Cell fusion enhances energy metabolism of mesenchymal tumor hybrid cells to sustain their proliferation and invasion

Abstract Background Cell-to-cell fusion is emerging as a key element of the metastatic process in various cancer types. We recently showed that hybrids made from the spontaneous merging of pre-malignant (IMR90 E6E7, i.e. E6E7) and malignant (IMR90 E6E7 RST, i.e. RST) mesenchymal cells recapitulate the main features of human undifferentiated pleomorphic sarcoma (UPS), with a highly rearranged genome and increased spreading capacities. To better characterize the intrinsic properties of these hybrids, we investigated here their metabolic energy profile compared to their parents. Results Our results unveiled that hybrids harbored a Warburg-like metabolism, like their RST counterparts. However, hybrids displayed a much greater metabolic activity, enhancing glycolysis to proliferate. Interestingly, modifying the metabolic environmental conditions through the use of 5-aminoimidazole-4-carbox-amide-1-β-D-ribofuranoside (AICAR), an activator of the 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK), specifically reduced the growth of hybrids, and also abrogated the invasive capacity of hybrids displaying enhanced glycolysis. Furthermore, AICAR efficiently blocked the tumoral features related to the aggressiveness of human UPS cell lines. Conclusion Altogether, our findings strongly suggest that hybrids rely on higher energy flux to proliferate and that a drug altering this metabolic equilibrium could impair their survival and be potentially considered as a novel therapeutic strategy

Germinal GLT8D1, GATAD2A and SLC25A39 mutations in a patient with a glomangiopericytal tumor and five different sarcomas over a 10-year period

International audienceSoft tissue sarcoma represents about 1% of all adult cancers. Occurrence of multiple sarcomas in a same individual cannot be fortuitous. A 72-year-old patient had between 2007 and 2016 a glomangiopericytal tumor of the right forearm and a succession of sarcomas of the extremities: a leiomyosarcoma of the left buttock, a myxofibrosarcoma (MFS) of the right forearm, a MFS of the left scapula, a left latero-thoracic MFS and two undifferentiated sarcomas on the left forearm. Pathological examination of the six locations was not in favor of disease with local/distant recurrences but could not confirm different diseases. An extensive molecular analysis including DNA-array, RNA-sequencing and DNA-Sanger-sequencing, was thus performed to determine the link between them. The genomic profile of the glomangiopericytal tumor and the six sarcomas revealed that five sarcomas were different diseases and one was the local recurrence of the glomangiopericytal tumor. While the chromosomal alterations in the six tumors were different, a common somatic CDKN2A/CDKN2B deletion was identified. RNA-sequencing of five tumors identified mutations in GLT8D1, GATAD2A and SLC25A39 in all samples. The germline origin of these mutations was confirmed by Sanger-sequencing. Innovative molecular analysis methods have made possible a better understanding of the complex tumorigenesis of multiple sarcomas

Cell-cell fusion of mesenchymal cells with distinct differentiations triggers genomic and transcriptomic remodelling toward tumour aggressiveness

Cell-cell fusion is a physiological process that is hijacked during oncogenesis and promotes tumour evolution. The main known impact of cell fusion is to promote the formation of metastatic hybrid cells following fusion between mobile leucocytes and proliferating tumour cells. We show here that cell fusion between immortalized myoblasts and transformed fibroblasts, through genomic instability and expression of a specific transcriptomic profile, leads to emergence of hybrid cells acquiring dissemination properties. This is associated with acquisition of clonogenic ability by fused cells. In addition, by inheriting parental properties, hybrid tumours were found to mimic the histological characteristics of a specific histotype of sarcomas: undifferentiated pleomorphic sarcomas with incomplete muscular differentiation. This finding suggests that cell fusion, as macroevolution event, favours specific sarcoma development according to the differentiation lineage of parent cells

Heterogeneity in sarcoma cell lines reveals enhanced motility of tetraploid versus diploid cells

International audienceSoft tissue sarcomas with complex genomics are very heterogeneous tumors lacking simple prognosis markers or targeted therapies. Overexpression of a subset of mitotic genes from a signature called CINSARC is of bad prognosis, but the significance of this signature remains elusive. Here we precisely measure the cell cycle and mitosis duration of sarcoma cell lines and we found that the mitotic gene products overexpression does not reflect variation in the time spent during mitosis or G2/M. We also found that the CINSARC cell lines, we studied, are composed of a mixture of aneuploid, diploid, and tetraploid cells that are highly motile in vitro. After sorting diploid and tetraploid cells, we showed that the tetraploid cell clones do not possess a proliferative advantage, but are strikingly more motile and invasive than their diploid counterparts. This is correlated with higher levels of mitotic proteins overexpression. Owing that mitotic proteins are almost systematically degraded at the end of mitosis, we propose that it is the abnormal activity of the mitotic proteins during interphase that boosts the sarcoma cells migratory properties by affecting their cytoskeleton. To test this hypothesis, we designed a screen for mitotic or cytoskeleton protein inhibitors affecting the sarcoma cell migration potential independently of cytotoxic activities. We found that inhibition of several mitotic kinases drastically impairs the CINSARC cell invasive and migratory properties. This finding could provide a handle by which to selectively inhibit the most invasive cells

Promoting role of cholecystokinin 2 receptor (CCK2R) in gastrointestinal stromal tumours pathogenesis

The cholecystokinin 2 receptor (CCK2R/CCKBR) is expressed in gastrointestinal stromal tumours (GIST). We sought to investigate the role of CCK2R in GIST pathogenesis. Molecular characterization of CCK2R was performed on a heterogeneous cohort of 50 GIST. In addition, CCK2R expression was evaluated by immunohistochemistry (IHC) using tissue microarray (TMA) containing 292 GIST, two cases of hyperplasia of interstitial Cajal's cells (ICC) and six gastric microscopic GIST. Mono-allelic loss of the CCK2R/11p15 allele was identified in 13.7% of GIST, having no impact on the level of CCK2R transcript expression. No CCK2R mutations were found. The CCK2Ri4sv, CCK2R splice variant with retention of intron 4, was detected in 6 out of 20 analysed tumours. Wild-type CCK2R transcripts were commonly expressed (57.1% of cases) and this expression highly correlated with gastric primary site of GIST (p<0.001). On protein level, expression of CCK2R in incidental ICC hyperplasia and early stages of gastric GIST development was documented, and its gastric association was confirmed on GIST-TMA by IHC. To explore the in vivo effect of CCK2R activation on tumour growth, gastrin versus placebo was administered intra-peritoneally in nude mice carrying human GIST xenografts. The tumour volume was followed up for ten weeks. The effect of this stimulation on tumour cell proliferation/apoptosis was assessed by IHC, and KIT/PKC-theta signalling was evaluated by Western blotting (WB). In vivo experiments showed a two-fold increase in the volume of tumours which were exposed to gastrin in comparison with non-exposed controls (p = 0.03), with a significant increase in mitotic activity (p= 0.04) and Ki-67 proliferation index (p = 0.008). By WB, gastrin stimulation resulted in hyper-activation of KIT and PKC-theta kinases, and in evident PI3K/AKT pathway over-activation. Our results indicate a promoting role of CCK2R on GIST tumourigenesis, particularly in tumours of gastric origin. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.status: publishe

Genomic index is a strong predictor of metastatic outcome in intermediate gastrointestinal stromal tumors and an inclusion criteria for imatinib adjuvant therapy

Abstract Gastrointestinal stroma tumors (GISTs) harboring a cKIT / PDGFRA activating mutation in 90% of cases benefit from the Imatinib mesylate (Glivec®) targeted therapy. Patients eligibility to imatinib adjuvant therapy is based, in Europe, on histological grading of tumor aggressiveness and no standard is currently available for tumor classified as intermediate risk (40% of patients). We recently validated that genomic index (GI), a measure of the number and type of genomic copy number alterations, constitutes a strong predictor of clinical outcome in GIST, making it a possible prognostic factor for the intermediate GIST subgroup. To definitively clarify whether this genomic grading system would permit to categorize intermediate-risk patients into good and poor prognosis, we selected a cohort of 82 intermediate patients based on the Armed Forces Institute of Pathology (AFIP) classification and performed genomic profiling from formalin-fixed, paraffin-embedded (FFPE) samples using a microarray-based comparative genomic hybridization (array CGH) approach. Data revealed that even if studied samples generally harbored a combination of the typical genetic aberrations found in GIST, i.e. 1p, 14q, 22q deletions and frequently lost CDKN2A locus on chromosome 9, they profoundly differed from each other on the total number of genomic changes and GI value, ranging, in the whole series, from 0 to 37 and 0 to 115.6, respectively. More interestingly, Kaplan-Meier analyses of metastatic-free survival unveiled that stratification of the tumors by the GI value at a cutoff of 10 (GI1&amp;lt;10 and GI2&amp;gt;10) separated the good (GI1) from the poor (GI2) prognosis patients (p&amp;lt;10-3), undoubtedly proven that metastatic-risk in GIST intermediate patients is strongly associated with high GI values and genome complexity. In conclusion, GI is validated here as a robust marker to predict the clinical outcome of patients diagnosed with GIST and classified as intermediate by the current histologic method for risk assessment. Applicable in numerous Pathology Laboratories already using aCGH with FFPE samples for routine diagnostic tests, this assay currently stands as the best tool to determine which intermediate GIST-patients are likely to benefit from imatinib therapy and constitutes as such a novel advance in the clinical management of GISTs. Citation Format: Lydia Lartigue, Pauline Lagarde, Céline Brulard, Agnès Neuville, Piotr Rutkowski, Paolo Dei Tos, Eva Waldermann, Maria Debiec-Rychter, Antoine Italiano, Jean-Michel Coindre, Frédéric Chibon. Genomic index is a strong predictor of metastatic outcome in intermediate gastrointestinal stromal tumors and an inclusion criteria for imatinib adjuvant therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3829. doi:10.1158/1538-7445.AM2014-3829</jats:p

Prognostic Value of <i>PLAGL1</i>-Specific CpG Site Methylation in Soft-Tissue Sarcomas

<div><p>Soft tissue sarcomas (STS) are rare, complex tumors with a poor prognosis. The identification of new prognostic biomarkers is needed to improve patient management. Our aim was to determine the methylation status of the 118 CpG sites in the <i>PLAGL1</i> tumor-suppressor gene P1 CpG island promoter and study the potential prognostic impact of <i>PLAGL1</i> promoter methylation CpG sites in STS. Training cohorts constituted of 28 undifferentiated sarcomas (US) and 35 leiomyosarcomas (LMS) were studied. <i>PLAGL1</i> mRNA expression was investigated by microarray analysis and validated by RT-qPCR. Pyrosequencing was used to analyze quantitative methylation of the <i>PLAGL1</i> promoter. Associations between global promoter or specific CpG site methylation and mRNA expression were evaluated using Pearson’s product moment correlation coefficient. Cox univariate and multivariate proportional hazard models were used to assess the predictive power of CpG site methylation status. Sixteen CpG sites associated with <i>PLAGL1</i> mRNA expression were identified in US and 6 in LMS. Statistical analyses revealed an association between CpG107 methylation status and both overall and metastasis-free survival in US, which was confirmed in a validation cohort of 37 US. The exhaustive study of P1 <i>PLAGL1</i> promoter methylation identified a specific CpG site methylation correlated with mRNA expression, which was predictive for both metastasis-free and overall survival and may constitute the first US-specific biomarker. Such a biomarker may be relevant for identifying patients likely to derive greater benefit from treatment. </p> </div

Correlation between <i>PLAGL1</i> genomic status assessed by CGHarray and <i>PLAGL1</i> mRNA expression assessed by Affymetrix HGU 133 plus 2.0 array in LMS (A) and US (B).

<p><i>PLAGL1</i> mRNA expression was evaluated by microarray (mean of the 3 Affimetrix probes) and plotted in log₂. CGH ratios between 0.8 and 1.2 (dotted lines) were considered as normal status, and ratios >1.2 and <0.8 were considered as gains and losses, respectively. </p

Pearson’s correlation between <i>PLAGL1</i> mRNA expression and the methylation percentage of CpGs in US and LMS.

<p>Correlation between <i>PLAGL1</i> mRNA expression and the mean methylation percentage of all CpG sites in undifferentiated sarcomas (US) (<b>Aa</b>) and leiomyosarcomas (LMS) (<b>Ba</b>). <b>Ab</b>: Correlation between <i>PLAGL1</i> mRNA expression and the mean methylation percentage of CpGs exhibiting positive correlation with <i>PLAGL1</i> mRNA expression in US. <b>Ac</b>: Correlation between <i>PLAGL1</i> mRNA expression and the mean methylation percentage of CpGs exhibiting negative correlation with <i>PLAGL1</i> mRNA expression in US; <b>Bb</b>: Correlation between <i>PLAGL1</i> mRNA expression and the mean methylation percentage of CpGs exhibiting negative correlation with <i>PLAGL1</i> mRNA expression in LMS. <i>PLAGL1</i> mRNA expression was evaluated by microarray (mean of the 3 Affimetrix probes) and plotted in log2. <i>P</i> ≤ 0.05 are considered significant.</p