Detection and Isolation of Circulating Tumor Cells from Breast Cancer Patients Using CUB Domain-Containing Protein 1.

In cancer metastasis, single circulating tumor cells (CTCs) in the blood and disseminated tumor cells (DTCs) in the bone marrow mediate cancer metastasis. Because suitable biomarker proteins are lacking, CTCs and DTCs with mesenchymal attributes are difficult to isolate from the bulk of normal blood cells. To establish a procedure allowing the isolation of such cells, we analyzed the cell line BC-M1 established from DTCs in the bone marrow of a breast cancer patient by stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry. We found high levels of the transmembrane protein CUB domain-containing protein 1 (CDCP1) in breast cancer cell lines with mesenchymal attributes. Peripheral blood mononuclear cells were virtually negative for CDCP1. Confirmation in vivo by CellSearch revealed CDCP1-positive CTCs in 8 of 30 analyzed breast cancer patients. Only EpCam-positive CTCs were enriched by CellSearch. Using the extracellular domain of CDCP1, we established a magnetic-activated cell sorting (MACS) approach enabling also the enrichment of EpCam-negative CTCs. Thus, our approach is particularly suited for the isolation of mesenchymal CTCs with downregulated epithelial cancer that occur, for example, in triple-negative breast cancer patients who are prone to therapy failure.

Circulating Cellular Communication Network Factor 1 Protein as a Sensitive Liquid Biopsy Marker for Early Detection of Breast Cancer.

BACKGROUND: Despite recent progress in liquid biopsy technologies, early blood-based detection of breast cancer is still a challenge. METHODS: We analyzed secretion of the protein cellular communication network factor 1 (CCN1, formerly cysteine-rich angiogenic inducer 61) in breast cancer cell lines by an enzyme-linked immunosorbent assay (ELISA). Soluble CCN1 in the plasma (2.5 µL) of 544 patients with breast cancer and 427 healthy controls was analyzed by ELISA. The breast cancer samples were acquired at the time of primary diagnosis prior to neoadjuvant therapy or surgery. A classifier was established on a training cohort of patients with breast cancer and age-adapted healthy controls and further validated on an independent cohort comprising breast cancer patients and healthy controls. Samples from patients with benign breast diseases were investigated as additional controls. Samples from patients with acute heart diseases (n = 127) were investigated as noncancer controls. The diagnostic accuracy was determined by receiver operating characteristic using the parameters area under the curve, sensitivity, and specificity. RESULTS: CCN1 was frequently secreted by breast cancer cell lines into the extracellular space. Subsequent analysis of clinical blood samples from patients with breast cancer and age-adjusted healthy controls revealed an overall specificity of 99.0% and sensitivity of 80.0% for cancer detection. Remarkably, 81.5% of small T1 cancers were already CCN1-positive, while CCN1 concentrations in patients with benign breast lesions were below the threshold for breast cancer detection. CONCLUSIONS: Circulating CCN1 is a potentially novel blood biomarker for the detection of breast cancer at the earliest invasive stage.

Sensitive Blood-Based Detection of Asbestos-Associated Diseases Using Cysteine-Rich Angiogenic Inducer 61 as Circulating Protein Biomarker.

BACKGROUND: Detection of asbestos-associated diseases like asbestosis or mesothelioma is still challenging. We sought to improve the diagnosis of benign asbestos-associated disease (BAAD) by detection of the protein cysteine-rich angiogenic inducer 61 (Cyr61) in human plasma. METHODS: Plasma Cyr61 was quantified using an enzyme-linked immunosorbent assay. Plasma samples from males diagnosed with BAAD, but without a malignant disease (n = 101), and malignant mesothelioma (n = 21; 15 males, 6 females), as well as nonasbestos-exposed healthy control participants (n = 150; 58 males, 92 females) were analyzed. Clinical sensitivity and specificity of Cyr61 were determined by receiver operating characteristic analysis. RESULTS: The median plasma Cyr61 concentration for healthy control participants was 0.27 ng/mL. Cytoplasmic Cyr61 in peripheral blood mononuclear cells from healthy control participants was evenly distributed, as detected by immunofluorescent staining. The increase in plasma Cyr61 concentrations in the BAAD study group was statistically significant compared to the healthy control participants (P < 0.0001). For the detection of BAAD vs male healthy control participants, clinical sensitivity was 88% and clinical specificity 95% with an area under the curve of 0.924 at maximal Youden Index. For a predefined clinical specificity of 100%, the clinical sensitivity was 76%. For male mesothelioma patients vs male healthy control participants, the clinical sensitivity at maximal Youden Index was 95% with a clinical specificity of 100% (area under the curve, 0.997) and for a predefined clinical specificity of 100%, the clinical sensitivity was 93%. CONCLUSIONS: In our study, plasma Cyr61 protein concentrations showed to be a new biomarker for asbestos-associated diseases like BAAD and mesothelioma in men, which deserves further investigation in large-scale cohort studies.

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.

Detection and clinical relevance of early disseminated breast cancer cells depend on their cytokeratin expression pattern.

The factors determining the clinical relevance of disseminated tumor cells (DTC) in breast cancer patients are largely unknown. Here we compared the specificity and clinical performance of two antibodies frequently used for DTC detection. Reactivities of antibodies A45-B/B3 (A45) and AE1/AE3 (AE) for selected cytokeratins (CK) were assessed by 2-DE Western Blot analysis. Using these antibodies bone marrow aspirates from 391 breast cancer patients (M(0), pT1-3, pN0-3) were screened for the presence of DTC. To obtain prognostic information, patients were followed up over a median of 83 months for time to relapse and 99 months for time to death. Among the analyzed CK, AE detected CK5, CK7, CK8, and CK19, whereas A45 recognized CK7 and CK18. In total, 24 of 391 patients (6.1%) were DTC-positive for A45, and 41 (10.5%) for AE. Although concordance between the two antibodies was 84.4%, overlap among positive cases was only 3.2%. DTC-positivity with AE and A45 was more frequent in patients of higher nodal status (P=0.019 and P=0.036, respectively). Nearly all patients with A45-positive DTC had hormone receptor-positive tumors (23/24), while detection of AE-positive DTC was more frequent among hormone receptor negative patients (P=0.006). Survival analyses of all patients revealed shorter distant disease-free survival (P=0.039) for patients with A45-positive DTC, whereas the prognostic relevance of AE-positive DTC was restricted to node-positive patients. The clinical utility of immunocytochemical (ICC) DTC detection depends on the anti-CK antibody used, which may reflect the complex CK composition of DTC.

Cysteine-Rich Angiogenic Inducer 61: Pro-Survival Function and Role as a Biomarker for Disseminating Breast Cancer Cells.

(1) Background: the early detection of cancer cells in the blood or bone marrow of breast cancer patients improves the understanding of metastasis. Disseminating tumor cells in the bone marrow with a pronounced manifestation of mesenchymal markers (mDTC) are difficult to detect by epithelial markers, but they are relevant in the initiation of metastasis. (2) Methods: the breast cancer mDTC cell line BC-M1 was analyzed by mass spectrometry, which revealed high levels of the protein-cysteine-rich angiogenic inducer 61 (Cyr61). The function of Cyr61 was investigated using shRNA and hypoxia. Peripheral blood samples from 35 breast cancer patients were investigated for CTCs defined as cytokeratin-positive/CD45-negative cells. (3) Results: the Cyr61 levels are elevated in mDTC lines from breast, lung, and prostate cancer patients. The loss of Cyr61 resulted in the diminished expression of hypoxia-inducible factor 1-alpha, and increased apoptosis. Cyr61 was present in 47 (43%) of the 109 detected circulating tumor cells (CTCs), while the blood and bone marrow cells from healthy controls were Cyr61-negative. (4) Conclusions: Cyr61 is expressed in mDTC lines, supports the viability of cancer cells, and classifies a new subset of cytokeratin-positive CTCs, which deserves further investigation.

Blood-based detection of lung cancer using cysteine-rich angiogenic inducer 61 (CYR61) as a circulating protein biomarker: a pilot study.

Lung cancer is the most often diagnosed cancer and the main cause of cancer deaths in the world compared with other tumor entities. To date, the only screening method for high-risk lung cancer patients is low-dosed computed tomography which still suffers from high false-positive rates and overdiagnosis. Therefore, there is an obvious need to identify biomarkers for the detection of lung cancer that could be used to guide the use of low-dosed computed tomography or other imaging procedures. We aimed to assess the performance of the protein cysteine-rich angiogenic inducer 61 (CYR61) as a circulating biomarker for the detection of lung cancer. CYR61 concentrations in plasma were significantly elevated in 87 lung cancer patients (13.7 ± 18.6 ng·mL-1 ) compared with 150 healthy controls (0.29 ± 0.22 ng·mL-1 ). Subset analysis stratified by sex revealed increased CYR61 concentrations for adenocarcinoma and squamous cell carcinoma in men compared with women. For male lung cancer patients versus male healthy controls, the sensitivity was 84% at a specificity of 100%, whereas for females, the sensitivity was 27% at a specificity of 99%. The determination of circulating CYR61 protein in plasma might improve the detection of lung cancer in men. The findings of this pilot study support further verification of CYR61 as a biomarker for lung cancer detection in men. Additionally, CYR61 is significantly elevated in women but sensitivity and specificity for CYR61 are too low for the improvement of the detection of lung cancer in women.

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.

Selective regain of egfr gene copies in CD44+/CD24-/low breast cancer cellular model MDA-MB-468.

BACKGROUND: Increased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies. METHODS: The basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique. RESULTS: Low and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies. CONCLUSION: Progressive genome modulation in the CD44+/CD24-/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression.

In Vitro Modeling of Reoxygenation Effects on mRNA and Protein Levels in Hypoxic Tumor Cells upon Entry into the Bloodstream.

BACKGROUND: Solid epithelial tumors like breast cancer are the most frequent malignancy in women. Circulating tumor cells (CTCs) are frequently released from hypoxic areas into the blood, where CTCs face elevated oxygen concentrations. This reoxygenation might challenge the use of CTCs for liquid biopsy. METHODS: We modeled this situation in vitro using the breast cancer cell lines-MCF-7, MDA-MB-468, MDA-MB-231-and the cell line BC-M1 established from DTCs in the bone marrow. Cells were cultured under hypoxia, followed by a reoxygenation pulse for 4 h, reflecting the circulation time of CTCs. Analyzed were gene products like EGFR, ErbB-2, EpCAM, PD-L1 on mRNA and protein level. RESULTS: mRNAs of erbb2 or pdl1 and protein levels of PD-L1 displayed significant changes, whereas ErbB-2 protein levels remained constant. The strongest discrepancy between protein and mRNA levels under hypoxia was observed for EGFR, supporting the idea of cap-independent translation of egfr mRNA. Analyses of the phosphorylation of AKT, Erk 1/2, and Stat3 revealed strong alterations after reoxygenation. CONCLUSIONS: CTCs reaching secondary sites faster than reoxygenation could alter the mRNA and protein levels in the cells. CTC and DTC with high PD-L1 levels might become quiescent under hypoxia but were easily reactivated by reoxygenation.

Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity.

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC-derived breast cancer cell line, designated CTC-ITB-01, established from a patient with metastatic estrogen receptor-positive (ER+ ) breast cancer, resistant to endocrine therapy. CTC-ITB-01 remained ER+ in culture, and copy number alteration (CNA) profiling showed high concordance between CTC-ITB-01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA-sequencing data indicate that CTC-ITB-01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER+ breast cancer. Downstream ER signaling was constitutively active in CTC-ITB-01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC-ITB-01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology.

miRNA-30 Family Members Inhibit Breast Cancer Invasion, Osteomimicry, and Bone Destruction by Directly Targeting Multiple Bone Metastasis-Associated Genes.

miRNAs are master regulators of gene expression that play key roles in cancer metastasis. During bone metastasis, metastatic tumor cells must rewire their biology and express genes that are normally expressed by bone cells (a process called osteomimicry), which endow tumor cells with full competence for outgrowth in the bone marrow. Here, we establish miR-30 family members miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e as suppressors of breast cancer bone metastasis that regulate multiple pathways, including osteomimicry. Low expression of miR-30 in primary tumors from patients with breast cancer were associated with poor relapse-free survival. In addition, estrogen receptor (ER)-negative/progesterone receptor (PR)-negative breast cancer cells expressed lower miR-30 levels than their ER/PR-positive counterparts. Overexpression of miR-30 in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivoIn vitro, miR-30 did not affect tumor cell proliferation, but did inhibit tumor cell invasion. Furthermore, overexpression of miR-30 restored bone homeostasis by reversing the effects of tumor cell-conditioned medium on osteoclastogenesis and osteoblastogenesis. A number of genes associated with osteoclastogenesis stimulation (IL8, IL11), osteoblastogenesis inhibition (DKK-1), tumor cell osteomimicry (RUNX2, CDH11), and invasiveness (CTGF, ITGA5, ITGB3) were identified as targets for repression by miR-30. Among these genes, silencing CDH11 or ITGA5 in ER-/PR-negative breast cancer cells recapitulated inhibitory effects of miR-30 on skeletal tumor burden in vivo Overall, our findings provide evidence that miR-30 family members employ multiple mechanisms to impede breast cancer bone metastasis and may represent attractive targets for therapeutic intervention.Significance: These findings suggest miR-30 family members may serve as an effective means to therapeutically attenuate metastasis in triple-negative breast cancer. Cancer Res; 78(18); 5259-73. ©2018 AACR.

PTEN mediates the cross talk between breast and glial cells in brain metastases leading to rapid disease progression.

Despite improvement of therapeutic treatments for breast cancer, the development of brain metastases has become a major limitation to life expectancy for many patients. Brain metastases show very commonly alterations in EGFR and HER2 driven pathways, of which PTEN is an important regulator. Here, we analyzed PTEN expression in 111 tissue samples of breast cancer brain metastases (BCBM). Loss of PTEN was found in a substantial proportion of BCBM samples (48.6%) and was significantly associated with triple-negative breast cancer (67.5%, p = 0.001) and a shorter survival time after surgical resection of brain metastases (p = 0.048). Overexpression of PTEN in brain-seeking MDA-MB-231 BR cells in vitro reduced activation of the AKT pathway, notably by suppression of Akt1 kinase activity. Furthermore, the migration of MDA-MB-231 BR cells in vitro was promoted by co-culturing with both astrocytes and microglial cells. Interestingly, when PTEN was overexpressed the migration was significantly inhibited. Moreover, in an ex vivo organotypic brain slice model, PTEN overexpression reduced invasion of tumor cells. This was accompanied by reduced astrocyte activation that was mediated by autocrine and paracrine activation of GM-CSF/ CSF2RA and AKT/ PTEN pathways. In conclusion, loss of PTEN is frequently detected in triple-negative BCBM patients and associated with poor prognosis. The findings of our functional studies suggest that PTEN loss promotes a feedback loop between tumor cells and glial cells, which might contribute to disease progression.

Functional studies on circulating and disseminated tumor cells in carcinoma patients.

Despite numerous clinical studies indicating the clinical relevance of circulating tumor cells (CTCs) in blood and disseminated tumor cells (DTCs) in the bone marrow of cancer patients, the functional properties of these cells are largely unknown. The focus of this review is to emphasize how functional studies on viable CTCs and DTCs can enlarge the spectrum of applications of "liquid biopsies". The low number of CTCs in the peripheral blood and DTCs in the bone marrow and the fact that carcinoma cells are difficult to culture are major challenges. Significant advances in the in vitro and in vivo expansion of CTCs and DTCs from cancer patients have been achieved, which enable us now to study the functional properties of these cells. Here, we discuss published data about functional studies on CTCs and DTCs using in vitro cultivation and in vivo xenograft models. Functional analyses on CTCs and DTCs offer the possibility to identify the metastasis-initiating cells. Moreover, CTC-derived cell lines and xenografts might point to new therapeutic targets and can be used for drug development.

Disseminated Tumor Cells Persist in the Bone Marrow of Breast Cancer Patients through Sustained Activation of the Unfolded Protein Response.

Disseminated tumor cells (DTC), which share mesenchymal and epithelial properties, are considered to be metastasis-initiating cells in breast cancer. However, the mechanisms supporting DTC survival are poorly understood. DTC extravasation into the bone marrow may be encouraged by low oxygen concentrations that trigger metabolic and molecular alterations contributing to DTC survival. Here, we investigated how the unfolded protein response (UPR), an important cytoprotective program induced by hypoxia, affects the behavior of stressed cancer cells. DTC cell lines established from the bone marrow of patients with breast cancer (BC-M1), lung cancer, (LC-M1), and prostate cancer (PC-E1) were subjected to hypoxic and hypoglycemic conditions. BC-M1 and LC-M1 exhibiting mesenchymal and epithelial properties adapted readily to hypoxia and glucose starvation. Upregulation of UPR proteins, such as the glucose-regulated protein Grp78, induced the formation of filamentous networks, resulting in proliferative advantages and sustained survival under total glucose deprivation. High Grp78 expression correlated with mesenchymal attributes of breast and lung cancer cells and with poor differentiation in clinical samples of primary breast and lung carcinomas. In DTCs isolated from bone marrow specimens from breast cancer patients, Grp78-positive stress granules were observed, consistent with the likelihood these cells were exposed to acute cell stress. Overall, our findings provide the first evidence that the UPR is activated in DTC in the bone marrow from cancer patients, warranting further study of this cell stress pathway as a predictive biomarker for recurrent metastatic disease.

Frequent expression of PD-L1 on circulating breast cancer cells.

Immune checkpoint regulators such as PD-L1 have become exciting new therapeutic targets leading to long lasting remissions in patients with advanced malignancies. However, in view of the remarkable costs and the toxicity profiles of these therapies, predictive biomarkers able to discriminate responders from non-responders are urgently needed. In the present paper, we provide evidence that PD-L1 is frequently expressed on metastatic cells circulating in the blood of hormone receptor-positive, HER2-negative breast cancer patients. We performed western blot, flow cytometry and immunocytochemical analyses to demonstrate the specificity of the PDL1 antibody used in our study and established immunoscores for PDL1 expression on single tumor cells. We then selected sixteen patients with circulating tumor cells (CTCs) using the CellSearch(®) system and found PD-L1((+)) CTCs in 11 patients (68.8%). The fraction of PD-L1((+)) CTCs varied from 0.2 to 100% in individual patients. This is the first report demonstrating the expression of PD-L1 on CTCs. The established CTC/PD-L1 assay can be used for liquid biopsy in future clinical trials for stratification and monitoring of cancer patients undergoing immune checkpoint blockade.

The interrelating dynamics of hypoxic tumor microenvironments and cancer cell phenotypes in cancer metastasis.

The interrelating dynamics of the primary tumor cells and their surrounding microenvironment might determine phenotypic characteristics of disseminated tumor cells and contribute to cancer metastasis. Cytoprotective mechanisms (e.g., energy metabolism control, DNA damage response, global translation control and unfolded protein response) exert selective pressure in the tumor microenvironment. In particular, adaptation to hypoxia is vital for survival of malignant cells in the tumor and at distant sites such as the bone marrow. In addition to the stress response, the ability of tumor cells to undergo certain cellular re-differentiation programmes like the epithelial-mesenchymal transition (EMT), which is linked to cancer stemness, appears to be important for successful cancer cell spread. Here we will discuss the selection pressures that eventually lead to the formation of overt metastases. We will focus the properties of the microenvironment including (i) metabolic and cytoprotective programs that ensure survival of disseminated tumor cells, (ii) blood vessel structure, and (iii) the hypoxia-normoxia switch as well as intrinsic factors affecting the evolvement of novel tumor cell populations.

Distinct functional roles of Akt isoforms for proliferation, survival, migration and EGF-mediated signalling in lung cancer derived disseminated tumor cells.

Single disseminated tumor cells (DTC) can be detected in the bone marrow (BM) from 20% to 60% of patients with various tumors including non-small cell lung cancer (NSCLC). Detection of DTC in the BM of NSCLC patients is associated with poor prognosis and may be responsible for metastatic relapse. However, the functional properties of DTC are widely unknown. Here, we performed the first functional analysis of DTC focusing on the activation of the PI3K/Akt signalling pathway and the functional roles of Akt isoforms. In vitro kinase assays revealed a high activity of Akt3 in NSCLC-derived DTC. Proliferation and survival of DTC was reduced by depletion of Akt3 and to a lesser extend by Akt1, but not after depletion of Akt2. The major effect of Akt3 on the proliferation of DTC was associated with an Akt3-mediated regulation of both, cyclin D1 and cyclin D3, whereas Akt1 regulated the expression of cyclin D1 only. In contrast all three Akt isoforms, especially Akt2, were involved in the regulation of migration. Analysis of signalling events downstream of distinct Akt isoforms revealed that expression levels of urokinase-type plasminogen activator and its receptor were decreased after knockdown of Akt1 and Akt3. In addition, EGF-stimulated proliferative and anti-apoptotic signals are mediated by Akt1 and Akt3 in DTC. Finally, by immunofluorescence staining of primary DTC from BM samples of lung cancer patients, pAkt(S473) and Akt3 positive DTC were detected in vivo. Our data demonstrate that Akt1 and notably Akt3 regulate proliferation, survival, migration and EGF-mediated signal transduction in NSCLC-derived DTC.

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.