An unconventional cancer-promoting function of methamphetamine in hepatocellular carcinoma

METH use is associated with a higher incidence of hepatocellular carcinoma (HCC) in METH abusers and rehabilitees. METH exposure promotes HCC progression by ROS-mediated activation of the Ras/MEK/ERK signaling pathway. Clearance of ROS by NAC abolishes METH-induced HCC progression.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** showed side effect of methamphetamine to promote liver cancer. The author needs to address the following concerns.
1. Only two cancer cell lines Huh7 and HepG2 are not enough to substantiate the conclusion of METH to promote tumor. Is the 1nM concentration biologically relevant to the dose used by human? 2. Brdu proliferation did not correlate with MTT assays. Brdu showed highly significant proliferation at 10nM OR 100nm in Huh7 or HepG2 in Fig 1.? It is important to show Ki67 in western blot to substantiate the data of MTT and Brdu assays.
3.pERK is not convincing about inducible phosphorylation upon METH treatment. Phosphoprotein (pERK)is more than total protein in Fig4.
Reviewer #2 (Comments to the Authors (Required)): Si, Yang et al., submitted the paper untitled "An unconventional cancer-promoting function of Methamphetamine in Hepatocellular carcinoma" for reviewing in Life Science Alliance. The authors state the effect of methamphetamine (METH) in hepatocellular Carcinoma (HCC) progression. METH are a really well-known substance causing oxidative stress, extensive DNA damage and inflammatory response. Literature have reported a correlation between METH uptake and hepatic failure or cancer. This study highlights the effect of METH up-taking on HCC through ROS-mediated Ras activation.
To do so, the authors build a robust clinical cohort for their studies and correlate METH uptake with liver cancer incidence. Combination of in vitro and in vivo experiments on Huh7 and HepG2 cell line show an effect of METH on tumour growth, cell proliferation, migration and invasion through ROS-regulated Ras/MEK/ERK signalling pathway. The authors highlight this study as the first one providing evidence between METH and HCC. Methamphetamine use can therefore be included as a new risk factors for HCC.
1. Meth use was associated with a higher incidence of liver cancer.
-The title must be written in the present tense: METH use is associate... -In Figure 1 and Table 1, the authors merge METH users and METH rehabilitees. Results are already significant but in order to strengthen the data and the analysis, can the authors provide METH rehabilitees data independently of METH users data? -Still in order to strengthen the results, the 2 non-METH users with HCC belong to the HBV/HCV subgroups, alcoholic subgroups or none? 2. METH promotes cancer-associated properties in human liver cancer cells.
General comment for Figure 2 and 3: All the experiments are performed with Huh7 and HepG2 cell line. These 2 cell lines are well-differentiated cells corresponding to early HCC stage. The authors should consider the possibility of repeating some key experiments with poorly differentiated HCC cell line corresponding to late HCC stage (by using for example HLE, HLE or SNU-449 cell line). These results can be added as supplemental figures.
-The authors base the mechanistical study on the increase of cell proliferation treated with METH. MTT assays and BrdU assays are complementary as MTT measure mitochondrial activity therefore viability and BrdU measure DNA synthesis, therefore cell proliferation. The authors need to stress the complementarity of the 2 tests in their manuscript. Viability is different that proliferation. -To increase the robustness of the data, apoptosis data should be added. If METH increase proliferation and viability, no apoptosis markers should be seen. The authors should check cleaved caspase 3 either by Western Blot or Immunofluorescence in the same conditions tested so far.
- Figure 2E and 2F: In addition of the colony number, the size of the colony should be measured. Representative picture of the colonies for each condition should be added. - Figure 2G: For invasion assay, authors used transwell chamber assay with serum gradient. This experiment trigger also chemotaxic behaviour. It is difficult to conclude only about invasion with this assay. In addition of the transwell assay, the authors should perform 3D invasion assay in Matrigel. The area of the spheroids will be measured overtime. Pictures of the spheroids will be added in addition of the quantification. -Migration assays: Transwell assay is not sufficient to analyse migration properties. Wound healing assays should be performed in the same conditions. Percentage of wound recovery will be reported, as well as representative pictures.
-Most importantly, the authors show in Figure 1, an increase of the proliferative capacities of the cells treated with METH. To confirm that the effect seen in Figure 2G,H,I and J are indeed invasion and migration and not proliferation, experiments needs to be performed in presence of a proliferation inhibitor like Mitomycine C.
- Figure 2I and J representing migration capacities should be put before the invasion results.
3. METH exposure activates Ras-ERK pathways in human liver cancer cells.
-The authors show by Western Blot an upregulation of Ras after treatment of the cells with METH. The quality of some revelations needs to be increase. For example, put better exposure of Ras Figure 3C and Ras Figure 3G. -The authors checked by Western Blot the activation of the downstream components ERK and MEK. The Ras/ERK pathway cannot be confirmed if alternative pathways are not checked. Among the several pathways that needs to be analysed, the authors should look at least by western blot: PI3K, p38, MAPkinase, ... -The quality of some western blot needs to be increase: Fig4A: MEK (less exposed), Fig 4C P-ERK and P-MEK (less exposed).
- Figure 4B and 4D: There are too many data on the same graphic. The authors should represent the graphic with 1 graphic per each protein checked.
4. ROS regulates Ras-ERK pathway in human liver cancer cells with METH exposure.
- Figure 5E: The quality of the western blot panel needs to be increase - Figure 5H and Figure 5I: As mentioned previously, invasion and migration should be performed in presence of an inhibitor of proliferation.
5. METH exposure promotes xenograft tumor formation through ROS-induced Ras activation in vivo.
-To validate the influence of METH on liver cancer progression, the authors injected HepG2 cells subcutaneously in nude mice. Due to the importance of tumour microenvironment in HCC and because of the sensitive parameters measured by the authors (Ros levels), the authors must in addition, inject the cells orthotopically to analyse ROS levels by Flow Cytometry. -After Figure 6B, a figure with tumour weight should be put to confirm the observation made in Figure 6B. -Panel of Figure 6 is difficult to follow. Some reorganisations of the panel should be done. Figure 6B should be the tumour weight of the mice presented in 6B. Not the tumour weight from mice treated with NAC. -As the tumour growth rapidly with METH 0,5 and 5µg/kg, we expect to see fibrosis appearance. The authors should document the tumours by performing tumours Eosin/hematoxylin and Masson's trichrome colorations on tumours sections in order to identify fibrosis events.

Reviewer #1:
Zizhen Sia et al manuscript"An Unconventional Cancer-Promoting function of Methamphetamine in Hepatocellular Carcinoma" showed side effect of methamphetamine to promote liver cancer.
The author needs to address the following concerns.

3.pERK is not convincing about inducible phosphorylation upon METH treatment.
Phosphoprotein (pERK)is more than total protein in Fig4.

REPLY:
Dear reviewer, thank you very much for your valuable suggestion. As suggested, we have put less exposed western blot in Figure 4 as follow:

Reviewer #2:
Si, Yang et al., submitted the paper untitled "An unconventional cancer-promoting function of Methamphetamine in Hepatocellular carcinoma" for reviewing in Life Science Alliance. 1. Meth use was associated with a higher incidence of liver cancer.
-The title must be written in the present tense: METH use is associate...

REPLY:
Dear reviewer, thank you for the precious suggestion. As suggested, we have corrected the title in the Results section in RED as follow: 3.1 METH use is associated with a higher incidence of liver cancer -In Figure 1 and Table 1, the authors merge METH users and METH rehabilitees. Results are already significant but in order to strengthen the data and the analysis, can the authors provide METH rehabilitees data independently of METH users data?
REPLY: Dear reviewer, thank you for the precious suggestion. As suggested, we have provided METH rehabilitees data independently of METH users data in the Supplementary Materials section in RED as follow: A total of 218 participants were included. Among these, 103 were diagnosed as METH users or METH rehabilitees (Table S1). -Still in order to strengthen the results, the 2 non-METH users with HCC belong to the HBV/HCV subgroups, alcoholic subgroups or none?
REPLY: Dear reviewer, thank you for the precious suggestion. As suggested, we have provided non-METH users data in the Supplementary Materials section in RED as follow and the results showed that 1 non-METH users with HCC belong to the HBV subgroups, and 2 non-METH users belong to alcoholic subgroups.
A total of 218 participants were included. Among these, 103 were diagnosed as METH users or METH rehabilitees (Table S1), and 115 were age and gender-matched normal subjects with no history of METH abuse (Table S2). 2. METH promotes cancer-associated properties in human liver cancer cells.
General comment for Figure  We repeated some key experiments with poorly differentiated HCC cell line corresponding to late HCC stage (by using HLE cell line) and the results were similar to that in HUH7 cells and HepG2 cells (Fig. S1). -The authors base the mechanistical study on the increase of cell proliferation treated with METH.
MTT assays and BrdU assays are complementary as MTT measure mitochondrial activity therefore viability and BrdU measure DNA synthesis, therefore cell proliferation. The authors need to stress the complementarity of the 2 tests in their manuscript. Viability is different that proliferation.

REPLY:
Dear reviewer, thank you for the precious suggestion. We have modified Result 3.2 to address this issue in RED, as shown below: Then, the cell viability was determined by MTT assays and cell proliferation ability was determined by BrdU assays. The MTT assay showed that treatment with low concentrations of METH significantly promoted cell viability in HUH7 cells (0.1, 1, or 10 nM) and HepG2 cells (1, 10 or 100nM) ( Fig. 2A, B). Then, HUH7 or HepG2 cells were exposed to METH for 72 hours and underwent the BrdU assay. As shown in Fig. 1C, D, low concentrations of METH promoted cell proliferation in HUH7 cells (0.1, 1, or 10 nM) and HepG2 cells (1, 10 or 100nM) (Fig. 2C, D).
-To increase the robustness of the data, apoptosis data should be added. If METH increase proliferation and viability, no apoptosis markers should be seen. The authors should check cleaved caspase 3 either by Western Blot or Immunofluorescence in the same conditions tested so far.  Western blot was used to detect the protein levels of cleaved caspase 3 in HepG2 cell. *P < 0.05.

REPLY
- Figure 2E and 2F: In addition of the colony number, the size of the colony should be measured.
Representative picture of the colonies for each condition should be added.

REPLY:
Dear reviewer, thank you for the precious suggestion. Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony size and representative picture for each condition have been added in the Results section in RED according to your suggestion as follow: Consistently, the colony formation assay showed that HUH7 cells and HepG2 cells treated with low concentrations of METH could form more colonies compared to the ddH2O control group - Figure 2G: For invasion assay, authors used transwell chamber assay with serum gradient. This experiment trigger also chemotaxic behaviour. It is difficult to conclude only about invasion with this assay. In addition of the transwell assay, the authors should perform 3D invasion assay in Matrigel. The area of the spheroids will be measured overtime. Pictures of the spheroids will be added in addition of the quantification.

REPLY:
Dear reviewer, thank you very much for your valuable suggestion. We also carefully considered the chemotaxis of the HUH7 and HepG2 cells when implementing the invasion assay.
To avoid this, for each condition the media in upper chamber and lower chamber both had same concentration of METH. In this situation, the METH gradient did not establish and chemotaxic behaviour was not induced. Thus, we believe that more invasive cells observed in METH groups compared to control groups was resulted from enhanced invasion capabilities stimulated by METH.
-Migration assays: Transwell assay is not sufficient to analyse migration properties. Wound healing assays should be performed in the same conditions. Percentage of wound recovery will be reported, as well as representative pictures.

REPLY:
Dear reviewer, thank you very much for your constructive comments to strengthen this manuscript. As suggested, wound healing assays were performed in the same conditions. The migration speed and representative picture for each condition have been added in the Results section in RED as follow: Further, the wound healing assay also showed the same result ( Fig. S4 A-D). (mean ± SD. of three independent experiments. *P < 0.05; **P < 0.01).
- Figure 2I and J representing migration capacities should be put before the invasion results.

REPLY:
Dear reviewer, thank you very much for your valuable suggestion. As suggested, the migration results have been put before the invasion results in Figure 2 as follow: (mean ± SD. of three independent experiments. *P < 0.05; **P < 0.01).
3. METH exposure activates Ras-ERK pathways in human liver cancer cells. -The quality of some western blot needs to be increase: Fig4A: MEK (less exposed), Fig 4C P-ERK and P-MEK (less exposed).

REPLY:
Dear reviewer, thank you very much for your valuable suggestion. As suggested, we have put less exposed western blot in Figure 4 as follow: - Figure 4B and 4D: There are too many data on the same graphic. The authors should represent the graphic with 1 graphic per each protein checked.

REPLY:
Dear reviewer, thank you very much for your valuable suggestion. As suggested, we have represented the graphic with 1 graphic per each protein checked in Figure 4 as follow: 4. ROS regulates Ras-ERK pathway in human liver cancer cells with METH exposure.
- Figure 5E: The quality of the western blot panel needs to be increase REPLY: Dear reviewer, thank you very much for your valuable suggestion. As suggested, we have put better western blot panels in Figure 5 as follow: - Figure 5H and Figure 5I: As mentioned previously, invasion and migration should be performed in presence of an inhibitor of proliferation.

REPLY:
Dear reviewer, thank you very much for your constructive comments to strengthen this manuscript. As suggested, 10 μg/mL Mitomycine C was applied to the cells and the results have been added in the Results section in RED as follow: We appreciate for that! -After Figure 6B, a figure with tumor weight should be put to confirm the observation made in Figure 6B.

REPLY:
Dear reviewer, thank you very much for your constructive comments to strengthen this manuscript. As suggested, a figure with tumor weight has been put to confirm the observation made in Figure 6B as follow. -Panel of Figure 6 is difficult to follow. Some reorganisations of the panel should be done. Figure   6B should be the tumour weight of the mice presented in 6B. Not the tumour weight from mice treated with NAC.

REPLY:
Dear reviewer, thank you very much for your constructive comments to strengthen this manuscript. As suggested, we have reorganized Figure 6 as follows. Cytometry was used to detect the ROS level in tumors. (F, G) NAC was applied to inhibit the level of METH induced-ROS in vivo, and tumor volume was measured every 7 days (mean ± SEM. n =5/group). *P < 0.05; **P < 0.01.
-As the tumour growth rapidly with METH 0,5 and 5µg/kg, we expect to see fibrosis appearance. Thank you for submitting your revised manuscript entitled "An Unconventional Cancer-Promoting function of Methamphetamine in Hepatocellular Carcinoma". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. Author addressed all the concerns.
Reviewer #2 (Comments to the Authors (Required)): The authors Sia, Yang et al. submitted the manuscript untitled "An unconventional cancer-promoting function of Methamphetamine in Hepatocellular Carcinoma" after performing the revisions asked by the reviewers.
In general, the authors have correctly answered the questions and additional experiments that were requested. The quality of the manuscript is now enhanced compared to the first submission. Nevertheless, the authors could have taken more time and performed the requested orthotopic injections. It is difficult to convince the readers of the effect of METH in tumour progression if the in-vivo experiments are not more extensive. In case the technique is not established in the lab, a collaboration could have been established with an expert research team. The data obtained after orthotopic injection of the cells and flow cytometric analysis of the ROS would have considerably reinforced the message of the paper.
-please upload your main and supplementary figures as single files; please upload your table files as separate editable doc or excel files, or make sure that they are in the doc file of your main manuscript.

REPLY:
Dear editor, thank you for the precious suggestion. We have uploaded our main and supplementary figures as single files and uploaded table files as separate editable excel files.
-please add the Twitter handle of your host institute/organization as well as your own or/and one of the authors in our system REPLY: Dear editor, thank you for the precious suggestion. We are sorry that our institute/organization do not have Twitter account. -please add a callout for Figure 4H and Figure S2 A,B to your main manuscript text REPLY: Dear editor, thank you for the precious suggestion. We have checked the callouts for Figure 4H and Figure S2 A,B in the main manuscript text in RED.
-please add sizes next to all blots REPLY: Dear editor, thank you for the precious suggestion. We have added sizes next to all blots.