Rationale: Angiogenesis is a fundamental process of tumorigenesis, growth, invasion and metastatic spread. Extracellular vesicles, especially exosomes, released by primary tumors promote angiogenesis and cancer progression. However, the mechanism underlying the pro-angiogenic potency of cancer cell-derived exosomes remains poorly understood. Methods: Exosomes were isolated from breast cancer cells with high metastatic potential (HM) and low metastatic potential (LM). The pro-angiogenic effects of these exosomes were evaluated by in vitro tube formation assays, wound healing assays, rat arterial ring budding assays and in vivo Matrigel plug assays. Subsequently, RNA sequencing, shRNA-mediated gene knockdown, overexpression of different EPHA2 mutants, and small-molecule inhibitors were used to analyze the angiogenesis-promoting effect of exosomal EPHA2 and its potential downstream mechanism. Finally, xenograft tumor models were established using tumor cells expressing different levels of EPHA2 to mimic the secretion of exosomes by tumor cells in vivo, and the metastasis of cancer cells were monitored using the IVIS Spectrum imaging system and Computed Tomography. Results: Herein, we demonstrated that exosomes produced by HM breast cancer cells can promote angiogenesis and metastasis. EPHA2 was rich in HM-derived exosomes and conferred the pro-angiogenic effect. Exosomal EPHA2 can be transferred from HM breast cancer cells to endothelial cells. Moreover, it can stimulate the migration and tube-forming abilities of endothelial cells in vitro and promote angiogenesis and tumor metastasis in vivo. Mechanistically, exosomal EPHA2 activates the AMPK signaling via the ligand Ephrin A1-dependent canonical forward signaling pathway. Moreover, inhibition of the AMPK signaling impairs exosomal EPHA2-mediated pro-angiogenic effects. Conclusion: Our findings identify a novel mechanism of exosomal EPHA2-mediated intercellular communication from breast cancer cells to endothelial cells in the tumor microenvironment to provoke angiogenesis and metastasis. Targeting the exosomal EPHA2-AMPK signaling may serve as a potential strategy for breast cancer therapy.

Recent evidence suggests that splicing factors (SFs) and alternative splicing (AS) play important roles in cancer progression. We constructed four SF-risk-models using 12 survival-related SFs. In Luminal-A, Luminal-B, Her-2, and Basal-Like BRCA, SF-risk-models for three genes (PAXBP1, NKAP, and NCBP2), four genes (RBM15B, PNN, ACIN1, and SRSF8), three genes (LSM3, SNRNP200, and SNU13), and three genes (SRPK3, PUF60, and PNN) were constructed. These models have a promising prognosis-predicting power. The co-expression and protein-protein interaction analysis suggest that the 12 SFs are highly functional-connected. Pathway analysis and gene set enrichment analysis suggests that the functional role of the selected 12 SFs is highly context-dependent among different BRCA subtypes. We further constructed four AS-risk-models with good prognosis predicting ability in four BRCA subtypes by integrating the four SF-risk-models and 21 survival-related AS-events. This study proposed that SFs and ASs were potential multidimensional biomarkers for the diagnosis, prognosis, and treatment of BRCA.

Emerging evidence suggests that the dysregulation of autophagy-related genes (ARGs) is coupled with the carcinogenesis and progression of breast cancer (BRCA). We constructed three subtype-specific risk models using differentially expressed ARGs. In Luminal, Her-2, and Basal-like BRCA, four- ( BIRC5 , PARP1 , ATG9B , and TP63 ), three- ( ITPR1 , CCL2 , and GAPDH ), and five-gene ( PRKN , FOS , BAX , IFNG , and EIF4EBP1 ) risk models were identified, which all have a receiver operating characteristic > 0.65 in the training and testing dataset. Multivariable Cox analysis showed that those risk models can accurately and independently predict the overall survival of BRCA patients. Comprehensive analysis showed that the 12 identified ARGs were correlated with the overall survival of BRCA patients; six of the ARGs ( PARP1 , TP63 , CCL2 , GAPDH , FOS , and EIF4EBP1 ) were differentially expressed between BRCA and normal breast tissue at the protein level. In addition, the 12 identified ARGs were highly interconnected and displayed high frequency of copy number variation in BRCA samples. Gene set enrichment analysis suggested that the deactivation of the immune system was the important driving force for the progression of Basal-like BRCA. This study demonstrated that the 12 ARG signatures were potential multi-dimensional biomarkers for the diagnosis, prognosis, and treatment of BRCA.

Background Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin-angiotensin system and a well-known functional receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells. The COVID-19 pandemic has brought ACE2 into the spotlight, and ACE2 expression in tumors and its relationship with SARS-COV-2 infection and prognosis of cancer patients have received extensive attention. However, the association between ACE2 expression and tumor therapy and prognosis, especially in breast cancer, remains ambiguous and requires further investigation. We have previously reported that ACE2 is elevated in drug-resistant breast cancer cells, but the exact function of ACE2 in drug resistance and progression of this malignant disease has not been explored. Methods The expression of ACE2 and HIF-1α in parental and drug-resistant breast cancer cells under normoxic and hypoxic conditions was analyzed by Western blot and qRT-PCR methods. The protein levels of ACE2 in plasma samples from breast cancer patients were examined by ELISA. The relationship between ACE2 expression and breast cancer treatment and prognosis was analyzed using clinical specimens and public databases. The reactive oxygen species (ROS) levels in breast cancer cells were measured by using a fluorescent probe. Small interfering RNAs (siRNAs) or lentivirus-mediated shRNA was used to silence ACE2 and HIF-1α expression in cellular models. The effect of ACE2 knockdown on drug resistance in breast cancer was determined by Cell Counting Kit 8 (CCK-8)-based assay, colony formation assay, apoptosis and EdU assay. Results ACE2 expression is relatively low in breast cancer cells, but increases rapidly and specifically after exposure to anticancer drugs, and remains high after resistance is acquired. Mechanistically, chemotherapeutic agents increase ACE2 expression in breast cancer cells by inducing intracellular ROS production, and increased ROS levels enhance AKT phosphorylation and subsequently increase HIF-1α expression, which in turn upregulates ACE2 expression. Although ACE2 levels in plasma and cancer tissues are lower in breast cancer patients compared with healthy controls, elevated ACE2 in patients after chemotherapy is a predictor of poor treatment response and an unfavorable prognostic factor for survival in breast cancer patients. Conclusion ACE2 is a gene in breast cancer cells that responds rapidly to chemotherapeutic agents through the ROS-AKT-HIF-1α axis. Elevated ACE2 modulates the sensitivity of breast cancer cells to anticancer drugs by optimizing the balance of intracellular ROS. Moreover, increased ACE2 is not only a predictor of poor response to chemotherapy, but is also associated with a worse prognosis in breast cancer patients. Thus, our findings provide novel insights into the spatiotemporal differences in the function of ACE2 in the initiation and progression of breast cancer.