Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk.

BACKGROUND: The biological phenomenon of cell fusion has been linked to several characteristics of tumour progression, including an enhanced metastatogenic capacity and an enhanced drug resistance of hybrid cells. We demonstrated recently that M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics spontaneously fused with MDA-MB-435-Hyg breast cancer cells, thereby giving rise to stable M13MDA435 hybrid cells, which are characterised by a unique gene expression profile and migratory behaviour. Here we investigated the involvement of the PLC-ß/γ1, PI3K/AKT and RAS-RAF-ERK signal transduction cascades in the EGF and SDF-1α induced migration of two M13MDA435 hybrid cell clones in comparison to their parental cells. RESULTS: Analysis of the migratory behaviour by using the three-dimensional collagen matrix migration assay showed that M13SV1-EGFP-Neo cells as well as M13MDA435 hybrid cells, but not the breast cancer cell line, responded to EGF stimulation with an increased locomotory activity. By contrast, SDF-1α solely stimulated the migration of M13SV1-EGFP-Neo cells, whereas the migratory activity of the other cell lines was blocked. Analysis of signal transduction cascades revealed a putative differential RAF-AKT crosstalk in M13MDA435-1 and -3 hybrid cell clones. The PI3K inhibitor Ly294002 effectively blocked the EGF induced migration of M13MDA435-3 hybrid cells, whereas the EGF induced locomotion of M13MDA435-1 hybrid cells was markedly increased. Analysis of RAF-1 S259 phosphorylation, being a major mediator of the negative regulation of RAF-1 by AKT, showed decreased pRAF-1 S259 levels in LY294002 treated M13MDA435-1 hybrid cells. By contrast, pRAF-1 S259 levels remained unaltered in the other cell lines. Inhibition of PI3K/AKT signalling by Ly294002 relieves the AKT mediated phosphorylation of RAF-1, thereby restoring MAPK signalling. CONCLUSIONS: Here we show that hybrid cells could evolve exhibiting a differential active RAF-AKT crosstalk. Because PI3K/AKT signalling has been chosen as a target for anti-cancer therapies our data might point to a possible severe side effect of AKT targeted cancer therapies. Inhibition of PI3K/AKT signalling in RAF-AKT crosstalk positive cancer (hybrid) cells could result in a progression of these cells. Thus, not only the receptor (activation) status, but also the activation of signal transduction molecules should be analysed thoroughly prior to therapy.

The interplay of HER2/HER3/PI3K and EGFR/HER2/PLC-γ1 signalling in breast cancer cell migration and dissemination.

HER2 signalling by heterodimerisation with EGFR and HER3 in breast cancer is associated with worst outcome of the afflicted patients, which is attributed not only to the aggressiveness of such tumours but also to therapy resistance. Thus, in the present study we investigated the role of EGFR, HER2 and HER3 lateral signalling in cell migration by applying the MDA-MB-468-HER2 (MDA-HER2) breast cancer cell line, representing a valid model system. Knockdown of HER3 expression by siRNA resulted in decreased phosphorylated AKT (pAKT) levels, abrogated epidermal growth factor (EGF)-mediated PLC-γ1 activation and a diminished EGF-induced migratory activity, depicting the interplay of EGF receptor (EGFR)/HER2/PLC-γ1 and HER2/HER3/PI3K signalling in mediating the migration of EGFR/HER2/HER3-expressing breast cancer cells. Since therapy failure usually arises from metastatic cells, we further investigated whether HER3 signalling was active in established breast cancer disseminated tumour cell (DTC) lines as well as in primary DTCs derived from breast cancer patients. EGF treatment of DTC lines resulted solely in increased pAKT S473 levels, whereas in MDA-HER2 cells both pAKT S473 and pAKT T308 levels were increased upon EGF stimulation. Moreover, despite active HER3 molecules, as indicated by pTyr1222 staining, about 90% of analysed breast cancer patient DTCs exhibited very low or even no detectable pAKT S473 levels, suggesting that these cells might have fallen into dormancy. In summary, our data indicate the important role in EGFR, HER2 and HER3 lateral signalling in breast cancer cell migration. Moreover, our data further show that primary tumour cells and DTCs can vary in their HER activation status, which is important to know in the context of cancer therapy.

Characterization of hybrid cells derived from spontaneous fusion events between breast epithelial cells exhibiting stem-like characteristics and breast cancer cells.

Several data of the past years clearly indicated that the fusion of tumor cells and tumor cells or tumor cells and normal cells can give rise to hybrids cells exhibited novel properties such as an increased malignancy, drug resistance, or resistance to apoptosis. In the present study we characterized hybrid cells derived from spontaneous fusion events between the breast epithelial cell line M13SV1-EGFP-Neo and two breast cancer cell lines: HS578T-Hyg and MDA-MB-435-Hyg. Short-tandem-repeat analysis revealed an overlap of parental alleles in all hybrid cells indicating that hybrid cells originated from real cell fusion events. RealTime-PCR-array gene expression data provided evidence that each hybrid cell clone exhibited a unique gene expression pattern, resulting in a specific resistance of hybrid clones towards chemotherapeutic drugs, such as doxorubicin and paclitaxel, as well as a specific migratory behavior of hybrid clones towards EGF. For instance, M13MDA435-4 hybrids showed a marked resistance towards etoposide, doxorubicin and paclitaxel, whereas hybrid clones M13MDA-435-1 and -2 were only resistant towards etoposide. Likewise, all investigated M13MDA435 hybrids responded to EGF with an increased migratory activity, whereas the migration of parental MDA-MB-435-Hyg cells was blocked by EGF, suggesting that M13MDA435 hybrids may have acquired a new motility pathway. Similar findings have been obtained for M13HS hybrids. We conclude from our data that they further support the hypothesis that cell fusion could give rise to drug resistant and migratory active tumor (hybrid) cells in cancer.

Discovery of a novel unfolded protein response phenotype of cancer stem/progenitor cells from the bone marrow of breast cancer patients.

Metastases arise from disseminated tumor cells (DTC) that colonize secondary organs. However, DTC survival strategies to start metastatic outgrowth are unclear. The hostile (hypoxic, hypoglycemic) microenvironmental conditions of the bone marrow serve as an ideal model environment for investigation of DTC survival strategies under environmental stress. We investigated the breast cancer DTC cell line BC-M1 established from the bone marrow of a cancer patient by 2-D DIGE and MS analysis. We observed specific overexpression of the unfolded protein response (UPR) proteins Grp78, Grp94, and protein disulfide-isomerase in breast, lung, and prostate cancer DTC cell lines from the bone marrow. The UPR contributes to survival under adverse environmental conditions including chemotherapy. We show in cellular models that Grp78 expression of the UPR is regulated by tyrosine 1248 of ErbB-2. The breast cancer DTC cell lines shared stem/progenitor cell cancer phenotypes (CD44(high)/CD24(low)). Immunocytochemical staining of bone marrow samples from breast cancer patients confirmed in situ high expression of Grp78 and Grp94 in DTC of breast cancer patients, indicating the potential of both proteins as novel markers for DTC detection. Our results suggest the presence of a previously not recognized stress resistant DTC population that combines stem/progenitor attributes with an UPR phenotype.

Two-dimensional differential gel electrophoresis of a cell line derived from a breast cancer micrometastasis revealed a stem/ progenitor cell protein profile.

Dissemination of primary cancer cells to distant sites is an early event in breast cancer. These cells can invade the bone marrow, rest there, and many years later disseminated tumor cells (DTC) can grow out to form overt metastases. Epithelium specific cytokeratins are commonly used as marker proteins for sensitive detection of metastatic lesions. However, due to difficulties in the detection of DTC, the question arises if DTC necessarily have the same protein expression profile as advanced tumors. On that account, we analyzed the previously uncharacterized breast cancer DTC cell line BC-M1 by 2-D DIGE. Special protein concentration and purification protocols for 2-DE were developed which resulted in high recovery rates and increased display of alkaline proteins. A broad range reference map of metastasis relevant proteins was compiled including the cytokeratins 5, 7, 8, 17, 18, and 19 and several classes of cytoskeleton proteins involved in metastasis like ezrin, gelsolin, vinculin, or vimentin. BC-M1 shows the rare and highly metastatic vimentin/cytokeratin 5 positive and cytokeratin 8/18 negative breast cancer phenotype and expresses Her-2, which is also found in stem cells/progenitor cells of primary tumors. Supported by the detection of several other epithelium-derived proteins, the example BC-M1 indicates that the protein expression profile of DTC might be reminiscent of the expression profile of the early tumor, which differs from the advanced tumor. Hence, DTC from breast cancer patients' bone marrow expressed cytokeratin 5, which further supports our hypothesis.

TOB1 is regulated by EGF-dependent HER2 and EGFR signaling, is highly phosphorylated, and indicates poor prognosis in node-negative breast cancer.

Clinical and animal studies have shown that coexpression of the receptor tyrosine kinases HER2 and epidermal growth factor (EGF) receptor (EGFR) indicates a highly metastatic phenotype of breast cancer. In a cellular model of this phenotype using differential gene expression analysis, we identified TOB1 to be up-regulated depending on EGF stimulation and transduction through phosphorylation of HER2 tyrosine 1248. mRNA expression analysis of breast cancers from a cohort of node-negative patients showed significantly shortened distant metastasis-free survival for patients with high TOB1 expression. In subsequent tissue microarray studies of 725 clinical samples, high HER2 and EGF protein levels were significantly correlated with TOB1 expression in breast cancer, whereas EGFR and EGF levels correlated with TOB1 phosphorylation. We did not observe a correlation between TOB1 expression and cyclin D1, which was previously suggested to mediate the antiproliferative effect of unphosphorylated TOB1. A positive correlation of TOB1 phosphorylation status with proliferation marker Ki67 suggests that elevated TOB1 phosphorylation might abrogate the antiproliferative effect of TOB1 in breast cancer. This suggests a new regulatory role for TOB1 in cancer progression with particular significance in HER2- and/or EGFR-positive breast cancers.

Options for visualizing metastatic disease in the living body.

Detection and observation of primary tumor growth and metastasis in living subjects is an important task in clinical and basic cancer research. Recently several approaches and techniques emerged which offer a huge variety of options with respect to the specific objectives and questions of a given study. Recent developments in the field of in vivo imaging not only allow the assessment of anatomic information but also functional processes with cellular resolution and molecular sensitivity. This chapter will provide an overview of the most common imaging techniques which are currently available for the detection and observation of metastasizing tumor cells. General capacities, advantages, limitations and drawbacks will be discussed. These techniques include computed tomography (CT), molecular resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), fluorescence imaging (FI), and bioluminescent imaging (BLI). The objective is to provide the cancer researcher with information that will help solve the dilemma of how best to apply the latest imaging tools for studying biological questions in the context of the living body.

First evidence supporting a potential role for the BMP/SMAD pathway in the progression of oestrogen receptor-positive breast cancer.

Oestrogen receptor expression is generally a sign of better tumour differentiation and comparatively good clinical outcome in invasive breast cancer. However, oestrogen receptor-positive, poorly differentiated carcinomas with a poor clinical outcome exist. The underlying genetic mechanisms and the genes involved remain obscure, even though chromosome 7p gains seem to be associated with these uncommon tumours. In this study, we compared two subsets of oestrogen receptor-positive breast cancers, which differed in tumour grade, cytogenetic instability, and tumour proliferation, for their differential gene expression in order to identify proteins involved in the progression of oestrogen receptor-positive breast cancers. We were able to show by means of subtractive suppression hybridization, real-time reverse transcriptase PCR, and tissue microarray analysis that expression of the bone morphogenetic protein receptor IB (BMPR-IB) is a major hallmark of the progression and dedifferentiation of breast cancer. Strong expression of BMPR-IB was associated with high tumour grade, high tumour proliferation, cytogenetic instability, and a poor prognosis in oestrogen receptor-positive carcinomas. Western blot analysis revealed that downstream signalling of this receptor is mainly mediated via phosphorylation of SMAD 1 in oestrogen receptor-positive breast cancer. Even though BMPR-IB was expressed in oestrogen receptor-positive and -negative breast cancers, an impact on tumour grade, proliferation, and cytogenetic instability, as parameters of tumour progression, could only be demonstrated in oestrogen receptor-positive carcinomas. This pro-proliferative effect was complemented by significant anti-apoptotic activity, indicated by XIAP and IAP-2 expression in BMPR-IB-positive carcinomas. These results show that the BMP/SMAD pathway is activated in breast cancer and may contribute to breast cancer progression and dedifferentiation in oestrogen receptor-positive breast cancer. The definition of this pathway characterizes a new potential target in the molecular treatment of invasive breast cancer.

Modified direct-double-differential PCR for gene dosage quantification of HER2.

HER2 amplification and/or overexpression in breast cancer are adverse prognostic factors and can predict the response to trastuzumab therapy. As assessment of HER2 status in breast cancer is of great importance for clinical decision-making, it is crucial to have reliable methods to quantify HER2. In the present project, we have developed a modification of the direct-double-differential PCR method (dddPCR) for gene dosage quantification of HER2 in breast cancer samples. The study was also undertaken to establish the potential application of the newly developed dddPCR method for HER2 testing in a diagnostic laboratory. The dddPCR validity for determining HER2 amplification in comparison to IHC-based HercepTest for the detection of protein levels was performed. Modified dddPCR was proven to be highly reproducible and reliable. A statistically significant correlation between protein expression and gene dosage of HER2 was found in the examined material. The obtained results indicate that dddPCR provides a fast and reliable diagnostic tool for HER2 quantification and may be considered as a supplementary method to verify equivocal results obtained with IHC-based HercepTest. Notably, dddPCR can be performed in every basic molecular biology laboratory, and is inexpensive and time-saving.

Molecular diagnostics of solid malignant tumours.

Three groups of genes have been identified which play a major role in determining the origin and spread of cancer: Oncogenes support the growth of tumour cells in a manner which does not depend on the tissue environment. They occur as proto-oncogenes in the genome of every cell in the body. If only one allele is excessively or inappropriately activated by mutation, they can change the phenotype of the cell lastingly and initiate malignant growth. Tumour suppressor genes hamper the growth of cells with DNA mutations. Both alleles must be mutated in the genome in order to block production of functional protein and to allow mutations to be passed on in their DNA to daughter cells. With an existing predisposition to cancer, one of the alleles is already mutated. The malignoma develops after a mutation occurs in the second allele. Mutator genes are responsible for maintaining the integrity of the genome and reliably transmitting the genetic code. A functional loss of both alleles increases error rates during DNA-replication. This can lead to mutations in oncogenes or tumour suppressor genes, and contribute to the origin or spreading of a malignoma. Tumour-modifier genes might turn out to be the genetic factors underlying the origin or even prevention of malignomas caused mainly by environmental factors. From experiments on mice it is known that these genes may be responsible for the incomplete penetrance of hereditary tumours. Demonstration of these genes using molecular diagnostic procedures will promote opportunities and knowledge in the field of oncology.

Induction of cancer cell migration by epidermal growth factor is initiated by specific phosphorylation of tyrosine 1248 of c-erbB-2 receptor via EGFR.

Induction of tumor cell migration is a key step in invasion and metastasis. Here we report that the epidermal growth factor (EGF)-induced cell migration of breast cancer cells is attributed to a transient, rather than a sustained, activation of phospholipase C (PLC)-gamma1 due to c-erbB-2 signaling. EGF stimulation of EGF receptor (EGFR) overexpressing cells resulted in long-term PLC-gamma1 tyrosine phosphorylation and sustained levels of inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG) producing sinusoidal calcium oscillations. In contrast, c-erbB-2/EGFR expressing cells displayed baseline transient calcium oscillations after EGF treatment due to short-term PLC-gamma1 tyrosine phosphorylation and short-term IP3 and DAG turnover. A third cell line expressing a point-mutated c-erbB-2 receptor that lacks the autophosphorylation Y1248 was generated to investigate whether the different PLC-gamma1 activation was attributed to this structure. Neither PLC-gamma1 tyrosine phosphorylation nor IP3 and DAG turnover and calcium oscillations were observed in this cell line, indicating the modulation of the PLC-g1 activation time course by c-erbB-2 signaling. Induction of cell migration was solely observable in the c-erbB-2-positive cell line as proved by the mode of actin reorganization and a cell migration assay, using a 3D-collagen lattice. In summary, c-erbB-2 up-regulation switches on the cell migration program by modulating the time course of PLC-gamma1 activation.

Trimodality treatment in Stage III nonsmall cell lung carcinoma: prrognostic impact of K-ras mutations after neoadjuvant therapy.

BACKGROUND: In a trimodality treatment approach for Stage III nonsmall cell lung carcinoma (NSCLC), the prognostic impact of the ras mutation status in resection specimens was evaluated. METHODS: Forty patients with Stage III NSCLC underwent tumor resection after neoadjuvant treatment with two cycles of chemotherapy (ifosfamide, carboplatin, and etoposide) and subsequent twice-daily radiotherapy (45 grays [Gy]; 2 x 1.5 Gy/day) with concurrent carboplatin and vindesine. Assessment of K-ras codon 12 mutation status was performed in the paraffin embedded resection specimens by a two-step polymerase chain reaction followed by restriction fragment length polymorphism analysis. RESULTS: K-ras mutation status could be assessed in 28 cases. A K-ras codon 12 point mutation was found in 13 of 28 resection specimens (46%). The mutation was found independently of gender, age, tumor stage, and clinical response status and occurred more frequently in adenocarcinomas. Even after complete resection, the presence of a K-ras mutation was a significant predictor for a poor progression free survival (P = 0.005). CONCLUSIONS: These data suggest that further evaluation of the K-ras codon 12 mutation status in trials on neoadjuvant and adjuvant therapy is warranted. This may contribute to the identification of stratification variables for future treatment approaches.

Cancer cell motility--on the road from c-erbB-2 receptor steered signaling to actin reorganization.

Cell migration depends mainly on actin polymerization and intracellular organization, which are influenced by a vast variety of actin binding proteins (ABPs). Regulation of ABP activity is mediated by second messengers such as phosphoinositides and calcium. Signaling via these second messengers is initiated and regulated by membrane receptors, e.g., receptor tyrosine kinases (RTKs), and by adhesion molecule interactions (e.g., integrins and selectins) and focal adhesion kinases. A major role in steering second-messenger signaling and thus in actin cytoskeleton reorganization and motility of cancer cells is played by the RTK c-erbB-2. This occurs through a number of signaling pathways which involve mainly enzymes, e.g., phospholipase Cgamma1 and GTPases, which modify signaling molecules. Furthermore large multiprotein complexes including actin-related protein 2/3, Wiskott-Aldrich syndrome protein, profilin, and capping protein among others play an important role in regulating actin reorganization. The complex picture of the mode of actin reorganization, which is involved in tumor cell migration, is slowly emerging from the mists of cellular signaling pathways, but this is still by no means a clear view.