Brittney S. Harrington

EGF inhibits constitutive internalization and palmitoylation-dependent degradation of membrane-spanning procancer CDCP1 promoting its availability on the cell surface.

Many cancers are dependent on inappropriate activation of epidermal growth factor receptor (EGFR), and drugs targeting this receptor can improve patient survival, although benefits are generally short-lived. We reveal a novel mechanism linking EGFR and the membrane-spanning, cancer-promoting protein CDCP1 (CUB domain-containing protein 1). Under basal conditions, cell surface CDCP1 constitutively internalizes and undergoes palmitoylation-dependent degradation by a mechanism in which it is palmitoylated in at least one of its four cytoplasmic cysteines. This mechanism is functional in vivo as CDCP1 is elevated and palmitoylated in high-grade serous ovarian tumors. Interestingly, activation of the EGFR system with EGF inhibits proteasome-mediated, palmitoylation-dependent degradation of CDCP1, promoting recycling of CDCP1 to the cell surface where it is available to mediate its procancer effects. We also show that mechanisms inducing relocalization of CDCP1 to the cell surface, including disruption of its palmitoylation and EGF treatment, promote cell migration. Our data provide the first evidence that the EGFR system can function to increase the lifespan of a protein and also promote its recycling to the cell surface. This information may be useful for understanding mechanisms of resistance to EGFR therapies and assist in the design of treatments for EGFR-dependent cancers.

Colonic microbiota can promote rapid local improvement of murine colitis by thioguanine independently of T lymphocytes and host metabolism.

Objective Mercaptopurine (MP) and pro-drug azathioprine are ‘first-line’ oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated lymphocytes homing to inflamed gut. We examined the role of host metabolism, lymphocytes and microbiome for the amelioration of colitis by the related thioguanine (TG). Design C57Bl/6 mice with or without specific genes altered to elucidate mechanisms responsible for TGs actions were treated daily with oral or intrarectal TG, MP or water. Disease activity was scored daily. At sacrifice, colonic histology, cytokine message, caecal luminal and mucosal microbiomes were analysed. Results Oral and intrarectal TG but not MP rapidly ameliorated spontaneous chronic colitis in Winnie mice (point mutation in Muc2 secretory mucin). TG ameliorated dextran sodium sulfate-induced chronic colitis in wild-type (WT) mice and in mice lacking T and B lymphocytes. Remarkably, colitis improved without immunosuppressive effects in the absence of host hypoxanthine (guanine) phosphoribosyltransferase (Hprt)-mediated conversion of TG to active drug, the thioguanine nucleotides (TGN). Colonic bacteria converted TG and less so MP to TGN, consistent with intestinal bacterial conversion of TG to so reduce inflammation in the mice lacking host Hprt. TG rapidly induced autophagic flux in epithelial, macrophage and WT but not Hprt−/− fibroblast cell lines and augmented epithelial intracellular bacterial killing. Conclusions Treatment by TG is not necessarily dependent on the adaptive immune system. TG is a more efficacious treatment than MP in Winnie spontaneous colitis. Rapid local bacterial conversion of TG correlated with decreased intestinal inflammation and immune activation.

Cyclin A/Cdk2 regulates Cdh1 and claspin during late S/G2 phase of the cell cycle

Whereas many components regulating the progression from S phase through G2 phase into mitosis have been identified, the mechanism by which these components control this critical cell cycle progression is still not fully elucidated. Cyclin A/Cdk2 has been shown to regulate the timing of Cyclin B/Cdk1 activation and progression into mitosis although the mechanism by which this occurs is only poorly understood. Here we show that depletion of Cyclin A or inhibition of Cdk2 during late S/early G2 phase maintains the G2 phase arrest by reducing Cdh1 transcript and protein levels, thereby stabilizing Claspin and maintaining elevated levels of activated Chk1 which contributes to the G2 phase observed. Interestingly, the Cyclin A/Cdk2 regulated APC/CCdh1 activity is selective for only a subset of Cdh1 targets including Claspin. Thus, a normal role for Cyclin A/Cdk2 during early G2 phase is to increase the level of Cdh1 which destabilises Claspin which in turn down regulates Chk1 activation to allow progression into mitosis. This mechanism links S phase exit with G2 phase transit into mitosis, provides a novel insight into the roles of Cyclin A/Cdk2 in G2 phase progression, and identifies a novel role for APC/CCdh1 in late S/G2 phase cell cycle progression.

CD169(+) macrophages mediate pathological formation of woven bone in skeletal lesions of prostate cancer.

Skeletal metastases present a major clinical challenge for prostate cancer patient care, inflicting distinctive mixed osteoblastic and osteolytic lesions that cause morbidity and refractory skeletal complications. Macrophages are abundant in bone and bone marrow and can influence both osteoblast and osteoclast function in physiology and pathology. Herein, we examined the role of macrophages in prostate cancer bone lesions, particularly the osteoblastic response. First, macrophage and lymphocyte distributions were qualitatively assessed in patients prostate cancer skeletal lesions by immunohistochemistry. Second, macrophage functional contributions to prostate tumour growth in bone were explored using an immune‐competent mouse model combined with two independent approaches to achieve in vivo macrophage depletion: liposome encapsulated clodronate that depletes phagocytic cells (including macrophages and osteoclasts); and targeted depletion of CD169+ macrophages using a suicide gene knock‐in model. Immunohistochemistry and histomorphometric analysis were performed to quantitatively assess cancer‐induced bone changes. In human bone metastasis specimens, CD68+ macrophages were consistently located within the tumour mass. Osteal macrophages (osteomacs) were associated with pathological woven bone within the metastatic lesions. In contrast, lymphocytes were inconsistently present in prostate cancer skeletal lesions and when detected, had varied distributions. In the immune‐competent mouse model, CD169+ macrophage ablation significantly inhibited prostate cancer‐induced woven bone formation, suggesting that CD169+ macrophages within pathological woven bone are integral to tumour‐induced bone formation. In contrast, pan‐phagocytic cell, but not targeted CD169+ macrophage depletion resulted in increased tumour mass, indicating that CD169− macrophage subset(s) and/or osteoclasts influenced tumour growth. In summary, these observations indicate a prominent role for macrophages in prostate cancer bone metastasis that may be therapeutically targetable to reduce the negative skeletal impacts of this malignancy, including tumour‐induced bone modelling. Copyright

Cell line and patient-derived xenograft models reveal elevated CDCP1 as a target in high-grade serous ovarian cancer

Background:Development of targeted therapies for high-grade serous ovarian cancer (HGSC) remains challenging, as contributing molecular pathways are poorly defined or expressed heterogeneously. CUB-domain containing protein 1 (CDCP1) is a cell-surface protein elevated in lung, colorectal, pancreas, renal and clear cell ovarian cancer.Methods:CUB-domain containing protein 1 was examined by immunohistochemistry in HGSC and fallopian tube. The impact of targeting CDCP1 on cell growth and migration in vitro, and intraperitoneal xenograft growth in mice was examined. Three patient-derived xenograft (PDX) mouse models were developed and characterised for CDCP1 expression. The effect of a monoclonal anti-CDCP1 antibody on PDX growth was examined. Src activation was assessed by western blot analysis.Results:Elevated CDCP1 was observed in 77% of HGSC cases. Silencing of CDCP1 reduced migration and non-adherent cell growth in vitro and tumour burden in vivo. Expression of CDCP1 in patient samples was maintained in PDX models. Antibody blockade of CDCP1 significantly reduced growth of an HGSC PDX. The CDCP1-mediated activation of Src was observed in cultured cells and mouse xenografts.Conclusions:CUB-domain containing protein 1 is over-expressed by the majority of HGSCs. In vitro and mouse model data indicate that CDCP1 has a role in HGSC and that it can be targeted to inhibit progression of this cancer.

Activation of membrane-bound proteins and receptor systems: a link between tissue kallikrein and the KLK-related peptidases

Abstract The 15 members of the kallikrein-related serine peptidase (KLK) family have diverse tissue-specific expression profiles and roles in a range of cellular processes, including proliferation, migration, invasion, differentiation, inflammation and angiogenesis that are required in both normal physiology as well as pathological conditions. These roles require cleavage of a range of substrates, including extracellular matrix proteins, growth factors, cytokines as well as other proteinases. In addition, it has been clear since the earliest days of KLK research that cleavage of cell surface substrates is also essential in a range of KLK-mediated cellular processes where these peptidases are essentially acting as agonists and antagonists. In this review we focus on these KLK-regulated cell surface receptor systems including bradykinin receptors, proteinase-activated receptors, as well as the plasminogen activator, ephrins and their receptors, and hepatocyte growth factor/Met receptor systems and other plasma membrane proteins. From this analysis it is clear that in many physiological and pathological settings KLKs have the potential to regulate multiple receptor systems simultaneously; an important issue when these peptidases and substrates are targeted in disease.

JIP4 is a PLK1 binding protein that regulates p38MAPK activity in G2 phase.

Cell cycle progression from G2 phase into mitosis is regulated by a complex network of mechanisms, all of which finally control the timing of Cyclin B/CDK1 activation. PLK1 regulates a network of events that contribute to regulating G2/M phase progression. Here we have used a proteomics approach to identify proteins that specifically bind to the Polobox domain of PLK1. This identified a panel of proteins that were either associated with PLK1 in G2 phase and/or mitosis, the strongest interaction being with the MAPK scaffold protein JIP4. PLK1 binding to JIP4 was found in G2 phase and mitosis, and PLK1 binding was self-primed by PLK1 phosphorylation of JIP4. PLK1 binding is required for JIP4-dependent p38MAPK activation in G2 phase during normal cell cycle progression, but not in either G2 phase or mitotic stress response. Finally, JIP4 is a target for caspase-dependent cleavage in mitotically arrested cells. The role for the PLK1-JIP4 regulated p38MAPK activation in G2 phase is unclear, but it does not affect either progression into or through mitosis.

Development of an enzyme-linked immunosorbent assay for detection of CDCP1 shed from the cell surface and present in colorectal cancer serum specimens

HIGHLIGHTSFirst report of an enzyme‐linked immunosorbent assay (ELISA) to detect fragments of the protein CDCP1 in human serum.The ELISA has a wide working range of 0.68–26.5 ng/ml, and a low limit of detection of 0.25 ng/ml.The ELISA has high intra‐assay (repeatability) and high inter‐assay (reproducibility) precision with all coefficients of variation ≤7%.The ELISA displays high accuracy detecting ShE‐CDCP1 levels at ≥94.8% of actual concentration.The findings will be relevant to investigators interested in CDCP1 as a cancer biomarker and potential therapeutic target. ABSTRACT CUB domain containing protein 1 (CDCP1) is a transmembrane protein involved in progression of several cancers. When located on the plasma membrane, full‐length 135 kDa CDCP1 can undergo proteolysis mediated by serine proteases that cleave after two adjacent amino acids (arginine 368 and lysine 369). This releases from the cell surface two 65 kDa fragments, collectively termed ShE‐CDCP1, that differ by one carboxyl terminal residue. To evaluate the function of CDCP1 and its potential utility as a cancer biomarker, in this study we developed an enzyme‐linked immunosorbent assay (ELISA) to reliably and easily measure the concentration of ShE‐CDCP1 in biological samples. Using a reference standard we demonstrate that the developed ELISA has a working range of 0.68–26.5 ng/ml, and the limit of detection is 0.25 ng/ml. It displays high intra‐assay (repeatability) and high inter‐assay (reproducibility) precision with all coefficients of variation ≤7%. The ELISA also displays high accuracy detecting ShE‐CDCP1 levels at ≥94.8% of actual concentration using quality control samples. We employed the ELISA to measure the concentration of ShE‐CDCP1 in human serum samples with our results suggesting that levels are significantly higher in serum of colorectal cancer patients compared with serum from individuals with benign conditions (p < 0.05). Our data also suggest that colorectal cancer patients with stage II‐IV disease have at least 50% higher serum levels of ShE‐CDCP1 compared with stage I cases (p < 0.05). We conclude that the developed ELISA is a suitable method to quantify ShE‐CDCP1 concentration in human serum.

Evidence that cell surface localization of serine protease activity facilitates cleavage of the protease activated receptor CDCP1

Abstract The cellular receptor CUB domain containing protein 1 (CDCP1) is commonly elevated and functionally important in a range of cancers. CDCP1 is cleaved by serine proteases at adjacent sites, arginine 368 (R368) and lysine 369 (K369), which induces cell migration in vitro and metastasis in vivo. We demonstrate that membrane localization of serine protease activity increases efficacy of cleavage of CDCP1, and that both secreted and membrane anchored serine proteases can have distinct preferences for cleaving at CDCP1-R368 and CDCP1-K369. Approaches that disrupt membrane localization of CDCP1 cleaving serine proteases may interfere with the cancer promoting effects of CDCP1 proteolysis.

EGF-induced recycling of the cancer promoting protein CDCP1

15th International Biennial Congress of the METASTASIS RESEARCH SOCIETY Heidelberg, Germany, June 28th–July 1st, 2014 Springer Science+Business Media Dordrecht 2015