Robert F. Shearer

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.

Tyrosine phosphorylation profiling reveals the signaling network characteristics of basal breast cancer cells

To identify therapeutic targets and prognostic markers for basal breast cancers, breast cancer cell lines were subjected to mass spectrometry-based profiling of protein tyrosine phosphorylation events. This revealed that luminal and basal breast cancer cells exhibit distinct tyrosine phosphorylation signatures that depend on pathway activation as well as protein expression. Basal breast cancer cells are characterized by elevated tyrosine phosphorylation of Met, Lyn, EphA2, epidermal growth factor receptor (EGFR), and FAK, and Src family kinase (SFK) substrates such as p130Cas. SFKs exert a prominent role in these cells, phosphorylating key regulators of adhesion and migration and promoting tyrosine phosphorylation of the receptor tyrosine kinases EGFR and Met. Consistent with these observations, SFK inhibition attenuated cellular proliferation, survival, and motility. Basal breast cancer cell lines exhibited differential responsiveness to small molecule inhibitors of EGFR and Met that correlated with the degree of target phosphorylation, and reflecting kinase coactivation, inhibiting two types of activated network kinase (e.g., EGFR and SFKs) was more effective than single agent approaches. FAK signaling enhanced both proliferation and invasion, and Lyn was identified as a proinvasive component of the network that is associated with a basal phenotype and poor prognosis in patients with breast cancer. These studies highlight multiple kinases and substrates for further evaluation as therapeutic targets and biomarkers. However, they also indicate that patient stratification based on expression/activation of drug targets, coupled with use of multi-kinase inhibitors or combination therapies, may be required for effective treatment of this breast cancer subgroup.

Hedgehog Overexpression Is Associated with Stromal Interactions and Predicts for Poor Outcome in Breast Cancer

Hedgehog (Hh) signaling plays an important role in several malignancies but its clinical significance in breast cancer is unclear. In a cohort of 279 patients with invasive ductal carcinoma of the breast, expression of Hh ligand was significantly associated with increased risk of metastasis, breast cancer-specific death, and a basal-like phenotype. A paracrine signature, encompassing high epithelial Hh ligand and high stromal Gli1, was an independent predictor for overall survival in multivariate analysis. In 2 independent histological progression series (n = 301), Hh expression increased with atypia. Hh ligand overexpression in a mouse model of basal breast cancer increased growth, induced a poorly differentiated phenotype, accelerated metastasis, and reduced survival. A stromal requirement for these effects was supported by the lack of similar Hh-mediated changes in vitro, and by stromal-specific expression of Hh target genes in vivo. Furthermore, inhibition of Hh ligand with a monoclonal antibody (5E1) inhibited tumor growth and metastasis. These data suggest that epithelial-stromal Hh signaling, driven by ligand expression in carcinoma cells, promotes breast cancer growth and metastasis. Blockade of Hh signaling to peritumoral stromal cells may represent a novel therapeutic approach in some basal-like breast cancers.

Adipocyte lipolysis links obesity to breast cancer growth: adipocyte-derived fatty acids drive breast cancer cell proliferation and migration

BackgroundObesity is associated with increased recurrence and reduced survival of breast cancer. Adipocytes constitute a significant component of breast tissue, yet their role in provisioning metabolic substrates to support breast cancer progression is poorly understood.ResultsHere, we show that co-culture of breast cancer cells with adipocytes revealed cancer cell-stimulated depletion of adipocyte triacylglycerol. Adipocyte-derived free fatty acids were transferred to breast cancer cells, driving fatty acid metabolism via increased CPT1A and electron transport chain complex protein levels, resulting in increased proliferation and migration. Notably, fatty acid transfer to breast cancer cells was enhanced from “obese” adipocytes, concomitant with increased stimulation of cancer cell proliferation and migration. This adipocyte-stimulated breast cancer cell proliferation was dependent on lipolytic processes since HSL/ATGL knockdown attenuated cancer cell responses.ConclusionsThese findings highlight a novel and potentially important role for adipocyte lipolysis in the provision of metabolic substrates to breast cancer cells, thereby supporting cancer progression.

Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

Basal breast cancer cells feature high expression of the Src family kinase Lyn that has been implicated in the pathogenicity of this disease. In this study, we identified novel Lyn kinase substrates, the most prominent of which was the atypical kinase SgK269 (PEAK1). In breast cancer cells, SgK269 expression associated with the basal phenotype. In primary breast tumors, SgK269 overexpression was detected in a subset of basal, HER2-positive, and luminal cancers. In immortalized MCF-10A mammary epithelial cells, SgK269 promoted transition to a mesenchymal phenotype and increased cell motility and invasion. Growth of MCF-10A acini in three-dimensional (3D) culture was enhanced upon SgK269 overexpression, which induced an abnormal, multilobular acinar morphology and promoted extracellular signal-regulated kinase (Erk) and Stat3 activation. SgK269 Y635F, mutated at a major Lyn phosphorylation site, did not enhance acinar size or cellular invasion. We show that Y635 represents a Grb2-binding site that promotes both Stat3 and Erk activation in 3D culture. RNA interference-mediated attenuation of SgK269 in basal breast cancer cells promoted acquisition of epithelial characteristics and decreased anchorage-independent growth. Together, our results define a novel signaling pathway in basal breast cancer involving Lyn and SgK269 that offers clinical opportunities for therapeutic intervention.

Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives

The use of third‐generation lentiviral vectors is now commonplace in most areas of basic biology. These systems provide a fast, efficient means for modulating gene expression, but experimental design needs to be carefully considered to minimize potential artefacts arising from off‐target effects and other confounding factors. This review offers a starting point for those new to lentiviral‐based vector systems, addressing the main issues involved with the use of lentiviral systems in vitro and outlines considerations which should be taken into account during experimental design. Factors such as selecting an appropriate system and controls, and practical titration of viral transduction are important considerations for experimental design. We also briefly describe some of the more recent advances in genome editing technology. TALENs and CRISPRs offer an alternative to lentivirus, providing endogenous gene editing with reduced off‐target effects often at the expense of efficiency.

Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers.

The BiCAP method reveals protein partner–specific signaling with implications for breast cancer. Learning the choreography of protein networks In addition to the many modifications, such as phosphorylation, that occur at the level of single molecules and regulate cell signaling, the formation of protein dimers and larger-order oligomers can alter the output of stimuli received by cells. The epidermal growth factor receptor (EGFR) family member ERBB2 dimerizes with itself and forms heterodimers with other family members. Croucher et al. developed a method called BiCAP that enables the specific isolation of any two interacting proteins by recognizing partner-specific conformations and demonstrated the utility of this approach to define partner-specific protein interaction networks for ERBB2, a receptor implicated in breast cancer. This revealed additional downstream mediators and signaling output for the ERBB3:ERBB2 heterodimer in breast cancer cells. Because changes in protein partners may contribute to disease or drug resistance, this method presents an approach to investigate how to exploit signals mediated by specific protein-protein interaction networks for patient therapy. The dynamic assembly of multiprotein complexes is a central mechanism of many cell signaling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive, response to rapidly changing cellular conditions. We describe a technique for the specific isolation and downstream proteomic characterization of any two interacting proteins, to the exclusion of their individual moieties and competing binding partners. We termed the approach bimolecular complementation affinity purification (BiCAP) because it combines the use of conformation-specific nanobodies with a protein-fragment complementation assay with affinity purification. Using BiCAP, we characterized the specific interactome of the epidermal growth factor receptor (EGFR) family member ERBB2 when in the form of a homodimer or when in the form of a heterodimer with either EGFR or ERBB3. We identified dimer-specific interaction patterns for key adaptor proteins and identified a number of previously unknown interacting partners. Functional analysis for one of these newly identified partners revealed a noncanonical mechanism of extracellular signal–regulated kinase (ERK) activation that is specific to the ERBB2:ERBB3 heterodimer and acts through the adaptor protein FAM59A in breast cancer cells.

Functional Roles of the E3 Ubiquitin Ligase UBR5 in Cancer.

The Ubiquitin-Proteasome System (UPS) is an important regulator of cell signaling and proteostasis, which are essential to a variety of cellular processes. The UPS is disrupted in many diseases including cancer, and targeting the UPS for cancer therapy is gaining wide interest. E3 ubiquitin ligases occupy a key position in the hierarchical UPS enzymatic cascade, largely responsible for determining substrate specificity and ubiquitin (Ub) chain topology. The E3 ligase UBR5 (aka EDD1) is emerging as a key regulator of the UPS in cancer and development. UBR5 expression is deregulated in many cancer types and UBR5 is frequently mutated in mantle cell lymphoma. UBR5 is highly conserved in metazoans, has unique structural features, and has been implicated in regulation of DNA damage response, metabolism, transcription, and apoptosis. Hence, UBR5 is a key regulator of cell signaling relevant to broad areas of cancer biology. However, the mechanism by which UBR5 may contribute to tumor initiation and progression remains poorly defined. This review synthesizes emerging insights from genetics, biochemistry, and cell biology to inform our understanding of UBR5 in cancer. These molecular insights indicate a role for UBR5 in integrating/coordinating various cellular signaling pathways. Finally, we discuss outstanding questions in UBR5 biology and highlight the need to systematically characterize substrates, and address limitations in current animal models, to better define the role of UBR5 in cancer. Mol Cancer Res; 13(12); 1523–32. ©2015 AACR.

SerpinB2 regulates stromal remodelling and local invasion in pancreatic cancer

Pancreatic cancer has a devastating prognosis, with an overall 5-year survival rate of ~8%, restricted treatment options and characteristic molecular heterogeneity. SerpinB2 expression, particularly in the stromal compartment, is associated with reduced metastasis and prolonged survival in pancreatic ductal adenocarcinoma (PDAC) and our genomic analysis revealed that SERPINB2 is frequently deleted in PDAC. We show that SerpinB2 is required by stromal cells for normal collagen remodelling in vitro, regulating fibroblast interaction and engagement with collagen in the contracting matrix. In a pancreatic cancer allograft model, co-injection of PDAC cancer cells and SerpinB2−/− mouse embryonic fibroblasts (MEFs) resulted in increased tumour growth, aberrant remodelling of the extracellular matrix (ECM) and increased local invasion from the primary tumour. These tumours also displayed elevated proteolytic activity of the primary biochemical target of SerpinB2—urokinase plasminogen activator (uPA). In a large cohort of patients with resected PDAC, we show that increasing uPA mRNA expression was significantly associated with poorer survival following pancreatectomy. This study establishes a novel role for SerpinB2 in the stromal compartment in PDAC invasion through regulation of stromal remodelling and highlights the SerpinB2/uPA axis for further investigation as a potential therapeutic target in pancreatic cancer.

Mitochondrial mutations and metabolic adaptation in pancreatic cancer

BackgroundPancreatic cancer has a five-year survival rate of ~8%, with characteristic molecular heterogeneity and restricted treatment options. Targeting metabolism has emerged as a potentially effective therapeutic strategy for cancers such as pancreatic cancer, which are driven by genetic alterations that are not tractable drug targets. Although somatic mitochondrial genome (mtDNA) mutations have been observed in various tumors types, understanding of metabolic genotype-phenotype relationships is limited.MethodsWe deployed an integrated approach combining genomics, metabolomics, and phenotypic analysis on a unique cohort of patient-derived pancreatic cancer cell lines (PDCLs). Genome analysis was performed via targeted sequencing of the mitochondrial genome (mtDNA) and nuclear genes encoding mitochondrial components and metabolic genes. Phenotypic characterization of PDCLs included measurement of cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a Seahorse XF extracellular flux analyser, targeted metabolomics and pathway profiling, and radiolabelled glutamine tracing.ResultsWe identified 24 somatic mutations in the mtDNA of 12 patient-derived pancreatic cancer cell lines (PDCLs). A further 18 mutations were identified in a targeted study of ~1000 nuclear genes important for mitochondrial function and metabolism. Comparison with reference datasets indicated a strong selection bias for non-synonymous mutants with predicted functional effects. Phenotypic analysis showed metabolic changes consistent with mitochondrial dysfunction, including reduced oxygen consumption and increased glycolysis. Metabolomics and radiolabeled substrate tracing indicated the initiation of reductive glutamine metabolism and lipid synthesis in tumours.ConclusionsThe heterogeneous genomic landscape of pancreatic tumours may converge on a common metabolic phenotype, with individual tumours adapting to increased anabolic demands via different genetic mechanisms. Targeting resulting metabolic phenotypes may be a productive therapeutic strategy.