Rut Klinger

Monounsaturated fatty acid enriched high fat-diets impede adipose NLRP3 inflammasome mediated IL-1β secretion and insulin resistance despite obesity

Saturated fatty acid (SFA) high-fat diets (HFDs) enhance interleukin (IL)-1β–mediated adipose inflammation and insulin resistance. However, the mechanisms by which different fatty acids regulate IL-1β and the subsequent effects on adipose tissue biology and insulin sensitivity in vivo remain elusive. We hypothesized that the replacement of SFA for monounsaturated fatty acid (MUFA) in HFDs would reduce pro-IL-1β priming in adipose tissue and attenuate insulin resistance via MUFA-driven AMPK activation. MUFA-HFD–fed mice displayed improved insulin sensitivity coincident with reduced pro-IL-1β priming, attenuated adipose IL-1β secretion, and sustained adipose AMPK activation compared with SFA-HFD–fed mice. Furthermore, MUFA-HFD–fed mice displayed hyperplastic adipose tissue, with enhanced adipogenic potential of the stromal vascular fraction and improved insulin sensitivity. In vitro, we demonstrated that the MUFA oleic acid can impede ATP-induced IL-1β secretion from lipopolysaccharide- and SFA-primed cells in an AMPK-dependent manner. Conversely, in a regression study, switching from SFA- to MUFA-HFD failed to reverse insulin resistance but improved fasting plasma insulin levels. In humans, high-SFA consumers, but not high-MUFA consumers, displayed reduced insulin sensitivity with elevated pycard-1 and caspase-1 expression in adipose tissue. These novel findings suggest that dietary MUFA can attenuate IL-1β–mediated insulin resistance and adipose dysfunction despite obesity via the preservation of AMPK activity.

SATB2 in Combination With Cytokeratin 20 Identifies Over 95% of all Colorectal Carcinomas

The special AT-rich sequence-binding protein 2 (SATB2), a nuclear matrix-associated transcription factor and epigenetic regulator, was identified as a tissue type-specific protein when screening protein expression patterns in human normal and cancer tissues using an antibody-based proteomics approach. In this respect, the SATB2 protein shows a selective pattern of expression and, within cells of epithelial lineages, SATB2 expression is restricted to glandular cells lining the lower gastrointestinal tract. The expression of SATB2 protein is primarily preserved in cancer cells of colorectal origin, indicating that SATB2 could function as a clinically useful diagnostic marker to distinguish colorectal cancer (CRC) from other types of cancer. The aim of this study was to further explore and validate the specific expression pattern of SATB2 as a clinical biomarker and to compare SATB2 with the well-known cytokeratin 20 (CK20). Immunohistochemistry was used to analyze the extent of SATB2 expression in tissue microarrays with tumors from 9 independent cohorts of patients with primary and metastatic CRCs (n=1882). Our results show that SATB2 is a sensitive and highly specific marker for CRC with distinct positivity in 85% of all CRCs, and that SATB2 and/or CK20 was positive in 97% of CRCs. In conclusion, the specific expression of SATB2 in a large majority of CRCs suggests that SATB2 can be used as an important complementary tool for the differential diagnosis of carcinoma of unknown primary origin.

Curcumin Affects Components of the Chromosomal Passenger Complex and Induces Mitotic Catastrophe in Apoptosis-Resistant Bcr-Abl-Expressing Cells

The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia and is a determinant of chemotherapy resistance occurring during the blast crisis phase of the disease. The aim of this article was to investigate the possibility of combating the resistance to apoptosis caused by Bcr-Abl by inducing an alternative cell death process. As a model of chronic myeloid leukemia, we employed Bcr-Abl-transfected mouse progenitor 32D cells with low and high Bcr-Abl expression levels corresponding to drug-sensitive and drug-resistant cells, respectively. The drug curcumin (diferuloylmethane), a known potent inducer of cell death in many cancer cells, was investigated for efficacy with Bcr-Abl-expressing cells. Curcumin strongly inhibited cell proliferation and affected cell viability by inducing apoptotic symptoms in all tested cells; however, apoptosis was a relatively late event. G2-M cell cycle arrest, together with increased mitotic index and cellular and nuclear morphology resembling those described for mitotic catastrophe, was observed and preceded caspase-3 activation and DNA fragmentation. Mitosis-arrested cells displayed abnormal chromatin organization, multipolar chromosome segregation, aberrant cytokinesis, and multinucleated cells—morphologic changes typical of mitotic catastrophe. We found that the mitotic cell death symptoms correlated with attenuated expression of survivin, a member of the chromosomal passenger complex, and mislocalization of Aurora B, the partner of survivin in the chromosomal passenger complex. Inhibition of survivin expression with small interfering RNA exhibited similar mitotic disturbances, thus implicating survivin as a major, albeit not the only, target for curcumin action. This study shows that curcumin can overcome the broad resistance to cell death caused by expression of Bcr-Abl and suggests that curcumin may be a promising agent for new combination regimens for drug-resistant chronic myeloid leukemia. (Mol Cancer Res 2006;4(7):457–69)

Cellular senescence induced by aberrant MAD2 levels impacts on paclitaxel responsiveness in vitro.

Background:The mitotic arrest deficiency protein 2 (MAD2) is a key component of the mitotic spindle assembly checkpoint, monitoring accurate chromosomal alignment at the metaphase plate before mitosis. MAD2 also has a function in cellular senescence and in a cells response to microtubule inhibitory (MI) chemotherapy exemplified by paclitaxel.Methods:Using an siRNA approach, the impact of MAD2 down-regulation on cellular senescence and paclitaxel responsiveness was investigated. The endpoints of senescence, cell viability, migration, cytokine expression, cell cycle analysis and anaphase bridge scoring were carried out using standard approaches.Results:We show that MAD2 down-regulation induces premature senescence in the MCF7 breast epithelial cancer cell line. These MAD2-depleted (MAD2↓) cells are also significantly replicative incompetent but retain viability. Moreover, they show significantly higher levels of anaphase bridges and polyploidy compared to controls. In addition, these cells secrete higher levels of IL-6 and IL-8 representing key components of the senescence-associated secretory phenotype (SASP) with the ability to impact on neighbouring cells. In support of this, MAD2↓ cells show enhanced migratory ability. At 72 h after paclitaxel, MAD2↓ cells show a significant further induction of senescence compared with paclitaxel naive controls. In addition, there are significantly more viable cells in the MAD2↓ MCF7 cell line after paclitaxel reflecting the observed increase in senescence.Conclusion:Considering that paclitaxel targets actively dividing cells, these senescent cells will evade cytotoxic kill. In conclusion, compromised MAD2 levels induce a population of senescent cells resistant to paclitaxel.

microRNAs: a new class of breast cancer biomarkers

MicroRNAs (miRNAs) are regulatory molecules known to be aberrantly expressed in cancer and contribute to numerous aspects of tumor biology including the initiation, growth and spread of the tumor. With such diverse roles, it is becoming apparent that some may also provide valuable information which may be of use in a clinical setting, demonstrating the potential to act as both screening tools for the stratification of high-risk patients, while informing the treatment decision-making process. There is mounting evidence to suggest that some miRNAs may even provide assistance in the diagnosis of patients with breast cancer. In addition, miRNAs may themselves be considered therapeutic targets, with inhibition or reintroduction of a particular miRNA capable of inducing a response in vivo. This review focuses on miRNAs that have prognostic, diagnostic or predictive potential in breast cancer as well as the possible challenges in the translation of such observations to the clinic.

Apelin: A putative novel predictive biomarker for bevacizumab response in colorectal cancer

Bevacizumab (bvz) is currently employed as an anti-angiogenic therapy across several cancer indications. Bvz response heterogeneity has been well documented, with only 10-15% of colorectal cancer (CRC) patients benefitting in general. For other patients, clinical efficacy is limited and side effects are significant. This reinforces the need for a robust predictive biomarker of response. To identify such a biomarker, we performed a DNA microarray-based transcriptional profiling screen with primary endothelial cells (ECs) isolated from normal and tumour colon tissues. Thirteen separate populations of tumour-associated ECs and 10 of normal ECs were isolated using fluorescence-activated cell sorting. We hypothesised that VEGF-induced genes were overexpressed in tumour ECs; these genes could relate to bvz response and serve as potential predictive biomarkers. Transcriptional profiling revealed a total of 2,610 differentially expressed genes when tumour and normal ECs were compared. To explore their relation to bvz response, the mRNA expression levels of top-ranked genes were examined using quantitative PCR in 30 independent tumour tissues from CRC patients that received bvz in the adjuvant setting. These analyses revealed that the expression of MMP12 and APLN mRNA was significantly higher in bvz non-responders compared to responders. At the protein level, high APLN expression was correlated with poor progression-free survival in bvz-treated patients. Thus, high APLN expression may represent a novel predictive biomarker for bvz unresponsiveness.

Dynamic and influential interaction of cancer cells with normal epithelial cells in 3D culture

BackgroundThe cancer microenvironment has a strong impact on the growth and dynamics of cancer cells. Conventional 2D culture systems, however, do not reflect in vivo conditions, impeding detailed studies of cancer cell dynamics. This work aims to establish a method to reveal the interaction of cancer and normal epithelial cells using 3D time-lapse.MethodsGFP-labelled breast cancer cells, MDA-MB-231, were co-cultured with mCherry-labelled non-cancerous epithelial cells, MDCK, in a gel matrix. In the 3D culture, the epithelial cells establish a spherical morphology (epithelial sphere) thus providing cancer cells with accessibility to the basal surface of epithelia, similar to the in vivo condition. Cell movement was monitored using time-lapse analyses. Ultrastructural, immunocytochemical and protein expression analyses were also performed following the time-lapse study.ResultsIn contrast to the 2D culture system, whereby most MDA-MB-231 cells exhibit spindle-shaped morphology as single cells, in the 3D culture the MDA-MB-231 cells were found to be single cells or else formed aggregates, both of which were motile. The single MDA-MB-231 cells exhibited both round and spindle shapes, with dynamic changes from one shape to the other, visible within a matter of hours. When co-cultured with epithelial cells, the MDA-MB-231 cells displayed a strong attraction to the epithelial spheres, and proceeded to surround and engulf the epithelial cell mass. The surrounded epithelial cells were eventually destroyed, becoming debris, and were taken into the MDA-MB-231 cells. However, when there was a relatively large population of normal epithelial cells, the MDA-MB-231 cells did not engulf the epithelial spheres effectively, despite repeated contacts. MDA-MB-231 cells co-cultured with a large number of normal epithelial cells showed reduced expression of monocarboxylate transporter-1, suggesting a change in the cell metabolism. A decreased level of gelatin-digesting ability as well as reduced production of matrix metaroproteinase-2 was also observed.ConclusionsThis culture method is a powerful technique to investigate cancer cell dynamics and cellular changes in response to the microenvironment. The method can be useful for various aspects such as; different combinations of cancer and non-cancer cell types, addressing the organ-specific affinity of cancer cells to host cells, and monitoring the cellular response to anti-cancer drugs.

Vitamin D receptor as a target for breast cancer therapy.

Considerable epidemiological evidence suggests that high levels of circulating vitamin D (VD) are associated with a decreased incidence and increased survival from cancer, i.e., VD may possess anti-cancer properties. The aim of this investigation was therefore to investigate the anti-cancer potential of a low calcaemic vitamin D analogue, i.e., inecalcitol and compare it with the active form of vitamin D, i.e., calcitriol, in a panel of breast cancer cell lines (n = 15). Using the MTT assay, IC50 concentrations for response to calcitriol varied from 0.12 µM to >20 µM, whereas those for inecalcitol were significantly lower, ranging from 2.5 nM to 63 nM (P = 0.001). Sensitivity to calcitriol and inecalcitol was higher in VD receptor (VDR)-positive compared to VDR-negative cell lines (P = 0.0007 and 0.0080, respectively) and in ER-positive compared to ER-negative cell lines (P = 0.043 and 0.005, respectively). Using RNA-seq analysis, substantial but not complete overlap was found between genes differentially regulated by calcitriol and inecalcitol. In particular, significantly enriched gene ontology terms such as cell surface signalling and cell communication were found after treatment with inecalcitol but not with calcitriol. In contrast, ossification and bone morphogenesis were found significantly enriched after treatment with calcitriol but not with inecalcitol. Our preclinical results suggest that calcitriol and inecalcitol can inhibit breast cancer cell line growth, especially in cells expressing ER and VDR. As inecalcitol is significantly more potent than calcitriol and has low calcaemic potential, it should be further investigated for the treatment of breast cancer.

THERAPEUTIC RATIONALE TO TARGET HIGHLY EXPRESSED CDK7 CONFERRING POOR OUTCOMES IN TRIPLE-NEGATIVE BREAST CANCER

Triple-negative breast cancer (TNBC) patients commonly exhibit poor prognosis and high relapse after treatment, but there remains a lack of biomarkers and effective targeted therapies for this disease. Here, we report evidence highlighting the cell-cycle-related kinase CDK7 as a driver and candidate therapeutic target in TNBC. Using publicly available transcriptomic data from a collated set of TNBC patients (n = 383) and the METABRIC TNBC dataset (n = 217), we found CDK7 mRNA levels to be correlated with patient prognosis. High CDK7 protein expression was associated with poor prognosis within the RATHER TNBC cohort (n = 109) and the METABRIC TNBC cohort (n = 203). The highly specific CDK7 kinase inhibitors, BS-181 and THZ1, each downregulated CDK7-mediated phosphorylation of RNA polymerase II, indicative of transcriptional inhibition, with THZ1 exhibiting 500-fold greater potency than BS-181. Mechanistic investigations revealed that the survival of MDA-MB-231 TNBC cells relied heavily on the BCL-2/BCL-XL signaling axes in cells. Accordingly, we found that combining the BCL-2/BCL-XL inhibitors ABT-263/ABT199 with the CDK7 inhibitor THZ1 synergized in producing growth inhibition and apoptosis of human TNBC cells. Collectively, our results highlight elevated CDK7 expression as a candidate biomarker of poor prognosis in TNBC, and they offer a preclinical proof of concept for combining CDK7 and BCL-2/BCL-XL inhibitors as a mechanism-based therapeutic strategy to improve TNBC treatment. Cancer Res; 77(14); 3834-45. ©2017 AACR.

Loss of Chromosome 18q11.2-q12.1 Is Predictive for Survival in Patients With Metastatic Colorectal Cancer Treated With Bevacizumab

Purpose Patients with metastatic colorectal cancer (mCRC) have limited benefit from the addition of bevacizumab to standard chemotherapy. However, a subset probably benefits substantially, highlighting an unmet clinical need for a biomarker of response to bevacizumab. Previously, we demonstrated that losses of chromosomes 5q34, 17q12, and 18q11.2-q12.1 had a significant correlation with progression-free survival (PFS) in patients with mCRC treated with bevacizumab in the CAIRO2 clinical trial but not in patients who did not receive bevacizumab in the CAIRO trial. This study was designed to validate these findings. Materials and Methods Primary mCRC samples were analyzed from two cohorts of patients who received bevacizumab as first-line treatment; 96 samples from the European multicenter study Angiopredict (APD) and 81 samples from the Italian multicenter study, MOMA. A third cohort of 90 samples from patients with mCRC who did not receive bevacizumab was analyzed. Copy number aberrations of tumor biopsy specimens were measured by shallow whole-genome sequencing and were correlated with PFS, overall survival (OS), and response. Results Loss of chromosome 18q11.2-q12.1 was associated with prolonged PFS most significantly in both the cohorts that received bevacizumab (APD: hazard ratio, 0.54; P = .01; PFS difference, 65 days; MOMA: hazard ratio, 0.55; P = .019; PFS difference, 49 days). A similar association was found for OS and overall response rate in these two cohorts, which became significant when combined with the CAIRO2 cohort. Median PFS in the cohort of patients with mCRC who did not receive bevacizumab and in the CAIRO cohort was similar to that of the APD, MOMA, and CAIRO2 patients without an 18q11.2-q12.1 loss. Conclusion We conclude that the loss of chromosome 18q11.2-q12.1 is consistently predictive for prolonged PFS in patients receiving bevacizumab. The predictive value of this loss is substantiated by a significant gain in OS and overall response rate.