The prognostic value of JUNB-positive CTCs in metastatic breast cancer: from bioinformatics to phenotypic characterization

BackgroundCirculating 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.MethodsBioinformatics 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/ΥWHΑΒ (14-3-3), and CK/TYROBP/PRDX1. A threshold value for every molecule was considered the mean expression in normal PBMCs.ResultsQuantification 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.ConclusionsCXCR4, 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.

[1]  L. Kenner,et al.  The oncoprotein NPM-ALK of anaplastic large-cell lymphoma induces JUNB transcription via ERK1/2 and JunB translation via mTOR signaling. , 2007, Blood.

[2]  Guangming Zhou,et al.  RAC2 promotes abnormal proliferation of quiescent cells by enhanced JUNB expression via the MAL-SRF pathway , 2018, Cell cycle.

[3]  Y. Benjamini,et al.  Controlling the false discovery rate in behavior genetics research , 2001, Behavioural Brain Research.

[4]  Susumu Goto,et al.  KEGG: Kyoto Encyclopedia of Genes and Genomes , 2000, Nucleic Acids Res..

[5]  R. Tibshirani,et al.  Significance analysis of microarrays applied to the ionizing radiation response , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[6]  P. Hall,et al.  Polymorphisms in oxidative stress-related genes and mortality in breast cancer patients--potential differential effects by radiotherapy? , 2013, Breast.

[7]  C. Wang,et al.  HMGB3 characterization in gastric cancer. , 2013, Genetics and molecular research : GMR.

[8]  D. Mavroudis,et al.  Predictive and prognostic value of peripheral blood cytokeratin-19 mRNA-positive cells detected by real-time polymerase chain reaction in node-negative breast cancer patients. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[9]  Michalis E. Zervakis,et al.  On the Identification of Circulating Tumor Cells in Breast Cancer , 2014, IEEE Journal of Biomedical and Health Informatics.

[10]  J. Šufliarský,et al.  CXCR4-SDF-1 interaction potentially mediates trafficking of circulating tumor cells in primary breast cancer , 2016, BMC Cancer.

[11]  K. Pienta,et al.  Apoptosis of circulating tumor cells in prostate cancer patients , 2004, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[12]  G. Kallergi,et al.  Evaluation of PD-L1/PD-1 on circulating tumor cells in patients with advanced non-small cell lung cancer , 2018, Therapeutic advances in medical oncology.

[13]  Jing Wang,et al.  WebGestalt 2017: a more comprehensive, powerful, flexible and interactive gene set enrichment analysis toolkit , 2017, Nucleic Acids Res..

[14]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Thomas R. Sutter,et al.  Epithelial to mesenchymal transition in human breast epithelial cells transformed by 17beta-estradiol. , 2007, Cancer research.

[16]  Ying Jiang,et al.  PDK1 induces JunB, EMT, cell migration and invasion in human gallbladder cancer , 2015, Oncotarget.

[17]  S. Digumarthy,et al.  Isolation of rare circulating tumour cells in cancer patients by microchip technology , 2007, Nature.

[18]  C. Sotiriou,et al.  HER2-Positive Circulating Tumor Cells in Breast Cancer , 2011, PloS one.

[19]  J. Russo,et al.  Epithelial to mesenchymal transition in human breast epithelial cells transformed by 17beta-estradiol. , 2007, Cancer research.

[20]  Y. Soini,et al.  Oxidative stress and counteracting mechanisms in hormone receptor positive, triple-negative and basal-like breast carcinomas , 2011, BMC Cancer.

[21]  S. Agelaki,et al.  Trastuzumab decreases the incidence of clinical relapses in patients with early breast cancer presenting chemotherapy-resistant CK-19mRNA-positive circulating tumor cells: results of a randomized phase II study. , 2012, Annals of oncology : official journal of the European Society for Medical Oncology.

[22]  Tanja Fehm,et al.  Circulating Tumor Cells in Patients with Breast Cancer Dormancy , 2004, Clinical Cancer Research.

[23]  Sameera R. Wijayawardana,et al.  Prognostic and predictive value of circulating tumor cells and CXCR4 expression as biomarkers for a CXCR4 peptide antagonist in combination with carboplatin-etoposide in small cell lung cancer: exploratory analysis of a phase II study , 2017, Investigational New Drugs.

[24]  Harris Markomanolaki,et al.  Apoptotic Circulating Tumor Cells in Early and Metastatic Breast Cancer Patients , 2013, Molecular Cancer Therapeutics.

[25]  F. Balkwill.  Cancer and the chemokine network , 2004, Nature Reviews Cancer.

[26]  G. Kallergi,et al.  Expression of truncated human epidermal growth factor receptor 2 on circulating tumor cells of breast cancer patients , 2015, Breast Cancer Research.

[27]  Gyan Bhanot,et al.  Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines , 2012, PloS one.

[28]  G. Kallergi,et al.  Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer. , 2010, Cancer letters.

[29]  Alison Stopeck,et al.  Circulating tumor cells, disease progression, and survival in metastatic breast cancer. , 2004, The New England journal of medicine.

[30]  Ivan Merelli,et al.  A multilevel data integration resource for breast cancer study , 2010, BMC Systems Biology.

[31]  Jocelyn Kaiser,et al.  Medicine. Cancer's circulation problem. , 2010, Science.

[32]  M. Karin,et al.  Rapid and preferential activation of the c-jun gene during the mammalian UV response , 1991, Molecular and cellular biology.

[33]  N. Selvamurugan,et al.  Transforming growth factor-β1 regulation of ATF-3, c-Jun and JunB proteins for activation of matrix metalloproteinase-13 gene in human breast cancer cells. , 2017, International journal of biological macromolecules.

[34]  Sridhar Ramaswamy,et al.  Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility , 2014, Science.

[35]  J. Muhlbauer,et al.  Peroxiredoxin proteins protect MCF-7 breast cancer cells from doxorubicin-induced toxicity. , 2014, International journal of oncology.

[36]  Mahmood Ayub,et al.  Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer , 2014, Nature Medicine.

[37]  Tim Holland-Letz,et al.  Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay , 2013, Nature Biotechnology.

[38]  R. Bravo,et al.  The jun and fos protein families are both required for cell cycle progression in fibroblasts , 1991, Molecular and cellular biology.

[39]  Alex E. Lash,et al.  Gene Expression Omnibus: NCBI gene expression and hybridization array data repository , 2002, Nucleic Acids Res..

[40]  Qiuping Hu,et al.  JUNB promotes the survival of Flavopiridol treated human breast cancer cells. , 2014, Biochemical and biophysical research communications.

[41]  Harris Markomanolaki,et al.  Hypoxia-inducible factor-1α and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients , 2009, Breast Cancer Research.

[42]  S. Agelaki,et al.  Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients , 2011, Breast Cancer Research.

[43]  G. Kallergi,et al.  Co-expression of putative stemness and epithelial-to-mesenchymal transition markers on single circulating tumour cells from patients with early and metastatic breast cancer , 2014, BMC Cancer.

[44]  H. Aburatani,et al.  JUNB governs a feed-forward network of TGFβ signaling that aggravates breast cancer invasion , 2017, Nucleic acids research.

[45]  T. Mcclanahan,et al.  Involvement of chemokine receptors in breast cancer metastasis , 2001, Nature.

[46]  G. Kallergi,et al.  Phenotypic characterization of circulating tumor cells in the peripheral blood of patients with small cell lung cancer , 2017, PloS one.

[47]  Ruud H. Brakenhoff,et al.  Detection, clinical relevance and specific biological properties of disseminating tumour cells , 2008, Nature Reviews Cancer.

[48]  Hilde van der Togt,et al.  Publisher's Note , 2003, J. Netw. Comput. Appl..

[49]  Da Li,et al.  LINC01016 promotes the malignant phenotype of endometrial cancer cells by regulating the miR-302a-3p/miR-3130-3p/NFYA/SATB1 axis , 2018, Cell Death & Disease.

[50]  S. Shyue,et al.  Increased expression of 14-3-3β promotes tumor progression and predicts extrahepatic metastasis and worse survival in hepatocellular carcinoma. , 2011, The American journal of pathology.

[51]  K. Pachmann,et al.  Quantification of circulating tumour cells for the monitoring of adjuvant therapy in breast cancer: an increase in cell number at completion of therapy is a predictor of early relapse. , 2007, Breast.

[52]  G. Kallergi,et al.  Phenotypic characterization of circulating tumor cells in triple negative breast cancer patients , 2016, Oncotarget.

[53]  J. Szade,et al.  Mesenchymal Phenotype of CTC-Enriched Blood Fraction and Lymph Node Metastasis Formation Potential , 2014, PloS one.

[54]  I. Vlachonikolis,et al.  Molecular detection of cytokeratin-19-positive cells in the peripheral blood of patients with operable breast cancer: evaluation of their prognostic significance. , 2002, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[55]  H. Stein,et al.  Aberrantly expressed c‐Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF‐κB , 2002, The EMBO journal.

[56]  Song Liu,et al.  Jcb: Article , 2022 .

[57]  Hiroyuki Ogata,et al.  KEGG: Kyoto Encyclopedia of Genes and Genomes , 1999, Nucleic Acids Res..

[58]  J. Teixidó,et al.  The good and bad faces of the CXCR4 chemokine receptor. , 2018, The international journal of biochemistry & cell biology.