Fine-tuned repression of Drp1-driven mitochondrial fission primes a ‘stem/progenitor-like state’ to support neoplastic transformation

  1. Brian Spurlock
  2. Danitra Parker
  3. Malay Kumar Basu
  4. Anita Hjelmeland
  5. Sajina GC
  6. Shanrun Liu
  7. Gene P Siegal
  8. Alan Gunter
  9. Aida Moran
  10. Kasturi Mitra

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    This paper is of interest for cell biologists studying mitochondrial fission as well as stem cell biologists studying neoplastic transformation. The work helps to clarify how variable levels of the master regulator of mitochondrial fission can have substantially different effects on gene regulation and mitochondrial network properties. A combination of complementary methods is used to support the key findings although aspects of data analysis and quantification could be improved.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Gene knockout of the master regulator of mitochondrial fission, Drp1, prevents neoplastic transformation. Also, mitochondrial fission and its opposing process of mitochondrial fusion are emerging as crucial regulators of stemness. Intriguingly, stem/progenitor cells maintaining repressed mitochondrial fission are primed for self-renewal and proliferation. Using our newly derived carcinogen transformed human cell model, we demonstrate that fine-tuned Drp1 repression primes a slow cycling ‘stem/progenitor-like state’, which is characterized by small networks of fused mitochondria and a gene-expression profile with elevated functional stem/progenitor markers (Krt15, Sox2 etc) and their regulators (Cyclin E). Fine tuning Drp1 protein by reducing its activating phosphorylation sustains the neoplastic stem/progenitor cell markers. Whereas, fine-tuned reduction of Drp1 protein maintains the characteristic mitochondrial shape and gene-expression of the primed ‘stem/progenitor-like state’ to accelerate neoplastic transformation, and more complete reduction of Drp1 protein prevents it. Therefore, our data highlights a ‘goldilocks’ level of Drp1 repression supporting stem/progenitor state dependent neoplastic transformation.

Article activity feed

  1. Version published to 10.7554/elife.68394 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    In their paper, Spurlock and colleagues look at the role of mitochondria fusion caused by Drp1 repression in driving the stem/progenitor-like state of skin stem cells. Prior work hinted at the possibility that mitochondrial fission/fusion activity is important in supporting neoplastic transformation, but it was unclear exactly what this role was. Here, the authors use an assay for neoplastic transformation induced by carcinogen treatment to demonstrate that diminution in mitochondrial fission activity (from increased phosphorylated Drp1 pools) can prime a stem/progenitor-like state in carcinogen-treated cells, leading to accelerated neoplastic transformation. Using genetic strategies and single cell RNAseq they additionally show that only partial repression of Drp1 is necessary for establishing the stem/progenitor-like state for driving neoplastic transformation, with too much or too little Drp1 repression having no effect. The data are therefore relevant for understanding the conditions for driving neoplastic transformation. Overall the results support the conclusions drawn by the authors and the work helps to clarify the mitochondria's role in neoplastic transformation. The paper is currrently overall difficult and in places confusing to read.

    We thank the Reviewer finding value in our manuscript and providing constructive comments to improve the quality of our manuscript. We have provided clarifications to all the comments and provided new experimental data to address concerns. We have also substantially improved the readability of our manuscript. The inclusion of new experiments and revisions provided has only strengthened the main conclusion of manuscript about fine tuning of Drp1 repression facilitating neoplastic transformation by enriching a stem cell state.

    Reviewer #2 (Public Review):

    The authors used a carcinogen to increase proliferation of the keratinocyte cell line HaCaT and to increase the capacity to form xenograft tumors in mice. They found that the levels of certain mitochondrial fission and fusion proteins (Drp1, Mfn1 and Opa1) were increased in the derived cell lines, but Fis1 levels was decreased in the most tumorigenic derivative as was the phosphorylation of Drp1 at position 616. Through single cell expression analysis, the author show that transformed cells have retained a subpopulation of slowly dividing cells with high expression of stem cell markers and reduced levels and phosphorylation of Drp1. This state could be mimicked by reducing Drp1 expression with shRNA. Cells with moderately reduced levels of Drp1 appeared to be more susceptible to enhanced proliferation caused by treatment with a carcinogen. The authors conclude that a moderate reduction in Drp1 levels causes an increase in proliferation and tumorigenesis of keratinocytes upon treatment with a carcinogen.

    The main strength of this paper is the use of single-cell analysis to identify a subpopulation of cells with increased stem cell gene expression and reduced levels of Drp1 and of Drp1 phosphorylation.

    A causal relation between tumorigenicity and Drp1 levels was tested by reducing levels of Drp1 with shRNA, but unfortunately, the data are very limited. The key contention that partial reduction in Drp1 levels increases proliferation is only supported by a single point and it contradicts results from other labs where it was shown that Drp1 phosphorylation and fission are increased with transformation.

    We thank the reviewer for this comment. Now, we provide 2 more lines of evidence in support of the main conclusion that a slow cycling ‘stem/progenitor-like [CyclinEhi-Sox2hi-Krt15hi] state’ is sustained by a fine-tuned “goldilocks” level of Drp1 activity that maintains small networks of fused mitochondria. The stem/progenitor state driven neoplastic transformation is supported by fine-tuned Drp1 repression maintained by reduced Drp1 protein levels, while the neoplastic stem/progenitor state is supported by fine-tuning Drp1 by reducing its S-616 phosphorylation that modulates mitochondrial potential. In the light of the new data, we have modified the title to: Fine-tuned repression of Drp1 driven mitochondrial fission primes a ‘stem/progenitor-like state’ to support neoplastic transformation. These new results are as follows:

    1. Now we show that lowering the knockdown efficacy for both the Drp1 shRNAs reduces abundance of cells with >80 Fusion1 metric (Figure 4-figure supplement 1C and its legend, Lines: 440-445), increases abundance of self-renewing cells and accelerates neoplastic transformation in Parental cells (Figure 4F, G and their legends, Lines: 402-409). Plotting the accelerated transformation efficacy with Drp1 protein levels remaining after knockdown predicts ~50% repression of Drp1 protein levels may maximally accelerate transformation within the experimental range (Figure 4-figure supplement 1A and its legend, Lines: 415-419) (such remnant Drp1 levels may remain overestimated due to the reduction of the Actin control with Drp1 knockdown, Figure 4A,E).

    2. Now we provide multiple analyses of the impact of overexpression of Drp1-wild type and the phospho-deficient Drp1-S616A mutant (Lines: 329-356). Our data suggest that elevated Sox2hi/Krt15hi sub-population is maintained by reducing Drp1-S616 phosphorylation of the elevated Drp1 protein levels in the TF-1 population, but not in the Parental (Figure 3I). We also confirmed Drp1-WT overexpression increases the [Fission] metric and reduces [Fusion] metrics, while the Drp1-S616A mutant remains attenuated in this ability in the Parental cells (Figure 3-figure supplement Figure 1C). But paradoxically in the TF-1 population, Drp1-WT overexpression enhances [Fusion] metrics that is not observed with the Drp1-S616A mutant, while not impacting the [Fission] metric (Figure 3-figure supplement 1C). We discuss this paradox in the light of the report that overexpression of certain Drp1 activators maintains mitochondrial fusion by unnaturally sequestering Drp1. Nonetheless, this data is consistent with our findings across various cell populations that moderate attenuation of [Fusion] metric happens with fine-tuned repression of Drp1, which supports enhanced Sox2-hi/Krt15-hi subpopulation (Figure 5F). The impact of the Drp1-WT and the Drp1-S616A mutant on TMRE also remains consistent (see Response in point 3), while that of on Cyclin E remains to be explored further (Figure 3-figure supplement 1D).

    It is unclear what mechanisms connect the proposed window of Drp1 activity to tumorigenesis. In previous studies the effects of different levels of fission and fusion proteins on metabolism and tumorigenesis were analyzed in detail, showing effects on metabolism that could lead to increased tumorigenesis. That is not done here and so one is left guessing as to what functions are affected by the proposed window of Drp1 expression and how that might affect tumorigenesis.

    We thank the Reviewer highlighting the strength of the manuscript and providing critical and constructive comments to improve the quality of our manuscript. We have provided clarifications to all the comments and provided new experimental data to address concerns.

    Reviewer #3 (Public Review):

    Spurlock et al. investigated how differential repression of Drp1, a master regulator of mitochondrial fission, affect neoplastic transformation of keratinocytes as well as key aspects of gene regulation and mitochondrial network dynamics. They find that "weak" repression of Drp1 in keratinocytes results in a gene expression profile reminiscent of a stem/progenitor like state, which is especially primed for neoplastic transformation. On the other hand, they show that "strong" repression of Drp1 has a very different effect and results in cells with hyperfused mitochondrial networks and less propensity towards transformation. They find that "weak" repression of Drp1 leads not to hyperfused networks but rather to small networks of fused mitochondria. These results are especially surprising as according to the authors analysis, there is less than 20% difference in the level of knockdown efficiency under the "weak" vs "strong" shRNA conditions. But the key findings in the weak vs strong knockdown conditions seem to be well supported by RNASeq analysis, mitochondrial network analysis, and immunofluorescence data (although quantification of specific data would likely strengthen their arguments).

    The authors relate these findings to those where they use differing levels of TCDD (1 nM vs 10nM) to transform HaCaT cells. While it is clear from the data that TF-1 has a different effect from TF-10 on gene expression, cell proliferation, and certain measures of stem/progenitor cell characteristics, the key findings concerning Drp1 levels that would directly relate TF-1/TF-10 to Drp1-shRNA weak/strong are not as well supported. In particular, the immunoblots of pDrp1 and Drp1 levels as well as the mitochondrial network analysis do not necessarily support the hypothesis that the differing characteristics of TF-1 vs parental or TF-10 results from Drp1/mitochondrial changes and not simply due to cell cycle or other effects of TCDD levels. Nevertheless, both sets of data are interesting and compelling and present a more nuanced view of how differing levels of transformation agents or shRNA-mediate depletion can have considerably different effects even within the same cell type. These data may also help to clarify differences seen in past studies between distinct cell types when Drp1 levels are manipulated but this remains to be tested and clarified.

    We thank the Reviewer highlighting the strength of the manuscript and providing constructive comments to improve the quality of our manuscript. We have provided clarifications to all the comments and provided new experimental data to address concerns.

    The individual conclusions of this paper are generally well supported by the data, but some aspects of data analysis need to be clarified and/or quantified.

    1. To better support the main link between the two sets of data, the levels of Drp1 (protein and activity) in TF-1 vs TF-10 conditions must be clarified and quantified (immunoblot analysis and/or in the immunofluorescence). Since the overall levels of Drp1 actually increase in both TF-1 and TF-10 compared to Parental but the authors suggest that pDrp1 decreases in TF-1, this must be quantified. Furthermore, the authors note that Drp1 is phosphorylated in a cell cycle dependent manner and go on to show significant differences in cell cycle dynamics between Parental, TF-1 and TF-10, and so the difference in pDrp1 levels could simply be a result of the cell cycle differences. While this would not change the conclusions about how differing levels of TCDD impact gene expression, transformation efficiency, and stem/progenitor cell like characteristics, it would call into question how related the effects from direct repression of Drp1 levels through shRNA are to the TCDD effects seen.

    We thank the reviewer for this comment. We have now quantitated all the previous and newly added blots (Figure 1D, 3A,D, 4A,E, Figure 1-figure supplements 1B, C). Key findings from immunoblots are consistent with data from single cell RNA-seq, immunofluorescence and microscopy analyses, as clarified in the manuscript (mentioned in relevant Result sections in the manuscript).

    Stemness is largely determined by the cell cycle modulation, while Cyclin E and other cyclins have been shown to sustain stemness. Given Drp1 knockdown modulates Cyclin E1 (Figure 4B,C) and other S phase genes (Figure 5C) (as expected from our previous work and others), we conclude that fine-tuned Drp1 repression modulates cell cycle towards enrichment of the stem cell state and facilitate the neoplastic transformation. Our gene expression data is consistent with the consensus that Drp1-S616 phosphorylation is driven by cell cycle in a CyclinB-CDK1 dependent manner. These data together support the working model that Drp1 gets modulated by certain cell cycle regulators to be able to impact other cell cycle regulators like Cyclin E to enrich a stem cell state supporting neoplastic transformation (Lines: 360-366, 530-534).

    1. There does not seem to be a big difference between the mitochondrial networks of TF-1 and parental line except possibly the spread of the Fusion5 metric. Is this statistically significant? Are any of the other measures of the mitochondrial network found to be different in Drp1-kd (W) similarly changed in TF-1? This could strengthen the connection between these data.

    We thank the reviewer for this comment. In the revised manuscript, we have provided a more thorough analyses supported by statistical test between parental, TF1 and TF-10 cells (Figure 1E and its legend, Lines: 134-165). Bivariate analyses of the [Fission] and [Fusion5] metrics, demonstrates that the TF-1 population, with minimum Drp1 activity, exhibits maximal enrichment of a cellular sub-population with defined mitochondrial [Fission] and [Fusion5] (Figure 1E). This same sub-population is also enriched in the Parental population with weak Drp1 repression, while more complete Drp1 repression expectedly increases the cell population with maximum mitochondrial fusion (hyperfused mitochondria). Therefore, our findings are consistent with our conclusion that TF-1 and Drp1-kd (W) share the uniqueness in the profile of mitochondrial morphology, as well as gene expression (Figure 5F and its legend).

  3. eLife

    Evaluation Summary:

    This paper is of interest for cell biologists studying mitochondrial fission as well as stem cell biologists studying neoplastic transformation. The work helps to clarify how variable levels of the master regulator of mitochondrial fission can have substantially different effects on gene regulation and mitochondrial network properties. A combination of complementary methods is used to support the key findings although aspects of data analysis and quantification could be improved.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In their paper, Spurlock and colleagues look at the role of mitochondria fusion caused by Drp1 repression in driving the stem/progenitor-like state of skin stem cells. Prior work hinted at the possibility that mitochondrial fission/fusion activity is important in supporting neoplastic transformation, but it was unclear exactly what this role was. Here, the authors use an assay for neoplastic transformation induced by carcinogen treatment to demonstrate that diminution in mitochondrial fission activity (from increased phosphorylated Drp1 pools) can prime a stem/progenitor-like state in carcinogen-treated cells, leading to accelerated neoplastic transformation. Using genetic strategies and single cell RNAseq they additionally show that only partial repression of Drp1 is necessary for establishing the stem/progenitor-like state for driving neoplastic transformation, with too much or too little Drp1 repression having no effect. The data are therefore relevant for understanding the conditions for driving neoplastic transformation. Overall the results support the conclusions drawn by the authors and the work helps to clarify the mitochondria's role in neoplastic transformation. The paper is currrently overall difficult and in places confusing to read.

  5. eLife

    Reviewer #2 (Public Review):

    The authors used a carcinogen to increase proliferation of the keratinocyte cell line HaCaT and to increase the capacity to form xenograft tumors in mice. They found that the levels of certain mitochondrial fission and fusion proteins (Drp1, Mfn1 and Opa1) were increased in the derived cell lines, but Fis1 levels was decreased in the most tumorigenic derivative as was the phosphorylation of Drp1 at position 616. Through single cell expression analysis, the author show that transformed cells have retained a subpopulation of slowly dividing cells with high expression of stem cell markers and reduced levels and phosphorylation of Drp1. This state could be mimicked by reducing Drp1 expression with shRNA. Cells with moderately reduced levels of Drp1 appeared to be more susceptible to enhanced proliferation caused by treatment with a carcinogen. The authors conclude that a moderate reduction in Drp1 levels causes an increase in proliferation and tumorigenesis of keratinocytes upon treatment with a carcinogen.

    The main strength of this paper is the use of single-cell analysis to identify a subpopulation of cells with increased stem cell gene expression and reduced levels of Drp1 and of Drp1 phosphorylation.

    A causal relation between tumorigenicity and Drp1 levels was tested by reducing levels of Drp1 with shRNA, but unfortunately, the data are very limited. The key contention that partial reduction in Drp1 levels increases proliferation is only supported by a single point and it contradicts results from other labs where it was shown that Drp1 phosphorylation and fission are increased with transformation.

    It is unclear what mechanisms connect the proposed window of Drp1 activity to tumorigenesis. In previous studies the effects of different levels of fission and fusion proteins on metabolism and tumorigenesis were analyzed in detail, showing effects on metabolism that could lead to increased tumorigenesis. That is not done here and so one is left guessing as to what functions are affected by the proposed window of Drp1 expression and how that might affect tumorigenesis.

  6. eLife

    Reviewer #3 (Public Review):

    Spurlock et al. investigated how differential repression of Drp1, a master regulator of mitochondrial fission, affect neoplastic transformation of keratinocytes as well as key aspects of gene regulation and mitochondrial network dynamics. They find that "weak" repression of Drp1 in keratinocytes results in a gene expression profile reminiscent of a stem/progenitor like state, which is especially primed for neoplastic transformation. On the other hand, they show that "strong" repression of Drp1 has a very different effect and results in cells with hyperfused mitochondrial networks and less propensity towards transformation. They find that "weak" repression of Drp1 leads not to hyperfused networks but rather to small networks of fused mitochondria. These results are especially surprising as according to the authors analysis, there is less than 20% difference in the level of knockdown efficiency under the "weak" vs "strong" shRNA conditions. But the key findings in the weak vs strong knockdown conditions seem to be well supported by RNASeq analysis, mitochondrial network analysis, and immunofluorescence data (although quantification of specific data would likely strengthen their arguments).

    The authors relate these findings to those where they use differing levels of TCDD (1 nM vs 10nM) to transform HaCaT cells. While it is clear from the data that TF-1 has a different effect from TF-10 on gene expression, cell proliferation, and certain measures of stem/progenitor cell characteristics, the key findings concerning Drp1 levels that would directly relate TF-1/TF-10 to Drp1-shRNA weak/strong are not as well supported. In particular, the immunoblots of pDrp1 and Drp1 levels as well as the mitochondrial network analysis do not necessarily support the hypothesis that the differing characteristics of TF-1 vs parental or TF-10 results from Drp1/mitochondrial changes and not simply due to cell cycle or other effects of TCDD levels. Nevertheless, both sets of data are interesting and compelling and present a more nuanced view of how differing levels of transformation agents or shRNA-mediate depletion can have considerably different effects even within the same cell type. These data may also help to clarify differences seen in past studies between distinct cell types when Drp1 levels are manipulated but this remains to be tested and clarified.

    The individual conclusions of this paper are generally well supported by the data, but some aspects of data analysis need to be clarified and/or quantified.

    1. To better support the main link between the two sets of data, the levels of Drp1 (protein and activity) in TF-1 vs TF-10 conditions must be clarified and quantified (immunoblot analysis and/or in the immunofluorescence). Since the overall levels of Drp1 actually increase in both TF-1 and TF-10 compared to Parental but the authors suggest that pDrp1 decreases in TF-1, this must be quantified. Furthermore, the authors note that Drp1 is phosphorylated in a cell cycle dependent manner and go on to show significant differences in cell cycle dynamics between Parental, TF-1 and TF-10, and so the difference in pDrp1 levels could simply be a result of the cell cycle differences. While this would not change the conclusions about how differing levels of TCDD impact gene expression, transformation efficiency, and stem/progenitor cell like characteristics, it would call into question how related the effects from direct repression of Drp1 levels through shRNA are to the TCDD effects seen.

    2. There does not seem to be a big difference between the mitochondrial networks of TF-1 and parental line except possibly the spread of the Fusion5 metric. Is this statistically significant? Are any of the other measures of the mitochondrial network found to be different in Drp1-kd (W) similarly changed in TF-1? This could strengthen the connection between these data.

  7. Version published to 10.1101/2021.03.05.434102v2 on bioRxiv
  8. Version published to 10.1101/2021.03.05.434102v1 on bioRxiv