Antonius W. M. Boersma

Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer

In this study, we quantified 249 mature micro-RNA (miRNA) transcripts in estrogen receptor-positive (ER+) primary breast tumors of patients with lymph node-negative (LNN) disease to identify miRNAs associated with metastatic capability. In addition, the prognostic value of the candidate miRNAs was determined in ER−/LNN breast cancer. Unsupervised analysis in a prescreening set of 38 patients identified three subgroups predominantly driven by three miRNA signatures: an ER-driven luminal B-associated miRNA signature, a stromal miRNA signature, and an overexpressed miRNA cluster located on chromosome 19q23, but these intrinsic miRNA signatures were not associated with tumor aggressiveness. Supervised analysis in the initial subset and subsequent analysis in additional tumors significantly linked four miRNAs (miR-7, miR-128a, miR-210, and miR-516–3p) to ER+/LNN breast cancer aggressiveness (n = 147) and one miRNA (miR-210) to metastatic capability in ER−/LNN breast cancer (n = 114) and in the clinically important triple-negative subgroup (n = 69) (all P < 0.05). Bioinformatic analysis coupled miR-210 to hypoxia/VEGF signaling, miR-7 and miR-516–3p to cell cycle progression and chromosomal instability, and miR-128a to cytokine signaling. In conclusion, our work connects four miRNAs to breast cancer progression and to several distinct biological processes involved therein.

Lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in testicular germ cell tumour cell lines

We investigated the role of p53 and of the Bcl‐2 family proteins in the apoptotic response of a panel of testicular tumour cell lines (NT2, NCCIT, S2 and 2102 EP). The p53 gene status and the capacity of the p53 protein to transactivate the p21/WAF/CIP gene were determined, and we examined the correlation between p53 status and the susceptibility to cisplatin‐induced apoptosis. In contrast to wild‐type p53‐containing NT2 and 2102 EP cells, NCCIT (mutant p53) and S2 (no p53 protein) cells were shown to be p53‐transactivation defective. However, NCCIT and S2 cells with non‐functional p53 were readily triggered into apoptosis by cisplatin, whereas p53‐transactivation competent 2102 EP cells failed to undergo cisplatin‐induced apoptosis. The defective apoptotic pathway in 2102 EP cells was reflected by a 4‐fold decreased sensitivity to cisplatin in the MTT assay. We further demonstrated that the p53‐independent differential cisplatin sensitivity among the testicular germ cell tumour (TGCT) cell lines was not due to differences in cellular cisplatin accumulation or DNA platination. The pattern of endogenous expression levels of Bax, Bcl‐2, Bcl‐x and Bak, which was not modulated by cisplatin treatment, demonstrated that these Bcl‐2 family proteins are not involved in drug‐induced apoptosis in the TGCT cell lines. Our results suggest a lack of correlation between cisplatin‐induced apoptosis, p53 status and expression of Bcl‐2 family proteins in our panel of TGCT cell lines. We conclude that the cisplatin‐induced apoptotic pathway in TGCT cell lines might be p53‐independent and is probably not associated with differences in the Bcl‐2/Bax rheostat. Int. J. Cancer 73:592–599, 1997.

Synergistic cytotoxicity of cisplatin and topotecan or SN-38 in a panel of eight solid-tumor cell lines in vitro.

Abstract The cytotoxicity of cisplatin alone and in combination with topotecan (TPT) or SN-38, two novel topoisomerase I (topo I) inhibitors, was determined in a panel of eight well-characterized human solid-tumor cell lines. Interactions between cisplatin and these topo I inhibitors were investigated using three different administration schedules: (1) simultaneous incubation (C + T and C + S), (2) cisplatin followed by TPT or SN-38 (C → T and C → S), and (3) TPT or SN-38 followed by cisplatin (T → C and S → C). Median-effect analysis revealed synergistic cytotoxicity in seven of the eight cell lines used. In addition, a significant schedule-dependent synergistic cytotoxicity was found in three of the cell lines used, with C → T (or C → S) being the most active schedule. The formation and repair of total cisplatin-DNA adducts in the IGROV-1 ovarian cancer cell line and its cisplatin-resistant subline IGROVCDDP was not significantly affected by TPT on simultaneous incubation. In contrast, the number of cisplatin-DNA interstrand cross-links detected in the IGROV-1 and IGROVCDDP lines at certain time points was significantly lower after coincubation of the cells with TPT. Assessment of the cell-cycle distribution revealed an accumulation of cells in the G2/M phase after exposure to cisplatin. After exposure to TPT a different pattern was observed that was cell-type-specific and dependent upon the TPT concentration. Although up to 4-fold differences in topo I activity were observed in this panel of cell lines, these differences did not appear to be related to the synergy observed between cisplatin and TPT or SN-38. The observed synergy may at least partly be explained by the increased retention of cisplatin-DNA interstrand cross-links in the presence of topo I inhibitors.

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

IntroductionBreast 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.MethodsUsing 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.ResultsUnsupervised 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.ConclusionLuminal-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.

Distinct p53-independent apoptotic cell death signalling pathways in testicular germ cell tumour cell lines

The induction of apoptosis by diverse apoptotic stimuli was studied in a panel of 6 testicular germ cell tumour(TGCT) cell lines with defined p53 status. Although the sensitivity to a particular stimulus varied considerably among the TGCT cell lines, the differences in response were not associated with the presence of functional p53. Mutant (mt) p53‐expressing NCCIT and S2 (no p53 protein) were both readily triggered into apoptosis by cisplatin and doxorubicin, while wild‐type(wt)‐p53‐transactivation‐competent 2102 EP cells failed to undergo drug‐induced apoptosis. Moreover, transactivation‐deficient NCCIT cells and wtp53‐expressing NT2 cells were equally sensitive to cisplatin, doxorubicin, gamma radiation, and cell‐permeable C2‐ceramide. Our p53 data suggest that, at least in this panel of non‐isogeneic TGCT cell lines, hypersensitivity to therapeutic agents is not associated with p53 status. Next, we examined the impact of p53 inactivation on apoptosis induction in isogeneic NT2 sublines expressing human papillomavirus E6 protein. Evidently, abrogation of p53 function did not affect the hypersensitivity to apoptotic stimuli. We noted that drug‐sensitive S2 cells were highly resistant to radiation‐induced apoptosis, indicating distinct signalling pathways for chemotherapy and irradiation. The impaired radiation‐induced apoptotic pathway in S2 and 2102 EP could not be restored by addition of cell‐permeable C2‐ceramide, suggesting that the blockade is downstream of ceramide generation. Ligation of Fas/APO‐1/CD95 by anti‐Fas effectively induced apoptosis in Fas‐antigen expressing S2, 2102 EP and 833 KE. The efficient Fas‐mediated activation of apoptosis in drug‐, radiation‐, and ceramide‐resistant 2102 EP cells further suggests that diverse apoptosis‐inducing factors may use distinct signalling pathways. In summary, we demonstrated the presence of distinct p53‐independent apoptotic pathways in TGCT cells. Int. J. Cancer 81:620–628, 1999.

Bax upregulation is an early event in cisplatin‐induced apoptosis in human testicular germ‐cell tumor cell line NT2, as quantitated by flow cytometry

Expression of the apoptosis-associated proteins Bcl-2 and Bax was quantitated by flow cytometry (FCM) in chemosensitive testicular germ-cell tumor NT2 cells, and the results were compared with those obtained by Western blotting. NT2 cells were incubated with cisplatin (3.1 microM for 2 h at 37 degrees C), and 24, 48, and 72 h later were analyzed for induction of apoptosis, and for modulation of the expression of cell death suppressing protein Bcl-2, as well as cell death promoting protein Bax. Apoptosis was quantitated by binding of annexin V conjugated with fluorescein isothiocyanate (FITC) to the cell membrane. Cisplatin-treatment induced apoptosis in NT2 cells. The apoptotic cell population increased in time, and at t = 72 h after drug incubation, about 90% of cells that were present in the cell culture were apoptotic. Subsequently, we determined the expression of the Bcl-2 and Bax proteins by FCM and Western blotting before and after drug treatment. NT2 cells had low constitutive expression levels of Bcl-2 and elevated constitutive expression levels of Bax protein, as determined by both methods. At t = 24 h and 48 h after drug treatment, no changes were observed in the expression of the Bcl-2 protein, as quantitated by FCM and Western blotting. Also, the expression of the Bax protein had not changed, based on Western blotting. However, FCM revealed that in a specific subpopulation of drug-treated NT2 cells, Bax expression was increased. On the basis of forward and perpendicular light-scatter this subpopulation, which consisted of large, early apoptotic, swollen cells with increased internal complexity, was sorted, and showed abundant Bax protein by FCM and Western blotting. Our results demonstrate that the chemosensitivity of NT2 cells is probably due to a low intrinsic threshold for drug-induced apoptosis that is accompanied by overexpression of the death-promoting Bax protein during the early stages of the apoptotic process. We conclude that FCM is superior to Western blotting for the detection of heterogeneous expression of Bax in a given cell population.

Expression of P53, P21/WAF/CIP, BCL-2, BAX, BCL-X, and BAK In Radiation-Induced Apoptosis in Testicular Germ Cell Tumor Lines

PURPOSE Testicular germ cell tumors (TGCTs) represent one of the few tumor types that are curable by antineoplastic therapy, probably due to the high sensitivity of this neoplasm to induction of apoptosis by chemotherapeutic agents and/or ionizing radiation. Here, we tested cell susceptibility to radiation-induced apoptosis in a panel of TGCT cell lines and attempted to correlate this with the known potentially relevant molecular determinants (p53 gene status and Bcl-2 family proteins) of apoptosis. METHODS AND MATERIALS Induction of apoptosis by gamma-radiation was morphologically recognized in NT2, NCCIT, S2, and 2102 EP using Hoechst/PI staining and additionally confirmed by Western blot analysis of PARP cleavage. The p53 gene status was estimated by sequence analysis. Expression of p21/WAF/CIP was determined by Northern blot analysis and immunoblotting was used to monitor p53, Bax, Bcl-2, Bcl-x, and Bak protein levels. In vitro colony formation was studied to establish clonogenic survival curves. RESULTS NT2 and NCCIT appeared to be susceptible for radiation-induced apoptosis, contrasting 2102 EP and S2 which were highly resistant. Sequence analysis showed that NT2, S2, and 2102 EP are homozygous for wild-type p53 (wtp53), whereas NCCIT contains mutant p53 (mtp53). NT2 and 2102 EP cells showed radiation-induced p53 upregulation, while NCCIT (mtp53) and S2 (no p53 protein) cells did not. Consistently, gamma-radiation-induced DNA damage resulted in a p53-dependent transactivation of the p21/WAF/CIP gene in NT2 and 2102 EP, but not in mtp53-containing NCCIT cells and p53 nonexpressing S2 cells. Constitutive expression of Bax, Bcl-2, Bcl-x, and Bak was not affected by radiation and showed no correlation with cell susceptibility to radiation-induced apoptosis. A discrepancy was found between apoptosis and reproductive death. CONCLUSIONS The present study revealed that: i) the presence of wtp53 may not be absolutely required for the hypersensitivity for radiation-induced apoptosis in TGCT cell lines, ii) the molecular mechanism underlying the unique radiosensitivity was independent of the expression of Bcl-2 family proteins, and iii) cell susceptibility to apoptosis induction is not sufficiently informative to predict intrinsic radiosensitivity as determined by clonogenic survival.

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.

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.

MiR-193b promotes autophagy and non-apoptotic cell death in oesophageal cancer cells

BackgroundSuccessful treatment of oesophageal cancer is hampered by recurrent drug resistant disease. We have previously demonstrated the importance of apoptosis and autophagy for the recovery of oesophageal cancer cells following drug treatment. When apoptosis (with autophagy) is induced, these cells are chemosensitive and will not recover following chemotherapy treatment. In contrast, when cancer cells exhibit only autophagy and limited Type II cell death, they are chemoresistant and recover following drug withdrawal.MethodsMicroRNA (miRNA) expression profiling of an oesophageal cancer cell line panel was used to identify miRNAs that were important in the regulation of apoptosis and autophagy. The effects of miRNA overexpression on cell death mechanisms and recovery were assessed in the chemoresistant (autophagy inducing) KYSE450 oesophageal cancer cells.ResultsMiR-193b was the most differentially expressed miRNA between the chemosensitive and chemoresistant cell lines with higher expression in chemosensitive apoptosis inducing cell lines. Colony formation assays showed that overexpression of miR-193b significantly impedes the ability of KYSE450 cells to recover following 5-fluorouracil (5-FU) treatment. The critical mRNA targets of miR-193b are unknown but target prediction and siRNA data analysis suggest that it may mediate some of its effects through stathmin 1 regulation. Apoptosis was not involved in the enhanced cytotoxicity. Overexpression of miR-193b in these cells induced autophagic flux and non-apoptotic cell death.ConclusionThese results highlight the importance of miR-193b in determining oesophageal cancer cell viability and demonstrate an enhancement of chemotoxicity that is independent of apoptosis induction.