Geminin overexpression induces mammary tumors via suppressing cytokinesis

Abstract

Aneuploidy plays an important role in the development of cancer. Here, we uncovered an oncogenic role for geminin in mitotic cells. In addition to chromatin, tyrosine phosphorylated geminin also localizes to centrosome, spindle, cleavage furrow and midbody during mitosis. Geminin binding to Aurora B prevents its binding to INCENP, and thus activation leading to lack of histone H3-(serine 10) phosphorylation, chromosome condensation failure, aborted cytokinesis and the formation of aneuploid, drug resistance cells. Geminin overexpressing human mammary epithelial cells form aneuploid, aggressive tumors in SCID mice. Geminin is overexpressed in more than half of all breast cancers analyzed. The current study reveals that geminin is a genuine oncogene that promotes cytokinesis failure and production of aneuploid, aggressive breast tumors when overexpressed and thus a worthy therapeutic target (oncotarget) for aggressive breast cancer.

Figure 1. Geminin localization in HME cells

Localization of geminin (green) and γ-tubulin (red) in interphase (A), metaphase (B) and in cytokinesis (C and D) in HME cells. Blue is DAPI-stained DNA. (E) Expression of Myc-tagged WT or Y-to-A geminin variants in HME cells. (F) Expression of His-tagged WT (Gem9) or Y-to-A mutant geminin variants in nuclear soluble or chromatin HME cells fractions. (G-V) Localization of endogenous geminin (green) and Myc-tagged exogenous geminin variants (red) in HME cells. Blue is DAPI stained DNA.

Figure 2. Analysis of histone H3 phosphorylation and ploidy in HME cells overexpressing WT or Y-to-A mutants geminin

(A-E) The p-(S10)-H3 expressing populations (percentages are shown, blue lines) in HME, and induced (96h) Gem9, GemY98A, GemY111A and GemY150A cells were measured by FACS. Data are compared to same IgG isotype (red lines). Results represent one of the experiments performed 3 times in triplicates. (F-J) Cell cycle analysis of uninduced Gem9 or induced (96h) Gem9, GemY98A, GemY111A and GemY150A cells as described in text. Tetraploid/aneuploid populations (percentages are shown) were measured by FACS (see red circles). R1=G₀/G₁, R3/R4/R5=early/mid/late S, R2=G₂/M and R6=>4N cells. Results represent one of the experiments performed 3 times in triplicates.

Figure 3. Tyrosine mutant geminin variants are apoptosis instead of tetraploidy/aneuploidy inducers

(A) Cell cycle analysis of HME and induced (96h) Gem9, GemY98A, GemY111A and GemY150A cells. HME and Gem9 cells were also transfected with luciferase or geminin siRNA during the last 72h. Cell cycle profiles (percentages are shown) were measured by FACS. Results represent the means ± SD of experiments performed 3 times in triplicates. Inset shows the knockdown effect of geminin siRNA in HME and induced Gem9 cells. (B) TUNEL analysis performed on HME and induced (96h) Gem9, GemY98A, GemY111A and GemY150A cells. (C) Number of apoptotic cells (TUNEL-positive) per field in cultures in (B). Results represent the means ± SD of experiments performed 3 times in triplicates, ** = p≤0.01. (D) Expression of geminin and pro-survival proteins in sonicates (total cellular proteins) of HME and induced (96) Gem9, GemY98A, GemY111A and GemY150A. (E) Phase contrast images of HME and induced (168h) Gem9, GemY98A, GemY111A and GemY150A. Images represent one of the experiments performed 3 times in triplicates.

Figure 4. The effect of geminin overexpression on chromosome condensation, centrosome number, cytokinesis, ploidy and transformation in HME cells, in vitro

(A) Representative images of PI stained metaphase spreads of uninduced (1) or induced (96h, 2) Gem9 cells. Experiments were performed 3 times in triplicates and at least 100 cells per culture were analyzed. (B) Representative images of γ-tubulin labeled cells of uninduced (1) or induced (96h, 2) Gem9 cells. Arrows show the number of centrosomes in each cell. Experiments were performed 3 times in triplicates and at least 100 cells per culture were counted. (C) Representative images of γ-tubulin labeled cells in uninduced (1) or induced (96h, 2) Gem9 cells. Arrows show the number of nuclei in each cell. Experiments were performed 3 times in triplicates and at least 100 cells per culture were counted. (D) Quantitative analysis of percentage of cells showing multiple centrosome (red bars), multi-nucleation (white bars) and cells aborting cytokinesis (black bars). Results represent the means ± SD of experiments performed 3 times in triplicates, ** = p≤0.01. (E) Time-lapse analysis of induced Gem9 cells (14 days). Cell “A” divided normally into 2 daughter cells, whereas cells “1, 2 and 3” attempted to undergo cytokinesis but failed and became tetraploid. Images correspond to Suppl. movie 1. (F) Giemsa stained metaphase chromosomes of Gem9 grown in the absence (1 and 3) or presence (2 and 4) or 2μg/ml of Dox for 8 weeks. Experiments were performed 3 times in triplicates and at least 100 cells per culture were counted. (G) Representative images of soft agar colony formation assay using uninduced (1) or induced Gem9 (2) cells. Experiments were performed 3 times in triplicates and at least 100 cells per culture were counted. (H) Quantitative analysis of the soft agar experiment described in (G). Data are represented as mean ± SD. *** = p<0.001.

Figure 5. Geminin overexpression inactivates AurB

Expression of several proteins in sonicates of HME and induced (96) Gem9, GemY98A, GemY111A and GemY150A cells. Note that because the same extracts were used to run the immunoblotting experiments described here and those in Figure 3D, geminin and actin blots are used in both figures. (B) Expression of several proteins in uninduced (−) or induced (+) Gem9 cells (left). Immunoprecipitated AurB from uninduced (-) or induced (+) Gem9 cells (right, upper) was used to in vitro phosphorylate GST-H3 (IVK, right). (C) Phosphorylation of survivin on T117 in uninduced (left) or induced (right) Gem9 cells (right, lower). (D) Immunoprecipitating of AurB from HME or induced Gem9 cells using AurB or INCENP specific antibody. (E) Immunoprecipitating AurB from G₁/S, G₂/M or M/G₁ HME cells using AurB or geminin specific antibodies. (F) The effect of AurB inhibitor ZM447493 on HME or induced Gem9 cell cycle progression. Results represent the means ± SD of experiments performed 3 times in triplicates, *= p<0.05 and ** = p<0.001.

Figure 6. Generation and analysis of geminin overexpression-induced tumors

(A) Volume of subcutaneous tumors developed in mice (10 mice/group) injected with HME/TERT, HME/TERT/LT, HME/TERT/geminin (i.e. Gem9), or HME/TERT/LT/geminin (i.e. Gem9/LT) cells. Representative H&E stained sections, in which Gem9/LT tumors invading mouse muscle (see M in B), bone (see B in C), skin (see S in D), nerve (see N in E) and sweet glands (see SG in F). (G and H) Representative H&E sections from Gem9/LT tumors showing different areas with high necrosis. N = necrotic cells and L = living cells. (I) Volume of mammary tumors developed in mice (10 mice/group) injected with HME/TERT, HME/TERT/LT, HME/TERT/geminin (i.e. Gem9), or HME/TERT/LT/geminin (i.e. Gem9/LT) cells. (J and K) Representative images showing the size (J) and the bloody appearance (K) of geminin overexpressing mammary tumors. Arrows show blood vessels. (L-N) Representative sections from geminin overexpressing mammary tumors stained with H&E showing normal (dashed arrows) and large (solid arrows) size nuclei. Sections also show increase blood vessels in these areas. (O) Density of blood vessels in areas containing tumor cells with normal size nuclei vs. areas with tumor cells with abnormal (large) size nuclei. (P) Volume of mammary tumors developed in mice (10 mice/group) injected with parental or geminin silenced MDAMB231 cells. Mice were kept on a Dox-supplemented water to induce geminin shRNAs. (Q) Representative Xenogene images at day 20 of subcutaneous tumors developed in mice injected with parental MDAMB231 (left) or MDAMB231/shGem1 (right) cells. Mice were kept on a Dox-supplemented water to induce geminin shRNA expression. Note the dramatic size difference in the luciferase signals.

Figure 7. Geminin overexpression in aggressive primary breast cancers

(A) Expression of geminin protein (upper) and mRNA (lower) in HME and several breast cancer cell lines. (B) Comparisons of geminin-positive (red bars) and -negative (green bars) in the test cohort (see text) using immunohistochemistry analysis. Blue bars show total number of tumors. (C and D) Representative images of geminin expression in normal (C) and invasive tumor (D) samples from the confirmation cohort. (D) Geminin mRNA level in breast cancer grade I (n=12) and grade III (n=78) samples. (E) Geminin mRNA level in metastasis < 5years and > 5years from diagnosis. Data in D and E were extrapolated from publicly available breast cancer gene expression microarray data set (vant' Veer et al., 2002). Data represented as mean ± SD and p values are shown.