Head and neck squamous cell carcinoma cell lines have an immunomodulatory effect on macrophages independent of hypoxia and toll-like receptor 9

Background: A low tissue oxygen level, Methods: Conditioned media (CMNOX or CMHOX) from cell lines UT-SCC-8, UT-SCC-74A, FaDu, MDA-MB-231 and HaCat cultured under normoxic (21% O-2) and hypoxic (1% O-2) conditions were used to polarize human monocyte-derived macrophages. Macrophage polarization was measured by flow cytometry and the production of cytokine mRNA using Taqman qPCR. To study the role of TLR9 in macrophage polarization, the lentiviral CRISPR/Cas9 method was used to establish a stable FaDu(TLR9def) clone.Results: Our results demonstrate that the soluble mediators produced by the cancer cells under normoxia polarize macrophages towards a hybridized M1/M2a/M2c phenotype. Furthermore, the results suggest that hypoxia has a limited role in altering the array of cancer-produced soluble factors affecting macrophage polarization and cytokine production. Our data also indicates that increased expression of TLR9 due to hypoxia in malignant cells does not markedly influence the polarization of macrophages. TLR9 transcriptional response to hypoxia is dissimilar to a HIF1-alpha-regulated LDH-A. This may indicate a context-dependent expression of TLR9 under hypoxia.Conclusions: HNSCC cell lines affect both macrophage activity (polarization) and functionality (cytokines), but with exception to iNOS expression, the effects appear independent of hypoxia and TLR9.</p

Luteinizing hormone and GATA4 action in the adrenocortical tumorigenesis of gonadectomized female mice

Background/Aims: Physiological role of luteinizing hormone (LH) and its receptor (LHCGR) in adrenal remains unknown. In inhibin-α/Simian Virus 40 T antigen (SV40Tag) (inhα/Tag) mice, gonadectomy-induced (OVX) elevated LH triggers the growth of transcription factor GATA4 (GATA4)-positive adrenocortical tumors in a hyperplasia-adenoma-adenocarcinoma sequence. Methods: We investigated the role of LHCGR in tumor induction, by crossbreeding inhα/Tag with Lhcgr knockout (LuRKO) mice. By knocking out Lhcgr and Gata4 in Cα1 adrenocortical cells (Lhcgr-ko, Gata4-ko) we tested their role in tumor progression. Results: Adrenal tumors of OVX inhα/Tag mice develop from the hyperplastic cells localized in the topmost layer of zona fasciculata. OVX inhα/Tag/LuRKO only developed SV40Tag positive hyperplastic cells that were GATA4 negative, cleaved caspase-3 positive and did not progress into adenoma. In contrast to Lhcgr-ko, Gata4-ko Cα1 cells presented decreased proliferation, increased apoptosis, decreased expression of Inha, SV40Tag and Lhcgr tumor markers, as well as up-regulated adrenal- and down-regulated sex steroid gene expression. Both Gata4-ko and Lhcgr-ko Cα1 cells had decreased expression of steroidogenic genes resulting in decreased basal progesterone production. Conclusion: Our data indicate that LH/LHCGR signaling is critical for the adrenal cell reprogramming by GATA4 induction prompting adenoma formation and gonadal-like phenotype of the adrenocortical tumors in inhα/Tag mice.</p

SORLA regulates endosomal trafficking and oncogenic fitness of HER2

The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.Peer reviewe

A feed-forward loop between SorLA and HER3 determines heregulin response and neratinib resistance

Current evidence indicates that resistance to the tyrosine kinase-type cell surface receptor (HER2)-targeted therapies is frequently associated with HER3 and active signaling via HER2-HER3 dimers, particularly in the context of breast cancer. Thus, understanding the response to HER2-HER3 signaling and the regulation of the dimer is essential to decipher therapy relapse mechanisms. Here, we investigate a bidirectional relationship between HER2-HER3 signaling and a type-1 transmembrane sorting receptor, sortilin-related receptor (SorLA; SORL1). We demonstrate that heregulin-mediated signaling supports SorLA transcription downstream of the mitogen-activated protein kinase pathway. In addition, we demonstrate that SorLA interacts directly with HER3, forming a trimeric complex with HER2 and HER3 to attenuate lysosomal degradation of the dimer in a Ras-related protein Rab4-dependent manner. In line with a role for SorLA in supporting the stability of the HER2 and HER3 receptors, loss of SorLA compromised heregulin-induced cell proliferation and sensitized metastatic anti-HER2 therapy-resistant breast cancer cells to neratinib in cancer spheroids in vitro and in vivo in a zebrafish brain xenograft model

SORLA regulates endosomal trafficking and oncogenic fitness of HER2

The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers

Molecular basis of essential thrombocythaemia in humans and dogs - a review

A potential cause of essential thrombocythaemia can be seen as the V617F point mutation within Janus kinase 2. This mutation occurs in 60-70% of patients with this disease and is located in the domain acting as an inhibitor. It increases the enzymatic activity of JAK2 kinase and induces intensified sensitivity of cells to cytokines. Identification of mutations in the JAK2 gene has made it possible to describe the molecular pathogenesis of myeloproliferative syndromes, which has enabled more accurate diagnosis and assisted in effective treatment. The significant similarity of the clinical, laboratory and morphological features of myeloproliferative syndromes (including essential thrombocythaemia) in animals and humans suggests that common signalling pathways within the JAK2 gene may be involved in the development of these diseases

Assessment of in vitro dynamics of pathogenic Acanthamoeba strains originating from contact lens wearers with infectious keratitis

Recently, incidents of Acanthamoeba keratitis, the vision-threatening eye disease, are reported with increasing frequency worldwide, particularly in contact lens wearers. In our study, the retrospective assessment of in vitro dynamics of subsequent pathogenic Acanthamoeba isolates cultured at 24°C, detected in Polish contact lens wearers with keratitis is presented and results compared with those of environmental A. castellanii Neff strain. There were delayed the proper diagnosis that influenced prolonged and severe course of this eye disease and treatment difficulties. The corneal material was examined directly to visualize developmental amoeba stages for diagnose verification, microbiologically tested for the specific identification of bacteriae and fungi, and in vitro grown in culture medium in temperature 24°C. Among twenty-six keratitis incidents analyzed, eleven were cases of Acanthamoeba keratitis; in the six of them, Acanthamoeba strains and concomitant bacterial and/or fungal infectious agents were detected. In vitro assays showed variability in population density of several clinical strains in the exponential growth phase expressed in various range of overall amoeba number and different proportion between trophozoites and cysts. The clear influence of temperature on the in vitro cultivation of the amoebae was observed: statistically significant lower population dynamics was revealed by most of pathogenic clinical isolates in comparison with those showed by environmental strain. The in vitro monitoring of dynamics of Acanthamoeba strains isolated from infected eyes may be helpful for diagnostics verification, especially in mixed infectious keratitis

Evaluation of in vitro effect of selected contact lens solutions conjugated with nanoparticles in terms of preventive approach to public health risk generated by Acanthamoeba strains

Introduction. Various Acanthamoeba species are free-living organisms widely distributed in the human environment. Amphizoic amoebae as facultative parasites may cause vision-threatening eye disease – Acanthamoeba keratitis, mostly among contact lens wearers. As the number of cases is increasing, and applied therapy often unsuccessful, proper hygienic measures and effective contact lenses disinfection are crucial for the prevention of this disease. Available contact lens solutions are not fully effective against amphizoic amoebae; there is a need to enhance their disinfecting activity to prevent amoebic infections. The use of developing nanotechnology methods already applied with success in the prevention, diagnostic and therapy of other infectious diseases might be helpful regarding amoebic keratitis. This study assesses the in vitro effect of selected contact lens solutions conjugated with nanoparticles against Acanthamoeba trophozoites. Materials and method. Three selected contact lens solutions conjugated with silver and gold nanoparticles in concentration of 0.25–2.5 ppm were used in vitro against the axenically cultured ATCC 30010 type Acanthamoeba castellanii strain. The anti-amoebic efficacy was examined based on the oxido-reduction of AlamarBlue. The cytotoxicity tests based on the measurement of lactate dehydrogenase (LDH) activity were performed using a fibroblast HS-5 cell line. Results. Enhancement of the anti-amoebic activity of contact lens solutions conjugated with selected nanoparticles expressed in the dose dependent amoebic growth inhibition with a low cytotoxicity profile was observed. Conclusions. Results of the study showed that conjugation of selected contact lens solutions with silver nanoparticles might be a promising approach to prevent Acanthamoeba keratitis among contact lens users