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This paper describes a dielectrophoretic method for selection of circulating melanoma cells (CMCs), which lack reliable identifying surface antigens and are extremely rare in blood. This platform captures CMCs individually by dielectrophoresis (DEP) at an array of wireless bipolar electrodes (BPEs) aligned to overlying nanoliter-scale chambers, which isolate each cell for subsequent on-chip single-cell analysis. To determine the best conditions to employ for CMC isolation in this DEP-BPE platform, the static and dynamic dielectrophoretic response of established melanoma cell lines, melanoma cells from patient-derived xenografts (PDX) and peripheral blood mononuclear cells (PBMCs) were evaluated as a function of frequency using two established DEP platforms. Further, PBMCs derived from patients with advanced melanoma were compared with those from healthy controls. The results of this evaluation reveal that each DEP method requires a distinct frequency to achieve capture of melanoma cells and that the distribution of dielectric properties of PBMCs is more broadly varied in and among patients versus healthy controls. Based on this evaluation, we conclude that 50 kHz provides the highest capture efficiency on our DEP-BPE platform while maintaining a low rate of capture of unwanted PBMCs. We further quantified the efficiency of single-cell capture on the DEP-BPE platform and found that the efficiency diminished beyond around 25% chamber occupancy, thereby informing the minimum array size that is required. Importantly, the capture efficiency of the DEP-BPE platform for melanoma cells when using optimized conditions matched the performance predicted by our analysis. Finally, isolation of melanoma cells from contrived (spike-in) and clinical samples on our platform using optimized conditions was demonstrated. The capture and individual isolation of CMCs, confirmed by post-capture labeling, from patient-derived samples suggests the potential of this platform for clinical application.

Introduction: An attractive, yet unrealized, goal in cancer therapy is repurposing psychiatric drugs that can readily penetrate the blood-brain barrier for the treatment of deadly primary brain tumors and brain metastasis. The phenothiazine (PTZ) class of anti-psychotic agents were originally developed to inhibit dopamine- and serotonin-receptors (DR/5-HTRs). PTZs have demonstrated anti-cancer properties in numerous studies through a variety of proposed mechanisms, including on-target (DR/5-HTR) and off-target (e.g. calmodulin (CaM) inhibition) effects. PTZs inhibit DR/5-HTRs in the low- to mid-nanomolar range whereas anti-cancer effects tend to be observed only at a higher (low micromolar) dose range. Thus, clinical efficacy of existing PTZs may be limited by toxicity of on-target effects (sedation) of these drugs as DR/5-HTR antagonists. However, it has been unclear whether anti-cancer effects of PTZs are entirely separate from their activity as DR/5-HTR antagonists. Here we evaluate the in vitro anti-cancer efficacy of a novel PTZ analog (CWHM-974, a N-benzyl-linked fluphenazine (FLU) derivative), originally developed to treat brain-resident fungal infections, that is a 100-1000-fold less potent DR/5-HTR antagonist than FLU. Experimental procedures: To evaluate the anti-cancer efficacy of FLU and CWHM-974, we assessed cytotoxicity following 72h exposure against a broad panel of cancer cell lines. To investigate CaM as a target for CWHM-974, we evaluated the correlation between the sensitivity to CMW-974 and sensitivity to the CaM inhibitor W-7 as well as CaM gene expression levels in a panel of cancer cell lines. We also tested the anti-cancer efficacy of a sulfoxide analog of CWHM-974 (SLU-0010894) which lacks CaM binding activity. Results: We confirmed the anti-cancer efficacy of FLU and demonstrated that CWHM-974 is consistently 2-10-fold more potent than FLU in a broad panel of cancer cell lines. Among the most sensitve cancer cell lines are melanoma, which is a cancer that frequently metastasizes to the brain. Consistent with the hypothesis that CaM is a relevant target for CWHM-974, we found that: 1) CaM mRNA (CALM1) levels are correlated with sensitivity to CWHM-974; 2) the activities of CWHM-974 and W7 are correlated, and 3) most importantly, SLU-0010894 does not exhibit anti-cancer activity. Conclusions: The anti-cancer efficacy of the PTZ derivative CWHM-974 is seperable from DR/5-HTR antagonism. Our data suggests that further optimization of this compound may preserve the desirable pharmacologic properties for targeting brain cancers while reducing toxicity due to DR/5-HTR antagonism. Moreover, we provide a variety of evidence suggesting that anti-cancer efficacy of CWHM-974 is associated with CaM binding. Ongoing efforts will characterize the activity of CWHM-974 in mouse models of brain metastasis. Citation Format: Marion Vanneste, Anita Venzke, Soumitra Guin, Damian Krysan, Marvin Meyers, Michael Henry. Anti-cancer efficacy of a novel phenothiazine derivative is independent of dopamine and serotonin receptor inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4881.

Introduction: Epithelial to Mesenchymal Transition (EMT) is a cellular phenotype which may contribute to metastasis and therapeutic resistance in cancer. Therefore, therapeutic targeting of EMT-like phenotypes in cancer cells may afford an approach to treating metastatic disease and/or thwarting therapeutic resistance. Recently, GPX4 inhibitors (GPX4i) became of interest in this field as they were identified as potent and selective compounds for cells in a mesenchymal state through induction of lipid peroxidation induced cell death also known as ferroptosis. The sensitivity to these inhibitors was also shown to be dependent on the master EMT regulator, ZEB1. However, the link between ZEB1 expression and ferroptosis susceptibility remains unclear. Methodology: We used PC-3E and TEM 4-18 cell lines, originally isolated from the PC3 prostate cancer cell line based on their differential ability to invade an endothelial monolayer, as our models of epithelial and EMT-like phenotypes respectively. To better understand the role of ZEB1 in ferroptosis susceptibility, we used ZEB1 KO TEM 4-18 cells as well as a panel of cancer cell lines that cover a broad spectrum of epithelial and EMT-like phenotypes. After evaluating the sensitivity to GPX4i in these cell lines, we examined different parameters affecting the lipid peroxidation pathway. We particularly focused on the regulation of iron metabolism by ZEB1. Results: The three GPX4i tested (ML210, ML162 and RSL3) displayed a greater toxicity against TEM 4-18 cells. Inhibitors of ferroptosis (ferrostatin-1, liproxstatin-1) completely reversed the effect of GPX4i indicating that ferroptosis was indeed triggered by GPX4i and responsible for their toxicity. Sensitivity to GPX4i correlated with ZEB1 expression in our panel of cancer cell lines and ZEB1 KO reversed toxicity of GPX4i in TEM 4-18 cells. Sensitivity to GPX4i correlated with a slightly higher labile iron pool in ZEB1 expressing cells. Expression of proteins with a previously established connection to iron metabolism such as Ferritin Light Chain (FtL), Ferritin Heavy Chain (FtH), Transferrin Receptor (TfR) and Prominin-2 (PROM2) were also dysregulated between resistant and sensitive cells. In particular, the ratio FtH:FtL and expression of PROM2 was positively correlated with resistance to GPX4i. In addition, using the Cancer Cell Line Encyclopedia (CCLE) database, we found that ZEB1 expression negatively correlated with PROM2 and Iron Responsive Element Binding Protein-2 (IREB2) suggesting these genes could be transcriptional targets of ZEB1. Conclusions: We validated GPX4i as potent EMT selective cytotoxic compounds and confirmed that the sensitivity to these inhibitors depends on ZEB1 expression. However, the mechanisms leading to sensitivity to GPX4i and how they connect to the EMT program have yet to be identified. As these mechanisms might be diverse, we hypothesize that ZEB1 regulates iron metabolism which is critical for ferroptosis induction. Citation Format: Marion Vanneste, Michael Darrin Henry, Akansha Jain. ZEB1 confers sensitivity to ferroptosis through regulation of iron metabolism [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A028.

Background and Purpose: Circulating tumor cells (CTCs) are exposed to mechanical and biochemical stresses including fluid shear stress (FSS), due to blood flow, and oxidative distress, from a lack of matrix attachment. Recently we have shown that viable CTCs actively resist destruction when exposed to FSS by a mechano-adaptive mechanism that depends on RhoA-actomyosin activity (PMID: 32187555). Since the RhoA-actomyosin axis is implicated in other cellular behaviors that might contribute to metastasis, here we explored the hypothesis that exposure to FSS alters the biology of viable CTCs to promote metastatic colonization. Methodology: To determine if exposure to FSS alters metastatic potential, we exposed PC-3 prostate cells to FSS prior to injection into the tail-vein of NCG mice. We then monitored metastasis formation by weekly bioluminescence imaging. We also examined whether FSS exposure altered the ability of PC-3 and MDA-MB-231 cells to invade through a collagen I matrix, as well as their ability to survive/proliferate under anchorage-independent conditions. We conducted GC/LC-MS metabolomic profiling immediately after FSS exposure. To investigate oxidative burden on FSS exposed cells, we used dihydroethidium (10µM, 30 minutes) to measure general ROS production and a lipid peroxide sensor (5µM, 20 minutes). Results: We found that FSS exposure shortens the time for metastasis formation by 20% (median of 42 vs 52.5 days metastasis free; p=0.0222, log-rank). For both PC-3 and MDA-MB-231 cells we found that FSS exposure leads to ~2x increase in invasion through collagen matrix, and that increase depends on RhoA. Moreover, FSS exposure resulted in increased proliferation (~25%) under anchorage independent conditions in both cell lines. Metabolomic profiling revealed that FSS exposure resulted increased glycolysis, reduced entry into the TCA cycle, and a decrease in the metabolites that feed the folate cycle. We blocked glycolysis in PC-3 and MDA-MB-231 cells with 2-deoxyglucose (25mM, 2 hours) and found that this increased the fraction of cells destroyed by FSS ~20-30%. We also investigated redox stress and found that compared to cells held in suspension, those exposed to FSS demonstrate lower ROS burden. Interestingly, cells exposed to FSS exhibit lower levels of lipid peroxides in a manner that depends on RhoA. Conclusion: Our findings demonstrate that brief pulses of FSS exposure can enhance metastatic potential, increase invasive and anchorage-independent proliferative capacity and rapidly alter metabolism. The phenotypic changes driven by exposure to FSS depend in part on RhoA activation but may also reflect other mechanisms by which cancer cells sense and respond to FSS. FSS rapidly alters cellular metabolism in a manner that may act to reduce oxidative distress while CTCs are in suspension. In summary, our data indicate that FSS exposure in the circulation not only represents a physical force that CTCs must overcome to survive, but it also provides instructive cues that may enhance the metastatic behavior of some CTCs. This abstract is being presented as a short talk in the scientific program. A full abstract is available in the Short Talks from Proffered Abstracts section (PR006) of the Conference Proceedings. Citation Format: Devon L. Moose, Amanda N. Pope, Marion Vanneste, Patrick Breheny, Eric B. Taylor, Michael D. HenryDevon L. Moose, Amanda N. Pope, Marion Vanneste, Patrick Breheny, Eric B. Taylor, Michael D. Henry. Fluid shear stress enhances the metastatic potential and rapidly alters metabolism of circulating tumor cells [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B047.

Epithelial-to-mesenchymal transition (EMT) is implicated in cancer metastasis and drug resistance. Specifically targeting cancer cells in an EMT-like state may have therapeutic value. In this study, we developed a cell imaging-based high-content screening protocol to identify EMT-selective cytotoxic compounds. Among the 2,640 compounds tested, salinomycin and monensin, both monovalent cation ionophores, displayed a potent and selective cytotoxic effect against EMT-like cells. The mechanism of action of monensin was further evaluated. Monensin (10 nM) induced apoptosis, cell cycle arrest, and an increase in reactive oxygen species (ROS) production in TEM 4-18 cells. In addition, monensin rapidly induced swelling of Golgi apparatus and perturbed mitochondrial function. These are previously known effects of monensin, albeit occurring at much higher concentrations in the micromolar range. The cytotoxic effect of monensin was not blocked by inhibitors of ferroptosis. To explore the generality of our findings, we evaluated the toxicity of monensin in 24 human cancer cell lines and classified them as resistant or sensitive based on IC50 cutoff of 100 nM. Gene Set Enrichment Analysis identified EMT as the top enriched gene set in the sensitive group. Importantly, increased monensin sensitivity in EMT-like cells is associated with elevated uptake of 3H-monensin compared to resistant cells.

Here we have improved an existing mouse model of prostate cancer based on prostate-specific deletion of Pten and Trp53 by incorporating a Cre-activatable luciferase reporter. By coupling the deletion of those genes to the activation of a luciferase reporter, we were able to monitor tumor burden non-invasively over time. We show that, consistent with previous reports, deletion of both Pten and Trp53 on a C57BL/6 background accelerates tumor growth and results in both the loss of androgen receptor expression and castrate resistant tumors as compared with loss of Pten alone. Loss of Trp53 results in the development of sarcomatoid histology and the expression of markers of epithelial-to-mesenchymal transition Zeb1 and vimentin, with kinetics and penetrance dependent on whether one or both alleles of Trp53 were deleted. Homozygous deletion of Trp53 and Pten resulted in uniformly lethal disease by 25 weeks. While we were able to detect locally invasive disease in the peritoneal cavity in aggressive tumors from the double knockout mice, we were unable to detect lymphatic or hematogenous metastatic disease in lymph nodes or at distant sites.

// Yousef Zakharia 1 , Varun Monga 1 , Umang Swami 1 , Aaron D. Bossler 4 , Michele Freesmeier 1 , Melanie Frees 1 , Mirza Khan 5 , Noah Frydenlund 2 , Rithu Srikantha 2 , Marion Vanneste 3 , Michael Henry 3 and Mohammed Milhem 1 1 Department of Hematology, Oncology and Blood and Marrow Transplantation and the Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, USA 2 The University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA 3 Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA 4 Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, USA 5 Department of Internal Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, USA Correspondence to: Mohammed Milhem, email: mohammed-milhem@uiowa.edu Keywords: vemurafenib, decitabine, epigenetics, BRAF, melanoma Received: June 16, 2017 Accepted: September 16, 2017 Published: September 26, 2017 ABSTRACT Introduction: Epigenetic modifications play an important role in progression and development of resistance in V600E BRAF positive metastatic melanoma. Therefore, we hypothesized that the action of vemurafenib (BRAF inhibitor) can be made more effective by combining with low dose decitabine (a DNA methyltransferase inhibitor). The primary objective of this phase lb study was to determine the dose limiting toxicity and maximum tolerated dose of combination of subcutaneous decitabine with oral vemurafenib in patients with V600E BRAF positive metastatic melanoma with or without any prior treatment. Experimental Design: The study employed 3+3 dose escalation combining subcutaneous decitabine at different doses and schedules (4 cohorts) with the standard oral dose of vemurafenib 960 mg twice daily. Preclinical assessment and further analysis were also performed in A375 melanoma cell line. Results: Fourteen patients received study treatment. No dose limiting toxicity was encountered and maximum tolerated dose was not reached. Important toxicities included fatigue, increased creatinine, neutropenia, leucopenia, hypophosphatemia, rash and hyperuricemia. Three patients achieved complete response, three had partial response and five had stable disease. Preclinical assessment demonstrated action of the combination which delayed the development of acquired resistance and improved duration of treatment sensitivity. Conclusions: The combination of oral vemurafenib with subcutaneous decitabine is safe and showed activity in V600E BRAF positive metastatic melanoma. Since most responses were seen in cohort 1, which utilized low-dose, long-term decitabine, future studies of this combination treatment should utilize longer duration of decitabine, at the lowest dose of 0.1 mg/kg.

Summary: During metastasis, cancer cells are exposed to potentially destructive hemodynamic forces including fluid shear stress (FSS) while en route to distant sites. However, prior work indicates that cancer cells are more resistant to brief pulses of high-level FSS in vitro relative to non-transformed epithelial cells. Herein, we identify a mechano-adaptive mechanism of FSS resistance in cancer cells. Our findings demonstrate that cancer cells activate RhoA in response to FSS, which protects them from FSS-induced plasma membrane damage. We show that cancer cells freshly isolated from mouse and human tumors are resistant to FSS, that formin and myosin II activity protects circulating tumor cells (CTCs) from destruction, and that short-term inhibition of myosin II delays metastasis in mouse models. Collectively, our data indicate that viable CTCs actively resist destruction by hemodynamic forces and are likely to be more mechanically robust than is commonly thought. : Moose et al. show that cancer cells exhibit a mechano-adaptive response to fluid shear stress through activation of the RhoA-actomyosin signaling axis. Utilizing in vivo models, they extend these findings to demonstrate that this axis maintains intravascular survival of circulating tumor cells (CTCs) that contributes to the development of metastasis. Keywords: fluid shear stress, circulating tumor cells, metastasis, RhoA, myosin II, formin

During metastasis cancer cells are exposed to potentially destructive hemodynamic forces including fluid shear stress (FSS) whileen routeto distant sites. However, prior work indicates that cancer cells are more resistant to brief pulses of high-level fluid shear stress (FSS)in vitrorelative to non-transformed epithelial cells. Herein we identify a mechanism of FSS resistance in cancer cells, and extend these findings to mouse models of circulating tumor cells (CTCs). We show that cancer cells acutely isolated from primary tumors are resistant to FSS. Our findings demonstrate that cancer cells activate the RhoA-myosin II axis in response to FSS, which protects them from FSS-induced plasma membrane damage. Moreover, we show that the myosin II activity is protective to CTCs in mouse models. Collectively our data indicate that viable CTCs actively resist destruction by hemodynamic forces and are likely to be more mechanically robust than is commonly thought.

Simple Summary Brain metastases are common in melanoma and are often associated with significant morbidity and mortality. Although many new treatments for melanoma have been approved in recent years, including immune checkpoint inhibitors and BRAF/MEK inhibitors, limited data are available for survival for patients with brain metastases treated with these novel therapies. The aim of this retrospective study was to evaluate current surgical, radiation, and systemic therapies over the past 10 years in melanoma patients with brain metastases. Our study noted increased overall survival in patients treated with craniotomy and CTLA-4 checkpoint inhibitors, while whole brain radiation was associated with poorer overall survival. Abstract Brain metastases commonly develop in melanoma and are associated with poor overall survival of about five to nine months. Fortunately, new therapies, including immune checkpoint inhibitors and BRAF/MEK inhibitors, have been developed. The aim of this study was to identify outcomes of different treatment strategies in patients with melanoma brain metastases in the era of checkpoint inhibitors. Patients with brain metastases secondary to melanoma were identified at a single institution. Univariate and multivariable analyses were performed to identify baseline and treatment factors, which correlated with progression-free and overall survival. A total of 209 patients with melanoma brain metastases were identified. The median overall survival of the cohort was 5.3 months. On multivariable analysis, the presence of non-cranial metastatic disease, poor performance status (ECOG 2–4), whole-brain radiation therapy, and older age at diagnosis of brain metastasis were associated with poorer overall survival. Craniotomy (HR 0.66, 95% CI 0.45–0.97) and treatment with a CTLA-4 checkpoint inhibitor (HR 0.55, 95% CI 0.32–0.94) were the only interventions associated with improved overall survival. Further studies with novel agents are needed to extend lifespan in patients with brain metastases in melanoma.

MAPK inhibition with targeted BRAF inhibitors has revolutionized the treatment of BRAF-mutant melanoma. However, in most cases, the effects of mutant BRAF inhibition are transient, owing to the acquisition of drug resistance. To resolve mechanisms that drive resistance to mutant BRAF inhibition, we performed a genome-scale shRNA screen capable of revealing genes whose loss of function confers resistance to vemurafenib (VEM). We validated ten of our top candidates with independent lentiviral shRNA constructs. We found that knockdown of NF1 and CUL3 confers resistance to VEM. This result is consistent with previous genome-scale loss-of-function screens that have also identified NF1 and CUL3 as drivers of resistance to mutant BRAF inhibition. NF1 encodes a protein that hydrolyzes Ras-GTP. Not surprisingly, it has been shown that loss of NF1 results in increased Ras-GTP levels that lead to reactivation of MAPK in the presence of VEM. However, little is known about the mechanistic changes that underlie CUL3-mediated resistance. Here, we show that unlike the classical reactivation of MAPK that is seen with the loss of NF1, loss of CUL3 results in increased signaling through a Rac1-dependent focal-adhesion-like process that is accompanied by sustained MAPK activity in the presence of VEM. Furthermore, we show that this mechanism of resistance can be abrogated by inhibiting the Src kinase family, a nine-member protein family that critically mediates integrin signaling. In relation to other known drivers of resistance to mutant BRAF inhibition, loss of NF1 reactivates MAPK in a manner much like RAS mutations or BRAF alterations/amplification. By contrast, loss of CUL3 leads to resistance reminiscent of a reversible therapy-resistant state that lacks the key genomic alterations in MAPK signaling proteins. Citation Format: Marion Vanneste, Eliot Zhu, Charlotte Feddersen, Afshin Varzavand, Tyler Foley, Lei Zhao, Rob Piper, Christopher Stipp, Adam Dupuy, Michael Henry. Genome-scale shRNA screen provides insight into the role of CUL3 in adaptive mechanisms of resistance to mutant BRAF inhibition [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B01.

Metastatic, drug-resistant cancer is lethal. Epithelial-to-mesenchymal transition (EMT) is a type of phenotypic plasticity that has been implicated in cancer metastasis and drug resistance. One potential therapeutic approach is to specifically target cancer cells in an EMT-like state. In this study, we have developed a cell-based high-throughput screening (HTS) protocol using the high content Operetta imaging platform to identify EMT cytotoxic compounds. This screen was performed on epithelial (PC-3E) and mesenchymal-like (TEM 4-18) cell lines, both isolated from the parental PC-3 prostate cancer cell line based on their differential ability to invade an endothelial monolayer. A library of 2,640 compounds was screened on co-cultured PC-3E GFP cells and TEM 4-18 mCherry cells. After 72h exposure to compounds, relative numbers of GFP- and mCherry-positive cells were quantitated. Dose-response curves were established for each compound exhibiting a greater toxicity against EMT-like cells. The mechanisms of action of one of the most potent and selective compound against EMT-like cells, monensin, were further evaluated. Among the 2,640 compounds tested, salinomycin and monensin, both monovalent cation ionophores, displayed a potent and selective cytotoxic effect against EMT-like cells. A closely related compound absent from the compound library, nigericin, was also evaluated and appeared to be the most potent of the three compounds tested. Monensin (10nM) induced apoptosis, cell cycle arrest, and an increase in ROS production in TEM 4-18 cells. In addition, monensin rapidly induced swelling of Golgi apparatus, a previously known effect of monensin, in multiple cell lines most likely resulting in a blockage of intracellular protein trafficking and cell death. We then evaluated the toxicity of monensin in 24 human cancer cell lines and classified them as resistant or sensitive based on IC50 cutoff of 100nM. Supporting these findings, Gene Set Enrichment Analysis identified EMT as the top of the gene sets enriched in the sensitive group. Increased monensin sensitivity in EMT-like cells is associated with elevated uptake of H3-monensin compared to resistant cells. In conclusion, using a high-throughput screening approach, we identified monensin as a potent and specific inhibitor of cancer cells in an EMT-like state where it rapidly disrupts Golgi function. At least part of selectivity of monensin for EMT-like cells is due to increased uptake of this compound. Citation Format: Marion Vanneste, Huang Qin, Devon Moose, Mengshi Li, Michael Schultz, Meng Wu, Michael Henry. Monensin is selectively cytotoxic to cancer cells in an EMT-like state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4186.

Background: Epithelial to mesenchymal transition (EMT) is a cellular phenotype which may contribute to metastasis and therapeutic resistance in cancer. Therapeutic targeting of EMT-like phenotypes in cancer cells, thus may afford an approach to treating metastatic disease and/or thwarting therapeutic resistance. Methods: We have developed a cell-based high-throughput screening protocol using the high content Operetta imaging platform to identify EMT cytotoxic compounds. This screen was performed on PC3E and TEM 4-18 cell lines which were originally isolated form a parental PC3 prostate cancer cell line based on their differential ability to invade an endothelial monolayer. A library of 2,640 compounds (Microsource) was screened on a co-culture of epithelial cells (PC3E GFP cells) and EMT-like cells (TEM 4-18 mCherry cells). After 72h exposure to compounds, relative numbers of GFP- and mCherry-positive cells were quantitated using the Operetta system. Dose-response curves were established for each compound exhibiting a greater toxicity against EMT-like cells. The results were validated on PC3E and TEM 4-18 cell lines cultured separately with the CellTiter Blue viability assay. Results: Among the 2,640 compounds tested, monensin and salinomycin, both monovalent cation ionophores, exhibited a relatively greater toxicity against EMT-like cells. Nigericin, compound closely related to monensin but absent from the Microsource library, was also evaluated. The highest potency and selectivity for the EMT-like cells was obtained with nigericin (IC50 357nM for PC3E vs 7nM for TEM 4-18), followed by monensin (IC50 457nM for PC3E vs 11nM for TEM 4-18) and salinomycin (IC50 1687nM for PC3E vs 278nM for TEM 4-18). Monensin and nigericin induced apoptosis, a cell cycle arrest, and an increase in ROS production in TEM 4-18 without apparent mitochondrial disruption. In addition, monensin rapidly induced swelling of golgi apparatus likely resulting in a blockage of intracellular protein trafficking and consequently cell death. To extend our findings, we evaluated the toxicity of these three ionophores in 15 cancer cell lines from different origins and classified them as resistant or sensitive. We analyzed publically available gene expression data for these cell lines and performed a Gene Set Enrichment Analysis to identify the gene sets differentially represented in the 2 groups. Supporting our hypothesis, the gene set involving EMT was the top gene sets enriched in the sensitive group. Conclusion: In this study we identified three chemically-related compounds (monensin, salinomycin and nigericin) exhibiting relatively greater toxicity against EMT-like cells. These compounds may serve as chemical probes for exploring the biology of EMT-like cells and point to strategies for selectively targeting these cells to disrupt metastasis and/or therapeutic resistance. Citation Format: Marion Vanneste, Qin Huang, Meng Wu, Michael D. Henry. Identification of EMT-selective cytotoxic compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1589.