Researchers at the German Cancer Research Center (DKFZ) and the MD Anderson Cancer Center used Cellecta's shRNA DECIPHER Libraries for an in vivo genetic screen that identified SALL1 as a breast cancer tumor suppressor that plays a role in E-cadherin regulation (Article).
E-cadherin (CDH1) has been shown to have a role in epithelial-to-mesenchymal (EMT) transition in breast cancer. Silencing of SALL1 via promoter hypermethylation has also been shown to be associated with breast cancer. In the current publication, researchers show that inhibition of SALL1 is correlated with decreased CDH1 expression, as well as changes in other gene expression associated with EMT.
Michael Boettcher's group at DKFZ transduced the SUM-149 breast cancer cell line with the DECIPHER short-hairpin RNA (shRNA) library Human Module 1, which targets approximately 5,000 human genes. Each gene is targeted by 5-6 shRNAs, and the sequences are barcoded for easy detection by NGS. They then injected the transduced cells into immunosuppressed mice and waited for tumors to develop. After harvesting the tumors they analyzed the genomic DNA using NGS to see which genes have tumor suppressor activities in vivo.
They identified 16 candidate tumor suppressor genes, and chose to focus the rest of their study on SALL1. SALL1 belongs to the zinc finger transcription factor family and has recently been implicated in breast cancer. In their RNAi screen, two shRNA constructs were identified that target SALL1. Each of these SALL1-targeting shRNAs was subcloned into an expression vector and transduced into the SUM-149 breast cancer cell line, as well as into several other breast cancer cell lines. The transduced SUM-149 cells were then injected into immunosuppressed mice. Expression of the shRNA to SALL1 decreased the tumor-free survival period in the immunosuppressed mice and increased the number of mitotically active tumor cells compared to control tumor cells.
To determine what other genes are affected by decreased SALL1 expression, the group analyzed SUM-149 cells expressing shSALL1 by microarray. Approximately 200 genes were significantly changed in the study. In particular, several genes involved in EMT were affected, including decreased CDH1, and increased CDH2, VIM, and MSN. They also found increased amounts of CTGF and CYR61, which are critical in breast cancer carcinogenesis, and decreased RARRES1 and RARRES3, which are known tumor suppressor genes.
The researchers discovered that decreased levels of SALL1 correlated with other changes typically associated with breast cancer. For example, SUM-149 cells expressing shSALL1 were shown to migrate more in wound healing assays, suggesting they were more metastatic, and had higher numbers of cells with the CD44+/CD24-/low phenotype—a marker associated with cancer stem cells. Also, patient breast cancer samples that had high amounts of SALL1 expressed correlated with higher relapse-free survival, overall survival, metastasis-free survival, and tumor-free survival.
This study provides an excellent example of how in vivo pooled RNAi screens can be used to elucidate cellular pathways associated with cancer pathology.
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