Antitumor Effects of PRMT5 Inhibition in Sarcomas.

Patients with advanced soft-tissue sarcomas (STS) have few therapeutic options. Protein arginine methyltransferase 5 (PRMT5), an anticancer target, has been extensively investigated in recent years in epithelial tumors. To date, no data related to the biological role of PRMT5 inhibition and its potential effect as a treatment in STS have been reported.To investigate the therapeutic potential of PRMT5 targeting in STS, we first evaluated the prognostic value of PRMT5 expression in two different cohorts of patients with STS. We then used the potent and selective GSK3326595 (GSK595) compound to investigate the antitumor effect of the pharmacologic inhibition of PRMT5 in vitro via MTT, apoptosis, cell cycle, clonogenicity, and proliferation assays. In vivo studies were performed with two animal models to evaluate the effects of GSK595 on tumor growth. The mechanisms of action were investigated by RNA sequencing, metabolic pathway analysis, Western blotting, and glucose uptake/lactate production assays.High PRMT5 gene expression levels were significantly associated with worsened metastasis-free survival of patients with STS. GSK595 decreased the global symmetric dimethylarginine level, the proliferation rate and clonogenicity of STS cell lines in vitro and tumor growth in vivo. Moreover, PRMT5 inhibition regulated aerobic glycolysis through downregulation of key enzymes of glycolysis as well as glucose uptake and lactate production.The current study demonstrated that PRMT5 regulates STS cell metabolism and thus represents a potential therapeutic target for STS. Additional studies in diverse sarcoma subtypes will be essential to confirm and expand upon these findings. SIGNIFICANCE: STSs have limited therapeutic options. We show here the poor prognostic value of high PRMT5 expression in STS. Moreover, we demonstrate that the pharmacologic inhibition of PRMT5 has significant antitumor activity through the downregulation of glycolysis. Our findings support the clinical investigation of PRMT5 inhibition in STSs.

High throughput profiling of undifferentiated pleomorphic sarcomas identifies two main subgroups with distinct immune profile, clinical outcome and sensitivity to targeted therapies.

BACKGROUND: Undifferentiated pleomorphic sarcoma (UPS) is the most frequent, aggressive and less-characterized sarcoma subtype. This study aims to assess UPS molecular characteristics and identify specific therapeutic targets. METHODS: High-throughput technologies encompassing immunohistochemistry, RNA-sequencing, whole exome-sequencing, mass spectrometry, as well as radiomics were used to characterize three independent cohorts of 110, 25 and 41 UPS selected after histological review performed by an expert pathologist. Correlations were made with clinical outcome. Cell lines and xenografts were derived from human samples for functional experiments. FINDINGS: CD8 positive cell density was independently associated with metastatic behavior and prognosis. RNA-sequencing identified two main groups: the group A, enriched in genes involved in development and stemness, including FGFR2, and the group B, strongly enriched in genes involved in immunity. Immune infiltrate patterns on tumor samples were highly predictive of gene expression classification, leading to call the group B 'immune-high' and the group A 'immune-low'. This molecular classification and its prognostic impact were confirmed on an independent cohort of UPS from TCGA. Copy numbers alterations were significantly more frequent in immune-low UPS. Proteomic analysis identified two main proteomic groups that highly correlated with the two main transcriptomic groups. A set of nine radiomic features from conventional MRI sequences provided the basis for a radiomics signature that could select immune-high UPS on their pre-therapeutic imaging. Finally, in vitro and in vivo anti-tumor activity of FGFR inhibitor JNJ-42756493 was selectively shown in cell lines and patient-derived xenograft models derived from immune-low UPS. INTERPRETATION: Two main disease entities of UPS, with distinct immune phenotypes, prognosis, molecular features and MRI textures, as well as differential sensitivity to specific anticancer agents were identified. Immune-high UPS may be the best candidates for immune checkpoint inhibitors, whereas this study provides rational for assessing FGFR inhibition in immune-low UPS. FUNDING: This work was partly founded by a grant from La Ligue.

MDM2 Antagonists Induce a Paradoxical Activation of Erk1/2 through a P53-Dependent Mechanism in Dedifferentiated Liposarcomas: Implications for Combinatorial Strategies.

The MDM2 gene is amplified in dedifferentiated liposarcoma (DDLPS). Treatment with MDM2 antagonists is a promising strategy to treat DDLPS; however, drug resistance is a major limitation when these drugs are used as a single agent. This study examined the impact of MDM2 antagonists on the mitogen-activated protein kinase (MAPK) pathway in DDLPS and investigated the potential synergistic activity of a MAPK kinase (MEK) inhibitor in combination with MDM2 antagonists. We identified a synergistic effect and identified the mechanism behind it. Combination effects of MDM2 antagonists and a MEK inhibitor were analyzed in a patient-derived xenograft mouse model and in DDLPS and leiomyosarcoma cell lines using different cell proliferation assays and immunoblot analysis. MDM2 antagonist (RG7388)-resistant IB115 [P4] cells and p53-silenced DDLPS cells were also established to understand the importance of functional p53. We found that MDM2 antagonists induced an upregulation of phosphorylated extracellular signal-regulated kinase (p-ERK) in DDLPS cells. The upregulation of p-ERK occurred due to mitochondrial translocation of p53, which resulted in increased production of reactive oxygen species, causing the activation of receptor tyrosine kinases (RTKs). Activated RTKs led to the activation of the downstream MEK/ERK signaling pathway. Treatment with a MEK inhibitor resulted in decreased expression of p-ERK, causing significant anti-tumor synergy when combined with MDM2 antagonists. Our results provide a framework for designing clinical studies of combination therapies in DDLPS patients.

ATR Inhibition Broadly Sensitizes Soft-Tissue Sarcoma Cells to Chemotherapy Independent of Alternative Lengthening Telomere (ALT) Status.

Only few drugs have shown activity in patients with advanced soft-tissue and the median overall survival is only 18 months. Alterations of genes involved in the DNA damage repair pathway have been associated with sarcoma risk and prognosis. ATR plays a crucial role in maintaining genomic integrity by responding to a large spectrum of DNA damage, including double strand breaks (DSBs) that interfere with replication. The objective of this study is to evaluate the pre-clinical activity of ATR inhibition in soft tissue sarcomas (STS). We explored the ability of the ATR inhibitor, VE-822, to prevent chemotherapy-induced intra-S-phase checkpoint activation and evaluated the antitumor potential of this combination in vitro and in vivo in STS cell lines and in a patient-derived xenograft model. The combination of VE-822 and gemcitabine in vitro was synergistic, inhibited cell proliferation, induced apoptosis, and accumulated in the S phase of the cell cycle with higher efficacy than either single agent alone. The combination also resulted in enhanced γH2AX intranuclear accumulation as a result of DNA damage induction. These effects were unrelated to the alternative lengthening of telomeres pathway. In vivo, the combination of VE-822 and gemcitabine significantly enhanced tumor growth inhibition and progression-free survival in an aggressive model of undifferentiated pleomorphic sarcoma. The combination of ATR inhibitor and chemotherapy is beneficial in pre-clinical models of soft-tissue sarcoma and deserves further exploration in the clinical setting.

Erratum to: Humanization of the mouse mammary gland by replacement of the luminal layer with genetically engineered preneoplastic human cells.

Due to a typesetting error, the labelling was changed and the figures in this article [1] were presented in the order 2, 4, 10, 6, 1, 3, 5, 7, 8, 9, 11, 12, 13, 14 and the supplementary figure links were inverted. The revised version has the figures in the correct order.

Humanization of the mouse mammary gland by replacement of the luminal layer with genetically engineered preneoplastic human cells.

INTRODUCTION: The cell of origin for estrogen receptor α-positive (ERα+) breast cancer is probably a luminal stem cell in the terminal duct lobular units. To model these cells, we have used the murine myoepithelial layer in the mouse mammary ducts as a scaffold upon which to build a human luminal layer. To prevent squamous metaplasia, a common artifact in genetically-engineered breast cancer models, we sought to limit activation of the epidermal growth factor receptor (EGFR) during in vitro cell culture before grafting the cells. METHODS: Human reduction mammoplasty cells were grown in vitro in WIT medium. Epidermal growth factor in the medium was replaced with amphiregulin and neuregulin to decrease activation of EGFR and increase activation of EGFR homologs 3 and 4 (ERBB3 and ERBB4). Lentiviral vectors were used to express oncogenic transgenes and fluorescent proteins. Human mammary epithelial cells were mixed with irradiated mouse fibroblasts and Matrigel, then injected through the nipple into the mammary ducts of immunodeficient mice. Engrafted cells were visualized by stereomicroscopy for fluorescent proteins and characterized by histology and immunohistochemistry. RESULTS: Growth of normal mammary epithelial cells in conditions favoring ERBB3/4 signaling prevented squamous metaplasia in vitro. Normal human cells were quickly lost after intraductal injection, but cells infected with lentiviruses expressing CCND1, MYC, TERT, BMI1 and a short-hairpin RNA targeting TP53 were able to engraft and progressively replace the luminal layer in the mouse mammary ducts, resulting in the formation of an extensive network of humanized ducts. Despite expressing multiple oncogenes, the human cells formed a morphologically normal luminal layer. Expression of a single additional oncogene, PIK3CA-H1047R, converted the cells into invasive cancer cells. The resulting tumors were ERα+, Ki67+ luminal B adenocarcinomas that were resistant to treatment with fulvestrant. CONCLUSIONS: Injection of preneoplastic human mammary epithelial cells into the mammary ducts of immunodeficient mice leads to replacement of the murine luminal layer with morphologically normal human cells. Genetic manipulation of the injected cells makes it possible to study defined steps in the transformation of human mammary epithelial cells in a more physiological environment than has hitherto been possible.

PLA2R1 mediates tumor suppression by activating JAK2.

Little is known about the physiological role of the phospholipase A2 receptor (PLA2R1). PLA2R1 has been described as regulating the replicative senescence, a telomerase-dependent proliferation arrest. The downstream PLA2R1 signaling and its role in cancer are currently unknown. Senescence induction in response to activated oncogenes is a failsafe program of tumor suppression that must be bypassed for tumorigenesis. We now present evidence that PLA2R1 functions in vitro as a tumor suppressor, the depletion of which is sufficient to escape oncogene-induced senescence (OIS), thereby facilitating oncogenic cell transformation. Furthermore, mice that are genetically deficient in PLA2R1 display increased sensitivity to RAS-induced tumorigenesis by facilitating OIS escape, highlighting its physiological role as a tumor suppressor. Unexpectedly, PLA2R1 activated JAK2 and its effector signaling, with PLA2R1-mediated inhibition of cell transformation largely reverted in JAK2-depleted cells. This finding was unexpected as the JAK2 pathway has been associated mainly with protumoral functions and several inhibitors are currently in clinical trials. Taken together, our findings uncover an unanticipated tumor suppressive role for PLA2R1 that is mediated by targeting downstream JAK2 effector signaling.

Extracellular cleavage of the p75 neurotrophin receptor is implicated in its pro-survival effect in breast cancer cells.

The p75 neurotrophin receptor (p75NTR) undergoes sequential proteolytic cleavages leading to the generation of a carboxyl-terminal fragment (p75NTR-CTF) and an intracellular domain (p75NTR-ICD) in many cellular models. We have previously shown that p75NTR is involved in the survival of breast cancer cells. Here, we demonstrated that p75NTR cleavage occurs also in these cells. Surprisingly, p75NTR-CTF increased cell survival, whereas p75NTR-ICD had no effect. The pro-survival effect of p75NTR-CTF was associated with a decrease of TNF-related apoptosis-inducing ligand (TRAIL)-induced PARP and caspase 3 cleavages. Finally, our findings indicate that p75NTR could favor cell survival via its carboxyl-terminal fragment, independently of PI3-kinase, NF-κB, or MAP kinase signaling pathways.

Potassium channel KCNA1 modulates oncogene-induced senescence and transformation.

Oncogene-induced senescence (OIS) constitutes a failsafe program that restricts tumor development. However, the mechanisms that link oncogenesis to senescence are not completely understood. We carried out a loss-of-function genetic screen that identified the potassium channel KCNA1 as a determinant of OIS escape that can license tumor growth. Oncogenic stress triggers an increase in KCNA1 expression and its relocation from the cytoplasm to the membrane. Mechanistically, this relocation is due to a loss of protein kinase A (PKA)-induced phosphorylation at residue S446 of KCNA1. Accordingly, sustaining PKA activity or expressing a KCNA1 phosphomimetic mutant maintained KCNA1 in the cytoplasm and caused escape from OIS. KCNA1 relocation to the membrane induced a change in membrane potential that invariably resulted in cellular senescence. Restoring KCNA1 expression in transformation-competent cells triggered variation in membrane potential and blocked RAS-induced transformation, and PKA activation suppressed both effects. Furthermore, KCNA1 expression was reduced in human cancers, and this decrease correlated with an increase in breast cancer aggressiveness. Taken together, our results identify a novel pathway that restricts oncogenesis through a potassium channel-dependent senescence pathway.

Brain-derived neurotrophic factor and neurotrophin-4/5 are expressed in breast cancer and can be targeted to inhibit tumor cell survival.

PURPOSE: Given that nerve growth factor has previously been shown to be involved in breast cancer progression, we have tested here the hypothesis that the other neurotrophins (NT) are expressed and have an influence in breast tumor growth. EXPERIMENTAL DESIGN: The expression of brain-derived neurotrophic factor (BDNF), NT-3 and NT-4/5, as well as the neurotrophin receptor p75(NTR), TrkB, and TrkC, was studied by RT-PCR, Western blotting, and immunohistochemistry in cell lines and tumor biopsies. The biological impacts of neurotrophins, and associated mechanisms, were analyzed in cell cultures and xenografted mice. RESULTS: BDNF and NT-4/5 were expressed and secreted by breast cancer cells, and the use of blocking antibodies suggested an autocrine loop mediating cell resistance to apoptosis. The corresponding tyrosine kinase receptor TrkB was only rarely observed at full length, whereas the expression of TrkB-T1, lacking the kinase domain, as well as p75(NTR), were detected in all tested breast cancer cell lines and tumor biopsies. In contrast, NT-3 and TrkC were not detected. SiRNA against p75(NTR) and TrkB-T1 abolished the antiapoptotic effect of BDNF and NT-4/5, whereas the pharmacological inhibitors K252a and PD98059 had no effect, suggesting the involvement of p75(NTR) and TrkB-T1, but not kinase activities from Trks and MAPK. In xenografted mice, anti-BDNF, anti-NT-4/5, anti-p75(NTR), or anti-TrkB-T1 treatments resulted in tumor growth inhibition, characterized by an increase in cell apoptosis, but with no change in proliferation. CONCLUSION: BDNF and NT-4/5 contribute to breast cancer cell survival and can serve as prospective targets in attempts to inhibit tumor growth.

Overexpression of p75(NTR) increases survival of breast cancer cells through p21(waf1).

The p75 neurotrophin receptor (p75(NTR)) plays a critical role in various neuronal and non-neuronal cell types by regulating cell survival, differentiation and proliferation. To evaluate the influence of p75(NTR) in breast cancer development, we have established and characterized breast cancer cells which stably overexpress p75(NTR). We showed that p75(NTR) overexpression per se promoted cell survival to apoptogens with a concomitant slowdown of cell growth. The pro-survival effect is associated with an increased expression of the inhibitor of apoptosis protein-1 (c-IAP1), a decrease of TRAIL-induced cleavage of PARP, procaspase 9 and procaspase 3, and a decrease of cytochrome C release from the mitochondria. The anti-proliferative effect is due to a cell accumulation in G0/G1, associated with a decrease of Rb phosphorylation and an increase of p21(waf1). Interestingly, inhibition of p21(waf1) with siRNA not only restores proliferation but also abolishes the pro-survival effect of p75(NTR), indicating the key role of p21(waf1) in the biological functions of p75(NTR). Finally, using a SCID mice xenograft model, we showed that p75(NTR) overexpression favors tumor growth and strongly increases tumor resistance to anti-tumoral treatment. Together, our findings suggest that p75(NTR) overexpression in breast tumor cells could favor tumor survival and contribute to tumor resistance to drugs. This provides a rationale to consider p75(NTR) as a potential target for the future design of innovative therapeutic strategies.