Letrozole-resistant Cell Research Articles

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

Contribution of Estrone Sulfate to Cell Proliferation in Aromatase Inhibitor (AI) -Resistant, Hormone Receptor-Positive Breast Cancer.

Aromatase inhibitors (AIs) effectively treat hormone receptor-positive postmenopausal breast cancer, but some patients do not respond to treatment or experience recurrence. Mechanisms of AI resistance include ligand-independent activation of the estrogen receptor (ER) and signaling via other growth factor receptors; however, these do not account for all forms of resistance. Here we present an alternative mechanism of AI resistance. We ectopically expressed aromatase in MCF-7 cells expressing green fluorescent protein as an index of ER activity. Aromatase-overexpressing MCF-7 cells were cultured in estrogen-depleted medium supplemented with testosterone and the AI, letrozole, to establish letrozole-resistant (LR) cell lines. Compared with parental cells, LR cells had higher mRNA levels of steroid sulfatase (STS), which converts estrone sulfate (E1S) to estrone, and the organic anion transporter peptides (OATPs), which mediate the uptake of E1S into cells. LR cells proliferated more in E1S-supplemented medium than did parental cells, and LR proliferation was effectively inhibited by an STS inhibitor in combination with letrozole and by ER-targeting drugs. Analysis of ER-positive primary breast cancer tissues showed a significant correlation between the increases in the mRNA levels of STS and the OATPs in the LR cell lines, which supports the validity of this AI-resistant model. This is the first study to demonstrate the contribution of STS and OATPs in E1S metabolism to the proliferation of AI-resistant breast cancer cells. We suggest that E1S metabolism represents a new target in AI-resistant breast cancer treatment.

Abstract P3-05-12: Combination of anti-progranulin (GP88/PGRN) antibody and letrozole inhibits tumor formation of letrozole resistant breast cancer cell lines

Abstract The 88 kDa glycoprotein GP88 (Progranulin, PCDGF, acrogranin) is the largest member of the granulin/epithelin family of growth modulators identified as a driver of tumorigenesis. GP88 (PGRN) was also shown to be overexpressed in invasive ductal carcinoma (IDC)whereas it was negative in benign tumors and normal mammry epithelial tissue, thereby establishing GP88 as a therapeutic and diagnostic target in breast cancer (BC). Our laboratory has developed validated tools to measure GP88 in tumor biopsies and biological fluids as well as blocking its action. WE showed that GP88 was secreted and detected in the serum of BC patients at an increased level when compared to healthy subjects. Pathological studies with 530 cases of ER+ IDC with clinical outcomes showed that GP88 tumor expression was an independent prognostic indicator of recurrence in early stage BC patients. Training study followed by an independent validation study demonstrated that high GP88 tissue expression (GP88 3+) was associated with a 4-fold increase in risk of recurrence at 5 years. A neutralizing anti-GP88 antibody AG1 was expressed in a high yield CHO cell line was developed. The present study examined the effect of AG1 in letrozole resistant cell line AGLetR developed by long term selection in letrozole supplemented medium. This cell line showed decreased letrozole responsiveness in vivo and therefore constituted an excellent model for investigating letrozole resistance in vitro as well as in vivo. Here we report the results of studies investigating the effect of various doses of AG1 on LetR tumor development in combination with letrozole for AGLetR. We show that treatment with AG1 (10 mg/kg i.p.) in combination with letrozole was efficient to maintain long term responsiveness and inhibit tumor growth. Letrozole alone (5mg/kg) was unable to inhibit tumor growth and showed a doubling of tumor volume. Interestingly, long term combination treatment lead to tumor regression and inhibited tumor growth. These data suggest that inhibiting GP88 could provide a novel and alternative therapeutic strategy for patients with resistance to anti-estrogen therapy, being tamoxifen or letrozole. This works is supported by 2R44CA124179, HHSN 261201200060C, and HHSN2612014400C from NCI. Citation Format: Serrero G, Dong J, Yue B, Hicks D, Hayashi J. Combination of anti-progranulin (GP88/PGRN) antibody and letrozole inhibits tumor formation of letrozole resistant breast cancer cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-12.

Abstract 3457: Evaluating estrogen receptor β agonists for the treatment of breast cancer

Abstract Breast cancer is the main cause of cancer-associated mortality in women worldwide. The estrogen receptors play a critical role in normal mammary gland development as well as breast cancer. Over two thirds of all breast cancers express high levels of Estrogen Receptor α (ERα) and are depend on estrogen for growth. Current endocrine therapies aim to decrease estrogen levels with the use aromatase inhibitors or to inhibit ERα signaling with the use of anti-estrogens. Although these treatment options are initially effective, a significant number of patients will present with recurrent disease. Therefore, there is a critical need to identify targeted therapies which will inhibit tumor progression and circumvent therapy resistance. In addition to focusing on ERα, emerging evidence has shed light on the importance of Estrogen Receptor β in breast cancer. Unlike ERα, ERβ has tumor suppressing activity, therefore targeting ERβ with selective agonists may provide therapeutic benefit in the treatment of breast cancer. The objective of this study was to investigate the effects of selective ERβ agonists in distinct breast cancer models. Liquiritigenin and S-Equol are selective plant-derived ERβ agonists currently in clinical trials for the relief of symptoms associated with menopause, and for the treatment of benign prostate hyperplasia. Additionally, LY500307, a synthetic ERβ agonist is currently in clinical trials and has been shown to be well tolerated. To test the effects of ERβ agonists on growth, we carried out cell growth assays using various cell lines. MCF7aro and ZR-75aro cells were used as models of post-menopausal breast cancer given their ability to synthesize their own estrogens. A letrozole resistant cell line was used to determine if ERβ agonists could inhibit the growth of therapy resistant cells. Additionally, we used a syngeneic tumor mouse model to investigate if ERβ agonist could inhibit tumor growth in a model with an intact immune system. Our results demonstrate that both, plant-derived and synthetic, ERβ agonists can inhibit the growth of breast cancer cells, including those that over express aromatase, and are resistant to letrozole. The synthetic ERβ agonist was able to inhibit growth at much lower doses, overcoming a critical barrier presented by the plant derived agonists. Our mechanistic studies show that ERβ agonists can alter ERα and ERβ levels, which shift the α/β ratio to a more tumor suppressive state. We also found that these compounds affect cell cycle through cyclin D1 and p27. Additionally, we also saw induction of apoptosis and increased p53 levels. Our studies evaluated the effects of ERβ agonists in various breast cancer cell lines, and our results suggest that targeting ERβ may be a valuable therapeutic option for the treatment of breast cancer. Citation Format: Cathy Samayoa, Naveen Krishnegowda, Ratna Vadlamudi, Rajeshwar Tekmal. Evaluating estrogen receptor β agonists for the treatment of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3457. doi:10.1158/1538-7445.AM2015-3457

New cell culture model for aromatase inhibitor-resistant breast cancer shows sensitivity to fulvestrant treatment and cross-resistance between letrozole and exemestane.

Aromatase inhibitor (AI) treatment is first-line systemic treatment for the majority of postmenopausal breast cancer patients with estrogen receptor (ER)-positive primary tumor. Although many patients benefit from treatment, some will develop resistance, and models mimicking acquired resistance will be valuable tools to unravel the resistance mechanisms and to find new treatments and biomarkers. Cell culture models for acquired resistance to the three clinically relevant AIs letrozole, anastrozole and exemestane were developed by selection and expansion of colonies of MCF-7 breast cancer cells surviving long-term AI treatment under conditions where endogenous aromatase-mediated conversion of androgen to estrogen was required for growth. Four cell lines resistant to each of the AIs were established and characterized. Maintenance of ER expression and function was a general finding, but ER loss was seen in one of twelve cell lines. HER receptor expression was increased, in particular EGFR expression in letrozole-resistant cell lines. The AI-resistant cell lines had acquired ability to grow without aromatase-mediated conversion of testosterone to estradiol, but upon withdrawal of AI treatment, testosterone induced minor growth stimulation. Letrozole, exemestane and tamoxifen were able to abrogate the testosterone stimulation but could not reduce growth to below the level in standard growth medium with AI, demonstrating cross-resistance between letrozole, exemestane and tamoxifen. In contrast, fulvestrant totally blocked growth of the AI resistant cell lines both after withdrawal of AI and with AI treatment. These data show that ER is the main driver of growth of the AI-resistant cell lines and indicate ligand-independent activation of ER. Fulvestrant is an efficient treatment option for these AI-resistant breast cancer cells, and the cell lines will be useful tools to disclose the underlying molecular mechanism for resistance to the different AIs.

Abstract P2-17-01: The PI3K inhibitor GDC-0032 is selectively potent against PIK3CA mutant breast cancer cell lines and tumors

Abstract Mutations in the phosphoinositide-3 kinase alpha isoform (PIK3CA) are frequent in breast cancer and activate the PI3K signaling pathway. We discovered GDC-0032, a selective, potent, orally bioavailable inhibitor of PI3Ka with a Ki = 0.2nM, and with reduced inhibitory activity against PI3Kβ. This selectivity profile, and excellent pharmacokinetic and pharmaceutical properties, allowed for greater efficacy in vivo at the maximum tolerated dose relative to a pan Class I PI3K inhibitor in PIK3CA mutant xenografts. Notably, GDC-0032 preferentially inhibited PIK3CA mutant cells relative to cells with wild-type PI3K. GDC-0032 potently inhibits signal transduction downstream of PI3K and induces apoptosis at low concentrations in breast cancer cell lines and xenograft models that harbor PIK3CA mutations. The mutant-bias of GDC-0032 is linked to unique properties of GDC-0032, including cellular potency against the mutant isoform and reduction of receptor tyrosine kinase (RTK) signaling. Endocrine therapies such as letrozole are commonly used treatment options for metastatic Hormone Receptor positive (HR+) breast cancer but many patients ultimately relapse. Due to the importance of PI3K in breast cancer, PI3K inhibitors such as GDC-0032 are attractive for combination with endocrine therapies. GDC-0032 was evaluated in breast cancer lines and models in combination with letrozole, and assayed for cellular viability, modulation of PI3K pathway, modulation of ER pathway markers, and apoptosis induction. The combination of GDC-0032 and letrozole decreased cellular viability and increased apoptosis relative to either single agent. We observed cross-talk between the PI3K and ER pathways that suggest a mechanism of action for the combination. In a secreted factor screen we found that multiple soluble factors render breast cancer cells non-responsive to letrozole. It was discovered that many of these factors signal through the PI3K pathway and GDC-0032 in combination with letrozole was able to overcome the growth inhibition caused by the soluble factor. We also established letrozole resistant cell lines that grow independently of any estrogen source. These letrozole resistant lines have elevated PI3K pathway signaling and are still sensitive to GDC-0032. Taken together, these data provide rationale for evaluating GDC-0032 in combination with endocrine therapies for ER+ breast cancer treatment in the clinic. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-17-01.

Abstract P5-09-16: Contribution of estrone-sulfate to cell proliferation in aromatase inhibitor resistance of estrogen receptor positive breast cancer

Abstract [Background] Estrogen receptor (ER) positive breast cancer was composed of about 70% of clinical breast cancer and therapy for the ER positive breast cancer have progressed, but some patients recur after endocrine therapy including aromatase inhibitors (AIs). The switch of proliferation drive to human epidermal growth factor receptor 2 was reported as one of AI resistance mechanisms, but ER status of many recurrent patients after AI treatment was maintained or slightly decreased. Moreover, plasma concentration of estrone-sulfate (E1S) is mostly unchanged after menopause and treatment of AI. Thus, we established AI resistant model with conserved ER expression and its activity from MCF-7 cell, and found that E1S contributed to proliferation of the AI-resistant cells. [Material and Method] We introduced plasmids carrying ERE-green fluorescent protein (GFP) gene to MCF-7 (E10), and then aromatase gene was additionally introduced to E10 (E10arom). E10arom was cultured in estrogen-depleted medium including testosterone and letrozole, monitoring GFP as ER activity for about three months. We finally established ER positive Letrozole-Resistant cell lines (LR cells). In addition, to confirm whether E1S-dependent growth in LR cells could be reproduced in ER positive breast cancer cell line, we also established human steroid sulfatase (STS) gene-transfected MCF-7 (MCF-STS) and organic anion transporter peptide (OATP) 4A1 gene-transfected MCF-7 (MCF-4A1). ER activity of these cell lines was examined by ERE-luciferase reporter assay, and response to each steroid and drug was investigated by cell proliferation assay. Quantitative PCR was used for comparing expression of mRNA in these cell lines. [Results] mRNA expression of STS and four OATPs including OATP4A1 increased in LR cells comparing to E10arom. Moreover, there was no change in mRNA expressions of efflux drug transporters, aromatase-metaboic enzyme and enzymes related with production for estrogenic androgen. ERE-luciferase assay with E1S showed higher ER activity of LR cells than E10arom, and LR cells were more proliferated by E1S in dose-dependent manner than the parental cell. STS inhibitor alone and its combination with letrozole were inhibitory to proliferation of LR cells. LR cells were also inhibited by selective estrogen receptor modulator (SERM) and downregulator (SERD). In proliferation assay of MCF-STS and MCF-4A1, we observed that these cell lines also proliferated by E1S in dose-dependent manner as LR cells did. [Discussion] Since ER status of recurrent patients after AI treatment was mostly unchanged, our model could be more practical for analysis of AI resistance in ER positive breast cancer. These results showed that combined induction of STS and OATP contributed to estradiol (E2) production from E1S in LR cells, suggesting that analysis of E1S as alternative E2 productive pathway needed investigation of both STS and transporter of E1S. The combination of STS inhibitor with letrozole was effective to LR cells, and SERM and SERD were also effective to these cells, suggesting that combined therapy of STS inhibitor with AI or switching AI to SERM or SERD was beneficial for AI-resistant breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-16.

Abstract P5-09-05: Progranulin (GP88) expression and letrozole resistance in breast cancer

Abstract The 88 kDa glycoprotein GP88 (Progranulin, PCDGF, acrogranin) is the largest member of the granulin/epithelin family of growth modulators. GP88 was originally characterized in our laboratory through a biological screen to identify drivers of tumorigenesis. Published studies have established that GP88 represents an ideal therapeutic and diagnostic target in breast cancer (BC) leading to the development of validated tools to measure GP88 in tumor biopsies and biological fluids as well as blocking its action. It was shown that: 1) GP88 expression increases with tumorigenesis; 2) in ER+ breast cancer cells, GP88 stimulates proliferation and its overexpression confers estrogen independence and resistance to several anti-estrogens and aromatase inhibitor; 3) inhibition of GP88 expression by antisense transfection inhibited proliferation in vitro and in vivo; 4) In Her-2 overexpressing breast tumors, GP88 stimulated Her-2 phosphorylation and conferred trastuzumab resistance; 5) GP88 is expressed in 80% invasive ductal carcinoma (IDC) and 60% of ductal carcinoma whereas it is negative in lobular carcinoma, benign lesions and normal mammary tissues; 6) GP88 is secreted and can be detected in the serum of BC patients at an increased level when compared to healthy subjects; 7) Pathological studies with 530 cases of ER+ IDC with clinical outcomes showed that GP88 tumor expression was an independent prognostic indicator of recurrence in early stage BC patients. Training study followed by an independent validation study demonstrated that high GP88 tissue expression (GP88 3+) was associated with a 4-fold increase in risk of recurrence at 5 years. Since GP88 displays not only diagnostic but also therapeutic potentials, we developed a neutralizing anti-GP88 antibody AG1 that inhibited GP88 biological effect (proliferation and migration) in a dose-dependent fashion in vitro. AG1 was expressed in a high yield CHO cell line and formulated. We have shown that in tamoxifen resistant cells, treatment with AG1 would inhibit tumor growth and restore tamoxifen sensitivity. The present study examined the effect of AG1 in letrozole resistant cells. We have developed from a letrozole sensitive cell ER+ BC cell line, a letrozole-resistant cell line by long term selection in letrozole-supplemented medium. This cell line (LetR) shows also decreased letrozole responsiveness in vivo and therefore constitutes an excellent model for investigating letrozole resistance in vivo as well as in vivo. Here we investigated the effect of various doses of AG1 on LetR tumor development alone or in combination with letrozole. Treatment with AG1 (10 mg/kg i.p.) in combination with letrozole was efficient to maintain long term responsiveness to letrozole and inhibited tumor growth. In addition to the mouse xenografts study, an IRB approved clinical study examines changes in GP88 circulating levels in patients with resistance to aromatase inhibitors. Preliminary data will be presented. In conclusion, inhibiting GP88 could provide a novel and alternative therapeutic strategy for patients with resistance to anti-estrogen therapy, being tamoxifen or letrozole. This work is supported by 2R44CA124179 and HHSN 261201200060C from NCI and 02- 2013-018 from the Avon Foundation for Women. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-05.

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.

Efficacy and mechanism of action of Proellex, an antiprogestin in aromatase overexpressing and Letrozole resistant T47D breast cancer cells

Aromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI. In this study, we determined the response of antiprogestin CDB-4124 (Proellex) on the aromatase overexpressing and Letrozole resistant cell lines and also studies its mechanism of action in inhibition of breast cancer cell proliferation. For these studies we generated aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines by stable transfection with plasmid containing CYP19A1 gene, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10μM Letrozole. Cell proliferation was determined by MTT or crystal violet assays. Gene expressions were quantified by QRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on the anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's ‘t’ test or ANOVA followed by Bonferroni's post hoc test. Results showed that T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA; 80–90 fold and protein) and as a result exhibited increased aromatization of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone (50–60%). In T47DaromLR cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex and other known aromatase inhibitors (Letrozole, Anastrozole, and Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. Cell growth inhibition was significantly greater when cells were treated with Proellex alone or in combination with other AIs as compared to AIs alone. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex is probably due to PR-B/EGFR modulation in ER+PR+, aromatase expressing cells. Overall these results suggest that antiprogestin, Proellex can be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for aromatase inhibitor resistant breast cancer patients.

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

P2-17-05: Antiprogestin Proellex Suppresses Proliferation of Aromatase Overexpressing and Letrozole Resistant T47D Breast Cancer Cells.

Abstract Background: Aromatase is a cytochrome P450 CYP19A1 enzyme responsible for the conversion of C19 androgens to C18 estrogen. Aromatase overexpression leads to increase in local estrogen concentration in post menopausal women diagnosed for breast cancer. Aromatase inhibitors (AI) are used as one of the first line therapies for the treatment of ER+PR+ breast cancers. However, many patients acquire resistance to AI treatment. Therefore alternative approaches are being sought for patients with AI resistance. Previous reports suggest that upregulation of progesterone receptor can lead to enhanced expression of EGFR/ERK/MAPK involved in acquiring resistance. In this study, we evaluated the effect of a selective progesterone receptor modulator, CDB4124 (Proellex) with low glucocorticoid activity on aromatase overexpressing and Letrozole resistant T47D cells. Methods: Aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines were generated by stable transfection with plasmid containing CYP19A1 coding region, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10μM Letrozole. Cell proliferation was determined by MTT or Crystal violet assays. Gene expressions were quantitated by qRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's ‘T’ test or ANOVA followed by Bonferroni's post hoc test. Results: T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA and protein) and as a result exhibited increased conversion of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone. In T47Darom cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex, Letrozole and other known AI (Anastrozole, Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. The inhibition of cell proliferation was significantly greater when cells were treated with Proellex in combination to other AIs. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex may be due to PR-B/EGFR modulation in ER+PR+, aromatase overexpressing cells. Conclusion: Overall these results suggest that antiprogestin, Proellex could be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for AI resistant breast cancer patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-17-05.

The role of microRNA-128a in regulating TGFbeta signaling in letrozole-resistant breast cancer cells

Resistance to endocrine therapy agents has presented a clinical obstacle in the treatment of hormone-dependent breast cancer. Our laboratory has initiated a study of microRNA regulation of signaling pathways that may result in breast cancer progression on aromatase inhibitors (AI). Microarray analysis of hormone refractory cell lines identified 115 differentially regulated microRNAs, of which 49 microRNAs were believed to be hormone-responsive. A group of microRNAs were inversely expressed in the AI-resistant lines versus LTEDaro and tamoxifen-resistant. We focused our work on hsa-miR-128a which was hormone-responsive and selectively up-regulated in the letrozole-resistant cell lines. Human miR-128a was predicted to target the TGFβ signaling pathway and indeed sensitivity to TGFβ was compromised in the letrozole-resistant cells, as compared to parental MCF-7aro. Human miR-128a was shown to negatively target TGFβRI protein expression by binding to the 3'UTR region of the gene. Inhibition of endogenous miR-128a resulted in resensitization of the letrozole-resistant lines to TGFβ growth inhibitory effects. These data suggest that the hormone-responsive miR-128a can modulate TGFβ signaling and survival of the letrozole-resistant cell lines. To our knowledge, this is the first study to address the role of microRNA regulation as well as TGFβ signaling in AI-resistant breast cancer cell lines. We believe that in addition to estrogen-modulation of gene expression, hormone-regulated microRNAs may provide an additional level of post-transcriptional regulation of signaling pathways critically involved in breast cancer progression and AI-resistance.