The transcription factor FOXM1 regulates the balance between proliferation and aberrant differentiation in head and neck squamous cell carcinoma

Sustained expression of FOXM1 is a hallmark of nearly all human cancers including squamous cell carcinomas of the head and neck (HNSCC). HNSCCs partially preserve the epithelial differentiation program, which recapitulates fetal and adult traits of the tissue of tumor origin but is deregulated by genetic alterations and tumor-supporting pathways. Using shRNA-mediated knockdown, we demonstrate a minimal impact of FOXM1 on proliferation and migration of HNSCC cell lines under standard cell culture conditions. However, FOXM1 knockdown in three-dimensional (3D) culture and xenograft tumor models resulted in reduced proliferation, decreased invasion, and a more differentiated-like phenotype, indicating a context-dependent modulation of FOXM1 activity in HNSCC cells. By ectopic overexpression of FOXM1 in HNSCC cell lines, we demonstrate a reduced expression of cutaneous-type keratin K1 and involucrin as a marker of squamous differentiation, supporting the role of FOXM1 in modulation of aberrant differentiation in HNSCC. Thus, our data provide a strong rationale for targeting FOXM1 in HNSCC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

AA-Amyloidosis Can Be Transferred by Peripheral Blood Monocytes

Spongiform encephalopathies have been reported to be transmitted by blood transfusion even prior to the clinical onset. Experimental AA-amyloidosis shows similarities with prion disease and amyloid-containing organ-extracts can prime a recipient for the disease. In this systemic form of amyloidosis N-terminal fragments of the acute-phase reactant apolipoprotein serum amyloid A are the main amyloid protein. Initial amyloid deposits appear in the perifollicular region of the spleen, followed by deposits in the liver. We used the established murine model and induced AA-amyloidosis in NMRI mice by intravenous injections of purified amyloid fibrils (‘amyloid enhancing factor’) combined with inflammatory challenge (silver nitrate subcutaneously). Blood plasma and peripheral blood monocytes were isolated, sonicated and re-injected into new recipients followed by an inflammatory challenge during a three week period. When the animals were sacrificed presence of amyloid was analyzed in spleen sections after Congo red staining. Our result shows that some of the peripheral blood monocytes, isolated from animals with detectable amyloid, contained amyloid-seed that primed for AA-amyloid. The seeding material seems to have been phagocytosed by the cells since the AA-precursor (SAA1) was found not be expressed by the monocytes. Plasma recovered from mice with AA amyloidosis lacked seeding capacity. Amyloid enhancing activity can reside in monocytes recovered from mice with AA-amyloidosis and in a prion-like way trigger amyloid formation in conjunction with an inflammatory disorder. Human AA-amyloidosis resembles the murine form and every individual is expected to be exposed to conditions that initiate production of the acute-phase reactant. The monocyte-transfer mechanism should be eligible for the human disease and we point out blood transfusion as a putative route for transfer of amyloidosis

Analysis of AA/SAA reactivity in peripheral blood monocytes by confocal microscopy showed immunoreactivity in 5% of the monocytes isolated from a mouse with AA-amyloidosis (A).

<p>There was no reactivity present in monocytes recovered from a mouse given one AgNO₃ injection 48 hrs prior to isolation (B) or in monocytes isolated from untreated mice (C). The used rabbit antiserum recognizes both protein AA and SAA and was visualized by goat anti rabbit Alexa488-cojugated IgG. Cell nuclei were labeled with TO-PRO3. Bar 10 um.</p

Analysis of AEF activity in peripheral blood monocytes isolated from mice with AA amyloidosis.

<p>The table presents detailed information on animals in group H 1–8. AA-amyloidosis was induced by i.v. injection of AEF and 0.2 ml 1% silver nitrate injections on day 1, 7, 14, 21 and 28. The animals were sacrificed on day 35. Blood was collected and amyloid was verified in spleen sections after Congo red staining. Isolated peripheral blood monocytes were injected into new animals, group H 1–8, and silver nitrate was given day 1, 7 and 14.The animals were sacrificed day 16 and the presence of amyloid was studied in spleen after Congo red staining.</p

SAA 1, SAA 2 and SAA 3 mRNA expression in peripheral blood monocytes were analysed with PCR.

<p>Cells were isolated from mice that developed AA-amyloid after AEF and AgNO₃ injections or from mice that received AEF or AgNO₃ injections only or from untreated mice. Expression of the amyloid-prone SAA 1 or non-amyloidogenic SAA 2 was absent in all monocyte preparations. SAA 3 mRNA was detected in all cells independent of treatment. Mouse liver cDNA was used as a positive control. The PCR products were separated on a 1.6% agarose gel.</p

Spleen amyloid deposits stained with Congo red.

<p>(A) The amyloid appears pink and is localized to the perifollicular zone. (B) The identical area exhibits green birefringence in polarized light. Amyloid is indicated by arrows.</p

Analysis of AEF activity in peripheral blood monocytes isolated from mice with AA-amyloid induced by AEF.

<p>AA-amyloid was induced in nine mice (G1–G9) by an i.v. injection of 0.1 ml AEF with concomitant s.c. injection of 0.2 ml 1% silver nitrate day 1, 7, 14, 21 and 28 and the mice were sacrificed on day 35. The presence of amyloid in the spleen was verified by Congo red staining. Peripheral blood monocytes were isolated, sonicated and re-introduced into the blood circulation of new groups of healthy mice (H1–H8). These mice received inflammatory stimuli day 1, 7 and 14 and were sacrificed day 16. The presence of amyloid was analysed in spleen sections after Congo red staining. Mice in group H9 received sonicated monocytes without subsequent inflammatory stimuli and group H10 received monocytes isolated from untreated mice and subsequent inflammatory stimuli on day 1, 7 and 14 and were sacrificed day 16 (group H10).</p

Lymphotoxin, but Not TNF, Is Required for Prion Invasion of Lymph Nodes

Neuroinvasion and subsequent destruction of the central nervous system by prions are typically preceded by a colonization phase in lymphoid organs. An important compartment harboring prions in lymphoid tissue is the follicular dendritic cell (FDC), which requires both tumor necrosis factor receptor 1 (TNFR1) and lymphotoxin β receptor (LTβR) signaling for maintenance. However, prions are still detected in TNFR1(-/-) lymph nodes despite the absence of mature FDCs. Here we show that TNFR1-independent prion accumulation in lymph nodes depends on LTβR signaling. Loss of LTβR signaling, but not of TNFR1, was concurrent with the dedifferentiation of high endothelial venules (HEVs) required for lymphocyte entry into lymph nodes. Using luminescent conjugated polymers for histochemical PrP(Sc) detection, we identified PrP(Sc) deposits associated with HEVs in TNFR1(-/-) lymph nodes. Hence, prions may enter lymph nodes by HEVs and accumulate or replicate in the absence of mature FDCs

Quality Matters: Biocuration Experts on the Impact of Duplication and Other Data Quality Issues in Biological Databases

Biological databases represent an extraordinary collective volume of work. Diligently built up over decades and comprising many millions of contributions from the biomedical research community, biological databases provide worldwide access to a massive number of records (also known as entries) [1]. Starting from individual laboratories, genomes are sequenced, assembled, annotated, and ultimately submitted to primary nucleotide databases such as GenBank [2], European Nucleotide Archive (ENA) [3], and DNA Data Bank of Japan (DDBJ) [4] (collectively known as the International Nucleotide Sequence Database Collaboration, INSDC). Protein records, which are the translations of these nucleotide records, are deposited into central protein databases such as the UniProt KnowledgeBase (UniProtKB) [5] and the Protein Data Bank (PDB) [6]. Sequence records are further accumulated into different databases for more specialized purposes: RFam [7] and PFam [8] for RNA and protein families, respectively; DictyBase [9] and PomBase [10] for model organisms; as well as ArrayExpress [11] and Gene Expression Omnibus (GEO) [12] for gene expression profiles. These databases are selected as examples; the list is not intended to be exhaustive. However, they are representative of biological databases that have been named in the “golden set” of the 24th Nucleic Acids Research database issue (in 2016). The introduction of that issue highlights the databases that “consistently served as authoritative, comprehensive, and convenient data resources widely used by the entire community and offer some lessons on what makes a successful database” [13]. In addition, the associated information about sequences is also propagated into non-sequence databases, such as PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) for scientific literature or Gene Ontology (GO) [14] for function annotations. These databases in turn benefit individual studies, many of which use these publicly available records as the basis for their own research

PrP^Sc is present both in and around MadCam1-positive HEVs in <i>TNFR1^−/−</i>-Ig mesenteric lymph nodes.

<p>TNFR1^−/− mice inoculated i.p. with 6 log LD₅₀ RML6 and treated weekly with control Ig were sacrificed at 60 d.p.i. Immunofluorescence (A–I) and histoblots (J & K) were then performed on frozen sections from prion-infected TNFR1^−/−-Ig mesenteric lymph nodes. Co-IF with anti-serum (XN) against PrP (green; A) and MadCam1 (red; B) showed points of intense PrP immunoreactivity localized to HEVs (C). Confocal co-IF with the amyloid-binding dye, p-FTAA (green; D) and MadCam1 (red; E,F) revealed some points of PrP^Sc association with HEVs (F); however much of the PrP^Sc was present outside of HEVs (I). Histoblots pre-stained with PNAd antibody and developed with AP (pink; J) also revealed some prion-infected HEVs (black arrows), some non-infected HEVs (white arrow), and some PrP^Sc deposits that were not HEV-associated (yellow arrow). (K) Total numbers of PNAd-positive HEVs in histoblot co-stains were counted and scored as PrP^Sc-positive (PrP^Sc+; black) or PrP^Sc-negative (PrP^Sc+; white), and total PrP^Sc deposits were counted and scored as PNAd-positive (PNAd+; black) or PNAd-negative (PNAd; white). 35% of HEVs were PrP^Sc-positive, and 58% of PrP^Sc deposits were PNAd-positive. Size bars in A–F = 50 µm. Size bars in G–I = 100 µm.</p