Xiaoding Hu

Background: The brain is a common site of relapse in inflammatory breast cancer (IBC), an E-cadherin-positive, aggressive form of breast cancer. Elevated serum levels of soluble E-cadherin (sEcad), an 80-kDa fragment, correlated with poorer outcomes and increased brain metastases in patients with metastatic IBC. We hypothesize that sEcad is a driver of brain metastasis in IBC. Methods: Serum sEcad levels from 348 IBC patients were quantified by ELISA. To examine sEcad function, we used recombinant sEcad protein and generated stable IBC cell lines by cloning and overexpressing Flag-tagged sEcad. Control and sEcad-overexpressing MDA-IBC3 and SUM149 cells were injected into SCID/Beige mice to evaluate brain metastasis burden and survival, and a brain-permeable CXCR2 inhibitor was also tested for efficacy in these models. Results: Higher serum sEcad levels correlated with poorer overall survival, earlier metastasis, and increased brain metastasis. In vitro, recombinant sEcad and stable sEcad overexpression in IBC cell lines promoted invasion, resistance to anoikis, and activation of pro-survival NF-κβ signaling. In vivo, mice injected with sEcad-overexpressing IBC cells had increased metastatic burden and reduced overall and brain metastasis-free survival. Further, sEcad induced reactive astrocytosis through the CXCL1/CXCL8-CXCR2 axis, and treatment with a brain-permeable CXCR2 antagonist reduced metastatic burden and prolonged survival in the brain metastasis models. Conclusion: sEcad drives brain metastasis by promoting invasion and anoikis resistance in cancer cells and inducing an inflammatory brain microenvironment via a targetable CXCL1/CXCL8-CXCR2 axis. These findings uncover a novel and critical role for sEcad and highlight CXCR2 as a therapeutic target in patients with metastatic IBC.

Supplementary Materials and Methods

Fig. S2 Venn diagram of the number of significant siRNAs identified with a 5% false discovery rate using different normalization methods (Z-score, b-score, and regional scaling) for comparison between untreated and treated samples.

Fig. S4 Effect of combination of drugs on SUM149-R cells. The clonogenic assay shows a significant dose-dependent decrease in the colony formation capability of SUM149-R cells after treatment with the combination of MEKK inhibitor AZD6244 and MCL-1 inhibitor S63845 at the same time.

Table S2 List of top 26 targets identified in the whole-genome siRNA screening.

Table S1 Quantitative PCR primers used in the study.

Fig. S3 CTB assay showing the effect of combination of drugs on SUM149-R cells. The SUM149-R cells showed a decrease in viability after treatment with the constant doses of AZD5991 (0.5, and 1µM) followed by the different doses of AZD6244.

Fig. S1 Comparative analysis of expression of MDR genes in the parental and resistant cells.

Summary: The brain is a common site of relapse in inflammatory breast cancer (IBC), an E-cadherin positive, aggressive form of breast cancer. We found that elevated serum levels of soluble E-cadherin (sEcad), an 80-kDa fragment of E-cadherin, in patients with metastatic IBC correlated with poorer outcomes and increased rates of brain metastases. In our effort to understand the underlying mechanism, we discovered that sEcad binds to XIAP, an inhibitor of cell death, activating the pro-survival NF-kβ signaling in tumor cells. We also discovered that sEcad affects the tumor cell microenvironment by enhancing cancer cell adhesion to endothelial cells and inducing reactive astrocytosis in the brain. In addition, we found that sEcad-mediated reactive astrocytosis relies on the CXCL1/CXCL8-CXCR2 axis and treatment with a brain-permeable CXCR2 antagonist reduced brain metastatic burden and prolonged survival. These findings implicate sEcad in brain metastasis and provide new insights into potential therapeutic targets for IBC. Graphical Highlights: High serum sEcad levels correlate clinically with poor survival outcomes and development of brain metastasis sEcad drives IBC brain metastasis growth in mouse models sEcad binds XIAP to activate NFkB and promote anoikis resistance and invasion of IBC cells sEcad activates reactive astrocytes and induces CXCR2 expression on tumor cells in vitro and in vivo CXCR2-IN-1, a brain-permeable CXCR2 antagonist, reduces metastasis and improves survival in IBC brain metastasis models

The MAPK pathway can drive resistance in highly aggressive breast cancers. Our previous work showed that the MEK inhibitor (MEKi) AZD6244 (selumetinib) prevented lung metastasis in a breast cancer xenograft model. In clinical studies, MEKis as single agents have had only modest activity against solid tumors due to the onset of resistance. Using synthetic lethality siRNA screening, we identified myeloid cell leukemia-1 (MCL-1) as a potential contributor to AZD6244 resistance. We hypothesized that MCL-1 promotes MEKi resistance in highly aggressive breast cancers and that MCL-1 inhibition overcomes AZD6244 resistance. We established two AZD6244-resistant cell lines: MDA-MB-231-R (triple-negative breast cancer) and SUM149-R (triple-negative inflammatory breast cancer). These resistant cells were characterized with respect to different parameters, and a combination of an MCL-1 inhibitor (MCL-1i) together with an MEKi was evaluated in vitro and in vivo to overcome the acquired resistance. Compared with their respective parental cells, MDA-MB-231-R and SUM149-R cells showed increased proliferation, colony formation, stemness, anchorage-independent growth, and MCL-1 expression levels. MCL-1 knockdown in resistant cells decreased cell proliferation and colony formation, increased apoptosis, and was associated with high expression of the proapoptotic proteins PUMA, NOXA, BAK, and BAX. MEKi resistance was overcome when resistant cells were treated with MCL-1i and MEKi combined. In an in vivo mouse model, inhibition of MCL-1 restored sensitivity to AZD6244. Our results suggest that MCL-1 is a driver of MEKi resistance and that combining an MCL-1i with an MEKi warrants further investigation in triple-negative and triple-negative inflammatory breast cancer.