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1.  Co-ordination of cell cycle, migration and stem cell-like activity in breast cancer 
Oncotarget  2014;5(17):7833-7842.
Migration, proliferation and stem cell-like activity are all key cellular characteristics which aid the formation and progression of breast cancer, in addition to involvement in treatment resistance. Many current therapies aim to target tumour proliferation, and although successful, mortality rates in breast cancer remain significant. Our main objectives were to investigate the relationship between proliferation, migration and stem cell-like activity in breast cancer.
We used a panel of cell lines and primary human breast cancer samples to assess the relationship between migration, proliferation and stem cells. We performed live cell sorting according to cell cycle (Hoechst-33324) and in combination with stem-cell markers (CD44/CD24/ESA) followed by assessment of migration and stem cell activity (mammosphere formation).
We identified an inverse relationship between proliferation and migration/stem cell-like activity. G0/1 cells showed increased migration and mammosphere formation. Furthermore we identified a subpopulation of low proliferative stem-like cells (CD44+/24lo/ESA+) with increased migration and mammosphere formation that are specifically inhibited by Dickkopf 1 (DKK1) and Dibenzazepine (DBZ) known stem-cell inhibitors.
These data show the co-ordination of migration, proliferation and stem cell activity in breast cancer, and has identified a sub-population of stem-like cells, greatly adding to our understanding of the complex nature of stem cell biology.
PMCID: PMC4202164  PMID: 25277201
Breast Cancer; Cellular proliferation; Cell migration; Cancer Stem cells
2.  Epithelial and Stromal MicroRNA Signatures of Columnar Cell Hyperplasia Linking Let-7c to Precancerous and Cancerous Breast Cancer Cell Proliferation 
PLoS ONE  2014;9(8):e105099.
Columnar cell hyperplasia (CCH) is the earliest histologically identifiable breast lesion linked to cancer progression and is characterized by increased proliferation, decreased apoptosis and elevated oestrogen receptor α (ERα) expression. The mechanisms underlying the initiation of these lesions have not been clarified but might involve early and fundamental changes in cancer progression. MiRNAs are key regulators of several biological processes, acting by influencing the post-transcriptional regulation of numerous targets, thus making miRNAs potential candidates in cancer initiation. Here we have defined novel epithelial as well as stromal miRNA signatures from columnar cell hyperplasia lesions compared to normal terminal duct lobular units by using microdissection and miRNA microarrays. Let-7c were among the identified downregulated epithelial miRNAs and its functions were delineated in unique CCH derived cells and breast cancer cell line MCF-7 suggesting anti-proliferative traits potentially due to effects on Myb and ERα. MiR-132 was upregulated in the stroma surrounding CCH compared to stoma surrounding normal terminal duct lobular units (TDLUs), and overexpression of miR-132 in immortalized fibroblasts and in fibroblasts co-cultured with epithelial CCH cells caused substantial expression changes of genes involved in metabolism, DNA damage and cell motility. The miRNA signatures identified in CCH indicate early changes in the epithelial and stromal compartment of CCH and could represent early key alterations in breast cancer progression that potentially could be targeted in novel prevention or treatment schedules.
PMCID: PMC4133372  PMID: 25122196
3.  Cell cycle regulators cyclin D1 and CDK4/6 have estrogen receptor-dependent divergent functions in breast cancer migration and stem cell-like activity 
Cell Cycle  2013;12(15):2384-2394.
Cyclin D1 and its binding partners CDK4/6 are essential regulators of cell cycle progression and are implicated in cancer progression. Our aim was to investigate a potential regulatory role of these proteins in other essential tumor biological characteristics. Using a panel of breast cancer cell lines and primary human breast cancer samples, we have demonstrated the importance of these cell cycle regulators in both migration and stem-like cell activity. siRNA was used to target cyclin D1 and CDK4/6 expression, having opposing effects on both migration and stem-like cell activity dependent upon estrogen receptor (ER) expression. Inhibition of cyclin D1 or CDK4/6 increases or decreases migration and stem-like cell activity in ER−ve (ER-negative) and ER+ve (ER-positive) breast cancer, respectively. Furthermore, overexpressed cyclin D1 caused decreased migration and stem-like cell activity in ER−ve cells while increasing activity in ER+ve breast cancer cells. Treatment of breast cancer cells with inhibitors of cyclin D1 and CDK4/6 (Flavopiridol/PD0332991), currently in clinical trials, mimicked the effects observed with siRNA treatment. Re-expression of ER in two ER−ve cell lines was sufficient to overcome the effects of either siRNA or clinical inhibitors of cyclin D1 and CDK4/6.
In conclusion, cyclin D1 and CDK4/6 have alternate roles in regulation of migration and stem-like cell activity. Furthermore, these effects are highly dependent upon expression of ER. The significance of these results adds to our general understanding of cancer biology but, most importantly, could be used diagnostically to predict treatment response to cell cycle inhibition in breast cancer.
PMCID: PMC3841318  PMID: 23839043
cell cycle; breast cancer; stem cell; migration; estrogen receptor
4.  Breastfeeding in relation to risk of different breast cancer characteristics 
BMC Research Notes  2014;7:216.
The aim of this present study was to examine duration of breastfeeding in relation to the risk of different subgroups of breast cancer. A prospective cohort, The Malmö Diet and Cancer study, including 14092 parous women, were followed during a mean of 10.2 years and a total of 424 incident breast cancers were diagnosed.
Tumours were classified regarding invasiveness, tumour size, axillary lymph node status, Nottingham grade, tumour proliferation (Ki67), HER2, cyclin D1 and p27, WHO histological type and hormone receptor status. Duration of breastfeeding was measured using total time of breastfeeding, categorized in quartiles using the lowest as the reference group (<4.0, ≥4.0- < 8.0, ≥8.0- < 13.0 and ≥13.0 months). Average duration of breastfeeding per child and breastfeeding duration of the first child were also used as exposures in separate analyses. Relative risks, with 95% confidence intervals, were obtained using a Cox’s proportional hazards analysis adjusted for potential confounders.
Overall risk for breast cancer was similar in all quartiles of breastfeeding. No strong results regarding breastfeeding duration and breast cancer subgroups were seen. A few results indicated an association between a relatively long duration of breastfeeding and tumours with high proliferation (Ki67) and grade III histological grade.
Breastfeeding duration was not associated with breast cancer risk and no strong results were seen with regard to breast cancer subgroups.
PMCID: PMC4022388  PMID: 24708573
Breast cancer risk; Breastfeeding; Stage; Characteristics
5.  Decreased expression of Yes-associated protein is associated with outcome in the luminal A breast cancer subgroup and with an impaired tamoxifen response 
BMC Cancer  2014;14:119.
Yes-associated protein (YAP1) is frequently reported to function as an oncogene in many types of cancer, but in breast cancer results remain controversial. We set out to clarify the role of YAP1 in breast cancer by examining gene and protein expression in subgroups of patient material and by downregulating YAP1 in vitro and studying its role in response to the widely used anti-estrogen tamoxifen.
YAP1 protein intensity was scored as absent, weak, intermediate or strong in two primary breast cancer cohorts (n = 144 and n = 564) and mRNA expression of YAP1 was evaluated in a gene expression dataset (n = 1107). Recurrence-free survival was analysed using the log-rank test and Cox multivariate analysis was used to test for independence. WST-1 assay was employed to measure cell viability and a luciferase ERE (estrogen responsive element) construct was used to study the effect of tamoxifen, following downregulation of YAP1 using siRNAs.
In the ER+ (Estrogen Receptor α positive) subgroup of the randomised cohort, YAP1 expression was inversely correlated to histological grade and proliferation (p = 0.001 and p = 0.016, respectively) whereas in the ER- (Estrogen Receptor α negative) subgroup YAP1 expression correlated positively to proliferation (p = 0.005). Notably, low YAP1 mRNA was independently associated with decreased recurrence-free survival in the gene expression dataset, specifically for the luminal A subgroup (p < 0.001) which includes low proliferating tumours of lower grade, usually associated with a good prognosis. This subgroup specificity led us to hypothesize that YAP1 may be important for response to endocrine therapies, such as tamoxifen, extensively used for luminal A breast cancers. In a tamoxifen randomised patient material, absent YAP1 protein expression was associated with impaired tamoxifen response which was significant upon interaction analysis (p = 0.042). YAP1 downregulation resulted in increased progesterone receptor (PgR) expression and a delayed and weaker tamoxifen in support of the clinical data.
Decreased YAP1 expression is an independent prognostic factor for recurrence in the less aggressive luminal A breast cancer subgroup, likely due to the decreased tamoxifen sensitivity conferred by YAP1 downregulation.
PMCID: PMC3937431  PMID: 24559095
Yes-associated protein; Breast cancer; Estrogen receptor; Luminal A; 11q deletion; Tamoxifen response; Independent prognostic factor
6.  Wnt Pathway Activity in Breast Cancer Sub-Types and Stem-Like Cells 
PLoS ONE  2013;8(7):e67811.
Wnt signalling has been implicated in stem cell regulation however its role in breast cancer stem cell regulation remains unclear.
We used a panel of normal and breast cancer cell lines to assess Wnt pathway gene and protein expression, and for the investigation of Wnt signalling within stem cell-enriched populations, mRNA and protein expression was analysed after the selection of anoikis-resistant cells. Finally, cell lines and patient-derived samples were used to investigate Wnt pathway effects on stem cell activity in vitro.
Wnt pathway signalling increased in cancer compared to normal breast and in both cell lines and patient samples, expression of Wnt pathway genes correlated with estrogen receptor (ER) expression. Furthermore, specific Wnt pathway genes were predictive for recurrence within subtypes of breast cancer. Canonical Wnt pathway genes were increased in breast cancer stem cell-enriched populations in comparison to normal breast stem cell-enriched populations. Furthermore in cell lines, the ligand Wnt3a increased whilst the inhibitor DKK1 reduced mammosphere formation with the greatest inhibitory effects observed in ER+ve breast cancer cell lines. In patient-derived metastatic breast cancer samples, only ER-ve mammospheres were responsive to the ligand Wnt3a. However, the inhibitor DKK1 efficiently inhibited both ER+ve and ER-ve breast cancer but not normal mammosphere formation, suggesting that the Wnt pathway is aberrantly activated in breast cancer mammospheres.
Collectively, these data highlight differential Wnt signalling in breast cancer subtypes and activity in patient-derived metastatic cancer stem-like cells indicating a potential for Wnt-targeted treatment in breast cancers.
PMCID: PMC3701602  PMID: 23861811
7.  Oestrogen increases the activity of oestrogen receptor negative breast cancer stem cells through paracrine EGFR and Notch signalling 
Although oestrogen is essential for the development of the normal breast, adult mammary stem cells are known to be oestrogen receptor alpha (ER) negative and rely on paracrine signals in the mammary epithelium for mediation of developmental cues. However, little is known about how systemic oestrogen regulates breast cancer stem cell (CSC) activity.
Here, we tested the effects of oestrogen on CSC activity in vitro and in vivo and investigated which paracrine signalling pathways locally mediate oestrogen effects.
CSC-enriched populations (ESA+CD44+CD24low) sorted from ER positive patient derived and established cell lines have low or absent ER expression. However, oestrogen stimulated CSC activity demonstrated by increased mammosphere and holoclone formation in vitro and tumour formation in vivo. This effect was abrogated by the anti-oestrogen tamoxifen or ER siRNA. These data suggest that the oestrogen response is mediated through paracrine signalling from non-CSCs to CSCs. We have, therefore, investigated both epidermal growth factor (EGF) and Notch receptor signals downstream of oestrogen. We demonstrate that gefitinib (epidermal growth factor receptor (EGFR) inhibitor) and gamma secretase inhibitors (Notch inhibitor) block oestrogen-induced CSC activity in vitro and in vivo but GSIs more efficiently reduce CSC frequency.
These data establish that EGF and Notch receptor signalling pathways operate downstream of oestrogen in the regulation of ER negative CSCs.
PMCID: PMC3672803  PMID: 23497505
8.  Low ERK Phosphorylation in Cancer-Associated Fibroblasts Is Associated with Tamoxifen Resistance in Pre-Menopausal Breast Cancer 
PLoS ONE  2012;7(9):e45669.
The aim of this study was to evaluate ERK phosphorylation as a stromal biomarker for breast cancer prognosis and tamoxifen treatment prediction within a randomized tamoxifen trial.
Patients and Methods
Tissue microarrays of two breast cancer cohorts including in total 743 invasive breast cancer samples were analyzed for ERK phosphorylation (pERK) and smooth muscle actin-alpha expression (SMAα) in cancer-associated fibroblasts (CAFs) and links to clinico-pathological data and treatment-predictive values were delineated.
By analyzing a unique randomized tamoxifen trial including breast cancer patients receiving no adjuvant treatment we show for the first time that patients low in ERK phosphorylation in CAFs did not respond to tamoxifen treatment despite having estrogen-receptor alpha (ERα-positive tumors compared to patients with high pERK levels in CAFs (P = 0.015, multivariate Cox regression interaction analysis). In both clinical materials we further show a significant association between pERK and SMAα, a characteristic marker for activated fibroblasts. SMAα expression however was not linked to treatment-predictive information but instead had prognostic qualities.
The data suggests that the presence of a subpopulation of CAFs, defined by minimal activated ERK signaling, is linked to an impaired tamoxifen response. Thus, this report illustrates the importance of the stroma for monitoring treatment effects in pre-menopausal breast cancer.
PMCID: PMC3454403  PMID: 23029174
9.  Genetic predisposition, parity, age at first childbirth and risk for breast cancer 
BMC Research Notes  2012;5:414.
Recent studies have identified several single-nucleotide polymorphisms (SNPs) associated with the risk of breast cancer and parity and age at first childbirth are well established and important risk factors for breast cancer. The aim of the present study was to examine the interaction between these environmental factors and genetic variants on breast cancer risk.
The Malmö Diet and Cancer Study (MDCS) included 17 035 female participants, from which 728 incident breast cancer cases were matched to 1448 controls. The associations between 14 SNPs and breast cancer risk were investigated in different strata of parity and age at first childbirth. A logistic regression analysis for the per allele risk, adjusted for potential confounders yielded odds ratios (OR) with 95% confidence intervals (CI).
Six of the previously identified SNPs showed a statistically significant association with breast cancer risk: rs2981582 (FGFR2), rs3803662 (TNRC9), rs12443621 (TNRC9), rs889312 (MAP3K1), rs3817198 (LSP1) and rs2107425 (H19). We could not find any statistically significant interaction between the effects of tested SNPs and parity/age at first childbirth on breast cancer risk after adjusting for multiple comparisons.
The results of this study are in agreement with previous studies of null interactions between tested SNPs and parity/age at first childbirth with regard to breast cancer risk.
PMCID: PMC3439270  PMID: 22867275
10.  Downregulation of miR-92a Is Associated with Aggressive Breast Cancer Features and Increased Tumour Macrophage Infiltration 
PLoS ONE  2012;7(4):e36051.
MicroRNAs are small non-coding RNAs involved in the regulation of gene expression on a posttranscriptional level. These regulatory RNAs have been implicated in numerous cellular processes and are further deregulated in different cancer types, including breast cancer. MiR-92a is part of the miR-17∼92 cluster, which was first reported to be linked to tumourigenesis. However, little is known about the expression of miR-92a in breast cancer and potential associations to tumour properties. The expression of miR-92a was therefore characterized in 144 invasive breast cancer samples using in situ hybridization and related to clinico-pathological data as well as to selected key properties of the tumour stroma, including the presence of macrophages (CD68) and cancer activated fibroblasts (alpha-SMA).
Methodology/Principal Findings
To measure miR-92a levels, an in situ hybridisation protocol was developed and validated using cell lines and miR-92a inhibitors. The expression in the tumour samples was objectively evaluated using digital image analysis program subtracting background activities. We found that the miR-92a expression varied between tumours and was inversely correlated to tumour grade (r = −0.276, p = 0.003) and recurrence-free survival (p = 0.008) and provided independent prognostic information in multivariate Cox analysis (HR: 0.375, CI: 0.145–0.972, p = 0.043). MiR-92a was moreover inversely correlated to the number of infiltrating macrophages in the tumour stroma (r = −0.357, p<0.001), and downregulation of miR-92a promoted cell migration (p<0.01).
This study demonstrates that downregulation of miR-92a in breast cancer is linked to key epithelial and stromal properties as well as clinical outcome.
PMCID: PMC3338574  PMID: 22563438
11.  Effects of cyclin D1 gene amplification and protein expression on time to recurrence in postmenopausal breast cancer patients treated with anastrozole or tamoxifen: a TransATAC study 
Gene amplification of CCND1 is observed in a subgroup of breast cancers with poor prognosis, whereas overexpression of the protein cyclin D1 has been linked to both worse and better clinical outcome. CCND1 amplification and protein overexpression have also been associated with resistance to treatment with tamoxifen or even to a potentially detrimental effect of tamoxifen.
To clarify these challenging and partly contrasting treatment predictive and prognostic links for cyclin D1 we analysed a large cohort of postmenopausal breast cancer patients randomised to receive either adjuvant anastrozole or tamoxifen, as part of the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial. The CCND1 amplification status and protein expression of cyclin D1 were assessed by chromogenic in situ hybridisation and immunohistochemistry, respectively, in 1,155 postmenopausal, oestrogen-receptor-positive breast cancer patients included in the TransATAC substudy.
Amplification of CCND1 was observed in 8.7% of the tumours and was associated with increased risk of disease recurrence (hazard ratio = 1.61; 95% confidence interval, 1.08 to 2.41) after adjustment for other clinicopathological parameters. In contrast, nuclear expression of cyclin D1 protein was associated with decreased recurrence rate (hazard ratio = 0.6; 95% confidence interval, 0.39 to 0.92). The intensity of nuclear or cytoplasmic expression was not of prognostic value. There was no significant interaction between cyclin D1 status and treatment efficacy, ruling out any major detrimental effect of tamoxifen in CCND1-amplified postmenopausal breast cancer.
In summary, CCND1 amplification and low nuclear expression of cyclin D1 predicted poor clinical outcome in postmenopausal breast cancer patients treated with either anastrozole or tamoxifen.
Trial Registration
Current Controlled Trials ISRCTN18233230.
PMCID: PMC3446392  PMID: 22475046
12.  Cyclin D1, Id1 and EMT in breast cancer 
BMC Cancer  2011;11:417.
Cyclin D1 is a well-characterised cell cycle regulator with established oncogenic capabilities. Despite these properties, studies report contrasting links to tumour aggressiveness. It has previously been shown that silencing cyclin D1 increases the migratory capacity of MDA-MB-231 breast cancer cells with concomitant increase in 'inhibitor of differentiation 1' (ID1) gene expression. Id1 is known to be associated with more invasive features of cancer and with the epithelial-mesenchymal transition (EMT). Here, we sought to determine if the increase in cell motility following cyclin D1 silencing was mediated by Id1 and enhanced EMT-features. To further substantiate these findings we aimed to delineate the link between CCND1, ID1 and EMT, as well as clinical properties in primary breast cancer.
Protein and gene expression of ID1, CCND1 and EMT markers were determined in MDA-MB-231 and ZR75 cells by western blot and qPCR. Cell migration and promoter occupancy were monitored by transwell and ChIP assays, respectively. Gene expression was analysed from publicly available datasets.
The increase in cell migration following cyclin D1 silencing in MDA-MB-231 cells was abolished by Id1 siRNA treatment and we observed cyclin D1 occupancy of the Id1 promoter region. Moreover, ID1 and SNAI2 gene expression was increased following cyclin D1 knock-down, an effect reversed with Id1 siRNA treatment. Similar migratory and SNAI2 increases were noted for the ER-positive ZR75-1 cell line, but in an Id1-independent manner. In a meta-analysis of 1107 breast cancer samples, CCND1low/ID1high tumours displayed increased expression of EMT markers and were associated with reduced recurrence free survival. Finally, a greater percentage of CCND1low/ID1high tumours were found in the EMT-like 'claudin-low' subtype of breast cancer than in other subtypes.
These results indicate that increased migration of MDA-MB-231 cells following cyclin D1 silencing can be mediated by Id1 and is linked to an increase in EMT markers. Moreover, we have confirmed a relationship between cyclin D1, Id1 and EMT in primary breast cancer, supporting our in vitro findings that low cyclin D1 expression can be linked to aggressive features in subgroups of breast cancer.
PMCID: PMC3192789  PMID: 21955753
Cyclin D1; Id1; EMT; breast cancer; migration; recurrence-free survival; claudin-low
13.  PKCα expression is a marker for breast cancer aggressiveness 
Molecular Cancer  2010;9:76.
Protein kinase C (PKC) isoforms are potential targets for breast cancer therapy. This study was designed to evaluate which PKC isoforms might be optimal targets for different breast cancer subtypes.
In two cohorts of primary breast cancers, PKCα levels correlated to estrogen and progesterone receptor negativity, tumor grade, and proliferative activity, whereas PKCδ and PKCε did not correlate to clinicopathological parameters. Patients with PKCα-positive tumors showed poorer survival than patients with PKCα-negative tumors independently of other factors. Cell line studies demonstrated that PKCα levels are high in MDA-MB-231 and absent in T47D cells which proliferated slower than other cell lines. Furthermore, PKCα silencing reduced proliferation of MDA-MB-231 cells. PKCα inhibition or downregulation also reduced cell migration in vitro.
PKCα is a marker for poor prognosis of breast cancer and correlates to and is important for cell functions associated with breast cancer progression.
PMCID: PMC2873434  PMID: 20398285
14.  Tensin3 Is a Negative Regulator of Cell Migration and All Four Tensin Family Members Are Downregulated in Human Kidney Cancer 
PLoS ONE  2009;4(2):e4350.
The Tensin family of intracellular proteins (Tensin1, -2, -3 and -4) are thought to act as links between the extracellular matrix and the cytoskeleton, and thereby mediate signaling for cell shape and motility. Dysregulation of Tensin expression has previously been implicated in human cancer. Here, we have for the first time evaluated the significance of all four Tensins in a study of human renal cell carcinoma (RCC), as well as probed the biological function of Tensin3.
Principal Findings
Expression of Tensin2 and Tensin3 at mRNA and protein levels was largely absent in a panel of diverse human cancer cell lines. Quantitative RT-PCR analysis revealed mRNA expression of all four Tensin genes to be significantly downregulated in human kidney tumors (50–100% reduction versus normal kidney cortex; P<0.001). Furthermore, the mRNA expressions of Tensins mostly correlated positively with each other and negatively with tumor grade, but not tumor size. Immunohistochemical analysis revealed Tensin3 to be present in the cytoplasm of tubular epithelium in normal human kidney sections, whilst expression was weaker or absent in 41% of kidney tumors. A subset of tumor sections showed a preferential plasma membrane expression of Tensin3, which in clear cell RCC patients was correlated with longer survival. Stable expression of Tensin3 in HEK 293 cells markedly inhibited both cell migration and matrix invasion, a function independent of putative phosphatase activity in Tensin3. Conversely, siRNA knockdown of endogenous Tensin3 in human cancer cells significantly increased their migration.
Our findings indicate that the Tensins may represent a novel group of metastasis suppressors in the kidney, the loss of which leads to greater tumor cell motility and consequent metastasis. Moreover, tumorigenesis in the human kidney may be facilitated by a general downregulation of Tensins. Therefore, anti-metastatic therapies may benefit from restoring or preserving Tensin expression in primary tumors.
PMCID: PMC2632886  PMID: 19194507
15.  Gene products of chromosome 11q and their association with CCND1 gene amplification and tamoxifen resistance in premenopausal breast cancer 
The amplification event occurring at chromosome locus 11q13, reported in several different cancers, includes a number of potential oncogenes. We have previously reported amplification of one such oncogene, namely CCND1, to be correlated with an adverse effect of tamoxifen in premenopausal breast cancer patients. Over-expression of cyclin D1 protein, however, confers tamoxifen resistance but not a tamoxifen-induced adverse effect. Potentially, co-amplification of an additional 11q13 gene, with a resulting protein over-expression, is required to cause an agonistic effect. Moreover, during 11q13 amplification a deletion of the distal 11q region has been described. In order to assess the potential impact of the deletion we examined a selected marker for this event.
Array comparative genomic hybridization analysis was employed to identify and confirm changes in the gene expression of a number of different genes mapping to the 11q chromosomal region, associated with CCND1 amplification. The subsequent protein expression of these candidate genes was then examined in a clinical material of 500 primary breast cancers from premenopausal patients who were randomly assigned to either tamoxifen or no adjuvant treatment. The protein expression was also compared with gene expression data in a subset of 56 breast cancer samples.
Cortactin and FADD (Fas-associated death domain) over-expression was linked to CCND1 amplification, determined by fluorescence in situ hybridization, but was not associated with a diminished effect of tamoxifen. However, deletion of distal chromosome 11q, defined as downregulation of the marker Chk1 (checkpoint kinase 1), was associated with an impaired tamoxifen response, and interestingly with low proliferative breast cancer of low grade. For Pak1 (p21-activated kinase 1) and cyclin D1 the protein expression corresponded to the gene expression data.
The results indicate that many 11q13 associated gene products are over-expressed in conjunction with cyclin D1 but not linked to an agonistic effect of tamoxifen. Finally, the deletion of distal 11q, linked to 11q13 amplification, might be an important event affecting breast cancer outcome and tamoxifen response.
PMCID: PMC2614516  PMID: 18823530
16.  C4b-Binding Protein Is Present in Affected Areas of Myocardial Infarction during the Acute Inflammatory Phase and Covers a Larger Area than C3 
PLoS ONE  2008;3(8):e2886.
During myocardial infarction reduced blood flow in the heart muscle results in cell death. These dying/dead cells have been reported to bind several plasma proteins such as IgM and C-reactive protein (CRP). In the present study we investigated whether fluid-phase complement inhibitor C4b-binding protein (C4BP) would also bind to the infarcted heart tissue.
Methods and Findings
Initial studies using immunohistochemistry on tissue arrays for several cardiovascular disorders indicated that C4BP can be found in heart tissue in several cardiac diseases but that it is most abundantly found in acute myocardial infarction (AMI). This condition was studied in more detail by analyzing the time window and extent of C4BP positivity. The binding of C4BP correlates to the same locations as C3b, a marker known to correlate to the patterns of IgM and CRP staining. Based on criteria that describe the time after infarction we were able to pinpoint that C4BP binding is a relatively early marker of tissue damage in myocardial infarction with a peak of binding between 12 hours and 5 days subsequent to AMI, the phase in which infiltration of neutrophilic granulocytes in the heart is the most extensive.
C4BP, an important fluid-phase inhibitor of the classical and lectin pathway of complement activation binds to jeopardized cardiomyocytes early after AMI and co-localizes to other well known markers such as C3b.
PMCID: PMC2483938  PMID: 18682851
17.  Diet and body constitution in relation to subgroups of breast cancer defined by tumour grade, proliferation and key cell cycle regulators 
Breast Cancer Research  2007;9(1):R11.
The general lack of clear associations between diet and breast cancer in epidemiological studies may partly be explained by the fact that breast cancer is a heterogeneous disease that may have disparate genetic associations and different aetiological bases.
A total of 346 incident breast cancers in a prospective cohort of 17,035 women enrolled in the Malmö Diet and Cancer study (Sweden) were subcategorized according to conventional pathology parameters, proliferation and expression of key cell cycle regulators. Subcategories were compared with prediagnostic diet and body measurements using analysis of variance.
A large hip circumference and high body mass index were associated with high grade tumours (P = 0.03 and 0.009, respectively), whereas low energy and unadjusted fat intakes were associated with high proliferation (P = 0.03 and 0.004, respectively). Low intakes of saturated, monounsaturated and polyunsaturated fatty acids were also associated with high proliferation (P = 0.02, 0.004 and 0.003, respectively). Low energy and unadjusted fat intakes were associated with cyclin D1 overexpression (P = 0.02 and 0.007, respectively), whereas cyclin E overexpression was positively correlated with fat intake. Oestrogen receptor status and expression of the tumour suppressor gene p27 were not associated with either diet or body constitution.
Low energy and low total fat (polyunsaturated fatty acids in particular) intakes, and high body mass index were associated with relatively more malignant breast tumours. Dietary behaviours and body constitution may be associated with specific types of breast cancer defined by conventional pathology parameters and cyclin D1 and cyclin E expression. Further studies including healthy control individuals are needed to confirm our results.
PMCID: PMC1851395  PMID: 17254341
18.  C4b-binding protein binds to necrotic cells and DNA, limiting DNA release and inhibiting complement activation 
The Journal of Experimental Medicine  2005;201(12):1937-1948.
After cell death, via apoptosis or necrosis, the uptake of dead cells by neighboring cells or phagocytes prevents the release of intracellular content. An array of molecules, including initiation molecules of the complement system, are involved in marking dead cells for uptake. After binding of these molecules, complement activation takes place, which when uncontrolled might result in a proinflammatory state. In the current study we demonstrate that complement inhibitor, C4b-binding protein (C4BP), binds strongly to necrotic cells, irrespective of the cell type used or the method of induction. After binding of the C4BP–protein S (PS) complex to necrotic cells via PS-phosphatidylserine and C4BP-DNA interactions, C4BP-PS inhibits complement activation on these cells. C4BP binds DNA via a patch of positively charged amino acids, mainly on the second complement control domain of the C4BP α-chain (affinity constant: 190 nM). Furthermore, C4BP limits DNA release from necrotic cells and inhibits DNA-mediated complement activation in solution. The C4BP–necrotic cell interaction also occurs in vivo as necrotic areas of arteriosclerotic plaques and of various cancers stain strongly positive for C4BP. This study describes a novel mechanism in which C4BP limits the inflammatory potential of necrotic cells.
PMCID: PMC2212022  PMID: 15967823

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