Dataset Information

Nuclear Localization of BRAF^V600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells.

ABSTRACT:

Background

Previously, we have demonstrated that nuclear BRAF^V600E is associated with melanoma aggressiveness and vemurafenib resistance. However, the underlying mechanisms of how nuclear localization of BRAF^V600E promotes cell aggressiveness have not yet been investigated. Despite therapeutic advancements targeting cutaneous melanoma, unknown cellular processes prevent effective treatment for this malignancy, prompting an urgent need to identify new biological targets. This study aims to explore the association of inducible heme oxygenase 1 (HMOX-1) with nuclear BRAF^V600E in promoting melanoma aggressiveness.

Methods

Proteomics analysis was performed to identify the interacting partner(s) of nuclear BRAF^V600E. Immunohistochemistry was applied to evaluate the levels of HMOX-1 and nuclear BRAF^V600E expression in melanoma and adjacent healthy tissues. Immunofluorescence assessed the nuclear localization of BRAF^V600E in vemurafenib-resistant A375R melanoma cells. Further study of HMOX-1 knockdown or BRAF^V600E overexpression in melanoma cells suggested a role for HMOX-1 in the regulation of cell proliferation in vivo and in vitro. Finally, Western blot analysis was performed to confirm the pathway by which HMOX-1 mediates Akt signaling.

Results

Proteomics results showed that HMOX-1 protein expression was 10-fold higher in resistant A375R cells compared to parental counterpart cells. In vitro and in vivo results illustrate that nuclear BRAF^V600E promotes HMOX-1 overexpression, whereas HMOX-1 reduction represses melanoma cell proliferation and tumor growth. Mechanistic studies revealed that HMOX-1 was associated with nuclear BRAF^V600E localization, thus promoting melanoma proliferation via a persistent activation of the AKT pathway.

Conclusions

Our results highlight a previously unknown mechanism in which the nuclear BRAF^V600E/HMOX-1/AKT axis plays an essential role in melanoma cell proliferation. Targeting HMOX-1 could be a novel method for treating melanoma patients who develop BRAF inhibitor resistance.

SUBMITTER: Zerfaoui M

PROVIDER: S-EPMC8773521 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BACKGROUND: In breast cancer cells, the metastatic cell state is strongly correlated to epithelial-to-mesenchymal transition (EMT) and the CD44+/CD24- stem cell phenotype. However, the MCF-7 cell line, which has a luminal epithelial-like phenotype and lacks a CD44+/CD24- subpopulation, has rare cell populations with higher Matrigel invasive ability. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression? METHODS: Throughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Comparative analysis of epithelial and mesenchymal marker expression was performed between parental MCF-7, selected MCF-7-14, and aggressive mesenchymal MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially expressed genes for their invasive potential, and performed pathway and network analysis to identify a set of interesting genes, which were evaluated by RT-PCR, flow cytometry or function-blocking antibody treatment. RESULTS: MCF-7-14 cells had enhanced migratory and invasive ability compared with MCF-7 cells. Although MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin but neither vimentin nor fibronectin, beta-catenin was expressed not only on the cell membrane but also in the nucleus. Furthermore, using gene expression profiles of MCF-7, MCF-7-14 and MDA-MB-231 cells, we demonstrated that MCF-7-14 cells have alterations in signaling pathways regulating cell migration and identified a set of genes (PIK3R1, SOCS2, BMP7, CD44 and CD24). Interestingly, MCF-7-14 and its invasive clone CL6 cells displayed increased CD44 expression and downregulated CD24 expression compared with MCF-7 cells. Anti-CD44 antibody treatment significantly decreased cell migration and invasion in both MCF-7-14 and MCF-7-14 CL6 cells as well as MDA-MB-231 cells. CONCLUSIONS: MCF-7-14 cells are a novel model for breast cancer metastasis without requiring constitutive EMT and are categorized as a "metastable phenotype", which can be distinguished from both epithelial and mesenchymal cells. The alterations and characteristics of MCF-7-14 cells, especially nuclear beta-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.

Project description:BackgroundSuperficial spreading melanomas (SSMs) are the most common type of melanoma and cause the majority of skin cancer deaths. More than 50% of cases harbor a mutation in the BRAF gene that activates the mitogen-activated protein kinase (MAPK) cancer signaling pathway. BRAFV600E is the most common BRAF mutation, and it represents an important biomarker that guides treatment selection. However, the relationship between the BRAFV600E gene expression and intratumoral protein distribution, on one side, and clinicopathological factors and patient outcomes, on the other, is not fully described. Additionally, whether MAPK cancer signaling activation in melanoma is due to increased biochemical activity of BRAFV600E, increased mRNA levels, or both requires further investigation. Here, we addressed these questions by examining expression patterns of BRAFV600E in primary treatment-naive melanomas and correlating them to clinicopathological factors and patient outcomes.MethodsIn 166 SSM cases, we performed immunohistochemical staining to investigate the protein expression of BRAFV600E, and we measured BRAF mRNA levels using NanoString nCounter system.ResultsNinety-seven (49%) melanomas stained positive for BRAFV600E, with nearly 100% intratumoral homogeneity observed. Positive BRAFV600E expression was significantly associated with nonrecurrent disease and was found to be an independent predictor of better prognosis in univariate and multivariable analyses. Furthermore, presence of tumor-infiltrating lymphocytes, sentinel lymph node biopsy negativity, and low Breslow thickness were all independent predictors of better prognosis. We observed no difference in the BRAF mRNA levels in BRAFV600E-negative and BRAFV600E-positive melanomas, respectively. Validation in a larger publicly available cohort confirmed that there is only a weak correlation (Spearman 0.4) between BRAFV600E mRNA and protein levels and no differences in mRNA between BRAFV600E mutated and non-mutated patients.ConclusionOur findings indicated that BRAFV600E is homogeneously present throughout the whole tumor and is associated with nonrecurrent disease and better survival in primary melanoma. We also showed that BRAFV600E mutation does not result in higher transcriptional levels, suggesting that activation of the MAPK signaling pathway in BRAFV600E mutated patients can be attributed to the increased biochemical activity caused by the mutation.

Project description:The Hedgehog signaling pathway is one of the most dysregulated pathways in human cancers. The glioma-associated oncogene homolog 1 (GLI1) transcription factor is the terminal effector of the Hedgehog pathway, frequently activated in human breast cancer and an emerging target of breast cancer therapy. While somatic mutations in the human GLI1 gene have never been reported in any cell or tumor type, we recently uncovered the existence of a novel alternatively spliced, truncated GLI1 (tGLI1) that has an in-frame deletion of 41 codons spanning the entire exon 3 and part of exon 4 of the GLI1 gene. Using glioblastoma models, we showed that tGLI1 has gained the ability to promote glioblastoma migration and invasion via its gain-of-function transcriptional activity. However, the pathological impact of tGLI1 on breast cancer remains undefined. Here, we report that tGLI1 is frequently expressed in human breast cancer cell lines and primary specimens we have examined to date, but is undetectable in normal breast tissues. We found for the first time that tGLI1, but not GLI1, binds to and enhances the human vascular endothelial growth factor-A (VEGF-A) gene promoter, leading to its upregulation. Consequently, tGLI1-expressing MDA-MB-231 breast cancer cells secret higher levels of VEGF-A and contain a higher propensity, than the isogenic cells with control vector and GLI1, to stimulate in vitro angiogenesis of human vascular endothelial cells. We further showed that tGLI1 has gained the ability to enhance the motility and invasiveness of breast cancer cells in a proliferation-independent manner and that this functional gain is associated with increased expression of migration/invasion-associated genes, CD24, MMP-2 and MMP-9. tGLI1 has also acquired the property to facilitate anchorage-independent growth of breast cancer cells. Collectively, our results define tGLI1 as a gain-of-function GLI1 transcription factor and a novel mediator of the behavior of clinically more aggressive breast cancer.

Dataset Information

Nuclear Localization of BRAF^V600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells.

Background

Methods

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Nuclear Localization of BRAFV600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells.

Background

Methods

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Nuclear Localization of BRAF^V600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells.