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Drug Delivery

Drug Delivery - Poster Session B

Poster J11 - Impact of Cancer Cachexia on Adipocyte BNIP3/L-Mediated Mitophagy

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Cancer-associated cachexia (CAC) is a multifactorial metabolic syndrome characterized by systemic inflammation and ongoing loss of muscle mass that cannot be reversed by nutritional supplementation. It is common in advanced cancers, associated with poor treatment response and reduced quality of life, and ultimately, is implicated in approximately 30% of cancer deaths. The incidence of CAC in common malignant tumors ranges from 20 to 90%. White adipose tissue (WAT) is one of the first organs to be affected and preserving WAT has shown to protect muscle in murine cachexia models. Despite the clinical relevance, the cellular mechanisms underlying AT loss in CAC remain unclear and human therapeutic interventions to preserve AT are lacking. Therefore, understanding the underlying mechanisms and developing new therapies that mitigate AT loss during CAC could help in preventing or lessening CAC progression.
Cancer cells spread signals by secreting intermediate metabolites such as lactate, succinate, and other oncometabolites, which AT can detect and respond to similarly to hypoxic or pseudohypoxic conditions. Indeed, circulating lactate levels positively correlate with the degree of body weight loss in CAC patients. BNIP3 (BCL2/adenovirus e1B 19 kDa protein interacting protein 3), an outer mitochondrial membrane protein, is a mitophagy receptor that mediates mitophagy under various stresses, particularly hypoxia, since BNIP3 is a hypoxia-responsive protein. We hypothesize that adipocytes sense oncometabolites as in pseudohypoxia conditions increasing BNIP3 so that future treatments can be developed to decrease BNIP3 levels in adipocytes.

Materials and

Methods:  We used human adipose stem cells (hASC) and differentiated them into mature adipocytes. Several growth factors and hormones were combined with growth media for 14 days to induce stem cell differentiation. After differentiation, mature adipocytes were treated with mouse Lewis lung cancer cell conditioned media (CM) in a 1:1 ratio for different time points (24h and 72h). Mature adipocytes were also treated with 10 mM of isolated oncometabolites sodium lactate (using both physical forms L and D enantiomers), sodium succinate, and sodium fumarate for different timepoints (24h and 72h). Real Time quantitative PCR was used to measure gene expression for BNIP3/L in adipocytes treated with CM and oncometabolites. 100 ng of isolated RNA was used for a 20uL cDNA reaction. 1uL of cDNA was added separately to 18S, BNIP3, and BNIP3L primers for a PCR reaction. RT-qPCR measured the relative gene expressions using 18S as a housekeeping gene. To confirm BNIP3 expression in adipocytes, cells were stained with a BNIP3 antibody (1:100, abcam) followed by Alexa Fluor 594 goat anti-rabbit second antibody (1:100, abcam). The stained cell slides were imaged with confocal microscopy, and the BNIP3 fluorescence intensity was quantified. To assess changes in functional metabolism of adipocytes, we are evaluating lipolysis of treated mature adipocytes using a colorimetric Lipolysis Assay Kit (abcam) quanitified by a BioTek microplate reader. Glycerol and fatty acids are the direct products of lipolysis. Using the colorimetric method, the color intensity is directly proportional to the amount of glycerol present.

Results, Conclusions, and Discussions: Adipocytes treated with CM for 24 h had a slight increase in BNIP3 gene expression compared to control cells. The BNIP3 gene expression was significantly increased when adipocytes were treated with lactate, indicating a link between exposure to lactate and modulating mitophagy that could lead to changes in metabolism and adipose tissue wasting. Interestingly, after treatment for 72 h, there was no increase in BNIP3 expression. Confocal images of fluorescently stained BNIP3 proteins were quantified to confirm higher numbers of BNIP3 in cells treated with enantiomers of sodium lactate versus only cancer CM. Images showed an increase in BNIP3 intensity in cells treated with lactate compared to cells treated with CM, confirming what we observed with RT-PCR. A lipolysis assay is expected to yield relatively more free glycerol from cells exposed to sodium lactate than untreated cells because mitophagy machinery modulation may lead to increased lipolysis, thus more free glycerol and fatty acids would be present. Overall, this data suggests that mature adipocytes exposed to sodium lactate have increased expression of BNIP3 and this could be the mechanism of adipose tissue waste during cancer cachexia. In future studies, we plan to use the drug delivery system developed in our lab that targets adipocytes to load molecules that can reduce BNIP3 levels. Treating adipocytes with an inhibitor of BNIP3 may decrease the pseudohypoxic response and combat adipose tissue depletion.

Acknowledgements (Optional):  UTHealth San Antonio Collaborators Dr. Pham, Vincent, and Dr. Huang, Gang