Mutant K-Ras in Pancreatic Cancer: An Insight on the Role of Wild-Type N-Ras and K-Ras-Dependent Cell Cycle Regulation

Round 1

Reviewer 1 Report

 Ferguson et al. intended to show the role of wild-type N-Ras protein that shields cancer cells against mutant K-Ras' cell cycle independent production of cyclins during carcinogenesis through their article "Wild-type N-Ras complements mutant K-Ras in pancreatic cancer cell lines but K-Ras has a specific role in cell cycle independent regulation of G2 cyclins."

 

This constitutes a comprehensive and interesting topic of work that is appreciated. However, there are several Major concerns that can be resolved to improve the quality of the manuscript, listed below.

1.      The Simple Summary, as well as abstract, is composed of different inferences from various observations, whereas the author should state the subsequent body of work, which should preferably flow from the question, rationale and address the question through the review.

2.      The introduction unquestionably needs to be revisited. This section currently contains mostly a cluster of information addressing K-Ras and its activities, which can be relocated with a more suitable subheading. The author also should address the importance and the need of their study in the introduction section.

3.      In Figure 1, any housekeeping marker should be added.

4.      Why the authors use Hela and Suit2 cell line, please give the justification in the result section as they have confined their research in pancreatic cancer. Additionally, Panc-1 cell has K-Ras mutation. Please correct the information (line:231)

5.   The writing structure throughout the whole work is quite poor. The majority of the lines do not adequately convey their message.

6.    Please revise the conclusion so that it conveys the message and key findings of your research more effectively.

7.      The quality of all the figures does not satisfy the standards. The author should improve the quality of the figurs.

8.  There are several recent articles that may be referred to as strong support for the author's work in their paper, and they are as follows:

a)      DOI: 10.1038/s41388-021-01951-x

b)     doi: 10.1101/cshperspect.a031435

c)      doi.org/10.3389/fonc.2021.638360

d)     doi.org/10.3390/ijms22094765

Author Response

We thank the reviewer for reading and reviewing our manuscript.

 

The reviewer comments that

1. The Simple Summary, as well as abstract, is composed of different inferences from various observations, whereas the author should state the subsequent body of work, which should preferably flow from the question, rationale and address the question through the review.

 

To address this we have made the following change to the simple summary

Most pancreatic cancers have a mutation in the gene that produces the protein K-Ras. The mutation allows the cancer cells to divide out of control and so the theory is that if we can stop the mutant protein working we will kill or at least stop the cancer growing. However, to date this has proved ineffective. This may be explained by the previous observation that pancreatic cancers can become independent of K-Ras (not needing the mutation to survive), this paper addresses the question of how cells with mutant K-Ras become independent of the oncogene. We show that cells which are independent of K-Ras have switched on another form of Ras protein and that this protein has taken on some (but not all) functions of K-Ras. Based on the pattern of functions replaced and not replaced we have developed a model for how pancreatic cells evolve into cancer cells using Ras proteins which may allow more effective targeting of Ras related pathways to overcome the disease.

 

The reviewer comments

2. The introduction unquestionably needs to be revisited. This section currently contains mostly a cluster of information addressing K-Ras and its activities, which can be relocated with a more suitable subheading. The author also should address the importance and the need of their study in the introduction section.

To address this we have added the following line at the end of the introduction

.., as this may inform strategies for treatment that not only target different (more commonly occuring) mutations of K-Ras[38], but also target specific downstream and upstream partners of pan-Ras such as SOS1[39].

The reviewer comments

3. In Figure 1, any housekeeping marker should be added.

We do not usually use a housekeeping gene as a control in a pull down experiment, believing that an input control is adequate, but we take the reviewer's point that a control to show that there is no non-specific pull down would be a good thing to have (which housekeeping gene to choose obviously being an issue). Nevertheless, we did not include such a control and cannot retrospectively add one.

The Reviewer asks

4. Why the authors use Hela and Suit2 cell line, please give the justification in the result section as they have confined their research in pancreatic cancer. Additionally, Panc-1 cell has K-Ras mutation. Please correct the information (line:231)

In our introduction we state that

Only one of the 9 K-Ras dependent cell lines tested by this group was reported to have an expression profile predictive of K-Ras independence[22]; this was the pancreatic line Suit-2, which is well known to be more mesenchymal than other pancreatic cell lines[37].

In the results we state

.. the K-Ras dependent cell line Suit-2 showed the most marked decrease in active Ras when K-Ras was knocked down. In the other cell lines any decline in active Ras following K-Ras depletion was marginal.  The non-pancreatic cell line HeLa showed the greatest decline in Ras activity when N-Ras was specifically targeted (Figure 1a).

 

To add further emphasis we have now added the text in parenthesis below

the K-Ras dependent cell line Suit-2 showed the most marked decrease in active Ras when K-Ras was knocked down (justifying our subsequent concentration on this specific cell line). 

PANC-1 does indeed have a K-Ras mutation and is assumed by us on the basis of the data in the paper to be K-Ras independent, but is also assumed in the previous literature to be K-Ras independent. I believe that all cell lines described as K-Ras independent (as well as all cell lines described as being dependent) have a K-Ras mutation. Therefore, we stand by the statement.

Comment 5,6,7 and 8 relate to the quality of out writing and presentation in the results and discussion. We apologise for any lack of quality. In response to specific comments by other reviewers we have made changes that we hope the reviewer will agree have improved the quality of the work. We have also added references that we think confirm that we have addressed relevant recent publications.

 

Reviewer 2 Report

Query#1

First, the title must be improved, although the title describes the content of the article adequately I find it long and not incisive and being the title the calling card of the article, I suggest to the authors to modify the title. For I instance, I should suggest the following title: “Mutant K-Ras in pancreatic cancer: an insight on the role of wild-type N-Ras and K-Ras dependent modulation of cell cycle.

Query#2

The introduction is well written and explains all the aspects concerning K-Ras mutation in pancreatic cancer, however I would suggest to the authors to introduce the different pancreatic cancer therapeutic approaches. Indeed, aside from standard chemotherapeutic drugs, different new therapies based on Immune Checkpoint Inhibitors (ICIs) and small molecules kinase inhibitors (SMKIs), are currently tested on pancreatic cancer cell lines. At this purpose, I suggest to the authors to cite the following updated literatures:

1.     Sun, J., Russell, C. C., Scarlett, C. J., McCluskey, A. (2020). Small molecule inhibitors in pancreatic cancer. RSC medicinal chemistry, 11(2), 164–183. https://doi.org/10.1039/c9md00447e

2.     Pecoraro, C., Parrino, B., Cascioferro, S., Puerta, A., Avan, A., Peters, G. J., Diana, P., Giovannetti, E., & Carbone, D. (2021). A New Oxadiazole-Based Topsentin Derivative Modulates Cyclin-Dependent Kinase 1 Expression and Exerts Cytotoxic Effects on Pancreatic Cancer Cells. Molecules (Basel, Switzerland), 27(1), 19. https://doi.org/10.3390/molecules27010019

3.      Jiang, H., Hegde, S., Knolhoff, B. L., Zhu, Y., Herndon, J. M., Meyer, M. A., Nywening, T. M., Hawkins, W. G., Shapiro, I. M., Weaver, D. T., Pachter, J. A., Wang-Gillam, A., DeNardo, D. G. (2016). Targeting focal adhesion kinase renders pancreatic cancers responsive to checkpoint immunotherapy. Nature medicine, 22(8), 851–860. https://doi.org/10.1038/nm.4123

4.     Pecoraro, C., De Franco, M., Carbone, D., Bassani, D., Pavan, M., Cascioferro, S., Parrino, B., Cirrincione, G., Dall'Acqua, S., Moro, S., Gandin, V., & Diana, P. (2023). 1,2,4-Amino-triazine derivatives as pyruvate dehydrogenase kinase inhibitors: Synthesis and pharmacological evaluation. European journal of medicinal chemistry, 249, 115134. https://doi.org/10.1016/j.ejmech.2023.115134

Query#3

Please, check the paragraph 2 Materials and Methods, section Western blot analysis line 128.

In general, the paper is well organized and supported by the experimental data obtained, I appreciated the fact that the role of K-Ras was analyzed in three different PDAC cell lines, including BXPC-3, SUIT-2 and Panc-1 and in non-pancreatic HeLa cell line. I fully recommend this publication after the revisions I suggested.

 

Author Response

 

Response to referees:

 

Referee 2

We thank the reviewer for their comments and advice

Query 1: We have changed the title to read 

Mutant K-Ras in pancreatic cancer: an insight on the role of wild-type N-Ras and K-Ras dependent cell cycle regulation

Query 2: We have added a brief discussion of other therapeutic approaches including the references suggested.

Novel therapeutic approaches based on Small Molecule Kinase Inhibitors[1, 2, 3] and Immune Checkpoint Inhibitors (ICIs)[4] do offer some hope for some patients, but the responsive populations have to date proved to be a very small minority of patients who are difficult to identify in advance of treatment (e.g. those patients who develop rash with erlotinib treatment[5] or the rare pancreatic cancer patients with microsatellite instability who respond to ICIs[6]). As well as the relatively low mutation burden in pancreatic cancer, that in itself makes pancreatic cancer less amenable to ICIs than other cancers, the most common mutations found in pancreatic cancer are mutations in the KRAS gene which in itself downregulates MHC-I further reducing the efficacy of this form of therapy[7].

 

References

1              Sun J, Russell CC, Scarlett CJ, McCluskey A. Small molecule inhibitors in pancreatic cancer. RSC Med Chem 2020;11:164-83.

2              Pecoraro C, Parrino B, Cascioferro S, Puerta A, Avan A, Peters GJ, et al. A New Oxadiazole-Based Topsentin Derivative Modulates Cyclin-Dependent Kinase 1 Expression and Exerts Cytotoxic Effects on Pancreatic Cancer Cells. Molecules 2021;27.

3              Pecoraro C, De Franco M, Carbone D, Bassani D, Pavan M, Cascioferro S, et al. 1,2,4-Amino-triazine derivatives as pyruvate dehydrogenase kinase inhibitors: Synthesis and pharmacological evaluation. European journal of medicinal chemistry 2023;249:115134.

4              Jiang H, Hegde S, Knolhoff BL, Zhu Y, Herndon JM, Meyer MA, et al. Targeting focal adhesion kinase renders pancreatic cancers responsive to checkpoint immunotherapy. Nat Med 2016;22:851-60.

5              Westphalen CB, Kukiolka T, Garlipp B, Hahn L, Fuchs M, Malfertheiner P, et al. Correlation of skin rash and overall survival in patients with pancreatic cancer treated with gemcitabine and erlotinib - results from a non-interventional multi-center study. BMC Cancer 2020;20:155.

6              Chida K, Kawazoe A, Suzuki T, Kawazu M, Ueno T, Takenouchi K, et al. Transcriptomic Profiling of MSI-H/dMMR Gastrointestinal Tumors to Identify Determinants of Responsiveness to Anti-PD-1 Therapy. Clin Cancer Res 2022;28:2110-7.

7              Yamamoto K, Iwadate D, Kato H, Nakai Y, Tateishi K, Fujishiro M. Targeting the Metabolic Rewiring in Pancreatic Cancer and Its Tumor Microenvironment. Cancers (Basel) 2022;14.

 Query 3: We have removed the text that had been struck through.

 

 

Reviewer 3 Report

I liked the article, I think that it will undoubtedly be of interest to readers. The design of the study is logical, the material is well and consistently presented, well-illustrated. There are a few small notes:

1. Figure 6 has unreadable inscriptions, corrections must be made.

2. In how many parallels was the study conducted? You need to add information to the Statistical Analysis section.

Author Response

We thank the reviewer for their comments.

 

1. Figure 6 has unreadable inscriptions, corrections must be made.

A higher quality version of Figure 6 has been included. On the version as we see it the inscriptions are all legible.

2. In how many parallels was the study conducted? You need to add information to the Statistical Analysis section.

We have added the following line to the statistical analysis section

All data were obtained with at least three parallel analyses.

Some analyses were carried out with more than three replicates depending on convenience, for example some protein samples were run on multiple SDS PAGE gels to allow comparison between gels, so in addition to the three minimum replicates with fully matched samples (for the specific query) there were additional replicates of partially matched samples. We think this is an unnecessarily confusing statement and the factually accurate summary statement is adequate to reassure the reader.

Round 2

Reviewer 1 Report

The authors need to consider about modifying the part of the paper that presents the seahorse figures.

Author Response

We thank the reviewer for the opportunity to add further clarity to the section showing that mutant K-Ras reduces both maximal respiration and efficiency of respiration which is compensated for by a mutant K-Ras dependent activity of N-Ras which increases efficiency of respiration and glycolysis. 

The modified text is as below

Role of wild type N-Ras in cell lines with mutant K-Ras

N-Ras has been shown to be active in all the human cell lines used in this study (Figure 1). Previous studies have shown that depleting wild type N and H-Ras in K-Ras mutant cell lines reduces EGFR responsiveness [34]. Mutant K-Ras is known to reduce oxidative phosphorylation in cancer cells relative to glycolysis[44]; this was confirmed by metabolic analysis shown in Figure 6. Depletion of K-Ras increases oxygen consumption rate (OCR) relative to extra cellular acidification rate (ECAR): in Figure 6a basal and maximum OCR is higher in the blue K-Ras depleted trace compared to the grey non-targeting trace, contrasting with higher ECAR for not-targeting in Figure 6b. OCR/ECAR is shown in Figure 6c. Although, N-Ras depletion alone had little effect, specifically a slight increase in OCR/ECAR ratio when compared to cells treated with control siRNA: OCR and ECAR are lower in the yellow N-Ras depleted traces than in the non-targeting traces in Figure 6a and 6b, resulting in rough equivalence of OCR/ECAR with non-targeting SiRNA treatment, although the N-Ras depletion does result in a slightly higher ratio after FCCP treatment. When both K and N-Ras are depleted the increase in OCR is significantly less than with depletion of K-Ras alone (Figure 6d). Further assessment of the data indicates that the increase in OCR/ECAR with K-Ras depletion seems to be due to an increase in maximum mitochondrial activity (Figure 6a) but with no increase in efficiency of respiration (Figure 6e), the OCR/ECAR increase at baseline being mainly due to a decrease in glycolysis rate (Figure 6b). In contrast N-Ras depletion causes a reduction in both maximal levels of mitochondrial activity (Figure 6a) and a decrease in the efficiency of respiration (Figure 6e). This suggests that mutant K-Ras reduces both maximal respiration and efficiency of respiration, compensated for in part by an increase in glycolysis, but also by a mutant K-Ras dependent activity of N-Ras which increases efficiency of respiration and glycolysis.