Single amino acid-based PROTACs trigger degradation of the oncogenic kinase BCR-ABL in chronic myeloid leukemia (CML).

Proteolysis-targeting chimera (PROTAC) that specifically targets harmful proteins for destruction by hijacking the ubiquitin-proteasome system is emerging as a potent anticancer strategy. How to efficiently modulate the target degradation remains a challenging issue. In this study, we employ a single amino acid-based PROTAC, which uses the shortest degradation signal sequence as the ligand of the N-end rule E3 ubiquitin ligases to degrade the fusion protein BCR (breakpoint cluster region)-ABL (Abelson proto-oncogene), an oncogenic kinase that drives the progression of chronic myeloid leukemia. We find that the reduction level of BCR-ABL can be easily adjusted by substituting different amino acids. Furthermore, a single PEG linker is found to achieve the best proteolytic effect. Our efforts have resulted in effective degradation of BCR-ABL protein by the N-end rule pathway and efficient growth inhibition of K562 cells expressing BCR-ABL in vitro and blunted tumor growth in a K562 xenograft tumor model in vivo. The PROTAC presented has unique advantages including lower effective concentration, smaller molecular size, and modular degradation rate. Demonstrating the efficacy of the N-end rule-based PROTACs in vitro and in vivo, our study further expands the limited degradation pathways currently available for PROTACs in vivo and is easily adapted for broader applications in targeted protein degradation.

How many authors does it take to publish a high profile or classic paper?

Although the process of publishing a scientific paper has gotten simpler, it is increasingly difficult to publish a paper in high profile journals. We have analyzed the publishing data in the cell biology field and found several alarming trends developing over the last two decades. There is an emerging divide between scientist-run journals and professional-run high profile journals. How did this happen? What should we do? The core issue is whether the current standard for high profile journals hurts rather than helps the scientific discovery process. In this regard, we suggest that the editors and scientists should direct their focus on the potential impact and rigor of the work instead of the "perfection" or "completeness" of the study.

The PRMT5-LSD1 axis confers Slug dual transcriptional activities and promotes breast cancer progression.

BACKGROUND: Downregulation of epithelial markers and upregulation of mesenchymal markers are the characteristics of the epithelial to mesenchymal transition (EMT) program, which provides the metastatic advantage of breast cancer. However, the mechanism underlying the switch of EMT markers remains poorly understood. METHODS: In this study, we used the affinity purification and mass spectrometry coupled approach to identify the interactome of Slug. CoIP, GST-pulldown, ChIP, Re-ChIP, qPCR and Immunoblot were used to investigate the underlying mechanism of Slug-PRMT5-LSD1 complex. The role of PRMT5 and LSD1 in breast cancer progression was evaluated both in vivo and in vitro. RESULTS: Here we found that the transcription factor Slug associates with PRMT5 and LSD1 in a complex and facilitates the breast cancer invasion in vitro. Mechanistically, PRMT5 and LSD1 work with Slug to exert dual transcriptional activities to inhibit E-cadherin expression by PRMT5-catalyzed H4R3me2s and LSD1-mediated demethylation of H3K4me2 on the E-cadherin (CDH1) promoter, and activate vimentin (VIM) expression via PRMT5-driven H3R2me2s and LSD1-mediated removal of H3K9me2. Importantly, PRMT5 and LSD1 are coordinately expressed in breast cancer patients and pharmacologic perturbation of both PRMT5 and LSD1 shows a synergetic effect on the inhibition of breast tumor growth and metastasis in vivo. CONCLUSIONS: Our study suggests that PRMT5 and LSD1 function as a dual epigenetic modifier to promote Slug induced EMT program, suggesting that the inhibition of PRMT5 and LSD1 presents a potential therapeutic strategy against cancer metastasis.

PCNA Ubiquitylation: Instructive or Permissive to DNA Damage Tolerance Pathways?

DNA lesions escaping from repair often block the DNA replicative polymerases required for DNA replication and are handled during the S/G2 phases by the DNA damage tolerance (DDT) mechanisms, which include the error-prone translesion synthesis (TLS) and the error-free template switching (TS) pathways. Where the mono-ubiquitylation of PCNA K164 is critical for TLS, the poly-ubiquitylation of the same residue is obligatory for TS. However, it is not known how cells divide the labor between TLS and TS. Due to the fact that the type of DNA lesion significantly influences the TLS and TS choice, we propose that, instead of altering the ratio between the mono- and poly-Ub forms of PCNA, the competition between TLS and TS would automatically determine the selection between the two pathways. Future studies, especially the single integrated lesion "i-Damage" system, would elucidate detailed mechanisms governing the choices of specific DDT pathways.

Circulating Tumor Cells (CTCs): A Unique Model of Cancer Metastases and Non-invasive Biomarkers of Therapeutic Response.

Late-stage cancer metastasis remains incurable in the clinic and is the major cause death in patients. Circulating tumor cells (CTCs) are thought to be metastatic precursors shed from the primary tumor or metastatic deposits and circulate in the blood. The molecular network regulating CTC survival, extravasation, and colonization in distant metastatic sites is poorly defined, largely due to challenges in isolating rare CTCs. Recent advances in CTC isolation and ex vivo culture techniques facilitates single-cell omics and the development of related animal models to study CTC-mediated metastatic progression. With these powerful tools, CTCs can potentially be used as non-invasive biomarkers predicting therapeutic response. These studies may open a new avenue for CTC-specific drug discoveries. In this short review, we aim to summarize recent progress in the characterization of CTCs and their clinical relevance in various cancers, setting the stage for realizing personalized therapies against metastases.

Positive feedback of SuFu negating protein 1 on Hedgehog signaling promotes colorectal tumor growth.

Hedgehog (Hh) signaling plays a critical role in embryogenesis and tissue homeostasis, and its deregulation has been associated with tumor growth. The tumor suppressor SuFu inhibits Hh signaling by preventing the nuclear translocation of Gli and suppressing cell proliferation. Regulation of SuFu activity and stability is key to controlling Hh signaling. Here, we unveil SuFu Negating Protein 1 (SNEP1) as a novel Hh target, that enhances the ubiquitination and proteasomal degradation of SuFu and thus promotes Hh signaling. We further show that the E3 ubiquitin ligase LNX1 plays a critical role in the SNEP1-mediated degradation of SuFu. Accordingly, SNEP1 promotes colorectal cancer (CRC) cell proliferation and tumor growth. High levels of SNEP1 are detected in CRC tissues and are well correlated with poor prognosis in CRC patients. Moreover, SNEP1 overexpression reduces sensitivity to anti-Hh inhibitor in CRC cells. Altogether, our findings demonstrate that SNEP1 acts as a novel feedback regulator of Hh signaling by destabilizing SuFu and promoting tumor growth and anti-Hh resistance.

The N-terminal domain of the non-receptor tyrosine kinase ABL confers protein instability and suppresses tumorigenesis.

Chromosome translocation can lead to chimeric proteins that may become oncogenic drivers. A classic example is the fusion of the BCR activator of RhoGEF and GTPase and the ABL proto-oncogene nonreceptor tyrosine kinase, a result of a chromosome abnormality (Philadelphia chromosome) that causes leukemia. To unravel the mechanism underlying BCR-ABL-mediated tumorigenesis, here we compared the stability of ABL and the BCR-ABL fusion. Using protein degradation, cell proliferation, 5-ethynyl-2-deoxyuridine, and apoptosis assays, along with xenograft tumor analysis, we found that the N-terminal segment of ABL, which is lost in the BCR-ABL fusion, confers degradation capacity that is promoted by SMAD-specific E3 ubiquitin protein ligase 1. We further demonstrate that the N-terminal deletion renders ABL more stable and stimulates cell growth and tumorigenesis. The findings of our study suggest that altered protein stability may contribute to chromosome translocation-induced cancer development.

Autophagy regulator Atg9 is degraded by the proteasome.

Autophagy is a highly conserved biological process essential to protein, cellular and organismal homeostasis. As autophagy plays a critical role in cellular responses to various external and internal stimuli, it is important to understand the mechanism underlying autophagy regulation. Here, we monitor the stability of 17 key autophagy factors in the yeast S. cerevisiae and show that Atg9 and Atg14 are degraded under normal growth conditions. Whereas Atg14 is regulated by both the proteasome and autophagy, Atg9 turnover is normally mediated by the proteasome but impeded upon starvation or rapamycin treatment. Interestingly, distinct segments of Atg9 confer instability, suggesting that multiple pathways are involved in Atg9 degradation. Our results provide the foundation to further elucidate the physiological significance of Atg9 turnover and also the interplay between two major proteolytic systems (i.e., autophagy and the proteasome).

A modular PROTAC design for target destruction using a degradation signal based on a single amino acid.

Proteolysis targeting chimeras (PROTACs) are bivalent molecules that bring a cellular protein to a ubiquitin ligase E3 for ubiquitination and subsequent degradation. Although PROTAC has emerged as a promising therapeutic means for cancers as it rewires the ubiquitin pathway to destroy key cancer regulators, the degradation signals/pathways for PROTACs remain underdeveloped. Here we append single amino acids, the simplest degradation signal, to a ligand specific for estrogen-related receptor α (ERRα) and demonstrate their utility in ERRα knockdown via the N-end rule pathway and also their efficiency in the growth inhibition of breast cancer cells. The modular design described offers unique advantages including smaller molecular size with shortest degradation sequences and degradation speed modulation with different amino acids. Our study expands the repertoire of limited ubiquitin pathways currently available for PROTACs and could be easily adapted for broad use in targeted protein degradation.

A Simple PCR-based Strategy for the Introduction of Point Mutations in the Yeast Saccharomyces cerevisiae via CRISPR/Cas9.

The methods currently employed for in vivo site-directed mutagenesis in yeast are laborious and/or inefficient. Recent developments of the CRISPR-based approaches hold great promise for genome editing, but its application in the yeast S. cerevisiae remains a time-consuming affair. The rate-limiting step in CRISPR-mediated genetic engineering in yeast is the incorporation of the guide sequences, which target Cas9 to relevant chromosomal locus, into the relevant yeast vectors. Here we present a PCR-based strategy to introduce specific point mutation into the yeast CDC48 gene via CRISPR. Our method eliminates the need for special dam- strain and markedly shortens the elaborate multi-step cloning process, leading to significant savings in time, labor and cost.

Multiple E3s promote the degradation of histone H3 variant Cse4.

The histone H3-like protein Cse4/CENP-A acts as a key molecular marker that differentiates the special centromeric chromatin structures from bulk nucleosomes. As altered Cse4/CENP-A activity leads to genome instability, it is pivotal to understand the mechanism underlying Cse4 regulation. Here, we demonstrate that four ubiquitin ligases (i.e., Ubr1, Slx5, Psh1, and Rcy1) work in parallel to promote Cse4 turnover in yeast. Interestingly, Cse4 overexpression leads to cellular toxicity and cell cycle delay in yeast cells lacking PSH1, but not in cells lacking UBR1, suggesting different roles of these two degradation pathways. Our findings suggest that various ubiquitin ligases collaborate to keep the Cse4 level in check, providing a basis for further delineating the intricate network involved in Cse4 regulation.

Arl13b Promotes Gastric Tumorigenesis by Regulating Smo Trafficking and Activation of the Hedgehog Signaling Pathway.

Inhibitors of the Hedgehog (Hh) pathway transducer Smoothened (Smo) have been approved for cancer treatment, but Smo mutations often lead to tumor resistance and it remains unclear how Smo is regulated. In this study, we identified the small GTPase Arl13b as a novel partner and regulator of Smo. Arl13b regulated Smo stability, trafficking, and localization, which are each crucial for Hh signaling. In gastric cancer cells, Arl13b stimulated proliferation, migration, and invasion in vitro and in vivo In clinical specimens of gastric cancer, Arl13b expression correlated strongly with tumor size and depth of invasion; patients with high levels of Arl13b had a poor prognosis. Our results show how Arl13b participates in Hh pathway activation in gastric cancer. Cancer Res; 77(15); 4000-13. ©2017 AACR.

Ubiquitylation of p62/sequestosome1 activates its autophagy receptor function and controls selective autophagy upon ubiquitin stress.

Alterations in cellular ubiquitin (Ub) homeostasis, known as Ub stress, feature and affect cellular responses in multiple conditions, yet the underlying mechanisms are incompletely understood. Here we report that autophagy receptor p62/sequestosome-1 interacts with E2 Ub conjugating enzymes, UBE2D2 and UBE2D3. Endogenous p62 undergoes E2-dependent ubiquitylation during upregulation of Ub homeostasis, a condition termed as Ub+ stress, that is intrinsic to Ub overexpression, heat shock or prolonged proteasomal inhibition by bortezomib, a chemotherapeutic drug. Ubiquitylation of p62 disrupts dimerization of the UBA domain of p62, liberating its ability to recognize polyubiquitylated cargoes for selective autophagy. We further demonstrate that this mechanism might be critical for autophagy activation upon Ub+ stress conditions. Delineation of the mechanism and regulatory roles of p62 in sensing Ub stress and controlling selective autophagy could help to understand and modulate cellular responses to a variety of endogenous and environmental challenges, potentially opening a new avenue for the development of therapeutic strategies against autophagy-related maladies.

Nek2A/SuFu feedback loop regulates Gli-mediated Hedgehog signaling pathway.

Suppressor of Fused (SuFu), one of the most conserved components of the Hedgehog (Hh) signaling, binds Gli transcription factors and impedes activation of target gene expression in mammalian cells. Despite the central importance of SuFu in the Hh pathway, little is known about SuFu regulation. In a previous study, we identified NIMA-related expressed kinase 2A (Nek2A) as a SuFu-interacting protein. Here, we show that Nek2A stabilizes SuFu through impairing ubiquitin/proteasome degradation of SuFu. In addition, Nek2A negatively regulates target genes of Hh signaling as well as Gli2 transcriptional activity. In turn, inhibition of Hh signaling by GANT61 diminishes mRNA and protein levels of Nek2A, and Hh agonist promotes transcription of NEK2A gene. Chromatin immunoprecipitation assays revealed that Gli1 and Gli2 directly bind to the promoter regions of NEK2A gene and induced its transcription. Thus, we uncovered one of the mechanisms by which Nek2A acts as a modulator of the Hh signaling pathway in the context of a novel negative-feedback loop, which may offer new insights into Gli-mediated Hh signaling regulation in development and human diseases.

Parameters of diffusional kurtosis imaging for the diagnosis of acute cerebral infarction in different brain regions.

Diffusional kurtosis imaging (DKI) is a new type diffusion-weighted sequence which measures the non-Gaussianity of water diffusion. The present study aimed to investigate whether the parameters of DKI could distinguish between differences in water molecule diffusion in various brain regions under the conditions of acute infarction and to identify the optimal DKI parameter for locating ischemic lesions in each brain region. A total of 28 patients with acute ischemic stroke in different brain regions were recruited for the present study. The relative values of DKI parameters were selected as major assessment indices, and the homogeneity of background image and contrast of adjacent structures were used as minor assessment indices. According to the brain region involved in three DKI parametric maps, including mean kurtosis (MK), axial kurtosis (Ka) and radial kurtosis (Kr), 112 groups of regions of interest were outlined in the following regions: Corpus callosum (n=17); corona radiata (n=26); thalamus (n=21); subcortical white matter (n=24); and cerebral cortex (n=24). For ischemic lesions in the corpus callosum and corona radiata, significant increases in relative Ka were detected, as compared with the other parameters (P<0.05). For ischemic lesions in the thalamus, subcortical white matter and cerebral cortices, an increase in the three parameters was detected, however this difference was not significant. Minor assessment indices demonstrated that Ka lacked tissue contrast and the background of Kr was heterogeneous; thus, MK was the superior assessment parameter for ischemic lesions in these regions. In conclusion, Ka is better suited for the diagnosis of acute ischemic lesions in highly anisotropic brain regions, such as the corpus callosum and corona radiate. MK may be appropriate for the lesions in low anisotropic or isotropic brain regions, such as the thalamus, subcortical white matter and cerebral cortices.

Nek2A phosphorylates and stabilizes SuFu: A new strategy of Gli2/Hedgehog signaling regulatory mechanism.

Suppressor of Fused (SuFu) plays a conservative role in the regulation of the Gli transcription factors within the Hedgehog (Hh) signaling pathway. Despite the central importance of SuFu in the Hh pathway, little is known about its regulation. Here, we performed a GAL4-based yeast two-hybrid screen using human SuFu as bait, and identified NIMA-related expressed kinase 2A (Nek2A) as a new SuFu-interacting protein, which was also confirmed by glutathione-S-transferase pull-down and co-immunoprecipitation assays. Intriguingly, Nek2A is found to stabilize SuFu at least partly depending on its kinase activity, thereby triggering phosphorylation of the SuFu protein. Moreover, the phosphorylated SuFu inhibits the nuclear localization and transcriptional activity of Gli2/Hh signaling. These findings reveal a new mechanism of mammalian SuFu regulation, and offers novel insights into Hh signaling regulation in development and human disease.

Evaluation of mean diffusion and kurtosis MRI mismatch in subacute ischemic stroke: Comparison with NIHSS score.

Neurological deterioration (ND) is a devastating complication following ischemic stroke. This study aimed to identify the differences in lesion characteristics in subacute ischemic stroke patients with and without ND using diffusional kurtosis imaging (DKI), as well as to confirm the responsible lesions that may lead to ND, as assessed by the National Institutes of Health Stroke Scale (NIHSS) score. Seventy-nine patients with subacute cerebral infarction were allocated to the ND (-) and ND (+) groups according to the NIHSS score and lesion number. The mean diffusion (MD) lesions were significantly larger than the mean kurtosis (MK) deficits in the ND (+) group (P<0.05); however, there was no significant difference in the ND (-) group (P>0.05). The MD and MK in the lesion recovered to normal levels over time; however, the recovery trends in the ND (+) group were substantially slower than the ND (-) group. The differences between the two groups were only significant regarding the MK (p<0.05). Furthermore, multiple infarction lesions exhibited good consistency in the ND (-) group, but were non-homogeneous in the ND (+) group. To the best of our knowledge, this is the first study to demonstrate that a significant MD/MK mismatch and heterogeneity of multiple ischemic lesions on MK in subacute ischemic stroke may represent a new expansion of an ischemic lesion or acute reinfarction, which is closely related to ND.

The F-box Protein Rcy1 Is Involved in the Degradation of Histone H3 Variant Cse4 and Genome Maintenance.

Cse4, a histone H3-like centromeric protein, plays critical functions in chromosome segregation. Cse4 level is tightly regulated, but the underlying mechanism remains poorly understood. We employed a toxicity-based screen to look for the degradation components involved in Cse4 regulation. Here, we show that the F-box containing protein Rcy1 is required for efficient Cse4 turnover as Cse4 degradation is compromised in yeast cells lacking RCY1 Excessive Cse4 accumulation in rcy1Δ cells leads to growth retardation. Furthermore, the deletion of RCY1 is tied to enhanced chromosome instability and temperature-sensitive cell growth. Our results reveal the involvement of Rcy1 in chromosome regulation and another regulatory pathway controlling the Cse4 level and activity.

Inhibition of Hedgehog signaling pathway impedes cancer cell proliferation by promotion of autophagy.

Multiple lines of evidence implicate that aberrant activation of Hedgehog (Hh) signaling is involved in a variety of human cancers. However, the molecular mechanisms underlying how cancer cells respond to Hh inhibition remain to be elucidated. In this study, we found that blockade of Hh signaling suppresses cell proliferation in human cancer cells. Microarray analysis revealed that differentially expressed genes (DEGs) in human cancer cells are enriched in autophagy pathway in response to the inhibition of Hh signaling. Interestingly, inhibition of Hh signaling induced autophagy, whereas activation of Hh signaling by ligand treatments prevented the induction of autophagy. In addition, inhibition of autophagy by 3-methyladenine (3-MA) partially suppressed cytotoxicity induced by inhibition of Hh signaling. Finally, in autophagy deficient cells, cytotoxic effect triggered by inhibition of Hh signaling was partially reversed, indicating the modulation of autophagy by Hh signaling is autophagy-specific. These results suggest that inhibition of Hh signaling impedes cancer cell proliferation in part through induction of autophagy.