SUM159 Tumors Research Articles

Energy and endoplasmic reticulum stress induction by gold(III) dithiocarbamate and 2-deoxyglucose synergistically trigger cell death in breast cancer

The elusiveness of triple-negative breast cancer from targeted therapy has redirected focus towards exploiting the metabolic shortcomings of these highly metastatic subtypes of breast cancer. Cueing from the metabolic heterogeneity of TNBC and the exposition of the dual dependence of some TNBCs on OXPHOS and glycolysis for ATP, we herein report the efficacy of cotreatment of TNBCs with an OXPHOS inhibitor, 2a and 2DG, a potent glycolysis inhibitor. 2a-2DG cotreatment inhibited TNBC cell proliferation with IC50 of ∼5 to 36 times lower than that of 2a alone and over 5000 times lower than IC50 of 2DG alone. 2a-2DG cotreatment suppressed mitochondrial ATP production and significantly induced AMPK activation. Mechanistic studies revealed the distinct yet synergistic contributions of 2a and 2DG to the antiproliferative effect of the cotreatment. While 2a induced apoptotic cell death, 2DG sensitized TNBCs to the antiproliferative effects of 2a via endoplasmic reticulum stress induction. Strikingly, the combination of 2a-2DG ablated SUM159 tumors in an orthotopic xenograft mouse model. This study highlights the synergistic effect of a gold-based complex with 2DG and the potential benefit of multi-metabolic pathways targeting as an effective therapeutic strategy against TNBCs.

Unlabeled aspirin as an activatable theranostic MRI agent for breast cancer.

Rationale: Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is emerging as an alternative to gadolinium-based contrast MRI. We have evaluated the possibility of CEST MRI of orthotopic breast tumor xenografts with unlabeled aspirin's conversion to salicylic acid (SA) through various enzymatic activities, most notably inhibition of cyclooxygenase (COX)-1/-2 enzymes.Methods: We measured the COX-1/-2 expression in four breast cancer cell lines by Western Blot analysis and selected the highest and lowest expressing cell lines. We then performed CEST MRI following aspirin treatment to detect SA levels and ELISA to measure levels of downstream prostaglandin E2 (PGE2). We also injected aspirin into the tail vein of mice growing orthotopic tumor xenografts which expressed high and low COX-1/-2 and acquired SA CEST MR images of these tumor xenografts for up to 70 minutes. Tumors were then harvested to perform Western Blot and ELISA experiments to measure COX-1/-2 expression and PGE2 levels, respectively.Results: Western Blots determined that SUM159 cells contained significantly higher COX-1/-2 expression levels than MDA-MB-231 cells, in line with higher levels of downstream PGE2. SA CEST MRI yielded similar contrast at approximately 3% for both cell lines, independent of COX-1/-2 expression level. PGE2 levels decreased by about 50% following aspirin treatment. Results from our mouse study aligned with cultured cells, the overall SA CEST MRI contrast in both MDA-MB-231 and SUM159 tumor xenograft models was 5~8% at one hour post injection. PGE2 levels were ten times higher in SUM159 than MDA-MB-231 and decreased by 50%. The CEST contrast directly depended on the injected dose, with ~6%, ~3% and ~1.5% contrast observed following injection of 100 µL of 300 mM, 200 mM and 150 mM aspirin, respectively.Conclusions: Our data demonstrate the feasibility of using aspirin as a noninvasive activatable CEST MRI contrast agent for breast tumor detection.

Triple-Negative Primary Breast Tumors Induce Supportive Premetastatic Changes in the Extracellular Matrix and Soluble Components of the Lung Microenvironment.

The lung is one of the deadliest sites of breast cancer metastasis, particularly in patients with triple-negative (TN) disease. We hypothesized that the presence of a TN primary breast tumor induces changes in the extracellular matrix (ECM) and soluble components of the lung microenvironment that support metastatic behavior. SUM159 (TN) and MCF7 (luminal A) breast cancer cells were injected into mice, and primary breast tumors were established prior to assessing metastatic niche changes. We observed increased CD117+ hematopoietic progenitor cells in the bone marrow of SUM159 mice versus MCF7 or control mice (p < 0.05). Relative to mice bearing MCF7 tumors and non-tumor controls, mice bearing SUM159 tumors demonstrated enhanced expression of ECM proteins in the lung (fibronectin, tenascin-c and periostin), with similar changes observed in lung fibroblasts treated with extracellular vesicles (EVs) from TN breast cancer cells (p < 0.05). Exposure to lung-conditioned media (LCM) from SUM159 tumor-bearing mice resulted in increased migration/proliferation of both SUM159 and MCF7 cells relative to the control (p < 0.05). In contrast, LCM from MCF-7 tumor-bearing mice had no such effect. LCM from SUM159 tumor-bearing mice contained 16 unique proteins relative to other LCM conditions, including the metastasis-associated proteins CCL7, FGFR4, GM-CSF, MMP3, thrombospondin-1 and VEGF. These findings suggest for the first time that the TN breast cancer molecular subtype may be an important determinant of premetastatic changes to both the ECM and soluble components of the lung, potentially mediated via breast cancer-derived EVs.

Subtype-Specific Radiation Response and Therapeutic Effect of FAS Death Receptor Modulation in Human Breast Cancer.

Breast cancer is the most common malignancy diagnosed among women and represents a heterogeneous group of subtypes. Radiation therapy is a critical component of treatment for breast cancer patients. However, little is known about radiation response among these intrinsic subtypes. In previous studies, we identified a significant induction of FAS after irradiation in biologically favorable breast cancer patients and breast cancer cell lines. Here, we expanded our study and investigated radiation response in a mouse model of breast cancer. MCF7 (luminal), HCC1954 (HER2+) or SUM159 (basal) cells were implanted orthotopically into the dorsal mammary fat pad of nude mice. These mice were then treated with different doses of radiation to assess tumor growth control. We further investigated the therapeutic effect of FAS modulation by silencing FAS in radiation-responsive tumors and injecting FAS agonist antibody into radiation-resistant tumors. Exposure to radiation inhibited MCF7, and to a lesser extent HCC1954 tumor growth in a dose-dependent manner. In contrast, SUM159 tumors were resistant to radiation. The estimated TCD50 values were 19.3 Gy for MCF7 and 44.9 Gy for SUM159. Radiation induced FAS expression in MCF7 tumors, but not SUM159 tumors. We found that silencing of FAS did not negatively impact radiation response in MCF7 tumors, possibly due to compensation by other apoptotic pathways. On the other hand, FAS activating antibody in combination with radiation treatment delayed SUM159 and HCC1954 tumor growth. However, it did not reach statistical significance compared to radiation treatment alone. Our results suggest that there is intrinsic variation in radiation response among breast cancer subtypes. FAS activation concurrent with radiation slows tumor growth in the radiation-resistant subtypes, but the effect was not significant. Alternative subtype-specific modulators of radiation response are under investigation.

Abstract 1656: Subtype-specific radiation response in human breast cancer and potential therapeutic effect of FAS death receptor modulation

Abstract Breast cancer is the most common malignancy diagnosed among women worldwide and represents a heterogeneous group of subtypes. Radiation therapy is an important component of multimodal treatment for women with breast cancer. However, little is known about radiation response among these subtypes. Recent clinical data suggests that distinct patterns of response to systemic therapy may exist in association with each phenotype. Therefore, understanding the intrinsic variation of radiation response in breast cancer subtypes is important in prescribing individualized radiotherapy that maximizes the therapeutic ratio. Our previous studies have identified marked changes in gene expression in breast patient tumors and breast tumor cell lines in response to radiation. Among candidate genes, FAS was consistently and significantly induced after radiation in luminal breast tumors and cell lines. Modulation of FAS in basal breast cancer cell lines favorably impacted radiation response. In this study, we further investigated the role of FAS modulation and radiation response in a mouse model of breast cancer. MCF7 (luminal), HCC1954 (HER2+) or SUM159 (basal) cells were implanted orthotopically into the dorsal mammary fat pad of nude mice. These mice were then treated with different doses of radiation. Our results showed that radiation treatment inhibited MCF7, and to a less extent HCC1954 tumor growth in a dose-dependent manner, while SUM159 tumors were resistant to radiation. The estimated TCD50 value was 19.30 Gy for MCF7 and 44.88 Gy for SUM159. FAS was induced in MCF7 tumors after radiation but showed no change in SUM159 tumors. To investigate whether FAS modulation can affect radiation response, we silenced FAS in MCF7 tumors and activated FAS in SUM159 tumors. We found that silencing of FAS did not negatively impact radiation response in MCF7 tumors, possibly due to compensation by other apoptotic pathways. On the other hand, FAS activating antibody in combination with radiation delayed SUM159 and HCC1954 tumor growth (p = NS). In conclusion, these results suggest that there is intrinsic variation in radiation response among breast cancer subtypes. FAS concurrent with radiation slows tumor growth in the radiation resistant subtypes, but the effect was not significant. Alternative subtype-specific modulators of radiation response are under investigation. Citation Format: Chen-Ting Lee, Yingchun Zhou, Kingshuk R. Choudhury, Sharareh Siamakpour Reihani, Mark W. Dewhirst, Janet K. Horton. Subtype-specific radiation response in human breast cancer and potential therapeutic effect of FAS death receptor modulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1656.

Antitumor activity of Cetuximab in combination with Ixabepilone on triple negative breast cancer stem cells.

BackgroundDeveloping novel strategies against treatment-resistant triple negative breast cancer (TNBC) cells remains a significant challenge. The ErbB family, including epidermal growth factor receptor (EGFR), plays key roles in metastasis, tumorigenesis, cell proliferation, and drug resistance. Recently, these characteristics have been linked to a small subpopulation of cells classified as cancer stem cells (CSC) which are believed to be responsible for tumor initiation and maintenance. Ixabepilone is a new generation microtubule-stabilizing agent, which has been expected to be more efficacious than conventional taxanes. Here we aim to investigate whether the EGFR monoclonal antibody Cetuximab, in combination with Ixabepilone, is more effective in eliminating CSC populations compared to chemotherapy alone in TNBC.MethodsRepresentative TNBC cell lines (MDA-MB-231 and SUM159) were used to evaluate breast CSC populations. We used fluorescence-activated cell sorter analysis (CD44+ and CD24-/low, or Aldefluor+) and a self-renewal assay called mammosphere formation efficiency (MSFE) to measure CSC population size after treatment with Cetuximab, or Cetuximab plus Ixabepilone in vitro.ResultsAlthough there was no significant decrease in cell viability, Cetuximab reduced MSFE and the CSC population in breast cancer cells in vitro and in vivo through inhibition of autophagy. Also, SUM159 and MDA-MB-231 orthotopic tumors demonstrated partial response to Centuximab or Ixabepilone monotherapy; however, the effect of the combination treatment was significant only in SUM159 tumors (p <0.0001), when compared to Ixabepilone alone.ConclusionsOverall, our findings demonstrate that EGFR-targeted therapy by Cetuximab effectively reduces the CSC population in TNBC tumors. However, combination therapy with Ixabepilone may be effective only in a small subset of TNBCs, warranting further investigation of alternative approaches to target multiple pathways for TNBC treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0662-4) contains supplementary material, which is available to authorized users.

Abstract 3302: Subtype-specific radiation response in a mouse model of human breast cancer

Abstract Radiation therapy is an important component of multimodal treatment for women with breast cancer. However, breast radiotherapy is currently delivered uniformly, regardless of breast cancer subtype. Recent clinical data have suggested that different subtypes of breast cancer may respond differently to radiation. Understanding the intrinsic variation of radiation response in breast cancer subtypes would lay the foundation for individualized radiotherapy designed to limit toxicity in those with radiation responsive cancers. Furthermore, identification of molecular targets can be used to modulate treatment outcomes in patients with radiation resistant tumors. Our previous in vitro studies have identified an association between breast cancer subtype and gene expression profiles in response to radiation in breast tumor cell lines. Cell lines with the greatest response to radiation were luminal subtype and exhibited preferential induction of genes involved in programmed cell death, such as FAS. In this study, we investigated tumor growth and FAS induction in response to radiation in a mouse model of breast cancer. MCF7 (luminal) or SUM159 (basal) cells were implanted orthotopically into the dorsal mammary fat pad of athymic nude mice. These mice were then treated with different doses of radiation. Our results showed that radiation treatment inhibited MCF7 tumor growth in a dose-dependent manner, while radiation treatment had little effect on SUM159 tumor growth. In addition, FAS was induced in MCF7 tumors 24 hour after radiation, but there was no change in FAS expression in SUM159 tumors. However, injection of FAS-stimulating antibody intratumorally led to downstream caspase 8 and PARP activation, suggesting that the FAS signaling pathway remains intact and can be used to modulate radiation response in the resistant SUM159 cells. In conclusion, these results suggest that there is intrinsic variation in radiation response among breast cancer subtypes and that stimulation of FAS may have the potential to modulate radiation response. Citation Format: Chen-Ting Lee, Yingchun Zhou, Sharareh Siamakpour-Reihani, Kingshuk R. Choudhury, Mark W. Dewhirst, Janet K. Horton. Subtype-specific radiation response in a mouse model of human 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 3302. doi:10.1158/1538-7445.AM2015-3302

Abstract 3681: Facile fabrication of MRI-capable and NIR-resonant core-satellite nanomediators for photothermal therapy

Abstract Although many techniques exist for fabricating near-infrared (NIR)-resonant and magnetic resonance imaging (MRI)-capable nanomediators for photothermal cancer therapy, preparing them in an efficient and scalable process remains a significant challenge. In this report, we develop two facile methods to fabricate both NIR-resonant and MRI-capable nanomediators utilizing well developed polysiloxane-containing polymer-coated iron oxide nanoparticles (IONPs). One of the methods is to attach multiple NIR-resonant nanoparticles onto a thiol-modified IONP core through one-step mixing with gold sulfide nanoparticles (Au2S NPs) to fabricate a core-satellite nanomediator. The other strategy is to covalently attach a NIR organic dye onto intact polysiloxane-containing polymer-coated IONPs through facile mixing with siloxane-modified IR820. The successful decoration with either Au2S NPs or IR820 is demonstrated for the resultant nanomediators by absorption spectra and transmission electron microscopy (TEM) images. It is estimated that 10 Au2S NPs and 2×104 dye molecules (loading capacity: 4.5 mg IR820/mg Fe) are attached onto the polymer-coated IONPs, respectively. The hydrodynamic sizes of these novel multifunctional nanomediators are ∼57.5 nm for IONP-Au2S NP and 28.2 nm for IONP-IR820, which are much smaller than traditional gold nanoshell-based designs. The photothermal efficiency of the IONP-Au2S NP and IONP-IR820 nanocomposite solutions under the irradiation of 885 nm NIR laser light is significantly enhanced compared to IONPs alone. The enhancement in photothermal efficiency by the newly developed nanomediators is also investigated in nude mice bearing SUM-159 tumor xenografts. The biodistribution data measured by inductively coupled plasma optical emission spectrometry (ICP-OES) reveal that ∼1% injection dose (ID) of IONP-Au2S NPs and ∼9% ID of IONP-IR820 accumulate into tumor tissue 24 h post intravenous injection at a dose of 20 mg Fe/Kg mouse body weight. The average tumor surface temperature recorded by an infrared camera is increased by 10.8 ± 1.2 °C for the mice administered with IONP-Au2S NPs and 25.7 ± 3.6 °C for the group injected with IONP-IR820, compared to 4.8 ± 0.5 °C for the PBS-injected control mice after laser irradiation with a power of 0.5 W for 10 min. Furthermore, our data reveal that the fabrication will not compromise the IONP core capability as an MRI contrast agent. Taken together, we demonstrate a facile technique to generate NIR-resonant and MRI-capable nanomediators from a polysiloxane-containing polymer-coated IONP platform. The as-developed dual functional nanomediators hold great promise for translatable MRI-guided photothermal cancer therapy. Citation Format: Hongwei Chen, Xiaoqing Ren, Hayley Paholak, Joseph Burnett, Feng Ni, Duxin Sun. Facile fabrication of MRI-capable and NIR-resonant core-satellite nanomediators for photothermal therapy. [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 3681. doi:10.1158/1538-7445.AM2015-3681

Metastatic breast cancer cells in lymph nodes increase nodal collagen density.

The most life-threatening aspect of breast cancer is the occurrence of metastatic disease. The tumor draining lymph nodes typically are the first sites of metastasis in breast cancer. Collagen I fibers and the extracellular matrix have been implicated in breast cancer to form avenues for metastasis. In this study, we have investigated extracellular matrix molecules such as collagen I fibers in the lymph nodes of mice bearing orthotopic human breast cancer xenografts. The lymph nodes in mice with metastatic MDA-MB-231 and SUM159 tumor xenografts and tumor xenografts grown from circulating tumor cell lines displayed an increased collagen I density compared to mice with no tumor and mice with non-metastatic T-47D and MCF-7 tumor xenografts. These results suggest that cancer cells that have metastasized to the lymph nodes can modify the extracellular matrix components of these lymph nodes. Clinically, collagen density in the lymph nodes may be a good marker for identifying lymph nodes that have been invaded by breast cancer cells.

Combination therapy with anti-DR5 antibody and tamoxifen for triple negative breast cancer

TRA-8, a monoclonal antibody targeting death receptor, has demonstrated high therapeutic effect for triple negative breast cancer (TNBC) in preclinical models. Tamoxifen, the standard of care for ERα-positive breast cancer, induces apoptosis via ERβ, which commonly presents in TNBC cells. The current study investigates the combination effects of TRA-8 and tamoxifen for TNBC. In vitro assays were implemented with two ERβ-positive TNBC cell lines, SUM159 and 2LMP, and in vivo therapy studies were followed using orthotopic breast tumor mouse models. IC50 of tamoxifen for SUM159 and 2LMP were 29 μM and 38 μM, respectively. Synergy between TRA-8 (0–1000 ng/mL) and tamoxifen (20 μM) was observed for both the cell lines. Tamoxifen (400 mg/kg diet) markedly suppressed the growth of SUM159 tumors for 6 weeks after therapy initiation, but it did not induce antitumor effect for 2LMP tumors. TRA-8 (0.1 mg, weekly, i.p.) successfully arrested the growth of both SUM159 and 2LMP tumors during therapy, but an antagonistic effect was observed when tamoxifen was combined. TRA-8 uptake into tumors was not changed by tamoxifen treatment. Histological analysis confirmed that caspase-3 activation induced by TRA-8 was significantly decreased when tamoxifen was used in combination. In conclusion, our findings suggest that the combined use of TRA-8 and tamoxifen may cause antagonistic effects for TNBC patients.

Transient Mild Hyperthermia Induces E-selectin Mediated Localization of Mesoporous Silicon Vectors in Solid Tumors

BackgroundHyperthermia treatment has been explored as a strategy to overcome biological barriers that hinder effective drug delivery in solid tumors. Most studies have used mild hyperthermia treatment (MHT) to target the delivery of thermo-sensitive liposomes carriers. Others have studied its application to permeabilize tumor vessels and improve tumor interstitial transport. However, the role of MHT in altering tumor vessel interfacial and adhesion properties and its relationship to improved delivery has not been established. In the present study, we evaluated effects of MHT treatment on tumor vessel flow dynamics and expression of adhesion molecules and assessed enhancement in particle localization using mesoporous silicon vectors (MSVs). We also determined the optimal time window at which maximal accumulation occur.ResultsIn this study, using intravital microscopy analyses, we showed that temporal mild hyperthermia (∼1 W/cm2) amplified delivery and accumulation of MSVs in orthotopic breast cancer tumors. The number of discoidal MSVs (1000×400 nm) adhering to tumor vasculature increased 6-fold for SUM159 tumors and 3-fold for MCF-7 breast cancer tumors. By flow chamber experiments and Western blotting, we established that a temporal increase in E-selectin expression correlated with enhanced particle accumulation. Furthermore, MHT treatment was shown to increase tumor perfusion in a time-dependent fashion.ConclusionsOur findings reveal that well-timed mild hyperthermia treatment can transiently elevate tumor transport and alter vascular adhesion properties and thereby provides a means to enhance tumor localization of non-thermally sensitive particles such as MSVs. Such enhancement in accumulation could be leveraged to increase therapeutic efficacy and reduce drug dosing in cancer therapy.

Early therapy assessment of combined anti-DR5 antibody and carboplatin in triple-negative breast cancer xenografts in mice using diffusion-weighted imaging and1H MR spectroscopy

To assess the early response of triple-negative breast-cancer (TNBC) following TRA-8 and carboplatin therapy using DWI and MRS in 2LMP and SUM159 mouse models. Four groups (n = 5/group) of each model were untreated or treated with carboplatin, TRA-8, and combination, respectively. DWI and MRS were applied on 0, 3, and 7 days after therapy initiation, and all tumors were collected thereafter for terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining. The changes in intratumoral apparent diffusion coefficient (ADC) and fat-water ratios (FWRs) were compared with tumor volume changes and apoptotic cell densities. Mean ADC values of 2LMP and SUM159 tumors significantly increased 4 ± 4% and 37 ± 11% during 7 days of combination therapy, respectively, as compared to control groups (P < 0.05). Similarly, mean FWRs of 2LMP and SUM159 tumors significantly increased 102 ± 30% and 126 ± 52%, respectively, for 7 days of combined treatment (P < 0.05). The changes of the mean ADC values for 3 days (or FWRs for 7 days) were linearly proportional to either the mean volume changes or apoptotic cell densities in both models. DWI and MRS assessed the early tumor response to TRA-8 and carboplatin in TNBC mouse models.

PD08-05: Stat3 Signaling in Human Breast Cancer Stem Cells.

Abstract Recent data suggest the existence of a unique subset of breast cancer cells capable of initiating tumor growth and giving rise to all other cells characteristic of a given tumor. These cells have been termed “cancer stem cells” or “tumor-initiating cells”. Breast cancer stem cells appear to be resistant to chemo- and radiotherapies and may be responsible for recurrence and metastasis in breast cancer patients. Identifying the cellular signaling pathways responsible for breast cancer stem cell maintenance and self-renewal represents a critical hurdle for developing effective therapeutics. The Stat3 pathway is a critical regulator of the function of normal stem cells, and shows altered expression in human breast cancers [4]. Moreover, IL-6, the Stat3 signaling agonist, is required for breast cancer stem cell function in human breast cancer cell lines. All these suggest an important role of Stat3 signaling in breast cancer stem cells. However, due to lack of method for pathway-activity-based live cell separation, whether Stat3 functions in the cancer stem cells themselves or whether it may function in surrounding niche cells remain unknown. We have constructed a lentiviral fluorescent reporter for Stat3 signaling which contains four copies of M67 Stat3 binding sites upstream of enhanced Green Fluorescent Protein. This reporter system enables FACS-sorting of cells with active Stat3 signaling and in vivo/in situ localization of Stat3 responsive cells. In addition, C188-9, the first Stat3 specific competitive inhibitor, has been developed in our collaborator's lab, which provides us an advanced tool in studying Stat3 function. We hypothesize that Stat3 signaling is preferentially active in stemlike subpopulation and depend on non-stem cancer cells to maintain its activation. Three aims to test this hypothesis include: 1. To test whether cancer cells with activated Stat3 signaling are enriched for breast cancer stem cells or whether they may serve as niche cells. 2. To test whether small molecule antagonists of Stat3 signaling can inhibit cancer stem cell function. 3. To identify novel targets of Stat3 signaling that may serve as indicators of responsiveness to Stat3 antagonist or predict treatment response. So far, our GFP reporters for Stat3 signaling effectively report Stat3 activity in both patient xenografts and human breast cancer cell lines in vivo and in vitro, which enables effective separation of Stat3+/Stat3- cells for functional Study. MDA231 and SUM159 tumors with reporters have been established. Tumor cells will be sorted according to Stat3 activity and perform limiting dilution and MSFE assays. Tumors with reporters will also be treated by C188-9, Chemo, or in combination to study synergistic effect of Stat3 inhibitors on tumor growth. Preliminary data based on inhibitor and limiting dilution studies suggested that Stat3 signaling is required for stem cell function. However, neither Stat3+ nor Stat3- cells are self-sufficient in performing these functions, indicating communications between subpopulations. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-05.

Abstract 3354: HDAC inhibitor SAHA suppressed beast cancer stem cells in vitro and in vivo

Abstract Although conventional chemotherapies eliminate differentiated cancer cells in the bulk of tumors, they are ineffective against cancer stem cells (CSCs), which initiate tumors and result in cancer recurrence. The purpose of this study is to investigate the efficacy of HDAC inhibition by SAHA on breast CSCs through epigenetic modifications in cancer stem cells. Two different breast cancer cell lines with basal (SUM159) and luminal (MCF7) subtypes were used in this study. In order to examine HDAC mediated CSC regulation, we utilized a number of in vitro and in vivo CSC assays including tumorsphere formation, Aldefluor assay, CD44+/CD24- phenotype and mouse xenografts. Breast cancer stem cells (CSC) were monitored after treatment of different concentrations of SAHA (0.5-2µM). The ability of HDAC inhibitor to suppress the self-renewal capability of CSCs was evaluated by tumorsphere assay from a single Aldefluor-positive cell in a 96-well Ultra-low attachment plate under the suspension condition. The tumorsphere formation rate was used to evaluate the self-renewal capability of CSCs. The mouse xenograft model bearing with SUM159 cells and primary breast tumors (MC1) was used to test the inhibition of SAHA on CSCs and impairment of their self renewal in vivo. Microarray gene expression analysis was performed to elucidate molecular mechanisms of HDAC underlying the CSC inhibition, following the treatment of SAHA. SAHA reduced SUM159 Aldefluor-positive cells in a concentration-dependent manner with significant reduction (&amp;gt;50%) at 1µM. SAHA dramatically reduced the CSC population (CD44+/CD24 low) by 90% in MCF7 cells. Since CSCs are able to expand its population through the self-renewal, a CSC-targeting drug to inhibit both self-renewal and to eliminate CSCs is highly desired. Tumorsphere formation assay showed that SAHA (0.5µM) reduced the tumorsphere formation rate by 77%. In addition, the sustaining tumorsphere reductions (∼80%) in second and third passages in the absence of SAHA suggest the the impaired self-renewal capability of breast CSCs by SAHA is not repairable. Furthermore, the size of SAHA-treated tumorspheres was substantially smaller than that of the control, suggesting that SAHA also inhibited the proliferation of progenitor cells. In SUM159 tumor xenograft model, SAHA significantly inhibited the tumor growth by 45% and 84% in SUM159 and MC1 tumor xenografts, respectively, compared to the vehicle-treated group. SAHA reduced Aldefluor-positive cells in SUM159 and MC1 tumors by 50-90% following 2-week treatment. Tumor reimplantation showed that SAHA was able to abolish the tumor engraftment ability of cells derived from drug-treated mice in secondary mice. These data suggest that SAHA was able to target breast CSCs in vitro and in vivo. SAHA not only diminished the CSC population but also attenuated their self-renewal ability of the residual CSCs. 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 3354. doi:10.1158/1538-7445.AM2011-3354