Tumorigenicity Of Breast Cancer Cells Research Articles

Transducin (β)-like 1 X-linked receptor 1 promotes proliferation and tumorigenicity in human breast cancer via activation of beta-catenin signaling.

IntroductionTransducin (β)-like 1 X-linked receptor 1(TBLR1) is an F-box-like and WD repeat-containing protein which functions as a switch in transcriptional activation, However, the clinical significance and biological role of TBLR1 in breast cancer remains largely unknown.MethodsWestern blotting, immunocytochemistry and real-time PCR were used to evaluate TBLR1 expression in normal breast epithelial cells and breast cancer cell lines, clinical tissue samples and adjacent nontumor tissues, and in 214 paraffin-embedded specimens. Statistical analyses were used to test for the prognostic and diagnostic associations. The biological role of TBLR1 -induced proliferation and tumorigenicity in breast cancer cells was explored in vitro and in vivo. The effect of TBLR1 on the expression of cyclin D1 and β-catenin signaling was examined by Western blotting, luciferase reporter assay and by several immunoprecipitation techniques.ResultsTBLR1 was significantly upregulated in breast cancer cells and tissues compared to normal control samples. Immunohistochemical analysis revealed high expression of TBLR1 in 113 of 214 (52.8%) paraffin-embedded archival breast cancer. The overall expression level of TBLR1 was significantly correlated with clinical stage (P <0.001), the tumor classification (P <0.001), node classification (P =0.024), and metastasis classification (P = 0.004), histological grade (P = 0.044), as well as with the expression level of c-erbB2 (P = 0.036) and Ki-67 (P <0.001). Patients with higher TBLR1 expression had shorter overall survival time, whereas patients with lower TBLR1 expression had better survival. Multivariate analysis suggested that TBLR1 expression might be an independent prognostic indicator for the survival of breast cancer patients. TBLR1 overexpression promoted, whereas TBLR1 silencing inhibited, proliferation and tumorigenicity in breast cancer cells both in vitro and in vivo. We found that TBLR1 expression was implicated in the upregulation of cyclin D1, phosphorylation of cell-cycle control protein Rb (pRb) and activation of β-catenin signaling in breast cancer.ConclusionsTBLR1 plays a key role in the development and progression of breast cancer cells via cyclin D1-transactivation and activation of the β-catenin signaling pathway. TBLR1 may be a novel prognostic marker and a potential therapeutic target in the treatment human breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0465-z) contains supplementary material, which is available to authorized users.

Surgery-induced wound response promotes stem-like and tumor-initiating features of breast cancer cells, via STAT3 signaling.

Inflammation is clinically linked to cancer but the mechanisms are not fully understood. Surgery itself elicits a range of inflammatory responses, suggesting that it could represent a perturbing factor in the process of local recurrence and/or metastasis. Post-surgery wound fluids (WF), drained from breast cancer patients, are rich in cytokines and growth factors, stimulate the in vitro growth of breast cancer cells and are potent activators of the STAT transcription factors. We wondered whether STAT signaling was functionally involved in the response of breast cancer cells to post-surgical inflammation. We discovered that WF induced the enrichment of breast cancer cells with stem-like phenotypes, via activation of STAT3. In vitro, WF highly stimulated mammosphere formation and self-renewal of breast cancer cells. In vivo, STAT3 signaling was critical for breast cancer cell tumorigenicity and for the formation of local relapse after surgery. Overall, we demonstrate here that surgery-induced inflammation promotes stem-like phenotypes and tumor-initiating abilities of breast cancer cells. Interfering with STAT3 signaling with a peri-surgical treatment was sufficient to strongly suppress this process. The understanding of the crosstalk between breast tumor-initiating cells and their microenvironment may open the way to successful targeting of these cells in their initial stages of growth and be eventually curative.

Acylglycerol kinase promotes cell proliferation and tumorigenicity in breast cancer via suppression of the FOXO1 transcription factor.

BackgroundAcylglycerol kinase (AGK) is reported to be overexpressed in multiple cancers. The clinical significance and biological role of AGK in breast cancer, however, remain to be established.MethodsAGK expression in breast cancer cell lines, paired patient tissues were determined using immunoblotting and Real-time PCR. 203 human breast cancer tissue samples were analyzed by immunochemistry (IHC) to investigate the relationship between AGK expression and the clinicopathological features of breast cancer. Functional assays, such as colony formation, anchorage-independent growth and BrdU assay, and a xenograft tumor model were used to determine the oncogenic role of AGK in human breast cancer progression. The effect of AGK on FOXO1 transactivity was further investigated using the luciferase reporter assays, and by detection of the FOXO1 downstream genes.ResultsHerein, we report that AGK was markedly overexpressed in breast cancer cells and clinical tissues. Immunohistochemical analysis showed that the expression of AGK significantly correlated with patients’ clinicopathologic characteristics, including clinical stage and tumor-nodule-metastasis (TNM) classification. Breast cancer patients with higher levels of AGK expression had shorter overall survival compared to patients with lower AGK levels. We gained valuable insights into the mechanism of AGK expression in breast cancer cells by demonstrating that overexpressing AGK significantly enhanced, whereas silencing endogenous AGK inhibited, the proliferation and tumorigenicity of breast cancer cells both in vitro and in vivo. Furthermore, overexpression of AGK enhanced G1-S phase transition in breast cancer cells, which was associated with activation of AKT, suppression of FOXO1 transactivity, downregulation of cyclin-dependent kinase inhibitors p21 Cip1 and p27 Kip1 and upregulation of the cell cycle regulator cyclin D1.ConclusionsTaken together, these findings provide new evidence that AGK plays an important role in promoting proliferation and tumorigenesis in human breast cancer and may serve as a novel prognostic biomarker and therapeutic target in this disease.

Enrichment of breast cancer stem-like cells by growth on electrospun polycaprolactone-chitosan nanofiber scaffolds

A small population of highly tumorigenic breast cancer cells has recently been identified. These cells, known as breast-cancer stem-like cells (BCSC), express markers similar to mammary stem cells, and are highly resistant to chemotherapy. Currently, study of BCSC is hampered by the inability to propagate these cells in tissue culture without inducing differentiation. Recently, it was reported that proliferation and differentiation can be modified by culturing cells on electrospun nanofibers. Here, we sought to characterize the chemoresistance and stem-like properties of breast cancer cell lines grown on nanofiber scaffolds. Cells cultured on three-dimensional templates of electrospun poly(ε-caprolactone)-chitosan nanofibers showed increases in mammary stem cell markers and in sphere-forming ability compared with cells cultured on polystyrene culture dishes. There was no increase in proliferation of stem cell populations, indicating that culture on nanofibers may inhibit differentiation of BCSC. The increase in stemness was accompanied by increases in resistance to docetaxel and doxorubicin. These data indicate that BCSC populations are enriched in cells cultured on electrospun poly(ε-caprolactone)-chitosan nanofibers, scaffolds that may provide a useful system to study BCSC and their response to anticancer drug treatment.

Chronic Oxidative Stress Increases Growth and Tumorigenic Potential of MCF-7 Breast Cancer Cells

Accumulating evidence suggests that exposures to elevated levels of either endogenous estrogen or environmental estrogenic chemicals are associated with breast cancer development and progression. These natural or synthetic estrogens are known to produce reactive oxygen species (ROS) and increased ROS has been implicated in both cellular apoptosis and carcinogenesis. Though there are several studies on direct involvement of ROS in cellular apoptosis using short-term exposure model, there is no experimental evidence to directly implicate chronic exposure to ROS in increased growth and tumorigenicity of breast cancer cells. Therefore, the objective of this study was to evaluate the effects of chronic oxidative stress on growth, survival and tumorigenic potential of MCF-7 breast cancer cells. MCF-7 cells were exposed to exogenous hydrogen peroxide (H2O2) as a source of ROS at doses of 25 µM and 250 µM for acute (24 hours) and chronic period (3 months) and their effects on cell growth/survival and tumorigenic potential were evaluated. The results of cell count, MTT and cell cycle analysis showed that while acute exposure inhibits the growth of MCF-7 cells in a dose-dependent manner, the chronic exposure to H2O2-induced ROS leads to increased cell growth and survival of MCF-7 cells. This was further confirmed by gene expression analysis of cell cycle and cell survival related genes. Significant increase in number of soft agar colonies, up-regulation of pro-metastatic genes VEGF, WNT1 and CD44, whereas down-regulation of anti-metastatic gene E-Cadherin in H2O2 treated MCF-7 cells observed in this study further suggests that persistent exposure to oxidative stress increases tumorigenic and metastatic potential of MCF-7 cells. Since many chemotherapeutic drugs are known to induce their cytotoxicity by increasing ROS levels, the results of this study are also highly significant in understanding the mechanism for adaptation to ROS-induced toxicity leading to acquired chemotherapeutic resistance in breast cancer cells.

Abstract B053: Phosphatidylinositol 3 kinase (PI3K) inhibitors as therapeutic agents for breast tumors overexpressing sfRon

Abstract Introduction: Receptor tyrosine kinases (RTKs) and their pleiotropic effectors are key factors in metastasis. Met and Ron are RTKs that have been shown to contribute to tumor growth and metastasis in both animal models and in patients. Ron is over-expressed in many epithelial cancers, but not usually amplified or mutated. Recently, an alternative Ron isoform known as “short-form” Ron (sfRon) was associated with aggressive phonotype of breast cancer. sfRon is a 55-kD N-terminally truncated form of Ron. It lacks most of the extracellular domain of Ron, but contains intact transmembrane and cytoplasmic domains. The expression of this alternative Ron isoform yields constitutively active protein. sfRon plays a crucial role in the aggressiveness of breast cancer; its expression significantly promotes tumor growth and spontaneous metastasis to distant organs. We observed that sfRon strongly signals through the PI3K pathway and that interaction between sfRon and PI3K is required for sfRon function. Blocking the interaction between sfRon and PI3K, or directly blocking PI3K activity, thoroughly abrogates the ability of sfRon to confer aggressive tumor behavior in vitro. The PI3K signaling cascade regulates many cellular functions that are mainly associated with cell proliferation, survival, and migration. PI3K is activated in a wide variety of human malignancies including breast cancer, resulting in tumor development and progression. The PI3K pathway has been identified as an important target in breast cancer research for a number of years. In our study we utilized NVP-BEZ235 and NVP-BKM120, compounds provided by Novartis. NVP-BEZ235 is a dual PI3K/mTOR inhibitor and NVP-BKM120 is a pan-PI3K inhibitor. Both compounds exhibit potent antiproliferative and proapoptotic activities in a variety of tumor cell lines as well as in animal models. Currently, NVP-BKM120 and NVP-BEZ235 compounds are undergoing phase I/II clinical trials in patients with cancer, including breast malignancy. For in vivo studies, we used highly tumorigenic and invasive MCF7 breast cancer cells. Our cell line overexpresses sfRon and is characterized by high levels of active PI3K. We have shown before that sfRon overexpression triggers activation of PI3K signaling, which is necessary for sfRon aggressive behavior. Our goal is to test whether inhibition of PI3K pathway, which signals downstream from sfRon, will have any effect on this aggressive tumor growth in vivo. Methods: Three groups of NOD/SCID female mice were injected orthotopically with 1x106 sfRon-overexpressing MCF7 cells suspended in Matrigel. Treatment was initiated when tumors reached 500 mm3 in size. Mice were treated for 3 weeks daily by oral gavage with 45 mg/kg of NVP-BEZ235, 60 mg/kg of NVP-BKM120, or vehicle. Tumor size was monitored every three days with caliper measurements. Treatment continued until mice had grown tumors up to the size limit. Results: The data showed that both drugs significantly inhibited mammary tumor growth in comparison to mice treated with vehicle. We observed the most dramatic inhibitory effect on tumor growth in the group of mice treated with NVP-BKM120. Three weeks after treatment initiation, NVP-BKM120-treated mice had an average tumor size of 405 mm3. Meanwhile, mice treated with NVP-BEZ235 and vehicle had tumors that averaged 1182 mm3 and 3007 mm3, respectively. Conclusion: Our data shows that PI3K pathway inhibitors are very well tolerated by mice and are excellent in the inhibition of tumor growth driven by sfRon, especially NVP-BKM120. NVP-BEZ235 and NVP-BKM120 compounds hold great promise in the treatment of breast cancer overexpressing sfRon with strongly activated PI3K. Citation Format: Parvathi Radhakrishnan, Magdalena Bieniasz, Alana Welm. Phosphatidylinositol 3 kinase (PI3K) inhibitors as therapeutic agents for breast tumors overexpressing sfRon. [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 B053.

Autocrine/paracrine mechanism of interleukin-17B receptor promotes breast tumorigenesis through NF-κB-mediated antiapoptotic pathway

Gain of function of membrane receptor was a good strategy exploited by cancer cells to benefit own growth and survival. Overexpression of HER2 has been found to serve as a target for developing trastuzumab to treat 20-25% of breast cancer. However, little or none of the other membrane receptor was found to be useful as a potential target for breast cancer treatment since then. Here, we showed that amplified signaling of interleukin-17 receptor B (IL-17RB) and its ligand IL-17B promoted tumorigenicity in breast cancer cells and impeded acinus formation in immortalized normal mammary epithelial cells. External signal transmitted through IL-17RB activated nuclear factor-κB to upregulate antiapoptotic factor Bcl-2 and induced etoposide resistance. Elevated expression of IL-17RB had a stronger correlation with poor prognosis than HER2 in breast cancer patients. Interestingly, breast cancer patients with high expression of IL-17RB and HER2 had the shortest survival rate. Depletion of IL-17RB in trastuzumab-resistant breast cancer cells significantly reduced their tumorigenic activity, suggesting that IL-17RB and HER2 have an independent role in breast carcinogenesis. Furthermore, treatment with antibodies specifically against IL-17RB or IL-17B effectively attenuated tumorigenicity of breast cancer cells. These results suggest that the amplified IL-17RB/IL-17B signaling pathways may serve as a therapeutic target for developing treatment to manage IL-17RB-associated breast cancer.

Treatment of cancer and cancer stem cells by blocking the Notch pathway.

2539 Background: Current treatments often fail to cure cancer. It has been shown (JCO, 26, June 10, 2008) that Cancer Stem Cells, (CSCs), are responsible for the initiation, metastasis and recurrence of many cancers and may be a key reason for the failure of current therapies.The NOTCH pathway is an important pathway in the development of many tumors ,and cancer stem cells in particular. Methods: We have generated genetically and synthetically a hybrid protein (Antp-DNMAML) .consisting of the truncated version of MastermindElike (MAML) that behaves in a dominant negative (DN) fashion inhibiting Notch activation, and the cell penetrating peptide Antennapedia (Antp). Results: It is demonstrated that Antp-DNMAML translocates into the nucleus and suppresses Notch activation. Attenuation of Notch signaling with AntpEDNMAML reverts the transformed phenotype, inhibits the anchorageEdependent growth, induces self contact inhibition and apoptosis in highly tumorigenic epithelial human breast cancer cells. More significantly, we provide direct evidence that inhibiting Notch signaling at the transcriptional level with the Antp-MAML protein ,suppresses the expression of downstream Notch targets, induces tumor cell apoptosis, and inhibits or eliminates human tumor growth in nude mice, without organ or systemic toxicity. Conclusions: Intracellular delivery of dominant-negative transcription complex proteins using the Antp platform is a new and specific approach for cancer therapy.

Abstract 530: Inhibition of amphiregulin-mediated EGFR signaling suppresses tumorigenicity in breast cancer cells.

Abstract Approximately half of all basal type breast cancers show evidence of autocrine activation of the epidermal growth factor receptor (EGFR) by amphiregulin (AREG). In addition, AREG expression is associated with aggressive and chemoresistant forms of breast cancer. Studies in our laboratory show that when AREG is the activating ligand, EGFR accumulates at the cell surface, resulting in altered receptor trafficking and downstream signaling. To investigate the effect of AREG-mediated activation of EGFR on the phenotype of AREG-expressing cancers, AREG expression was knocked-down in SUM149 cells, a highly tumorigenic and metastatic cell line isolated from a basal subtype inflammatory breast cancer, which expresses high levels of AREG and overexpresses EGFR. Microarray analysis of AREG knockdown cells revealed a number of relevant pathways affected by AREG-EGFR signaling. These pathways include Notch signaling, MAPK activation, IL-1 signaling and multiple components of the Wnt signaling pathway. In particular, knockdown of AREG or inhibition of EGFR signaling in AREG stimulated cells resulted in the upregulation of DKK-1, an antagonist of canonical Wnt signaling, and the expression of several WNT target genes. Flow cytometric analysis of these cells demonstrated that AREG knockdown resulted in a significant reduction in CD44+ cells while simultaneously increasing the non-stem cell fraction of CD44-/CD24+ cells. Because CD44hi/CD24lo is characteristic of undifferentiated cells that have the potential to initiate tumor formation, the tumorigenicity of these cells was investigated by transplanting parental and AREG knock-down SUM149 cells into the mammary fat pads of NOD-SCID mice. The tumorigenic activity of the AREG knock-down derivatives was significantly reduced compared to the parental SUM149 cells. In particular, the AREG knockdown clone that expressed the lowest level of AREG expression and the highest level of DKK-1 expression failed to form tumors in NOD-SCID mice. To investigate the link between AREG-mediated activation of EGFR, receptor trafficking, and downstream signaling pathways that are responsible for changes in expression of WNT pathway genes, we are investigating differences in EGFR phosphorylation and downstream signaling stimulated by AREG versus EGF. In conjunction with our previous findings that AREG-mediated EGFR signaling results in activation of NF-κβ and the upregulation of IL1α/β, our current results suggest a link between these pathways, WNT signaling, and tumorigenicity in SUM-149 cells. Citation Format: Christiana S. Kappler, Stephen P. Ethier. Inhibition of amphiregulin-mediated EGFR signaling suppresses tumorigenicity in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 530. doi:10.1158/1538-7445.AM2013-530

Abstract 234: Inhibition of the NOTCH transcription factor complex by a novel hybrid protein leads to effective tumor therapy.

Abstract The Notch pathway has been implicated in the generation and propagation of cancer stem cells and is thus an attractive target for therapy. We have generated a hybrid protein (Antp-DNMAML) consisting of the truncated version of Mastermind-like (MAML) that behaves in a dominant negative (DN) fashion inhibiting Notch, and the cell penetrating peptide Antennapedia (Antp). Results indicate that the Antp-DNMAML translocates into the nucleus, suppresses Notch activation, reverts the transformed phenotype, inhibits the anchorage-dependent growth and induces self contact inhibition and apoptosis in tumorigenic human breast cancer cells. More significantly, there is direct evidence that inhibiting Notch signaling at the transcriptional level with the Antp-DNMAML protein, suppresses the expression of downstream Notch targets and inhibits tumor growth in nude mice, without organ or systemic toxicity. In summary, intracellular delivery of dominant-negative transcription complex proteins using the Antp platform is a new and specific approach for cancer therapy . Citation Format: Agamemnon A. Epenetos, Christina Kousparou, Spyros Stylianou, Mahendra Deonarain, Aleksandra Filipovic. Inhibition of the NOTCH transcription factor complex by a novel hybrid protein leads to effective tumor therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 234. doi:10.1158/1538-7445.AM2013-234

Abstract P6-04-26: Tamoxifen may block estrogen induced secretion of certain cytokines to interrupt tumor associated macrophage infiltration in breast cancer

Abstract The role of tumor microenvironment during the initiation and progression of breast cancer is now realized to be of critical importance, both for understanding of the fundamental cancer biology and exploiting relatively new mechanism for breast cancer metastasis. Macrophage is a one of the most common components in the breast cancer microenvironment. Normally, the macrophage in tumor microenvironment is referred to tumor associated macrophage (TAM), which shares some attributes of alternatively activated macrophage. Many pre-clinical and clinical studies demonstrate an inverse correlation between TAM infiltration and patients' prognosis indicating a macrophage supporting role for tumor progression. It is well documented that selective patients with breast cancer can benefit from anti-estrogen therapy. However, the mechanisms involved are still not fully elucidated. Our previous study indicates that highly tumorigenic breast cancer cell line MDA-MB-231 can educate monocyte differentiation into alternatively activated macrophage, while weakly tumorigenic cell line MCF-7 (without estrogen supplementation) can not. In the present study, we explored the effects of estrogen and tamoxifen on the secreting of certain cytokines, which are required for monocyte chemotaxis and differentiation. Breast cancer cell lines with different estrogen receptor status were applied to estimate the level of cytokines (IL4, IL6, M-CSF, GM-CSF, and MCP-1) before and after cultured with 17β-estradiol, tamoxifen or both. We found that weakly invasive breast cancer cell lines (MCF-7, T47D, MDA-MB-468, and SKBR3) generally expressed lower levels of such cytokines compared with highly invasive breast cancer cell lines (MDA-MB-231, MDA-MB-231-BO, MDA-MB-231 HM, Bcap 37, BT549, and Hs578T) at baseline. However, the discrepancies could be compensated partially by exposure to 10nM 17β-estradiol in estrogen receptor (ER) positive cell lines (MCF-7 and T47D). Moreover, the compensation was substantially blocked by 2μM tamoxifen. On the contrary, tamoxifen, alone, didn't affect the secretion of such cytokines mentioned above in vitro. Based on these findings, we tentatively concluded that some patients with ER positive breast cancers benefiting from anti-estrogen therapy may partially attributes to blocking monocyte differentiation into TAM, which destroyed the tumor microenvironment. These findings suggest the future possibility of using TAM as a novel therapeutic target in patients with anti-estrogen resistance and primary triple-negative breast cancers (TNBC) with no effectively therapeutic measures nowadays, which are defined by lack of estrogen receptor, progesterone receptor and ERBB2 gene amplification, representing approximately 16% of all breast cancers. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-26.

Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo

Nicastrin (NCT) is a crucial component of the γ-secretase (GS) enzyme, which prompted investigations into its biological role in cancer. We have previously shown that nicastrin is overexpressed in breast cancer (BC), conferring worse overall survival in invasive, ERα negative patients. Here, we used 2D and 3D Matrigel, anchorage-independent growth conditions and a breast cancer xenograft mouse model to assess the impact of nicastrin on breast cancer stem cell (BCSC) propagation and invasion in vitro and tumor growth in vivo. Stable knockdown of nicastrin in HCC1806 breast cancer cells reduced cell invasion by 51.4 ± 1.7%, accompanied by a morphological change to a rounded cell phenotype and down-regulation of vimentin, Snail, Twist, MMP2, and MMP9. We observed a reduction of the pool of CD44(+)/CD24(-) and ALDH1 high breast cancer stem cells by threefold and twofold, respectively, and a reduction by 2.6-fold of the mammospheres formation. Nicastrin overexpression in nontransformed MCF10A cells caused an induction of epithelial to mesenchymal regulators, as well as a fivefold increased ALDH1 activity, a threefold enrichment for CD44(+)/CD24(-) stem cells, and a 3.2-fold enhanced mammosphere-forming capacity. Using the γ-sescretase inhibiton, Notch1/4 siRNA, and Akt inhibition, we show that nicastrin regulates breast cancer stem cells partly through Notch1 and the Akt pathway. Exploiting serial dilution transplantation of the HCC1806 cells expressing nicastrin and HCC1806 stably depleted of nicastrin, in vivo, we demonstrate that nicastrin inhibition may be relevant for the reduced tumorigenicity of breast cancer cells. These data could serve as a benchmark for development of nicastrin-targeted therapies in breast cancer.

Overexpression of GOLPH3 Promotes Proliferation and Tumorigenicity in Breast Cancer via Suppression of the FOXO1 Transcription Factor

Golgi phosphoprotein 3 (GOLPH3) has been reported to be involved in various biologic processes. The clinical significance and biologic role of GOLPH3 in breast cancer, however, remains unknown. Expression of GOLPH3 in normal breast cells, breast cancer cells, and 6-paired breast cancer and adjacent noncancerous tissues were quantified using real-time PCR and Western blotting. GOLPH3 protein expression was analyzed in 258 archived, paraffin-embedded breast cancer samples using immunohistochemistry. The role of GOLPH3 in breast cancer cell proliferation and tumorigenicity was explored in vitro and in vivo. Western blotting and luciferase reporter analyses were used to investigate the effect of GOLPH3 overexpression and silencing on the expression of cell-cycle regulators and FOXO1 transcriptional activity. GOLPH3 was significantly upregulated in breast cancer cells and tissues compared with normal cells and tissues. Immunohistochemical analysis revealed high expression of GOLPH3 in 133 of 258 (51.6%) breast cancer specimens. Statistical analysis showed a significant correlation of GOLPH3 expression with advanced clinical stage and poorer survival. Overexpression and ablation of GOLPH3 promoted and inhibited, respectively, the proliferation and tumorigenicity of breast cancer cells in vitro and in vivo. GOLPH3 overexpression enhanced AKT activity and decreased FOXO1 transcriptional activity, downregulated cyclin-dependent kinase (CDK) inhibitor p21(Cip1), p27(Kip1), and p57(Kip2), and upregulated the CDK regulator cyclin D1. Our results suggest that high GOLPH3 expression is associated with poor overall survival in patients with breast cancer and that GOLPH3 overexpression increases the proliferation and tumorigenicity of human breast cancer cells.

Abstract 4669: A combination of raloxifene and gefitinib potentiates the cytotoxicity and induces apoptosis in estrogen receptor negative breast cancer cell lines

Abstract In recent years, numerous studies have shown that the selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene reduce the risk of invasive breast carcinoma. However, while SERMs do prevent the development of many estrogen-receptor (ER)-positive breast cancers, few studies suggest a potential effect of these drugs in the prevention of the development of ER-negative breast cancers. Although, the therapeutic efficacy of raloxifene in cancer therapy is thought to arise primarily from its ability to compete with estrogens; raloxifene has been shown to have effects through estrogen receptor (ER)-independent mechanisms. Recently, a study perform in our laboratory demonstrated that raloxifene decreased the level of EGFR expression in xenograft tumors. Based on these results, we hypothesized that a combination of raloxifene and a tyrosine kinase inhibitor, gefitinib may improve cytotoxicity towards ER-negative breast cancers. In the present study, we show that raloxifene (5µM) in combination with gefitinib (10µM) further decrease cell viability of two human ER-negative breast cancer cell lines, MDA-MB-468 and MDA-MB-231, compare to a single treatment from either of these two drugs. The combination potentiates apoptosis in MDA-MB-231 by 6 fold and in MDA-MB-468 by 3 fold after 36 h. Expression and phosphorylation pattern of proteins involved in cell proliferation such as NFκB, β-catenin, epidermal growth factor (EGFR) were also markedly affected by the combination treatment. Furthermore, evidence of apoptosis activation was also observed by an increase of the cleaved form of caspase-3. Additional studies demonstrated that the combination decreased cell migration and tumorigenicity of breast cancer cells. Moreover, the combined treatment decreased the ability of neovascularization as assessed by tube formation of endothelial cells. All together, these results suggested the potential of the combination of gefitinib and raloxifene for the prevention of tumor growth and appearance of metastatic events. In conclusion, these results provide us with the basis for future studies on the mechanism involved in raloxifene-gefitinib inhibition of ER-negative tumor growth. 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 4669. doi:1538-7445.AM2012-4669

Abstract 5314: An oncogenic splice variant of the KLF6 tumor suppressor gene is associated with poor survival and is a potent driver of breast cancer metastasis

Abstract Breast cancer is a leading causes of cancer death in women worldwide. It is a heterogeneous and genetically complex disease has left a significant proportion of patients with inadequate treatment options. Since metastasis and drug resistance pose significant challengea in breast cancer treatment, elucidating the mechanisms of these processes is critical for understanding the key drivers of disease progression and for the development of targeted therapies. Accumulating evidence from our laboratory and other groups suggest that the tumor suppressor gene, KLF6, and its oncogenic splice variant, KLF6-SV1, play a role in breast cancer progression, dissemination, and chemoresistance. Here we demonstrate that in multiple independent clinical cohorts of over 1200 breast cancer patients with defined clinical outcome, high KLF6-SV1 mRNA levels in the primary tumor associated with poor survival and disease recurrence. Specifically, upregulated KLF6-SV1 expression in the primary tumors correlated with poor survival independent of disease stage and grade. Thus, we hypothesized that KLF6-SV1 is an early driver/molecular determinant of invasive breast cancer. In order to investigate the functional/biological relevance of KLF6-SV1 in breast cancer development and progression, we performed a series of experiments using retroviral-based overexpression in multiple non-tumorigenic, tumorigenic, and metastatic breast cancer cell lines. We demonstrated that overexpression of KLF6-SV1 increased mesenchymal marker gene expression, cellular survival, invasion, as well as the migratory potential of KLF6-SV1 transduced cells. Interestingly, KLF6-SV1 did not increase growth rate of these mammary epithelial cell lines, similar to the clinical data which showed an absence of a correlation between KLF6-SV1 expression and primary tumor size. In a 3D model KLF6-SV1 upregulation disrupted mammary acinar morphogenesis promoting complex multiacinar structures. To assay whether the KLF6-SV1-induced EMT phenotype conferred increased metastatic potential in vivo, we injected tumorigenic cells expressing high levels of KLF6-SV1 subcutaneously into immunodeficient mice. Strikingly, KLF6-SV1 overexpression alone drove the entire metastatic cascade resulting in dissemination to many organs including the liver, kidney, heart, lung, and spleen. Furthermore, KLF6-SV1 overexpression in a metastatic breast cancer cell line increased metastasis to the lungs and liver in an orthotopic model of the disease. This is consistent with our clinical data in which high KLF6-SV1 expression was correlated with decreased overall survival and metastasis free survival. Together these findings suggest a role for the KLF6-SV1 splice variant as a driver of breast cancer metastasis and validate its potential utility as a novel biomarker and therapeutic target for breast cancer. 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 5314. doi:1538-7445.AM2012-5314

Antiproliferative effects of artemisinin on human breast cancer cells requires the downregulated expression of the E2F1 transcription factor and loss of E2F1-target cell cycle genes

Artemisinin, a sesquiterpene phytolactone derived from Artemisia annua, is a potent antimalarial compound with promising anticancer properties, although the mechanism of its anticancer signaling is not well understood. Artemisinin inhibited proliferation and induced a strong G1 cell cycle arrest of cultured MCF7 cells, an estrogen-responsive human breast cancer cell line that represents an early-stage cancer phenotype, and effectively inhibited the in-vivo growth of MCF7 cell-derived tumors from xenografts in athymic nude mice. Artemisinin also induced a growth arrest of tumorigenic human breast cancer cell lines with preneoplastic and late stage cancer phenotypes, but failed to arrest the growth of a nontumorigenic human mammary cell line. Concurrent with the cell cycle arrest of MCF7 cells, artemisinin selectively downregulated the transcript and protein levels of the CDK2 and CDK4 cyclin-dependent kinases, cyclin E, cyclin D1, and the E2F1 transcription factor. Analysis of CDK2 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK2 gene expression was accounted for by the loss of CDK2 promoter activity. Chromatin immunoprecipitation revealed that artemisinin inhibited E2F1 interactions with the endogenous MCF7 cell CDK2 and cyclin E promoters. Moreover, constitutive expression of exogenous E2F1 prevented the artemisinin-induced cell cycle arrest and downregulation of CDK2 and cyclin E gene expression. Taken together, our results demonstrate that the artemisinin disruption of E2F1 transcription factor expression mediates the cell cycle arrest of human breast cancer cells and represents a critical transcriptional pathway by which artemisinin controls human reproductive cancer cell growth.

Hsp27 participates in the maintenance of breast cancer stem cells through regulation of epithelial-mesenchymal transition and nuclear factor-κB

IntroductionHeat shock proteins (HSPs) are normally induced under environmental stress to serve as chaperones for maintenance of correct protein folding but they are often overexpressed in many cancers, including breast cancer. The expression of Hsp27, an ATP-independent small HSP, is associated with cell migration and drug resistance of breast cancer cells. Breast cancer stem cells (BCSCs) have been identified as a subpopulation of breast cancer cells with markers of CD24-CD44+ or high intracellular aldehyde dehydrogenase activity (ALDH+) and proved to be associated with radiation resistance and metastasis. However, the involvement of Hsp27 in the maintenance of BCSC is largely unknown.MethodsMitogen-activated protein kinase antibody array and Western blot were used to discover the expression of Hsp27 and its phosphorylation in ALDH + BCSCs. To study the involvement of Hsp27 in BCSC biology, siRNA mediated gene silencing and quercetin treatment were used to inhibit Hsp27 expression and the characters of BCSCs, which include ALDH+ population, mammosphere formation and cell migration, were analyzed simultaneously. The tumorigenicity of breast cancer cells after knockdown of Hsp27 was analyzed by xenograftment assay in NOD/SCID mice. The epithelial-mesenchymal transition (EMT) of breast cancer cells was analyzed by wound-healing assay and Western blot of snail, vimentin and E-cadherin expression. The activation of nuclear factor kappa B (NF-κB) was analyzed by luciferase-based reporter assay and nuclear translocation.ResultsHsp27 and its phosphorylation were increased in ALDH+ BCSCs in comparison with ALDH- non-BCSCs. Knockdown of Hsp27 in breast cancer cells decreased characters of BCSCs, such as ALDH+ population, mammosphere formation and cell migration. In addition, the in vivo CSC frequency could be diminished in Hsp27 knockdown breast cancer cells. The inhibitory effects could also be observed in cells treated with quercetin, a plant flavonoid inhibitor of Hsp27, and it could be reversed by overexpression of Hsp27. Knockdown of Hsp27 also suppressed EMT signatures, such as decreasing the expression of snail and vimentin and increasing the expression of E-cadherin. Furthermore, knockdown of Hsp27 decreased the nuclear translocation as well as the activity of NF-κB in ALDH + BCSCs, which resulted from increasing expression of IκBα. Restored activation of NF-κB by knockdown of IκBα could reverse the inhibitory effect of Hsp27 siRNA in suppression of ALDH+ cells.ConclusionsOur data suggest that Hsp27 regulates the EMT process and NF-κB activity to contribute the maintenance of BCSCs. Targeting Hsp27 may be considered as a novel strategy in breast cancer therapy.

Nuclear delivery of a therapeutic peptide by long circulating pH-sensitive liposomes: Benefits over classical vesicles

The purpose of this study is to propose a suitable vector combining increased circulation lifetime and intracellular delivery capacities for a therapeutic peptide. Long circulating classical liposomes [SPC:CHOL:PEG-750-DSPE (47:47:6 molar% ratio)] or pH-sensitive stealth liposomes [DOPE:CHEMS:CHOL:PEG 750-DSPE (43:21:30:6 molar% ratio)] were used to deliver a therapeutic peptide to its nuclear site of action. The benefit of using stealth pH-sensitive liposomes was investigated and formulations were compared to classical liposomes in terms of size, shape, charge, encapsulation efficiency, stability and, most importantly, in terms of cellular uptake. Confocal microscopy and flow cytometry were used to evaluate the intracellular fate of liposomes themselves and of their hydrophilic encapsulated material. Cellular uptake of peptide-loaded liposomes was also investigated in three cell lines: Hs578t human epithelial cells from breast carcinoma, MDA-MB-231 human breast carcinoma cells and WI-26 human diploid lung fibroblast cells. The difference between formulations in terms of peptide delivery from the endosome to the cytoplasm and even to the nucleus was investigated as a function of time. Characterization studies showed that both formulations possess acceptable size, shape and encapsulation efficiency but cellular uptake studies showed the important benefit of the pH-sensitive formulation over the classical one, in spite of liposome PEGylation. Indeed, stealth pH-sensitive liposomes were able to deliver hydrophilic materials strongly to the cytoplasm. Most importantly, when encapsulated in pH-sensitive stealth liposomes, the peptide was able to reach the nucleus of tumorigenic and non tumorigenic breast cancer cells.

Knockdown of FLOT1 Impairs Cell Proliferation and Tumorigenicity in Breast Cancer through Upregulation of FOXO3a

Lipid rafts, specialized domains in cell membranes, function as physical platforms for various molecules to coordinate a variety of signal transduction processes. Flotinllin-1 (FLOT1), a marker of lipid rafts, is involved in the progression of cancer, but the precise mechanism remains unclear. The aim of the present study was to examine the role of FLOT1 on the tumorigenesis of breast cancer cells and its clinical significance in progression of the disease. FLOT1 expression was analyzed in 212 paraffin-embedded, archived clinical breast cancer samples by using immunohistochemistry (IHC). The effect of FLOT1 on cell proliferation and tumorigenesis was examined in vitro and in vivo. Western blotting and luciferase reporter analyses were carried out to identify the effects of downregulating FLOT1 on expression of cell cycle regulators and transcriptional activity of FOXO3a. IHC analysis revealed high expression of FLOT1 in 129 of the 212 (60.8%) paraffin-embedded archived breast cancer specimens. The overall expression level of FLOT1 significantly correlated with clinical staging and poor patient survival of breast cancer. Strikingly, we found that silencing FLOT1 inhibited proliferation and tumorigenicity of breast cancer cells both in vitro and in vivo, which was further shown to be mechanistically associated with suppression of Akt activity, enhanced transcriptional activity of FOXO3a, upregulation of cyclin-dependent kinase inhibitor p21(Cip1) and p27(Kip1), and downregulation of the CDK regulator cyclin D1. FLOT1 plays an important role in promoting proliferation and tumorigenesis of human breast cancer and may represent a novel prognostic biomarker and therapeutic target for the disease.

Abstract 1210: Activation of heterogeneous nuclear ribonucleoprotein K (HNRNPK) by ERBB2/ERBB3

Abstract ERBB2 (HER2/neu) amplification constitute one of the most prominent oncogenic amplification event in breast cancer, and ERBB2 overexpression is well established as a predominantly proliferation and metastasis favoring signaling input. It is consequently the object of different targeted therapies (e.g. Trastuzumab, Pertuzumab or Lapatinib). We carried out 2-D difference gel electrophoresis based proteomics studies (DIGE) of early posttranslational protein modifications in MCF7 breast cancer cells in response to ligand induced activation. These studies have identified heterogeneous nuclear ribonucleoprotein K (HNRNPK) as a target for rapid tyrosine phosphorylation after ERBB2/ERBB3 activation by neuregulin β1. Upregulation of protein levels coupled with cytoplasmic accumulation of HNRNPK has been found to be associated with its oncogenic role in several types of cancer, and HNRNPK overexpression enhances the tumorigenicity of breast cancer cells in cell culture models. Previous studies have shown that HNRNPK levels are reduced in breast cancer cells after prolonged treatment with EGFR or ERBB2 directed therapeutic antibodies. However, while molecular mechanistic data exist on HNRNPK from a variety of model systems, we currently lack a coherent mechanistic framework of the crosstalk of ERBB2 and HNRNPK signaling in breast cancer. Following our DIGE analysis, we found that ligand induced and ERBB2/ERBB3 mediated tyrosine phosphorylation of HNRNPK in MCF7 cells is mediated by members of the SRC kinase family. Following ligand dependent stimulation, activation of HNRNPK coincides with a cytoplasmic relocalization of a relatively small portion of the predominantly nuclear HNRNPK pool. We compared our observations of ligand induced activation with the status of HNRNPK in ERBB2 over expressing breast cancer cell lines in presence or absence of ERBB2 targeted inhibitors. These studies are aimed at elucidating the interplay of HNRNPK and constitutive ERBB2 signaling in breast cancer cells and its role in cellular transformation. 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 1210. doi:10.1158/1538-7445.AM2011-1210