Uev1A counteracts oncogenic Ras stimuli in both polyploid and diploid cells

Reviewer #1 (Public review):

Summary:

This study uncovers a protective role of the ubiquitin-conjugating enzyme variant Uev1A in mitigating cell death caused by over-expressed oncogenic Ras in polyploid Drosophila nurse cells and by RasK12 in diploid human tumor cell lines. The authors previously showed that overexpression of oncogenic Ras induces death in nurse cells, and now they perform a deficiency screen for modifiers. They identified Uev1A as a suppressor of this Ras-induced cell death. Using genetics and biochemistry, the authors found that Uev1A collaborates with the APC/C E3 ubiquitin ligase complex to promote proteasomal degradation of Cyclin A. This function of Uev1A appears to extend to diploid cells, where its human homologs UBE2V1 and UBE2V2 suppress oncogenic Ras-dependent phenotypes in human colorectal cancer cells in vitro and in xenografts in mice.

Strengths:

(1) Most of the data is supported by a sufficient sample size and appropriate statistics.
(2) Good mix of genetics and biochemistry.
(3) Generation of new transgenes and Drosophila alleles that will be beneficial for the community.

Weaknesses:

(1) Phenotypes are based on artificial overexpression. It is not clear whether these results are relevant to normal physiology.

(2) The phenotype of "degenerating ovaries" is very broad, and the study is not focused on phenotypes at the cellular level. Furthermore, no information is provided in the Materials and Methods on how degenerating ovaries are scored, despite this being the most important assay in the study.

(3) In Figure 5, the authors want to conclude that uev1a is a tumor-suppressor, and so they over-express ubev1/2 in human cancer cell lines that have RasK12 and find reduced proliferation, colony formation, and xenograft size. However, genes that act as tumor suppressors have loss-of-function phenotypes that allow for increased cell division. The Drosophila uev1a mutant is viable and fertile, suggesting that it is not a tumor suppressor in flies. Additionally, they do not deplete human ubev1/2 from human cancer cell lines and assess whether this increases cell division, colony formation, and xenograph growth.

(4) A critical part of the model does not make sense. CycA is a key part of their model, but they do not show CycA protein expression in WT egg chambers or in their over-expression models (nos.RasV12 or bam>RasV12). Based on Lilly and Spradling 1996, Cyclin A is not expressed in germ cells in region 2-3 of the germarium; whether CycA is expressed in nurse cells in later egg chambers is not shown but is critical to document comprehensively.

(5) The authors should provide more information about the knowledge base of uev1a and its homologs in the introduction.

Reviewer #2 (Public review):

Summary:

The authors performed a genetic screen using deficiency lines and identified Uev1a as a factor that protects nurse cells from RasG12V-induced cell death. According to a previous study from the same lab, this cell death is caused by aberrant mitotic stress due to CycA upregulation (Zhang et al.). This paper further reveals that Uev1a forms a complex with APC/C to promote proteasome-mediated degradation of CycA.

In addition to polyploid nurse cells, the authors also examined the effect of RasG12V-overexpression in diploid germline cells, where RasG12V-overexpression triggers active proliferation, not cell death. Uev1a was found to suppress its overgrowth as well.

Finally, the authors show that the overexpression of the human homologs, UBE2V1 and UBE2V2, suppresses tumor growth in human colorectal cancer xenografts and cell lines. Notably, the expression of these genes correlates with the survival of colorectal cancer patients carrying the Ras mutation.

Strength:

This paper presents a significant finding that UBE2V1/2 may serve as a potential therapy for cancers harboring Ras mutations. The authors propose a fascinating mechanism in which Uev1a forms a complex with APC/C to inhibit aberrant cell cycle progression.

Weakness:

The quantification of some crucial experiments lacks sufficient clarity.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study uncovers a protective role of the ubiquitin-conjugating enzyme variant Uev1A in mitigating cell death caused by over-expressed oncogenic Ras in polyploid Drosophila nurse cells and by RasK12 in diploid human tumor cell lines. The authors previously showed that overexpression of oncogenic Ras induces death in nurse cells, and now they perform a deficiency screen for modifiers. They identified Uev1A as a suppressor of this Ras-induced cell death. Using genetics and biochemistry, the authors found that Uev1A collaborates with the APC/C E3 ubiquitin ligase complex to promote proteasomal degradation of Cyclin A. This function of Uev1A appears to extend to diploid cells, where its human homologs UBE2V1 and UBE2V2 suppress oncogenic Ras-dependent phenotypes in human colorectal cancer cells in vitro and in xenografts in mice.

Strengths:

(1) Most of the data is supported by a sufficient sample size and appropriate statistics.

(2) Good mix of genetics and biochemistry.

(3) Generation of new transgenes and Drosophila alleles that will be beneficial for the community.

We greatly appreciate these comments.

Weaknesses:

(1) Phenotypes are based on artificial overexpression. It is not clear whether these results are relevant to normal physiology.

Downregulation of Uev1A, Ben, and Cdc27 together significantly increased the incidence of dying nurse cells in normal ovaries (Figure 2-figure supplement 4), indicating that the mechanism we uncovered also protects nurse cells from death during normal oogenesis.

(2) The phenotype of "degenerating ovaries" is very broad, and the study is not focused on phenotypes at the cellular level. Furthermore, no information is provided in the Materials and Methods on how degenerating ovaries are scored, despite this being the most important assay in the study.

Thanks for pointing out this issue. We quantified the phenotype of nurse cell death using “degrading/total egg chambers per ovary”, not “degenerating ovaries” (see all quantification data in our manuscript). Notably, this phenotype ranges from mild to severe. In normal nurse cells, nuclei exhibit a large, round morphology in DAPI staining (see the first panel in Figure 1D). During early death, nurse cell nuclei become disorganized and begin to condense and fragment (see the third panel in Figure 2-figure supplement 2E). In late-stage death, the nuclei are completely fragmented into small, condensed spherical structures (see the second panel in Figure 1D), making cellular-level phenotypic quantification impossible. Since all nurse cells within the same egg chamber are interconnected, their death process is synchronous. Thus, quantifying the phenotype at the egg-chamber level is more practical than at the cellular level. To improve clarity, we will provide a detailed description of the phenotype and integrate this explanation into the main text of the revised manuscript.

(3) In Figure 5, the authors want to conclude that uev1a is a tumor-suppressor, and so they over-express ubev1/2 in human cancer cell lines that have RasK12 and find reduced proliferation, colony formation, and xenograft size. However, genes that act as tumor suppressors have loss-of-function phenotypes that allow for increased cell division. The Drosophila uev1a mutant is viable and fertile, suggesting that it is not a tumor suppressor in flies. Additionally, they do not deplete human ubev1/2 from human cancer cell lines and assess whether this increases cell division, colony formation, and xenograph growth.

We apologize for our misleading description. In Figure 5, we aimed to demonstrate that UBE2V1/2, like Uev1A in Drosophila “nos>Ras^G12V+bam-RNAi” germline tumors (Figure 4), suppress oncogenic KRAS-driven overgrowth in diploid human cancer cells. Importantly, this function of Uev1A and UBE2V1/2 is dependent on Ras-driven tumors; there is no evidence that they act as broad tumor suppressors in the absence of oncogenic Ras. Drosophila uev1a mutants were lethal, not viable (see Lines 131-133), and germline-specific knockdown of uev1a (nos>uev1a-RNAi) caused female sterility without inducing tumors. These findings suggest that Uev1A lacks tumor-suppressive activity in the Drosophila female germline in the absence of Ras-driven tumors. We will revise the manuscript to prevent misinterpretation. Furthermore, we will investigate whether depletion of UBE2V1, UBE2V2, or both promotes oncogenic KRAS-driven overgrowth in human cancer cells.

(4) A critical part of the model does not make sense. CycA is a key part of their model, but they do not show CycA protein expression in WT egg chambers or in their over-expression models (nos.RasV12 or bam>RasV12). Based on Lilly and Spradling 1996, Cyclin A is not expressed in germ cells in region 2-3 of the germarium; whether CycA is expressed in nurse cells in later egg chambers is not shown but is critical to document comprehensively.

We appreciate this critical comment. CycA is a key cyclin that partners with Cdk1 to promote cell division (Edgar and Lehner, 1996). Notably, nurse cells are post-mitotic endocycling cells (Hammond and Laird, 1985) and typically do not express CycA (Lilly and Spradling, 1996) (see the last sentence, page 2518, paragraph 3). However, their death induced by oncogenic Ras^G12V is significantly suppressed by monoallelic deletion of either cycA or cdk1 (Zhang et al., 2024). Conversely, ectopic CycA expression in nurse cells triggers their death (Figure 2C, 2D). These findings suggest that polyploid nurse cells exhibit high sensitivity to aberrant division-promoting stress, which may represent a distinct form of cellular stress unique to polyploid cells. To further test our model, we will compare CycA expression levels in normal nurse cells versus those undergoing oncogenic Ras^G12V-induced cell death.

(5) The authors should provide more information about the knowledge base of uev1a and its homologs in the introduction.

Thanks for this suggestion. We will include this information in the introduction of the revised manuscript.

Reviewer #2 (Public review):

Summary:

The authors performed a genetic screen using deficiency lines and identified Uev1a as a factor that protects nurse cells from Ras^G12V-induced cell death. According to a previous study from the same lab, this cell death is caused by aberrant mitotic stress due to CycA upregulation (Zhang et al.). This paper further reveals that Uev1a forms a complex with APC/C to promote proteasome-mediated degradation of CycA.

In addition to polyploid nurse cells, the authors also examined the effect of Ras^G12V-overexpression in diploid germline cells, where Ras^G12V-overexpression triggers active proliferation, not cell death. Uev1a was found to suppress its overgrowth as well.

Finally, the authors show that the overexpression of the human homologs, UBE2V1 and UBE2V2, suppresses tumor growth in human colorectal cancer xenografts and cell lines. Notably, the expression of these genes correlates with the survival of colorectal cancer patients carrying the Ras mutation.

Strength:

This paper presents a significant finding that UBE2V1/2 may serve as a potential therapy for cancers harboring Ras mutations. The authors propose a fascinating mechanism in which Uev1a forms a complex with APC/C to inhibit aberrant cell cycle progression.

We greatly appreciate these comments.

Weakness:

The quantification of some crucial experiments lacks sufficient clarity.

Thanks for highlighting this issue. We will provide requested details regarding these quantification data in the revised manuscript.

References

Edgar, B.A., and Lehner, C.F. (1996). Developmental control of cell cycle regulators: a fly's perspective. Science 274, 1646-1652.

Hammond, M.P., and Laird, C.D. (1985). Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster. Chromosoma 91, 267-278.

Lilly, M.A., and Spradling, A.C. (1996). The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev 10, 2514-2526.

Zhang, Q., Wang, Y., Bu, Z., Zhang, Y., Zhang, Q., Li, L., Yan, L., Wang, Y., and Zhao, S. (2024). Ras promotes germline stem cell division in Drosophila ovaries. Stem Cell Reports 19, 1205-1216.