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  1. eLife

    Author Response:

    Reviewer #1 (Public Review):

    In this manuscript Rao et al. describe an interesting relationship between KSR1 and the translation regulation of EPSTI1 (a regulator of EMT). They identified this relationship by polysome RNAseq of CRC cells in the context of KSR1 knockdown (KD) which they confirm by polysome QPCR. They then go on to show that KSR KD and add back influences EPSTI1 expression at the protein but not mRNA level and impacts cell viability, anchorage-independent growth, and possibly cell migration. They focus on the cell migration phenotype and show that it is associated with changes in EMT-related genes including E-cad and N-cad. Interestingly, add back of EPSTI1 can reverse the phenotype elicited by KSR1 deletion. Overall, this story is interesting and translation regulation by KSR1 has not been described previously. However, Rao et al. do not provide a mechanism for how KSR1 regulates the translation of EPSTI1, and it is unclear whether this occurs through eIF4E, as the authors suggest.

    We agree completely that our observation that KSR1-dependent ERK regulation of EPSTI1 to promote an EMT-like phenotype raises new questions regarding how the translation of EPSTI1 mRNA is regulated. An additional intriguing question that arises from out work is how this relatively nondescript protein enhances the E- to N-cadherin switch in the colon cancer cells. Multiple possibilities (e.g., altered RNA processing or ribosome heterogeneity) may mediate ERK-dependent regulation of EPSTI1 translation and induction of the cadherin switch. RNA-binding proteins affect discrete cell behaviors, including motility and invasion, by selectively regulating pre-mRNA splicing, mRNA stability, and localization. However, it is hard to imagine a general mechanism involving ERK-mediated regulation of 4E-BP1 and eIF4E, which would affect global mRNA translation, as responsible for a selective effect EPSTI1 mRNA translation and discrete components of EMT-like behavior. Indeed, while KSR1 disruption and ERK inhibition potently suppressed EPSTI1 translation, robust inhibition of mTOR signaling had little effect on EPSTI1. Further development of the detailed cellular mechanisms and critical regulators mediating translation- dependent EMT-like behavior should now be possible.

    Reviewer #2 (Public Review):

    KSR1 functions as a critical rheostat to fine-tune MAPK signalling, and identifying modes by which its over-expression promotes tumor progression is clinically important and potentially druggable. Ras is highly mutated in CRC and unfortunately inhibitors of Ras have been challenging to develop. However, small molecules which stabilize an inactive form of the KSR are actively being developed in an attempt to repress RAS signaling. Thus, this study, which seeks to identify how KSR1 promotes oncogenic mRNA translation, is potentially highly clinically relevant, as it may identify novel druggable targets.

    In this manuscript the authors performed polysome profiling in colorectal cancer (CRC) cells and proposed that KSR1 and ERK regulate the translation of EPSTI1 mRNA. They go on to characterize the phenotypes associated with knock-down or knock-out of KSR1 in CRC, and show that their defects in invasion, anchorage-independent growth and switch to a less EMT-like phenotype are all EPSTI1-dependent.

    The authors succeeded in providing ample in vitro data that KSR1 and EPSTI1 are potential therapeutic targets in CRC. However, the data demonstrating that KSR1 and ERK regulate EPSTI1 mRNA translation is tenuous. Although the authors state that "EPSTI1 is necessary and sufficient for EMT in CRC cells", the data presented are consistent with a more restrained conclusion of a partial-EMT and not EMT per se. Finally, without an in vivo model it is difficult to glean novel insight into the mechanism by which KSR1 and/or EPSTI1 control the invasive and metastatic behaviour of cells.

    We greatly appreciate your comments and are excited about the implications of KSR1-EPSTI1 signaling in promoting the EMT-like phenotype in colon cancer cell lines. We have corrected the use of term ‘EMT’ to ‘EMT-like phenotype’ within the text of the manuscript. We recognize the limitations of using only in vitro data to demonstrate the role of KSR1 and EPSTI1 in promoting motility and invasion in colon cancer cells. In vivo studies will be invaluable to our future efforts to determine the extent to which EPSTI1 promotes metastatic behavior in colon tumors.

    Reviewer #3 (Public Review):

    It is established that Kinase suppressor of Ras 1 (KSR1) contributes to the oncogenic actions of Ras by promoting ERK activation. However, the downstream actions of this pathway are poorly understood. Here Rao et al. demonstrate that this KSR1-dependent pathway increases translation of Epithelial-Stromal Interaction-1 (EPSTI1) mRNA and expression of EPSTI1 protein. This is significant because EPSTI1 drives aspects of EMT, including expression of ZEB1, SLUG, and N-Cadherin. The analysis is thorough and includes both loss-of-function and gain-of-function studies. Overall, the conclusions of this study are convincing and advance our understanding of cancer development.

    We appreciate the positive feedback, and we are excited on implications of our findings on translation regulation of KSR1 on EPSTI1.

  2. eLife

    Reviewer #3 (Public Review):

    It is established that Kinase suppressor of Ras 1 (KSR1) contributes to the oncogenic actions of Ras by promoting ERK activation. However, the downstream actions of this pathway are poorly understood. Here Rao et al. demonstrate that this KSR1-dependent pathway increases translation of Epithelial-Stromal Interaction-1 (EPSTI1) mRNA and expression of EPSTI1 protein. This is significant because EPSTI1 drives aspects of EMT, including expression of ZEB1, SLUG, and N-Cadherin. The analysis is thorough and includes both loss-of-function and gain-of-function studies. Overall, the conclusions of this study are convincing and advance our understanding of cancer development.

  3. eLife

    Reviewer #2 (Public Review):

    KSR1 functions as a critical rheostat to fine-tune MAPK signalling, and identifying modes by which its over-expression promotes tumor progression is clinically important and potentially druggable. Ras is highly mutated in CRC and unfortunately inhibitors of Ras have been challenging to develop. However, small molecules which stabilize an inactive form of the KSR are actively being developed in an attempt to repress RAS signaling. Thus, this study, which seeks to identify how KSR1 promotes oncogenic mRNA translation, is potentially highly clinically relevant, as it may identify novel druggable targets.

    In this manuscript the authors performed polysome profiling in colorectal cancer (CRC) cells and proposed that KSR1 and ERK regulate the translation of EPSTI1 mRNA. They go on to characterize the phenotypes associated with knock-down or knock-out of KSR1 in CRC, and show that their defects in invasion, anchorage-independent growth and switch to a less EMT-like phenotype are all EPSTI1-dependent.

    The authors succeeded in providing ample in vitro data that KSR1 and EPSTI1 are potential therapeutic targets in CRC. However, the data demonstrating that KSR1 and ERK regulate EPSTI1 mRNA translation is tenuous. Although the authors state that "EPSTI1 is necessary and sufficient for EMT in CRC cells", the data presented are consistent with a more restrained conclusion of a partial-EMT and not EMT per se. Finally, without an in vivo model it is difficult to glean novel insight into the mechanism by which KSR1 and/or EPSTI1 control the invasive and metastatic behaviour of cells.

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript Rao et al. describe an interesting relationship between KSR1 and the translation regulation of EPSTI1 (a regulator of EMT). They identified this relationship by polysome RNAseq of CRC cells in the context of KSR1 knockdown (KD) which they confirm by polysome QPCR. They then go on to show that KSR KD and add back influences EPSTI1 expression at the protein but not mRNA level and impacts cell viability, anchorage-independent growth, and possibly cell migration. They focus on the cell migration phenotype and show that it is associated with changes in EMT-related genes including E-cad and N-cad. Interestingly, add back of EPSTI1 can reverse the phenotype elicited by KSR1 deletion. Overall, this story is interesting and translation regulation by KSR1 has not been described previously. However, Rao et al. do not provide a mechanism for how KSR1 regulates the translation of EPSTI1, and it is unclear whether this occurs through eIF4E, as the authors suggest.

  5. eLife

    Evaluation Summary:

    This paper demonstrates the involvement of Kinase Suppressor of Ras 1, a protein that acts as a scaffold in the mitogen activated protein kinase (MAPK) signaling cascade, in translational control of epithelial-to-mesenchymal transition. The analysis is thorough and includes both loss-of-function and gain-of-function studies. This study advances our understanding of cancer development.

    (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. Reviewer #3 agreed to share their name with the authors.)

  6. Version 1 published on bioRxiv