PTK6 Regulates Survival with other oncogenes suppress anoikis through any or all of these mechanisms [4]. As a complement to these gain-of-function screens, loss-of- function screens also provide insight into mechanisms that are necessary for anoikis suppression and identify potential targets for therapeutic intervention. Screens utilizing small molecule inhibi- tors have previously been reported [5], [6]; these studies have highlighted multiple ways in which anoikis resistance may be overcome, including manipulation of the extrinsic cell death pathway and hypoosmotic stress. Here we present a novel siRNA screen designed to identify regulators of IGF-1 receptor (IGF-1R)- driven anoikis resistance of breast epithelial cells. IGF-1R has been shown to be expressed in the majority of human breast cancers with evidence of sporadic amplification in a small proportion of these cases [7], [8]. Although initially thought to correlate with estrogen receptor (ER) expression, IGF-1R has recently been implicated in multiple breast cancer subtypes and its expression correlates with poor prognoses [9], [10]. IGF-1 stimulation directly induces anoikis resistance of several different epithelial cell types (including breast, prostate, and colon) by activating downstream signaling molecules such as Ras/MAPK and PI3K/Akt [11]. IGF-1R is also required for transformation and anoikis suppression induced by other oncogenes, such as Ras, c-Src, SV40 Large T antigen and the chimeric ETV6-NTRK3 (reviewed in [12], [13]). Based on these findings, we utilized mammary epithelial cells expressing elevated levels of IGF-1R for a siRNA screen to identify mediators of anoikis protection. In addition, use of cells in which a known gene drives protection from anoikis greatly facilitates mechanistic follow-up studies. This siRNA screen led to the identification of PTK6, a member of the Src family of tyrosine kinases that is frequently overexpressed in a variety of tumor types [14–17]. Here we demonstrate a critical role for PTK6 in anchorage-independent survival of specific subtypes of breast cancer cells. We also present evidence for IGF-1 receptor regulation as a novel mechanism for survival regulation by PTK6. Results Loss-of-function screen to identify genes involved in anchorage-independent survival induced by IGF-1R stimulation In contrast to non-transformed MCF-10A mammary epithelial cells, MCF-10A cells overexpressing IGF-1 receptor (IGF-1R cells) exhibit enhanced survival in suspension cultures with IGF-1 stimulation (Figure 1A). To identify genes that are required for Figure 1. siRNA screen identifies regulators of IGF-1-induced anchorage-independent survival. A) IGF-1R hyperstimulation enhances anchorage-independent survival of MCF-10A cells. MCF-10A cells overexpressing vector control or IGF-1R (IGF-1R) were cultured on adherent plates or in suspension cultures for 48 hours. The cells were collected and cell death was assessed by measuring cytoplasmic histone and DNA content in triplicate wells (Roche Diagnostics). Higher values indicate more death. The fold change is the average of three independent experiments. Error bars indicate standard deviation. Western blot analysis shows relative IGF-1R levels in the cell lines. B) Candidate siRNAs that are critical for IGF-1R-induced anchorage-independent survival were identified by a loss-of-function screen. In the primary screen, a library of siRNA oligonucleotides targeting all human kinases was screened using the IGF1R overexpressing MCF10A line. Alamar blue reduction was assayed. Positives from the primary screen were counter-screened in MCF10A cells cultured in 2% Matrigel in suspension. The screen positives that showed a differential effect in IGF1R- overexpressing cells compared to the MCF10A cells in Matrigel were selected for validation using additional sequences. doi:10.1371/journal.pone.0011729.g001 PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11729