Emerging Mechanisms and Therapies in Endocrine-Related Tumor Biology

Darjan Duraki, BS Biochemistry

University of Illinois at Urbana-Champaign, IL, United States

Abstract

Metastatic estrogen receptor α (ERα) positive breast cancer is presently incurable and most patients die within 7 years. From a medicinal chemistry program, we identified a novel small molecule that acts through ERα to kill breast cancer cells and often induces complete regression without recurrence of large, therapy-resistant primary breast tumors and of lung, bone, and liver metastases. We exploited our finding that estrogen-ERα activates an extranuclear tumor-protective, signaling pathway, the anticipatory unfolded protein response (UPR). We repurposed this tumor protective pathway by targeting it with the small molecule, ErSO. ErSO kills cancer cells by acting non-competitively through ERα to induce lethal hyperactivation of the anticipatory UPR (a-UPR), triggering rapid necrotic cell death. Using luciferase to image primary tumors and metastases containing lethal ERαD538G and ERαY537S mutations seen in metastatic breast cancer, oral and injected ErSO exhibited unprecedented antitumor activity. In mouse xenografts bearing large breast tumors, oral and injected ErSO induced complete regression (>115,000 fold mean regression) in about 45% of mice (18/39). Although durable response without treatment for 4-6 months was common, tumors that did recur remained fully sensitive to ErSO re-treatment. Consistent with the essential nature of the a-UPR pathway targeted by ErSO, in more than 100 tumor-bearing mice, we have never seen an ErSO-resistant tumor. In just 7 days, oral ErSO induced complete regression of most lung, bone, and liver metastases. ErSO is well-tolerated in mice and blood-brain-barrier penetrant. Injected ErSO induced profound regression of challenging brain tumors. On average, ErSO-treated tumors were >180-fold smaller than vehicle-treated tumors. Moreover, use of ErSO is not limited to breast cancer. With its unique mechanism of action through the a-UPR, ErSO eradicated orthotoptic ERα positive ovarian tumors that do not require estrogen for growth. These xenograft studies used human cancer cells in immune compromised mice and therefore did not exploit the known ability of inducers of necrotic cell death to activate immune cells and induce immunogenic cell death. Notably, medium from breast cancer cells killed by ErSO contained high levels of the established immune cell activators, HMGB1 and ATP, robustly activated mouse and human macrophages and increased macrophage migration. ErSO’s potent activity against advanced primary and metastatic ERα-positive breast cancers represents a paradigm shift in leveraging ERα for anticancer efficacy.

Erin Gibbons, MS

UNIVERSITY OF ROCHESTER, NY, United States

Abstract

Lymphangioleiomyomatosis (LAM) is an estrogen-sensitive lung disease found almost exclusively in women. LAM is characterized by the hyperproliferation of smooth muscle cells creating small tumors throughout the lungs, resulting in the formation of large cysts that replace normal alveolar space. Growth of these tumors and progression of the cyst development leads to loss of pulmonary function, and sometimes subsequent lung transplantation. LAM tumor cells contain mutations in one of the tuberous sclerosis genes (TSC1 or TSC2), leading to activation of the mTORC1 pathway. In fact, mTOR inhibitors are commonly used to treat LAM; however, these drugs are not always effective and have significant side effects, suggesting the need for new therapeutic targets. Additionally, tumors recur even after lung transplantation and LAM cells are found in circulating body fluids, suggesting a metastatic nature of LAM, and a question of the origin of the LAM cell. Due to LAM’s estrogen sensitivity, female specificity, and metastatic nature, we previously proposed that LAM cells originate from the uterine myometrium. We therefore designed a uterine-specific TSC2-null mouse model where all the mice generate uterine tumors characteristic of LAM and half develop lung metastases. Using RNASeq analysis of uterine tissue from this mouse model, when focusing on genes regulated by estrogen and TSC2, we discovered significant upregulation of inflammatory proteases such as Neutrophil Elastase (NE). NE is secreted by myeloid cells such as polymorphonuclear cells (PMNs) and has been reported to promote invasion, migration, and proliferation in various cancers. We found this to be true in LAM as well, as depleting myeloid cells with an antibody directed against PMNs, or inhibiting NE with the NE inhibitor, sivelestat, markedly decreased TSC2-null uterine tumor growth. NE is released when PMNs undergo Neutrophil Extracellular Trap release, or NETosis. NETosis has been shown to have a pro-tumorigenic role in various cancers and we are investigating the effects of NETosis in LAM. We have also generated a novel uterine-specific TSC2-null mouse in the background of no NE to determine whether uterine tumor burden and lung metastases are reduced in NE-null mice and if these mice have PMNs capable of undergoing NETosis in the absence of NE. Overall, our results suggest that NE release from PMNs is critical for LAM tumor development and may be a novel target for its treatment.

Jay Gertz, PhD

University of Utah, UT, United States

Abstract

One of the oldest and most common therapies for endometrial complex atypical hyperplasia (CAH) and low-stage, low-grade endometrioid endometrial carcinoma (EEC) is the use of progestins. Importantly, the use of progestins remains the only fertility-sparing treatment available. Despite frequent initial response to progestins, relapse rates are high (35-50%). Currently, there are no biomarkers available for predicting a woman’s likelihood of successful progestin therapy. Primary samples (n = 63) were obtained from a total of 31 patients with either CAH or EEC who underwent progestin therapy and were acquired pre- and post-treatment with progestins. Pathological review of the FFPE samples was performed to identify regions of high hyperplastic or neoplastic content for core punches and RNA extraction. RNA-seq was then performed on the FFPE RNA using the TruSeq RNA Exome approach, a method that uses targeted capture to improve sequencing from fragmented samples. Differential expression analysis was performed using two methods: DESeq2 a parametric method and Noiseq a non-parametric method. Both methods were used to obtain an overlapping subset of genes to reduce spurious results due to samples with outlier expression. Analysis of all samples identified 137 genes significantly associated with outcome. These 137 genes were largely increased in post-treatment samples from progestin responders and were highly enriched for progestogen and estrogen responsive genes, indicating a strong hormonal gene expression response to progestin therapy. Importantly, post-treatment samples from non-responding patients did not show this expression pattern, demonstrating that this set of genes may indicate successful hormone response in post-treatment samples. We also identified a 61 gene signature that remains high in non-responders after treatment compared to responders. Overall, we find that responders show a coordinated change in expression during progestin therapy that is missing from non-responders and this signature could be used in the early evaluation of progestin treatment success. Focusing solely on pre-treatment samples, we identified more variable expression differences across tumors, suggesting multiple reasons for progestin success/failure. We found that the combined expression of estrogen receptor alpha and progesterone receptor was predictive of progestin therapy success. In addition, non-responding tumors had increased expression of several immune-related genes that we are currently exploring. Overall, these results show that progestin therapy response could be predicted using gene expression signatures and that multiple factors may underlie progestin success/failure.

Rebecca B Riggins, PhD

Georgetown University, DC, United States

Abstract

Reema S Wahdan-Alaswad, PhD, MS

University of Colorado Anschutz Medical Campus, CO, United States

Abstract

Background: Breast cancer (BC) and thyroid disease are well-recognized comorbidities. Hyperthyroidism and supraphysiologic thyroid hormone (TH) have been shown to promote BC incidence. We recently reported that thyroid hormone replacement therapy (THRT) was significantly and independently associated with shortened disease-free and overall survival, as well as endocrine resistance only in patients with steroid receptor-positive (SR+) BC (1). TH markedly upregulated estrogen and cell cycle signaling in vivo and in vitro and promoted dedifferentiation to basaloid and pre-stem phenotypes. Metformin (Met) attenuated this shift. Mechanisms of TH-mediated endocrine therapy resistance in ER+ BC are the focus of this report. Design: Two clinical cohorts of early-stage lymph node-negative (LN-) SR+ BC patients (n=820 and n=160) were used to determine the effect of THRT on overall survival using Kaplan-Meier methods. Bi-directional cross-talk between TH and E2 was tested using different BC cell lines, ER+ PDX in vivo models, in vitro methods, and publically available in silico data for modeling. Results: Our results show that E2+TH increases cell proliferation, enhances cell cycle, and hormone-associated oncogenic signaling in SR+/ER+ BC. Given that high expression of THRA is associated with poor prognosis in SR+ BC, knockdown of THRA and ESR1 reduced cell proliferation in ER+ BC cells. ER+ PDX tumors were implanted into NSG mice containing E2 pellet and subsequently treated with TH, Tamoxifen (Tam), Fulvestrant (ICI) or Met. Our data show that TH-mediated endocrine resistance only in the E2+TH+Tam treated tumors (P<0.0001 vs E2+Tam alone). Both ICI and Met provided significant attenuation of tumor growth in vivo. RNAseq analysis of E2+TH+Tam tumors show an increase in pro-oncogenic signaling (Wnt/Fizzled, MMPs, and TCL/LEFT). Our data suggest that the use of Tam did not dampen tumor growth whereas a full ER-antagonist (ICI) or Met attenuated E2-TH mediated cross-talk and tumor growth. Conclusions: These findings suggest that TH+Tam may enhance oncogenic signaling and is associated with a significantly increase in mortality risk in ER+/SR+ BC tumors. Exogenous TH adversely affects SR+ BC and not SR- BC. Understanding the mechanism of cross-talk between TH and E2 allows us to define novel therapeutic strategies that will facilitate rapid clinical application for ER+ BC patients currently taking THRT and anti-estrogen treatments.Reference: (1) Wahdan-Alaswad et. al. Clin Cancer Res October 23 2020 DOI: 10.1158/1078-0432.CCR-20-264.