between PTK6 copy number gain and cyclin D1 amplification was also observed (Figure 7A). However, in contrast to a previous report [23], a correlation with ErbB2 amplification was not observed in the set of tumors analyzed for this study. We examined the relative expression of PTK6 mRNA in different breast tumor subtypes (Figure 7C). In previously published microarray analyses of breast tumor specimens downloaded from Oncomine, PTK6 transcript levels were elevated in nearly all breast tumor subtypes with the exception of basal-like tumors. PTK6 expression was the highest in both Her2+ and high-grade ER+ (Luminal B) tumors. Many Luminal B tumors also express high levels of IGF-1R transcript (data not shown). Elevated levels of PTK6 mRNA expression are associated with prognostic significance. In two large breast cancer patient cohorts with long-term follow up, tumor specimens were stratified based on PTK6 expression levels. Patients with tumors with the highest expression levels of PTK6 mRNA transcript in each cohort were less likely to be recurrence-free over long-term follow up (Figure 7D, top). This prognostic significance was observed independent of estrogen receptor or nodal status in multivariate analyses (data not shown). High PTK6 transcript levels were also associated with adverse outcomes for the subset of patients with ER+ tumors in both cohorts (Figure 7D, bottom). This prognostic significance was observed in multiple other cohorts that utilized different array platforms (Figure S6). Discussion In contrast to normal epithelial cells, most cancer cells have acquired the ability to grow in an anchorage-independent manner. In this report, we identified PTK6 as a gene that is critically involved in IGF-1R-stimulated anchorage-independent survival using a highly validated siRNA screen. PTK6 was also found to be required for anchorage independence of breast and ovarian cancer cells. These findings were supported by gain-of-function studies showing that PTK6 is an effective ‘‘collaborative oncogene’’, efficiently enhancing transformation in combination with other activated oncogenes. Insights into the mechanisms whereby PTK6 induces anoikis resistance were provided by high throughput protein arrays and subsequent analyses, which demonstrated that loss of PTK6 had broad effects on the major signaling pathways activated by IGF-1R as well as IGF-1R itself. Subsequent studies confirmed that PTK6 regulates the expression and the activity of IGF-1R. Effects of PTK6 on anchorage-independence could contribute to the poor outcomes associated with high PTK6 transcript levels that we found in multiple cohorts of breast cancer patients. Our studies indicate that PTK6 regulates anchorage-indepen- dent survival through suppression of caspase-mediated apoptosis, as downregulation of PTK6 increased PARP cleavage and resulted in cell death that was inhibited by ZVAD-fmk. Interestingly, Harvey et al. recently reported that downregulation of PTK6 in the matrix-deprived T47-D breast cancer cells induces autophagy, but not apoptosis [31]; however, they did not assess whether autophagy was responsible for cell death. Matrix detachment has been shown to cause metabolic impairment [32] and induce autophagy associated with activation of AMPK, an enzyme that senses metabolic stress [33]. Interestingly, inhibition of autophagy enhances apoptotic cell death in MCF-10A cells [33], suggesting that autophagy promotes epithelial cell survival, rather than cell death, in matrix-detached MCF-10A cells. RPPA analyses in this study demonstrated that AMPK and its target protein ACC were activated by PTK6 downregulation, PTK6 Regulates Survival indicating that loss of PTK6 also causes metabolic stress. In addition, although cell death induced by PTK6 downregulation was inhibited by ZVAD-fmk, the metabolic impairment was not (data not shown). While autophagy can be an adaptive survival mechanism for cells under stress it can also progress to cell death (reviewed in [34], [35]). It is possible that both autophagy and apoptosis play roles in cell death resulting from PTK6 downreg- ulation and the relative contribution may differ depending on cellular context, levels of anti-apoptotic proteins and duration of matrix deprivation. Our studies together with previous reports [36], [37] indicating that PTK6 may regulate multiple signaling molecules infer that there may be multiple mechanisms by which PTK6 regulates cell growth and survival. Overexpression of PTK6 enhanced anchorage-independent survival. Although PTK6 is distantly related to the Src family of tyrosine kinases with an SH3, SH2 and catalytic domain, it lacks a native myristylation signal shared by most members of this class [14], [38]. The ability of myristylated PTK6 to effectively enhance anchorage-independent survival points to a potentially critical role for membrane localization in this oncogenic function of PTK6. During the preparation of this manuscript, Kim and Lee also reported that myristylated PTK6, but not nuclear localized PTK6, promoted proliferation, migration, and colony formation of HEK293 cells [39]. Membrane localization is not a general requirement for all src-family kinases, as a related kinase Frk, which also lacks a native myristylation sequence, failed to enhance soft agar colony growth when targeted to the membrane (unpublished data). Thus subcellular localization likely determines access to specific substrates and interacting partners that modulate PTK6’s role in tumorigenesis. While the ability of PTK6 to enhance signaling downstream of EGF receptor family kinases is well established [24], [28], our studies are the first to highlight the ability of PTK6 to regulate other growth factor receptor kinases, namely IGF-1R. Comple- mentary protein arrays performed on cells with either overex- pression or downregulation of PTK6 revealed an effect of PTK6 on IGF-1 receptor phosphorylation and function. This ability of PTK6 could underpin its critical role in a subset of breast tumors which do not express substantial levels of ErbB receptor family kinases, but rather express high levels of both IGF-1R and PTK6. Specifically, we observed a trend for high- grade ER+ (i.e. Luminal B) tumors to express high levels of both of these transcripts (data not shown); therefore, PTK6 may contribute to the growth and survival of these tumors by modulating IGF-1R signaling. Whether the effects of PTK6 on IGF-1R function involve direct interactions with the receptor remain to be addressed. The ability of PTK6 to regulate the IGF-1 receptor does not appear to be dependent on a secreted factor as conditioned media from cells in which PTK6 expression was modulated failed to induce any change in IGF-1R (data not shown). The ability of PTK6 to co- precipitate with both the IGF-1 receptor and IRS-1 suggests that PTK6 is able to associate with the receptor complex. Interestingly, v-Src has been reported to directly phosphorylate the beta chain of the IGF-1 receptor at the corresponding autophosphorylation sites in rat fibroblasts with subsequent activation of IGF-1R kinase, and IGF-1R is required for v-Src transforming activity [40]. PTK6, a Src family member, may similarly phosphorylate IGF-1R directly following recruitment to the membrane. Although both gain- and loss-of-function approaches used in this report highlighted a positive role for PTK6 in anchorage- independent survival, other studies utilizing mouse intestinal cells and rat fibroblasts suggest that PTK6 may be required for cell death triggered by specific stimuli such as DNA damage [41], [42]. There may be tissue-specific functions of PTK6 depending on PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11729