Lyndsay Noel-Vanhoy Rhodes

Silver ultrasmall nanoparticles (Ag UNPs) (size < 5 nm) were used as biosensing probes to analyze the efflux kinetics contributing to multidrug resistance (MDR) in single live triple-negative breast cancer (TNBC) cells by using dark-field optical microscopy to follow their size-dependent localized surface plasmon resonance. TNBC cells lack expression of estrogen (ER−), progesterone (PR−), and human epidermal growth factor 2 (HER2−) receptors and are more likely to acquire resistance to anticancer drugs due to their ability to transport harmful substances outside the cell. The TNBC cells displayed greater nuclear and cytoplasmic efflux, resulting in less toxicity of Ag UNPs in a concentration-independent manner. In contrast, more Ag UNPs and an increase in cytotoxic effects were observed in the receptor-positive breast cancer cells that have receptors for ER+, PR+, and HER2+ and are known to better respond to anticancer therapies. Ag UNPs accumulated in receptor-positive breast cancer cells in a time-and concentration-dependent mode and caused decreased cellular growth, whereas the TNBC cells due to the efflux were able to continue to grow. The TNBC cells demonstrated a marked increase in survival due to their ability to have MDR determined by efflux of Ag UNPs outside the nucleus and the cytoplasm of the cells. Further evaluation of the nuclear efflux kinetics of TNBC cells with Ag UNPs as biosensing probes is critical to gain a better understanding of MDR and potential for enhancement of cancer drug delivery.

Abstract Triple-negative breast cancer (TNBC) is a highly aggressive breast subtype of breast cancer that lacks targeted therapies. We have previously identified several novel stilbene compounds that effectively decreased viability of TNBC. Stilbenes are natural defense compounds produced by plants and have been popularized in recent years due to general observed health benefits. Resveratrol, the most widely studied stilbene, has been revealed to possess significant antioxidant and anti-cancer effects. Many stilbenes are classified as phytoestrogens, but unlike estrogens, stilbenes do not appear to negatively impact normal breast tissue or reproductive organ health. Thus, these compounds represent new alternatives for cancer therapeutic development. The focus of this research was to identify stilbene compounds with anti-migration and/or anti-invasive properties in TNBC cells and to determine the mechanism of these inhibitory effects. Two TNBC cell lines, MDA-MB-157 and BT-549, were treated with stilbene compounds at sub-lethal doses and assessed for effects on migration and invasion using transwell assays. Cell morphology changes were evaluated with results suggestive of reversal of epithelial-to-mesenchymal transition (EMT), a key contributor in the metastatic process and a common characteristic of many TNBC cells. Gene expression analysis confirmed stilbene-induced changes of EMT-related genes. These data indicate that stilbenes, like resveratrol and related analogues, reduce cell motility and invasion through regulation of EMT gene expression in TNBC cells. Development of synthetic analogs of natural compounds remains a viable drug development opportunity for new therapeutics. Citation Format: Lyndsay V. Rhodes, Kaelyn Julmeus. Novel stilbene compounds inhibit triple-negative breast cancer invasive potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 949.

Purpose The transcription factors ZEB1 and ZEB2 mediate epithelial-to-mesenchymal transition (EMT) and metastatic progression in numerous malignancies including breast cancer. ZEB1 and ZEB2 drive EMT through transcriptional repression of cell-cell junction proteins and members of the tumor suppressive miR200 family. However, in estrogen receptor positive (ER +) breast cancer, the role of ZEB2 as an independent driver of metastasis has not been fully investigated. Methods In the current study, we induced exogenous expression of ZEB2 in ER + MCF-7 and ZR-75-1 breast cancer cell lines and examined EMT gene expression and metastasis using dose-response qRT-PCR, transwell migration assays, proliferation assays with immunofluorescence of Ki-67 staining. We used RNA sequencing to identify pathways and genes affected by ZEB2 overexpression. Finally, we treated ZEB2-overexpressing cells with 17 beta-estradiol (E2) or ICI 182,780 to evaluate how ZEB2 affects estrogen response. Results Contrary to expectation, we found that ZEB2 did not increase canonical epithelial nor decrease mesenchymal gene expressions. Furthermore, ZEB2 overexpression did not promote a mesenchymal cell morphology. However, ZEB1 and ZEB2 protein expression induced significant migration of MCF-7 and ZR-75-1 breast cancer cells in vitro and MCF-7 xenograft metastasis in vivo. Transcriptomic (RNA sequencing) pathway analysis revealed alterations in estrogen signaling regulators and pathways, suggesting a role for ZEB2 in endocrine sensitivity in luminal A breast cancer. Expression of ZEB2 was negatively correlated with estrogen receptor complex genes in luminal A patient tumors. Furthermore, treatment with 17 beta-estradiol (E2) or the estrogen receptor antagonist ICI 182,780 had no effect on growth of ZEB2-overexpressing cells. Conclusion ZEB2 is a multi-functional regulator of drug sensitivity, cell migration, and metastasis in ER + breast cancer and functions through non-canonical mechanisms.

Chemokine receptor 4 (CXCR4) and its ligand stromal-derived factor 1 (SDF-1) have well-characterized functions in cancer metastasis; however, the specific mechanisms through which CXCR4 promotes a metastatic and drug-resistant phenotype remain widely unknown. The aim of the present study was to demonstrate the application of a phenotypic screening approach using a small molecule inhibitor library to identify potential CXCR4-mediated signaling pathways. The present study demonstrated a new application of the Published Kinase Inhibitor Set (PKIS), a library of small molecule inhibitors from diverse chemotype series with varying levels of selectivity, in a phenotypic medium-throughput screen to identify potential mechanisms to pursue. Crystal violet staining and brightfield microscopy were employed to evaluate relative cell survival and changes to cell morphology in the screens. 'Hits' or lead active compounds in the first screen were PKIS inhibitors that reversed mesenchymal morphologies in CXCR4-activated breast cancer cells without the COOH-terminal domain (MCF-7-CXCR4-Delta CTD) and in the phenotypically mesenchymal triple-negative breast cancer cells (MDA-MB-231, BT-549 and MDA-MB-157), used as positive controls. In a following screen, the phenotypic and cell viability screen was used with a positive control that was both morphologically mesenchymal and had acquired fulvestrant resistance. Compounds within the same chemotype series were identified that exhibited biological activity in the screens, the 'active' inhibitors, were compared with inactive compounds. Relative kinase activity was obtained using published datasets to discover candidate kinase targets responsible for CXCR4 activity. MAP4K4 and MINK reversed both the mesenchymal and drug-resistant phenotypes, NEK9 and DYRK2 only reversed the mesenchymal morphology, and kinases, including ROS, LCK, HCK and LTK, altered the fulvestrant-resistant phenotype. Oligoarray experiments revealed pathways affected in CXCR4-activated cells, and these pathways were compared with the present screening approach to validate our screening tool. The oligoarray approach identified the integrin-mediated, ephrin B-related, RhoA, RAC1 and ErbB signaling pathways to be upregulated in MCF-7-CXCR4-Delta CTD cells, with ephrin B signaling also identified in the PKIS phenotypic screen. The present screening tool may be used to discover potential mechanisms of targeted signaling pathways in solid cancers.

Resveratrol, a plant‑derived stilbene compound, has exhibited anticancerous properties, including breast cancer. Stilbenes have a molecular structure highly similar to estrogen and have the ability to bind estrogen receptors and regulate activity. Numerous studies have demonstrated the effectiveness of resveratrol in estrogen receptor‑positive (ER‑positive) subtypes of breast cancer, yet the effects in ER‑negative subtypes, including triple‑negative breast cancer (TNBC), have been limited. In the present study, resveratrol and 28 analogues were tested on a panel of ER‑positive and TNBC cell lines to determine effects on cell viability. Several compounds exhibited significant impacts on cell viability and suggested changes in cell morphology, with high potency of select compounds compared to resveratrol observed in a dose‑dependent manner. Due to the lack of estrogen receptors in TNBC and the estrogenic nature of stilbenes, regulation of breast cancer‑associated cellular pathways was assessed for five analogues shown to significantly inhibit cell viability. Top regulated pathways included apoptosis (confirmed by caspase assay) and DNA damage repair. Overall, our results indicated several resveratrol analogues to be active in ER‑negative phenotypes, acting through an ER receptor‑independent manner, supporting further investigation into their mechanism of action and use as potential chemotherapeutics in higher‑risk breast cancer cases.

Of the 200,000 new cases of breast cancer diagnosed each year in the U.S, ~80% of the cancers are estrogen receptor alpha (ER) positive. Therefore, most first line treatments are endocrine therapies that target the ER. Selective estrogen receptor modulators (SERMs) and selective estrogen receptor down-regulators (SERDs) are used in premenopausal women, and aromatase inhibitors (AIs) are often used in postmenopausal women. Many of these cancers revert to an endocrine resistant, metastatic phenotype after one or more successful first line treatment(s). Patients who present with endocrine refractory disease have no good option for therapy other than chemotherapy regimens, which ultimately fail with high rates of mortality. Interestingly, approximately 25% of patients with endocrine resistant, metastatic disease harbor a mutated ER within the cancer cells, a mutation that was not detected in the primary tumor. During metastasis, some cells from the primary tumor are shed into the bloodstream and undergo hemodynamic shear stress on route to the secondary site of metastasis. Some of these cells, known as circulating tumor cells (CTCs), can seed in secondary organs and produce metastatic lesions. Several studies have recently linked fluid shear stress (FSS) with DNA instability and tumor metastasis. To explore this possibility, we used an in vitro model to test the effect of FSS on breast cancer cells, with the ultimate aim of understanding how FSS changes genomic dynamics, possibly leading to increased mutation frequency. We have preliminary results that physiological levels of FSS induce activation of the ATR DNA damage response pathway in both MDA-MB231 and MCF-7 breast cancer cells. Whole exome sequencing results support this data by showing sequential exposure of MCF-7 cells to FSS results in both de novo somatic mutations as well as an increase in mutation frequency at several common SNP sites. These data suggest FSS may play a role in downstream genomic instability and mutagenesis. Furthermore, Western blot and migration data of sheared cells show that FSS alters the phenotype of these cells, potentially rendering them more drug resistant and/or metastatically competent. In conclusion, we have shown that FSS may play a direct role in altering genomic instability as well as the phenotypic signature in breast cancer cells, resulting in cells that may be more metastatic and aggressive. The ultimate goal of this research is to utilize this in vitro approach to identify molecular targets that can be pursued to either prevent or delay metastatic breast disease. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Of the 200,000 new cases of breast cancer diagnosed each year in the U.S, ~80% of the cancers are estrogen receptor alpha (ER) positive. Therefore, most first line treatments are endocrine therapies that target the ER. Selective estrogen receptor modulators (SERMs) and selective estrogen receptor down‐regulators (SERDs) are used in premenopausal women, and aromatase inhibitors (AIs) are often used in postmenopausal women. Unfortunately, many of these cancers revert to an endocrine resistant, metastatic phenotype after one or more successful first line treatment(s). Patients who present with endocrine refractory disease have no good option for therapy other than chemotherapy regimens, which ultimately fail with high rates of mortality. Interestingly, approximately 25% of patients with endocrine resistant, metastatic disease harbor a mutated ER within the cancer cells, a mutation that was not detected in the primary tumor. During metastasis, cells from the primary tumor are shed into the bloodstream and undergo hemodynamic shear stress on route to the secondary site of metastasis. Some of these cells, known as circulating tumor cells (CTCs), will seed in secondary organs and produce metastatic lesions. Several studies have recently linked fluid shear stress (FSS) with DNA instability and tumor metastasis. To explore this possibility further, we used an in vitro model to test the effect of FSS on breast cancer cells, with the ultimate aim of understanding how FSS changes genomic dynamics, possibly leading to increased mutation frequency. Using this system, we have preliminary results that physiological levels of FSS induces activation of the ATR DNA damage response pathway in both MDA‐MB231 and MCF‐7 breast cancer cells. Whole exome sequencing results support this data by showing sequential exposure of MCF‐7 cells to FSS results in both de novo somatic mutations as well as an increase in mutation frequency at several common SNP sites. These data suggest FSS may play a role in downstream genomic instability and mutagenesis. Furthermore, initial microscopy and Western blot data of sheared cells show that FSS alters the phenotype of these cells, potentially rendering them more drug resistant and/or metastatically competent. In conclusion, we have shown, using an in vitro model, that FSS plays a direct role in altering genomic instability as well as the phenotypic signature in breast cancer cells, resulting in cells that may be more metastatic and aggressive. The ultimate goal of this research is to utilize this in vitro approach to identify molecular targets that can be pursued to either prevent or delay metastatic breast disease. Support or Funding Information This work was made possible by funding from the NIMHD‐RCMI grant 5G12MD007595 from the National Institute on Minority Health and Health Disparities and the NIGMS‐BUILD grant number 8UL1GM118967. This work was also made possible by the Louisiana Cancer Research Consortium. This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.

Background: Triple-negative breast cancer (TNBC) represents an aggressive subtype with limited therapeutic options. Experimental preclinical models that recapitulate their tumors of origin can accelerate target identification, thereby potentially improving therapeutic efficacy. Patient-derived xenografts (PDXs), due to their genomic and transcriptomic fidelity to the tumors from which they are derived, are poised to improve the preclinical testing of drug-target combinations in translational models. Despite the previous development of breast and TNBC PDX models, those derived from patients with demonstrated health-disparities are lacking.

Methods: We use an aggressive TNBC PDX model propagated in SCID/Beige mice that was established from an African-American woman, TU-BcX-2 K1, and assess its metastatic potential and drug sensitivities under distinct in vitro conditions. Cellular derivatives of the primary tumor or the PDX were grown in 2D culture conditions or grown in mammospheres 3D culture. Flow cytometry and fluorescence staining was used to quantify cancer stem cell-like populations. qRT-PCR was used to describe the mesenchymal gene signature of the tumor. The sensitivity of TU-BcX-2 K1-derived cells to anti-neoplastic oncology drugs was compared in adherent cells and mammospheres. Drug response was evaluated using a live/dead staining kit and crystal violet staining.

Results: TU-BcX-2 K1 has a low propensity for metastasis, reflects a mesenchymal state, and contains a large burden of cancer stem cells. We show that TU-BcX-2 K1 cells have differential responses to cytotoxic and targeted therapies in 2D compared to 3D culture conditions insofar as several drug classes conferred sensitivity in 2D but not in 3D culture, or cells grown as mammospheres.

Conclusions: Here we introduce a new TNBC PDX model and demonstrate the differences in evaluating drug sensitivity in adherent cells compared to mammosphere, or suspension, culture.

Keywords: Triple-negative breast cancer, Patient-derived xenograft, Mammosphere, Chemoresistance

Triple-negative breast cancer (TNBC) subtypes are clinically aggressive and cannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derived xenograft methods.
We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0.
Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole and intact breast tumor, specific cell populations within the tumor, and the extracellular matrix.
Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.

Estrogen receptor alpha (ERα) signaling pathways are frequently disrupted in breast cancer and contribute to disease progression. ERα signaling is multifaceted and many ERα regulators have been identified including transcription factors and growth factor pathways. More recently, microRNAs (miRNAs) are shown to deregulate ERα activity in breast carcinomas, with alterations in both ERα and miRNA expression correlating to cancer progression. In this study, we show that a high expression of Argonaute 2 (AGO2), a translation regulatory protein and mediator of miRNA function, correlates with the luminal B breast cancer subtype. We further demonstrate that a high expression of AGO2 in ERα+ tumors correlates with a poor clinical outcome. MCF-7 breast cancer cells overexpressing AGO2 (MCF7-AGO2) altered ERα downstream signaling and selective ERα variant expression. Enhanced ERα-36, a 36 kDa ERα isoform, protein and gene expression was observed in vitro. Through quantitative polymerase chain reaction (qPCR), we demonstrate decreased basal expression of the full-length ERα and progesterone receptor genes, in addition to loss of estrogen stimulated gene expression in vitro. Despite the loss, MCF-7-AGO2 cells demonstrated increased estrogen stimulated tumorigenesis in vivo. Together with our clinical findings on AGO2 expression and the luminal B subtype, we suggest that AGO2 is a regulator of altered ERα signaling in breast tumors.