Mammosphere Research Articles

Abstract P5-11-01: Identification of novel ER and ER-NFκB driven stem-like cell populations in ER+ breast cancer

Abstract Approximately 70% of breast cancers are estrogen receptor (ER) positive and can be treated with endocrine therapy. Unfortunately, 30-40% of tumors will recur, often as therapy-resistant metastatic disease. One contributor to disease recurrence is a population of cells with stem-like features (i.e. breast cancer stem cells, BCSCs). Previously, we have shown that positive crosstalk between two critical transcription factors, ER and NFκB, can contribute to the expansion of ER+ BCSCs, as indicated by increased mammosphere (MS) forming efficiency and BCSC markers. NFκB has been previously described as a major player in BCSC biology. However, the role of ER, as well as the interaction between ER and NFκB, in driving BCSCs remains unclear. We found in MS that both ER and NFκB are endogenously active and required for MS formation. To investigate whether ER and NFκB work together or independently, we made dual ERE and NFκB-RE reporter cell lines. We confirmed activation of both ER and NFκB in MS, but with a high degree of heterogeneity as indicated by 4 different cell populations (ERE+/NFκB-RE+, ERE+/NFκB-RE-, ERE-/NFκB-RE+ and ERE-/NFκB-RE-). Interestingly, we found >90% of MS arose from ERE+ cells while NFκB-RE+ cells failed to from MS, suggesting that ER but not NFκB activity was sufficient to initiate MS development. Moreover, ERE+ cells, with or without NFκB-RE activity, formed more secondary MS and expressed higher levels of stem cell-associated genes than ERE- cells, indicating ERE+ cells are more stem-like. To study ER and NFκB function in more detail, we utilized single cell RNA sequencing of MS. We identified 4 clusters with clusters 1 and 2 enriched for stem cell gene signatures. Bioinformatic analysis revealed Cluster 2 to be enriched for ER, proliferation, and DREAM complex signatures, and we confirmed these genes were enriched in ERE+ cells. However, we also found that ER activity is associated with activation of the DREAM complex in standard cell culture. This suggests that ER regulation of proliferation via the DREAM complex may not be specific for BCSCs. In contrast to Cluster 2, Cluster 1 was enriched with ER and NFκB signatures, as well as an ER-NFκB crosstalk signature. In addition, Cluster 1 was enriched with pathways related to adaptive stress response such as: oxidative phosphorylation, unfolded protein response, and protein secretion. As some studies have reported activation of stress pathways is associated with maintenance of stemness, we hypothesize that ER and NFκB may work together to promote stem features through activation of multiple stress pathways to increase survival and growth in an anchorage-independent environment. To determine whether either Cluster 1 or Cluster 2 derived signatures are associated with clinical outcome, we first interrogated single cell RNA sequencing data from a metastatic ER+ PDX model. The Cluster 2 signature was enriched in proliferative cell populations found in both primary and metastatic tumors. In contrast, the Cluster 1 signature was enriched only in a metastatic tumor cell population. We also showed that expression of signatures derived from Cluster 1 or 2 in ER+ breast cancer patient tumors predict poor relapse free survival in the METABRIC dataset. However, only co-expression of both signatures predicted poor disease-free survival. Taken together, our findings suggest there are two unique subpopulations of stem-like cells, one of which is dependent on ER driven proliferation, whereas the other population relies on an ER-NFkB mediated stress response for survival and self-renewal. Furthermore, both of these population signatures in patients’ tumors are associated with more aggressive disease and poor outcome. Citation Format: Svetlana E. Semina, Mark Maienschein-Cline, Elaine T. Alarid, Carol A. Sartorius, Jonna Frasor. Identification of novel ER and ER-NFκB driven stem-like cell populations in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-11-01.

Abstract PS16-04: Estrogen receptor (ER) and NFkB activity determines cancer stem cell properties in ER positive breast cancer

Abstract Approximately 50% of estrogen receptor (ER) positive breast cancer patients will experience relapse. One explanation for this is the presence of breast cancer stem cells (BCSCs), which have self-renewal capability and can contribute to therapy resistance, metastasis, and relapse. Previously, we have shown that cells with active ER and NFκB gain stem cell properties. They are characterized by higher mammosphere (MS) forming efficiency and increased expression of stem-associated markers. NFκB has been previously described as a player in BCSC biology. However, the role of ER remains unclear since some studies have suggested that BCSCs are ER negative. In order to study the role of ER and NFκB in BCSCs in more detail, we utilized single cell RNA sequencing of MS. Dimensionality reduction of single cells identified 4 clusters with unique transcriptional signatures. In order to determine which cluster of these 4 represents BCSCs, we ran functional enrichment analysis (FEA) using MsigDB as a source of various stem cell signatures. Results of FEA showed 1 of 9 stem cell signatures enriched in Cluster 0. Whereas clusters 1, 2 and 3 demonstrated increasing enrichment of different stem cell signatures, respectively. Further analysis of stem cell signatures and genes revealed that cluster 1 included not only classical stem markers (ALDH1A3, KRT8, CD36), but also was enriched for PI3K and p53 signaling pathways. The cluster 2 stem cell signature was associated with DREAM complex activity and BRCA1 mutation. Cluster 3 was enriched for expression of NANOG and SOX2 targets. We also ran FEA to determine ER and NFκB activity in each population. Cluster 0 showed no activity of either ER or NFκB, whereas Cluster 1 was enriched for both, cluster 2 was enriched for ER only, and cluster 3 was enriched for NFκB only. Taken together this data suggests that ER and NFκB may play different roles in promoting BCSC properties. In order to determine how both pathways effect BCSC features, we made a dual reporter cell line expressing ERE-mCherry and NFκB-RE-GFP. It was found ERE activity was constant throughout MS formation, whereas NFκB-RE activity increased proportionally to MS growth over time, suggesting these pathways have different dynamics and further supporting unique roles for each in MS development. To determine which cell population has the capacity to seed MS, an indication of stemness, we sorted cells by reporter activity and tested MS forming efficiency. We discovered that ERE+ cells, either with or without NFκB activity, are more stem-like, whereas activation of NFκB alone is not sufficient to drive MS development. This finding was verified by stem cell-associated gene expression. These data suggest that ER activity is a key driver for BCSC properties and that NFκB may play a supporting role. Furthermore, a novel mechanism of ER regulating the DREAM complex, which is known to be involved in cancer progression and used to evaluate the risk of recurrence in breast cancer, is suggested as a potential ER-mediated mechanism for driving BCSCs. Taken together, our findings suggest novel roles for ER and NFκB in BCSC biology, which could be exploited to target BCSCs therapeutically in ER+ breast cancer patients in order to improve their outcome. Citation Format: Svetlana E. Semina, Irida Kastrati, Luis Alejo Cruz, Tara McCray, Mark Maienschein-Cline, Elaine T. Alarid, Jonna Frasor. Estrogen receptor (ER) and NFkB activity determines cancer stem cell properties in ER positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-04.

ALDH1+ stem cells demonstrate more stem cell-like characteristics than CD44+/CD24-/low stem cells in different molecular subtypes of breast cancer.

BackgroundCD44+/CD24–/low and ALDH1+ are both markers of breast cancer stem cell (BCSC), we compared the biological characteristics of CD44+/CD24–/low and ALDH1+ BCSCs in the four molecular subtypes of breast cancer.MethodsFour fresh blocks of breast cancer tissue were obtained from patients with Luminal A, Luminal B, human epidermal receptor-2 (HER2)-overexpression and triple-negative breast cancer, respectively. Flow cytometry was used to sort CD44+/CD24–/low and ALDH1+ BCSCs. The mammosphere (MS) formation experiment and NOD/SCID mouse xenograft experiment were performed to compare the self-renewal and tumorigenic abilities of CD44+/CD24–/low with those of ALDH1+ BCSCs.ResultsThe proportions of CD44+/CD24–/low BCSC and ALDH1+ BCSC in Luminal A, Luminal B, HER2 overexpression and triple-negative subtypes were 12.1% and 8.7%, 2.7% and 5.7%, 0.8% and 8.7%, 0.7% and 4.5%, respectively. The MS formation experiment demonstrated that ALDH1+ BCSC formed significantly more MSs than CD44+/CD24–/low BCSC in the four molecular subtypes (P<0.001). The NOD/SCID mouse xenograft experiment demonstrated that the sizes of grafted tumors formed by ALDH1+ BCSC were larger than those formed by CD44+/CD24–/low BCSC in the four molecular subtypes (P<0.05).ConclusionsIn the four molecular subtypes of breast cancer, ALDH1+ BCSC demonstrated better self-renewal and tumorigenic abilities than CD44+/CD24–/low BCSC.

Abstract 1853: Ovarian steroid hormones promote progression of DCIS by increasing cancer stem cell self-renewal through IL-6 signaling

Abstract Introduction: The ability of the ovarian steroid hormones estradiol (E) and progesterone (P) to influence cancer stem cell (CSC) self-renewal and progression of human ductal carcinoma in situ (DCIS) is a critical but understudied area or research. The objective of this study is to characterize steroid hormone signaling involved in CSC self-renewal and invasion in ER+PR+ DCIS. We have found that these hormones can increase CSC self-renewal in a subset of DCIS patients, and that this may be facilitated by activation of the IL-6 pathway. Methods: Mammosphere (MS) assays (an in vitro assessment of CSC self-renewal) and the mouse intraductal (MIND) xenograft model were used to characterize hormonal regulation of the CSC population and invasion potential of primary human ER+PR+ DCIS cells. Additionally, ovarectomized mice were used to create MIND xenografts of ER+PR+ DCIS, treated with E+P or vehicle for 8 weeks, and used for RNA sequencing and immunofluorescent staining. Results: Only a subset of cases responded to E+P in vitro by increasing MS efficiency, indicating increased CSC self-renewal. RNA sequencing of MSs identified genes that differed between vehicle and E+P treatment only in DCIS cases that respond to E+P by increasing MS efficiency. Many of these changes were associated with epithelial mesenchymal transition (EMT), including Twist1, Snai1, MMP9. In E+P-treated MIND xenografts, DCIS cases that respond to E+P by increasing MS efficiency also had higher IL-8 and vimentin (markers of EMT) compared to non-responders (P&amp;lt;0.05). This suggests that the cases which responded to E+P by increasing CSC self-renewal may have undergone EMT. Additionally, IL-6 and IL-8 gene expression and protein increased (P&amp;lt;0.05) in a DCIS case which also responded to E+P treatment by increasing MS efficiency. In an ER+PR+ DCIS case which did not increase MS efficiency in response to E+P, IL-6 and IL-8 secretion remained unchanged. Finally, RNA sequencing analysis of E+P vs. control-treated mammary glands from ovarectomized MIND-transplanted mice indicated the IL-6 signaling pathway was one of the top signaling pathways to be activated. Expression of IL-8 and vimentin in E+P compared to control-treated mice also increased (P&amp;lt;0.05). Conclusions: Our results indicate that steroid hormone treatment can enrich the CSC population by increasing MS efficiency in some but not all DCIS cases, and that IL-6 may be involved in this selective ability. Therefore, DCIS cases with higher MS efficiency in response to E+P treatment may also show higher invasion potential in vivo. Our current model is that steroid hormones induce increased secretion of IL-6, leading to increased self-renewal and invasion potential. Further characterization of the signaling pathway(s) associated with this selective effect may allow us to predict which DCIS lesions have the ability to progress to invasive ductal carcinoma and allow development of novel therapeutic targets. Citation Format: Kelli E. Valdez, Yan Hong, Fang Fan, Lisa May, Therese Cusick, Marc Inciardi, Mark Redick, Jason Gatewood, Fariba Behbod. Ovarian steroid hormones promote progression of DCIS by increasing cancer stem cell self-renewal through IL-6 signaling. [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 1853. doi:10.1158/1538-7445.AM2015-1853

Abstract 4086: HMG Box Domain Containing 3 (HMGXB3), a novel TGFβ-induced cancer gene that influences cancer stem cells and metastasis in triple negative breast cancer

Abstract The role of HMGXB3 (HMG Box Domain Containing 3), member of the non-canonical high mobility group (HMG) genes, in cancer remains largely unknown. A preliminary “mutual exclusivity” analysis of the Breast Invasive TCGA database showed a significant association between HMGXB3 and TGFβ (Transforming Growth Factor β). TGFβ plays a significant role in metastasis and increases stem-like properties of breast cancer cells. This work aimed at elucidating whether there is a relationship between HMGXB3 and TGFβ, and their role on breast cancer stem cells (BCSC) and metastasis in triple negative breast cancer (TNBC). Changes in HMGXB3 protein levels (Abgent) were assessed by Western blot in MCF10A cells treated with human recombinant (hr) TGFβ1 (R&amp;D) with/without the TGFβR1 kinase inhibitor, LY2157299. Knockdown was performed in MCF10A and two TNBC cell lines, SUM159 and MDA-MB-231. Cells transfected with two different siRNAs against HMGXB3, siRNA#1 and siRNA#2, and one negative control (siRNA-SCR) were assayed for proliferation as well as primary and secondary mammosphere (MS) formation for 48h with hrTGFβ1 (10ng/ml). Cell migration was determined in transfected TNBC cell lines treated with 10ng/ml hrTGFβ1 for 72h. For in vivo experiments, MDA-MB-231, stably transfected with a luciferase/GFP reporter plasmid, were injected in the right mammary fat pad of female SCID Beige mice (n = 10). Tumor-bearing mice were treated with DOPC (dioleoyl phosphatidylcholine) liposomes loaded with 5μg of either siRNA-SCR or HMGXB3-siRNA#2 with/without docetaxel (20mg/kg) for 21 days. Tumors were digested and cells isolated. Single cells were assayed for aldefluor (ALDF+) by flow cytometry and MS formation. Lung metastases were determined in lungs by ex vivo imaging of luciferase positive cells. Our results show a concentration- and time-dependent induction of HMGXB3 protein levels under treatment with hrTGFβ1 in MCF10A cells. Such an enhancement was efficiently abrogated by the addition of LY2157299. Knockdown of HMGXB3 in presence of hrTGFβ1 significantly reduced cell proliferation, migration and MS formation in the TNBC cell lines tested herein. In vivo experiments demonstrated that silencing of HMGXB3 correlates with reduced tumor growth, ALDF+ BCSC subpopulation, and formation of secondary MS compared to siRNA-SCR. Finally, silencing HMGXB3 significantly abrogated progression of metastasis from primary tumor to lungs in contrast to siRNA-SCR with/without chemotherapy. In conclusion, we are the first to identify HMGXB3 as a TGFβ-induced cancer gene that impacts tumor cell proliferation, migration, BCSC self-renewal and lung metastases in TNBC. Our preliminary results suggest HMGXB3 as a novel target for prevention of metastasis. Future experiments will elucidate the role and mechanism of HMGXB3 in cancer. This work will give rise to novel therapeutic targets for the treatment of breast cancer. Citation Format: Sergio Granados-Principal, Yi Liu, Cristian Rodriguez-Aguayo, Anil K. Sood, Jenny C. Chang. HMG Box Domain Containing 3 (HMGXB3), a novel TGFβ-induced cancer gene that influences cancer stem cells and metastasis in triple negative 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 4086. doi:10.1158/1538-7445.AM2015-4086

Abstract P2-06-02: Breast cancer stem-like cell activity correlates with tumour progression to metastasis but not with clinical or tumour characteristics

Abstract Introduction: Breast cancers exhibit cellular heterogeneity, containing both stem-like and more differentiated cells. The activity of cancer stem cells (CSC) is likely to be dependent on the microenvironment or niche. Using 158 patient tumour samples, correlations between niche-independent breast CSC activity and clinical and tumour characteristics were tested. Methods: 104 early breast cancer surgical samples and 54 unrelated metastatic samples from pleural or ascitic fluid were harvested. To test CSC activity, isolated cells were grown in both primary (formation) and secondary (self-renewal) mammosphere (MS) culture. Tumour initiating activity was also tested by transplanting breast cancer fragments or cells into the sub-cutaneous flanks of NSG mice (n=84 early and n=10 metastatic). Results: No correlation was found between MS growth, MS formation (%), MS self-renewal (%) or in vivo tumour initiation and breast cancer sub-type, grade, node status or Nottingham prognostic index. 33% of the samples that formed MS in vitro initiated tumours in vivo while only 9% that failed to form MS initiated tumour growth. Metastatic compared to early BC samples grew MS more frequently (53/54 compared to 81/104), and had a higher primary MS formation efficiency (1% vs 0.6%; P&amp;lt;0.001) although rates of MS self-renewal were similar. Tumour initiation in vivo was also more frequent in metastatic than early breast cancer samples (7/10 versus 25/84; P&amp;lt;0.02). Conclusions: In summary, niche-independent breast CSC activity measured in vitro by MS assay and in vivo by xenograft growth is not directly correlated with standard clinical parameters. However, both in vitro and in vivo CSC activity are increased in metastatic samples. These results suggest that breast CSC activity is independent of other prognostic indicators but may predict for poor outcome tumours. Relapse free survival data are maturing and will be presented with analysis of primary tumour ALDH1 expression. Citation Format: Sacha J Howell, Denis Alferez, Katherine Spence, Rachel Eyre, Fran Shaw, Bruno Simoes, Angelica Santiago-Gomez, Maria Bramley, Mohamed Absar, Zahida Saad, Sumohan Chatterjee, Cliona Kirwan, Ashu Gandhi, Anne C Armstrong, Andrew M Wardley, Gillian Farnie, Robert B Clarke. Breast cancer stem-like cell activity correlates with tumour progression to metastasis but not with clinical or tumour characteristics [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-02.

Abstract P1-06-05: Expansion of tumor initiating cells is mediated by tumor microenvironment in breast cancer metastasis

Abstract Background: Breast cancer metastasis which ultimately results in breast cancer death, is an event believed to be initiated by the migration of tumor initiating cells (TIC) from the primary tumor to niches for micrometastatic disease. Recent data suggests the tumor microenvironment promotes TIC. The clinical relevance of secreted factors from the microenvironment on TIC surrogate, mammosphere (MS) formation and MS sensitivity to drug therapy was investigated using breast cancer patient fluids inherently conditioned by the tumor microenvironment: post-operative seromas and malignant pleural effusions. Methods: Fluids from 48 patients with breast cancer (15 seromas and 33 pleural effusions) and mesenchymal stem cells (MSC) from healthy donors were collected on IRB approved protocols. Cellular components were eliminated from patient-derived fluids using density-gradient centrifugation. MSC conditioned media (MSC-CM) was collected from 3D cultures of primary MSC. Luminex multiplex array platform was used to characterize 79 cytokine and growth factor components of all fluids. In addition, MSC-CM and patient-derived fluids were added to cultures of breast cancer cell lines: MCF-7, an estrogen receptor (ER)-positive cell line; SUM149, a triple-negative inflammatory breast cancer cell line; and SUM159, a triple-negative metaplastic breast cancer cell line and MS forming efficiency was examined. Results: Our results show that pleural effusions and seromas are enriched for factors also secreted by MSC such as MCP-1, GRO, IL-6, and VEGF-A. We found remarkable similarities regarding the cytokines and growth factors profile in pleural effusions and seromas. Both patient-derived fluids have comparable amount of Angiopoetin-2, Leptin, TNF-beta, VEGF, IL-2, IL-3, IL-4 and IL-10. EGF, TNF-alpha, IL-1, IL-6, IL-8 and IL-16 were significantly different between pleural effusions and seromas. Seroma fluid from bilateral drains in a patient with an invasive cancer and a contralateral benign mastectomy had very similar cytokine concentrations. Moreover, MSC-CM and pleural fluids from ER-positive and ER-negative patients increased the MS formation efficiency of both triple-negative cell lines while seroma fluids from ER-positive and ER-negative patients increased the MS formation efficiency of ER-positive cell line MCF-7. Finally, we evaluated the impact of a panel of drugs (simvastatin, pravastatin and erlotinib) on cell cultures grown with MSC-CM and patient-derived fluids. We found that the effect of chemotherapies on MS formation can be attenuated by patient-derived fluids. Conclusions: Seroma and pleural effusion fluids from breast cancer patients have similar cytokine profiles, change MS formation efficiency of standard breast cancer cell models, and mediate sensitivity to therapy. Here we demonstrate that host and microenvironmental factors are critical for determining resistance to therapy and may be independent of obvious tumor related factors. Future studies will investigate the prognostic implications of factors that promote TIC survival in the fluid tumor microenvironment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-05.

Abstract C41: Ovarian steroid hormones influence progression of premalignant breast cancer by disregulated cancer stem cell self-renewal

Abstract Introduction: Ductal carcinoma in situ (DCIS), the premalignant form of human breast cancer, only progresses to invasive breast cancer (IBC) in an estimated 50% of diagnosed cases. Elucidation of the mechanisms underlying this selective event could prevent unnecessary treatment and increase the accuracy of diagnosis. Progression to an invasive phenotype has been associated with an increase in mammary cancer stem cells (CSCs). The ability of the ovarian steroid hormones estradiol and progesterone to influence mammary stem cell self-renewal has been demonstrated in models of both normal and pathological mammary gland growth. The common theme appears to involve estradiol and progesterone acting on the ER, PR-expressing luminal cells, which in turn through paracrine signaling can induce proliferation and increased self-renewal of both normal and CSC. However, none of these studies have examined the ability of these steroid hormones to influence CSC self-renewal and progression of DCIS. Purpose: The objective of this study is to characterize steroid hormone paracrine signaling involved in aberrant stem cell self-renewal and invasion in human primary DCIS. Our hypothesis is that those DCIS cases which respond to hormonal treatment by increasing CSC self-renewal also have the greatest invasive potential and risk of malignant progression. Experimental design: Mammosphere assays, an in vitro assessment of self-renewal, and the in vivo mouse intraductal (MIND) xenograft model are being used to characterize hormonal regulation of the stem cell population and invasion potential of primary human DCIS cells obtained from patient biopsies. Results: Cells from primary human DCIS, atypical hyperplasia, and DCIS with microinvasion were cultured under non-adherent conditions to allow primary mammosphere (MS) formation in the presence of vehicle, 10 nM estradiol, 100 nM progesterone, or both. After 14 days, cells were passaged to allow secondary MS formation. We found that only a subset of cases responded to the steroid hormone treatment by increasing MS forming efficiency, indicating increased self-renewal ability. Using human growth factor antibody arrays to assay the cell culture media, we found that insulin-like growth factor 2 (IGF2) was selectively up-regulated in response to steroid hormone treatment only in those cases which also demonstrated increased CSC self-renewal. Changes in gene expression in response to treatment are also being examined by next generation RNA sequencing. RNA from vehicle and steroid-treated DCIS cells was isolated from cases that either did or did not respond to E+P treatment. Differential expression analysis was used to identify genes that differed between vehicle and E+P treatment only in DCIS cases that respond to E+P by increasing mammosphere-forming efficiency. Ingenuity Pathway Analysis was used to characterize the biological functions and disease states associated with gene changes observed. The top results included cellular movement, cancer, and reproductive system disease. Many of the gene changes observed were associated with changes that appear during epithelial mesenchymal transition (EMT). This supports our hypothesis that the cases which respond to steroid hormone treatment by increasing stem cell self- renewal may have greater invasive potential. Additionally, MIND xenografts in ovariectomized mice are being created and treated with steroid hormones using cells from these same DCIS cases to determine if these cases also have greater growth and invasion potential in vivo. Differences in gene expression of Wnt4, RankL, FGF9, IGF2, and amphiregulin will also be examined, as these have been shown to be involved in the signaling pathways regulating steroid hormone-induced stem cell proliferation. Conclusions: Our results indicate that steroid hormone treatment can enrich the stem cell population by increasing MS efficiency in some but not all DCIS cases, and that IGF2 may be involved in this selective ability. Based on these data, we expect that DCIS cases with higher MS efficiency in response to steroid hormone treatment will also show higher invasion potential in vivo and in vitro. Further characterization of the signaling pathway(s) associated with this selective effect may allow us to predict which DCIS lesions have the ability to progress to invasive ductal carcinoma. Citation Format: Kelli E. Valdez, Yan Hong, Fang Fan, Therese Cusick, Lisa May, William Smith, Fariba Behbod. Ovarian steroid hormones influence progression of premalignant breast cancer by disregulated cancer stem cell self-renewal. [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 C41.

Abstract B038: Ovarian steroid hormones regulate cancer stem cell self-renewal through aberrant signaling pathways in primary human ductal carcinoma in situ

Abstract Introduction: The ability of the ovarian steroid hormones estradiol and progesterone to influence mammary stem cell self-renewal has been demonstrated in several prominent studies using models of both normal and pathological mammary gland growth. The common theme appears to involve estradiol and progesterone acting on the ER, PR-expressing luminal cells, which in turn through paracrine signaling can induce proliferation and increased self-renewal of both normal and cancer stem cells (CSC). However, none of these studies have examined the ability of these steroid hormones to influence CSC self-renewal and progression of ductal carcinoma in situ (DCIS). Purpose: The objective of this study is to characterize steroid hormone paracrine signaling involved in aberrant stem cell self-renewal and invasion in human primary DCIS. Our hypothesis is that those DCIS cases which respond to hormonal treatment by increasing CSC self-renewal also have the greatest invasive potential and risk of malignant progression. Experimental design: Mammosphere assays, an in vitro assessment of self-renewal, and the in vivo mouse intraductal (MIND) xenograft model are being used to characterize hormonal regulation of the stem cell population and invasion potential of primary human DCIS cells obtained from patient biopsies. Results: Cells from primary human DCIS, atypical hyperplasia, and DCIS with microinvasion were cultured under non-adherent conditions to allow primary mammosphere (MS) formation in the presence of vehicle, 10 nM estradiol, 100 nM progesterone, or both. After 14 days, cells were passaged to allow secondary MS formation. We found that only a subset of cases responded to the steroid hormone treatment by increasing MS forming efficiency, indicating increased self-renewal ability. Using human growth factor antibody arrays to assay the cell culture media, we found that insulin-like growth factor 2 (IGF2) was selectively up-regulated in response to steroid hormone treatment only in those cases which also demonstrated increased CSC self-renewal. Changes in gene expression in response to treatment are also being examined by next generation RNA sequencing. RNA from vehicle and steroid-treated DCIS cells was isolated from cases that either did or did not respond to E+P treatment. Differential expression analysis was used to identify genes that differed between vehicle and E+P treatment only in DCIS cases that respond to E+P by increasing mammosphere-forming efficiency. Ingenuity Pathway Analysis was used to characterize the biological functions and disease states associated with gene changes observed. The top results included cellular movement, cancer, and reproductive system disease. Many of the gene changes observed were associated with changes that appear during epithelial mesenchymal transition (EMT). This supports our hypothesis that the cases which respond to steroid hormone treatment by increasing stem cell self- renewal may have greater invasive potential. Characterization of MIND xenografts created using cells from these same cases will allow us to determine if these cases also have greater growth and invasion potential in vivo. Conclusions: Our results indicate that steroid hormone treatment can enrich the stem cell population by increasing MS efficiency in some but not all DCIS cases, and that IGF2 may be involved in this selective ability. Based on these data, we expect that DCIS cases with higher MS efficiency in response to steroid hormone treatment will also show higher invasion potential in vivo and in vitro. Further characterization of the signaling pathway(s) associated with this selective effect may allow us to predict which DCIS lesions have the ability to progress to invasive ductal carcinoma. Citation Format: Kelli E. Valdez, Yan Hong, Fang Fan, Therese Cusick, Lisa S. May, William P. Smith, Fariba Behbod. Ovarian steroid hormones regulate cancer stem cell self-renewal through aberrant signaling pathways in primary human ductal carcinoma in situ. [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 B038.

Abstract 5210: Impact of tumor microenvironment on expansion of tumor initiating cells in breast cancer metastasis

Abstract Breast cancer metastasis which ultimately results in breast cancer death, is an event believed to be initiated by the migration of tumor initiating cells (TIC) from the primary tumor to niches for micrometastatic disease. Recent data suggests the tumor microenvironment promotes TIC. The clinical relevance of secreted factors from the microenvironment on TIC surrogate, mammosphere (MS) formation and MS sensitivity to drug therapy was investigated using breast cancer patient fluids inherently conditioned by the tumor microenvironment: post-operative seromas and malignant pleural effusions. Fluids from 20 patients with breast cancer (8 seromas and 13 pleural effusions) and mesenchymal stem cells (MSC) from healthy donors were collected on IRB approved protocols. Cellular components were eliminated from patient-derived fluids using density-gradient centrifugation. MSC conditioned media (MSC-CM) was collected from 3D cultures of primary MSC. Luminex multiplex array platform was used to characterize 79 cytokine and growth factor components of all fluids. In addition, MSC-CM and patient-derived fluids were added to cultures of breast cancer cell lines: MCF-7, an estrogen receptor (ER)-positive cell line; SUM149, a triple-negative inflammatory breast cancer cell line; and SUM159, a triple-negative metaplastic breast cancer cell line and MS forming efficiency was examined. Our results show that pleural effusions and seromas are enriched for factors also secreted by MSC such as MCP-1, GRO, IL-6 and IL-8. We found remarkable similarities regarding the cytokines and growth factors profile in pleural effusions and seromas. Both patient-derived fluids have comparable amount of IL-2, IL-3, IL-4 and IL-10, as well as, VEGF, EGF and FGF-b. IL-1β and IL-16 were significantly different between pleural effusions and seromas. Seroma fluid from bilateral drains in a patient with an invasive cancer and a contralateral benign mastectomy had very similar cytokine concentrations. Moreover, MSC-CM and pleural fluids from ER+ and ER- patients increased the MS formation efficiency of both triple-negative cell lines while seroma fluids from ER+ and ER- patients increased the MS formation efficiency of ER-positive cell line MCF-7. Finally, we evaluated the impact of a panel of drugs on cell cultures grown with MSC-CM and patient-derived fluids. We found that the effect of chemotherapies on MS formation can be attenuated by patient-derived fluids. Seroma and pleural effusion fluids from breast cancer patients have similar cytokine profiles, change MS formation efficiency of standard breast cancer cell models, and mediate sensitivity to therapy. Here we demonstrate that host and microenvironmental factors are critical for determining resistance to therapy. Future studies will investigate the prognostic implications of factors that promote TIC survival in the fluid tumor microenvironment. 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 5210. doi:1538-7445.AM2012-5210

P1-03-02: The Norton-Simon Hypothesis and Cancer Stem Cells: How Cancer Stem Cells May Explain the Effectiveness of Dose-Dense Chemotherapy.

Abstract Background: Systemic therapies are effective initially in controlling and reversing tumor growth; however, residual cancers will invariably re-grow despite this initial response. We have published data from paired human breast cancer samples that standard therapy every three weeks kills dividing daughter cells but not tumor-initiating cells (TICs), so that samples obtained after therapy are enriched for CD44+/CD24−/low putative “tumor-initiating” or “cancer stem” cells, indicating that standard treatment regimens are missing the critical targets, TICs. Interestingly, we have recent data in human breast tumors that indicate that TICs may in fact be chemosensitive initially, with a decrease in TICs observed within two days of chemotherapy, but shortly thereafter, TICS are actually induced by chemotherapy. Materials and Methods: To evaluate TICs response to chemotherapy, mice with human breast tumor xenograft lines BCM-2665a and BCM-2147 were treated with vehicle, 10-, or 33-mg/kg docetaxel, and then tumors were collected for TIC assays and molecular analysis at both 48 and 72h after treatment. Using Affymetrix gene expression microarrays and reverse phase protein array (RPPA) analysis with 119 different validated antibodies, we identified pathways involved in regulation of TICs. Results: According to flow cytometric analysis for TIC markers and mammosphere (MS) formation efficiency, BCM-2665 TICs were reduced by docetaxel treatment compared to vehicle-treated at 48h post 10 mg/kg docetaxel (4-fold decrease) and 48 and 72h post 33 mg/kg docetaxel compared to control (14- and 2-fold decrease, respectively). Although the BCM-2147 TICs did not significantly decrease at any time points or doses tested, they were clearly induced at 72h post-treatment compared to control. Additionally, BCM-2665 TICs were increased within 72h post 10 mg/kg docetaxel, indicating that these time points are ideal for defining the mechanisms responsible for induction of TICs. Ingenuity Pathway Analysis of Affymetrix microarray data for both BCM-2665 and BCM-2147 revealed induction of inflammatory pathways, suggesting leukocyte infiltration associated with induction of TICs. Furthermore, RPPA analysis confirmed gene expression changes from the microarray data, and implicated apoptosis and inflammatory pathways. Sixteen of 28 proteins significantly changed with activation of CSC are involved in development of leukocytes. Discussion: These findings are consistent with the Norton-Simon Hypothesis in that chemotherapy regimens given more frequently may in fact eliminate TICs, thereby explaining the proven increased effectiveness of dose-dense chemotherapy. Based on when TICs became chemoresistant, we are comparing dose-dense treatment (4 mg/kg docetaxel every 3 days) to a traditional single dose of 32 mg/kg, in an effort to eliminate the tumor cells that cause tumor recurrence. Furthermore, our analysis of gene expression at both the RNA and protein level implicated the immune cells as TICs inducers. Since our immunocompromised mice lack T- and B- cells but have active macrophages, macrophages are indicated as inducers of TICs. We are focusing our current efforts at identifying how immune cells activate TICs and thus enhance tumorigenesis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-02.

Abstract LB-100: The Norton-Simon hypothesis and cancer stem cells: how cancer stem cells may explain the effectiveness of dose-dense chemotherapy

Abstract Systemic therapies are effective initially in controlling and reversing tumor growth; however, residual cancers will invariably re-grow despite this initial response. We have published data from paired human breast cancer samples that standard therapy every three weeks kills dividing daughter cells but not tumor-initiating cells (TICs), so that samples obtained after therapy are enriched for CD44+/CD24−/low putative “tumor-initiating” or “cancer stem” cells. These data indicate that standard treatment regimens are missing the critical targets, TICS; thus, we are trying to identify novel therapeutic combinations and regimens that will eliminate these TICS that ultimately cause breast tumor recurrence and metastasis. Interestingly, we have recent data in human breast tumors that indicate that TICs may in fact be initially chemosensitive, with a decrease in TICs observed within three days of chemotherapy, but shortly thereafter, TICS are actually induced by chemotherapy. To evaluate TIC response to chemotherapy, mice with xenografted human breast tumors were treated with docetaxel (20 mg/kg) or vehicle, and then tumors were collected and dissociated for TICs assays either 3d or 14d after treatment. According to multiple assays for TICs, TICs were reduced compared to vehicle-treated at 3d post-docetaxel, and increased at 14d. This was evident in both primary and secondary mammosphere (MS) formation. Flow cytometric analyses for TICs markers (CD44+/CD24−/low and ALDH+) further corroborated these data. These results indicate TICs were initially responsive but became chemoresistant, or even activated by chemotherapy. To further define TICs response to chemotherapy, mice with xenografted human breast tumors were treated with vehicle, 10 mg/kg docetaxel, or 33 mg/kg docetaxel, and then tumors were collected and dissociated for TICs assays either 48h or 72h after treatment. Although BCM-2147 TICs did not significantly decrease at any time points or doses tested, they were induced by 72h post-treatment compared to control (ALDH+ 2- to 4-fold increased). In contrast, according to TIC markers and MS formation efficiency, BCM-2665 TICS were reduced by docetaxel compared to vehicle at 48h post 10 mg/kg docetaxel (4-fold decrease), and 48h and 72h post 33 mg/kg docetaxel compared to control (14- and 2-fold decrease, respectively). Furthermore, BCM-2665 ALDH+ cells showed 6-fold increased proliferation 72h post cisplatin compared to control, indicating that chemotherapy may inducing proliferation of TICs. When TICs are induced to proliferate, they may be more susceptible to traditional proliferative targets such as chemotherapy. These findings are consistent with the Norton-Simon Hypothesis in that chemotherapy regimens given more frequently may in fact eliminate TICs, thereby explaining the proven increased effectiveness of dose-dense chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-100. doi:10.1158/1538-7445.AM2011-LB-100

Abstract S5-7: Estrogen Receptor Alpha Negative (ER-) Breast Cancer Stem Cells in ER+ Cell Lines and Primary Cancers Are Enriched by Tamoxifen Therapy

Abstract Introduction: Resistance to endocrine therapy in ER+ breast cancers is associated with tumour relapse and poor prognosis. Breast cancer stem cells (BCSCs), characterised by the CD44hi/CD24lo/ESA+ phenotype and mammosphere initiating capacity (MIC), show treatment resistance to chemo-/radiotherapy. We hypothesised that BCSCs may also be resistant to endocrine therapies due to absent ER expression in vitro and in vivo. Methods: Three ER+ cell lines (MCF7, T47D and BT474) and six treatment naïve ER+ early breast cancers (5 IDC, 1 ILC) were used for in vitro and in vivo assays of BCSC activity. Putative CSC number was assessed by the non-adherent mammosphere (MS) assay, and by Fluorescence Activated Cell Sorting (FACS) for the markers CD44+/CD24-/low/epithelial specific antigen(ESA)+ in the presence and absence of tamoxifen (TAM), and also by limiting dilution implantation of cells into NOD/SCID IL2gammaR-/- (NSG) mice. Furthermore the effect of acquired tamoxifen resistance on BCSC activity was investigated in two independent Tamoxifen-resistant (TAM-R) MCF-7 variants, from Cardiff (TAM-RCARD) and Copenhagen (TAM-RCOP) and five (ER+) endocrine resistant metastatic cancers (3 pleural effusion, 2 ascites). ER alpha expression was assessed before and after BCSC enrichment in cell lines, primary and metastatic cancers by immunohistochemistry. Results: Putative BCSCs, (CD44+/CD24-/low/ESA or MIC phenotype) were highly enriched for ER- cells (eg. MCF7 cell line; CD44+/CD24-/low/ESA 73.2 ± 4.6% vs. 6.1 ± 1.4% p= 0.0007 or MIC 45.7 ± 1.7 vs. 6.1 ± 1.4 P&amp;lt;0.001). There was a significant increase in the proportion of ER-cells in TAM-R cell lines (TAM-RCARD 11.6 ± 1.4% p=0.01 and TAM-RCOP 22 ± 4.8% p=0.02 respectively) compared to the parental TAM-S controls. Similarly, the enriched BCSC population of primary and metastatic ER+ breast cancers were enriched for ER-cells (eg. BB7 metastatic cancer 91.6 ± 4.8% vs. 47.4 ± 9.5% p = 0.02). ER+ cell lines, treatment naïve primary samples and treatment resistant metastatic samples all demonstrated BCSC activity, which correlated with tumour grade and stage. BCSC activity was significantly increased in MCF7 models of acquired TAM resistance compared to parental TAM sensitive controls (mammosphere assay TAM-RCARD 4% vs. 2.5% p=0.03, and TAM-RCOP 8% vs. 3% p=0.007). A similar trend was observed in endocrine resistant (End-R) patient samples compared to treatment naïve (TN) controls (End-R 0.46 vs. TN 0.10 p = 0.1). Tumour take rates in NSG mice were significantly increased in endocrine resistant cell lines and metastatic samples. Importantly, TAM pre-treatment increased mammosphere forming efficiency in MCF7, T47D and BT474, ER+ primary and ER+ metastatic samples, suggesting BCSCs are relatively resistant to TAM compared to more differentiated cells. Conclusions: The putative BCSC populations in ER+ cell lines and breast cancers are enriched for ER-cells and this finding is further exaggerated in acquired endocrine resistance. TAM treatment and acquired tamoxifen resistance enriches for BCSC activity in in vitro and in vivo models, presumably by preferentially inhibiting more differentiated cells. These findings establish a novel mechanism of TAM resistance in ER+ breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S5-7.

Differentiation therapy: targeting breast cancer stem cells to reduce resistance to radiotherapy and chemotherapy

Studies have shown that cancer stem-like cells (CSCs) from solid cancers are resistant to both radiotherapy and chemotherapy. We have shown that primary breast cancers (n = 8) and a breast cancer cell line (MCF7) enriched for breast cancer stem cells (BCSC) using mammosphere (MS) clonogenic culture can preferentially survive radiotherapy and chemotherapy treatment in vitro, showing ≥ 50% increase in MS survival compared with non-BCSC enriched cells. The BCSC enriched population, defined by the cell surface markers ESA+/CD44+/CD24-/low, had reduced levels of DNA damage (measured by γH2AX) after 4 Gy irradiation or doxorubicin (1 μM) treatment. This suggests that the BCSC enriched population avoids or repairs the DNA damage significantly more than the whole population. Differentiating agents have been used to re-sensitise breast cancers to endocrine treatment but effects on BCSC are unknown. All-trans-retinoic acid (ATRA), tricostatin A and vorinostat caused a dose-dependent decrease in the BCSC population using MS culture and FACS analysis after 72 hours of treatment in a monolayer. Vorinostat (100 nM) showed the greatest effect, with 80% reduction in the ESA+/CD44+/CD24-/low population and a 50% reduction in MS formation. Our data suggest that in vitro treatment with differentiating agents reduces the number of the BCSC within the MCF7 cell line. Combination of ATRA (2 μM) or vorinostat with 6 Gy irradiation caused a significant reduction in MS survival showing a 30% and 70% decrease compared with an irradiated control. Similarly, in combination with paclitaxel (0.5 μM) ATRA and vorinostat caused a significant reduction in MS survival, showing 70% and 60% decrease compared with paclitaxel alone. In primary breast cancers (n = 3), combination of ATRA and 6 Gy irradiation significantly decreased MS formation by ≥ 25% respectively compared with irradiation alone. These observations suggest that targeting BCSC with agents that eliminate or differentiate BCSC is a promising strategy to overcome resistance to radiotherapy and chemotherapy in the clinic.

Abstract 4213: Lentiviral shRNA screen testing a gene signature of breast cancer stem cells using high throughput mammosphere assays

Abstract One in 8 women in the US suffer from breast cancer and current treatments have reduced the rate of cancer dependent deaths, the major problem facing this field is the high likelihood of relapse within 5-10 years of initial therapy. Relapse might be caused by the presence of cancer stem cells in the tumor which are not inhibited by traditional therapies and thus may require therapies that target this subpopulation of cells. We have recently published a gene signature which is derived from an overlap between CD44+/CD24-/low vs. all other cell subpopulations, and cancer-derived Mammosphere (MS) vs. bulk tumor which we reasoned will best represent the tumor-initiating or “cancer stem cells. We found that 477 genes were differentially expressed in the combination group of which 185 of these were highly expressed in CD44+/CD24-/low cells and in MS, a highly significant overlap (p&amp;lt;1.0E-9, one-sided Fisher's exact). The rest of the 292 genes demonstrated a reduction in expression in the CD44+/CD24-/low cells and cancer-derived MS vs. all other cells and bulk tumors, respectively (p&amp;lt;5.0E-5, one-sided Fisher's exact). We then proceed to test our gene signature by screening this entire list of genes for its role in MS. We created a library comprises 14 plates containing approximately 1193unique shRNA constructs, with 3-4 shRNA constructs per gene, thereby targeting 493 unique genes. The library is designed and constructed on a “one gene, one well” structure, such that each sequence validated lentiviral shRNA construct targeting an individual gene is located in a unique well of a 96-well plate. Using SUM159 cells that were transduced with individual lentiviruses representing our previously identified genes (efficiency ∼80%), along with a positive control lentivirus targeting Bmi1, a polycomb group gene known to be required for self-renewal of many stem/progenitor cell types, a non-specific negative control lentivirus targeting firefly luciferase, as well as untreated cells. In each of the transduced population cells will be plated in a 96 well dish at a density of 2,000 cells/well in serum-free medium supplemented with B27 nutrients, bFGF, EGF. MSs were allowed to form for 4 days, and then counted using Gelcount (Oxford Optornix). The MSs were trypsinized and replated in the same plate to form secondary mammospheres and primary and secondary mammosphere formation efficiency was calculated. The primary &amp; secondary MSFE was determined for all the shRNAs and we identified 151 shRNAs while the secondary MS formation identified 63 shRNA's of which 13 shRNAs were common that demonstrated significant change (p&amp;lt;0.05) in MS formation with respect to control. This gives us a set of targets that need to be validated by testing them in another cell line, which in turn will provide pharmaceutical targets to eliminate cancer stem cells. Top 5 targets will be tested in tumor xenografts to test clinical relevance of these therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4213.

Tamoxifen Resistance in Estrogen Receptor Positive (ER+) Breast Cancer Is Driven by Estrogen Receptor Negative (ER-) Cancer Stem-Like Cells.

Abstract Introduction: Intrinsic and acquired resistance to endocrine therapy significantly reduces survival rates of women with ER+ breast cancer. In the normal breast, multipotent stem cells are ER-. We hypothesised that tumourigenic breast cancer stem-like cells (CSCs) are ER- and therefore function independently of estrogen, representing a novel mechanism of resistance to endocrine therapy.Methods: Three ER+ cell lines, MCF-7, T47D and BT474 were assessed for ER expression by immunocytochemistry (ICC) in monolayer and after CSC enrichment. Putative CSC number was assessed by the non-adherent mammosphere (MS) assay, in the presence and absence of tamoxifen, and by Fluorescence Activated Cell Sorting (FACS) for the markers CD44+/CD24-/low/epithelial specific antigen(ESA)+. The effect of acquired Tamoxifen resistance on the CSC population of two independent Tamoxifen-resistant (TAM-R) MCF-7 variants, from Cardiff (TAM-RCARD) and Copenhagen (TAM-RCOP) was compared to parental, tamoxifen sensitive (TAM-S) controls. In the MS assay both the number and size of colonies were assessed.Results: A small sub-population of ER_ cells were demonstrated in MCF7 (6.1 ± 1.4%), T47D (6.8± 0.5) and BT474 (3.7±1.3) cells by ICC. However, the majority of putative breast CSCs (CD44+/CD24-/low/ESA-) in MCF7 cells were ER_ (73.2 ± 4.6 % p= 0.0007). There was a significant increase in the proportion of ER- cells in TAM-R cell lines (TAM-RCARD 11.6 ± 1.4% p=0.01 and TAM-RCOP 22 ± 4.8% p=0.02 respectively) compared to the parental TAM-S controls. Both TAM-R cell lines demonstrated increased mammosphere forming efficiency (MFE) compared to TAM-S cells (TAM-RCARD 4% vs. 2.5% p=0.03, and TAM-RCOP 8% vs. 3% p=0.007). The putative CSC population (CD44+/CD24-/low/ESA+) was enriched more than 2.5 fold in TAM-R compared to TAM-S parental controls. Compared with controls, the absolute MFE was not affected by Tamoxifen treatment, although mammospheres formed were significantly smaller.Conclusions: The majority of the putative CSC populations in ER+ breast cancer do not express ER. There is an increase in the proportion of putative ER- CSCs in TAM-R MCF7 cell lines. The reduction in MS size suggests that Tamoxifen targets transit amplifying cells but does not reduce the absolute number of CSC in ER+ breast cancers. These findings demonstrate a novel mechanism of endocrine resistance in ER+ breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5125.

Therapeutic resistance and tumor-initiation: Molecular pathways involved in breast cancer stem cell self-renewal

528 Background: Recent evidence supports the existance of a rare subpopulation of ‘cancer stem cells‘ (CSCs) which is chemoresistant and capable of self-renewal and tumor-initiation, resulting in relapse and metastases. We hypothesized that residual breast tumors after conventional chemotherapy (CTx) are enriched for CSCs bearing CD44+/CD24- markers, and show increased self-renewal as demonstrated by mammosphere (MS) forming assays. Molecular pathways like Notch, Wnt, and the polycomb family that regulate normal mammary self-renewal may be in aberrant in CSCs. Methods: Paired breast cancer biopsies from 35 patients were obtained before and after 12 weeks of neoadjuvant CTx (docetaxel 100 mg/m2 or Adriamycin/Cytoxan 60/600 mg/m2, 4 cycles, q3weeks), digested by collagenase, stained with CD24/CD44/lineage antibodies, and analyzed by flow cytometry. MS assays were performed to measure self-renewal ability. Gene expression, using the Affymetrix U133 GeneChip platform, of cancer cells bearing CD44+/CD24- markers vs. all other sorted cells, and between secondary cancer MS vs. the primary bulk invasive cancers were analyzed. Results: CD44+/CD24- cells increased from a median of 4.8% to 14.8% after CTx (p&lt;0.005). Increased self-renewal was demonstrated by an increase in MS capacity after CTx (p=0.03), with a positive correlation between the number of CD44+/24- cells and MS assays (R=0.8, p&lt;0.05). Common molecular pathways shared by CD44+/CD24- cells and MS show increased expression in normal self-renewal pathways - polycomb family (PCGF5), Notch (MAML2), FOXP1, and BBX. In addition, genes governing alternative splicing were increased, including a non-coding RNA (MALAT1) of unknown function, and RNA splicing factors (SFRS3, SFRS21P, SFRS4). Conclusions: Our results with an increase in cells bearing stem cell markers, and increased MS formation of residual tumors provide the first strong clinical evidence for the existance of therapy-resistant cancer stem cells. Post-transcriptional regulation may play a crucial role in modifying gene function involved in cancer stem cell self-renewal. Clinical trials targeting these newly identified pathways may eradicate residual disease and improved cure rates for many breast cancer patients. [Table: see text]