Cancer cells differentially modulate mitochondrial respiration to alter redox state and enable biomass synthesis in nutrient-limited environments

Reviewer #1 (Public review):

Summary:

This manuscript investigates how cellular NAD/NADH ratios are controlled in cancer cell lines in vitro. The authors build on previous work, which shows that serine synthesis is sensitive to NAD/NADH ratios and PHGDH expression. Here, the authors demonstrate that serine synthesis is variable across a panel of cell lines, even when controlling for expression of serine synthesis enzymes such as PHGDH. The authors show that cellular NAD/NADH ratios correlate with the ability to synthesize serine and grow in serine-deprived environments when PHGDH levels remain constant. Investigating this variability in NAD/NADH ratios, the authors find that the cells that can positively respond to serine deprivation are able to increase oxygen consumption and cellular NAD/NADH ratios. Cells that do not increase oxygen consumption in response to serine deprivation do not increase NAD/NADH ratios and cannot grow well without serine. The authors go on to show that in cells with the ability to increase oxygen consumption upon serine deprivation, PHGDH expression alone is sufficient to fully restore growth-serine; in cells that cannot increase oxygen consumption, both PHGDH expression and interventions to increase NAD/NADH ratios are required to increase growth. Thus, cells need both PHGDH and NAD/NADH increases to maximize serine synthesis in response to serine deprivation. The authors previously showed that lipid synthesis likewise requires NAD regeneration. Interestingly, one cell line that does not increase oxygen consumption in response to serine limitation tends to increase oxygen consumption in response to lipid deprivation; accordingly, depriving this cell line of lipids increases the synthesis of serine. Together, these findings show that how cells respond to nutrient deprivation is highly variable and that the response to nutrient deprivation (for example, whether or not oxygen consumption is increased) will determine how well cells tolerate depletion of nutrients with related biosynthetic constraints. This work sheds light on the complexity of cancer cell metabolism and helps to explain why it is difficult to predict which nutrients will be limiting to any cancer cell type or environment.

Strengths:

(1) The authors use multiple interventions to manipulate NAD/NADH ratios in cells.

(2) Experiments are well controlled and appropriately interpreted.

Weaknesses:

Overall the data support the conclusions of the manuscript. I have only two minor comments and suggestions:.

(1) Figure 2B/C: data are presented as relative to +serine, which shows how some cells respond to -serine, but may also be of interest to see how absolute (not relative) NAD/NADH levels correlate with serine synthesis and serine-independent proliferation. In other words, is it the dynamic increase in the ratio that is most important, or the absolute level of the ratio?

(2) Line 177-178: the authors write, "We hypothesized that the elevated NAD+/NADH ratio represented a cellular response to make the NAD+/NADH ratio more oxidized to enable serine synthesis". I recommend modest edits to avoid anthropomorphizing. It is possible that the ratio responds for reasons yet to be determined and not necessarily because the cell is deliberately trying to enable serine synthesis.

Reviewer #2 (Public review):

In the manuscript "Cancer cells differentially modulate mitochondrial respiration to alter redox state and enable biomass synthesis in nutrient-limited environments", Chang et al investigate how cancer cells respond to the limitation of certain environmental nutrients by regulating the cellular NAD+/NADH ratio. They focus on serine and lipid metabolism, pathways known to be controlled by the NAD+/NADH ratio, and propose that changes in mitochondrial respiration in response to deprivation of these nutrients can influence the NAD+/NADH ratio, thereby impacting biomass synthesis.

While the study is descriptive in nature and does not investigate specific molecular mechanisms that explain the crosstalk between nutrient availability and mitochondrial redox changes, the experimental component is robust, and the conclusions are well supported by the results. Some suggestions could further refine the conclusions and enhance the quality of the manuscript.

Main critiques:

(1) Throughout the manuscript, the authors utilise the number of cell doublings per day as an endpoint readout of cell proliferation. It would be advisable to include a quantification of the cell number and to display the proliferation rate over time. This would provide valuable insights into the timeline of cellular responses and avoid potential confounding effects associated with the use of Sulforhodamine B dye, an indirect measure of cell proliferation based on protein content, which may be influenced by some of the interventions. Furthermore, it will help determine whether specific treatments reduce cellular doublings resulting from cell death. This concern is particularly evident in treatments with rotenone, e.g., Fig. 1G, where the increase in doublings could be attributed to cell death.

(2) The authors propose a model in which the deprivation of extracellular nutrients impacts mitochondrial respiration, which in turn increases the NAD+/NADH ratio and ultimately affects metabolic biosynthetic pathways that occur in the cytosol, such as serine biosynthesis. The mechanism by which nutrient availability is sensed and transmitted across different cellular compartments to regulate mitochondrial redox status remains unclear. This concern is particularly relevant for serine metabolism, as its synthesis occurs in the cytosol, but the authors connect it to mitochondrial respiration. Compartment-specific measurements of NAD+/NADH ratio would help to understand to what extent the redox state is affected by nutrients in the mitochondria and in the cytoplasm (see also minor critiques point 2). Moreover, the use of the genetic tool LbNox could be employed to manipulate the NAD+/NADH ratio in a compartment-specific manner, while also avoiding the toxicity of certain compounds, such as rotenone. This set of experiments would add depth to the investigation, which might otherwise appear too descriptive.

Reviewer #3 (Public review):

Summary:

The manuscript by Chang and colleagues provides new insights into how cancer cells adapt their metabolism under nutrient-deprived conditions. They find cells respond differentially to serine and lipid deprivation via oxidising the cell redox state, which enables biomass synthesis and cell proliferation. They identified mitochondrial respiration as the major mechanism that dictates the endogenous NAD+/NADH ratio. By incorporating a dual stress paradigm, serine and lipid deprivation, the study further suggests that the NAD+/NADH ratio can serve as a link to orchestrate the complex interplay between multiple nutrient changes in the tumour microenvironment.

Strengths:

A novel aspect of this study is the idea that cancer cells are not uniformly passive victims of nutrient limitation; some can actively invoke endogenous NAD+ regeneration to combat nutrient stress. The conclusion is well-supported by comparing multiple cell lines from different tissues and genetic backgrounds, which improves generalizability. While most of the smaller conclusions align with common reasoning and expectations, the step-by-step deduction that leads to a novel 'big picture' is commendable. Another notable strength is the integration of dual stress (lipid and serine deprivation), which better mimics the complex tumor microenvironment with multiple nutrient fluctuations, raising the translational potential of these findings. The observation that lipid-deprived cells can stimulate serine synthesis and support proliferation in a subset of cancer cell lines offers a novel perspective on metabolic plasticity under starvation conditions.

Weaknesses:

Although the authors derive a novel and valuable overarching concept, the presentation of this "big picture" is not clearly articulated, making it less accessible to readers outside the immediate field. It would greatly enhance the manuscript to include a clearer summary of the overarching model and its implications. Additionally, discussing the potential clinical significance and applications of the findings would increase the relevance and broader impact of the work. Finally, the manuscript's clarity and credibility are undermined by inconsistent figure labeling and the lack of statistical analysis, particularly for the Western blot data.

While this study identifies changes in serine synthesis, mitochondrial respiration, PHGDH protein levels, and NAD+/NADH ratio in different cell lines, some of these relationships appear correlative rather than causally established (Figure 2; Figure 5; Figure 6). Some claims are thus overinterpreted. For example, the co-occurrence of increased NAD+/NADH ratio and citrate levels under lipid deprivation in A549 cells does not establish causality (Figure 5). Direct perturbation experiments that manipulate NAD+/NADH and assess downstream effects on citrate synthesis would substantially strengthen the conclusions.

The study focuses predominantly on mitochondrial respiration as a source of NAD+ regeneration. However, it will also be interesting to check other significant pathways, such as NAD+ salvage, which have been implicated in supporting serine biosynthesis. In addition, the subcellular distribution of NAD+ may distinguish whether some cells are truly redox-unresponsive. Mitochondrial NAD+ regeneration might counteract the cytosolic NAD+ consumption, rendering a relatively stable intracellular NAD+/NADH ratio. The malate-aspartate shuttle can be an interesting aspect.

The authors should acknowledge the limitations of short-term isotope tracing in their experimental design. Differences in metabolic rates across cell lines can affect the kinetics of metabolite labeling, limiting the direct comparability of metabolic fluxes between them. As a result, observed changes may reflect transient adaptations rather than stable metabolic reprogramming. It is important to clarify that the study primarily captures short-term responses, and the conclusions may not extrapolate to longer-term adaptations or protein-level changes under sustained nutrient stress.