CXCR4 and JUNB double-positive disseminated tumor cells are detected frequently in breast cancer patients at primary diagnosis.
ABSTRACT: Background:The chemokine receptor CXCR4 and the transcription factor JUNB, expressed on a variety of tumor cells, seem to play an important role in the metastatic process. Since disseminated tumor cells (DTCs) in the bone marrow (BM) have been associated with worse outcomes, we evaluated the expression of CXCR4 and JUNB in DTCs of primary, nonmetastatic breast cancer (BC) patients before the onset of any systemic treatment. Methods:Bilateral BM (10?ml) aspirations of 39 hormone receptor (HR)-positive, HER2-negative BC patients were assessed for the presence of DTCs using the following combination of antibodies: pan-cytokeratin (A45-B/B3)/CXCR4/JUNB. An expression pattern of the examined proteins was created using confocal laser scanning microscopy, Image J software and BC cell lines. Results:CXCR4 was overexpressed in cancer cells and DTCs, with the following hierarchy of expression: SKBR3?>?MCF7?>?DTCs?>?MDA-MB231. Accordingly, the expression pattern of JUNB was: DTCs?>?MDA-MB231?>?SKBR3?>?MCF7. The mean intensity of CXCR4 (6411?±?334) and JUNB (27725.64?±?470) in DTCs was statistically higher compared with BM hematopoietic cells (2009?±?456, p?=?0.001; and 11112.89?±?545, p?=?0.001, respectively). The (CXCR4+JUNB+CK+) phenotype was the most frequently detected [90% (35/39)], followed by the (CXCR4-JUNB+CK+) phenotype [36% (14/39)]. However, (CXCR4+JUNB-CK+) tumor cells were found in only 5% (3/39) of patients. Those patients harboring DTCs with the (CXCR4+JUNB+CK+) phenotype revealed lower overall survival (Cox regression: p?=?0.023). Conclusions:(CXCR4+JUNB+CK+)-expressing DTCs, detected frequently in the BM of BC patients, seem to identify a subgroup of patients at higher risk for relapse that may be considered for close follow up.
Project description:BACKGROUND:Circulating tumor cells (CTCs) are important for metastatic dissemination of cancer. They can provide useful information, regarding biological features and tumor heterogeneity; however, their detection and characterization are difficult due to their limited number in the bloodstream and their mesenchymal characteristics. Therefore, new biomarkers are needed to address these questions. METHODS:Bioinformatics functional enrichment analysis revealed a subgroup of 24 genes, potentially overexpressed in CTCs. Among these genes, the chemokine receptor CXCR4 plays a central role. After prioritization according to the CXCR4 corresponding pathways, five molecules (JUNB, YWHAB, TYROBP, NFYA, and PRDX1) were selected for further analysis in biological samples. The SKBR3, MDA-MB231, and MCF7 cell lines, as well as PBMCs from normal (n?=?10) blood donors, were used as controls to define the expression pattern of all the examined molecules. Consequently, 100 previously untreated metastatic breast cancer (mBC) patients (n?=?100) were analyzed using the following combinations of antibodies: CK (cytokeratin)/CXCR4/JUNB, CK/NFYA/?WH?? (14-3-3), and CK/TYROBP/PRDX1. A threshold value for every molecule was considered the mean expression in normal PBMCs. RESULTS:Quantification of CXCR4 revealed overexpression of the receptor in SKBR3 and in CTCs, following the subsequent scale (SKBR3>CTCs>Hela>MCF7>MDA-MB231). JUNB was also overexpressed in CTCs (SKBR3>CTCs>MCF7>MDA-MB231>Hela). According to the defined threshold for each molecule, CXCR4-positive CTCs were identified in 90% of the patients with detectable tumor cells in their blood. In addition, 65%, 75%, 14.3%, and 12.5% of the patients harbored JUNB-, TYROBP-, NFYA-, and PRDX-positive CTCs, respectively. Conversely, none of the patients revealed YWHAB-positive CTCs. Interestingly, JUNB expression in CTCs was phenotypically and statistically enhanced compared to patients' blood cells (p?=?0.002) providing a possible new biomarker for CTCs. Furthermore, the detection of JUNB-positive CTCs in patients was associated with poorer PFS (p?=?0.015) and OS (p?=?0.002). Moreover, JUNB staining of 11 primary and 4 metastatic tumors from the same cohort of patients revealed a dramatic increase of JUNB expression in metastasis. CONCLUSIONS:CXCR4, JUNB, and TYROBP were overexpressed in CTCs, but only the expression of JUNB was associated with poor prognosis, providing a new biomarker and a potential therapeutic target for the elimination of CTCs.
Project description:Dormant or slow-cycling disseminated tumor cells (DTCs) in bone marrow (BM) are resistant to conventional therapy in various cancers including head and neck squamous cell carcinoma (HNSCC), although the molecular mechanisms remain largely unknown. This study aimed to identify the intrinsic molecular mechanisms underlying drug resistance in BM-DTCs. We used in vivo selection of the human HNSCC cell line HEp3, which mimics non-proliferative BM-DTCs in mice, to establish BM-DTC-derived (BM-HEp3) and lung metastases-derived (Lu-HEp3) sublines. Both sublines had higher migration activity and shortened survival in a murine xenograft model compared with parental (P-HEp3) cells. Slow-cycling BM-HEp3 cells had intrinsically enhanced cisplatin resistance compared with Lu-HEp3 cells, which also manifested this resistance but proliferated rapidly. The drug resistance and slow-cycling state of BM-HEp3 cells depended on enhanced positive feedback of the signaling axis of stromal cell-derived factor-1 (SDF-1)-C-X-C chemokine receptor-4 (CXCR4) via their overexpression. Interestingly, BM-DTCs highly expressed transforming growth factor-beta 2 (TGF-?2) to maintain SDF-1-CXCR4 overexpression. Inhibition of SDF-1-CXCR4 signaling by down-regulating TGF-?2 fully reversed the drug resistance of BM-HEp3 cells via reactivation of cell proliferation. These data suggest that the intrinsic TGF-?2-triggered SDF-1-CXCR4 signaling axis is crucial for drug resistance dependent on a slow-cycling state in BM-DTCs.
Project description:Circulating tumor cells (CTCs) are the major players in the metastatic process. A potential mechanism of cell migration and invasion is the formation of microtentacles in tumor cells. These structures are supported by ?-tubulin (TUB), detyrosinated ?-tubulin (GLU), and vimentin (VIM). In the current study, we evaluated the expression of those cytoskeletal proteins in CTCs.Forty patients with breast cancer (BC) (16 early and 24 metastatic) were enrolled in the study. CTCs were isolated using the ISET platform and stained with the following combinations of antibodies: pancytokeratin (CK)/VIM/TUB and CK/VIM/GLU. Samples were analyzed with the ARIOL platform and confocal laser scanning microscopy.Fluorescence quantification revealed that the ratios CK/TUB, CK/VIM, and CK/GLU were statistically increased in MCF7 compared with more aggressive cell lines (SKBR3 and MDA-MB-231). In addition, all of these ratios were statistically increased in MCF7 cells compared with metastatic BC patients' CTCs (p?=?0.0001, p?=?0.0001, and p?=?0.003, respectively). Interestingly, intercellular connections among CTCs and between CTCs and blood cells through cytoskeleton bridges were revealed, whereas microtentacles were increased in patients with CTC clusters. These intercellular connections were supported by TUB, VIM, and GLU. Quantification of the examined molecules revealed that the median intensity of TUB, GLU, and VIM was significantly increased in patients with metastatic BC compared with those with early disease (TUB, 62.27 vs 11.5, p?= 0.0001; GLU, 6.99 vs 5.29, p?= 0.029; and VIM, 8.24 vs 5.38, p?= 0.0001, respectively).CTCs from patients with BC aggregate to each other and to blood cells through cytoskeletal protrusions, supported by VIM, TUB, and GLU. Quantification of these molecules could potentially identify CTCs related to more aggressive disease.
Project description:Real-time monitoring of biologic changes in tumors may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in cancer patients pose major hurdles. The goal of this study was to determine whether breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes.CTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counterstaining of red blood cell-lysed blood and bone marrow fractions, respectively. The rate of lung metastases (LM) was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher's Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed by using the expression arrays of primary tumors from different PDX lines and subsequently overlapped to identify common genes.In total, 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present as either single cells or clusters, were detected in 83% (15 of 18) and 62.5% (10 to16) of the lines, respectively. A positive association was noted between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 of 18 (50%) lines, of which all nine had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the two genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified four genes (HLA-DP1A, GJA1, PEG3, and XIST). This four-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients.This study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases.
Project description:In the bone marrow (BM) microenvironment, where breast cancer (BC) disseminated tumour cells (DTCs) can remain dormant for decades, NG2<sup>+</sup>/Nestin<sup>+</sup> mesenchymal stem cells (MSCs) promote hematopoietic stem cell quiescence. Here, we reveal that periarteriolar BM-resident NG2<sup>+</sup>/Nestin<sup>+</sup> MSCs can also instruct BC DTCs to enter dormancy. NG2<sup>+</sup>/Nestin<sup>+</sup> MSCs produce TGFβ2 and BMP7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which <i>via</i> p38-kinase result in p27 induction. Genetic depletion of MSCs or conditional knock-out of TGFβ2 in MSCs using an NG2-Cre<sup>ER</sup> driver led to bone metastatic outgrowth of otherwise dormant p27<sup>+</sup>/Ki67<sup>-</sup> DTCs. Also ER<sup>+</sup> BC patients without systemic recurrence displayed higher frequency of TGFβ2 and BMP7 detection in the BM. Our results provide a direct proof that HSC dormancy niches control BC DTC dormancy and suggest that aging or extrinsic factors that affect the NG2<sup>+</sup>/Nestin<sup>+</sup> MSC niche homeostasis may result in a break from dormancy and BC bone relapse.
Project description:<h4>Purpose</h4>Molecular characterization of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients has been hindered by their rarity. To enrich for these cells using an antigen-independent methodology, we have evaluated a size-based microfiltration device in combination with several downstream biomarker assays.<h4>Methods</h4>BM aspirates were collected from healthy volunteers or BC patients. Healthy BM was mixed with a specified number of BC cells to calculate recovery and fold enrichment by microfiltration. Specimens were pre-filtered using a 70 ?m mesh sieve and the effluent filtered through CellSieve microfilters. Captured cells were analyzed by immunocytochemistry (ICC), FISH for HER-2/neu gene amplification status, and RNA in situ hybridization (RISH). Cells eluted from the filter were used for RNA isolation and subsequent qRT-PCR analysis for DTC biomarker gene expression.<h4>Results</h4>Filtering an average of 14×106 nucleated BM cells yielded approximately 17-21×103 residual BM cells. In the BC cell spiking experiments, an average of 87% (range 84-92%) of tumor cells were recovered with approximately 170- to 400-fold enrichment. Captured BC cells from patients co-stained for cytokeratin and EpCAM, but not CD45 by ICC. RNA yields from 4 ml of patient BM after filtration averaged 135ng per 10 million BM cells filtered with an average RNA Integrity Number (RIN) of 5.3. DTC-associated gene expression was detected by both qRT-PCR and RISH in filtered spiked or BC patient specimens but, not in control filtered normal BM.<h4>Conclusions</h4>We have tested a microfiltration technique for enrichment of BM DTCs. DTC capture efficiency was shown to range from 84.3% to 92.1% with up to 400-fold enrichment using model BC cell lines. In patients, recovered DTCs can be identified and distinguished from normal BM cells using multiple antibody-, DNA-, and RNA-based biomarker assays.
Project description:Breast cancer (BC) is the most common cancer in women. Estrogen, epidermal growth factor receptor 2 (ERBB2, HER2), and oxidative stress represent critical mechanistic nodes associated with BC. RLIP76 is a major mercapturic acid pathway transporter whose expression is increased in BC. In the quest of a novel molecule with chemopreventive and chemotherapeutic potential, we evaluated the effects of 2'-Hydroxyflavanone (2HF) in BC. 2HF enhanced the inhibitory effects of RLIP76 depletion and also inhibited RLIP76-mediated doxorubicin transport in BC cells. RNA-sequencing revealed that 2HF induces strong reversal of the gene expression pattern in ER+MCF7, HER2+ SKBR3 and triple-negative MDA-MB-231 BC cells with minimal effects on MCF10A normal breast epithelial cells. 2HF down regulated ER? and enhanced inhibitory effects of imatinib mesylate/Gleevec in MCF7 cells. 2HF also down regulated ER? and HER2 gene networks in MCF7 and SKBR3 cells, respectively. 2HF activated TP53 and inhibited TGF?1 canonical pathway in MCF7 and MDA-MB-231 BC cells. 2HF also regulated the expression of a number of critical prognostic genes of MammaPrint panel and their upstream targets including TP53, CDKN2A and MYC. The collective findings from this study provide a comprehensive, direct and integrated evidence for the benefits of 2HF in targeting major and clinically relevant mechanistic regulators of BC.
Project description:Increased expression of Yes-associated protein-1 (YAP1) was shown to correlate with reduced survival in breast cancer (BC) patients. However, the exact mechanism of YAP1 regulation in BC cells remains ambiguous. Genomic sequence search showed that the promoter region of the YAP1 gene contains CpG Islands, hence the likelihood of epigenetic regulation by DNA methylation. To address this possibility, the effect of estrogen (17? estradiol; E2) on YAP1 gene expression and YAP1 promoter methylation status was evaluated in BC cells. The functional consequences of E2 treatment in control and YAP1-silenced BC cells were also investigated. Our data showed that E2 modulates YAP1 expression by hypomethylation of its promoter region via downregulation of DNA methyltransferase 3B (DNMT3B); an effect that seems to facilitate tumor progression in BC cells. Although the effect of E2 on YAP1 expression was estrogen receptor (ER) dependent, E2 treatment also upregulated YAP1 expression in MDA-MB231 and SKBR3 cells, which are known ER-negative BC cell lines but expresses ER?. Functionally, E2 treatment resulted in increased cell proliferation, decreased apoptosis, cell cycle arrest, and autophagic flux in MCF7 cells. The knockdown of the YAP1 gene reversed these carcinogenic effects of E2 and inhibited E2-induced autophagy. Lastly, we showed that YAP1 is highly expressed and hypomethylated in human BC tissues and that increased YAP1 expression correlates negatively with DNMT3B expression but strongly associated with ER expression. Our data provide the basis for considering screening of YAP1 expression and its promoter methylation status in the diagnosis and prognosis of BC.
Project description:C-X-C chemokine receptor type 4 (CXCR4) is known to regulate lung, pancreatic and prostate cancer stem cells. In breast cancer, CXCR4 signalling has been reported to be a mediator of metastasis, and is linked to poor prognosis. However its role in normal and malignant breast stem cell function has not been investigated. Anoikis resistant (AR) cells were collected from immortalised (MCF10A, 226L) and malignant (MCF7, T47D, SKBR3) breast cell lines and assessed for stem cell enrichment versus unsorted cells. AR cells had significantly higher mammosphere forming efficiency (MFE) than unsorted cells. The AR normal cells demonstrated increased formation of 3D structures in Matrigel compared to unsorted cells. In vivo, SKBR3 and T47D AR cells had 7- and 130-fold enrichments for tumour formationrespectively, compared with unsorted cells. AR cells contained significantly elevated CXCR4 transcript and protein levels compared to unsorted cells. Importantly, CXCR4 mRNA was higher in stem cell-enriched CD44+/CD24- patient-derived breast cancer cells compared to non-enriched cells. CXCR4 stimulation by its ligand SDF-1 reduced MFE of the normal breast cells lines but increased the MFE in T47D and patient-derived breast cancer cells. CXCR4 inhibition by AMD3100 increased stem cell activity but reduced the self-renewal capacity of the malignant breast cell line T47D. CXCR4+ FACS sorted MCF7 cells demonstrated a significantly increased MFE compared with CXCR4- cells. This significant increase in MFE was further demonstrated in CXCR4 over-expressing MCF7 cells which also had an increase in self-renewal compared to parental cells. A greater reduction in self-renewal following CXCR4 inhibition in the CXCR4 over-expressing cells compared with parental cells was also observed. Our data establish for the first time that CXCR4 signalling has contrasting effects on normal and malignant breast stem cell activity. Here, we demonstrate that CXCR4 signalling specifically regulates breast cancer stem cell activities and may therefore be important in tumour formation at the sites of metastases.
Project description:<h4>Background</h4>ERBB-2 is overexpressed in about 20% of breast cancers (BCs), indicating poor prognosis. The receptor activator of nuclear factor-?B (RANK) pathway is implicated in ERBB-2 (+) BC. The purpose of this study was to elucidate the underlying molecular mechanism of this interaction and the beneficial impact of dual targeting of RANK and ERBB-2 pathways.<h4>Methods</h4>We used SKBR3, MCF7, MDA-MB-453, and BT-474 human BC cell lines. We examined RANK and RANKL expression using RT-PCR, Western blot, and immunofluorescence. The evaluation of RANK expression in a cohort of BC patients was performed using immunohistochemistry. The interaction between RANK and ERBB family members was detected using proximity ligation assay (PLA), which enables the visualization of interacting proteins. We used inhibitors of both pathways [trastuzumab (T), pertuzumab (P), denosumab (D)]. NF-?B pathway activation was studied using Western blot. Cell growth and viability was evaluated using XTT, flow cytometry, and clonogenic assay. For cell migration evaluation, scratch assay was performed. Data were analyzed by one-way ANOVA.<h4>Results</h4>Cell lines express RANK and RANKL. RANK immunostaining was also detected in human BC tissue samples. RANK receptor dimerizes with ERBB family members. RANK/ERBB-2 dimer number seems to be associated with ERBB-2 expression (SKBR3, 5.4; BT-474, 8.2; MCF7, 0.7; MDA-MB-453, 0.3). RANK/ERBB-2 dimers were decreased in the presence of the inhibitors D, T, and P, while they were increased after RANKL (R) treatment in SKBR3 (m, 5.4; D, 1.2; T, 1.9; DT, 0.6; TP, 1; DTP, 0.4; R, 11.8) and BT-474 (m, 8.2; D, 3.1; T, 4.3; DT, 0.7; TP, 3.4; DTP, 3.2; R, 11.6). Combination targeting of SKBR3 further decreased NF-?B pathway activation compared to single targeting. In SKBR3, RANKL and ERBB-2 blockage resulted in reduced cell proliferation, increased apoptosis, and lower metastatic potential compared to mock cells (m) and reversed values in RANKL presence. The combination treatment of SKBR3 with D, T, and P had an advantage in functional traits compared to single targeting. Denosumab suppressed NF-?B signaling and diminished proliferation rate in MDA-MB-453 cells. MCF7 did not correspond to inhibitors.<h4>Conclusions</h4>The results indicate a novel physical and molecular association between ERBB-2 and RANK pathways that affects ERBB-2 (+) BC growth. We also present data suggesting that the combination of anti-ERBB-2 agents and RANKL inhibitors have a potential direct anti-tumor effect and should be further tested in certain BC patients.