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. METHODS: Primary normal and DCIS-associated MECs, and normal (N-1089) and DCIS-modified myoepithelial (ß6-1089) cell lines, were used to assess MMP-8 expression and function. ß6-1089 lacking MMP-8 were transfected with MMP-8 WT and catalytically inactive MMP-8 EA, and MMP-8 in N-1089 MEC was knocked down with siRNA. The effect on adhesion and migration to extracellular matrix (ECM), localisation of α6ß4 integrin to hemidesmosomes (HD), TGF-ß signalling and gelatinase activity was measured. The effect of altering MEC MMP-8 expression on tumour cell invasion was investigated in 2D and 3D organotypic models. RESULTS: Assessment of primary cells and MEC lines confirmed expression of MMP-8 in normal MEC and its loss in DCIS-MEC. Over-expression of MMP-8 WT but not MMP-8 EA in ß6-1089 cells increased adhesion to ECM proteins and reduced migration. Conversely, knock-down of MMP-8 in N-1089 reduced adhesion and increased migration. Expression of MMP-8 WT in ß6-1089 led to greater localisation of α6ß4 to HD and reduced retraction fibre formation, this being reversed by MMP-8 knock-down in N-1089. Over-expression of MMP-8 WT reduced TGF-ß signalling and gelatinolytic activity. MMP-8 knock-down enhanced TGF-ß signalling and gelatinolytic activity, which was reversed by blocking MMP-9 by knock-down or an inhibitor. MMP-8 WT but not MMP-8 EA over-expression in ß6-1089 reduced breast cancer cell invasion in 2D and 3D invasion assays, while MMP-8 knock-down in N-1089 enhanced cancer cell invasion. Staining of breast cancer cases for MMP-8 revealed a statistically significant loss of MMP-8 expression in DCIS with invasion versus pure DCIS (p = 0.001). CONCLUSIONS: These data indicate MMP-8 is a vital component of the myoepithelial tumour-suppressor function. It restores MEC interaction with the matrix, opposes TGF-ß signalling and MMP-9 proteolysis, which contributes to inhibition of tumour cell invasion. Assessment of MMP-8 expression may help to determine risk of DCIS progression.

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. Cell binding to fibronectin through either α5ß1 integrins (constitutively expressed) or αvß6 integrins (selectively expressed in cancer and development) adapts force generation, actin flow and integrin recruitment to rigidities associated with healthy or malignant tissue, respectively. In vitro experiments and theoretical modelling further demonstrate that this behaviour is explained by the different binding and unbinding rates of both integrin types to fibronectin. Moreover, rigidity sensing through differences in integrin bond dynamics applies both when integrins bind separately and when they compete for binding to fibronectin.

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. In a second pathway, the plastid FeSOD is preferentially retained over other abundant Fe proteins, heme-containing cytochrome f, diiron magnesium protoporphyrin monomethyl ester cyclase, and Fe2S2-containing ferredoxin, demonstrating prioritized allocation of Fe within the chloroplast. Maintenance of FeSOD occurs, after an initial phase of degradation, by de novo resynthesis in the absence of extracellular Fe, suggesting the operation of salvage mechanisms for intracellular recycling and reallocation.

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. We found that PKA activity was essential for this oscillatory circuit and was capable of not only initiating the signaling oscillations but also modulating their frequency, thereby diversifying the spatiotemporal control of downstream signaling. Our findings suggest that exquisite temporal control of kinase activity, mediated via signaling circuits resulting from cross-regulation of signaling pathways, can encode diverse inputs into temporal parameters such as oscillation frequency, which in turn contribute to proper regulation of complex cellular functions in a context-dependent manner.

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). To gain insight into the mechanism of persistent signaling, we compared proximal steps of inositolmonophosphate (IP1) signaling by TRH-Rs. Persistent signaling was not caused by slower dissociation of TRH from TRH-R2 (t(1/2)=77 ± 8.1 min) compared with TRH-R1 (t(1/2)=82 ± 12 min) and was independent of internalization, as inhibition of internalization did not affect persistent signaling (115% of control), but required continuously activated receptors, as an inverse agonist decreased persistent signaling by 60%. Gα(q/11) knockdown decreased persistent signaling by TRH-R2 by 82%, and overexpression of Gα(q/11) induced persistent signaling in cells expressing TRH-R1. Lastly, persistent signaling was induced in cells expressing high levels of TRH-R1. We suggest that persistent signaling by TRHRs is exhibited when sufficient levels of agonist/receptor/G-protein complexes are established and maintained and that TRH-R2 forms and maintains these complexes more efficiently than TRH-R1.

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. Somewhat paradoxically, the neoplastic cells in DCIS are genetically comparable to those in invasive disease, suggesting the tumour microenvironment is the driving force for progression. Clinical and mechanistic studies highlight the complex DCIS microenvironment, with multiple cell types competing to regulate progression. Here, we examine recent studies detailing distinct aspects of the DCIS microenvironment and discuss how these may inform more effective care.

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. Using a heterocellular spheroid model of breast cancer progression, we demonstrate the repressive function of myoepithelial ADAMTS3, with its loss directing myoepithelial-led invasion of luminal cells through a physiologically relevant matrix. Degradomic analysis, using terminal amine isotopic labelling of substrates (TAILS), combined with functional assays, implicate ADAMTS3 as a mediator of fibronectin degradation. We show further that loss of ADAMTS3 enhances levels of fibronectin in the microenvironment, promoting invasion through canonical integrin α5ß1 activation. Our data highlight a tumour suppressive role for ADAMTS3 in early stage breast cancer, and contribute to the growing evidence that proteases can restrain cancer progression.

TGFß-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. To understand the role of the normally tumour suppressive myoepithelial cell in disease progression we present a 3D in vitro model incorporating both luminal and myoepithelial cells in physiomimetic conditions. We demonstrate that DCIS-associated myoepithelial cells promote striking myoepithelial-led invasion of luminal cells, mediated by the collagenase MMP13 through a non-canonical TGFß - EP300 pathway. In vivo, MMP13 expression is associated with stromal invasion in a murine model of DCIS progression and is elevated in myoepithelial cells of clinical high-grade DCIS cases. Our data identify a key role for myoepithelial-derived MMP13 in facilitating DCIS progression and point the way towards a robust marker for risk stratification in DCIS patients.

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. Dose responses for TSH-stimulated dissociation of prebound (125)I-TSH (negative cooperativity; EC(50)=70 mU/ml), which requires TSH binding to both sites of the TSH receptor (TSHR) homodimer, and TSH-stimulated IP1 production (EC(50)=50 mU/ml) were indistinguishable. Fluorescence resonance energy transfer (FRET) using tagged receptors showed that TSHR formed homodimers and heterodimers with two binding-deficient mutant TSHRs, L252P and C41S. When L252P or C41S was expressed with TSHR, that is, when TSHR/L252P or TSHR/C41S heterodimers could only bind one TSH, TSH-stimulated IP1 production was decreased relative to cAMP production. The slopes of linear regression analyses comparing fold stimulation by TSH of IP1 vs. cAMP production were 0.044 ± 0.0047, 0.0043 ± 0.0041, and 0.0059 ± 0.0014 for cells expressing TSHR alone, TSHR and L252P, or TSHR and C41S, respectively. We suggest that TSHR coupling to phosphoinositide signaling is dependent on binding 2 molecules of TSH to TSHR homodimer, causing a conformational change allowing coupling to G(q/11).

Clinical and functional significance of α9ß1 integrin expression in breast cancer: a novel cell-surface marker of the basal phenotype that promotes tumour cell invasion.

Integrin α9ß1 is a receptor for ECM proteins, including Tenascin-C and the EDA domain of fibronectin, and has been shown to transduce TGFß signalling. This study has examined the expression pattern of α9ß1 in 141 frozen breast carcinoma samples and related expression to prognostic indices, molecular subtype and patient outcome. Effects of α9ß1 on tumour cell migration and invasion were assessed using blocking antibody and gene transduction approaches. Integrin α9ß1 localized to myoepithelial cells in normal ducts and acini, a pattern maintained in DCIS. A subset (17%) of invasive carcinomas exhibited tumour cell expression of α9ß1, which related significantly to the basal-like phenotype, as defined by either CK5/6 or CK14 expression. Tumour expression of α9ß1 showed a significant association with reduced overall patient survival (p < 0.0001; HR 5.94, 95%CI 3.26-10.82) and with reduced distant-metastasis-free survival (p < 0.0001; HR 6.37, CI 3.51-11.58). A series of breast cancer cell lines was screened for α9ß1 with the highly invasive basal-like GI-101 cell line expressing significant levels. Both migration and invasion of this line were reduced significantly in the presence of α9-blocking antibody and following α9-knockdown with siRNA. Conversely, migratory and invasive behaviour of α9-negative MCF7 cells and α9-low MDA MB468 cells was enhanced significantly by over-expression of α9. Thus, α9ß1 acts as a novel marker of the basal-like breast cancer subtype and expression is associated with reduced survival, while its ability to promote breast cancer cell migration and invasion suggests that it contributes to the aggressive clinical behaviour of this tumour subtype.

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. By analysing DCIS duct size in sections of human breast tumour samples, we identified an associated upregulation of integrin ß6 and an increase in periductal fibronectin deposition with increased DCIS duct size that associated with the progression of DCIS to invasion. Our modelling of the mechanical stretching myoepithelial cells undergo during DCIS progression confirmed the upregulation of integrin ß6 and fibronectin expression in isolated primary and cell line models of normal myoepithelial cells. Our studies reveal that this mechanostimulated DCIS myoepithelial cell phenotype enhances invasion in a TGFß-mediated upregulation of MMP13. Immunohistochemical analysis identified that MMP13 was specifically upregulated in DCIS, and it was associated with progression to invasion. These findings implicate tissue mechanics in altering the myoepithelial cell phenotype in DCIS, and that these alterations may be used to stratify DCIS patients into low and high risk for invasive progression.

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. A small-molecule agonist and a thyroid-stimulating antibody also caused persistent IP1 and cAMP signaling. A small-molecule inverse agonist and a neutral antagonist inhibited TSH-stimulated persistent IP1 production, whereas the inverse agonist but not the neutral antagonist inhibited persistent cAMP production. As with persistent cAMP production, persistent IP1 production was not affected when TSHR internalization was inhibited or enhanced. Moreover, Alexa546-TSH-activated TSHR internalization was not accompanied by Gα(q) coupling protein internalization. Thus, transient exposure to high concentrations of TSH causes persistent phosphoinositide and cAMP signaling that is not dependent on internalization. To our knowledge, this is the first demonstration of persistent activation by any G protein-coupled receptor (GPCR) via the Gα(q) pathway and of two G protein-mediated pathways by any GPCR.

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. Specifically, we demonstrate that bimolecular switches designed to sense protein kinase A (PKA) or protein kinase C (PKC) activities can turn on FRET as well as bioluminescence signals. Notably, the FRET-based sensors gain larger dynamic ranges in comparison with their unimolecular counterparts; the novel bioluminescence-based reporters for PKA and PKC show high sensitivity and a unique capability to detect basal kinase activities and should enable new applications in in vivo imaging of kinase activity and high-throughput compound screening. Thus, this generalizable design advances the molecular toolkit of kinase activity detection and provides a means for versatile and sensitive detection of kinase activity in various biological systems.

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). METHODS: The present study has analysed the functional significance of this altered TNC isoform profile in breast cancer. TNC-16 and TNC-14/16 splice variants were generated using PCR-ligation and over-expressed in breast cancer cells (MCF-7, T47D, MDA-MD-231, MDA-MB-468, GI101) and human fibroblasts. The effects of these variants on tumour cell invasion and proliferation were measured and compared with the effects of the large (TNC-L) and fully spliced small (TNC-S) isoforms. RESULTS: TNC-16 and TNC-14/16 significantly enhanced tumour cell proliferation (P < 0.05) and invasion, both directly (P < 0.01) and as a response to transfected fibroblast expression (P < 0.05) with this effect being dependent on tumour cell interaction with TNC, because TNC-blocking antibodies abrogated these responses. An analysis of 19 matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases 1 to 4 (TIMP 1 to 4) revealed that TNC up-regulated expression of MMP-13 and TIMP-3 two to four fold relative to vector, and invasion was reduced in the presence of MMP inhibitor GM6001. However, this effect was not isoform-specific but was elicited equally by all TNC isoforms. CONCLUSIONS: These results demonstrate a dual requirement for TNC and MMP in enhancing breast cancer cell invasion, and identify a significant role for the tumour-associated TNC-16 and TNC-14/16 in promoting tumour invasion, although these isoform-specific effects appear to be mediated through MMP-independent mechanisms.

FER1 and FER2 encoding two ferritin complexes in Chlamydomonas reinhardtii chloroplasts are regulated by iron.

Two unlinked genes FER1 and FER2 encoding ferritin subunits were identified in the Chlamydomonas genome. An improved FER2 gene model, built on the basis of manual sequencing and incorporation of unplaced reads, indicated 49% identity between the ferritin subunits. Both FER1 and FER2 transcripts are increased in abundance as iron nutrition is decreased but the pattern for each gene is distinct. Using subunit-specific antibodies, we monitored expression at the protein level. In response to low iron, ferritin1 subunits and the ferritin1 complex are increased in parallel to the increase in FER1 mRNA. Nevertheless, the iron content of the ferritin1 complex is decreased. This suggests that increased expression results in increased capacity for iron binding in the chloroplast of iron-limited cells, which supports a role for ferritin1 as an iron buffer. On the other hand, ferritin2 abundance is decreased in iron-deprived cells, indicative of the operation of iron-nutrition-responsive regulation at the translational or post-translational level for FER2. Both ferritin subunits are plastid localized but ferritin1 is quantitatively recovered in soluble extracts of cells while ferritin2 is found in the particulate fraction. Partial purification of the ferritin1 complex indicates that the two ferritins are associated in distinct complexes and do not coassemble. The ratio of ferritin1 to ferritin2 is 70:1 in iron-replete cells, suggestive of a more dominant role of ferritin1 in iron homeostasis. The Volvox genome contains orthologs of each FER gene, indicating that the duplication of FER genes and potential diversification of function occurred prior to the divergence of species in the Volvocales.

Regulation and localization of isoforms of the aerobic oxidative cyclase in Chlamydomonas reinhardtii.

Tetrapyrrole synthesis is complex and is regulated at several points. The aerobic oxidative cyclase catalyzes the oxidative closure of the fifth ring characteristic of all chlorophylls. Chlamydomonas reinhardtii encodes two paralogous protein components of the cyclase, which are differentially accumulated based on copper nutrition and oxygen supply. CRD1 is accumulated in copper-deficient conditions, whereas CTH1 is present in copper replete conditions. Here, we show that CRD1 expression is regulated through a copper-responsive element located upstream of the transcription start site. The differential production of CRD1 transcript accounts for the differential accumulation of the corresponding polypeptide. The CTH1 locus produces two transcripts: a 2.1 kb one under copper-replete conditions and a 3.1 kb one under copper-deficient conditions. We show that the 2.1 kb transcript can be translated into protein in vitro whereas the 3.1 kb transcript cannot. Differential accumulation of the 2.1 vs the 3.1 kb transcript therefore accounts for the copper-responsive accumulation of CTH1. Biochemical fractionation reveals that both CRD1 and CTH1 are localized to chloroplast envelope as well as thylakoid membranes.

Between a rock and a hard place: trace element nutrition in Chlamydomonas.

Photosynthetic organisms are among the earliest life forms on earth and their biochemistry is strictly dependent on a wide range of inorganic nutrients owing to the use of metal cofactor-dependent enzymes in photosynthesis, respiration, inorganic nitrogen and sulfur assimilation. Chlamydomonas reinhardtii is a photosynthetic eukaryotic model organism for the study of trace metal homeostasis. Chlamydomonas spp. are widely distributed and can be found in soil, glaciers, acid mines and sewage ponds, suggesting that the genus has significant capacity for acclimation to micronutrient availability. Analysis of the draft genome indicates that metal homeostasis mechanisms in Chlamydomonas represent a blend of mechanisms operating in animals, plants and microbes. A combination of classical genetics, differential expression and genomic analysis has led to the identification of homologues of components known to operate in fungi and animals (e.g., Fox1, Ftr1, Fre1, Fer1, Ctr1/2) as well as novel molecules involved in copper and iron nutrition (Crr1, Fea1/2). Besides activating iron assimilation pathways, iron-deficient Chlamydomonas cells re-adjust metabolism by reducing light delivery to photosystem I (to avoid photo-oxidative damage resulting from compromised FeS clusters) and by modifying the ferredoxin profile (perhaps to accommodate preferential allocation of reducing equivalents). Up-regulation of a MnSOD isoform may compensate for loss of FeSOD. Ferritin could function to buffer the iron released from programmed degradation of iron-containing enzymes in the chloroplast. Some metabolic adjustments are made in anticipation of deficiency while others occur only with sustained or severe deficiency. Copper-deficient Chlamydomonas cells induce a copper assimilation pathway consisting of a cell surface reductase and a Cu(+) transporter (presumed CTR homologue). There are metabolic adaptations in addition: the synthesis of "back-up" enzymes for plastocyanin in photosynthesis and the ferroxidase in iron assimilation plus activation of alternative oxidase to handle the electron "overflow" resulting from reduced cytochrome oxidase function. Oxygen-dependent enzymes in the tetrapyrrole pathway (coproporphyrinogen oxidase and aerobic oxidative cyclase) are also increased in expression and activity by as much as 10-fold but the connection between copper nutrition and tetrapyrroles is not understood. The copper-deficiency responses are mediated by copper response elements that are defined by a GTAC core sequence and a novel metalloregulator, Crr1, which uses a zinc-dependent SBP domain to bind to the CuRE. The Chlamydomonas model is ideal for future investigation of nutritional manganese deficiency and selenoenzyme function. It is also suited for studies of trace nutrient interactions, nutrition-dependent metabolic changes, the relationship between photo-oxidative stress and metal homeostasis, and the important questions of differential allocation of limiting metal nutrients (e.g., to respiration vs. photosynthesis).

FRET-based biosensors for protein kinases: illuminating the kinome.

Protein kinases are crucial components of intracellular signaling pathways which transmit signals by phosphorylation of downstream targets, altering their function. Efficient signal transduction requires precise kinase regulation within specific biological contexts, making tools that allow study of their dynamics in situ critical for understanding kinase function. Highlighted in this article is the design of genetically-encodable, FRET-based kinase biosensors with examples of their implementation to study kinase regulation in live biological contexts with high spatial and temporal resolution.

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. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct, and this prevents cells from engaging senescence arrest. Our data show that the key driver of this phenotype is repression of CDKN2A transcript p16 where myoepithelial cells harbour cancer-like gene expression but do not exhibit anchorage-independent growth. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, which may facilitate tumour initiation.

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. Recent successes of PD-1 blockade in treating multiple cancers, and new developments with other immune targets such as CTLA-4 and CSF-1 inhibitors, highlight that it is becoming ever more important that we understand the complexity of the immune and inflammatory systems in the development and progression of breast cancer. With this knowledge it may be possible to not only target therapy but also more accurately predict those patients that truly need it. This review summarises some of the most significant findings for the role of the immune system and inflammatory response in breast cancer progression. Focusing on how the inflammatory microenvironment may be involved in the progression of pre-invasive ductal carcinoma in situ to invasive breast cancer. It will also discuss the use of immune markers as diagnostic and prognostic tools and summarise the state of the art of immune-therapeutics in breast cancer treatment.