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MSLN-mediated activation of EGFR-ERK1/2 signaling drives liver metastasis in breast cancer [HCC1806-HM3]

ABSTRACT: Introduction: Breast cancer (BC) is the most prevalent malignant disease affecting female patients globally, with triple-negative breast cancer (TNBC) being the subtype linked to the poorest clinical outcome. The liver is a frequent metastatic site of breast cancer. Therefore, elucidating the mechanism underlying liver metastasis in TNBC is crucial for identifying effective diagnostic and therapeutic targets, which holds significant potential for guiding clinical treatment. Objectives: To identify the key gene driving liver metastasis in breast cancer, investigate its mechanism of action, and advance breast cancer treatment. Methods: RNA sequencing was conducted on 4T1-HM3 and its corresponding primary tumor cell 4T1-Pri to identify and screen genes involved in liver metastasis. The enhanced MSLN, a gene encoding mesothelin, was identified in metastatic TNBC cells and tissues using qRT-PCR, Western blotting, and immunohistochemistry. Dual-luciferase reporter assays, chromatin immunoprecipitation (ChIP) analysis, and bioinformatics were employed to identify transcription factors regulating MSLN. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), co-immunoprecipitation (co-IP), and immunofluorescence (IF) co-localization were utilized to investigate the mechanism of MSLN action in BC cells. Cellular viability was assessed using a colony formation assay. Cell proliferation and chemosensitivity were assessed using the CCK8 assay. Results: MSLN exhibits aberrantly overexpression in liver-metastatic cells. Elevated MSLN levels are significantly associated with liver metastasis compared to metastases in other organs. MSLN promotes cell proliferation and survival in vitro and TNBC liver metastasis in vivo. Mechanistically, MSLN binds to epidermal growth factor receptor (EGFR) and activates the EGFR-ERK1/2 signaling axis, thereby promoting TNBC cell survival and proliferation during metastasis. Notably, targeting MSLN with a paclitaxel/carboplatin combination can efficiently inhibit liver metastasis of hepatotropic TNBC in a mouse model.

ORGANISM(S): Homo sapiens

PROVIDER: GSE306126 | GEO | 2026/01/14

REPOSITORIES: GEO

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Project description:Metabolic rewiring is essential for breast tumor growth and progression to metastatic disease, yet little is known regarding how cancer cells modify their acquired metabolic programs in response to different metastatic microenvironments. Transcriptional and metabolomic analysis have previously shown that liver-metastatic breast cancer cells adopt an intrinsic metabolic profile characterized by increased HIF-1α activity and dependence on glycolysis. Stable isotope tracing analysis (SITA) performed in vivo confirmed that the glycolytic nature of liver-metastatic breast cancer cells is retained when these cells are grown as primary tumors or as liver metastases. However, our data also reveal that unique metabolic adaptations are specifically induced by the liver microenvironment. Indeed, liver metastases display elevated expression of genes involved in glutathione (GSH) biosynthesis and reactive oxygen species (ROS) detoxification when compared to primary tumors. Moreover, breast cancer liver metastases rely strongly on glucose and glutamine-derived carbons to support de novo GSH synthesis. Glutathione is a tripeptide that acts as a major scavenger for reactive oxygen species (ROS). Liver metastases display reduced levels of 8-Oxo-2'-deoxyguanosine, confirming their increased capacity to buffer ROS. We demonstrated the dependence of liver metastases formation on these distinct metabolic adaptations by depleting glutamate-cysteine ligase (Gcl), the rate-limiting enzyme in glutathione biosynthesis. Gcl depletion, and decreased GSH levels, strongly reduced the capacity of liver-metastatic cells to form distant metastasis within the liver. We performed gene expression analysis of liver metastasis and primary tumors from liver-metastatic breast cancer cells derived from 4T1 cells. 4T1-2776 (76) and 4T1-2792 (92) cells were injected in the mammary fat pads of BALB/c and grown as primary tumors or were grown as liver metastasis after splenic injection. Tissues were harvested from primary tumors and liver metastasis and submitted to laser-capture microdissection (LCM). For liver metastasis, tumour tissue was harvested at 10 days, 2 weeks and 3 weeks after splenic injection. For each time point, tissue was isolated from the tumor centre (core), a peripheral area of the tumor (margin), a region of the liver proximal to the tumor (adjacent) and a region of the liver far from the tumor (distant). For primary tumors from 4T1-2776 cells (1034, 1036, 1043) and 4T1-2792 (MFP2L, MFP3L, MFP5L) tissue was collected from the tumor core and margin, at the experimental endpoint.

Project description:Background: More than 90% mortality of triple negative breast cancer (TNBC) patients are attributed to cancer metastasis with organotropism. Lung is the frequent site of TNBC metastasis. However, the precise mechanism of lung-specific metastasis of TNBC, particularly the driving factors, is not well understood. Methods: RNA sequencing was performed to identify patterns of gene expression associated with lung metastatic behavior using 4T1-LM3, 231-LM3 and their parental cells (4T1-P, 231-P). Expressions of RGCC, encoding the completion 32 protein response protein, were detected in TNBC cells and tissues by qRT-PCR, western blotting and IHC. Kinase activity assay were performed to evaluated PLK1 kinase activity and the amount of phosphorylated AMP-activated protein kinase α2 (AMPKα2). RGCC-mediated metabolism was determined by UHPLC system. Oxidative phosphorylation was evaluated by JC-1 staining and oxygen consumption rate (OCR) assay. Fatty acid oxidation assay, NADPH and ROS was applied to measure the status of RGCC-mediated fatty acid oxidation. The chemical sensitivity of cells was evaluated by CCK8 assay. Results: RGCC is aberrantly upregulated in pulmonary metastatic cells. High level of RGCC is significantly related with lung metastasis rather than other organ metastases. RGCC can effectively promote the kinase activity of PLK1, and activated PLK1 phosphorylates AMPKα2 to facilitate TNBC lung metastasis. Mechanistically, Survival and colonization of TNBC cells in the lungs by upregulating oxidative phosphorylation of mitochondria to obtain more energy and promoting fatty acid oxidation to produce abundant NADPH and sustains redox homeostasis, thus prevents excessive accumulation of potentially detrimental ROS in metastatic tumor cells, ultimately establishing metastases. Importantly, targeting RGCC in combination with paclitaxel/carboplatin effectively suppresses pulmonary metastases of TNBC in a mouse model. Conclusions: The enhanced RGCC is closely associated with lung-specific metastasis of TNBC. RGCC leads to activation of AMPKα2 and downstream signaling through RGCC-driven PLK1 activity to facilitate TNBC lung metastasis, which may determine a potential value of RGCC-driven OXPHOS and fatty acid oxidation as an important therapeutic target.

Project description:Abstract Background. Epidermal growth factor receptor (EGFR) is a targetable molecule in basal-like breast cancer, which comprises most “triple negative” breast cancer (TNBC), the only breast cancer subtype without established targeted therapy. Methods. In this randomized phase II trial, metastatic TNBC patients received the anti-EGFR antibody cetuximab (250mg/kg/week iv) with carboplatin (AUC2/wk iv) added on progression, or concomitant cetuximab + carboplatin. Molecular subtyping was done on archival specimens and those with accessible tumors provided fresh tissue, before and after 7-14 days of therapy, for microarray analyses to explore EGFR pathway inhibition. Results. Of 102 TNBC patients 74% were of the basal-like molecular subtype. Response rate to cetuximab was 6% (2/31), and was 16% (4/25) to cetuximab + carboplatin after progression. Upfront cetuximab + carboplatin produced responses in 17% (12/71); 31% responded or had prolonged disease stabilization. Time to progression was 2.1 months (95% CI 1.8-5.5) and overall survival 10.4 months (95% CI 7.7-13.1) for those treated with the combination regimen. Among 16 patients with evaluable serial biopsies, genomic patterns of the EGFR pathway showed activated status in 13 and inhibition by therapy in 5. Conclusions. While most TNBC were basal-like, a significant proportion were different subtypes. The aggressive nature of metastatic TNBC leads to limited survival. Despite a promising preclinical rationale and evidence of EGFR pathway activation in most, targeted treatment with cetuximab as a single agent had marginal activity and cetuximab added to carboplatin demonstrated modest activity. Serial biopsies as part of metastatic breast cancer studies are feasible, and this study confirmed that the EGFR pathway was inhibited by therapy in only a minority suggesting ligand-independent activation in most tumors. Tissue acquisition and drug selection based upon individualized pathway activation status should be an important part of future studies of TNBC. This series contains 36 microarrays that are from 18 patients with fresh frozen tissue available from the metastatic site. Pretreatment samples (Bx0) are available for two patients. Pretreatment and post single agent treatment (7-14 days after start of treatment with cetuximab; BxSingle) are available for three patients. Pretreatment and post combination treatment (7-14 days after start of treatment with cetuximab plus carboplatin; BxCombo) are available for eight patients. Two patients have a pretreatment sample, a sample after 7-14 days of treatment with single agent cetuximab, and a third sample 7-14 days on cetuximab plus carboplatin after switching to the combination arm upon progression on the single agent arm. Samples were hybridized with Stratagene common reference spiked with RNA from two breast cancer cell lines (ME16C and MCF-7) on Custom 1x44K Agilent microarrays. Arrays were scanned on an Axon 4000B scanner and analyzed with GenePix Pro software.

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MSLN-mediated activation of EGFR-ERK1/2 signaling drives liver metastasis in breast cancer [HCC1806-HM3]

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