scholarly journals Cancer of unknown primary stem-like cells model multi-organ metastasis and unveil liability to MEK inhibition

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Federica Verginelli ◽  
Alberto Pisacane ◽  
Gennaro Gambardella ◽  
Antonio D’Ambrosio ◽  
Ermes Candiello ◽  
...  
Keyword(s):  
Unknown Primary ◽  
Fatal Disease ◽  
Mek Inhibition ◽  
Organ Metastasis ◽  
Transcriptomic Signature ◽  
Primary Stem ◽  
Shed Light ◽  
Serial Transplantation

AbstractCancers of unknown primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, are a biological enigma and a fatal disease. We propose that CUPs are a distinct, yet unrecognized, pathological entity originating from stem-like cells endowed with peculiar and shared properties. These cells can be isolated in vitro (agnospheres) and propagated in vivo by serial transplantation, displaying high tumorigenicity. After subcutaneous engraftment, agnospheres recapitulate the CUP phenotype, by spontaneously and quickly disseminating, and forming widespread established metastases. Regardless of different genetic backgrounds, agnospheres invariably display cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis, which confers sensitivity to MEK inhibitors in vitro and in vivo. Such sensitivity is associated with a transcriptomic signature predicting that more than 70% of CUP patients could be eligible to MEK inhibition. These data shed light on CUP biology and unveil an opportunity for therapeutic intervention.

Stem-like cells from Cancer of Unknown Primary are endowed with distinctive hypermetastatic properties and unveil liability to MEK inhibition

2020 ◽  
Author(s):  
Federica Verginelli ◽  
Alberto Pisacane ◽  
Gennaro Gambardella ◽  
Antonio D'Ambrosio ◽  
Marco Ferrio ◽  
...  
Keyword(s):  
Cancer Of Unknown Primary ◽  
Unknown Primary ◽  
Fatal Disease ◽  
Self Renewal ◽  
Mek Inhibition ◽  
Transcriptomic Signature ◽  
Shed Light ◽  
Serial Transplantation

Abstract Cancers of Unknown Primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, are a biological enigma and a fatal disease. We propose that CUPs are a distinct, yet unrecognized, pathological entity originating from stem-like cells endowed with unique properties. These cells can be isolated in vitro (agnospheres) and propagated in vivo by serial transplantation, displaying exceptionally high tumorigenicity. After subcutaneous engraftment, agnospheres recapitulated the CUP phenotype, by showing the ability to spontaneously and quickly disseminate, and form widespread established metastases. Regardless of different genetic backgrounds, agnospheres invariably displayed cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis, which confers sensitivity to MEK inhibitors in vitro and in vivo. Such sensitivity is associated with a transcriptomic signature predicting that 75% of CUP patients could be eligible to MEK inhibition. These data shed light on CUP biology and unveil an opportunity for therapeutic intervention.

scholarly journals Using the Microwell-mesh to culture microtissues in vitro and as a carrier to implant microtissues in vivo into mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Melissa E. Monterosso ◽  
Kathryn Futrega ◽  
William B. Lott ◽  
Ian Vela ◽  
Elizabeth D. Williams ◽  
...  
Keyword(s):  
Hair Follicles ◽  
Live Animal ◽  
Nsg Mice ◽  
Animal Imaging ◽  
Organ Specific ◽  
Bioluminescence Signal ◽  
Serial Transplantation ◽  
Live Animal Imaging

AbstractProstate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of “pieces” of tumour in mice, but the cellular composition of pieces is not standardised. Herein, we optimised a microwell platform, the Microwell-mesh, to aggregate precise numbers of cells into arrays of microtissues, and then implanted the Microwell-mesh into NOD-scid IL2γ−/− (NSG) mice to study microtissue growth. First, mesh pore size was optimised using microtissues assembled from bone marrow-derived stromal cells, with mesh opening dimensions of 100×100 μm achieving superior microtissue vascularisation relative to mesh with 36×36 μm mesh openings. The optimised Microwell-mesh was used to assemble and implant PCa cell microtissue arrays (hereafter microtissues formed from cancer cells are referred to as microtumours) into mice. PCa cells were enriched from three different PDX lines, LuCaP35, LuCaP141, and BM18. 3D microtumours showed greater in vitro viability than 2D cultures, but neither proliferated. Microtumours were successfully established in mice 81% (57 of 70), 67% (4 of 6), 76% (19 of 25) for LuCaP35, LuCaP141, and BM18 PCa cells, respectively. Microtumour growth was tracked using live animal imaging for size or bioluminescence signal. If augmented with further imaging advances and cell bar coding, this microtumour model could enable greater resolution of PCa PDX drug response, and lead to the more efficient use of animals. The concept of microtissue assembly in the Microwell-mesh, and implantation in vivo may also have utility in implantation of islets, hair follicles or other organ-specific cells that self-assemble into 3D structures, providing an important bridge between in vitro assembly of mini-organs and in vivo implantation.

scholarly journals Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

2016 ◽  
Vol 113 (17) ◽  
pp. 4788-4793 ◽  
Author(s):  
Monica Markovski ◽  
Jessica L. Bohrhunter ◽  
Tania J. Lupoli ◽  
Tsuyoshi Uehara ◽  
Suzanne Walker ◽  
...  
Keyword(s):  
Polymerase Activity ◽  
Activation Mechanism ◽  
Phenotypic Analysis ◽  
Outer Membranes ◽  
Division Site ◽  
Physiological Relevance ◽  
Membrane Lipoproteins ◽  
Shed Light

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a ofEscherichia colirequire the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b–LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.

Serial Transplantation of a Human Acute T Lymphoblastic Leukemia into Nude Mice

1986 ◽  
Vol 72 (6) ◽  
pp. 553-558 ◽  
Author(s):  
Maria Giovanna Martinotti ◽  
Roberto Arione ◽  
Roberto Foà ◽  
Luigi Pegoraro ◽  
Cristina Jemma ◽  
...  
Keyword(s):  
Nude Mice ◽  
Lymphoblastic Leukemia ◽  
Experimental System ◽  
Leukemic Cells ◽  
Immune Reactivity ◽  
Chromosomal Markers ◽  
Total Body ◽  
Serial Transplantation

A human acute T lymphoblastic leukemia line (PF-382) was serially transplanted into nude mice. No takes were observed in untreated nude mice, whereas solid tumors were observed in splenectomized and total body, sublethally irradiated mice. The minimal tumor-inducing dose and the latency time remained unchanged after the third and fifth serial transplants. Moreover, leukemic cells recovered from the 8th in vivo passages displayed the same differentiation antigens and chromosomal markers as the in vitro PF-382 cell line used for the first transplant. This stable and well-characterized experimental system could be a new model for T-lymphocyte differentiation and immune-reactivity against human leukemias.

scholarly journals Structural analysis reveals features of the spindle checkpoint kinase Bub1–kinetochore subunit Knl1 interaction

2012 ◽  
Vol 196 (4) ◽  
pp. 451-467 ◽  
Author(s):  
Veronica Krenn ◽  
Annemarie Wehenkel ◽  
Xiaozheng Li ◽  
Stefano Santaguida ◽  
Andrea Musacchio
Keyword(s):  
Crystal Structure ◽  
Structural Analysis ◽  
Convex Surface ◽  
Point Mutations ◽  
Tetratricopeptide Repeats ◽  
Checkpoint Protein ◽  
Checkpoint Kinases ◽  
Shed Light

The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR–KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1.

scholarly journals Tumor growth of neurofibromin-deficient cells is driven by decreased respiration and hampered by NAD+ and SIRT3

2021 ◽  
Author(s):  
Ionica Masgras ◽  
Giuseppe Cannino ◽  
Francesco Ciscato ◽  
Carlos Sanchez-Martin ◽  
Marco Pizzi ◽  
...  
Keyword(s):  
Succinate Dehydrogenase ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Chain Complex ◽  
Respiratory Chain Complex ◽  
Succinate Dehydrogenase Activity ◽  
Nadh Ratio ◽  
Shed Light

Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here, we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion. This provides cells with resistance to pro-oxidants targeting complex I and decreases both respiration and intracellular NAD+. Expression of the alternative NADH dehydrogenase NDI1 raises NAD+/NADH ratio, enhances the activity of the mitochondrial NAD+-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. This anti-neoplastic effect is mimicked both in vitro and in vivo by administration of NAD+ precursors or by rising expression of the NAD+ deacetylase SIRT3, and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes of these cells. These findings shed light on chemotherapeutic resistance and on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD+/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.

scholarly journals Proteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Antonella Roveri ◽  
Mattia Zaccarin ◽  
Andrea Pagetta ◽  
Elisa Tramentozzi ◽  
Paola Finotti
Keyword(s):  
Heavy Chain ◽  
Mass Analysis ◽  
Control Subjects ◽  
N Terminus ◽  
Proteomic Investigation ◽  
Healthy Control ◽  
Shed Light

The glucose-regulated protein94 (Grp94) has been found in complexes with IgG in plasma of Type 1 (T1) diabetic subjects; however, the pathogenetic meaning of Grp94-IgG complexes has not yet been elucidated. To shed light on the nature and structure of these complexesin vivo, we conducted a proteomic analysis on plasma of both T1 diabetic subjects and healthy control subjects. IgG purified from plasma was submitted to 2D PAGE followed by Western blotting and mass analysis. Grp94 was detected in plasma of all diabetic but not control subjects and found linked with its N-terminus to the IgG heavy chain. Mass analysis of heavy chain of IgG that binds Grp94 alsoin vitro, forming stable complexes with characteristics similar to those of native ones, permitted identifying CH2 and CH3 regions as those involved in binding Grp94. At the electron microscopy, IgG from diabetic plasma appeared as fibrils of various lengthes and dimensions, suggestive of elevated aggregating tendency conferred to IgG by Grp94. The nonimmune nature of complexes turned out to be responsible for the particular stability and structure adopted by complexes in plasma of diabetic subjects. Results are of relevance to understanding the pathogenetic mechanisms underlying diabetes and its complications.

scholarly journals The Biological Activity of Alkaloids from the Amaryllidaceae: From Cholinesterases Inhibition to Anticancer Activity

2016 ◽  
Vol 11 (10) ◽  
pp. 1934578X1601101 ◽  
Author(s):  
Klára Habartová ◽  
Lucie Cahlíková ◽  
Martina Řezáčová ◽  
Radim Havelek
Keyword(s):  
Molecular Level ◽  
Plant Family ◽  
Amaryllidaceae Alkaloids ◽  
Medicinal Properties ◽  
The Many ◽  
Small Chemical ◽  
Shed Light

Modern research has shown that Amaryllidaceae alkaloids represent a rich reservoir of potential small chemical molecules exhibiting several medicinal properties through various mechanisms. Among the many Amaryllidaceae compounds, galanthamine has been given a great amount of attention due to the fact that it possesses potent acetylcholinesterase inhibitory activity. In spite of the amount of evidence indicating the potential usefulness of Amaryllidaceae alkaloids in therapy, research groups have focused their attention on the other alkaloids present in this plant family. New investigations have shed light on many aspects of the structure of Amaryllidaceae alkaloids and on their semisynthetic modification, function, and mechanisms underlying in vitro and in vivo activity. In addition, Amaryllidaceae alkaloids have frequently been identified as having promising cytotoxic properties against cancer cell lines. While follow-up studies have repeatedly shown that Amaryllidaceae alkaloids and their derivatives demonstrate antiproliferative, cytotoxic and apoptosis-inducing activity, the mechanisms remain unclear. This review addresses the most important Amaryllidaceae alkaloids with anticancer potential, particularly those that have been studied for the purpose of gaining a better understanding of the basis of the activity at the cellular and molecular level.

scholarly journals SCIDOT-27. TGFβ SIGNALING-INDUCED miRNA PARTICIPATES IN AUTOPHAGIC REGULATION BY TARGETING PRAS40 IN MESENCHYMAL SUBTYPE OF GLIOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi277-vi277
Author(s):  
Yingbin Xie ◽  
Luyue Chen ◽  
Junhu Zhou ◽  
Qixue Wang ◽  
Yunfei Wang ◽  
...  
Keyword(s):  
Cell Fate ◽  
Therapeutic Interventions ◽  
Functional Verification ◽  
Tgfβ Signaling ◽  
Signature Genes ◽  
Dismal Prognosis ◽  
Tgfβ Receptor ◽  
Transcriptomic Signature

Abstract Mesenchymal subtype of glioblastoma (mesGBM) is a refractory disease condition characterized by therapeutic failure and tumor recurrence, and for the past decade, no treatment option has improved the dismal prognosis of this disease. In Cancer Genome Atlas (TCGA) GBM datasets, two of the 840 transcriptomic signature genes for GBM subtyping, TGFβ1 and TGFβ receptor type II (TβRII), were exclusively upregulated in mesGBM, indicating hyperactive TGFβ signaling. In the present study, dysregulated microRNAs (miRNAs) were identified after LY2109761 (a TβRI/II inhibitor) treatment in a mesGBM-derived cell line. Importantly, several novel miRNAs that may be missing pieces of the puzzle of TGFβ signaling-mediated pathogenesis in mesGBM were identified. Novel_miR26/56/93/97/119 were validated by quantitative PCR to correlate with TGFβ signaling activity, and novel_miR56 was selected as a promising candidate for further functional verification. A proline-rich akt substrate of 40 kDa (PRAS40), which regulates autophagy initiation by targeting mTORC1, was confirmed to be a functional target of novel_miR56. Autophagy plays dichotomous roles in the determination of GBM cell fate, and in this study, TGFβ signaling-induced novel_miR56 seemed to inhibit autophagic activity in GBM and its mesenchymal subtype, resulting in the maintenance of proliferative capability. The pro-survival effect of novel_miR56 can be impeded by intentionally downregulating its expression both in vivo and in vitro. In summary, we provide novel insight into TGFβ signaling-mediated pathogenesis and targets for the development of novel therapeutic interventions for mesGBM.

scholarly journals Yorkie and JNK revert syncytial muscles into myoblasts during Org-1–dependent lineage reprogramming

2019 ◽  
Vol 218 (11) ◽  
pp. 3572-3582 ◽  
Author(s):  
Christoph Schaub ◽  
Marcel Rose ◽  
Manfred Frasch
Keyword(s):  
Transcription Factors ◽  
Transcriptional Activators ◽  
Direct Reprogramming ◽  
Research Attention ◽  
Lineage Reprogramming ◽  
Adult Drosophila ◽  
Alary Muscles ◽  
Shed Light

Lineage reprogramming has received increased research attention since it was demonstrated that lineage-restricted transcription factors can be used in vitro for direct reprogramming. Recently, we reported that the ventral longitudinal musculature of the adult Drosophila heart arises in vivo by direct lineage reprogramming from larval alary muscles, a process that starts with the dedifferentiation and fragmentation of syncytial muscle cells into mononucleate myoblasts and depends on Org-1 (Drosophila Tbx1). Here, we shed light on the events occurring downstream of Org-1 in this first step of transdifferentiation and show that alary muscle lineage-specific activation of Yorkie plays a key role in initiating the dedifferentiation and fragmentation of these muscles. An additional necessary input comes from active dJNK signaling, which contributes to the activation of Yorkie and furthermore activates dJun. The synergistic activities of the Yorkie/Scalloped and dJun/dFos transcriptional activators subsequently initiate alary muscle fragmentation as well as up-regulation of Myc and piwi, both crucial for lineage reprogramming.

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