The co-existence of transcriptional activator and transcriptional repressor MEF2 complexes influences tumor aggressiveness

The contribution of MEF2 TFs to the tumorigenic process is still mysterious. Here we clarify that MEF2 can support both pro-oncogenic or tumor suppressive activities depending on the interaction with co-activators or co-repressors partners. Through these interactions MEF2 supervise histone modifications associated with gene activation/repression, such as H3K4 methylation and H3K27 acetylation. Critical switches for the generation of a MEF2 repressive environment are class IIa HDACs. In leiomyosarcomas (LMS), this two-faced trait of MEF2 is relevant for tumor aggressiveness. Class IIa HDACs are overexpressed in 22% of LMS, where high levels of MEF2, HDAC4 and HDAC9 inversely correlate with overall survival. The knock out of HDAC9 suppresses the transformed phenotype of LMS cells, by restoring the transcriptional proficiency of some MEF2-target loci. HDAC9 coordinates also the demethylation of H3K4me3 at the promoters of MEF2-target genes. Moreover, we show that class IIa HDACs do not bind all the regulative elements bound by MEF2. Hence, in a cell MEF2-target genes actively transcribed and strongly repressed can coexist. However, these repressed MEF2-targets are poised in terms of chromatin signature. Overall our results candidate class IIa HDACs and HDAC9 in particular, as druggable targets for a therapeutic intervention in LMS

Tumor genotype, location and malignant potential shape the immunogenicity of primary, untreated Gastrointestinal Stromal Tumors

Intratumoral immune infiltrate was recently reported in Gastrointestinal Stromal Tumors (GIST). However, what tumor-intrinsic factors dictate GIST immunogenicity is still largely undefined. To shed light on this issue a large cohort (82 samples) of primary untreated GIST, representative of major clinicopathological variables, was investigated by an integrated immunohistochemical, transcriptomic and computational approach. Our results indicate that tumor genotype, location and malignant potential concur to shape the immunogenicity of primary na\uefve GIST. Immune infiltration was greater in overt GIST than in lesions with limited malignant potential (miniGIST), in KIT/PDGFRA mutated than in KIT/PDGFRA wild-type tumors and in PDGFRA versus KIT mutated GIST. Within the KIT mutated subset, a higher degree of immune colonization was detected in the intestine. Immune hot tumors showed expression patterns compatible with a potentially proficient but curbed antigen-specific immunity, hinting at sensitivity to immunomodulatory treatments. Poorly infiltrated GIST, primarily KIT/PDGFRA wild-type intestinal tumors, showed activation of Hedgehog and WNT/\u3b2-catenin immune excluding pathways. This finding discloses a potential therapeutic vulnerability, as the targeting of these pathways might prove effective by both inhibiting pro-oncogenic signals and fostering anti-tumor immune responses. Finally, an intriguing anticorrelation between immune infiltration and ANO1/DOG1 expression was observed, suggesting an immunomodulatory activity for anoctamin-1

YAP1 acts as oncogenic target of 11q22 amplification in multiple cancer subtypes

The transcriptional coactivator YAP1 is a critical effector of the human Salvador-Warts-Hippo pathway. Literature data report apparently discrepant results on the carcinogenic role of YAP1, which acts either as oncogene or as tumor suppressor in different in vitro and in vivo models. Furthermore, genomic amplification events of 11q22 locus encompassing YAP1 gene have been detected in multiple tumor types but there is limited direct evidence about the oncogenic role of endogenous YAP1 within in the amplicon. We screened a panel of human tumor samples and cancer cell lines and identified that the YAP1 amplification event is actually present in up to 23% of the cases. We exploited EKVX (lung cancer), CaSki (cervical cancer) and RO82 (thyroid cancer) cell lines harboring both genomic YAP1 amplification and YAP1 protein overexpression, in order to study the effects of downregulation of endogenous YAP1 by RNA-interference strategies. Class comparison analysis of gene expression profiling data identified 707 statistically significantly modulated genes (multivariable global test p-value = 0.002) that were functionally annotated for cell proliferation and cellular movement ontologies. Mechanistic studies of the identified perturbed pathways revealed that YAP1 silencing significantly decreased cell proliferation and cell cycle perturbation associated with upregulation of p21 and p27 cell-cycle inhibitors, reduced cell migration (p<0.048) and anchorage-independent growth (p<0.02). In CaSki cell line, YAP1 silencing induced significantly increased sensitivity and cell-death response to cisplatin treatment (p=0.011) as well as reduction of in-vivo tumorigenic potential (p=0.027). Overall, these results establish that YAP1 is a direct oncogenic target of the 11q22 amplicon in previously unreported cancer types and support the relevance of such genetic aberration in carcinogenesis in a fraction of multiple tumor types

Case report: Long-term survival of an infant syndromic patient affected by atypical teratoid-rhabdoid tumor

Background: Atypical teratoid rhabdoid tumor (ATRT) patients display a dismal median overall survival of less than 1 year. A consistent fraction of cases carries de-novo SMARCB1/INI1 constitutional mutations in the setting of the " rhabdoid tumor predisposition syndrome" and the outcome is worst in infant syndromic ATRT patients. Case presentation: We here describe a patient affected by mosaic Klinefelter syndrome and by rhabdoid tumor predisposition syndrome caused by constitutional SMARCB1/INI1 heterozygous mutation c.118C>T (Arg40X). Patient's ATRT primary tumor occurred at 2 years of age concurrent with metastatic lesions. The patient was rendered without evidence of disease by combined surgery, high-dose poli-chemotherapy and craniospinal irradiation, followed by autologous hematopoietic stem cell transplantation. At the onset of a spinal lesion 5.5 years later, both tumors were pathologically and molecularly evaluated at the national central pathology review board and defined as ATRT in a syndromic patient, with strong evidence of a clonal origin of the two lesions. The patient was then treated according to SIOP guidelines and is now alive without evidence of disease 24 months after the detection of metastatic disease and 90 months after the original diagnosis. Conclusion: The report underscores the current utility of multiple comprehensive approaches for the correct diagnosis and clinical management of patients affected by rare and atypical brain neoplasms. Successful local control of disease and achievement of long-term survival is possible in ATRT patients even in the setting of rhabdoid tumor predisposition syndrome, infant age at diagnosis and metastatic spread of disease, thus justifying the efforts for the management of this severe condition. © 2013 Modena et al; licensee BioMed Central Ltd

In vivo and in vitro assessment of pathways involved in contrast media-induced renal cells apoptosis.

Contrast-induced nephropathy accounts for 410% of all causes of hospital-acquired renal failure, causes a prolonged in- hospital stay and represents a powerful predictor of poor early and late outcome. Mechanisms of contrast-induced nephropathy are not completely understood. In vitro data suggests that contrast media (CM) induces a direct toxic effect on renal tubular cells through the activation of the intrinsic apoptotic pathway. It is unclear whether this effect has a role in the clinical setting. In this work, we evaluated the effects of CM both in vivo and in vitro. By analyzing urine samples obtained from patients who experienced contrast-induced acute kidney injury (CI-AKI), we verified, by western blot and immunohistochemistry, that CM induces tubular renal cells apoptosis. Furthermore, in cultured cells, CM caused a dose–response increase in reactive oxygen species (ROS) production, which triggered Jun N-terminal kinases (JNK1/2) and p38 stress kinases marked activation and thus apoptosis. Inhibition of JNK1/2 and p38 by different approaches (i.e. pharmacological antagonists and transfection of kinase- death mutants of the upstream p38 and JNK kinases) prevented CM-induced apoptosis. Interestingly, N-acetylcysteine inhibited ROS production, and thus stress kinases and apoptosis activation. Therefore, we conclude that CM-induced tubular renal cells apoptosis represents a key mechanism of CI-AKI

Evolution of dermatofibrosarcoma protuberans to DFSP-derived fibrosarcoma: An event marked by epithelial-mesenchymal transition-like process and 22q loss

Dermatofibrosarcoma protuberans (DFSP) is a rare and indolent cutaneous sarcoma. At times, a fibrosarcomatous transformation marked by a more aggressive clinical behavior may be present. We investigated the natural history and the molecular bases of progression from classic DFSP to the fibrosarcomatous form (FS-DFSP), looking, retrospectively, at the outcome of all patients affected by primary DFSP treated at our institution from 1993 to 2012 and analyzing the molecular profile of 5 DFSPs and 5 FS-DFSPs by an integrated genomics approach (whole transcriptome sequencing, copy number analysis, FISH, qRT-PCR, IHC). The presence of fibrosarcomatous features was identified in 20 (7.6%) patients out of 263 DFSP. All cases were treated with macroscopic complete surgery. A local relapse occurred in 4 of 23 patients who received a microscopic marginal surgery (2 classic DFSP, 2 FS-DFSP), while metastasis affected 2 patients, both FS-DFSP (10% of FS-DFSP), being the first event. DFSP evolution to FS-DFSP was paralleled by a transcriptional reprogramming. The recurrent loss of chromosome 22q appeared to contribute to this phenomenon by promoting the expression of epigenetic regulators, such as EZH2. Loss of the p16/CDKN2A/INK4A locus at 9p was also observed in two FS-DFSP metastatic cases. Implications: FS-DFSP is a rare subgroup among DFSP, with a 10% metastatic risk, that was independent from local recurrence and that was not observed in DFSP, that were all cured by wide surgery. Chromosome 22q deletion might play a role in FS-DFSP, and p16 loss may convey a poor outcome. EZH2 dysregulation was also found and represents a druggable target

A Pediatric Intra-Axial Malignant SMARCB1-Deficient Desmoplastic Tumor Arising in Meningioangiomatosis

SMARCB1 inactivation is a well-established trigger event in atypical teratoid/rhabdoid tumor. Recently, a role for SMARCB1 inactivation has emerged as a mechanism of clonal evolution in other tumor types, including rare brain tumors. We describe an unusual malignant intra-axial SMARCB1-deficient spindle cell desmoplastic neoplasm, occurring in a 6-year-old child with meningioangiomatosis and a long history of seizures. Striking features of the tumor were a storiform pattern and strong CD34 expression. Undifferentiated round cell areas with isolated rhabdoid cells showing high mitotic index and focal necrosis with INI1 expression loss were present. The meningioangiomatosis component showed few chromosomal imbalances, including chromosomal 22 monosomy (where SMARCB1 maps) and gain at 6q14.3. In addition to these abnormalities, the spindle cell desmoplastic neoplasm and its dedifferentiated SMARCB1-deficient component shared several other aberrations, including homozygous deletion at 9p21.3, losses at 1p, 3p, 3q, 10p, and 13q, gains and losses at 5p and 11p. In line with INI1 loss, the dedifferentiated component showed remarkably decreased levels of SMARCB1 transcript. The residual SMARCB1 allele was wildtype. Our findings suggest progression from the meningioangiomatosis to the malignant desmoplastic neoplasm through the occurrence of complex chromosomal abnormalities, and point to functional silencing of SMARCB1 in the dedifferentiation component

Analysis of MEF2D-HDAC4 repressive complexes in LMS cells.

<p>A) Quantitative analysis of the immunofluorescence studies. LMS cells were treated or not for 2 hours with 5ng/ml leptomycin B (LC Laboratories). After fixation of the cells, immunofluorescence analysis was performed to visualize HDAC4. Nuclei were stained with Hoechst 33342. Data are presented as mean ± SD (n = 3). B) MEF2D-HDAC4 complexes were immunoprecipitated using 1μg of anti-HDAC4, or anti-USP33, as a control, antibodies. Immunoblotting using an anti-MEF2D antibody was next used for the detection. The same amounts of cellular lysates were immunoprecipitated and the immunoblot were developed under the same circumstances. C) Chromatin was immunoprecipitated from SK-LMS-1 or SK-UT-1 cells using the anti-MEF2D and the anti-HDAC4 antibodies. Anti-FLAG antibody was used as control. <i>TK</i> promoter was used as negative control. The MEF2 binding site, the amplified region and the TSS are indicated for each tested gene, respectively with a vertical arrow, two arrowheads and a horizontal arrow. The TK promoter was used as negative control.</p

HDAC4 and HDAC9 KO in SK-UT-1 cells.

<p>A) Immunoblot analysis of HDAC4, HDAC9 and MEF2D in the indicated SK-UT-1 clones. Two different HDAC9 KO clones were selected. Actin was used as loading control. B) mRNA expression levels of the indicated atypical and classical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC9. Data are presented as mean ± SD; n = 3. C) Turkey box-plots illustrating the mRNA expression levels of classical and atypical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC9. Dunn's Multiple Comparison Test was applied to test the significance. D) Immunoblot analysis of HDAC4 and HDAC9 in the indicated SK-UT-1 clones. Two different HDAC4 KO clones generated by different sgRNAs were selected. Actin was used as loading control. E) mRNA expression levels of the indicated atypical and classical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC4. Data are presented as mean ± SD; n = 3. F) Turkey box-plots illustrating the mRNA expression levels of classical and atypical MEF2-target genes in SK-UT-1 cells WT or KO for HDAC4. Dunn's Multiple Comparison Test was applied to test the significance. G) Invasion properties of the SK-UT-1 cells WT, KO for HDAC4 or KO for HDAC9, as indicated. Data are presented as mean ± SD; n = 4. H) Example of growth in soft agar of SK-UT-1 cells WT or KO for HDAC9. Foci were stained with MTT. I) Quantitative results of colony formation assay for SK-UT-1 cells WT, KO for HDAC4 or KO for HDAC9, as indicated. Data are presented as mean ± SD; n ≥ 3. * p < 0.05, ** p < 0.01, *** p < 0.001</p