ADAMTS3 restricts cancer invasion in models of early breast cancer progression through enhanced fibronectin degradation.

Proteases have long been associated with cancer progression, due to their ability to facilitate invasion upon matrix remodelling. However, proteases are not simply degraders of the matrix, but also play fundamental roles in modulating cellular behaviour through the proteolytic processing of specific substrates. Indeed, proteases can elicit both pro- and anti- tumorigenic effects depending on context.

TGFβ-mediated MMP13 secretion drives myoepithelial cell dependent breast cancer progression.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. Virtually all women with DCIS are treated, despite evidence suggesting up to half would remain with stable, non-threatening, disease. Overtreatment thus presents a pressing issue in DCIS management.

Everybody needs good neighbours: the progressive DCIS microenvironment.

Ductal carcinoma in situ (DCIS) is a pre-invasive form of breast cancer where neoplastic luminal cells are confined to the ductal tree. While as many as 70% of DCIS cases will remain indolent, most women are treated with surgery, often combined with endocrine and radiotherapies. Overtreatment is therefore a major issue, demanding new methods to stratify patients.

Mechanostimulation of breast myoepithelial cells induces functional changes associated with DCIS progression to invasion.

Women with ductal carcinoma in situ (DCIS) have an increased risk of progression to invasive breast cancer. Although not all women with DCIS will progress to invasion, all are treated as such, emphasising the need to identify prognostic biomarkers. We have previously shown that altered myoepithelial cells in DCIS predict disease progression and recurrence.

Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors.

Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue.

Loss of MMP-8 in ductal carcinoma in situ (DCIS)-associated myoepithelial cells contributes to tumour promotion through altered adhesive and proteolytic function.

Background: Normal myoepithelial cells (MECs) play an important tumour-suppressor role in the breast but display an altered phenotype in ductal carcinoma in situ (DCIS), gaining tumour-promoter functions. Matrix metalloproteinase-8 (MMP-8) is expressed by normal MECs but is lost in DCIS. This study investigated the function of MMP-8 in MECs and the impact of its loss in DCIS.

The role of inflammation in progression of breast cancer: Friend or foe? (Review).

There is a growing interest in the role of the microenvironment in cancer, however, it has been known for over one hundred years that the immune system plays a prominent role in cancer. Recent decades have revealed more and more data on how our own host response to cancer cells can help or hinder progression of the disease. Despite all this work it is surprising how little is known about the role of the immune system in human breast cancer development, as compared to other cancers.

αvβ6 Expression in myoepithelial cells: a novel marker for predicting DCIS progression with therapeutic potential.

The tumor microenvironment dynamically regulates the progression of cancer. In the breast, a unique component of the microenvironment is the myoepithelial cell. Normal myoepithelial cells act as "natural tumor suppressors"; however, more recent evidence suggests that these cells develop phenotypic changes, which may contribute to loss of tumor suppressor activity.

Rigidity sensing and adaptation through regulation of integrin types.

Tissue rigidity regulates processes in development, cancer and wound healing. However, how cells detect rigidity, and thereby modulate their behaviour, remains unknown. Here, we show that sensing and adaptation to matrix rigidity in breast myoepithelial cells is determined by the bond dynamics of different integrin types.

Prognostic and therapeutic impact of argininosuccinate synthetase 1 control in bladder cancer as monitored longitudinally by PET imaging.

Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion.

Altered microenvironment promotes progression of preinvasive breast cancer: myoepithelial expression of αvβ6 integrin in DCIS identifies high-risk patients and predicts recurrence.

Purpose: This study investigated the functional and clinical significance of integrin αvβ6 upregulation in myoepithelial cells of ductal carcinoma in situ (DCIS).

Experimental Design: Archival samples of DCIS and DCIS with associated invasion (n = 532) were analyzed for expression of αvβ6 by immunohistochemistry and ability to predict recurrence and progression assessed in an independent, unique cohort of DCIS cases with long-term follow-up. Primary myoepithelial cells and myoepithelial cell lines, with and without αvβ6 expression, were used to measure the effect of αvβ6 on growth and invasion of tumor cell lines in vitro and in a xenograft mouse model.

GATA3 mutations found in breast cancers may be associated with aberrant nuclear localization, reduced transactivation and cell invasiveness.

Somatic and germline mutations in the dual zinc-finger transcription factor GATA3 are associated with breast cancers expressing the estrogen receptor (ER) and the autosomal dominant hypoparathyroidism-deafness-renal dysplasia syndrome, respectively. To elucidate the role of GATA3 in breast tumorigenesis, we investigated 40 breast cancers that expressed ER, for GATA3 mutations. Six different heterozygous GATA3 somatic mutations were identified in eight tumors, and these consisted of: a frameshifting deletion/insertion (944_945delGGinsAGC), an in-frame deletion of a key arginine residue (991_993delAGG), a seven-nucleotide frameshifting insertion (991_992insTGGAGGA), a frameshifting deletion (1196_1197delGA), and two frameshifting single nucleotide insertions (1224_1225insG found in three tumors and 1224_1225insA).

Fe sparing and Fe recycling contribute to increased superoxide dismutase capacity in iron-starved Chlamydomonas reinhardtii.

Fe deficiency is one of several abiotic stresses that impacts plant metabolism because of the loss of function of Fe-containing enzymes in chloroplasts and mitochondria, including cytochromes, FeS proteins, and Fe superoxide dismutase (FeSOD). Two pathways increase the capacity of the Chlamydomonas reinhardtii chloroplast to detoxify superoxide during Fe limitation stress. In one pathway, MSD3 is upregulated at the transcriptional level up to 10(3)-fold in response to Fe limitation, leading to synthesis of a previously undiscovered plastid-specific MnSOD whose identity we validated immunochemically.

Persistent signaling by thyrotropin-releasing hormone receptors correlates with G-protein and receptor levels.

G-protein-coupled receptors with dissociable agonists for thyrotropin, parathyroid hormone, and sphingosine-1-phosphate were found to signal persistently hours after agonist withdrawal. Here we show that mouse thyrotropin-releasing hormone (TRH) receptors, subtypes 2 and 1(TRH-R2 and TRH-R1), can signal persistently in HEK-EM293 cells under appropriate conditions, but TRH-R2 exhibits higher persistent signaling activity. Both receptors couple primarily to Gα(q/11).

Spatiotemporally regulated protein kinase A activity is a critical regulator of growth factor-stimulated extracellular signal-regulated kinase signaling in PC12 cells.

PC12 cells exhibit precise temporal control of growth factor signaling in which stimulation with epidermal growth factor (EGF) leads to transient extracellular signal-regulated kinase (ERK) activity and cell proliferation, whereas nerve growth factor (NGF) stimulation leads to sustained ERK activity and differentiation. While cyclic AMP (cAMP)-mediated signaling has been shown to be important in conferring the sustained ERK activity achieved by NGF, little is known about the regulation of cAMP and cAMP-dependent protein kinase (PKA) in these cells. Using fluorescence resonance energy transfer (FRET)-based biosensors localized to discrete subcellular locations, we showed that both NGF and EGF potently activate PKA at the plasma membrane, although they generate temporally distinct activity patterns.

Small-molecule thyrotropin receptor agonist activates naturally occurring thyrotropin-insensitive mutants and reveals their distinct cyclic adenosine monophosphate signal persistence.

Background: Subclinical hypothyroidism (SHT), characterized by normal thyroid hormone levels maintained by elevated thyrotropin (TSH), predisposes patients to health problems as they age. Some cases arise from mutations of the TSH receptor (TSHR) that confer TSH resistance. This resistance might be circumvented by TSHR agonists with different modes of binding compared with TSH.

Occupancy of both sites on the thyrotropin (TSH) receptor dimer is necessary for phosphoinositide signaling.

The thyroid-stimulating hormone (TSH) receptor signals via G(s) to produce cAMP and via G(q/11) to produce inositol-1,4,5-trisphosphate, which is degraded to inositol monophosphate (IP1; phosphoinositide signaling). The potency of TSH for cAMP signaling is higher than for phosphoinositide signaling, and it was suggested that there are "spare receptors" for cAMP signaling. In a human embryonic kidney macrophage scavenger receptor-expressing (HEK-EM) 293 model system, there are no spare receptors, but the cells still exhibited 100-fold differences in potencies.

Thyrotropin receptor stimulates internalization-independent persistent phosphoinositide signaling.

The thyrotropin [thyroid-stimulating hormone (TSH)] receptor (TSHR) is known to acutely and persistently stimulate cAMP signaling and at higher TSH concentrations to acutely stimulate phosphoinositide signaling. We measured persistent signaling by stimulating TSHR-expressing human embryonic kidney-EM293 cells with TSH and measuring cAMP or inositol monophosphate (IP1) production, a measure of phosphoinositide signaling, 60 min or longer after TSH removal. In contrast to persistent cAMP production, persistent IP1 production increased progressively when TSH exposure was increased from 1 to 30 min, whereas the rates of decay of persistent signaling were similar.

Luminescent kinase activity biosensors based on a versatile bimolecular switch.

Real-time tracking of kinase activity in living systems has revealed new modes of encoding signaling information into spatiotemporal activity patterns and opened new avenues for screening kinase modulators. However, the sensitivity of kinase activity detection, which is commonly coupled to a fluorescence resonance energy transfer (FRET)-based readout, has often been a limiting factor. Here we show that a kinase-inducible bimolecular switch consisting of a substrate for the kinase of interest and a phosphoamino acid binding domain can be designed to sense different kinase activities and coupled to various readouts, thereby allowing for examination of dynamic kinase activity with increased sensitivity and versatility.

Clinical and functional significance of α9β1 integrin expression in breast cancer: a novel cell-surface marker of the basal phenotype that promotes tumour cell invasion.

Integrin α9β1 is a receptor for ECM proteins, including Tenascin-C and the EDA domain of fibronectin, and has been shown to transduce TGFβ signalling. This study has examined the expression pattern of α9β1 in 141 frozen breast carcinoma samples and related expression to prognostic indices, molecular subtype and patient outcome. Effects of α9β1 on tumour cell migration and invasion were assessed using blocking antibody and gene transduction approaches.

Signaling diversity of PKA achieved via a Ca2+-cAMP-PKA oscillatory circuit.

Many protein kinases are key nodal signaling molecules that regulate a wide range of cellular functions. These functions may require complex spatiotemporal regulation of kinase activities. Here, we show that protein kinase A (PKA), Ca(2+) and cyclic AMP (cAMP) oscillate in sync in insulin-secreting MIN6 beta cells, forming a highly integrated oscillatory circuit.

Visualization of PKA activity in plasma membrane microdomains.

Membrane rafts are sphingolipid- and cholesterol-rich microdomains that contain dynamic arrangements of signaling proteins. Notably, various components of the ubiquitous cAMP/Protein Kinase A (PKA) pathway, including β-adrenergic receptors (β-ARs), G proteins, and adenylyl cyclases (ACs), have been shown to localize differentially between membrane rafts and non-raft regions of the plasma membrane. As PKA participates in regulating diverse fundamental cellular functions, a number of which require membrane rafts, it is important to understand how PKA activity is specifically regulated in these membrane microdomains.

The cAMP-dependent protein kinase inhibitor H-89 attenuates the bioluminescence signal produced by Renilla Luciferase.

Background: Investigations into the regulation and functional roles of kinases such as cAMP-dependent protein kinase (PKA) increasingly rely on cellular assays. Currently, there are a number of bioluminescence-based assays, for example reporter gene assays, that allow the study of the regulation, activity, and functional effects of PKA in the cellular context. Additionally there are continuing efforts to engineer improved biosensors that are capable of detecting real-time PKA signaling dynamics in cells.

Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms.

Introduction: The stromal microenvironment has a profound influence on tumour cell behaviour. In tumours, the extracellular matrix (ECM) composition differs from normal tissue and allows novel interactions to influence tumour cell function. The ECM protein tenascin-C (TNC) is frequently up-regulated in breast cancer and we have previously identified two novel isoforms - one containing exon 16 (TNC-16) and one containing exons 14 plus 16 (TNC-14/16).