MCF10CA1a Cells Research Articles

Abstract 781: Impact of vitamin D and pyruvate carboxylase on hypoxia inducible lipid droplet associated (HILPDA) protein in metastatic breast cancer cells

Abstract Metastatic breast cancer, a critical health issue for women globally, is closely linked to aberrant lipid metabolism. One feature of this dysregulated metabolism is the increase in lipid storage in the form of cytoplasmic lipid droplets (CLDs), although what drives their accumulation or their role in metastasis is not clear. In prior studies, we demonstrated that the active form of vitamin D (1,25-dihydroxyvitamin D, 1,25(OH)2D) downregulates pyruvate carboxylase (PC), a key metabolic enzyme, thereby reducing lipid accumulation in metastatic breast cancer cells (MCF10CA1a). Additionally, we have demonstrated that both 1,25(OH)2D treatment and PC-inhibition reduce breast cancer cell migration. Therefore, the goal of the current research is to identify the proteins associated with CLDs in metastatic MCF10CA1a breast cancer cells following 1,25(OH)2D treatment or reduction in PC expression (shPC). Proteins from CLDs and whole cell lysates (WCL) were processed for untargeted proteomic analysis. In the CLD fractions, 1562 identified enriched proteins were common to both MCF10CA1a and 1,25(OH)2D treated cells, while 160 proteins were higher in the MCF10CA1a cells, and 550 proteins were higher in the 1,25(OH)2D-treated cells compared to MCF10CA1a CLD fraction. In the CLD fractions, MCF10CA1a and shPC cells had a similar level of 380 enriched proteins, although 65 were higher in the shPC compared with MCF10CA1a, whereas 1268 were more enriched in the MCF10CA1a CLD fraction. Hypoxia Inducible Lipid Droplet Associated (HILPDA, known to inhibit triacylglycerol lipolysis) was only enriched in the CLD fraction of the metastatic MCF10CA1a cells. HILPDA was not identified in the CLDs of the 1,25(OH)2D treated or the shPC cells, nor in any WCL fraction. Further investigation of HILPDA is important to determine if HILPDA plays a role in dysregulated cellular metabolism, including CLD accumulation, or migration, and may potentially be a target for future therapies to prevent breast cancer progression. Citation Format: Yazhen Song, Chaylen Andolino, Dorothy Teegarden. Impact of vitamin D and pyruvate carboxylase on hypoxia inducible lipid droplet associated (HILPDA) protein in metastatic breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 781.

Abstract 365: Proteomic analysis of cytoplasmic lipid droplets and whole cell lysates reveal ferroptosis regulation by fatty acid synthase-derived lipid droplets in metastatic breast cancer

Abstract Breast cancer remains a serious public health concern, where metastasis accounts for the majority of patient mortality. Lipid accumulation in metastases is associated with reduced treatment response and poorer patient outcomes, although the mechanisms linking lipid accumulation and metastasis are not clear. We previously demonstrated that metastatic MCF10CA1a breast cancer cells have higher de novo lipogenesis and cytoplasmic lipid droplets (CLDs) compared to non-metastatic MCF10A-ras cells. Additionally, fatty acid synthase (FASN)-derived TAG stores sustained key metastatic processes in the MCF10CA1a cells. The proteome of CLDs is diverse and plastic, reflecting differing CLD functions; therefore, we sought to identify proteins differentially abundant on CLDs from vehicle-treated and FASN-inhibited MCF10CA1a breast cancer cells. We performed untargeted, global proteomics analysis on whole cell lysates (WCLs) and CLDs from vehicle-treated and FASN-inhibited (TVB-3166) MCF10CA1a cells to determine protein pathways differentially enriched on CLDs. Together, 2,416 and 3,539 proteins were identified within CLD fractions and WCLs, respectively. Of the proteins identified in CLD fractions, ~30% (717) were commonly identified within both conditions. Interestingly, 1,571 proteins were unique to the CLD fraction from vehicle-treated cells, whereas only 19 proteins were unique to the CLD fraction from FASN-inhibited cells. Proteins unique to CLDs from vehicle-treated cells include those known to reduce cancer cell survival and progression, such as proteins involved in cadherin-binding and cell cycle regulation. Proteins known to reduce cancer progression that associate with CLDs may prevent them from functioning at their normal site of action. Interestingly, an inhibitor of ferroptosis, glutathione peroxidase 4 (GPX4), was only detected in the vehicle-treated CLD and WCL samples, whereas proteins that promote ferroptosis were more abundant on CLDs of the more migratory cells, although their levels between WCLs were similar. Taken together, these data suggested that the machinery for ferroptosis may be mislocalized in TAG-rich cells, thereby protecting them from ferroptosis. Indeed, FASN-inhibited cells had nearly 30% more intracellular iron compared to the vehicle-treated cells, an important prerequisite for ferroptosis, thus indicating higher levels of ferroptotic stress. Functionally, the vehicle-treated cells were 25% more readily rescued by N-acetylcysteine from ferroptosis induced by erastin than were FASN-inhibited cells, despite comparable sensitivity to erastin. These preliminary findings suggest that FASN-derived CLDs in metastatic cells may aid in protecting against ferroptosis to promote breast cancer progression. Citation Format: Chaylen Andolino, Kimberly K. Buhman, Michael F. Coleman, Stephen D. Hursting, Marjorie Layosa, Michael K. Wendt, Dorothy Teegarden. Proteomic analysis of cytoplasmic lipid droplets and whole cell lysates reveal ferroptosis regulation by fatty acid synthase-derived lipid droplets in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 365.

Increased Fatty Acid Synthesis and Catabolism Supports Metastatic Breast Cancer Cell Migration

Lipid accumulation is positively associated with breast cancer aggressiveness and poorer clinical outcomes. Despite this observation, mechanisms underlying the increase and role of stored lipid in breast cancer progression remain incompletely understood. The accumulation of triacylglycerol (TAG) is dependent on the balance of uptake, synthesis, and utilization of fatty acids (FAs). In the current study, we utilized non‐metastatic MCF10A‐ras and metastatic MCF10CA1a human breast cancer cells to determine differences in TAG storage and catabolism for sustaining migration—a critical step in the metastatic cascade. Our results demonstrate that MCF10CA1a cells have 90% more TAG than MCF10A‐ras (TAG Assay and Transmission Electron Microscopy), although no significant difference in 14C‐palmitate uptake. MCF10CA1a cells have greater FA synthesis as shown by greater incorporation of 13C‐acetate (60%), 13C‐glucose (65%), and 13C‐glutamine (50%) into palmitate compared to the MCF10A‐ras cells (LC‐MS/MS), as well as higher protein levels of FA synthase (FASN). Additionally, MCF10CA1a cells display greater flux of uniformly‐labeled 13C‐glucose and 13C‐glutamine to the FA synthesis precursor, citrate, and lower intracellular citrate pool size compared to MCF10A‐ras. In addition to increased FA anabolism, MCF10CA1a cells rely on FA oxidation (FAO) for cellular migration compared to MCF10A‐ras cells, as determined by inhibiting the rate‐limiting enzyme of FAO, carnitine palmitoyltransferase 1, with etomoxir (transwell and wound‐healing assays). Pretreatment with the FASN inhibitor TVB‐3166 (3 d) significantly reduced TAG levels and subsequent migration by 67% compared to vehicle‐treated cells. Similarly, pretreatment with inhibitors of the TAG‐synthesizing enzymes diacylglycerol O‐acyltransferase 1 and 2 reduced TAG accumulation and subsequent migration (75%) of MCF10CA1a cells compared to vehicle. Together, the data suggest that FA synthesis may contribute to TAG storage necessary to sustain breast cancer migration. To test this hypothesis, we inhibited the initial enzyme of the TAG lipolysis pathway, adipose triacylglycerol lipase (ATGL; inhibitor ATGListatin), and measured migration. Inhibition of ATGL alone reduced MCF10CA1a cell migration (20% decrease compared to vehicle), and addition of the FAO inhibitor (etomoxir) provided no further effect. Our study indicates that metastatic MCF10CA1a cells accumulate FAs by increasing de novo lipogenesis, store them as TAG, and that catabolism of these stores drives FAO‐dependent migration.

Pterostilbene Changes Epigenetic Marks at Enhancer Regions of Oncogenes in Breast Cancer Cells.

Epigenetic aberrations are linked to sporadic breast cancer. Interestingly, certain dietary polyphenols with anti-cancer effects, such as pterostilbene (PTS), have been shown to regulate gene expression by altering epigenetic patterns. Our group has proposed the involvement of DNA methylation and DNA methyltransferase 3B (DNMT3B) as vital players in PTS-mediated suppression of candidate oncogenes and suggested a role of enhancers as target regions. In the present study, we assess a genome-wide impact of PTS on epigenetic marks at enhancers in highly invasive MCF10CA1a breast cancer cells. Following chromatin immunoprecipitation (ChIP)-sequencing in MCF10CA1a cells treated with 7 μM PTS for 9 days, we discovered that PTS leads to increased binding of DNMT3B at enhancers of 77 genes, and 17 of those genes display an overlapping decrease in the occupancy of trimethylation at lysine 36 of histone 3 (H3K36me3), a mark of active enhancers. We selected two genes, PITPNC1 and LINC00910, and found that their enhancers are hypermethylated in response to PTS. These changes coincided with the downregulation of gene expression. Of importance, we showed that 6 out of 17 target enhancers, including PITPNC1 and LINC00910, are bound by an oncogenic transcription factor OCT1 in MCF10CA1a cells. Indeed, the six enhancers corresponded to genes with established or putative cancer-driving functions. PTS led to a decrease in OCT1 binding at those enhancers, and OCT1 depletion resulted in PITPNC1 and LINC00910 downregulation, further demonstrating a role for OCT1 in transcriptional regulation. Our findings provide novel evidence for the epigenetic regulation of enhancer regions by dietary polyphenols in breast cancer cells.

Abstract 2326: Differential lipid metabolism in mammary cancer cell progression

Abstract There is an association between increased lipid accumulation and aggressive breast cancer phenotypes, and therefore poorer clinical outcomes. The mechanism by which dysregulated lipid metabolism may promote metastasis remains incompletely understood. Several potential mechanisms by which the neutral lipid storage organelles, cytoplasmic lipid droplets, may promote metastasis include utilization of fatty acids (FAs) for oxidation, protection against lipotoxcity, and production of lipid signaling mediators. We have previously demonstrated that metastatic MCF10CA1a mammary cancer cells rely on fatty acid oxidation (FAO) compared to non-metastatic MCF10A-ras cells, using etomoxir to inhibit the rate-limiting enzyme of FAO, carnitine palmitoyltransferase 1 (CPT1). Previous literature indicates that there is an increase in various lipid metabolism pathways, including FA uptake, de novo lipogenesis, and FA storage in neutral lipid. We found that there is no significant difference between palmitic acid uptake between the non-metastatic and metastatic cells. In the current study, we show that MCF10CA1a cells have significantly higher levels of carbon incorporation into palmitic and oleic FAs from 13C-acetate (25% and 20% greater), 13C-glucose (21% and 11% greater), and 13C-lactate (29% and 22% greater) respectively, compared to the MCF10A-ras cells, as detected by LC-MS/MS. In order to determine the source of FAs that contributes to the migration-dependent FAO, we assessed MCF10CA1a cell migration following inhibition of lipolysis (adipose triglyceride lipase—ATGL; ATGListatin) alone or in combination with the FAO inhibitor, etomoxir. We determined that inhibition of ATGL, the initial enzyme of triglyceride lipolysis pathway, significantly reduces MCF10CA1a cell migration (20% decrease compared to vehicle). Inhibition of FAO and lipolysis did not induce additional reduction in cell migration. We also showed that MCF10CA1a cells are more dependent on FA metabolism than the non-metastatic MCF10A-ras cells, demonstrated by MCF10CA1a cells having a greater decrease in cell viability compared to the MCF10A-ras cells given each FA metabolism inhibition (p<0.05: 22% greater decrease for FAO inhibition and 16% greater decrease for dual FAO and lipolysis inhibition, p=0.05: 17% greater decrease for FA synthesis inhibition). These data suggest that metastatic MCF10CA1a cells rely on their increased FA metabolism to survive and migrate, and specifically that the catabolism of FAs from triglyceride supports the FAO-driven migration of metastatic cancer cells. Citation Format: Chaylen Andoline, Parik Pawar, Dorothy Teegarden. Differential lipid metabolism in mammary cancer cell progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2326.

Abstract 2873: Comparison of metastatic and nonmetastatic breast cancer cell survival in glutamine depleted extracellular matrix detached conditions

Abstract For women, breast cancer is the most diagnosed cancer, with metastasis being the primary cause of most breast cancer-related deaths. During metastasis, cells detach from the extracellular matrix (ECM) to move to a secondary site. Unlike normal cells, metastatic cells are resistant to cell death upon ECM detachment. The amino acid glutamine is used as an energy source during metastatic progression via replenishing the TCA cycle. Breast cancer cells have decreased viability in the absence of glutamine, but the effect of glutamine in ECM detached conditions in metastatic cells is not known. We hypothesize that metastatic and non-metastatic cells have a decreased viability in ECM detachment in glutamine deprived conditions. In this study, metastatic MCF10CA1a cells and non-metastatic Harvey-ras oncogene transfected MCF10A-ras cells were employed. As expected, in ECM detached conditions, the MCF10A-ras cells had a greater decrease in viability (58.4%) in comparison to the MCF10CA1a cells (32.7%). However, in glutamine deprived conditions, the MCF10CA1a cells decrease in viability was 15.2% greater compared to the MCF10A-ras cells. mRNA expression of the major glutamine transporter, SLC1A5, was decreased in detached conditions in both cell lines, but to a greater extent in the MCF10A-ras cells (70.04%) compared to the MCF10CA1a cells (20.15%). Additionally, mRNA expression in detached conditions of glutamate dehydrogenase, the enzyme that metabolizes the downstream glutamine metabolite glutamate to α-ketoglutarate (αKG), was decreased by 77% in the non-metastatic cells, but increased by 23% in the metastatic cells. Additionally, mRNA expression of GOT1, a gene which encodes for the transaminase enzyme which synthesizes αKG and aspartate from glutamate and oxaloacetate, decreased 73% in detached conditions in the non-metastatic cells but was unchanged in the metastatic cells. Moreover, following detachment the glutamine synthesizing enzyme, glutamine synthetase mRNA expression was increased by 81.92% in the non-metastatic cells and 88.43% in the metastatic cells. In summary, the greater effect of glutamine depletion on viability, and the maintained expression of glutamine metabolizing enzymes in the metastatic cells suggest greater dependence on glutamine metabolism in ECM detached conditions compared to non-metastatic cells. Comparison of metastatic and non-metastatic breast cancer cells will provide insights to direct the development of new targets for the prevention of breast cancer metastasis. Citation Format: Kanika K. Garg, Madeline Sheeley, Dorothy Teegarden. Comparison of metastatic and nonmetastatic breast cancer cell survival in glutamine depleted extracellular matrix detached conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2873.

Proteomic Characterization of Cytoplasmic Lipid Droplets in Human Metastatic Breast Cancer Cells.

One of the characteristic features of metastatic breast cancer is increased cellular storage of neutral lipid in cytoplasmic lipid droplets (CLDs). CLD accumulation is associated with increased cancer aggressiveness, suggesting CLDs contribute to metastasis. However, how CLDs contribute to metastasis is not clear. CLDs are composed of a neutral lipid core, a phospholipid monolayer, and associated proteins. Proteins that associate with CLDs regulate both cellular and CLD metabolism; however, the proteome of CLDs in metastatic breast cancer and how these proteins may contribute to breast cancer progression is unknown. Therefore, the purpose of this study was to identify the proteome and assess the characteristics of CLDs in the MCF10CA1a human metastatic breast cancer cell line. Utilizing shotgun proteomics, we identified over 1500 proteins involved in a variety of cellular processes in the isolated CLD fraction. Interestingly, unlike other cell lines such as adipocytes or enterocytes, the most enriched protein categories were involved in cellular processes outside of lipid metabolism. For example, cell-cell adhesion was the most enriched category of proteins identified, and many of these proteins have been implicated in breast cancer metastasis. In addition, we characterized CLD size and area in MCF10CA1a cells using transmission electron microscopy. Our results provide a hypothesis-generating list of potential players in breast cancer progression and offers a new perspective on the role of CLDs in cancer.

Fibronectin-Expressing Mesenchymal Tumor Cells Promote Breast Cancer Metastasis.

Simple SummaryFor cancer to metastasize, tumor cells must not only invade the local tissue but must also grow and proliferate once they arrive. Tumor cell heterogeneity, the presence of multiple types of cancer cells within a tumor, can increase cell proliferation and invasion through cooperative interactions, increasing the potential for metastasis. We recently found that pro-invasion cancer cells express the protein fibronectin and increase metastasis for pro-growth cancer cells. We investigated this interaction by analyzing these two cell types’ migration and survival, both alone and in co-cultures. We find that pro-invasion cells have a protective effect on pro-growth cells, which otherwise die after two days in nutrient-starved conditions. Further, we find that adding soluble fibronectin to pro-growth cells in culture was sufficient to improve survival. However, their survival was higher for co-culturing conditions. These studies highlight the importance of cancer cell heterogeneity and the role of fibronectin in metastasis.Tumor metastasis is connected to epithelial-mesenchymal heterogeneity (EMH) and the extracellular matrix (ECM) within the tumor microenvironment. Mesenchymal-like fibronectin (FN) expressing tumor cells enhance metastasis within tumors that have EMH. However, the secondary tumors are primarily composed of the FN null population. Interestingly, during tumor cell dissemination, the invasive front has more mesenchymal-like characteristics, although the outgrowths of metastatic colonies consist of a more epithelial-like population of cells. We hypothesize that soluble FN provided by mesenchymal-like tumor cells plays a role in supporting the survival of the more epithelial-like tumor cells within the metastatic niche in a paracrine manner. Furthermore, due to a lower rate of proliferation, the mesenchymal-like tumor cells become a minority population within the metastatic niche. In this study, we utilized a multi-parametric cell-tracking algorithm and immunoblotting to evaluate the effect of EMH on the growth and invasion of an isogenic cell series within a 3D collagen network using a microfluidic platform. Using the MCF10A progression series, we demonstrated that co-culture with FN-expressing MCF10CA1h cells significantly enhanced the survival of the more epithelial MCF10CA1a cells, with a two-fold increase in the population after 5 days in co-culture, whereas the population of the MCF10CA1a cells began to decrease after 2.5 days when cultured alone (p < 0.001). However, co-culture did not significantly alter the rate of proliferation for the more mesenchymal MCF10CA1h cells. Epithelial tumor cells not only showed prolonged survival, but migrated significantly longer distances (350 µm compared with 150 µm, respectively, p < 0.01) and with greater velocity magnitude (4.5 µm/h compared with 2.1 µm/h, respectively, p < 0.001) under co-culture conditions and in response to exogenously administered FN. Genetic depletion of FN from the MCF10CA1h cells resulted in a loss of survival and migration capacity of the epithelial and mesenchymal populations. These data suggest that mesenchymal tumor cells may function to support the survival and outgrowth of more epithelial tumor cells within the metastatic niche and that inhibition of FN production may provide a valuable target for treating metastatic disease.

Abstract 5130: Proteomic characterization of cytoplasmic lipid droplets in human metastatic breast cancer cells

Abstract One of the characteristic features of metastatic breast cancer is altered lipid metabolism, including increased cellular storage of neutral lipid in cytoplasmic lipid droplets (CLDs). CLD accumulation is associated with increased cancer aggressiveness, suggesting CLDs contribute to metastasis. Although multiple hypotheses exist for how CLDs may promote cancer progression, how exactly CLDs contribute to breast cancer metastasis is not clear. CLDs are composed of a neutral lipid core, a phospholipid monolayer, and associated proteins. Proteins that associate with CLDs are thought to regulate CLD metabolism; however, the proteome of CLDs in metastatic breast cancer and how these proteins may contribute to altered lipid metabolism and breast cancer progression is unknown. Therefore, we utilized untargeted shotgun proteomics and transmission electron microscopy to identify the proteome and assess the characteristics of CLDs in the MCF10CA1a human metastatic breast cancer cell line. We identified a total number of 1537 proteins in the CLD fraction isolated from MCF10CA1a cells, and these proteins were associated with a variety of cellular processes. Interestingly, unlike other cell lines such as adipocytes or enterocytes, the most enriched protein categories were involved in cellular processes outside of lipid metabolism. For example, proteins involved in cell-cell adhesion were the most enriched in the CLD fraction, and some of these proteins had associated terms in cell migration and signaling. In addition, we characterized CLD size and area in MCF10CA1a cells using transmission electron microscopy. Our results provide a hypothesis-generating list of potential players in breast cancer progression and aggressiveness, making them possible therapeutic targets for prevention or treatment of this disease. Citation Format: Alyssa S. Zembroski, Chaylen Andolino, Kimberly K. Buhman, Dorothy Teegarden. Proteomic characterization of cytoplasmic lipid droplets in human metastatic breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5130.

RUNX1 and RUNX2 transcription factors function in opposing roles to regulate breast cancer stem cells.

Breast cancer stem cells (BCSCs) are competent to initiate tumor formation and growth and refractory to conventional therapies. Consequently BCSCs are implicated in tumor recurrence. Many signaling cascades associated with BCSCs are critical for epithelial-to-mesenchymal transition (EMT). We developed a model system to mechanistically examine BCSCs in basal-like breast cancer using MCF10AT1 FACS sorted for CD24 (negative/low in BCSCs) and CD44 (positive/high in BCSCs). Ingenuity Pathway Analysis comparing RNA-seq on the CD24-/low versus CD24+/high MCF10AT1 indicates that the top activated upstream regulators include TWIST1, TGFβ1, OCT4, and other factors known to be increased in BCSCs and during EMT. The top inhibited upstream regulators include ESR1, TP63, and FAS. Consistent with our results, many genes previously demonstrated to be regulated by RUNX factors are altered in BCSCs. The RUNX2 interaction network is the top significant pathway altered between CD24-/low and CD24+/high MCF10AT1. RUNX1 is higher in expression at the RNA level than RUNX2. RUNX3 is not expressed. While, human-specific quantitative polymerase chain reaction primers demonstrate that RUNX1 and CDH1 decrease in human MCF10CA1a cells that have grown tumors within the murine mammary fat pad microenvironment, RUNX2 and VIM increase. Treatment with an inhibitor of RUNX binding to CBFβ for 5 days followed by a 7-day recovery period results in EMT suggesting that loss of RUNX1, rather than increase in RUNX2, is a driver of EMT in early stage breast cancer. Increased understanding of RUNX regulation on BCSCs and EMT will provide novel insight into therapeutic strategies to prevent recurrence.

Regulation of Vitamin D Receptor by 1,25-dihydroxyvitamin D in Breast Cancer (P05-013-19)

ObjectivesLoss of vitamin D receptor (VDR) is reported during tumor progression in cancer cells, including breast cancer, with the implication that vitamin D may not be effective in inhibiting metastasis. The purpose of these studies was to investigate the expression and activity of the VDR in breast cancer cells at different stages of progression and the response to treatment with the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D). We hypothesized that although constitutive expression of VDR is downregulated during cancer progression, 1,25(OH)2D induces an increase in VDR transcriptional activity and VDR expression in metastatic breast cancer cells. MethodsWe employed a series of human cells representing different stages of breast cancer, the MCF-10A series which includes untransformed MCF10A, Harvey-ras oncogene transfected early progression model (MCF10A-ras), and metastatic MCF10CA1a cells. Cells were treated with 1,25(OH)2D (10 nM) or vehicle for 2 and 5 days. VDR mRNA expression was measured by PCR and VDR transcriptional activity by mRNA expression of CYP24. ResultsConstitutive VDR mRNA expression was reduced by 76% in metastatic MCF10CA1a compared to untransformed MCF10A. Treatment with 1,25(OH)2D for 5 days decreased VDR mRNA expression in MCF10A (43%) and MCF10A-ras (37%) cells compared to vehicle treatment. In contrast, treatment with 1,25(OH)2D for 5 days increased VDR mRNA expression by 61% in metastatic MCF10CA1a cells. In addition, VDR transcriptional specific activity increased in MCF10A-ras cell and trended towards an increase (P = 0.06) in MCF10CA1a cells compared to MCF10A, as assessed by measuring the ratio of CYP24/VDR mRNA expression. ConclusionsTaken together, these results suggest that although constitutive expression of VDR is reduced in metastatic cells, vitamin D may exert inhibitory effects through 1,25(OH)2D upregulation of VDR expression and activity in breast cancer. Funding SourcesSupport from National Institute of Health and USDA.

Abstract P2-01-13: The splicing factor PHD finger protein 5A inhibits apoptosis to promote breast cancer progression

Abstract Background: All the widely accepted hallmarks of cancer are known to be affected by aberrant splicing (AS), and splicing dysregulation itself is considered a valuable therapeutic target. Understanding the AS that promote cancer progression is crucial for the development of effective strategies for treating breast cancer. Methods: An in vivo CRISPR screen targeting RNA-binding proteins (RBPs) was performed to reveal the key splicing modulator (PHD finger protein 5A, PHF5A) of breast tumor progression. Immunohistochemistry method and survival analysis were performed using a tissue microarray (TMA) containing 450 breast carcinoma. Proliferation, transwell migration and in vivo tumor formation assays were utilized to assess the biological role of PHF5A. RNA sequencing and RT-PCR assay were used to identify PHF5A-regulated AS events in breast cancer cells. Biological functions and molecular pathways of the affected genes were investigated through a gene ontology (GO) analysis. Flow cytometry and Western blot analysis were used for apoptotic assessments. The correlation between PHF5A expression and AS events was further analysed using mRNA-Seq data of 40 paired breast cancer and adjacent normal breast tissues. And the correlation between the levels of PHF5A and cleaved caspase-3 were evaluated in the TMA. Results: According to RNA sequencing analysis of MCF10 cell series (MCF10A, MCF10AT, MCF10DCIS and MCF10CA1a), 159 RBPs were found to be up-regulated in cancer cells compared with non-cancer cells. And the CRISPR screen targeting these 159 RBPs systematically identified highest-ranking genes including PHF5A. In TMA cohort, high PHF5A expression was correlated with poor disease-free survival. PHF5A is frequently up-regulated in breast cancer and is essential for cancer cell proliferation, migration and tumor formation. Knockdown of PHF5A induces genome-wide AS events. The RT-PCR assay of MCF10CA1a cells showed that splicing changes of nine arbitrarily selected target genes were all modulated by PHF5A. GO analysis showed that PHF5A-regulated AS events were involved in apoptotic and anti-apoptotic pathways, among which FAS-activated serine/threonine kinase (FASTK) AS showed significant PSI (percent spliced in) difference. PHF5A knockdown appeared to switch full-length FASTK (FASTK-L) to an intron 5-retained variant (herein termed FASTK short, FASTK-S) in MCF10CA1a cells. The knockdown of PHF5A resulted in cleavage of caspase-3 and poly-ADP-ribose polymerase and conversion of the FASTK-L (61 kDa) and FASTK-S (42 kDa) proteins. Intriguingly, cells transduced with exogenous FASTK-S showed the most significant apoptotic effect, whereas the FASTK-L group presented a decreased apoptotic effect. The PHF5A ratios of paired non-tumor to tumor tissue were negatively correlated with the FASTK PSI differences between non-tumor and tumor tissues. A strong negative correlation was found between the PHF5A and cleaved caspase-3 levels in TMA. Conclusions: PHF5A depletion sensitizes cancer cells to apoptotic signaling partially through AS-mediated FASTK isoform conversion. This apoptotic suppressor plays a key role in breast cancer progression and acts as a prognostic indicator, and should be critically considered for optimization of the current therapeutic strategy. Citation Format: Zheng Y-Z, Xue M-Z, Shen H-J, Li X-G, Ma D, Gong Y, Hu X, Shao Z-M. The splicing factor PHD finger protein 5A inhibits apoptosis to promote breast cancer progression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-13.

NON‐CONTACT ELECTRIC FIELDS POTENTLY HINDER EGF PROMOTED BREAST CANCER MOTILITY BY DOWNREGULATING EGFR PHOSPHORYLATION

The objective of the proposed study is to assess the therapeutic potential of non‐contact inductive E‐fields (iEFs) in inhibiting breast cancer motility. Normal breast ducts are lined with monolayer of epithelial cells that have asymmetric distribution of ion channels resulting in a charge separation between the luminal and the basal sides giving rise to trans‐epithelial potentials (TEP). These TEPs are responsible for endogenous E‐fields that are directed radially from luminal to basal side. Consequently, the sites of invasion in breast tumors are depolarized, which suggest that that cell migration in late‐stage tumors occurs in absence of endogenous E‐fields. Furthermore, breast cancer cells in‐vitro have been shown to migrate towards the positive electrodes under the influence of externally applied direct current electric fields (1–10 V/cm). Therefore, we hypothesize that E‐fields applied in the direction of migrating breast cancer cells potently inhibit cell motility. To test our hypothesis, we used a panel of three breast cell lines, 1) normal MCF10A, 2) MCF10CA1a(MIV) (a malignant variant of MCF10A cells with H‐Ras mutation), and 3) triple negative breast cancer (TNBC) cells MDA‐MB‐231 in conjunction with a custom Bi‐directional Microtrack Assay (BMA). The BMA consists of an array of parallel‐aligned tracks that measure 700 μm in length and with a cross‐section of 20 μm × 20 μm. These microtracks mimic the topological cues encountered by migrating tumor cells that have detached from the primary tumor site during invasion. Inductive Electric Fields (iEFs ~ 60 μV/cm), which mimic the endogenous E‐fields in the breast duct, were applied using a custom‐designed air‐cored Helmholtz coils. iEFs applied anti‐parallel to the direction of migration and in absence of a pro‐migratory epidermal growth factor (EGF) gradient on cancer cells significantly increased migration speeds (Fig A) but had no effect on the control epithelial cells which maintained their non‐migratory phenotype. In presence of a stable EGF gradient, iEFs applied parallel in the direction of migration significantly reduce migration speeds for both the cancerous cell lines whereas it has no effect on migration speeds of MCF10A cells. Actin aggregation at the leading and/or trailing edges is a characteristic of migratory cells and for cancer cells that showed significant speed reduction the intracellular actin was uniformly distributed (Fig B) rather aggregating on the tips indicating a possible mechanism controlled through the EGFR/EGF pathway. Furthermore, we verified using Western Blotting techniques that iEFs significantly reduced phosphorylation of EGFR in EGF‐treated MDA‐MB‐231 cells (Fig C) whereas they downregulated the expression of EGFR in EGF‐treated MCF10CA1a(MIV) cells. For each case, the protein levels had a one‐to‐one correlation with the migration speeds for each cell line. Moreover, we also found that a combinatorial treatment of MK2206 (Akt inhibitor) with iEFs significantly reduced migration speeds of these breast cancer cells (Fig A) below the level of their respective controls. Thus, iEFs selectively hindered cancer cells with no adverse effects on normal epithelial cells, making them a potential candidate for cancer control and intervention. From a therapeutic standpoint, iEFs may help augment current therapeutic approaches to hinder the metastasis of breast cancer and confer survival benefit, including in TNBC, which currently lack any targeted therapies.Support or Funding InformationFunding for JWS: American Cancer Society and Institute for Materials Research at The Ohio State University. JWS acknowledge support from Pelotonia.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 5534: Runx1 possesses anti-tumor activity and inhibits stemness in breast cancer cells

Abstract Background: Despite advances in detection and therapies, breast cancer is still the most common malignant disease in women worldwide. Cancer stem cells (CSC) play an important role in formation, growth, drug-resistance and recurrence of tumors. Transcription factor Runx1 has received recent attention for its potential tumor suppressor activity in solid tumors. We previously showed that Runx1 represses the epithelial to mesenchymal transition in breast cancer cells. The objective of the present study is to investigate Runx1 functional effects on cellular activities, and its contribution to inhibiting CSC-like properties in breast cancer cells. Methods: We generated Runx1 overexpression and Runx1 knockdown stable cell lines in both malignant MCF10AT1 and metastatic MCF10CA1a cells. Cells were analyzed for tumorsphere formation, protein (Western) and RNA (RT-qPCR) expression analysis. Cellular activity assays (cell proliferation, migration, and invasion) were performed to measure the tumorigenic potential of the cells. Xenograft studies were used to determine whether Runx1 overexpression in breast cancer cells represses tumor growth in vivo. Results: Upon overexpressing Runx1, both MCF10AT1 and MCF10CA1a cells reduce their proliferation, migration and invasion, which support the concept that Runx1 acts as a tumor suppressor in breast cancer cells. Notably, overexpressing Runx1 inhibited the formation of tumorspheres and effectively reduced the stem-like population in these two breast cancer cells. These observations are consistent with the results of Runx1 knock-down in early stage ER positive MCF7 breast cancer cells which showed increased tumorsphere formation efficiency. Furthermore, Runx1 tumor suppressor activity was confirmed in mouse xenograft by showing that Runx1 overexpression inhibits tumor growth. Conclusion: Our studies show that Runx1 inhibits proliferation, migration, and invasion, reflecting its tumor suppressor activity. Runx1 mediated reduction of tumorsphere formation suggests that Runx1 also contributes significantly to inhibit the population of the breast cancer stem cells. Therefore these studies offer Runx1 as a novel bio-therapeutic molecule for breast cancer intervention. Citation Format: Deli Hong, Andrew J, Fritz, Kristiaan Finstad, Mark P. Fitzgerald, Janet L. Stein, Jane Lian, Gary S. Stein. Runx1 possesses anti-tumor activity and inhibits stemness in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5534. doi:10.1158/1538-7445.AM2017-5534

Abstract 4353: Polyphenol-mediated epigenetic reactivation of tumor suppressor gene SEMA3A in breast cancer cells

Abstract Alterations in DNA methylation occur in cancer, and may underlie silencing of genes with tumor suppressor functions. Reversing DNA methylation, thus potentially reactivating genes that combat cancer, constitutes a promising anti-cancer strategy. Interestingly, studies have indicated that certain dietary polyphenols, such as resveratrol present in grapes, exert anti-cancer effects through epigenetic regulation of gene expression. However, studies have been limited to candidate genes, and comprehensive and mechanistic insights are missing. In the present study, following genome-wide DNA methylation analysis with Illumina 450K BeadChip array, we identified CpG sites within regulatory regions of tumor suppressor genes that are hypomethylated upon treatment of breast cancer cells with resveratrol. Non-invasive MCF10CA1h and invasive MCF10CA1a human breast cancer cell lines were used as an experimental model. Pyrosequencing and QPCR were performed to assess respectively methylation and expression of selected genes. Chromatin immunoprecipitation (ChIP) was applied to assess binding events. We identified 990 hypomethylated CpG sites in MCF10CA1h and 1,146 hypomethylated CpG sites in MCF10CA1a cells upon 9-day treatment with 15µM resveratrol as compared with control untreated cells (differential methylation ≤-0.05, nominal p&amp;lt;0.05, limma t-test). Those CpG sites corresponded to approximately 650 genes that were predominantly associated with tumor suppressor function in cancer. We selected tumor suppressor gene SEMA3A as resveratrol target for further investigation. As array data indicated, resveratrol led to reduction in methylation of SEMA3A promoter region. The 18% decrease in methylation of SEMA3A was confirmed by pyrosequencing in MCF10CA1a breast cancer cells. This coincided with 23% up-regulation of SEMA3A expression. In addition, analysis of the effects on DNA methyltransferases (DNMTs) demonstrated that resveratrol decreases expression of DNMT3A. Furthermore, ChIP indicated decreased occupancy of DNMT3A at the SEMA3A promoter, suggesting that down-regulation and lower affinity of this de novo DNA methylating enzyme to DNA may mediate decrease in methylation within tumor suppressor genes upon resveratrol exposure. In support of reactivation of SEMA3A, active histone mark, H3K9ac, was significantly increased and repressive histone mark, H3K27me3, was significantly decreased after resveratrol treatment, indicating an open, more transcriptionally active chromatin state. These results demonstrate a role for polyphenol-mediated epigenetic modifications in reactivation of tumor suppressor genes in breast cancer and pave the way for further studies on the mechanism driving these changes. This study was supported by the PCCR, Indiana CTSI (UL1TR001108), Women’s Global Health Institute, and USDA National Institute of Food and Agriculture (Hatch project 1005656) granted to BS. Citation Format: Megan Beetch, Katarzyna Lubecka, Lucinda Kurzava, Kirsty Flower, James M. Flanagan, Barbara Stefanska. Polyphenol-mediated epigenetic reactivation of tumor suppressor gene SEMA3A in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4353. doi:10.1158/1538-7445.AM2017-4353

Intranuclear and higher-order chromatin organization of the major histone gene cluster in breast cancer.

Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher-order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA-seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal-like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub-clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub-clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in-between them, suggesting that the histone sub-clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub-nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher-order chromatin organization of the major HLB and its regulation during breast cancer progression.

Selective expression of long non-coding RNAs in a breast cancer cell progression model.

Long non-coding RNAs (lncRNAs) are acknowledged as regulators of cancer biology and pathology. Our goal was to perform a stringent profiling of breast cancer cell lines that represent disease progression. We used the MCF-10 series, which includes the normal-like MCF-10A, HRAS-transformed MCF-10AT1 (pre-malignant), and MCF-10CA1a (malignant) cells, to perform transcriptome wide sequencing. From these data, we have identified 346 lncRNAs with dysregulated expression across the progression series. By comparing lncRNAs from these datasets to those from an additional set of cell lines that represent different disease stages and subtypes, MCF-7 (early stage, luminal), and MDA-MB-231 (late stage, basal), 61 lncRNAs that are associated with breast cancer progression were identified. Querying breast cancer patient data from The Cancer Genome Atlas, we selected a lncRNA, IGF-like family member 2 antisense RNA 1 (IGFL2-AS1), of potential clinical relevance for functional characterization. Among the 61 lncRNAs, IGFL2-AS1 was the most significantly decreased. Our results indicate that this lncRNA plays a role in downregulating its nearest neighbor, IGFL1, and affects migration of breast cancer cells. Furthermore, the lncRNAs we identified provide a valuable resource to mechanistically and clinically understand the contribution of lncRNAs in breast cancer progression.

Effect of NIMA-related kinase 2B on the sensitivity of breast cancer to paclitaxel in vitro and vivo.

NIMA-related kinase 2B has been known to be an important centrosome regulatory factor. The aim of this study was to investigate the effect of NIMA-related kinase 2B on the sensitivity of breast cancer to paclitaxel. We detected the expression of NIMA-related kinase 2B messenger RNA in MCF-10 cells, including MCF-10A, MCF-10AT, MCF-10DCIS.com , and MCF-10CA1a. The influence of NIMA-related kinase 2B in nude mouse was also detected. The association between NIMA-related kinase 2B and clinicopathological factors was explored in invasive ductal carcinoma tissues. NIMA-related kinase 2B was lowly expressed in the precancerous cells, MCF-10A and MCF-10AT, and it was highly expressed in carcinomatous cells, MCF-10DCIS.com and MCF-10CA1a. The upregulation of NIMA-related kinase 2B can introduce the growth of MCF-10AT cells, knockdown of NIMA-related kinase 2B could remarkably inhibit cell proliferation in MCF-10DCIS.com and MCF-10 CA1a cells. Comparing the volume of the xenografts in nude mouse, we found that the tumors treated by NIMA-related kinase 2B small interfering RNA associated with paclitaxel were the smallest among all the groups. Expression of NIMA-related kinase 2B messenger RNA was associated with higher histological grades, positive lymph node, and high Ki67 index (>20%). The partial response rates were 75.0% in NIMA-related kinase 2B negative (NIMA-related kinase 2B-) patients and 15.8% in NIMA-related kinase 2B++ patients. The progressive disease rates were 10.0% in NIMA-related kinase 2B- patients and 52.6% in NIMA-related kinase 2B++ patients ( p = 0.002). Our findings suggested that NIMA-related kinase 2B could play a role in the development and progression of breast cancer. Combination treatment using NIMA-related kinase 2B small interfering RNA and paclitaxel might be a novel potential therapy method for breast cancer.

1α,25‐dihydroxyvitamin D3 Inhibits Matrix Metalloprotease 2 Expression in Metastatic Breast Cancer Cells

Breast cancer remains the second leading cause of cancer death among women in the United States. When the tumor is localized, 99% of breast cancer patients survive five years after diagnosis. However, if the tumor has metastasized to distant secondary organs, the survival rate drastically decreases to 22%. Epidemiological and animal studies support the hypothesis that vitamin D may inhibit breast cancer metastasis, but the mechanisms remain unknown. The purpose of the present studies was to investigate the effect of the bioactive vitamin D metabolite, 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D) on expression of Matrix Metalloproteinases (MMP) in MCF10CA1a breast epithelial cells. MMPs are a family of endopeptidases implicated in both cancer cell invasion and migration. First, the pattern of MMP expression in MCF10CA1a cells was investigated focusing on MMPs 1,2,3,9 and 13. MCF10CA1a cells expressed only MMP2 mRNA at a detectable level. Treatment with 1,25(OH)2D (10 nM,) reduced the expression of MMP2 by 32%±10 and 35%±7 at 2 and 5 days, respectively. Furthermore, the effect of 1,25(OH)2D on migratory capability of MCF10CA1a cells was assessed using a wound heal assay. Pretreatment with 1,25(OH)2D for 48 hours decreased cell migration by 44%±9.6 relative to vehicle. Further studies are needed to evaluate a causal link between vitamin D mediated downregulation of MMP2 and the nutrient's inhibition of breast cancer cell migration.Support or Funding InformationThis work was supported by a Project Development Team within the ICTSI NIH/NCRR (Grant Number UL1TR001108) and the National Institutes of Health, National Cancer Institute (R25CA128770) Cancer Prevention Internship Program. Additional support was received from the Indiana Elks Charities and the SIRG grant, both administered through the Purdue University Center for Cancer Research (Purdue University, West Lafayette, IN).

1α,25‐dihydroxyvitamin D3 Inhibits Adipocyte Mediated Metastatic Capability of Breast Cancer Cells

Excess adiposity is thought to be associated with approximately 20% of all cancers, including breast cancer. However the mechanism underlying this relationship remains controversial. Adipocytes secrete a wide array of bioactive molecules which act as key mediators in several obesity‐associated diseases. Further, the active form of vitamin D, 1α,25dihydroxyvitamin D (1,25(OH)2D) regulates multiple aspects of adipocyte biology, including adipogenesis and adipocyte apoptosis, as well as modulating the release of factors associated with cancer promotion. The purpose of these studies was to investigate 1,25(OH)2D‐mediated regulation of differentiated adipocytes to determine the impact on breast cancer progression. Differentiated 3T3‐L1 adipocytes were treated with 1,25(OH)2D (10 nM) for 2 days, followed by harvesting the adipocytes and collection of adipocyte conditioned media (ACM). ACM from adipocytes treated with 1,25(OH)2D decreased cell proliferation in metastatic MDA‐MB‐231 and MCF10CA1a cells compared to vehicle treated ACM, (42% and 37% of vehicle ACM, respectively), as measured by an MTT assay. In addition, ACM from adipocytes treated with 1,25(OH)2D inhibited cell migration and wound healing in MDA‐MB‐231 cells (37% and 30% respectively relative to vehicle ACM). In order to explore the mechanism underlying these effects on breast cancer metastatic capability, the mRNA expression of IGF‐1, IL‐6 and leptin was measured in adipocytes treated with either vehicle or 1,25(OH)2D. Treatment of adipocytes with 1,25(OH)2D decreased mRNA expression of IGF‐1, IL‐6 and leptin by 23%, 85%, and 39%, respectively. Further studies aim at demonstrating the mechanism underlying the impact of 1,25(OH)2D on adipocytes to inhibit their capability of promoting breast cancer metastasis. In summary, these results suggest that 1,25(OH)2D alters adipocyte secretions to inhibit breast cancer metastasis.Support or Funding InformationThis work was supported in part by the Higher Education Challenge Grant from the National Institute of Food and Agriculture, United States Department of Agriculture (#2013‐70003‐20922).