Genetic inactivation of the Fanconi anemia gene FANCC identified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents

Background: Inactivation of the Fanconi anemia (FA) pathway through defects in one of 13 FA genes occurs at low frequency in various solid cancer entities among the general population. As FA pathway inactivation confers a distinct hypersensitivity towards DNA interstrand-crosslinking (ICL)-agents, FA defects represent rational targets for individualized therapeutic strategies. Except for pancreatic cancer, however, the prevalence of FA defects in gastrointestinal (GI) tumors has not yet been systematically explored. Results: A panel of GI cancer cell lines was screened for FA pathway inactivation applying FANCD2 monoubiquitination and FANCD2/RAD51 nuclear focus formation and a newly identified FA pathway-deficient cell line was functionally characterized. The hepatocellular carcinoma (HCC) line HuH-7 was defective in FANCD2 monoubiquitination and FANCD2 nuclear focus formation but proficient in RAD51 focus formation. Gene complementation studies revealed that this proximal FA pathway inactivation was attributable to defective FANCC function in HuH-7 cells. Accordingly, a homozygous inactivating FANCC nonsense mutation (c.553C > T, p.R185X) was identified in HuH-7, resulting in partial transcriptional skipping of exon 6 and leading to the classic cellular FA hypersensitivity phenotype; HuH-7 cells exhibited a strongly reduced proliferation rate and a pronounced G2 cell cycle arrest at distinctly lower concentrations of ICL-agents than a panel of non-isogenic, FA pathway-proficient HCC cell lines. Upon retroviral transduction of HuH-7 cells with FANCC cDNA, FA pathway functions were restored and ICL-hypersensitivity abrogated. Analyses of 18 surgical HCC specimens yielded no further examples for genetic or epigenetic inactivation of FANCC, FANCF, or FANCG in HCC, suggesting a low prevalence of proximal FA pathway inactivation in this tumor type. Conclusions: As the majority of HCC are chemoresistant, assessment of FA pathway function in HCC could identify small subpopulations of patients expected to predictably benefit from individualized treatment protocols using ICL-agents

USP9X stabilizes XIAP to regulate mitotic cell death and chemoresistance in aggressive B-cell lymphoma

The mitotic spindle assembly checkpoint (SAC) maintains genome stability and marks an important target for antineoplastic therapies. However, it has remained unclear how cells execute cell fate decisions under conditions of SAC‐induced mitotic arrest. Here, we identify USP9X as the mitotic deubiquitinase of the X‐linked inhibitor of apoptosis protein (XIAP) and demonstrate that deubiquitylation and stabilization of XIAP by USP9X lead to increased resistance toward mitotic spindle poisons. We find that primary human aggressive B‐cell lymphoma samples exhibit high USP9X expression that correlate with XIAP overexpression. We show that high USP9X/XIAP expression is associated with shorter event‐free survival in patients treated with spindle poison‐containing chemotherapy. Accordingly, aggressive B‐cell lymphoma lines with USP9X and associated XIAP overexpression exhibit increased chemoresistance, reversed by specific inhibition of either USP9X or XIAP. Moreover, knockdown of USP9X or XIAP significantly delays lymphoma development and increases sensitivity to spindle poisons in a murine Eμ‐Myc lymphoma model. Together, we specify the USP9X–XIAP axis as a regulator of the mitotic cell fate decision and propose that USP9X and XIAP are potential prognostic biomarkers and therapeutic targets in aggressive B‐cell lymphoma

Hof1 and Rvs167 Have Redundant Roles in Actomyosin Ring Function during Cytokinesis in Budding Yeast

This is an open-access article distributed under the terms of the Creative Commons Attribution License.The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously. © 2013 Nkosi et al.The authors are grateful to Cancer Research United Kingdom who funded this work. ASD joined the University of Cantabria as a recipient of a Ramón y Cajal contract (call 2010) and now receives funding from the Cantabria International Campus and via grant BFU2011-23193 from the Spanish "Ministerio de Economía y Competitividad" (co-funded by the European Regional Development Fund).Peer Reviewe

Hof1 and Rvs167 Have Redundant Roles in Actomyosin Ring Function during Cytokinesis in Budding Yeast

The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously.Funding: The authors are grateful to Cancer Research United Kingdom who funded this work. ASO joined the University of Cantabria as a recipient of a Ramón y Cajal contract (call 2010) and now receives funding from the Cantabria lnternational Campus and via grant BFU2011-23193 from the Spanish "Ministerio de Economía y Competitividad" (co-funded by the European Regional Development Fund). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Recruitment of Iqg1 to the bud-neck is defective in cells lacking Hof1 and Rvs167.

<p>(<b>A</b>) Cells were processed as described above for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057846#pone-0057846-g004" target="_blank">Figure 4</a>, so that the recruitment of GFP-Iqg1 to the bud-neck (i) could be monitored as cells passed through mitosis (ii). (<b>B</b>) Images from the experiment described in (A). The scale bars correspond to 2 µm.</p

Actin ring assembly still occurs following inactivation of Arp2/3.

<p>(<b>A</b>) The indicated strains were arrested in G2-M phase with nocodazole at 24°C, before expression of <i>GAL-UBR1</i> and incubation at 37°C for 60’ to inactivate Hof1-td and Arp2-2. Cells were then released at 37°C into fresh medium lacking nocodazole, and processed as described above for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057846#pone-0057846-g005" target="_blank">Figure 5</a>. (<b>B</b>) Images of cells with actin rings (marked by arrows), from the 30-minute timepoint in the experiment in (A). The scale bars correspond to 2 µm.</p

Physical interactions between Rvs167 and Inn1.

<p>(<b>A</b>) Truncated alleles of Rvs167 and Inn1 were used to show that the region of Rvs167 after the BAR domain (Rvs167 241-482) can interact in a 2-hybrid assay with the Proline-rich region of Inn1 after its C2 domain (Inn1 135-409). (<b>B</b>) Scheme explaining how the indicated protein fragments were expressed in cultures of <i>E. coli</i> cells, and then mixed to allow the purification of protein complexes. After induction with IPTG, pairs of cultures were mixed as indicated in (D) below, and used to purify protein complexes between the induced proteins, via Strep-Tactin Superflow and Ni-NTA agarose resins (see Methods). (<b>C</b>) Immunoblots showing induction of the various protein fragments listed in (B). The tagged proteins were detected with anti-Streptag or anti-His antibodies. In each case, a non-specific band corresponding to an unknown <i>E. coli</i> protein is included to provide a loading control. (<b>D</b>) Inn1 135-409 can interact directly to form a stable complex with the SH3 domains of Hof1 and Cyk3, as well as with Rvs167 241-482. Pairs of <i>E. coli</i> cell cultures expressing the indicated protein fragments were mixed and used to purify putative protein complexes as shown in (B). The final purified fractions were analysed by SDS-PAGE and the gels were stained with colloidal Coomassie blue.</p

Inn1 can still be recruited to the bud-neck in the absence of the SH3 domains of Rvs167 and Hof1.

<p>(<b>A</b>) The indicated strains were released from G1-arrest at 24°C and allowed to progress through the cell cycle. The proportion of binucleate cells was monitored in parallel with recruitment of Inn1 to the bud-neck. (<b>B</b>) Examples of cells with Inn1-GFP rings at the bud-neck are shown for the 90’ time-point in (A). The scale-bars indicate 2 µm.</p

Recruitment of Inn1 to the bud-neck is defective in the complete absence of Hof1 and Rvs167.

<p>(<b>A</b>) The indicated strains were synchronised in G1 phase at 24°C, before expression of <i>GAL-UBR1</i> (Ubr1 is the E3 ligase for N-end rule pathway that mediates ubiquitylation of the heat inducible degron) and degradation of Hof1-td and Cyk3-td at 37°C. Cells were then released from G1 arrest and samples taken at the indicated times to determine the proportion of bi-nucleate cells (i) and the percentage of cells with rings or spots of Inn1 at the bud-neck (ii), as cells completed the cell cycle. (<b>B</b>) Images from the experiment described in (A). The Inn1-GFP rings in <i>hof1-td cyk3-td</i> were frequently less bright than those observed in control cells. The scale bars correspond to 2 µm.</p