ATP1A1 is a promising new target for melanoma treatment and can be inhibited by its physiological ligand bufalin to restore targeted therapy efficacy.

Despite advancements in treating metastatic melanoma, many patients exhibit resistance to targeted therapies. Our study focuses on ATP1A1, a sodium pump subunit associated with cancer development. We aimed to assess ATP1A1 prognostic value in melanoma patients and examine the impact of its ligand, bufalin, on melanoma cell lines in vitro and in vivo. High ATP1A1 expression (IHC) correlated with reduced overall survival in melanoma patients. Resistance to BRAF inhibitor was linked to elevated ATP1A1 levels in patient biopsies (IHC, qPCR) and cell lines (Western blot, qPCR). Additionally, high ATP1A1 mRNA expression positively correlated with differentiation/pigmentation markers based on data from The Cancer Genome Atlas (TCGA) databases and Verfaillie proliferative gene signature analysis. Bufalin specifically targeted ATP1A1 in caveolae, (proximity ligation assay) and influenced Src phosphorylation (Western blot), thereby disrupting multiple signaling pathways (phosphokinase array). In vitro, bufalin induced apoptosis in melanoma cell lines by acting on ATP1A1 (siRNA experiments) and, in vivo, significantly impeded melanoma growth using a nude mouse xenograft model with continuous bufalin delivery via an osmotic pump. In conclusion, our study demonstrates that ATP1A1 could serve as a prognostic marker for patient survival and a predictive marker for response to BRAF inhibitor therapy. By targeting ATP1A1, bufalin inhibited cell proliferation, induced apoptosis in vitro, and effectively suppressed tumor development in mice. Thus, our findings strongly support ATP1A1 as a promising therapeutic target, with bufalin as a potential agent to disrupt its tumor-promoting activity.

Macrophage Profiling in Head and Neck Cancer to Improve Patient Prognosis and Assessment of Cancer Cell-Macrophage Interactions Using Three-Dimensional Coculture Models.

Tumor-associated macrophages are key components of the tumor microenvironment and play important roles in the progression of head and neck cancer, leading to the development of effective strategies targeting immune cells in tumors. Our study demonstrated the prognostic potential of a new scoring system (Macroscore) based on the combination of the ratio and the sum of the high and low densities of M1 (CD80+) and M2 (CD163+) macrophages in a series of head and neck cancer patients, including a training population (n = 54) and a validation population (n = 19). Interestingly, the Macroscore outperformed TNM criteria and p16 status, showing a significant association with poor patient prognosis, and demonstrated significant predictive value for overall survival. Additionally, 3D coculture spheroids were established to analyze the crosstalk between cancer cells and monocytes/macrophages. Our data revealed that cancer cells can induce monocyte differentiation into protumoral M2 macrophages, creating an immunosuppressive microenvironment. This coculture also induced the production of immunosuppressive cytokines, such as IL10 and IL8, known to promote M2 polarization. Finally, we validated the ability of the macrophage subpopulations to induce apoptosis (M1) or support proliferation (M2) of cancer cells. Overall, our research highlights the potential of the Macroscore as a valuable prognostic biomarker to enhance the clinical management of patients and underscores the relevance of a spheroid model in gaining a better understanding of the mechanisms underlying cancer cell-macrophage interactions.

Bufalin for an innovative therapeutic approach against cancer.

Bufalin is an endogenous cardiotonic steroid, first discovered in toad venom but also found in the plasma of healthy humans, with anti-tumour activities in different cancer types. The current review is focused on its mechanisms of action and highlights its very large spectrum of effects both in vitro and in vivo. All leads to the conclusion that bufalin mediates its effects by affecting all the hallmarks of cancer and seems restricted to cancer cells avoiding side effects. Bufalin decreases cancer cell proliferation by acting on the cell cycle and inducing different mechanisms of cell death including apoptosis, necroptosis, autophagy and senescence. Bufalin also moderates metastasis formation by blocking migration and invasion as well as angiogenesis and by inducing a phenotype switch towards differentiation and decreasing cancer cell stemness. Regarding its various mechanisms of action in cancer cells, bufalin blocks overactivated signalling pathways and modifies cell metabolism. Moreover, bufalin gained lately a huge interest in the field of drug resistance by both reversing various drug resistance mechanisms and affecting the immune microenvironment. Together, these data support bufalin as a quite promising new anti-cancer drug candidate.

Neutrophil-to-lymphocyte ratio as a prognostic marker for head and neck cancer with lung metastasis: a retrospective study.

PURPOSE: The neutrophil-to-lymphocyte ratio (NLR) is the most widely biomarker used to assess the inflammatory system in various solid cancers. An elevated NLR has been reported to be associated with worse outcomes in head and neck squamous cell cancers (HNSCC). However, questions remain about the prognostic value of these findings in HNSCC patients with lung metastasis. This study aims to quantify the prognostic impact of NLR on HNSCC patients with lung metastasis. METHODS: A retrospective chart review of 169 HNSCC patients was performed at the Otorhinolaryngology and the Stomatology and Maxillofacial Surgery Department (Saint-Pierre Hospital), between 2000 and 2017. All patients were divided into two subgroups. Patients who developed lung involvement were assigned to the lung-metastasis-group (LM-group) in contrast to no-lung-metastasis-group patients (NLM-group). The prognostic significance of NLR was evaluated using multivariable analysis adjusting for overall-survival (OS) and lung-metastasis-free-survival (LMFS). RESULTS: 95 patients were enrolled in the NLM-group while 74 were in the LM-group. Multivariable analysis highlights that patients with a higher NLR value had shortened OS in the NLM subgroup (HR 1.3; p = 0.024). However, this association was not found in the LM subgroup. When considering both subgroups, an elevated NLR was reported as a prognostic factor of poor LMFS (HR 1.65; p = 0.047). CONCLUSION: Our data revealed that pretreatment NLR is an independent prognostic factor of mortality and lung metastasis development. However, the prognostic value of NLR is not confirmed in patients who suffered from lung metastasis. Physicians should integrate these findings in their treatment algorithm approach.

Dealing with Macrophage Plasticity to Address Therapeutic Challenges in Head and Neck Cancers.

The head and neck tumor microenvironment (TME) is highly infiltrated with macrophages. More specifically, tumor-associated macrophages (TAM/M2-like) are one of the most critical components associated with poor overall survival in head and neck cancers (HNC). Two extreme states of macrophage phenotypes are described as conducting pro-inflammatory/anti-tumoral (M1) or anti-inflammatory/pro-tumoral (M2) activities. Moreover, specific metabolic pathways as well as oxidative stress responses are tightly associated with their phenotypes and functions. Hence, due to their plasticity, targeting M2 macrophages to repolarize in the M1 phenotype would be a promising cancer treatment. In this context, we evaluated macrophage infiltration in 60 HNC patients and demonstrated the high infiltration of CD68+ cells that were mainly related to CD163+ M2 macrophages. We then optimized a polarization protocol from THP1 monocytes, validated by specific gene and protein expression levels. In addition, specific actors of glutamine pathway and oxidative stress were quantified to indicate the use of glutaminolysis by M2 and the production of reactive oxygen species by M1. Finally, we evaluated and confirmed the plasticity of our model using M1 activators to repolarize M2 in M1. Overall, our study provides a complete reversible polarization protocol allowing us to further evaluate various reprogramming effectors targeting glutaminolysis and/or oxidative stress in macrophages.

Development of a Method for Producing oxLDL: Characterization of Their Effects on HPV-Positive Head and Neck Cancer Cells.

Cardiovascular diseases (CVD) and cancers are the two main causes of death worldwide. The initiation and progression of atherosclerosis is, in large part, caused by oxidized low-density lipoproteins (oxLDL); interestingly, oxLDL may also play a role in cancer cell metabolism and migration. As oxLDL are generally obtained by tedious ultracentrifugation procedures, "home-made" oxLDL were obtained by (i) applying a purification kit to isolate LDL and VLDL from human plasma; (ii) isolating LDL from VLDL by gel permeation chromatography (GPC); and (iii) oxidating LDL through CuSO4 incubation. On three HPV-positive head and neck cancer cells (HNCC) (93VU-147T, UM-SCC47, and UPCI-SCC154), cell migration was assessed using Boyden chambers, the Wnt/ß-catenin pathway was analyzed by Western Blotting, and the expression of two oxLDL receptors, LOX-1 and CD36, in response to oxLDL exposure, was analysed by immunofluorescence. Our data indicate: (a) a non-significant difference between reference and "home-made" oxLDL; (b) a decreased migration, parallel to an inhibition of the ß-catenin pathway; and (c) an increase of CD36 and LOX-1 expression in all HNCC. In conclusion, we successfully produced oxLDL. Our results demonstrate a decrease in HNCC migration after oxLDL exposure, and an increased expression of LOX-1 and CD36 associated with lipid uptake.

Epidemiological, otolaryngological, olfactory and gustatory outcomes according to the severity of COVID-19: a study of 2579 patients.

OBJECTIVE: To investigate prevalence and epidemiological and clinical factors associated with olfactory dysfunction (OD) and gustatory dysfunction (GD) in COVID-19 patients according to the disease severity. STUDY DESIGN: Cross-sectional study. METHODS: A total of 2579 patients with a positive diagnosis of COVID-19 were identified between March 22 and June 3, 2020 from 18 European hospitals. Epidemiological and clinical data were extracted. Otolaryngological symptoms, including OD and GD, were collected through patient-reported outcome questionnaire and Sniffin'Sticks tests were carried out in a subset of patients. RESULTS: A total of 2579 patients were included, including 2166 mild (84.0%), 144 moderate (5.6%) and 269 severe-to-critical (10.4%) patients. Mild patients presented an otolaryngological picture of the disease with OD, GD, nasal obstruction, rhinorrhea and sore throat as the most prevalent symptoms. The prevalence of subjective OD and GD was 73.7 and 46.8%, and decreases with the severity of the disease. Females had higher prevalence of subjective OD and GD compared with males. Diabetes was associated with a higher risk to develop GD. Among the subset of patients who benefited from psychophysical olfactory evaluations, there were 75 anosmic, 43 hyposmic and 113 normosmic patients. The prevalence of anosmia significantly decreased with the severity of the disease. Anosmia or hyposmia were not associated with any nasal disorder, according to SNOT-22. CONCLUSION: OD and GD are more prevalent in patients with mild COVID-19 compared with individuals with moderate, severe or critical diseases. Females might have a higher risk of developing OD and GD compared with males.

Survival and treatment outcome of head and neck cancer patients with pulmonary oligometastases.

OBJECTIVES: The purpose of this study was to determine the outcome of head and neck squamous cell carcinoma (HNSCC) patients developing lung metastasis. DESIGN: Retrospective study. PARTICIPANTS: HNSCC patients with lung metastasis treated between 2001 and 2018 were included. MEAN OUTCOMES MEASURES: Statistical analyses described the relationship between patient survival, treatment efficacy and pulmonary metastasis occurrence. RESULTS: One hundred HNSCC patients were included in the study. The median overall survival (OS) was 21 months. The median recurrence-free survival (RFS) was seven months. Patient survival with only lung metastases was significantly longer compared to patients with lung metastases and lymph nodes involvement or other metastases. Moreover, patients with a single metastasis had longer post-RFS and OS than patients with multiple metastases. The local control of metastasis was better when patients presented only lung metastases, and it was more effective in single metastasis. The surgery allowed better metastases local control than supportive care or radio and/or chemotherapy. In case of specific therapy, pulmonary resection was associated with a longer post-RFS and a longer OS compared to supportive care or radio and/or chemotherapy. CONCLUSIONS: We confirmed, in the current study, the significant survival benefit for HNSCC patients treated by surgery for their pulmonary metastasis. While treatment of multiple metastases required palliative chemotherapy or best supportive care in most of the cases, specific surgical treatment in selected HNSCC patients should be considered.

Olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (COVID-19): a multicenter European study.

OBJECTIVE: To investigate the occurrence of olfactory and gustatory dysfunctions in patients with laboratory-confirmed COVID-19 infection. METHODS: Patients with laboratory-confirmed COVID-19 infection were recruited from 12 European hospitals. The following epidemiological and clinical outcomes have been studied: age, sex, ethnicity, comorbidities, and general and otolaryngological symptoms. Patients completed olfactory and gustatory questionnaires based on the smell and taste component of the National Health and Nutrition Examination Survey, and the short version of the Questionnaire of Olfactory Disorders-Negative Statements (sQOD-NS). RESULTS: A total of 417 mild-to-moderate COVID-19 patients completed the study (263 females). The most prevalent general symptoms consisted of cough, myalgia, and loss of appetite. Face pain and nasal obstruction were the most disease-related otolaryngological symptoms. 85.6% and 88.0% of patients reported olfactory and gustatory dysfunctions, respectively. There was a significant association between both disorders (p < 0.001). Olfactory dysfunction (OD) appeared before the other symptoms in 11.8% of cases. The sQO-NS scores were significantly lower in patients with anosmia compared with normosmic or hyposmic individuals (p = 0.001). Among the 18.2% of patients without nasal obstruction or rhinorrhea, 79.7% were hyposmic or anosmic. The early olfactory recovery rate was 44.0%. Females were significantly more affected by olfactory and gustatory dysfunctions than males (p = 0.001). CONCLUSION: Olfactory and gustatory disorders are prevalent symptoms in European COVID-19 patients, who may not have nasal symptoms. The sudden anosmia or ageusia need to be recognized by the international scientific community as important symptoms of the COVID-19 infection.

Breast cancer cells and bone marrow mesenchymal stromal cells: a regulated modulation of the breast tumor in the context of immune response.

OBJECTIVE: The role of direct cell-cell interactions mediating selective bone metastasis by breast cancer cells (BCCs) niche is still mostly unknown. MATERIALS AND METHODS: Conditioned medium and direct cell-cell contacts experiments were used to investigate the effect of bone marrow-derived mesenchymal stromal cells (MSCs), osteoprogenitor-like cells (MG-63) and osteosarcoma cells (SaOS-2) on luminal-like (MCF-7) and basal-like (MDA-MB-231) BCCs flow cytometry was used to assess the purity of isolated BCCs and osteoblasts. Expression of osteoblastic markers was investigated by semi-quantitative RT-PCR. RANKL and OPG levels were measured by ELISA. RESULTS: Conditioned medium from MSCs and osteoblasts induced the expression of osteoblastic markers in BCCs. While co-culture assays with SaOS-2 increased the expression of osteoblastic markers in MCF-7 cells, SaOS-2 cell conditioned medium increased the expression of RANKL, PTHrP, VEGF and NOGGIN in MCF-7 cells. Co-cultures with either MG-63 cells or MSCs induced OPG and MMP-2 in both tumor cell lines. Interestingly, conditioned medium from co-cultures of MSCs and MDA-MB-231 cells significantly decreased the proliferation of activated T lymphocytes which was reversed by addition of anti-OPG antibodies to the co-cultures. CONCLUSION: Our data suggest that MSCs strongly contribute to the adaptation and invasiveness of breast cancer cells in skeletal tissues.

Galectins and Carcinogenesis: Their Role in Head and Neck Carcinomas and Thyroid Carcinomas.

Head and neck cancers are among the most frequently occurring cancers worldwide. Of the molecular drivers described for these tumors, galectins play an important role via their interaction with several intracellular pathways. In this review, we will detail and discuss this role with specific reference to galectins-1, -3, and -7 in angiogenesis, cell proliferation, and invasion as well as in cell transformation and cancer progression. Furthermore, we will evaluate the prognostic value of galectin expression in head and neck cancers including those with oral cavity, salivary gland, and nasopharyngeal pathologies. In addition, we will discuss the involvement of these galectins in thyroid cancers where their altered expression is proposed as a new diagnostic biomarker.

The Interplay between Atherosclerosis and Cancer: Breast Cancer Cells Increase the Expression of Endothelial Cell Adhesion Markers.

Cardiovascular diseases are the leading causes of death worldwide, closely followed by cancer. To investigate the impact of breast cancer cell lines (SKBR3, MCF-7, and MDA-MB-231) on endothelial cell adhesion, a blended medium containing 30% breast-cancer-conditioned medium was prepared. This medium was then exposed to human umbilical vein endothelial cells (HUVECs) and monocytes (THP-1) for 48 h. Homemade oxidized low-density lipoproteins (oxLDL) were optionally added to the blended medium. Immunofluorescence was performed to assess the expression of E-selectin, connexin-43, and ICAM-1 on HUVECs, as well as LOX-1, CD36, and CD162 on THP-1. Additionally, unoxidized LDL was exposed to the three breast cancer cell lines for 48 h, and the formation of oxLDL was quantified. Our results revealed an upregulation of all six adhesion markers involved in the initiation of atherosclerosis when HUVECs and THP-1 were exposed to the breast-cancer-conditioned medium. Furthermore, this expression was further increased by exposure to oxLDL. We also observed a significant elevation in oxLDL levels when LDL was exposed to breast cancer cells. In conclusion, our findings successfully demonstrate an increased LDL oxidation in the presence of breast cancer cells, accompanied by an augmented expression of receptors involved in atherosclerosis initiation. These findings shed new light on the clinically observed interplay between atherosclerosis and cancer.

Targeting Prohibitins to Inhibit Melanoma Growth and Overcome Resistance to Targeted Therapies.

Despite important advances in the treatment of metastatic melanoma with the development of MAPK-targeted agents and immune checkpoint inhibitors, the majority of patients either do not respond to therapies or develop acquired resistance. Furthermore, there is no effective targeted therapy currently available for BRAF wild-type melanomas (approximately 50% of cutaneous melanoma). Thus, there is a compelling need for new efficient targeted therapies. Prohibitins (PHBs) are overexpressed in several types of cancers and implicated in the regulation of signaling networks that promote cell invasion and resistance to cell apoptosis. Herein, we show that PHBs are highly expressed in melanoma and are associated with not only poor survival but also with resistance to BRAFi/MEKi. We designed and identified novel specific PHB inhibitors that can inhibit melanoma cell growth in 3D spheroid models and a large panel of representative cell lines with different molecular subtypes, including those with intrinsic and acquired resistance to MAPKi, by significantly moderating both MAPK (CRAF-ERK axis) and PI3K/AKT pathways, and inducing apoptosis through the mitochondrial pathway and up-regulation of p53. In addition, autophagy inhibition enhances the antitumor efficacy of these PHB ligands. More important, these ligands can act in synergy with MAPKi to more efficiently inhibit cell growth and overcome drug resistance in both BRAF wild-type and mutant melanoma. In conclusion, targeting PHBs represents a very promising therapeutic strategy in melanoma, regardless of mutational status.

The benefit of co-targeting PARP-1 and c-Met on the efficacy of radiotherapy in wild type BRAF melanoma.

Melanoma is known to be a radioresistant cancer. Melanoma radioresistance can be due to several factors such as pigmentation, antioxidant defenses and high Deoxyribonucleic acid (DNA) repair efficacy. However, irradiation induces intracellular translocation of RTKs, including cMet, which regulates response to DNA damage activating proteins and promotes DNA repair. Accordingly, we hypothesized that co-targeting DNA repair (PARP-1) and relevant activated RTKs, c-Met in particular, may radiosensitize wild-type B-Raf Proto-Oncogene, Serine/Threonine Kinase (WTBRAF) melanomas where RTKs are often upregulated. Firstly, we found that PARP-1 is highly expressed in melanoma cell lines. PARP-1 inhibition by Olaparib or its KO mediates melanoma cell sensitivity to radiotherapy (RT). Similarly, specific inhibition of c-Met by Crizotinib or its KO radiosensitizes the melanoma cell lines. Mechanistically, we show that RT causes c-Met nuclear translocation to interact with PARP-1 promoting its activity. This can be reversed by c-Met inhibition. Accordingly, RT associated with the inhibition of both c-Met and PARP-1 resulted in a synergistic effect not only on tumor growth inhibition but also on tumor regrowth control in all animals following the stop of the treatment. We thus show that combining PARP and c-Met inhibition with RT appears a promising therapeutic approach in WTBRAF melanoma.

Correction: Krayem et al. The Benefit of Reactivating p53 under MAPK Inhibition on the Efficacy of Radiotherapy in Melanoma. Cancers 2019, 11, 1093.

In the original article [...].

Bioconjugation studies of an EGF-R targeting ligand on dendronized iron oxide nanoparticles to target head and neck cancer cells.

A major challenge in nanomedicine is designing nanoplatforms (NPFs) to selectively target abnormal cells to ensure early diagnosis and targeted therapy. Among developed NPFs, iron oxide nanoparticles (IONPs) are good MRI contrast agents and can be used for therapy by hyperthermia and as radio-sensitizing agents. Active targeting is a promising method for selective IONPs accumulation in cancer tissues and is generally performed by using targeting ligands (TL). Here, a TL specific for the epidermal growth factor receptor (EGFR) is bound to the surface of dendronized IONPs to produce nanostructures able to specifically recognize EGFR-positive FaDu and 93-Vu head and neck cancer cell lines. Several parameters were optimized to ensure a high coupling yield and to adequately quantify the amount of TL per nanoparticle. Nanostructures with variable amounts of TL on the surface were produced and evaluated for their potential to specifically target and be thereafter internalized by cells. Compared to the bare NPs, the presence of the TL at the surface was shown to be effective to enhance their internalization and to play a role in the total amount of iron present per cell.

Preventive and Therapeutic Effect of Metformin in Head and Neck Cancer: A Concise Review.

BACKGROUND: Head and neck cancer (HNC) is a complex affection. Nowadays, conventional treatments are associated with many side effects, reducing the patient's quality of life. Recent studies suggest that metformin, a first-line treatment for diabetes, could decrease cancer incidence and improve cancer-related survival rates. METHODS: This systematic review summarizes important data from studies evaluating metformin's contribution to preventing and treating HNC. RESULTS: The results suggest a protective effect of metformin in HNC. However, no consensus has been found on its therapeutic effects. Metformin seems to confer an improved cancer-related survival rate in a diabetic population, but compared to a non-diabetic population, the review could not identify any advantages. Nevertheless, no studies presented a negative impact. CONCLUSION: In conclusion, the results of this systematic review suggest that HNC patients may benefit from metformin. Indeed, it would reduce the HNC incidence. However, more studies are required to evaluate the effect on cancer-related survival rates.

Effect of the Size and Shape of Dendronized Iron Oxide Nanoparticles Bearing a Targeting Ligand on MRI, Magnetic Hyperthermia, and Photothermia Properties-From Suspension to In Vitro Studies.

Functionalized iron oxide nanoparticles (IONPs) are increasingly being designed as a theranostic nanoplatform combining specific targeting, diagnosis by magnetic resonance imaging (MRI), and multimodal therapy by hyperthermia. The effect of the size and the shape of IONPs is of tremendous importance to develop theranostic nanoobjects displaying efficient MRI contrast agents and hyperthermia agent via the combination of magnetic hyperthermia (MH) and/or photothermia (PTT). Another key parameter is that the amount of accumulation of IONPs in cancerous cells is sufficiently high, which often requires the grafting of specific targeting ligands (TLs). Herein, IONPs with nanoplate and nanocube shapes, which are promising to combine magnetic hyperthermia (MH) and photothermia (PTT), were synthesized by the thermal decomposition method and coated with a designed dendron molecule to ensure their biocompatibility and colloidal stability in suspension. Then, the efficiency of these dendronized IONPs as contrast agents (CAs) for MRI and their ability to heat via MH or PTT were investigated. The 22 nm nanospheres and the 19 nm nanocubes presented the most promising theranostic properties (respectively, r2 = 416 s-1·mM-1, SARMH = 580 W·g-1, SARPTT = 800 W·g-1; and r2 = 407 s-1·mM-1, SARMH = 899 W·g-1, SARPTT = 300 W·g-1). MH experiments have proven that the heating power mainly originates from Brownian relaxation and that SAR values can remain high if IONPs are prealigned with a magnet. This raises hope that heating will maintain efficient even in a confined environment, such as in cells or in tumors. Preliminary in vitro MH and PTT experiments have shown the promising effect of the cubic shaped IONPs, even though the experiments should be repeated with an improved set-up. Finally, the grafting of a specific peptide (P22) as a TL for head and neck cancers (HNCs) has shown the positive impact of the TL to enhance IONP accumulation in cells.

Cytokine Landscape in Central Nervous System Metastases.

The central nervous system is the location of metastases in more than 40% of patients with lung cancer, breast cancer and melanoma. These metastases are associated with one of the poorest prognoses in advanced cancer patients, mainly due to the lack of effective treatments. In this review, we explore the involvement of cytokines, including interleukins and chemokines, during the development of brain and leptomeningeal metastases from the epithelial-to-mesenchymal cell transition and blood-brain barrier extravasation to the interaction between cancer cells and cells from the brain microenvironment, including astrocytes and microglia. Furthermore, the role of the gut-brain axis on cytokine release during this process will also be addressed.