ACCUMULATION AND ROLE OF CYTOPLASMIC LIPID DROPLETS IN THE PROGRESSION OF METASTATIC BREAST CANCER

The goal of this research is two-fold: to determine pathways that contribute to triacylglycerol (TAG) accumulation in metastatic breast cancer, and to discover how TAG accumulation within lipid droplets (LDs) may drive steps within the metastatic cascade.

For these studies we utilized an in vitro model of breast cancer progression that was derived from the human, non-cancerous, breast epithelial MCF10A cells. These cells were transfected with the Harvey-ras oncogene (MCF10A-ras) and serial passaged in vivo to generate more aggressive cell lines, including the non-metastatic MCF10CA1h cells, which form undifferentiated tumors, and the metastatic-capable MCF10CA1a cells. We have demonstrated that this model has increasing TAG accumulation as the series progresses to metastasis, which complements clinical findings. Specifically, the MCF10A-ras cells have minimal TAG accumulation, the non-metastatic MCF10CA1h cells have significantly more TAG, and the metastatic MCF10CA1a cells contain the most TAG. Therefore, we employed these cell lines to interrogate what role TAG within these LDs may play in promoting breast cancer metastasis, as well as what pathways are dysregulated in the metastatic MCF10CA1a compared to the non-metastatic cell line MCF10A-ras to account for the higher observed levels of TAG.

Since the function of LDs is often reflected by their associated proteins, and that the metastatic MCF10CA1a cells accumulate TAG, we conducted the first shotgun, untargeted proteomics analysis on LDs isolated from metastatic breast cancer cells and characterized the protein enrichment from these organelles (Chapter 2). By implementing functional annotation analysis using online databases that categorize proteins into biological functions and pathways, we discovered that the proteins associated with LDs from the MCF10CA1a metastatic breast cancer cells are markedly different than LDs of other cell types, such as adipocytes and enterocytes. Specifically, proteins involved in lipid metabolism constituted only 3% of the total LD proteome, while other novel categories, like proteins involved in cell-cell adhesion, were the most enriched term. Many of these proteins enriched on the LD have been previously implicated in breast cancer metastasis; therefore, this study resulted in a hypothesis-generating list of potential proteins involved breast cancer progression that can be applied to future studies to elucidate the role LDs may play in promoting breast cancer metastasis. 

Further, we successfully isolated LDs from the non-metastatic MCF10CA1h cell line to compare to the LD proteome of the metastatic MCF10CA1a cells (Chapter 3). Additionally, the WCL from these samples were collected and analyzed to inform the findings from the LD proteome analysis. Results from this analysis indicate that the metastatic breast cancer cells, which we show have larger LDs than the non-metastatic cells, have greater protein diversity (enrichment of different proteins) than the non-metastatic cell line. Further, 363 proteins from the LD fraction isolated from MCF10CA1a cells had an average fold change greater than two compared to proteins from the LDs of MCF10CA1h cells. Only 14 proteins from the non-metastatic LD fraction had an average fold change greater than two compared to the metastatic cells’ LDs. Categories involved in proteasome function, protein folding, RNA splicing, and nucleobase metabolism were most enriched in LDs from both the non-metastatic and metastatic cell lines. These categories are distinct from the top categories identified from their WCLs; therefore, we propose that the localization of these proteins to the LD may be necessary for early-stage progression of breast cancer, but are not required for metastasis. Interestingly, this analysis revealed that LDs from the MCF10CA1a cells are enriched in proteins involved in protein localization within the cell, including DNA binding, as well as LD organization. Therefore, results from this work provided a list of proteins involved in lipid metabolism that may contribute to the differences in lipid accumulation in metastatic compared to non-metastatic breast cancer cells. We also identified a unique potential function of the LDs in metastatic cells, which is to regulate transcription factor localization, and specifically to sequester nuclear factor kappa B (NFκB). This study provides a narrow list of proteins that may be involved in regulating LD accumulation in the metastatic cells, as well as lists of other proteins that associate with metastatic, but not non-metastatic LDs, which warrant further investigation to identify their potential functional roles in promoting progression of breast cancer to metastatic disease. 

The next set of experiments described in Chapter 4 provide evidence that stored TAG in MCF10CA1a cancer cells sustain the metastatic processes of migration and survival in detached conditions. Further, we identified differences in lipid metabolism pathways that contribute to the discrepancy in TAG accumulation in the non-metastatic MCF10A-ras and metastatic MCF10CA1a cells. Briefly, we show that metastatic MCF10CA1a cells have more, and larger, LDs than the MCF10A-ras cells, in accordance with their higher levels of TAG. We demonstrate that de novo lipogenesis of glucose, glutamine and acetate is higher in the metastatic MCF10CA1a cells compared to the MCF10A-ras cells, and that inhibition of FASN or the final enzymes in the TAG synthesis pathway reduces TAG accumulation and subsequent migration and survival in detached conditions. Further, we identified that though FA uptake is similar in the non-metastatic and metastatic cells, the MCF10A-ras cells have higher levels of exogenous FA oxidation than MCF10CA1a cells, indicating that the metastatic cells may store exogenous FAs prior to oxidation. Further, the oxidation of endogenous FA stores is greater in the MCF10CA1a cells. We demonstrate that FAO is necessary to sustain MCF10CA1a, but not MCF10A-ras, cell migration, and that this FAO-dependent metastatic cell migration requires lipolysis of endogenous TAG stores. Finally, we also propose several future directions from these studies, including investigation of the potential role of HIF1α protein signaling in sustaining the pro-migratory, lipogenic phenotype of MCF10CA1a cells, as well as exploration into whether LD and mitochondrial relocalization to the leading edge of migrating cells is required for cell movement. In sum, this work identified cell metabolism pathways necessary to sustain TAG accumulation and catabolism, both of which are needed to support MCF10CA1a cell metastatic capacity. Specifically, we demonstrate that FASN for de novo lipogenesis and the first enzyme in the TAG lipolysis pathway, adipose triglyceride lipase (ATGL), are both needed for FAO-dependent migration. Results from these studies propose that dysregulated lipid metabolism, including simultaneous FA synthesis and storage within TAG, and subsequent TAG lipolysis and FA oxidation, occurs in the metastatic cells, and supports their migratory capacity. 

Lastly, we conducted untargeted proteomic analysis of LDs and WCLs from the metastatic MCF10CA1a cells treated with vehicle (high TAG, high migratory rate) or the FASN-inhibitor, TVB-3166 (less TAG, lower migratory rate) to identify other novel potential candidate proteins on FASN-derived LDs that may contribute to breast cancer progression. Similar the comparison of the LD proteome of non-metastatic and metastatic cells, we identified far fewer proteins on LDs from the FASN-inhibited cells. The LD fraction from the more aggressive, TAG-rich cell line (vehicle-treated) contains a greater diversity of proteins, and of the proteins identified in both samples, the vehicle-treated cells have more proteins with a significantly higher relative abundance compared to the LDs from less aggressive (FASN-inhibited) cells. Functional annotation of these samples revealed that ferroptosis and cell adhesion related proteins on MCF10CA1a LDs become less enriched following FASN-inhibition and TAG depletion. Thus, this study identified a new potential pathway to further investigate, ferroptosis, as well as a similar set of cell adhesion proteins identified from the MCF10CA1a LD characterization (Chapter 2).  It is plausible that these proteins may be sequestered to the LD and away from their typical sites of function, so that they cannot exert anti-cancer effects, thereby increasing metastatic potential.    

Overall, research from this dissertation contributes to the existing field of cancer cell metabolism by determining pathways necessary for TAG accumulation in metastatic breast cancer cells and identifying how these lipid stores support processes required for metastasis. Additionally, this work contains the first LD proteomic studies in breast cancer cells, and importantly, the first comparisons among LD proteomes across different stages of cancer progression, including cells with less TAG and lower metastatic potential (MCF10CA1h and FASN-inhibited MCF10CA1a cells) versus cells with more TAG and higher metastatic potential (MCF10CA1a cells alone or with vehicle treatment). We identified that lipid metabolism is dysregulated in the MCF10CA1a cells, where FA synthesis and storage into TAG, as well as TAG lipolysis and FA oxidation, likely occur simultaneously. Further, we highlight several pathways of proteins that are unique to metastatic LDs that warrant additional assessment in order to determine their potential functional role in LD accumulation and promoting breast cancer progression to metastatic disease.