Pharmacological inhibition of BAP1 recruits HERC2 to competitively dissociate BRCA1-BARD1, suppresses DNA repair and sensitizes CRC to radiotherapy.

Radiotherapy is widely used in the management of advanced colorectal cancer (CRC). However, the clinical efficacy is limited by the safe irradiated dose. Sensitizing tumor cells to radiotherapy via interrupting DNA repair is a promising approach to conquering the limitation. The BRCA1-BARD1 complex has been demonstrated to play a critical role in homologous recombination (HR) DSB repair, and its functions may be affected by HERC2 or BAP1. Accumulated evidence illustrates that the ubiquitination-deubiquitination balance is involved in these processes; however, the precise mechanism for the cross-talk among these proteins in HR repair following radiation hasn't been defined. Through activity-based profiling, we identified PT33 as an active entity for HR repair suppression. Subsequently, we revealed that BAP1 serves as a novel molecular target of PT33 via a CRISPR-based deubiquitinase screen. Mechanistically, pharmacological covalent inhibition of BAP1 with PT33 recruits HERC2 to compete with BARD1 for BRCA1 interaction, interrupting HR repair. Consequently, PT33 treatment can substantially enhance the sensitivity of CRC cells to radiotherapy in vitro and in vivo. Overall, these findings provide a mechanistic basis for PT33-induced HR suppression and may guide an effective strategy to improve therapeutic gain.

Caged Luciferase Inhibitor-Based Bioluminescence Switching Strategy Enables Efficient Detection of Serum APN Activity and the Identification of Its Roles in Metastasis of Non-Small Cell Lung Cancer.

Bioluminogenic probes emerged as powerful tools for imaging and analysis of various bioanalyses, but traditional approaches would be limited to the low sensitivity during determine the low activity of protease in clinical specimens. Herein, we proposed a caged luciferase inhibitor-based bioluminescence-switching strategy (CLIBS) by using a cleavable luciferase inhibitor to modulate the activity of luciferase reporter to amplify the detective signals, which led to the enhancement of detection sensitivity, and enabled the determination of circulating Aminopeptidase N (APN) activity in thousands of times diluted serum. By applying the CLIBS to serum samples in non-small cell lung cancer (NSCLC) patients from two clinical cohorts, we revealed that, for the first time, higher circulating APN activities but not its concentration, were associated with more NSCLC metastasis or higher metastasis stages by subsequent clinical analysis, and can serve as an independent factor for forecasting NSCLC patients' risk of metastasis.

Mitochondrial creatine kinase 1 regulates the cell cycle in non-small cell lung cancer via activation of cyclin-dependent kinase 4.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the main type of the most common malignant tumor in the world. Previous studies have shown that the expression level of mitochondrial creatine kinase 1 (CKMT1) is abnormal in NSCLC, but the mechanism of its effect remains unclear. Therefore, in this study, we intend to clarify the potential mechanism of CKMT1 in NSCLC and provide the theoretical basis for the clinical application of CKMT1. METHODS: The function of CKMT1 in NSCLC was identified by analyzing the GEO dataset and evaluating using in vitro and in vivo models. Protein mass spectrometry was used to find proteins interacting with CKMT1, and Co-immunoprecipitation (Co-IP) and GST-pull down experiments were used to verify the interaction between proteins. The immunofluorescence (IF) assay was used to explore the functional position of CKMT1 in cells. The effect of CKMT1 expression level on the efficacy of paclitaxel (TAX) in the treatment of NSCLC was analyzed by a combined TAX experiment in vivo and in vitro. RESULTS: CKMT1 expression was increased in NSCLC and CKMT1 promoted the proliferation of NSCLC cells in vitro and in vivo. CKMT1 knockdown resulted in a significantly increased G0/G1 fraction and decreased S phase cell fraction, indicating G1 phase arrest. Mechanically, the cyclin-dependent kinase 4 (CDK4) was identified to interact with CKMT1, and the crucial binding areas were focused on the DH domain of CKMT1 and the N- and C-terminal of CDK4. A fraction of the CDK4 proteins colocalize and interact with the CKMT1 at mitochondria, the level of phosphorylated CDK4 was regulated by CKMT1. Hence, the decrease in CKMT1 expression level could increase the antitumor effect of G2/M cell cycle antagonist-TAX in NSCLC in vitro and in vivo. CONCLUSIONS: CKMT1 could interact with CDK4 in mitochondria and regulate the phosphorylated level of CDK4, thus contributing to the proliferation and cell cycle transition of NSCLC cells. And CKMT1 could be a potential target to improve the sensitivity of chemotherapy based on TAX.

Stabilization of DEPTOR sensitizes hypopharyngeal cancer to radiotherapy via targeting degradation.

The use of radiotherapy for hypopharyngeal cancer (HC) treatment is increasing, and it is currently the primary treatment option for this cancer. However, radioresistance occurs in a proportion of patients. Here, we found that radiation increased proteasomal gene expression and that proteasome assembly was dependent on the induction of transcription factor NRF1 in HC. Through screening assays, we identified a mechanism by which proteasome-mediated degradation of DEP domain-containing mTOR-interacting protein (DEPTOR) contributes to the elevation of mTORC1 signaling after radiation. Therefore, after treatment with proteasome inhibitors (PIs), stabilization of DEPTOR inhibited mTORC1 signaling elevated by radiation and ultimately sensitized HC to radiotherapy. Mechanically, PIs not only interrupted the deubiquitination and degradation of DEPTOR but also suppressed the ubiquitination of DEPTOR mediated by ß-TrCP. Clinically, the high levels of DEPTOR in HC cells were associated with sensitivity to radiotherapy and favorable prognosis. Stabilizing DEPTOR through targeting proteasome-mediated degradation is a potential strategy for sensitizing HC to radiotherapy.

Genome-wide RNAi Screening Identifies RFC4 as a Factor That Mediates Radioresistance in Colorectal Cancer by Facilitating Nonhomologous End Joining Repair.

PURPOSE: Neoadjuvant chemoradiotherapy (neoCRT) is a standard treatment for locally advanced rectal cancer (LARC); however, resistance to chemoradiotherapy is one of the main obstacles to improving treatment outcomes. The goal of this study was to identify factors involved in the radioresistance of colorectal cancer and to clarify the underlying mechanisms. EXPERIMENTAL DESIGN: A genome-wide RNAi screen was used to search for candidate radioresistance genes. After RFC4 knockdown or overexpression, colorectal cancer cells exposed to X-rays both in vitro and in a mouse model were assayed for DNA damage, cytotoxicity, and apoptosis. Moreover, the regulatory effects and mechanisms of RFC4 in DNA repair were investigated in vitro. Finally, the relationships between RFC4 expression and clinical parameters and outcomes were investigated in 145 patients with LARC receiving neoCRT. RESULTS: RFC4, NCAPH, SYNE3, LDLRAD2, NHP2, and FICD were identified as potential candidate radioresistance genes. RFC4 protected colorectal cancer cells from X-ray-induced DNA damage and apoptosis in vitro and in vivo. Mechanistically, RFC4 promoted nonhomologous end joining (NHEJ)-mediated DNA repair by interacting with Ku70/Ku80 but did not affect homologous recombination-mediated repair. Higher RFC4 expression in cancer tissue was associated with weaker tumor regression and poorer prognosis in patients with LARC treated with neoCRT, which likely resulted from the effect of RFC4 on radioresistance, not chemoresistance. CONCLUSIONS: RFC4 was identified as a radioresistance factor that promotes NHEJ-mediated DNA repair in colorectal cancer cells. In addition, the expression level of RFC4 predicted radiotherapy responsiveness and the outcome of neoadjuvant radiotherapy in patients with LARC.

Adipose­derived mesenchymal stem cells exhibit tumor tropism and promote tumorsphere formation of breast cancer cells.

Mesenchymal stem cells reportedly have a marked effect on tumor growth or suppression. However, it remains uncertain whether adipose­derived mesenchymal stem cells (ADSCs) from grafted fat can contribute to breast cancer growth and recurrence. In the present study, interactions between ADSCs and MCF­7 breast cancer cells were evaluated in a Matrigel co­culture system and in an in vivo nude mouse model. Results suggested that MCF­7 cells exerted tumor tropism effects on ADSCs and this may be regulated by chemokines, such as the macrophage inflammatory protein (MIP)­1δ and MIP­3α. Additionally, ADSCs significantly induced tumorsphere formation in vitro and promoted tumorigenicity in vivo. RT­qPCR analysis indicated that tumorsphere formation by MCF­7 cells was associated with the induction of stem­like properties, which was mediated by epithelial­-mesenchymal transition. Together, the present findings indicated that ADSCs exhibit tropism and induce tumorsphere formation of MCF­7 cells.

Pretreatment TACC3 expression in locally advanced rectal cancer decreases the response to neoadjuvant chemoradiotherapy.

Chemoradiotherapy combined with surgical resection is the standard treatment for locally advanced rectal cancer, but not all the patients respond to neoadjuvant treatment. Transforming acidic coiled-coil protein-3 (TACC3) is frequently aberrantly expressed in rectal cancer tissue. In this study, we investigated whether TACC3 could serve as a biomarker predictive of the efficacy of chemoradiotherapy. In all, 152 rectal cancer patients with tumor tissue collected at biopsy and set aside before treatment were enrolled in this study. All patients received chemoradiotherapy and surgical resection. Immunohistochemically detected tumoral TACC3 expression significantly decreased sensitivity to chemoradiotherapy [risk ratio (RR) = 2.236, 95% confidence interval (CI): 1.447-3.456; P = 0.001] and thus the pathological complete response rate (P = 0.001). TACC3 knockdown using specific siRNA enhanced radiotherapy-induced decreases in proliferation and colony formation by HCT116 and SW480 cells and increased the incidence of radiotherapy-induced apoptosis. Cox multivariate analysis showed that TACC3 was a significant prognostic factor for overall survival (P = 0.017) and disease-free survival (P = 0.020). These findings suggest TACC3 expression may be predictive of chemoradiotherapy sensitivity and prognosis in locally advanced rectal cancer.

Polycaprolactone nanofibrous mesh reduces foreign body reaction and induces adipose flap expansion in tissue engineering chamber.

Tissue engineering chamber technique can be used to generate engineered adipose tissue, showing the potential for the reconstruction of soft tissue defects. However, the consequent foreign body reaction induced by the exogenous chamber implantation causes thick capsule formation on the surface of the adipose flap following capsule contracture, which may limit the internal tissue expansion. The nanotopographical property and architecture of nanofibrous scaffold may serve as a promising method for minimizing the foreign body reaction. Accordingly, electrospinning porous polycaprolactone (PCL) nanofibrous mesh, a biocompatible synthetic polymer, was attached to the internal surface of the chamber for the reducing local foreign body reaction. Adipose flap volume, level of inflammation, collagen quantification, capsule thickness, and adipose tissue-specific gene expression in chamber after implantation were evaluated at different time points. The in vivo study revealed that the engineered adipose flaps in the PCL group had a structure similar to that in the controls and normal adipose tissue structure but with a larger flap volume. Interleukin (IL)-1ß, IL-6, and transforming growth factor-ß expression decreased significantly in the PCL group compared with the control. Moreover, the control group had much more collagen deposition and thicker capsule than that observed in the PCL group. These results indicate that the unique nanotopographical effect of electrospinning PCL nanofiber can reduce foreign body reaction in a tissue engineering chamber, which maybe a promising new method for generating a larger volume of mature, vascularized, and stable adipose tissue.

Molecular mechanism of oxidative damage of lung in mice following exposure to lanthanum chloride.

Exposure to lanthanoids (Ln) elicits an adverse response such as oxidative injury of lung in animals and human. The molecular targets of Ln remain unclear. In the present study, the function and signal pathway of nuclear factor erythroid 2 related factor 2 (Nrf2) in LaCl3 -induced oxidative stress in mouse lung were investigated. Mice were exposed to 2, 5, and 10 mg/kg body weight by nasal administration for 6 consecutive months. With increased doses, La was markedly accumulated and promoted the reactive oxygen species (ROS) production in the lung, which in turn resulted in peroxidation of lipids, proteins and DNA, and severe pulmonary damages. Furthermore, LaCl3 exposure could significantly increase levels of Nrf2, heme oxygenase 1 (HO-1) and glutamate-cysteine ligase catalytic subunit (GCLC) expressions in the LaCl3 -exposed lung. These findings imply that the induction of Nrf2 expression is an adaptive intracellular response to LaCl3 -induced oxidative stress in mouse lung, and that Nrf2 may regulate the LaCl3 -induced pulmonary damages.