The G1 phase Cdks regulate the centrosome cycle and mediate oncogene-dependent centrosome amplification

Because centrosome amplification generates aneuploidy and since centrosome amplification is ubiquitous in human tumors, a strong case is made for centrosome amplification being a major force in tumor biogenesis. Various evidence showing that oncogenes and altered tumor suppressors lead to centrosome amplification and aneuploidy suggests that oncogenes and altered tumor suppressors are a major source of genomic instability in tumors, and that they generate those abnormal processes to initiate and sustain tumorigenesis. We discuss how altered tumor suppressors and oncogenes utilize the cell cycle regulatory machinery to signal centrosome amplification and aneuploidy

Cdk4 and Nek2 Signal Binucleation and Centrosome Amplification in a Her2+ Breast Cancer Model

<div><p>Centrosome amplification (CA) is a contributor to carcinogenesis, generating aneuploidy, and chromosome instability. Previous work shows that breast adenocarcinomas have a higher frequency of centrosome defects compared to normal breast tissues. Abnormal centrosome phenotypes are found in pre-malignant lesions, suggesting an early role in breast carcinogenesis. However, the role of CA in breast cancers remains elusive. Identification of pathways and regulatory molecules involved in the generation of CA is essential to understanding its role in breast tumorigenesis. We established a breast cancer model of CA using Her2-positive cells. Our goal was to identify centrosome cycle molecules that are deregulated by aberrant Her2 signaling and the mechanisms driving CA. Our results show some Her2+ breast cancer cell lines harbor both CA and binucleation. Abolishing the expression of Cdk4 abrogated both CA and binucleation in these cells. We also found the source of binucleation in these cells to be defective cytokinesis that is normalized by downregulation of Cdk4. Protein levels of Nek2 diminish upon Cdk4 knockdown and vice versa, suggesting a molecular connection between Cdk4 and Nek2. Knockdown of Nek2 reduces CA and binucleation in this model while its overexpression further enhances centrosome amplification. We conclude that CA is modulated through Cdk4 and Nek2 signaling and that binucleation is a likely source of CA in Her2+ breast cancer cells.</p></div

Centrosome aberrations and chromosome instability contribute to tumorigenesis and intra-tumor heterogeneity

Centrosomes serve as the major microtubule organizing centers in cells and thereby contribute to cell shape, polarity, and motility. Also, centrosomes ensure equal chromosome segregation during mitosis. Centrosome aberrations arise when the centrosome cycle is deregulated, or as a result of cytokinesis failure. A long-standing postulate is that centrosome aberrations are involved in the initiation and progression of cancer. However, this notion has been a subject of controversy because until recently the relationship has been correlative. Recently, it was shown that numerical or structural centrosome aberrations can initiate tumors in certain tissues in mice, as well as invasion. Particularly, we will focus on centrosome amplification and chromosome instability as drivers of intra-tumor heterogeneity and their consequences in cancer. We will also discuss briefly the controversies surrounding this theory to highlight the fact that the role of both centrosome amplification and chromosome instability in cancer is highly context-dependent. Further, we will discuss single-cell sequencing as a novel technique to understand intra-tumor heterogeneity and some therapeutic approaches to target chromosome instability

Binucleation and CA in Her2+ cells are mediated by Nek2.

<p>(a) Protein lysates from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065971#pone-0065971-g002" target="_blank">Figure 2a</a> were used in western blots to detect levels of Nek2 in MCF10A and Her2+ breast cancer cell lines; β-actin was used as a loading control. Western blot results show two separate gels; different exposures are commensurate with protein abundance. (b) Western blotting was done in lysates collected from proliferating SKBr3 and JIMT1 cells transfected with siCdk4 constructs. Antibodies against Cdk4 and Nek2 were used; β-actin was used as a loading control. Western blot results show two separate gels; different exposures are commensurate with protein abundance. (c) Western blotting was done in lysates collected from serum arrested HCC1954 expressing shRNAs against Cdk4. Antibodies against Cdk4, Nek2, phospho-NPM, and NPM protein were used; β-actin was used as a loading control. (d) Transient transfection of siNek2 was performed in target cell lines; scrambled siRNA was used as a control. Knockdown was determined by western blotting using an antibody against Nek2; β-actin was used as a loading control. CA was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065971#pone-0065971-g001" target="_blank">Figure 1a</a>. Western blot results show two separate gels; different exposures are commensurate with protein abundance. (e) Lentiviral shPLKO.1 control and shNek2 vectors were used to infect SKBr3 and HCC1954 cells and create stable cell lines via puromycin selection. Independent lentiviral clones were screened in each cell line; knockdown was confirmed by western blot using an antibody against Nek2; β-actin was used as a loading control. The percentage of binucleation was compared in SKBr3 parental, shPLKO.1, and two independent shNek2 cell lines, and HCC1954 parental, shPLKO.1 control, and two independent shNek2 cell lines as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065971#pone-0065971-g003" target="_blank">Figure 3a</a>. Statistical significance was addressed using a T-test (* = p≤0.05; ** = p≤0.01).</p

Her2+ cells display CA.

<p>(a) Centrosome amplification (CA) was measured by staining proliferating cells plated in four-chambered microscopy slides with an antibody against pericentrin and counterstaining with DAPI. Independent experiments were done three times using 200 cells per experiment. Graphs show the percent of cells with CA. Statistical significance was addressed using a T-test (* = p≤0.05). (b) Protein lysate was collected under starvation conditions. MCF10A and Her2+ breast cancer cell lines were probed with antibodies against Her2, and cyclin D1; β-actin was used as a loading control. Western blot results show two separate gels; different exposures are commensurate with protein abundance.</p

CA in Her2+ cells is mediated by Cdk4.

<p>(a) siRNAs against Cdk2 and Cdk4 were transfected into target cell lines; scrambled siRNA was used as a control. siRNA knockdown was confirmed by western blot using antibodies against Cdk2 and Cdk4; β-actin was used as a loading control. Western blot results show three separate gels; different exposures are commensurate with protein abundance. The number of centrosomes in proliferating cells was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065971#pone-0065971-g001" target="_blank">Figure 1a</a>. Statistical significance was addressed using a T-test (* = p≤0.05; ** = p≤0.01). (b) Lentiviral shPLKO.1 control and shCdk4 vectors were used to infect MCF10A, SKBr3, and HCC1954 and create stable cell lines via puromycin selection. Independent lentiviral clones were screened in each cell line; knockdown was confirmed by western blot using an antibody against Cdk4; β-actin was used as a loading control. Western blot results show three separate gels; different exposures are commensurate with protein abundance. Centrosome amplification was measured in cell lines where knockdown was successful as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065971#pone-0065971-g001" target="_blank">Figure 1a</a>. Statistical significance was addressed using a T-test (** = p≤0.01).</p

Her2+ breast cancer cells display elevated percentages of binucleation and cytokinesis defects.

<p>(a) Binucleation was measured in MCF10A, SKBr3, and HCC1954 parental cell lines by fixing, processing, and staining proliferating cells with an antibody against α-tubulin and counterstaining with DAPI. Arrows indicate binucleated cells. Independent experiments were done three times using 200 cells per experiment. Graphs show the percent of binucleated cells. Statistical significance was addressed using a T-test (* = p≤0.05; ** = p≤0.01). (b) The percentage of CA in binucleated cells was measured by fixing, processing, and staining proliferating cells with antibodies against pericentrin and α-tubulin and counterstaining with DAPI. Independent experiments were done two times using 200 cells per experiment. The percentage of binucleation was measured as described in (a). (c) Binucleation was measured in HCC1954 parental, HCC1954 shPLKO.1 control, and HCC1954 shCdk4 cells as described in (a). (d) Still panels were captured from live cell imaging video of HCC1954 shPLKO.1 and shCdk4-1 to analyze the formation of binucleates in a proliferating population. Arrow indicates a binucleate resulting from failed cytokinesis.</p

Working model.

<p>Our working model proposes that in a Her2+ breast cancer model overexpression of cyclin D1/Cdk4 leads to an abundance of Nek2. Based on our results and the results of others, overexpression of Nek2 could drive binucleation through failed cytokinesis, leading to CA and potentially transformation and mammary tumorigenesis.</p

Knockdown of Cdk4 does not affect the fraction of cells in S phase.

<p>Results are from three independent experiments.</p>*<p>p-value is calculated by T-test.</p

Knockdown of Cdk4 does not affect cell cycle profiles.

<p>Results are pooled from two independent experiments.</p>*<p>p-value is calculated by T-test.</p