CUEDC1 promotes glycolytic metabolism reprogramming through the CUEDC1/CACNG4/PI3K axis to promote ER-positive breast cancer growth

Background The reprogramming of energy metabolism, particularly glycolysis, upholds the malignancy of tumors. The relationship between CUE Domain Containing protein-1 (CUEDC1) and glycolysis, along with its influence on the development of ER-positive breast cancer (ER+ BRCA), is not well defined. This investigation explores the functional involvement of CUEDC1 in glycolysis regulation and uncovers a previously unidentified pathway contributing to the progression of ER+ BRCA. Methods Immunohistochemistry, western blotting and qRT-PCR were used to detect the expression of CUEDC1 in ER+ BRCA tissues and cell lines. A series of molecular analyses, including dual-luciferase reporter assays, RNA-seq, and ChIP, were performed to elucidate the potential mechanisms underlying CUEDC1's involvement in ER+ BRCA progression. Metabolic assays focusing on glycolysis were employed to investigate the functional roles of CUEDC1 and Calcium Voltage-Gated Channel Auxiliary Subunit Gamma 4 (CACNG4). The Connectivity Map (CMap) database was utilized to screen CUEDC1 inhibitors. Results CUEDC1 is aberrantly upregulated in ER+ BRCA tissues and cells. Increased CUEDC1 can promote enhanced tumor growth and lead to poor clinical outcomes in ER+ BRCA patients. The MYC associated zinc finger protein (MAZ) can up-regulate CUEDC1 gene transcription in ER+ BRCA cells by directly binding to its promoter. We determined that CUEDC1 directly modulated CACNG4 to enhance PI3K/AKT pathway activation, thereby facilitating tumor growth of ER+ BRCA. Clinical observations also revealed a positive correlation between CACNG4 expression and CUEDC1, with both factors being strongly associated with poor prognosis in ER+ BRCA patients. Mechanistically, The CUEDC1/CACNG4/PI3K signal axis enhanced glycolysis through upregulating GLUT1, a crucial protein in glucose metabolism, thereby supporting tumor growth of ER+ BRCA. Furthermore, methotrexate was identified as a potential inhibitor of CUEDC1. Importantly, the combination of Ipatasertib (a PI3K/AKT pathway inhibitor), with methotrexate effectively suppressed growth of ER+ BRCA in a mouse model. Conclusions Our research reveals that the enhanced CUEDC1 is intricately linked to the proliferation of ER+ BRCA. CUEDC1-driven CACNG4 expression stimulates PI3K/AKT pathway activity and its downstream signaling, which facilitates tumor progression and positions CUEDC1-driven glycolysis as a key therapeutic target in addressing this malignancy. Overall design: I. Sample Information for Transcriptome Sequencing (RNA-seq) This study performed transcriptome sequencing on the estrogen receptor-positive breast cancer (ER+ BRCA) cell line T47D. The specific sample grouping and design are as follows: Sample Type: In vitro cultured ER+ BRCA cell line (T47D). The core reason for selecting this cell line is its high endogenous CUEDC1 expression level (verified by Western blotting and qRT-PCR), which makes it suitable for investigating the downstream regulatory mechanism of CUEDC1. Grouping Setup: A total of 2 groups, with 3 biological replicates in each group (to ensure data reproducibility and reliability): Control Group (shNC Group): T47D cells stably transfected with non-targeting short hairpin RNA (shRNA) lentivirus, serving as the negative control. CUEDC1 Knockdown Group (shCUEDC1 Group): T47D cells stably transfected with CUEDC1-specific shRNA lentivirus to reduce CUEDC1 expression. The knockdown efficiency was verified by qRT-PCR and Western blotting, showing that the levels of CUEDC1 mRNA and protein were reduced by > 70%. II. Analysis Content Based on Transcriptome Sequencing Data Focusing on the transcriptomic differences between CUEDC1-knockdown and control T47D cells, the study conducted the following analyses: 1. Screening of Differentially Expressed Genes (DEGs) Differential analysis of the sequencing data was performed using the DESeq2 tool. With the screening criteria of |log2 fold change (FC)| > 1 and adjusted P-value < 0.05, a total of 154 differentially expressed genes were identified: upregulated genes accounted for 50, while downregulated genes accounted for 104. 2. Identification of Key Downstream Target Genes of CUEDC1 Core target genes were identified through "intersection analysis of differentially expressed genes and CUEDC1-related genes": first, a gene set related to CUEDC1 expression in breast cancer was extracted from the UALCAN database; then, overlap analysis was conducted between this gene set and the aforementioned 154 DEGs. Finally, CACNG4 (Calcium Voltage-Gated Channel Auxiliary Subunit ?4) was determined as the key downstream target gene of CUEDC1. After CUEDC1 knockdown, the mRNA and protein levels of CACNG4 were significantly downregulated; conversely, CUEDC1 overexpression significantly upregulated CACNG4. 3. Functional Pathway Enrichment Analysis (KEGG Analysis) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on the differentially expressed genes using the clusterProfiler tool. Key findings are as follows: the PI3K/AKT signaling pathway was significantly enriched (P < 0.05), suggesting that CUEDC1 may affect the biological functions of ER+ BRCA by regulating this pathway; combined with subsequent verification experiments (detection of PI3K, AKT, and phosphorylated AKT (p-AKT) levels by Western blotting), it was further confirmed that CUEDC1 activates the PI3K/AKT pathway through its target gene CACNG4. 4. Correlation Analysis of Glycolysis-Related Genes Based on the research background that "tumor metabolic reprogramming (especially enhanced glycolysis) promotes the malignant progression of ER+ BRCA", genes related to glucose metabolism among the differentially expressed genes were analyzed: first, key genes involved in glucose metabolism (such as GLUT1, LDHA, HK2, etc.) were screened out; correlation analysis revealed that GLUT1 (Glucose Transporter 1) had the strongest positive correlation with CACNG4 expression (verified by the TCGA database and confirmed by qRT-PCR and Western blotting experiments); combined with functional experiments (detection of glucose uptake, lactate production, and extracellular acidification rate (ECAR)), it was confirmed that the CUEDC1/CACNG4/PI3K axis promotes the glycolysis process of ER+ BRCA cells by upregulating GLUT1.