Indiana University-Purdue University Indianapolis (IUPUI) / Cachexia is a devastating muscle wasting syndrome affecting multiple biochemical pathways and is a comorbidity of many diseases including pancreatic ductal adenocarcinoma (PDAC). PDAC patients with cachexia commonly experience systemic inflammation, progressive loss of lean muscle and adipose tissue, and cardiac dysfunction. The present workflow identifies proteins and their post-translational modifications extracted from both cardiac and skeletal muscle tissue isolated from a murine model of PDAC-associated cachexia. Reported here are differentially occurring post-translational modifications found on the most abundant contractile proteins. Tissue from mouse muscle samples were collected two weeks after either receiving a sham surgery or orthotopically implanted with PDAC tumor cells, with or without a follow-up chemotherapy treatment of the standard of care agent gemcitabine with nab-Paclitaxel. Whole tissue blocks of gastrocnemius or heart were either flash frozen and pulverized or homogenized in denaturing lysis buffer and then sonicated to facilitate protein extraction. After disulfide bond reduction, cysteine alkylation, and trypsin digestion, the resultant peptides were subjected to molecular barcoding using tandem mass tag isobaric labeling reagents to facilitate multiplexing. The first and second dimension of peptide separation in the multiplexed sample is accomplished with an offline, high pH, reverse phase (RP)-LC fractionation followed by an online RP-LC at lower pH. The use of high-field asymmetric-waveform ion mobility spectrometry provided a last dimension of separation before MSn analyses. ​This novel, proteomic workflow enables deep proteome profiling in the progression of cancer-induced cachexia. The use of multi-dimensional chromatographic separation and differential ion mobility technique have allowed us to identify almost 4,500 proteins groups of gastrocnemius muscle tissue and nearly 7,100 protein groups of myocardium taken from the murine PDAC model of pancreatic cancer. A comprehensive analysis of the data collected from this workflow was used to calculate differential post-translational modifications on major contractile proteins isolated from PDAC model muscle tissue, with or without chemotherapy, when compared to sham surgery controls. Differential post-translational modifications and protein expression changes found to contribute to cancer cachexia may elucidate novel molecular mechanisms and cellular signaling that drive cachexia progression. / 2022-08-23

Cancer-associated cachexia is a condition defined by a sustained net-negative energy imbalance. Although the different types of adipose tissue – white, beige, and brown – have been implicated in contributing to cancer-associated cachexia, the mechanisms of these maladaptive changes and their impact on whole-body energy expenditure have not been fully elucidated. Using breast cancer as our model, we demonstrate white adipose tissue browning in murine and human breast cancer; furthermore, we demonstrate that this effect is extremely localized and takes place early in tumor progression. We utilized in vitro cell culture techniques and demonstrate that cancer secreted factors and cross-talk with white adipocytes decrease expression of classic white adipose tissue-related genes. We also demonstrate in murine and human culture models that cancer secreted factors reduce white adipocyte lipid droplet size, and cross-talk between cancer cells and adipocytes results in an increase in lipolysis-related gene expression. Interestingly, our results strongly suggest that in mice, neither cancer secreted factors nor cross talk with adipocytes can induce white adipose tissue browning, indicate that this process likely occurs independently of direct cancer interactions with local white adipocytes. We demonstrate that interleukin 6, a cytokine with previous implications in white adipose tissue browning, induces interleukin 6-mediated signaling; however, that signaling alone is not enough to directly induce white adipose tissue browning. We present preliminary data suggesting that immune cell population shifts within the white adipose tissue of mice with breast cancer tumors may be source of white adipose tissue browning. We show that the Virginia Commonwealth University Health System has an identifiable population of patients with cancer with what we hypothesize as maladaptive thermogenic adipose tissue activity, and discuss ongoing experiments aimed at understanding the implications of these changes on whole body energy expenditure in human patients. Lastly, in a case of autoimmune diabetes mellitus in the setting of an extra-adrenal paraganglioma, we demonstrate that the interaction between cancer and whole-body metabolism is multifaceted. Together, these experiments demonstrate that adipose tissue plasticity occurs in breast cancer (and other cancers), and that different drivers for individual changes exist within the tumor microenvironment. We predict that further exploration of the exact mechanisms and translational implications will provide useful information to lead to new therapeutic treatments for patients with cancer-associated cachexia.