Darren N. Saunders

TRIP12 and UBR5 Suppress Spreading of Chromatin Ubiquitylation at Damaged Chromosomes

Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis.

Revisiting the biological roles of PAI2 (SERPINB2) in cancer

Tumour expression of the urokinase plasminogen activator correlates with invasive capacity. Consequently, inhibition of this serine protease by physiological inhibitors should decrease invasion and metastasis. However, of the two main urokinase inhibitors, high tumour levels of the type 1 inhibitor actually promote tumour progression, whereas high levels of the type 2 inhibitor decrease tumour growth and metastasis. We propose that the basis of this apparently paradoxical action of two similar serine protease inhibitors lies in key structural differences controlling interactions with components of the extracellular matrix and endocytosis–signalling co-receptors.

Membranous Expression of Secreted Frizzled-Related Protein 4 Predicts for Good Prognosis in Localized Prostate Cancer and Inhibits PC3 Cellular Proliferation in Vitro

Purpose: Activation of the Wnt-signaling pathway is implicated in aberrant cellular proliferation in a variety of cancers. Secreted frizzled-related protein 4 (sFRP4) is a secreted protein with putative inhibitory activity of the Wnt-signaling cascade through binding and sequestering Wnt ligands. Because sFRP4 mRNA is overexpressed in prostate cancers (PCs), the aim of this study was to define the pattern of sFRP4 protein expression in normal and malignant human prostate tissue and to determine whether changes in expression were associated with disease progression and prognosis, as well as to define the phenotype of sFRP4-overexpression in an in vitro model of PC. Experimental Design: Polyclonal antibodies were raised against a COOH-terminal peptide of sFRP4, characterized and used to assess sFRP4 protein expression in benign prostate tissue and 229 patients with clinically localized PC (median follow-up 77 months, range 1–156). In vitro studies of the function of sFRP4 overexpression were performed using PC3 cells transfected with sFRP4. Results: Benign and malignant prostate tissue demonstrated cytoplasmic sFRP4 immunoreactivity, but there was a decrease in the expression of membranous sFRP4 in PCs compared with the hyperplastic lesions (P < 0.0001). Kaplan-Meier analysis revealed that patients whose PC expressed membranous sFRP4 in >20% of cells had improved relapse-free survival compared with those with ≤20% membranous expression (P = 0.002). Moreover, membranous sFRP4 expression (P = 0.04) was an independent predictor of relapse when modeled with Gleason score (P = 0.006), pathological stage (P = 0.002), and pre-operative prostate-specific antigen levels (P = 0.004). In addition, in vitro studies demonstrated a decrease in the proliferation rate of PC3 cells transfected with sFRP4 when compared with the control PC3-empty vector cells (P < 0.0001). Decreased levels of phosphorylated glycogen synthase kinase 3β in PC3-sFRP4 cells suggested that this phenotype is mediated by the “Wnt/β-catenin” pathway. Conclusions: These data suggest that sFRP4 expression may be prognostic for localized PC, potentially as a consequence of an inhibitory effect on PC cell proliferation.

Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling

The dimerisation of Raf kinases involves a central cluster within the kinase domain, the dimer interface (DIF). Yet, the importance of the DIF for the signalling potential of wild‐type B‐Raf (B‐Raf wt) and its oncogenic counterparts remains unknown. Here, we show that the DIF plays a pivotal role for the activity of B‐Raf wt and several of its gain‐of‐function (g‐o‐f) mutants. In contrast, the B‐Raf V600E, B‐Raf insT and B‐Raf G469A oncoproteins are remarkably resistant to mutations in the DIF. However, compared with B‐Raf wt, B‐Raf V600E displays extended protomer contacts, increased homodimerisation and incorporation into larger protein complexes. In contrast, B‐Raf wt and Raf‐1wt mediated signalling triggered by oncogenic Ras as well as the paradoxical activation of Raf‐1 by kinase‐inactivated B‐Raf require an intact DIF. Surprisingly, the B‐Raf DIF is not required for dimerisation between Raf‐1 and B‐Raf, which was inactivated by the D594A mutation, sorafenib or PLX4720. This suggests that paradoxical MEK/ERK activation represents a two‐step mechanism consisting of dimerisation and DIF‐dependent transactivation. Our data further implicate the Raf DIF as a potential target against Ras‐driven Raf‐mediated (paradoxical) ERK activation.

Walking the tightrope: proteostasis and neurodegenerative disease.

A characteristic of many neurodegenerative diseases, including Alzheimers disease (AD), Parkinsons disease (PD), Huntingtons disease (HD), and amyotrophic lateral sclerosis (ALS), is the aggregation of specific proteins into protein inclusions and/or plaques in degenerating brains. While much of the aggregated protein consists of disease specific proteins, such as amyloid‐β, α‐synuclein, or superoxide dismutase1 (SOD1), many other proteins are known to aggregate in these disorders. Although the role of protein aggregates in the pathogenesis of neurodegenerative diseases remains unknown, the ubiquitous association of misfolded and aggregated proteins indicates that significant dysfunction in protein homeostasis (proteostasis) occurs in these diseases. Proteostasis is the concept that the integrity of the proteome is in fine balance and requires proteins in a specific conformation, concentration, and location to be functional. In this review, we discuss the role of specific mechanisms, both inside and outside cells, which maintain proteostasis, including molecular chaperones, protein degradation pathways, and the active formation of inclusions, in neurodegenerative diseases associated with protein aggregation.

Germline mutations in CDH1 are infrequent in women with early-onset or familial lobular breast cancers

Background Germline mutations in CDH1 are associated with hereditary diffuse gastric cancer; lobular breast cancer also occurs excessively in families with such condition. Method To determine if CDH1 is a susceptibility gene for lobular breast cancer in women without a family history of diffuse gastric cancer, germline DNA was analysed for the presence of CDH1 mutations in 318 women with lobular breast cancer who were diagnosed before the age of 45 years or had a family history of breast cancer and were not known, or known not, to be carriers of germline mutations in BRCA1 or BRCA2. Cases were ascertained through breast cancer registries and high-risk cancer genetic clinics (Breast Cancer Family Registry, the kConFab and a consortium of breast cancer genetics clinics in the United States and Spain). Additionally, Multiplex Ligation-dependent Probe Amplification was performed for 134 cases to detect large deletions. Results No truncating mutations and no large deletions were detected. Six non-synonymous variants were found in seven families. Four (4/318 or 1.3%) are considered to be potentially pathogenic through in vitro and in silico analysis. Conclusion Potentially pathogenic germline CDH1 mutations in women with early-onset or familial lobular breast cancer are at most infrequent.

Plasminogen binding and activation at the breast cancer cell surface: the integral role of urokinase activity

IntroductionThe regulation of extracellular proteolytic activity via the plasminogen activation system is complex, involving numerous activators, inhibitors, and receptors. Previous studies on monocytic and colon cell lines suggest that plasmin pre-treatment can increase plasminogen binding, allowing the active enzyme to generate binding sites for its precursor. Other studies have shown the importance of pre-formed receptors such as annexin II heterotetramer. However, few studies have used techniques that exclusively characterise cell-surface events and these mechanisms have not been investigated at the breast cancer cell surface.MethodsWe have studied plasminogen binding to MCF-7 in which urokinase plasminogen activator receptor (uPAR) levels were upregulated by PMA (12-O-tetradecanoylphorbol-13-acetate) stimulation, allowing flexible and transient modulation of cell-surface uPA. Similar experiments were also performed using MDA-MB-231 cells, which overexpress uPAR/uPA endogenously. Using techniques that preserve cell integrity, we characterise the role of uPA as both a plasminogen receptor and activator and quantify the relative contribution of pre-formed and cryptic plasminogen receptors to plasminogen binding.ResultsCell-surface plasminogen binding was significantly enhanced in the presence of elevated levels of uPA in an activity-dependent manner and was greatly attenuated in the presence of the plasmin inhibitor aprotinin. Pre-formed receptors were also found to contribute to increased plasminogen binding after PMA stimulation and to co-localise with uPA/uPAR and plasminogen. Nevertheless, a relatively modest increase in plasminogen-binding capacity coupled with an increase in uPA led to a dramatic increase in the proteolytic capacity of these cells.ConclusionWe show that the majority of lysine-dependent plasminogen binding to breast cancer cells is ultimately regulated by plasmin activity and is dependent on the presence of significant levels of active uPA. The existence of a proteolytic positive feedback loop in plasminogen activation has profound implications for the ability of breast cancer cells expressing high amounts of uPA to accumulate a large proteolytic capacity at the cell surface, thereby conferring invasive potential.

EDD Mediates DNA Damage-induced Activation of CHK2

EDD, the human orthologue of Drosophila melanogaster “hyperplastic discs,” is overexpressed or mutated in a number of common human cancers. Although EDD has been implicated in DNA damage signaling, a definitive role has yet to be demonstrated. Here we report a novel interaction between EDD and the DNA damage checkpoint kinase CHK2. EDD and CHK2 associate through a phospho-dependent interaction involving the CHK2 Forkhead-associated domain and a region of EDD spanning a number of putative Forkhead-associated domain-binding threonines. Using RNA interference, we demonstrate a critical role for EDD upstream of CHK2 in the DNA damage signaling pathway. EDD is necessary for the efficient activating phosphorylation of CHK2 in response to DNA damage following exposure to ionizing radiation or the radiomimetic, phleomycin. Cells depleted of EDD display impaired CHK2 kinase activity and an inability to respond to DNA damage. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasize the potential importance of EDD in cancer.

Synthetic Lethality Screens Reveal RPS6 and MST1R as Modifiers of Insulin-like Growth Factor-1 Receptor Inhibitor Activity in Childhood Sarcomas

The insulin-like growth factor-1 receptor (IGF1R) is emerging as a promising therapeutic target in human cancers. In the high-risk childhood sarcomas Ewing family tumor and rhabdomyosarcoma, IGF1R-blocking antibodies show impressive antitumor activity in some but not all patients, and acquired resistance is observed. Because tumor IGF1R mutations are not described, the basis of IGF1R inhibitor resistance remains unknown. We hypothesized that compensatory signaling cascades bypassing targeted IGF1R inhibition might be involved. To test this systematically, we performed small interfering RNA (siRNA) screens in sarcoma cell lines to identify IGF1R pathway components or related protein tyrosine kinase (PTK) networks that modulate the antitumor efficacy of the BMS-536924 IGF1R kinase inhibitor. This strategy revealed (a) that sarcoma cells are exquisitely sensitive to loss of distal rather than proximal IGF1R signaling components, such as ribosomal protein S6 (RPS6); (b) that BMS-536924 fails to block RPS6 activation in resistant sarcoma cell lines; and (c) that siRNA knockdown of the macrophage-stimulating 1 receptor tyrosine kinase (MST1R; also known as RON) restores BMS-536924 efficacy, even in highly drug-resistant cell lines. We confirmed MST1R expression across a broad panel of childhood sarcomas, and found that loss of MST1R by RNA interference blocks downstream RPS6 activation when combined with BMS-536924 in vitro. These findings underscore the importance of fully understanding PTK networks for successful clinical implementation of kinase inhibitor strategies.

The Urokinase/PAI-2 Complex A NEW HIGH AFFINITY LIGAND FOR THE ENDOCYTOSIS RECEPTOR LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN

The efficient inactivation of urokinase plasminogen activator (uPA) by plasminogen activator inhibitor type 2 (PAI-2) at the surface of carcinoma cells is followed by rapid endocytosis of the uPA-PAI-2 complex. We now show that one pathway of this receptor-mediated endocytosis is mediated via the low density lipoprotein receptor-related protein (LRP) in prostate cancer cells. Detailed biochemical analyses using ligand binding assays and surface plasmon resonance revealed a novel and distinct interaction mechanism between native, human LRP and uPA-PAI-2. As reported previously for PAI-1, inhibition of uPA by PAI-2 significantly increased the affinity of the complex for LRP (KD of 36 nm for uPA-PAI-2 versus 200 nm for uPA). This interaction was maintained in the presence of uPAR, confirming the validity of this interaction at the cell surface. However, unlike PAI-1, no interaction was observed between LRP and PAI-2 in either the stressed or the relaxed conformation. This suggests that the uPA-PAI-2-LRP interaction is mediated by site(s) within the uPA molecule alone. Thus, as inhibition of uPA by PAI-2 resulted in accelerated clearance of uPA from the cell surface possibly via its increased affinity for LRP, this represents a mechanism through which PAI-2 can clear proteolytic activity from the cell surface. Furthermore, lack of a direct interaction between PAI-2 and LRP implies that downstream signaling events initiated by PAI-1 may not be activated by PAI-2.