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Nuclear Respiratory Factor 1 Acting as an Oncoprotein Drives Estrogen-Induced Breast Carcinogenesis
Received: 3 September 2018 / Accepted: 18 November 2018 / Published: 27 November 2018
De 5 forskarna är Jayanta K. Das,Quentin Felty,Robert Poppiti,Robert M. Jackson,Deodutta Roy.
Abstract:
We have previously shown nuclear respiratory factor 1
(NRF1)-mediated transcriptional programming of mitobiogenesis
contributes to estrogen-induced breast cancer through modulating cell
cycle progression.
In this study, we report a new role of NRF1 that goes
beyond that of programming mitobiogenesis. Specifically, we report a
novel oncogenic function of NRF1 supporting its causative role in breast
cancer development and progression. The gain of NRF1 and/or treatment
with 17β-estradiol (E2) produced heterogeneous breast cancer stem cell
(BCSC)-like subsets composed of more than 10 distinct cell
sub-populations. Flow sorting combined with confocal imaging of markers
for pluripotency, epithelial mesenchymal transition (EMT), and BCSCs
phenotypically confirmed that the BCSC-like subset arise from cell
re-programming. Thus, we determined the molecular actions of NRF1 on its
target gene CXCR4 because of its known role in the acquisition of the
BCSC-like subset through EMT. CXCR4 was activated by NRF1 in a
redox-dependent manner during malignant transformation. An NRF1-induced
BCSC-like subset was able to form xenograft tumors in vivo, while
inhibiting transcription of CXCR4 prevented xenograft tumor growth.
Consistent with our observation of NRF1-driven breast tumorigenesis in
the experimental model, higher protein levels of NRF1 were also found in
human breast cancer tissue specimens. This highly novel role of NRF1 in
the stochastic acquisition of BCSC-like subsets and their progression
to a malignant phenotype may open an entirely new research direction
targeting NRF1 signaling in invasive breast cancer. Our discovery of
targeting transcriptional activation of CXCR4 to inhibit NRF1-induced
oncogenic transformation provides a mechanistic explanation for
estrogen-dependent breast carcinogenesis and opens new avenues in
strategic therapeutics to fight breast cancer.
1. Introduction
Nuclear respiratory factor 1 (NRF1) is widely recognized for regulating genes encoding mitochondrial biogenesis.
Recent evidence also indicates that the NRF1 protein interacts with a
broad spectrum of transcription factors; its unique DNA binding
recognition site is one of seven transcription factor binding sites most
frequently found in the proximal promoters of ubiquitous genes.
The NRF1 motif is found on the promoters of genes regulating the cell
cycle, chromatin structure, cell apoptosis, cell adhesion/invasion, DNA
repair, DNA methylation and transcriptional repression signaling, and
epithelial adherens junctions.
These findings suggest that NRF1 is a multifunctional protein with
roles in diverse cellular functions. The actual role NRF1 plays in
breast cancer remains the least studied of all transcription factors. We
have previously reported the role of NRF1 in transcriptional
programming of mitochondrial biogenesis in estrogen-induced growth of
breast cancer cells.
Estrogen treatment increases mitochondrial mass, the DNA-binding
activity of NRF1, a regulator of TFAM, and the level of TFAM, and TFAM
shRNA inhibits colony formation in E2-treated breast cancer cells. These
findings suggest that NRF1-mediated transcriptional programming of
mitobiogenesis contributes to estrogen-induced cell cycle progression Meta-analysis of 18 published breast cancer microarray data showed that NRF1 is elevated in high-grade breast tumors.
Our findings were validated by a recent study using TGCI normal and
breast tumor specimens in which it was shown that NRF1 activity was
significantly higher in human breast cancers compared to adjacent
surrounding control breast tissue.
Furthermore, we have also shown that reactive oxygen species
(ROS)-mediated activation of NRF1 is critical for the growth of
estrogen-induced breast cancer cells and estrogen-induced malignant
breast cell transformation.
Whether NRF1 contributes to estrogen carcinogenesis in breast cancer is
not fully understood. Life time exposure to elevated levels of estrogen
is a major risk factor for breast cancer.
Estrogen is a breast carcinogen; however, the molecular mechanisms
responsible for estrogen-induced breast tumor initiation remain poorly
understood. Although several signaling pathways may be targeted by
estrogen in human mammary epithelial cells (HMECs) during the induction
of a pre-malignant phenotype, our focus is on the NRF1 signal
transduction pathway because DNA sequence motifs bound by NRF1
positively correlate with malignant breast cancer progression.
Despite
tremendous progress in understanding breast cancer, gaps remain in our
knowledge of the molecular basis underlying the aggressiveness of breast
cancer. Breast tumor-initiating cells (BTICs) or breast cancer stem
cells (BCSCs) are considered to be responsible for estrogen-induced
initiation and aggressive progression of breast tumors.
We have recently shown that activation of the NRF1 pathway may
participate in the development of breast tumors; however, its
contribution to the acquisition of cancer stem cells remains unexplored
in breast cancer.
This malignancy may occur via transformation of adult stem cells into
cancer stem cells that give rise to the tumor. There are several key
genes related to cell growth, cell transformation, cell
adhesion/motility, and tumor suppression that are regulated by NRF1.
Some of these genes, including CXCR4, are upregulated by estrogen
treatment. The contribution of CXCR4 to reprogramming breast cancer
cells to cancer stem cells is of particular interest to our research on
NRF1. CXCR4 has an established role in the acquisition of BCSCs through
epithelial mesenchymal transition (EMT).
Thus, it is biologically plausible that high NRF1 activity in breast
tissue increases susceptibility to estrogen-induced breast
carcinogenesis via upregulation of the CXCR4 gene. This NRF1-regulated
gene alone or in concert with others may contribute to the
estrogen-induced malignant phenotype. The purpose of this study was to
investigate whether NRF1-modulated CXCR4 expression drives
estrogen-induced malignant transformation of breast epithelial cells to
BCSCs and whether this NRF1 activity plays a major role in breast cancer
development and progression. Our findings demonstrate a new oncogenic
role of NRF1 that goes beyond that of programming mitobiogenesis.
Overexpression of NRF1 combined with exposure to a carcinogenic dose of
17β-estradiol (E2) through regulating CXCR4-generated BTICs that formed
tumors in vivo. Further clinical validation of this finding may lead to
new avenues for NRF1 targeted therapeutic strategies to fight breast
cancer.
2. Materials and Methods
(Utvalda delar)
2.8. Tumorigenic Spheroid Assays
For
tumorigenic spheroid formation, approximately 100 cells per well were
seeded in an ultra-low attachment 96-well plate (Corning Inc., Lowell,
MA, USA). The tumorigenic spheroids were photographed with the
HoloMonitoring and confocal microscopy as live images. Cells were
suspended in serum-free DMEM/F12 (1:1) culture medium supplemented with
B27. Approximately 100–150 cells per well were seeded in an ultra-low
attachment 96-well plate (Corning Inc., Lowell, MA, USA). The effect of
carcinogenic regimen of 17β-estradiol (E2) was evaluated by E2 treatment
(100 pg/mL) on the day of seeding cells. Spheroids were grown for 27
days in liquid culture. A total of 15 spheroids with a minimum diameter
of 50 mm were counted in each experimental group. Data were analyzed by
ANOVA; Tukey’s HSD test was used for multiple comparisons. Cells
obtained from spheroids were analyzed by immunofluorescence, FACS, or
immunoblotting, as described previously.
2.10. Cell Migration Assay
Cells
were cultured in 6-well plates and a sterile plastic 1 mL micropipette
tip was used to scratch in the middle area of the well as a line. Then
cells were incubated in growth medium for 48 h.
The scoring wounds were
photographed with the HoloMonitoring and confocal microscopy as live
images.
Figure 4.
Large tumor spheroids were observed by HoloMonitoring and confocal
microscopy, for live images of NRF1 and NRF1+E2 BTIC clones compared to
vector control did not form tumor spheroids at 5 and 15 days (Figure 4C,D).
Phenotypic characteristics of CD44+CD49f+ALDH+CXCR4+NRF1+ BCSCs subtype
assessed by live imaging of tumorosphere formation, the migration and
proliferation potential, and xenograft tumor growth assays; large tumor
spheroids were observed by HoloMonitoring (C) and confocal microscopy (D), as live images of NRF1 and NRF1+E2 BTIC clones compared to the vector control did not form tumor spheroids at 5 and 15 days.
Figure 6.
.......and the representative HoloMonitor microscopy live cells images, showing
transfection of SiRNA CXCR4 inhibiting NRF1-induced tumor spheroids (H) (** p < 0.01 vs. control).
Min kommentar
Av respekt för forskarna och deras omfattande studier tänker bloggen denna gång inte ge sig på att försöka översätta och förklara innehållet då studien är på alldeles för hög nivå för undertecknad.
Men det kan konstateras att man forskat kring stamceller och DNA där NRF1 (se under Introduction) utforskats som en möjlig väg att påverka en bröstcancerutveckling.
Forskarna kunde dock vara tydligare med att skriva ut användandet av PHI´s HoloMonitor M4 är bloggens åsikt.
Nu använder man dess namn i studien som ett vedertaget begrepp, HoloMonitoring, för att berätta att man långtidsstuderat celler i en inkubatormiljö. Det är förvisso hedrande för PHI om forskare på den allra högsta nivån skapar ett nytt "forskarord" baserat på bolagets teknik.
För att bloggen skulle bli 100% säker på att man verkligen använt sig av en M4 så fick spaden gräva lite djupare i myllan.
Florida International University and PHI collaborate to fight cancer
Florida International University (FIU) and PHI have entered a
collaboration agreement. To determine the scope of a long-term
collaboration, PHI will initially provide FIU with a HoloMonitor
instrument during 4 months.
The instrument will be used to characterize breast cancer stem cells.
Additional information about the collaboration has been published on the university’s website, breakthroughs.fiu.edu.
The instrument will be used to characterize breast cancer stem cells.
Additional information about the collaboration has been published on the university’s website, breakthroughs.fiu.edu.
" Most recently, the college has entered into a research agreement with
Sweden’s Phase Holographic Imaging (PHI) for evaluation of
HoloMonitorM4® technology to characterize the behavior of breast cancer
stem cells.
The evaluation will be spearheaded by Deodutta Roy, a professor in the college’s Department of Environmental & Occupational Health,
whose current projects include investigation of the role of reactive
oxygen species (ROS) and redox-sensitive transcription factor – nuclear
respiratory factor (NRF1) that contribute to cancer stem development,
and accelerate the progression of the disease. PHI will support Roy’s
research by providing a holographic imaging cytometry platform
HoloMonitorM4 as well as training and support for the purpose of
enabling the successful evaluation. The research group will provide
laboratory space, lab equipment, personnel and biological test models
for evaluation.
“We are excited about the potential of collaboration with PHI to
evaluate the use of HoloMonitor technology in our research focused on
the role of NRF1 in the generation of breast cancer stem cells. We’re
particularly encouraged by HoloMonitor’s real-time multiplexing
capabilities to study cell cycle, cell motility, cell survival and 3D
morphological analysis of living normal and breast cancer stem cells,”
says Roy.
 |
| Professor Deodutta Roy |
Peter Egelberg, CEO of PHI, adds, “This new alliance reflects our
emphasis on collaborative research and expansion of the international
network of scientists using HoloMonitor technology. We are enthusiastic
about the evaluation and hope that our technology will contribute
significantly to Roy’s research in the understanding of mechanisms of
breast cancer progression and development on novel noninvasive
diagnostic and prognostic biomarkers.”
Voilá, säcken är därmed ihopknuten.
Av detta kan man anta att den då lånade utrustningen gjorde avtryck och behövdes för denna omfattande studie 3 år senare vilket ledde till inköp.
Florida International University finns inte med på Bolagets User lista idag.
Florida International University finns inte med på Bolagets User lista idag.
Mvh the99
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