Frequent genetic alterations in EGFR- and HER2-driven pathways in breast cancer brain metastases.

Current standard systemic therapies for treating breast cancer patients with brain metastases are inefficient. Targeted therapies against human epidermal growth factor receptors are of clinical interest because of their alteration in a subset of breast cancers (BCs). We analyzed copy number, mutation status, and protein expression of epidermal growth factor receptor (EGFR), human epidermal growth factor 2 (HER2), phosphatase and tensin homologue (PTEN), and PI3K catalytic subunit (PIK3CA) in 110 ductal carcinoma in situ, primary tumor, and metastatic BC samples. Alterations in EGFR, HER2, and PTEN, alone or in combination, were found in a significantly larger fraction of breast cancer brain metastases tumor tissue compared with samples from primary tumors with good prognosis, bone relapse, or other distant metastases (all P < 0.05). Primary tumor patients with a subsequent brain relapse showed almost equally high frequencies of especially EGFR and PTEN alteration as the breast cancer brain metastases patients. PIK3CA was not associated with an increased risk of brain metastases. Genetic alterations in both EGFR and PTEN were especially common in triple-negative breast cancer patients and rarely were seen among HER2-positive patients. In conclusion, we identified two independent high-risk primary BC subgroups for developing brain metastases, represented by genetic alterations in either HER2 or EGFR/PTEN-driven pathways. In contrast, none of these pathways was associated with an increased risk of bone metastasis. These findings highlight the importance of both pathways as possible targets in the treatment of brain metastases in breast cancer.

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.

Quantitative high-resolution genomic analysis of single cancer cells.

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.

Cellular and tumor radiosensitivity is correlated to epidermal growth factor receptor protein expression level in tumors without EGFR amplification.

PURPOSE: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. METHODS AND MATERIALS: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. RESULTS: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. CONCLUSIONS: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

Expression Regulation of the Metastasis-Promoting Protein InsP3-Kinase-A in Tumor Cells.

Under physiologic conditions, the inositol-1,4,5-trisphosphate (InsP(3))-metabolizing, F-actin-bundling InsP(3)-kinase-A (ITPKA) is expressed only in neurons. Tumor cells that have gained the ability to express ITPKA show an increased metastatic potential due to the migration-promoting properties of ITPKA. Here we investigated the mechanism how tumor cells have gained the ability to reexpress ITPKA by using a breast cancer cell line (T47D) with no expression and a lung carcinoma cell line (H1299) with ectopic ITPKA expression. Cloning of a 1,250-bp ITPKA promoter fragment revealed that methylation of CpG islands was reduced in H1299 as compared with T47D cells, but DNA demethylation did not alter the expression of ITPKA. Instead, we showed that the repressor-element-1-silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF), which suppresses expression of neuronal genes in nonneuronal tissues, regulates expression of ITPKA. Knockdown of REST/NRSF induced expression of ITPKA in T47D cells, whereas its overexpression in H1299 cells strongly reduced the level of ITPKA. In T47D cells, REST/NRSF was bound to the RE-1 site of the ITPKA promoter and strongly reduced its activity. In H1299 cells, in contrast, expressing comparable REST/NRSF levels as T47D cells, REST/NRSF only slightly reduced ITPKA promoter activity. This reduced suppressor activity most likely results from expression of a dominant-negative isoform of REST/NRSF, REST4, which impairs binding of REST/NRSF to the RE-1 site. Thus, ITPKA may belong to the neuronal metastasis-promoting proteins whose ectopic reexpression in tumor cells is associated with impaired REST/NRSF activity. Mol Cancer Res; 9(4); 1-10. ©2011 AACR.

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.

Mechanisms of egfr gene transcription modulation: relationship to cancer risk and therapy response.

The epidermal growth factor receptor (EGFR) plays a crucial role in growth, differentiation, and motility of normal as well as cancer cells. For predictive cancer diagnostics and therapeutic targeting of EGFR, it is important to know how the expression level of EGFR is controlled and related to receptor signaling. A novel transcriptional regulation mechanism has been described that depends on the length of a CA repeat in intron 1 [CA simple sequence repeat 1 (CA SSR I)] of the EGFR gene. Thereby, the number of CA repeats is inversely correlated to pre-mRNA synthesis. Indirect evidence for the importance of this mechanism includes the preferential occurrence of amplifications in cancer tissue harboring short CA repeats in this sequence and the discovery of distinct alleles in young breast cancer patients with a family history of the disease and in Japanese breast cancer patients. It can be postulated that the length of the CA repeat influences DNA bendability and, in consequence, the binding of repressor proteins. In summary, it seems that the CA SSR I represents an inherited variable for response to anti-EGFR therapies that could be determined before therapy. Moreover, the potential for synergistic effects with other polymorphism [e.g., EGFR R497K (HER-1 497K) and CCND1 A870G] leading to a simultaneous increase of EGFR signaling activity and expression should be investigated. From a practical perspective, assessment of the CA SSR I number of CA dinucleotide repeats as a predictor for clinical outcome is very attractive because it is a constant feature that does not change over time and can be easily measured in normal and cancer tissues (blood cells, skin, and tumor biopsies) in an assay that is technically simple, objective, and even quantitative.