differential substrate/binding partner availability. In addition, given the critical role that subcellular localization may play in oncogenic vs. non-oncogenic activity, the ability of PTK6 to be recruited to the membrane by any mechanism may determine its ultimate role in cell survival. PTK6 is expressed in multiple human tumor types, including breast and ovarian tumors [14–16,18]. PTK6 maps to a chromosomal region (20q13.3) that is frequently amplified in breast tumors and amplicons spanning this region variably contain other suspected oncogenes, such as TDE1, NCoA3, BCAS4, and ZNF217. The PTK6 copy number gain detected in our study is relatively modest. However, it is possible that low levels are sufficient to contribute to tumorigenic phenotypes collaboratively, as we have shown that PTK6 effectively collaborates with other oncogenic signaling pathways. In multiple microarray studies, elevated PTK6 mRNA expression was most highly associated with specific breast cancer subtypes (Her2+ and Luminal B/high grade + ER ). Although we have shown that PTK6 expression is critical for anchorage-independent survival of representative cell lines of these subtypes, the full range of functions attributable to PTK6 expression in these specific subtypes (e.g. regulation of sensitivity to chemotherapy or endocrine therapy, proliferation) remains to be addressed. In our study we analyzed the prognostic significance of elevated PTK6 transcript expression in multiple large cohorts of patients with breast cancer. Our finding that elevated expression of PTK6 transcript correlates with adverse outcomes for patients with breast cancer in all cohorts analyzed is consistent with a recently reported correlation between PTK6 mRNA levels and tumor grade based on analyses of 44 tumors [31]. However, this association of PTK6 with adverse outcome contrasts with findings of another group who reported (1) no correlation between PTK6 mRNA expression levels and disease-free survival, and (2) a positive correlation between PTK6 protein expression (determined by immunohisto- chemistry) and long-term disease-free survival. [43–45]. The exact basis for this discrepancy with our findings for PTK6 mRNA levels is not clear, although the analyses of Aubele et al. were all performed on a single cohort. Their finding with respect to PTK6 protein expression is intriguing; the lack of correlation between immunohistochemical signal intensity and either PTK6 FISH signals or transcript levels suggests that PTK6 protein levels may be regulated post-transcriptionally. Further complicating the studies of prognostic significance is the finding that subcellular localization of PTK6 protein may critically regulate oncogenic activity; in studies of prostate tumor cells, cytoplasmic localization of PTK6, in contrast to nuclear expression, was associated with poor differentiation and androgen independence, which are adverse prognostic factors [46]. Thus, immunohistochemical analyses of breast tumor specimens with attention to PTK6 expression in specific subcellular compartments could be infor- mative. PTK6 is an attractive potential therapeutic target for breast and ovarian cancer for several reasons. The rather restricted expression of PTK6 in normal tissues and the prevalence of PTK6 copy number gain and high expression in some breast and ovarian tumors improve the chance of selectively targeting malignant cells. PTK6 overexpression could contribute to resistance to currently available therapies, such as those targeting Her2 amplified tumors [23]. Inhibition or downregulation of PTK6 may also prove to be an effective strategy for enhancing the response of high-grade ER+/Luminal B breast cancers to endocrine therapy or overcoming acquired resistance to these agents. Validation of PTK6 as a viable target will depend on identifying those tumors in which PTK6 is functionally active and PTK6 Regulates Survival determining the specific substrates and domains of PTK6 that are required for its oncogenic activity. Materials and Methods Reagents, cells and cell culture MCF-10A, ZR751, MCF-7, BT474, DOV-13, Hey C2 and human immortalized mammary epithelial cells (HMEC) were obtained from ATCC (Manassas, Virginia). MCF-10A cells overexpressing IGF-1R receptor (IGF-1R) have been previously described [21]. MCF-10A cells and IGF-1R cells were cultured, as previously described [21], [47]. ZR-751 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. MCF-7 cells were cultured in DMEM supplemented with 10% FBS. Ovarian cancer lines were cultured in RPMI-0 supplemented with 10% fetal bovine serum and penicillin/streptomycin. Three- dimensional (3D) MatrigelTM cultures were performed as described previously [21]. ZVAD-fmk, a pan-caspase inhibitor, was purchased from BD Biosciences. IGF-1 receptor kinase inhibitor (BMS-536924) was purchased from Selleck and solubi- lized in DMSO. Antiserum or monoclonal antibodies directed against the following proteins were obtained from the indicated suppliers: Cell Signaling Technologies: phospho-IGF-1R (tyrosine 1131/1135/1136), phospho-Akt (Ser 473), Akt, cleaved PARP, phospho-STAT3, EGFR and Akt1; Santa Cruz Biotechnology: IGF-1R, PTK6, Erk2, tubulin; Biosource: phospho-Erk1/2; Millipore: IRS-1 and phospho-Brk; and Chemicon: Laminin 5,c2 subunit (LAMC2). Alamar Blue was purchased from Biosource. Constructs, virus production and stable cell line generation The constructs for PTK6 (kinase-active and kinase-inactive) were generated by cloning into pMSCV-puro. The construct encoding short hairpin RNA sequences targeting PTK6 was obtained from Open Biosystems (Catalog # TRCN0000021552). pBP-MF-PTK6 and pBP-F-PTK6 were obtained from the Harvard Institute of Proteomics (gift of Haley Hieronymus and Jesse Boehm). Viral packaging 293T or GPG-293T cells were transfected according to standard protocols. Viral supernatant was collected 36 and 60 hrs post-transfection. Immortalized HMECs or MCF-10A cells were infected in the presence of 2mg/ml polybrene with the viral supernatant or were spin-infected for 30 minutes at 2250 rpm. After three hours, media was changed on the target cells, which were then allowed to recover overnight, followed by another round of infection. Forty-eight hours post- infection, the target cells were exposed to puromycin (0.5–1 mg/ml for 48 hrs) to select for infected cells. Anchorage-independent viability screen A library consisting of two individual siRNA sequences targeting human kinases were obtained from Qiagen (v.1.0). MCF-10A cells overexpressing IGF-1R were trypsinized and plated in 96 well plates pre-coated with polyhema (Sigma). Cells were transfected using Oligofectamine in quadruplicate wells with individual siRNA duplexes from the Qiagen kinase siRNA library. Cells were cultured for an additional 72 hours in suspension and assayed for Alamar reducing potential on a multiplate reader according to the manufacturer’s protocol. In the counter screen, parental MCF-10A cells cultured on polyhema plates were transfected with siRNA duplexes. After four hours the media was supplemented with MatrigelTM to a final concentration of 2%. After an additional 72 hour incubation, Alamar blue reduction was assayed as above. The ratio between PLoS ONE | www.plosone.org 11 July 2010 | Volume 5 | Issue 7 | e11729