Nuclear fascin regulates cancer cell survival
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This work explores a topic of high interest to cell and cancer biologists - the role of actin polymerization, and here specifically the role of fascin, in the nucleus. The authors show that fascin regulates nuclear actin, chromatin organization, response to DNA damage, and demonstrate the need for control of steady-state nuclear levels to avoid cell death. Studying nuclear actin is technically challenging, and the authors deploy some novel technologies towards this goal. There are some very elegant experiments in this paper that suggest fascin has an important role in regulating nuclear actin and other important aspects of cancer cell behaviour. The work could be enhanced by the authors considering adding some additional experiments and providing clarifications and some further details or discussion.
(This preprint has been reviewed by eLife. We include the joint public review from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. All three Reviewers agreed to share their names with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Cancer Biology (eLife)
Abstract
Fascin is an important regulator of F-actin bundling leading to enhanced filopodia assembly. Fascin is also overexpressed in most solid tumours where it supports invasion through control of F-actin structures at the periphery and nuclear envelope. Recently, fascin has been identified in the nucleus of a broad range of cell types but the contributions of nuclear fascin to cancer cell behaviour remain unknown. Here, we demonstrate that fascin bundles F-actin within the nucleus to support chromatin organisation and efficient DDR. Fascin associates directly with phosphorylated Histone H3 leading to regulated levels of nuclear fascin to support these phenotypes. Forcing nuclear fascin accumulation through the expression of nuclear-targeted fascin-specific nanobodies or inhibition of Histone H3 kinases results in enhanced and sustained nuclear F-actin bundling leading to reduced invasion, viability, and nuclear fascin-specific/driven apoptosis. These findings represent an additional important route through which fascin can support tumourigenesis and provide insight into potential pathways for targeted fascin-dependent cancer cell killing.
Article activity feed
-
-
Evaluation Summary:
This work explores a topic of high interest to cell and cancer biologists - the role of actin polymerization, and here specifically the role of fascin, in the nucleus. The authors show that fascin regulates nuclear actin, chromatin organization, response to DNA damage, and demonstrate the need for control of steady-state nuclear levels to avoid cell death. Studying nuclear actin is technically challenging, and the authors deploy some novel technologies towards this goal. There are some very elegant experiments in this paper that suggest fascin has an important role in regulating nuclear actin and other important aspects of cancer cell behaviour. The work could be enhanced by the authors considering adding some additional experiments and providing clarifications and some further details or discussion.
(This preprint …
Evaluation Summary:
This work explores a topic of high interest to cell and cancer biologists - the role of actin polymerization, and here specifically the role of fascin, in the nucleus. The authors show that fascin regulates nuclear actin, chromatin organization, response to DNA damage, and demonstrate the need for control of steady-state nuclear levels to avoid cell death. Studying nuclear actin is technically challenging, and the authors deploy some novel technologies towards this goal. There are some very elegant experiments in this paper that suggest fascin has an important role in regulating nuclear actin and other important aspects of cancer cell behaviour. The work could be enhanced by the authors considering adding some additional experiments and providing clarifications and some further details or discussion.
(This preprint has been reviewed by eLife. We include the joint public review from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. All three Reviewers agreed to share their names with the authors.)
-
Joint Public Review:
While the presence of fascin in the nucleus, and its function at the cytoplasmic side of the nuclear envelope, have been shown previously, the role of fascin in the nucleus is not known. This important new study reveals that nuclear fascin regulates nuclear actin, likely actin bundling, DNA damage response, and too much nuclear fascin promotes apoptosis. The authors begin by using biochemical fractionation and imaging (a strength of this group) to show that fascin can localise to the nucleus of two human cancer cell lines. Mutation of a putative nuclear export sequence in fascin, or treatment with an exportin-1 inhibitor, results in nuclear accumulation of fascin, demonstrating that it shuttles between the cytoplasm and the nucleus. Imaging experiments clearly show the colocalisation of fascin with tagged nuclear …
Joint Public Review:
While the presence of fascin in the nucleus, and its function at the cytoplasmic side of the nuclear envelope, have been shown previously, the role of fascin in the nucleus is not known. This important new study reveals that nuclear fascin regulates nuclear actin, likely actin bundling, DNA damage response, and too much nuclear fascin promotes apoptosis. The authors begin by using biochemical fractionation and imaging (a strength of this group) to show that fascin can localise to the nucleus of two human cancer cell lines. Mutation of a putative nuclear export sequence in fascin, or treatment with an exportin-1 inhibitor, results in nuclear accumulation of fascin, demonstrating that it shuttles between the cytoplasm and the nucleus. Imaging experiments clearly show the colocalisation of fascin with tagged nuclear actin; in combination with fascin-knockdown cells and expression of a non-bundling fascin mutant, this implies a requirement of fascin for nuclear actin bundling.
To explore the molecular complexes that may be regulating nuclear fascin function, the authors examined potential nuclear fascin-interacting proteins using mass spectrometry (MS)-based affinity proteomics. A smart approach exploited GFP-tagged fascin-specific nanobodies that contained nuclear localisation or nuclear export signals, which targeted fascin to the nucleus or cytoplasm, respectively. Proteomic analysis identified histones H3 and H4 as hits enriched in nuclear fascin nanobody pull-downs over non-nuclear fascin nanobody pull-downs. There are some deficiencies in the reporting of the MS data that would benefit from expansion to ensure the results of these experiments are clear, such as hit selection threshold criteria and any statistical analyses used. The potential interaction of fascin with histone H3 was suggested further using FRET between GFP-tagged histone H3 and mCherry-tagged nuclear fascin nanobody, although additional controls would improve interpretation of these data. While they are clearly present in the same complex, the imaging and FRET experiments stop short of showing the interaction is direct. While the use of FRET can be a very powerful means to show interaction, the authors require further controls, for example, a negative control would be important.
The authors identified reduced focal staining of the DNA damage response factor γH2AX in the first hour after DNA damage induction in fascin-knockdown cells. The role of fascin in the DDR is interesting, but the way the images are presented/analysed - the data are not as convincing as they might be. The differences look quite subtle due to relatively large variance and/or heterogeneity. Chromatin compaction was then tested using histone H2B-H2B FRET. Some statistical tests need to be clarified to ensure that comparisons between groups were tested appropriately, particularly for the interpretation of the chromatin compaction results upon the addition of DNA damaging agents to fascin-knockdown cells. Perhaps for discussion, but what role do the authors propose for fascin in chromatin organisation?
Driving fascin to the nucleus using the nuclear-targeted fascin nanobody resulted in substantially reduced filopodia formation, 2D migration speed, and invasion into 3D collagen gel. The alignment of representative confocal z-stacks in the presentation of the invasion assay (nuclear nanobody and fascin-knockdown cells compared to the other conditions) should be clarified. Longer-term nuclear targeting of fascin with the nanobody induced cell cycle arrest and caspase-3 cleavage, implicating nuclear fascin dynamics in loss of cancer cell viability. The phenotypic screening was well performed, including a dose-response analysis of hits and a secondary screen, to identify compounds that could induce nuclear localisation of fascin and promote apoptosis. Very useful supplementary tables have dose-response curves built in to enable interrogation of the screening datasets. The screening identified three compounds that regulate histone phosphorylation; interestingly, two of the compounds reduced histone phosphorylation and reduced histone pulldown in nuclear fascin nanobody affinity purifications in the cancer cells tested. The most potent histone H3 phosphorylation inhibitor also increased γH2AX staining, which appeared to correlate with fascin localisation in the nucleus. Can the authors make, or comment on, further evidence that Haspin-induced effects, for example, increased γH2X (was this at DNA-damage-associated foci in the nucleus?), are due to nuclear localization of fascin and/or resultant F-actin polymerization? Some follow-up data on Haspin could help to enhance the impact of the final part of the paper.
Although further delineation of the role of phospho-histone H3 in modulating nuclear fascin function would help to corroborate the ideas derived from the final figure of the paper, particularly to distinguish correlation from causation, this study demonstrates that nuclear fascin associates with histone H3, promotes nuclear actin, likely bundling, promotes DNA damage response and can induce apoptosis in cancer cell lines. The subcellular localisation of fascin, and its dynamic nuclear localisation, therefore appear important for regulating cancer cell behaviour. The idea that previously described nuclear envelope-localised fascin could serve as a pool of fascin for rapid nuclear import in response to cellular stress, discussed here, is very interesting. Given that fascin is upregulated in many solid tumours, questions around whether the spatiotemporal dynamics of fascin can inform prognostic assessments or can be targeted/modulated therapeutically in tumours will be exciting to discuss or address later. Overall, the quantitative characterisation of nuclear fascin functions will be of interest to cancer cell biologists, particularly those curious about the regulation of nuclear actin and its role in controlling cell behaviour.
-
