Estrogen positive breast cancer accounts for nearly 70% of breast cancer-related deaths, and often is treated with estrogen receptor antagonists such as tamoxifen. Unfortunately, ERα targeted therapies can result in resistance, subsequent metastasis, and death. Reprogramming of lipid metabolism is an established hallmark of cancer and is associated with drug-resistance, including resistance to anti-endocrine therapies. The rate-limiting enzyme for endogenous lipid biosynthesis, fatty acid synthase (FASN), is overexpressed in numerous cancers and is associated with drug-resistance. The FASN inhibitor TVB-2640 has shown preliminary evidence of activity in a phase 1 clinical trial dose expansion arm for metastatic breast cancer. In this study, we illustrate that FASN inhibitor, TVB-3166 (preclinical version of TVB-2640), significantly reduces growth and proliferation of tamoxifen resistant breast cancer in in vitro, ex-vivo and in vivo models by inducing ERα degradation through increased endoplasmic reticulum stress.
The inhibition of FASN has several consequences that result in either apoptosis or stasis in cell progression [13]. The primary product of FASN includes the long-chain fatty acid, palmitate, that can post-translationally modify oncoproteins, such as Wnt and epidermal growth factor receptor (EGFR) that facilitate their intracellular localization and membrane hydrophobicity [32]. Moreover, palmitate itself undergoes subsequent alterations to generate phospholipids for membrane synthesis, which is essential for rapidly proliferating tissue [33]. A phase 1 study demonstrated a decrease in circulating palmitate metabolites in patients treated with TVB-2640 [19]. FASN is transcriptionally and post-transcriptionally regulated through PI3K/Akt/mTOR pathway downstream of receptor tyrosine kinases (RTKs), such as EGFR [34]. Thus, FASN-induced palmitoylation of RTKs could mediate a feedforward loop to support lipid metabolic programming in anti-endocrine resistance. Additionally, we have also observed an increase in malonyl-carnitine, a more stable derivative of the FASN substrate malonyl-CoA [19]. Malonyl-CoA has a short half-life and is therefore difficult to measure. Thus, the decreased cellular pool of palmitate for membrane synthesis and post-translational modifications could explain the growth inhibitory effects of FASN inhibition observed.
In response to treatment with tamoxifen, TVB-3166, or in combination, RNA sequencing and phospho-receptor tyrosine kinase arrays demonstrated a significant increase in the receptor tyrosine kinase-like orphan receptor-2 (ROR2) when treated with the combination of TVB and tamoxifen compared to tamoxifen treatment alone in tamoxifen-resistant cells. Classically, both ROR1 and ROR2, are involved in neurogenesis and embryonic development. Moreover, expression of ROR1 and ROR2 is associated with increased invasion and poor prognosis in numerous cancers [35, 36]. Interestingly, in certain cancers, ROR2 expression exhibits tumor-suppressing characteristics. Recent studies have illustrated ROR2 hypermethylation in nasopharyngeal, esophageal, and breast cancer cell lines promoted EMT and proliferation [35]. Additionally, ROR2 overexpression resulted in reduced Akt and β-catenin signaling in multiple cancer cell lines [35]. Not only does ROR2 possess tumor-suppressing traits but is also implicated in inducing apoptosis through the EnRS pathway. Recently, ROR2 promoted apoptosis in ovarian carcinoma through the induction EnRS sensing protein, inositol requiring enzyme-1α (IRE1α) and downstream C/EBP homologous protein (CHOP) [35, 36]. Thus, ROR2 overexpression observed in response to TVB and tamoxifen treatment could be illustrating tumor-suppressing characteristics.
Additionally, we demonstrate that treatment with TVB-3166 leads to a decrease in ERα protein that was specific to tamoxifen-resistant breast cancer compared to the tamoxifen sensitive. Several studies have elucidated an increase in cap-dependent mRNA translation through mTOR and ERK pathways as a mechanism of acquired tamoxifen resistance. Moreover, FASN inhibition has been demonstrated to attenuate Akt and mTORC1 signaling in multiple studies. In our lab, we observed an inhibition in the phosphorylation of Akt, which is upstream of mTORC1. Pathways upregulated in tamoxifen resistance, such as mTOR, involve the phosphorylation of translational inhibitory complexes such as 4E binding proteins (4E-BP1) leading to an alleviation of eIF4E cap-binding protein. The mRNA translational initiation complex is composed of several eIF4F family members that include an RNA helicase (eIF4A), scaffolding protein (eIF4G), and the rate-limiting cap-binding protein (eIF4E) [30, 31]. Moreover, many oncoproteins and growth factors such as cyclin D1 and vascular endothelial growth factor (VEGF) are encoded by mRNAs with longer than average 5’ untranslated regions (UTRs) [30]. Translational complexes, such as eIF4E, have an enhanced affinity for longer UTRs. More specifically, using a mutated eIF4E that renders it unable to become phosphorylated resulted in an attenuation in proliferation in response to tamoxifen in tamoxifen-resistant cell lines [31]. This upregulation in mRNA cap-dependent translation pathways in tamoxifen- resistance holds the premise for an increased sensitivity to FASN inhibition in tamoxifen-resistant compared to tamoxifen sensitive breast cancer.
We observed that FASN inhibition induced ERα degradation is mediated through the EnRS pathway. Historically, higher ratios of phosphatidylcholine to phosphoethanolamine as well as increases in intracellular cholesterol lead to EnRS [23]. Studies have shown alterations in intracellular membrane lipids as well as a compensatory increase in cholesterol biosynthesis in response to FASN inhibition. Recent studies, conducted by Zadra et al., 2019, illustrated that FASN inhibition in prostate cancer leads to an alteration in phospholipids within the EnR membrane [22]. The initiation of protein translation involves both the recognition of the start codon in mRNA by the preinitiation complex as well as the recruitment of various eukaryotic initiation factors (eIFs) [31]. eIFs 1, 1A, 2, 3, 4 and 5 are all involved in translation[31]. Moreover, the eIF2 complex is the site of translational control in periods of starvation, stress, or viral infection[31, 37]. EnRS through the PKR like endoplasmic reticulum kinase (PERK) results in the phosphorylation of eukaryotic initiation factor 2α [23]. This results in protein translation inhibition, which could explain the decrease in ERα protein levels observed upon FASN inhibition. This EnRS response to FASN inhibition was only observed in tamoxifen-resistant breast cancer; however, the specific response could be due to the increased reliance upon translational machinery that has been recorded previously in acquired tamoxifen resistance [30, 31]. Intriguingly, the increased expression of ROR2 recorded in the phospho-RTK assay could be a potential mediator of endoplasmic reticulum stress. Previous studies connecting ROR2 expression to CHOP induced apoptosis highlight a potential connection to the PERK/p-eIF2α EnRS pathway [36]. Not only does p-eIF2α lead to a decrease in protein translation, but also allows the initiation complex to continue down the mRNA until it reaches the alternate reading frame [31]. The alternate reading frame of mRNA is the location of translation for EnRS response proteins, such as CHOP and ATF-4 [30, 31]. Thus, the overexpression of ROR2 from treatment with TVB and tamoxifen could potentially be contributing to endoplasmic reticulum stress.
We previously preliminarily demonstrated in a phase 1 study that FASN inhibition is a viable treatment option for ER + breast cancer patients that have become resistant to anti-endocrine therapies [19]. In this study, we explore the potential mechanism and have shown that FASN inhibitor, TVB-3166, targets ERα protein for degradation in tamoxifen-resistant breast cancer. This loss in ERα protein was found to be as a result of increased endoplasmic reticulum stress. Future studies should investigate the connection to lipid metabolic programming and the mRNA translational network. Moreover, more specific mechanisms linking FASN expression directly to acquired anti-estrogen resistance should be conducted. Finally, more investigation should be done illustrating the role of ROR2 in tamoxifen-resistant breast cancer. With more targeted and less toxic therapy options, patients are extending not only their life, but their quality of life. FASN inhibition, either as a monotherapy or in combination, could be a potential novel treatment for anti-endocrine therapy-resistant breast cancer.