PTK6 Regulates Survival Figure 3. Effects of PTK6 overexpression on anchorage-independent survival. A) Overexpression of PTK6 enhances anchorage- independent survival. MCF-10A cells overexpressing vector control or FLAG-tagged PTK6, with or without a myristylation sequence (MF-PTK6 or F- PTK6, respectively), were cultured in suspension for 48 hours. Cell death was assessed as in Figure 1. The average of three independent experiments is shown. Error bars indicate standard deviation. Western blot analysis shows relative expression levels of PTK6 for each cell line. B) Overexpression of kinase inactive PTK6 does not rescue MCF-10A cells from anoikis. MCF-10A cells overexpressing vector control, MF-PTK6 or a kinase inactive PTK6 (KD- MFPTK6) were cultured in suspension for 48 hours. Cell death was assessed as in Figure 1. The average of three independent experiments is shown. Error bars indicate standard deviation. Relative expression and activity of PTK6 in each cell line was assessed by Western blot analyses. C) Overexpression of PTK6 enhances soft agar growth in collaboration with other oncogenes. HMLE cells expressing MEKDD (HMLE-M) or myristylated Akt (HMLE-A) were engineered to express vector control, F-PTK6 or MF-PTK6. Soft agar colony growth was assessed after four weeks. A representative experiment of three replicate experiments using triplicate wells for each cell line is shown. Error bars indicate standard deviation. Western blot analysis shows levels of expression of PTK6 in each cell line. doi:10.1371/journal.pone.0011729.g003 regulation of PTK6 was associated with increased anoikis of BT474 cells and ErbB2 overexpressing MCF-10A cells (NeuN), lines in which anchorage-independent survival is induced via the ErbB2 growth factor receptor, supporting a role for PTK6 in survival of Her2+ cells (Figures 4A and 4B). PTK6 was also highly expressed in several Her22, ER+ cell lines, including MCF-7 (Figure S3A). MCF-7 cells express high levels of endogenous IGF- 1R, thereby providing another context for evaluating the role of PTK6 in IGF-1 induced, anchorage-independent survival. Stim- ulation of MCF-7 cells with IGF-1 rescued cells from anoikis, which was significantly reversed by downregulation of PTK6 (Figure 4C). This death was inhibited by the caspase inhibitor ZVAD-fmk, again supporting a role for PTK6 in modulating apoptosis of breast cancer cells under matrix-detached conditions. These results support an expanded role for PTK6 in growth factor receptor-induced anoikis resistance of breast cancer cells. Downregulation of PTK6 also prevented IGF-1 stimulated anoikis resistance of DOV-13 ovarian cancer cells, which co- express PTK6 and IGF-1R (Figure 4D). There is increasing evidence supporting a role for IGF-1 signaling in ovarian tumor progression [26], [27]. Furthermore, PTK6 has previously been reported to be amplified and overexpressed in a significant number of ovarian cancers although its functions in ovarian tumorigenesis have not been studied [18]. In contrast to normal human ovarian surface epithelial (HOSE) cells which do not express detectable levels of PTK6, DOV-13 cells express higher levels of PTK6 protein ([18] and Figure S3B). While DOV-13 cells are relatively sensitive to anoikis when compared to HOSE cells, IGF-1 stimulation enhances survival in suspension (Figure 4D). Downregulation of PTK6 reverses this IGF-1 induced anchorage-independent survival. Interestingly downreg- ulation of PTK6 also enhanced basal levels of death, suggesting that it may also play a critical role in anchorage-independent survival of DOV-13 cells distinct from its role in IGF-1- stimulated anoikis resistance. Regulation of IGF-1R signaling by PTK6 To begin to elucidate the mechanisms by which PTK6 modulates anchorage-independent survival, we utilized reverse- phase protein arrays (RPPA) to identify signaling molecules that are altered by downregulation or overexpression of PTK6 (Figures 5A, 6A). IGF-1R cells overexpressing Bcl2 (IGF/Bcl2) were transfected with multiple different siRNA oligonucleotides targeting PTK6 in suspension cultures in order to minimize changes in phosphoprotein signatures resulting secondarily from apoptosis. Decreased expression of PTK6 was associated with PLoS ONE | www.plosone.org 5 July 2010 | Volume 5 | Issue 7 | e11729