Jayanta Das

Redox signalling to nuclear regulatory proteins by reactive oxygen species contributes to oestrogen-induced growth of breast cancer cells

Background:17β-Oestradiol (E2)-induced reactive oxygen species (ROS) have been implicated in regulating the growth of breast cancer cells. However, the underlying mechanism of this is not clear. Here we show how ROS through a novel redox signalling pathway involving nuclear respiratory factor-1 (NRF-1) and p27 contribute to E2-induced growth of MCF-7 breast cancer cells.Methods:Chromatin immunoprecipitation, qPCR, mass spectrometry, redox western blot, colony formation, cell proliferation, ROS assay, and immunofluorescence microscopy were used to study the role of NRF-1.Results:The major novel finding of this study is the demonstration of oxidative modification of phosphatases PTEN and CDC25A by E2-generated ROS along with the subsequent activation of AKT and ERK pathways that culminated in the activation of NRF-1 leading to the upregulation of cell cycle genes. 17β-Oestradiol-induced ROS by influencing nuclear proteins p27 and Jab1 also contributed to the growth of MCF-7 cells.Conclusions:Taken together, our results present evidence in the support of E2-induced ROS-mediated AKT signalling leading to the activation of NRF-1-regulated cell cycle genes as well as the impairment of p27 activity, which is presumably necessary for the growth of MCF-7 cells. These observations are important because they provide a new paradigm by which oestrogen may contribute to the growth of breast cancer.

PCB153-Induced Overexpression of ID3 Contributes to the Development of Microvascular Lesions

Microvascular lesions resulting from endothelial cell dysfunction are produced in the brain, lung, kidney, and retina of patients of complex chronic diseases. The environmental and molecular risk factors which may contribute in the development of microvascular damage are unclear. The mechanism(s) responsible for initiating microvascular damage remain poorly defined, although several inciting factors have been proposed, including environmental toxicants-induced oxidative stress. Enhanced neovascularization has been implicated in either the development or progression of proliferative vascular lesions. Here, we present evidence for how PCB-induced ROS may contribute to the development of a neovascular phenotype with the aim of elucidating the role of environmental toxicants in endothelial dysfunction with a specific focus on the inhibitor of differentiation protein ID3. We used a combination of phenotype and immunohistochemical analysis followed by validating with protein expression and post-translational modifications with Western Blot and MALDI-TOF/TOF analysis. We also looked for a correlation between ID3 expression in vascular tissue. Our results showed that PCB-induced ROS mediated a highly tube branched neovascular phenotype that also depended on ID3 and Pyk2; and PCB153 treatment increased the size of endothelial spheroids under conditions typically used for clonal selection of stem cell spheroids. High ID3 protein expression correlated with a greater degree of malignancy and oxidative DNA damage marker 8-OHdG in blood vessels from human subjects. PCB153 treatment increased both serine and tyrosine phosphorylation of endothelial ID3. Stable ID3 overexpression increased cell survival of human microvascular endothelial cell line hCMEC/D3. In summary, our data provide evidence that ID3 may play a critical role in regulating vascular endothelial cell survival and development of microvascular lesions induced by persistent environmental pollutants such as PCB153. Findings of this study are important because they provide a new paradigm by which PCBs may contribute to the growth of microvascular lesions.

Microvascular Lesions by Estrogen-Induced ID3: Its Implications in Cerebral and Cardiorenal Vascular Disease

Severe symptoms of cerebral and cardiorenal vascular diseases can be triggered when cerebral, coronary, or glomerular arterioles grow inappropriately as a result of abnormal cell proliferation. The risk factor(s) and molecular mechanisms responsible for microvascular lesion formation are largely unknown. Although controversial, both animal and epidemiological studies have shown that estrogen increases the risk of stroke which may be due to microvascular lesions. Since microvascular diseases are characterized by excessive vessel growth, it is plausible that estrogen-induced neovascularization contributes to the growth of microvascular lesions. We present evidence for how ID3 overexpression in endothelial cells contributes to the development of an estrogen-induced neovascular phenotype with an additional focus on Pyk2 kinase. Our data showed that ID3 overexpression increased neovascularization, cell migration, and spheroid growth of human cerebral microvascular endothelial cells, hCMEC/D3. ID3-overexpressing cells showed significant estrogen-induced G2/M phase transition. Estrogen treatment increased both ID3 phosphorylation; total protein that was inhibited by tamoxifen, and Pyk2-mediated estrogen-induced ID3 mRNA expression. These findings suggest that Pyk2 signals ID3 expression and ID3 is necessary for estrogen-induced neovascularization in hCMEC/D3 cells. A better understanding of how microvascular lesions depend on ID3 may open new avenues for prevention and treatment of neurological diseases.

ID3 Contributes to the Acquisition of Molecular Stem Cell-Like Signature in Microvascular Endothelial Cells: Its implication for understanding microvascular diseases

While significant progress has been made to advance our knowledge of microvascular lesion formation, yet the investigation of how stem-like cells may contribute to the pathogenesis of microvascular diseases is still in its infancy. We assessed whether the inhibitor of DNA binding and differentiation 3 (ID3) contributes to the acquisition of a molecular stem cell-like signature in microvascular endothelial cells. The effects of stable ID3 overexpression and SU5416 treatment - a chemical inducer of microvascular lesions, had on the stemness signature were determined by flow cytometry, immunoblot, and immunohistochemistry. Continuous ID3 expression produced a molecular stemness signature consisting of CD133(+) VEGFR3(+) CD34(+) cells. Cells exposed to SU5416 showed positive protein expression of ID3, VEGFR3, CD34 and increased expression of pluripotent transcription factors Oct-4 and Sox-2. ID3 overexpressing cells supported the formation of a 3-D microvascular lesion co-cultured with smooth muscle cells. In addition, in vivo microvascular lesions from SuHx rodent model showed an increased expression of ID3, VEGFR3, and Pyk2 similar to SU5416 treated human endothelial cells. Further investigations into how normal and stem-like cells utilize ID3 may open up new avenues for a better understanding of the molecular mechanisms which are underlying the pathological development of microvascular diseases.

Abstract 3312: Transcriptional regulation of chemokine receptor 4 (CXCR4) by nuclear respiratory factor 1 (NRF1) controls estrogen-induced malignant transformation of breast epithelial cells to breast cancer stem cells

Chemokine receptor 4 (CXCR4) is involved in the maintenance normal stem cells and metastasis of cancer stem cells. NRF1 is one of the major transcription factor that controls transcription of CXCR4. The functional regulation of transcription of NRF1 target genes has not been explored in breast cancer. In this study, we have investigated whether transcriptional regulation of CXCR4 by NRF1 regulates estrogen-induced malignant transformation of breast epithelial cells to breast cancer stem cells (CSCs). 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 remains unknown. In this study we showed that NRF1 and E2 jointly contributed in reprogramming of breast epithelial cells to CSCs. 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. For morphological details we used a holographic microscope (HoloMonitor M4), we observed that E2 generated breast cancer stem cells with CD44+CD24+ALDH1+CD133+ markers produced large live spheroids in NRF1 overexpressing cells compared to cells transfected with vector receiving E2. 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. The suppression of NRF-1 activity induced growth arrest and apoptosis, and reduced stemness, growth and tumorigenic property of cancer stem cells. 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. Citation Format: Jayanta Kumar Das, Deodutta Roy. Transcriptional regulation of chemokine receptor 4 (CXCR4) by nuclear respiratory factor 1 (NRF1) controls estrogen-induced malignant transformation of breast epithelial cells to breast cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3312.

NRF1 motif sequence-enriched genes involved in ER/PR −ve HER2 +ve breast cancer signaling pathways

Nuclear respiratory factor 1 (NRF1) transcription factor has recently been shown to control breast cancer progression. However, mechanistic aspects by which NRF1 may contribute to susceptibility to different breast tumor subtypes are still not fully understood. Since transcriptional control of NRF1 seems to be dependent on epidermal growth factor receptor signaling, herein, we investigated the role of NRF1 in estrogen receptor/progesterone receptor negative, but human epidermal growth factor receptor 2-positive (ER/PR −ve HER2 +ve) breast cancer. We found that both mRNA and protein levels of NRF1 and its transcriptional activity were significantly higher in ER/PR −ve HER2 +ve breast cancer samples compared to normal breast tissues. This was consistent with our observation of higher NRF1 protein expression in the experimental model of HER2+ breast cancer brain metastasis. To identify network-based pathways involved in the susceptibility to the ER/PR −ve HER2 +ve breast cancer subtype, the NRF1 transcriptional regulatory genome-wide landscape was analyzed using the approach consisting of a systematic integration of ChIP DNA-seq, RNA-Microarray, NRF1 protein-DNA motif binding, signal pathway analysis, and Bayesian machine learning. Our findings show that a high percentage of known HER2+ breast cancer susceptibility genes, including EGFR, IGFR, and E2F1, are under transcriptional control of NRF1. Promoters of several genes from the KEGG HER2+ breast cancer pathway and 11 signaling pathways linked to 6 hallmarks of cancer contain the NRF1 motif. By pathway analysis, key breast cancer hallmark genes of epithelial-mesenchymal transition, stemness, cell apoptosis, cell cycle regulation, chromosomal integrity, and DNA damage/repair were highly enriched with NRF1 motifs. In addition, we found using Bayesian network-based machine learning that 30 NRF1 motif-enriched genes including growth factor receptors—FGFR1, IGF1R; E2Fs transcription factor family—E2F1, E2F3; MAPK pathway—SHC2, GRB2, MAPK1; PI3K-AKT-mTOR signaling pathway—PIK3CD, PIK3R1, PIK3R3, RPS6KB2; WNT signaling pathway—WNT7B, DLV1, DLV2, GSK3B, NRF1, and DDB2, known for its role in DNA repair and involvement in early events associated with metastatic progression of breast cancer cells, were associated with HER2-amplified breast cancer. Machine learning search further revealed that the likelihood of HER2-positive breast cancer is almost 100% in a patient with the high NRF1 expression combined with expression patterns of high E2F3, GSK3B, and MAPK1, low or no change in E2F1 and FGFR1, and high or no change in PIK3R3. In summary, our findings suggest novel roles of NRF1 and its regulatory networks in susceptibility to the ER/PR −ve HER2 +ve aggressive breast cancer subtype. Clinical confirmation of our machine learned Bayesian networks will have significant impact on our understanding of the role of NRF1 as a valuable biomarker for breast cancer diagnosis and prognosis as well as provide strong rationale for future studies to develop NRF1 signaling-based therapeutics to target HER2+ breast cancer.

Abstract 4061: Origanum majorana organic extract induces senescence and autophagic cell death in breast cancer cells through influencing mitochondrial metabolism

Excess 17β estradiol (E2) is a risk factor of breast cancer. Previously, we have reported that E2 through influencing mitochondria by unknown mechanism contribute to the estrogen induced breast carcinogenesis. The aim of this study was to evaluate the mitotoxic and cytotoxic effects of normal O. Majorana organic extract (OME) as well as PEGylated nanoconjugate of OME with triphenylphosphonium (P-OME) against human breast epithelial and cancer cell lines. Origanum majorana, commonly known as marjoram, is a perennial herb, which is widely used in the Middle East as a spice. It has been shown to possess extensive range of biological activity, including antioxidant, anti-inflammatory, and anti-tumor growth effects. Interestingly, the anticancer potential of O. Majorana against breast cancer remains largely unexplored. Here, we investigated the anticancer effect of O. Majorana on three breast cell lines. We also used triphenylphosphonium (TPP) cation to check whether an imbalance in mitochondrial bioenergetics, in part, may be responsible for estrogen induced growth of breast cancer. Determination of cell density (cell survival) by SRB and mitochondrial metabolic activity by MTT assays showed that both OME and P-OME reduced growth of MDA-MB-231 and MCF-7 cells, but no effect on normal breast epithelial MCF-10A cells. OME and TPP blocked E2-induced increases of cell survival, metabolic activity and proliferation of breast cancer MCF-7 cells. E2 treatment increased MitoTracker® Red 580 labeling, indicating E2 treatment increased MCF7 cells mitochondrial mass. We observed a several fold increase in mitochondrial transcription factor A (TFAM) DNA binding activity as early as 3 hrs in treated MCF7 cells. DNA synthesis was inhibited in E2-exposed MCF7 cells by the silencing of TFAM. To discern whether a decrease in ATP production may be responsible for the E2-induced growth signaling, we measured the ATP present in the MCF7 cells. Our data showed that the ATP levels in E2 treated cells were very similar to control cells. E2 treatment of MCF-7 cells increased mRNA and protein levels of BNIP3 involved in mitophagy/autophagy. Together these data suggest that a carcinogenic concentration of E2 may modify mitochondrial dynamics, mitophagy, biogenesis and metabolism. In summary, our results demonstrated for the first time that OME was able to inhibit estrogen-induced growth of MCF-7 cells in a time- and concentration-dependent manner. Our results also demonstrated that P-OME nanoconjugate compared to OME was far more effective in exerting its cytotoxic effect through the induction of growth arrest, mitochondrial metabolic activity, senescence, apoptosis and autophagic cell death in the highly metastatic triple negative MDA-MB-231 cells. Our findings offer a new perspective on the utility of O. Majorana plant extract to be developed as a new alternative medicinal therapy against breast tumors. Citation Format: Mohannad Garoub, Jayanta Das, Stanislaw Wnuk, Deodutta Roy. Origanum majorana organic extract induces senescence and autophagic cell death in breast cancer cells through influencing mitochondrial metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4061.

Abstract 803: 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

The actual role nuclear respiratory factor-1 (NRF-1) plays in breast cancer remains the least studied of all transcription factors. A growth role for the NRF1 through regulation of cell cycle genes during estrogen-induced malignant transformation has recently been reported by us; however, the underlying mechanism of its contribution to estrogen-induced breast carcinogenesis is undefined. In this study we provide new mechanistic insight into the mode by which NRF1 may contribute in estrogen-induced neoplastic conversion of cells by suppressing anoikis; identified BNIP3 as a key transcriptional target of NRF1; and survival signals conferred by NRF1 seems to impinge on the hypoxia-inducible death factor BNIP3. ChIP, qPCR, mass spectrometry, redox Western blot, colony formation, cell proliferation, ROS assay, immunofluorescence microscopy were used to study the role of NRF-1. Our data showed that higher expresion of NRF-1 in MCF10A produced large sizes of spheroids with increased level of stem cell markers -CD44+CD24+CD49f+ compared to cells transfected with vector alone. E2-induced increase of spheroid formation, suppression of cell death and cell survival were amplified by expression of NRF1 and these effects were diminished by loss of NRF1 function through expression of dominant negative NRF1. Breast cancer cell lines - MCF7 and MDA-MB231 overexpressed both NRF1 and BNIP3, and formed larger tumor spheroids compared to the MCF10A or vector-transformed breast cancer cells. E2 treatment as well as overexpression of NRF1 increased mRNA and protein levels of BNIP3. In addition, we found the increased transcriptional interaction of NRF-1 transcription factor in the BNIP3 promoter region by ChIP-qPCR assay in cells treated with E2. We also found the increased functional activity of the BNIP3 promoter in cells treated with E2 and this was decreased with co-treatment of ROS scavenger-Ebselen. NRF1 spheroids have the ability to micro-invasion. In summary, overexpression of NRF1 altered the cell morphology towards mesenchymal stem-like shape and promoted cancer stem cell-like phenotype. Findings of this study unveiled the underlying mechanism of NRF1 action through the activation of BNIP3 which is regulated by NRF1 and is presumably responsible for the estrogen-induced malignant transformation of MCF10A cells, including anoikis resistance, anchorage-independent cell growth, and increased cell migration and invasion. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR. Citation Format: Jayanta Das, Deodutta Roy. 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. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 803. doi:10.1158/1538-7445.AM2015-803

Abstract 4179: Id3 generated cancer stem-like cells: A microvascular niche for the development of glioma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Pro-angiogenic vascular stem cells and angiogenesis are key events in the process of glioma formation. Vascular lesions that arise from a focal budding of endothelial cells have been associated with a poor prognosis in glioblastoma multiforme. Hyper-proliferative endothelial lesions are common in patients with severe brain tumors, however, little is known about the relevance of vascular stem cells in the development of these lesions. We tested the hypothesis that the overexpression of the inhibitor of DNA binding and differentiation protein 3 (ID3) reprograms the molecular signature of endothelial cells to stem-like cells; and we hypothesize that these stem-like cells participate in the development of vascular lesion formation in glioma. We generated ID3 overexpressing cells using the human cerebral microvascular endothelial cell line, hCMEC/D3; and determined the effect of ID3 on molecular stem-like signature and vascular lesion formation. ID3 overexpressing cells resided more in the G0/G1 phase and showed a significantly higher level of stem cell markers CD34, CD133, Oct-4, and Sox-2 than wild-type cells. ID3 overexpressing cells also showed a significant increase in VEGFR3, an endothelial growth factor receptor that has been previously reported to correlate with tumor grade of malignant gliomas. In addition, ID3 overexpressing cells showed increased levels of Pyk2, a kinase that has been shown to control glioma cell migration. In the 3-D culture model, we observed that ID3 was essential for the growth of endothelial spheroids. Our findings demonstrate that ID3 generated vascular stem-like cells have the capacity to participate in vascular lesion formation and that vascular stem-like molecular signatures are regulated in part by ID3. Furthermore, the correlation of ID3 overexpression with an increase in VEGFR3 and Pyk2 suggests the importance of ID3 in malignant glioma endothelium. Assessing ID3 in malignant gliomas on a functional level may enable a better understanding for how the microvascular niche contributes to the development of glioma. A better understanding of how microvascular lesions depend on ID3 may open new avenues for the prevention and treatment of glioma. Citation Format: Jayanta Das, Quentin H. Felty. Id3 generated cancer stem-like cells: A microvascular niche for the development of glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4179. doi:10.1158/1538-7445.AM2015-4179

Abstract 4559: Phosphodeficient NRF1 mutant suppresses the susceptibility of the breast epithelial cells to develop tumors when exposed to estrogen - a major breast cancer risk factor

Although several nuclear regulatory proteins may be targeted by estrogen, our efforts have focused on the redox nuclear respiratory factor-1 (NRF-1), because our recent study showed that NRF-1 gene expression significantly increases with the progression of breast tumor grades. NRF-1 overexpression supported in vitro tumor formation. To determine if NRF-1 is required for 17 beta estradiol (E2)-induced neoplastic phenotype, we have generated NRF-1 mutants by site directed mutagenesis. We have generated stable clones that express NRF-1 mutant. The in vitro tumor formation was detected by anchorage independent growth and 3D spheroid assays. NRF-1 overexpression enhanced in vitro tumor spheroid formation, cell migration and cell invasion. Our flow cytometry analysis showed that overexpression of wild-type NRF-1 increased the percent of transformed MCF-10A cells in S phase compared to vector alone. Dominant negative NRF-1 protein showed reduction in the tumor formation. In addition, shRNA targeting NRF-1 resulted in the inhibition of anchorage independent growth of MCF-10A cells in both vehicle control and E2-treated cells. Since NRF-1 is a substrate of the kinase AKT, we determined whether NRF-1 phosphorylation was increased in MCF-10A cells after treatment with a carcinogenic regimen of E2. We observed more than a 2-fold increase in phospho-NRF-1 in E2 treated (100pg/ml for 30 min) MCF-10A cells and phosphorylation of NRF-1 was inhibited by co-treatment with either biological (CAT or MnSOD) or chemical (20μM ebselen) ROS modifiers. These results suggest that E2-induced phosphorylation of NRF-1 is influenced by ROS signaling messengers. We found that E2 treated MCF-10A cells showed increased NRF-1 binding to the promoters of Cdc2, PRC1, PCNA, Cyclin B1, and CDC25C genes. NRF-1 binding induced by E2 treatment was inhibited by the overexpression of CAT and MnSOD. NRF-1 phosphorylation site specific to kinase AKT, Thr-109, was mutated to Asp (D) or Ala (A), and the NRF-1 acetylation site specific to acetyl-transferase PCAF, Lys- 89, was mutated to Gln (glutamine)-Q or Ala (A). We examine the effect of one of the NRF-1 mutants on E2-induced in vitro tumor formation. We observed that the expression of NRF-1 (T109A) phosphodeficient mutant (NRF-1PMT) significantly inhibited E2-induced cell transformation, whereas empty vector did not influence E2-induced colony formation. Together these findings support the idea that NRF-1 may play an important role in E2-induced malignant transformation of breast epithelial cells. Thus, our data is consistent with the hypothesis that in addition to the estrogen receptor activity, NRF-1 activation contributes to the susceptibility to develop malignant phenotype in response to exposure to estrogen. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR Citation Format: Lazaro Mesa, Jayanta Das, Alok Deoraj, Victor Okoh, Deodutta Roy. Phosphodeficient NRF1 mutant suppresses the susceptibility of the breast epithelial cells to develop tumors when exposed to estrogen - a major breast cancer risk factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4559. doi:10.1158/1538-7445.AM2015-4559