The T-cell inhibitory molecule PD-L1 (B7-H1, CD274) is expressed on tumor cells of a subset of breast cancer patients. However, the mechanism that regulates PD-L1 expression in this group of patients is still not well-identified. We have used loss and gain of function gene manipulation approach, multi-parametric flow cytometry, large scale gene expression dataset analysis and immunohistochemistry of breast cancer tissue sections. Induction of epithelial to mesenchymal transition (EMT) in human mammary epithelial cells upregulated PD-L1 expression, which was dependent mainly on the activation of the PI3K/AKT pathway. Interestingly, gene expression signatures available from large cohort of breast tumors showed a significant correlation between EMT score and the PD-L1 mRNA level (p < 0.001). Strikingly, very strong association (p < 0.0001) was found between PD-L1 expression and claudin-low subset of breast cancer, which is known to have high EMT score. On the protein level, significant correlation was found between PD-L1 expression and standard markers of EMT (p = 0.005) in 67 breast cancer patients. Importantly, specific downregulation of PD-L1 in claudin-low breast cancer cells showed signs of EMT reversal as manifested by CD44 and Vimentin downregulation and CD24 upregulation. We have demonstrated a bidirectional effect between EMT status and PD-L1 expression especially in claudin-low subtype of breast cancer cells. Our findings highlights the potential dual benefit of anti-PD-L1 particularly in this subset of breast cancer patients that will likely benefit more from anti-PD-L1 targeted therapy as well as in monitoring biological changes upon treatment.

Introduction: B7-H1 (PD-L1, CD274) is a T cell inhibitory molecule expressed in many types of cancer, leading to immune escape of tumor cells. Indeed, in previous reports we have shown an association of B7-H1 expression with high-risk breast cancer patients. Methods: In the current study, we used immunohistochemistry, immunofluorescence and Western blot techniques to investigate the effect of neoadjuvant chemotherapy on the expression of B7-H1 in breast cancer cells. Results: Among tested chemotherapeutic agents, doxorubicin was the most effective in downregulating cell surface expression of B7-H1 in vitro. These results were validated in vivo in a xenograft mouse model, as well as in murine heart tissue known to constitutively express B7-H1. The doxorubicin-dependent cell surface downregulation of B7-H1 was accompanied by an upregulation of B7-H1 in the nucleus. This re-distribution of B7-H1 was concurrent with a similar translocation of phosphorylated AKT to the nucleus. Inhibition of the PI3K/AKT pathway abrogated the doxorubicin-mediated nuclear up-regulation of B7-H1, suggesting an involvement of PI3K/AKT pathway in the nuclear up-regulation of B7-H1. Interestingly, siRNA knock down of B7-H1 lead to an increase in spontaneous apoptosis, as well as doxorubicin-induced apoptosis, which indicates an anti-apoptotic role for B7-H1 in breast cancer cells. The novel discovery of B7-H1 expression in the nuclei of breast cancer cells suggests that B7-H1 has functions other than inhibition of T cells. Conclusions: Our findings explain the previously reported immunomodulatory effect of anthracyclines on cancer cells, and provide a link between immunoresistance and chemoresistance. Finally these results suggest the use of dual combinatorial agents to inhibit B7-H1 beside chemotherapy, in breast cancer patients.

The actin-bundling protein, fascin, is a member of the cytoskeletal protein family that has restricted expression in specialized normal cells. However, many studies have reported the induction of this protein in various transformed cells including breast cancer cells. While the role of fascin in the regulation of breast cancer cell migration has been previously shown, the underlying molecular mechanism remained poorly defined. We have used variety of immunological and functional assays to study whether fascin regulates breast cancer metastasis-associated molecules. In this report we found a direct relationship between fascin expression in breast cancer patients and; metastasis and shorter disease-free survival. Most importantly, in vitro interference with fascin expression by loss or gain of function demonstrates a central role for this protein in regulating the cell morphology, migration and invasion potential. Our results show that fascin regulation of invasion is mediated via modulating several metastasis-associated genes. We show for the first time that fascin down-regulates the expression and nuclear translocation of a key metastasis suppressor protein known as breast cancer metastasis suppressor-1 (BRMS1). In addition, fascin up-regulates NF-kappa B activity, which is essential for metastasis. Importantly, fascin up-regulates other proteins that are known to be critical for the execution of metastasis such as urokinase-type plasminogen activator (uPA) and the matrix metalloproteases (MMP)-2 and MMP-9. This study demonstrates that fascin expression in breast cancer cells establishes a gene expression profile consistent with metastatic tumors and offers a potential therapeutic intervention in metastatic breast cancer treatment through fascin targeting.

Background: A major therapeutic challenge for breast cancer is the ability of cancer cells to evade killing of conventional chemotherapeutic agents. We have recently reported the actin-bundling protein (fascin) as a major regulator of breast cancer metastasis and survival. Methods: Survival of breast cancer patients that received chemotherapy and xenograft tumour model was used to assess the effect of chemotherapy on fascin-positive and -negative breast cancer cells. Molecular and cellular assays were used to gain in-depth understanding of the relationship between fascin and chemoresistance. Results: We showed a significant correlation between fascin expression and shorter survival in breast cancer patients who received chemotherapy. In xenograft experiments, fascin-positive cancer cells displayed significantly more resistance to chemotherapy-mediated apoptotic cell death than fascin-negative counterparts. This increased chemoresistance was at least partially mediated through PI3K/Akt signalling, and was paralleled by increased FAK phosphorylation, enhanced expression of the inhibitor of apoptosis proteins (XIAP and Livin) and suppression of the proapoptotic markers (caspase 9, caspase 3 and PARP). Conclusions: This is the first report to demonstrate fascin involvement in breast cancer chemotherapeutic resistance, supporting the development of fascin-targeting drugs for better treatment of chemoresistance breast cancer.

An emerging dogma shows that tumors are initiated and maintained by a subpopulation of cancer cells that hijack some stem cell features and thus referred to as “cancer stem cells” (CSCs). The exact mechanism that regulates the maintenance of CSC pool remains largely unknown. Fascin is an actin-bundling protein that we have previously demonstrated to be a major regulator of breast cancer chemoresistance and metastasis, two cardinal features of CSCs. Here, we manipulated fascin expression in breast cancer cell lines and used several in vitro and in vivo approaches to examine the relationship between fascin expression and breast CSCs. Fascin knockdown significantly reduced stem cell-like phenotype (CD44^hi/CD24^lo and ALDH⁺) and reversal of epithelial to mesenchymal transition. Interestingly, expression of the embryonic stem cell transcriptional factors (Oct4, Nanog, Sox2, and Klf4) was significantly reduced when fascin expression was down-regulated. Functionally, fascin-knockdown cells were less competent in forming colonies and tumorspheres, consistent with lower basal self-renewal activity and higher susceptibility to chemotherapy. Fascin effect on CSC chemoresistance and self-renewability was associated with Notch signaling. Activation of Notch induced the relevant downstream targets predominantly in the fascin-positive cells. Limiting-dilution xenotransplantation assay showed higher frequency of tumor-initiating cells in the fascin-positive group. Collectively, our data demonstrated fascin as a critical regulator of breast CSC pool at least partially via activation of the Notch self-renewal signaling pathway and modification of the expression embryonic transcriptional factors. Targeting fascin may halt CSCs and thus presents a novel therapeutic approach for effective treatment of breast cancer. Stem Cells 2016;34:2799–2813 Video Highlight: https://youtu.be/GxS4fJ_Ow-o

Breast cancer remains the second cause of tumor‐related mortality in women worldwide mainly due to chemoresistance and metastasis. The chemoresistance and metastasis are attributed to a rare subpopulation with enriched stem‐like characteristics, thus called Cancer Stem Cells (CSCs). We have previously reported aberrant expression of the actin‐bundling protein (fascin) in breast cancer cells, which enhances their chemoresistance, metastasis and enriches CSC population. The intracellular mechanisms that link fascin with its downstream effectors are not fully elucidated. Here, loss and gain of function approaches in two different breast cancer models were used to understand how fascin promotes disease progression. Importantly, findings were aligned with expression data from actual breast cancer patients. Expression profiling of a large breast cancer dataset (TCGA, 530 patients) showed statistically significant correlation between fascin expression and a key adherence molecule, β1 integrin (ITGB1).In vitromanipulation of fascin expression in breast cancer cells exhibited its direct effect on ITGB1 expression. Fascin‐mediated regulation of ITGB1 was critical for several breast cancer cell functions including adhesion to different extracellular matrix, self‐renewability and chemoresistance. Importantly, there was a significant relationship between fascin and ITGB1 co‐expression and short disease‐free as well as overall survival in chemo‐treated breast cancer patients. This novel role of fascin effect on ITGB1 expression and its outcome on cell self‐renewability and chemoresistance strongly encourages for dual targeting of fascin‐ITGB1 axis as a therapeutic approach to halt breast cancer progression and eradicate it from the root.