Abstract PS2-11-12: Ccl5 and endoglin as a therapeutic target in er positive breast cancer

<div>Abstract<p>Resistance to endocrine therapy represents a major concern for patients with estrogen receptor α positive (ERα+) breast cancer. Endocrine therapy resistance is commonly mediated by activated E2F signaling. A better understanding of the mechanisms governing E2F1 activity in resistant cells could reveal strategies for overcoming resistance. Here, we identified the long non-coding RNA (lncRNA) actin gamma 1 pseudogene 25 (AGPG) as a regulator of E2F1 activity in endocrine resistant breast cancer. Expression of EGPG was increased in endocrine-resistant breast cancer cells, which was driven by epigenomic activation of an enhancer. AGPG was also abnormally upregulated in patient breast tumors, especially in the luminal B subtype, and high AGPG expression was associated with poor survival of ERα+ breast cancer patients receiving endocrine therapy. The upregulation of AGPG mediated resistance to endocrine therapy and CDK4/6 inhibition in breast cancer cells. Mechanistically, AGPG physically interacted with PURα, thus releasing E2F1 from PURα and leading to E2F1 signaling activation in ERα+ breast cancer cells. In breast cancer patients, E2F1 target genes were positively and negatively correlated with expression of AGPG and PURα, respectively. Co-administration of chemically modified AGPG siRNA and tamoxifen strongly suppressed tumor growth in endocrine resistant cell line-derived xenografts. Together, these results demonstrate that AGPG can drive endocrine therapy resistance and indicate that it is a promising biomarker and potential therapeutic target in breast cancer.</p></div>

Lipid metabolism may be involved in the development of endocrine drug resistance in ER-positive (ER+) breast cancer (BC). This study aimed to investigate the relationship between serum lipid levels, risk stratification of dyslipidemia, and endocrine resistance. We collected the data from 166 ER + breast cancer patients who received endocrine therapy (ET). 73 of 166 patients (44.0%)developed endocrine resistance. Univariate and multivariate COX regression were conducted to explore the potential factors affecting endocrine resistance in BC. The clinical T stage, mean serum lipid levels in ET progression-free-survival (total cholesterol, triglycerides, low-density lipoprotein cholesterol, apolipoprotein A, and triglycerides/high-density lipoprotein cholesterol) were correlated with endocrine resistance (R = 0.214, P = .006; R = 0.268, P < .001; R = 0.182, P = .019;R = 0.197, P = .011; R = 0.211, P = .006; R = 0.159, P < .041). Clinical stage, triglycerides (TG) in endocrine therapy progression-free-survival (ePFS) and low-density lipoprotein cholesterol (LDL-C) in ePFS were independent predictors of endocrine resistance (P < .05; OR = 1.406, CI 1.108-1.783, P < .05; OR = 1.309, CI 1.026-1.669, P < .05, respectively). Moreover, in clinical stage III, the ePFS was worse in patients with in the high-risk and extremely high-risk group the median ePFS time was 8.0 months (95% CI: 1.140-14.860, P < .05). Clinical stage, TG in ePFS and LDL-C in ePFS may act as a new predictive biomarker for endocrine resistance in BC. The lipid levels of BC patients should be closely monitored throughout the treatment process, and patients with dyslipidemia should receive treatment immediately.

Steroid receptor coactivator-1: A versatile regulator and promising therapeutic target for breast cancer

To study anticancer effects, underlying mechanism and safety of ethoxy mansonone G (EMG) which is the potent derivative of mansonone G (MG) in breast cancer cells. Anticancer, antimigration, anti-invasion effects and anchorage-independent growth were investigated by MTT, scratch, matrigel invasion and soft agar assays. Estrogen receptor (ER)-targeted genes and endocrine-resistant genes were assessed by RT-PCR and Western blot. Ethoxy mansonone G is the most potent MG derivative and has anticancer effects in ER-positive, endocrine-resistant and ER-negative breast cancer cells. Our results demonstrated that EMG can significantly inhibit estrogen-induced cell proliferation and the expression of ER-targeted genes in ER-positive breast cancer cells, suggesting the anti-estrogenic property of EMG which is consisting with the virtual molecular docking that EMG could possibly bind to the ERα. Moreover, EMG has synergistic effect with tamoxifen in endocrine-resistant cells. EMG also inhibited cell proliferation, invasion and anchorage-independent growth by reducing expression of genes involved in endocrine resistance and invasive factors during the metastatic process. Ethoxy mansonone G has an anticancer effect in breast cancer cells and is possible to use as a therapeutic agent in patients with breast cancer.

Deleted in Liver Cancer-1 (DLC1), a member of the RhoGAP family of proteins, functions as a tumor suppressor in several cancers including breast cancer. However, its clinical relevance is unclear in breast cancer. In this study, expression of DLC1 was correlated with prognosis using publicly available breast cancer gene expression datasets and quantitative Reverse Transcription PCR in cohorts of Estrogen Receptor-positive (ER+) breast cancer. Low expression of DLC1 correlates with poor prognosis in patients with ER+ breast cancer with further decrease in metastatic lesions. The Cancer Genome Atlas (TCGA) data showed that down regulation of DLC1 is not due to methylation or mutations. To seek further insights in understanding the role of DLC1 in ER+ breast cancer, we stably overexpressed DLC1-full-length (DLC1-FL) in T-47D breast cancer cells; this inhibited cell colony formation significantly in vitro compared to its control counterpart. Label-free global proteomic and TiO2 phosphopeptide enrichment assays (ProteomeXchange identifier PXD008220) showed that 205 and 122 phosphopeptides were unique to DLC1-FL cells and T-47D-control cells, respectively, whereas 6,726 were quantified by phosphoproteomics analysis in both conditions. The top three significant clusters of differentially phosphopeptides identified by DAVID pathway analysis represent cell-cell adhesion, mRNA processing and splicing, and transcription regulation. Phosphoproteomics analysis documented an inverse relation between DLC1 expression and several phosphopeptides including epithelial cell transforming sequence 2 (ECT2). Decreased phosphorylation of ECT2 at the residue T359, critical for its active conformational change, was validated by western blot. In addition, the ECT2 T359-containing phosphopeptide was detected in both basal and luminal patient-derived breast cancers breast cancer phosphoproteomics data on the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Assay portal. Together, for the first time, this implicates ECT2 phosphorylation in breast cancer, which has been proposed as a therapeutic target in lung cancer. In conclusion, this data suggests that low expression of DLC1 is associated with poor prognosis. Targeting ECT2 phosphopeptides could provide a promising mechanism for controlling poor prognosis seen in DLC1low ER+ breast cancer.

Targeting endocrine resistant breast cancer cells has been successfully accomplished by the use of mTORC1 inhibitors in combination with either aromatase inhibitors or tamoxifen. While there was preclinical data suggesting that targeting of the type I IGF receptor (IGF1R) might also be a target for endocrine resistant cells, clinical trials using IGF1R monoclonal antibodies showed no advantage and even suggested potential harm for the combination. IGF1R inhibition results in the disruption of an endocrine feedback loop and results in elevation of the pituitary growth factor growth hormone (GH). GH is involved in normal human growth, development, metabolism, and longevity. Growth hormone released from the pituitary stimulates production of IGF-I from the liver. The GH/IGF-1 axis is known to promote mammary gland hyperplasia and breast cancer carcinogenesis. Previous studies have shown that GH promotes carcinogenesis independently of insulin-like growth factor. The role of GH in specific subtypes of breast carcinoma remains to be defined. Our laboratory has found that estrogen receptor positive breast cancer cell lines up regulated oncogenic STAT5, Akt, and MAPK signaling pathways in response to GH. Tamoxifen resistant (TamR) and long-term estrogen deprived (LTED) breast cancer cell lines derived from MCF-7 and T47D cells demonstrated increased oncogenic signaling in response to GH compared to the corresponding parental cell lines from which they were derived. In contrast, GH stimulation of a triple negative breast cancer cell line (MDA-MB-231) failed to induce signaling via these oncogenic pathways. Although GH treatment of parental, TamR, and LTED breast cancer cells had minimal effect on cell proliferation, cell cycle progression, or migration; GH signaling protected cells from cytotoxic chemotherapy induced apoptosis. This data shows that GH signaling is found in endocrine sensitive and resistant cells. Furthermore, GH may function in protecting cells from cell death induced by either endocrine or cytotoxic drugs. GH receptor blockade may be a valid treatment option for endocrine resistant breast cancer. Citation Format: Heather C Beckwith, Douglas Yee. Growth hormone induction of oncogenic signaling promotes survival of endocrine resistant breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-06-06.

Background: Gene fusions resulting from genomic rearrangements are important drivers for cancer initiation and progression. Estrogen receptor-positive (ER+) breast cancer is the most common type of breast cancer, and luminal B confers a more aggressive phenotype and higher risk of early relapse and prone to resistance to endocrine therapy. Identify new druggable genetic driver will be critical to improve clinical outcome of aggressive luminal B tumors. Our lab has identified a recurrent rearrangement between the estrogen receptor gene ESR1 and its neighbor gene CCDC170, in 6-8% of luminal B tumors. The exon 2 of ESR1 fused with the exon 6, 7, 8 or 10 of CCDC170, which enables the expression of different-sized N-terminally truncated CCDC170 (ΔCCDC170) under ESR1 promoter. Ectopic expression of ΔCCDC170 led to malignant transformation phenotypes in ER+ breast cancer cells, such as increase the cell migration, invasion and anchor-independent cell growth, as well as enhance xenograft tumor growth in mice. Methods: The effect of ESR1-CCDC170 on endocrine resistance was examined by cologenic assay in vitro, and by xenograft mouse model in vivo. Bioinformatics approaches, Reverse Phase Protein Array (RPPA) analysis and Bimolecular Fluorescence Complementation assay were used to elucidate the potential molecular mechanism. Results: This study aimed to determine the role of ESR1-CCDC170 in breast cancer endocrine resistance and elucidate the potential mechanisms as well as the therapeutic effect thereof. Our results show that ectopic expression of fusion variants in ER+ T47D cell line sustains the cell proliferation ability under estrogen deprivation (ED), and decreases the sensitivity to 4-OH Tamoxifen treatment. While silencing of ESR1-CCDC170 in fusion positive HCC1428 cell line increases the sensitivity to 4-OHTamoxifen and Fulvestrant treatment. Results from T47D xenograft tumor models show that ESR1-CCDC170 fusion induces endocrine resistance in vivo. Further mechanism studies show that ESR1-CCDC170 possesses a potential ATP binding pocket, has the ability to form homodimers. Conclusion: These results demonstrate the important role of ESR1-CCDC170 fusions in breast cancer endocrine resistance, and suggest that ESR1-CCDC170 fusion could be a potential therapeutic target for the treatment of breast cancer. Citation Format: Li Li, Jamunarani Veeraraghavan, Yiheng Hu, Xian Wang, Ying Tan, Rachel Schiff, Xiaosong Wang. Therapeutic role of ESR1-CCDC170 gene fusion in breast cancer endocrine resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 376.

Abstract #3027 Background: Acquired endocrine resistance in breast cancer cells is accompanied by altered growth factor receptor signalling and a highly migratory cell phenotype. In tamoxifen-resistant ('TamR') MCF7 cells, our microarray analysis has demonstrated elevated levels of CD44, a transmembrane glycoprotein, known to interact with and modulate the function of a number of growth factor receptors and plays a major role in tumour metastasis. Here, we have explored the role of CD44 as a modulator of erbB family activity in TamR cells and evaluated the expression of CD44 in a series of breast cancer patients on endocrine therapy.&amp;#x2028; Methods: RT-PCR was performed to confirm overexpression of CD44 at the gene level in TamR cells. Western blotting and immunocytochemistry were used to investigate expression of CD44 at the protein level together with the activity of erbB signalling pathways following stimulation of CD44 by its ligand, hyaluronan (HA), or following stimulation with erbB ligands (TGFα, heregulin β1) after knockdown of CD44 expression using siRNA. Immunofluorescence microscopy was used to determine association between CD44 and erbB family members. Cell migration was assessed by seeding cells onto fibronectin-coated microporous inserts for 24 hours. CD44 expression levels were evaluated using paraffin-embedded tissue sections in a series of primary breast cancer tissues (n=103) from patients receiving endocrine therapy by immunohistochemical staining.&amp;#x2028; Results: CD44 was overexpressed at the gene and protein level in TamR cells versus their endocrine sensitive counterparts. siRNA-mediated suppression of CD44 expression reduced basal EGFR/Her2 activity in TamR cells together with suppressing their migratory phenotype. siRNA-mediated inhibition of CD44 expression greatly attenuated the ability of erbB ligands to activate erbB receptors and inhibited TGFα and heregulin β1-induced migration. Conversely, stimulation of TamR, but not MCF7 cells, with HA promoted activation of erbB family members together with their downstream signalling intermediates ERK1/2, Src and AKT. Analysis of CD44 expression in clinical tissue revealed an association between elevated CD44 expression and a shortened response to endocrine therapy.&amp;#x2028; Conclusions: These data demonstrate that overexpression of CD44 accompanies endocrine resistance where it enhances the sensitivity of these cells to factors (erbB ligands, HA) found within the tumour microenvironment. This may implicate CD44 in loss of endocrine response clinically and, therefore, provide a novel therapeutic target in endocrine resistant breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3027.

Cancer cells undergo dynamic and frequently reversible modifications and this cellular plasticity permits reprogramming in response to long term endocrine treatment. A well-established theory, the cancer stem cell theory; suggests that within an ER positive tumour cell population are a small number of stem cells which are capable of infinite self-renewal and are insensitive to treatment with standard endocrine regimes. However, emerging reports that pluripotent cells can be generated from adult somatic cells (Takahashi, 2007) alludes to an alternate mechanism for cancer cells to reprogramme and evade endocrine treatment. To investigate the role of tamoxifen in promoting cellular reprogramming and endocrine resistance in breast cancer, we isolated single luminal A breast cancer CD24+CD44- clones from endocrine sensitive MCF-7 cells. The clones were expanded in the presence and absence of tamoxifen. Extensive profiling of cells following long-term exposure to tamoxifen revealed a resistant phenotype similar to that reported in other established models. Steroid receptor co-activator 1 (SRC-1) is a master regulatory protein which has been shown to be central to the development of endocrine resistance. The mechanisms of which are still poorly understood (Walsh et al., 2012). Utilising genome-wide transcriptomic sequencing this study investigated whether SRC-1 can regulate transcriptional networks which mediate reprogramming in individual cells to induce survival adaptability and drug resistance in breast cancer. RNA-sequencing was carried out in a model of endocrine resistance (shNon-Targeting versus shSRC-1). There were 1,731 genes up-regulated by SRC-1, of which 153 were identified as transcription factors (TFs)/chromatin remodellers. Combining the transcriptomic profiling with ChIP-sequencing we identified a transcriptional network pertinent to SRC-1. Molecular characterisation identified E2F7, NFIA, DEK, SMAD2, SMARCA1, ASCL1 and TRPS1 as key drivers of SRC-1 mediated endocrine resistance. Each TF was confirmed as a direct target of SRC-1, via promoter specific binding and were found to be drivers of endocrine resistant cell migration. Furthermore, all TFs were necessary in mammosphere formation and their promotion of cellular de-differentiation was observed by 3D acinar organisation and flow cytometry. To elucidate the core TFs required for cancer cell reprogramming, their effect on the pioneer reprogramming TFs (OCT4, SOX2, cMYC and KLF4) was investigated. SMAD2, SMARCA1, ASCL1 and TRPS1 have emerged as pivotal regulators of endocrine resistant cell reprogramming. This study has unravelled an SRC-1-mediated TF network responsible for promoting the cellular reprogramming of breast cancer cells. Concerted activity of this network is responsible for driving de-differentiation of cells and enhancing their stem-like, highly migratory and proliferative tumour initiation population. This study provides important information regarding the mechanism of cellular reprogramming in ER positive endocrine resistant cancer and may lead to potential novel therapeutic targets. Citation Format: Browne A, Fagan A, Vareslija D, Ward E, Hill A, Young L. Steroid receptor co-activator 1 mediation of cancer cell reprogramming in endocrine resistant breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-16.

Inhibition of RAC1 activity in cancer associated fibroblasts favours breast tumour development through IL-1β upregulation.

Resistance to endocrine and chemotherapies remains the primary cause of breast cancer treatment failure. We have synthesized four novel D: -erythro N-octanoyl sphingosine analogs and catalogued their activity in drug-sensitive (MCF-7), endocrine-resistant (MDA-MB-231) and chemoresistant (MCF-7TN-R) breast cancer cells. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell viability; colony assay was performed to determine effects on clonogenic survival and (1)H NMR, (13)C NMR, HPLC spectra and elemental analytical data analyses were used to determine analog identity and purity. All four analogs inhibited both viability and clonogenic survival, with analog C exhibiting a log-fold improvement in anti-survival activity compared to the parent compound. With resistance to current breast cancer chemotherapies on the rise, the development of novel therapeutic targets is of growing importance. Our results show that lipid analogs have therapeutic potential in treating chemo- and endocrine-resistant breast cancer.

&lt;div&gt;AbstractPurpose:&lt;p&gt;Endocrine resistance remains a major clinical challenge in estrogen receptor (ER)–positive breast cancer. Despite the encouraging results from clinical trials for the drugs targeting known survival signaling, relapse is still inevitable. There is an unmet need to discover new drug targets in the unknown escape pathways. Here, we report Nemo-like kinase (NLK) as a new actionable kinase target that endows previously uncharacterized survival signaling in endocrine-resistant breast cancer.&lt;/p&gt;Experimental Design:&lt;p&gt;The effects of NLK inhibition on the viability of endocrine-resistant breast cancer cell lines were examined by MTS assay. The effect of VX-702 on NLK activity was verified by kinase assay. The modulation of ER and its coactivator, SRC-3, by NLK was examined by immunoprecipitation, kinase assay, luciferase assay, and RNA sequencing. The therapeutic effects of VX-702 and everolimus were tested on cell line- and patient-derived xenograft (PDX) tumor models.&lt;/p&gt;Results:&lt;p&gt;NLK overexpression endows reduced endocrine responsiveness and is associated with worse outcome of patients treated with tamoxifen. Mechanistically, NLK may function, at least in part, via enhancing the phosphorylation of ERα and its key coactivator, SRC-3, to modulate ERα transcriptional activity. Through interrogation of a kinase profiling database, we uncovered and verified a highly selective dual p38/NLK inhibitor, VX-702. Coadministration of VX-702 with the mTOR inhibitor, everolimus, demonstrated a significant therapeutic effect in cell line-derived xenograft and PDX tumor models of acquired or &lt;i&gt;de novo&lt;/i&gt; endocrine resistance.&lt;/p&gt;Conclusions:&lt;p&gt;Together, this study reveals the potential of therapeutic modulation of NLK for the management of the endocrine-resistant breast cancers with active NLK signaling.&lt;/p&gt;&lt;/div&gt;

Introduction Brain metastasis (BrM) occurs in 10-30% of patients with advanced breast cancer (BC). BrM is increasing in incidence and confers a poor prognosis. We aimed to investigate the contribution of global epi-transcriptomic alterations in N6-methyladenosine (m6A) RNA-methylation as a therapeutic target in brain metastatic breast cancer. Method In preliminary studies we have demonstrated m6A demethylase – FTO as the main contributor to the progression of ER+ breast cancer. Furthermore an association between FTO and reduced disease-free-survival (n=870, p=0.018) was observed. Here we conducted an epigenetic inhibitor screen using two therapeutic agents, ethyl-ester-meclofenamic acid (MA2) and FB23-2 on matched 2D cell line, 3D organoid cultures and patient-derived xenografts (PDX) explant models of brain metastasis. Result Upon integration of mapped global RNA methylation landscape with matched proteomic analysis, we observed genome-wide RNA hypo-methylation of key pluripotency genes, including SOX2 and KLF4, as key players underlying tumour progression to the brain. Genetic and pharmacological inhibition of FTO in novel ex vivo models of BrM significantly reduced protein expression levels of KLF4 and SOX2. Moreover, pharmacological inhibition of FTO with MA2 and FB23-2, inhibited cell proliferation in endocrine-resistant BC and patient BrM cells. We translate our findings to the clinic by demonstrating the efficacy of anti-FTO therapies in several unique PDX and 3D organoid BrM models. Conclusion Our results reveal epi-transcriptional remodelling events as a key mechanism in BrM. This study establishes an early role for targeting RNA methylation in the management of disease progression and presents FTO as a potential therapeutic target in BrM. Take-home message This study establishes an early role for targeting RNA methylation in the management of disease progression and presents FTO as a potential therapeutic target in brain metastatic breast cancer.

e14550 Background: Endocrine therapy is the standard of care treatment for patients with estrogen-receptor positive advanced breast cancer, owing to improved tolerability and comparable efficacy to that of cytotoxic chemotherapy. Half of such cancers will progress through first line therapy (primary endocrine resistance) and half will progress after an initial period of disease control (secondary or acquired endocrine resistance). A significant challenge is to test for and identify biomarkers which can guide the likely success of endocrine therapy as a single agent or in combination with small molecule inhibitors.This is particularly challenging where metastatic deposits reside at sites where biopsy is difficult. Potential biomarkers indicative of resistance to endocrine therapy have been identified and can be detected in circulating tumour DNA (ctDNA). CtDNA is shed from tumours and is detectable in a cancer patient’s bloodstream. Information on mutational profiles can guide an oncologist in the selection of targeted therapy in addition to hormonal therapy. Methods: We have analysed formalin-fixed paraffin-embedded(FFPE) tumour samples and longitudinal liquid biopsies from 19 patients who were treated with fulvestrant in combination with a novel inhibitor of the PIK3CA/AKT pathway with next-generation sequencing using a targeted 44-gene panel. Mutations identified using this technique were tracked during the course of treatment using droplet-digital PCR(ddPCR). Results: 57 samples were tested using a 44-gene panel; 19 FFPE tumour samples and matched ctDNA samples were obtained prior therapy and ctDNA samples at disease progression. Mutations detected in PIK3CA, AKT1, ESR1and TP53 genes were trackable in longitudinal ctDNA samples using ddPCR. The association between ctDNA dynamics and outcome will be presented. Conclusions: Multiple mutations that enable the early detection of treatment failure and resistance can be tracked in ctDNA. Investigating the clinical utility of ctDNA is paramount.

Evaluation of: Turner N, Pearson A, Sharpe R et al.FGFR1 amplification drives endocrine therapy resistance and is a therapeutic target in breast cancer. Cancer Res. 70(5), 2085–2094 (2010).FGF receptor (FGFR) family members are aberrantly activated during carcinogenesis due to gene amplification, chromosomal translocation and missense mutation. FGFR1 is preferentially amplified in estrogen receptor-positive breast cancer, whereas FGFR2 is amplified in triple-negative breast cancer and diffuse-type gastric cancer. Gene amplification of FGFRs results in ligand-independent FGFR signaling to RAS–ERK, PI3K–AKT and JAK–STAT cascades due to the overexpression of wild-type or C-terminally deleted FGFRs. Cediranib, TKI258, Ki23057, MK-2461 and brivanib are broad-range tyrosine kinase inhibitors targeting FGFRs and other receptors. Clinical application of small-molecule FGFR inhibitors could improve the prognosis of FGFR-driven cancer patients. Diagnostic detection of tumors with FGFR genetic alterations in primary lesion, peritoneal effusion, pleural effusion and bone marrow is necessary to select patients for FGFR-targeted therapeutics.