microRNAs are small non-coding RNA molecules involved in the regulation of gene expression and play critical roles in human malignancies. Next generation sequencing analysis of the MCF-7 breast cancer cell line overexpressing miR-335-5p and miR-335-3p demonstrated that the miRNA duplex repressed genes involved in the ERα signaling pathway, and enhanced resistance of MCF-7 cells to the growth inhibitory effects of tamoxifen. These data suggest that despite its conventional role in tumor suppression, the miR-335 transcript can also play an oncogenic role in promoting agonistic estrogen signaling in a cancerous setting.

Estrogen receptor alpha (ERα) signaling pathways are frequently disrupted in breast cancer and contribute to disease progression. ERα signaling is multifaceted and many ERα regulators have been identified including transcription factors and growth factor pathways. More recently, microRNAs (miRNAs) are shown to deregulate ERα activity in breast carcinomas, with alterations in both ERα and miRNA expression correlating to cancer progression. In this study, we show that a high expression of Argonaute 2 (AGO2), a translation regulatory protein and mediator of miRNA function, correlates with the luminal B breast cancer subtype. We further demonstrate that a high expression of AGO2 in ERα+ tumors correlates with a poor clinical outcome. MCF-7 breast cancer cells overexpressing AGO2 (MCF7-AGO2) altered ERα downstream signaling and selective ERα variant expression. Enhanced ERα-36, a 36 kDa ERα isoform, protein and gene expression was observed in vitro. Through quantitative polymerase chain reaction (qPCR), we demonstrate decreased basal expression of the full-length ERα and progesterone receptor genes, in addition to loss of estrogen stimulated gene expression in vitro. Despite the loss, MCF-7-AGO2 cells demonstrated increased estrogen stimulated tumorigenesis in vivo. Together with our clinical findings on AGO2 expression and the luminal B subtype, we suggest that AGO2 is a regulator of altered ERα signaling in breast tumors.

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Rationale: We have previously shown that cell-free hemoglobin (CFH) increases endothelial cell permeability in vitro and in the ex vivo isolated, perfused human lung. However, it remains unclear whether CFH can also augment the host immune response. This study was designed to determine if CFH can function as a damage-associated molecular pattern (DAMP) by increasing endothelial cell adhesion molecule expression and cytokine release. Objective: To test whether human derived primary lung microvascular endothelial cells exposed to CFH can modulate the innate immune response by upregulating expression of active adhesion molecules and cytokines. Methods: Primary human lung microvascular endothelial cells (HPLMVECs) were grown in culture to confluence then treated with 0.5mg/mL of purified human CFH (Fe 3+ , ferric state) at 1, 3, 6, and 24 hours. Expression levels of ICAM-1, ICAM-2, VCAM-1, P-Selectin, E-selectin, PECAM-1, pro-IL1β, IL-6, and IL-8 were measured via RT-PCR. Release of IL-6 was measured by ELISA in conditioned media. Results CFH exposure led to a significant increase in transcription of E-Selectin at 24 hours (86.56 fold change relative expression, p<0.0002) as measured by RT-PCR. No change in transcription was seen for P-selectin (1.96 fold change relative expression, p=0.07). Integrins were also upregulated in a time dependent manner with increased relative transcription seen at 3 hours for VCAM-1 (98.3 fold change relative expression, p<0.0001), ICAM-1 (256.0 fold change relative expression, p<0.0001), ICAM-2 (6.11 fold change relative expression, p<0.0001). PECAM-1 transcription did not change in response to CFH. Cytokine transcription was also upregulated at 3 hours of CFH exposure for IL-6 (1532 fold change relative expression, P<0.0001) and IL-8 (147.2 fold change relative expression, p= 0.0083). A significant increase in transcription was seen for pro-IL1β (74.8 fold change relative expression, P= 0.0086) at the 6 hour time point. Conditioned media demonstrated a significant increase in IL-6 concentration at 6 hours (70.96 ng/mL vs 210.5 ng/mL, p= 0.0035). Conclusions: In response to CFH exposure, lung microvascular endothelial cells increase expression of adhesion molecules and cytokines which could lead to leukocyte activation, adhesion, and migration into the lung, increasing localized inflammation and tissue injury. This data suggests that CFH is acting as a DAMP and may help to explain the association between elevated CFH and poor clinical outcomes in sepsis and acute respiratory distress syndrome.