Proteasomal control of anti-CRISPRs for the regulation of CRISPR/Cas9 activity using Cas9-ACROBAT.

Small molecule-mediated proteasomal degradation of proteins is a powerful tool for synthetic regulation of biological activity. To control Cas9 activity in cells, we engineered an anti-CRISPR protein, AcrIIA4, fused to a degradation (dTAG) or small molecule assisted shutoff (SMASh) tag. Co-expression of the tagged AcrIIA4 along with Cas9 and riboswitch-regulated sgRNAs enables precise tunable control of CRISPR activity by small molecule addition.

The adaptive immune system is a major driver of selection for tumor suppressor gene inactivation.

During tumorigenesis, tumors must evolve to evade the immune system and do so by disrupting the genes involved in antigen processing and presentation or up-regulating inhibitory immune checkpoint genes. We performed in vivo CRISPR screens in syngeneic mouse tumor models to examine requirements for tumorigenesis both with and without adaptive immune selective pressure. In each tumor type tested, we found a marked enrichment for the loss of tumor suppressor genes (TSGs) in the presence of an adaptive immune system relative to immunocompromised mice. Nearly one-third of TSGs showed preferential enrichment, often in a cancer- and tissue-specific manner. These results suggest that clonal selection of recurrent mutations found in cancer is driven largely by the tumor's requirement to avoid the adaptive immune system.

Genome-Wide CRISPR Screen Identifies Semaphorin 6A and 6B as Receptors for Paeniclostridium sordellii Toxin TcsL.

The exotoxin TcsL is a major virulence factor in Paeniclostridium (Clostridium) sordellii and responsible for the high lethality rate associated with P. sordellii infection. Here, we present a genome-wide CRISPR-Cas9-mediated screen using a human lung carcinoma cell line and identify semaphorin (SEMA) 6A and 6B as receptors for TcsL. Disrupting SEMA6A/6B expression in several distinct human cell lines and primary human endothelial cells results in reduced TcsL sensitivity, while SEMA6A/6B over-expression increases their sensitivity. TcsL recognizes the extracellular domain (ECD) of SEMA6A/6B via a region homologous to the receptor-binding site in Clostridioides difficile toxin B (TcdB), which binds the human receptor Frizzled. Exchanging the receptor-binding interfaces between TcsL and TcdB switches their receptor-binding specificity. Finally, administration of SEMA6A-ECD proteins protects human cells from TcsL toxicity and reduces TcsL-induced damage to lung tissues and the lethality rate in mice. These findings establish SEMA6A and 6B as pathophysiologically relevant receptors for TcsL.

Proteomic Analysis Identifies Membrane Proteins Dependent on the ER Membrane Protein Complex.

The endoplasmic reticulum (ER) membrane protein complex (EMC) is a key contributor to biogenesis and membrane integration of transmembrane proteins, but our understanding of its mechanisms and the range of EMC-dependent proteins remains incomplete. Here, we carried out an unbiased mass spectrometry (MS)-based quantitative proteomic analysis comparing membrane proteins in EMC-deficient cells to wild-type (WT) cells and identified 36 EMC-dependent membrane proteins and 171 EMC-independent membrane proteins. Of these, six EMC-dependent and six EMC-independent proteins were further independently validated. We found that a common feature among EMC-dependent proteins is that they contain transmembrane domains (TMDs) with polar and/or charged residues. Mutagenesis studies demonstrate that EMC dependency can be converted in cells by removing or introducing polar and/or charged residues within TMDs. Our studies expand the list of validated EMC-dependent and EMC-independent proteins and suggest that the EMC is involved in handling TMDs with residues challenging for membrane integration.

Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets.

BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.

Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation.

Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific "activator" for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity.

Preclinical Study of Novel Curcumin Analogue SSC-5 Using Orthotopic Tumor Xenograft Model for Esophageal Squamous Cell Carcinoma.

PURPOSE: Tumor xenograft model is an indispensable animal cancer model. In esophageal squamous cell carcinoma (ESCC) research, orthotopic tumor xenograft model establishes tumor xenograft in the animal esophagus, which allows the study of tumorigenesis in its native microenvironment. MATERIALS AND METHODS: In this study,we described two simple and reproducible methods to develop tumor xenograft at the cervical or the abdominal esophagus in nude mice by direct injection of ESCC cells in the esophageal wall. RESULTS: In comparing these two methods, the cervical one presented with more clinically relevant features, i.e., esophageal stricture, body weight loss and poor survival. In addition, the derived tumor xenografts accompanied a rapid growth rate and a high tendency to invade into the surrounding structures. This model was subsequently used to study the anti-tumor effect of curcumin, which is known for its potential therapeutic effects in various diseases including cancers, and its analogue SSC-5. SSC-5 was selected among the eight newly synthesized curcumin analogues based on its superior anti-tumor effect demonstrated in an MTT cell proliferation assay and its effects on apoptosis induction and cell cycle arrest in cultured ESCC cells. Treatment of orthotopic tumor-bearing mice with SSC-5 resulted in an inhibition in tumor growth and invasion. CONCLUSION: Taken together, we have established a clinically relevant orthotopic tumor xenograft model that can serve as a preclinical tool for screening new anti-tumor compounds, e.g., SSC-5, in ESCC.

A Role for Mitochondrial Translation in Promotion of Viability in K-Ras Mutant Cells.

Activating mutations in the KRAS oncogene are highly prevalent in tumors, especially those of the colon, lung, and pancreas. To better understand the genetic dependencies that K-Ras mutant cells rely upon for their growth, we employed whole-genome CRISPR loss-of-function screens in two isogenic pairs of cell lines. Since loss of essential genes is uniformly toxic in CRISPR-based screens, we also developed a small hairpin RNA (shRNA) library targeting essential genes. These approaches uncovered a large set of proteins whose loss results in the selective reduction of K-Ras mutant cell growth. Pathway analysis revealed that many of these genes function in the mitochondria. For validation, we generated isogenic pairs of cell lines using CRISPR-based genome engineering, which confirmed the dependency of K-Ras mutant cells on these mitochondrial pathways. Finally, we found that mitochondrial inhibitors reduce the growth of K-Ras mutant tumors in vivo, aiding in the advancement of strategies to target K-Ras-driven malignancy.

Neuropilin-2 promotes tumourigenicity and metastasis in oesophageal squamous cell carcinoma through ERK-MAPK-ETV4-MMP-E-cadherin deregulation.

Oesophageal squamous cell carcinoma (ESCC) is the most common histological subtype of oesophageal cancer. The disease is particularly prevalent in southern China. The incidence of the disease is on the rise and its overall survival rate remains dismal. Identification and characterization of better molecular markers for early detection and therapeutic targeting are urgently needed. Here, we report levels of transmembrane and soluble neuropilin-2 (NRP2) to be significantly up-regulated in ESCC, and to correlate positively with advanced tumour stage, lymph node metastasis, less favourable R category and worse overall patient survival. NRP2 up-regulation in ESCC was in part a result of gene amplification at chromosome 2q. NRP2 overexpression promoted clonogenicity, angiogenesis and metastasis in ESCC in vitro, while NRP2 silencing by lentiviral knockdown or neutralizing antibody resulted in a contrary effect. This observation was extended in vivo in animal models of subcutaneous tumourigenicity and tail vein metastasis. Mechanistically, overexpression of NRP2 induced expression of ERK MAP kinase and the transcription factor ETV4, leading to enhanced MMP-2 and MMP-9 activity and, as a consequence, suppression of E-cadherin. In summary, NRP2 promotes tumourigenesis and metastasis in ESCC through deregulation of ERK-MAPK-ETV4-MMP-E-cadherin signalling. NRP2 represents a potential diagnostic or prognostic biomarker and therapeutic target for ESCC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

14-3-3σ confers cisplatin resistance in esophageal squamous cell carcinoma cells via regulating DNA repair molecules.

Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer in Asia. Cisplatin is commonly used in chemoradiation for unresectable ESCC patients. However, the treatment efficacy is diminished in patients with established cisplatin resistance. To understand the mechanism leading to the development of cisplatin resistance in ESCC, we compared the proteomes from a cisplatin-resistant HKESC-2R cell line with its parental-sensitive counterpart HKESC-2 to identify key molecule involved in this process. Mass spectrometry analysis detected 14-3-3σ as the most abundant molecule expressed exclusively in HKESC-2R cells, while western blot result further validated it to be highly expressed in HKESC-2R cells when compared to HKESC-2 cells. Ectopic expression of 14-3-3σ increased cisplatin resistance in HKESC-2 cells, while its suppression sensitized SLMT-1 cells to cisplatin. Among the molecules involved in drug detoxification, drug transportation, and DNA repair, the examined DNA repair molecules HMGB1 and XPA were found to be highly expressed in HKESC-2R cells with high 14-3-3σ expression. Subsequent manipulation of 14-3-3σ by both overexpression and knockdown approaches concurrently altered the expression of HMGB1 and XPA. 14-3-3σ, HMGB1, and XPA were preferentially expressed in cisplatin-resistant SLMT-1 cells when compared to those more sensitive to cisplatin. In ESCC patients with poor response to cisplatin-based chemoradiation, their pre-treatment tumors expressed higher expression of HMGB1 than those with response to such treatment. In summary, our results demonstrate that 14-3-3σ induces cisplatin resistance in ESCC cells and that 14-3-3σ-mediated cisplatin resistance involves DNA repair molecules HMGB1 and XPA. Results from this study provide evidences for further work in researching the potential use of 14-3-3σ and DNA repair molecules HMGB1 and XPA as biomarkers and therapeutic targets for ESCC.

Oxygen carrier YQ23 can enhance the chemotherapeutic drug responses of chemoresistant esophageal tumor xenografts.

PURPOSE: Adjunct chemoradiation is offered to unresectable esophageal squamous cell carcinoma (ESCC) patients, while its use is limited in tumors with strong resistance. Oxygen carriers or anti-hypoxic drugs belong to an emerging class of regulators that can alleviate tumor hypoxia. METHODS: We investigate the potential use of a novel oxygen carrier YQ23 in sensitizing chemoresistant ESCC in a series of subcutaneous tumor xenograft models developed using ESCC cell lines with different strengths of chemosensitivities. RESULTS: Tumor xenografts were developed using SLMT-1 and HKESC-2 ESCC cell lines with different strengths of resistance to two chemotherapeutic drugs, 5-fluorouracil and cisplatin. More resistant SLMT-1 xenografts responded better to YQ23 treatment than HKESC-2, as reflected by the induced tumor oxygen level. YQ23 sensitized SLMT-1 xenografts toward 5-fluorouracil via its effect on reducing the level of a hypoxic marker HIF-1α. Furthermore, a derangement of tumor microvessel density and integrity was demonstrated with a concurrent decrease in the level of a tumor mesenchymal marker vimentin. Similar to the 5-fluorouracil sensitizing effect, YQ23 also enhanced the response of SLMT-1 xenografts toward cisplatin by reducing the tumor size and the number of animals with invasive tumors. Chemosensitive HKESC-2 xenografts were irresponsive to combined YQ23 and cisplatin treatment. CONCLUSIONS: In all, YQ23 functions selectively on chemoresistant ESCC xenografts, which implicates its potential use as a chemosensitizing agent for ESCC patients.

Desacetyluvaricin induces S phase arrest in SW480 colorectal cancer cells through superoxide overproduction.

Annonaceous acetogenins (ACGs) are a group of fatty acid-derivatives with potent anticancer effects. In the present study, we found desacetyluvaricin (Dau) exhibited notable in vitro antiproliferative effect on SW480 human colorectal carcinoma cells with IC50 value of 14 nM. The studies on the underlying mechanisms revealed that Dau inhibited the cancer cell growth through induction of S phase cell cycle arrest from 11.3% (control) to 33.2% (160 nM Dau), which was evidenced by the decreased protein expression of cyclin A Overproduction of superoxide, intracellular DNA damage, and inhibition of MEK/ERK signaling pathway, were also found involved in cells exposed to Dau. Moreover, pre-treatment of the cells with ascorbic acid significantly prevented the Dau-induced overproduction of superoxide, DNA damage and cell cycle arrest. Taken together, our results suggest that Dau induces S phase arrest in cancer cells by firstly superoxide overproduction and subsequently the involvement of various signaling pathways.

Overexpression of transferrin receptor CD71 and its tumorigenic properties in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer in endemic Asian regions. In the present study, we investigated the clinical implication and role of transferrin receptor CD71 in ESCC. CD71 has a physiological role in cellular iron intake and is implicated in the carcinogenesis of various types of tumors. In our cohort, more than a 2-fold upregulation of the CD71 transcript was detected in 61.5% of patients using quantitative polymerase chain reaction. Immunohistochemical analysis also showed strong membranous and cytoplasmic localization of CD71 in paraffin-embedded tumors. Staining parallel tumor sections with the proliferative marker Ki-67 revealed that the pattern of Ki-67 staining was associated with CD71 expression. Analysis of clinicopathological data indicated that CD71 overexpression can be used as an indicator for advanced T4 stage (p=0.0307). These data suggested a strong link between CD71 and ESCC. Subsequent in vitro assays using short interfering RNA (siRNA) to suppress CD71 expression confirmed the tumorigenic properties of CD71 in ESCC; cell growth inhibition and cell cycle arrest at S phase were observed in CD71-suppressed cells. The underlying mechanism involved activation of the MEK/ERK pathway. In summary, the present study provides evidence showing the tumorigenic properties of CD71 in ESCC with clinical correlations and suggests targeting CD71 as a strategy for the treatment of ESCC.

Cyanidin reverses cisplatin-induced apoptosis in HK-2 proximal tubular cells through inhibition of ROS-mediated DNA damage and modulation of the ERK and AKT pathways.

Cyanidin is an anthocyanin widely distributed in food diet with novel antioxidant activity. Herein, we investigated the protective effects of cyanidin on HK-2 proximal tubular cells against cisplatin-induced apoptosis and elucidated the underlying mechanisms. The results showed that cisplatin-induced apoptosis in HK-2 cells was significantly attenuated by cyanidin. The cleavage of caspases and PARP, activation of p53 and mitochondrial-mediated apoptosis pathways induced by cisplatin were effectively blocked by cyanidin. Moreover, cyanidin significantly suppressed the overproduction of ROS, and activation of ERK and AKT pathways triggered by cisplatin. Our results indicate that cyanidin exhibits therapeutic potential in prevention of cisplatin-induced nephrotoxicity.