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.

Mouse model of postsurgical primary tumor recurrence and regional lymph node metastasis progression in HPV-related head and neck cancer.

HPV-positive head and neck squamous cell carcinoma (HNSCC) is increasingly frequent. Management is particularly debated in the case of postsurgical high-risk features, that is, positive surgical margins and extracapsular spread (ECS). In this increasingly complex emerging framework of HNSCC treatment, representative preclinical models are needed to support future clinical trials and advances in personalized medicine. Here, we present an immunocompetent mouse model based on the implantation of mouse tonsil epithelial HPV16-E6/E7-expressing cancer cells into the submental region of the floor-of-the-mouth. Primary tumors were found to replicate the patterns of human HNSCC local invasion and lymphatic dissemination. To study disease progression after surgery, tumors were removed likely leaving behind residual disease. Surgical resection of tumors was followed by a high rate of local recurrences (>90%) within the first 2-3 weeks. While only 50% of mice had lymph node metastases (LNM) at time of primary tumor excision, all mice with recurrent tumors showed evidence of LNM. To study the consecutive steps of LNM progression and distant metastasis development, LNs from tumor-bearing mice were transplanted into naïve recipient mice. Using this approach, transplanted LNs were found to recapitulate all stages and relevant histological features of regional metastasis progression, including ECS and metastatic spread to the lungs. Altogether, we have developed an immunocompetent HPV-positive HNSCC mouse model of postsurgical local recurrence and regional and distant metastasis progression suitable for preclinical studies.

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 ß receptor (LTßR) 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 LTßR signaling. Loss of LTßR 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.

Efficient amyloid A clearance in the absence of immunoglobulins and complement factors.

Amyloid A amyloidosis is a protein misfolding disease characterized by deposition of extracellular aggregates derived from the acute-phase reactant serum amyloid A protein. If untreated, amyloid A amyloidosis leads to irreversible damage of various organs, including the kidneys, liver, and heart. Amyloid A deposits regress upon reduction of serum amyloid A concentration, indicating that the amyloid can be efficiently cleared by natural mechanisms. Clearance was proposed to be mediated by humoral immune responses to amyloid. Here, we report that amyloid clearance in mice lacking complement factors 3 and 4 (C3C4(-/-)) was equally efficient as in wild-type mice (C57BL/6), and was only slightly delayed in agammaglobulinemic mice (J(H-/-)). Hence, antibodies or complement factors are not necessary for natural amyloid clearance, implying the existence of alternative physiological pathways for amyloid removal.

Mitochondrial uncoupling protein 2 gene transcript levels are elevated in maturating erythroid cells.

Mitochondrial uncoupling protein 2 (UCP2) is abundant in developing monocyte/macrophage cells and may affect hematopoiesis by reducing formation of reactive oxygen species. The aims of this study were to further characterize the involvement of UCP2 in hematopoiesis. In situ hybridization in mouse embryos identified UCP2-positive cells in liver and inside primitive blood vessels from 10.5 days of prenatal development. High UCP2 transcript levels were detected in reticulocytes and other maturating erythroid cells in peripheral blood of mice exposed to hypoxia, and in umbilical cord blood of human neonates and peripheral blood of adults. Our results suggest involvement of UCP2 in erythropoiesis.

Involvement of AMP-activated protein kinase in fat depot-specific metabolic changes during starvation.

The mechanisms controlling fat depot-specific metabolism are poorly understood. During starvation of mice, downregulation of lipogenic genes, suppression of fatty acid synthesis, and increases in lipid oxidation were all more pronounced in epididymal than in subcutaneous fat. In epididymal fat, relatively strong upregulation of uncoupling protein 2 and phosphoenolpyruvate carboxykinase genes was found. In mice maintained both at 20 and 30 degrees C, AMP-activated protein kinase was activated in epididymal but did not change in subcutaneous fat. Our results suggest that AMPK may have a role in the different response of various fat depots to starvation.

Unusual binding mode of an HIV-1 protease inhibitor explains its potency against multi-drug-resistant virus strains.

Protease inhibitors (PIs) are an important class of drugs for the treatment of HIV infection. However, in the course of treatment, resistant viral variants with reduced sensitivity to PIs often emerge and become a major obstacle to successful control of viral load. On the basis of a compound equipotently inhibiting HIV-1 and 2 proteases (PR), we have designed a pseudopeptide inhibitor, QF34, that efficiently inhibits a wide variety of PR variants. In order to analyze the potency of the inhibitor, we constructed PR species harboring the typical (signature) mutations that confer resistance to commercially available PIs. Kinetic analyses showed that these mutated PRs were inhibited up to 1,000-fold less efficiently by the clinically approved PIs. In contrast, all PR species were effectively inhibited by QF34. In a clinical study, we have monitored 30 HIV-positive patients in the Czech Republic undergoing highly active antiretroviral therapy, and have identified highly PI resistant variants. Kinetic analyses revealed that QF34 retained its subnanomolar potency against multi-drug resistant PR variants. X-ray crystallographic analysis and molecular modeling experiments explained the wide specificity of QF34: this inhibitor binds to the PR in an unusual manner, thus avoiding contact sites that are mutated upon resistance development, and the unusual binding mode and consequently the binding energy is therefore preserved in the complex with a resistant variant. These results suggest a promising route for the design of second-generation PIs that are active against a variety of resistant PR variants.

Possible involvement of AMP-activated protein kinase in obesity resistance induced by respiratory uncoupling in white fat.

The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy charge that promotes catabolic and inhibits anabolic pathways. However, the role of AMPK in adipocytes is poorly understood. We show that transgenic expression of mitochondrial uncoupling protein 1 in white fat, which induces obesity resistance in mice, is associated with depression of cellular energy charge, activation of AMPK, downregulation of adipogenic genes, and increase in lipid oxidation. Activation of AMPK may explain the complex metabolic changes in adipose tissue of these animals and our results support a role for adipocyte AMPK in the regulation of storage of body fat.

Modulation of lipid metabolism by energy status of adipocytes: implications for insulin sensitivity.

It is becoming evident that insulin resistance of white adipose tissue is a major factor underlying the cardiovascular risk of obesity. Impaired fat storage rather than altered glucose metabolism in adipocytes probably contributes to development of insulin resistance in muscle and other tissues, in particular via increased delivery of nonesterified fatty acids into circulation. Lipid metabolism of adipose tissue is affected by the energy status of fat cells. In vitro experiments indicated the dependence of both lipogenesis and lipolysis on ATP levels in adipocytes. Thus, respiratory uncoupling in adipocytes that results in stimulation of energy dissipation and depression of ATP synthesis may contribute to the control of lipid metabolism, adiposity, and insulin sensitivity. This notion is supported by the expression of UCPs in adipocytes, for example, UCP2, UCP5, as well as some protonophoric anion transporters, and by induction of UCP1 and UCP3 in white fat by pharmacological treatments that reduce adiposity. A negative correlation between expression of UCPs in adipocytes and accumulation of white fat was also found. Expression of UCP1 from the adipose-specific promoter in the aP2-Ucp1 transgenic mice mitigated obesity induced by genetic or dietary factors. The obesity resistance, accompanied by respiratory uncoupling in adipocytes and increased energy expenditure, resulted from ectopic expression of UCP1 in white, but not brown fat. Probably due to depression of the ATP/ADP ratio, both fatty acid synthesis and lipolytic action of norepinephrine in adipocytes of transgenic mice were relatively low. Expression of regulatory G-proteins, which are essential for both catecholamine and insulin signaling in adipocytes, was also altered by ectopic UCP1. These results support the role of protonophoric proteins in adipocytes in the control of adiposity and insulin sensitivity. Antidiabetic effects of thiazolidinediones, fibrates, beta(3)-adrenoreceptor agonists, dietary n-3 PUFAs, and leptin may be explained at least partially by their effects on the energy and hence also the lipid metabolism of fat cells.

Omega-3 PUFA of marine origin limit diet-induced obesity in mice by reducing cellularity of adipose tissue.

Omega-3 PUFA of marine origin reduce adiposity in animals fed a high-fat diet. Our aim was to learn whether EPA and DHA could limit development of obesity and reduce cellularity of adipose tissue and whether other dietary FA could influence the effect of EPA/DHA. Weight gain induced by composite high-fat diet in C57BL/6J mice was limited when the content of EPA/DHA was increased from 1 to 12% (wt/wt) of dietary lipids. Accumulation of adipose tissue was reduced, especially of the epididymal fat. Low ratio of EPA to DHA promoted the effect. A higher dose of EPA/DHA was required to reduce adiposity when admixed to diets that did not promote obesity, the semisynthetic high-fat diets rich in EFA, either alpha-linolenic acid (ALA, 18:3 n-3, the precursor of EPA and DHA) or linoleic (18:2 n-6) acid. Quantification of adipose tissue DNA revealed that except for the diet rich in ALA the reduction of epididymal fat was associated with 34-50% depression of tissue cellularity, similar to the 30% caloric restriction in the case of the high-fat composite diet. Changes in plasma markers and adipose gene expression indicated improvement of lipid and glucose metabolism due to EPA/DHA even in the context of the diet rich in ALA. Our results document augmentation of the antiadipogenic effect of EPA/DHA during development of obesity and suggest that EPA/DHA could reduce accumulation of body fat by limiting both hypertrophy and hyperplasia of fat cells. Increased dietary intake of EPA/DHA may be beneficial regardless of the ALA intake.

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.

Impaired noradrenaline-induced lipolysis in white fat of aP2-Ucp1 transgenic mice is associated with changes in G-protein levels.

In vitro experiments suggest that stimulation of lipolysis by catecholamines in adipocytes depends on the energy status of these cells. We tested whether mitochondrial uncoupling proteins (UCPs) that control the efficiency of ATP production could affect lipolysis and noradrenaline signalling in white fat in vivo. The lipolytic effect of noradrenaline was lowered by ectopic UCP1 in white adipocytes of aP2-Ucp1 transgenic mice, overexpressing the UCP1 gene from the aP2 gene promoter, reflecting the magnitude of UCP1 expression, the impaired stimulation of cAMP levels by noradrenaline and the reduction of the ATP/ADP ratio in different fat depots. Thus only subcutaneous but not epididymal fat was affected. UCP1 also down-regulated the expression of hormone-sensitive lipase and lowered its activity, and altered the expression of trimeric G-proteins in adipocytes. The adipose tissue content of the stimulatory G-protein alpha subunit was increased while that of the inhibitory G-protein alpha subunits decreased in response to UCP1 expression. Our results support the idea that the energy status of cells, and the ATP/ADP ratio in particular, modulates the lipolytic effects of noradrenaline in adipose tissue in vivo. They also demonstrate changes at the G-protein level that tend to overcome the reduction of lipolysis when ATP level in adipocytes is low. Therefore, respiratory uncoupling may exert a broad effect on hormonal signalling in adipocytes.