No abstract

ΔNp63 has been recently involved in self-renewal potential of breast cancer stem cells. Although the p63 transcriptional profile has been extensively characterized, our knowledge of the p63-binding partners potentially involved in the regulation of breast tumour progression is limited. Here, we performed the yeast two hybrid approach to identify p63α interactors involved in breast tumorigenesis and we found that SETDB1, a histone lysine methyl transferases, interacts with ΔNp63α and that this interaction contributes to p63 protein stability. SETDB1 is often amplified in primary breast tumours, and its depletion confers to breast cancer cells growth disadvantage. We identified a list of thirty genes repressed by ΔNp63 in a SETDB1-dependent manner, whose expression is positively correlated to survival of breast cancer patients. These results suggest that p63 and SETDB1 expression, together with the repressed genes, may have diagnostic and prognostic potential.

Fused in sarcoma: DNA damage‐inducible transcript 3 protein ( FUS : DDIT 3) is a chimeric fusion oncoprotein present in 90% of human myxoid liposarcomas ( MLS ). The study by Trautmann et al in this issue of EMBO Molecular Medicine utilizes a drop‐out RNA i screen to establish hyperactive Yes‐associated protein 1 ( YAP 1), a major downstream nuclear effector of the Hippo signaling pathway, as a selectively essential transcript promoting viability and growth of MLS . These observations add to a growing body of evidence underscoring the importance of dysregulation of Hippo signaling in soft‐tissue sarcomas expressing fusion oncoproteins and identify a novel target for therapeutic intervention in MLS . Comprehensive molecular characterization pipelines are needed to screen patients with advanced soft‐tissue sarcomas for the presence of druggable alterations, including but not limited to nuclear YAP 1 expression in MLS , to facilitate treatment decisions and advance therapy.

No abstract

Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Both primary human RMS cultures and low-passage Myf6Cre,Pax3:Foxo1,p53 mouse RMS cell lines, which express the fusion oncoprotein Pax3:Foxo1 and lack the tumor suppressor Tp53 (Myf6Cre,Pax3:Foxo1,p53), exhibit marked heterogeneity in PAX3:FOXO1 (P3F) expression at the single cell level. In mouse RMS cells, P3F expression is directed by the Pax3 promoter and coupled to eYFP. YFPlow/P3Flow mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFPlow/P3Flow compared with YFPhigh/P3Fhigh cells. Both YFPlow/P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFPlow/P3Flow RMS cells. Heterogeneous expression of PAX3:FOXO1 at the single cell level may provide a critical advantage during tumor progression.

Amino acids are integral components of cancer metabolism. The non-essential amino acid asparagine supports the growth and survival of various cancer cell types. Here, different mass spectrometry approaches were employed to identify lower aspartate levels, higher aspartate/glutamine ratios and lower tricarboxylic acid (TCA) cycle metabolite levels in asparagine-deprived sarcoma cells. Reduced nicotinamide adenine dinucleotide (NAD+)/nicotinamide adenine dinucleotide hydride (NADH) ratios were consistent with redirection of TCA cycle flux and relative electron acceptor deficiency. Elevated lactate/pyruvate ratios may be due to compensatory NAD+ regeneration through increased pyruvate to lactate conversion by lactate dehydrogenase. Supplementation with exogenous pyruvate, which serves as an electron acceptor, restored aspartate levels, NAD+/NADH ratios, lactate/pyruvate ratios and cell growth in asparagine-deprived cells. Chemicals disrupting NAD+ regeneration in the electron transport chain further enhanced the anti-proliferative and pro-apoptotic effects of asparagine depletion. We speculate that reductive stress may be a major contributor to the growth arrest observed in asparagine-starved cells.

Functional genomic screening of KRAS-driven mouse sarcomas was previously employed to identify proliferation-relevant genes. Genes identified included Ubiquitin-conjugating enzyme E2 (Ube2c), Centromere Protein E (Cenpe), Hyaluronan Synthase 2 (Has2), and CAMP Responsive Element Binding Protein 3 Like 2 (Creb3l2). This study examines the expression and chemical inhibition of these candidate genes, identifying variable levels of protein expression and significant contributions to rhabdomyosarcoma (RMS) cell proliferation. Chemical treatment of human and murine RMS cell lines with bortezomib, UA62784, latrunculin A and sorafenib inhibited growth with approximate EC50 concentrations of 15-30nM for bortezomib, 25-80nM for UA62784 and 80-220nM for latrunculin A. The multikinase inhibitor sorafenib increased in vitro proliferation of 4 of 6 sarcoma cell lines tested. Latrunculin A was further associated with disruption of the actin cytoskeleton and reduced ERK1/2 phosphorylation. Together, this work advances opportunities for developing therapies to block progression of soft-tissue sarcomas and demonstrates that disruption of the actin cytoskeleton in sarcoma cells by latrunculin A is associated with a reduction in RMS cell growth. (167 words).

Background: Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Expression of the fusion oncogene PAX3:FOXO1 (P3F) was previously shown to differ between individual tumor cells and fluctuate over time. Methods: In mouse Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- RMS tumors, expression of P3F is directed by the Pax3 promoter and coupled to an eYFP fluorescent marker, which is activated as a second cistron downstream from an encephalomyocarditis virus-derived internal ribosome entry site. Low passage Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- mouse RMS cell lines and primary human RMS cultures were used to study the functional impact of variable P3F expression at the cellular level in RMS. Results: The Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- mouse RMS cell pool contains cells expressing different levels of YFP, correlating with variable P3F expression. Single-cell PCR was used to demonstrate substantial cell-to-cell variability in P3F expression in the human RMS cell pool. Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- mouse RMS cells were then sub-fractionated by fluorescence-activated cell sorting (FACS) to discriminate YFPhigh/P3Fhigh and YFPlow/P3Flow cell subsets. YFPlow/P3Flow mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFPlow/P3Flow compared to YFPhigh/P3Fhigh cells after injection into the extremity muscles of immunocompromised mice. We also observed higher clonal activity of YFPlow/P3Flow compared to YFPhigh/P3Fhigh cells in vitro. Both YFPlow/P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Finally, exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFPlow/P3Flow RMS cells. Conclusions: Our observations indicate that therapies aimed at eliminating P3Fhigh cells by targeting the fusion oncogene may not cure the disease. Moreover, dynamic expression of PAX3:FOXO1 at the single cell level may result in adaptive plasticity, allow RMS cells to adapt to environmental challenges and provide them with a critical advantage during tumor progression. Citation Format: Carla Regina, Geoffroy Andriuex, Sina Angenendt, Michaela Schneider, Manching Ku, Marie Follo, Marco Wachtel, Eugene Ke, Ken Kikuchi, Anton G. Henssen, Beat W. Schäfer, Melanie Boerries, Amy J. Wagers, Charles Keller, Simone Hettmer. Negative correlation of single-cell PAX3:FOXO1 expression with tumorigenicity in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3122.