Final Report Summary - COP1-DRP1 (Exploring the role of mitochondrial dynamics in tumor regulation by Cop1)
Drp1 affects cell proliferation and cell cycle
Loss of cop1 in p53 deficient Mouse Embryonic Fibroblasts (MEFs) leads to increased proliferation rates. Preliminary data had demonstrated that Drp1 levels and subsequently, mitochondrial morphology were altered in p53, cop1 double mutant cells. In order to provide mechanistic insights, we have designed experiments to evaluate the impact of Drp1 inactivation on cell growth. To do that we have infected double mutant MEFs with retroviral constructs expressing Drp1 and its dominant negative isoform Drp1K38A. Selected transductant have been tested for enhanced Drp1 expression and afterwards analysed for their proliferative capacity.
We first performed a proliferation curve by monitoring cell numbers over time. Wild type cells, constitutively overexpressing Drp1, grew in number significantly more rapidly than control. Cop1 deficient cells instead did not change their proliferative regime in response to higher levels of Drp1. Interestingly loss of Drp1 activity, as prompted by ectopic expression of the mutant Drp1K38A isoform, strongly compromised cell growth with a more marked effect on wild type cells.
To further analyse their growth capacity we tested MEFs seeding efficiency in low density (colony assay). In this experiment an increasing number of cells is plated at low-density measuring, after some time, the amount and the morphology of colonies derived from a single cell. As for the proliferation curve, forced expression of Drp1 did improve seeding efficiency in low density only in WT cells. Fibroblasts expressing Drp1 dominant negative isoform (Drp1K38A) instead, grew in fewer colonies, smaller in size.
The impact on proliferation scored by ectopic expression of WT and Drp1 mutant isoforms could derive from a specific effect on cell cycle progression of the infected cells. We therefore analysed cell cycle phases by Fluorescence-Activated Cell Sorting (FACS), after marking the DNA content with Propidium Iodide (P.I.). Surprisingly Cop1 loss did not affect the fraction of cells in S-Phase as compared to WT controls. Moreover enhanced Drp1 expression did not change the relative proportion of cell cycle phases to both cop1 mutant and WT cells, as compared to empty vector infected cells. Nevertheless, forced expression of the dominant negative Drp1 isoform (Drp1K38A) dramatically increased the G2/M-Phase fraction in WT cells, having no significant effect on mutant cells, in line with the strong effect observed in the proliferation curve.
Since our previous data demonstrated that Cop1 loss of function alters mitochondrial morphology, we decided to determine the impact of Drp1 loss of activity in the infected cells, by staining for the mitochondrial importing factor Tom20. While Drp1 overexpression did not show any measurable effect, significantly, Drp1K38A increased the fraction of cells with normally elongated, not clustered, organelle morphology.
All together these data suggest that Drp1 is a main driver of proliferation regulating cell cycle progression. Moreover, we provided evidence that the increased capacity of cop1 mutant cells to grow in numbers requires Drp1 activity. In addition our data indicate that the acquired proliferative capacity of cop1 mutant cells is sustained by Drp1 alteration of mitochondrial morphology.
We provided evidence of Drp1 regulation of proliferation and cell cycle progression in MEFs, inhibiting its activity using a dominant negative isoform. To further prove our hypothesis we decided to specifically abolish Drp1 expression by knockdown experiments. We have therefore generated MEFs cell lines carrying constitutive (G0; G9; G4) or doxycycline inducible (Tz1) expression of specific short-hairpin RNAs (shRNAs) targeting Drp1, by lentiviral infection. Both lines reported the expression of the short hairpin coupling GFP (constitutive) or RFP (inducible) expression with the shRNAs. In both cases we have obtained cell lines carrying sensibly efficient down regulation of Drp1 steady state levels. Analysis of the biological behaviour of these MEFs is now on-going.
Regulation and analysis of Drp1 protein levels in Cop1 mutant background
With the aim to confirm the correlation between Drp1 levels and Cop1 loss, we measured the amount of Drp1 protein in independently isolated primary MEFs, during steady state conditions or after treatment with the proteasome inhibitor MG132.
We could observe, in six different cell lines (p53-/-; cop1h/h), that Drp1 protein levels were systematically affected in double mutant cells compared to single mutant p53-/- MEFs; in addition, we also noticed an apparent shift in its characteristic doublet-like western blot band pattern. Drp1 antibody recognizes specifically two bands, migrating around 80 kDa, the double mutant cells reproducibly showed a decrement in the relative ratio between the lower band and the higher band. Interestingly, upon MG132 treatment the ratio was instead close to 1 and no differences were detected between p53-/- and double mutant cells.
Since we have always analysed cells, immortalized via p53 loss, we asked whether the absence of p53 could play a role in the phenotype observed. Primary WT and Cop1 mutant cells were therefore isolated and protein extracted at early passages, before the on set of proliferative senescence. Surprisingly Drp1 protein levels and its western migration pattern were not affected by the absence of Cop1 in non-immortalized p53 WT cells, indicating that loss of p53 is required for the onset of the described phenotype.
Ectopic expression of the constitutively active h-RasV12G point mutant isoform of h-Ras in immortalized MEFs leads to in vitro transformation. These cells change their morphology and acquire tumour behaviour, as such as growing under the skin of nude mice or independence from substrate. To evaluate the impact of transformation on Drp1 we analysed the protein levels and band pattern in p53-/- and double mutant MEFs, in vitro transformed by h-RasV12G ectopic expression. Interestingly both tumour cell lines showed an apparent redistribution of Drp1 band pattern, as seen in immortalized cop1 mutant cells non-transformed although Drp1 protein levels where still sensibly higher in absence of Cop1.
All together, these observations suggest that altered Drp1 levels and activity may play a role in the process of tumour formation in vitro, since the more the cells acquire a transformed phenotype the more Drp1 levels are affected and concomitantly, its western blot band pattern is altered.
Loss of cop1 in immortalized MEFs does not affect Drp1 levels
To further measure the effect of Drp1 on the process of in vitro tumour formation we used Drp1f/f Mefs, immortalized by large T antigen constitutive over-expression. First of all we could show that we could efficiently delete the floxed allele therefore abolishing Drp1 expression. Afterwards, in this genetic background, we measure the effect, upon loss of cop1, on Drp1 protein levels and band pattern.
Immortalized Drp1f/f cells have been treated with three specific small interference RNAs (siRNAs) for Cop1 and with a scrambled siRNA coupled with a fluorescent dye (siRNA-Glo). Surprisingly, although we managed to reach very high levels of knock-down efficiency, no differences in Drp1 levels or band-pattern were observed.
We speculate that the process of immortalization may have provided a strong impact on the overall regulatory network of this specific cell line. We therefore decided that it was not a suitable reagent for further experiments.
Cop1 overexpression in 293T cell does not affect Drp1 levels
After we have established that Cop1 deficiency alters Drp1 levels we decided to test whether Cop1 forced over-expression could destabilize Drp1 steady state protein. To do so, we have transfected 293 cells with increasing amounts of a plasmid expressing a myc-tagged Cop1 isoform. These conditions did not affect Drp1 levels or Drp1 band-pattern. Since also c-Jun protein levels (an established Cop1 target protein) were also unaffected in this context, we decided to discard these experimental conditions to answer our question.
Generation of a suitable mouse line for in vivo tumour formation analysis
Cop1 mutant mice develop spontaneously T-cell lymphoma, whose latency and incidence is markedly implemented by whole body -irradiation treatment (4Gy). We therefore considered testing the in vivo relevance of Drp1 in tumour formation in this mouse model. With this aim we generated cop1 mutant mice deficient for drp1 in the thymus, combining the cop1 mutation with the drp1 floxed allele and a cre transgenic line whose expression is restricted to thymocites (lckCre).
Unfortunately we had faced some problems to achieve this genetic combination. The lckCre allele is a low frequency germ line deleter, provoking a marked delay obtaining the necessary experimental genotypes, since Drp1 full mutant mice are lethal. Moreover cop1 mutant mice, that had already in pure C57/Bl6 background strain a marked lethality (30% of penetrance) during embryonic development, showed an increment of this effect, when the genetic background was mixed during the breeding required to obtain the experimental genotypes, producing very low numbers of mice suitable for the experiments.
Nevertheless we obtained a cohort of mice that we will later on analyse for tumour formation.
Future perspectives
In the next phase of the project, we aim to clarify and further analyse the role of Drp1 in the process of in vivo and in vitro transformation.
Since p53 mutant cells seem to be necessary to unravel the impact of Drp1 in cell proliferation and transformation, we will isolate MEFs deficient for Cop1 and for the p53 regulator p19Arf. In this genetic background we will be able to confirm our previous data in a different and independent experimental setting and the analysed cell lines will not be affected by the intrinsic mutagenic background determined by the complete abolishment of the p53 DNA damage surveillance pathway.
The obtainment of these cells will also provide an experimental system suitable to evaluate the impact of Drp1 loss during in vitro transformation following RasV12 ectopic expression.
Because of the unexpected difficulties to evaluate Drp1 role in the context of Cop1 deficiency, we aim to obtain and analyse for spontaneous or -irradiation induced tumour formation, p19Arf knock-out mice deficient for Drp1 in the thymus.
In both in vivo and in vitro cases, we will evaluate the impact of Drp1 on mitochondrial function both considering apoptosis, ROS formation and respiration activity.
Loss of cop1 in p53 deficient Mouse Embryonic Fibroblasts (MEFs) leads to increased proliferation rates. Preliminary data had demonstrated that Drp1 levels and subsequently, mitochondrial morphology were altered in p53, cop1 double mutant cells. In order to provide mechanistic insights, we have designed experiments to evaluate the impact of Drp1 inactivation on cell growth. To do that we have infected double mutant MEFs with retroviral constructs expressing Drp1 and its dominant negative isoform Drp1K38A. Selected transductant have been tested for enhanced Drp1 expression and afterwards analysed for their proliferative capacity.
We first performed a proliferation curve by monitoring cell numbers over time. Wild type cells, constitutively overexpressing Drp1, grew in number significantly more rapidly than control. Cop1 deficient cells instead did not change their proliferative regime in response to higher levels of Drp1. Interestingly loss of Drp1 activity, as prompted by ectopic expression of the mutant Drp1K38A isoform, strongly compromised cell growth with a more marked effect on wild type cells.
To further analyse their growth capacity we tested MEFs seeding efficiency in low density (colony assay). In this experiment an increasing number of cells is plated at low-density measuring, after some time, the amount and the morphology of colonies derived from a single cell. As for the proliferation curve, forced expression of Drp1 did improve seeding efficiency in low density only in WT cells. Fibroblasts expressing Drp1 dominant negative isoform (Drp1K38A) instead, grew in fewer colonies, smaller in size.
The impact on proliferation scored by ectopic expression of WT and Drp1 mutant isoforms could derive from a specific effect on cell cycle progression of the infected cells. We therefore analysed cell cycle phases by Fluorescence-Activated Cell Sorting (FACS), after marking the DNA content with Propidium Iodide (P.I.). Surprisingly Cop1 loss did not affect the fraction of cells in S-Phase as compared to WT controls. Moreover enhanced Drp1 expression did not change the relative proportion of cell cycle phases to both cop1 mutant and WT cells, as compared to empty vector infected cells. Nevertheless, forced expression of the dominant negative Drp1 isoform (Drp1K38A) dramatically increased the G2/M-Phase fraction in WT cells, having no significant effect on mutant cells, in line with the strong effect observed in the proliferation curve.
Since our previous data demonstrated that Cop1 loss of function alters mitochondrial morphology, we decided to determine the impact of Drp1 loss of activity in the infected cells, by staining for the mitochondrial importing factor Tom20. While Drp1 overexpression did not show any measurable effect, significantly, Drp1K38A increased the fraction of cells with normally elongated, not clustered, organelle morphology.
All together these data suggest that Drp1 is a main driver of proliferation regulating cell cycle progression. Moreover, we provided evidence that the increased capacity of cop1 mutant cells to grow in numbers requires Drp1 activity. In addition our data indicate that the acquired proliferative capacity of cop1 mutant cells is sustained by Drp1 alteration of mitochondrial morphology.
We provided evidence of Drp1 regulation of proliferation and cell cycle progression in MEFs, inhibiting its activity using a dominant negative isoform. To further prove our hypothesis we decided to specifically abolish Drp1 expression by knockdown experiments. We have therefore generated MEFs cell lines carrying constitutive (G0; G9; G4) or doxycycline inducible (Tz1) expression of specific short-hairpin RNAs (shRNAs) targeting Drp1, by lentiviral infection. Both lines reported the expression of the short hairpin coupling GFP (constitutive) or RFP (inducible) expression with the shRNAs. In both cases we have obtained cell lines carrying sensibly efficient down regulation of Drp1 steady state levels. Analysis of the biological behaviour of these MEFs is now on-going.
Regulation and analysis of Drp1 protein levels in Cop1 mutant background
With the aim to confirm the correlation between Drp1 levels and Cop1 loss, we measured the amount of Drp1 protein in independently isolated primary MEFs, during steady state conditions or after treatment with the proteasome inhibitor MG132.
We could observe, in six different cell lines (p53-/-; cop1h/h), that Drp1 protein levels were systematically affected in double mutant cells compared to single mutant p53-/- MEFs; in addition, we also noticed an apparent shift in its characteristic doublet-like western blot band pattern. Drp1 antibody recognizes specifically two bands, migrating around 80 kDa, the double mutant cells reproducibly showed a decrement in the relative ratio between the lower band and the higher band. Interestingly, upon MG132 treatment the ratio was instead close to 1 and no differences were detected between p53-/- and double mutant cells.
Since we have always analysed cells, immortalized via p53 loss, we asked whether the absence of p53 could play a role in the phenotype observed. Primary WT and Cop1 mutant cells were therefore isolated and protein extracted at early passages, before the on set of proliferative senescence. Surprisingly Drp1 protein levels and its western migration pattern were not affected by the absence of Cop1 in non-immortalized p53 WT cells, indicating that loss of p53 is required for the onset of the described phenotype.
Ectopic expression of the constitutively active h-RasV12G point mutant isoform of h-Ras in immortalized MEFs leads to in vitro transformation. These cells change their morphology and acquire tumour behaviour, as such as growing under the skin of nude mice or independence from substrate. To evaluate the impact of transformation on Drp1 we analysed the protein levels and band pattern in p53-/- and double mutant MEFs, in vitro transformed by h-RasV12G ectopic expression. Interestingly both tumour cell lines showed an apparent redistribution of Drp1 band pattern, as seen in immortalized cop1 mutant cells non-transformed although Drp1 protein levels where still sensibly higher in absence of Cop1.
All together, these observations suggest that altered Drp1 levels and activity may play a role in the process of tumour formation in vitro, since the more the cells acquire a transformed phenotype the more Drp1 levels are affected and concomitantly, its western blot band pattern is altered.
Loss of cop1 in immortalized MEFs does not affect Drp1 levels
To further measure the effect of Drp1 on the process of in vitro tumour formation we used Drp1f/f Mefs, immortalized by large T antigen constitutive over-expression. First of all we could show that we could efficiently delete the floxed allele therefore abolishing Drp1 expression. Afterwards, in this genetic background, we measure the effect, upon loss of cop1, on Drp1 protein levels and band pattern.
Immortalized Drp1f/f cells have been treated with three specific small interference RNAs (siRNAs) for Cop1 and with a scrambled siRNA coupled with a fluorescent dye (siRNA-Glo). Surprisingly, although we managed to reach very high levels of knock-down efficiency, no differences in Drp1 levels or band-pattern were observed.
We speculate that the process of immortalization may have provided a strong impact on the overall regulatory network of this specific cell line. We therefore decided that it was not a suitable reagent for further experiments.
Cop1 overexpression in 293T cell does not affect Drp1 levels
After we have established that Cop1 deficiency alters Drp1 levels we decided to test whether Cop1 forced over-expression could destabilize Drp1 steady state protein. To do so, we have transfected 293 cells with increasing amounts of a plasmid expressing a myc-tagged Cop1 isoform. These conditions did not affect Drp1 levels or Drp1 band-pattern. Since also c-Jun protein levels (an established Cop1 target protein) were also unaffected in this context, we decided to discard these experimental conditions to answer our question.
Generation of a suitable mouse line for in vivo tumour formation analysis
Cop1 mutant mice develop spontaneously T-cell lymphoma, whose latency and incidence is markedly implemented by whole body -irradiation treatment (4Gy). We therefore considered testing the in vivo relevance of Drp1 in tumour formation in this mouse model. With this aim we generated cop1 mutant mice deficient for drp1 in the thymus, combining the cop1 mutation with the drp1 floxed allele and a cre transgenic line whose expression is restricted to thymocites (lckCre).
Unfortunately we had faced some problems to achieve this genetic combination. The lckCre allele is a low frequency germ line deleter, provoking a marked delay obtaining the necessary experimental genotypes, since Drp1 full mutant mice are lethal. Moreover cop1 mutant mice, that had already in pure C57/Bl6 background strain a marked lethality (30% of penetrance) during embryonic development, showed an increment of this effect, when the genetic background was mixed during the breeding required to obtain the experimental genotypes, producing very low numbers of mice suitable for the experiments.
Nevertheless we obtained a cohort of mice that we will later on analyse for tumour formation.
Future perspectives
In the next phase of the project, we aim to clarify and further analyse the role of Drp1 in the process of in vivo and in vitro transformation.
Since p53 mutant cells seem to be necessary to unravel the impact of Drp1 in cell proliferation and transformation, we will isolate MEFs deficient for Cop1 and for the p53 regulator p19Arf. In this genetic background we will be able to confirm our previous data in a different and independent experimental setting and the analysed cell lines will not be affected by the intrinsic mutagenic background determined by the complete abolishment of the p53 DNA damage surveillance pathway.
The obtainment of these cells will also provide an experimental system suitable to evaluate the impact of Drp1 loss during in vitro transformation following RasV12 ectopic expression.
Because of the unexpected difficulties to evaluate Drp1 role in the context of Cop1 deficiency, we aim to obtain and analyse for spontaneous or -irradiation induced tumour formation, p19Arf knock-out mice deficient for Drp1 in the thymus.
In both in vivo and in vitro cases, we will evaluate the impact of Drp1 on mitochondrial function both considering apoptosis, ROS formation and respiration activity.