Congressing kinetochores progressively load Ska complexes to prevent force-dependent detachment

Kinetochores mediate chromosome congression by either sliding along the lattice of spindle microtubules or forming end-on attachments to their depolymerizing plus-ends. By following the fates of individual kinetochores as they congress in live cells, we reveal that the Ska complex is required for a distinct substep of the depolymerization-coupled pulling mechanism. Ska depletion increases the frequency of naturally occurring, force-dependent P kinetochore detachment events, while being dispensable for the initial biorientation and movement of chromosomes. In unperturbed cells, these release events are followed by reattachment and successful congression, whereas in Ska-depleted cells, detached kinetochores remain in a futile reattachment/detachment cycle that prevents congression. We further find that Ska is progressively loaded onto bioriented kinetochore pairs as they congress. We thus propose a model in which kinetochores mature through Ska complex recruitment and that this is required for improved load-bearing capacity and silencing of the spindle assembly checkpoint

Abstract 1062: Overexpression of miRNAs 181a and 222 play a role in triple negative breast cancer, and are targeted by entinostat

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer in women. Clinically, this subtype is characterized by high recurrence rates, poor prognosis, and lack of targeted therapies. Therefore, there is considerable need to identify TNBC-specific biomarkers that can serve as a diagnositc markers and therapeutic targets. MIcroRNAs (miRNAs), may be such biomarkers. miRNAs are short, non-coding regulatory RNA molecules that modulate the expression of specific proteins by binding to target messenger RNAs (mRNAs) and causing either degradation of the mRNAs or inhibition of their translation to protein. Thus, they play an important role in a variety of normal cellular processes (e.g., differentiation, cell growth, cell death, etc.), and in diseases, such as cancer. MiRNAs have been implicated in breast cancer, but there is not consistent agreement as to which miRNAs are involved in TNBCs, nor have molecular targeting drugs been identified that could treat TNBCs by affecting miRNAs. Previous studies by our lab and others indicate that miRNAs 181a and 222 are involved in estrogen-receptor independence, cancer stem cells, and drug resistance (ex. letrozole resistance). Thus, in this study, the expression of miRNAs 181 and 222 in TNBCs, the effect of inhibiting each miRNA on cell viability and cancer stem cells, and the effect of histone deactylase inhibitor entinostat on miRNA expression are explored. RT-PCR analysis of miRNA expression in both HS578T and BT547 TNBCs and MCF-7 cells (represents the least aggressive subtype), and in representative breast cancer patient biopsy samples indicates that both miRNA 181a and 222 are upregulated by at least 15-fold in TNBCs compared to MCF-7 (least aggressive subtype). Specific inhibition of miRNA 181a via siRNA/miRNA inhibitor in HS578T cells significantly decreased cell viability by at least 80%, as determined by MTT assay, and cancer stem cells by 50%, as determined by mammosphere assays. In addition, inhibition of miRNA 181a produced morphological changes in HS578T cells, in which cells lost their protrusions, became more round, and grew in colonies. Lastly, treatment of HS578T cells or of patient derived xenografts with entinostat, which is currently being explored as a TNBC-targeting drug, decreased miRNA 181a and 222 expression and produced similar results as miRNA inhibition on cell viability, cancer stem cells, and morphology. Overall, these results suggest that miRNAs 181a and 222 are overexpressed in TNBCs and may play a role in regulation of cell viability and cancer stem cells. They also indicate that HDAC inhibitor, entinostat, may be an effective treatment for TNBCs through their action on miRNAs 181a and 222. Citation Format: Armina A. Kazi, Alexa Giammarino, Nicholas Musacchio, Gauri Sabnis, Amanda Schech, Angela Brodie. Overexpression of miRNAs 181a and 222 play a role in triple negative breast cancer, and are targeted by entinostat. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1062.</jats:p

Long Noncoding RNA DANCR Activates Wnt/β-Catenin Signaling through MiR-216a Inhibition in Non-Small Cell Lung Cancer

Long noncoding RNA differentiation antagonizing nonprotein coding RNA (lncRNA-DANCR) is associated with poor prognosis in multiple cancers, and promotes cancer stemness and invasion. However, the exact mechanisms by which DANCR promotes non-small cell lung cancer (NSCLC) remain elusive. In this study, we determined that DANCR knockdown (KD) impeded cell migration and reduced stem-like characteristics in two NSCLC cell lines, A549 and H1755. Wnt signaling was shown to promote NSCLC proliferation, stemness, and invasion; therefore, we hypothesized that DANCR may regulate these activities through induction of the Wnt/β-catenin pathway. DANCR KD reduced β-catenin signaling and protein expression, and decreased the expression of β-catenin gene targets c-Myc and Axin2. One of the well-defined functions of lncRNAs is their ability to bind and inhibit microRNAs. Through in silico analysis, we identified tumor suppressor miR-216a as a potential binding partner to DANCR, and confirmed this binding through coimmunoprecipitation and luciferase-reporter assays. Furthermore, we show that DANCR-induced β-catenin protein expression may be blocked with miR-216a overexpression. Our findings illustrate a role of DANCR in NSCLC migration and stemness, and suggest a novel DANCR/miR-216a signaling axis in the Wnt/β-catenin pathway.</jats:p

A Photoredox-Catalyzed Approach for Formal Hydride Abstraction to Enable Csp3–H Functionalization with Nucleophilic Partners

While a great number of C–H functionalization methods have been developed in recent years, new mechanistic paradigms to deconstruct such bonds have been comparatively rare. Amongst possible strategies for breaking a C&lt;i&gt;&lt;sub&gt;sp&lt;/sub&gt;&lt;sup&gt;3&lt;/sup&gt;&lt;/i&gt;–H bond are deprotonation, oxidative addition with a metal catalyst, direct insertion via a nitrene intermediate, hydrogen atom transfer (HAT) with both organic and metal-based abstractors, and lastly, hydride abstraction. The latter is a relatively unexplored approach due to the unfavorable thermodynamics of such an event, and thus has not been developed as a general way to target both activated and unactivated C&lt;i&gt;&lt;sub&gt;sp&lt;/sub&gt;&lt;sup&gt;3&lt;/sup&gt;&lt;/i&gt;–H bonds on hydrocarbon substrates. Herein, we report our successful efforts in establishing a catalytic C–H functionalization manifold for accessing an intermediate carbocation by formally abstracting hydride from unactivated C&lt;i&gt;&lt;sub&gt;sp&lt;/sub&gt;&lt;sup&gt;3&lt;/sup&gt;&lt;/i&gt;–H bonds. The novel catalytic design relies on a stepwise strategy driven by visible light photoredox catalysis and is demonstrated in the context of a C–H fluorination employing nucleophilic fluorine sources. Difluorination of methylene groups is also demonstrated, and represents the first C–H difluorination with nucleophilic fluoride. Additionally, the formal hydride abstraction is shown to be amenable to several other classes of nucleophiles, allowing for the construction of C–C or C–heteroatom bonds.</jats:p

A Photoredox-Catalyzed Approach for Formal Hydride Abstraction to Enable Csp3–H Functionalization with Nucleophilic Partners

While a great number of C–H functionalization methods have been developed in recent years, new mechanistic paradigms to deconstruct such bonds have been comparatively rare. Amongst possible strategies for breaking a Csp3–H bond are deprotonation, oxidative addition with a metal catalyst, direct insertion via a nitrene intermediate, hydrogen atom transfer (HAT) with both organic and metal-based abstractors, and lastly, hydride abstraction. The latter is a relatively unexplored approach due to the unfavorable thermodynamics of such an event, and thus has not been developed as a general way to target both activated and unactivated Csp3–H bonds on hydrocarbon substrates. Herein, we report our successful efforts in establishing a catalytic C–H functionalization manifold for accessing an intermediate carbocation by formally abstracting hydride from unactivated Csp3–H bonds. The novel catalytic design relies on a stepwise strategy driven by visible light photoredox catalysis and is demonstrated in the context of a C–H fluorination employing nucleophilic fluorine sources. Difluorination of methylene groups is also demonstrated, and represents the first C–H difluorination with nucleophilic fluoride. Additionally, the formal hydride abstraction is shown to be amenable to several other classes of nucleophiles, allowing for the construction of C–C or C–heteroatom bonds