Signalling Networks in Cell Shape and Motility (Novartis Foundation Symposium 269)

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Cancers that have spread from their original location to another part of the body are particular challenging to treat, and cause the vast majority of cancer deaths every year. Treatments that can recognize and eradicate cancer cells, while leaving nearby healthy cells untouched, are still needed—and so there has been a lot of research into identifying the key differences between healthy cells and cancer cells.

For several decades, researchers have been aware that cancer cells have more proteins coated with modified sugars on their cell surfaces than healthy cells. This is caused by the enzymes that add these sugars to the proteins relocating from one location within the cell, the Golgi apparatus, to another, called the endoplasmic reticulum. Now Chia et al. ERK8 is shown to prevent the relocation of the sugar-adding enzymes from the Golgi to the endoplasmic reticulum, thereby restricting the production of sugar-coated proteins that help the cancer cells to spread within the body.

By identifying 12 potential targets for new therapeutics aimed at preventing the spread of cancer, the work of Chia et al. GalNAc-type O-linked glycans are polysaccharides present on secreted and membrane-inserted proteins Tran and Ten Hagen, Traditionally associated with mucin-like proteins, recent advances in mass spectrometric analysis have revealed O-glycosylation on hundreds of different proteins Steentoft et al. Recent results have also highlighted specific functional roles for O-glycans; for instance, in regulating the secretion of the phosphatemia regulator FGF23 Kato et al.

The addition of GalNAc on proteins generates the Tn antigen, with antigenicity being lost upon the addition of other sugar residues. Earlier work demonstrated that carcinomas stain prominently with antibodies and lectins such as the Helix Pomatia Lectin HPL , which binds to Tn antigens Springer, The high prevalence and specificity of this cancer glycophenotype is remarkable, with matching normal tissues and benign tumours expressing much lower levels. In breast carcinoma, however, high Tn levels seem to be caused by a different mechanism: GalNAc-Ts are massively relocated from the Golgi apparatus to the endoplasmic reticulum ER with Tn staining largely located in the ER Gill et al.

Further, in some cancer cells, O-glycosylation initiation in the ER has also been reported Egea et al. It is surmised that glycosylation of ER-resident proteins likely explains this observed increase in Tn staining, as several of these proteins bear O-GalNAc in mass spectrometric analyses Steentoft et al.

Although it is unclear which specific proteins are modified, O-glycosylation in the ER results in a marked stimulatory effect on cell adhesion and cell migration Gill et al. This suggests that ER O-glycosylation promotes the invasive and metastatic potential of malignant tumour cells.

Tn levels are consistently higher in higher grade, more aggressive breast tumours. Conversely, ER-specific inhibition of O-glycosylation reduced drastically lung metastasis in a mice model Gill et al. COPI is a multimeric protein complex required for the formation of transport carriers and functions in the retrograde traffic between the Golgi and the ER Beck et al. To better understand the mechanisms regulating Tn expression in cancer, we performed an RNAi screen targeting genes presumed to be involved in signal transduction. The loss of ERK8 activity results in increased O-glycosylation and increased cell motility.

We find that ERK8 expression is also frequently downregulated in lung carcinomas, which may partly explain the high Tn phenotype and invasiveness of these tumours. We recently reported the results from a screen for regulators of Golgi morphology and organisation using various markers including fluorescently labelled HPL Chia et al. In a pilot screen using HeLa cells, which targeted 63 known players of membrane traffic Chia et al. None of the gene depletions seemed to significantly reduce the basal levels of Tn in HeLa cells Figure 1C. The knockdown effects were reproduced in independent replicates Figure 1—figure supplement 1A , and the trend was mostly independent of the analysis algorithm used, although the fold increase was higher with one method than with the other Figure 1—figure supplement 1B.

A mask was generated for both HPL and nuclei Hoechst staining to classify the region of measurement lower panels.

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  6. B Schematic overview of the screening process. Images from the RNAi screen in Chia et al.

    Primary hits were selected based on a threshold of a nine-fold increase red dashed line and the final validated genes are labelled in red Hit genes. To focus our analysis, we used a stringent cut-off of a ninefold increase in HPL staining intensity, which resulted in 19 genes Figure 1B,C.

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    It should be noted that knock-down was not optimised for the other 18 genes and thus Tn levels may reflect partly the extent of target depletion. To exclude the possibility of off-target effects in the 19 hits, we repeated the screen using the individual siRNAs that were used in each siRNA pool in the primary screen.

    Using a threshold of 4. To verify that the effects observed were not specific to the detection method used, knockdown cells were also stained with a different lectin, Vicia Villosa Lectin VVL , which revealed a highly consistent pattern Figure 1—figure supplement 1E. To validate that these genes indeed led to the up-regulation in GalNAc protein O-glycosylation, we sought to downregulate the responsible enzymes. Co-depletion of the two enzymes also reduced significantly the Tn increase from the knockdown of ERK8 as well as that from other Tn regulators Figure 1E , Figure 1—figure supplement 1F.

    This effect was not caused by inefficient ERK8 knockdown, as other co-knockdown experiments did not have such an effect. In addition, ERK8 levels were still significantly reduced in the triple knockdown configuration Figure 1—figure supplement 1G. Overall, our screen revealed several negative regulators of the O-GalNAc glycosylation process, which thus appears to be tightly controlled by signalling mechanisms. To distinguish between the two possibilities, PNA staining was quantified upon depletion of each of the 12 Tn regulators. Therefore, none of the Tn-regulating genes we identified appears to be required for core 1-forming activity and thus do not regulate Tn levels by inhibiting O-GalNAc extension.

    Cells were analysed using MetaXpress Translocation-Enhanced analysis module. This suggests that the entire content of the Golgi apparatus becomes redistributed to the ER, and is consistent with our previous findings, where PI4KA depletion induced a redistribution of TGN46, a trans golgi marker Chia et al. Next, we used a quantitative approach to determine the extent of ER relocalisation that occurs in response to depletion of the other Tn-regulating genes, measuring the degree of co-localisation between GalNAc-T1 and Calreticulin staining.


    Collectively, our results suggest that signalling genes influence Tn levels through the subcellular distribution of GalNAc-Ts and that, with the exception of PI4KA, they affect the trafficking of these enzymes specifically. To explore how the products of these genes might be functioning, we retrieved data pertaining to their subcellular localisation and protein—protein interactions from Protein Atlas, GeneCards and STRING Jensen et al. Since Golgi-to-ER retrograde traffic depends on microtubule tracks Palmer et al. Finally, several other interactions, either direct or with one intermediate, were found between the Tn-regulating genes.

    Altogether, this analysis suggests that the Tn-regulating genes are acting at the Golgi level, perhaps part of a regulatory network controlling the subcellular localisation of GalNAc-Ts Figure 2E. Further experiments are required to confirm the reality of this network and its precise connectivity. We next sought to understand the mechanistic basis of Tn level regulation by ERK8. ERK8 displays high basal activity in resting cells and is not stimulated by growth factor activity Abe et al. HPL intensities were quantified for transfected GFP-expressing and non-transfected non-GFP-expressing cell populations, each comprising hundreds of cells.

    Ro inhibits the kinase activity of ERK8 Klevernic et al. A significant increase in Tn was observed as early as 1 hr after treatment and peaked at a nearly eightfold increase after 3. After that, we observed some cell death, which possibly explains the accompanying decrease in Tn levels. This result indicated that the increase in Tn staining is a relatively rapid phenomenon and that O-GalNAc glycosylated protein accumulation can be achieved in a few hours.

    Our findings also suggest that these changes were not caused by expression changes of the O-glycoproteins or the O-glycosylation machinery.

    After pulldown with HPL-conjugated beads, we found a significant increase in the glycosylation of this reporter upon treatment with Ro for 3. Recently, several ER-resident proteins were shown to be O-glycosylated Steentoft et al. This suggests that ERK8 controls the O-glycosylation status of several proteins, which probably includes ER residents. The effects of Ro offered the possibility to test how quickly Tn levels can return to baseline levels after drug washout.

    High Tn levels obtained after 3. Consistently, staining for GalNAc-Ts showed a similar trend, with a significant increase in enzyme localisation at the Golgi within 2 hr of washout Figure 3—figure supplement 1F,G. This suggests that reactivation of ERK8 slows the continuous retrograde flow of GalNAc-Ts and that anterograde traffic shifts their distribution back to the Golgi. Thus, relocation of these glycosylation enzymes to the ER upon ERK8 depletion could result either from an enhanced export from the Golgi or an inhibition of exit from the ER.

    To address this, we first analysed the subcellular localisation of ERK8 protein by immunofluorescence and observed a predominantly cytosolic pattern in wild-type HeLa cells. However, prolonged permeabilisation 2 hr clearly revealed positive Golgi staining, suggesting that a fraction of ERK8 is associated with this organelle Figure 4A. This is consistent with the perinuclear pattern reported in A cells by the Protein Atlas project Uhlen et al. Combined knockdowns of Arfs further increased the rescue, suggesting functional redundancy amongst the Arf proteins.

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    Proteinuria is a type of nephropathy caused by the injury of three cell types including mesangial cells, podocytes, and endothelial cells in glomerular units. Degeneration of podocytes is a primary event in the development of chronic renal failure and proteinuria. The features of cancers include uncontrollable growth and distant dissemination. They are important index for design ofcancer therapeutic strategies. The actin cytoskeletal reorganization is involved in these phenotypes, and several toxins targeting on the networks of actin filaments have been reported to efficiently inhibit cell cycle progression and induce apoptosis in cancer cells [74, 75].

    In addition, a series of actin toxins that display highly antitumor effects have been used for anticancer researches by interfering the actin polymerization and depolymerization. For instance, latrunculin A LA has been used for inhibition of gastric cancers [81]; cytochalasin B CB is able to control the growth of lung cancers [82], and jasplakinolide JP can inhibit the prostate cancer and Lewis lung cancer [83]. LA can induce cofilin-1 phosphorylation, but the mechanisms are largely unknown [84]. In our lab, we have demonstrated that overexpressed cofilin-1 enhances radiosensitivity and G1 phase arrest in human lung cancer cells [39, 85].

    Overexpressed cofilin-1 is normally phosphorylated and is able to repress the expression of several DNA repair proteins including Rad51, Ku70, and Ku For the G1 phase arrest, cofilin-1 may upregulate the cell cycle inhibitor p27kip1 through transcriptional and translational mechanisms [9].

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    Whether cofilin-1 can be used for gene therapy to avoid high toxicity of actin toxins is of interest to further investigate. The distant metastasis of tumor cells is the primary cause of mortality. Thus, targeting on these signaling pathways would be one of the important strategies for cancer treatment. Toshima, J. Toshima, T.