Free Fatty Acids Rewire Cancer Metabolism in Obesity-Associated Breast Cancer via Estrogen Receptor and mTOR Signaling

Obesity is a risk factor for postmenopausal estrogen receptor alpha (ERα)-positive (ER+) breast cancer. Molecular mechanisms underlying factors from plasma that contribute to this risk and how these mechanisms affect ERα signaling have yet to be elucidated. To identify such mechanisms, we performed whole metabolite and protein profiling in plasma samples from women at high risk for breast cancer, which led us to focus on factors that were differentially present in plasma of obese versus nonobese postmenopausal women. These studies, combined with in vitro assays, identified free fatty acids (FFA) as circulating plasma factors that correlated with increased proliferation and aggressiveness in ER+ breast cancer cells. FFAs activated both the ERα and mTOR pathways and rewired metabolism in breast cancer cells. Pathway preferential estrogen-1 (PaPE-1), which targets ERα and mTOR signaling, was able to block changes induced by FFA and was more effective in the presence of FFA. Collectively, these data suggest a role for obesity-associated gene and metabolic rewiring in providing new targetable vulnerabilities for ER+ breast cancer in postmenopausal women. Furthermore, they provide a basis for preclinical and clinical trials where the impact of agents that target ERα and mTOR signaling cross-talk would be tested to prevent ER+ breast cancers in obese postmenopausal women

The role of metabolic rewiring in endocrine resistance

Breast cancer is the most common cancer type in women population affecting a large number of women worldwide. Due to its highly heterogenous molecular characteristics, there is not a default treatment strategy for breast cancer. Almost two third of breast cancer cases are estrogen receptor positive ER (+). Majority of breast cancer specific deaths in women with ERα (+) tumor occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to prevent or delay recurrence of ERα (+) tumors. The overall objective of this dissertation project is to define the interaction among endocrine resistance, nuclear export system, and cellular metabolic pathways, and to evaluate the metabolic impact of combinational ER and XPO1 targeting by using combination of endocrine therapy agents and a XPO1-specific small molecule inhibitor (Selinexor-SEL) in different endocrine resistant ER (+) breast cancer cell lines. The long term goal is to develop effective treatment strategies to overcome endocrine resistance in ER (+) breast cancer. In the first part of this project, we focused on the characterization of XPO1 protein in tamoxifen resistance. In this part, we used endocrine-sensitive and -resistant breast cancer cell lines (in vitro) and tumor xenograft models (in vivo) to test our hypothesis. Our results showed that expression profile of XPO1 protein has a pivotal role in the progression of tamoxifen resistance in ER (+) breast cancer cell models. We found out that XPO1 protein modulates tamoxifen resistance due to its function to determine subcellular localization of important kinases. In the second part of the project, we investigated how nuclear export system contribute to tamoxifen resistance development by testing with a novel combinational targeting strategy using 4-OHT and SEL in Luminal B type breast cancer cell lines, and to investigate the metabolic outcomes of this novel therapy. To test our hypotheses, we used actual patient tumor samples and endocrine-sensitive and -resistant breast cancer cell lines (in vitro). Our findings indicated that XPO1 expression is significantly higher in Luminal B subtype compared to other molecular subtypes. We demonstrated that combined targeting of XPO1 and ERα rewires metabolic pathways (e.g. Akt pathway), and shuts down both glycolytic and mitochondrial pathways that would eventually lead to autophagy. Lastly, we assessed the impact of other endocrine therapy options alone or in combination with Selinexor on metabolism in different endocrine resistant breast cancer cell lines and possible metastatic organ sites. We used 3-D cell culture models and endocrine-sensitive and -resistant breast cancer cell lines (in vitro). Using a combination of transcriptomics, kinase arrays, metabolomics and metabolic flux experiments, we identified glutamine metabolism pathways to be rewired during endocrine resistance. In limited media conditions mimicking nutrient deprived tumor microenvironment, endocrine resistant cells were more dependent on mitochondria for energy production. Their glucose and fatty acid dependency decreased in the presence of SEL and cells were more dependent on glutamine. The effect of glutamine was dependent on conversion of the glutamine to glutamate and mitochondrial complex 1 activity. In order to examine metabolites that might result in the observed phenotype we performed GC/MS whole metabolite profiling and identified amino acid metabolism pathways to be upregulated when cells were treated with SEL. In conclusion, our study indicated that remodeling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and given the need for better strategies for improving therapy response of relapsed ERα(+) tumors, our findings show great promise for uncovering the role ERα-XPO1 crosstalk plays in reducing cancer recurrences.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste

Figure S1 from FGFR1 Signaling Facilitates Obesity-Driven Pulmonary Outgrowth in Metastatic Breast Cancer

Pemigatinib blocks FGF2-induced signaling and 3D outgrowth of D2.OR cells via inhibition of FGFR1.</p

Figure S2 from FGFR1 Signaling Facilitates Obesity-Driven Pulmonary Outgrowth in Metastatic Breast Cancer

Paracrine FGF2 can induce the outgrowth of D2.OR cells in 3D culture.</p

Supplementary Figure Legends from FGFR1 Signaling Facilitates Obesity-Driven Pulmonary Outgrowth in Metastatic Breast Cancer

These are the figure legends for Supplementary figures 1 and 2.</p

Combined Targeting of Estrogen Receptor Alpha and XPO1 Prevent Akt Activation, Remodel Metabolic Pathways and Induce Autophagy to Overcome Tamoxifen Resistance

A majority of breast cancer specific deaths in women with ER&#945; (+) tumors occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to resensitize recurrent ER&#945; (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ER&#945; and the nuclear transport protein XPO1 in overcoming endocrine resistance. Selinexor (SEL), an XPO1 antagonist, has been evaluated in multiple late stage clinical trials in patients with relapsed and/or refractory hematological and solid tumor malignancies. Our transcriptomics analysis showed that 4-Hydroxytamoxifen (4-OHT), SEL alone or their combination induced differential Akt signaling- and metabolism-associated gene expression profiles. Western blot analysis in endocrine resistant cell lines and xenograft models validated differential Akt phosphorylation. Using the Seahorse metabolic profiler, we showed that ER&#945;-XPO1 targeting changed the metabolic phenotype of TAM-resistant breast cancer cells from an energetic to a quiescent profile. This finding demonstrated that combined targeting of XPO1 and ER&#945; rewired the metabolic pathways and shut down both glycolytic and mitochondrial pathways that would eventually lead to autophagy. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and given the need for better strategies to improve therapy response in relapsed ER&#945; (+) tumors, our findings show great promise for uncovering the role that ER&#945;-XPO1 crosstalk plays in reducing cancer recurrences

CRY1‐CBS binding regulates circadian clock function and metabolism

Circadian rhythms are generated by interlocked transcription-translation feedback loops that establish cell-autonomous biological timing of ∼24 h. Mutations in core clock genes that alter their stability or affinity for one another lead to changes in circadian period. The human CRY1Δ11 mutant lengthens circadian period to cause delayed sleep phase disorder (DSPD), characterized by a very late onset of sleep. CRY1 is a repressor that binds to the transcription factor CLOCK:BMAL1 to inhibit its activity and close the core feedback loop. We previously showed how the PHR (photolyase homology region) domain of CRY1 interacts with distinct sites on CLOCK and BMAL1 to sequester the transactivation domain from coactivators. However, the Δ11 variant alters an intrinsically disordered tail in CRY1 downstream of the PHR. We show here that the CRY1 tail, and in particular the region encoded by exon 11, modulates the affinity of the PHR domain for CLOCK:BMAL1. The PHR-binding epitope in exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and the subunit CLOCK. Moreover, PHR-tail interactions are conserved in the paralog CRY2 and reduced when either CRY is bound to the circadian corepressor PERIOD2. Discovery of this autoregulatory role for the mammalian CRY1 tail and conservation of PHR-tail interactions in both mammalian cryptochromes highlights functional conservation with plant and insect cryptochromes, which also utilize PHR-tail interactions to reversibly control their activity