IGF-1 induced anchorage-independent survival, IGF-1R cells were transfected in suspension cultures with a library of individual siRNA oligonucleotides targeting all human kinases and related proteins (Figure 1B). The ability of individual siRNAs to modulate or reverse IGF-1dependent survival was initially assessed by monitoring changes in Alamar Blue reduction signal. Candidate siRNAs identified in the primary screen were rescreened using parental MCF-10A cells cultured in suspension with reconstituted basement membrane proteins to prioritize those siRNAs that more specifically inhibited IGF-1 driven survival. Lastly we screened additional siRNAs targeting the IGF-1R specific hits to identify those genes that were targeted by three or more distinct siRNA sequences. PTK6 modulates IGF-1 dependent anoikis resistance PTK6 was identified as a ‘‘high-confidence’’ candidate gene because multiple siRNA oligonucleotides caused a significant, preferential inhibition of IGF-1R-driven anchorage-independent viability when compared to parental MCF-10A cells. PTK6 was also considered an attractive candidate for further study because it is highly expressed in multiple tumor types, including breast and ovarian tumors [14–16], [18]. Low level expression of PTK6 protein was detected in parental MCF-10A cells grown in suspension cultures; this expression was enhanced in IGF-1R cells (Figure S1A) All three oligonucleotides identified in the phenotypic screen downregulated PTK6 protein expression effectively, as did a pool of siRNA olgonucleotides (OTP) from an independent vendor (Figure S1B). We also identified a lentivirally-delivered shRNA vector that effectively downregulated PTK6 protein expression (Figure 2A, left). To more specifically determine whether PTK6 downregulation induces cell death of IGF-1R cells in suspension cultures, we utilized Cell Death ELISA assays and monitored cleavage of PARP, a caspase target. Downregulating PTK6 by either transfecting siRNA oligonucleotides or expressing an shRNA vector enhanced death of IGF-1R cells in suspension with enhanced detection of cleaved PARP product (Figure 2A and Figure S1C). The effect of PTK6 downregulation on apoptosis is specific for matrix-deprived cells as downregulation of PTK6 had no effect on attached IGF-1R cells (Figure 2A), supporting the specificity of the screen in identifying genes that modulate survival only in matrix-detached conditions. This death was inhibited by ZVAD-fmk, a pan-caspase inhibitor, further supporting a role for PTK6 in suppressing anoikis induced by matrix detachment (Figure 2A). Downregulation of PTK6 inhibits luminal filling induced by IGF-1R hyperstimulation Acquisition of anchorage-independent survival is believed to play a role in the abnormal accumulation of cells in the lumen of terminal breast acinar structures, a property that is commonly observed in human breast lesions like ductal carcinoma in situ (DCIS). Consistent with this, we have found that filling of the matrix-free lumen of mammary acinar structures in 3D culture models requires induction of both hyperproliferation and anti- apoptotic activity [19]. We and others have previously reported that IGF-1R hyperstimulation of breast epithelial cells results in the formation of abnormally large structures with filled lumen due to hyperproliferation and suppression of death of the centrally localized cells in the presumptive luminal space [20], [21]. To address whether PTK6 plays a critical role in modulating the survival of cells in the luminal space, we examined the effects of PTK6 downregulation on IGF-1-induced acinar formation and PTK6 Regulates Survival luminal filling. PTK6 shRNA expression in IGF-IR/MCF-10A cells suppressed the formation of large, hyperproliferative structures (Figure 2B left). While many PTK6 shRNA-expressing structures resembled parental MCF-10A acini, some were aborted in early stages of outgrowth. These results indicate that PTK6 is also critical for IGF-1R-induced hyperproliferation in 3D culture. In contrast, downregulation of PTK6 had no significant effect on basal morphogenesis of parental MCF-10A cells (Figure 2B left). We then examined the effect of PTK6 downregulation on IGF- 1R-induced luminal filling in 3D cultures. Compared to acini expressing vector control, PTK6 downregulation resulted in a significantly higher percentage of structures with clear lumen (Figure 2B right). Thus, PTK6 is critical for anchorage- independent survival of cells in suspension and in the luminal space of IGF-1R overexpressing acinar structures. The combined effects of PTK6 downregulation on proliferation and anchorage- independent survival contribute to inhibition of abnormal acinar formation induced by IGF-1R stimulation. Overexpression of PTK6 enhances anchorage- independent survival To complement the loss-of-function studies, we examined the effect of overexpressing PTK6 in both MCF-10A cells and immortalized human mammary epithelial cells (HMLE). Overex- pression of PTK6 in parental MCF-10A cells using either a FLAG-tagged wild type PTK6 (F-PTK6) or myristylated, membrane targeted PTK6 (MF-PTK6), was sufficient for a modest, but significant, suppression of cell death upon detachment from matrix (Figure 3A). MCF-10A cells overexpressing MF- PTK6 consistently displayed a greater degree of anoikis resistance when compared to cells overexpressing F-PTK6. PTK6 kinase activity is important for suppression of anoikis of MCF-10A cells, as overexpression of a kinase-inactive MF-PTK6 (KD-MFPTK6) did not significantly rescue these cells from matrix detachment- induced cell death (Figure 3B). Overexpression of PTK6 alone (MF-PTK6 or F-PTK6) in HMLE was not sufficient to significantly promote colony formation in soft agar, another measure of anchorage-independent survival (Figure S2). However, PTK6 was able to enhance soft agar colony growth induced by other oncogenes that activate the PI3-K or Erk pathways (Figure 3C). Utilizing a cell system similar to that described previously in which activated Akt and MEK are used to substitute for Ras activation in transformation of immortalized HMEC [22], we observed that overexpression of PTK6 in immortalized HMLE expressing either activated MEK (HMLE-M) or myristylated Akt (HMLE-A) significantly enhanced colony formation (Figure 3B). Interestingly, membrane-targeted PTK6 was as effective, if not more effective, than wild type PTK6 in enhancing colony formation. Although PTK6 lacks a native myristylation sequence (in contrast to other Src family kinases), PTK6 has been reported to localize to membrane ruffles and associate with membrane-bound receptors such as EGFR and ErbB2 [23–25]. Membrane-localization may therefore play a critical role in the collaborative transforming functions of PTK6. PTK6 modulates anoikis sensitivity of breast and ovarian cancer cells expressing IGF-1R We sought to determine whether PTK6 plays a critical role in modulating anchorage-independent survival of breast and ovarian cancer cells in which PTK6 is highly expressed, particularly those which co-express IGF-1R. Examination of a panel of cell lines revealed that PTK6 was abundantly expressed in several Her2+ breast cancer lines, as previously reported (Figure S3A). Down- PLoS ONE | www.plosone.org 3 July 2010 | Volume 5 | Issue 7 | e11729