Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of therapeutic targets.

BACKGROUND: The dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels. RESULTS: In order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells. CONCLUSION: The unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation.

Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival.

Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imaging-based experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.

The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide.

There is a great demand for the improvement of mammalian cell production systems such that they can compete economically with their prokaryotic counterparts. Of a number of parameters that need to be explored to accomplish this we have tested the effects of different signal peptides on the synthesis and secretion of Gaussia princeps luciferase in mammalian cells. A series of plasmids were transfected into CHO cells where the coding region for the marine luciferase was fused to the signal peptide coding regions derived from different sources. Both cell extracts and medium samples were analysed for luciferase activity. When the native Gaussia luciferase signal sequence in the vector was substituted by that from human interleukin-2 or albumin then the amount of active recombinant protein produced was substantially reduced, both in transiently and stably transfected cells. Western blotting showed that enzyme activity and protein levels mirrored one another. The major decrease in luciferase activity was shown not to be a result of decreased mRNA levels, indicating the involvement of a post-transcriptional event. When the coding region of human endostatin was fused to that of the Gaussia luciferase signal peptide then an elevated level of secreted endostatin was observed compared to when that of the albumin signal peptide was used. Stable transfection of HepG2 cells with the different signal peptide constructs gave essentially the same results as seen in CHO cells. The overall results indicate that the choice of signal peptide can be imperative to ensure an optimal synthesis and secretion of a recombinant protein in a mammalian cell culture system.