Delivery of GalNAc-conjugated splice-switching ASOs to non-hepatic cells through ectopic expression of asialoglycoprotein receptor

Mapping Intimacies ◽

10.1101/382606 ◽

2018 ◽

Author(s):

Juergen Scharner ◽

Sabrina Qi ◽

Frank Rigo ◽

C. Frank Bennett ◽

Adrian R. Krainer

Keyword(s):

Cancer Cells ◽

Mouse Models ◽

Genetic Diseases ◽

Ectopic Expression ◽

Asialoglycoprotein Receptor ◽

Viable Solution ◽

Subcutaneous Xenograft ◽

Therapeutic Tools

AbstractSplice-switching antisense oligonucleotides (ASOs) are promising therapeutic tools to target various genetic diseases, including cancer. However,in vivodelivery of ASOs to orthotopic tumors in cancer mouse models or to certain target tissues remains challenging. A viable solution already in use is receptor-mediated uptake of ASOs via tissue-specific receptors. For example, the asialoglycoprotein receptor (ASGP-R) is exclusively expressed in hepatocytes. Triantennary GalNAc (GN3)-conjugated ASOs bind to the receptor and are efficiently internalized by endocytosis, enhancing ASO potency in the liver. Here we explore the use of GalNAc-mediated targeting to deliver therapeutic splice-switching ASOs to cancer cells that ectopically express ASGP-R, bothin vitroand in tumor mouse models. We found that ectopic expression of the major isoform ASGP-R1 H1a is sufficient to promote uptake and increase GN3-ASO potency to various degrees in all tested cancer cells. We show that cell-type specific glycosylation of the receptor does not affect its activity.In vivo, GN3-conjugated ASOs specifically target subcutaneous xenograft tumors that ectopically express ASGP-R1, and modulate splicing significantly more strongly than unconjugated ASOs. Our work shows that GN3-targeting is a useful tool for proof-of-principle studies in orthotopic cancer models, until endogenous receptors are identified and exploited for efficiently targeting cancer cells.

Download Full-text

Branched-chain amino acid aminotransferase 2 regulates ferroptotic cell death in cancer cells

Cell Death and Differentiation ◽

10.1038/s41418-020-00644-4 ◽

2020 ◽

Author(s):

Kang Wang ◽

Zhengyang Zhang ◽

Hsiang-i Tsai ◽

Yanfang Liu ◽

Jie Gao ◽

...

Keyword(s):

Cell Death ◽

Amino Acid ◽

Cancer Cells ◽

Ectopic Expression ◽

Branched Chain Amino Acid ◽

Pancreatic Cancer Cells ◽

Key Enzyme ◽

Branched Chain

Abstract Ferroptosis, a form of iron-dependent cell death driven by cellular metabolism and iron-dependent lipid peroxidation, has been implicated as a tumor-suppressor function for cancer therapy. Recent advance revealed that the sensitivity to ferroptosis is tightly linked to numerous biological processes, including metabolism of amino acid and the biosynthesis of glutathione. Here, by using a high-throughput CRISPR/Cas9-based genetic screen in HepG2 hepatocellular carcinoma cells to search for metabolic proteins inhibiting ferroptosis, we identified a branched-chain amino acid aminotransferase 2 (BCAT2) as a novel suppressor of ferroptosis. Mechanistically, ferroptosis inducers (erastin, sorafenib, and sulfasalazine) activated AMPK/SREBP1 signaling pathway through iron-dependent ferritinophagy, which in turn inhibited BCAT2 transcription. We further confirmed that BCAT2 as the key enzyme mediating the metabolism of sulfur amino acid, regulated intracellular glutamate level, whose activation by ectopic expression specifically antagonize system Xc– inhibition and protected liver and pancreatic cancer cells from ferroptosis in vitro and in vivo. On the contrary, direct inhibition of BCAT2 by RNA interference, or indirect inhibition by blocking system Xc– activity, triggers ferroptosis. Finally, our results demonstrate the synergistic effect of sorafenib and sulfasalazine in downregulating BCAT2 expression and dictating ferroptotic death, where BCAT2 can also be used to predict the responsiveness of cancer cells to ferroptosis-inducing therapies. Collectively, these findings identify a novel role of BCAT2 in ferroptosis, suggesting a potential therapeutic strategy for overcoming sorafenib resistance.

Download Full-text

The plasma membrane transporter SLC5A8 suppresses tumour progression through depletion of survivin without involving its transport function

Biochemical Journal ◽

10.1042/bj20121248 ◽

2013 ◽

Vol 450 (1) ◽

pp. 169-178 ◽

Cited By ~ 27

Author(s):

Veena Coothankandaswamy ◽

Selvakumar Elangovan ◽

Nagendra Singh ◽

Puttur D. Prasad ◽

Muthusamy Thangaraju ◽

...

Keyword(s):

Plasma Membrane ◽

Cancer Cells ◽

Histone Deacetylase Inhibitors ◽

Tumour Progression ◽

Ectopic Expression ◽

Membrane Transporter ◽

Transport Function ◽

Plasma Membrane Transporter

SLC5A8 (solute carrier gene family 5A, member 8) is a sodium-coupled transporter for monocarboxylates. Among its substrates are the HDAC (histone deacetylase) inhibitors butyrate, propionate and pyruvate. Expression of SLC5A8 is silenced in cancers via DNA methylation, and ectopic expression of SLC5A8 in cancer cells induces apoptosis in the presence of its substrates that are HDAC inhibitors. In the present study we show that ectopic expression of SLC5A8 in cancer cells translocates the anti-apoptotic protein survivin to the plasma membrane through protein–protein interaction resulting in depletion of nuclear survivin and also decreases cellular levels of survivin through inhibition of transcription. These SLC5A8-induced changes in the location and levels of survivin result in cell-cycle arrest, disruption of the chromosome passenger complex involved in mitosis, induction of apoptosis and enhancement in chemosensitivity. These effects are seen independently of the transport function of SLC5A8 and histone acetylation status of the cell; in the presence of pyruvate, a SLC5A8 substrate and also an HDAC inhibitor, these effects are amplified. Ectopic expression of SLC5A8 in the breast cancer cell line MB231 inhibits the ability of cells to form colonies in vitro and to form tumours in mouse xenografts in vivo. The suppression of survivin transcription occurs independently of HDAC inhibition, and the underlying mechanism is associated with decreased phosphorylation of STAT3 (signal transducer and activator of transcription 3). The observed effects are specific for survivin with no apparent changes in expression of other inhibitor-of-apoptosis proteins. The present study unravels a novel, hitherto unrecognized, mechanism for the tumour-suppressive role of a plasma membrane transporter independent of its transport function.

Download Full-text

Branched chain amino acid aminotransferase 2 Regulates Ferroptotic Cell Death in Cancer Cells

10.1101/2020.02.17.952754 ◽

2020 ◽

Author(s):

Kang Wang ◽

Zhengyang Zhang ◽

Tsai Hsiang-i ◽

Yanfang Liu ◽

Ming Wang ◽

...

Keyword(s):

Amino Acid ◽

Cancer Cells ◽

Therapeutic Strategy ◽

Ectopic Expression ◽

Branched Chain Amino Acid ◽

Pancreatic Cancer Cells ◽

Branched Chain

AbstractFerroptosis has been implicated as a tumor-suppressor function for cancer therapy. Recently the sensitivity to ferroptosis was tightly linked to numerous biological processes, including metabolism of amino acid. Here, using a high-throughput CRISPR/Cas9 based genetic screen in HepG2 cells to search for metabolic proteins inhibiting ferroptosis, we identified branched chain amino acid aminotransferase 2 (BCAT2) as a novel suppressor of ferroptosis. Mechanistically, ferroptosis inducers (erastin, sorafenib and sulfasalazine) activated AMPK/SREBP1 signaling pathway through ferritinophagy, which in turn inhibited BCAT2 transcription. We further confirmed that BCAT2 mediating the metabolism of sulfur amino acid, regulated intracellular glutamate level, whose activation by ectopic expression specifically antagonize system Xc– inhibition and protected liver and pancreatic cancer cells from ferroptosis in vitro and in vivo. Finally, our results demonstrate the synergistic effect of sorafenib and sulfasalazine in downregulating BCAT2 expression and dictating ferroptotic death, where BCAT2 can also be used to predict the responsiveness of cancer cells to ferroptosis-inducing therapies. Collectively, these findings identify a novel role of BCAT2 in ferroptosis, suggesting a potential therapeutic strategy for overcoming sorafenib resistance.

Download Full-text

HPV E7-mediated NCAPH ectopic expression regulates the carcinogenesis of cervical carcinoma via PI3K/AKT/SGK pathway

Cell Death and Disease ◽

10.1038/s41419-020-03244-9 ◽

2020 ◽

Vol 11 (12) ◽

Author(s):

Meng Wang ◽

Xiaowen Qiao ◽

Tamara Cooper ◽

Wei Pan ◽

Liang Liu ◽

...

Keyword(s):

Gene Expression ◽

Cervical Cancer ◽

Cancer Cells ◽

Molecular Mechanisms ◽

Ectopic Expression ◽

Depth Of Invasion ◽

Mesenchymal Transition ◽

Hpv E7

AbstractCervical cancer is one of the most common gynecological tumors in the world, and human papillomavirus (HPV) infection is its causative agent. However, the molecular mechanisms involved in the carcinogenesis of cervical cancer still require clarification. Here we found that knockdown of Non-SMC (Structural Maintenance of Chromosomes) condensin I complex subunit H (NCAPH) gene expression significantly inhibited the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) of cervical cancer cells in vitro, and restrained xenograft tumor formation in vivo. Intriguingly, HPV E7 could form a positive feedback loop with NCAPH. E7 upregulated NCAPH gene expression via E2F1 which initiated NCAPH transcription by binding to its promoter directly. Silencing of NCAPH reduced E7 transcription via promoting the transition of AP-1 heterodimer from c-Fos/c-Jun to Fra-1/c-Jun. Moreover, the E7-mediated NCAPH overexpression was involved in the activation of the PI3K/AKT/SGK signaling pathway. In vivo, NCAPH expression in cervical cancer tissues was significantly higher than which in normal cervix and high-grade squamous intraepithelial lesion (HSIL) tissues, and its expression was significantly correlated with tumor size, depth of invasion and lymph node metastasis. Patients with high NCAPH expression had a significantly better survival outcomes than those with low-expression, suggesting that NCAPH-induced cell proliferation might sensitize cancer cells to adjuvant therapy. In conclusion, our results revealed the role of NCAPH in the carcinogenesis of cervical cancer in vitro and in vivo. The interaction between E7 and NCAPH expands the mechanism of HPV induced tumorigenesis and that of host genes regulating HPV E7.

Download Full-text

Phage display screening identifies a novel peptide to suppress ovarian cancer cells in vitro and in vivo in mouse models

BMC Cancer ◽

10.1186/s12885-015-1891-8 ◽

2015 ◽

Vol 15 (1) ◽

Cited By ~ 7

Author(s):

Cong Zhou ◽

Jiali Kang ◽

Xiaoxia Wang ◽

Wei Wei ◽

Wenyan Jiang

Keyword(s):

Ovarian Cancer ◽

Phage Display ◽

Cancer Cells ◽

Mouse Models ◽

Ovarian Cancer Cells

Download Full-text

Ephrin-A2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT

10.21203/rs.3.rs-78710/v1 ◽

2020 ◽

Author(s):

Yao Zhao ◽

Chenchen Cai ◽

Lubing Shi ◽

Jiwei Wang ◽

Miaomiao Zhang ◽

...

Keyword(s):

Prostate Cancer ◽

Cancer Cells ◽

Cancer Metastasis ◽

Ectopic Expression ◽

Migration And Invasion ◽

Prostate Cancer Cells ◽

Eph Receptor ◽

Prostate Cancer Metastasis

Abstract Background Ephrin-A2, a member of the Eph receptor subgroup, is used in diagnosing and determining the prognosis of prostate cancer. However, the role of ephrin-A2 in prostate cancer is still unclear. Methods We established stable clones overexpressing or silencing ephrin-A2 from prostate cancer cells. Then, CCK-8 was used in analyzing the proliferation ability of cells. CD31 staining was used in evaluating angiogenesis. Migration and invasion assay were conducted in vivo and in vitro. The expression of EMT-related markers was evaluated in prostate cancer cells through Western blotting. Results We revealed that the ectopic expression of ephrin-A2 in prostate cancer cells facilitated cell migration and invasion in vitro and promoted tumor metastasis and angiogenesis in vivo and that the silencing of ephrin-A2 completely reversed this effect. Although ephrin-A2 did not affect tumor cell proliferation in vitro, ephrin-A2 significantly promoted primary tumor growth in vivo. Furthermore, to determine the biological function of ephrin-A2, we assayed the expression of EMT-related markers in stable established cell lines. Results showed that the overexpression of ephrin-A2 in prostate cancer cells down-regulated the expression of epithelial markers (ZO-1, E-cadherin, and claudin-1) and up-regulated the expression of mesenchymal markers (N-cadherin, β-catenin, vimentin, Slug, and Snail), but the knocking out of ephrin-A2 opposed the effects on the expression of EMT markers. Conclusions These findings indicate that ephrin-A2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT and may be a potentially therapeutic target in metastatic prostate cancer.

Download Full-text

ZIP10 is a negative determinant for anti-tumor effect of mannose in thyroid cancer by activating phosphate mannose isomerase

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02195-z ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Sharui Ma ◽

Na Wang ◽

Rui Liu ◽

Rui Zhang ◽

Hui Dang ◽

...

Keyword(s):

Enzyme Activity ◽

Thyroid Cancer ◽

Cancer Cells ◽

Cellular Response ◽

Ectopic Expression ◽

Underlying Mechanism ◽

Zinc Ion ◽

Thyroid Cancer Cells

Abstract Background Mannose, a natural hexose existing in daily food, has been demonstrated to preferentially inhibit the progression of tumors with low expression of phosphate mannose isomerase (PMI). However, its function in thyroid cancer still remains elusive. Methods MTT, colony formation and flow cytometry assays were performed to determine the response of thyroid cancer cells to mannose. Meanwhile, mouse models of subcutaneous xenograft and primary papillary thyroid cancer were established to determine in vivo anti-tumor activity of mannose. The underlying mechanism of mannose selectively killing thyroid cancer cells was clarified by a series of molecular and biochemical experiments. Results Our data demonstrated that mannose selectively suppressed the growth of thyroid cancer cells, and found that enzyme activity of PMI rather than its protein expression was negatively associated with the response of thyroid cancer cells to mannose. Besides, our data showed that zinc ion (Zn2+) chelator TPEN clearly increased the response of mannose-insensitive cells to mannose by inhibiting enzyme activity of PMI, while Zn2+ supplement could effectively reverse this effect. Further studies found that the expression of zinc transport protein ZIP10, which transport Zn2+ from extracellular area into cells, was negatively related to the response of thyroid cancer cells to mannose. Knocking down ZIP10 in mannose-insensitive cells significantly inhibited in vitro and in vivo growth of these cells by decreasing intracellular Zn2+ concentration and enzyme activity of PMI. Moreover, ectopic expression of ZIP10 in mannose-sensitive cells decrease their cellular response to mannose. Mechanistically, mannose exerted its anti-tumor effect by inhibiting cellular glycolysis; however, this effect was highly dependent on expression status of ZIP10. Conclusion The present study demonstrate that mannose selectively kills thyroid cancer cells dependent on enzyme activity of PMI rather than its expression, and provide a mechanistic rationale for exploring clinical use of mannose in thyroid cancer therapy.

Download Full-text

Zyxin promotes colon cancer tumorigenesis in a mitotic phosphorylation-dependent manner and through CDK8-mediated YAP activation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1800621115 ◽

2018 ◽

Vol 115 (29) ◽

pp. E6760-E6769 ◽

Cited By ~ 11

Author(s):

Jiuli Zhou ◽

Yongji Zeng ◽

Lian Cui ◽

Xingcheng Chen ◽

Seth Stauffer ◽

...

Keyword(s):

Colon Cancer ◽

Cancer Cells ◽

Critical Role ◽

Ectopic Expression ◽

Tumor Formation ◽

Dependent Manner ◽

Colon Cancer Cells ◽

Mitotic Phosphorylation

Zyxin is a member of the focal adhesion complex and plays a critical role in actin filament polymerization and cell motility. Several recent studies showed that Zyxin is a positive regulator of Yki/YAP (Yes-associated protein) signaling. However, little is known about the mechanisms by which Zyxin itself is regulated and how Zyxin affects Hippo–YAP activity. We first showed that Zyxin is phosphorylated by CDK1 during mitosis. Depletion of Zyxin resulted in significantly impaired colon cancer cell proliferation, migration, anchorage-independent growth, and tumor formation in xenograft animal models. Mitotic phosphorylation is required for Zyxin activity in promoting growth. Zyxin regulates YAP activity through the colon cancer oncogene CDK8. CDK8 knockout phenocopied Zyxin knockdown in colon cancer cells, while ectopic expression of CDK8 substantially restored the tumorigenic defects of Zyxin-depletion cells. Mechanistically, we showed that CDK8 directly phosphorylated YAP and promoted its activation. Fully activated YAP is required to support the growth in CDK8-knockout colon cancer cells in vitro and in vivo. Together, these observations suggest that Zyxin promotes colon cancer tumorigenesis in a mitotic-phosphorylation-dependent manner and through CDK8-mediated YAP activation.

Download Full-text