FOXP2 confers oncogenic effects in prostate cancer

Article activity feed

1. 

   Version published to 10.7554/elife.81258 on eLife

   Sep 5, 2023
2. 

   eLife

   Apr 6, 2023

   Author Response

   Reviewer #1 (Public Review):

   The authors start the study with an interesting clinical observation, found in a small subset of prostate cancers: FOXP2-CPED1 fusion. They describe how this fusion results in enhanced FOXP2 protein levels, and further describe how FOXP2 increases anchorageindependent growth in vitro, and results in pre-malignant lesions in vivo. Intrinsically, this is an interesting observation. However, the mechanistic insights are relatively limited as it stands, and the main issues are described below.

   Main issues:

   1. While the study starts off with the FOXP2 fusion, the vast majority of the paper is actually about enhanced FOXP2 expression in tumorigenesis. Wouldn't it be more logical to remove the FOXP2 fusion data? These data seem quite interesting and novel but they are underdeveloped within the …

   Author Response

   Reviewer #1 (Public Review):

   The authors start the study with an interesting clinical observation, found in a small subset of prostate cancers: FOXP2-CPED1 fusion. They describe how this fusion results in enhanced FOXP2 protein levels, and further describe how FOXP2 increases anchorageindependent growth in vitro, and results in pre-malignant lesions in vivo. Intrinsically, this is an interesting observation. However, the mechanistic insights are relatively limited as it stands, and the main issues are described below.

   Main issues:

   1. While the study starts off with the FOXP2 fusion, the vast majority of the paper is actually about enhanced FOXP2 expression in tumorigenesis. Wouldn't it be more logical to remove the FOXP2 fusion data? These data seem quite interesting and novel but they are underdeveloped within the current manuscript design, which is a shame for such an exciting novel finding. Along the same lines, for a study that centres on the prostate lineage, it's not clear why the oncogenic potential of FOXP2 in mouse 3T3 fibroblasts was tested.

   We thank the reviewer very much for the comment. We followed the suggestion and added a set of data regarding the newly identified FOXP2 fusion in Figure 1 to make our manuscript more informative. We tested the oncogenic potential of FOXP2 in NIH3T3 fibroblasts because NIH3T3 cells are a widely used model to demonstrate the presence of transformed oncogenes2,3. In our study, we observed that when NIH3T3 cells acquired the exogenous FOXP2 gene, the cells lost the characteristic contact inhibition response, continued to proliferate and eventually formed clonal colonies. Please refer to "Answer to Essential Revisions #1 from the Editors” for details.

   1. While the FOXP2 data are compelling and convincing, it is not clear yet whether this effect is specific, or if FOXP2 is e.g. universally relevant for cell viability. Targeting FOXP2 by siRNA/shRNA in a non-transformed cell line would address this issue.

   We appreciate these helpful comments. Please refer to the "Answer to Essential Revisions #1 from the Editors” for details.

   1. Unfortunately, not a single chemical inhibitor is truly 100% specific. Therefore, the Foretinib and MK2206 experiments should be confirmed using shRNAs/KOs targeting MEK and AKT. With the inclusion of such data, the authors would make a very compelling argument that indeed MEK/AKT signalling is driving the phenotype.

   We thank the reviewer for highlighting this point and we agree with the reviewer’s point that no chemical inhibitor is 100% specific. In this study, we used chemical inhibitors to provide further supportive data indicating that FOXP2 confers oncogenic effects by activating MET signaling. We characterized a FOXP2-binding fragment located in MET and HGF in LNCaP prostate cancer cells by utilizing the CUT&Tag method. We also found that MET restoration partially reversed oncogenic phenotypes in FOXP2-KD prostate cancer cells. All these data consistently supported that FOXP2 activates MET signaling in prostate cancer. Please refer to the "Answer to Essential Revisions #2 from the Editors” and to the "Answer to Essential Revisions #7 from the Editors” for details.

   1. With the FOXP2-CPED1 fusion being more stable as compared to wild-type transcripts, wouldn't one expect the fusion to have a more severe phenotype? This is a very exciting aspect of the start of the study, but it is not explored further in the manuscript. The authors would ideally elaborate on why the effects of the FOXP2-CPED1 fusion seem comparable to the FOXP2 wildtype, in their studies.

   We thank the reviewer very much for the comment. We had quantified the number of colonies of FOXP2- and FOXP2-CPED1-overexpressing cells, and we found that both wildtype FOXP2 and FOXP2-CPED1 had a comparable putative functional influence on the transformation of human prostate epithelial cells RWPE-1 and mouse primary fibroblasts NIH3T3 (P = 0.69, by Fisher’s exact test for RWPE-1; P = 0.23, by Fisher’s exact test for NIH3T3). We added the corresponding description to the Results section in Line 487 on Page 22 in the tracked changes version of the revised manuscript. Please refer to the "Answer to Essential Revisions #5 from the Editors” for details.

   1. The authors claim that FOXP2 functions as an oncogene, but the most-severe phenotype that is observed in vivo, is PIN lesions, not tumors. While this is an exciting observation, it is not the full story of an oncogene. Can the authors justifiably claim that FOXP2 is an oncogene, based on these results?

   We appreciate the comment, and we made the corresponding revision in the revised manuscript. Please refer to the "Answer to Essential Revisions #3 from the Editors” for details.

   1. The clinical and phenotypic observations are exciting and relevant. The mechanistic insights of the study are quite limited in the current stage. How does FOXP2 give its phenotype, and result in increased MET phosphorylation? The association is there, but it is unclear how this happens.

   We appreciate this valuable suggestion. In the current study, we used the CUT&Tag method to explore how FOXP2 activated MET signaling in LNCaP prostate cancer cells, and we identified potential FOXP2-binding fragments in MET and HGF. Therefore, we proposed that FOXP2 activates MET signaling in prostate cancer through its binding to MET and METassociated gene. Please refer to the "Answer to Essential Revisions #2 from the Editors” for details.

   Reviewer #2 (Public Review):

   1. The manuscript entitled "FOXP2 confers oncogenic effects in prostate cancer through activating MET signalling" by Zhu et al describes the identification of a novel FOXP2CPED1 gene fusion in 2 out of 100 primary prostate cancers. A byproduct of this gene fusion is the increased expression of FOXP2, which has been shown to be increased in prostate cancer relative to benign tissue. These data nominated FOXP2 as a potential oncogene. Accordingly, overexpression of FOXP2 in nontransformed mouse fibroblast NIH-3T3 and human prostate RWPE-1 cells induced transforming capabilities in both cell models. Mechanistically, convincing data were provided that indicate that FOXP2 promotes the expression and/or activity of the receptor tyrosine kinase MET, which has previously been shown to have oncogenic functions in prostate cancer. Notably, the authors create a new genetically engineered mouse model in which FOXP2 is overexpressed in the prostatic luminal epithelial cells. Overexpression of FOXP2 was sufficient to promote the development of prostatic intraepithelial neoplasia (PIN) a suspected precursor to prostate adenocarcinoma and activate MET signaling.

   Strengths:

   This study makes a convincing case for FOXP2 as 1) a promoter of prostate cancer initiation and 2) an upstream regulator of pro-cancer MET signaling. This was done using both overexpression and knockdown models in cell lines and corroborated in new genetically engineered mouse models (GEMMs) of FOXP2 or FOXP2-CPED1 overexpression in prostate luminal epithelial cells as well as publicly available clinical cohort data.

   Major strengths of the study are the demonstration that FOXP2 or FOXP2-CPED1 overexpression transforms RWPE-1 cells to now grow in soft agar (hallmark of malignant transformation) and the creation of new genetically engineered mouse models (GEMMs) of FOXP2 or FOXP2-CPED1 overexpression in prostate luminal epithelial cells. In both mouse models, FOXP2 overexpression increased the incidence of PIN lesions, which are thought to be a precursor to prostate cancer. While FOXP2 alone was not sufficient to cause prostate cancer in mice, it is acknowledged that single gene alterations causing prostate cancer in mice are rare. Future studies will undoubtedly want to cross these GEMMs with established, relatively benign models of prostate cancer such as Hi-Myc or Pb-Pten mice to see if FOXP2 accelerates cancer progression (beyond the scope of this study).

   We appreciate these positive comments from the reviewer. We agree with the suggestion from the reviewer that it is worth exploring whether FOXP2 is able to cooperate with a known disease driver to accelerate the progression of prostate cancer. Therefore, we are going to cross Pb-FOXP2 transgenic mice with Pb-Pten KO mice to assess if FOXP2 is able to accelerate malignant progression.

   1. Weaknesses: It is unclear why the authors decided to use mouse fibroblast NIH3T3 cells for their transformation studies. In this regard, it appears likely that FOXP2 could function as an oncogene across diverse cell types. Given the focus on prostate cancer, it would have been preferable to corroborate the RWPE-1 data with another prostate cell model and test FOXP2's transforming ability in RWPE-1 xenograft models. To that end, there is no direct evidence that FOXP2 can cause cancer in vivo. The GEMM data, while compelling, only shows that FOXP2 can promote PIN in mice and the lone xenograft model chosen was for fibroblast NIH-3T3 cells.

   To determine the oncogenic activity of FOXP2 and the FOXP2-CPDE1 fusion, we initially used mouse primary fibroblast NIH3T3 for transformation experiments, because NIH3T3 cells are a widely used cell model to discover novel oncogenes2,3,10,11. Subsequently, we observed that overexpression of FOXP2 and its fusion variant drove RWPE-1 cells to lose the characteristic contact inhibition response, led to their anchorage-independent growth in vitro, and promoted PIN in the transgenic mice. During preparation of the revised manuscript, we tested the transformation ability of FOXP2 and FOXP2-CPED1 in RWPE1 xenograft models. We subcutaneously injected 2 × 106 RWPE-1 cells into the flanks of NOD-SCID mice. The NODSCID mice were divided into five groups (n = 5 mice in each group): control, FOXP2overexpressing (two stable cell lines) and FOXP2-CPED1- overexpressing (two cell lines) groups. The experiment lasted for 4 months. We observed that no RWPE-1 cell-injected mice developed tumor masses. We propose that FOXP2 and its fusion alone are not sufficient to generate the microenvironment suitable for RWPE-1-xenograft growth. Collectively, our data suggest that FOXP2 has oncogenic potential in prostate cancer, but is not sufficient to act alone as an oncogene.

   1. There is a limited mechanism of action. While the authors provide correlative data suggesting that FOXP2 could increase the expression of MET signaling components, it is not clear how FOXP2 controls MET levels. It would be of interest to search for and validate the importance of potential FOXP2 binding sites in or around MET and the genes of METassociated proteins. At a minimum, it should be confirmed whether MET is a primary or secondary target of FOXP2. The authors should also report on what happened to the 4-gene MET signature in the FOXP2 knockdown cell models. It would be equally significant to test if overexpression of MET can rescue the anti-growth effects of FOXP2 knockdown in prostate cancer cells (positive or negative results would be informative).

   We appreciate all the valuable comments. As suggested, we performed corresponding experiments, please refer to the " Answers to Essential Revisions #2 from the Editors”, to the "Answer to Essential Revisions #6 from the Editors”, and to the "Answer to Essential Revisions #7 from the Editors” for details.

   Reviewer #3 (Public Review):

   1. In this manuscript, the authors present data supporting FOXP2 as an oncogene in PCa. They show that FOXP2 is overexpressed in PCa patient tissue and is necessary and sufficient for PCa transformation/tumorigenesis depending on the model system. Overexpression and knock-down of FOXP2 lead to an increase/decrease in MET/PI3K/AKT transcripts and signaling and sensitizes cells to PI3K/AKT inhibition.

   Key strengths of the paper include multiple endpoints and model systems, an over-expression and knock-down approach to address sufficiency and necessity, a new mouse knock-in model, analysis of primary PCa patient tumors, and benchmarking finding against publicly available data. The central discovery that FOXP2 is an oncogene in PCa will be of interest to the field. However, there are several critically unanswered questions.

   1. No data are presented for how FOXP2 regulates MET signaling. ChIP would easily address if it is direct regulation of MET and analysis of FOXP2 ChIP-seq could provide insights.

   1. Beyond the 2 fusions in the 100 PCa patient cohort it is unclear how FOXP2 is overexpressed in PCa. In the discussion and in FS5 some data are presented indicating amplification and CNAs, however, these are not directly linked to FOXP2 expression.

   1. There are some hints that full-length FOXP2 and the FOXP2-CPED1 function differently. In SF2E the size/number of colonies between full-length FOXP2 and fusion are different. If the assay was run for the same length of time, then it indicates different biologies of the overexpressed FOXP2 and FOXP2-CPED1 fusion. Additionally, in F3E the sensitization is different depending on the transgene.

   We appreciate these valuable comments and constructive remarks. As suggested, we performed the CUT&Tag experiments to detect the binding of FOXP2 to MET, and to examine the association of CNAs of FOXP2 with its expression. Please refer to the " Answer to Essential Revisions #2 from the Editors" and the " Answer to Essential Revisions #4 from the Editors" for details. We also added detailed information to show the resemblance observed between FOXP2 fusion- and wild-type FOXP2-overexpressing cells. We added the corresponding description to the Results section in Line 487 on Page 22 in the tracked changes version of the revised manuscript. Please refer to the “Answer to Essential Revisions #5 from the Editors” for details.

   1. The relationship between FOXP2 and AR is not explored, which is important given 1) the critical role of the AR in PCa; and 2) the existing relationship between the AR and FOXP2 and other FOX gene members.

   We thank the reviewer very much for highlighting this point. We agree that it is important to examine the relationship between FOXP2 and AR. We therefore analyzed the expression dataset of 255 primary prostate tumors from TCGA and observed that the expression of FOXP2 was significantly correlated with the expression of AR (Spearman's ρ = 0.48, P < 0.001) (Figure 1. a). Next, we observed that both FOXP2- and FOXP2-CPED1overexpressing 293T cells had a higher AR protein abundance than control cells (Figure 1. b). In addition, shRNA-mediated FOXP2 knockdown in LNCaP cells resulted in a decreased AR protein level compared to that in control cells (Figure 1. c). However, we analyzed our CUT&Tag data and observed no binding of FOXP2 to AR (Figure 1. d). Our data suggest that FOXP2 might be associated with AR expression.

   

   Figure 1. a. AR expression in a human prostate cancer dataset (TCGA, Prostate Adenocarcinoma, Provisional; n = 493) classified by FOXP2 expression level (bottom 25%, low expression, n = 120; top 25%, high expression, n = 120; negative expression, n = 15). P values were calculated by the MannWhitney U test. The correlation between FOXP2 and AR expression was evaluated by determining the Spearman's rank correlation coefficient. b. Immunoblot analysis of the expression levels of AR in 293T cells with overexpression of FOXP2 or FOXP2-CPED1. c. Immunoblot analysis of the expression levels of AR in LNCaP cells with stable expression of the scrambled vector or FOXP2 shRNA. d. CUT&Tag analysis of FOXP2 association with the promoter of AR. Representative track of FOXP2 at the AR gene locus is shown.

   Reference

   1. Mayr C, Bartel DP. Widespread shortening of 3'UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell. 2009 Aug 21;138(4):673-84.
   2. Gara SK, Jia L, Merino MJ, Agarwal SK, Zhang L, Cam M et al., Germline HABP2 Mutation Causing Familial Nonmedullary Thyroid Cancer. N Engl J Med. 2015 Jul 30;373(5):448-55.
   3. Kohno T, Ichikawa H, Totoki Y, Yasuda K, Hiramoto M, Nammo T et al., KIF5B-RET fusions in lung adenocarcinoma. Nat Med. 2012 Feb 12;18(3):375-7.
   4. Chen F, Byrd AL, Liu J, Flight RM, DuCote TJ, Naughton KJ et al., Polycomb deficiency drives a FOXP2-high aggressive state targetable by epigenetic inhibitors. Nat Commun. 2023 Jan 20;14(1):336.
   5. Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Bryson TD, Henikoff JG et al., CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun. 2019 Apr 29;10(1):1930.
   6. Spiteri E, Konopka G, Coppola G, Bomar J, Oldham M, Ou J et al., Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain. Am J Hum Genet. 2007 Dec;81(6):1144-57.
   7. Lai CS, Fisher SE, Hurst JA, Vargha-Khadem F, Monaco AP. A forkhead-domain gene is mutated in a severe speech and language disorder. Nature. 2001 Oct 4;413(6855):519-23.
   8. Hannenhalli S, Kaestner KH. The evolution of Fox genes and their role in development and disease. Nat Rev Genet. 2009 Apr;10(4):233-40.
   9. Shu W, Yang H, Zhang L, Lu MM, Morrisey EE. Characterization of a new subfamily of winged-helix/forkhead (Fox) genes that are expressed in the lung and act as transcriptional repressors. J Biol Chem. 2001 Jul 20;276(29):27488-97.
   10. Wang C, Liu H, Qiu Q, Zhang Z, Gu Y, He Z. TCRP1 promotes NIH/3T3 cell transformation by over-activating PDK1 and AKT1. Oncogenesis. 2017 Apr 24;6(4):e323.
   11. Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D et al., Cell transformation by the superoxide-generating oxidase Mox1. Nature. 1999 Sep 2;401(6748):79-82.

   Read the original source
3. 

   eLife

   Sep 26, 2022

   Evaluation Summary:

   The authors identify a new FOXP2-CPED1 gene fusion in prostate cancer that leads to the increased expression of FOXP2 and subsequent transformation of non-cancer cells. Increased FOXP2 was shown to promote prostate cancer in part through the increased expression and activation of the receptor tyrosine kinase MET, a known driver of prostate cancer. Notably, the authors created new genetically engineered mouse models of FOXP2 and FOXP2-CPED1 overexpression in prostate luminal epithelial cells which was sufficient to cause prostatic intraepithelial neoplasia in these mice with lesions that confirmed increased MET signaling. Oncogenes are typically interesting drug targets or interact with possible drug targets, and the manuscript could thus have a significant societal impact on better understanding drivers of the disease.

   Evaluation Summary:

   The authors identify a new FOXP2-CPED1 gene fusion in prostate cancer that leads to the increased expression of FOXP2 and subsequent transformation of non-cancer cells. Increased FOXP2 was shown to promote prostate cancer in part through the increased expression and activation of the receptor tyrosine kinase MET, a known driver of prostate cancer. Notably, the authors created new genetically engineered mouse models of FOXP2 and FOXP2-CPED1 overexpression in prostate luminal epithelial cells which was sufficient to cause prostatic intraepithelial neoplasia in these mice with lesions that confirmed increased MET signaling. Oncogenes are typically interesting drug targets or interact with possible drug targets, and the manuscript could thus have a significant societal impact on better understanding drivers of the disease.

   (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.)

   Read the original source
4. 

   eLife

   Sep 26, 2022

   Reviewer #1 (Public Review):

   The authors start the study with an interesting clinical observation, found in a small subset of prostate cancers: FOXP2-CPED1 fusion. They describe how this fusion results in enhanced FOXP2 protein levels, and further describe how FOXP2 increases anchorage-independent growth in vitro, and results in pre-malignant lesions in vivo. Intrinsically, this is an interesting observation. However, the mechanistic insights are relatively limited as it stands, and the main issues are described below.

   Main issues:

   1. While the study starts off with the FOXP2 fusion, the vast majority of the paper is actually about enhanced FOXP2 expression in tumorigenesis. Wouldn't it be more logical to remove the FOXP2 fusion data? These data seem quite interesting and novel but they are underdeveloped within the current manuscript …

   Reviewer #1 (Public Review):

   The authors start the study with an interesting clinical observation, found in a small subset of prostate cancers: FOXP2-CPED1 fusion. They describe how this fusion results in enhanced FOXP2 protein levels, and further describe how FOXP2 increases anchorage-independent growth in vitro, and results in pre-malignant lesions in vivo. Intrinsically, this is an interesting observation. However, the mechanistic insights are relatively limited as it stands, and the main issues are described below.

   Main issues:

   1. While the study starts off with the FOXP2 fusion, the vast majority of the paper is actually about enhanced FOXP2 expression in tumorigenesis. Wouldn't it be more logical to remove the FOXP2 fusion data? These data seem quite interesting and novel but they are underdeveloped within the current manuscript design, which is a shame for such an exciting novel finding.

   Along the same lines, for a study that centres on the prostate lineage, it's not clear why the oncogenic potential of FOXP2 in mouse 3T3 fibroblasts was tested.

   2. While the FOXP2 data are compelling and convincing, it is not clear yet whether this effect is specific, or if FOXP2 is e.g. universally relevant for cell viability. Targeting FOXP2 by siRNA/shRNA in a non-transformed cell line would address this issue.

   3. Unfortunately, not a single chemical inhibitor is truly 100% specific. Therefore, the Foretinib and MK2206 experiments should be confirmed using shRNAs/KOs targeting MEK and AKT. With the inclusion of such data, the authors would make a very compelling argument that indeed MEK/AKT signalling is driving the phenotype

   4. With the FOXP2-CPED1 fusion being more stable as compared to wild-type transcripts, wouldn't one expect the fusion to have a more severe phenotype? This is a very exciting aspect of the start of the study, but it is not explored further in the manuscript. The authors would ideally elaborate on why the effects of the FOXP2-CPED1 fusion seem comparable to the FOXP2 wildtype, in their studies.

   5. The authors claim that FOXP2 functions as an oncogene, but the most-severe phenotype that is observed in vivo, is PIN lesions, not tumors. While this is an exciting observation, it is not the full story of an oncogene. Can the authors justifiably claim that FOXP2 is an oncogene, based on these results?

   6. The clinical and phenotypic observations are exciting and relevant. The mechanistic insights of the study are quite limited in the current stage. How does FOXP2 give its phenotype, and result in increased MET phosphorylation? The association is there, but it is unclear how this happens.

   Read the original source
5. 

   eLife

   Sep 26, 2022

   Reviewer #2 (Public Review):

   The manuscript entitled "FOXP2 confers oncogenic effects in prostate cancer through activating MET signalling" by Zhu et al describes the identification of a novel FOXP2-CPED1 gene fusion in 2 out of 100 primary prostate cancers. A byproduct of this gene fusion is the increased expression of FOXP2, which has been shown to be increased in prostate cancer relative to benign tissue. These data nominated FOXP2 as a potential oncogene. Accordingly, overexpression of FOXP2 in nontransformed mouse fibroblast NIH-3T3 and human prostate RWPE-1 cells induced transforming capabilities in both cell models. Mechanistically, convincing data were provided that indicate that FOXP2 promotes the expression and/or activity of the receptor tyrosine kinase MET, which has previously been shown to have oncogenic functions in …

   Reviewer #2 (Public Review):

   The manuscript entitled "FOXP2 confers oncogenic effects in prostate cancer through activating MET signalling" by Zhu et al describes the identification of a novel FOXP2-CPED1 gene fusion in 2 out of 100 primary prostate cancers. A byproduct of this gene fusion is the increased expression of FOXP2, which has been shown to be increased in prostate cancer relative to benign tissue. These data nominated FOXP2 as a potential oncogene. Accordingly, overexpression of FOXP2 in nontransformed mouse fibroblast NIH-3T3 and human prostate RWPE-1 cells induced transforming capabilities in both cell models. Mechanistically, convincing data were provided that indicate that FOXP2 promotes the expression and/or activity of the receptor tyrosine kinase MET, which has previously been shown to have oncogenic functions in prostate cancer. Notably, the authors create a new genetically engineered mouse model in which FOXP2 is overexpressed in the prostatic luminal epithelial cells. Overexpression of FOXP2 was sufficient to promote the development of prostatic intraepithelial neoplasia (PIN) a suspected precursor to prostate adenocarcinoma and activate MET signaling.

   Strengths:

   This study makes a convincing case for FOXP2 as 1) a promoter of prostate cancer initiation and 2) an upstream regulator of pro-cancer MET signaling. This was done using both overexpression and knockdown models in cell lines and corroborated in new genetically engineered mouse models (GEMMs) of FOXP2 or FOXP2-CPED1 overexpression in prostate luminal epithelial cells as well as publicly available clinical cohort data.

   Major strengths of the study are the demonstration that FOXP2 or FOXP2-CPED1 overexpression transforms RWPE-1 cells to now grow in soft agar (hallmark of malignant transformation) and the creation of new genetically engineered mouse models (GEMMs) of FOXP2 or FOXP2-CPED1 overexpression in prostate luminal epithelial cells. In both mouse models, FOXP2 overexpression increased the incidence of PIN lesions, which are thought to be a precursor to prostate cancer. While FOXP2 alone was not sufficient to cause prostate cancer in mice, it is acknowledged that single gene alterations causing prostate cancer in mice are rare. Future studies will undoubtedly want to cross these GEMMs with established, relatively benign models of prostate cancer such as Hi-Myc or Pb-Pten mice to see if FOXP2 accelerates cancer progression (beyond the scope of this study).

   Weaknesses:

   It is unclear why the authors decided to use mouse fibroblast NIH-3T3 cells for their transformation studies. In this regard, it appears likely that FOXP2 could function as an oncogene across diverse cell types. Given the focus on prostate cancer, it would have been preferable to corroborate the RWPE-1 data with another prostate cell model and test FOXP2's transforming ability in RWPE-1 xenograft models. To that end, there is no direct evidence that FOXP2 can cause cancer in vivo. The GEMM data, while compelling, only shows that FOXP2 can promote PIN in mice and the lone xenograft model chosen was for fibroblast NIH-3T3 cells.

   There is a limited mechanism of action. While the authors provide correlative data suggesting that FOXP2 could increase the expression of MET signaling components, it is not clear how FOXP2 controls MET levels. It would be of interest to search for and validate the importance of potential FOXP2 binding sites in or around MET and the genes of MET-associated proteins. At a minimum, it should be confirmed whether MET is a primary or secondary target of FOXP2. The authors should also report on what happened to the 4-gene MET signature in the FOXP2 knockdown cell models. It would be equally significant to test if overexpression of MET can rescue the anti-growth effects of FOXP2 knockdown in prostate cancer cells (positive or negative results would be informative).

   Read the original source
6. 

   eLife

   Sep 26, 2022

   Reviewer #3 (Public Review):

   In this manuscript, the authors present data supporting FOXP2 as an oncogene in PCa. They show that FOXP2 is overexpressed in PCa patient tissue and is necessary and sufficient for PCa transformation/tumorigenesis depending on the model system. Over-expression and knock-down of FOXP2 lead to an increase/decrease in MET/PI3K/AKT transcripts and signaling and sensitizes cells to PI3K/AKT inhibition.

   Key strengths of the paper include multiple endpoints and model systems, an over-expression and knock-down approach to address sufficiency and necessity, a new mouse knock-in model, analysis of primary PCa patient tumors, and benchmarking finding against publicly available data. The central discovery that FOXP2 is an oncogene in PCa will be of interest to the field.

   However, there are several critically unanswered …

   Reviewer #3 (Public Review):

   In this manuscript, the authors present data supporting FOXP2 as an oncogene in PCa. They show that FOXP2 is overexpressed in PCa patient tissue and is necessary and sufficient for PCa transformation/tumorigenesis depending on the model system. Over-expression and knock-down of FOXP2 lead to an increase/decrease in MET/PI3K/AKT transcripts and signaling and sensitizes cells to PI3K/AKT inhibition.

   Key strengths of the paper include multiple endpoints and model systems, an over-expression and knock-down approach to address sufficiency and necessity, a new mouse knock-in model, analysis of primary PCa patient tumors, and benchmarking finding against publicly available data. The central discovery that FOXP2 is an oncogene in PCa will be of interest to the field.

   However, there are several critically unanswered questions.

   • No data are presented for how FOXP2 regulates MET signaling. ChIP would easily address if it is direct regulation of MET and analysis of FOXP2 ChIP-seq could provide insights.
   • Beyond the 2 fusions in the 100 PCa patient cohort it is unclear how FOXP2 is overexpressed in PCa. In the discussion and in FS5 some data are presented indicating amplification and CNAs, however, these are not directly linked to FOXP2 expression.
   • There are some hints that full-length FOXP2 and the FOXP2-CPED1 function differently. In SF2E the size/number of colonies between full-length FOXP2 and fusion are different. If the assay was run for the same length of time, then it indicates different biologies of the over-expressed FOXP2 and FOXP2-CPED1 fusion. Additionally, in F3E the sensitization is different depending on the transgene.
   • The relationship between FOXP2 and AR is not explored, which is important given 1) the critical role of the AR in PCa; and 2) the existing relationship between the AR and FOXP2 and other FOX gene members.

   Read the original source
7. 

   Version published to 10.1101/2022.07.06.498943v1 on bioRxiv

   Jul 6, 2022