Individual-specific variation in the respiratory activities of HMECs and their bioenergetic response to IGF1 and TNFα.

Grace Makari‐Judson,Sallie S. Schneider,Holly S. Mason,Giovanna M. Crisi,Nazneen Sultana,Elizabeth M. Henchey,Nagendra Yadava,Melissa Johnson,Richard B. Arenas,D. Joseph Jerry,Stephanie M. Morin

doi:10.1002/jcp.25932

Abstract

Metabolic reprograming is a hallmark of cancer cells. However, the roles of pre‐existing differences in normal cells metabolism toward cancer risk is not known. In order to assess pre‐existing variations in normal cell metabolism, we have quantified the inter‐individual variation in oxidative metabolism of normal primary human mammary epithelial cells (HMECs). We then assessed their response to selected cytokines such as insulin growth factor 1 (IGF1) and tumor necrosis factor alpha (TNFα), which are associated with breast cancer risk. Specifically, we compared the oxidative metabolism of HMECs obtained from women with breast cancer and without cancer. Our data show considerable inter‐individual variation in respiratory activities of HMECs from different women. A bioenergetic parameter called pyruvate‐stimulated respiration (PySR) was identified as a key distinguishing feature of HMECs from women with breast cancer and without cancer. Samples showing PySR over 20% of basal respiration rate were considered PySR+ve and the rest as PySR−ve. By this criterion, HMECs from tumor‐affected breasts (AB) and non‐tumor affected breasts (NAB) of cancer patients were mostly PySR−ve (88% and 89%, respectively), while HMECs from non‐cancer patients were mostly PySR+ve (57%). This suggests that PySR−ve/+ve phenotypes are individual‐specific and are not caused by field effects due to the presence of tumor. The effects of IGF1 and TNFα treatments on HMECs revealed that both suppressed respiration and extracellular acidification. In addition, IGF1 altered PySR−ve/+ve phenotypes. These results reveal individual‐specific differences in pyruvate metabolism of normal breast epithelial cells and its association with breast cancer risk.

Highlights

These results reveal individual-specific differences in pyruvate metabolism of normal breast epithelial cells and its association with breast cancer risk
The analyses demonstrate that cytokines can alter breast epithelial cells metabolism
The 20% cut-off for pyruvate-stimulated respiration (PySR)+ve phenotype was arbitrarily set above the average standard error of controls, IGF1and TNFα-treated rNAB-human mammary epithelial cells (HMECs), because mostly they showed the PySR+ve phenotype and they were from women who did not have cancer

Summary

| MATERIALS AND METHODS

Metabolic individuality and its relevance to health risks are currently being explored (Suhre et al, 2011a, 2011b). The third involved pNAB- versus rNAB-HMECs from breast cancer and reduction mammoplasty patients, respectively Bioenergetics of both AB- and NAB-HMECs with and without IGF1 and TNFα treatments were assessed by in situ respirometry. Respirometry data were analyzed to quantify (i) interindividual variation, (ii) bioenergetic differences in AB- versus NAB-HMECs, and (iii) their responses to IGF1 and TNFα treatments. Prefix p in pAB and pNAB indicates paired AB- and NAB-HMECs obtained from the tumor affected (AB) and non-affected breasts (NAB) of cancer patients. Pilot experiments were performed to optimize seeding cell density and compare OCR and extracellular acidification rates (ECAR) of HMECs from the same subject at P2 and P4. Cells (10,000/well) were seeded in 50 μl FIGURE 2 A representative respiratory profile It shows the experimental scheme, pyruvate-stimulated respiration (PySR) and selected bioenergetic parameters. The 20% cut-off for PySR+ve phenotype was arbitrarily set above the average standard error of controls, IGF1and TNFα-treated rNAB-HMECs, because mostly they showed the PySR+ve phenotype and they were from women who did not have cancer

| RESULTS

Findings

| DISCUSSION