BRCA2 mediates centrosome cohesion via an interaction with cytoplasmic dynein

<p>BRCA2 is responsible for familial breast and ovarian cancer and has been linked to DNA repair and centrosome duplication. Here we analyzed the mechanism by which the centrosomal localization signal (CLS) of BRCA2 interacts with cytoplasmic dynein 1 to localize BRCA2 to the centrosome. <i>In vitro</i> pull-down assays demonstrated that BRCA2 directly binds to the cytoplasmic dynein 1 light intermediate chain 2. A dominant-negative HA-CLS-DsRed fusion protein, the depletion of dynein by siRNA, and the inactivation of dynein by EHNA, inhibited the localization of BRCA2 at centrosomes and caused the separation of centrosome pairs during the S-phase. The double depletion of BRCA2 and C-Nap1 caused a larger dispersion of centrosome distances than the silencing of C-Nap1. These results suggest that cytoplasmic dynein 1 binds to BRCA2 through the latter's CLS and BRCA2 mediates the cohesion between centrosomes during the S phase, potentially serving as a cell-cycle checkpoint.</p

Genome-Wide Association Study of Breast Cancer in the Japanese Population

<div><p>Breast cancer is the most common malignancy among women in worldwide including Japan. Several studies have identified common genetic variants to be associated with the risk of breast cancer. Due to the complex linkage disequilibrium structure and various environmental exposures in different populations, it is essential to identify variants associated with breast cancer in each population, which subsequently facilitate the better understanding of mammary carcinogenesis. In this study, we conducted a genome-wide association study (GWAS) as well as whole-genome imputation with 2,642 cases and 2,099 unaffected female controls. We further examined 13 suggestive loci (<i>P</i><1.0×10⁻⁵) using an independent sample set of 2,885 cases and 3,395 controls and successfully validated two previously-reported loci, rs2981578 (combined <i>P</i>-value of 1.31×10⁻¹², OR = 1.23; 95% CI = 1.16–.30) on chromosome 10q26 (<i>FGFR2</i>), rs3803662 (combined <i>P</i>-value of 2.79×10⁻¹¹, OR = 1.21; 95% CI = 1.15–.28) and rs12922061 (combined <i>P</i>-value of 3.97×10⁻¹⁰, OR = 1.23; 95% CI = 1.15–.31) on chromosome 16q12 (<i>TOX3-LOC643714</i>). Weighted genetic risk score on the basis of three significantly associated variants and two previously reported breast cancer associated loci in East Asian population revealed that individuals who carry the most risk alleles in category 5 have 2.2 times higher risk of developing breast cancer in the Japanese population than those who carry the least risk alleles in reference category 1. Although we could not identify additional loci associated with breast cancer, our study utilized one of the largest sample sizes reported to date, and provided genetic status that represent the Japanese population. Further local and international collaborative study is essential to identify additional genetic variants that could lead to a better, accurate prediction for breast cancer.</p></div

Demographic data of patients recruited for this study.

*<p>Family members who have breast and/or ovarian cance.</p

The Manhattan plot for GWAS of breast cancer in the Japanese population.

<p>This plot is based on –log₁₀ (<i>P</i>-value) from GWAS and imputation analysis against chromosome position, each color represents different chromosome. Blue line indicate suggestive association threshold, <i>P</i> = 1×10⁻⁵ while red line indicate genome-wide significant threshold <i>P</i><5×10⁻⁸.</p

Association of previously reported to be breast cancer susceptibility loci in current Japanese GWAS dataset.

<p>CHR: chromosome, SNP: single nucleotide polymorphism, Chr.loci/Gene: Chromosome location/Gene, BP: SNP genomic location, Ref: reference, Case_N: Number of cases, Ctrl_N: Number of controls, RAF: risk allele frequency, P_value: <i>P</i>-value from logistic regression analysis after age adjustment, OR: odds ratio, L95: lower 95% confidence interval, U95: upper 95% confidence interval.</p

Regional association plots for two significantly associated loci with breast cancer in Japanese population, (a) chromosome 10q26.13 (<i>FGFR2</i>) and (b) chromosome 16q21.1 (<i>TOX3-LOC643714</i>).

<p>SNPs from the GWAS are plotted as circles; imputed SNPs are plotted as crosses. The color intensity reflects the extent of LD with the marker SNP: red, (r²≥ 0.8), orange (0.6≤r²≤0.8), green (0.4≤r²≤0.6), light blue (0.2≤r²≤0.4) and dark blue (r²<0.2). Purplish blue lines represent local recombination rates. The SNP position is based on NCBI build 37.</p

Keratin 17 Is Induced in Oral Cancer and Facilitates Tumor Growth

<div><p>Keratin subtypes are selectively expressed depending on the cell type. They not only provide structural support, but regulate the metabolic processes and signaling pathways that control the growth of the epithelium. KRT17 (keratin 17) is induced in the regenerative epithelium and acts on diverse signaling pathways. Here, we demonstrate that KRT17 is invariably and permanently induced in oral squamous cell carcinoma (OSCC), as revealed by immunohistochemistry and cDNA microarray analysis. Two representative OSCC cell lines; KRT17-weakly expressing Ca9-22 and KRT17-highly expressing HSC3 were used to establish KRT17-overexpressing Ca9-22 and KRT17-knockdown HSC3 cells. Analysis of these cells revealed that KRT17 promoted cell proliferation and migration by stimulating the Akt/mTOR pathway. KRT17 also upregulated the expression of SLC2A1 (solute carrier family 2 member 1/Glut1) and glucose uptake. To further investigate the effect of KRT17 on tumorigenesis, KRT17-knockout HSC3 cells were established and were transplanted to the cephalic skin of nude mice. The tumors that developed from KRT17-knockout HSC3 cells had a lower Ki-67 labeling index and were significantly smaller compared to the controls. These results indicate that KRT17 stimulates the Akt/mTOR pathway and glucose uptake, thereby facilitating tumor growth. We could not confirm the relationship between KRT17 and SFN (stratifin) in the cells examined in this study. However, our study reinforces the concept that the cellular properties of cancer are regulated by a series of molecules similar to those found in wound healing. In OSCC, KRT17 acts as a pathogenic keratin that facilitates tumor growth through the stimulation of multiple signaling pathways, highlighting the importance of KRT17 as a multifunctional promoter of tumorigenesis.</p></div

KRT17 upregulates SLC2A1 and glucose uptake.

<p>(A) Representative images of western blot analysis of Ca9/K17+ (C-4), Ca9, HSC3/K17- (Kd-4) and HSC3 for detecting KRT17, SLC2A1, and beta1-tubulin (TUBB). Representative images of three assays. (B) Expression of SLC2A1 in Ca9/K17+ (C-1, C-2, C-3, and C-4) and HSC3/K17- (Kd-1, Kd-2, Kd-3, and Kd-4) compared with Ca9 and HSC3, respectively, as revealed by densitometric analysis of the western blots. Expression level of protein X in a clone was normalized against TUBB and then against the control cells using the formula (Value of protein X in the clone / Value of TUBB in the clone) / (Value of protein X in the control cells / Value of TUBB in the control cells). The normalized expression levels were plotted on a log scale. Representative results of three independent assays. (C) Glucose uptake assay of Ca9 and Ca9/K17+ (C-4) cells as measured by fluorescence microscopy. The cells were treated with a fluorescent glucose analogue for 2 h. The fluorescence image was digitally analyzed and the signal in individual cells was depicted as a brightness unit. The box plot illustrates the maximum, third quartile, median, first quartile and minimum signals of 100 cells. Representative results of three assays. (D) Glucose uptake assay as measured by flow cytometry. The cells were treated with the fluorescent glucose analogue for 2 h and were harvested for flow cytometric analysis (n = 1000). (E) cDNA microarray analysis of <i>KRT17</i> and <i>SLC2A1</i> in 43 oral squamous cell carcinomas (OSCCs) and 7 normal controls. <i>SLC2A1</i> was significantly upregulated in OSCC (P < 0.001), with an approximate three-fold increase both in the mean and the median value. There was a positive correlation between <i>SLC2A1</i> and <i>KRT17</i> expression (r = 0.46; ***P < 0.001). The scales on the horizontal and vertical axes represent absolute signal values. Crosses denote each OSCC case and filled circles denote normal controls. (E) Immunohistochemical expression of SLC2A1 in normal tongue epithelium and tongue cancer. Expression was greatest in lymphocytes (arrows in left upper and lower panels). In the normal oral epithelium, SLC2A1 was weakly expressed in the basal and spinous cells (left upper panel). In OSCC, SLC2A1 was upregulated, showing a level of expression comparable with lymphocytes (left and right lower panels). Scale bar, 100 μm.</p

KRT17 expression was induced in the regenerative epithelium and oral squamous cell carcinoma (OSCC).

<p>(A) KRT17 expression in regenerative epithelium. Hematoxylin and eosin (HE) staining (left panel) and immunohistochemical staining using the anti-KRT17 antibody (right panel). KRT17 expression was induced in the regenerative epithelium at the edge of the traumatic ulcer of the oral mucosa. Scale bar, 100 μm. (B) KRT17 expression in hyperplastic epithelium. Immunohistochemical staining using the anti-KRT17 antibody. In this buccal mucosa with nonspecific inflammation, KRT17 expression was induced in the basal cells. Note the scattered distribution of KRT17-positive cells in the basal layer. Scale bar, 100 μm. (C) cDNA microarray analysis of <i>KRT16</i> and <i>KRT17</i> in 43 OSCCs and 7 normal oral epithelia. The scales on the horizontal and vertical axes represent absolute signal values. Crosses denote each OSCC case and filled circles denote normal controls. The dashed lines were placed at the maximum values in the normal epithelium. (D-G) Immunohistochemical expression of KRT17 in OSCC. (D) KRT17 was expressed in the carcinoma <i>in situ</i> (left side), whereas KRT17 was not expressed in the normal epithelium (right side). Scale bar, 100 μm. (E, F, G) HE staining (upper panels) and immunohistochemical staining using the anti-KRT17 antibody (lower panels) in OSCC specimens. The upper and lower panels are serial sections from one sample. KRT17 expression was constantly induced in OSCC regardless of the histology. The levels and distributions of expression showed little variation across cases. Scale bar, 100 μm. (E) Keratinizing tumor nests and (F) small tumor islands of OSCC, both showing similar levels of ubiquitous KRT17 expression. Scale bars, 200 μm (E) and 100 μm (F). (G) Condylomatous tumor that showed highly differentiated histology was also positive for KRT17. Normal epithelium was present at the left side, showing negative staining. Scale bar, 1 mm. (H) Summary of immunohistological KRT17 expression in 50 OSCC cases. +, strong staining; -, weak or no staining. (I) Comparison between <i>KRT17</i> expression and histological grade of differentiation in 43 OSCCs. G1, well differentiated; G2, moderately differentiated; G3, poorly differentiated. Bars depict the median. N is the total number of cases analyzed. (J) Comparison between the <i>KRT17</i> expression and presence of lymph node metastasis in 43 OSCCs. Bars depict the median. N is the total number of cases analyzed.</p

KRT17, pAKT1, MTOR, phosphorylated EIF4EBP1 (pEIF4EBP1) and SLC2A1 were concurrently upregulated in oral squamous cell carcinoma (OSCC).

<p>(A) Representative images of immunohistochemical expression of KRT17, MTOR, pEIF4EBP1, SLC2A1 and pAKT1 in normal oral epithelium (normal) and OSCC (cancer). HE, hematoxylin and eosin staining. Scale bar, 100 μm. (B) Schematic representation of the immunohistochemical expression of KRT17, MTOR, pEIF4EBP1, SLC2A1 and pAKT1 in 50 OSCC cases. A blue square denotes increased expression in the individual OSCC case compared with the normal epithelium as an internal control. A white square denotes no immunohistochemical expression. A light-blue square denotes weak staining comparable to that in the normal epithelium. There was not a single case of downregulation of these proteins in OSCC.</p