The Sphingosine Kinase 2 Inhibitor ABC294640 Restores the Sensitivity of BRAFV600E Mutant Colon Cancer Cells to Vemurafenib by Reducing AKT-Mediated Expression of Nucleophosmin and Translationally-Controlled Tumour Protein

Vemurafenib (PLX4032), small-molecule inhibitor of mutated BRAFV600E protein, has emerged as a potent anti-cancer agent against metastatic melanoma harboring BRAFV600E mutation. Unfortunately, the effect of PLX4032 in the treatment of metastatic BRAF mutated colorectal cancer (CRC) is less potent due to high incidence of fast-developing chemoresistance. It has been demonstrated that sphingolipids are important mediators of chemoresistance to various therapies in colon cancer. In this study, we will explore the role of major regulators of sphingolipid metabolism and signaling in the development of resistance to vemurafenib in BRAF mutant colon cancer cells. The obtained data revealed significantly increased expression levels of activated sphingosine kinases (SphK1 and SphK2) in resistant cells concomitant with increased abundance of sphingosine-1-phosphate (S1P) and its precursor sphingosine, which was accompanied by increased expression levels of the enzymes regulating the ceramide salvage pathway, namely ceramide synthases 2 and 6 and acid ceramidase, especially after the exposure to vemurafenib. Pharmacological inhibition of SphK1/SphK2 activities or modulation of ceramide metabolism by exogenous C6-ceramide enhanced the anti-proliferative effect of PLX4032 in resistant RKO cells in a synergistic manner. It is important to note that the inhibition of SphK2 by ABC294640 proved effective at restoring the sensitivity of resistant cells to vemurafenib at the largest number of combinations of sub-toxic drug concentrations with minimal cytotoxicity. Furthermore, the obtained findings revealed that enhanced anti-proliferative, anti-migratory, anti-clonogenic and pro-apoptotic effects of a combination treatment with ABC294640 and PLX4032 relative to either drug alone were accompanied by the inhibition of S1P-regulated AKT activity and concomitant abrogation of AKT-mediated cellular levels of nucleophosmin and translationally-controlled tumour protein. Collectively, our study suggests the possibility of using the combination of ABC294640 and PLX4032 as a novel therapeutic approach to combat vemurafenib resistance in BRAF mutant colon cancer, which warrants additional preclinical validation studies.     

V600EBRAF Inhibition Induces Cytoprotective Autophagy through AMPK in Thyroid Cancer Cells

The dysregulation of autophagy is important in the development of many cancers, including thyroid cancer, where ^V600EBRAF is a main oncogene. Here, we analyse the effect of ^V600EBRAF inhibition on autophagy, the mechanisms involved in this regulation and the role of autophagy in cell survival of thyroid cancer cells. We reveal that the inhibition of ^V600EBRAF activity with its specific inhibitor PLX4720 or the depletion of its expression by siRNA induces autophagy in thyroid tumour cells. We show that ^V600EBRAF downregulation increases LKB1-AMPK signalling and decreases mTOR activity through a MEK/ERK-dependent mechanism. Moreover, we demonstrate that PLX4720 activates ULK1 and increases autophagy through the activation of the AMPK-ULK1 pathway, but not by the inhibition of mTOR. In addition, we find that autophagy blockade decreases cell viability and sensitize thyroid cancer cells to ^V600EBRAF inhibition by PLX4720 treatment. Finally, we generate a thyroid xenograft model to demonstrate that autophagy inhibition synergistically enhances the anti-proliferative and pro-apoptotic effects of ^V600EBRAF inhibition in vivo. Collectively, we uncover a new role of AMPK in mediating the induction of cytoprotective autophagy by ^V600EBRAF inhibition. In addition, these data establish a rationale for designing an integrated therapy targeting ^V600EBRAF and the LKB1-AMPK-ULK1-autophagy axis for the treatment of ^V600EBRAF-positive thyroid tumours.     

In Vitro Treatment of Melanoma Brain Metastasis by Simultaneously Targeting the MAPK and PI3K Signaling Pathways

Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAF^V600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway [1]. In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN [2]. Here, we treated two melanoma brain metastasis cell lines, H1_DL2, harboring a BRAF^V600E mutation and PTEN loss, and H3, harboring WT (wild-type) BRAF and PTEN loss, with the MAPK (BRAF) inhibitor vemurafenib and the PI3K pathway associated mTOR inhibitor temsirolimus. Combined use of the drugs inhibited tumor cell growth and proliferation in vitro in H1_DL2 cells, compared to single drug treatment. Treatment was less effective in the H3 cells. Furthermore, a strong inhibitory effect on the viability of H1_DL2 cells, when grown as 3D multicellular spheroids, was seen. The treatment inhibited the expression of pERK1/2 and reduced the expression of pAKT and p-mTOR in H1_DL2 cells, confirming that the MAPK and PI3K pathways were inhibited after drug treatment. Microarray experiments followed by principal component analysis (PCA) mapping showed distinct gene clustering after treatment, and cell cycle checkpoint regulators were affected. Global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment. In conclusion, we demonstrate for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro. The presented results highlight the potential of combined treatment to overcome treatment resistance that may develop after vemurafenib treatment of melanomas.     

BRAF and MEK Inhibitors Affect Dendritic-Cell Maturation and T-Cell Stimulation

BRAF and MEK inhibitor (BRAFi/MEKi) combinations are currently the standard treatment for patients with BRAF^V600 mutant metastatic melanoma. Since the RAS/RAF/MEK/ERK-pathway is crucial for the function of different immune cells, we postulated an effect on their function and thus interference with anti-tumor immunity. Therefore, we examined the influence of BRAFi/MEKi, either as single agent or in combination, on the maturation of monocyte-derived dendritic cells (moDCs) and their interaction with T cells. DCs matured in the presence of vemurafenib or vemurafenib/cobimetinib altered their cytokine secretion and surface marker expression profile. Upon the antigen-specific stimulation of CD8⁺ and CD4⁺ T cells with these DCs or with T2.A1 cells in the presence of BRAFi/MEKi, we detected a lower expression of activation markers on and a lower cytokine secretion by these T cells. However, treatment with any of the inhibitors alone or in combination did not change the avidity of CD8⁺ T cells in peptide titration assays with T2.A1 cells. T-helper cell/DC interaction is a bi-directional process that normally results in DC activation. Vemurafenib and vemurafenib/cobimetinib completely abolished the helper T-cell-mediated upregulation of CD70, CD80, and CD86 but not CD25 on the DCs. The combination of dabrafenib/trametinib affected DC maturation and activation as well as T-cell activation less than combined vemurafenib/cobimetinib did. Hence, for a potential combination with immunotherapy, our data indicate the superiority of dabrafenib/trametinib treatment.     

Establishment of Canine Transitional Cell Carcinoma Cell Lines Harboring BRAF V595E Mutation as a Therapeutic Target

Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract and tends to have a poor prognosis due to its invasive potential. Recent studies have reported that up to 80% of canine urothelial carcinoma has the BRAF V595E mutation, which is homologous to the human V600E mutation. Activating the BRAF mutation is an actionable target for developing effective therapeutic agents inhibiting the BRAF/mitogen-activated protein kinase (MAPK) pathway in canine cancer as well as human cancer. We established novel canine TCC cell lines from two tumor tissues and one metastatic lymph node of canine TCC patients harboring the BRAF V595E mutation. Tumor tissues highly expressed the BRAF mutant and phosphorylated extracellular signal-related kinases (ERK)1/2 proteins. The derived cell lines demonstrated activated MAPK pathways. We also evaluated the cell lines for sensitivity to BRAF inhibitors. Sorafenib, a multiple kinase inhibitor targeting RAF/vascular endothelial growth factor receptor (VEGFR), successfully inhibited the BRAF/MAPK pathway and induced apoptosis. The established canine TCC cell lines responded with greater sensitivity to sorafenib than to vemurafenib, which is known as a specific BRAF inhibitor in human cancer. Our results demonstrated that canine TCC cells showed different responses compared to human cancer with the BRAF V600E mutation. These cell lines would be valuable research materials to develop therapeutic strategies for canine TCC patients.     

Tracking of Glycans Structure and Metallomics Profiles in BRAF Mutated Melanoma Cells Treated with Vemurafenib

Nearly half of patients with advanced and metastatic melanomas harbor a BRAF mutation. Vemurafenib (VEM), a BRAF inhibitor, is used to treat such patients, however, responses to VEM are very short-lived due to intrinsic, adaptive and/or acquired resistance. In this context, we present the action of the B-Raf serine-threonine protein kinase inhibitor (vemurafenib) on the glycans structure and metallomics profiles in melanoma cells without (MeWo) and with (G-361) BRAF mutations. The studies were performed using α1-acid glycoprotein (AGP), a well-known acute-phase protein, and concanavalin A (Con A), which served as the model receptor. The detection of changes in the structure of glycans can be successfully carried out based on the frequency shifts and the charge transfer resistance after interaction of AGP with Con A in different VEM treatments using QCM-D and EIS measurements. These changes were also proved based on the cell ultrastructure examined by TEM and SEM. The LA-ICP-MS studies provided details on the metallomics profile in melanoma cells treated with and without VEM. The studies evidence that vemurafenib modifies the glycans structures and metallomics profile in melanoma cells harboring BRAF mutation that can be further implied in the resistance phenomenon. Therefore, our data opens a new avenue for further studies in the short-term addressing novel targets that hopefully can be used to improve the therapeutic regiment in advanced melanoma patients. The innovating potential of this study is fully credible and has a real impact on the global patient society suffering from advanced and metastatic melanomas.     

Oncogenic Mutation BRAF V600E Changes Phenotypic Behavior of THLE-2 Liver Cells through Alteration of Gene Expression

The accumulation of mutations in cancer driver genes, such as tumor suppressors or proto-oncogenes, affects cellular homeostasis. Disturbances in the mechanism controlling proliferation cause significant augmentation of cell growth and division due to the loss of sensitivity to the regulatory signals. Nowadays, an increasing number of cases of liver cancer are observed worldwide. Data provided by the International Cancer Genome Consortium (ICGC) have indicated many alterations within gene sequences, whose roles in tumor development are not well understood. A comprehensive analysis of liver cancer (virus-associated hepatocellular carcinoma) samples has identified new and rare mutations in B-Raf proto-oncogene (BRAF) in Japanese HCC patients, as well as BRAF V600E mutations in French HCC patients. However, their function in liver cancer has never been investigated. Here, using functional analysis and next generation sequencing, we demonstrate the tumorigenic effect of BRAF V600E on hepatocytes (THLE-2 cell line). Moreover, we identified genes such as BMP6, CXCL11, IL1B, TBX21, RSAD2, MMP10, and SERPIND1, which are possibly regulated by the BRAF V600E-mediated, mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signaling pathway. Through several functional assays, we demonstrate that BRAF L537M, D594A, and E648G mutations alone are not pathogenic in liver cancer. The investigation of genome mutations and the determination of their impact on cellular processes and functions is crucial to unraveling the molecular mechanisms of liver cancer development.     

Immunomodulatory Properties of BRAF and MEK Inhibitors Used for Melanoma Therapy—Paradoxical ERK Activation and Beyond

The advent of mitogen-activated protein kinase (MAPK) inhibitors that directly inhibit tumor growth and of immune checkpoint inhibitors (ICI) that boost effector T cell responses have strongly improved the treatment of metastatic melanoma. In about half of all melanoma patients, tumor growth is driven by gain-of-function mutations of BRAF (v-rat fibrosarcoma (Raf) murine sarcoma viral oncogene homolog B), which results in constitutive ERK activation. Patients with a BRAF mutation are regularly treated with a combination of BRAF and MEK (MAPK/ERK kinase) inhibitors. Next to the antiproliferative effects of BRAF/MEKi, accumulating preclinical evidence suggests that BRAF/MEKi exert immunomodulatory functions such as paradoxical ERK activation as well as additional effects in non-tumor cells. In this review, we present the current knowledge on the immunomodulatory functions of BRAF/MEKi as well as the non-intended effects of ICI and discuss the potential synergistic effects of ICI and MAPK inhibitors in melanoma treatment.     

Integrated Genomics Identifies miR-181/TFAM Pathway as a Critical Driver of Drug Resistance in Melanoma

MicroRNAs (miRNAs) are attractive therapeutic targets and promising candidates as molecular biomarkers for various therapy-resistant tumors. However, the association between miRNAs and drug resistance in melanoma remains to be elucidated. We used an integrative genomic analysis to comprehensively study the miRNA expression profiles of drug-resistant melanoma patients and cell lines. MicroRNA-181a and -181b (miR181a/b) were identified as the most significantly down-regulated miRNAs in resistant melanoma patients and cell lines. Re-establishment of miR-181a/b expression reverses the resistance of melanoma cells to the BRAF inhibitor dabrafenib. Introduction of miR-181 mimics markedly decreases the expression of TFAM in A375 melanoma cells resistant to BRAF inhibitors. Furthermore, melanoma growth was inhibited in A375 and M14 resistant melanoma cells transfected with miR-181a/b mimics, while miR-181a/b depletion enhanced resistance in sensitive cell lines. Collectively, our study demonstrated that miR-181a/b could reverse the resistance to BRAF inhibitors in dabrafenib resistant melanoma cell lines. In addition, miR-181a and -181b are strongly down-regulated in tumor samples from patients before and after the development of resistance to targeted therapies. Finally, melanoma tissues with high miR-181a and -181b expression presented favorable outcomes in terms of Progression Free Survival, suggesting that miR-181 is a clinically relevant candidate for therapeutic development or biomarker-based therapy selection.     

The Interplay between HGF/c-met Axis and Nox4 in BRAF Mutated Melanoma

Background: Melanoma is the leading cause of death due to cutaneous malignancy and its incidence is on the rise. Several signaling pathways, including receptor tyrosine kinases, have a role in the development and progression of melanocytic lesions and malignant melanoma. Among those, the hepatocyte growth factor (HGF)/c-met axis is emerging as a critical player because it can play a role in drug resistance. Indeed, 50% of melanoma patients present BRAF mutations, however, all responders develop resistance to the inhibitors typically within one year of treatment. Interestingly, BRAF inhibitors induce reactive oxygen species (ROS) in melanoma cells, therefore, the aim of this study was to investigate a possible interplay between HGF/c-met and ROS sources, such as NADPH oxidases (Nox). Methods: The expression of c-met and Nox were quantified in 60 patients with primary cutaneous melanoma. In vitro experiments on melanoma primary cells and the cell line were performed to dissect the underpinned molecular mechanism. Results: The outcome of interest was the correlation between the high positivity for both Nox4 and c-met and metastasis occurring at least 1 year later than melanoma diagnosis in BRAF mutated patients, in contrast to nonmutated. In vitro experiments demonstrated that the axis HGF/c-met/Nox4/ROS triggers the epithelial-mesenchymal transition. Conclusions: The observed correlation suggests an interplay between c-met and Nox4 in promoting the onset of metastasis. This study suggests that Nox4 inhibitors could be associated to the current therapy used to treat melanoma patients with BRAF mutations.     

Matrine Activates PTEN to Induce Growth Inhibition and Apoptosis in V600EBRAF Harboring Melanoma Cells

Here, we report a natural chemical Matrine, which exhibits anti-melanoma potential with its PTEN activation mechanism. Matrine effectively inhibited proliferation of several carcinoma cell lines, including melanoma ^V600EBRAF harboring M21 cells. Flow cytometry analysis showed Matrine induced G₀/G₁ cell cycle arrest in M21 cells dose-dependently. Apoptosis in M21 cells induced by Matrine was identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis and Annexin-V/FITC staining. Molecular mechanistic study suggested that Matrine upregulated both mRNA level and protein expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN), leading to inhibition of the PI3K/Akt pathway. Downregulation of phosphor-Akt^ser473 by Matrine activated p21 and Bax, which contributed to G₀/G₁ cell cycle and apoptosis. Besides, Matrine enhanced the PI3K/Akt inhibition effects to inhibit the cell proliferation with PI3K inhibitor, LY2940002. In summary, our findings suggest Matrine is a promising antitumor drug candidate with its possible PTEN activation mechanisms for treating cancer diseases, such as melanomas.     

Immunomodulatory Properties of PI3K/AKT/mTOR and MAPK/MEK/ERK Inhibition Augment Response to Immune Checkpoint Blockade in Melanoma and Triple-Negative Breast Cancer

Hyperactivation of PI3K/AKT/mTOR and MAPK/MEK/ERK signaling pathways is commonly observed in many cancers, including triple-negative breast cancer (TNBC) and melanoma. Moreover, the compensatory upregulation of the MAPK/MEK/ERK pathway has been associated with therapeutic resistance to targeted inhibition of the PI3K/AKT/mTOR pathway, and vice versa. The immune-modulatory effects of both PI3K and MAPK inhibition suggest that inhibition of these pathways might enhance response to immune checkpoint inhibitors (ICIs). ICIs have become the standard-of-care for metastatic melanoma and are recently an option for TNBC when combined with chemotherapy, but alternative options are needed when resistance develops. In this review, we present the current mechanistic understandings, along with preclinical and clinical evidence, that outline the efficacy and safety profile of combinatorial or sequential treatments with PI3K inhibitors, MAPK inhibitors, and ICIs for treatment of malignant melanoma and metastatic TNBC. This approach may present a potential strategy to overcome resistance in patients who are a candidate for ICI therapy with tumors harboring either or both of these pathway-associated mutations.     

EGFR-Mutated Non-Small Cell Lung Cancer and Resistance to Immunotherapy: Role of the Tumor Microenvironment

Lung cancer is a leading cause of cancer-related deaths worldwide. About 10–30% of patients with non-small cell lung cancer (NSCLC) harbor mutations of the EGFR gene. The Tumor Microenvironment (TME) of patients with NSCLC harboring EGFR mutations displays peculiar characteristics and may modulate the antitumor immune response. EGFR activation increases PD-L1 expression in tumor cells, inducing T cell apoptosis and immune escape. EGFR-Tyrosine Kinase Inhibitors (TKIs) strengthen MHC class I and II antigen presentation in response to IFN-γ, boost CD8+ T-cells levels and DCs, eliminate FOXP3+ Tregs, inhibit macrophage polarization into the M2 phenotype, and decrease PD-L1 expression in cancer cells. Thus, targeted therapy blocks specific signaling pathways, whereas immunotherapy stimulates the immune system to attack tumor cells evading immune surveillance. A combination of TKIs and immunotherapy may have suboptimal synergistic effects. However, data are controversial because activated EGFR signaling allows NSCLC cells to use multiple strategies to create an immunosuppressive TME, including recruitment of Tumor-Associated Macrophages and Tregs and the production of inhibitory cytokines and metabolites. Therefore, these mechanisms should be characterized and targeted by a combined pharmacological approach that also concerns disease stage, cancer-related inflammation with related systemic symptoms, and the general status of the patients to overcome the single-drug resistance development.     

CrOx/SiO2 catalysts prepared using chromium recovered from tanning sewage

Catalysts at different Cr loading (x, in the range 0.18–3.8 wt% Cr) were prepared by (i) impregnation of SiO₂ Aerosil directly with the waste water (SCx(I) samples); or (ii) mixing Aerosil with chromium oxo-hydroxides, precipitated from the waste water to eliminate the sulphate and sodium ions (SCx(II) and SCx(III) samples). Preparations (II) and (III) only differ for the accuracy in sulphate and sodium ions elimination. Catalyst surface characterization was performed by FT-IR spectroscopy using CO, adsorbed at room temperature, as test molecule, and by UV-Vis-NIR DR spectroscopy. Their activity towards ethylene polymerization were tested by FT-IR spectroscopy, monitoring the time dependence of the CH₂ stretching modes intensity. The obtained results were compared with those obtained for simplified Phillips catalysts with comparable chromium contents, SCx(R). SCx(I) catalysts were not active in the ethylene polymerization, while the SCx(II) and SCx(III) ones showed lower activity (1/10 at maximum) compared to that of simplified Phillips catalysts with comparable chromium contents. SCx(II) and SCx(III) catalysts were active in the isobutane dehydrogenation reaction and showed activity in the range of those of standard catalysts, while SCx(I) ones showed activity at 873 K, but very low at the usual temperatures (773–823 K).     

Insights into the Mechanisms of Action of MDA-7/IL-24: A Ubiquitous Cancer-Suppressing Protein

Melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24), a secreted protein of the IL-10 family, was first identified more than two decades ago as a novel gene differentially expressed in terminally differentiating human metastatic melanoma cells. MDA-7/IL-24 functions as a potent tumor suppressor exerting a diverse array of functions including the inhibition of tumor growth, invasion, angiogenesis, and metastasis, and induction of potent “bystander” antitumor activity and synergy with conventional cancer therapeutics. MDA-7/IL-24 induces cancer-specific cell death through apoptosis or toxic autophagy, which was initially established in vitro and in preclinical animal models in vivo and later in a Phase I clinical trial in patients with advanced cancers. This review summarizes the history and our current understanding of the molecular/biological mechanisms of MDA-7/IL-24 action rendering it a potent cancer suppressor.     

Comparison of activities in selective catalytic reduction of NOx by C3H8 over Co/MFI, Fe/MFI, and H/MFI zeolite catalysts

The Co/MFI(SiO₂/Al₂O₃ = 30) were prepared by a precipitation method with NaOCl in alkali solutions exhibited high activities to N₂ at 250 °C for the selective catalytic reduction (SCR) of NO_x. These catalysts showed two UV–vis bands at 700 and 400 nm, indicating the presence of octahedral Co(III) as well as tetrahedral Co(II). The high SCR activity over such Co(III, II)/MFI(30) seems to come from Co(III)---O moieties. The Co(II)MFI(30) catalysts prepared from Co(II)Cl₂ exhibited low SCR activities due to the presence of tetrahedral Co(II) ions in MFI. Less CO formation occurred over Co/MFI catalysts. The Fe/MFI(30) catalyst exhibited high activity due to the presence of some Fe---O species in MFI but more amount of CO were produced during SCR. H/MFI(30) catalyst exhibited a good SCR activity. However, more amount of carbonaceous deposits were produced on it. The correlation between acid concentration and SCR activity was discussed over H/MFIs.     

The photocatalytic efficiency of the metal doped TiO2 with ceramic foam as catalyst carriers

The metal doped TiO₂ was prepared with Fe(III), Co(II), Ni(II), Cu(II), Ag(I), La (III), Nd(III), Ho(III), and Y(III) as doped catalysts. These catalysts were carried by ceramic foams to enhance their photocatalytic efficiency, which was later studied with methylene blue (MB) and Escherichia coli (E. coli) as targets. The results suggested that the photocatalytic activities of TiO₂ were enhanced when ceramic foams were used as catalyst carriers and that the photocatalytic efficiency could also be significantly increased by the dopants, especially by Ag(I) and rare earth. In the bactericidal activity testing, the inhibitory effect of TiO₂ on E. coli was enhanced significantly when ceramic foams were used as carriers. Ag(I) doped TiO₂ showed the greatest inhibition on E. coli. As to the E. coli cells treated by Ag(I) doped TiO₂, the observation with a Scanning Electronic Microscope (SEM) suggested that the cells could no longer maintain their morphology and the spheroplasts were formed after the treatment.