Breast Cancer-derived Cell Lines Research Articles

Hormone receptor-independent CXCL10 production is associated with the regulation of cellular factors linked to breast cancer progression and metastasis

Breast cancer (BC) is a major health problem for women around the world. Although advances in the field of molecular therapy have been achieved, the successful therapeutic management of BC, particularly metastatic disease, remains a challenge for patients and clinicians. One of the areas of current investigation is the circulating tumor cells (CTCs), which have a determinant role in the development of distant metastasis. At the present, many of the available treatment strategies for metastatic disease are of limited benefit. However, the elucidation of the mechanisms of tumor progression and metastasis may help to identify key molecules/components that may function as therapeutic targets in the future. In the present study, the functional analysis of CTCs revealed their ability to grow and proliferate to form colonies. Immunofluorescence staining of the CTCs' colonies exhibits elevated expression of cell growth and survival associated proteins such as, survivin, ERK and Akt1. More importantly, the functional screening of the chemokine profile in BC patients' sera revealed an HR-independent elevation of the chemokine CXCL10 when compared to healthy controls. The analysis of chemokines CXCL9 and CXCL11 demonstrated an HR-dependent production pattern. The levels of both CXCL9 and CXCL11 were markedly high in HR+ patients' sera when compared to HR− patients and healthy controls. The functional analysis of HR+ and HR− BC derived cell lines when cultivated in media supplemented with patients' sera demonstrated the alteration of tumor progression and metastasis related proteins. We noted the induction of survivin, β-catenin, MKP-1, pERK, CXCR4 and MMP-1 both at the protein and mRNA levels. The induction of those proteins was in keeping with patients' sera induced cell proliferation as measured by the MTT assay. In conclusion, our data emphasizes the role of chemokines, especially CXCL10, in BC progression and metastasis via the induction of signaling pathways, which mainly involve survivin, β-catenin, MKP-1 and MMP-1.

Potential signaling molecules involved in the progression of metastatic prostate, breast and colorectal cancer (TUM3P.1051)

Abstract Metastases and their complications lead to death in up to 90% of cancer patients. Hematogenous metastasis is associated with loss of cell adhesion, migration in the blood (as individual circulating tumor cells, (CTC) or clusters), adhesion to the target organ and proliferation to form metastases. The present investigation was designed to demonstrate key molecules in the sera of metastatic cancer patients that induce ERK 1/2 and Survivin protein expression. We showed that CTCs grown in media supplemented with patient sera express the potential to form colonies and spheres followed by senescence. The cultured CTCs overexpressed ERK and Survivin, which reflects their aggressiveness. We demonstrated by immunofluorescence and western blot, with confirmation by qRT-PCR (in progress), that prostate, breast and colorectal cancer-derived cell lines cultured in media supplemented with sera from patients with CTCs, induced expression of ERK 1/2 and Survivin proteins. Using the MTT assay, we demonstrated that cancer-derived cell lines treated with sera from CTC positive patients showed accelerated proliferation compared to controls. It is anticipated that correlation with clinical data might reveal future potential targets for adjuvant therapy to minimize the risk of metastasis.

Abstract P2-16-01: Discovery of fully human monoclonal antibodies as therapeutic candidates for the treatment of HER2-negative breast cancer

Abstract Unlike HER2-positive breast cancer, there are limited treatment options for patients diagnosed with triple negative and endocrine resistant HER2-negative breast cancer, and as such HER2-negative breast cancer represents a significant unmet medical need. The goal of this work is to use Theraclone’s proprietary I-STAR platform to mine the memory B cell immune repertoire of breast cancer patients for the discovery of therapeutically relevant monoclonal antibodies (mAbs) and targets that may be exploited as candidates for treatment of HER2-negative breast cancer. Matched serum and PBMC samples were collected from breast cancer patients at multiple clinical sites. The selected patient populations included, but were not limited to, patients who were treatment naïve, those who had received immunotherapy or adjuvant chemotherapy, and patients who had an exceptional clinical response to treatment and/or disease. Serum antibody binding to a diverse panel of 5 well characterized breast cancer-derived cell lines of luminal and basal sub-types was determined. Utilizing this approach, patients with a robust serological profile across multiple breast cancer cell lines were identified and prioritized for memory B cell repertoire analysis via the I-STAR platform. Using a high throughput and miniaturized, multiplex flow cytometry assay, the secreted IgG antibodies from over 85,000 individually enriched and expanded B cell clones were screened for binding to the tumor cell line panel and a large number of positive B cell clones were identified. The screening hits can be binned into several unique binding profiles, many of which were confirmed to be shared across multiple patient samples. Deep sequence analysis of the variable regions of the antibodies produced by the B cell clones demonstrated that several of the screening hits were derived from clonally related B cells; the majority of the screening hits represented antibodies derived from unique B cell clones. A representative set of these antibodies was expressed recombinantly for further in vitro characterization. Citation Format: Christy Boozer, Paul Algate, Aurelio Bonavia, Mark Branum, Po-Ying Chan-Hui, Alison Fitch, Brad Greenfield, Claire Sutherland, Kristine Swiderek. Discovery of fully human monoclonal antibodies as therapeutic candidates for the treatment of HER2-negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-16-01.

KLF15 in breast cancer: a novel tumor suppressor?

Krüppel-like factor 15 (KLF15) is a transcription factor that is involved in various biological processes, including cellular proliferation, differentiation and death. In addition, KLF15 has recently been implicated in the development of several human malignancies, including breast cancer. In vitro breast cancer studies have pointed at a putative role in the regulation of cell proliferation. As yet, however, KLF15 expression analyses in primary human breast cancers have not been reported. Here, we set out to investigate the clinical and biological significance of KLF15 expression in human breast cancers. KLF15 expression was evaluated by immunohistochemistry in 54 primary invasive ductal breast carcinomas, and its status was correlated with various clinicopathological parameters. We also assessed KLF15 expression in vitro in 4 breast cancer-derived cell lines using Western blotting, and examined the effects of exogenous KLF15 expression on cell cycle progression using flow cytometry. Concomitant (changes in) p21, p27 and TOPO2A expression levels were examined using real-time RT-PCR and immunocytochemistry, respectively. In ~90% of the primary breast carcinoma tissues tested, KLF15 was found to be expressed and localized in either the cytoplasm, the nucleus or both. Predominant nuclear immunoreactivity was found to be associated with clinicopathological factors predicting a better clinical outcome (i.e., ER positive, HER2 negative, low grade, low Ki-67 expression). The breast cancer-derived cell lines tested showed a low KLF15 expression with a predominant cytoplasmic localization. Subsequent exogenous KLF15 over-expression resulted in a predominant nuclear localization and a concomitant decreased cellular proliferation and an arrest at the G0/G1 phase of the cell cycle. In addition, we found that nuclear KLF15 expression results in up-regulation of p21, a pivotal suppressor of the G1 to S phase transition of the cell cycle. Our results indicate that nuclear KLF15 expression suppresses breast cancer cell proliferation at least partially through p21 up-regulation and subsequent cell cycle arrest. This is a first study addressing the role of KLF15 in breast cancer development.

Combined enzyme/prodrug treatment by genetically engineered AT-MSC exerts synergy and inhibits growth of MDA-MB-231 induced lung metastases.

BackgroundMetastatic spread of tumor cells remains a serious problem in cancer treatment. Gene-directed enzyme/prodrug therapy mediated by tumor-homing genetically engineered mesenchymal stromal cells (MSC) represents a promising therapeutic modality for elimination of disseminated cells. Efficacy of gene-directed enzyme/prodrug therapy can be improved by combination of individual systems. We aimed to define the combination effect of two systems of gene therapy mediated by MSC, and evaluate the ability of systemically administered genetically engineered mesenchymal stromal cells to inhibit the growth of experimental metastases derived from human breast adenocarcinoma cells MDA-MB-231/EGFP.MethodsHuman adipose tissue-derived mesenchymal stromal cells (AT-MSC) were retrovirally transduced with fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT) or with Herpes simplex virus thymidine kinase (HSVtk). Engineered MSC were cocultured with tumor cells in the presence of prodrugs 5-fluorocytosin (5-FC) and ganciclovir (GCV). Combination effect of these enzyme/prodrug approaches was calculated. SCID/bg mice bearing experimental lung metastases were treated with CD::UPRT-MSC, HSVtk-MSC or both in combination in the presence of respective prodrug(s). Treatment efficiency was evaluated by EGFP-positive cell detection by flow cytometry combined with real-time PCR quantification of human cells in mouse organs. Results were confirmed by histological and immunohistochemical examination.ResultsWe demonstrated various extent of synergy depending on tested cell line and experimental setup. The strongest synergism was observed on breast cancer-derived cell line MDA-MB-231/EGFP. Systemic administration of CD::UPRT-MSC and HSVtk-MSC in combination with 5-FC and GCV inhibited growth of MDA-MB-231 induced lung metastases.ConclusionsCombined gene-directed enzyme/prodrug therapy mediated by MSC exerted synergic cytotoxic effect and resulted in high therapeutic efficacy in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0149-2) contains supplementary material, which is available to authorized users.

In-silico identification and functional validation of allele-dependent AR enhancers.

Androgen Receptor (AR) and Estrogen Receptors (ERs) are key nuclear receptors that can cooperate in orchestrating gene expression programs in multiple tissues and diseases, targeting binding elements in promoters and distant enhancers. We report the unbiased identification of enhancer elements bound by AR and ER-α whose activity can be allele-specific depending on the status of nearby Single Nucleotide Polymorphisms (SNP). ENCODE data were computationally mined to nominate genomic loci with: (i) chromatin signature of enhancer activity from activation histone marks, (ii) binding evidence by AR and ER-α, (iii) presence of a SNP. Forty-one loci were identified and two, on 1q21.3 and 13q34, selected for characterization by gene reporter, Chromatin immunoprecipitation (ChIP) and RT-qPCR assays in breast (MCF7) and prostate (PC-3) cancer-derived cell lines. We observed allele-specific enhancer activity, responsiveness to ligand-bound AR, and potentially influence on the transcription of closely located genes (RAB20, ING1, ARHGEF7, ADAM15). The 1q21.3 variant, rs2242193, showed impact on AR binding in MCF7 cells that are heterozygous for the SNP. Our unbiased genome-wide search proved to be an efficient methodology to discover new functional polymorphic regulatory regions (PRR) potentially acting as risk modifiers in hormone-driven cancers and overall nominated SNPs in PRR across 136 transcription factors.

Calcitriol and its analogues enhance the antiproliferative activity of gefitinib in breast cancer cells

Coexpression of EGFR and HER2 has been associated with poor disease outcome, high rates of metastasis and resistance to conventional treatments in breast cancer. Gefitinib, a tyrosine kinase inhibitor, reduces both cell proliferation and tumor growth of breast cancer cells expressing EGFR and/or HER2. On the other hand, calcitriol and some of its synthetic analogs are important antineoplastic agents in different breast cancer subtypes. Herein, we evaluated the effects of the combined treatment of gefitinib with calcitriol or its analogs on cell proliferation in breast cancer cells.The presence of EGFR, HER2 and vitamin D receptor were evaluated by Western blot in two established breast cancer cell lines: SUM-229PE, SKBR3 and a primary breast cancer-derived cell line. The antiproliferative effects of gefitinib alone or in combination with calcitriol and its analogs, calcipotriol and EB1089, were assessed by growth assay using a DNA content-based method. Inhibitory concentrations on cell proliferation were calculated by non-linear regression analysis using sigmoidal fitting of dose-response curves. Pharmacological effects of the drug combinations were calculated by the Chou–Talalay method. Phosphorylation of ERK1/2 MAPK was evaluated by Western blot. Gene expression of EGFR, HER2 and BIM was assessed by real time PCR. BIM protein levels were analyzed in cells by flow cytometry. The effects of the drugs alone or combinated on cell cycle phases were determined using propidium iodide. Apoptosis was evaluated by detection of subG1 peak and determination of active caspase 3 by flow cytometry.Gefitinib, calcitriol, calcipotriol and EB1089 inhibited cell proliferation in a dose dependent manner. The combinations of gefitinib with calcitriol or its analogs were more effective to inhibit cell growth than each compound alone in all breast cancer cells studied. The gene expression of EGFR and HER2 was downregulated and not affected, respectively, by the combined treatment. Furthermore, phosphorylation of ERK 1/2 was inhibited a greater extent in co-treated cells than in the cells treated with alone compounds. The combination of gefitinib with calcitriol or their synthetic analogs induced apoptosis in SUM-229PE cells, this was shown by the significant upregulation of BIM protein levels, higher percentages of cells in subG1 peak and increase of caspase 3-positive cells.The combination of gefitinib with calcitriol or their synthetic analogs resulted in a greater antiproliferative effect than with either of the agents alone in EGFR and HER2 positive breast cancer cells. The mechanistic explanation for these results includes downregulation of MAPK signaling pathway, decrease of cells in G2/M phase and induction of apoptosis mediated by upregulation of BIM and activation of caspase 3.This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’.

JHDM1B expression regulates ribosome biogenesis and cancer cell growth in a p53 dependent manner.

Tumors characterized by an intense ribosome biogenesis often display a more aggressive behavior. Ribosomal RNA (rRNA) synthesis is controlled at several levels, including the epigenetic regulation of the condensation of chromatin portions containing rRNA genes. JHDM1B (Jumonji C histone demethylase 1B) is a histone demethylase able to regulate the accessibility of rRNA genes. In this study, we aimed to define the contribution of JHDM1B expression to the features of breast cancer, a tumor type whose behavior is related to the rate of ribosome biogenesis. We show that, in breast cancer-derived cell lines, the increase in rRNA transcription that follows JHDM1B knock-down is mirrored by an augmented cell proliferation only in p53 compromised cells, while p53 competent cells undergo cellular senescence and death. The latter effect appears to be mediated by a p38-dependent phosphorylation of p53, inducing the expression of p15(Ink4b) and p21(Waf1). In breast cancers, lower JHDM1B expression correlates with an increased size of specifically stained nucleolar organized regions, a morphological parameter directly related to the rate of ribosome biogenesis and with a poorer prognosis. In addition, in tumors lacking the controller function of p53, a lower expression of JHDM1B is associated with an increased tumor size at diagnosis. Altogether, our data indicate that epigenetic activation of rDNA genes induced by JHDM1B depletion is associated with a p53-dependent growth arrest, but may promote cancer cell growth when p53 is lacking.

Abstract 5145: KDM2B expression regulates ribosome biogenesis and cancer cell growth in a p53-dependent manner

Abstract Ribosome biogenesis is a cellular process requiring a fine-tuned control, as its deregulation is linked to cancer progression. Indeed, tumors characterized by an intense ribosome biogenesis often display a more aggressive biological and clinical behavior. Ribosomal RNA (rRNA) synthesis is controlled at several levels, the higher one being the epigenetic regulation of chromatin portions containing rRNA genes. KDM2B (Lysine (K)-specific demethylase 2B) is a histone demethylase modulating the accessibility of rRNA genes, thereby repressing their transcription. In particular KDM2B is known to specifically act on trimethylated K4 of histone H3 (H3K4) associated with rDNA. In this study we aimed to define the contribution of KDM2B expression to the biological features of breast cancer, a tumor type whose clinical behavior is known to be related to the rate of ribosome biogenesis. We show that, in primary breast carcinomas, lower KDM2B expression correlates with an increased size of specifically stained nucleolar organizer regions, a morphological parameter directly related to the rate of ribosome biogenesis, and with a poorer prognosis. In vitro, in breast cancer-derived cell lines, siRNA mediated KDM2B knock-down (KD) induced an increase in H3K4 trimethylation and in rRNA transcription. However, this is mirrored by an augmented cell proliferation only in p53 compromised cells, while p53 competent cells undergo cellular senescence and death. The latter effect, appears to be mediated by a p38-mediated phosphorylation of p53, inducing the expression of p15Ink4b and p21Waf1, and is not linked to p53 stabilization. In the long term, stable KDM2B KD in p53-compromised breast cancer cells induced a series of additional biological features including their augmented cellular clonogenic potential and invasive capability. Altogether our data indicate that abnormal JHDM1B-mediated epigenetic activation of rDNA genes elicits a p53-mediated growth arrest, but may promote cancer cell growth when p53 is lacking. Citation Format: Marianna Penzo, Lucia Casoli, Laura Sicuro, Alice Galibiati, Daniela Pollutri, Marzia Govoni, Claudio Ceccarelli, Donatella Santini, Mario Taffurelli, Davide Treré, Lorenzo Montanaro. KDM2B expression regulates ribosome biogenesis and cancer cell growth in a p53-dependent manner. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5145. doi:10.1158/1538-7445.AM2014-5145

ARID3B expression in primary breast cancers and breast cancer-derived cell lines.

ARID3B (AT-rich interaction domain 3) is a member of the family of ARID proteins, which constitutes evolutionarily conserved transcription factors implicated in normal development, differentiation, cell cycle regulation and chromatin remodeling. In addition, ARID3B has been linked to cellular immortalization, epithelial-mesenchymal transition (EMT) and tumorigenesis. Given the emerging role of ARID3B in tumor development, we examined its expression in primary patient-derived breast cancer samples and breast cancer-derived cell lines. Immunohistochemistry (IHC) was used to detect ARID3B expression in 63 formalin-fixed paraffin-embedded (FFPE) invasive breast cancer samples. In addition, a panel of 6 (estrogen receptor-positive and -negative, ERBB2-positive and -negative) breast cancer-derived cell lines and immortalized non-tumorigenic epithelial breast cells were used for ARID3B expression analysis using RT-PCR. Specific primers and Western blotting were used to detect ARID3B isoforms. Using IHC, nuclear, cytoplasmic and low levels of membranous ARID3B staining were detected in all 63 primary invasive breast tumors. Nuclear ARID3B staining positively correlated with estrogen receptor (ER) status and negatively correlated with tumor grade, mitotic index and ERBB2 status of the patients. Increased nuclear expression of ARID3B was confirmed in breast cancer-derived cell lines expressing ERα. In addition, two out of three ERBB2-positive breast cancer cell lines were found to lack full length ARID3B. Three ARID3B isoforms were found to be present in normal breast epithelial cells as well as in breast cancer cells. We report a positive correlation between ER positivity and nuclear ARID3B expression in primary breast cancers, along with a negative correlation with the ERBB2 status. Very similar correlations were noted in breast cancer-derived cell lines. Since in the recent past ARID3B expression has increasingly been related to cancer-associated proteins and microRNAs, knowledge on ARID3B expression and function may be instrumental for gaining further insight into potentially important cancer-related networks.

Translational suppression of HIF-1α by miconazole through the mTOR signaling pathway.

Miconazole is an imidazole antifungal agent that has amply been used in the treatment of superficial mycosis. Preliminary data indicate that miconazole may also induce anticancer effects. As yet, however, little is known about the therapeutic efficacy of miconazole on cancer and the putative mechanism(s) involved. Here, we show that miconazole suppresses hypoxia inducible factor-1α (HIF-1α) protein translation in different cancer-derived cells. The effect of miconazole on HIF-1α expression was examined by Western blotting and reverse transcriptase polymerase chain reaction assays in human U87MG and MCF-7 glioma and breast cancer-derived cell lines, respectively. The transcriptional activity of the HIF-1 complex was confirmed using a luciferase assay. To assess whether angiogenic factors are increased under hypoxic conditions in these cells, vascular endothelial growth factor (VEGF) levels were measured by ELISA. Metabolic labeling was performed to examine HIF-1α protein translation and global protein synthesis. The role of the mammalian target of rapamycin (mTOR) signaling pathway was examined to determine translation regulation of HIF-1α after miconazole treatment. Miconazole was found to suppress HIF-1α protein expression through post-transcriptional regulation in U87MG and MCF-7 cells. The suppressive effect of HIF-1α protein synthesis was found to be due to inhibition of mTOR. Miconazole significantly inhibited the transcriptional activity of the HIF-1 complex and the expression of its target VEGF. Moreover, miconazole was found to suppress global protein synthesis by inducing phosphorylation of the translation initiation factor 2α (eIF2α). Our data indicate that miconazole plays a role in translational suppression of HIF-1α. We suggest that miconazole may represent a novel therapeutic option for the treatment of cancer.

Antiproliferative activity and induction of apoptotic by ethanolic extract of Alpinia galanga rhizhome in human breast carcinoma cell line

BackgroundWe investigated the potential of galangal rhizomes to induce cytotoxic and apoptotic effects in the cultured human breast carcinoma cell line, (MCF-7) in compare with the non-malignant (MRC-5) cells.MethodsBoth cells were cultured in DMEM medium and treated with galangal rhizomes for three consecutive days. The percentage of apoptotic cells was determined by flow cytometry using Annexin-V fluorescein isothiocyanate.ResultsThe results showed that the ethanolic extract of galangal rhizomes decreased cell viability in the malignant cells as a concentration- and time- dependent manner. The IC50 values against MCF-7 were determined at 400.0 ± 11.7 and 170.0 ± 5.9 μg/ml after 48 and 72 h respectively. The morphology of MCF-7 cells treated with the ethanolic extract confirmed the cell proliferation assay results. Alpinia galanga induced apoptosis in MCF-7 cells, as determined by flow cytometry.ConclusionsWe concluded that the extract of Alpinia galanga exerts pro-apoptotic effects in a breast cancer-derived cell line and could be considered as a potential chemotherapeutic agent in breast cancer.

Development of a Human Breast-Cancer Derived Cell Line Stably Expressing a Bioluminescence Resonance Energy Transfer (BRET)-Based Phosphatidyl Inositol-3 Phosphate (PIP3) Biosensor

Stimulation of tyrosine kinase receptors initiates a signaling cascade that activates PI3K. Activated PI3K uses PIP2 to generate PIP3, which recruit Akt to the plasma membrane through its pleckstrin homology (PH) domain, permitting its activation by PDKs. Activated Akt controls important biological functions, including cell metabolism, proliferation and survival. The PI3K pathway is therefore an attractive target for drug discovery. However, current assays for measurement of PIP3 production are technically demanding and not amenable to high-throughput screening. We have established a MCF-7-derived breast cancer cell line, that stably co-expresses the PH domain of Akt fused to Renilla luciferase and YFP fused to a membrane localization signal. This BRET biosensor pair permits to monitor, in real time, in living cells, PIP3 production at the plasma membrane upon stimulation by different ligands, including insulin, the insulin analogue glargine, IGF1, IGF2 and EGF. Moreover, several known inhibitors that target different steps of the PI3K/Akt pathway caused inhibition of ligand-induced BRET. Cetuximab, a humanized anti-EGF receptor monoclonal antibody used for the treatment of cancer, completely inhibited EGF-induced BRET, and the tyrosine kinase inhibitor tyrphostine AG1024 inhibited insulin effect on PIP3 production. Moreover, the effects of insulin and IGF1 were inhibited by molecules that inhibit PI3K catalytic activity or the interaction between PIP3 and the PH domain of Akt. Finally, we showed that human serum induced a dose-dependent increase in BRET signal, suggesting that this stable clone may be used as a prognostic tool to evaluate the PI3K stimulatory activity present in serum of human patients. We have thus established a cell line, suitable for the screening and/or the study of molecules with stimulatory or inhibitory activities on the PI3K/Akt pathway that will constitute a new tool for translational research in diabetes and cancer.

Sprouty4 interferes with cell proliferation and migration of breast cancer-derived cell lines

Sprouty proteins are modulators of mitogen-induced signal transduction processes and therefore can influence the process of cancerogenesis. In particular, Sprouty2 has been shown to have an important role in cancer development of many tumor entities including breast cancer. In this report, we investigated the role of Sprouty4 in breast cancer-derived cell lines. We have found that ectopic Sprouty4 expression inhibits cell proliferation of breast cancer cell lines independently of their endogenous expression levels. Corroborating Sprouty4 downregulation causes accelerated growth. Furthermore, we demonstrate that an increase in Sprouty4 content interferes with serum-induced activation of mitogen-activated protein kinase pathway. Additionally, Sprouty4 expression negatively influences cell migration. These data suggest that Sprouty4 is a possible candidate for a tumor suppressor in breast cancer.

Impact of SOX18 expression in cancer cells and vessels on the outcome of invasive ductal breast carcinoma

SOX18 is a transcription factor known to be involved in hair follicle, blood and lymphatic vessel development, as well as wound healing processes (together with SOX7 and SOX17). In addition, it has been reported that SOX18 may affect the growth of cancer cells in vitro. Until now, the exact role of SOX18 expression in invasive ductal breast carcinoma (IDC) has remained unknown. In this study, we have investigated SOX18 expression in cancer cells and endothelial cells in 122 IDC samples using immunohistochemistry (IHC). SOX18 expression was also determined using real-time PCR and Western blotting in a series of breast cancer-derived cell lines (i.e., MCF-7, BT-474, SK-BR-3, MDA-MB-231, BO2). Using IHC, we observed SOX18 nuclear expression in cancer cells, as well as in blood and lymphatic vessels of the IDC samples tested. SOX18 expression in the IDC samples correlated with a higher malignancy grade (Grade 2 and Grade 3 versus Grade 1; p = 0.02 and p = 0.009, respectively) and VEGF-D expression (r = 0.27, p = 0.007). SOX18 expression was also associated with HER2 positivity (p = 0.02). A significantly higher SOX18 expression was found in the HER2-positive cell line BT-474, and a significantly lower expression in the triple negative cell lines MDA-MB-231 and BO2. Laser capture microdissection of IDC samples revealed significantly higher mRNA SOX7, SOX17 and SOX18 expression levels in the vessels as compared to the cancer cells (p = 0.02 and p = 0.0002, p < 0.0001, respectively). SOX18 positive intratumoral and peritumoral microvessel counts (MVC) were associated with higher malignancy grades (p = 0.04 and p = 0.02, respectively). Moreover, peritumoral SOX18 positive MVC were found to act as an independent marker for a poor prognosis (p = 0.04). SOX18 expression may serve as a marker for a poor prognosis in IDC.

Doxorubicin induces cell death in breast cancer cells regardless of Survivin and XIAP expression levels

Breast cancer is the leading cause of deaths in women around the world. Resistance to therapy is the main cause of treatment failure and still little is known about predictive biomarkers for response to systemic therapy. Increasing evidence show that Survivin and XIAP overexpression is closely associated with chemoresistance and poor prognosis in breast cancer. However, their impact on resistance to doxorubicin (dox), a chemotherapeutic agent widely used to treat breast cancer, is poorly understood. Here, we demonstrated that dox inhibited cell viability and induced DNA fragmentation and activation of caspases-3, -7 and -9 in the breast cancer-derived cell lines MCF7 and MDA-MB-231, regardless of different p53 status. Dox exposure resulted in reduction of Survivin and XIAP mRNA and protein levels. However, when we transfected cells with a Survivin-encoding plasmid, we did not observe a cell death-resistant phenotype. XIAP and Survivin silencing, either alone or in combination, had no effect on breast cancer cells sensitivity towards dox. Altogether, we demonstrated that breast cancer cells are sensitive to the chemotherapeutic agent dox irrespective of Survivin and XIAP expression levels. Also, our findings suggest that dox-mediated modulation of Survivin and XIAP might sensitize cells to taxanes when used in a sequential regimen.

Abstract 3962: SGN-LIV1A: a development stage antibody drug-conjugate targeting LIV-1 for the treatment of metastatic breast cancer.

Abstract LIV-1, also known as SLC39A6 or ZIP6, is a member of the zinc transporter family and was first identified as an estrogen-inducible gene in breast cancer derived cell lines. LIV-1, as a downstream target of STAT3, promotes the epithelial to mesenchymal transition that is important in the malignant progression to metastasis. Consistent with its role in cancer, we determined by immunohistochemical (IHC) analysis that LIV-1 is expressed in subtypes of metastatic breast cancers (ER+/HER2-, HER2+ and triple negative). In healthy human tissues, LIV-1 expression is limited to four hormonally-regulated organs. The broad expression of LIV-1 in metastatic breast cancer in combination with the limited expression in vital organs makes LIV-1 an excellent target for an antibody-drug conjugate (ADC). SGN-LIV1A is an ADC consisting of a humanized anti-LIV-1 mAb conjugated to the microtubule-disrupting agent, monomethyl auristatin E, via a protease-cleavable linker. In vitro, SGN-LIV1A shows target specific internalization and cytotoxic activity against a breast cancer cell line. In vivo studies also demonstrate antitumor activity of SGN-LIV1A in preclinical xenograft models with significant delay of tumor growth compared to control groups. These findings support further evaluation and development of SGN-LIVA as a therapeutic for the treatment of metastatic breast cancer. Citation Format: Django Sussman, Leia M. Smith, Martha E. Anderson, Steve Duniho, Joshua H. Hunter, Heather Kostner, Jamie B. Miyamoto, Albina Nesterova, Lori Westendorf, Heather A. Van Epps, Nancy Whiting, Dennis R. Benjamin. SGN-LIV1A: a development stage antibody drug-conjugate targeting LIV-1 for the treatment of metastatic 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 3962. doi:10.1158/1538-7445.AM2013-3962

Synthesis and biological evaluation of guanidino analogues of roscovitine

A series of 2,9-substituted 6-guanidinopurines, structurally related to the cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine, has been synthesized and characterized. A new copper-catalyzed method for the synthesis of 2-substituted 6-guanidino-9-isopropylpurines under mild reaction conditions has been developed. All prepared compounds were screened for their CDK1 and CDK2 inhibitory activities, cytotoxicity and antiproliferative effects in the breast cancer-derived cell line MCF7. The most active derivative 16g possessed an identical side chain in the C2 position to roscovitine; this compound displayed approximately five fold higher inhibitory activity towards CDK2/cyclin E and more than ten fold increase in cytotoxicity in MCF7 cells. Interestingly and in contrast to previously described findings, (S)-6-guanidinopurine derivatives were generally more active than their (R)-counterparts. Kinase selectivity profiling of (R)- and (S)-enantiomers 16e and 16g, respectively, revealed that introduction of a guanidino group at the C6 position of the purine moiety decreased selectivity towards protein kinases compared to roscovitine. Nevertheless, increased inhibitory activity and decreased selectivity offer a good starting point for further development of new protein kinase inhibitors.

Understanding the Role of Keratins 8 and 18 in Neoplastic Potential of Breast Cancer Derived Cell Lines

BackgroundBreast cancer is a complex disease which cannot be defined merely by clinical parameters like lymph node involvement and histological grade, or by routinely used biomarkers like estrogen receptor (ER), progesterone receptor (PGR) and epidermal growth factor receptor 2 (HER2) in diagnosis and prognosis. Breast cancer originates from the epithelial cells. Keratins (K) are cytoplasmic intermediate filament proteins of epithelial cells and changes in the expression pattern of keratins have been seen during malignant transformation in the breast. Expression of the K8/18 pair is seen in the luminal cells of the breast epithelium, and its role in prognostication of breast cancer is not well understood.Methodology/Principal FindingsIn this study, we have modulated K8 expression to understand the role of the K8/18 pair in three different breast epithelium derived cell lines: non-transformed MCF10A, transformed but poorly invasive MDA MB 468 and highly invasive MDA MB 435. The up-regulation of K8 in the invasive MDA MB 435 cell line resulted in a significant decrease in proliferation, motility, in-vitro invasion, tumor volume and lung metastasis. The down-regulation of K8 in MDA MB 468 resulted in a significant increase in transformation potential, motility and invasion in-vitro, while MCF10A did not show any changes in cell transformation assays.Conclusions/SignificanceThese results indicate the role of K8/18 in modulating invasion in breast cancer -its presence correlating with less invasive phenotype and absence correlating with highly invasive, dedifferentiated phenotype. These data may have important implications for prognostication of breast cancer.

In vitro Antitumour Activity, Genotoxicity, and Antiproliferative Effects of Aminophosphonic Acid Diesters and their Synthetic Precursors

The Schiff bases N-furfurylidene-p-toluidine and N-(4-dimethylaminobenzilidene)-p-toluidine, and the recently synthesized aminophosphonic acid diesters p-[N-methyl-(diethoxyphosphonyl)-(2-furyl)]toluidine and p-[N-methyl(diethoxyphosphonyl)-(4-dimethylaminophenyl)]toluidine were tested for in vitro antitumour activity on six human epithelial cancer cell lines. The genotoxicity and antiproliferative activity of these compounds were tested in mice. The aminophosphonates showed high in vitro antitumour activity towards the breast cancer-derived cell lines (MCF-7 and MDA-MB-231), the cervical carcinoma cell line (HeLa), and the human colon adenocarcinoma cell line (HT-29). In addition, the Schiff base N-furfurylidene-p-toluidine significantly inhibited the growth of bladder carcinoma cells (647-V) and the hepatocellular carcinoma line HepG2, and U-shaped dose-response curves were observed after treatment of 647-V and MCF-7 cells. All studied compounds had a moderate genotoxic and antiproliferative activity in vivo.