Organization of mammary epithelial cells into 3D acinar structures requires glucocorticoid and JNK signaling

Mammary epithelial cells cultured on a concentrated laminin-rich extracellular matrix formed 3D acinar structures that matured to polarized monolayers surrounding a lumen. In the absence of glucocorticoids mature acinus formation failed and the expression of an acinus-associated, activator protein 1 (AP1) and nuclear factor κB transcription factor DNA-binding profile was lost. Treatment with the JNK inhibitor, SP600125, caused similar effects, whereas normal organization of the mammary epithelial cells as acini caused JNK activation in a glucocorticoid-dependent manner. The forming acini expressed BRCA1, GADD45β, MEKK4, and the JNK activating complex GADD 45β−MEKK4 in a glucocorticoid-dependent fashion. JNK catalyzed phosphorylation of c-Jun was also detected in the acini. In addition, expression of β4 integrin and in situ occupation of its promoter by AP1 components, c-Jun and Fos, was glucocorticoid dependent. These results suggest that glucocortocoid signaling regulates acinar integrity through a pathway involving JNK regulation of AP1 transcription factors and β4 integrin expression

Deletion of Irs2 causes reduced kidney size in mice: role for inhibition of GSK3β?

<p>Abstract</p> <p>Background</p> <p>Male <it>Irs2</it>^-/-mice develop fatal type 2 diabetes at 13-14 weeks. Defects in neuronal proliferation, pituitary development and photoreceptor cell survival manifest in <it>Irs2</it>^-/-mice. We identify retarded renal growth in male and female <it>Irs2</it>^-/-mice, independent of diabetes.</p> <p>Results</p> <p>Kidney size and kidney:body weight ratio were reduced by approximately 20% in <it>Irs2</it>^-/-mice at postnatal day 5 and was maintained in maturity. Reduced glomerular number but similar glomerular density was detected in <it>Irs2</it>^-/-kidney compared to wild-type, suggesting intact global kidney structure. Analysis of insulin signalling revealed renal-specific upregulation of PKBβ/Akt2, hyperphosphorylation of GSK3β and concomitant accumulation of β-catenin in <it>Irs2</it>^-/-kidney. Despite this, no significant upregulation of β-catenin targets was detected. Kidney-specific increases in Yes-associated protein (YAP), a key driver of organ size were also detected in the absence of <it>Irs2</it>. YAP phosphorylation on its inhibitory site Ser127 was also increased, with no change in the levels of YAP-regulated genes, suggesting that overall YAP activity was not increased in <it>Irs2</it>^-/-kidney.</p> <p>Conclusions</p> <p>In summary, deletion of <it>Irs2 </it>causes reduced kidney size early in mouse development. Compensatory mechanisms such as increased β-catenin and YAP levels failed to overcome this developmental defect. These data point to <it>Irs2 </it>as an important novel mediator of kidney size.</p

Annexin A8 identifies a subpopulation of transiently quiescent c-kit positive luminal progenitor cells of the ductal mammary epithelium

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ~15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G0/G1 arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin

TGFβ and CCN2/CTGF mediate actin related gene expression by differential E2F1/CREB activation

BACKGROUND: CCN2/CTGF is an established effector of TGFβ driven responses in diabetic nephropathy. We have identified an interaction between CCN2 and TGFβ leading to altered phenotypic differentiation and inhibited cellular migration. Here we determine the gene expression profile associated with this phenotype and define a transcriptional basis for differential actin related gene expression and cytoskeletal function. RESULTS: From a panel of genes regulated by TGFβ and CCN2, we used co-inertia analysis to identify and then experimentally verify a subset of transcription factors, E2F1 and CREB, that regulate an expression fingerprint implicated in altered actin dynamics and cell hypertrophy. Importantly, actin related genes containing E2F1 and CREB binding sites, stratified by expression profile within the dataset. Further analysis of actin and cytoskeletal related genes from patients with diabetic nephropathy suggests recapitulation of this programme during the development of renal disease. The Rho family member Cdc42 was also found uniquely to be activated in cells treated with TGFβ and CCN2; Cdc42 interacting genes were differentially regulated in diabetic nephropathy. CONCLUSIONS: TGFβ and CCN2 attenuate CREB and augment E2F1 transcriptional activation with the likely effect of altering actin cytoskeletal and cell growth/hypertrophic gene activity with implications for cell dysfunction in diabetic kidney disease. The cytoskeletal regulator Cdc42 may play a role in this signalling response

Association Testing of Previously Reported Variants in a Large Case-Control Meta-analysis of Diabetic Nephropathy

We formed the GEnetics of Nephropathy–an International Effort (GENIE) consortium to examine previously reported genetic associations with diabetic nephropathy (DN) in type 1 diabetes. GENIE consists of 6,366 similarly ascertained participants of European ancestry with type 1 diabetes, with and without DN, from the All Ireland-Warren 3-Genetics of Kidneys in Diabetes U.K. and Republic of Ireland (U.K.-R.O.I.) collection and the Finnish Diabetic Nephropathy Study (FinnDiane), combined with reanalyzed data from the Genetics of Kidneys in Diabetes U.S. Study (U.S. GoKinD). We found little evidence for the association of the EPO promoter polymorphism, rs161740, with the combined phenotype of proliferative retinopathy and end-stage renal disease in U.K.-R.O.I. (odds ratio [OR] 1.14, P = 0.19) or FinnDiane (OR 1.06, P = 0.60). However, a fixed-effects meta-analysis that included the previously reported cohorts retained a genome-wide significant association with that phenotype (OR 1.31, P = 2 × 10−9). An expanded investigation of the ELMO1 locus and genetic regions reported to be associated with DN in the U.S. GoKinD yielded only nominal statistical significance for these loci. Finally, top candidates identified in a recent meta-analysis failed to reach genome-wide significance. In conclusion, we were unable to replicate most of the previously reported genetic associations for DN, and significance for the EPO promoter association was attenuated

Homeobox transcription factor muscle segment homeobox 2 (Msx2) correlates with good prognosis in breast cancer patients and induces apoptosis in vitro

Introduction: The homeobox-containing transcription factor muscle segment homeobox 2 (Msx2) plays an important role in mammary gland development. However, the clinical implications of Msx2 expression in breast cancer are unclear. The aims of this study were to investigate the potential clinical value of Msx2 as a breast cancer biomarker and to clarify its functional role in vitro. Methods: Msx2 gene expression was first examined in a well-validated breast cancer transcriptomic dataset of 295 patients. Msx2 protein expression was then evaluated by immunohistochemistry in a tissue microarray (TMA) containing 281 invasive breast tumours. Finally, to assess the functional role of Msx2 in vitro, Msx2 was ectopically expressed in a highly invasive breast tumour cell line (MDA-MB-231) and an immortalised breast cell line (MCF10a), and these cell lines were examined for changes in growth rate, cell death and cell signalling. Results: Examination of Msx2 mRNA expression in a breast cancer transcriptomic dataset demonstrated that increased levels of Msx2 were associated with good prognosis (P = 0.011). Evaluation of Msx2 protein expression on a TMA revealed that Msx2 was detectable in both tumour cell nuclei and cytoplasm. Cytoplasmic Msx2 expression was associated with low grade tumours (P = 0.012) and Ki67 negativity (P = 0.018). Nuclear Msx2 correlated with low-grade tumours (P = 0.015), estrogen receptor positivity (P = 0.038), low Ki67 (P = 0.005) and high cyclin D1 expression (P = 0.037). Increased cytoplasmic Msx2 expression was associated with a prolonged breast cancer-specific survival (P = 0.049), recurrence-free survival (P = 0.029) and overall survival (P = 0.019). Ectopic expression of Msx2 in breast cell lines resulted in radically decreased cell viability mediated by induction of cell death via apoptosis. Further analysis of Msx2-expressing cells revealed increased levels of p21 and phosphorylated extracellular signal-regulated kinase (ERK) and decreased levels of Survivin and the 'split ends' (SPEN) protein family member RBM15. Conclusions: We conclude that increased Msx2 expression results in improved outcome for breast cancer patients, possibly by increasing the likelihood of tumour cell death by apoptosis

Linking Symptom Inventories using Semantic Textual Similarity

An extensive library of symptom inventories has been developed over time to measure clinical symptoms, but this variety has led to several long standing issues. Most notably, results drawn from different settings and studies are not comparable, which limits reproducibility. Here, we present an artificial intelligence (AI) approach using semantic textual similarity (STS) to link symptoms and scores across previously incongruous symptom inventories. We tested the ability of four pre-trained STS models to screen thousands of symptom description pairs for related content - a challenging task typically requiring expert panels. Models were tasked to predict symptom severity across four different inventories for 6,607 participants drawn from 16 international data sources. The STS approach achieved 74.8% accuracy across five tasks, outperforming other models tested. This work suggests that incorporating contextual, semantic information can assist expert decision-making processes, yielding gains for both general and disease-specific clinical assessment

Key signalling nodes in mammary gland development and cancer. Mitogen-activated protein kinase signalling in experimental models of breast cancer progression and in mammary gland development

Seven classes of mitogen-activated protein kinase (MAPK) intracellular signalling cascades exist, four of which are implicated in breast disease and function in mammary epithelial cells. These are the extracellular regulated kinase (ERK)1/2 pathway, the ERK5 pathway, the p38 pathway and the c-Jun N-terminal kinase (JNK) pathway. In some forms of human breast cancer and in many experimental models of breast cancer progression, signalling through the ERK1/2 pathway, in particular, has been implicated as being important. We review the influence of ERK1/2 activity on the organised three-dimensional association of mammary epithelial cells, and in models of breast cancer cell invasion. We assess the importance of epidermal growth factor receptor family signalling through ERK1/2 in models of breast cancer progression and the influence of ERK1/2 on its substrate, the oestrogen receptor, in this context. In parallel, we consider the importance of these MAPK-centred signalling cascades during the cycle of mammary gland development. Although less extensively studied, we highlight the instances of signalling through the p38, JNK and ERK5 pathways involved in breast cancer progression and mammary gland development