK pathway. Our data suggest a link between mitotic regulation and the p38 MAPK pathway, in which p38γ prevents chromosomal instability and supports mitotic cell viability.

The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis.

The spindle assembly checkpoint (SAC) is a conserved mechanism that ensures the fidelity of chromosome distribution in mitosis by preventing anaphase onset until the correct bipolar microtubule-kinetochore attachments are formed. Errors in SAC function may contribute to tumorigenesis by inducing numerical chromosome anomalies (aneuploidy). On the other hand, total disruption of SAC can lead to massive genomic imbalance followed by cell death, a phenomena that has therapeutic potency. We performed a cell-based high-throughput screen with a compound library of 2000 bioactives for novel SAC inhibitors and discovered a plant-derived phenolic compound eupatorin (3',5-dihydroxy-4',6,7-trimethoxyflavone) as an anti-mitotic flavonoid. The premature override of the microtubule drug-imposed mitotic arrest by eupatorin is dependent on microtubule-kinetochore attachments but not interkinetochore tension. Aurora B kinase activity, which is essential for maintenance of normal SAC signaling, is diminished by eupatorin in cells and in vitro providing a mechanistic explanation for the observed forced mitotic exit. Eupatorin likely has additional targets since eupatorin treatment of pre-mitotic cells causes spindle anomalies triggering a transient M phase delay followed by impaired cytokinesis and polyploidy. Finally, eupatorin potently induces apoptosis in multiple cancer cell lines and suppresses cancer cell proliferation in organotypic 3D cell culture model.

Ovarian Cancers with Low CIP2A Tumor Expression Constitute an APR-246-Sensitive Disease Subtype.

Identification of ovarian cancer patient subpopulations with increased sensitivity to targeted therapies could offer significant clinical benefit. We report that 22% of the high-grade ovarian cancer tumors at diagnosis express CIP2A oncoprotein at low levels. Furthermore, regardless of their significantly lower likelihood of disease relapse after standard chemotherapy, a portion of relapsed tumors retain their CIP2A-deficient phenotype. Through a screen for therapeutics that would preferentially kill CIP2A-deficient ovarian cancer cells, we identified reactive oxygen species inducer APR-246, tested previously in ovarian cancer clinical trials. Consistent with CIP2A-deficient ovarian cancer subtype in humans, CIP2A is dispensable for development of MISIIR-Tag-driven mouse ovarian cancer tumors. Nevertheless, CIP2A-null ovarian cancer tumor cells from MISIIR-Tag mice displayed APR-246 hypersensitivity both in vitro and in vivo. Mechanistically, the lack of CIP2A expression hypersensitizes the ovarian cancer cells to APR-246 by inhibition of NF-κB activity. Accordingly, combination of APR-246 and NF-κB inhibitor compounds strongly synergized in killing of CIP2A-positive ovarian cancer cells. Collectively, the results warrant consideration of clinical testing of APR-246 for CIP2A-deficient ovarian cancer tumor subtype patients. Results also reveal CIP2A as a candidate APR-246 combination therapy target for ovarian cancer.

High frequency of TTK mutations in microsatellite-unstable colorectal cancer and evaluation of their effect on spindle assembly checkpoint.

Frameshift mutations frequently accumulate in microsatellite-unstable colorectal cancers (MSI CRCs) typically leading to downregulation of the target genes due to nonsense-mediated messenger RNA decay. However, frameshift mutations that occur in the 3' end of the coding regions can escape decay, which has largely been ignored in previous works. In this study, we characterized nonsense-mediated decay-escaping frameshift mutations in MSI CRC in an unbiased, genome wide manner. Combining bioinformatic search with expression profiling, we identified genes that were predicted to escape decay after a deletion in a microsatellite repeat. These repeats, located in 258 genes, were initially sequenced in 30 MSI CRC samples. The mitotic checkpoint kinase TTK was found to harbor decay-escaping heterozygous mutations in exon 22 in 59% (105/179) of MSI CRCs, which is notably more than previously reported. Additional novel deletions were found in exon 5, raising the mutation frequency to 66%. The exon 22 of TTK contains an A(9)-G(4)-A(7) locus, in which the most common mutation was a mononucleotide deletion in the A(9) (c.2560delA). When compared with identical non-coding repeats, TTK was found to be mutated significantly more often than expected without selective advantage. Since TTK inhibition is known to induce override of the mitotic spindle assembly checkpoint (SAC), we challenged mutated cancer cells with the microtubule-stabilizing drug paclitaxel. No evidence of checkpoint weakening was observed. As a conclusion, heterozygous TTK mutations occur at a high frequency in MSI CRCs. Unexpectedly, the plausible selective advantage in tumourigenesis does not appear to be related to SAC.

Compressive stress-mediated p38 activation required for ERα + phenotype in breast cancer.

Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ERα + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ERα-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ERα + breast cancer models. The ERα + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ERα is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ERα signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ERα phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK.

Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres.

Incenp is an essential mitotic protein that, together with Aurora B, Survivin, and Borealin, forms the core of the chromosomal passenger protein complex (CPC). The CPC regulates various mitotic processes and functions to maintain genomic stability. The proper subcellular localization of the CPC and its full catalytic activity require the presence of each core subunit in the complex. We have investigated the mitotic tasks of the CPC using a function blocking antibody against Incenp microinjected into cells at different mitotic phases. This method allowed temporal analysis of CPC functions without perturbation of complex assembly or activity prior to injection. We have also studied the dynamic properties of Incenp and Aurora B using fusion protein photobleaching. We found that in early mitotic cells, Incenp and Aurora B exhibit dynamic turnover at centromeres, which is prevented by the anti-Incenp antibody. In these cells, the loss of centromeric CPC turnover is accompanied by forced mitotic exit without the execution of cytokinesis. Introduction of anti-Incenp antibody into early anaphase cells causes abnormalities in sister chromatid separation through defects in anaphase spindle functions. In summary, our data uncovers new mitotic roles for the CPC in anaphase and proposes that CPC turnover at centromeres modulates spindle assembly checkpoint signaling.

Assessment of the WAP-Myc mouse mammary tumor model for spontaneous metastasis.

Breast cancer is the most common form of cancer in women. Despite significant therapeutic advances in recent years, breast cancer also still causes the greatest number of cancer-related deaths in women, the vast majority of which (> 90%) are caused by metastases. However, very few mouse mammary cancer models exist that faithfully recapitulate the multistep metastatic process in human patients. Here we assessed the suitability of a syngrafting protocol for a Myc-driven mammary tumor model (WAP-Myc) to study autochthonous metastasis. A moderate but robust spontaneous lung metastasis rate of around 25% was attained. In addition, increased T cell infiltration was observed in metastatic tumors compared to donor and syngrafted primary tumors. Thus, the WAP-Myc syngrafting protocol is a suitable tool to study the mechanisms of metastasis in MYC-driven breast cancer.

Dietary flavonoid fisetin induces a forced exit from mitosis by targeting the mitotic spindle checkpoint.

Fisetin is a natural flavonol present in edible vegetables, fruits and wine at 2-160 microg/g concentrations and an ingredient in nutritional supplements with much higher concentrations. The compound has been reported to exert anticarcinogenic effects as well as antioxidant and anti-inflammatory activity via its ability to act as an inhibitor of cell proliferation and free radical scavenger, respectively. Our cell-based high-throughput screen for small molecules that override chemically induced mitotic arrest identified fisetin as an antimitotic compound. Fisetin rapidly compromised microtubule drug-induced mitotic block in a proteasome-dependent manner in several human cell lines. Moreover, in unperturbed human cancer cells fisetin caused premature initiation of chromosome segregation and exit from mitosis without normal cytokinesis. To understand the molecular mechanism behind these mitotic errors, we analyzed the consequences of fisetin treatment on the localization and phoshorylation of several mitotic proteins. Aurora B, Bub1, BubR1 and Cenp-F rapidly lost their kinetochore/centromere localization and others became dephosphorylated upon addition of fisetin to the culture medium. Finally, we identified Aurora B kinase as a novel direct target of fisetin. The activity of Aurora B was significantly reduced by fisetin in vitro and in cells, an effect that can explain the observed forced mitotic exit, failure of cytokinesis and decreased cell viability. In conclusion, our data propose that fisetin perturbs spindle checkpoint signaling, which may contribute to the antiproliferative effects of the compound.

Integrin beta 1 inhibition alleviates the chronic hyperproliferative dermatitis phenotype of SHARPIN-deficient mice.

SHARPIN (Shank-Associated RH Domain-Interacting Protein) is a component of the linear ubiquitin chain assembly complex (LUBAC), which enhances TNF-induced NF-κB activity. SHARPIN-deficient (Sharpincpdm/cpdm) mice display multi-organ inflammation and chronic proliferative dermatitis (cpdm) due to TNF-induced keratinocyte apoptosis. In cells, SHARPIN also inhibits integrins independently of LUBAC, but it has remained enigmatic whether elevated integrin activity levels in the dermis of Sharpincpdm/cpdm mice is due to increased integrin activity or is secondary to inflammation. In addition, the functional contribution of increased integrin activation to the Sharpincpdm/cpdm phenotype has not been investigated. Here, we find increased integrin activity in keratinocytes from Tnfr1-/- Sharpincpdm/cpdm double knockout mice, which do not display chronic inflammation or proliferative dermatitis, thus suggesting that SHARPIN indeed acts as an integrin inhibitor in vivo. In addition, we present evidence for a functional contribution of integrin activity to the Sharpincpdm/cpdm skin phenotype. Treatment with an integrin beta 1 function blocking antibody reduced epidermal hyperproliferation and epidermal thickness in Sharpincpdm/cpdm mice. Our data indicate that, while TNF-induced cell death triggers the chronic inflammation and proliferative dermatitis, absence of SHARPIN-dependent integrin inhibition exacerbates the epidermal hyperproliferation in Sharpincpdm/cpdm mice.

SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras.

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes.

Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2.

Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible ß-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through ß subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-µ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions.