Many cancer cells display a CIN (Chromosome Instability) phenotype, by which they exhibit high rates of chromosome loss or gain at each cell cycle. Over the years, a number of different mechanisms, including mitotic spindle multipolarity, cytokinesis failure, and merotelic kinetochore orientation, have been proposed as causes of CIN. However, a comprehensive theory of how CIN is perpetuated is still lacking. We used CIN colorectal cancer cells as a model system to investigate the possible cellular mechanism(s) underlying CIN. We found that CIN cells frequently assembled multipolar spindles in early mitosis. However, multipolar anaphase cells were very rare, and live-cell experiments showed that almost all CIN cells divided in a bipolar fashion. Moreover, fixed-cell analysis showed high frequencies of merotelically attached lagging chromosomes in bipolar anaphase CIN cells, and higher frequencies of merotelic attachments in multipolar vs. bipolar prometaphases. Finally, we found that multipolar CIN prometaphases typically possessed γ-tubulin at all spindle poles, and that a significant fraction of bipolar metaphase/early anaphase CIN cells possessed more than one centrosome at a single spindle pole. Taken together, our data suggest a model by which merotelic kinetochore attachments can easily be established in multipolar prometaphases. Most of these multipolar prometaphase cells would then bi-polarize before anaphase onset, and the residual merotelic attachments would produce chromosome mis-segregation due to anaphase lagging chromosomes. We propose this spindle pole coalescence mechanism as a major contributor to chromosome instability in cancer cells.

Centrosome separation can be completed either before or after nuclear envelope breakdown (NEB). A combination of experimental and computational approaches shows that incomplete centrosome separation at NEB decreases the accuracy of chromosome segregation and thus represents a severe threat to genome stability.

SUMMARY Centromeres are specialized chromatin domains specified by the centromere-specific CENP-A nucleosome. The stable inheritance of vertebrate centromeres is an epigenetic process requiring deposition of new CENP-A nucleosomes by HJURP. We show HJURP is recruited to centromeres through a direct interaction between the HJURP centromere targeting domain and the Mis18α-β C-terminal coiled-coil domains. We demonstrate Mis18α and Mis18β form a heterotetramer through their C-terminal coiled-coil domains. Mis18α-β heterotetramer formation is required for Mis18BP1 binding and centromere recognition. S. pombe contains a single Mis18 isoform that forms a homotetramer, showing tetrameric Mis18 is conserved from fission yeast to humans. HJURP binding disrupts the Mis18α-β heterotetramer and removes Mis18α from centromeres. We propose stable binding of Mis18 to centromeres in telophase licenses them for CENP-A deposition. Binding of HJURP deposits CENP-A at centromeres and facilitates the removal of Mis18, restricting CENP-A deposition to a single event per cell cycle.

The mitotic spindle self-assembles in prometaphase by a combination of centrosomal pathway, in which dynamically unstable microtubules search in space until chromosomes are captured, and a chromosomal pathway, in which microtubules grow from chromosomes and focus to the spindle poles. Quantitative mechanistic understanding of how spindle assembly can be both fast and accurate is lacking. Specifically, it is unclear how, if at all, chromosome movements and combining the centrosomal and chromosomal pathways affect the assembly speed and accuracy. We used computer simulations and high-resolution microscopy to test plausible pathways of spindle assembly in realistic geometry. Our results suggest that an optimal combination of centrosomal and chromosomal pathways, spatially biased microtubule growth, and chromosome movements and rotations is needed to complete prometaphase in 10 -20 min while keeping erroneous merotelic attachments down to a few percent. The simulations also provide kinetic constraints for alternative error correction mechanisms, shed light on the dual role of chromosome arm volume, and compare well with experimental data for bipolar and multipolar HT-29 colorectal cancer cells.assembly speed and accuracy ͉ merotelic attachments ͉ microtubules ͉ search and capture T he mitotic spindle is a complex molecular machine segregating chromosomes (1, 2). Molecular inventory and general principles of the spindle dynamics are becoming clear (3), but quantitative understanding of spindle mechanics in general and its self-assembly in particular is lacking. The first hypothesis of how the spindle assembles, elegantly called ''search and capture'' (Fig. 1A), was put forward in ref. 4 after the discovery of the dynamic instability phenomenon: Microtubules (MTs) grow and shorten rapidly and repeatedly from the centrosomes in random directions ''searching'' for the kinetochores (KTs), specialized chromosome structures that function as an interface between the chromosomes and the mitotic spindle. Whenever a growing MT plus end runs into a KT, this MT is stabilized; the assembly is complete when all KTs are thus captured transforming two MT asters into a typical bipolar spindle. Capture of a single astral MT by a KT has been visualized directly in newt lung cell cultures (5).How can hundreds of MTs turning over in tens of seconds capture tens of chromosomes within 10-20 min (6) is one of the fundamental questions of mitosis. Mathematical modeling has been instrumental in attempts to answer this question, because it is very hard to experimentally resolve individual MTs, follow their formation, and perturb their dynamics (7). First applications of modeling were the analyses (8, 9) suggesting that the dynamic instability parameters have to be optimized to ensure fast assembly, so that a MT switches from growth to shortening when it is as long as the distance between the centrosome and the chromosome. This analysis was extended (10) to simulate hundreds of MTs searching for tens of KTs in realistic geometry. The simulation...

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The Mis18 complex specifies the site of new CENP-A nucleosome assembly by recruiting the CENP-A specific assembly factor HJURP (Holliday junction recognition protein). The human Mis18 complex consists of Mis18α, Mis18β and Mis18 binding protein 1 (Mis18BP1/hsKNL2). Although Mis18α and Mis18β are highly homologous proteins, we find that their conserved YIPPEE domains mediate distinct interactions that are essential to link new CENP-A deposition to existing centromeres. We find that Mis18α directly interacts with the N-terminus of Mis18BP1; whereas, Mis18β directly interacts with CENP-C during G1 phase, revealing that these proteins have evolved to serve distinct functions in centromeres of higher eukaryotes. The N-terminus of Mis18BP1, containing both the Mis18α and CENP-C binding domains, is necessary and sufficient for centromeric localization. Therefore, the Mis18 complex contains dual CENP-C recognition motifs that are combinatorially required to generate robust centromeric localization that leads to CENP-A deposition.

Loss of monoubiquitination of histone H2B (H2Bub1) was found to be associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma. This study investigated the association and impact of the ubiquitin-specific peptidase 22 (USP22), an H2Bub1 deubiquitinase, on stem cell-like characteristics and cisplatin resistance in cancer-initiating cells (CIC) from primary lung adenocarcinoma. CICs were isolated, enriched, and characterized from patient-derived cancer tissues using both tumorsphere formation and xenograft assays. USP22 was determined to be predominantly expressed in CICs, a subpopulation of cells with high expression of the stem cell biomarkers, CD133 and CD44. The expression of USP22 in CICs is markedly reduced upon FBS/retinoic acid-induced differentiation. Moreover, knockdown of USP22 significantly suppressed tumorsphere formation and xenograft growth in NOD-SCID gamma (NSG) mice. Notably, USP22 and aldehyde dehydrogenase (ALDH) activity were elevated in tumorsphere cells that survived cisplatin treatment, whereas knockdown of USP22 significantly sensitizes tumorsphere cells to cisplatin. Interestingly, ALDH1A3, a predominant ALDH isozyme implicated in enhancing cisplatin resistance in lung adenocarcinoma, is significantly downregulated upon knockdown of USP22 in tumorsphere cells. Furthermore, knockdown of ALDH1A3 significantly sensitizes tumorsphere cells to cisplatin. Combined, these data demonstrate that USP22, predominantly expressed in CD133 CICs, plays a critical role in tumorigenicity and cisplatin resistance in lung adenocarcinoma. Targeting USP22 represents a potential therapeutic approach to suppress CICs in lung adenocarcinoma partially through downregulation of ALDH1A3 expression. .

In the last decade, it has become clear that epigenetic changes act together with genetic mutations to promote virtually every stage of tumorigenesis and cancer progression. This knowledge has triggered searches for "epigenetic drugs" that can be developed into new cancer therapies. Here we report that triptolide reduced lung cancer incidence from 70% to 10% in a Fen1 E160D transgenic mouse model and effectively inhibited cancer growth and metastasis in A549 and H460 mouse xenografts. We found that triptolide induced lung cancer cell apoptosis that was associated with global epigenetic changes to histone 3 (H3). These global epigenetic changes in H3 are correlated with an increase in protein expression of five Wnt inhibitory factors that include WIF1, FRZB, SFRP1, ENY2, and DKK1. Triptolide had no effect on DNA methylation status at any of the CpG islands located in the promoter regions of all five Wnt inhibitory factors. Wnt expression is implicated in promoting the development and progression of many lung cancers. Because of this, the potential to target Wnt signaling with drugs that induce epigenetic modifications provides a new avenue for developing novel therapies for patients with these tumor types.