PTK6 Regulates Survival role in anchorage-independent survival via regulation of IGF-1R and IGF-1 stimulated signaling. PTK6 is amplified and expressed in specific subtypes of human breast tumors PTK6 may promote human cancer growth by modulating signaling via growth factors receptors, such as IGF-1R and ErbB2. PTK6 has been reported to be expressed in multiple tumor types, including breast and ovarian cancer [14–16], [18]. This expression pattern may be in part due to copy number gain as PTK6 maps to Chromosome 20q13.3, a region frequently amplified in breast tumors. Recently, Xiang et al. reported coamplification of PTK6 in Her2+ breast tumors [23]. We examined PTK6 copy number in a cohort of 93 breast tumors. Genomic DNA extracted from a series of breast tumors was hybridized to SNP (Single Nucleotide Polymorphism) arrays [30] (Figure 7A). PTK6 copy number gain (copy number greater than or equal to 3.0) was observed in 15 of 93 breast tumors (15%) in SNP analysis. Most PTK6 copy number gains are modest and with the exception of one tumor, are broad (,2.8Mbp) gains (Figure S5). Fluorescence in situ hybridization of tumors selected on the basis of elevated PTK6 signal intensity in microarray analyses showed an increase in the number of hybridized signals with the PTK6-specific probe (Figure 7B). For some tumor cells within a sample, there was also an observed increase in the number of hybridized signals with the Chromo- some 20 centromere-specific probe, suggesting that in some cases apparent PTK6 copy number gains may be due to increased numbers of chromosome 20. A statistically significant correlation Figure 7. PTK6 copy number gain and overexpression in primary human breast tumor samples. A) (Top) SNP array analyses of 93 breast tumors demonstrates PTK6 copy number gain in a subet of tumors.The x-axis represents individual breast tumor specimens; the y-axis represents copy number. The red line indicates copy number = 3 (Bottom) Correlation analysis with other amplified genes is shown. The table lists the number of tumors with PTK6 copy number gain (copy number$3.0), the Pearson product-moment correlation for each pair of variables and the associated P- values. B) FISH analysis demonstrates PTK6 copy number gain. Two human breast tumors were selected for FISH analyses based on high PTK6 transcript expression levels in microarray analyses. FISH analysis was performed using a probe specific for PTK6 (green) or a control chromosome 20 centromeric probe (pink). C) PTK6 mRNA is differentially expressed in human breast cancer subtypes. Publicly available datasets were used to compare levels of PTK6 mRNA in breast cancer subtypes (basal, HER2/ERBB2+, Luminal A, Luminal B) and normal samples. P-values were calculated using ANOVA in JMP 7.0 software. D) PTK6 mRNA level is a predictor of prognosis in human breast cancer. Kaplan-Meier curves depict the probability of breast cancer recurrence based on relative levels of PTK6 transcript expression. Probabilities for the entire cohort (top) as well as the ER+ subgroup (bottom) were determined. The Cox regression test evaluates the association of PTK6 level with patient outcome treating the level of PTK6 as a continuous variable. The log-rank test evaluates whether there are significant differences between any of the three groups. Data sets from Van’t Veer et al. (Van’t Veer et al., 2002) and Wang et al.(Wang et al., 2005) were analyzed. doi:10.1371/journal.pone.0011729.g007 PLoS ONE | www.plosone.org 9 July 2010 | Volume 5 | Issue 7 | e11729