SKAP interacts with Aurora B to guide end-on capture of spindle microtubules via phase separation.

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complex and spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internal dynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP comets in vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP-Aurora B interaction via phase separation constitutes a dynamic pool of Aurora B activity during the lateral to end-on conversion of kinetochore-microtubule attachments to achieve faithful cell division.

Feedback control of PLK1 by Apolo1 ensures accurate chromosome segregation.

Stable transmission of genetic material during cell division requires accurate chromosome segregation. PLK1 dynamics at kinetochores control establishment of correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the regulatory mechanism responsible for PLK1 activity in prometaphase has not yet been affirmatively identified. Here we identify Apolo1, which tunes PLK1 activity for accurate kinetochore-microtubule attachments. Apolo1 localizes to kinetochores during early mitosis, and suppression of Apolo1 results in misaligned chromosomes. Using the fluorescence resonance energy transfer (FRET)-based PLK1 activity reporter, we found that Apolo1 sustains PLK1 kinase activity at kinetochores for accurate attachment during prometaphase. Apolo1 is a cognate substrate of PLK1, and the phosphorylation enables PP1γ to inactivate PLK1 by dephosphorylation. Mechanistically, Apolo1 constitutes a bridge between kinase and phosphatase, which governs PLK1 activity in prometaphase. These findings define a previously uncharacterized feedback loop by which Apolo1 provides fine-tuning for PLK1 to guide chromosome segregation in mitosis.

Cisplatin combined with laser­activated nanotherapy as an adjuvant therapy for head and neck cancer.

Head and neck cancers (HNCs), in general, have a poor prognosis with a worldwide 5­year survival rate of <50%. Numerous HNC patients with locoregionally advanced, difficult­to­treat, inoperable, recurrent and drug­resistant tumors may require additional treatment options when the standard of care surgery, chemotherapy and radiation are not viable. The poor outcomes justify exploring strategies to increase the efficacy of lower doses of drugs, such as cisplatin, by combining these drugs with other treatment modalities and manipulating the dosing schedule. Cisplatin is a standard and effective anticancer drug; however, some patients cannot tolerate the side­effects or exhibit drug resistance. Adjuvant therapies may lower the effective dose, decrease side­effects, address drug resistance and improve overall survival outcomes, particularly for patients with difficult­to­treat tumors. The present study focuses on combining cisplatin with laser­activated nanotherapy (LANT), as an adjuvant HNC therapy, with the aim of enhancing the therapeutic efficacy of lower doses of cisplatin and decreasing treatment times. The results demonstrate the potential of cisplatin and LANT co­therapy as a possible addition to the adjuvant therapy options for HNC using 3 cell lines: Detroit 562, FaDu and CAL 27 cells. Combining cisplatin with LANT demonstrated up to a 5.4­fold greater therapeutic efficacy than with cisplatin monotreatment. The most effective combination in the present study was 1 µM Cis + 5 nM LANT, which demonstrated cell death comparable to 5.9, 4.2 and 5.3 µM of Cis monotreatment, in Detroit 562, FaDu and CAL 27 cells, respectively. This result suggests that a lower cisplatin dose may be combined with LANT to achieve the same therapeutic efficacy as that obtained with higher doses of cisplatin monotreatment. The combination of LANT and cisplatin suggests that LANT may enhance the therapeutic efficiency of low doses of cisplatin, decrease treatment times and improve patient outcomes.

Acetylation of ezrin regulates membrane-cytoskeleton interaction underlying CCL18-elicited cell migration.

Ezrin, a membrane-cytoskeleton linker protein, plays an essential role in cell polarity establishment, cell migration, and division. Recent studies show that ezrin phosphorylation regulates breast cancer metastasis by promoting cancer cell survivor and promotes intrahepatic metastasis via cell migration. However, it was less characterized whether there are additional post-translational modifications and/or post-translational crosstalks on ezrin underlying context-dependent breast cancer cell migration and invasion. Here we show that ezrin is acetylated by p300/CBP-associated factor (PCAF) in breast cancer cells in response to CCL18 stimulation. Ezrin physically interacts with PCAF and is a cognate substrate of PCAF. The acetylation site of ezrin was mapped by mass spectrometric analyses, and dynamic acetylation of ezrin is essential for CCL18-induced breast cancer cell migration and invasion. Mechanistically, the acetylation reduced the lipid-binding activity of ezrin to ensure a robust and dynamic cycling between the plasma membrane and cytosol in response to CCL18 stimulation. Biochemical analyses show that ezrin acetylation prevents the phosphorylation of Thr567. Using atomic force microscopic measurements, our study revealed that acetylation of ezrin induced its unfolding into a dominant structure, which prevents ezrin phosphorylation at Thr567. Thus, these results present a previously undefined mechanism by which CCL18-elicited crosstalks between the acetylation and phosphorylation on ezrin control breast cancer cell migration and invasion. This suggests that targeting PCAF signaling could be a potential therapeutic strategy for combating hyperactive ezrin-driven cancer progression.

Acetylation of ACAP4 regulates CCL18-elicited breast cancer cell migration and invasion.

Tumor metastasis represents the main causes of cancer-related death. Our recent study showed that chemokine CCL18 secreted from tumor-associated macrophages regulates breast tumor metastasis, but the underlying mechanisms remain less clear. Here, we show that ARF6 GTPase-activating protein ACAP4 regulates CCL18-elicited breast cancer cell migration via the acetyltransferase PCAF-mediated acetylation. CCL18 stimulation elicited breast cancer cell migration and invasion via PCAF-dependent acetylation. ACAP4 physically interacts with PCAF and is a cognate substrate of PCAF during CCL18 stimulation. The acetylation site of ACAP4 by PCAF was mapped to Lys311 by mass spectrometric analyses. Importantly, dynamic acetylation of ACAP4 is essential for CCL18-induced breast cancer cell migration and invasion, as overexpression of the persistent acetylation-mimicking or non-acetylatable ACAP4 mutant blocked CCL18-elicited cell migration and invasion. Mechanistically, the acetylation of ACAP4 at Lys311 reduced the lipid-binding activity of ACAP4 to ensure a robust and dynamic cycling of ARF6-ACAP4 complex with plasma membrane in response to CCL18 stimulation. Thus, these results present a previously undefined mechanism by which CCL18-elicited acetylation of the PH domain controls dynamic interaction between ACAP4 and plasma membrane during breast cancer cell migration and invasion.

Phosphorylation of PP1 Regulator Sds22 by PLK1 Ensures Accurate Chromosome Segregation.

During cell division, accurate chromosome segregation is tightly regulated by Polo-like kinase 1 (PLK1) and opposing activities of Aurora B kinase and protein phosphatase 1 (PP1). However, the regulatory mechanisms underlying the aforementioned hierarchical signaling cascade during mitotic chromosome segregation have remained elusive. Sds22 is a conserved regulator of PP1 activity, but how it regulates PP1 activity in space and time during mitosis remains elusive. Here we show that Sds22 is a novel and cognate substrate of PLK1 in mitosis, and the phosphorylation of Sds22 by PLK1 elicited an inhibition of PP1-mediated dephosphorylation of Aurora B at threonine 232 (Thr232) in a dose-dependent manner. Overexpression of a phosphomimetic mutant of Sds22 causes a dramatic increase in mitotic delay, whereas overexpression of a non-phosphorylatable mutant of Sds22 results in mitotic arrest. Mechanistically, the phosphorylation of Sds22 by PLK1 strengthens the binding of Sds22 to PP1 and inhibits the dephosphorylation of Thr232 of Aurora B to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling hierarchy that orchestrates dynamic protein phosphorylation and dephosphorylation of critical mitotic regulators during chromosome segregation to guard chromosome stability.

A histological evaluation and in vivo assessment of intratumoral near infrared photothermal nanotherapy-induced tumor regression.

PURPOSE: Nanoparticle (NP)-enabled near infrared (NIR) photothermal therapy has realized limited success in in vivo studies as a potential localized cancer therapy. This is primarily due to a lack of successful methods that can prevent NP uptake by the reticuloendothelial system, especially the liver and kidney, and deliver sufficient quantities of intravenously injected NPs to the tumor site. Histological evaluation of photothermal therapy-induced tumor regression is also neglected in the current literature. This report demonstrates and histologically evaluates the in vivo potential of NIR photothermal therapy by circumventing the challenges of intravenous NP delivery and tumor targeting found in other photothermal therapy studies. METHODS: Subcutaneous Cal 27 squamous cell carcinoma xenografts received photothermal nanotherapy treatments, radial injections of polyethylene glycol (PEG)-ylated gold nanorods and one NIR 785 nm laser irradiation for 10 minutes at 9.5 W/cm(2). Tumor response was measured for 10-15 days, gross changes in tumor size were evaluated, and the remaining tumors or scar tissues were excised and histologically analyzed. RESULTS: The single treatment of intratumoral nanorod injections followed by a 10 minute NIR laser treatment also known as photothermal nanotherapy, resulted in ~100% tumor regression in ~90% of treated tumors, which was statistically significant in a comparison to the average of all three control groups over time (P<0.01). CONCLUSION: Photothermal nanotherapy, or intratumoral nanorod injections followed by NIR laser irradiation of tumors and tumor margins, demonstrate the potential of NIR photothermal therapy as a viable localized treatment approach for primary and early stage tumors, and prevents NP uptake by the reticuloendothelial system.

Nanodiamonds enhance therapeutic efficacy of doxorubicin in treating metastatic hormone-refractory prostate cancer.

Enhancing therapeutic efficacy is essential for successful treatment of chemoresistant cancers such as metastatic hormone-refractory prostate cancer (HRPC). To improve the efficacy of doxorubicin (DOX) for treating chemoresistant disease, the feasibility of using nanodiamond (ND) particles was investigated. Utilizing the pH responsive properties of ND, a novel protocol for complexing NDs and DOX was developed using a pH 8.5 coupling buffer. The DOX loading efficiency, loading on the NDs, and pH responsive release characteristics were determined utilizing UV-Visible spectroscopy. The effects of the ND-DOX on HRPC cell line PC3 were evaluated with MTS and live/dead cell viability assays. ND-DOX displayed exceptional loading efficiency (95.7%) and drug loading on NDs (23.9 wt%) with optimal release at pH 4 (80%). In comparison to treatment with DOX alone, cell death significantly increased when cells were treated with ND-DOX complexes demonstrating a 50% improvement in DOX efficacy. Of the tested treatments, ND-DOX with 2.4 µg mL(-1) DOX exhibited superior efficacy (60% cell death). ND-DOX with 1.2 µg mL(-1) DOX achieved 42% cell death, which was comparable to cell death in response to 2.4 µg mL(-1) of free DOX, suggesting that NDs aid in decreasing the DOX dose necessary to achieve a chemotherapeutic efficacy. Due to its enhanced efficacy, ND-DOX can be used to successfully treat HRPC and potentially decrease the clinical side effects of DOX.

Nanodiamond-DGEA peptide conjugates for enhanced delivery of doxorubicin to prostate cancer.

The field of nanomedicine has emerged as an approach to enhance the specificity and efficacy of cancer treatments as stand-alone therapies and in combination with standard chemotherapeutic treatment regimens. The current standard of care for metastatic cancer, doxorubicin (DOX), is presented with challenges, namely toxicity due to a lack of specificity and targeted delivery. Nano-enabled targeted drug delivery systems can provide an avenue to overcome these issues. Nanodiamonds (ND), in particular, have been researched over the past five years for use in various drug delivery systems but minimal work has been done that incorporates targeting capability. In this study, a novel targeted drug delivery system for bone metastatic prostate cancer was developed, characterized, and evaluated in vitro. NDs were conjugated with the Asp-Gly-Glu-Ala (DGEA) peptide to target α2ß1 integrins over-expressed in prostate cancers during metastasis. To facilitate drug delivery, DOX was adsorbed to the surface of the ND-DGEA conjugates. Successful preparation of the ND-DGEA conjugates and the ND-DGEA+DOX system was confirmed with transmission electron microscopy, hydrodynamic size, and zeta potential measurements. Since traditional DOX treatment regimens lack specificity and increased toxicity to normal tissues, the ND-DGEA conjugates were designed to distinguish between cells that overexpress α2ß1 integrin, bone metastatic prostate cancers cells (PC3), and cells that do not, human mesenchymal stem cells (hMSC). Utilizing the ND-DGEA+DOX system, the efficacy of 1 µg/mL and 2 µg/mL DOX doses increased from 2.5% to 12% cell death and 11% to 34% cell death, respectively. These studies confirmed that the delivery and efficacy of DOX were enhanced by ND-DGEA conjugates. Thus, the targeted ND-DGEA+DOX system provides a novel approach for decreasing toxicity and drug doses.