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.

MicroRNA expression profiles distinguish liposarcoma subtypes and implicate miR-145 and miR-451 as tumor suppressors.

Liposarcomas are rare, heterogeneous and malignant tumors that can be divided into four histological subtypes with different characteristics and clinical behavior. Treatment consists of surgery in combination with systemic chemotherapy, but nevertheless mortality rates are high. More insight into the biology of liposarcoma tumorigenesis is needed to devise novel therapeutic approaches. MicroRNAs (miRNAs) have been associated with carcinogenesis in many tumors and may function as tumor suppressor or oncogene. In this study we examined miRNA expression in an initial series of 57 human liposarcomas (including all subtypes), lipomas and normal fat by miRNA microarrays. Supervised hierarchical clustering of the most differentially expressed miRNAs (p < 0.0002) distinguished most liposarcoma subtypes and control tissues. The distinction between well differentiated liposarcomas and benign lipomas was blurred, suggesting these tumor types may represent a biological continuum. MiRNA signatures of liposarcoma subtypes were established and validated in an independent series of 58 liposarcomas and control tissues. The expression of the miR-143/145 and miR-144/451 cluster members was clearly reduced in liposarcomas compared to normal fat. Overexpression of miR-145 and miR-451 in liposarcoma cell lines decreased cellular proliferation rate, impaired cell cycle progression and induced apoptosis. In conclusion, we show that miRNA expression profiling can be used to discriminate liposarcoma subtypes, which can possibly aid in objective diagnostic decision making. In addition, our data indicate that miR-145 and miR-451 act as tumor suppressors in adipose tissue and show that re-expression of these miRNAs could be a promising therapeutic strategy for liposarcomas.

MicroRNA response to hypoxic stress in soft tissue sarcoma cells: microRNA mediated regulation of HIF3α.

BACKGROUND: Hypoxia is often encountered in solid tumors and known to contribute to aggressive tumor behavior, radiation- and chemotherapy resistance resulting in a poor prognosis for the cancer patient. MicroRNAs (miRNAs) play a role in the regulation of the tumor cell response to hypoxia, however, not much is known about the involvement of miRNAs in hypoxic signalling pathways in soft tissue sarcomas (STS). METHOD: A panel of twelve STS cell lines was exposed to atmospheric oxygen concentrations (normoxia) or 1% oxygen (hypoxia) for up to 48 h. Hypoxic conditions were verified and miRNA expression profiles were assessed by LNA™ oligonucleotide microarrays and RT-PCR after 24 h. The expression of target genes regulated by hypoxia responsive miRNAs is examined by end-point PCR and validated by luciferase reporter constructs. RESULTS: Exposure of STS cell lines to hypoxic conditions gave rise to upregulation of Hypoxia Inducible Factor (HIF) 1α protein levels and increased mRNA expression of HIF1 target genes CA9 and VEGFA. Deregulation of miRNA expression after 24 h of hypoxia was observed. The most differentially expressed miRNAs (p<0.001) in response to hypoxia were miR-185-3p, miR-485-5p, miR-216a-5p (upregulated) and miR-625-5p (downregulated). The well-known hypoxia responsive miR-210-3p could not be reliably detected by the microarray platform most likely for technical reasons, however, its upregulation upon hypoxic stress was apparent by qPCR. Target prediction algorithms identified 11 potential binding sites for miR-485-5p and a single putative miR-210-3p binding site in the 3'UTR of HIF3α, the least studied member of the HIF family. We showed that HIF3α transcripts, expressing a 3'UTR containing the miR-485-5p and miR-210-3p target sites, are expressed in all sarcoma cell lines and upregulated upon hypoxia. Additionally, luciferase reporter constructs containing the 3'UTR of HIF3α were used to demonstrate regulation of HIF3α by miR-210-3p and miR-485-5p. CONCLUSION: Here we provide evidence for the miRNA mediated regulation of HIF3α by hypoxia responsive miRNAs in STS, which may help to tightly regulate and fine-tune the hypoxic response. This provides a better insight into the mechanisms underlying the hypoxic response in STS and may ultimately yield information on novel prognostic and predictive markers or targets for treatment.

MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation.

MicroRNAs (miRNAs) are non-protein coding molecules with important regulatory functions; many have tissue-specific expression patterns. Their very small size in principle makes them less prone to degradation processes, unlike messenger RNAs (mRNAs), which were previously proposed as molecular tools for forensic body fluid identification. To identify suitable miRNA markers for forensic body fluid identification, we first screened total RNA samples derived from saliva, semen, vaginal secretion, and venous and menstrual blood for the expression of 718 human miRNAs using a microarray platform. All body fluids could be easily distinguished from each other on the basis of complete array-based miRNA expression profiles. Results from quantitative reverse transcription PCR (RT-PCR; TaqMan) assays for microarray candidate markers confirmed strong over-expression in the targeting body fluid of several miRNAs for venous blood and several others for semen. However, no candidate markers from array experiments for other body fluids such as saliva, vaginal secretion, or menstrual blood could be confirmed by RT-PCR. Time-wise degradation of venous blood and semen stains for at least 1 year under lab conditions did not significantly affect the detection sensitivity of the identified miRNA markers. The detection limit of the TaqMan assays tested for selected venous blood and semen miRNA markers required only subpicogram amounts of total RNA per single RT-PCR test, which is considerably less than usually needed for reliable mRNA RT-PCR detection. We therefore propose the application of several stable miRNA markers for the forensic identification of blood stains and several others for semen stain identification, using commercially available TaqMan assays. Additional work remains necessary in search for suitable miRNA markers for other forensically relevant body fluids.

Deregulated microRNAs in neurofibromatosis type 1 derived malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNST) are aggressive cancers that occur spontaneously (sporadic MPNST) or from benign plexiform neurofibromas in neurofibromatosis type 1 (NF1) patients. MPNSTs metastasize easily, are therapy resistant and are frequently fatal. The molecular changes underlying the malignant transformation in the NF1 setting are incompletely understood. Here we investigate the involvement of microRNAs in this process. MicroRNA expression profiles were determined from a series of archival, paired samples of plexiform neurofibroma and MPNST. Ninety differentially expressed microRNAs were identified between the paired samples. Three downregulated microRNAs (let-7b-5p, miR-143-3p, miR-145-5p) and two upregulated microRNAs (miR135b-5p and miR-889-3p) in MPNST were selected for functional characterization. In general, their differential expression was validated in a relevant cell line panel but only partly in a series of unpaired, fresh frozen tumor samples. As part of the validation process we also analyzed microRNA expression profiles of sporadic MPNSTs observing that microRNA expression discriminates NF1-associated and sporadic MPNSTs. The role of microRNAs in cancer progression was examined in NF1-derived MPNST cell lines by transiently modulating microRNA levels. Our findings indicate that some microRNAs affect migratory and invasive capabilities and Wnt signaling activity but the effects are distinct in different cell lines. We conclude that miRNAs play essential regulatory roles in MPNST facilitating tumor progression.

MicroRNA expression and DNA methylation profiles do not distinguish between primary and recurrent well-differentiated liposarcoma.

Approximately one-third of the patients with well-differentiated liposarcoma (WDLPS) will develop a local recurrence. Not much is known about the molecular relationship between the primary tumor and the recurrent tumor, which is important to reveal potential drivers of recurrence. Here we investigated the biology of recurrent WDLPS by comparing paired primary and recurrent WDLPS using microRNA profiling and genome-wide DNA methylation analyses. In total, 27 paired primary and recurrent WDLPS formalin-fixed and paraffin-embedded tumor samples were collected. MicroRNA expression profiles were determined using TaqMan® Low Density Array (TLDA) cards. Genome-wide DNA methylation and differentially methylated regions (DMRs) were assessed by methylated DNA sequencing (MeD-seq). A supervised cluster analysis based on differentially expressed microRNAs between paired primary and recurrent WDLPS did not reveal a clear cluster pattern separating the primary from the recurrent tumors. The clustering was also not based on tumor localization, time to recurrence, age or status of the resection margins. Changes in DNA methylation between primary and recurrent tumors were extremely variable, and no consistent DNA methylation changes were found. As a result, a supervised clustering analysis based on DMRs between primary and recurrent tumors did not show a distinct cluster pattern based on any of the features. Subgroup analysis for tumors localized in the extremity or the retroperitoneum also did not yield a clear distinction between primary and recurrent WDLPS samples. In conclusion, microRNA expression profiles and DNA methylation profiles do not distinguish between primary and recurrent WDLPS and no putative common drivers could be identified.

Molecular Comparison of Imatinib-Naïve and Resistant Gastrointestinal Stromal Tumors: Differentially Expressed microRNAs and mRNAs.

Despite the success of imatinib in advanced gastrointestinal stromal tumor (GIST) patients, 50% of the patients experience resistance within two years of treatment underscoring the need to get better insight into the mechanisms conferring imatinib resistance. Here the microRNA and mRNA expression profiles in primary (imatinib-naïve) and imatinib-resistant GIST were examined. Fifty-three GIST samples harboring primary KIT mutations (exon 9; n = 11/exon 11; n = 41/exon 17; n = 1) and comprising imatinib-naïve (IM-n) (n = 33) and imatinib-resistant (IM-r) (n = 20) tumors, were analyzed. The microRNA expression profiles were determined and from a subset (IM-n, n = 14; IM-r, n = 15) the mRNA expression profile was established. Ingenuity pathway analyses were used to unravel biochemical pathways and gene networks in IM-r GIST. Thirty-five differentially expressed miRNAs between IM-n and IM-r GIST samples were identified. Additionally, miRNAs distinguished IM-r samples with and without secondary KIT mutations. Furthermore 352 aberrantly expressed genes were found in IM-r samples. Pathway and network analyses revealed an association of differentially expressed genes with cell cycle progression and cellular proliferation, thereby implicating genes and pathways involved in imatinib resistance in GIST. Differentially expressed miRNAs and mRNAs between IM-n and IM-r GIST were identified. Bioinformatic analyses provided insight into the genes and biochemical pathways involved in imatinib-resistance and highlighted key genes that may be putative treatment targets.

Expression and inhibition of BRD4, EZH2 and TOP2A in neurofibromas and malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNST) are rare, highly aggressive sarcomas that can occur spontaneously or from pre-existing plexiform neurofibromas in neurofibromatosis type1 (NF1) patients. MPNSTs have high local recurrence rates, metastasize easily, are generally resistant to therapeutic intervention and frequently fatal for the patient. Novel targeted therapeutic strategies are urgently needed. Standard treatment for patients presenting with advanced disease is doxorubicin based chemotherapy which inhibits the actions of the enzyme topoisomerase IIα (TOP2A). Recent molecular studies using murine models and cell lines identified the bromodomain containing protein 4 (BRD4) and enhancer of zeste homolog 2 (EZH2) as novel targets for MPNST treatment. We investigated the expression and potential use of BRD4, EZH2 and TOP2A as therapeutic targets in human NF1-derived MPNSTs. The transcript levels of BRD4, EZH2 and TOP2A were determined in paired formalin-fixed paraffin-embedded (FFPE) neurofibroma/MPNST samples derived from the same NF1 patient and in a set of plexiform neurofibromas, atypical neurofibromas and MPNST. We further examined the effect on cell viability of genetic or pharmacological inhibition of BRD4, EZH2 and TOP2A in an MPNST cell line panel. Our results indicated that in MPNST samples BRD4 mRNA levels were not upregulated and that MPNST cell lines were relatively insensitive to the bromodomain inhibitor JQ1. We corroborated that EZH2 mRNA expression is increased in MPNST but failed to confirm its reported pivotal role in MPNST pathogenesis as EZH2 knockdown by siRNA did not interfere with cellular proliferation and viability. Finally, the relation between TOP2A levels and sensitivity for doxorubicin was examined, confirming reports that TOP2A mRNA levels were overexpressed in MPNST and showing that MPNST cell lines exhibited relatively high TOP2A protein levels and sensitivity to doxorubicin. We tentatively conclude that the potential for effective therapeutic intervention in MPNST by targeting BRD4, EZH2 and TOP2A individually, may be limited. Clinical studies are necessary to ultimately prove the relevance of BRD4 and EZH2 inhibition as novel therapeutic strategies for MPNST.

Mismatch repair and treatment resistance in ovarian cancer.

BACKGROUND: The treatment of ovarian cancer is hindered by intrinsic or acquired resistance to platinum-based chemotherapy. The aim of this study is to determine the frequency of mismatch repair (MMR) inactivation in ovarian cancer and its association with resistance to platinum-based chemotherapy. METHODS: We determined, microsatellite instability (MSI) as a marker for MMR inactivation (analysis of BAT25 and BAT26), MLH1 promoter methylation status (methylation specific PCR on bisulfite treated DNA) and mRNA expression of MLH1, MSH2, MSH3, MSH6 and PMS2 (quantitative RT-PCR) in 75 ovarian carcinomas and eight ovarian cancer cell lines RESULTS: MSI was detected in three of the eight cell lines i.e. A2780 (no MLH1 mRNA expression due to promoter methylation), SKOV3 (no MLH1 mRNA expression) and 2774 (no altered expression of MMR genes). Overall, there was no association between cisplatin response and MMR status in these eight cell lines. Seven of the 75 ovarian carcinomas showed MLH1 promoter methylation, however, none of these showed MSI. Forty-six of these patients received platinum-based chemotherapy (11 non-responders, 34 responders, one unknown response). The resistance seen in the eleven non-responders was not related to MSI and therefore also not to MMR inactivation. CONCLUSION: No MMR inactivation was detected in 75 ovarian carcinoma specimens and no association was seen between MMR inactivation and resistance in the ovarian cancer cell lines as well as the ovarian carcinomas. In the discussion, the results were compared to that of twenty similar studies in the literature including in total 1315 ovarian cancer patients. Although no association between response and MMR status was seen in the primary tumor the possible role of MMR inactivation in acquired resistance deserves further investigation.