Assessment of gene expression of intracellular calcium channels, pumps and exchangers with epidermal growth factor-induced epithelial-mesenchymal transition in a breast cancer cell line

Background Epithelial-mesenchymal transition (EMT) is a process implicated in cancer metastasis that involves the conversion of epithelial cells to a more mesenchymal and invasive cell phenotype. In breast cancer cells EMT is associated with altered store-operated calcium influx and changes in calcium signalling mediated by activation of cell surface purinergic receptors. In this study, we investigated whether MDA-MB-468 breast cancer cells induced to undergo EMT exhibit changes in mRNA levels of calcium channels, pumps and exchangers located on intracellular calcium storing organelles, including the Golgi, mitochondria and endoplasmic reticulum (ER). Methods Epidermal growth factor (EGF) was used to induce EMT in MDA-MB-468 breast cancer cells. Serum-deprived cells were treated with EGF (50 ng/mL) for 12 h and gene expression was assessed using quantitative RT-PCR. Results and conclusions These data reveal no significant alterations in mRNA levels of the Golgi calcium pump secretory pathway calcium ATPases (SPCA1 and SPCA2), or the mitochondrial calcium uniporter (MCU) or Na+/Ca2+ exchanger (NCLX). However, EGF-induced EMT was associated with significant alterations in mRNA levels of specific ER calcium channels and pumps, including (sarco)-endoplasmic reticulum calcium ATPases (SERCAs), and inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) calcium channel isoforms. The most prominent change in gene expression between the epithelial and mesenchymal-like states was RYR2, which was enriched 45-fold in EGF-treated MDA-MB-468 cells. These findings indicate that EGF-induced EMT in breast cancer cells may be associated with major alterations in ER calcium homeostasis.

Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden

Over 80% of women diagnosed with advanced-stage ovarian cancer die as a result of disease recurrence due to failure of chemotherapy treatment. In this study, using two distinct ovarian cancer cell lines (epithelial OVCA 433 and mesenchymal HEY) we demonstrate enrichment in a population of cells with high expression of CSC markers at the protein and mRNA levels in response to cisplatin, paclitaxel and the combination of both. We also demonstrate a significant enhancement in the sphere forming abilities of ovarian cancer cells in response to chemotherapy drugs. The results of these in vitro findings are supported by in vivo mouse xenograft models in which intraperitoneal transplantation of cisplatin or paclitaxel-treated residual HEY cells generated significantly higher tumor burden compared to control untreated cells. Both the treated and untreated cells infiltrated the organs of the abdominal cavity. In addition, immunohistochemical studies on mouse tumors injected with cisplatin or paclitaxel treated residual cells displayed higher staining for the proliferative antigen Ki67, oncogeneic CA125, epithelial E-cadherin as well as cancer stem cell markers such as Oct4 and CD117, compared to mice injected with control untreated cells. These results suggest that a short-term single treatment of chemotherapy leaves residual cells that are enriched in CSC-like traits, resulting in an increased metastatic potential. The novel findings in this study are important in understanding the early molecular mechanisms by which chemoresistance and subsequent relapse may be triggered after the first line of chemotherapy treatment.

Automatic classification of human epithelial type 2 cell indirect immunofluorescence images using cell pyramid matching

This paper describes a novel system for automatic classification of images obtained from Anti-Nuclear Antibody (ANA) pathology tests on Human Epithelial type 2 (HEp-2) cells using the Indirect Immunofluorescence (IIF) protocol. The IIF protocol on HEp-2 cells has been the hallmark method to identify the presence of ANAs, due to its high sensitivity and the large range of antigens that can be detected. However, it suffers from numerous shortcomings, such as being subjective as well as time and labour intensive. Computer Aided Diagnostic (CAD) systems have been developed to address these problems, which automatically classify a HEp-2 cell image into one of its known patterns (eg. speckled, homogeneous). Most of the existing CAD systems use handpicked features to represent a HEp-2 cell image, which may only work in limited scenarios. We propose a novel automatic cell image classification method termed Cell Pyramid Matching (CPM), which is comprised of regional histograms of visual words coupled with the Multiple Kernel Learning framework. We present a study of several variations of generating histograms and show the efficacy of the system on two publicly available datasets: the ICPR HEp-2 cell classification contest dataset and the SNPHEp-2 dataset.

Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices

Background The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. Results We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.

Differentiation state and invasiveness of human breast cancer cell lines

Eighteen breast cancer cell lines were examined for expression of markers of epithelial and fibroblastic differentiation: E-cadherin, desmoplakins, ZO- 1, vimentin, keratin and β1 and β4 integrins. The cell lines were distributed along a spectrum of differentiation from epithelial to fibroblastic phenotypes. The most well-differentiated, epithelioid cell lines contained proteins characteristic of desmosomal, adherens and tight junctions, were adherent to one another on plastic and in the basement membrane matrix Matrigel and were keratin-positive and vimentin-negative. These cell lines were all weakly invasive in an in vitro chemoinvasion assay. The most poorly-differentiated, fibroblastic cell lines were E-cadherin-, desmoplakin- and ZO-1-negative and formed branching structures in Matrigel. They were vimentin-positive, contained only low levels of keratins and were highly invasive in the in vitro chemoinvasion assay. Of all of the markers analyzed, vimentin expression correlated best with in vitro invasive ability and fibroblastic differentiation. In a cell line with unstable expression of vimentin, T47D(CO), the cells that were invasive were of the fibroblastic type. The differentiation markers described here may be useful for analysis of clinical specimens and could potentially provide a more precise measure of differentiation grade yielding more power for predicting prognosis.

Sphingosine-1-phosphate, a novel second messenger involved in cell growth regulation and signal transduction, affects growth and invasiveness of human breast cancer cells

This review will focus on the role of sphingosine and its phosphorylated derivative sphingosine-1-phosphate (SPP) in cell growth regulation and signal transduction. We will show that many of the effects attributed to sphingosine in quiescent Swiss 3T3 fibroblasts are mediated via its conversion to SPP. We propose that SPP has appropriate properties to function as an intracellular second messenger based on the following: it elicits diverse cellular responses; it is rapidly produced from sphingosine by a specific kinase and rapidly degraded by a specific lyase; its concentration is low in quiescent cells but increases rapidly and transiently in response to the growth factors, fetal calf serum (FCS) and platelet derived growth factor (PDGF); it releases Ca2+ from internal sources in an InsP3-independent manner; and finally, it may link sphingolipid signaling pathways to cellular ras-mediated signaling pathways by elevating phosphatidic acid levels. The effects of this novel second messenger on growth, differentiation and invasion of human breast cancer cells will be discussed. © 1994 Kluwer Academic Publishers.

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.

SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines

Activation of the matrix metalloproteinase 2 (MMP-2) has been shown to play a major role in the proteolysis of extracellular matrix (ECM) associated with tumor invasion. Although the precise mechanism of this activation remains elusive, levels of the membrane type 1-MMP (MT1-MMP) at the cell surface and of the tissue inhibitor of MMP-2 (TIMP-2) appear to be two important determinants. Induction of MMP-2 activation in cells cultivated on collagen type I gels indicated that the ECM is important in the regulation of this process. In this study, we show that SPARC/osteonectin, a small ECM- associated matricellular glycoprotein, can induce MMP-2 activation in two invasive breast cancer cell lines (MDA-MB-231 and BT549) but not in a noninvasive counterpart (MCF7), which lacks MT1-MMP. Using a set of peptides from different regions of SPARC, we found that peptide 1.1 (corresponding to the NH2-terminal region of the protein) contained the activity that induced NIMP-2 activation. Despite the requirement for MT1-MMP, seen in MCF-7 cells transfected with MT1-MMP, the activation of MMP-2 by SPARC peptide 1.1 was not associated with increased steady-state levels of MT1-MMP mRNA or protein in either MT1-MMP-transfected MCF-7 cells or constitutively expressing MDA- MB-231 and BT549 cells. We did, however, detect decreased levels of TIMP-2 protein in the media of cells incubated with peptide 1.1 or recombinant SPARC; thus, the induction of MMP-2 activation by SPARC might be due in part to a diminution of TIMP-2 protein. We conclude that SPARC, and specifically its NH2-terminal domain, regulates the activation of MMP-2 at the cell surface and is therefore likely to contribute to the proteolytic pathways associated with tumor invasion.

The heat shock protein 90 inhibitor, 17-allylamino-17- demethoxygeldanamycin, enhances osteoclast formation and potentiates bone metastasis of a human breast cancer cell line

Breast cancer metastasis to the bone occurs frequently, causing numerous complications including severe pain, fracture, hypercalcemia, and paralysis. Despite its prevalence and severity, few effective therapies exist. To address this, we examined whether the heat shock protein 90 (Hsp90) inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), would be efficacious in inhibiting breast cancer metastasis to bone. Utilizing the human breast cancer subline, MDA-MB-231SA, previously in vivo selected for its enhanced ability to generate osteolytic bone lesions, we determined that 17-AAG potently inhibited its in vitro proliferation and migration. Moreover, 17-AAG significantly reduced MDA-MB-231SA tumor growth in the mammary-fat pad of nude mice. Despite these findings, 17-AAG enhanced the incidence of bone metastasis and osteolytic lesions following intracardiac inoculation in the nude mouse. Consistent with these findings, 17-AAG enhanced osteoclast formation 2- to 4-fold in mouse bone marrow/osteoblast cocultures, receptor activator of nuclear factor κB ligand (BANKL)-stimulated bone marrow, and RAW264.7 cell models of in vitro osteoclastogenesis. Moreover, the drug enhanced osteoclastogenesis in human cord blood progenitor cells, demonstrating that its effects were not limited to mouse models. In addition to 17-AAG, other Hsp90 inhibitors, such as radicicol and herbimycin A, also enhanced osteoclastogenesis. A pro-osteolytic action of 17-AAG independent of tumor presence was also determined in vivo, in which 17-AAG-treated tumor-naive mice had reduced trabecular bone volume with an associated increase in osteoclast number. Thus, HSP90 inhibitors can stimulate osteoclast formation, which may underlie the increased incidence of osteolysis and skeletal tumor incidence causedby 17-AAG in vivo. These data suggest an important contraindication to the Hsp90 targeted cancer therapy currently undergoing clinical trial.

Epidermal growth factor promotes MDA-MB-231 breast cancer cell migration through a phosphatidylinositol 3'-kinase and phospholipase C-dependent mechanism

Epidermal growth factor receptor (EGFR) levels predict a poor outcome in human breast cancer and are most commonly associated with proliferative effects of epidermal growth factor (EGF), with little emphasis placed on motogenic responses to EGF. We found that MDA-MB-231 human breast cancer cells elicited a potent chemotactic response despite their complete lack of a proliferative response to EGF. Antagonists of EGFR ligation, the EGFR kinase, phosphatidylinositol 3'-kinase, and phospholipase C, but not the mitogen- activated protein kinases (extracellular signal-regulated protein kinase 1 and 2), blocked MDA-MB-231 chemotaxis. These findings suggest that EGF may influence human breast cancer progression via migratory pathways, the signaling for which appears to be dissociated, at least in part, from the proliferative pathways.

Loss of epithelial markers and acquisition of vimentin expression in adriamycin- and vinblastine-resistant human breast cancer cell lines

We have previously observed that breast cancer cell lines could exhibit either epithelial or fibroblastic phenotypes as reflected by their morphologies and intermediate filament protein expression (C. L. Sommers, D. Walker-Jones, S. E. Heckford, P. Worland, E. Valverius, R. Clark, M. Stampfer, and E. P. Gelmann, Cancer Res., 49: 4258-4263, 1989). Fibroblastoid, vimentin-expressing breast cancer cell lines are more invasive in vitro and in vivo (E. W. Thompson, S. Paik, N. Brunner, C. L. Sommers, G. Zugmaier, R. Clarke, T. B. Shima, J. Torri, S. Donahue, M. E. Lippman, G. R. Martin, and R. B. Dickson, J. Cell. Physiol., 150: 534-544, 1992). We hypothesized that a breast cancer cell with an epithelial phenotype could undergo a transition to a fibroblastic phenotype, possibly resulting in more invasive capacity. We now show that two Adriamycin-resistant MCF-7 cell lines and a vinblastine-resistant ZR-75-B cell line have undergone such a transition. Adriamycin-resistant MCF-7 cells express vimentin, have diminished keratin 19 expression, have lost cell adhesion molecule uvomorulin expression, and have reduced formation of desmosomes and tight junctions as determined by reduced immunodetection of their components desmoplakins I and II and zonula occludens (ZO)-1. Other MCF-7 cell lines selected for resistance to vinblastine and to Adriamycin and verapamil did not have these characteristics, indicating that drug selection does not invariably cause these phenotypic changes. In addition, to determine if vimentin expression in MCF-7 cells alone could manifest a fibroblastic phenotype, we transfected the full-length human vimentin complementary DNA into MCF-7 cells. Although vimentin expression was achieved in MCF-7 cells, it did not affect the phenotype of the cells in terms of the distribution of keratins, desmoplakins I and II, ZO-1, or uvomorulin or in terms of in vitro invasiveness. We conclude that vimentin expression is a marker for a fibroblastic and invasive phenotype in breast cancer cells but does not by itself give rise to this phenotype.

ICI 164,384, a pure antagonist of estrogen-stimulated MCF-7 cell proliferation and invasiveness

Estrogen is known to stimulate the proliferation and basement membrane invasiveness of the MCF-7 human breast cancer cell line. We have compared the new steroidal antiestrogen ICI 164,384, the triphenylethylene 4-hydroxytamoxifen (OHT), and the benzothiophene LY 117018, for their effects on the proliferation and invasiveness of the MCF-7 cell line and its antiestrogen-resistant variant LY-2. While all three antiestrogens blocked the proliferative effects of 17β-estradiol on MCF-7 cells, OHT and LY 117018, but not ICI 164,384 stimulated their proliferation in the absence of estrogen. The proliferative effects of OHT and LY 117018 were blocked by ICI 164,384. Basement membrane invasiveness of MCF-7 cells was stimulated by 17β-estradiol and OHT, but not LY 117018 or ICI 164,384. Both ICI 164,384 and Ly 117018 were able to block the invasiveness induced by either 17β-estradiol or OHT. The LY-2 antiestrogen-resistant variant of the MCF-7 cell line showed increased basal proliferation, and responded only slightly to estrogen. ICI 164,384, but not OHT or LY 117018 antagonized the effects of 17β-estradiol, but did not reduce proliferation below control levels. The LY-2 line was not resistant to the antiestrogenic effects of LY 117018 or ICI 164,384 on invasiveness, and was stimulated by LY 117018 for this parameter. Thus, ICI 164,384 is a pure antiestrogen for MCF-7 cell proliferation and invasiveness, and may offer clinical advantage over nonsteroidal antiestrogens which can stimulate these activities in tumor models in vitro.

Supernatants of acquired immunodeficiency syndrome-related Kaposi's sarcoma cells induce endothelial cell chemotaxis and invasiveness

Kaposi's sarcoma (KS) in general, and acquired immunodeficiency syndrome-related KS (AIDS-KS) in particular, is a highly invasive and intensely angiogenic neoplasm of unknown cellular origin. We have recently established AIDS-KS cells in long term culture and reported the development of KS-like lesions in nude mice inoculated with these cells. Here, we have examined the in vitro invasiveness of basement membrane by AIDS-KS cells, as well as the effect(s) of their supernatants on the migration and invasiveness of human vascular endothelial cells. AIDS-KS cells were highly invasive in the Boyden chamber invasion assay and formed invasive, branching colonies in a 3-dimensional gel (Matrigel). Normal endothelial cells form tube-like structures on Matrigel. AIDS-KS cell-conditioned media induced endothelial cells to form invasive clusters in addition to tubes. KS-cell-conditioned media, when placed in the lower compartment of the Boyden chamber, stimulated the migration of human and bovine vascular endothelial cells across filters coated with either small amounts of collagen IV (chemotaxis) or a Matrigel barrier (invasion). Basic fibroblast growth factor could also induce endothelial cell chemotaxis and invasion in these assays. However, when antibodies to basic fibroblast growth factor were used the invasive activity induced by the AIDS-KS-cell-conditioned media was only marginally inhibited, suggesting that the large quantities of basic fibroblast growth factor-like material released by the AIDS-KS cells are not the main mediators of this effect. Specific inhibitors of laminin and collagenase IV action, which represent critical determinants of basement membrane invasion, blocked the invasiveness of the AIDS-KS cell-activated endothelial cells in these assays. These data indicate that KS cells appear to be of smooth muscle origin but secrete a potent inducer of endothelial cell chemotaxis and invasiveness which could be responsible for angiogenesis and the resulting highly vascularized lesions. These assays appear to be a model to study the invasive spread and angiogenic capacity of human AIDS-related KS and should prove useful in the identification of molecular mediators and potential inhibitors of neoplastic neovascularization.

Epithelial mesenchymal transition traits in human breast cancer cell lines

Epithelial mesenchymal transition (EMT) has long been associated with breast cancer cell invasiveness and evidence of EMT processes in clinical samples is growing rapidly. Genome-wide transcriptional profiling of increasingly larger numbers of human breast cancer (HBC) cell lines have confirmed the existence of a subgroup of cell lines (termed Basal B/Mesenchymal) with enhanced invasive properties and a predominantly mesenchymal gene expression signature, distinct from subgroups with predominantly luminal (termed Luminal) or mixed basal/luminal (termed Basal A) features (Neve et al Cancer Cell 2006). Studies providing molecular and cellular analyses of EMT features in these cell lines are summarised, and the expression levels of EMT-associated factors in these cell lines are analysed. Recent clinical studies supporting the presence of EMT-like changes in vivo are summarised. Human breast cancer cell lines with mesenchymal properties continue to hold out the promise of directing us towards key mechanisms at play in the metastatic dissemination of breast cancer.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.