TEM8 marks neovasculogenic tumor-initiating cells in triple-negative breast cancer

Enhanced neovasculogenesis, especially vasculogenic mimicry (VM), contributes to the development of triple-negative breast cancer (TNBC). Breast tumor-initiating cells (BTICs) are involved in forming VM; however, the specific VM-forming BTIC population and the regulatory mechanisms remain undefined. We find that tumor endothelial marker 8 (TEM8) is abundantly expressed in TNBC and serves as a marker for VM-forming BTICs. Mechanistically, TEM8 increases active RhoC level and induces ROCK1-mediated phosphorylation of SMAD5, in a cascade essential for promoting stemness and VM capacity of breast cancer cells. ASB10, an estrogen receptor ERα trans-activated E3 ligase, ubiquitylates TEM8 for degradation, and its deficiency in TNBC resulted in a high homeostatic level of TEM8. In this work, we identify TEM8 as a functional marker for VM-forming BTICs in TNBC, providing a target for the development of effective therapies against TNBC targeting both BTIC self-renewal and neovasculogenesis simultaneously.


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MRI-based neuroimaging
The data from TCGA analyzed in the study are available in a public repository from the National Cancer Institute Cancer Genome Atlas website (https://tcga-data. nci.nih.gov/tcga/). The RNA-seq data profiles analyzed in the study have been deposited in the National Center for Biotechnology Information under the accession code PRJNA739366 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA739366/). The publicly available dataset used in this study can be accessed under the GEO accession codes-GSE43742, GSE86788, GSE5327, GSE2603, GSE2034, GSE22133. All other data supporting the findings of the study are available in this article and its supplementary information files. All other relevant data are available from the corresponding author on request.
For in vitro tumor cell tube formation assay, the number of implanted cells on growth factor reduced matrigel was based on prior experience of HUVEC cell tube formation. For in vitro mammosphere formation assay, the number of implanted cells into low attachment surface plate was depended on the size of plate. For in vitro invasion assay and MTT assay, the number of implanted cells was based on prior experience. For in vitro human kinase array assay and dual luciferase reporter assay, the sample size was determined based on the manufacturer's protocol. For in vivo validation experiments, the number of mice used in each experimental group was determined by power analysis and on the basis of prior experience with animal models of xenografts. Animals were randomly assigned to treatment groups. All sample sizes are listed in the corresponding figure legends or on the figures.
No data was excluded from the analyses All experiments were conducted at least three times independently, and similar results were adopted for further analysis to guarantee reproducibility.
For in vitro studies, the samples/cells were randomized into different groups prior to treatment. For animal experiments, all mouse were randomly divided into each group before receiving different treatment.
Human breast cancer cell lines SUM149 and SUM159 were got from Asterland Bioscience; human breast cancer cell lines ( MDA-MB-231, MDA-MB-231lung, and MCF-7) and 293T cell lines were purchased from ATCC (Manassas, Virginia),

STR testing
All cell lines were tested to be mycoplasma negative.
All cell lines used in this study are not commonly misidentified cell lines.
Three to four-week-old female Nude mice and NOD/SCID mice were purchased from Charles river (Beijing, China) and housed in standard animal cages under a Specific-pathogen-free (SPF) facility at 23-25°C on a 12-h light/dark cycle in the Department of Laboratory Animal Science of Fudan University.
No wild animals was used in this study.
No field collected samples were used in the study.