A Ubiquitination Cascade Regulating the Integrated Stress Response and Survival in Carcinomas.

Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy. SIGNIFICANCE: We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells. See related commentary Leli and Koumenis, p. 535. This article is highlighted in the In This Issue feature, p. 517.

Redirecting Vesicular Transport to Improve Nonviral Delivery of Molecular Cargo.

Cell engineering relies heavily on viral vectors for the delivery of molecular cargo into cells due to their superior efficiency compared to nonviral ones. However, viruses are immunogenic and expensive to manufacture, and have limited delivery capacity. Nonviral delivery approaches avoid these limitations but are currently inefficient for clinical applications. This work demonstrates that the efficiency of nonviral delivery of plasmid DNA, mRNA, Sleeping Beauty transposon, and ribonucleoprotein can be significantly enhanced through pretreatment of cells with the nondegradable sugars (NDS), such as sucrose, trehalose, and raffinose. The enhancement is mediated by the incorporation of the NDS into cell membranes, causing enlargement of lysosomes and formation of large (>500 nm) amphisome-like bodies (ALBs). The changes in subcellular structures redirect transport of cargo to ALBs rather than to lysosomes, reducing cargo degradation in cells. The data indicate that pretreatment of cells with NDS is a promising approach to improve nonviral cargo delivery in biomedical applications.

The Long Noncoding RNA NEAT1 Promotes Sarcoma Metastasis by Regulating RNA Splicing Pathways.

Soft-tissue sarcomas (STS) are rare malignancies showing lineage differentiation toward diverse mesenchymal tissues. Half of all high-grade STSs develop lung metastasis with a median survival of 15 months. Here, we used a genetically engineered mouse model that mimics undifferentiated pleomorphic sarcoma (UPS) to study the molecular mechanisms driving metastasis. High-grade sarcomas were generated with Cre recombinase technology using mice with conditional mutations in Kras and Trp53 (KP) genes. After amputation of the limb bearing the primary tumor, mice were followed for the development of lung metastasis. Using RNA-sequencing of matched primary KP tumors and lung metastases, we found that the long noncoding RNA (lncRNA) Nuclear Enriched Abundant Transcript 1 (Neat1) is significantly upregulated in lung metastases. Furthermore, NEAT1 RNA ISH of human UPS showed that NEAT1 is upregulated within a subset of lung metastases compared with paired primary UPS. Remarkably, CRISPR/Cas9-mediated knockout of Neat1 suppressed the ability of KP tumor cells to colonize the lungs. To gain insight into the underlying mechanisms by which the lncRNA Neat1 promotes sarcoma metastasis, we pulled down Neat1 RNA and used mass spectrometry to identify interacting proteins. Interestingly, most Neat1 interacting proteins are involved in RNA splicing regulation. In particular, KH-Type Splicing Regulatory Protein (KHSRP) interacts with Neat1 and is associated with poor prognosis of human STS. Moreover, depletion of KHSRP suppressed the ability of KP tumor cells to colonize the lungs. Collectively, these results suggest that Neat1 and its interacting proteins, which regulate RNA splicing, are involved in mediating sarcoma metastasis. IMPLICATIONS: Understanding that lncRNA NEAT1 promotes sarcoma metastasis, at least in part, through interacting with the RNA splicing regulator KHSRP may translate into new therapeutic approaches for sarcoma.

Current Progress in Electrotransfection as a Nonviral Method for Gene Delivery.

Electrotransfection (ET) is a nonviral method for delivery of various types of molecules into cells both in vitro and in vivo. Close to 90 clinical trials that involve the use of ET have been performed, and approximately half of them are related to cancer treatment. Particularly, ET is an attractive technique for cancer immunogene therapy because treatment of cells with electric pulses alone can induce immune responses to solid tumors, and the responses can be further enhanced by ET of plasmid DNA (pDNA) encoding therapeutic genes. Compared to other gene delivery methods, ET has several unique advantages. It is relatively inexpensive, flexible, and safe in clinical applications, and introduces only naked pDNA into cells without the use of additional chemicals or viruses. However, the efficiency of ET is still low, partly because biological mechanisms of ET in cells remain elusive. In previous studies, it was believed that pDNA entered the cells through transient pores created by electric pulses. As a result, the technique is commonly referred to as electroporation. However, recent discoveries have suggested that endocytosis plays an important role in cellular uptake and intracellular transport of electrotransfected pDNA. This review will discuss current progresses in the study of biological mechanisms underlying ET and future directions of research in this area. Understanding the mechanisms of pDNA transport in cells is critical for the development of new strategies for improving the efficiency of gene delivery in tumors.

Enhancing Electrotransfection Efficiency through Improvement in Nuclear Entry of Plasmid DNA.

The nuclear envelope is a physiological barrier to electrogene transfer. To understand different mechanisms of the nuclear entry for electrotransfected plasmid DNA (pDNA), the current study investigated how manipulation of the mechanisms could affect electrotransfection efficiency (eTE), transgene expression level (EL), and cell viability. In the investigation, cells were first synchronized at G2-M phase prior to electrotransfection so that the nuclear envelope breakdown (NEBD) occurred before pDNA entered the cells. The NEBD significantly increased the eTE and the EL while the cell viability was not compromised. In the second experiment, the cells were treated with a nuclear pore dilating agent (i.e., trans-1,2-cyclohexanediol). The treatment could increase the EL, but had only minor effects on eTE. Furthermore, the treatment was more cytotoxic, compared with the cell synchronization. In the third experiment, a nuclear targeting sequence (i.e., SV40) was incorporated into the pDNA prior to electrotransfection. The incorporation was more effective than the cell synchronization for enhancing the EL, but not the eTE, and the effectiveness was cell type dependent. Taken together, the data described above suggested that synchronization of the NEBD could be a practical approach to improving electrogene transfer in all dividing cells.

Distinct effects of endosomal escape and inhibition of endosomal trafficking on gene delivery via electrotransfection.

A recent theory suggests that endocytosis is involved in uptake and intracellular transport of electrotransfected plasmid DNA (pDNA). The goal of the current study was to understand if approaches used previously to improve endocytosis of gene delivery vectors could be applied to enhancing electrotransfection efficiency (eTE). Results from the study showed that photochemically induced endosomal escape, which could increase poly-L-lysine (PLL)-mediated gene delivery, decreased eTE. The decrease could not be blocked by treatment of cells with endonuclease inhibitors (aurintricarboxylic acid and zinc ion) or antioxidants (L-glutamine and ascorbic acid). Chemical treatment of cells with an endosomal trafficking inhibitor that blocks endosome progression, bafilomycin A1, resulted in a significant decrease in eTE. However, treatment of cells with lysosomotropic agents (chloroquine and ammonium chloride) had little effects on eTE. These data suggested that endosomes played important roles in protecting and intracellular trafficking of electrotransfected pDNA.

Clinical and demographic variables related to pain in HIV-infected individuals treated with effective, combination antiretroviral therapy (cART).

OBJECTIVE: With widespread use of combination antiretroviral therapy (cART), this study tested the hypotheses that: 1) pain would be reported less frequently than in earlier studies; 2) pain would correlate less with markers of disease progression (declining cluster of differentiation 4 [CD4+] count), than with age; and 3) pain would be associated inversely with adherence to cART. DESIGN: Retrospective data analysis. SETTING: Outpatient center of a university teaching hospital. PATIENTS: Forty-one consecutive human immunodeficiency virus (HIV)-infected persons receiving cART. OUTCOME MEASURES: Self-reported pain scale data were retrospectively gathered by their treating physician, along with data regarding gender, age, CD4+ count, self-reported cART adherence, and receipt of pain medication. In addition, data on pain location, duration, and etiology, and on specific cART agents utilized were available for 26 of these subjects. Blinded data were submitted to the investigator, and associations between self-reported pain scores and other variables were calculated. RESULTS: Pain was less prevalent than reported prior to cART (39% vs 60-80%), and pain scale scores were lower (2.0 vs 7.4). Patients reporting more intense pain were more likely to be receiving medication for pain than those reporting less severe pain (87.5% vs 25.0%). Pain was transient in 73% patients and chronic in 27%. Pain scores did not differ by gender, nor did they correlate with adherence scores, disease progression, or age. No patients reported neuropathic pain. CONCLUSIONS: In this cohort treated with cART, pain was less prevalent and less likely to be associated with HIV disease progression or treatment than indicated by studies conducted prior to the widespread use of cART.