Search Results (234)

Background/Objectives: RAF pathway aberrations are one of the hallmarks of lung cancer. Sorafenib is a multi-kinase inhibitor targeting the RAF pathway and is FDA-approved for several cancers, yet its efficacy in lung cancer is controversial. Previous clinical research showed that a ARAF p.S214C mutation exhibited exceptional responsiveness to sorafenib in lung adenocarcinoma. Methods: Considering this promising clinical potential, the oncogenic potential and sorafenib response of the ARAF p.S214C mutation were investigated using lung cancer models. ARAF p.S214C mutant, ARAF wild-type (WT), and EGFP control genes were ectopically expressed in lung adenocarcinoma cell lines retroviral transduction. In vitro and in vivo sorafenib sensitivity studies were performed, followed by transcriptomics and proteomics analyses. Results: Compared to the ARAF-WT and EGFP-engineered cells, the ARAF p.S214C-engineered cells activated Raf-MEK-ERK signaling and exhibited enhanced oncogenic potential in terms of in vitro cell proliferation, colony and spheroid formation, migration, and invasion abilities, as well as in vivo tumorigenicity. The ARAF p.S214C-engineered cells also displayed heightened sensitivity to sorafenib in vitro and in vivo. RNA sequencing and reverse-phase protein array analyses demonstrated elevated expression of genes and proteins associated with tumor aggressiveness in the ARAF p.S214C mutants, and its sorafenib sensitivity was likely moderated through inhibition of the cell cycle and DNA replication. The ERK and PI3K signaling pathways were also significantly deregulated in the ARAF p.S214C mutants regardless of sorafenib treatment. Conclusions: This study demonstrates the oncogenicity and sorafenib sensitivity of the ARAF p.S214C mutation in lung cancer cells, which may serve as a biomarker for predicting the sorafenib response in lung cancer patients. Importantly, investigating the gene–drug sensitivity pairs in clinically exceptional responders may guide and accelerate personalized cancer therapies based on specific tumor mutations. Full article

Background/Objectives: Melanoma is one of the most aggressive forms of skin cancer and is frequently associated with the B-Raf⁶⁰⁰E mutation, which constitutively activates the MAPK signaling pathway. Although selective inhibitors such as Vemurafenib offer clinical benefits, their long-term efficacy is often hindered by resistance mechanisms and adverse effects. In this study, twelve phytochemicals from Brazilian green propolis were evaluated for their potential as selective B-Raf⁶⁰⁰E inhibitors using a computational approach. Methods: Physicochemical, ADME, and electronic properties were assessed, followed by molecular docking using the B-Raf⁶⁰⁰E crystal structure (PDB ID: 3OG7). Redocking validation and 500 ns molecular dynamics simulations were performed to investigate the stability of the ligand-protein complexes, and free energy calculations were then computed. Results: Among the tested compounds, Artepillin C exhibited the strongest binding affinity (−8.17 kcal/mol) in docking and maintained stable interactions with key catalytic residues throughout the simulation, also presenting free energy of binding ΔG of −20.77 kcal/mol. HOMO-LUMO and electrostatic potential analyses further supported its reactivity and selectivity. Notably, Artepillin C remained bound within the ATP-binding site, mimicking several critical interactions observed with Vemurafenib. Results: Among the tested compounds, Artepillin C exhibited the strongest binding affinity (−8.17 kcal/mol) and maintained stable interactions with key catalytic residues throughout the simulation. HOMO-LUMO and electrostatic potential analyses further supported its reactivity and selectivity. Notably, Artepillin C remained bound within the ATP-binding site, mimicking several critical interactions observed with Vemurafenib. Conclusions: These findings indicate that Artepillin C is a promising natural compound for further development as a selective B-Raf⁶⁰⁰E inhibitor and suggest its potential utility in melanoma treatment strategies. This study reinforces the value of natural products as scaffolds for targeted drug design and supports continued experimental validation. Full article

Reciprocal signaling between acute myeloid leukemia (AML) cells and the surrounding bone-marrow microenvironment (BMME) promotes AML progression through several mechanisms. One of the most important mechanisms is the induction of Galectin-3 (Gal-3) expression by AML cells and bone marrow mesenchymal stromal cells (BM-MSCs). Emerging evidence indicates that Gal-3 upregulation in the BMME promotes AML cell adhesion and survival, leading to the development of chemotherapy resistance, relapse, and poor prognosis. Identifying the biological function and critical signaling pathways of Gal-3 may contribute to overcoming acquired drug resistance and preventing post-treatment relapse. Gal-3 is involved in several molecular signaling pathways, including PI3K/AKT/mTOR, Ras/Raf/MEK/ERK, JAK/STAT, JNK, Wnt/β-catenin, PLC/PKC and NF-κB, which are interconnected to promote AML cell survival and resistance to chemotherapy. This review focuses on the biological effects, molecular mechanisms of action and regulation of Gal-3 in the pathogenesis and progression of AML. The therapeutic potential of potent synthetic small-molecule Gal-3 inhibitors in high-risk patients with AML is also discussed based on preclinical and clinical evidence from several human diseases. Currently, the effect of these Gal-3 inhibitors in AML has not been investigated either in vitro or in vivo. The findings provide a rationale for targeting Gal-3 that may be a very promising therapeutic approach, especially for patients with relapsed/refractory AML, and may enhance the efficacy of conventional chemotherapeutic drugs and/or immune checkpoint inhibitors. Full article

Pediatric gliomas encompass the most common brain tumor in children and are subdivided into pediatric low-grade gliomas (pLGGs) and pediatric high-grade gliomas (pHGGs). The era of molecular diagnosis has shifted the treatment paradigms and management of these patients. RAS/MAPK pathway alterations serve as the driver in the majority of pLGGs, a subset of pHGG and NF1-related plexiform neurofibromas (PNs). The role of small molecule inhibitors in the treatment of these tumors has evolved in the past decade, facilitated through multiple clinical trials and moving into earlier stages of treatment. Although these developments hold promise, questions remain regarding targeted therapy, the long-term toxicities, the duration of treatment and the potential effects on the natural history of the tumor behavior. Full article

Colorectal cancer (CRC) is one of the most common cancers worldwide, with KRAS mutations occurring in approximately 40% of cases. These mutations drive tumorigenesis through the constitutive activation of key signaling pathways, such as RAS-RAF-MEK-ERK (MAPK) and PI3K-AKT-mTOR, contributing to therapeutic resistance and poor prognosis. Advances in molecular biology have led to significant breakthroughs, including the development of KRAS G12C inhibitors, such as sotorasib and adagrasib, which have shown promise in clinical trials. However, their efficacy is limited to a small subset of KRAS-mutant CRC, and resistance mechanisms often emerge through compensatory pathway activation. Combination strategies, including KRAS inhibitors with anti-EGFR agents, have been explored in trials like KRYSTAL-1 and CodeBreaK 300. Emerging research highlights the role of the tumor microenvironment in immune evasion and therapeutic resistance, offering opportunities for novel immunotherapy approaches, including KRAS neoantigen vaccines and adoptive T-cell therapy. Despite these advancements, challenges such as intratumoral heterogeneity, limited immune infiltration, and non-G12C KRAS mutations remain significant hurdles. This review provides a comprehensive overview of the molecular mechanisms, current advances and challenges, and future prospects in the management of KRAS-mutant CRC. Full article

Resistance to HER2 tyrosine-kinase inhibitor Lapatinib (Lap) is one of the leading causes of cancer treatment failure in HER2+ breast cancer (BC), associated with an aggressive tumor phenotype. Cryptotanshinone (Cry) is a natural terpene molecule that could function as a chemosensitizer by disturbing estrogen receptor (ERα) signaling and inhibiting the protein translation factor-4A, eIF4A. Therefore, we evaluated Cry dual regulation on eIF4A and ERα. This study aimed to elucidate the underlying mechanisms of Lap chemoresistance and the impact of Cry on them. We generated two Lap-resistant BT474 cell HER2+ variants named BT474^LapRV1 and BT474^LapRV2 with high chemoresistance levels, with 7- and 11-fold increases in EC₅₀, respectively, compared to BT474 parental cells. We found a PDCD4-p70S6Kβ axis association with Lap chemoresistance. However, a concomitant down-regulation of the RAF-MEK-ERK cell survival pathway and NF-κB was found in the chemoresistant cell variants; this phenomenon was exacerbated by joint treatment of Cry and Lap under a Lap plasmatic reported concentration. Optimized calcium management was identified as a compensatory mechanism contributing to chemoresistance, as determined by the higher expression of calcium pumps PMCA1/4 and SERCA2. Contrary to expectations, a combination of Lap and Cry did not affect the chemoresistance despite the ERα down-regulation; Cry-eIF4A binding possibly dampens this condition. Results indicated the pro-survival eIF4A/STAT/Bcl-xl pathway and that the down-regulation of the MAPK-NF-κB might function as an adaptive mechanism; this response may be compensated by calcium homeostasis in chemoresistance, highlighting new adaptations in HER2+ cells that lead to chemoresistance. Full article

Background: Oncogenic KRAS mutations are nearly ubiquitous in pancreatic ductal adenocarcinoma (PDAC), yet therapeutic attempts to target KRAS, as well as downstream MAPK pathway effectors, have shown limited clinical success. While KRAS canonically drives MAPK signaling via RAF-MEK-ERK, it is also known to play a role in PI3K-AKT signaling. Methods: Our therapeutic study targeted the PI3K pathway with the drug Omipalisib (p110α/β/δ/γ and mTORC1/2 inhibitor) in combination with two different MAPK pathway inhibitors: Trametinib (MEK1/2 inhibitor) or SHP099-HCL (SHP099; SHP2 inhibitor). Western blot analysis demonstrated that the application of Trametinib or SHP099 alone selectively blocked ERK phosphorylation (pERK) but failed to suppress phosphorylated AKT (pAKT). Conversely, Omipalisib alone successfully inhibited pAKT but failed to suppress pERK. Therefore, we hypothesized that a combination therapeutic comprised of Omipalisib with either Trametinib or SHP099 would inhibit two prominent mitogenic pathways, MAPK and PI3K-AKT, and effectively suppress PDAC growth. Results: In vitro studies demonstrated that, in several cell lines, both Omipalisib/Trametinib and Omipalisib/SHP099 combination therapeutic strategies were more effective than treatment with each drug individually at reducing proliferation, colony formation, and cell migration compared to vehicle controls. In vivo oral administration of combined Omipalisib/Trametinib treatment was significantly more effective than Omipalisib/SHP099 in reducing implanted tumor growth, and the Omipalisib/Trametinib treatment more effectively reduced tumor progression and prolonged survival in an aggressive genetically engineered mouse model of PDAC than either Omipalisib or Trametinib alone. Conclusions: Altogether, our data support a rationale for a dual treatment strategy targeting both PI3K and MAPK pathways in pancreatic cancers. Full article

B-RAF is a serine/threonine kinase that plays a crucial role in the MAPK signaling pathway, regulating cell proliferation and survival. Mutations in B-RAF, particularly V600E, are associated with several malignancies, including melanoma, colorectal cancer, and non-small cell lung cancer, making it a key therapeutic target. The development of B-RAF inhibitors, such as Vemurafenib, Dabrafenib, and second-generation inhibitors like Encorafenib, has led to significant advancements in targeted cancer therapy. However, acquired resistance, driven by MAPK pathway reactivation, RAF dimerization, and alternative signaling pathways, remains a major challenge. This review explores the molecular mechanisms of B-RAF inhibitors, their therapeutic efficacy, and resistance mechanisms, emphasizing the importance of combination strategies to enhance treatment outcomes. The current standard of care involves B-RAF and MEK inhibitors, with additional therapies such as EGFR inhibitors and immune checkpoint blockades showing potential in overcoming resistance. Emerging pan-RAF and brain-penetrant inhibitors offer new opportunities for treating refractory cancers, while precision medicine approaches, including genomic profiling and liquid biopsies, are shaping the future of B-RAF-targeted therapy. Full article

Gastrodin (gas) has been shown to promote neuroprotection and reverse Alzheimer’s disease (AD) pathology. However, its high effective dose limits its potential in treating AD. In this study, a bioassay system using PC12 cells and the nerve growth factor (NGF)-mimic effect was employed to investigate the structure–activity relationship of gas derivatives. Among the synthesized compounds, GAD037 demonstrated the highest NGF-mimic activity, surpassing gas. Additionally, GAD037 exhibited significant neuroprotective effects, reducing reactive oxygen species (ROS) and malondialdehyde (MDA) levels, thereby improving the survival of PC12 cells under oxidative stress. It also protected cells from Aβ-induced toxicity. Target protein identification and mechanistic studies revealed that insulin receptor (INSR) and alpha-actinin-4 (ACTN4) are potential targets of GAD037, confirmed through specific inhibitors, small interfering RNA (siRNA) analysis, a cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS). Moreover, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and rat sarcoma (Ras)/protooncogene serine–threonine protein kinase (Raf)/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways were found to be involved in the NGF-mimic activity of GAD037. In conclusion, GAD037 exhibits superior NGF-mimic and neuroprotective activities compared to gas, suggesting its potential as a lead compound for anti-AD applications. Full article

Colorectal cancer (CRC) is the third-most common cancer globally and a leading cause of cancer-related deaths. While the prognostic and predictive roles of RAS mutations in advanced CRC are well-established, their significance in early-stage CRC remains a topic of debate. Studies have been conducted for many years on clinical and pathological parameters that may be associated with RAS mutation, and there are inconsistent results in this regard. Currently, the only biomarker used in early-stage CRC is microsatellite status. KRAS mutations are detected in 40–50% of patients with colorectal cancer. RAS activating mutations cause loss of EGFR regulation by acting on the RAS/RAF/MAPK signaling pathways. In advanced colorectal cancer, these mechanisms cause a decrease in the effectiveness of EGFR inhibitors. However, studies on patients with early-stage colorectal cancer have inconsistent results. This review highlights the prognostic and clinical significance of KRAS mutations in early-stage CRC, particularly in MSS tumors. In the MSS group, KRAS mutations were associated with shorter TTR and OS compared to DWT patients. In contrast, in the MSI-H group, KRAS mutations showed no prognostic effect in TTR and OS. However. KRAS mutations were associated with shorter SAR in both MSI-H and MSS groups of patients. The findings underscore the need for routine molecular profiling, including KRAS and MSI status, to refine risk stratification and guide adjuvant therapy decisions. Further studies are warranted to explore targeted therapeutic approaches for KRAS-mutant CRC in the adjuvant setting. Full article

Multiple signaling pathways are dysregulated in melanoma, notably the Ras/RAF/MAPK/ERK and PI3K/AKT/mTOR pathways, which can be targeted therapeutically. The high immunogenicity of melanoma has been exploited using checkpoint inhibitors. Whilst targeted therapies and immune checkpoint inhibitors have improved the survival of patients with advanced melanoma, treatment resistance, their side effect profiles, and the prohibitive cost remain a challenge, and the survival outcomes remain suboptimal. Treatment resistance has been attributed to the presence of cancer stem cells (CSCs), a small subpopulation of pluripotent, highly tumorigenic cells proposed to drive cancer progression, recurrence, metastasis, and treatment resistance. CSCs reside within the tumor microenvironment (TME) regulated by the immune system, and the paracrine renin–angiotensin system, which is expressed in many cancer types, including melanoma. This narrative review discusses the role of CSCs and the paracrine renin–angiotensin system in the melanoma TME, and its implications on the current treatment of advanced melanoma with targeted therapy and immune checkpoint blockers. It also highlights the regulation of the Ras/RAF/MAPK/ERK and PI3K/AKT/mTOR pathways by the renin–angiotensin system via pro-renin receptors, and how this may relate to CSCs and treatment resistance, underscoring the potential for improving the efficacy of targeted therapy and immunotherapy by concurrently modulating the renin–angiotensin system. Full article

As a scaffolding protein, Raf kinase binding protein (RKIP) is involved in a variety of cellular pathways, including the Raf–MEK–ERK-cascade. It acts as a negative regulator by binding to its partners, making it an attractive target in the development of therapeutic strategies for cancer. Despite its structural stability as a monomer, RKIP may form a dimer, resulting in the switching of binding partners. It is still unclear how RKIP switches between monomeric and dimeric forms. Here, we identified the role of cysteine 133 in RKIP structural dynamics using recombinant human RKIP (rhRKIP) proteins purified from Escherichia coli BL21(DE3) cells. Mutation of alanine or serine instead of cysteine in RKIP proteins did not affect the biochemical characteristics, while dynamic light scattering and liquid chromatography (LC) quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) suggested distinct peaks in solution, which were identified via LC–MS/MS analyses, and further clarified the role of cysteine in RKIP dimerization. rhRKIP dimer formation was abrogated by a 32-aa peptide mimicking the region between two RKIP proteins for dimerization. In addition, the 32-aa peptide and its short derivatives were investigated for effects on cancer cell viability. Taken together, our findings suggest that it may be possible to regulate RKIP function by controlling its dynamics with reducing agents, which could aid the targeting of cancer cells. Full article

Background/Objectives: All 11 metallothionein protein-coding genes are located on human chromosome 16q13. It is unique among human genetics to have an entire pathway’s genes clustered in a short chromosomal region. Since solid tumors, particularly high-grade serous ovarian cancer (HGSC), exhibit high rates of monoallelic aneuploidy, this region is commonly lost. Studies have not yet been performed to determine what vulnerability may be created in cancer cells with low metallothionein expression. Here, a screen of FDA-approved cancer small molecule drugs for those best targeting low metallothionein ovarian cancer was completed. Methods: Screening methods were tested and compared using vehicle-treated negative controls and cadmium chloride, a positive control for cell loss selective for low metallothionein cells. CAOV3 cells, which are unique in their expression of only two metallothionein isoforms, were used, with or without shRNA knockdown of the predominantly expressed MT2A gene. A library of FDA-approved molecules was then screened. Results: The optimal assay utilized Hoechst 33342 nuclear staining and mechanized fluorescent microscope counting of cell content. Encorafenib, an RAF inhibitor, was identified as the most selective for enhanced cytotoxicity in MT2A knockdown cells compared to scrambled controls. Conclusions: The nuclear stain Hoechst 33342, assessed by fluorescence microscopy, provides a low variance, moderate throughput platform for cancer cell loss screens. Low metallothionein ovarian cancer cells exhibit a vulnerability to the RAF inhibitor encorafenib. Full article

Neutrophil extracellular traps (NETs) formation is a key process in inflammatory diseases like gout, but the underlying molecular mechanisms remain incompletely understood. This study aimed to establish a model to examine the formation of NETs induced by monosodium urate (MSU) and phorbol 12-myristate 13-acetate (PMA) and to elucidate their molecular pathways. Laser confocal microscopy was used to visualize NET formation, while flow cytometry was employed to detect reactive oxygen species (ROS) production. The microstructure of neutrophils was observed by transmission electron microscopy, and the expression of key proteins was determined by Western blotting. Additionally, the effect of various inhibitors targeting the MAPK signaling pathway on NET formation was evaluated. They include the Ras inhibitor Salirasib, Raf inhibitor Vemurafenib, ERK inhibitor PD98059, and p38 MAPK inhibitor SB203580, as well as NADPH oxidase inhibitor DPI and neutrophil elastase inhibitor Alvelestat. The results showed that MSU and PMA triggered significant NET formation, which was accompanied by increased ROS levels, lactate dehydrogenase release, dsDNA, and IL-8. Notably, selective MAPK pathway inhibitors and DPI and Alvelestat, except for SB203580, effectively down-regulated these indicators. These data indicated that the activation of a signaling pathway involving Ras-Raf-ERK, which is dependent on ROS, is crucial for the induction of NET formation by MSU and PMA. Given the involvement of NETs in multiple pathologies, our findings could potentially serve as molecular targets for the intervention and treatment of crystal-related diseases, especially for gout. Full article

Cancer cells undergo metabolic rewiring to support rapid proliferation and survival in challenging environments. Glutamine is a preferred resource for cancer metabolism, as it provides both carbon and nitrogen for cellular biogenesis. Recent studies suggest the potential anticancer activity of amino acid analogs. Some of these analogs disrupt cellular nucleotide synthesis, thereby inhibiting the formation of DNA and RNA in cancer cells. In the present study, we investigated the anticancer properties of Acivicin and Azaserine in the breast cancer MCF-7 cell line, comparing their effects to those on the non-tumorigenic MCF-10 epithelial cell line in vitro. Interestingly, at lower concentrations, both Acivicin and Azaserine showed potent inhibition of MCF-7 cell proliferation, as assessed by the MTT assay, without detectable toxicity to normal cells. In contrast, Sorafenib (Nexavar), a commonly used drug for solid tumors, showed harmful effects on normal cells, as indicated by increased lactate dehydrogenase (LDH) production in treated cells. Furthermore, unlike Sorafenib, treatment with Acivicin and Azaserine significantly affected apoptotic signaling in treated cells, indicating the role of both amino acid analogs in activating programmed cell death (PCD), as assessed by the Annexin-V assay, DAPI staining, and the relative expression of tumor suppressor genes PTEN and P53. ELISA analysis of MCF-7 cells revealed that both Acivicin and Azaserine treatments promoted the production of anti-inflammatory cytokines, including IL-4 and IL-10, while significantly reducing the production of tumor necrosis factor alpha (TNF-α). Mechanistically, both Acivicin and Azaserine treatment led to a significant reduction in the expression of glutamine synthetase (GS) at both the RNA and protein levels, resulting in a decrease in intracellular glutamine concentrations over time. Additionally, both treatments showed comparable effects on Raf-1 gene expression and protein phosphorylation when compared with Sorafenib, a Raf-1 inhibitor. Moreover, docking studies confirmed the strong binding affinity between Acivicin, Azaserine, and glutamine synthetase, as evidenced by their docking scores and binding interactions with the enzyme crystal. Collectively, these findings provide evidence for the anticancer activity of the two amino acid analogs Acivicin and Azaserine as antagonists of glutamine synthetase, offering novel insights into potential therapeutic strategies for breast cancer. Full article