Negative Human Breast Cancer Cell Research Articles

Abstract P5-08-05: The influence of doxorubicin on PTEN and PI3K-Akt-mTOR signaling in human breast cancer

Abstract Background: De novo as well as acquired resistance are common features of anthracycline therapy in breast cancer. The PTEN tumor suppressor gene is frequently inactivated in human breast cancer, leading to induction of PI3K-Akt-mTOR signaling, which facilitates cell growth, survival, invasion and ultimately tumor progression. Upregulation of the PI3K-Akt-mTOR pathway has been suggested as a possible mechanism of anthracycline resistance, but there is limited data in the clinical setting, including its influence on PTEN expression. Methods: The early PTEN response to doxorubicin was assessed in vitro in MCF7 (estrogen receptor positive) and MDA-MB231 (triple negative) human breast cancer cell lines 24 hours after the initiation of chemotherapy. Tumor samples from thirty patients with locally advanced breast cancer were analyzed for PTEN expression levels and PI3K-Akt-mTOR signaling before and after neoadjuvant anthracycline therapy. The early effect of anthracyclines on PTEN expression is currently being evaluated in human breast cancer samples 24 hours after the initiation of chemotherapy. Mice bearing MCF7 and MDA-MB231 xenografts will be used to assess the potential of PI3K-Akt-mTOR signal transduction inhibitors in preventing anthracycline resistance. Results: PTEN expression was found to be profoundly downregulated in both MCF7 and MDA-MB231 cell lines, 24 hours after exposure to doxorubicin at their respective IC50. An increase in Akt1 protein levels in both cell lines was observed in the same setting. In 19 out of 30 human breast cancers PTEN levels increased after a median of 16 weeks neoadjuvant doxorubicin treatment. No difference between estrogen receptor positive and negative breast cancers was observed. The increase in PTEN was accompanied by an increase in fibrotic tissue in the tumor interior. There was no correlation between PTEN level or treatment-induced alterations in PTEN expression and response rates or recurrence-free survival (RFS). The results from our currently ongoing experiments will be presented. Conclusion: PTEN expression increases in the majority of human breast cancers during long-term exposure to doxorubicin. However, PTEN is greatly downregulated, with corresponding Akt1 increase, 24 hours after exposure to high doses of doxorubicin in vitro. These results indicate different effects of short- and long-term exposure to doxorubicin, which may be of importance to the development of anthracycline resistance, and to understand at what time a combinational approach with inhibitors of the PI3K-Akt-mTOR pathway will be beneficial. Current efforts are aimed at evaluating the potential of such inhibitors to counteract doxorubicin resistance in this setting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-05.

Abstract B30: 15-Methylene-Eburnamonine: A preventative agent against breast to brain metastases by targeting circulating tumor cells and cancer stem cells through induction of oxidative stress

Abstract One in eight women in the United States will develop invasive breast cancer during their lifetime. Of these women, a significant portion (10-16%) will develop metastases to the brain, resulting in significant morbidity and poor prognosis. Symptoms of brain metastasis are devastating and include dizziness, nausea, vertigo, headaches, impaired vision and cognitive functions. Brain metastases arise from highly aggressive triple negative (estrogen, progesterone, and Her2 receptor negative) or Her2 positive (estrogen and progesterone receptor negative, and Her2 positive) mammary tumors and often afflict young, premenopausal women. Less than 2% of women with such lesions survive past 2 years. Since there are currently no effective therapies against brain metastases, the best approach is to prevent their formation by targeting circulating tumor cells before they engraft at distant sites. It has been suggested that cancer stem cells (CSCs) contribute to the metastatic spread in many solid tumors, including those of the breast. A successful preventative agent must kill circulating tumor cells and CSCs before they have a chance to colonize a secondary site while having minimal toxicity against normal tissue. Eburnamonine (EBN) is a pharmacologically active compound derived from the flowering plant Periwinkle with vasoregulatory and anti-hypoxic properties. It has been found to cross the blood-brain-barrier and has been used as a treatment for a number of cerebrovascular disorders, including ischemia and anoxia. EBN has no known neurotoxicity, in fact it has been found to be neuroprotective. A derivative of EBN, 15-methylene-(-)-eburnamonine (15-M-EBN), with increased cytotoxity against cancer cells has been synthesized by our group. We found that EBN is cytotoxic against triple negative human breast cancer cells, MDA-MB-231 (LC50=42.0 μM). Our derivative, 15-M-EBN, is even more effective against triple negative human breast cancer cells, MDA-MB-231 (LC50=14.0 μM), as well as against triple negative breast to brain metastatic cells, MDA-MB-231BR (LC50=26.4 μM); and Her2 positive breast to brain metastatic cells, MDA-MB-231BR-Her2 (LC50=27.2 μM). Mammosphere assay indicated that 15-M-EBN kills CSCs found in MDA-MB-231, triple negative breast cancer cells (LC50=10.3 μM), as well as those found in triple negative breast to brain metastatic cells, MDA-MB-231BR, (LC50=26.2 μM) and Her2 positive breast to brain metastatic cells, MDA-MB-231BR-Her2 (LC50=19.8 μM). Also, by using 3D cultures and coatings of various extracellular matrix component proteins, we established that 15-M-EBN is active under the conditions of environmental-mediated drug-resistance. Staining with Annexin V showed that 15-M-EBN induces apoptosis that we demonstrate to be mediated through the activation of caspases 3, 6, and 7 and cleavage of cytokeratin 18. Addition of reducing agents (N-Acetyl-L-cysteine, glutathione, or DTT) to 15-M-EBN treated cultures blocked its cytotoxic effects, demonstrating that 15-M-EBN acts through the induction of oxidative stress. Furthermore, to establish that the methylene group on the fifteenth carbon in the structure of 15-M-EBN is the reactive site, our group synthesized a compound with a methyl group in place of the methylene (15-methyl-EBN). It was tested in cultures of breast cancer cells and showed that replacing the methylene with the methyl group abrogates the cytotoxic effects of this compound. Taken together our data show that 15-M-EBN kills triple negative and Her2 positive breast cancer cells and CSCs derived from these tumors through an induction of oxidative stress, overcomes the pro-tumorigenic effects of the tumor microenvironment while exhibiting minimal toxicity against normal tissue. These findings suggest that 15-M-EBN has a high potential to become a preventative agent against breast to brain metastases. Citation Format: Mary Minyi Zheng, Britney Harris, James Woods, David Colby, Julia Kirshner. 15-Methylene-Eburnamonine: A preventative agent against breast to brain metastases by targeting circulating tumor cells and cancer stem cells through induction of oxidative stress. [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 B30.

Role of mitogen‐activated protein kinases and Mcl‐1 in apoptosis induction by withaferin A in human breast cancer cells

Withaferin A (WA), a bioactive constituent of Ayurvedic medicine plant Withania somnifera, is a potent apoptosis inducer in cancer cells but the mechanism of cell death induction is not fully characterized. The present study was undertaken to determine the role of mitogen-activated protein kinases (MAPK), including c-jun NH2 -terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK, and anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) in regulation of WA-induced apoptosis using human breast cancer cells. Exposure of MCF-7 (estrogen responsive) and SUM159 (triple negative) human breast cancer cells to WA resulted in increased phosphorylation of ERK, JNK, and p38 MAPK, but these effects were relatively more pronounced in the former cell line than in SUM159. Overexpression of manganese-superoxide dismutase conferred partial protection against WA-mediated hyperphosphorylation of ERK, but not JNK or p38 MAPK. Cell death resulting from WA treatment in MCF-7 cells was significantly augmented by pharmacological inhibition of ERK and p38 MAPK. Interestingly, the WA-induced apoptosis in MCF-7 cells was partially but significantly blocked in the presence of a JNK-specific inhibitor. Pharmacological inhibition of ERK or JNK had no effect on WA-induced apoptosis in SUM159 cells. The WA-treated cells exhibited induction of long and short forms of Mcl-1. RNA interference of Mcl-1 alone triggered apoptosis. Furthermore, the WA-induced cell death in MCF-7 cells was modestly but significantly augmented by knockdown of the Mcl-1 protein. These observations indicate that: MAPK have cell line-specific role in cell death by WA, and Mcl-1 induction confers modest protection against WA-induced apoptosis.

Equol as a potent radiosensitizer in estrogen receptor-positive and -negative human breast cancer cell lines

Breast cancer is the most common cause of cancer death among women worldwide, and diet plays an important role in its prevention and progression. Radiotherapy has a limited but important role in the management of nearly every stage of breast cancer. We studied whether equol, the major metabolite of the soybean isoflavone daidzein, could enhance radiosensitivity in two human breast cancer cell lines (T47D and MDA-MB-231). MTT assay was used to examine equol's effect on cell viability. Sensitivity of cells to equol, radiation and a combination of both was determined by colonogenic assays. Induction of apoptosis by equol, radiation and the combination of both was also determined by acridine orange/ethidium bromide double staining fluorescence microscopy. DNA strand breaks were assessed by Comet assay. MTT assay showed that equol (0.1-350 μM) inhibited MDA-MB-231 and T47D cell growth in a time- and dose-dependent manner. Treatment of cells with equol for 72 h (MDA-MB-231) and 24 h (T47D) was found to inhibit cell growth with IC50 values of 252 μM and 228 μM, respectively. Furthermore, pretreatment of cells with 50 μM equol for 72 h (MDA-MB-231) and 24 h (T47D) sensitized the cells to irradiation. Equol was also found to enhance radiation-induced apoptosis. Comet assay results showed that the radiosensitizing effect of equol was accompanied by increased radiation-induced DNA damages. These results suggest for the first time that equol can be considered as a radiosensitizing agent and its effects may be due to increasing cell death following irradiation, increasing the remaining radiation-induced DNA damage and thus reducing the surviving fraction of irradiated cells.

P53siRNA therapy reduces cell proliferation, migration and induces apoptosis in triple negative breast cancer cells

p53 protein is probably the best known tumor suppressor. Earlier reports proved that human breast cancer cells expressing mutant p53 displayed resistance to apoptosis. This study is intended to investigate, the potential applications of RNA interference (RNAi) to block p53 expression, as well as its subsequent effect on cell growth, apoptosis and migration on a triple negative human breast cancer cell line (Hs578T). p53siRNA significantly reduced cell index (CI) compared to the control and we observed an inhibition of cellular migration in the interval of time between 0 and 30 h, as shown in the data obtained by dynamic evaluation using the xCELLigence System. Also, by using PCR-array technology, a panel of 84 key genes involved in apoptosis was investigated. Our studies indicate that the knockdown of p53 expression by siRNA modulates several genes involved in cell death pathways and apoptosis, showing statistically significant gene expression differences for 22 genes, from which 18 were upregulated and 4 were downregulated. The present research also emphasizes the important role of BCL-2 pro-apoptotic family of genes (Bim, Bak, and Bax) in activating apoptosis and reducing cell proliferation by p53siRNA treatment. Death receptors cooperate with BCL-2 pro-apoptotic genes in reducing cell proliferation. The limited success may be due to the activation of the antiapoptotic gene Mcl-1, and it may be associated with the resistance of triple negative breast cancer cells to cancer treatment. Thus, targeting p53siRNA pathways using siRNA may serve as a promising therapeutic strategy for the treatment of breast cancers.

Highlights of This Issue

Using a chemosensitivity RNA interference screen against 133 genes with known somatic mutations in breast cancer, Singel and colleagues identified the kinesin family member 14 (KIF14) and talin 1 as modulators of response to docetaxel in 4 triple-negative breast cancer cell lines but not in 3 hormonereceptor positive cell lines. Knockdown of these genes did not affect chemosensitivity to normal mammary epithelial cells. Their results show that high KIF14 expression is prognostic of poor relapse-free survival (946 patients) and poor overall survival (652 patients). Therefore, decreasing expression of these genes is an attractive target to sensitize triple negative human breast cancer cells to taxane treatment.Induction of the viral lytic cycle is a promising strategy for treatment of Epstein–Barr virus (EBV)–driven malignancies because viral lytic replication is associated with death of EBV-carrying tumor cells. Giunco and colleagues show that inhibition of human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase, promotes induction of the viral lytic cycle and triggers apoptosis of EBV-immortalized and fully transformed B cells. These results provide the rationale to activate clinical studies based on the combination of antiviral drugs with inhibitors of hTERT expression/activity for the treatment of EBV-driven malignancies.The multikinase inhibitor sorafenib successfully displayed antitumor activity in preclinical models of metastatic osteosarcoma. Unfortunately, in the clinical setting tumor shrinkage and disease stabilization with sorafenib were short-lived, owing to drug resistance. Pignochino and colleagues showed mTOR complex 2 (mTORC2) upregulation in sorafenib-treated osteosarcoma models, thereby suggesting a potential escape mechanism. The combination of sorafenib with the mTOR inhibitor everolimus achieved total blockage of both mTORC1 and mTORC2 through an increase of reactive oxygen species and the activation of AMP-dependent kinase. This complete inhibition eventually enhances the antitumor, antimetastatic, and antiangiogenic effects in osteosarcoma xenograft models.In a phase II clinical study in metastatic colorectal cancer, Amatu and colleagues show for the first time that dacarbazine activity is confined to patients with tumors defective in O6-methylguanine-DNA-methyltransferase function due to promoter CpG hypermethylation. In the context of cancer science, these data therefore highlight a previously unidentified subgroup of patients with colorectal cancer who benefit from treatment with methylating agents based on a specific epigenetic alteration in individual tumors. This is a step in the direction of personalized cancer medicine.

Abstract 290: EMT and cancer stem cells: role in radiation resistance of breast cancer.

Abstract Epithelial-Mesenchymal Transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. During EMT, epithelial cells lose epithelial characteristics, such as E-cadherin expression and gain mesenchymal properties, such as vimentin expression. EMT cells have stem cells properties and possess the characteristics of cell motility, invasiveness and chemotherapy resistance and these cancer stem cells may be responsible for mediating tumor metastasis and resistance to cancer treatments. However, very little is known about the role of EMT and cancer stem cells in modulating radiation response of human breast cancer cells. We compared expression levels of E-cadherin and other EMT related markers in ER-a negative (MDA-MB-231 and Hs578t) and ER-a positive (MCF-7) human breast cancer cells. Clonogenic cell survival assays showed that the cell lines expressing estrogen receptor (MCF-7) were more sensitive to increasing doses of radiation and had high expression of E-cadherin. In contrast, ER negative cells (MDA-MB-231 and Hs578t) had no detectable expression of E-cadherin and were more radioresistant. We transfected MDA-MB-231 and Hs578t cells with a CDH1-expression vector and isolated stable clones. These clones were selected and tested for radiosensitivity. MDA-MB-231 and Hs578t cells transfected with a control vector (pCMV) served as controls. Restoring E-cadherin expression radiosensitized the cells compared to the control vector cell line, suggesting that restoration of E-cadherin expression in mesenchymal-like cells produces a radiosensitizing effect. Our preliminary data demonstrates that EMT, detected as the loss of E-cadherin expression, may regulate tumor cell radiosensitivity, i.e. cells that have undergone EMT are relatively radioresistant compared to the lines that have retained the epithelial phenotype, which are relatively radiosensitive. Thus, there was a general correlation between EMT, based on loss of E-cadherin expression, and radioresistance. Overall, our results suggest that E-cadherin interacts with radiation and enhances the radioresponse of human breast cancer cells. Since it has been demonstrated that the process of EMT contributes to drug resistance and results in cells with CSC-like characteristics, we compared ALDH1 expression in ER positive (MCF-7) and ER negative (MDA-MB-231 and Hs578t) cell lines. Our data shows that ALDH1 expression correlates with the ER status of the breast cancer cells, with a higher number of ALDH1+ cells in ER negative compared to ER positive cell lines. Our preliminary investigation leads us to believe that potentially resistant breast cancer stem cell populations appear to be overrepresented in ER negative breast cancer cell lines. Our observation concurs with clinical data that ER negative cancers are resistant to radiation therapy compared to ER positive breast cancers. Citation Format: Anupama Munshi, Meghna Mehta, Yuhui Yuan, Huifeng Liu, Raymond E. Meyn, Rajagopal Ramesh. EMT and cancer stem cells: role in radiation resistance of breast cancer. [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 290. doi:10.1158/1538-7445.AM2013-290

Abstract 518: Kisspeptin stimulates invasiveness of ERα -negative human mammary epithelial and breast cancer cells.

Abstract Recently, we have shown that KP-10 stimulates invasion of estrogen receptor (ERα)-negative breast cancer cells via transactivation of the epidermal growth factor receptor (EGFR) [Zajac et al. (2011) PLoS ONE 6(6): e21599]. Here we report that KP-10 stimulated cell motility and invasiveness of ERα-negative non-malignant mammary epithelial MCF10A cells that endogenously expressing KISS1R, and MCF10A cells stably expressing KISS1R using three-dimensional (3D) Matrigel cultures. Additionally, exogenous expression of KISS1R in ERα-negative SKBR3 breast cancer cells stimulated invasion in 3D cultures and induced extravasation in vivo using chorioallantoic membrane (CAM) assays. Exogenous KISS1R expression in MCF10A and SKBR3 cells also induced a partial epithelial-to-mesenchymal transition like phenotype as evidenced by the acquisition of a spindle-shaped morphology, intracellular localization of the epithelial marker E-cadherin, increased stress fibre formation and elevated levels of the mesenchymal markers, Snail/Slug, vimentin and N-cadherin. In contrast, KP-10 had no effect on migration and invasion of the ERα-positive T47D and MCF7 breast cancer cells and failed to transactivate EGFR in the ERα-positive cells. This suggested that ERα negatively regulates KISS1R-dependent breast cancer cell migration, invasion and EGFR transactivation. In support of this, we found KP-10-stimulated cell migration, invasion and EGFR transactivation were ablated upon stably expressing ERα in the ERα-negative MDA-MB-231 cells. Lastly, we found that KISS1R was localized at the leading edge of motile cells, where it co-localized with the actin scaffolding protein, IQGAP1. We have identified IQGAP1 as a novel binding partner of KISS1R and have demonstrated that KISS1R regulates EGFR transactivation in breast cancer cells in an IQGAP1-dependent manner. Overall, our data strongly suggest that the ERα status of mammary cells will dictate whether the KISS1R signaling pathway may be a novel clinical target for the treatment of breast cancer metastasis. Citation Format: Moshmi M. Bhattacharya, Dragana Cvetkovic, Magdalena Dragan, Hon Sing Leong, Andy Babwah. Kisspeptin stimulates invasiveness of ERα -negative human mammary epithelial and 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 518. doi:10.1158/1538-7445.AM2013-518

Abstract 4385: The green tea polyphenol EGCG induces mesenchymal to epithelial transition (MET) and tumor regression in triple negative breast cancer (TNBC) cells and mouse xenograft model: involvement of CCN5.

Abstract Background: Epithelial to Mesenchymal transition (EMT) is an important and coordinated series of events associated with tumor metastasis and invasion. Recent studies had shown the importance of CCN5 (also known as WISP-2,Wnt-1-induced signaling protein-2) in the regulation of various carcinomas including the breast cancer. Recent studies had showed that ectopic expression of CCN5 can reverse Epithelial-Mesenchymal transition (EMT) and inhibit cancer metastasis. Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, has been extensively studied as a bioactive dietary component against various types of carcinomas through multiple mechanisms such as anti-oxidation, induction of apoptosis, inhibition of angiogenesis and metastasis. However, the mechanism of action of EGCG in breast carcinoma is uncertain. Objective: The objective of the present study is to determine whether CCN5 plays any significant role in EGCG-mediated cytotoxicity in triple negative breast cancer cells. Results: Exposure of triple negative human breast cancer (TNBC) cells, MDA-MB-231 and HCC70 to EGCG resulted in a dose-dependent inhibition of proliferation after 72h and the IC50 was observed around 75μM for MDA-MB-231 cells and 50μM for HCC70 respectively. We found EGCG-treatment effectively induces MET and inhibits the in vitro migration parallel with the induction of CCN5 expression in TNBC cells in a dose-dependent fashion. Furthermore, consistent with in vitro findings, tumor progression was drastically inhibited in EGCG-treated MDA-MB-231-tumor xenograft in nude mouse model. Conclusion: EGCG imparts its anti-cancer activity in both TNBC cells as well as MB-231-tumor xenografts via induction of CCN5. Citation Format: Amlan Das, Snigdha Banerjee, Archana De, Inamul Haque, Gargi Maity, Matt McEwen, Sushanta K. Banerjee. The green tea polyphenol EGCG induces mesenchymal to epithelial transition (MET) and tumor regression in triple negative breast cancer (TNBC) cells and mouse xenograft model: involvement of CCN5. [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 4385. doi:10.1158/1538-7445.AM2013-4385

Ethanolic extracts of California mugwort (Artemisia douglasiana Besser) are cytotoxic against normal and cancerous human cells

California mugwort (Artemisia douglasiana Besser) is used by many tribes throughout California to treat a variety of conditions, including colds, allergies, and pain. California mugwort is also utilized as women's medicine. Its use is on the rise outside of Native communities, often without the guidance of a traditional healer or experienced herbalist. Because it has been shown to have antiproliferative activity against plant and animal cells, we investigated whether California mugwort extracts have an effect on normal human cells as well as estrogen receptor positive (ER+) and estrogen receptor negative (ER-) human breast cancer cells. Ethanolic and aqueous extracts of A. douglasiana leaves were tested for cytotoxicity against unstimulated normal human peripheral blood mononuclear cells (hPBMC), as well as against an ER+ human breast cancer cell line (BT-474) and an ER- human breast cancer cell line (MDA-MB-231). An ethanolic leaf extract killed hPBMC, BT-474, and MDA-MB-231 cells with IC50 values of 23.6 ± 0.3, 27 ± 5, and 37 ± 4 μg/ml, respectively. An aqueous extract killed hPBMC with an IC50 value of 60 ± 10 μg/ml, but had no effect on the two cancer cell lines at concentrations up to 100 μg/ml. The results of this study indicate that the cytotoxicity of California mugwort extends to normal human cells, as well as cancerous cells. Therefore, until further is known about the safety of this medicine, caution should be taken when consuming extracts of California mugwort, whether as a tincture or as a tea.

The green tea polyphenol EGCG induces Mesenchymal to Epithelial Transition (MET) and tumor regression in Triple Negative Breast Cancer (TNBC) cells and mouse‐xenograft model: Involvement of CCN5

BackgroundEpithelial to Mesenchymal transition (EMT) is an important and coordinated series of events associated with tumor metastasis and invasionRecent studies had shown the importance of CCN5 in the regulation of various carcinomas including the breast cancer. EGCG, the most abundant green tea polyphenol, has been extensively studied as a bioactive dietary component against various types of carcinomas. However, the mechanism of action of EGCG in breast carcinoma is uncertain.ObjectiveThe objective of the present study is to determine whether CCN5 plays any significant role in EGCG‐mediated cytotoxicity in triple negative breast cancer cells.SummaryExposure of triple negative human breast cancer (TNBC) cells, MDA‐MB‐231 and HCC70 to EGCG resulted in a dose‐dependent inhibition of proliferation after 72h and the IC50 was observed around 75μM for MDA‐MB‐231 cells and 50μM for HCC70 respectively. We found EGCG‐treatment effectively induces MET and inhibits the in vitro migration parallel with the induction of CCN5 expression in TNBC cells in a dose‐dependent fashion. Furthermore, consistent with in vitro findings, tumor progression was drastically inhibited in EGCG‐treated MDA‐MB‐ 231‐tumor xenograft in nude mouse model.ConclusionEGCG imparts its anti‐cancer activity via induction of CCN5.(Support: VA Merit Grants, S. Banerjee and S.K. Banerjee)

Effects of steroidal aromatase inhibitors on sensitive and resistant breast cancer cells: Aromatase inhibition and autophagy

Several therapeutic approaches are used in estrogen receptor positive (ER+) breast cancers, being one of them the use of aromatase inhibitors (AIs). Although AIs demonstrate higher efficacy than tamoxifen, they can also exhibit de novo or acquired resistance after prolonged treatment. Recently, we have described the synthesis and biochemical evaluation of four steroidal AIs, 3β-hydroxyandrost-4-en-17-one (1), androst-4-en-17-one (12), 4α,5α-epoxyandrostan-17-one (13a) and 5α-androst-2-en-17-one (16), obtained from modifications in the A-ring of the aromatase substrate, androstenedione. In this study, it was investigated the biological effects of these AIs in different breast cancer cell lines, an ER+ aromatase-overexpressing human breast cancer cell line (MCF-7aro cells), an estrogen-receptor negative (ER−) human breast cancer cell line (SK-BR-3 cells), and a late stage of acquired resistance cell line (LTEDaro cells). The effects of an autophagic inhibitor (3-methyladenine) plus AIs 1, 12, 13a or exemestane in LTEDaro cells were also studied to understand the involvement of autophagy in AI acquired resistance. Our results showed that these steroids inhibit aromatase of MCF-7aro cells and decrease cell viability in a dose- and time-dependent manner. The new AI 1 is the most potent inhibitor, although the AI 12 demonstrates to be the most effective in decreasing cell viability. Besides, and in advantage over exemestane, AIs 12 and 13a also reduced LTEDaro cells viability. The use of the autophagic inhibitor allowed AIs to diminish viability of LTEDaro cells, presenting a similar behavior to the sensitive cells. Thus, inhibition of autophagy may sensitize hormone-resistant cancer cells to anti-estrogen therapies.

A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

IntroductionTumor cell migration and invasion are critical initiation steps in the process of breast cancer metastasis, the primary cause of breast cancer morbidity and death. Here we investigated the role of p21Cip1 (p21), a member of the core cell cycle machinery, in transforming growth factor-beta (TGFβ)-mediated breast cancer cell migration and invasion.MethodsA mammary fat pad xenograft mouse model was used to assess the mammary tumor growth and local invasion. The triple negative human breast cancer cell lines MDA-MB231 and its sub-progenies SCP2 and SCP25, SUM159PT, SUM149PT, SUM229PE and SUM1315MO2 were treated with 5 ng/ml TGFβ and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFβ transcriptional activity was measured by a TGFβ/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFβ downstream target genes. The interactions among p21, p/CAF and Smad3 were performed by co-immunoprecipitation. In addition, Smad3 on DNA binding ability was measured by DNA immunoprecipitation using biotinylated Smad binding element DNA probes. Finally, the association among active TGFβ/Smad signaling, p21 and p/CAF with lymph node metastasis was examined by immunohistochemistry in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node positive.ResultsWe found p21 expression to correlate with poor overall and distant metastasis free survival in breast cancer patients. Furthermore, using xenograft animal models and in vitro studies, we found p21 to be essential for tumor cell invasion. The invasive effects of p21 were found to correlate with Smad3, and p/CAF interaction downstream of TGFβ. p21 and p/CAF regulates TGFβ-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGFβ/Smad signaling correlates with high p21 and p/CAF expression levels and lymph node involvement using tissue microarrays from breast cancer patients.ConclusionsTogether these results highlight an important role for p21 and p/CAF in promoting breast cancer cell migration and invasion at the transcriptional level and may open new avenues for breast cancer therapy.

Hypoxia Regulates CD44 and Its Variant Isoforms through HIF-1α in Triple Negative Breast Cancer

BackgroundThe CD44 transmembrane glycoproteins play multifaceted roles in tumor progression and metastasis. CD44 expression has also been associated with stem-like breast cancer cells. Hypoxia commonly occurs in tumors and is a major cause of radiation and chemo-resistance. Hypoxia is known to inhibit differentiation and facilitates invasion and metastasis. Here we have investigated the effect of hypoxia on CD44 and two of its isoforms in MDA-MB-231 and SUM-149 triple negative human breast cancer cells and MDA-MB-231 tumors using imaging and molecular characterization.Methods and FindingsThe roles of hypoxia and hypoxia inducible factor (HIF) in regulating the expression of CD44 and its variant isoforms (CD44v6, CD44v7/8) were investigated in human breast cancer cells, by quantitative real-time polymerase chain reaction (qRT-PCR) to determine mRNA levels, and fluorescence associated cell sorting (FACS) to determine cell surface expression of CD44, under normoxic and hypoxic conditions. In vivo imaging studies with tumor xenografts derived from MDA-MD-231 cells engineered to express tdTomato red fluorescence protein under regulation of hypoxia response elements identified co-localization between hypoxic fluorescent regions and increased concentration of 125I-radiolabeled CD44 antibody.ConclusionsOur data identified HIF-1α as a regulator of CD44 that increased the number of CD44 molecules and the percentage of CD44 positive cells expressing variant exons v6 and v7/8 in breast cancer cells under hypoxic conditions. Data from these cell studies were further supported by in vivo observations that hypoxic tumor regions contained cells with a higher concentration of CD44 expression.

Abstract 139: The role of miRNAs in cyclooxygenase-2-mediated breast cancer progression

Abstract We had shown that over-expression of cyclo-oxygenase (COX)-2 in human, as well as murine breast cancer cells, promotes tumor progression and metastasis by multiple mechanisms: host immune cell inactivation and a stimulation of cancer cell migration, invasion, tumor-associated angiogenesis and lymphangiogenesis, which support blood-borne and lymph-borne metastasis. Most of these events resulted from activation of the prostanoid receptor EP4 by endogenous PGE2. Recently, by stable transfection of COX-2 cDNA into a non-metastatic, COX-2 negative human breast cancer cell line MCF-7, we showed that COX-2 induces all the phenotypic properties of stem-like or “tumor initiating cells” (TIC) in MCF-7-COX-2 cells, as defined by in vitro studies and validated in vivo. Through combined gene expression and microRNA (miRNA) micro array analysis, we identified two miRNAs (miR-526b and miR-655) that are up-regulated in MCF-7-COX-2 cells, associated with a down-regulation of 14 target genes linked with tumor-suppressor functions. We hypothesize that these miRNAs are important for COX-2 mediated TIC associated functions in human breast cancer. As a first step, we validated their expression in several COX-2 disparate human breast cancer cell lines: MCF-7 (COX-2 negative), MCF-7-COX-2, SKBR-3 (HER-2 over-expressing but COX-2 negative) and SKBR-3-COX-2 (COX-2 over-expressing following stable transfection of COX-2). The expression levels of miR-655 were strongly correlated with COX-2 mRNA (quantified RT-PCR) expression in these cell lines. Furthermore, the migratory and invasive capacities of the cell lines went hand in hand with miR-655 expression. Expression of miR-655 was markedly inhibited by treating MCF-COX-2 cells with a COX-2 inhibitor NS398 or an EP4 antagonist ONO-AE3-208, indicating that the expression depends on both COX-2 and EP4 activity. Finally, we discovered that cells derived from tumorspheres (in vitro correlate of TIC growth) produced by various human breast cancer cell lines (Hs578T, T47D, MDA-MB-231, SKBR-3, SKBR-3-COX-2, and MCF-7-COX-2) grown on low attachment plates, exhibited a dramatic increase in COX-2 expression in comparison to the cells grown as monolayer. We also found that the tumorspheres over-express miR-655. These findings, taken together, fortify the notion that COX-2, EP4 and COX-2 induced miR-655 expression, play important roles in promoting and maintaining the TIC phenotype in breast cancer cells. In support, COX-2 inhibitors and EP4 antagonists were highly effective in abrogating tumor growth, tumor- associated angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in our mouse breast cancer model. Currently we are testing whether EP4 and miR-655 serve as useful prognostic markers and therapeutic targets in human breast cancer. (Supported by the CBCF, Ontario chapter and the OICR funds to PKL. LD is a TBCRU scholar.) 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 139. doi:1538-7445.AM2012-139

Growth of Triple Negative Human Breast Cancer Cells is Regulated by the OGF‐OGFr Axis

Breast cancer is the most common cancer, and the leading cause of cancer death, in females worldwide. Triple negative breast cancer (TNBC) accounts for 10–20% of all breast cancers. There are no targeted therapies for TNBC. The opioid growth factor (OGF) and its receptor (OGFr) is a tonically active inhibitory pathway that regulates normal and neoplastic cell proliferation, and we hypothesize that the OGF‐OGFr axis is a determinant of the progression of TNBC. Both OGF and OGFr were detected in TNBC human cell line MDA‐MB‐231 by immunohistochemistry. Receptor binding assays revealed specific and saturable binding (Bmax = 8.5 ± 1.8 fmol/mg protein; Kd = 4.1 ± 1.1 nM). Exogenous OGF depressed growth by 19 to 28% beginning at 24 hr and persisting to 120 hr. Growth inhibition was dose‐dependent (10−4M to 10−10 M), reversible, and receptor‐mediated, and suppression of cell proliferation was dependent on RNA and protein synthesis. Other endogenous and synthetic opioids, including those targeting μ, δ, and κ opioid receptors, did not alter growth at a concentration of 10−6 M. Cells with a knockdown of OGFr using siRNA did not respond to OGF. A complete blockade of the OGF‐OGFr axis with the opioid antagonist naltrexone increased cell number, indicating that this peptide‐receptor complex is critical in maintaining homeostasis of cell proliferation. These data show that the OGF‐OGFr system is present and functioning as a native biological regulator of human breast cancer, and will be important in designing treatment strategies for this group of deadly neoplasias. Supported by the Paul K. and Anna E. Shockey Family Foundation.

Anticancer Activity of Proapoptotic Peptides is Highly Improved by Thermal Targeting using Elastin-like Polypeptides

Inducing apoptosis in cancer cells is an effective strategy for cancer therapy. The cationic α-helix forming KLAKLAKKLAKLAK peptide (KLAK) has been known to induce apoptosis by disrupting the mitochondria. In the present study, we have designed a thermally targeted KLAK peptide by genetically engineering the KLAK sequence to the carboxy terminus of the heat responsive biopolymer elastin-like polypeptide (ELP). The cellular internalization of ELP-KLAK was made possible by engineering a cell penetrating peptide sequence (SynB1) to the amino terminus of ELP. The SynB1-ELP1-KLAK fusion polypeptide was cytotoxic against both estrogen receptor positive and negative human breast cancer cell lines. The potency of SynB1-ELP1-KLAK was further enhanced when mild hyperthermia was added to the treatment. In response to hyperthermia, SynB1-ELP1-KLAK selectively triggered apoptosis, which was associated with disruption of the mitochondria. The thermally responsive SynB1-ELP-KLAK polypeptide can have improved tumor targeting by the application of mild hyperthermia. Furthermore, the pharmacokinetic properties of ELP can prevent degradation of KLAK in vivo, and the use of SynB1 can mediate tumor cell uptake, thereby augmenting the effect of KLAK.

P2-03-05: Attenuation of TGF-beta Signaling Suppresses Premature Senescence in a p21-Dependent Manner and Promotes Oncogenic ras-Mediated Metastatic Transformation in hTERT-Immortalized Basal-Like Human Mammary Epithelial Cells.

Abstract Triple negative, basal-like breast cancer is highly aggressive and has a poor prognosis. The molecular mechanisms that drive its progression are elusive and no molecular target has been identified for its prevention or treatment. Here, we demonstrate that triple negative human breast cancer cells and clinical samples show an attenuated transforming growth factor-beta (TGF-β) signaling. Therefore, we developed a series of isogenic basal-like human mammary epithelial cells (HMECs) with altered TGF-β sensitivity and different malignancy, resembling a full spectrum of basal-like breast carcinogenesis, and determined the molecular mechanisms that contribute to oncogene-induced transformation of basal-like HMECs when TGF-β signaling is attenuated. We found that expression of a dominant-negative RII (DNRII) receptor of TGF-β abrogated autocrine TGF-β signaling in telomerase-immortalized HMECs and suppressed H-ras-V12-induced senescence-like growth arrest (SLGA). Furthermore, co-expression of DNRII and H-ras-V12 rendered HMECs to become highly tumorigenic and metastatic in vivo in comparison with H-ras-V12-transformed HMECs that spontaneously escaped H-ras-V12-induced SLGA. By using microarray analysis, we found that p21 is the major player mediating Ras-induce SLGA and attenuated or loss of p21 expression contributed to the escape from SLGA when autocrine TGF-β signaling is blocked in HMECs. Furthermore, knockdown of p21 also suppressed H-ras-V12-induced SLGA. Our results identifies that autocrine TGF-β signaling is an integral part of cellular antitransformation network by suppressing the expression of a host of genes including p21-regulated genes that mediate oncogene-induced transformation in basal-like breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-03-05.

P1-02-08: Modulation of EMT by Targeting E-Cadherin Restores Radiation Sensitivity in Human Breast Cancer Cells.

Abstract E-cadherin is the major adhesion protein associated with epithelial cells and loss of its expression is diagnostic of the epithelial to mesenchymal transition (EMT). Findings from our laboratory, suggest that EMT, detected as the loss of E-cadherin expression, may regulate tumor cell radiosensitivity, i.e. cells that have undergone EMT are relatively radioresistant compared to the lines that have retained the epithelial phenotype, which typically are relatively radiosensitive. To definitively test this hypothesis we compared expression levels of E-cadherin and other EMT related markers in ER-a negative (MDA-MB-231 and Hs578t) and ER-a positive (MCF-7) human breast cancer cells. We observed that many ER-a negative lines, were also E-cadherin negative, and the ER-a positive lines were positive for E-cadherin expression. Clonogenic cell survival assays showed that the cell lines expressing estrogen receptor (MCF-7) and had high expression of E-cadherin, were also more sensitive to increasing doses of radiation. Treatment of MCF-7 cells with 100nM siRNA to E-cadherin for 72 hrs made the cells more resistant to radiation with the surviving fraction changing from 42.3% in control cells to 56.4% in siRNA treated cells. In contrast, ER negative cells (MDA-MB-231 and Hs578t) had no detectable expression of E-cadherin and were more radioresistant. We transfected MDA-MB-231 and Hs578t cells with a CDH1 -expression vector and isolated stable clones, which were then tested for radiosensitivity. Cells transfected with the backbone vector (pCMV) served as controls. Restoring E-cadherin expression radiosensitized both, MDA-MB-231 and Hs578t cells, compared to the control vector cell line, suggesting that restoration of E-cadherin expression in mesenchymal-like cells produces a radiosensitizing effect. These observations suggest that ER-a signaling might regulate E-cadherin and implied that this regulation might influence EMT and radioresistance. Overall, our results suggest that the EMT status of breast cancer cells governs their response to radiation. A detailed investigation of the mechanism is ongoing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-08.

Abstract A101: Metformin prevents mammary tumors in p53-null mammary gland mice

Abstract Background: Breast cancer prevention is now possible using antiestrogen drugs such as tamoxifen and raloxifene; however, antiestrogens are ineffective against estrogen receptor (ER)-negative breast cancers. Since ER-negative breast cancers are highly aggressive and particularly difficult to treat, preventive agents against these life-threatening cancers are urgently needed. Metformin, a first line treatment for adult-onset diabetes, recently was shown to be associated with reduced incidence of cancer (including breast cancer) in epidemiologic studies. Metformin activates AMP-dependent kinase (AMPK) and stimulation of AMPK by metformin reduces cell proliferation in both estrogen receptor alpha (ERa)-positive and -negative human breast cancer cell lines. We hypothesized that the metformin would effectively prevent the growth and development of ER-negative breast cancer. To test this hypothesis, we tested metformin's ability to prevent mammary tumors in two mouse models: the MMTV-erbB2 transgenic mouse model that develops ER-negative mammary tumors and the p53-null mammary gland mouse model that develops both ER-positive and ER-negative mammary tumors. Methods: MCF7, BT474, MDA MB 468 and MDA MB 453 cells were treated with10mM metformin and cell proliferation was measured using an MTS assay. Virgin female MMTV-erbB2 mice (Jackson Lab) were housed in the institutional animal facilities and fed a purified diet (AIN-76A; Harlan Teklad). Mice were separated into 2 treatment groups: 1) Vehicle control (PBS via gastric gavage daily), 2) Metformin (250mg/kg via gastric gavage daily; from 3 months to 12 months of age). P53-null Balb/C mammary glands from donor mice were transplanted into both cleared inguinal mammary fat pads of Balb/C p53 wild-type mice. Mice were observed daily for tumor formation and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was visualized using Kaplan Meier curves and analyzed using the generalized Wilcoxon test. Results: Metformin (at 10mM) significantly reduced proliferation of breast cancer cells irrespective to their ER, Her2 and p53 status. In the MMTV-erbB2 mice, metformin treatment did not prevent or delay mammary tumor formation. However, in p53-null mammary gland mice, metformin completely prevented mammary tumor formation. Conclusions: Metformin treatment totally prevented mammary tumor development in p53-null mammary gland mice. These results suggest that metformin will be most useful in preventing breast cancers with P53 mutations such as occurs in Li-Fraumeni syndrome individuals or individuals with ERa-negative or “triple-negative” breast cancer. These results support testing metformin in clinical trials for the prevention of ER-negative and p53-mutant breast cancers. This research was supported by the NIH/NCI R01 CA78480 grant (PHB). Citation Information: Cancer Prev Res 2011;4(10 Suppl):A101.