Embryonic stem cells are devoid of macropinocytosis, a trafficking pathway for activin A in differentiated cells.

Ligand-receptor complexes formed at the plasma membrane are internalised via various endocytic pathways that influence the ultimate signalling output by regulating the selection of interaction partners by the complex along the trafficking route. We report that, in differentiated cells, activin A-receptor complexes are internalised via clathrin-mediated endocytosis (CME) and macropinocytosis (MP), whereas in human embryonic stem cells (hESCs) internalisation occurs via CME. We further show that hESCs are devoid of MP, which becomes functional upon differentiation towards endothelial cells through mesoderm mediators. Our results reveal, for the first time, that MP is an internalisation route for activin A in differentiated cells, and that MP is not active in hESCs and is induced as cells differentiate.

Inhibitors in AKTion: ATP-competitive vs allosteric.

Aberrant activation of the PI3K pathway is one of the commonest oncogenic events in human cancer. AKT is a key mediator of PI3K oncogenic function, and thus has been intensely pursued as a therapeutic target. Multiple AKT inhibitors, broadly classified as either ATP-competitive or allosteric, are currently in various stages of clinical development. Herein, we review the evidence for AKT dependence in human tumours and focus on its therapeutic targeting by the two drug classes. We highlight the future prospects for the development and implementation of more effective context-specific AKT inhibitors aided by our increasing knowledge of both its regulation and some previously unrecognised non-canonical functions.

A systematic molecular and pharmacologic evaluation of AKT inhibitors reveals new insight into their biological activity.

BACKGROUND: AKT, a critical effector of the phosphoinositide 3-kinase (PI3K) signalling cascade, is an intensely pursued therapeutic target in oncology. Two distinct classes of AKT inhibitors have been in clinical development, ATP-competitive and allosteric. Class-specific differences in drug activity are likely the result of differential structural and conformational requirements governing efficient target binding, which ultimately determine isoform-specific potency, selectivity profiles and activity against clinically relevant AKT mutant variants. METHODS: We have carried out a systematic evaluation of clinical AKT inhibitors using in vitro pharmacology, molecular profiling and biochemical assays together with structural modelling to better understand the context of drug-specific and drug-class-specific cell-killing activity. RESULTS: Our data demonstrate clear differences between ATP-competitive and allosteric AKT inhibitors, including differential effects on non-catalytic activity as measured by a novel functional readout. Surprisingly, we found that some mutations can cause drug resistance in an isoform-selective manner despite high structural conservation across AKT isoforms. Finally, we have derived drug-class-specific phosphoproteomic signatures and used them to identify effective drug combinations. CONCLUSIONS: These findings illustrate the utility of individual AKT inhibitors, both as drugs and as chemical probes, and the benefit of AKT inhibitor pharmacological diversity in providing a repertoire of context-specific therapeutic options.

Primary Cilia Mediate Diverse Kinase Inhibitor Resistance Mechanisms in Cancer.

Primary cilia are microtubule-based organelles that detect mechanical and chemical stimuli. Although cilia house a number of oncogenic molecules (including Smoothened, KRAS, EGFR, and PDGFR), their precise role in cancer remains unclear. We have interrogated the role of cilia in acquired and de novo resistance to a variety of kinase inhibitors, and found that, in several examples, resistant cells are distinctly characterized by an increase in the number and/or length of cilia with altered structural features. Changes in ciliation seem to be linked to differences in the molecular composition of cilia and result in enhanced Hedgehog pathway activation. Notably, manipulating cilia length via Kif7 knockdown is sufficient to confer drug resistance in drug-sensitive cells. Conversely, targeting of cilia length or integrity through genetic and pharmacological approaches overcomes kinase inhibitor resistance. Our work establishes a role for ciliogenesis and cilia length in promoting cancer drug resistance and has significant translational implications.

Vessel co-option is common in human lung metastases and mediates resistance to anti-angiogenic therapy in preclinical lung metastasis models.

Anti-angiogenic therapies have shown limited efficacy in the clinical management of metastatic disease, including lung metastases. Moreover, the mechanisms via which tumours resist anti-angiogenic therapies are poorly understood. Importantly, rather than utilizing angiogenesis, some metastases may instead incorporate pre-existing vessels from surrounding tissue (vessel co-option). As anti-angiogenic therapies were designed to target only new blood vessel growth, vessel co-option has been proposed as a mechanism that could drive resistance to anti-angiogenic therapy. However, vessel co-option has not been extensively studied in lung metastases, and its potential to mediate resistance to anti-angiogenic therapy in lung metastases is not established. Here, we examined the mechanism of tumour vascularization in 164 human lung metastasis specimens (composed of breast, colorectal and renal cancer lung metastasis cases). We identified four distinct histopathological growth patterns (HGPs) of lung metastasis (alveolar, interstitial, perivascular cuffing, and pushing), each of which vascularized via a different mechanism. In the alveolar HGP, cancer cells invaded the alveolar air spaces, facilitating the co-option of alveolar capillaries. In the interstitial HGP, cancer cells invaded the alveolar walls to co-opt alveolar capillaries. In the perivascular cuffing HGP, cancer cells grew by co-opting larger vessels of the lung. Only in the pushing HGP did the tumours vascularize by angiogenesis. Importantly, vessel co-option occurred with high frequency, being present in >80% of the cases examined. Moreover, we provide evidence that vessel co-option mediates resistance to the anti-angiogenic drug sunitinib in preclinical lung metastasis models. Assuming that our interpretation of the data is correct, we conclude that vessel co-option in lung metastases occurs through at least three distinct mechanisms, that vessel co-option occurs frequently in lung metastases, and that vessel co-option could mediate resistance to anti-angiogenic therapy in lung metastases. Novel therapies designed to target both angiogenesis and vessel co-option are therefore warranted. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Vessel co-option mediates resistance to anti-angiogenic therapy in liver metastases.

The efficacy of angiogenesis inhibitors in cancer is limited by resistance mechanisms that are poorly understood. Notably, instead of through the induction of angiogenesis, tumor vascularization can occur through the nonangiogenic mechanism of vessel co-option. Here we show that vessel co-option is associated with a poor response to the anti-angiogenic agent bevacizumab in patients with colorectal cancer liver metastases. Moreover, we find that vessel co-option is also prevalent in human breast cancer liver metastases, a setting in which results with anti-angiogenic therapy have been disappointing. In preclinical mechanistic studies, we found that cancer cell motility mediated by the actin-related protein 2/3 complex (Arp2/3) is required for vessel co-option in liver metastases in vivo and that, in this setting, combined inhibition of angiogenesis and vessel co-option is more effective than the inhibition of angiogenesis alone. Vessel co-option is therefore a clinically relevant mechanism of resistance to anti-angiogenic therapy and combined inhibition of angiogenesis and vessel co-option might be a warranted therapeutic strategy.

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis.

Expression of the initiator methionine tRNA (tRNAi(Met)) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi(Met) expression levels influence tumor progression. We have found that tRNAi(Met) expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi(Met) in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi(Met) contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi(Met) gene (2+tRNAi(Met) mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi(Met) mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi(Met) mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi(Met) significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi(Met)-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi(Met)-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi(Met) mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi(Met) levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis.

Direct binding of Bcl-2 family proteins by quercetin triggers its pro-apoptotic activity.

Bcl-2 family proteins are important regulators of apoptosis and its antiapoptotic members, which are overexpressed in many types of cancer, are of high prognostic significance, establishing them as attractive therapeutic targets. Quercetin, a natural flavonoid, has drawn much attention because it exerts anticancer effects, while sparing normal cells. A multidisciplinary approach has been employed herein, in an effort to reveal its mode of action including dose-response antiproliferative activity and induced apoptosis effect, biochemical and physicochemical assays, and computational calculations. It may be concluded that, quercetin binds directly to the BH3 domain of Bcl-2 and Bcl-xL proteins, thereby inhibiting their activity and promoting cancer cell apoptosis.

SARA and RNF11 at the crossroads of EGFR signaling and trafficking.

The classical view that endocytosis serves only for growth factor receptor degradation and signaling termination has recently been challenged by an increasing number of reports showing that various growth factor receptors such as epidermal growth factor receptor (EGFR) continue to activate downstream signaling molecules en route to lysosomes prior to their degradation. Moreover, the trafficking route that the ligand-receptor complexes follow to enter the cell is mutually interconnected with the final signaling output. Endosomal resident effector proteins are compartmentalized and regulate the signaling and trafficking of the ligand-bound receptor complexes. Smad anchor for receptor activation (SARA) is an early endosomal protein facilitating TGF-ß signaling cascade. Even though SARA was identified as an adaptor protein that regulates SMAD2 activation and TGF-ß signal propagation, an increasing number of reports in various systems describe SARA as a trafficking regulator. Recently, SARA has been shown to interact with the E3 ubiquitin ligase RNF11 (RING finger protein 11) and members of the ESCRT-0 (endosomal sorting complex required for transport) complex functionally participating in the degradation of EGFR.

ERBIN is a new SARA-interacting protein: competition between SARA and SMAD2 and SMAD3 for binding to ERBIN.

SARA, an early endosomal protein, plays a key role in TGFß signalling, as it presents SMAD2 and SMAD3 for phosphorylation by the activated TGFß receptors. Here, we show that ERBIN is a new SARA-interacting protein that can be recruited by SARA to early endosomes. ERBIN was recently shown to bind and segregate phosphorylated SMAD2 and SMAD3 (SMAD2/3) in the cytoplasm, thereby inhibiting SMAD2/3-dependent transcription. SARA binds to ERBIN using a new domain, which we have called the ERBID (ERBIN-binding domain), whereas ERBIN binds to SARA using a domain (amino acids 1208-1265) that also interacts with SMAD2 and SMAD3, which we have called the SSID (SARA- and SMAD-interacting domain). We additionally show that SARA competes with SMAD2/3 for binding to ERBIN. In agreement, overexpression of SARA or the ERBID peptide reverses the inhibitory effect of ERBIN on SMAD2/3-dependent transcription. Taken together, these data suggest that the response of cells to TGFß and activin A can be influenced by the relative concentrations of SARA, ERBIN and SMAD2/3.