Results: 5

1.
Figure 2

Figure 2. Western blot and Immunohistochemical (IHC) analyses of IRX2 and TBL1XR1. From: Identification of novel gene amplifications in breast cancer and coexistence of gene amplification with an activating mutation of PIK3CA.

A) Protein expression, analyzed by Western blot, showed that both IRX2 and TBL1XR1 protein expression increased progressively from MCF10A to MCF10AT to the malignant cells, MCF10CA1h and MCF10CA1a. B) IHC with antibodies against IRX2 on tissue microarray (TMA) slides is shown. Two tumor cores for each negative (IRX2-), weak-positive (IRX2+) and strong-positive (IRX2++) are shown together with numbers and percentage of each staining category among the 85 tumor samples.

Mitsutaka Kadota, et al. Cancer Res. ;69(18):7357-7365.
2.
Figure 5

Figure 5. Concomitant activation mutation and gene amplification of PIK3CA in breast cancer. From: Identification of novel gene amplifications in breast cancer and coexistence of gene amplification with an activating mutation of PIK3CA.

A) Examples of PIK3CA amplification in five tumors are illustrated. Formatting of Figure 5A is the same as for . B) The mutation analysis for these five tumors is depicted. Two exons (10 and 21) of PIK3CA showed mutations. The tumors in Figures 5A and 5B are matched and displayed in the same order from top to bottom. The first tumor (at the top), which exhibits high-level copy number gain, lacks a PIK3CA mutation in both exons 10 and 21. The arrowheads in the first tumor mark the bases with the mutations, E545K (exon 10), H1047R (exon 21), and G1049R (exon 21), identified in other tumors. The positions of the mutations in these last four tumors are highlighted by black boxes, with the mutations labeled by the amino acid substitutions. C) Association of PIK3CA mutation and gene amplification are summarized here. On the left side, it displays a 2×2 contingency table showing the number of tumors in each of the four categories: copy number gain only; PIK3CA mutation only; copy number gain plus PIK3CA mutation; neither copy number gain nor PIK3CA mutation. On the right side, it displays a 2×2 contingency table showing only tumors with a PIK3CA mutation. Copy number gain is depicted in relation to type of PIK3CA mutation. The following four categories are included: copy number gain with an H1047R mutation; copy number gain with a non-H1047R mutation; an H1047R mutation without copy number gain: a non-H1047R mutation without copy number gain.

Mitsutaka Kadota, et al. Cancer Res. ;69(18):7357-7365.
3.
Figure 4

Figure 4. From: Identification of novel gene amplifications in breast cancer and coexistence of gene amplification with an activating mutation of PIK3CA.

Figure 4A. Clustering analysis of gene amplification in 161 primary breast tumors. The log2ratios of the 17 genes listed in for the 161 tumors were used to perform clustering analysis, generating the dendrogram at the top of the figure and the heat map at the bottom. The tested genes from are listed to the left of the heat map. The labels to the left of the middle portion of the figure are: node (red for lymph node positive and blue for negative), invasion (red for invasive breast cancer and blue for noninvasive, or DCIS), size (red for tumor size over 5 cm and blue for less than 5 cm), and mutation status for PIK3CA, TP53, and AKT1 (red for the presence of mutation and blue for absence of mutation). Figure 4B. Survival plots calculated by Kaplan-Meier analysis using expression data of 4 genes POLD3, CCND1, FGFR1, and FGFR2. The four publicly available gene expression datasets from the GEO database are GSE4922, GSE2034, GSE1456, and GSE3494. The red and blue curves represent good and poor survival patient groups, respectively. The two patient groups were determined by hierarchical clustering analysis based on gene expression values of these 4 genes as described in Results and Discussion section.

Mitsutaka Kadota, et al. Cancer Res. ;69(18):7357-7365.
4.
Figure 1

Figure 1. DNA copy number analysis of 161 primary breast tumors identified novel gene amplification events. From: Identification of novel gene amplifications in breast cancer and coexistence of gene amplification with an activating mutation of PIK3CA.

We used the Affymetrix SNP arrays to identify potential oncogenes in regions of focal amplification. A) Copy number estimation of ERBB2 by SNP array and qPCR were conducted using 76 tumors that are the subset of the 161 tumors. The regression line is described by y = 0.1635x + 0.7479 with R2 = 0.7965. B) Examples of three loci exhibiting gene amplification listed in are shown here, including TBL1XR1, IRX2, and NOTCH3/BRD4. Two tumor samples are shown for each locus. The graphs were generated by the Partek Genomics Suite. Genomic position is displayed on x-axis and log2ratio (tumor hybridization intensity divided by normal reference samples from HapMap project) is on y-axis. The red lines highlight gene amplification regions. Gene annotation encompassing each amplification region is provided at the top of the graphs. C) Gene expression measured by RT-qPCR is shown. Gene expression was measured in 14 tumors. For 7 of the tumors, adjacent normal samples were also analyzed. The expression values of tumors are normalized by the average value of the seven normal samples; hence, gene expression is indicated on the y-axis relative to this average value. The matched normal sample is connected to each corresponding tumor from the same patient by a straight line. The matched normal and tumor samples are represented by filled diamonds, whereas tumors without matched normal are indicated by open squares. The tumors showing amplification and their adjacent normal samples are indicated by red symbols and lines.

Mitsutaka Kadota, et al. Cancer Res. ;69(18):7357-7365.
5.
Figure 3

Figure 3. Characterization of cell transformation in breast cancer cell lines with shRNA knockdown of the TBL1XR1 gene. From: Identification of novel gene amplifications in breast cancer and coexistence of gene amplification with an activating mutation of PIK3CA.

TBL1XR1 knockdown in MCF10CA1h cells resulted in reduction of cell migration and loss of cell invasion examined by an in vitro cell culture system. A) Western blot analysis showed reduced TBL1XR1 protein in TBL1XR1-shRNA-transduced cells (lane labeled “TBL1XR1-shRNA”). B) In vitro scratch assays showed reduction of cell migration in TBL1XR1-shRNA-containing cells. Pictures were taken at 24 hours after scratching. In the image labeled “control-shRNA”, the inset (higher-magnification) shows individual migrating cells at the front edge of the cell mass; in contrast, the edge of the scratched area appears smooth in the TBL1XR1-shRNA-transduced cells. The width of the scratched area was measured at 12 and 24 hours after scratching. Each point represents a mean width with SD (standard deviation) based on 6 measurements. C) Cancer cell invasion was assayed using the Matrigel Matrix system at 60 hours after cell plating. Numbers of cells were counted in 10 randomly selected areas under the microscope. The histogram illustrates the mean of these cell numbers +/- SD. D) Knockdown of TBL1XR1 inhibited breast carcinoma development in mouse xenografts. MCF10CA1h cells begin forming detectable tumors as early as 14 days after injection, with tumor volumes increasing rapidly afterwards. A similar tumor growth curve was observed for control-shRNA. In contrast, the mice injected with the cells containing TBL1XR1-shRNA showed marked reduction in tumor growth (t-test, p-value<0.001) compared to control-shRNA (marked by *).

Mitsutaka Kadota, et al. Cancer Res. ;69(18):7357-7365.

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