Mutant Kras- and p16-regulated NOX4 activation overcomes metabolic checkpoints in development of pancreatic ductal adenocarcinoma

Kras activation and p16 inactivation are required to develop pancreatic ductal adenocarcinoma (PDAC). However, the biochemical mechanisms underlying these double alterations remain unclear. Here we discover that NAD(P)H oxidase 4 (NOX4), an enzyme known to catalyse the oxidation of NAD(P)H, is upregulated when p16 is inactivated by looking at gene expression profiling studies. Activation of NOX4 requires catalytic subunit p22phox, which is upregulated following Kras activation. Both alterations are also detectable in PDAC cell lines and patient specimens. Furthermore, we show that elevated NOX4 activity accelerates oxidation of NADH and supports increased glycolysis by generating NAD+, a substrate for GAPDH-mediated glycolytic reaction, promoting PDAC cell growth. Mechanistically, NOX4 was induced through p16-Rb-regulated E2F and p22phox was induced by KrasG12V-activated NF-κB. In conclusion, we provide a biochemical explanation for the cooperation between p16 inactivation and Kras activation in PDAC development and suggest that NOX4 is a potential therapeutic target for PDAC.

In this manuscript the authors report on the role of KRas activation and the role of NOX4 in malignant transformation of HPNE cells. They report on the dole of the increased NOX4 activity that appears linked to overcoming metabolic checkpoints, resulting in formation of pancreatic ductal carcinomas. This is an interesting paper that needs some additional work, as indicated below. The authors show that Kras results in increased p16, and that this protein needs to be silenced for the HPNE cells to undergo malignant transformation. If this is so, how would Kras result in malignant transformation in patients? Do they have silenced p16? Or, is it known to be silenced in models of malignant transformation of normal cells? What is the pathophysiological context of the current research? It would be interesting (and should be checked) whether p16 is 'silenced' in premalignant lesions. More realistic models should be used, the HPNE cells with silenced p16, albeit providing interesting results, are an artificial system. Although there are references to the link of p16 suppression and malignant transformation, the authors should show this in the context of their research. On a more general level, the authors had shown before that KRas causes a switch in cells from respiration to glycolysis. They should refer here to work by others, since this topic has been covered extensively in the past, and is rather controversial (e.g. Weinberg et al PNAS 2010, 1078. There is no attempt in the report to characterize respiration of the manipulated cells. That is, HPNE KRas-transfected and p16-sulenced cells. This should be included. The results in Fig. 6 concerning tumour formation are insufficient. There is only the endpoint results, tumour weight, documenting the differences. The authors should show kinetics of tumour progression. They should also analyse the tumours derived from control and shNOX4 cells for glycolysis and respiration. Reviewer #2: Expert in PDAC and Ras signalling (Remarks to the Author): The current manuscript by Dr Chiao and colleagues addresses the role of Nox4 in pancreatic cancer. Using HPNE and HPDE cells, they show that Nox4 expression is up-regulated upon activation of oncogenic Kras and concomitant loss of the tumor suppressor p16. Further, they show that the catalytic subunit of Nox4, p22PHOX , is expressed downstream of oncogenic Kras through activation of NFkappaB. They further show that Nox4 overexpression is detected in human pancreatic cancer samples, and that it mediates metabolic reprogramming that is characteristic of pancreatic cancer. Finally, the authors provide evidence that Nox4 inhibition is cytostatic in pancreatic cancer cell lines, and that the Nox4 inhibitor DPI increases survival in the iKras mouse model of pancreatic cancer. The manuscript investigates a timely topic, namely metabolic reprogramming in pancreatic cancer and its potential therapeutic implications. Some additional analysis could strengthen the undelying message.
1) The authors propose that Nox4 overexpression requires concomitant loss of p16 and activation of oncogenic Kras. They should determine whether this holds true in the panel of human cell lines and tissue samples that are included in Figure 2A, B. In addition, they should investigate the expression of Nox4 in iKras mouse tumors ( Figure 6), which have Kras expression combined with inactivation of p53, rather than p16. 2) In Figure 4, the authors should determine whether modulation of Kras and its downstream effectors alters the expression of Nox4, in addition to p22PHOX.
3) The data indicating reduced tumor growth upon inactivation of Nox4 by shRNA could be strengthened by the use of an inducible shRNA, which would allow to determine whether the effect of Nox4 inactivation is due to a defect at the implantation stage or in the maintenance/growth of established tumors. 4) The increased survival in iKras mice ( Figure 6G) is impressive, but would benefit from further analysis. It appears that the survival curve is pushed to the right, yet once the mice start dying the slope is similar as to the control. At the very least histological analysis of the tumor should be performed to determine whether resistance mechanisms have been established. An easy readout would be to determine whether the proliferation index has rebounded. Another possibility Is that the tumor grow more slowly, thus reach the critical point later. Understanding a potential mechanism of resistance would be important to assess any therapeutic outcome. 5) On a different note, related to the iKras experiment, the authors should comment on the specificity of DPI as a Nox4 inhibitor.

Reviewers' comments:
Reviewer #1: Expert in cancer metabolism (Remarks to the Author): In this manuscript the authors report on the role of KRas activation and the role of NOX4 in malignant transformation of HPNE cells. They report on the dole of the increased NOX4 activity that appears linked to overcoming metabolic checkpoints, resulting in formation of pancreatic ductal carcinomas. This is an interesting paper that needs some additional work, as indicated below.
Response: Thank you very much for reviewing our manuscript. We are very glad to know that the reviewers thought our findings are interesting and significant. We appreciate the reviewers' questions and comments, which have been replied with a point by point response. All changes are identified by page and paragraph locations, and noted by highlight or strikethrough in the text. Our detailed responses are as follows.   "Recent studies by our group and others showed that Kras activation led to suppression of mitochondrial respiratory activity and rendered the cell more dependent on glycolysis [1,2]. Conversely, others reported that mitochondrial ROS generation is essential for Krasinduced cell proliferation and Kras-mediated tumorigenicity [3]. Dysfunctional mitochondria and increased aerobic glycolysis are two important biochemical characteristics observed frequently in cancer cells [1,4,5]. A metabolic switch from oxidative phosphorylation in the mitochondria to glycolysis in the cytosol in cancer cells has been well known as "Warburg effect" for decades [6,7]." To strengthen our experimental evidence, we performed metabolite isotope tracing experiments with 13 carbon labeled glucose (U-13 C 6 Glu). We found that overexpression of NOX4 and Kras G12V /shp16 in HPNE cells increased pyruvate and lactate level, thereby confirming the increased glucose to lactate conversion or glycolysis. Although the data from the described tracing experiments are not requested directly by reviewers, the results strengthen our finding and conclusion.

1) The authors show that
So, these results are added in our revised Figure

Reviewer #2: Expert in PDAC and Ras signalling (Remarks to the Author):
The Response: Thank you very much for your review of our manuscript. Thank you for your comments and the following suggestion to improve our study. All changes are identified by page and paragraph, and noted by strikethrough or highlight in the text.
Below are our detailed point by point responses. Figure   2A, B. In addition, they should investigate the expression of Nox4 in iKras mouse tumors (Figure 6), which have Kras expression combined with inactivation of p53, rather than p16. Supplementary Figure 1 (b) The expression of p16 in PDAC cell lines and tissues was analyzed by qPCR assay. Data in B are presented as mean ± SD (n=3). Supplementary  Figure 2 (b) Representative IHC staining with H&E or anti-NOX4 antibodies in sections of formalin-fixed PDAC from transgenic iKras; p53 L/+ mice.

Response:
That is a good point and well taken. As suggested, we firstly detected the expression profile of p16 in 12 PDAC cell lines and 21 human PDAC tissues by qPCR assay. We found that the mRNA level of p16 was only detected in one of 21 patient PDAC samples, and only expressed in AsPc-1, Colo357 and Capan-2 cell lines ( Supplementary Fig. 1b). These results demonstrated that p16 is mutational inactivated in most PDAC. However, even p16 is high expressed in the Colo357 and Canpan-2 cells, the NOX4 expression level was still high. Considering that gene transcription is a complicated process, it is likely that other transcription factors and co-activators are involved in the regulation of NOX4. P53 are reported to regulate NOX4 expression in lung and breast cancer (Br J Cancer. 2014 May 13;110 (10):2569-82). As known, p53-regulated p21 expression is decreased when p53 is knocked out. As the result, CDK4 is activated and phosphorylates Rb, thus, leading to activation of E2F. Indeed, IHC staining indicated that the expression of NOX4 was substantially higher in these PDAC from iKras; p53 L/+ mouse model than in histologically normal pancreata from control mice (Supplementary Fig. 2b). These above results are shown in Supplementary Fig. 1b and Fig. 2c and described in our revised manuscript (Page 5, paragraph 2; Page 8, paragraph 2; Page 13, paragraph 1). Figure 4

Response:
We thank the reviewer for the suggestion. We firstly established the inducible NOX4-konckdown cell line (AsPc-1/i-shNOX4). As shown, addition of doxycycline to the culture medium induced the knockdown of NOX4 protein in AsPc-1/i-shNOX4 cells detected by immunoblotting (Supplementary Fig. 5D). Then, ten mice were orthotopically injected with AsPc-1/i-shNOX4 cells and randomly assigned to two groups (Dox/on and Dox/off). The mice of "Dox/on" group were fed with doxycontaining water from 2 weeks after inoculation. Compared with the control group after 4 weeks of dox treatment, the "Dox/on" group mice showed a significant reduction in tumor burden ( Figure 6I). These results indicated that NOX4 play key roles in maintaining the tumor growth in an orthotopic xenograft nude mouse model.
These results are shown in Supplementary Figure 5D and Figure 6I as below and described in our revised manuscript (Page 15, paragraph 1). Figure 6G)

5) On a different note, related to the iKras experiment, the authors should comment
on the specificity of DPI as a Nox4 inhibitor.
Response: Actually, diphenyleneiodonium (DPI) is non-specific inhibitor for NOX4. It is a chemical inhibitor of flavoprotein-containing enzymes, including NOX oxidases.
Given that inhibition of NOX4 has proven potential for PDAC treatment and DPI just has moderate effects, the development of potent and specific drugs that target NOX4 deserves further exploration. Following the reviewer's suggestion, we have discussed this point in our revised manuscript (page 17, paragraph 2).