Replication stress inducing ELF3 upregulation promotes BRCA1-deficient breast tumorigenesis in luminal progenitors

Reviewer #1 (Public Review):

The authors set out to define the molecular basis for LP as the origin of BRCA1-deficient breast cancers. They showed that LPs have the highest level of replicative stress, and hypothesise that this may account for their tendency to transform. They went on to identify ELF3 as a candidate driver of LP transformation and showed that ELF3 expression is up-regulated in response to replicative stress as well as BRCA1 deficiency. They went on to show that ELF3 inactivation led to a higher level of DNA damage, which may result from compromised replicative stress responses.

While the manuscript supports the interesting idea wherein ELF3 may fuel LP cell transformation, it remains obscure how ELF3 promotes cell tolerance to DNA damage. Interestingly the authors proposed that ELF3 suppresses excessive genomic instability, but in my opinion, I do not see any evidence that supports this claim. In fact, one might think that genomic instability is key to cell transformation.

Reviewer #2 (Public Review):

Summary:
The manuscript focuses on a persistent question of why germline mutations in BRCA1 which impair homology-directed repair of DNA double-strand breaks predispose to primarily breast and ovarian cancers but not other tissues. The authors propose that replication stress is elevated in the luminal progenitor (LP) cells and apply the gene signature from Dreyer et al as a measure of replication stress in populations of cells selected by FACS previously (published by Lim et al.) and suggest an enrichment of replication stress among the LP cells. This is followed by single-cell RNA seq data from a small number of breast tissues from a small number of BRCA1 mutation carriers but the pathogenic variants are not listed. The authors perform an elegant analysis of the effects of BRCA1 knockdown in MCF10A cells, but these cells are not considered a model of LP cells.

Overall, the manuscript suffers from significant gaps and leaps in logic among the datasets used. The connection to luminal progenitor cells is not adequately established because the models used are not representative of this population of cells. Therefore, the central hypothesis is not sufficiently justified.

Strengths:
The inducible knockdown of BRCA1 provided compelling data pointing to an upregulation of ELF3 in this setting as well as a small number of other genes. It would be useful to discuss the other genes for completeness and explain the logic for focusing on ELF3. Nonetheless, the connection with ELF 3 is reasonable. The authors provide significant data showing a role for ELF3 in breast epithelial cells and its role in cell survival.

Weaknesses:
The initial observations in primary breast cells have small sample sizes. The mutations in BRCA1 seem to be presumed to be all the same, but we know that pathogenic variants differ among individuals and range from missense mutations affecting interactions with one critical partner to large-scale truncations of the protein.

The figure legends are missing critical details that make it difficult for the reader to evaluate the data. The data support the notion that ELF3 may participate in relieving replication stress, but does not appear to be limited to LP cells as proposed in the hypothesis.

Author Response

We would like to express our gratitude to the reviewers for their insightful comments and suggestions on our manuscript. We appreciate the time and effort they have devoted to evaluating our work. In response to their valuable feedback, we will undertake a comprehensive revision of our manuscript to address their concerns and enhance the clarity of our findings.

Reviewer #1 has raised the important point of the need for a more thorough exploration of how ELF3 promotes cell tolerance to DNA damage.

Just as mentioned by the reviewer, we totally agreed that genomic instability is key to cell transformation. In the original manuscript, we proposed that ELF3 might be an important factor for cells to tolerate the lethal genomic instability caused by BRCA1 deficiency, keeping an “appropriate” level of genomic instability, thus fueling cell transformation. And we acknowledge the limitation that the mechanism of how ELF3 promotes cell to tolerate DNA damage remains further exploration. To address this, ELF3 overexpression and knockdown experiments in more BRCA1 wildtype or deficient breast cell lines are planned. In addition, since ELF3 is an inherent transcription factor, we suspect the function of ELF3 to promote cell tolerance to DNA damage is mediated by transcription, and more downstream genes of ELF3 will be explored as well.

Regarding the concerns raised by Reviewer #2, we acknowledge that our manuscript may have contained gaps and limitations of the datasets used.

We appreciate the reviewer's feedback regarding the limitations of our cell models and their representativeness of LP cells. While we have utilized MCF10A cells for the knockdown experiments, we understand that these may not be a perfect representation of LP cells. To address this concern, we will incorporate a discussion on the limitations of our cell models and their relevance to LP cells, along with potential plans in LP cells that may be included in future studies.

We will also clarify the rationale for focusing on ELF3 and discuss the other genes identified in our analysis for completeness. Regarding to ELF3 functions in cells other than LP, in our analysis, ELF3 is highly expressed in LPs compared to other cell populations in mammary gland, making ELF3 a previously undefined LP gene. Thus, we suspect that ELF3 functions may be more significant in LP cells. We are also interested in ELF3 functions in cells other than LP cells and will further explore

We agree that different pathogenic variants of BRCA1 may cause diverse impacts on its function and tumorigenesis. We will add detailed information and discussion about BRCA1 pathogenic variants of patients in our single-cell RNA-seq. Also, to enhance the overall clarity of our manuscript, we will revise the figure legends to include critical details that were previously omitted. This will ensure that readers can better evaluate the presented data.