Cell-type-specific role of CHK2 in mediating DNA damage-induced G2 cell cycle arrest.

Cancer is a life-threatening disease that affects one in three people. Although most cases are sporadic, cancer risk can be increased by genetic factors. It remains unknown why certain genes predispose for specific forms of cancer only, such as checkpoint protein 2 (CHK2), in which gene mutations convey up to twofold higher risk for breast cancer but do not increase lung cancer risk. We have investigated the role of CHK2 and the related kinase checkpoint protein 1 (CHK1) in cell cycle regulation in primary breast and lung primary epithelial cells. At the molecular level, CHK1 activity was higher in lung cells, whereas CHK2 was more active in breast cells. Inhibition of CHK1 profoundly disrupted the cell cycle profile in both lung and breast cells, whereas breast cells were more sensitive toward inhibition of CHK2. Finally, we provide evidence that breast cells require CHK2 to induce a G2-M cell cycle arrest in response of DNA damage, whereas lung cells can partially compensate for the loss of CHK2. Our results provide an explanation as to why CHK2 germline mutations predispose for breast cancer but not for lung cancer.

MicroRNAs as possible indicators of drug sensitivity in breast cancer cell lines.

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally. In this way they might influence whether a cell is sensitive or resistant to a certain drug. So far, only a limited number of relatively small scale studies comprising few cell lines and/or drugs have been performed. To obtain a broader view on miRNAs and their association with drug response, we investigated the expression levels of 411 miRNAs in relation to drug sensitivity in 36 breast cancer cell lines. For this purpose IC50 values of a drug screen involving 34 drugs were associated with miRNA expression data of the same breast cancer cell lines. Since molecular subtype of the breast cancer cell lines is considered a confounding factor in drug association studies, multivariate analysis taking subtype into account was performed on significant miRNA-drug associations which retained 13 associations. These associations consisted of 11 different miRNAs and eight different drugs (among which Paclitaxel, Docetaxel and Veliparib). The taxanes, Paclitaxel and Docetaxel, were the only drugs having miRNAs in common: hsa-miR-187-5p and hsa-miR-106a-3p indicative of drug resistance while Paclitaxel sensitivity alone associated with hsa-miR-556-5p. Tivantinib was associated with hsa-let-7d-5p and hsa-miR-18a-5p for sensitivity and hsa-miR-637 for resistance. Drug sensitivity was associated with hsa-let-7a-5p for Bortezomib, hsa-miR-135a-3p for JNJ-707 and hsa-miR-185-3p for Panobinostat. Drug resistance was associated with hsa-miR-182-5p for Veliparib and hsa-miR-629-5p for Tipifarnib. Pathway analysis for significant miRNAs was performed to reveal biological roles, aiding to find a potential mechanistic link for the observed associations with drug response. By doing so hsa-miR-187-5p was linked to the cell cycle G2-M checkpoint in line with this checkpoint being the target of taxanes. In conclusion, our study shows that miRNAs could potentially serve as biomarkers for intrinsic drug resistance and that pathway analyses can provide additional information in this context.

Tissue-Specific Chk1 Activation Determines Apoptosis by Regulating the Balance of p53 and p21.

The DNA damage response (DDR) protects cells against genomic instability. Surprisingly, little is known about the differences in DDR across tissues, which may affect cancer evolutionary trajectories and chemotherapy response. Using mathematical modeling and quantitative experiments, we found that the DDR is regulated differently in human breast and lung primary cells. Equal levels of cisplatin-DNA lesions caused stronger Chk1 activation in lung cells, leading to resistance. In contrast, breast cells were more resistant and showed more Chk2 activation in response to doxorubicin. Further analyses indicate that Chk1 activity played a regulatory role in p53 phosphorylation, whereas Chk2 activity was essential for p53 activation and p21 expression. We propose a novel "friction model," in which the balance of p53 and p21 levels contributes to the apoptotic response in different tissues. Our results suggest that modulating the balance of p53 and p21 dynamics could optimize the response to chemotherapy.

Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance.

Although chemotherapy is designed to eradicate tumor cells, it also has significant effects on normal tissues. The platinum-induced fatty acid 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) induces systemic resistance to a broad range of DNA-damaging chemotherapeutics. We show that 16:4(n-3) exerts its effect by activating splenic F4/80+/CD11blow macrophages, which results in production of chemoprotective lysophosphatidylcholines (LPCs). Pharmacologic studies, together with analysis of expression patterns, identified GPR120 on F4/80+/CD11blow macrophages as the relevant receptor for 16:4(n-3). Studies that used splenocytes from GPR120-deficient mice have confirmed this conclusion. Activation of the 16:4(n-3)-GPR120 axis led to enhanced cPLA2 activity in these splenic macrophages and secretion of the resistance-inducing lipid mediator, lysophosphatidylcholine(24:1). These studies identify a novel and unexpected function for GPR120 and suggest that antagonists of this receptor might be effective agents to limit development of chemotherapy resistance.-Houthuijzen, J. M., Oosterom, I., Hudson, B. D., Hirasawa, A., Daenen, L. G. M., McLean, C. M., Hansen, S. V. F., van Jaarsveld, M. T. M., Peeper, D. S., Jafari Sadatmand, S., Roodhart, J. M. L., van de Lest, C. H. A., Ulven, T., Ishihara, K., Milligan, G., Voest, E. E. Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance.

miR-634 restores drug sensitivity in resistant ovarian cancer cells by targeting the Ras-MAPK pathway.

BACKGROUND: Drug resistance hampers the efficient treatment of malignancies, including advanced stage ovarian cancer, which has a 5-year survival rate of only 30 %. The molecular processes underlying resistance have been extensively studied, however, not much is known about the involvement of microRNAs. METHODS: Differentially expressed microRNAs between cisplatin sensitive and resistant cancer cell line pairs were determined using microarrays. Mimics were used to study the role of microRNAs in drug sensitivity of ovarian cancer cell lines and patient derived tumor cells. Luciferase reporter constructs were used to establish regulation of target genes by microRNAs. RESULTS: MiR-634 downregulation was associated with cisplatin resistance. Overexpression of miR-634 affected cell cycle progression and enhanced apoptosis in ovarian cancer cells. miR-634 resensitized resistant ovarian cancer cell lines and patient derived drug resistant tumor cells to cisplatin. Similarly, miR-634 enhanced the response to carboplatin and doxorubicin, but not to paclitaxel. The cell cycle regulator CCND1, and Ras-MAPK pathway components GRB2, ERK2 and RSK2 were directly repressed by miR-634 overexpression. Repression of the Ras-MAPK pathway using a MEK inhibitor phenocopied the miR-634 effects on viability and chemosensitivity. CONCLUSION: miR-634 levels determine chemosensitivity in ovarian cancer cells. We identify miR-634 as a therapeutic candidate to resensitize chemotherapy resistant ovarian tumors.

Lysophospholipids secreted by splenic macrophages induce chemotherapy resistance via interference with the DNA damage response.

Host responses to systemic anti-cancer treatment play important roles in the development of anti-cancer drug resistance. Here we show that F4/80(+)/CD11b(low) splenocytes mediate the resistance to DNA-damaging chemotherapeutics induced by two platinum-induced fatty acids (PIFAs), 12-S-keto-5,8,10-heptadecatrienoic acid and 4,7,10,13-hexadecatetraenoic acid (16:4(n-3)) in xenograft mouse models. Splenectomy or depletion of splenic macrophages by liposomal clodronate protects against PIFA-induced chemoresistance. In addition, we find that 12-S-HHT, but not 16:4(n-3), functions via leukotriene B4 receptor 2 (BLT2). Genetic loss or chemical inhibition of BLT2 prevents 12-S-HHT-mediated resistance. Mass spectrometry analysis of conditioned medium derived from PIFA-stimulated splenic macrophages identifies several lysophosphatidylcholines as the resistance-inducing molecules. When comparing cisplatin and PIFA-treated tumours with cisplatin alone treated tumours we found overall less γH2AX, a measure for DNA damage. Taken together, we have identified an intricate network of lysophospholipid signalling by splenic macrophages that induces systemic chemoresistance in vivo via an altered DNA damage response.

DNA damage responsive microRNAs misexpressed in human cancer modulate therapy sensitivity.

The DNA damage response (DDR) is activated upon DNA damage and prevents accumulation of mutations and chromosomal rearrangements, both driving carcinogenesis. Tumor cells often have defects in the DDR, which in combination with continuous cell proliferation are exploited by genotoxic cancer therapies. Most cancers, overcome initial sensitivity and develop drug resistance, e.g. by modulation of the DDR. Not much is known, however, about DNA damage responsive microRNAs in cancer therapy resistance. Therefore, we mapped temporal microRNA expression changes in primary breast epithelial cells upon low and high dose exposure to the DNA damaging agents ionizing radiation and cisplatin. A third of all DDR microRNAs commonly regulated across all treatments was also misexpressed in breast cancer, indicating a DDR defect. We repeated this approach in primary lung epithelial cells and non-small cell lung cancer samples and found that more than 40% of all DDR microRNAs was deregulated in non-small cell lung cancer. Strikingly, the microRNA response upon genotoxic stress in primary breast and lung epithelial cells was markedly different, although the biological outcome of DNA damage signaling (cell death/senescence or survival) was similar. Several DDR microRNAs deregulated in cancer modulated sensitivity to anti-cancer agents. In addition we were able to distinguish between microRNAs that induced resistance by potentially inducing quiescence (miR-296-5p and miR-382) or enhancing DNA repair or increased DNA damage tolerance (miR-21). In conclusion, we provide evidence that DNA damage responsive microRNAs are frequently misexpressed in human cancer and can modulate chemotherapy sensitivity.

miRNA expression profiling of 51 human breast cancer cell lines reveals subtype and driver mutation-specific miRNAs.

INTRODUCTION: Breast cancer is a genetically and phenotypically complex disease. To understand the role of miRNAs in this molecular complexity, we performed miRNA expression analysis in a cohort of molecularly well-characterized human breast cancer cell lines to identify miRNAs associated with the most common molecular subtypes and the most frequent genetic aberrations. METHODS: Using a microarray carrying LNA™ modified oligonucleotide capture probes), expression levels of 725 human miRNAs were measured in 51 breast cancer cell lines. Differential miRNA expression was explored by unsupervised cluster analysis and was then associated with the molecular subtypes and genetic aberrations commonly present in breast cancer. RESULTS: Unsupervised cluster analysis using the most variably expressed miRNAs divided the 51 breast cancer cell lines into a major and a minor cluster predominantly mirroring the luminal and basal intrinsic subdivision of breast cancer cell lines. One hundred and thirteen miRNAs were differentially expressed between these two main clusters. Forty miRNAs were differentially expressed between basal-like and normal-like/claudin-low cell lines. Within the luminal-group, 39 miRNAs were associated with ERBB2 overexpression and 24 with E-cadherin gene mutations, which are frequent in this subtype of breast cancer cell lines. In contrast, 31 miRNAs were associated with E-cadherin promoter hypermethylation, which, contrary to E-cadherin mutation, is exclusively observed in breast cancer cell lines that are not of luminal origin. Thirty miRNAs were associated with p16INK4 status while only a few miRNAs were associated with BRCA1, PIK3CA/PTEN and TP53 mutation status. Twelve miRNAs were associated with DNA copy number variation of the respective locus. CONCLUSION: Luminal-basal and epithelial-mesenchymal associated miRNAs determine the subdivision of miRNA transcriptome of breast cancer cell lines. Specific sets of miRNAs were associated with ERBB2 overexpression, p16INK4a or E-cadherin mutation or E-cadherin methylation status, which implies that these miRNAs may contribute to the driver role of these genetic aberrations. Additionally, miRNAs, which are located in a genomic region showing recurrent genetic aberrations, may themselves play a driver role in breast carcinogenesis or contribute to a driver gene in their vicinity. In short, our study provides detailed molecular miRNA portraits of breast cancer cell lines, which can be exploited for functional studies of clinically important miRNAs.

The kinase RSK2 modulates the sensitivity of ovarian cancer cells to cisplatin.

Platinum-based chemotherapy (e.g. cisplatin, carboplatin) is standard of care for many types of cancer including ovarian cancer, however, the efficacy of treatment is hampered by the development of therapy resistance. The mechanisms behind platinum resistance are not completely understood. Here, we have investigated the role of the family of p90 Ribosomal S6 kinases (RSK), important downstream mediators of ERK1/2, in the response to cisplatin chemotherapy. Strikingly, whereas treatment with cisplatin did not alter the levels of RSK1 in response to cisplatin treatment, the structurally related RSK2 protein was downregulated in an ovarian cancer cell line (A2780). Furthermore, we found that knockdown of RSK2, in contrast to knockdown of RSK1, gave rise to enhanced cisplatin sensitivity in a cisplatin sensitive as well as a cisplatin-resistant A2780 cell line. These results indicate that RSK2 is regulated in response to cisplatin treatment, and this downregulation may contribute to the cytotoxic action of cisplatin. Since RSK2 is frequently amplified in a growing number of cancers, this may have implications for the sensitivity of these tumours to platinum-based cytotoxics.

Defining genetic factors that modulate intergenerational CAG repeat instability in Drosophila melanogaster.

Trinucleotide repeat instability underlies >20 human hereditary disorders. These diseases include many neurological and neurodegenerative situations, such as those caused by pathogenic polyglutamine (polyQ) domains encoded by expanded CAG repeats. Although mechanisms of instability have been intensely studied, our knowledge remains limited in part due to the lack of unbiased genome-wide screens in multicellular eukaryotes. Drosophila melanogaster displays triplet repeat instability with features that recapitulate repeat instability seen in patients with disease. Here we report an enhanced fly model with substantial instability based on a noncoding 270 CAG (UAS-CAG(270)) repeat construct under control of a germline-specific promoter. We find that expression of pathogenic polyQ protein modulates repeat instability of CAG(270) in trans, indicating that pathogenic-length polyQ proteins may globally modulate repeat instability in the genome in vivo. We further performed an unbiased genetic screen for novel modifiers of instability. These studies indicate that different aspects of repeat instability are under independent genetic control, and identify CG15262, a protein with a NOT2/3/5 conserved domain, as a modifier of CAG repeat instability in vivo.

MicroRNAs in ovarian cancer biology and therapy resistance.

Epithelial ovarian cancer is the most common cause of death from gynecological malignancies in the Western world. The overall 5-year survival is only 30% due to late diagnosis and development of resistance to chemotherapy. There is, therefore, a strong need for prognostic and predictive markers to help optimize and personalize treatment hence ameliorating the prognosis of ovarian cancer patients. Since 2006, an increasing number of studies have indicated an essential role for microRNAs in ovarian cancer tumorigenesis. In this review, we provide an overview of the microRNAs that have been associated with different aspects of ovarian cancer, such as tumor subtype, stage, histological grade, germline mutations in BRCA genes, prognosis and therapy resistance. We highlight the role of the let-7 and miR-200 families, two major microRNA families that are frequently dysregulated in ovarian cancer and have been associated with poor prognosis. Interestingly, both have been implicated in the regulation of the epithelial-to-mesenchymal transition, a cellular transition associated with tumor aggressiveness, tumor invasion and chemoresistance. Furthermore, we discuss several other microRNAs that have been associated with chemotherapy resistance, such as miR-214, miR-130a, miR-27a and miR-451. In the final section, we speculate on the possibilities of microRNA-based therapies and the use of microRNAs as diagnostic tools.