Luminal Breast Cancer Models Research Articles

Evolution of Cancer Stem-like Cells in Endocrine-Resistant Metastatic Breast Cancers Is Mediated by Stromal Microvesicles.

The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ERlo/Notchhi feed-forward loop responsible for the generation of CD133hi CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6-pStat3 pathway promoted the biogenesis of onco-miR-221hi CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133hi cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133hi CSCs. Conversely, in models of CD133neg, HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133hi CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927-41. ©2017 AACR.

Les inhibiteurs du cycle cellulaire et cancer du sein hormonodépendant

Dysregulation of cellular cycle is a key component of carcinogenesis and its targeting represents an interesting approach. Recently, the development of selective inhibitors of the cycle targeting the cyclin-dependent kinases (CDK) 4and 6revived interest in this therapeutic class after the failure of pan-inhibitors. Palbociclib, ribociclib, and abemaciclib are the 3drugs with the most advanced development. They demonstrated preclinical activity in luminal breast cancer models and are under clinical evaluation. The first available studies demonstrate the value of these compounds with an improved prognosis of metastatic patients in combination with endocrine therapy (palbociclib, ribociclib) or in monotherapy (abemaciclib). The results of ongoing studies will clarify the role of these agents in our new strategies and the individualisation of biomarkers will help to define patients who benefit most from this approach.

β-Catenin haploinsufficiency promotes mammary tumorigenesis in an ErbB2-positive basal breast cancer model

Aberrant activation of β-catenin through its activity as a transcription factor has been observed in a large proportion of human malignancies. Despite the improved understanding of the β-catenin signaling pathway over the past three decades, attempts to develop therapies targeting β-catenin remain challenging, and none of these targeted therapies have advanced to the clinic. In this study, we show that part of the challenge in antagonizing β-catenin is caused by its dual functionality as a cell adhesion molecule and a signaling molecule. In a mouse model of basal ErbB2 receptor tyrosine kinase 2 (ErbB2)-positive breast cancer (ErbB2KI), which exhibits aberrant β-catenin nuclear signaling, β-catenin haploinsufficiency induced aggressive tumor formation and metastasis by promoting the disruption of adherens junctions, dedifferentiation, and an epithelial to mesenchymal transition (EMT) transcriptional program. In contrast to the accelerated tumor onset observed in the haploid-insufficient ErbB2 tumors, deletion of both β-catenin alleles in the ErbB2KI model had only a minor impact on tumor onset that further correlated with the retention of normal adherens junctions. We further showed that retention of adherens junctional integrity was caused by the up-regulation of the closely related family member plakoglobin (γ-catenin) that maintained both adherens junctions and the activation of Wnt target genes. In contrast to the ErbB2KI basal tumor model, modulation of β-catenin levels had no appreciable impact on tumor onset in an ErbB2-driven model of luminal breast cancer [murine mammary tumor virus promoter (MMTV-NIC)]. These observations argue that the balance of junctional and nuclear β-catenin activity has a profound impact on tumor progression in this basal model of ErbB2-positive breast cancer.

Abstract IA07: Systemic regulation of breast cancer progression

Abstract Recurrent disease affects approximately 30% of breast cancer patients but, unfortunately, by the time overt metastases are detected, it is no longer possible to treat breast cancer patients with curative intent. Clinical evidence indicates that in women who are diagnosed with metastatic breast cancer, tumor cells disseminate from the primary tumor prior to surgical resection but can remain indolent for varying periods of time before the patient becomes symptomatic. The reasons why certain disseminated cells remain indolent while others erupt into malignant tumors remain unknown; it is therefore currently difficult to predict with any degree of accuracy which patients will experience disease relapse. Our lab uses various mouse and xenograft models of breast cancer progression to study the early phases of metastatic disease when patients harbor indolent disseminated tumor cells in the periphery at the time of their primary diagnosis. Using triple-negative models, we previously showed that certain tumor systemically support the outgrowth of distant, otherwise indolent disseminated tumors, doing so by secreting cytokines into the host circulation and activating bone marrow hematopoietic cells to become pro-tumorigenic (McAllister, et al., Cell, 2008; Elkabets, et al., JCI, 2011; Castano, et al, Ca Disc, 2013). Using models of luminal breast cancer, we discovered that certain tumors promote the outgrowth of distant tumors by supporting angiogenesis (Kuznetsov, et al., Ca Disc, 2012). More recently, we have begun to identify the properties of disseminated tumor cells that are poised to respond to systemic factors and importantly, the phenotype of those that do not respond to systemic regulation. We have learned that certain systemic factors (cytokines and hematopoietic cells) elicited by primary breast tumors can sustain tumor-initiating properties of disseminated cells. Our findings highlight the systemic environment as an important component of disease progression that can be exploited to more accurately identify patients who would benefit from neoadjuvant or adjuvant therapy. Moreover, we hope that this work will pave the way to designing new therapies that can be delivered at a time when what has previously been an inexorable progression to terminal metastatic disease may be prevented. Citation Format: Sandra S. McAllister. Systemic regulation of breast cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA07.

Abstract A063: Cancer cell expression of CCR2 regulates the PD-L1/ PD-1 immune checkpoint in breast cancer

Abstract C-C chemokine receptor type 2 (CCR2) is highly expressed in human breast cancer tissue by both cancer cells and infiltrating immune cells. High expression of CCR2 is correlated with advanced tumor stage, metastasis, and early relapse. Studies in mice have suggested that CCR2 expression on infiltrating inflammatory monocytes promotes primary tumor growth and metastasis. However, the role of CCR2 expression on cancer cells has not been well studied. To determine how CCR2 directly impacts tumor growth and metastasis, we used a murine model of luminal breast cancer, the mouse mammary tumor virus (MMTV) promoter polyoma middle T antigen (PyMT) model. We crossed MMTV-PyMT mice to Ccr2-/- mice and found that loss of CCR2 did not alter tumor onset. However, primary tumors of MMTV-PyMT;Ccr2+/+ mice grew significantly faster than tumors of MMTV-PyMT;Ccr2-/- mice. Lung metastases were also larger in MMTV-PyMT;Ccr2+/+ mice than in MMTV-PyMT;Ccr2-/- mice. We determined by RNA in situ hybridization that CCR2 was indeed expressed by both host cells and cancer cells in MMTV-PyMT tumors. To differentiate between the contributions of CCR2 expression in the tumor and host, we transplanted primary cancer cells derived from MMTV-PyMT;Ccr2+/+ and MMTV-PyMT;Ccr2-/- mice to syngeneic Ccr2+/+ and Ccr2-/- hosts. Surprisingly, CCR2 expression in the host had no significant effect on tumor growth. However, deletion of CCR2 from cancer cells resulted in delayed tumor growth and increased survival. Furthermore, when Ccr2+/+ and Ccr2-/- cancer cells were transplanted to athymic nude mice (lacking T cells), the resulting tumors no longer showed a delayed growth. Consistent with this finding, flow cytometry of infiltrating leukocytes in tumors transplanted to syngeneic mice revealed increased levels of CD8+ cytotoxic T cells and antigen-presenting CD103+ dendritic cells into Ccr2-/- tumors compared to wild type tumors. These data suggest that CCR2 expression on cancer cells regulates an immune response to the tumors. Supporting this conclusion, expression of programmed cell death ligand 1 (PD-L1) on Ccr2-/- cancer cells was reduced to just 30% of the level on wild type cancer cells, indicating that CCR2 expression on cancer cells suppresses the anti-tumor immune response. Together, our results establish a novel role for cancer cell CCR2 expression in breast cancer progression by suppression of cytotoxic T cells through an immune checkpoint. Citation Format: Miriam R. Fein, Ana S. Almeida, Anaïs Eberhardt, Mikala Egeblad. Cancer cell expression of CCR2 regulates the PD-L1/ PD-1 immune checkpoint in breast cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A063.

ELF5 Drives Lung Metastasis in Luminal Breast Cancer through Recruitment of Gr1+ CD11b+ Myeloid-Derived Suppressor Cells.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis–free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

CCL5-Mediated Th2 Immune Polarization Promotes Metastasis in Luminal Breast Cancer.

The tumor-promoting chemokine CCL5 has been implicated in malignant transformation of breast epithelial cells, with studies to date focusing mainly on basal-type breast cancers. In this study, we investigated the consequences of CCL5 deletion in the MMTV-PyMT transgenic mouse model of luminal breast cancer. In this model, primary tumor burden and pulmonary metastases were reduced significantly in CCL5-deficient subjects, an effect found to be associated with a deficit of Th2 (IL4⁺CD4⁺ T) cells. Mechanistic investigations revealed that CCL5 activates CCR3, a highly expressed chemokine receptor on CD4⁺ T cells, and also boosts Gfi1 expression to promote the differentiation of Th2 cells, which enhance the prometastatic activity of tumor-associated myeloid cells. Clinically, polarization toward this immunosuppressive Th2 phenotype was also evident in patients with advanced luminal breast cancer. Thus, our findings showed that CCL5/CCR3 signaling promotes metastasis by inducing Th2 polarization of CD4⁺ T cells, with implications for prognosis and immunotherapy of luminal breast cancer.

Abstract 1322: Novel CDK4/6 inhibitors provide robust antitumor activity in a murine model of luminal breast cancer

Abstract Introduction: Selective CDK4/6 inhibitors have emerged as attractive antineoplastic agents due to the importance of CDK4/6 activity in regulating cell proliferation. We have identified a series of orally available, potent and selective dual CDK4/6 inhibitors (G1T100182, G1T100183, G1T101551). Preclinical characterization of these compounds indicates that they inhibit CDK4 and CDK6 with an IC50 of 0.7-1.0 nM and 5-6 nM, respectively. In Rb competent tumor cells, these compounds potently inhibit Rb phosphorylation resulting in a G1 arrest. Here we assess the in vivo efficacy of these compounds in a genetically engineered mouse model of luminal breast cancer. Methods: MMTV-Neu mice were housed and therapeutic response assessed in the UNC Lineberger Comprehensive Cancer Center's Mouse Phase I Unit (MP1U). Compounds were supplied by G1 Therapeutics, Inc. Tumors were serially assessed weekly using caliper measurements. Therapeutic intervention began once tumors reached 40-64mm3 and continued for 28 consecutive days. Tumor volume was calculated using the formula ((Width2) x Length)/2). All compounds were administered orally via medicated diets. RECIST criteria were used to assess objective response rates. Results: Significant objective responses were noted in mice treated with G1T100182, G1T100183, G1T101551. Treatments produced modest reversible myelosuppression, but where otherwise well tolerated with no other clinical signs of toxicity or deaths associated with toxicity. A linear regression t-test was used to determine statistical significance of tumor volume changes over time. All treatment cohorts demonstrated statistically significant improvement when compared to un-treated mice; G1T100182, p<0.0001; G1T100183, p=0.0001; G1T101551, p<0.0001. Conclusions: In summary, we have identified a series of orally active, potent, and selective small molecule inhibitors of CDK4/6 that may provide therapeutic benefit to cancer patients with tumors that have functional Rb protein. Complete ResponsePartial ResponseStable DiseaseProgressive DiseaseORR % (CR+PR+SD/PD)Mean % Change in Tumor VolNo Treatment00090 (0/9)500G1T1001821510100 (7/0)-70G1T100183141185 (6/1)-67G1T1015511250100 (8/0)-40 Citation Format: Patrick J. Roberts, David B. Darr, John E. Bisi, Jay C. Strum. Novel CDK4/6 inhibitors provide robust antitumor activity in a murine model of luminal 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 1322. doi:10.1158/1538-7445.AM2014-1322

Flaxseed oil enhances the effectiveness of trastuzumab in reducing the growth of HER2-overexpressing human breast tumors (BT-474)

Flaxseed oil (FSO) reduces breast tumorigenesis and HER2 expression in animal models of luminal breast cancer. The primary treatment for HER2-overexpressing tumors is trastuzumab (TRAS). We aimed to determine the effect of 4% FSO alone and combined with TRAS on HER2-overexpressing tumor (BT-474) growth and to explore potential mechanisms with a specific focus on HER2, mitogen-activated protein kinase (MAPK) and Akt signaling and fatty acid profile. Athymic mice with established tumors were fed the basal diet (control) or 4% FSO diet, with or without TRAS (1 or 2.5mg/kg) treatment for 4weeks. Tumor growth, HER2 signaling biomarkers (mRNA and protein) and fatty acid profile were measured. Tumors treated with FSO alone showed no difference in tumor growth compared to control; however, compared to TRAS2.5 and other groups, FSO+TRAS2.5 caused significantly lower tumor growth and cell proliferation and higher apoptosis and the greatest lowering of signaling biomarker expressions (MAPK2, HER2 mRNA; pHER2 protein). Both TRAS and FSO had main effects of reducing the phosphorylated/total expression of Akt and MAPK protein expression. Dietary FSO altered the tumor fatty acid profile. In conclusion, 4% dietary FSO alone does not affect BT-474 tumor growth but enhances the tumor-reducing effect of TRAS (2.5mg/kg). FSO×TRAS interactive effect may be modulated by their combined reductions of HER2 signaling through the Akt and MAPK pathways leading to reduced cell proliferation and increased apoptosis. FSO alters tumor fatty acid profile that likely contributes to effects on signaling pathways. This supports FSO as a complementary treatment for HER2+ breast cancer treated with TRAS.

Acquired resistance to endocrine treatments is associated with tumor-specific molecular changes in patient-derived luminal breast cancer xenografts.

Patients with luminal breast cancer (LBC) often become endocrine resistant over time. We investigated the molecular changes associated with acquired hormonoresistances in patient-derived xenografts of LBC. Two LBC xenografts (HBCx22 and HBCx34) were treated with different endocrine treatments (ET) to obtain xenografts with acquired resistances to tamoxifen (TamR) and ovariectomy (OvaR). PI3K pathway activation was analyzed by Western blot analysis and IHC and responses to ET combined to everolimus were investigated in vivo. Gene expression analyses were performed by RT-PCR and Affymetrix arrays. HBCx22 TamR xenograft was cross-resistant to several hormonotherapies, whereas HBCx22 OvaR and HBCx34 TamR exhibited a treatment-specific resistance profile. PI3K pathway was similarly activated in parental and resistant xenografts but the addition of everolimus did not restore the response to tamoxifen in TamR xenografts. In contrast, the combination of fulvestrant and everolimus induced tumor regression in vivo in HBCx34 TamR, where we found a cross-talk between the estrogen receptor (ER) and PI3K pathways. Expression of several ER-controlled genes and ER coregulators was significantly changed in both TamR and OvaR tumors, indicating impaired ER transcriptional activity. Expression changes associated with hormonoresistance were both tumor and treatment specific and were enriched for genes involved in cell growth, cell death, and cell survival. PDX models of LBC with acquired resistance to endocrine therapies show a great diversity of resistance phenotype, associated with specific deregulations of ER-mediated gene transcription. These models offer a tool for developing anticancer therapies and to investigate the dynamics of resistance emerging during pharmacologic interventions. Clin Cancer Res; 20(16); 4314-25. ©2014 AACR.

Isolation and Immortalization of Fibroblasts from Different Tumoral Stages

Tumour microenvironment and cancer-associated fibroblasts in particular exhibit tumour promoting abilities that are not present in their normal counterparts (Calvo et al., 2013; Hanahan and Coussens, 2012). Therefore, functional and molecular characterization of the modifications occurring in fibroblasts during tumour progression is essential to fully understand their role in tumour progression. Previous studies have addressed this issue using human fibroblasts and comparing normal and adjacent fibroblasts to tumour-associated fibroblasts (Kalluri and Zeisberg, 2006). However, these studies are hampered by the intrinsic variability of human samples (e.g. pairing, age, genomic landscape, etc). In order to overcome these issues, we used a fully characterised mouse breast cancer model, MMTV-PyMT (Guy et al., 1992; Lin et al., 2003). MMTV-PyMT transgenic mice express the Polyoma Virus middle T antigen under the direction of the mouse mammary tumor virus promoter/enhancer. This is a multifocal luminal breast cancer model that goes through well defined and characterised stages (namely, hyperplasia, adenoma, carcinoma and invasive carcinoma). Interestingly, this model has a 100% incidence, is very desmoplastic (presenting high concentration of fibroblasts) and gives raise to spontaneous metastasis in the lung with 80-94% incidence. Importantly, at least for the inguinal mammary glands (glands 4 and 9), the different tumoral stages are well correlated to the age of the mouse: hyperplasia arising at 6 weeks of age, adenoma between 6-8 weeks of age, carcinoma and invasive carcinoma from 8 weeks onwards. This model allowed us to confidently isolate fibroblasts from different tumoral stages and carefully characterise their functional and molecular properties (Calvo et al., 2013).

Abstract P5-04-05: Preclinical modeling of luminal breast cancer: Recapitulating progression to lethal and tamoxifen-resistant lung metastases in novel patient-derived xenotransplant models in prolactin-humanized mice

Abstract Seventy to eighty percent of newly diagnosed breast cancer cases are estrogen receptor(ER)-positive and are classified as luminal. Despite hormone therapy, 25-30% of luminal breast cancers will recur within 15 years of surgical removal of the primary tumor, and many of these patients will die from currently incurable distant metastases. Lung and bone are the most common organ sites for distant breast cancer metastases, with either site affected in approximately 70% of patients based on autopsy studies. A major hurdle for therapeutic progress with luminal breast cancer is the historical difficulty to establish xenograft models of human luminal breast cancer in mice. In particular, there is a need for experimental ER-positive breast cancer models that recapitulate distant metastasis formation from orthotopic tumor implants in mammary glands. Such experimental models will allow surgical resection of the primary tumor followed by clinically relevant testing of targeted adjuvant agents against distant residual disease. We have engineered prolactin-humanized mice that have been backcrossed for ten generations into the Nod-SCOD-IL2Rgamma (NSG) immunodeficient background. These prolactin-humanized mice display improved take rates of patient-derived luminal breast cancer. Using prolactin-humanized mice we have established patient-derived serially transplantable luminal breast cancer models that metastasize to distant sites when grown as primary tumors in the mammary gland. One of the ER-positive lines, PDX2, effectively metastasizes to lungs in 100% of animals within 55 days of grafting into mammary glands. PDX2 lung metastases retain ER and progesterone receptor (PR) expression as well as expression of the luminal marker, GATA3, and display high degree of Ki67 positivity indicating rapidly proliferative lesions. Importantly, metastatic PDX2 lesions show only limited dormancy. In fact, when primary PDX2 tumors are surgically removed at Day 55, mice will die from lung metastases around Day 150. Estrogen supplementation is required for establishment of PDX2 tumors in mice. After primary PDX2 tumors are established in mammary glands in the presence of estradiol, primary tumors respond to tamoxifen with growth suppression but do not undergo tumor regression, and develop resistance to tamoxifen. Treatment of mice with adjuvant tamoxifen following surgical removal of primary PDX2 tumors led to extensive regression of existing lung metastases to barely detectable levels within 30 days of surgery. However, tamoxifen-refractory PDX2 lung metastases regrew during the next 30 day-period in the continued exposure to tamoxifen. Molecular phenotyping of the PDX2 model and other new luminal breast cancer models are ongoing, with the goal of characterizing tamoxifen-responsive and tamoxifen-refractory primary and metastatic lesions. Long-term, our intent is to use the PDX2 and other preclinical xenograft models in prolactin-humanized mice to systematically explore agents for synergy with anti-estrogens to establish curative combination treatments for metastatic luminal breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-05.

Abstract P5-09-07: Identification of resistance-specific gene expression signatures in a breast cancer patient-derived xenograft with acquired resistance to different endocrine therapies

Abstract Background: Acquired resistance to endocrine treatments (ET) occurs in more than 70% of cases of luminal breast cancer (LBC). We used patient derived xenografts (PDX) models of LBC to study molecular changes associated with acquired resistance to different ET modalities. Methods: A PDX model of LBC, established from an early stage BRCA2-mutated breast cancer, was treated with different ET (tamoxifen, fulvestrant, oophorectomy and letrozole) during several months. Tumors escaping to therapies were re-engrafted and maintained under therapy. ET-resistant and parental hormono-responders tumors were analyzed with immunohistochemistry (IHC), RT-PCR and Affymetrix Gene Expression Arrays. Hormono-resistant tumors were additionally studied for their in vivo response to ET, mTOR and PARP inhibitors. Results: From the initially ET sensitive HBCx22 xenograft model (Cottu, BCRT 2012) two resistant models were obtained respectively to tamoxifen (HBCx22-TamR) and to estrogen deprivation (HBCx22-OvaR). Unsupervised clustering of gene expression showed a clear cut separation between parental, TamR and OvaR tumors. Genes differentially expressed in TamR and OvaR tumors compared to parental HBCx22 were only partially overlapping. Up-Regulated genes in both TamR and OvaR tumors (n = 302) were involved in response to wounding, nucleotide metabolism, immune system, adhesion and cell growth. Biological Processes (BP) specifically deregulated in OvaR tumors (n = 380) included embryonic development, antigen presentation, amino acid and lipid metabolism. The top BP specifically regulated in TamR tumors (n = 1059) were response to estrogen and steroid hormones, TGF-b signaling, hypoxia, regulation of cell proliferation, with several strongly up-regulated genes of the histone clusters 1 and 3. Ingenuity Transcription Factor Analysis predicted activation of NFKB, SP1, AP-1 and JUN, and inhibition of ESR1. RT-PCR and IHC analyses confirmed the down regulation of ER controlled genes in the TamR tumors. Expression of ER co-regulators determined by RT-PCR showed that GREB1 was strongly reduced in TamR, while PBX1, GATA3 and FOXA1 were inhibited in OvaR. IHC analysis showed a loss of PTEN expression in HBCx22, with high levels of p-AKT and p-RPS6 in both parental and TamR and OvaR tumors. In vivo ET showed that the TamR xenograft was resistant to all modalities of ET, while OvaR was resistant to estrogen deprivation while retaining some sensitivity to tamoxifen and fulvestrant. Treatment with the mTOR inhibitor RAD001 arrested tumor growth but did not show any additive effect when combined to ET in TamR or OvaR tumors. Conversely, the combination of RAD001 with Olaparib was highly synergistic and induced complete tumor response in 70% of mice. Conclusions: According to the therapeutic selection, tumors derived from a PDX model of ER+ breast cancer show specific resistance patterns and gene expression profiles including disruption in the ER transcriptional program. The analysis of additional resistant tumors established from a second ER+ PDX will be presented at the meeting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-07.

Abstract A03: Tumorigenesis involving expansion of CD24 high CD61 cells is abrogated by tamoxifen in mouse models of luminal breast cancer

Abstract The cells within the mammary gland that are altered by tamoxifen chemoprevention and correlate with abrogation of tumorigenesis are unknown. We have documented that a 60 day tamoxifen chemopreventive regimen reduces tumor incidence in NRL-TGFalpha and MMTV-c-neu mouse models of luminal breast cancer. For this study our goal was to identify the aberrant and/or altered mammary cell subpopulations typically found in the mammary gland of TGFalpha and c-neu mice and determine the effect of tamoxifen on their relative quantity. Mammary cell subpopulations were evaluated using flow cytometric analysis by labeling lineage negative cells with CD24, CD61 and CD49f fluorescently tagged antibodies. TGFalpha mice developed an expanded CD24high CD61positive fraction of cells coinciding with hyperplasia and tumor presence. Prior to tumor development c-neu mice had an expanded CD24medium CD49f high cell fraction and by the time tumors developed, both nontumorous glands and tumors also displayed an expanded CD24high CD61positive cell fraction. Mammary glands from mice that received a 60 day tamoxifen chemopreventive regimen did not display expanded cell fractions compared to placebo treated mice in both models. Our data indicate that tamoxifen chemoprevention abrogates tumorigenesis that involves expansion of CD24high CD61positive cells. Further investigations into tamoxifen effects on mammary cell subpopulations is critical for a clearer understanding of the utility of tamoxifen and other estrogen receptor modulators as chemopreventives. Citation Format: Olga Zhdankin, Melanie Bomier, Donna Parke, Jon Holy, Teresa Rose-Hellekant. Tumorigenesis involving expansion of CD24 high CD61 cells is abrogated by tamoxifen in mouse models of luminal breast cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A03.

Abstract C174: Establishment of luminal breast cancer patient-derived xenografts with acquired resistance to both hormonotherapy and everolimus.

Abstract The addition of everolimus (Rad) to endocrine therapies (ET) in luminal advanced breast cancers has shown to increase overall survival in randomized studies. However drug resistance is an emerging problem and most patients eventually experience progression of disease. We aimed to establish and characterize patient-derived xenografts (PDX) of luminal breast cancers with acquired resistance in vivo to both ET and Rad. Two PDX xenografts (HBCx22 and HBCx34), established from early stage ER+ breast cancers, were treated in vivo with different ET (tamoxifen, fulvestrant, ovariectomy and letrozole) during 12 months and tumors escaping to therapies were re-engrafted and maintained under treatment. Four hormono-resistant (HR) xenografts were established, 2 resistant to Tamoxifen (HBCx22TamR and HBCx34TamR) and 2 resistant to ovariectomy (HBCx22OvaR and HBCx34OvaR). Expression of estrogen receptor (ER) was maintained, while expression of several ER-controlled genes (MYB, PS2 and PR) was decreased in HR tumors indicating modifications of ER transcriptional activity. The expression of ER co-regulators was additionally studied: GREB1 was reduced specifically in TamR, while FOXA1 were increased specifically in TamR. Expression analysis of P-AKT, P-mTOR and P-S6 showed similar levels of PI3 kinase pathway activation between parental and HR xenografts. Gene expression profiling, performed with Affymetrix Gene Expression Arrays, showed that genes differentially expressed in TamR and OvaR tumors were only partially overlapping, suggesting treatment-specific mechanisms of hormono-resistance. In addition, TamR and OvaR signatures were tumor-specific. Gene ontology enrichment and pathway analyses of gene expression datasets will be presented at the meeting. HR xenografts were then treated with Rad alone and combined to ET. In HBCx22OvaR and TamR, resistant to all ET, treatment with Rad arrested tumor growth in vivo but did not show any additive effect when combined to ET. Conversely, the HBCx34TamR model was still responding to fulvestrant, and the combination Rad+Fulvestrant resulted in tumor regressions. In the HBCx34TamR xenograft, tumor escape to Rad treatment occurred in one mouse after 8 months of continuous treatment. The tumor was re-engrafted and maintained as tumorgraft resistant to both tamoxifen and Rad (HBCx34Tam-RadR). Western blot and IHC studies showed that mTOR signaling was still inhibited in this tumor, but it was associated to MAP kinase activation and increased IGF-1R expression. In conclusion, we have developed PDX models of luminal breast cancer with cross-resistance to ET and everolimus in vivo. These models provide a valuable preclinical tool to investigate molecular mechanisms of acquired resistance and to test new targeted therapies and new combinations in patients’ tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C174. Citation Format: Rania El Botty, Paul Cottu, Franck Assayag, Pierre de la Grange, David Gentien, Aurélie Thuleau, Sophie Vacher, Sophie Chateau-Joubert, Ivan Bièche, Elisabetta Marangoni. Establishment of luminal breast cancer patient-derived xenografts with acquired resistance to both hormonotherapy and everolimus. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C174.

Abstract B004: Expansion of lineage negative, CD24high, CD61+ mammary cells is an early step in mammary tumorigenesis in NRL-TGFalpha and MMTV-c-neu but not MMTV-c-myc models of luminal breast cancer

Abstract Our goal was to define the balance of normal, healthy mammary epithelial cell subpopulations, including putative stem, progenitor and mature cells, in mouse models of luminal breast cancer prior to tumor development. We isolated mammary cells using flow cytometric analysis, first by removing immune cells through negative lineage selection followed by labeling of remaining cells with CD24, CD61 and CD49f fluorescently tagged antibodies. We distinguished the presence of cell subpopulations and determined relative fractions of cells with expression patterns of enriched presumptive luminal progenitor cells (CD24 high, CD61positive), mature luminal cells (CD24high, CD61positive) and mammary stem cells (CD24 medium, CD49f high). Mammary glands from 3-4 month TGFalpha mice contained similar fractions of mammary cell subpopulations as wild type mice but at 7-8 month TGFalpha mice had an expanded CD24 high CD61positive fraction associated with hyperplasias and tumors. Four-month-old c-neu mice had expanded CD24 medium, CD49f high cell fractions and by the time tumors developed, both nontumorous glands and tumors also displayed an expanded CD24 high, CD61+ cell fraction. The cell surface marker expression pattern of mammary cells was unique in c-myc mice in that CD24 high (luminal) versus CD24 medium (myoepithelial) populations were not distinguishable at any time point. Our results indicate that early phases of tumorigenesis can be recognized by the disproportion presence of otherwise “normal” epithelial cell subpopulations or by the presence of novel subpopulations. Citation Format: Olga Zhdankin, Melanie Bomier, Donna Parke, Jon Holy, Teresa Rose-Hellekant. Expansion of lineage negative, CD24high, CD61+ mammary cells is an early step in mammary tumorigenesis in NRL-TGFalpha and MMTV-c-neu but not MMTV-c-myc models of luminal breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B004.

Abstract A005: MMP FRET ACPP imaging of mammary tumors in a mouse model of postmenopausal obesity

Abstract Obesity in postmenopausal women is a risk factor for breast cancer. Since 25-30% of women in the USA are obese, the public health implications of obesity and breast cancer are immense. Activation of inflammatory pathways is one plausible mechanism underlying the association between obesity and increased breast cancer risk. We have used a novel immunocompetent mouse model of luminal breast cancer to demonstrate that luminal tumor progression is enhanced in ovariectomized mice fed a high fat diet (HFD). The obese mammary fat pads are proinflammatory as measured by an increase in M1 polarized macrophages, increased TNF-α production and increased matrix metalloproteinase (MMP)-2 and -9 expression. To evaluate activatable cell penetrating peptide (ACPP) targeting of mammary tumors in the obese fat pad we utilized a version of the MMP cleavable ACPP that has a Cy5 fluorophore attached to the polycationic domain of the peptide and a Cy7 conjugated to the polyanionic domain. Upon cleavage of the peptide by MMPs, the fluorescence resonance energy transfer (FRET) between the two dyes is disrupted and the Cy5 emission increases while the Cy7 re-emission diminishes. This change in emission spectra is measured and the ratio of Cy5 to Cy7 fluorescence is used as a measure of peptide cleavage. The ACPP was administered at a dose of 0.4nmol/g and allowed to circulate for 2 hours prior to harvesting of the fat pads and imaging. The Cy5/Cy7 ratio was 24% higher in the obese fat pads, with a 45% increase in fluorescence when examining just the Cy5 emission. To determine if tumors could be identified in the obese fat pads with the higher background resulting from increased macrophage recruitment and MMP activity, normal chow (NC) and HFD fed C57Bl/6 mice were injected with syngeneic luminal Py230 and claudin low EMT Py8119 cell lines derived from spontaneous polyomavirus middle T (PyVmT) tumor homogenates. Mice harboring both Py230 and Py8119 tumors in their thoracic mammary fat pads were dosed with either the MMP cleavable FRET ACPP or an uncleavable control ACPP. Two hours after peptide injection, mice were sacrificed and the skin was removed to image the tumors in the context of the fat pad. In concordance with our previous results, tumor growth of the Py230 cells was significantly greater in the HFD mice. Interestingly, growth of the more aggressive Py8119 tumors was not enhanced in the HFD microenvironment. For both cell lines, the tumors were clearly delineated using the Cy5/Cy7 ratio. The Py8119 tumors had a ratio of 6.1±0.7 in NC mice and 5.4±0.3 in the HFD mice, and a similar trend was observed in the less aggressive luminal Py230 tumors, with ratios of 4.9±0.8 for NC and 3.8±0.4 for HFD fed mice. This was consistent with analysis showing increased macrophage recruitment in the Py8119 tumors compared with the Py230 tumors, but no difference in recruitment between tumors of either type in mice fed NC or HFD. Our results indicate that although inflammation associated with obesity results in higher background fluorescence in the mammary fat pad, this is not a barrier to visualizing the tumor tissue in the fat pad. Since a large amount of adipose tissue can be problematic during surgery, MMP FRET ACPPs may be useful for identifying and resecting breast cancers in obese patients leading to improved survival. Citation Format: Jessica L. Crisp, Elamprakash N. Savariar, Jonathan P. Hasselmann, Heekyung Chung, Roger Y. Tsien, Lesley G. Ellies. MMP FRET ACPP imaging of mammary tumors in a mouse model of postmenopausal obesity. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A005.

Abstract IA24: How tumors regulate their microenvironment in primary sites and metastasis

Abstract IA24: How tumors regulate their microenvironment in primary sites and metastasis

PIK3CA and AKT1 mutations have distinct effects on sensitivity to targeted pathway inhibitors in an isogenic luminal breast cancer model system.

Activating mutations in the phosphoinositide-3-kinase (PI3K)/AKT/mTOR pathway are present in the majority of breast cancers and therefore are a major focus of drug development and clinical trials. Pathway mutations have been proposed as predictive biomarkers for efficacy of PI3K-targeted therapies. However, the precise contribution of distinct PI3K pathway mutations to drug sensitivity is unknown. We describe the creation of a physiologic human luminal breast cancer cell line model to study the phenotype of these mutations using the MCF-7 cell line. We used somatic cell gene targeting to "correct" PIK3CA E545K-mutant alleles in MCF-7 cells to wild-type sequence. The AKT1 E17K hotspot mutation was knocked in on this wild-type background. Loss of mutant PIK3CA dramatically reduced phosphorylation of AKT proteins and several known AKT targets, but other AKT target proteins and downstream effectors of mTOR were not affected. PIK3CA wild-type cells exhibited reduced proliferation in vitro and in vivo. Knockin of the AKT1 E17K hotspot mutation on this PIK3CA wild-type background restored pathway signaling, proliferation, and tumor growth in vivo. PIK3CA, but not AKT1 mutation, increased sensitivity to the PI3K inhibitor GDC-0941 and the allosteric AKT inhibitor MK-2206. AKT1 E17K is a bona fide oncogene in a human luminal breast cancer context. Distinct PI3K pathway mutations confer differential sensitivity to drugs targeting the pathway at different points and by distinct mechanisms. These findings have implications for the use of tumor genome sequencing to assign patients to targeted therapies.

Vandetanib increases the efficacy of tamoxifen in a preclinical luminal breast cancer model

RESULTS: Tumor was distinguishable from normal tissue using both SPY and Pearl systems, with both platforms being able to detect tumor as small as 0.5 mg. Serial surgical resections demonstrated that real-time fluorescence can differentiate subclinical segments of disease. Pathologic examination of samples using histology, Odyssey (LI-COR), and immunohistochemistry confirmed that the bioconjugates were specific for tumor cells and were able to accurately distinguish these malignant areas from normal tissue.