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.

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 [...].

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.

Disturbing the Redox Balance Using Buthionine Sulfoximine Radiosensitized Somatostatin Receptor-2 Expressing Pre-Clinical Models to Peptide Receptor Radionuclide Therapy with 177Lu-DOTATATE.

Peptide receptor radionuclide therapy with 177Lu-DOTATATE improves the outcome of patients with somatostatin receptor (SSTR)-expressing neuroendocrine tumours. Nevertheless, stable disease has been the main response pattern observed, with some rare complete responses. Lu-177 exerts about two-thirds of its biological effects via the indirect effects of ionizing radiation that generate reactive oxygen species, eventually leading to oxidative damage and cell death. This provides a rationale for targeting the antioxidant defence system in combination with 177Lu-DOTATATE. In the present study, the radiosensitizing potential and the safety of depleting glutathione (GSH) levels using buthionine sulfoximine (BSO) during 177Lu-DOTATATE therapy were assessed in vitro and in vivo using a xenograft mouse model. In vitro, the combination resulted in a synergistic effect in cell lines exhibiting a BSO-mediated GSH decrease. In vivo, BSO neither influenced 177Lu-DOTATATE biodistribution nor induced liver, kidney or bone marrow toxicity. In terms of efficacy, the combination resulted in reduced tumour growth and metabolic activity. Our results showed that disturbing the cell redox balance using a GSH synthesis inhibitor increased 177Lu-DOTATATE efficacy without additional toxicity. Targeting the antioxidant defence system opens new safe treatment combination opportunities with 177Lu-DOTATATE.

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.

Regorafenib Induces Senescence and Epithelial-Mesenchymal Transition in Colorectal Cancer to Promote Drug Resistance.

Potential intrinsic resistance mechanisms to regorafenib were explored after short exposure (3 days) on five CRC cell lines (HCT-116, SW1116, LS-1034, SW480, Caco-2). The observation of senescence-like features led to the investigation of a drug-initiated phenotype switch. Following long-term exposure (12 months) of HCT-116 and SW480 cell lines to regorafenib, we developed resistant models to explore acquired resistance. SW480 cells demonstrated senescent-like properties, including a cell arrest in the late G2/prophase cell cycle stage and a statistically significant decrease in the expression of G1 Cyclin-Dependent Kinase inhibitors and key cell cycle regulators. A specific senescence-associated secretome was also observed. In contrast, HCT-116 treated cells presented early senescent features and developed acquired resistance triggering EMT and a more aggressive phenotype over time. The gained migration and invasion ability by long-exposed cells was associated with the increased expression level of key cellular and extracellular EMT-related factors. The PI3K/AKT pathway was a significant player in the acquired resistance of HCT-116 cells, possibly related to a PI3KCA mutation in this cell line. Our findings provide new insights into the phenotypic plasticity of CRC cells able, under treatment pressure, to acquire a stable TIS or to use an early senescence state to undergo EMT.

Understanding the Radiobiological Mechanisms Induced by 177Lu-DOTATATE in Comparison to External Beam Radiation Therapy.

Radionuclide Therapy (RNT) with 177Lu-DOTATATE targeting somatostatin receptors (SSTRs) in neuroendocrine tumours (NET) has been successfully used in routine clinical practice, mainly leading to stable disease. Radiobiology holds promise for RNT improvement but is often extrapolated from external beam radiation therapy (EBRT) studies despite differences in these two radiation-based treatment modalities. In a panel of six human cancer cell lines expressing SSTRs, common radiobiological endpoints (i.e., cell survival, cell cycle, cell death, oxidative stress and DNA damage) were evaluated over time in 177Lu-DOTATATE- and EBRT-treated cells, as well as the radiosensitizing potential of poly (ADP-ribose) polymerase inhibition (PARPi). Our study showed that common radiobiological mechanisms were induced by both 177Lu-DOTATATE and EBRT, but to a different extent and/or with variable kinetics, including in the DNA damage response. A higher radiosensitizing potential of PARPi was observed for EBRT compared to 177Lu-DOTATATE. Our data reinforce the need for dedicated RNT radiobiology studies, in order to derive its maximum therapeutic benefit.

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.

Development of fluorizoline analogues as prohibitin ligands that modulate C-RAF signaling, p21 expression and melanogenesis.

Fluorizoline is a cytotoxic trifluorothiazoline that targets the scaffold proteins prohibitins-1 and -2 (PHB1/2) to inhibit the kinase C-RAF and promote the expression of the cyclin-dependent kinase inhibitor p21 to induce cancer cell death. In melanocytes, fluorizoline also induces the synthesis of melanin. Herein we report the first structural requirement of fluorizoline analogues for these activities. We identified in particular some compounds that display enhanced anti-C-RAF and anti-MEK activities, and a higher cytotoxicity in HeLa cells compared to fluorizoline. These results provide a foundation for further optimization of PHB ligands for the treatment of cancers. We also discovered an analogue of fluorizoline that displays pharmacological effects opposed to those of fluorizoline and that can be used as a chemical tool to explore PHB signaling in cancers and other diseases.

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.

Understanding Molecular Mechanisms of Phenotype Switching and Crosstalk with TME to Reveal New Vulnerabilities of Melanoma.

Melanoma cells are notorious for their high plasticity and ability to switch back and forth between various melanoma cell states, enabling the adaptation to sub-optimal conditions and therapeutics. This phenotypic plasticity, which has gained more attention in cancer research, is proposed as a new paradigm for melanoma progression. In this review, we provide a detailed and deep comprehensive recapitulation of the complex spectrum of phenotype switching in melanoma, the key regulator factors, the various and new melanoma states, and corresponding signatures. We also present an extensive description of the role of epigenetic modifications (chromatin remodeling, methylation, and activities of long non-coding RNAs/miRNAs) and metabolic rewiring in the dynamic switch. Furthermore, we elucidate the main role of the crosstalk between the tumor microenvironment (TME) and oxidative stress in the regulation of the phenotype switching. Finally, we discuss in detail several rational therapeutic approaches, such as exploiting phenotype-specific and metabolic vulnerabilities and targeting components and signals of the TME, to improve the response of melanoma patients to treatments.

Recent advances in radiosensitivity determinants in melanoma.

PURPOSE OF REVIEW: Radiotherapy has been proven to be useful but insufficient in melanoma management due to the intrinsic radioresistance of melanoma cells. Elucidation of the molecular mechanisms and pathways related to resistance/sensitivity to radiotherapy in melanoma is of paramount importance. In this review, we will summarize and discuss the recent 'discoveries' and advances in radiosensitivity determinants in melanoma. RECENT FINDINGS: The different levels of radiosensitivity among the various melanoma tumors could be attributed to the DNA damage signaling and repair proteins, tumor microenvironment, hypoxia, cell metabolism, glutathione and redox balance, protein kinase signaling pathways as well as pigmentation and melanin content. SUMMARY: It is therapeutically important to elucidate the factors involved in radiation resistance/sensitivity of melanoma. More importantly, improving radiosensitivity may 'widen the clinical utility' in melanoma of this important therapeutic modality.

Tyrosine-Dependent Phenotype Switching Occurs Early in Many Primary Melanoma Cultures Limiting Their Translational Value.

The use of patient-derived primary cell cultures in cancer preclinical assays, including drug screens and genotoxic studies, has increased in recent years. However, their translational value is constrained by several limitations, including variability that can be caused by the culture conditions. Here, we show that the medium composition commonly used to propagate primary melanoma cultures has limited their representability of their tumor of origin and their cellular plasticity, and modified their sensitivity to therapy. Indeed, we established and compared cultures from different melanoma patients propagated in parallel in low-tyrosine (Ham's F10) or in high-tyrosine (Ham's F10 supplemented with tyrosine or RPMI1640 or DMEM) media. Tyrosine is the precursor of melanin biosynthesis, a process particularly active in differentiated melanocytes and melanoma cells. Unexpectedly, we found that the high tyrosine concentrations promoted an early phenotypic drift towards either a mesenchymal-like or senescence-like phenotype, and prevented the establishment of cultures of melanoma cells harboring differentiated features, which we show are frequently present in human clinical biopsies. Moreover, the invasive phenotype emerging in these culture conditions appeared irreversible and, as expected, associated with intrinsic resistance to MAPKi. In sharp contrast, differentiated melanoma cell cultures retained their phenotypes upon propagation in low-tyrosine medium, and importantly their phenotypic plasticity, a key hallmark of melanoma cells. Altogether, our findings underline the importance of culturing melanoma cells in low-tyrosine-containing medium in order to preserve their phenotypic identity of origin and cellular plasticity.

Humanized Mice as a Valuable Pre-Clinical Model for Cancer Immunotherapy Research.

Immunotherapy with checkpoint inhibitors opened new horizons in cancer treatment. Clinical trials for novel immunotherapies or unexplored combination regimens either need years of development or are simply impossible to perform like is the case in cancer patients with limited life expectancy. Thus, the need for preclinical models that rapidly and safely allow for a better understanding of underlying mechanisms, drug kinetics and toxicity leading to the selection of the best regimen to be translated into the clinic, is of high importance. Humanized mice that can bear both human immune system and human tumors, are increasingly used in recent preclinical immunotherapy studies and represent a remarkably unprecedented tool in this field. In this review, we describe, summarize, and discuss the recent advances of humanized mouse models used for cancer immunotherapy research and the challenges faced during their establishment. We also highlight the lack of preclinical studies using this model for radiotherapy-based research and argue that it can be a great asset to understand and answer many open questions around radiation therapy such as its presumed associated "abscopal effect".

Apoptotic and Non-Apoptotic Modalities of Thymoquinone-Induced Lymphoma Cell Death: Highlight of the Role of Cytosolic Calcium and Necroptosis.

Targeting non-apoptotic modalities might be therapeutically promising in diffuse large B cell lymphoma (DLBCL) patients with compromised apoptotic pathways. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells, but little is known about its effect on non-apoptotic pathways. This work investigates TQ selectivity against DLBCL cell lines and the cell death mechanisms. TQ reduces cell viability and kills cell lines with minimal toxicity on normal hematological cells. Mechanistically, TQ promotes the mitochondrial caspase pathway and increases genotoxicity. However, insensitivity of most cell lines to caspase inhibition by z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) pointed to a critical role of non-apoptotic signaling. In cells dying through non-apoptotic death, TQ increases endoplasmic reticulum (ER) stress markers and substantially increases cytosolic calcium ([Ca2+]c) through ER calcium depletion and activation of store-operated calcium entry (SOCE). Chelation of [Ca2+]c, but not SOCE inhibitors, reduces TQ-induced non-apoptotic cell death, highlighting the critical role of calcium in a non-apoptotic effect of TQ. Investigations showed that TQ-induced [Ca2+]c signaling is primarily initiated by necroptosis upstream to SOCE, and inhibition necroptosis by necrostatin-1 alone or with z-VAD-fmk blocks the cell death. Finally, TQ exhibits an improved selectivity profile over standard chemotherapy agents, suggesting a therapeutic relevance of the pro-necroptotic effect of TQ as a fail-safe mechanism for DLBCL therapies targeting apoptosis.

Management of the Most Common Functional Gastrointestinal Disorders in Infancy: The Middle East Expert Consensus.

The occurrence of functional gastrointestinal disorders (FGIDs) is a formidable challenge for infants, parents, and healthcare professionals. Although data from the Middle East are scarce, experts consider FGIDs a prevalent condition in everyday clinical practice. The new Rome IV criteria revisited the definitions from a clinical perspective to provide a practical and consistent diagnostic protocol for FGIDs. However, the treatment practices for functional disorders vary considerably among Middle Eastern countries, often resulting in mismanagement with unnecessary investigations and treatments. In addition, the role of various treatment modalities, including probiotics such as Lactobacillus reuteri DSM 17938, in FGIDs requires further discussion and evaluation. During a consensus meeting, a locally relevant approach for treating common FGIDs such as infant regurgitation, infant colic, and functional constipation was discussed and approved by regional experts. The participants suggested a simplified treatment plan and protocol for general pediatricians and other primary care physicians managing FGIDs. This easy-to-follow standardized protocol will help streamline the initial management of this complex disorder in the Middle East region and even globally.