Loss of HAS2 Confers Acquired Antiestrogen Resistance By Upregulating Ezrin Expression In ER-Positive Breast Cancer

Background: Resistance to endocrine therapy is a major challenge for estrogen receptor-positive (ER+) breast cancer patients, but the underlying mechanisms remain unclear. Methods: Loss of hyaluronan synthase 2 (Has2) in adaptive resistant cells to tamoxifen and fulvestrant was observed by immunblotting assay. CRISPR/Cas9 technology was used to knock out Has2 in MCF7 cells to verify the effect of Has2 on the expression of ER and Ezrin and Akt and MAPK/ERK signaling routes. We utilized an Ezrin small-interfering RNA and Ezrin inhibitor to inhibit Ezrin expression for evaluating Has2 and ERα expression and the Akt/MAPK signaling cascade upon tamoxifen or fulvestrant treatment.Results: In this work, we showed that a Has2-loss state was acquired from adaptive resistance to tamoxifen and fulvestrant in luminal BrCas. Notably, the adapted loss of Has2 induced acquired resistance to antiestrogens in estrogen receptor (ER)-positive breast cancer cells through up-regulating the expression of Ezrin. Furthermore, we found that the loss of Has2 promoted while the consequent increase of Ezrin inhibited ERα expression/activity through the Akt and MAPK/ERK signaling routes, indicating an opposite effect on ERα expression during the development of antiestrogens-resistance. Inhibition of Ezrin reversed Has2 and ERα expression and the Akt/MAPK signaling cascade upon tamoxifen or fulvestrant, suggesting a Has2-Ezrin-ER negative-feedback loop in governing cellular sensitivity to tamoxifen or fulvestrant in luminal-like breast cancer cells. Finally, Knockdown or inhibition of Ezrin restored sensitivity to antiestrogens, implying that Ezrin could be a potential therapeutic target to tackle endocrine resistance. Conclusions: Taken together, our findings provide a direct relationship between ERα and Has2 implicated in resistance to endocrine therapy and a new insight into how ERα-signaling is regulated upon antiestrogens treatment, suggesting a novel therapeutic target for ER-positive breast cancer.

Loss of HAS2 Confers Acquired Antiestrogen Resistance by Upregulating Ezrin Expression in ER-Positive Breast Cancer

Background: Resistance to endocrine therapy is a major challenge for estrogen receptor-positive (ER+) breast cancer patients, but the underlying mechanisms remain unclear. Methods: Loss of hyaluronan synthase 2 (Has2) in adaptive resistant cells to tamoxifen and fulvestrant was observed by immunblotting assay. CRISPR/Cas9 technology was used to knock out Has2 in MCF7 cells to verify the effect of Has2 on the expression of ER and Ezrin and Akt and MAPK/ERK signaling routes. We utilized an Ezrin small-interfering RNA and Ezrin inhibitor to inhibit Ezrin expression for evaluating Has2 and ERα expression and the Akt/MAPK signaling cascade upon tamoxifen or fulvestrant treatment.Results: In this work, we showed that a Has2-loss state was acquired from adaptive resistance to tamoxifen and fulvestrant in luminal BrCas. Notably, the adapted loss of Has2 induced acquired resistance to antiestrogens in estrogen receptor (ER)-positive breast cancer cells through up-regulating the expression of Ezrin. Furthermore, we found that the loss of Has2 promoted while the consequent increase of Ezrin inhibited ERα expression/activity through the Akt and MAPK/ERK signaling routes, indicating an opposite effect on ERα expression during the development of antiestrogens-resistance. Inhibition of Ezrin reversed Has2 and ERα expression and the Akt/MAPK signaling cascade upon tamoxifen or fulvestrant, suggesting a Has2-Ezrin-ER negative-feedback loop in governing cellular sensitivity to tamoxifen or fulvestrant in luminal-like breast cancer cells. Finally, Knockdown or inhibition of Ezrin restored sensitivity to antiestrogens, implying that Ezrin could be a potential therapeutic target to tackle endocrine resistance. Conclusions: Taken together, our findings provide a direct relationship between ERα and Has2 implicated in resistance to endocrine therapy and a new insight into how ERα-signaling is regulated upon antiestrogens treatment, suggesting a novel therapeutic target for ER-positive breast cancer.

Bidirectional Tumor-Promoting Activities of Macrophage Ezrin

Ezrin links the cytoskeleton to cell surface integrins and plasma membrane receptors, contributing to the proliferative and metastatic potential of cancer cells. Elevated ezrin expression in several cancers is associated with poor outcomes. Tumor cell ezrin expression and function have been investigated in depth; however, its role in macrophages and other tumor microenvironment cells remains unexplored. Macrophages profoundly influence tumorigenesis, and here we explore ezrin’s influence on tumor-promoting macrophage functions. Ezrin knockdown in THP-1 macrophages reveals its important contribution to adhesion to endothelial cells. Unexpectedly, ezrin is essential for the basal and breast cancer cell-stimulated THP-1 expression of ITGAM mRNA that encodes integrin CD11b, critical for cell adhesion. Ezrin skews the differentiation of THP-1 macrophages towards the pro-tumorigenic, M2 subtype, as shown by the reduced expression of FN1, IL10, and CCL22 mRNAs following ezrin knockdown. Additionally, macrophage ezrin contributes to the secretion of factors that stimulate tumor cell migration, invasion, and clonogenic growth. Lastly, THP-1 ezrin is critical for the expression of mRNAs encoding vascular endothelial growth factor (VEGF)-A and matrix metalloproteinase (MMP)-9, consistent with pro-tumorigenic function. Collectively, our results provide insight into ezrin’s role in tumorigenesis, revealing a bidirectional interaction between tumor-associated macrophages and tumor cells, and suggest myeloid cell ezrin as a target for therapeutic intervention against cancer.

Expression of Pinopodes in the Endometrium from Recurrent Pregnancy Loss Women. Role of Thrombomodulin and Ezrin

Background: Pinopode expression has been suggested as a marker of endometrial receptivity. Methods: We set up an experimental study comparing endometrial tissue from recurrent pregnancy loss (RPL, n = 30) and fertile control (CTR, n = 20) women in terms of pinopode expression/morphology; expression of thrombomodulin (TM) and ezrin; cytoskeletal organization. Endometrial samples were collected during implantation window and evaluated by scanning electron microscopy, western blot, and immunofluorescence. Results: We found that RPL endometrial tissue showed: (i) increased pinopodes density (* p < 0.05); (ii) a reduced diameter of pinopodes (* p < 0.05); (iii) a decreased TM and ezrin expression (p < 0.05). Additionally, confocal images showed a significantly reduced expression of phosphorylated (p)-ezrin, confirming the results obtained through immunoblot analysis. Immunofluorescence staining showed that in CTR samples, junctions between cells are intact and clearly visible, whereas actin filaments appear completely lost in RPL endometrial samples; this suggests that, due to the impaired expression and activity of TM and ezrin, actin does not bind to plasma membrane in order to orchestrate the cytoskeletal actin filaments. Conclusions: Our findings suggest that an impaired expression of TM and expression/activation of ezrin may affect the connection between the TM and actin cytoskeleton, impairing the organization of cytoskeleton and, eventually, the adequate pinopode development.

Knockdown of Ezrin inhibited migration and invasion of cervical cancer cells in vitro

Cervical cancer is the fourth most common malignancy in women. The aim of this study was to investigate the functions of Ezrin in cervical cancer cells. Two cervical cancer cell lines, SiHa and CaSki, were cultured in vitro. Following the knockdown of Ezrin using siRNA, real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were applied to analyze Ezrin expression at the messenger RNA (mRNA) and protein levels. Subsequently, wound healing assay, transwell assay, and sulforhodamine B (SRB) assay were used to detect the migration, invasion, and viability of cervical cancer cells, respectively. Results revealed that Ezrin siRNA can notably inhibit the migration and invasion of SiHa and CaSki cells ( P < 0.05). However, knockdown of Ezrin shows no effects on the viability of SiHa and CaSki cells ( P < 0.05). It is indicated that Ezrin plays a possible role in promoting the migration and invasion of cervical cancer cells and may be a therapeutic target to prevent metastasis of cervical cancer.