Exosome-Mediated Paracrine Signaling Unveils miR-1246 as a Driver of Aggressiveness in Fusion-Negative Rhabdomyosarcoma.

Rhabdomyosarcoma is a pediatric cancer associated with aggressiveness and a tendency to develop metastases. Fusion-negative rhabdomyosarcoma (FN-RMS) is the most commonly occurring subtype of RMS, where metastatic disease can hinder treatment success and decrease survival rates. RMS-derived exosomes were previously demonstrated to be enriched with miRNAs, including miR-1246, possibly contributing to disease aggressiveness. We aimed to decipher the functional impact of exosomal miR-1246 on recipient cells and its role in promoting aggressiveness. Treatment of normal fibroblasts with FN-RMS-derived exosomes resulted in a significant uptake of miR-1246 paired with an increase in cell proliferation, migration, and invasion. In turn, delivery of miR-1246-mimic lipoplexes promoted fibroblast proliferation, migration, and invasion in a similar manner. Conversely, when silencing miR-1246 in FN-RMS cells, the resulting derived exosomes demonstrated reversed effects on recipient cells' phenotype. Delivery of exosomal miR-1246 targets GSK3ß and promotes ß-catenin nuclear accumulation, suggesting a deregulation of the Wnt pathway, known to be important in tumor progression. Finally, a pilot clinical study highlighted, for the first time, the presence of high exosomal miR-1246 levels in RMS patients' sera. Altogether, our results demonstrate that exosomal miR-1246 has the potential to alter the tumor microenvironment of FN-RMS cells, suggesting its potential role in promoting oncogenesis.

The Intricate Interplay between the ZNF217 Oncogene and Epigenetic Processes Shapes Tumor Progression.

The oncogenic transcription factor ZNF217 orchestrates several molecular signaling networks to reprogram integrated circuits governing hallmark capabilities within cancer cells. High levels of ZNF217 expression provide advantages to a specific subset of cancer cells to reprogram tumor progression, drug resistance and cancer cell plasticity. ZNF217 expression level, thus, provides a powerful biomarker of poor prognosis and a predictive biomarker for anticancer therapies. Cancer epigenetic mechanisms are well known to support the acquisition of hallmark characteristics during oncogenesis. However, the complex interactions between ZNF217 and epigenetic processes have been poorly appreciated. Deregulated DNA methylation status at ZNF217 locus or an intricate cross-talk between ZNF217 and noncoding RNA networks could explain aberrant ZNF217 expression levels in a cancer cell context. On the other hand, the ZNF217 protein controls gene expression signatures and molecular signaling for tumor progression by tuning DNA methylation status at key promoters by interfering with noncoding RNAs or by refining the epitranscriptome. Altogether, this review focuses on the recent advances in the understanding of ZNF217 collaboration with epigenetics processes to orchestrate oncogenesis. We also discuss the exciting burgeoning translational medicine and candidate therapeutic strategies emerging from those recent findings connecting ZNF217 to epigenetic deregulation in cancer.

Non-coding RNA in rhabdomyosarcoma progression and metastasis.

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma of skeletal muscle differentiation, with a predominant occurrence in children and adolescents. One of the major challenges facing treatment success is the presence of metastatic disease at the time of diagnosis, commonly associated with the more aggressive fusion-positive subtype. Non-coding RNA (ncRNA) can regulate gene transcription and translation, and their dysregulation has been associated with cancer development and progression. MicroRNA (miRNA) are short non-coding nucleic acid sequences involved in the regulation of gene expression that act by targeting messenger RNA (mRNA), and their aberrant expression has been associated with both RMS initiation and progression. Other ncRNA including long non-coding RNA (lncRNA), circular RNA (circRNA) and ribosomal RNA (rRNA) have also been associated with RMS revealing important mechanistic roles in RMS biology, but these studies are still limited and require further investigation. In this review, we discuss the established roles of ncRNA in RMS differentiation, growth and progression, highlighting their potential use in RMS prognosis, as therapeutic agents or as targets of treatment.

Staple Line Intrathoracic Migration After Sleeve Gastrectomy: Correlation between Symptoms, CT Three-Dimensional Stomach Analysis, and 24-h pH Monitoring.

PURPOSE: Migration of the staple line is the definition of sliding hiatus hernia in sleeve gastrectomy patients. The main aim was to determine the frequency and measurement of intrathoracic staple line migration and its correlation with GERD symptoms and pH monitoring. MATERIALS AND METHODS: This was a prospective clinical trial including all patients who underwent sleeve gastrectomy more than 1 year previously. All the patients underwent computed tomography (CT) imaging, and migration of the proximal end of the suture above the level of the hiatus was measured in mm. All the patients with symptoms suggestive of GERD were assessed using the GERD impact scale (GIS), and wireless 24-h esophageal pH and symptom association monitoring (SAP) were carried out. Analysis of risk factors for postoperative staple line migration was performed. RESULTS: Between March 2018 and December 2018, 194 patients were evaluated (mean age 45.1 ± 11.2 years; 161 females); 88/194 (45.4%) presented an average intrathoracic migration of 16.2 ± 6.9 mm. Thirty-eight of 194 (19.5%) patients presented symptoms suggestive of gastroesophageal reflux. There was a significant relationship between staple line intrathoracic migration and postsleeve GERD symptomatology (p = 0.0004, OR = 4.25 [1.92-9.39]). However, there was no significant correlation between positive 24-h pH monitoring and intrathoracic migration of the staple line (p = 0.1). CONCLUSION: A migration greater than 17 mm was strongly correlated with postsleeve GERD symptoms but not with positive 24-h pH monitoring.

Exploring the Significance of the Exon 4-Skipping Isoform of the ZNF217 Oncogene in Breast Cancer.

Oncogene alternative splicing events can create distinct functional transcripts that offer new candidate prognostic biomarkers for breast cancer. ZNF217 is a well-established oncogene but its exon 4-skipping isoform (ZNF217-ΔE4) has never been investigated in terms of clinical or biological relevance. Using in silico RNA-seq and RT-qPCR analyses, we demonstrated for the first time the existence of ZNF217-ΔE4 transcripts in primary breast tumors, and a positive correlation between ZNF217-ΔE4 mRNA levels and those of the wild-type oncogene (ZNF217-WT). A pilot retrospective analysis revealed that, in the Luminal subclass, the combination of the two ZNF217 variants (the ZNF217-ΔE4-WT gene-expression signature) provided more information than the mRNA expression levels of each isoform alone. Ectopic overexpression of ZNF217-ΔE4 in breast cancer cells promoted an aggressive phenotype and an increase in ZNF217-WT expression levels that was inversely correlated with DNA methylation of the ZNF217 gene. This study provides new insights into the possible role of the ZNF217-ΔE4 splice variant in breast cancer and suggests a close interplay between the ZNF217-WT and ZNF217-ΔE4 isoforms. Our data suggest that a dual signature combining the expression levels of these two isoforms may serve as a novel prognostic biomarker allowing better stratification of breast cancers with good prognosis and aiding clinicians in therapeutic decisions.

Low phospholipid-associated cholelithiasis syndrome: A rare cause of acute pancreatitis that should not be neglected.

BACKGROUND: Low phospholipid-associated cholelithiasis (LPAC) syndrome is a very particular form of biliary lithiasis with no excess of cholesterol secretion into bile, but a decrease in phosphatidylcholine secretion, which is responsible for stones forming not only in the gallbladder, but also in the liver. LPAC syndrome may be underreported due to a lack of testing resulting from insufficient awareness among clinicians. AIM: To describe the clinical and radiological characteristics of patients with LPAC syndrome to better identify and diagnose the disease. METHODS: We prospectively evaluated all patients aged over 18 years old who were consulted or hospitalized in two hospitals in Paris, France (Bichat University Hospital and Croix-Saint-Simon Hospital) between January 1, 2017 and August 31, 2018. All patients whose profiles led to a clinical suspicion of LPAC syndrome underwent a liver ultrasound examination performed by an experienced radiologist to confirm the diagnosis of LPAC syndrome. Twenty-four patients were selected. Data about the patients' general characteristics, their medical history, their symptoms, and their blood tests results were collected during both their initial hospitalization and follow-up. Cytolysis and cholestasis were expressed compared to the normal values (N) of serum aspartate and alanine transaminase activities, and to the normal value of alkaline phosphatase level, respectively. The subjects were systematically reevaluated and asked about their symptoms 6 mo after inclusion in the study through an in-person medical appointment or phone call. Genetic testing was not performed systematically, but according to the decision of each physician. RESULTS: Most patients were young (median age of 37 years), male (58%), and not overweight (median body mass index was 24). Many had a personal history of acute pancreatitis (54%) or cholecystectomy (42%), and a family history of gallstones in first-degree relatives (30%). LPAC syndrome was identified primarily in patients with recurring biliary pain (88%) or after a new episode of acute pancreatitis (38%). When present, cytolysis and cholestasis were not severe (2.8N and 1.7N, respectively) and disappeared quickly. Interestingly, four patients from the same family were diagnosed with LPAC syndrome. At ultrasound examination, the most frequent findings in intrahepatic bile ducts were comet-tail artifacts (96%), microlithiasis (83%), and acoustic shadows (71%). Computed tomography scans and magnetic resonance imaging were performed on 15 and three patients, respectively, but microlithiasis was not detected. Complications of LPAC syndrome required hospitalizing 18 patients (75%) in a conventional care unit for a mean duration of 6.8 d. None of them died. Treatment with ursodeoxycholic acid (UDCA) was effective and well-tolerated in almost all patients (94%) with a rapid onset of action (3.4 wk). Twelve patients' (67%) adherence to UDCA treatment was considered "good." Five patients (36%) underwent cholecystectomy (three of them were treated both by UDCA and cholecystectomy). Despite UDCA efficacy, biliary pain recurred in five patients (28%), three of whom adhered well to treatment guidelines. CONCLUSION: LPAC syndrome is easy to diagnose and treat; therefore, it should no longer be overlooked. To increase its detection rate, all patients who experience recurrent biliary symptoms following an episode of acute pancreatitis should undergo an ultrasound examination performed by a radiologist with knowledge of the disease.

Long-Term Exposure of Early-Transformed Human Mammary Cells to Low Doses of Benzo[a]pyrene and/or Bisphenol A Enhances Their Cancerous Phenotype via an AhR/GPR30 Interplay.

It is of utmost importance to decipher the role of chronic exposure to low doses of environmental carcinogens on breast cancer progression. The early-transformed triple-negative human mammary MCF10AT1 cells were chronically (60 days) exposed to low doses (10-10 M) of Benzo[a]pyrene (B[a]P), a genotoxic agent, and/or Bisphenol A (BPA), an endocrine disruptor. Our study revealed that exposed MCF10AT1 cells developed, in a time-dependent manner, an acquired phenotype characterized by an increase in cancerous properties (anchorage independent growth and stem-like phenotype). Co-exposure of MCF10AT1 cells to B[a]P and BPA led to a significantly greater aggressive phenotype compared to B[a]P or BPA alone. This study provided new insights into the existence of a functional interplay between the aryl hydrocarbon receptor (AhR) and the G protein-coupled receptor 30 (GPR30) by which chronic and low-dose exposure of B[a]P and/or BPA fosters the progression of MCF10AT1 cells into a more aggressive substage. Experiments using AhR or GPR30 antagonists, siRNA strategies, and RNAseq analysis led us to propose a model in which AhR signaling plays a "driver role" in the AhR/GPR30 cross-talk in mediating long-term and low-dose exposure of B[a]P and/or BPA. Retrospective analysis of two independent breast cancer cohorts revealed that the AhR/GPR30 mRNA expression signature resulted in poor breast cancer prognosis, in particular in the ER-negative and the triple-negative subtypes. Finally, the study identified targeting AhR and/or GPR30 with specific antagonists as a strategy capable of inhibiting carcinogenesis associated with chronic exposure to low doses of B[a]P and BPA in MCF10AT1 cells. Altogether, our results indicate that the engagement of both AhR and GPR30 functions, in particular in an ER-negative/triple-negative context of breast cells, favors tumor progression and leads to poor prognosis.

The Bone Morphogenetic Protein Signaling Inhibitor LDN-193189 Enhances Metastasis Development in Mice.

Breast cancer with bone metastasis is essentially incurable with current anticancer therapies. The bone morphogenetic protein (BMP) pathway is an attractive therapeutic candidate, as it is involved in the bone turnover and in cancer cell formation and their colonization of distant organs such as the bone. We previously reported that in breast cancer cells, the ZNF217 oncogene drives BMP pathway activation, increases the metastatic growth rate in the bone, and accelerates the development of severe osteolytic lesions in mice. In the present study, we aimed at investigating the impact of the LDN-193189 compound, a potent inhibitor of the BMP type I receptor, on metastasis development in vivo. ZNF217-revLuc cells were injected into the left ventricle of nude mice (n = 16) while control mice (n = 13) were inoculated with control pcDNA6-revLuc cells. Mice from each group were treated or not with LDN-193189 for 35 days. We found that systemic LDN-193189 treatment of mice significantly enhanced metastasis development, by increasing both the number and the size of metastases. In pcDNA6-revLuc-injected mice, LDN-193189 also affected the kinetics of metastasis emergence. Altogether, these data suggest that in vivo, LDN-193189 might affect the interaction between breast cancer cells and the bone environment, favoring the emergence and development of multiple metastases. Hence, our report highlights the importance of the choice of drugs and therapeutic strategies used in the management of bone metastases.

Oestrogen Non-Genomic Signalling is Activated in Tamoxifen-Resistant Breast Cancer.

Endocrine therapies targeting oestrogen signalling have significantly improved breast cancer management. However, their efficacy is limited by intrinsic and acquired resistance to treatment, which remains a major challenge for oestrogen receptor α (ERα)-positive tumours. Though many studies using in vitro models of endocrine resistance have identified putative actors of resistance, no consensus has been reached. We demonstrated previously that oestrogen non-genomic signalling, characterized by the formation of the ERα/Src/PI3K complex, is activated in aggressive breast cancers (BC). We wondered herein whether the activation of this pathway is also involved in resistance to endocrine therapies. We studied the interactions between ERα and Src or PI3K by proximity ligation assay (PLA) in in-vitro and in-vivo endocrine therapy-resistant breast cancer models. We reveal an increase in ERα/Src and ERα/PI3K interactions in patient-derived xenografts (PDXs) with acquired resistance to tamoxifen, as well as in tamoxifen-resistant MCF-7 cells compared to parental counterparts. Moreover, no interactions were observed in breast cancer cells resistant to other endocrine therapies. Finally, the use of a peptide inhibiting the ERα-Src interaction partially restored tamoxifen sensitivity in resistant cells, suggesting that such components could constitute promising targets to circumvent resistance to tamoxifen in BC.

The ZNF217 Biomarker Predicts Low- and High-Risk Oncotype DX® Recurrence Score in ER-Positive Invasive Breast Cancers.

We assessed mRNA and protein expression levels of the ZN217 oncogene in 17 clinical FFPE ER-positive invasive breast cancer specimens with known (low or high) Oncotype DX® Recurrence Scores. This study shows that mRNA or nuclear protein levels of the ZNF217 significantly correlate with Oncotype DX® Recurrence Score.

Evaluating ZNF217 mRNA Expression Levels as a Predictor of Response to Endocrine Therapy in ER+ Breast Cancer.

ZNF217 is a candidate oncogene with a wide variety of deleterious functions in breast cancer. Here, we aimed at investigating in a pilot prospective study the association between ZNF217 mRNA expression levels and the clinical response to neoadjuvant endocrine therapy (ET) in postmenopausal ER-positive (ER+) breast cancer patients. Core surgical biopsy samples before treatment initiation and post-treatment were obtained from 68 patients, and Ki-67 values measured by immunohistochemistry (IHC) were used to identify responders (n = 59) and non-responders (n = 9) after 4 months of ET. We report for the first time that high ZNF217 mRNA expression level measured by RT-qPCR in the initial tumor samples (pre-treatment) is associated with poor response to neoadjuvant ET. Indeed, the clinical positive response rate in patients with low ZNF217 expression levels was significantly higher than that in those with high ZNF217 expression levels (P = 0.027). Additionally, a retrospective analysis evaluating ZNF217 expression levels in primary breast tumor of ER+/HER2-/LN0 breast cancer patients treated with adjuvant ET enabled the identification of poorer responders prone to earlier relapse (P = 0.013), while ZNF217 did not retain any prognostic value in the ER+/HER2-/LN0 breast cancer patients who did not receive any treatment. Altogether, these data suggest that ZNF217 expression might be predictive of clinical response to ET.

The critical role of the ZNF217 oncogene in promoting breast cancer metastasis to the bone.

Bone metastasis affects >70% of patients with advanced breast cancer. However, the molecular mechanisms underlying this process remain unclear. On the basis of analysis of clinical datasets, and in vitro and in vivo experiments, we report that the ZNF217 oncogene is a crucial mediator and indicator of bone metastasis. Patients with high ZNF217 mRNA expression levels in primary breast tumours had a higher risk of developing bone metastases. MDA-MB-231 breast cancer cells stably transfected with ZNF217 (MDA-MB-231-ZNF217) showed the dysregulated expression of a set of genes with bone-homing and metastasis characteristics, which overlapped with two previously described 'osteolytic bone metastasis' gene signatures, while also highlighting the bone morphogenetic protein (BMP) pathway. The latter was activated in MDA-MB-231-ZNF217 cells, and its silencing by inhibitors (Noggin and LDN-193189) was sufficient to rescue ZNF217-dependent cell migration, invasion or chemotaxis towards the bone environment. Finally, by using non-invasive multimodal in vivo imaging, we found that ZNF217 increases the metastatic growth rate in the bone and accelerates the development of severe osteolytic lesions. Altogether, the findings of this study highlight ZNF217 as an indicator of the emergence of breast cancer bone metastasis; future therapies targeting ZNF217 and/or the BMP signalling pathway may be beneficial by preventing the development of bone metastases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Contribution of Computed Tomographic Imaging to the Management of Acute Abdominal Pain after Gastric Bypass: Correlation Between Radiological and Surgical Findings.

BACKGROUND: We evaluated the diagnostic accuracy of first interpretations of computed tomographic (CT) images and blind interpretation using predefined CT signs in patients with previous Roux-en-Y gastric bypass (RYGBP) and acute abdominal pain. METHODS: We performed a retrospective chart review of patients with RYGBP who underwent surgical exploration from January 2009 to December 2014 for acute abdominal pain in our university institution, excluding patients without CT scan and comparing initial CT imaging interpretation with surgical findings. Two blinded radiologist specialists in bariatric imaging evaluated the CT images for seven previously reported CT signs. We then calculated the sensitivity and specificity of these signs and Cohen's kappa inter-observer agreement for diagnosing internal hernia. RESULTS: Sixty-four patients had a recorded CT scan. The original CT interpretation showed that 26/64 (40%) patients had an accurate diagnosis. Cohen's kappa coefficient for concordance between surgical exploration and first interpretation was 0.26. The image review showed an accurate diagnosis was obtained in 51/64 patients (79.6%) and 48/64 (75%) patients for the first and second reader, respectively (Cohen's kappa coefficient = 0.67; 95% confidence interval = 0.52-0.76). The most prevalent sign indicating internal hernia was whirling of the mesentery (sensitivity = 82-91%; specificity = 79-93.1%). CONCLUSIONS: CT is an important diagnostic tool for skilled readers for managing acute abdominal pain in patients with previous RYGBP. Experience in the abdominal and bariatric imaging and the use of predetermined CT image signs provided a high degree of accuracy and confidence. A low threshold for surgical exploration remains the gold standard of appropriate treatment.

Long-term exposure to bisphenol A or benzo(a)pyrene alters the fate of human mammary epithelial stem cells in response to BMP2 and BMP4, by pre-activating BMP signaling.

Bone morphogenetic protein 2 (BMP2) and BMP4 are key regulators of the fate and differentiation of human mammary epithelial stem cells (SCs), as well as of their niches, and are involved in breast cancer development. We established that MCF10A immature mammary epithelial cells reliably reproduce the BMP response that we previously identified in human primary epithelial SCs. In this model, we observed that BMP2 promotes luminal progenitor commitment and expansion, whereas BMP4 prevents lineage differentiation. Environmental pollutants are known to promote cancer development, possibly by providing cells with stem-like features and by modifying their niches. Bisphenols, in particular, were shown to increase the risk of developing breast cancer. Here, we demonstrate that chronic exposure to low doses of bisphenol A (BPA) or benzo(a)pyrene (B(a)P) alone has little effect on SCs properties of MCF10A cells. Conversely, we show that this exposure affects the response of immature epithelial cells to BMP2 and BMP4. Furthermore, the modifications triggered in MCF10A cells on exposure to pollutants appeared to be predominantly mediated by altering the expression and localization of type-1 receptors and by pre-activating BMP signaling, through the phosphorylation of small mothers against decapentaplegic 1/5/8 (SMAD1/5/8). By analyzing stem and progenitor properties, we reveal that BPA prevents the maintenance of SC features prompted by BMP4, whereas promoting cell differentiation towards a myoepithelial phenotype. Inversely, B(a)P prevents BMP2-mediated luminal progenitor commitment and expansion, leading to the retention of stem-like properties. Overall, our data indicate that BPA and B(a)P distinctly alter the fate and differentiation potential of mammary epithelial SCs by modulating BMP signaling.

LIM Kinase Inhibitor Pyr1 Reduces the Growth and Metastatic Load of Breast Cancers.

LIM kinases (LIMK) are emerging targets for cancer therapy, and they function as network hubs to coordinate actin and microtubule dynamics. When LIMKs are inhibited, actin microfilaments are disorganized and microtubules are stabilized. Owing to their stabilizing effect on microtubules, LIMK inhibitors may provide a therapeutic strategy to treat taxane-resistant cancers. In this study, we investigated the effect of LIMK inhibition on breast tumor development and on paclitaxel-resistant tumors, using a novel selective LIMK inhibitor termed Pyr1. Treatment of breast cancer cells, including paclitaxel-resistant cells, blocked their invasion and proliferation in vitro and their growth in vivo in tumor xenograft assays. The tumor-invasive properties of Pyr1 were investigated in vivo by intravital microscopy of tumor xenografts. A striking change of cell morphology was observed with a rounded phenotype arising in a subpopulation of cells, while other cells remained elongated. Notably, although Pyr1 decreased the motility of elongated cells, it increased the motility of rounded cells in the tumor. Pyr1 administration prevented the growth of metastasis but not their spread. Overall, our results provided a preclinical proof of concept concerning how a small-molecule inhibitor of LIMK may offer a strategy to treat taxane-resistant breast tumors and metastases. Cancer Res; 76(12); 3541-52. ©2016 AACR.

The dark side of ZNF217, a key regulator of tumorigenesis with powerful biomarker value.

The recently described oncogene ZNF217 belongs to a chromosomal region that is frequently amplified in human cancers. Recent findings have revealed that alternative mechanisms such as epigenetic regulation also govern the expression of the encoded ZNF217 protein. Newly discovered molecular functions of ZNF217 indicate that it orchestrates complex intracellular circuits as a new key regulator of tumorigenesis. In this review, we focus on recent research on ZNF217-driven molecular functions in human cancers, revisiting major hallmarks of cancer and highlighting the downstream molecular targets and signaling pathways of ZNF217. We also discuss the exciting translational medicine investigating ZNF217 expression levels as a new powerful biomarker, and ZNF217 as a candidate target for future anti-cancer therapies.