The Effect of Tobacco Smoke N-Nitrosamines, NNK and NDEA, and Nicotine, on DNA Mismatch Repair Mechanism and miRNA Markers, in Hypopharyngeal Squamous Cell Carcinoma: An In Vivo Model and Clinical Evidence

Round 1

Reviewer 1 Report

1. Line 46 - Tobacco smoking cannot be the cause of death from cancer, it can be a factor in the occurrence of cancer, which in turn leads to death. Rephrase, please. 2. Why were the NNK (0.2 mmol/L) and NDEA (0.004 mmol/L) exposure groups not studied separately? 3. It seems to me that from the point of view of methodology, it would be more correct to take groups with different concentrations of NNK and NDEA, so that the real level of exposure would be in the middle of the concentration range. When the authors look at people who have an average smoking history of 37 pack-years, their exposure to nitrosamines is higher. 4. Figure 1B, 2B are very small, it is impossible to make out the inscriptions. Please correct. 5. Figure 5 is also of poor quality. Please correct. 6. In table 3, should there be something else besides arrows? It is not clear why it is needed in the article. 7. To assess the correctness of the conclusions, we would like to see if there is a dose-dependent effect of the identified changes on the concentration of nitrosamines and nicotine.

Author Response

Reviewer #1

 

Comments and Suggestions for Authors

#1-Comment: Line 46 - Tobacco smoking cannot be the cause of death from cancer, it can be a factor in the occurrence of cancer, which in turn leads to death. Rephrase, please.

#1-Response: Thank you for the comment. We rephrased line 46 as follows: “Tobacco smoking remains a leading risk factor of devastating diseases, such as cancer.”

 

#2-Comment: Why were the NNK (0.2 mmol/L) and NDEA (0.004 mmol/L) exposure groups not studied separately?

#2-Response: We would like to thank the reviewer for this comment giving us the opportunity to further discuss our prior studies. Using an in vitro model, we have previously shown that (i) the NNK alone group, at either low or high dose, could deregulate the MMR mechanism and increase the survival of lung and head and neck cancer cells (Doukas SG, et al. Cells 2019), as well as (ii) the combination of NNK (0.2 mmol/L) and NDEA (0.004 mmol/L) could promote pre-neoplastic lesions in the in vivo exposed murine hypopharyngeal mucosa and activation of key-oncogenic pathways, like NF-kB (Vageli DP, et al. Cancer Prev Res 2022). These findings inspired us to aim this study and further explore whether this particular combination of N-Nitrosamines could cause deregulations of MMR and miRNAs expression profiles. Our current in vivo findings are in line with our previous in vitro data, showing that direct exposure to TS-related NNK can lead to lower expression of MSH2 and MLH1 (17).

We agree with the reviewer’s suggestion that further investigation on whether the individual NDEA alone group could affect the MMR mechanism or miRNA profiles at a different level. We revised our manuscript as follows:

 

Discussion (first paragraph): “We recently showed using an in vitro study that exposure of hypopharyngeal or lung cancer cells to N-Nitrosamines, like NNK, at either low or high dose, can similarly lead to deregulation of MMR genes likely due to miRNA dysregulation [17]. Based on these in vitro findings, we concluded that NNK even at a low dose can contribute to dysregulated MMR mechanism and increased cell survival rates, …... We have also recently shown that the combination of N-Nitrosamines, NNK and NDEA, at concentrations based on values previously measured in the mainstream of TS [24, 25, 26] and used in in vivo models [6, 28, 29, 30], could promote pre-neoplastic lesions in exposed murine hypopharyngeal mucosa, possibly through the activation of NF-κB and its related oncogenic pathways [6]. …...

Here, we used an in vivo murine experimental approach involving long time treatment (14 weeks) of the hypopharynx to TS-related N-Nitrosamines, NNK, and NDEA with or without nicotine, investigating for the first time whether chronic exposure to TS components can affect the MMR mechanism and miRNA markers, previously linked to carcinogenesis of the upper aerodigestive tract [19, 35, 36, 41]. We used a mixture of NNK and NDEA with or without nicotine at concentrations based on our prior in vivo model and other studies [6, 24, 25, 26, 28, 29, 30]. Our novel in vivo experimental data provides direct evidence that chronic exposure of the murine hypopharyngeal epithelium to TS components can induce a defective MMR mechanism and deregulation of cancer-related regulatory miRNA molecules in premalignant murine HM, providing new insights into the mechanism of TS-related carcinogenesis of the upper aerodigestive tract. Our current study revealed significant molecular and pathological changes after 14 weeks of treatment, especially in the groups treated with N-Nitrosamines and nicotine, encouraging us to extensively investigate the effect of individual N-Nitrosamines, NNK and NDEA, and their combination with nicotine at variable concentrations and multiple time points to reveal the step-by-step progression to carcinogenesis...”

 

Conclusions: “…Our data encourage future studies with longer exposure or variable concentrations to further elucidate a possible in vivo dose-dependent effect of individual or combined N-nitrosamines, NNK and/or NDEA, and nicotine, on the MMR mechanism….”

 

#3-Comment: It seems to me that from the point of view of methodology, it would be more correct to take groups with different concentrations of NNK and NDEA, so that the real level of exposure would be in the middle of the concentration range. When the authors look at people who have an average smoking history of 37 pack-years, their exposure to nitrosamines is higher.

#7-Comment: To assess the correctness of the conclusions, we would like to see if there is a dose-dependent effect of the identified changes on the concentration of nitrosamines and nicotine.

 

3 # 7-Responses: We provided together our responses for comment #3 [to take groups with different (lower) concentrations of N-Nitrosamines] and comment #7 (to explore a possible dose-dependent effect of nitrosamines and nicotine), as follows:

 

#3 # 7-Responses: We would like to thank again the reviewer for these suggestions. We have revised our conclusions.  We have mentioned above (2# Response) that we have previously shown in vitro that NNK, which is known tobacco smoke-carcinogen, at either low or high dose, can similarly deregulate the MMR gene expression profiles in both lung and head and neck cancer cells, concluding that even low dose of NNK can contribute to MMR deregulation and increased cell survival rates (Doukas SG, et al. Cells 2019). These data indicate a non-dose-dependent effect of TS-related N-Nitrosamines, such as NNK.

A previous study by our team also showed that the chronic effect of N-nitrosamines at concentrations based on values previously measured in the mainstream of TS [refs: 24, 25, 26] and used in in vivo models [refs: 6, 28, 29, 30], could promote precancerous lesions in the in vivo exposed murine hypopharyngeal mucosa, while the combination of N-Nitrosamines with other risk factors, such as bile reflux, is particularly noxious, as it could accelerate the carcinogenic process by causing invasive cancer (Vageli DP, et al. Cancer prev Res 2022). Here we explored for the first time, the in vivo chronic effect of N-Nitrosamines (NNK and NDEA), based on our prior in vivo model and other studies. We showed that N-Nitrosamines at a total dose similar to those of a 37-pack-year tobacco smoker could induce dysplastic changes, reduction of MMR gene expression, and upregulation of “oncomirs”, while the combination of this mixture of N-Nitrosamines with nicotine could promote more severe dysplastic lesions and a stronger reduction of MMR gene expression or “oncomirs” upregulation, compared to N-nitrosamines alone.

Based on our current and prior data a possible additive effect of N-Nitrosamines with nicotine or other risk factors could be suggested, while the dose-dependent effect of NNK is not clear. We agree that our current findings strongly suggest large-scale in vivo investigations to further clarify whether variable doses of N-Nitrosamines with or without nicotine or other risk factors could differentially affect MMR gene expression profiles and produce similar histopathological results. Following your insightful suggestion, we added in the “Discussion” (first paragraph) and “Conclusions” sections the following:

 

 

Discussion (first paragraph): “…We have recently shown using an in vitro study that exposure of hypopharyngeal or lung cancer cells to N-Nitrosamines, like NNK, at either low or high dose, can similarly lead to deregulation of MMR genes likely due to miRNA dysregulation [17]. Based on these in vitro findings, we concluded that NNK even at a low dose can contribute to dysregulated MMR mechanism and increased cell survival rates, indicating a non-dose-dependent effect of TS-related N-Nitrosamines (Doukas SG, et al. Cells 2019). We have also recently shown that the combination of N-Nitrosamines, NNK and NDEA, at concentrations based on values previously measured in the mainstream of TS [24, 25, 26] and used in in vivo models [6, 28, 29, 30], could promote pre-neoplastic lesions in the in vivo exposed murine hypopharyngeal mucosa, possibly through the activation of NF-κB and its related oncogenic pathways (6). On the other hand, we have shown that the combination of tobacco smoke N-Nitrosamines with other risk factors, such as bile reflux, was found particularly noxious, as it could accelerate the carcinogenic process by causing invasive cancer [6].  Here, we used an in vivo murine experimental approach involving long time treatment (14 weeks) of the hypopharynx to TS-related N-Nitrosamines, NNK and NDEA with or without nicotine, investigating for the first time whether chronic exposure to TS components can affect the MMR mechanism and miRNA markers, previously linked to carcinogenesis of the upper aerodigestive tract [19, 35, 36, 41]. We used a mixture of NNK and NDEA with or without nicotine at concentrations based on our prior in vivo model and other studies (6). Our novel in vivo experimental data provide direct evidence that chronic exposure of the murine hypopharyngeal epithelium to TS components can induce a defective MMR mechanism and deregulation of cancer-related regulatory miRNA molecules in premalignant murine HM, providing new insights into the mechanism of TS-related carcinogenesis of the upper aerodigestive tract (Fig. 8). Our current study revealed significant molecular and pathological changes after 14 weeks of treatment, especially in the groups treated with N-Nitrosamines and nicotine, encouraging us to extensively investigate the effect of individual N-Nitrosamines, NNK and NDEA, and their combination with nicotine at variable concentrations and multiple time points to reveal the step-by-step progression to carcinogenesis...”

 

Conclusion: “…Our data encourage future studies with longer exposure or variable concentrations to further elucidate a possible in vivo dose-dependent effect of individual or combined N-nitrosamines, NNK and/or NDEA, and nicotine, on the MMR mechanism.”

 

#4-Comment: Figure 1B, 2B are very small, it is impossible to make out the inscriptions. Please correct.

#4-Response: We appreciate the suggestion. We revised Figures 1B and 2B, accordingly, providing larger graphs to better present our data.

 

#5-Comment: Figure 5 is also of poor quality. Please correct.

#5-Response: Following the reviewer’s suggestion, we present our data in a revised Figure 5 and in a new Figure 6, providing graphs of higher quality.

 

#6-Comment: In table 3, should there be something else besides arrows? It is not clear why it is needed in the article.

#6-Response: Thank you for noticing that. We have removed the arrows and replaced them with values to illustrate better our data.

 

 

 

Reviewer 2 Report

 

Doukas et al. investigated the effects of some tabaco components on DNA Mismatch repair mechanism and miRNA markers and found the similar phenomenon in human species. This manuscript is very interesting and supported by solid data. however, some data presentation and minor comments should be carefully addressed before publication.

The detailed comments are listed as follows:

1/Table 1. Experimental and control groups of wt-C57BL/6J. The definition for the symbols “-/*” in this table should be explained.

2/The pattern for figure 1-2 can be modified to make it look beautiful.

3/The ratio value in figure 4-5 should be included as “Value + SD”.

4/The table 3 should be changed.

5/The protein levels (like western blot analysis) for some cancer-related miRNA markers should be detected.

6/A scheme should be provided for this manuscript.

7/I wonder whether the longer exposure (or higher concentration) of NNK lead to the cancer development in mice. Did author observe the real cancer development on mice via biopsy except for gene lever change?

Author Response

Reviewer #2 

Doukas et al. investigated the effects of some tobacco components on DNA Mismatch repair mechanism and miRNA markers and found the similar phenomenon in human species. This manuscript is very interesting and supported by solid data. however, some data presentation and minor comments should be carefully addressed before publication.

The detailed comments are listed as follows:

#1-Comment: /Table 1. Experimental and control groups of wt-C57BL/6J. The definition for the symbols “-/*” in this table should be explained.

#1-Response: Thank you for the comment. The definition for the symbols “-/*” in Table 1 has been explained, as follows:

“-/*, component of the experimental or control fluid.”

 

#2-Comment: /The pattern for figure 1-2 can be modified to make it look beautiful.

#2-Response: We appreciate the suggestion. We revised Figure 1B and 2B, accordingly providing larger graphs to better present our data.

 

#3-Comment: /The ratio value in figure 4-5 should be included as “Value + SD”.

#3-Response: Following the reviewer’s suggestion we have included in Figs 3,4 and 5 ratio values of mRNA or miRNA expression levels as “mean ± SD”.

 

#4-Comment: /The table 3 should be changed.

#4-Response: Thank you for the suggestion. We have revised Table 3, removing the arrows and replacing by values to better illustrate our data.

 

#5-Comment: /The protein levels (like western blot analysis) for some cancer-related miRNA markers should be detected.

#5-Response: Following the reviewer’s suggestion, we have included a new Figure 7, which shows the protein levels of MSH2 (by Image scope quantified as total protein levels) for “oncomir” miR-21, as was found to have an inverse correlation (r=-9856, p=0.0072). We would like to note that microRNA markers are small non-coding RNA molecules that regulate gene expression, hence they are analyzed by qPCR analysis rather than quantitative protein analysis.        

 

#6-Comment: /A scheme should be provided for this manuscript.

#6-Response: Following the reviewer’s suggestion we provided a scheme in Figure 8.

 

#7-Comment: /I wonder whether the longer exposure (or higher concentration) of NNK lead to the cancer development in mice. Did author observe the real cancer development on mice via biopsy except for gene lever change?

#7-Response: We would like to thank the reviewer for the insightful comment. (i) We have previously shown in vitro that NNK at either low or high doses can similarly deregulate MMR gene expression profiles and increased survival rates of both lung and head and neck cancer cells, concluding that TS-related N-Nitrosamines. (Doukas SG, et al. Cells 2019). (ii) We have also previously shown that 12-week exposure to NNK and NDEA at concentrations based on values previously measured in the mainstream of TS  [refs: 24, 25, 26] and used in in vivo models [refs: 6, 28, 29, 30] can promote precancerous lesions in murine exposed hypopharyngeal mucosa and transcriptional activation of oncogenic factors, such as NF-kB, STAT3, IL6 etc, while the combination of NNK-NDEA with other risk factors, such as bile reflux is particularly noxious, as it could accelerate the carcinogenic process by causing invasive cancer (Vageli DP, et al. Cancer prev Res 2022). Here, using similar concentrations of NNK and NDEA, we showed for the first time in vivo that the 14-week exposure of hypopharyngeal mucosa to N-nitrosamines can dysregulate the MMR mechanism and produce premalignant lesions.

 

In view of the cancer development on tissue biopsies, we have included in Supplementary Fig S1 histologic examination of tissue sections from biopsies of murine hypopharyngeal mucosa with 14-week exposure to N-nitrosamines (NNK and NDEA) in dose similarly found in a 37-pack-year tobacco smoker. These findings revealed a dysregulated MMR mechanism in premalignant murine hypopharyngeal mucosa, while their combination with nicotine promoted more severe dysplastic lesions and a stronger reduction of MMR gene expression or “oncomirs” upregulation, compared to N-nitrosamines alone.

 

Our current and prior data support a possible additive effect of N-Nitrosamines with nicotine or other risk factors while a dose-dependent effect is not clear. We believe that future large-scale studies with longer exposure or variable concentrations would further clarify a possible time or dose-dependent effect of NNK and/or NDEA, with or without nicotine, on the MMR mechanism in vivo.

 

 

Discussion (first paragraph): “…We have recently shown using an in vitro study that exposure of hypopharyngeal or lung cancer cells to N-Nitrosamines, like NNK, at either low or high dose, can similarly lead to deregulation of MMR genes likely due to miRNA dysregulation [17]. Based on these in vitro findings, we concluded that NNK even at a low dose can contribute to dysregulated MMR mechanism and increased cell survival rates, indicating a non-dose-dependent effect of TS-related N-Nitrosamines. We have also recently shown that the combination of N-Nitrosamines, NNK and NDEA, at concentrations based on values previously measured in the mainstream of TS [24, 25, 26] and used in in vivo models [6, 28, 29, 30], could promote pre-neoplastic lesions in the in vivo exposed murine hypopharyngeal mucosa, possibly through the activation of NF-κB and its related oncogenic pathways [6]. On the other hand, we have shown that the combination of tobacco smoke N-Nitrosamines with other risk factors, such as bile reflux, was found particularly noxious, as it could accelerate the carcinogenic process by causing invasive cancer [6].  Here, we used an in vivo murine experimental approach involving long time treatment (14 weeks) of the hypopharynx to TS-related N-Nitrosamines, NNK and NDEA with or without nicotine, investigating for the first time whether chronic exposure to TS components can affect the MMR mechanism and miRNA markers, previously linked to carcinogenesis of the upper aerodigestive tract [19, 35, 36, 41]. We used a mixture of NNK and NDEA with or without nicotine at concentrations based on our prior in vivo model and other studies [6, 24, 25, 26, 28, 29, 30]. Our novel in vivo experimental data provides direct evidence that chronic exposure of the murine hypopharyngeal epithelium to TS components can induce a defective MMR mechanism and deregulation of cancer-related regulatory miRNA molecules in premalignant murine HM, providing new insights into the mechanism of TS-related carcinogenesis of the upper aerodigestive tract. Our current study revealed significant molecular and pathological changes after 14 weeks of treatment, especially in the groups treated with N-Nitrosamines and nicotine, encouraging us to extensively investigate the effect of individual N-Nitrosamines, NNK and NDEA, and their combination with nicotine at variable concentrations and multiple time points to reveal the step-by-step progression to carcinogenesis...”

 

Conclusion: “Our study provides first-time insights that NNK-NDEA combined with or without nicotine produces downregulation of MSH2 and MLH1 MMR genes likely via miRNA dysregulation in dysplastic hypopharyngeal mucosa, and further support the role of NF-κB in TS-associated MMR dysfunction and carcinogenesis in the upper aerodigestive tract. Our data encourage future studies with longer exposure or variable concentrations to further elucidate a possible in vivo dose-dependent effect of individual or combined N-nitrosamines, NNK and/or NDEA, and nicotine, on the MMR mechanism.”

 

 

Round 2

Reviewer 1 Report

The authors gave detailed responses to the reviewer's comments and substantially revised the manuscript. I think that in its present form the article can be recommended for publication.