Identification of prognostic cancer-associated fibroblast markers in luminal breast cancer using weighted gene co-expression network analysis.

Background: Cancer-associated fibroblasts (CAFs) play a pivotal role in cancer progression and are known to mediate endocrine and chemotherapy resistance through paracrine signaling. Additionally, they directly influence the expression and growth dependence of ER in Luminal breast cancer (LBC). This study aims to investigate stromal CAF-related factors and develop a CAF-related classifier to predict the prognosis and therapeutic outcomes in LBC. Methods: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were utilized to obtain mRNA expression and clinical information from 694 and 101 LBC samples, respectively. CAF infiltrations were determined by estimating the proportion of immune and cancer cells (EPIC) method, while stromal scores were calculated using the Estimation of STromal and Immune cells in MAlignant Tumors using Expression data (ESTIMATE) algorithm. Weighted gene co-expression network analysis (WGCNA) was used to identify stromal CAF-related genes. A CAF risk signature was developed through univariate and least absolute shrinkage and selection operator method (LASSO) Cox regression model. The Spearman test was used to evaluate the correlation between CAF risk score, CAF markers, and CAF infiltrations estimated through EPIC, xCell, microenvironment cell populations-counter (MCP-counter), and Tumor Immune Dysfunction and Exclusion (TIDE) algorithms. The TIDE algorithm was further utilized to assess the response to immunotherapy. Additionally, Gene set enrichment analysis (GSEA) was applied to elucidate the molecular mechanisms underlying the findings. Results: We constructed a 5-gene prognostic model consisting of RIN2, THBS1, IL1R1, RAB31, and COL11A1 for CAF. Using the median CAF risk score as the cutoff, we classified LBC patients into high- and low-CAF-risk groups and found that those in the high-risk group had a significantly worse prognosis. Spearman correlation analyses demonstrated a strong positive correlation between the CAF risk score and stromal and CAF infiltrations, with the five model genes showing positive correlations with CAF markers. In addition, the TIDE analysis revealed that high-CAF-risk patients were less likely to respond to immunotherapy. Gene set enrichment analysis (GSEA) identified significant enrichment of ECM receptor interaction, regulation of actin cytoskeleton, epithelial-mesenchymal transition (EMT), and TGF-ß signaling pathway gene sets in the high-CAF-risk group patients. Conclusion: The five-gene prognostic CAF signature presented in this study was not only reliable for predicting prognosis in LBC patients, but it was also effective in estimating clinical immunotherapy response. These findings have significant clinical implications, as the signature may guide tailored anti-CAF therapy in combination with immunotherapy for LBC patients.

Cytotoxic indole alkaloids and polyketides produced by a marine-derived fungus Aspergillus flavipes DS720.

Marine-derived microorganisms possess the unique metabolic pathways to produce structurally novel secondary metabolites with potent biological activities. In this study, bioactivity-guided isolation of the marine deep-sea-derived fungus Aspergillus flavipes DS720 led to the characterization of four indole alkaloids (compounds 1-4) and four polyketides (compounds 5-8), such as two new indoles, flavonoids A (1) and B (2) with a C-6 reversed prenylation, and a new azaphilone, flaviazaphilone A (5). Their chemical structures were unambiguously established by an extensive interpretation of spectroscopic data, such as 1D/2D NMR and HRESIMS data. The absolute configurations of the new compound 5 were solved by comparing the experimental and calculated Electronic Circular Dichroism (ECD) spectra. Since sufficient amount of flavonoids A (1) was obtained, 1 was subjected to a large-scale cytotoxic activity screening against 20 different human tumor cell lines. The results revealed that 1 showed broad-spectrum cytotoxicities against HeLa, 5637, CAL-62, PATU8988T, A-375, and A-673 cell lines, with the inhibition rates of more than 90%. This study indicated that the newly discovered indole alkaloid 1 may possess certain potential for the development of lead compounds in the future.

Characterization and prognosis of estrogen receptor-positive/progesterone receptor-negative male breast cancer: a population-based study.

BACKGROUND: The aim of this study was to explore the characteristics and prognostic information of estrogen receptor-positive/progesterone receptor-negative (ER+/PR-) male breast cancer. METHODS: Using the US National Cancer Institute's Surveillance, Epidemiology, and End Results database, we compared the demographics, clinical characteristics, and outcome of estrogen receptor-positive/progesterone receptor-positive (ER+/PR+) patients with ER+/PR- male breast cancer patients from 1990 to 2010. Two thousand three hundred twenty-two patients with ER+/PR+ tumors and 355 patients with ER+/PR- tumors were included in our study. RESULTS: ER+/PR- patients were younger (P = 0.008) and more likely to be African American (P < 0.001) while presented with higher histological grade (P < 0.001), larger tumor size (P = 0.010), and more invasion to the lymph nodes (P = 0.034) and distant sites (P < 0.001), thus later stage (P = 0.001). Despite higher chance of receiving chemotherapy (51.0% vs 36.5%, P < 0.001), ER+/PR- patients experienced significantly worse breast cancer-specific survival (BSCC) (P < 0.001) and shorter overall survival (OS) (P = 0.003). Multivariate Cox model confirmed that tumor size, lymph node invasion, metastasis, and surgery were independent prognostic factors of both BSCC and OS for ER+/PR- male breast cancer. Age at diagnosis and chemotherapy were significantly associated with OS but not with BSCC. CONCLUSION: ER+/PR- male breast cancer was more aggressive and experienced shorter survival than ER+/PR+ patients. The prognosis was mainly associated with tumor size, lymph node invasion, metastasis, and surgery.

Decreased expression of SOX17 is associated with tumor progression and poor prognosis in breast cancer.

The SOX17 (SRY-related HMG-box) transcription factor is involved in a variety of biological processes and is related to the tumorigenesis and progression of multiple tumors. However, the clinical application of SOX17 for breast cancer prognosis is currently limited. The aim of this study was to investigate the clinicopathologic and prognostic significance of SOX17 expression in human breast cancer. qPCR and western blot assays were performed to measure the expression of SOX17 in breast cancer cell lines and 30 matched pairs of breast cancer and corresponding noncancerous tissues. A SOX17 overexpression cell model was used to examine changes in cell growth in vitro. Immunohistochemical analyses were performed to retrospectively examine the prognostic impact of SOX17 expression in 187 additional breast cancer patients. Our results showed that SOX17 expression was decreased at both the messenger RNA (mRNA) and protein levels in the breast cancer cell lines and tissues, and that SOX17 overexpression could strongly suppress cell growth in vitro. Furthermore, the lack of SOX17 protein expression was strongly correlated with higher tumor grade (P = 0.002), lymph node metastasis (P < 0.001), and tumor node metastasis (TNM) stage (P = 0.001) and had poorer disease-free survival (DFS) and overall survival (OS) compared to normal SOX17 expression (P = 0.002 and 0.001, respectively). Univariate and multivariate analyses indicated that lower SOX17 expression was an independent prognostic factor for DFS (P = 0.007; HR = 2.854; 95 % CI 1.326-6.147) and OS (P = 0.005; HR = 5.035; 95 % CI 1.648-15.385) for breast cancer. Our findings indicate that SOX17 expression is a useful prognostic biomarker for breast cancer.

Sox17, the canonical Wnt antagonist, is epigenetically inactivated by promoter methylation in human breast cancer.

SRY-box 17 (Sox17) is a transcription factor which involved in a variety of developmental processes and can act as an antagonist of canonical Wnt/beta-catenin signaling pathway. However, the relationship between Sox17 gene expression, methylation status, and beta-catenin in breast cancer has not been established. Here we report that the expression level of Sox17 mRNA was dramatically decreased in five different breast cancer cell lines and 23 of 31 primary breast tumor samples, which significantly correlated with its methylation status. After treated with 5-aza-2'-deoxycytidine (5-aza-dC, a demethylation agent), the expression levels of Sox17 mRNA and protein were obviously increased. Restored expression of Sox17 by 5-aza-dC treatment decreased the expression level of beta-catenin in breast cancer cell lines. Furthermore, small interfering RNA (siRNA)-mediated knockdown of Sox17 in SKBR-3 and Bacp-37 cells enhanced beta-catenin expression. In 31 paired tissue samples, a significant difference between the expression level of Sox17 and beta-catenin was also observed (P < 0.001). Clinically, Sox17 methylation was detected in 74.3% breast tumors (84/113) and 31.9% (36/113) paired normal tissues, respectively (P < 0.0001). Sox17 methylation was also associated with tumor stage (P = 0.028) and lymph node metastasis (P = 0.013). These findings indicate that silencing of Sox17 due to promoter hypermethylation is a frequent event and may contribute to aberrant activation of Wnt signaling in breast cancer. Sox17 may be a valuable biomarker for the study of breast cancer carcinogenesis and progression.

Frequent epigenetic inactivation of the receptor tyrosine kinase EphA5 by promoter methylation in human breast cancer.

EphA5 is a member of the Eph receptor tyrosine kinase family, which plays a critical role in the regulation of carcinogenesis. Our previous DNA methylation microarray results suggested that the CpG islands in the EphA5 promoter exhibited higher methylation levels in breast cancer tissues. In this study, we further analyzed EphA5 gene expression profiles, methylation status, and clinical implications in breast cancer. We found that the level of EphA5 mRNA was dramatically decreased in 5 different breast cancer cell lines. After treating the cell lines with 5-aza-2'-deoxycytidine (5-aza-dC, a demethylation agent), the levels of EphA5 mRNA and protein were significantly increased. Bisulfite sequencing and methylation-specific polymerase chain reaction detection showed that decreased expression of EphA5 was associated with its methylation status. We also found a significant correlation (P = .017) between the reduction of EphA5 mRNA levels and aberrant methylation of EphA5 in 31 paired tissue samples. In clinical samples, EphA5 methylation was detected in 64.1% (75/117) of breast tumors and 28.2% (33/117) of paired normal tissues (P < .001), which was associated with higher tumor grade (P = .024), lymph node metastasis (P = .004), and progesterone receptor-negative status (P = .008). Our data indicate that EphA5 might be a potential target for epigenetic silencing in primary breast cancer and a valuable molecular marker for breast cancer carcinogenesis and progression.