LTLT-Ca Cells Research Articles

Abstract 5030: Treatment of aromatase inhibitor-resistant cells with polyisoprenylated cysteinyl amide inhibitors stimulates the mitogen-activated protein kinase pathway enzymes, impedes cell proliferation and causes cell death

Abstract Overexpression and hyperactivity of the estrogen receptor drives 67-80% and 90% of breast cancer in women and men, respectively. Resistance to aromatase inhibitor (AI) therapies necessitates the continuous search for novel therapies. Previous studies demonstrate AI-resistance is associated with hormone-independence, enhanced motility, and increased growth factor signaling. Here, long-term letrozole-treated (LTLT-Ca) breast cancer cells were used to evaluate polyisoprenylated cysteinyl amide inhibitors (PCAIs) as potential alternative therapies for the treatment of aromatase inhibitor-resistant breast cancer. We determined the potency of PCAIs as anticancer agents by evaluating their effects on cell viability, phosphorylation of MAPK pathway enzymes, G-proteins levels, cell migration and apoptosis. Among the PCAIs tested, NSL-YHJ-2-27 showed significant potency against cell viability with an EC50 of 3.6 µM. MEK (p-MEK1/2), ERK (p-ERK1/2), and p90RSK (p-p90RSK) phosphorylation were significantly increased by 178, 119 and 125%, respectively, over controls. In addition, of the seven G-proteins evaluated, only KRAS and NRAS showed significant increases of 49% and 97%, respectively. Cell proliferation and colony formation were impeded by 82% and 74%, respectively, after PCAIs treatment. Migration of the LTLT-Ca cells was inhibited by 80% following treatment with 5 µM of NSL-YHJ-2-27. Lastly, the PCAIs also caused the degeneration of the spheroids after 10 µM PCAIs treatment, dead cells were very apparent after AO/EB staining. Our findings suggest that the PCAIs’ suppression of cell viability and activation of the MAPK pathway causes apoptosis possibly through the activation of the proapoptotic p-p90RSK isoforms. The results reveal the ability of the PCAIs to effectively cause a negative impact on several cancer hallmarks suggesting their potency as anticancer agents. These findings also support a potential role for PCAIs’ use in treating breast cancers that have become resistant to aromatase inhibitor therapies. Citation Format: Jassy Mary S. Lazarte, Syreeta L. Tilghman, Nazarius S. Lamango. Treatment of aromatase inhibitor-resistant cells with polyisoprenylated cysteinyl amide inhibitors stimulates the mitogen-activated protein kinase pathway enzymes, impedes cell proliferation and causes cell death. [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 5030.

Novel Therapeutic Combination Targets the Growth of Letrozole-Resistant Breast Cancer through Decreased Cyclin B1.

As breast cancer cells transition from letrozole-sensitive to letrozole-resistant, they over-express epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and human epidermal growth factor receptor 2 (HER2) while acquiring enhanced motility and epithelial-to-mesenchymal transition (EMT)-like characteristics that are attenuated and reversed by glyceollin treatment, respectively. Interestingly, glyceollin inhibits the proliferation and tumor progression of triple-negative breast cancer (TNBC) and estrogen-independent breast cancer cells; however, it is unlikely that a single phytochemical would effectively target aromatase-inhibitor (AI)-resistant metastatic breast cancer in the clinical setting. Since our previous report indicated that the combination of lapatinib and glyceollin induced apoptosis in hormone-dependent AI-resistant breast cancer cells, we hypothesized that combination therapy would also be beneficial for hormone independent letrozole-resistant breast cancer cells (LTLT-Ca) compared to AI-sensitive breast cancer cells (AC-1) by decreasing the expression of proteins associated with proliferation and cell cycle progression. While glyceollin + lapatinib treatment caused comparable inhibitory effects on the proliferation and migration in both cell lines, combination treatment selectively induced S and G2/M phase cell cycle arrest of the LTLT-Ca cells, which was mediated by decreased cyclin B1. This phenomenon may represent a unique opportunity to design novel combinatorial therapeutic approaches to target hormone-refractory breast tumors.

Mammospheres of letrozole-resistant breast cancer cells enhance breast cancer aggressiveness.

Aromatase inhibitors (AIs), such as letrozole, are considered as first-line treatment for estrogen receptor-positive breast cancer in postmenopausal women. Despite the successful use of letrozole, resistance to therapy, tumor relapse and metastasis remain principal causes of patient mortality. Although there is no therapy currently available for AI-resistant breast cancer, previous reports have demonstrated that AI resistance is associated with hormone independence, increased growth factor signaling, enhanced cellular motility and epithelial to mesenchymal transition (EMT). This suggests a convergence of EMT and cancer stem cells (CSCs) in endocrine resistance. The present study evaluated the contribution of mammospheres in letrozole-resistant breast cancer by characterizing mammospheres and their potential impact on cellular motility. Ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with either letrozole-resistant MCF-7 cells (LTLT-Ca) or letrozole-sensitive MCF-7 cells (AC-1). Subsequently, intratumoral CSC marker expression was assessed by immunohistochemistry. The results indicated that LTLT-Ca tumors were CD44+/CD24+, while AC-1 tumors presented low CD44/CD24 expression. Since mammosphere formation depends on CSCs, both cell lines were cultured either adherently (2D) or as mammospheres (3D) to assess the CD44/CD24 protein expression profile. When 3D culturing both cell lines, higher expression levels of CD44 and CD24 were observed when compared with their adherent counterparts, with the most robust change observed in the LTLT-Ca cell line. To quantitate the breast cancer stem cell activity, mammosphere formation assays were performed, and the LTLT-Ca cells formed mammospheres at a 3.4-fold higher index compared with AC-1 cells. Additionally, targeted gene expression arrays were conducted to compare the LTLT-Ca 3D and 2D cells, revealing that LTLT-Ca 3D cells displayed decreased expression levels of genes involved in cell adhesion and tumor suppression (e. g., E-cadherin, caveolin 1 and β-catenin). To validate this finding, wound healing assays were performed, and LTLT-Ca mammospheres exhibited a 70% wound closure, whereas AC-1 mammospheres exhibited a 39% wound closure. Collectively, the present findings demonstrated a strong association between AI-resistant mammospheres and an increased propensity for migration, which may be indicative of a poor prognosis.

Abstract 3794: Mammary cancer stem cells promote an aggressive phenotype in letrozole resistant breast cancer

Abstract Aromatase inhibitors (AI), like letrozole, are the first-line treatment for ER+ breast cancer in post-menopausal women. Despite widespread successful usage of letrozole, resistance to therapy, tumor relapse, and metastasis remain the principal causes of death for breast cancer patients. While there are no cures for AI-resistant breast cancer, previous reports demonstrate AI resistance is associated with hormone independence, increased growth factor signaling, enhanced cellular motility and the epithelial to mesenchymal transition (EMT). Given recent evidence suggesting a convergence of EMT and cancer stem cells (CSC) and their combined implications in endocrine resistance, we hypothesized that breast cancer stem cells contribute to letrozole resistant breast cancer cells by increasing cellular motility. To test this hypothesis, ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with either MCF-7 cells stably transfected with the human aromatase gene that were long term letrozole-treated (LTLT-Ca) or letrozole sensitive, MCF-7 cells stably transfected with the human aromatase gene (AC-1). Afterwards, intratumoral putative CSC marker expression was assessed by immunohistochemistry. Results indicate LTLT-Ca tumors were CD44+/CD24+ while AC-1 tumors were CD44low/CD24low. Both cell lines were cultured either adherently (2D) or as mammospheres (3D) and CD44/CD24 protein expression was assessed by immunoblots. When both cell lines were cultured three dimensionally, they expressed higher levels of CD44 and CD24 compared to their adherent counterparts, with the most robust change occurring within the LTLT-Ca cells. In order to quantitate the breast cancer stem cell activity mammosphere formation assay was performed and LTLT-Ca cells formed mammospheres at a 3.4-fold higher index compared to AC-1 cells. Additionally, we performed targeted gene expression arrays and our results demonstrated decreased expression of genes involved in cell adhesion and tumor suppression (ie., E-cadherin, caveolin 1 and β catenin). Taken together our study demonstrated a strong correlation between AI resistance, increased cancer stem cell expression, an invasive phenotype that may be associated with a poor prognosis. This work was supported by an NIH grant awarded to SL Tilghman (1SC1GM125617). Citation Format: Karen Melissa Gallegos, Jankiben Patel, Rashidra R. Walker, A Michael Davidson, Ian R. Davenport, Syreeta L. Tilghman. Mammary cancer stem cells promote an aggressive phenotype in letrozole resistant breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3794.

Abstract P3-11-15: A novel phytoalexin, glyceollins, trigger anti-proliferative effects in aromatase inhibitor resistant breast cancer cells

Abstract Aromatase inhibitors (AIs) are currently the standard treatment for postmenopausal women with estrogen-dependent metastatic breast cancer. While effective, patients develop resistance leading to tumor relapse, metastasis, and more aggressive phenotypes. Previous research suggested that AI resistance is due to upregulation of growth factor pathways (i.e., HER2 and EGFR) and increased cellular motility. Our lab previously demonstrated that a novel phytoalexin, glyceollins, inhibits proliferation, survival, and migration of hormone independent letrozole-resistant breast cancer cells (LTLT-Ca). However, many postmenopausal women with AI-resistance tumors remain hormone dependent. Therefore, there is a need to understand distinctions between estrogen receptor (ER+) positive and ER negative (ER-) AI resistant tumors and their response to therapy. We hypothesize that treating ER+ letrozole-resistant breast cancer using a combination approach of glyceollin and lapatinib (a dual EGFR/HER2 inhibitor) will reduce growth factor signaling, inhibit proliferation and motility. To test this hypothesis, T47Darom cells (T47D cells which stably overexpress aromatase) and T47DaromLR cells (T47Darom cells that are letrozole resistant) were treated with lapatinib, glyceollin, or the combination. To evaluate viability, resazurin assays were performed and results demonstrated that glyceollin and/or lapatinib had no effect on proliferation in the T47Darom cells. However, glyceollin alone and glyceollin + lapatinib inhibited proliferation of T47DaromLR cells by 46% and 59%, respectively. Cell survival was assessed by colony formation assays and glyceollin caused a 33% reduction in cell survival and glyceollin + lapatinib caused a 60% reduction in the colony formation of T47Darom cells. Interestingly, in T47DaromLR cells glyceollin and the combination significantly inhibited cell survival by 94% alone and 96%. To further evaluate changes that occur as AI sensitive cells transition to AI resistance, a quantitative proteomic analysis of the whole cell lysates of T47DaromLR (AI-resistant) versus T47Darom cells (AI-sensitive) was performed to identify significant protein expression changes. Results demonstrated a 3.195-fold-increase (p<0.01) in protein disulfide isomerase (P4HB) and a 0.484-fold decrease (p<0.01) in peptidyl-prolyl cis-trans isomerase (FKBP4) which was validated by immunoblotting. While the molecular mechanism by which P4HB contributes to tumorigenesis and metastasis is unknown, it has been demonstrated to be involved in the proliferation, survival, and metastasis of several types of cancer cells. FKBP4 is also of interest as it has been shown to be involved in mitotic arrest and apoptotic signaling. To interrogate the effect of combination therapy on HER effector signaling cascades, western blot analyses were performed to establish the endogenous expression of various proteins. Results demonstrated ERα expression was increased while HER2 was decreased in T47Darom cells compared to T47DaromLR cells. The protein expression findings from the T47D variant cell lines followed a similar trend as those previously reported in LTLT-Ca cells and their AI-sensitive counterparts. Taken together, these results suggest that dual inhibition using glyceollins and lapatinib may have potential as a novel combination therapy approach for postmenopausal patients with hormone-dependent, letrozole-resistant breast tumors. This work was also supported by NIH grant 1SC1GM126617 awarded to SL Tilghman. Citation Format: Rashidra R Walker, Jankiben Patel, A. Michael Davidson, Karen Gallegos, Syreeta L Tilghman. A novel phytoalexin, glyceollins, trigger anti-proliferative effects in aromatase inhibitor resistant breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-15.

Abstract 1014: Characterization of breast cancer cells resistant to letrozole and palbociclib combination therapy and the potential of dinaciclib to treat such resistance

Abstract Treatment of estrogen receptor positive (ER+) breast cancer have included single-agent endocrine therapy, such as with aromatase inhibitors (AIs) and, more recently, AIs in combination with the cyclin-dependent kinase 4/6 inhibitor palbociclib. While combination therapy is effective, drug resistance is a well-known phenomenon and breast cancer cells can acquire resistance to aromatase inhibitor and palbociclib individually. In anticipation of patient who may become resistant to combination therapy with aromatase inhibitor (letrozole) and palbociclib, this current study has 1) developed MCF7Ca cells resistant to both letrozole and palbociclib (MCF7Calet+palb); 2) characterized them in comparison to drug naïve (MCF7Ca) and letrozole alone-resistant (LTLTCa) cells; and 3) explored potential treatments. Characterization of MCF7Calet+palb cells by phase contrast microscopy, mammosphere assays, and invasion assays, indicate that they have more basal/mesenchymal morphology, produce more mammospheres (an indicator of cancer stem cells; 150±10 vs. 80±10 in MCF7Ca and 123± 8 in LTLTCa cells), and are more invasive (1200±50 cells vs. 791±15 in MCF7Ca cells). The more aggressive phenotype of MC7Calet+palb correlated with changes in protein expression. Compared to the other cell lines, MCF7Calet+palb expressed significantly lower fold expression levels of cell cycle cyclin D1 (0.2±0.1), phosphorylated CDK9 (0.5±0.1), and phosphoRb and total Rb (0.4±0.2), but higher levels of phosphoCDK1 and CDK2 (1.4±0.1 and 3.1±0.6). Regarding growth pathway proteins, MCF7Calet+palb comparably had increased HER2 and phosphorylated Akt (6.2±1.3 and 3.3±1.2, resp.) as LTLTCa vs. MCF7Ca cells, but had even more reduced levels of ERα (0.04 vs. 0.1±0.4 in LTLTCa and 1±0.01 in MCF7Ca). Cancer stem cell marker breast cancer resistance protein (BCRP) was increased further in MCF7Calet+palb cells (2.11-fold). Based on these results, the effect of dinaciclib, a broader range CDK inhibitor, was studied. In MTT assays, dinaciclib was shown to be effective in inhibiting cell viability in all three cell types. Dinaclicib’s IC50s were 1.4 nM (MC7Ca) 9.5 nM (LTLTCa) and 12.4 nM in MCF7Calet+palb cells, while palbociclib IC50s were 1.5 μM, 11.2 μM, and 118 μM, respectively. Dinaciclib also significantly reduced mammosphere formation 0.01-fold and cell invasion/migration by 0.3-fold vs. untreated. Lastly, preliminary exome sequencing data suggested highly mutated FGFRL1, ARL11, and FCGBP genes, among others, when MCF7Calet+palb cells were compared to MCF7Ca cells. Overall, these results suggest that acquired resistance to combination therapy with letrozole and palbociclib induces genetic, molecular, and phenotypic changes in breast cancer cells, and that dinaciclib may be an effective treatment to either treat and/or prevent this resistance. Citation Format: Armina Kazi, Emily Shukdinas, Saranya Chumsri, Gauri J. Sabnis. Characterization of breast cancer cells resistant to letrozole and palbociclib combination therapy and the potential of dinaciclib to treat such 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 1014.

Retraction: Aromatase Inhibitor-mediated Downregulation of INrf2 (Keap1) Leads to Increased Nrf2 and Resistance in Breast Cancer.

Aromatase inhibitors (AIs) are effective drugs that reduce or eliminate hormone sensitive breast cancer. However, despite their efficacy, resistance to these drugs can occur in some patients. The INrf2 (Keap1):Nrf2 complex serves as a sensor of drug/radiation-induced oxidative/electrophilic stress. INrf2 constitutively suppresses Nrf2 by functioning as an adapter protein for the Cul3/Rbx1-mediated ubiquitination/degradation of Nrf2. Upon stress, Nrf2 dissociates from INrf2, is stabilized, translocates to the nucleus, and coordinately induces a battery of cytoprotective gene expression. Current studies investigated the role of Nrf2 in AI resistance. RT-PCR and immunoblot assays showed that AI-resistant breast cancer LTLTCa and AnaR cells express lower INrf2 and higher Nrf2 protein levels, as compared to drug sensitive MCF-7Ca and AC1 cells, respectively. The increase in Nrf2 was due to lower ubiquitination/degradation of Nrf2 in AI-resistant cells. Higher Nrf2-mediated levels of biotransformation enzymes, drug-transporters and anti-apoptotic proteins contributed to reduced efficacy of drugs and aversion to apoptosis that led to drug resistance. shRNA inhibition of Nrf2 in LTLTCa (LTLTCa-Nrf2KD) cells reduced resistance and sensitized cells to AI exemestane. Interestingly, LTLTCa-Nrf2KD cells also showed reduced levels of aldehyde dehydrogenase, a marker of Tumor-Initiating Cells and significantly decreased mammosphere formation, as compared to LTLTCa-Vector control cells. The results together suggest that persistent AI treatment down-regulated INrf2 leading to higher expression of Nrf2 and Nrf2 regulated cytoprotective proteins that resulted in increased AI drug resistance. These findings provide a rationale for the development of Nrf2 inhibitors to overcome resistance and increase efficacy of AI.

Abstract 3183: Proteomic characterization of aromatase inhibitor resistant mammospheres reveal the presence of a novel nuclear chaperone

Abstract Postmenopausal women with early-stage metastatic estrogen-dependent breast cancer are generally treated with aromatase inhibitors (AIs) (e.g., letrozole). However, acquired resistance remains a major clinical obstacle. Previously, our group revealed a global proteomic signature of a letrozole-resistant cell line (LTLT-Ca) associated with hormone independence, enhanced cell motility and epithelial to mesenchymal (EMT). Given recent evidence suggesting a convergence of EMT and cancer stem cells (CSC), we chose to utilize a two-dimensional (2D) vs three-dimensional (3D) culture system to compare the proteome of LTLT-Ca cells, as 3D culture not only enriches for CSC, but more accurately recapitulates the tumor microenvironment, morphology, function and response to therapy compared to conventional 2D culture. We hypothesize utilizing a novel systems biology approach may reveal previously unconsidered molecular changes that could aid in understanding complex signaling networks and be exploited as therapeutic targets. To address this hypothesis ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with LTLT-Ca or letrozole sensitive cells (AC-1) and intratumoral putative CSC marker expression was assessed by immunohistochemistry. Results indicate LTLT-Ca tumors were CD44high/CD24low while AC-1 tumors were CD44-/CD24low. Mammosphere formation assays were conducted and LTLT-Ca cells formed mammospheres at a 3.4-fold higher index than AC-1 cells. A quantitative proteomic analysis of whole cell lysates from LTLT-Ca (2D adherent cells) versus LTLT-Ca (3D mammospheres) was conducted. Results identified significant protein expression changes within a panel of 1010 proteins; 173 were upregulated and 186 downregulated (p<0.05, fold change >1.20). Additionally, functional enrichment analyses were performed and 19 gene ontology (GO) terms and one KEGG pathway (hsa03010:Ribosome) were over-represented (BH adjusted p-value < 0.01) by the cognate genes. Notably, there was a 35.04-fold increase in midasin (MDN1), a nuclear chaperone protein required for maturation and nuclear export of pre-60S ribosome subunit. Increased MDN1 expression was strongly correlated with highly tumorigenic breast cancer spheres. Additionally, Kaplan-Meier survival plots demonstrate that increased MDN1 levels were positively correlated with decreased relapse free survival in estrogen receptor negative breast cancer, and is constitutively expressed as breast tumors progress from atypical ductal hyperplasia to ductal carcinoma in situ to invasive, metastatic breast cancer. The TGCA database was interrogated and MDN1 was frequently amplified or mutated in breast tumors. Taken together our study for the first time implicates a role for ribosomal assembly in AI resistant cells enriched for CSCs and underscores a potential for MDN1 in the progression and responsiveness to therapy. Citation Format: Syreeta L. Tilghman, Jamal Pratt, Shawn D. Llopis, A. Michael Davidson, Rashidra R. Walker, Patrick Carriere, Ian R. Davenport, Wensheng Zhang, Karen Zhang. Proteomic characterization of aromatase inhibitor resistant mammospheres reveal the presence of a novel nuclear chaperone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3183. doi:10.1158/1538-7445.AM2017-3183

Abstract 2053: Mechanisms of resistance to palbociclib and aromatase inhibitors in hormone receptor positive breast cancer

Abstract Estrogen receptor-positive (HR+) breast cancer is the most common form of breast cancer that is accountable for the majority of breast cancer mortality. Currently, the cyclin dependent kinase inhibitor palbociclib in combination with endocrine therapy represents the new standard first and second line therapy for patients with metastatic HR+ breast cancer. While palbociclib has been shown to significantly improved progression free survival in combination with aromatase inhibitor (AI) and fulvestrant, resistance will inevitably occur in patients with metastatic breast cancer. However, the mechanisms of resistance to the combination of palbociclib and AI remain largely unknown. It is also unclear whether resistance mechanisms would be the same if palbociclib was given as first line treatment in combination with letrozole vs. if palbociclib was given as second line treatment after letrozole resistance had already occurred. To address this, previously well established and well characterizied letrozole-sensitive MCF7Ca cells and letrozole-resistant LTLTCa cells were subjected to continuous, long term treatment with increasing doses of letrozole and palbociclib until resistance to both drugs was achieved (MCF7Calet+palb and LTLTCapalb, respectively). Preliminary MTT cell viability assays indicate palbociclib IC50s of 750 nM in MCF7Ca and LTLTCa cells vs. 21 uM in palbociclib and letrozole-resistant cells. First line and second line palbociclib resistance correlated with changes in morphology, protein expression, and cancer stem cell characteristics. Under phase contrast microscopy, first line and second line palbociclib-resistant MCF7Ca and LTLTCa cells were larger in size, more irregular in shape, and tended not to grow in epithelial cell-like groups compared to palbociclib-sensitive MCF7Ca and letrozole cells, with second-line palbociclib resistant cells exhibiting these characteristics the most. Western blot showed that ER protein expression in ER+/HER2- MCF7Ca cells decreased with first line palbociclib and letrozole resistance, and that both ER and HER2 protein expression were decreased with second line palbociclib resistance in ERlow/HER2+ LTLTCa cells. Lastly, mammosphere assays demonstrated increasing percentage of cancer stem cells with letrozole resistance alone (4 per 1000 cells plated MCF7Ca vs. 58 per 1000 planted LTLTCa) and with palbociclib and palbociclib resistance (317 per 1000 cells plated MCF7Ca and 202 per 1000 cells plated LTLTCa cells). Overall, these results indicate that 1) resistance to palbociclib, whether as first line or second line treatment has significant effects on breast cancer cells that may be relevant to patient diagnosis and treatment. Citation Format: Armina A. Kazi, Antony Sare, Saranya Chumsri, Angela Brodie. Mechanisms of resistance to palbociclib and aromatase inhibitors in hormone receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2053. doi:10.1158/1538-7445.AM2017-2053

Glyceollin I Reverses Epithelial to Mesenchymal Transition in Letrozole Resistant Breast Cancer through ZEB1.

Although aromatase inhibitors are standard endocrine therapy for postmenopausal women with early-stage metastatic estrogen-dependent breast cancer, they are limited by the development of drug resistance. A better understanding of this process is critical towards designing novel strategies for disease management. Previously, we demonstrated a global proteomic signature of letrozole-resistance associated with hormone-independence, enhanced cell motility and implications of epithelial mesenchymal transition (EMT). Letrozole-resistant breast cancer cells (LTLT-Ca) were treated with a novel phytoalexin, glyceollin I, and exhibited morphological characteristics synonymous with an epithelial phenotype and decreased proliferation. Letrozole-resistance increased Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression (4.51-fold), while glyceollin I treatment caused a −3.39-fold reduction. Immunofluorescence analyses resulted of glyceollin I-induced increase and decrease in E-cadherin and ZEB1, respectively. In vivo studies performed in ovariectomized, female nude mice indicated that glyceollin treated tumors stained weakly for ZEB1 and N-cadherin and strongly for E-cadherin. Compared to letrozole-sensitive cells, LTLT-Ca cells displayed enhanced motility, however in the presence of glyceollin I, exhibited a 68% and 83% decrease in invasion and migration, respectively. These effects of glyceollin I were mediated in part by inhibition of ZEB1, thus indicating therapeutic potential of glyceollin I in targeting EMT in letrozole resistant breast cancer.

Histone deacetylase inhibitor entinostat in combination with a retinoid downregulates HER2 and reduces the tumor initiating cell population in aromatase inhibitor-resistant breast cancer.

Resistance to aromatase inhibitors (AIs) involves increased HER2. One mechanism by which HER2 may mediate resistance is through expansion of the tumor initiating cell (TIC) population. This study investigates whether combining all-trans retinoic acid (ATRA) and histone deacetylase inhibitor entinostat (ENT) can inhibit TICs and HER2 in AI-resistant cells and tumors. Modulation of cell viability and HER2 expression were assessed in AI-resistant cells treated with ATRA + ENT. Letrozole-resistant LTLT-Ca cells treated with ATRA + ENT were assayed for changes in TIC characteristics, such as TIC markers (BCRP, ALDH, and BMI-1), side population (SP), and mammosphere formation. Xenograft tumors of MCF-7Ca cells made resistant to letrozole were treated with ATRA, ATRA + letrozole, ATRA + ENT, or ATRA + ENT + letrozole. Resulting tumors were assayed for changes in TIC characteristics. Patient samples taken pre- and post-AI treatment were analyzed for changes in ERα and HER2 protein expression. Treatment with ATRA + ENT reduced HER2 expression and viability (P < 0.001) in AI-resistant cells, as well as decreased SP (P < 0.0001), mammosphere formation (P < 0.01), and expression of TIC molecular markers (P < 0.01) in LTLT-Ca. A reduction in tumor growth rate was observed in mice treated with ENT + ATRA + letrozole when compared to mice treated with single agents (P < 0.0001) or ENT + ATRA (P = 0.02). Decreased TIC characteristics, including mammosphere formation (P < 0.05), were observed in tumors from the triple combination. An increase in HER2 and downregulation in ERα protein expression was observed in patients upon resistance to AI (P < 0.005). These studies indicate that the combination of ATRA and ENT inhibits the TIC population of AI-resistant cells and may be effective in reducing tumor recurrence.

Aromatase Inhibitor-Mediated Downregulation of INrf2 (Keap1) Leads to Increased Nrf2 and Resistance in Breast Cancer.

Aromatase inhibitors are effective drugs that reduce or eliminate hormone-sensitive breast cancer. However, despite their efficacy, resistance to these drugs can occur in some patients. The INrf2 (Keap1):Nrf2 complex serves as a sensor of drug/radiation-induced oxidative/electrophilic stress. INrf2 constitutively suppresses Nrf2 by functioning as an adapter protein for the Cul3/Rbx1-mediated ubiquitination/degradation of Nrf2. Upon stress, Nrf2 dissociates from INrf2, is stabilized, translocates to the nucleus, and coordinately induces a battery of cytoprotective gene expression. Current studies investigated the role of Nrf2 in aromatase inhibitor resistance. RT-PCR and immunoblot assays showed that aromatase inhibitor-resistant breast cancer LTLTCa and AnaR cells express lower INrf2 and higher Nrf2 protein levels, as compared with drug-sensitive MCF-7Ca and AC1 cells, respectively. The increase in Nrf2 was due to lower ubiquitination/degradation of Nrf2 in aromatase inhibitor-resistant cells. Higher Nrf2-mediated levels of biotransformation enzymes, drug transporters, and antiapoptotic proteins contributed to reduced efficacy of drugs and aversion to apoptosis that led to drug resistance. shRNA inhibition of Nrf2 in LTLTCa (LTLTCa-Nrf2KD) cells reduced resistance and sensitized cells to aromatase inhibitor exemestane. Interestingly, LTLTCa-Nrf2KD cells also showed reduced levels of aldehyde dehydrogenase, a marker of tumor-initiating cells and significantly decreased mammosphere formation, as compared with LTLTCa-Vector control cells. The results together suggest that persistent aromatase inhibitor treatment downregulated INrf2 leading to higher expression of Nrf2 and Nrf2-regulated cytoprotective proteins that resulted in increased aromatase inhibitor drug resistance. These findings provide a rationale for the development of Nrf2 inhibitors to overcome resistance and increase efficacy of aromatase inhibitors.

Abstract 1471: HER2 regulated miRNA expression in letrozole resistant breast cancer

Abstract MicroRNAs (miRNAs) are short non-coding regulatory RNA molecules that modulate the expression of specific proteins by binding to target messenger RNAs (mRNAs) and causing either degradation of the mRNAs or inhibition of their translation to protein. Thus, miRNAs play an important role in a variety of normal cellular processes (e.g., differentiation, cell growth, cell death, etc.), and in diseases, such as cancer. MiRNAs have been implicated in breast cancer, but very little is known about their role in aromatase-inhibitor (AI)-resistant breast cancer. Studies by us and others suggest that acquired resistance to AIs (e.g., letrozole, let), which inhibit the aromatase enzyme from converting androgens (e.g., androstenedione) to estrogen, involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as epidermal growth factor receptor (EGFR)/HER2. Recent work in our lab has further indicated that HER2's effects on resistance are mediated by hypoxia inducible factor 1 (HIF-1) and cancer stem cells. The aim of this current study was to determine if miRNAs were also involved in this mechanism. Initial miRNA microarray results by Zhou et al. identified a number of miRNAs that were either upregulated or down regulated in ER-/HER2+, let-resistant LTLTCa cells vs. ER+/HER2- let-sensitive MCF-7Ca cells. Of particular interest, was data indicating increased expression of miRNA181a and miRNA222 in LTLTCa cells. MiRNA181a has been linked to HIF-1 and cancer stem cells. MiRNA222 has been linked to drug resistance in breast cancer cells. In vitro miRNA isolation and RT-PCR analyses confirmed the microarray results: miRNA181a and miRNA22 levels were at least 13-fold and 10-fold higher in LTLTCa cells vs. MCF-7Ca cells, respectively. MiRNA expression was also analyzed in mouse tumor xenografts. Compared to control (androstenedione-supplemented) tumors, let-treated tumors had increased miRNA181a and 222 expression, starting at 2 weeks of let treatment, when HER2 has previously been shown to be expressed, and further increasing through 16 weeks of letrozole treatment, when tumors are letrozole-resistant. Since HER2 is associated with let resistance, the effect of inhibiting HER2 expression and/or activity on miRNA expression was determined. Lapatinib and trastuzumab either alone or in combination, significantly decreased miRNA 181a expression (0.1-fold vs. vehicle). MiRNA 181a and 222 expression was also analyzed in another ER-/HER2+ breast cancer cell line, SKBR3. Compared to MCF-7Ca cells, miRNA 181a, but not miRNA 222, was significantly upregulated 16-fold in SKBR3 cells. Lastly, preliminary studies were conducted to look at miRNA181a expression in patient tumor samples. Significant and comparable levels of miRNA181a were detected in both ER+/HER2+, ER-/HER2+ samples Overall, these results suggest that miRNAs 181a and 222 are regulated by HER2 and may mediate its effects on AI-resistance. Note: This abstract was not presented at the meeting. Citation Format: Armina A. Kazi, Gauri Sabnis, Qun Zhou, Saranya Chumsri, Amanda Schech, Preeti Shah, Angela Brodie. HER2 regulated miRNA expression in letrozole resistant breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1471. doi:10.1158/1538-7445.AM2014-1471

Proteomic Signatures of Acquired Letrozole Resistance in Breast Cancer: Suppressed Estrogen Signaling and Increased Cell Motility and Invasiveness

Aromatase inhibitors, such as letrozole, have become the first-line treatment for postmenopausal women with estrogen-dependent breast cancer. However, acquired resistance remains a major clinical obstacle. Previous studies demonstrated constitutive activation of the MAPK signaling, overexpression of HER2, and down-regulation of aromatase and ERα in letrozole-resistant breast cancer cells. Given the complex signaling network involved in letrozole-refractory breast cancer and the lack of effective treatment for hormone resistance, further investigation of aromatase inhibitor resistance by a novel systems biology approach may reveal previously unconsidered molecular changes that could be utilized as therapeutic targets. This study was undertaken to characterize for the first time global proteomic alterations occurring in a letrozole-resistant cell line. A quantitative proteomic analysis of the whole cell lysates of LTLT-Ca (resistant) versus AC-1 cells (sensitive) was performed to identify significant protein expression changes. A total of 1743 proteins were identified and quantified, of which 411 were significantly up-regulated and 452 significantly down-regulated (p < 0.05, fold change > 1.20). Bioinformatics analysis revealed that acquired letrozole resistance is associated with a hormone-independent, more aggressive phenotype. LTLT-Ca cells exhibited 84% and 138% increase in migration and invasion compared with the control cells. The ROCK inhibitor partially abrogated the enhanced migration and invasion of the letrozole-resistant cells. Flow cytometric analyses also demonstrated an increase in vimentin and twist expression in letrozole-resistance cells, suggesting an onset of epithelial to mesenchymal transition (EMT). Moreover, targeted gene expression arrays confirmed a 28-fold and sixfold up-regulation of EGFR and HER2, respectively, whereas ERα and pS2 were dramatically reduced by 28-fold and 1100-fold, respectively. Taken together, our study revealed global proteomic signatures of a letrozole-resistant cell line associated with hormone independence, enhanced cell motility, EMT and the potential values of several altered proteins as novel prognostic markers or therapeutic targets for letrozole resistant breast cancer.

Abstract 95: Inhibiiton of non-hypoxic HIF-1 expression in letrozole-resistant breast cancer cells reduces their cancer stem cell characteristics.

Abstract Although aromatase inhibitors (AIs) have been shown to be highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Studies suggest that acquired resistance to AIs (i.e., letrozole) involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as epidermal growth factor receptor (EGFR)/HER2. Recent work in our lab has also linked hypoxia inducible factor 1 (HIF-1) and its target gene breast cancer resistance protein (BCRP, a stem cell marker) to HER2 and AI resistance. Other studies have associated each of these factors with cancer stem cells (CSCs),, which have been implicated in drug resistance. Thus, the purpose of this study is to 1) determine whether HIF-1 expression is enriched in the CSC subpopulation of letrozole-resistant breast cancer cells; and 2) determine the effects of inhibiting HIF-1 on CSC characteristics. The in vitro model used for acquired letrozole resistant breast cancer is the LTLTCa cell line, which was obtained through long-term letrozole treatment of MCF-7Ca xenograft tumors. The CSC subpopulation in LTLTCa cells was isolated by flow cytometry based on their aldehyde dehydrogenase (ALDH) expression, and the CSC characteristics studied include mammosphere formation, side population percentage, and CD44+/CD24+ expression. Inhibition of HIF-1 expression was accomplished using either commercially available HIF-1α siRNA or EZN-2968, a specific RNA antagonist against HIF-1α currently in clinical trials. HIF-1α is the inducible subunit of HIF-1. RT-PCR indicates that mRNA expression of HIF-1α and stem cell markers, such as BCRP, BMI-1, and Nanog, were significantly higher (p&amp;lt;0.05) in ALDHhigh (CSC) vs. ALDHlow (non-CSC) cells. HIF-1 siRNA and EZN-2968 decreased HIF-1α mRNA and protein expression in LTLTCa cells within 48h (0.4 to 0.01-fold vs. negative control siRNA/RNA antagonist). This correlated with decreases in mRNA expression of stem cell markers BCRP and BMI-1, and TWIST, an EMT marker and regulator of BMI-1. HIF-1α inhibition also reduced mammosphere formation by 33% (p&amp;lt;0.05). Interestingly, preliminary results indicate that while EZN-2968 decreased the ratio of CD44+/24+ expression (28% vs. 52% in negative control RNA antagonist) and side population percentage (8% vs. 16% in negative control RNA antagonist) in LTLTCa cells, the commercially available HIF-1α siRNA had no effect. These results suggest that nonhypoxic HIF-1 is involved in regulating cancer stem cell characteristics in letrozole-resistant breast cancer cells, and that EZN-2968 should be further explored in the prevention and/or treatment of AI-resistant breast cancer. EZN-2968 kindly provided by Enzon Pharmaceuticals. This work is funded by DOD Breast Cancer Research Program Postdoctoral Award (BC1039031). Citation Format: Armina A. Kazi, Preeti Shah, Amanda Schech, Gauri Sabnis, Saranya Chumsri, Angela Brodie. Inhibiiton of non-hypoxic HIF-1 expression in letrozole-resistant breast cancer cells reduces their cancer stem cell characteristics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 95. doi:10.1158/1538-7445.AM2013-95

Abstract 1314: HDAC inhibitor entinostat reverses letrozole resistance in MCF-7Ca xenografts through modulation of Her-2.

Abstract Development of aromatase inhibitors (AIs) has significantly improved the treatment outcome of hormone responsive post-menopausal breast cancer. However, not all tumors respond initially and others eventually acquire resistance. Using our hormone sensitive human aromatase expressing MCF-7Ca xenograft model, we also saw that although AI letrozole provides a longer control over tumor growth, tumors eventually began to grow. Furthermore, we observed that Her-2 protein levels within the tumors were upregulated as early as 4 weeks (as tumors were responding). Her-2 levels increased further as the tumors became resistant. A cell line was isolated from these Long-Term Letrozole Treated tumors (LTLT-Ca), which showed increased Her-2 and reduction in ERα and aromatase. The increase in Her-2 protein levels was not accompanied by gene amplification but we observed that Her-2 protein in LTLT-Ca cells was more stable and had a longer half-life than that in MCF-7Ca cells. In addition, when MCF-7Ca cells were deprived of estrogen (E2W) or treated with letrozole, stability of Her-2 protein was immediately increased, suggesting that lower estrogen levels stabilize Her-2. In order to identify novel approaches to restoring sensitivity to AIs, we treated letrozole resistant MCF-7Ca tumors with histone deacetylase (HDAC) inhibitor entinostat (ENT). Addition of ENT to either letrozole or exemestane in letrozole resistant MCF-7Ca tumors led to increased ERα expression, aromatase activity and significantly inhibited further tumor growth, along with downregulation of Her-2, p-Her-2, p-MAPK and p-Akt. Based on the observations made in letrozole resistant tumors treated with entinostat (with or without letrozole), we hypothesized that entinostat reverses letrozole resistance due to modulation of Her-2. Entinostat reduced interaction between HSP-90 and Her-2 in entinostat treated tumors, resulting in degradation of Her-2. Based on the findings that Her-2 is upregulated even when the tumors are responding, we evaluated the effect of adding entinostat to letrozole from the start of treatment in the MCF-7Ca xenografts. Tumors of MCF-7Ca cells were allowed to form in the presence of androstenedione (Δ4A), aromatizable substrate for estrogen. When the tumors reached ∼300mm3, the mice were grouped to receive Δ4A 100μg/day (control), letrozole (1μg/day), entinostat (50μg/day) or combination of entinostat plus letrozole. Mice receiving letrozole treatment had significantly lower tumor growth rate than control (p=0.0001), so did mice treated with the combination of entinostat plus letrozole (p&amp;lt;0.0001). The growth rate of tumors treated with entinostat + letrozole was similar to the growth rate of letrozole alone suggesting that in the absence of acquired or de novo resistance pathways the addition of entinostat did not provide benefit beyond the aromatase inhibitor alone. Citation Format: Gauri J. Sabnis, Armina Kazi, Olga Goloubeva, Saranya Chumsri, Angela Brodie. HDAC inhibitor entinostat reverses letrozole resistance in MCF-7Ca xenografts through modulation of Her-2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1314. doi:10.1158/1538-7445.AM2013-1314

Abstract 1312: The estrogen receptor β agonists, Liquiritigenin and S-equol, inhibit breast cancer cell proliferation through the activation of tumor-suppressor and other pathways.

Abstract Estrogen and estrogen receptors (ERα and ERβ) play an important role in breast cancer. Currently, antiestrogen (AE) targeted to ERα and aromatase inhibitors (AI), which block local and systemic estrogen production, are used in the treatment of breast cancer. Both treatments improve outcomes for about 50% of patients with early or advanced ERα positive breast cancer. Unfortunately, in women with advanced breast cancer, essentially all breast cancers develop resistance to these two classes of agents. Moreover, the side effects associated with currently used AI limit the long-term utility of them as chemopreventative agents. Therefore there is a critical need to develop effective alternate strategies to prevent or delay the development of resistance to endocrine therapy and the resulting tumor progression. Recent studies have identified several natural compounds that have the potential to function as ERβ agonists. Plant-derived ERβ-agonists, Liquiritigenin (LIQ), from the plant Glycyrrhizae uralensis and an active component of MF101, and S-equol, from the soy isoflavone daidzein, are currently being tested to treat vasomotor hot flashes associated with menopause. In addition, S-equol is also being tested as a treatment for BPH in men. However, the exact mechanism of action of these compounds is not clear. In this study we sought to determine the mechanism by which ERβ acts as a tumor suppressor, and whether these ERβ agonists could resensitize Letrozole-resistant breast cancer cells. MCF7aro and LTLT-ca cells were used as models of hormone-responsive and hormone-resistant breast cancer respectively. MTT assay was used to determine the half maximal inhibitory concentrations of the ERβ agonists. Upon treatment, our results show changes in the levels of p53, NFkB, cyclin D1, KLF5 and others using qRT-PCR and western blot analysis. The effects of ERβ agonists on the activation of these genes were confirmed using specific gene promoter-luciferase reporter gene constructs. Specificity of ERβ-mediated actions was confirmed using a siRNA approach in addition to other methods and knockdown efficiency was confirmed through RT-PCR. Interestingly, our results show that the plant-derived ERβ-agonists, LIQ and S-equol, not only affect the growth of breast cancer cells and tumors but treatment increases the expression of p53 and others, and upon ERβ knockdown this effect is ablated. This study suggests that ERβ acts as a tumor suppressor by regulating the expression of p53 and others involved in cell proliferation. In conclusion, this study demonstrates the potential role for ERβ agonists to improve adjuvant endocrine therapy to treat both hormone-responsive and hormone-resistant breast cancers, as well as provides rationale for new preventive approaches to decrease the incidence of breast cancer. Citation Format: Cathy Samayoa, Anil Kotha, Naveen K. Krishnegowda, Ratna K. Vadlamudi, Rajeshwar Rao Tekmal. The estrogen receptor β agonists, Liquiritigenin and S-equol, inhibit breast cancer cell proliferation through the activation of tumor-suppressor and other pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1312. doi:10.1158/1538-7445.AM2013-1312

Abstract 787: Involvement of HER2, HIF-1, and BCRP in cancer stem cell characteristics of letrozole-resistant breast cancer cells

Abstract Studies by us and others suggest that acquired resistance to aromatase inhibitors (AIs; i.e., letrozole) involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as epidermal growth factor receptor (EGFR)/HER2. Recent work in our lab has also linked hypoxia inducible factor 1 (HIF-1) and breast cancer resistance protein (BCRP, a stem cell marker) to HER2 and AI resistance. Other studies have associated each of these factors with cancer stem cells (CSCs) and CSCs have been implicated in drug resistance. Therefore, this current study aimed to 1) compare the CSC characteristics of letrozole-resistant (LTLTCa) and parental letrozole-sensitive (MCF-7Ca) breast cancer cells; 2) determine the roles of HER2, BCRP, and HIF-1 on CSC characteristics; and 3) compare the effects of HER2 inhibition and of differentiating agent all-trans retinoic acid (ATRA) on CSC population of the LTLTCa cells. LTLTCa cells were found to have higher expression of CSC characteristics compared to parental MCF-7Ca cells: 9.17%±2.0% vs. 0.02% ±0.01% (p &amp;lt;0.01) measured by side population, and 3.08% vs. 0.53% (p&amp;lt;0.05) by aldehyde dehydrogenase (ALDH) activity. Consistent with previous results, inhibition of HER2, HIF-1, or BCRP resulted in decreased CSC characteristics in LTLTCa cells. Inhibition of HER2 by treatment with 1 μM lapatinib (Lap) decreased side population (from 7.91%±2.10 to 0.76%±0.20) and mammosphere formation (from &amp;gt;200 mammospheres/10,000 cells to ∼50 mammospheres/10,000 cells). Flow cytometry also demonstrated that LTLTCa cells high in both ALDH activity and CD44 positivity, were also high in HER2. Inhibition of HIF-1 (by siRNA) or BCRP (by pharmacological inhibitors or siRNA) significantly decreased mammosphere formation from 406 mammospheres/80,000 cells to 93 and 81 mammospheres/80,000 cells, respectively (p&amp;lt;0.01). Pharmacological inhibition of BCRP also decreased side population. Comparing the effects of Lap and ATRA on LTLTCa cells, both drugs decreased mammosphere formation after 48 h and cell viability after 7 days of treatment (p&amp;lt;0.05 for each). Both drugs also increased ERα and decreased BCRP expression in LTLTCa cells. Lastly, Lap and ATRA each increased the expression ratio of epithelial markers versus mesenchymal markers, but they did this by affecting different proteins. Lap increased expression of epithelial marker E-cadherin, but had no effect on mesenchymal marker N-cadherin. ATRA decreased N-cadherin but had no effect on E-cadherin. Overall, the results from this study suggest that HER2-HIF-1-BCRP signaling in contributes to acquired breast cancer cell resistance to letrozole by 1) increasing the cancer stem cell population, and possibly by 2) promoting epithelial mesenchymal transition (EMT), an indicator of cancer cell invasive and metastatic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 787. doi:1538-7445.AM2012-787

Abstract 1063: The ErbB3/4 receptor ligand Heregulin negatively regulates ErbB2 and ErbB3 receptor levels

Abstract ErbB2 is overexpressed in 30% of breast cancers and its overexpression is associated with poor prognosis. In addition, the ErbB2/3 heterodimer forms the most potent mitogenic receptor in vitro and is key to the proliferation of breast cancer cells. Despite the importance of this heterodimer in breast cancer progression, the negative regulation of these ErbB tyrosine kinases is still poorly understood. We demonstrate here for the first time that the ErbB3/4 ligand Heregulin (HRG) reduces mRNA and protein levels of both ErbB2 and ErbB3 in human breast cancer cell lines. We have found that after 24h of HRG treatment, ErbB2 expression decreased at both the RNA and protein levels in ErbB2 overexpressing AU565, BT474 and LTLT-Ca breast cancer cell lines. In contrast, EGFR levels were unaffected by HRG treatment. An initial decline in steady state mRNA levels was first noted at 1 hour after HRG treatment. ErbB3 levels also decreased in LTLT-Ca cells after HRG treatment. Lapatinib, which inhibits ErbB2 and EGFR kinase activity and HRG induced-ErbB3 phosphorylation, diminished the HRG- induced decrease in ErbB2 and ErbB3 in AU565 and LTLT-Ca cells. This finding suggests that the kinase activity of EGFR/ErbB2 may be involved in the HRG-induced down-regulation. HRG did not change the activity of ErbB2 promoter reporter constructs spanning 215 kb to 6007 kb upstream of the ErbB2 transcription start site as indicated by luciferase assays. An ongoing study suggests that HRG could be involved in decreasing ErbB2 mRNA stability. Our findings suggest that resistance to Lapatinib may be due in part to the fact that inhibition of ErbB2/ EGFR kinase activity dampens the negative effect of HRG on ErbB2 expression, resulting in elevated levels of ErbB2 in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1063. doi:1538-7445.AM2012-1063

Abstract LB-415: Global proteomic signature of letrozole-resistance in breast cancer cells

Abstract Postmenopausal women with early-stage metastatic estrogen-dependent breast cancer are generally treated with aromatase inhibitors (AIs) (e.g., anastrozole and letrozole). However, acquired resistance remains a major clinical obstacle. Previous studies demonstrated letrozole-resistant breast cancer cells undergo adaptive changes resulting in constitutive activation of the MAPKinase signaling pathway and increased expression of HER2 and IGFR with decreased expression of aromatase and ERα. Given the complex signaling network involving letrozole-refractory breast cancer and the lack of effective treatment for hormone resistance, further investigation of AI resistance by a novel systems biology approach may reveal previously unconsidered molecular changes that could be utilized as therapeutic targets. The objective of this study was to characterize for the fist time global proteomic alterations occurring in the LTLT-Ca letrozole-resistant cell line. A quantitative proteomic analysis of the whole cell lysates of LTLT-Ca cells vs. AC1 cells (letrozole-sensitive) was performed to identify significant protein expression changes. Protein extracts were digested with trypsin and the resulting peptides were labeled with tandem mass tags. Fractionation and nanoflow HPLC-MS/MS was used for peptide separation, identification and quantification. Proteomic data were analyzed by bioinformatics tools to gain insight into global adaptive signaling modulations as a result of acquired resistance to letrozole. A total of 1743 proteins were identified and quantified that were present in both cell lines, of which 411 were significantly upregulated and 452 were significantly downregulated (p&amp;lt;0.05, fold change &amp;gt; 1.20). Bioinformatics analysis of these altered proteins revealed that acquired letrozole resistance is associated with a hormone-independent, more motile phenotype. In particular, overexpression of multiple proteins was implicated in the enhanced migration and invasion in the resistant cell line, including ROCK, RAC1, S100 family proteins and the actin bundling protein, fascin. Subsequent migration and invasion assays demonstrated more than a 25% increase in the motility of the LTLT-Ca cells. Treatment with pharmacologic inhibitors of fascin, ROCK, and RAC1 partially abrogated both invasion and migration of the letrozole-resistant cells. Moreover, targeted gene expression arrays confirmed a 28-fold and 6-fold increase in the expression of EGFR and HER2, respectively, while ERα and pS2 were dramatically suppressed by 28-fold and 1100-fold, respectively. Taken together, our study for the first time revealed global proteomic signatures of a letrozole-resistant cell line associated with hormone independence and enhanced cell motility and the potential values of several altered proteins as novel therapeutic targets for treatment of acquired letrozole resistance in postmenopausal breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-415. doi:1538-7445.AM2012-LB-415