Lene Rask

High expression of miR-21 in tumor stroma correlates with increased cancer cell proliferation in human breast cancer

Rask L, Balslev E, Jørgensen S, Eriksen J, Flyger H, Møller S, Høgdall E, Litman T, Nielsen BS. High expression of miR‐21 in tumor stroma correlates with increased cancer cell proliferation in human breast cancer. APMIS 2011; 119: 663–73.

Differential expression of miR-139, miR-486 and miR-21 in breast cancer patients sub-classified according to lymph node status

PurposeTherapeutic decisions in breast cancer are increasingly guided by prognostic and predictive biomarkers. Non-protein-coding microRNAs (miRNAs) have recently been found to be deregulated in breast cancers and, in addition, to be correlated with several clinico-pathological features. One of the most consistently up-regulated miRNAs is miR-21. Here, we specifically searched for differentially expressed miRNAs in high-risk breast cancer patients as compared to low-risk breast cancer patients. In the same patients, we also compared miR-21 expression with the expression of its presumed target PTEN.MethodsBoth microarray and RT-qPCR techniques were used to assess miRNA expression levels in lymph node-positive and -negative human invasive ductal carcinoma tissues. Simultaneously, PTEN protein expression levels were assessed using immunohistochemistry.ResultsmiR-486-5p and miR-139-5p were found to be down-regulated in patients with lymph node metastases, whereas miR-21 was found to be up-regulated in patients with a positive lymph node status. miR-21 expression levels were found to significantly correlate with tumour size (r = 0.403, p = 0.009; Spearman’s rank), whereas no relation was found between miR-21 and PTEN expression levels (Kruskal-Wallis test).ConclusionDown-regulation of miR-486-5p and miR-139-5p, in conjunction with up-regulation of miR-21, may represent a useful signature for the identification of high-risk breast cancer patients.

MicroRNA expression profiles associated with development of drug resistance in Ehrlich ascites tumor cells.

Multidrug resistance (MDR) poses a major obstacle to successful chemotherapeutic treatment of cancer, and often involves multiple genes, which may be regulated post-transcriptionally by microRNAs (miRNAs). The purpose of the present study was therefore to identify any resistance-associated changes in miRNA expression in a sensitive and five increasingly drug-resistant Ehrlich ascites tumor (EAT) cell lines, representing different steps in the development of resistance. We used an LNA-enhanced microarray platform to study the global miRNA expression profiles in the six murine EAT cell lines, and identified growth-, hypoxia-, and resistance-specific miRNA patterns. Among the differentially expressed miRNAs, we found the two clusters miR-183∼miR-96∼miR-182 and miR-200b∼miR-200a∼miR-429 as well as miR-141 to be consistently upregulated in the MDR cell lines, while miR-125b-5p and the two clusters miR-30d∼miR-30b and miR-23b∼miR-27b∼miR-24-1 were downregulated in most of the resistant EAT cells. Several of the target genes for these miRNAs-including Zeb1/Zeb2 and members of the Fox gene family-could contribute to the drug-resistant phenotype, although we did not find that the degree of resistance was directly correlated to any specific changes in miRNA expression. Probably, the observed miRNA expression patterns reflect the underlying genomic instability of the tumor cells, and further studies are needed to explore how the highly complex regulatory miRNA networks contribute to the development of MDR.

N-linked deglycosylated melanopsin retains its responsiveness to light.

Melanopsin is an opsin expressed in the plasma membrane of retinal ganglion cells that mainly project to the circadian clock and thus is important for nonvisual responses to light. Rat melanopsin contains two potential sites (Asn31 and Asn35) for N-linked glycosylation in the N-terminal extracellular part. To investigate if melanopsin is N-linked glycosylated and whether N-bound glycans influence the response of melanopsin to light as evidenced by Fos mRNA induction, we transfected PC12 cells to stably express rat wild-type melanopsin or mutant melanopsin lacking both N-linked glycosylation sites. Immunoblotting for membrane-bound melanopsin from the PC12 cells transfected to express wild-type melanopsin disclosed two immunoreactive bands of 62 and 49 kDa. Removal of N-linked glycosylation by tunicamycin or PNGase F changed the 62 kDa band to a 55 kDa band, while the 49 kDa band corresponding to the core melanopsin protein was unaffected. Likewise, mutation of the two extracellular N-linked glycosylation sites gave a melanopsin size comparable to that of PNGase F or tunicamycin treatment (55 kDa). Further in vitro O-linked deglycosylation of wild-type or mutant melanopsin with O-glycosidase and neuraminidase converted the 55 kDa band to a 49 kDa band. Finally, neither in vivo N-linked deglycosylation nor mutations of the two N-linked glycosylation sites significantly affected melanopsin function measured by Fos induction after light stimulation. In conclusion, we have shown that heterologously expressed rat melanopsin is both N-linked and O-linked glycosylated and that N-linked glycosylation is not crucial for the melanopsin response to light.

Stressful life events and leucocyte telomere length: Do lifestyle factors, somatic and mental health, or low grade inflammation mediate this relationship? Results from a cohort of Danish men born in 1953

Exposure to psychosocial stress is associated with increased risk of a number of somatic and mental disorders with relation to immune system functioning. We aimed to explore whether stressful events in early and recent life was associated with leucocyte telomere length (TL), which is assumed to reflect the accumulated burden of inflammation and oxidative stress occurring during the life course. We specifically aimed to address whether childhood constitutes a sensitive period and how much of the relation between stressful life events and TL is mediated through somatic and mental health, lifestyle, and markers of low-grade inflammation. A cohort of Danish men born in 1953 has been followed since birth in the Metropolit Cohort. These men underwent a health examination including blood sampling in 2010 and a subset of 324 also had a quantitative PCR-based measurement of TL. The relation between stressful life events and TL was analysed using structural equation modelling, which also provided an estimate of the proportion of the total effect mediated by somatic and mental health (cardiovascular disease, body mass and depressive mood), lifestyle factors, and low grade inflammation (C-reactive protein (CRP), interleukin (IL)-6 and IL-10). Total number of stressful events experienced during the life course was not associated with TL. In terms of sensitive periods, we found that number of stressful events in childhood was associated with shorter TL (βper number stressful events in childhood=-0.02(SE=-0.02); P=0.05). This relation was particularly strong for being placed away from home (β=-0.16; P<0.000). Thirty percent of the total effect of stressful events in childhood on TL was mediated by the included variables, with the largest proportion being mediated through depressive mood (16%) and CRP (9%). This study suggests that stressful events in childhood are associated with shorter TL in middle-aged men and that part of this relation is explained by depressive mood and low grade inflammation.

Development of a metastatic fluorescent Lewis Lung carcinoma mouse model: Identification of mRNAs and microRNAs involved in tumor invasion

Cancer metastasis is the foremost cause of death in cancer patients. A series of observable pathological changes takes place during progression and metastasis of cancer, but the underlying genetic changes remain unclear. Therefore, new approaches are required, including insights from cancer mouse models. To examine the mechanisms involved in tumor metastasis, we first generated a stably transfected Lewis Lung carcinoma cell line expressing a far-red fluorescent protein, called Katushka. After in vivo growth in syngeneic mice, two fluorescent Lewis Lung cancer subpopulations were isolated from primary tumors and lung metastases. The metastasis-derived cells exhibited a significant improvement in in vitro invasive activity compared to the primary tumor-derived cells, using a quantitative invasion chamber assay. Moreover, expression levels of 84 tumor metastasis-related mRNAs, 88 cancer-related microRNAs as well as Dicer and Drosha were determined using RT-qPCR. Compared to the primary Lewis Lung carcinoma subculture, the metastasis-derived cells exhibited statistically significantly increased mRNA levels for several matrix metalloproteinases as well as hepatocyte growth factor (HGF) and spleen tyrosine kinase (SYK). A modest decrease in Drosha and Dicer mRNA levels was accompanied by significant downregulation of ten microRNAs, including miR-9 and miR-203, in the lung metastatic Lewis Lung carcinoma cell culture. Thus, a tool for cancer metastasis studies has been established and the model is well suited for the identification of novel microRNAs and mRNAs involved in malignant progression. Our results suggest that increases in metalloproteinase expression and impairment of microRNA processing are involved in the acquirement of metastatic ability.

Cognitive Change during the Life Course and Leukocyte Telomere Length in Late Middle-Aged Men

Importance: Cognitive skills are known to decline through the lifespan with large individual differences. The molecular mechanisms for this decline are incompletely understood. Although leukocyte telomere length provides an index of cellular age that predicts the incidence of age-related diseases, it is unclear whether there is an association between cognitive decline and leukocyte telomere length. Objective: To examine the association between changes in cognitive function during adult life and leukocyte telomere length after adjusting for confounding factors such as education, mental health and life style. Design, Setting, and Participants: Two groups of men with negative (n = 97) and positive (n = 93) change in cognitive performance were selected from a birth cohort of 1985 Danish men born in 1953. Cognitive performance of each individual was assessed at age ~20 and 56 years. Leukocyte telomere length at age ~58 was measured using qPCR. Linear regression models were used to investigate the association between cognitive function and leukocyte telomere length. Results: Men with negative change in cognitive performance during adult life had significantly shorter mean leukocyte telomere length than men with positive change in cognitive performance (unadjusted difference β = −0.09, 95% CI −0.16 to −0.02, p = 0.02). This association remained significant after adjusting for smoking, alcohol consumption, leisure time activity, body mass index (BMI) and cholesterol (adjusted difference β = −0.09, 95% CI −0.17 to −0.01, p = 0.02) but was non-significant after adjusting for smoking, alcohol consumption, leisure time activity, BMI, cholesterol, current cognitive function, depression and education (adjusted difference β = −0.07, 95% CI −0.16 to −0.01, p = 0.08). Conclusion and Relevance: Preclinical cognitive changes may be associated with leukocyte telomere length.

The Light-Induced FOS Response in Melanopsin Expressing HEK-293 Cells is Correlated with Melanopsin Quantity and Dependent on Light Duration and Irradiance

We established a cell line (HEK‐hMel) expressing melanopsin in a tetracycline dependent manner to elucidate new aspects of melanopsins light response. Different light stimuli were evaluated using FOS expression as response parameter. Immunoblotting was used to evaluate expression of melanopsin and FOS and qPCR to quantify FOS mRNA responses. The magnitude of the FOS response was found to correlate with the amount of melanopsin expressed by the cells, and a transient FOS mRNA induction followed by FOS protein still elevated after 24 h of illumination was revealed. Exposing the cells to darkness after light resulted in reduction of the response compared to exposure to light solely showing dependency on continuous light. Increasing irradiances of blue light (480 nm) up to 1011 quanta cm−2 s−1 elicited steep increases in FOS mRNA, while increases between 1012 and 5 × 1013 quanta cm−2 s−1 resulted in equally high FOS expression. The HEK‐hMel cells were used to characterize facets of melanopsins light‐induced FOS response not approachable in vivo. Novel information such as dependency of the FOS response on both melanopsin amount and light intensity in addition to a detailed time‐course of both FOS mRNA and protein were revealed.

Visual steady state in relation to age and cognitive function

Neocortical gamma activity is crucial for sensory perception and cognition. This study examines the value of using non-task stimulation-induced EEG oscillations to predict cognitive status in a birth cohort of healthy Danish males (Metropolit) with varying cognitive ability. In particular, we examine the steady-state VEP power response (SSVEP-PR) in the alpha (8Hz) and gamma (36Hz) bands in 54 males (avg. age: 62.0 years) and compare these with 10 young healthy participants (avg. age 27.6 years). Furthermore, we correlate the individual alpha-to-gamma difference in relative visual-area power (ΔRV) with cognitive scores for the older adults. We find that ΔRV decrease with age by just over one standard deviation when comparing young with old participants (p<0.01). Furthermore, intelligence is significantly negatively correlated with ΔRV in the older adult cohort, even when processing speed, global cognition, executive function, memory, and education (p<0.05). In our preferred specification, an increase in ΔRV of one standard deviation is associated with a reduction in intelligence of 48% of a standard deviation (p<0.01). Finally, we conclude that the difference in cerebral rhythmic activity between the alpha and gamma bands is associated with age and cognitive status, and that ΔRV therefore provide a non-subjective clinical tool with which to examine cognitive status in old age.

Tetranectin positive expression in tumour tissue leads to longer survival in Danish women with ovarian cancer. Results from the 'Malova' ovarian cancer study

The primary objective of this study was to analyse Tetranectin (TN) expression in tumour tissues and TN serum concentration in 758 women with epithelial ovarian tumours. The second was to evaluate, whether TN tissue expression levels correlate with clinico‐pathological parameters and prognosis of the disease. Using tissue arrays we analysed the expression levels in tissues from 166 women with borderline ovarian tumours (BOTs) and 592 women with ovarian cancer (OC). A panel of three antibodies was used for immunohistochemistry: a polyclonal and two monoclonal antibodies. Serum TN was measured using the polyclonal antibody A‐371. Univariate survival analyses stratified for chemotherapy showed that positive tissue TN as demonstrated by the polyclonal antibody indicated a significantly longer overall survival (OS) (p = 0.0001) as well as cancer specific survival (CSS) (p < 0.0001). High serum TN was likewise found to imply longer OS (p < 0.0001) and CSS (p < 0.0001), whereas tissue staining with the two monoclonal antibodies failed to demonstrate any significant correlation with either survival type. Univariate Kaplan–Meier survival analysis performed on all OC cases showed a significantly longer OS (p = 0.0009) and CSS (p = 0.0006) for women with TN positive tumour tissue and in women with high serum TN levels (p < 0.0001 for both). However, in the multivariate Cox regression analysis, only serum TN was found to be an independent prognostic factor for OS (p = 0.01) and not for CSS (p = 0.08). In conclusion, our results predict that a positive TN expression of both tumour tissue and serum points to a more favourable outcome for OC patients.