Breast Cancer Cell Secretome Analysis to Decipher miRNA Tumor Biology and Discover Potential Biomarkers

Feser, Riley; Opperman, Reid Morgan; Nault, Braydon; Maiti, Sujit; Chen, Vincent; Majumder, Mousumi

doi:10.21203/rs.3.rs-1328838/v1

Cited by 3 publications

(5 citation statements)

References 42 publications

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“…A data curation pipeline was applied, consisting of a ± 1.5 log2 threshold, exclusion of proteins not found in the breast proteome, and secretome prediction methods. We previously reported two members of the 14-3-3 family, YWHAB (14-3-3 Beta) and SFN (14-3-3 Sigma), high in miRNA-high cell secretions, however, we did not validate the function of either in breast cancer (9).…”

Section: -3-3 Protein Family Expression In Mcf7-mir526b and Mcf7-mir6...contrasting

confidence: 82%

“…Using previously generated liquid chromatography-mass spectrometry (LC-MS) data (9), secretory protein expression of all isoforms within the 14-3-3 family of proteins was isolated. Comparisons were made between low miRNA MCF7-Parental and miRNA overexpressed cell lines MCF7-miR526b and MCF7-miR655.…”

Section: Mass-spec Datamentioning

confidence: 99%

“…To further investigate how these oncogenic miRNAs can alter the tumour microenvironment to promote metastasis, a secretome analysis was conducted by comparing miRNA-high and miRNA-low cells. This identi ed several secretory proteins differentially regulated that could drive these phenotypes (9). One of the identi ed proteins, tyrosine 3monooxygenase/tryptophan 5-monooxygenase activation protein beta (YWHAB), was upregulated in the miRNA-high cell secretome.…”

Section: Introductionmentioning

confidence: 99%

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Investigating the Roles of YWHAB in Breast Cancer

Winstone,

Gatien,

GOPAUL

et al. 2024

Preprint

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Background: miR-526b and miR-655 have been shown to promote aggressive breast cancer phenotypes. Analysis of cell-free secretions of MCF7-miRNA-high cells identified eight differentially secreted proteins, including 14-3-3 Beta (YWHAB). Here, we investigated the roles of YWHAB in breast cancer and tested its potential as a biomarker. Methods: Breast tumor cell lines MCF7, SKBR3, Hs578T, MCF7-COX2 and stable miRNA-overexpressing MCF7-miR526b, MCF7-miR655, SKBR3-526b cells were used in vitro assays including mRNA, protein expression, and functional assays. In silico data was used to support our findings by identifying potential links between miRNAs and YWHAB and to test diagnostic and prognostic biomarker potential. Biomarker potential was validated using breast tumor biopsy tissue and plasma samples. Results: YWHAB expression is significantly upregulated in miRNA-overexpressing cell lines both at total RNA and secretory RNA levels. These miRNAs have previously been shown to increase cell migration. Following YWHAB-KD cell migration and proliferation decreased, E-Cad expression increased, and Vimentin decreased, evidently showing YWHAB involved in EMT. In silico data showed increased expression of YWHAB mRNA in breast cancer biopsy tissue and blood plasma and increased YWHAB protein in breast tumors. High expression of YWHAB is associated with poor breast cancer patient survival. YWHAB expression was measured in breast tissues and blood plasma and found to be significantly high in all advanced stages and hormonal subtypes of tumors, compared to control tissue. YWHABshowed high sensitivity as a tumour biomarker (AUC of 0.7340, p = 0.0012) and in combination with pri-miR526b showed strong potential as a blood biomarker (AUC of 0.711, p = 0.032). Conclusion: High expression of YWHAB is associated with poor survival. It can be used as a prognostic marker, and therapeutic target in aggressive cancers to mitigate cell migration. In combination with pri-miR526b, YWHAB is a promising blood biomarker for breast cancer detection.

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Section: -3-3 Protein Family Expression In Mcf7-mir526b and Mcf7-mir6...contrasting

confidence: 82%

Section: Mass-spec Datamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Investigating the Roles of YWHAB in Breast Cancer

Winstone,

Gatien,

GOPAUL

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…(13,26) Finally, we discovered markers linked with cell metabolism, such as ATP5A1, TXNDC12, and SFN, in the miRNA-high tumor cell secretion. (11) Therefore, miR-526b could be linked with increased cellular metabolism and might be a key regulator of metabolic plasticity in breast cancer to sustain growth and survival. We conducted this research to understand how miR-526b promotes glucose metabolism and metabolic exibility and how these processes t within the complex and heterogeneous tumor microenvironment.…”

Section: Discussionmentioning

confidence: 99%

“…When normal endothelial cells were treated with miRNA-high cell secretions, they showed pre-metastatic phenotypes. (9,11) The primary transcript of miR-526b (pri-miR-526b) can be detected in human blood plasma. It is a proven biomarker that can distinguish malignant tumor plasma from benign tumor plasma as early as stage I.…”

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confidence: 99%

miR-526b dysregulates glucose metabolism via the COX2/EP4 pathway

Nault,

Majumder

2024

Preprint

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Introduction: Breast cancer (BRCA) remains a primary global health concern, with ongoing research focused on early detection and improving treatment methods. Moving forward, it is crucial to understand cancer cell metabolism and its impact on tumor growth and metastasis. This study aims to identify potential BRCA markers related to glucose metabolism for targeted therapy, focusing on the role of miR-526b. miR-526b promotes BRCA phenotypes, including migration, invasion, hypoxia, angiogenesis, and metastasis. Further, cell-free secretion of miR-526b-high BRCA tumor cells can alter the tumor microenvironment. This study will investigate the role of miR-526b in the dysregulation of glucose metabolism. Methods: We used two immortalized BRCA cell lines, MCF7 and SKBR3, and stable miR-526b overexpressed cells MCF7-miR526b and SKBR3-miR526b and a naturally miR-526b high cell line MCF7-COX2 for in vitro assays. We measured ATP production, oxygen consumption rate, and extracellular acidification rate. We used glycolysis and OXPHOS inhibitors to measure metabolic plasticity induced by miR-526b. A COX-2 inhibitor and EP4 antagonist were used to alter miR-526b-induced functions. For RNA and protein measurement, we used qRT-PCR and western blots. In silico analysis with online datasets validated our findings in human BRCA. Results: In silico analysis showed that genes related to glycolysis and oxidative phosphorylation (OXPHOS) are enriched in human breast cancer tissues. Overexpression of miR-526b promotes cell proliferation and ATP production. It also contributes to the upregulation of LDHA and PDHA1, which determine the fate of the glycolytic product pyruvate, either producing lactate or entering the TCA cycle to promote OXPHOS. miR-526b overexpressing cells demonstrated increased metabolic plasticity and decreased adverse effects following treatment with glycolysis and OXPHOS inhibitors, showing increased survival and proliferation. The metabolic dysregulation induced by miR-526b, including increased proliferation, ATP production, and marker expression, can be reversed using a COX2 inhibitor and EP4 antagonist. Conclusion: miR-526b promotes increased glucose metabolism and ATP production, supporting increased growth and division of BRCA cells. It also increases metabolic plasticity, improving cells' ability to thrive in a complex and heterogeneous tumor microenvironment. The dysregulation observed with miR-526b can be reversed by targeting the COX2/EP4 pathway.

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