Download Mitochondria-driven cancer pathways in triple negative breast cancer

Mitochondria-driven cancer pathways in triple negative breast cancer
Junhyoung Park, Vadiraja B. Bhat Santhosh Kumar, Sajna Vithayathil, Kavisha Arora, Efrosini Tsouko, Taraka R Donti, Daniel E Frigo, Chad J Creighton, Nagireddy Putluri,
Michael T Lewis, Arun Sreekumar, Lee-Jun Wong & Benny Abraham Kaipparettu
Department of Molecular and Human Genetics and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX-77030; Agilent Technologies, Inc. Wilmington DE, 19808
Abstract: Driver pathways of triple negative breast cancer (TN-BCa) are still poorly
Mitochondrial ATP regulate Src activity
Dasatinib
N
O
SUM159/SUM159
MDA231/SUM159
Src
MCF10A/143B
N. Loss of pSrc (Y416) in cybrids after treatment with Src
family kinases inhibitor, Dasatinib. O. Colony formation
assay suggesting a dose dependent decrease of colonies
in MDA231/SUM159 cybrids after Dasatinib treatment.
MDA468/143B
143B/143B
P
Q
R
Cybrids are generated by fusing enucleated mitochondrial donor cells with mtDNA depleted
nuclear donor cells (ρ0 cells)
Results
Proteomics analysis
140000
Unique Peptides
100000
Protein Groups
I
H
132845
120000
S
T
U
80000
13466
8528
44466
33048 7252
32430 7196
32037 7094
8614
46636
30612 6982
7899
35703 7407
36193 7479
38249
27161 6393
28877 6362
29364 6650
8672
46231
30060 7070
8617
32813 7201
36360 7512
44675
28969 6887
31378 7031
35989 7519
8852
46680
31823 7307
36380 7656
31010 7190
8199
42051
32439 7085
20000
28753 6706
29738 6734
40000
31829 7235
34671 7583
60000
Introduction
Mitochondria is critical in Src autophosphorylation
F
G
Cybrids
MCF10A/SUM159
A1N4/SUM159
8962
46170
33034 7416
understood. Thus, it is important to identify the underlying mechanisms of TN-BCa
progression. Mitochondria-nuclear crosstalk is known to affect tumor properties and
metastasis. Here we have generated transmitochondrial-cybrid models to understand
mitochondria-regulated cancer pathways and performed different experiments to compare
the tumor properties of the cybrids. All OMICs approach was used to study mitochondrial
regulation of tumor pathways in transmitochondrial-cybrid models.
Transmitochondrial-cybrids were generated under common nuclear backgrounds with
mitochondria from benign breast and metastatic TN-BCa cells. Colony formation assay,
wound healing assay and in vivo tumor mammary gland transplantation were used to
understand tumor properties of cybrids. Mitochondrial electron-transport chain activity,
mtDNA genomic variations, reactive oxygen species levels, respiration and mitochondrial
ATP levels were analyzed. Gene expression profile was performed by established
protocols. Shotgun Jetstream proteomics was performed on UHPLC/AJS-iFunnel Q-TOF.
MRM experiments were performed on QQQ LC-MS for metabolomics analysis.
In vivo and in vitro analyses confirmed that mitochondria-nuclear crosstalk regulates
tumor property of cancer cell. Microarray data suggested that several cancer-related
genes are up and down-regulated in cybrids with cancer and benign mitochondria.
Pathway analysis suggested that Src oncogenic pathway is one of the critical pathways
involved. We performed pathway-based approach to understand mitochondria-mediated
regulation of Src in TN-BCa and we observed autophosphorylation at Y419, which is
significantly increased in cancer cells and their cybrids. Further mitochondria respiratory
complexes inhibitors suggested that mitochondrial electron-transport chain is playing
critical role in the regulation of Src autophosphorylation. Shotgun Jetstream proteomics
data suggested that several proteins related to oxidoreductase activity, mitochondrion
and lipid metabolic process have altered in cybrids and parental cells.
Cybrid models with breast cancer mitochondria
V
W
X
0
 Nearly a century of research has revealed that mitochondrial dysfunction is one
of the most common and consistent phenotypes of cancer cells
 Mitochondria is the only organelle that have their own genome (mtDNA) other
than the nucleus of the cell
Total Protein Groups (n=72)
SUM-159 (n=9)
SUM-159-3
SUM-159-2
SUM-159-1
MDA-MB231 (n=9)
MDA-MB231-3
MDA-MB231-2
MDA-MB231-1
MCF-10A (n=9)
MCF-10A-3
MCF-10A-2
MCF-10A-1
A1N4-159B (n=9)
A1N4-159B-3
A1N4-159B-2
A1N4-159B-1
A1N4 (n=9)
A1N4-3
A1N4-2
A1N4-1
231-159B (n=9)
231-159B-3
231-159B-2
231-159B-1
159-159NT (n=9)
159-159NT-3
159-159NT-2
159-159NT-1
10A-159B (n=9)
10A-159B-3
10A-159B-2
10A-159B-1
ATP
pSrc (Y416)
J
K
pSrc (Y527)
Src
Technical and biological triplicate analysis of Cybrid models with breast cancer mitochondria cell lysate digest (25μg
on-column) analyzed on an Agilent AdvanceBio Peptide Mapping 2.1 × 250 mm column with a 100 minute gradient.
Barr graph showing the number of unique proteins and peptides identified (1 % FDR) in each triplicate analysis of
biological triplicates as well as the total number for all three biological triplicate runs demonstrating excellent
reproducibility. 25mg of tryptic peptides from different types of cell lysates were analyzed in triplicate on UHPLC AJS
6550 interfaces coupled to an iFunnel Q-TOF using a 100min gradient. The data was processed by Spectrum Mill
software (Agilent Technologies) and summarized with 1% FDR at the protein level.
L
M
Mitochondria maintain oncogenic properties
 mtDNA is highly susceptible to mutations due to lack of protective histones and
increased exposure to reactive oxygen species (ROS)
Conclusions
Mitochondria-nuclear crosstalk in cancer cells can regulate
pathways and tumor properties
Mitochondrial retrograde regulation (MRR) is a bi-directional pathway of
communication between the mitochondria and the nucleus and influences many
cellular activities
Transmitochondrial cybrid technology is an excellent tool to understand
mitochondria-nuclear cross-talk in a defined nuclear background
Unlike other subgroups of patients with breast cancer, for Triple Negative (TN)
breast cancer, there is a current lack of understanding of driver pathways and hence
are often managed using more generic therapies
The c-Src kinase activity are frequently over-expressed in TN breast cancer and is
associated with metastatic disease progression
Further understanding of the mechanism regulation of Src is necessary to use Src
inhibitors as its monotherapy in TN breast cancer patients that resulted in only
partial response
Src (Y416) is regulated by mitochondrial ATP: Minimal alteration of Src (Y416) phosphorylation
status observed in parental cells and cybrids after treatment with ROS scavenger NAC (P) or
glycolysis inhibitor 3-Bromopyruvate (3BP) (Q). When parental cells forced to depend on
mitochondrial respiration by culturing in Galactose medium (R) an increase Src (Y416) observed.
However, treatment with mitochondrial ETC complex-1 (rotenone) (S), complex-III (Antimycine) (T)
or complex-V (oligomycin) (U) inhibitors dose dependently inhibited pSrc (Y416)
autophosphorylation suggesting its critical regulation through ATP from mitochondrial ETC. V.
Oxygen consumption rate (OCR) analyzed by Seahorse Extracellular Flux Analyzers suggest that
aggressive metastatic cells have high respiration. W. ATP analysis suggested increased ATP
levels in cybrids with metastatic mitochondria compared to cybrids with benign mitochondria. X.
In vitro phosphorylation assay suggested that autophosphorylation of Src at Y416 site is directly
depending on the ATP concentration. However, no such correlation was observed with Src Y527
phosphorylation site.
A. SUM159 TN BCa cybrid model with mitochondria from benign (MCF10A & A1N4), moderately metastatic
(SUM159) and highly metastatic (MDA231) TN cells under a defined nuclear background of SUM159 mitochondria
depleted ρ0 cells. Soft agar colony formation in parental mitochondrial donor cells (left panel) and cybrids (right
panel).
F. Pathway analysis from microarray gene expression data of 143B cybrids suggested Src as one of the
major pathways affected by benign mitochondria. G. Analysis of TCGA data suggested that Src (Y416) is
significantly up regulated in basal subtype of tumors compared to Hormone regulated breast cancers. H.
Western Blot analysis of total Src and phosphorylated Src (Y416) Src in ER positive and TN BCa cell shows
significantly higher phosphorylation in TN breast cancer cells. I. Analysis of patient-derived triple negative
xenografts models suggested that high levels of pSrc (Y416) expression favors distant metastasis (DM) to
lung and brain. J. Src (Y416) analysis in parental cells and their cybrids suggest that Src Tyr416 but not total
Src is highly activated in cancer cells and their cybrids. K. Analysis of cybrids and their mtDNA depleted
Rho0 cells (ρ0) suggest that Src (Y416) and their target pFak but not Src (Y527) is abolished in ρ0 cells.
Western Blot of whole cell lysate (W), mitochondrial (M) and cytoplasmic (C) fractions shows mitochondrial
localization Src (Y416). Total Src, Tim23, α-Tublin expressions were used as controls for whole cell extract,
mitochondrial and cytoplasmic fractions. M. Confocal analysis confirm co-localization of pSrc (Y416) with
mitochondria localized protein (Tim23). DAPI was used for nuclear staining.
several oncogenic
Mitochondrial tumor characteristics is critical in the regulation of Src (Y416)
autophosphorylation that promote oncogenic and metastatic potential of TN breast
cancer.
Mitochondrial ATP is responsible for the phosphorylation of Src (Y416) and the
regulation of its down-stream oncogenic pathways.
These novel findings will have significant impact on the treatment of breast cancer
and the development of new drug targets for currently non-targetable Src-dependant
cancer subtypes like the TN breast cancer.
Our findings suggest mitochondrial targets as promising combination therapy for
the management of Src-dependant TN breast cancer
Further studies are in progress to explore the clinical and therapeutic application of
this finding.