Intrabiliary growth by metastatic colorectal carcinoma (CRC) is an unusual finding that can clinically mimic cholangiocarcinoma. We evaluated prevalence of intrabiliary growth by retrospective review of 1596 diagnostic reports and by prospective evaluation of 223 hepatectomies. Positive cases were scored for extent of intrabiliary growth (major vs. minor duct involvement), architectural pattern (colonization of biliary epithelium and/or intrabiliary tumor plugs), and secondary sclerosing cholangitis in non-neoplastic parenchyma. By retrospective review, we identified intrabiliary growth in 41 (3.6%) of 1144 metastatic CRCs but only 3 (0.7%) of 452 noncolorectal tumors (P<0.001). Prospectively, we found intrabiliary growth in 18 (10.6%) of 170 metastatic CRCs and 1 (1.9%) of 53 other tumors (P=0.05). Among our final population of 43 CRCs with intrabiliary growth, 24 (56%) had major and 19 (44%) had minor duct involvement, 35 (81%) showed colonization of biliary epithelium, and 35 (81%) showed intrabiliary tumor plugs. Compared with minor duct involvement and 51 controls without intrabiliary growth, major duct involvement was more likely to produce obstructive liver chemistries (P=0.004), radiographic evidence of biliary disease (P<0.0001), and sclerosing cholangitis in non-neoplastic liver (P<0.0001). However, there was no impact on overall survival. Clinically, 5 (21%) cases of major duct involvement resulted in diagnostic uncertainty between metastatic CRC and cholangiocarcinoma. These findings underscore the frequency of intrabiliary growth by metastatic CRCs and its rarity with other metastases. Major duct involvement should be recognized because of its distinctive clinical features, which can overlap with cholangiocarcinoma.

Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C), initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes.

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome-microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element-binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.

The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin-conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome. UbcH10 is overexpressed in many human cancer types and is associated with tumor progression. However, whether UbcH10 overexpression causes tumor formation is unknown. To address this central question and to define the molecular and cellular consequences of UbcH10 overexpression, we generated a series of transgenic mice in which UbcH10 was overexpressed in graded fashion. In this study, we show that UbcH10 overexpression leads to precocious degradation of cyclin B by the APC/C, supernumerary centrioles, lagging chromosomes, and aneuploidy. Importantly, we find that UbcH10 transgenic mice are prone to carcinogen-induced lung tumors and a broad spectrum of spontaneous tumors. Our results identify UbcH10 as a prominent protooncogene that causes whole chromosome instability and tumor formation over a wide gradient of overexpression levels.

The mitotic checkpoint protein Bub1 is essential for embryogenesis and survival of proliferating cells, and bidirectional deviations from its normal level of expression cause chromosome missegregation, aneuploidy, and cancer predisposition in mice. To provide insight into the physiological significance of this critical mitotic regulator at a modular level, we generated Bub1 mutant mice that lack kinase activity using a knockin gene-targeting approach that preserves normal protein abundance. In this paper, we uncover that Bub1 kinase activity integrates attachment error correction and mitotic checkpoint signaling by controlling the localization and activity of Aurora B kinase through phosphorylation of histone H2A at threonine 121. Strikingly, despite substantial chromosome segregation errors and aneuploidization, mice deficient for Bub1 kinase activity do not exhibit increased susceptibility to spontaneous or carcinogen-induced tumorigenesis. These findings provide a unique example of a modular mitotic activity orchestrating two distinct networks that safeguard against whole chromosome instability and reveal the differential importance of distinct aneuploidy-causing Bub1 defects in tumor suppression.

RanBP2/Nup358, the major component of the cytoplasmic filaments of the nuclear pore complex (NPC), is essential for mouse embryogenesis and is implicated in both macromolecular transport and mitosis, but its specific molecular functions are unknown. Using RanBP2 conditional knockout mouse embryonic fibroblasts and a series of mutant constructs, we show that transport, rather than mitotic, functions of RanBP2 are required for cell viability. Cre-mediated RanBP2 inactivation caused cell death with defects in M9- and classical nuclear localization signal (cNLS)-mediated protein import, nuclear export signal-mediated protein export, and messenger ribonucleic acid export but no apparent mitotic failure. A short N-terminal RanBP2 fragment harboring the NPC-binding domain, three phenylalanine-glycine motifs, and one Ran-binding domain (RBD) corrected all transport defects and restored viability. Mutation of the RBD within this fragment caused lethality and perturbed binding to Ran guanosine triphosphate (GTP)-importin-β, accumulation of importin-β at nuclear pores, and cNLS-mediated protein import. These data suggest that a critical function of RanBP2 is to capture recycling RanGTP-importin-β complexes at cytoplasmic fibrils to allow for adequate cNLS-mediated cargo import.

The BubR1 gene encodes for a mitotic regulator that ensures accurate segregation of chromosomes through its role in the mitotic checkpoint and the establishment of proper microtubule-kinetochore attachments. Germline mutations that reduce BubR1 abundance cause aneuploidy, shorten lifespan and induce premature ageing phenotypes and cancer in both humans and mice. A reduced BubR1 expression level is also a feature of chronological ageing, but whether this age-related decline has biological consequences is unknown. Using a transgenic approach in mice, we show that sustained high-level expression of BubR1 preserves genomic integrity and reduces tumorigenesis, even in the presence of genetic alterations that strongly promote aneuplodization and cancer, such as oncogenic Ras. We find that BubR1 overabundance exerts its protective effect by correcting mitotic checkpoint impairment and microtubule-kinetochore attachment defects. Furthermore, sustained high-level expression of BubR1 extends lifespan and delays age-related deterioration and aneuploidy in several tissues. Collectively, these data uncover a generalized function for BubR1 in counteracting defects that cause whole-chromosome instability and suggest that modulating BubR1 provides a unique opportunity to extend healthy lifespan.

Aging is a highly complex biological process that is believed to involve multiple mechanisms. Mice that have small amounts of the mitotic checkpoint protein BubR1 age much faster than normal mice, but whether other mitotic checkpoint genes function to prevent the early onset of aging is unknown. In this study, we show that several aging-associated phenotypes appear early in mice that are double haploinsufficient for the mitotic checkpoint genes Bub3 and Rae1 but not in mice that are single haploinsufficient for these genes. Mouse embryonic fibroblasts (MEFs) from Bub3/Rae1 haploinsufficient mice undergo premature senescence and accumulate high levels of p19, p53, p21, and p16, whereas MEFs from single haploinsufficient mice do not. Furthermore, although BubR1 hypomorphic mice have less aneuploidy than Bub3/Rae1 haploinsufficient mice, they age much faster. Our findings suggest that early onset of aging-associated phenotypes in mice with mitotic checkpoint gene defects is linked to cellular senescence and activation of the p53 and p16 pathways rather than to aneuploidy.

Neuroendocrine carcinoma (NEC) of the breast is a rare type of carcinoma that has not been well studied or characterized. Of the limited number of studies reported in the literature, most are case reports. A few small retrospective series studies have been reported.

The physiological role of the mitotic checkpoint protein Bub1 is unknown. To study this role, we generated a series of mutant mice with a gradient of reduced Bub1 expression using wild-type, hypomorphic, and knockout alleles. Bub1 hypomorphic mice are viable, fertile, and overtly normal despite weakened mitotic checkpoint activity and high percentages of aneuploid cells. Bub1 haploinsufficient mice, which have a milder reduction in Bub1 protein than Bub1 hypomorphic mice, also exhibit reduced checkpoint activity and increased aneuploidy, but to a lesser extent. Although cells from Bub1 hypomorphic and haploinsufficient mice have similar rates of chromosome missegregation, cell death after an aberrant separation decreases dramatically with declining Bub1 levels. Importantly, Bub1 hypomorphic mice are highly susceptible to spontaneous tumors, whereas Bub1 haploinsufficient mice are not. These findings demonstrate that loss of Bub1 below a critical threshold drives spontaneous tumorigenesis and suggest that in addition to ensuring proper chromosome segregation, Bub1 is important for mediating cell death when chromosomes missegregate.