Abstract: Chemokine receptor CXCR4 is involved in the maintenance of stemness property of stem cells, aging and metastasis of cancer. NRF1 is one of the major transcription factors that controls transcription of CXCR4. In this study, we have investigated whether transcription regulation of CXCR4 by NRF1 controls estrogen-induced malignant transformation of breast epithelial cells to breast cancer stem cells. The functional regulation of transcription of NRF1 target genes has not been explored in breast cancer. We have previously shown that NRF1 may be involved in 17 β-estradiol (E2) induced malignant transformation of breast epithelial cells, however, the mechanism of transcriptional regulation of the NRF1 target genes, such as CXCR4 remain unknown. In this study we showed that NRF1 and estrogen jointly contributed in reprogramming of breast epithelial cells to breast cancer stem cells. Levels of breast cancer stem cell markers (CD44+CD24+ALDH1+CD133+) were significantly increased by E2 treatment in NRF1 overexpressing cells compared to cells transfected with vector receiving E2. E2-induced increases of spheroid formation, cell survival and growth of cancer stem cells were modulated by functional gain or loss of NRF1. Overexpression of NRF1 promoted the transition of E2-treated breast epithelial MCF-10A cells to mesenchymal stem cell-like phenotype. The ChIP qPCR, RT-qPCR, Western blotting and immunofluorescence microscopic assays showed that NRF1 mediated transcriptional changes of its target genes -CXCR4, BNIP3, and DJ-1 correlated with malignant phenotypic changes. In summary, our findings for the first time showed that transcriptional regulation of CXCR4 by NRF1 may contribute in the induction of a pre-malignant phenotype by estrogen presumably by promoting generation of breast cancer stem cells. These data suggest NRF1 as an emerging potential target for therapeutic intervention against breast cancer. Jayanta Kumar Das 1, 2 and Deodutta Roy 1,2 1 Department of Environmental & Occupational Health, Florida International University, Miami, FL 33199, 2 Miami VA Medical Center, 1201 NW 16th St, Miami, FL 33125. Transcription regulation of chemokine receptor CXCR4 by nuclear respiratory factor 1 (NRF1) controls estrogen-induced malignant transformation of breast epithelial cells to breast cancer stem cells Results Acknowledgments This work was in part supported by a VA MERIT Review (VA BX001463) grant to Dr. Roy. This work is also in part supported by Phase Holographic Imaging (PHI),Lund, Sweden by providing a holographic imaging cytometry platform - HoloMonitorM4. E2 Increased the Proportion of CD44+CD24+CD133+ALDH+ cels. Figure 2. (A) & (B).The representative flow sorted data of the cells for CSC markers CD44+CD24-CD133+ALDH1+. (C) Histogram represents the flow sorted data of CD44+CD24-CD133+ALDH1+. MCF10A NRF1E2 CSC markers cells were approximately 2 fold by treatment of E2 compare to control of NRF1. Error bars represent the mean ± SD. * p<0.05 vs. NRF1 at 14D. NRF1 induced expression of 8-oxo-dG, CXCR4, & BNIP3 as a result of reactive oxygen species (ROS) production with the treatment of E2. NRF1–dependent activation of CXCR4 mRNA. Figure 8. The levels of CXCR4mRNA of NRF1+ cells were 2 fold higher (A) & BNIP3 mRNA of NRF1+ showed 1 fold increase (B) with E2, . **p<0.01 vs. E2. Statistics by ANOVA; Tukey HSD test. Summary of Key Findings NRF1 might influence estrogen-induced breast cancer risk by generating tumor-initiating breast cancer stem cells. NRF1 through regulating CXCR4 seems to contribute in the estrogen-induced malignant transformation of MCF10A cells, including anchorage-independent cell growth, and increased cell migration and invasion. NRF1-mediated transcription of CXCR4 is dependent on DNA oxidation and ROS-dependent redox signaling. Live imaging by HoloMonitor showed that siRNA of CXCR4 inhibited NRF1-dependent E2-induced tumorphere formation. References Okoh V., Garba N., Penney R., Das JK., Deoraj A., Singh K., Sarkar S., Felty Q., Yoo C., Jackson R., Roy D. (2015). Redox Signaling to Nuclear Regulatory Proteins by Reactive Oxygen Species Contributes to Estrogen- Induced Growth of Breast Cancer Cells. Br J Cancer; 112, 1687–1702, doi: 10.1038/bjc.2014.586 Das JK, Roy D. (2015). Overexpression of NRF1 leads to the generation of cancer stem-like cells and resistance to anoikis pathways to anchorage- independent growth during estrogen-induced malignant transformation, Cancer Res, 75:803; doi:10.1158/1538-445.AM2015-803 (A) (B) (A) Introduction: Treatment of metastatic breast cancer is still unsuccessful due to development of breast cancer stem cells (BCSCs) which are resistant to antiestrogen/chemo-/radio-therapy. The chemokine receptor CXCR4 has been found to be a prognostic marker in various types of cancer, including breast cancer. In this study, we have investigated whether transcription regulation of CXCR4 by NRF1 controls estrogen-induced malignant transformation of breast epithelial cells to breast cancer stem cells. We found increased expression level of breast cancer stem cell markers (CD44+CD24+/24-ALDH1+CD133+) when treated with E2. The ChIP qPCR, RT-qPCR, Western blotting and immunofluorescence microscopic assays confirmed that NRF1 mediated transcriptional changes of its target genes -CXCR4, BNIP3, and DJ-1 . VectorE2 NRF1 NRF1E2 Figure 1. (A) & (B).The representative flow sorted data of the cells for co-expression of CSC markers CD44+CD24+CD133+ALDH1+. (C) Histogram represents the flow sorted data of CD44+CD24+CD133+ALDH1+ from MCF10A and MDAMB231 cells with and without NRF1 exposed to E2 (100 pg/ml). Error bars represent the mean ± SD. . **p<0.01 or * p<0.05 vs. NRF1 at 14D. Statistics by ANOVA; Tukey HSD test. (B) (A) MCF10A MDA-MB231 0 6 12 VectorE2 NRF1 NRF1E2 % of CD44+CD24+CD133+A LDH+ cells MCF10A MDAMB231 (C) ** * E2 also Increased the Proportion of CD44+CD24- CD133+ALDH+ cells. VectorE2 NRF1 NRF1E2 MDA-MB231 * * * * MCF10A (c) 0 10 20 Vector NRF1+ NRF1- CXCR4 mRNA Levels (fold) -+ -+ -+ E2 0 10 20 Vector NRF1+ NRF1- BNIP3 mRNA Levels (fold) -+ -+ -+ E2 ** ** Alician Blue NRF1+ Vector Chondrogenic differentiation Smooth muscle differentiation α-SMA+/DRAQ5® Neuron like cell differentiation β-tubulin III+/DRAQ5® Differentiation of Tumor Initiating Stem Cells into Other Cells). Figure 6. Differentiation of tumor initiating stem cells (TIS or CSC) to chondrocytes, neurons and smooth muscle cells. JRK (57 kDa) ß-actin (42 kDa) ß-actin (42 kDa) PINK1 (63 kDa) Parkin/PARK2 (52 kDa) LC3B (15 kDa) ß-actin (42 kDa) Park-7/DJ-1 (21 kDa) E2 Vector NRF1+ NRF1- - + -+ -+ CD133+ E2 Vehicle MCF10A WT SOX2+ E2 Vehicle MCF10A NRF1+ Nanog+ Oct4+ Cd49f+ CD44+ E2 induced NRF1 dependent expression of CXCR4, BNIP3 and DJ1 proteins 6h Figure 4. (A) The representative HoloMonitor images showing the area scratched (wounds) and the areas healed after 6hrs. The images were captured by holographic live imagining system of TIS cells. (B) Graph shows the relative changes of healing areas at 6h. Error bars represent the mean ± SD. **p<0.01 or * p<0.05 vs. vector or NRF1. HoloMonitoring showed that E2 treatment increased the healing area of tumor initiating CD44+24+CD133+ALDH1+ MCF10A stem cells . (A) (B) 0h CSC (E2) Vector ( WT) NRF1 +CSC (No treatment) NRF1 +CSC +E2 Wound Heal 0 50 100 Vector control VectorCS CE2 NRF1CS CControl NRF1CS CE2 Healing % * ** Figure 3. Increased sizes of spheroids were observed in NRF1 with E2 treatment where as NRF1 mutant 109 showed inhibition of spheroid formation at 15D. 200X Both E2 (100pg/mL) and NRF1 jointly increased spheroid sizes and Mutant NRF1 109 inhibited the spheroid formation. Vector NRF1 Mut 109 Mut 221 Vehicle E2 MCF10A MDA- MB 231 E2 Vehicle Vector (WT) NRF1+ csc (CD44+24- 133+ALDH1) Vehicle E2 Tumorpheres (15D) Figure 7. ChIP assays (A, B) were performed using anti-NRF1 antibodies. CXCR4, & BNIP3 promoter regions of the NRF1 precipitated chromatin were amplified by real-time PCR using Epitech Chip qPCR primer assay. Analysis of E2-induced NRF-1 binding to promoters of CXCR4 (A), & BNIP3 (B). **p<0.01 vs. E2. Statistics by ANOVA; Tukey HSD test. --- E2 + + + E2 (A) NRF1 protein binds to the promoter of CXCR4 and BNIP3 genes. (B) 0 100 200 300 400 500 NRF1- NRF1+ Vector NRF1- NRF1+ Vector NRF1/CXCR4 Promoter (fold) 0 100 200 300 400 500 NRF1- NRF1+ Vector NRF1- NRF1+ Vector NRF1/BNIP3 Promoter (fold) ** ** ** ** --- + + + -+ E2 Vector NRF1+ NRF1- - + E-cadherin E2 Vehicle MCF10A WT N-cadherinVimentin E2 Vehicle MCF10A NRF1+ Expression of Epithelial-Mesenchymal Transition Markers E- and N- Cadherin, and Vimentin and Pluripotency Markers in Tumor Initiating Stem Cells. Figure 10. EMT markers detected by confocal microscopy and Western blotting. N-cadherin (130 kDa) Nanog (42 kDa) Vimentin (55 kDa) ß-actin (42 kDa) E-cadherin (120 kDa) ß-actin (42 kDa) ALDH1 (56 kDa) -+ Figure 9. Immunofluorescence staining of 8-oxo-dG as a red color, CXCR4 as green color and BNIP3 as a blue color in vector, NRF1 & NRF1- of MCF10A cells. E2 treatment increased the expression level of 8- oxo-dG, BNIP3 & CXCR4 with increased ROS level. ROS scavenger (NAC, Eb, & Peg-Cat) reduced these levels. Photomicrographs were captured with Nikon Confocal microscopy. Scale bar=70um E2 (367.1 pM) Vehicle MCF10A Vector MCF10A NRF1+ MCF10A NRF1- NAC (1mM) H2O2(600uM) NAC+E2 H2O2+PEG-Cat (500ug/mL) Eb (20uM) Eb+E2 CXCR 4 8-Oxo- dG BNIP3 CXCR4 8-Oxo- dG BNIP3 CXCR4 8-Oxo- dG BNIP3 Vector (WT) NRF1+ csc (CD44+24- 133+ALDH1) Scratch assay confirmed the findings of HoloMonitoring sowing that E2 treatment increases the healing area of NRF1 TIS or CSC (CD44+24-CD133+ALDH1+) MCF10A cells . 0h 6h (B) (A) Figure 5. (A) The representative photomicrographs shows the different groups with wounds and the areas healed after 6hrs. Wound healing was higher in both NRF1 as well as NRF1+E2 treated cancer stem like cells (CSCs). The images were captured by confocal microscopy. Magnification 200X. GFP+ cells showed green fluorescence.(B) Histogram shows the relative changes of healing areas at 6h. (C) Histogram shows the migrating cells in the healing areas. Error bars represent the mean ± SD. **p<0.01 or * p<0.05 vs. vector or NRF1. CSC (E2) Vector ( WT) NRF1 +CSC (No treatment) NRF1 +CSC +E2 Wound 0 60 120 Vector control VectorC SCE2 NRF1CS CControl NRF1CS CE2 Healing % 0 60 120 Vector control VectorC SCE2 NRF1CS CControl NRF1CS CE2 Migrating cells count (c) * ** * ** Both E2 and NRF1 jointly increased the expression of mRNA level of CXCR4 & BNIP3, and ROS scavenger (NAC, Eb, & Peg-Cat) reduced E2 or NRF1-induced the mRNA levels of CXCR4 in MCF10A. 0 20 40 Vector NRF1+ NRF1- CXCR4 mRNA Levels (Fold) Control E2 H2O2 NAC NAC+E2 Eb Eb+E2 H2O2+PegCat ** (B) 0 20 40 Vector NRF1+ NRF1- BNIP3 mRNA level (fold) Control E2 H2O2 NAC NAC+E2 Eb Eb+E2 H2O2+PegCat ** Figure11. Histograms represents the mRNA level of CXCR4 & BNIP3 at 24h. **p<0.01 vs. vector. Statistics by ANOVA; Tukey HSD test. siRNA CXCR4 Scrambled RNA E2 Vehicle NRF1 NRF1+siRNA CXCR4 NRF1- dependent E2 induced protein expression of CXCR4, BNIP3 and Park- 7/D-J1 E2 CXCR4 + NRF1+ BNIP3 Park-7 E2 Vehicle MCF10A WT Vehicle MCF10A NRF1+ Tumorpheres (15D) 3D 15D