Altered Retrograde Signaling Patterns in Breast Cancer Cells Cybrids with H and J Mitochondrial DNA Haplogroups

The aim of this study was to determine the role of retrograde signaling (mitochondria to nucleus) in MCF7 breast cancer cells. Therefore, in the present study, MCF7-H and MCF7-J cybrids were produced using the mitochondria from the same H and J individuals that were already used in our non-diseased retinal pigment epithelium (ARPE19) cybrids. MCF7 cybrids were treated with cisplatin and analyzed for cell viability, mitochondrial membrane potential, ROS, and expression levels of genes associated with the cGAS-STING and cancer-related pathways. Results showed that unlike the ARPE19-H and ARPE19-J cybrids, the untreated MCF7-H and MCF7-J cybrids had similar levels of ATP, lactate, and OCR: ECAR ratios. After cisplatin treatment, MCF7-H and MCF7-J cybrids showed similar (a) decreases in cell viability and ROS levels; (b) upregulation of ABCC1, BRCA1 and CDKN1A/P21; and (c) downregulation of EGFR. Cisplatin-treated ARPE19-H and ARPE19-J cybrids showed increased expression of six cGAS-STING pathway genes, while two were increased for MCF7-J cybrids. In summary, the ARPE19-H and ARPE19-J cybrids behave differentially from each other with or without cisplatin. In contrast, the MCF7-H and MCF7-J cybrids had identical metabolic/bioenergetic profiles and cisplatin responses. Our findings suggest that cancer cell nuclei might have a diminished ability to respond to the modulating signaling of the mtDNA that occurs via the cGAS-STING pathway.


Introduction
The mitochondria (mt) are unique organelles in that they possess their own DNA. The mtDNA is maternally inherited and can be classified into haplogroups which represent ancestral origins of populations. The haplogroups are defined by an accumulation of specific single nucleotide polymorphisms (SNPs) that represent a geographic origin for a population. The most common European haplogroup is H, representing over 40% of the population [1]. The J haplogroup, representing populations that originated in Northern Europe, account for approximately 9% of the population [1]. The mitochondria are critical for oxidative phosphorylation (OXPHOS) and bioenergetics of all cells.
Belizzi et al. compared oxygen consumption rates of test subjects with different European haplogroups and reported that the H haplogroup subjects had higher O 2 consumption rates while the J haplogroup rates were lower, suggesting higher glycolysis level in the latter group [2]. Similarly, Larsen et al. found that test subjects with the H haplogroup mtDNA had increased oxygen consumption rates compared to those with the U haplogroup [3]. subjects as the ARPE cybrids, the MCF7-J cybrids would exhibit differential properties compared to the MCF7-H cybrids, similar to the non-pathologic RPE-J versus RPE-H cybrids, which would have implications in personalized treatment of cancer patients with respect to cisplatin.

Cellular Metabolism and Survival in Cisplatin-Treated MCF&-H and MCF7-J Cybrids
We initiated our study by measuring the IC50 values for cisplatin in order to determine the concentration of cisplatin required to inhibit cell survival by 50% in MCF7-H and MCF7-J cybrids ( Figure 1A,B). The R 2 value for the MCF7-H cybrids was 0.8851 and the IC50 value was 32.62 μm (95% confidence interval 27.02 to 39.37 μM). MCF7-J cybrids had an R 2 value of 0.94938 and an IC50 of 24.25 μM (95% confidence interval: 22.05 to 26.66 μM).  20,40,60,80, 100, or 120 μM) and then incubated for another 48 h. Cell viabilities of the cybrids were measured using the colorimetric MTT assay and normalized to the untreated MCF7-H cybrid sample. An IC-50 analysis was done to determine the concentration at which viability decreased by 50%. For the MCF7-H cybrids the IC-50 was 32.62 μM (R 2 = 0.8851) and the MCF7-J cybrid had a value of 24.25 μM (R 2 = 0.9493). (C) Both the MCF7-H and MCF7-J cybrids had significant but similar decreases in survival in response to cisplatin. At 20 μM of cisplatin, viability for the MCF7-H and MCF7-J cybrids decreased by 13% (**** p < 0.0001) and 22% (**** p < 0.0001) and (*** p <0.001), respectively. There was also a significant decrease in response to 40 μM of cisplatin of 34% (p < 0.0001) and 40% (p < 0.0001) for MCF7-H and MCF7-J cybrids respectively. Each MCF7-H and MCF7-J haplogroup had four different biological cell lines each, with 12 samples for each treatment and cell line group.
Then, we assessed the survival of MCF7-H and -J cybrids treated with 20, and 40 μM cisplatin. Using the MTT assay, we determined the levels of metabolic activity indicative  20,40,60,80, 100, or 120 µM) and then incubated for another 48 h. Cell viabilities of the cybrids were measured using the colorimetric MTT assay and normalized to the untreated MCF7-H cybrid sample. An IC-50 analysis was done to determine the concentration at which viability decreased by 50%. For the MCF7-H cybrids the IC-50 was 32.62 µM (R 2 = 0.8851) and the MCF7-J cybrid had a value of 24.25 µM (R 2 = 0.9493). (C) Both the MCF7-H and MCF7-J cybrids had significant but similar decreases in survival in response to cisplatin. At 20 µM of cisplatin, viability for the MCF7-H and MCF7-J cybrids decreased by 13% (**** p < 0.0001) and 22% (**** p < 0.0001) and (*** p <0.001), respectively. There was also a significant decrease in response to 40 µM of cisplatin of 34% (p < 0.0001) and 40% (p < 0.0001) for MCF7-H and MCF7-J cybrids respectively. Each MCF7-H and MCF7-J haplogroup had four different biological cell lines each, with 12 samples for each treatment and cell line group.

ATP and Lactate Production in MCF7-H and MCF7-J Cybrids
Next, we determined whether MCF7-H and -J cybrids have distinct metabolic profiles. To study this phenomenon, we measured the concentrations of ATP and lactate in the respective cybrids. The MCF7-H and MCF7-J cybrids were cultured for 24 h and the relative amounts of ATP were measured ( Figure 2C). Similar levels of ATP production were found in the MCF7-H (100% ± 4.7%) and the MCF7-J cybrids (92.9% ± 3%; p = 0.21). The lactate levels were measured in both the 1:2 dilution and the 1:4 dilution samples ( Figure 2D). At the 1:2 ratio, the MCF7-H cybrids showed a lactate concentration of 0.25 ± 0.02 mM compared to the MCF7-J cybrids with 0.28 ± 0.02 mM, p = 0.17. The 1:4 ratio samples demonstrated the MCF7-H and MCF7-J cybrids had similar lactate levels (0.20 ± 0.01 mM and 0.22 ± 0.01 mM, respectively, p = 0.28). Our findings indicate that the MCF7-H and MCF7-J cybrids showed similar ATP and lactate levels suggesting that the H versus J mtDNA haplogroups were not influencing the mode of energy production.

Gene Expression for the MCF7-J and MCF7-H Cybrids after Cisplatin Treatment
Next, we want to examine the differential gene expression levels associated with cellular stress in response to cisplatin treatment in the cybrids derived from MCF7 and ARPE cells. One of the critical pathways for assessing cellular stress is the accumulation of mitochondrial DNA (mtDNA), which is regulated by the cGAS-STING signaling axis. This axis disrupts mitochondrial homeostasis and mtDNA efflux into the cytosol, activating the IRF3 or NF-B pathway in the process [16][17][18]. Therefore, we proceed by assessing the relative expression levels of the cGAS-STING genes in cybrids generated from MCF7 and ARPE cells in response to cisplatin treatment.

Gene Expression for the MCF7-J and MCF7-H Cybrids after Cisplatin Treatment
Next, we want to examine the differential gene expression levels associated with cellular stress in response to cisplatin treatment in the cybrids derived from MCF7 and ARPE cells. One of the critical pathways for assessing cellular stress is the accumulation of mitochondrial DNA (mtDNA), which is regulated by the cGAS-STING signaling axis. This axis disrupts mitochondrial homeostasis and mtDNA efflux into the cytosol, activating the IRF3 or NF-B pathway in the process [16][17][18]. Therefore, we proceed by assessing the relative expression levels of the cGAS-STING genes in cybrids generated from MCF7 and ARPE cells in response to cisplatin treatment.
Following that, we examined the differential expression of cancer-related genes in cybrids generated from the ARPE and MCF7 cell lines in response to cisplatin treatment.
Following that, we examined the differential expression of cancer-related genes in cybrids generated from the ARPE and MCF7 cell lines in response to cisplatin treatment.

Discussion
There are many studies that have demonstrated crosstalk between the mitochondria and nuclear gene expression [6,11,19,20]. The importance of this retrograde signaling has become clearer using the transmitochondrial cybrid model where all cell lines have identical nuclei but contain mitochondria from individuals of different mitochondrial haplogroups (i.e., H and J cybrids). Our previous studies comparing the ARPE19-H cybrids versus the ARPE19-J cybrids showed that the retrograde signaling from different haplogroups modulated the different cell lines differently [4,6,11], which made them of great interest in cancer studies. The ARPE19-H cybrids preferentially used OXPHOS, had higher levels of ROS and ATP production, lower levels of lactate, and increased rates of cell growth compared to the J cybrids [11,21]. When mitochondria from the identical subjects were placed into the MCF7 Rho0 cells, the responses were significantly different than those seen in the ARPE19 cybrids. The MCF7-H and MCF7-J cybrids had similar levels of ATP, lactate, ROS formation, mitochondrial membrane potential, OCR:ECAR ratios, and Seahorse ® bioenergetic values. These findings suggest that the nuclei in ARPE19 cybrids are receptive to the retrograde signaling originating from the H and J mitochondria. In contrast, the MCF7 nuclei are resistant to the retrograde signaling and therefore, the MCF7-H and MCF7-J cybrids exhibit similar behaviors ( Figure 5).

Discussion
There are many studies that have demonstrated crosstalk between the mitochondria and nuclear gene expression [6,11,19,20]. The importance of this retrograde signaling has become clearer using the transmitochondrial cybrid model where all cell lines have identical nuclei but contain mitochondria from individuals of different mitochondrial haplogroups (i.e., H and J cybrids). Our previous studies comparing the ARPE19-H cybrids versus the ARPE19-J cybrids showed that the retrograde signaling from different haplogroups modulated the different cell lines differently [4,6,11], which made them of great interest in cancer studies. The ARPE19-H cybrids preferentially used OXPHOS, had higher levels of ROS and ATP production, lower levels of lactate, and increased rates of cell growth compared to the J cybrids [11,21]. When mitochondria from the identical subjects were placed into the MCF7 Rho0 cells, the responses were significantly different than those seen in the ARPE19 cybrids. The MCF7-H and MCF7-J cybrids had similar levels of ATP, lactate, ROS formation, mitochondrial membrane potential, OCR:ECAR ratios, and Seahorse ® bioenergetic values. These findings suggest that the nuclei in ARPE19 cybrids are receptive to the retrograde signaling originating from the H and J mitochondria. In contrast, the MCF7 nuclei are resistant to the retrograde signaling and therefore, the MCF7-H and MCF7-J cybrids exhibit similar behaviors ( Figure 5). It has been documented that the cGAS-STING pathway is capable of sensing damaged mitochondria through cytosolic mtDNA and activation following this signal. Therefore, this pathway was determined to be possibly responsible for the retrograde signaling that was seen in this study. When expression levels of STING genes were measured, the cisplatin-treated ARPE19-H and ARPE19-J cybrids showed significant increases in expression levels of seven STING genes, including the downstream IFN-a, similar to the results It has been documented that the cGAS-STING pathway is capable of sensing damaged mitochondria through cytosolic mtDNA and activation following this signal. Therefore, this pathway was determined to be possibly responsible for the retrograde signaling that was seen in this study. When expression levels of STING genes were measured, the cisplatintreated ARPE19-H and ARPE19-J cybrids showed significant increases in expression levels of seven STING genes, including the downstream IFN-a, similar to the results found in the study from Parkes, et al. [22]. After cisplatin treatment, the responses of the ARPE19 cybrids differed from each other with increases for the STING, TBK1, and NFkB1 expression in ARPE19-J-treated cells and higher levels of IFN-α and IRF3 in the ARPE19-H-treated cybrids compared to their respective untreated controls. Since all cybrid cell lines had identical nuclear genomes, the differential responses of the STING pathway genes are due to the modulation by the H versus J mtDNA haplogroups. In contrast, the cisplatin-treated MCF7-H cybrids did not show any significant changes in STING genes and the cisplatintreated MCF7-J cybrids showed upregulation for only the cGAS and IRF3 genes. This leads to the conclusion that the MCF7 cybrids failed to activate its STING pathway in the presence of s-phase-specific DNA damage caused by cisplatin, which would normally be expected [22]. While the exact mechanism behind this is unclear, it could be hypothesized that the cGAS-STING cytosolic DNA sensing system is interfered with by the cancerous nucleus, obstructing the cGAS-STING response that would normally follow cisplatininduced damage. Another possibility may be that the DNA repair mechanisms inherent in the MCF7 cell line are robust enough that minimal cytosolic DNA is produced from cisplatin-induced damage, eliminating the need for the cGAS-STING response.
In the untreated MCF7 cybrids, the expression levels for the BRCA1 gene were significantly higher in the MCF7-J cybrids compared to the MCF7-H cybrids (p = 0.0076). If similar responses were found in cancer patients, then individuals with the mtDNA J haplogroup might have higher levels of BRCA1 that might provide protection since it maintains genomic stability by repairing DNA breaks and yields tumor suppression. Moreover, after cisplatin treatment, the MCF7-H and MCF7-J cybrids showed 3.15-fold and 2.78-fold increase in BRCA1 transcription, respectively, which may contribute to the inhibition of tumor growth from the treatment. Although the ARPE19 and MCF7 cybrids contained mitochondria from identical subjects, the responses to cisplatin were different in the MCF7 cybrids compared to the ARPE cybrids [6]. After cisplatin treatment, the cisplatin-treated ARPE19 cybrids showed differential expression for CDKN1A/P21 (ARPE19-H was upregulated, ARPE19-J had no change) and CYP51A (ARPE19-J was upregulated, APRE19-H showed no change). There were no significant changes in gene expression levels for the seven other cancer genes (ABCC1, ALK1, BRCA1, DHRS2/HEP27, EGFR, ERBB2, and ERCC1). In contrast, both MCF7-H and MCF7-J cybrids showed parallel levels of upregulation in ABCC1, BRCA1, and CDKN1A/P21, along with lower transcription of the EGFR gene.
After cisplatin treatment, the MCF7-H and MCF7-J cybrids exhibited a five-fold and four-fold upregulation of the ABCC1 gene, respectively, which is also known as "multidrug resistance protein 1" and has been associated with cisplatin resistance. Tommasi S et al., reported that the H haplogroup was associated with breast cancer tumor grade differentiation and with BRCA2 mutations [23], suggesting that mtDNA variants may act as a genetic modifier for breast cancer. While the mtDNA U haplogroup has been overrepresented in control subjects [24], the mtDNA I haplogroup has higher association with the breast cancer group [25], although there is still some controversy of the association between European mtDNA haplogroups and breast cancer risk [26]. Multiple studies have speculated that in Asian, African-American, and European-American populations, there may be an increased association between the m.10398G > A polymorphism and breast cancer [27][28][29][30]. The m.10398G > A is a non-synonymous SNP in the MT-ND3 gene, and it has been speculated that this alternation in Complex I would result in higher ROS production and increased apoptosis of normal cells, thereby allowing proliferation of cancer cells.
Another Chinese study using cybrids created in MDAMB-231 cells showed those with the D5 haplogroup mtDNA had lower mitochondrial respiration, ATP production, and mitochondrial membrane potential along with tumorigenic behavior compared to non-D5 cybrids [31]. The difference in these studies showing that the D5 haplogroup modulated the cell behavior which was absent in our MCF7 cells may be due to the fact that the MDAMB-231 cells are metastatic mammary adenocarcinoma 1 while the MCF7 cell line is also from a metastatic differentiated mammary epithelial cell that can possess estrogen receptors and can process estradiol.

ARPE19-H, ARPE19-J, MCF7-H, and MCF7-J Cybrids
All subjects read and signed an informed consent (IRB #2003-3131) from the Institutional Review Board of the University of California, Irvine. All clinical investigations and protocols were conducted according to the principles of the Declaration of Helsinki and approved by the appropriate investigational review boards (University of California, Irvine). Cybrids were generated as described previously [4,6,12]. H and J cybrids were created by polyethylene glycol fusion of platelets with the Rho0 (mtDNA free) ARPE-19 or MCF-7 cells, which had been treated by low dosage ethidium bromide as described by Miceli [19]. H and J cybrids were cultured to the fifth passage using DMEM-F12 containing 10% dialyzed fetal bovine serum, 100 unit/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL fungizone, 50 µg/mL gentamycin, and 17.5 mM glucose. Cybrid haplogroups were verified by PCR, restriction enzyme digestion, and/or mtDNA sequencing. According to PhyloTree.org, the J haplogroup was defined by the SNPs at m.295C > T, m.489T > C, m.12612A > G, m.13708G > A, and m.16069C > T. The specific J cybrids used in this study included those with haplogroups J1d1a, J1c1, J1b1a, and J1c1a. As for the H cybrids, the cell lines included those with the H4a1a4b2, H1e1a, H1j, and H1 haplogroups. The defining SNPs for the H haplogroup according to PhyloTree.org were m.7028T > C and m.2706G > A. Further specific haplotyping was also done according to the haplotyping tree on PhyloTree.org.

IC 50 Analysis of Cisplatin Titration Curve Measuring Cell Survival
MCF7-H and MCF7-J cybrids were plated in 96-well plates (10,000 cells/well), incubated for 24 h, and then treated with 0, 20, 40, 60, 80, 100, or 120 µM of cisplatin. The cybrids were incubated for another 48 h before having their cell viabilities measured with an MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Cat. # 30006, Biotium, Inc., Fremont, CA, USA). In this assay, the cybrids were incubated for two hours with the MTT reagent and absorbance was measured with the ELx808 spectrophotometer (BioTek Instruments/Agilent, Winooski, VT, USA) at 570 nm with reference wavelength at 630 nm. The background absorbance was subtracted from the signal absorbance and values were normalized to the untreated specimen of each cell line. Each treatment was analyzed with eight replicates. An IC 50 analysis was done to determine the concentration of cisplatin required to inhibit the cell viability by 50% (GraphPad Prism Software, Inc., Version 5.0, San Diego, CA, USA).

Cell Survival Assay
ARPE19-H, ARPE19-J, MCF7-H, and MCF7-J cybrids were plated in 96-well plates (10,000 cells/well) and incubated for 24 h. Then cisplatin was added to the media at concentrations of 0, 20, or 40µM and incubated for another 48 h. MTT reagent (Cat. # 30006, Biotium, CA, USA) was added to the cultures for two hours and absorbance was measured using the ELx808 spectrophotometer (BioTek, Winooski, VT, USA) at 570 nm with reference wavelength at 630 nm. The background absorbance was subtracted from the signal absorbance and values were normalized to H untreated and analyzed using the two tailed t-test (GraphPad Software Inc., San Diego, CA, USA). Experiments analyzed in triplicate and the entire experiment was repeated twice. The data results were normalized to the untreated MCF-H group and normalized to their respective cell viability values to account for cell number differences. Differences in cisplatin-treated cells compared to untreated cells were analyzed (Prism, GraphPad Software Inc., San Diego, CA, USA) and were considered to be statistically significant when p ≤ 0.05. Experiments were analyzed in quadruplicate and the entire experiment repeated three separate times.

ATP Assay
ARPE19-H, ARPE19-J, MCF7-H, and MCF7-J cybrids were plated in 96-well plates (10,000 cells/well) and incubated for 24 h. The cells were then lysed with a lysis solution and lightly shaken for five minutes. A substrate solution containing luciferase and luciferin (Abcam, Cambridge, UK) was then added, and the cells were lightly shaken for another five minutes. The cells were then stored in the dark for 10 min before the luminescence was read using the Gemini XPS Microplate Reader (Molecular Devices, San Jose, CA, USA) at 560 nm. Values were normalized to H untreated and analyzed using the two tailed T-test (Prism, GraphPad Software Inc., Version 5.0, San Diego, CA, USA). Experiments were analyzed in triplicate and the entire experiment was repeated twice.

Lactate Assay
The lactate levels were measured using the Lactate Assay Kit (Eton Bioscience Inc., San Diego, CA, USA) and according to the manufacturer's protocol. MCF7-H and MCF7-J cybrids were plated at 10,000 cells/well in 96-well plates, incubated overnight and lactate concentrations were analyzed. The solutions were diluted 1:2 and 1:4 to verify the repeatability of the assay. Standards and samples were set up as duplicates and quadruplicates and experiments were repeated twice. All data from XF24 assays were collected using the XF Reader software from Seahorse Bioscience. The oxygen consumption rate (OCR) was determined by measuring the drop in O 2 partial pressure over time followed by linear regression to find the slope. The extracellular acidification rate (ECAR) was determined by measuring the change in pH levels over time followed by linear regression to find the slope of the line, which represents ECAR. The percentage ATP Turnover Rate is calculated by the following formula: 100 − (ATP coupler response/basal respiration × 100). The percentage Spare Respiratory Capacity represents a bioenergetic value for cells needing high amounts of ATP in response to demands placed upon them. This is calculated by the formula: Electron transport chain (ETC) accelerator response/basal respiration × 100. The percentage Proton Leak equals the ATP coupler response-non-mitochondrial respiration. Data from these experiments were exported to GraphPad Prism 5 (GraphPad Software, Version 5.0, San Diego, CA, USA) where they were analyzed, normalized, and graphed. Statistical significance was determined by performing two-tailed Student t tests and p ≤ 0.05 was considered significant in all experiments.  Table 2 shows the GenBank Accession numbers and functions for 18 genes that were investigated. The six genes, related to the STING pathway, are cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), nuclear factor kappa B subunit 2 (NFkB2), and interferon alpha (IFN-a). The nine cancer-related genes were excision repair cross-complementation group 1 (ERCC1), dehydrogenase/reductase member 2 (DHRS2/HEP27), ATP-binding cassette, sub-family C (ABCC1), breast cancer type 1 susceptibility protein (BRCA1), epidermal growth factor receptor 2 (ERBB2), activin receptor-like kinase 1 (ALK1), and epidermal growth factor receptor (EGFR), cyclin-dependent kinase inhibitor 1A/p21 (CDKN1A/P21) and cytochrome P450, family 51, subfamily A, polypeptide 1 (CYP51A). We also examined BCL2-associated X protein (BAX), caspase-3 (CASP3), and caspase-9 (CASP9), which show pro-apoptosis. Target cycle thresholds (Ct) values were initially compared to the Ct values of reference genes and subsequently, comparisons between untreated and cisplatin-treated values (∆∆Ct) were evaluated for statistical significance. Fold differences were calculated using the equation 2 (∆∆Ct) .  Associates and forms a heterodimer with BCL2. Functions in apoptotic behavior by opening the mitochondrial voltage dependent anion channel, leading to loss of membrane potential and opening of cytochrome C.
GenBank Accession Numbers and Functions from Uniprot.org.

Statistical Analyses
Statistical analysis of the data was performed by ANOVA (GraphPad Prism, version 5.0). Newman-Keuls multiple-comparison or the two-tailed t-tests were used to compare the data within each experiment. p < 0.05 was considered statistically significant. Error bars in the graphs represent standard error of the mean (SEM).

Conclusions
To conclude, this is the first study showing the altered pattern of the retrograde signaling pathway in MCF7-H and MCF-J mitochondrial cybrids. Our findings support that the retrograde signaling processes may differ depending upon the specific mtDNA haplogroup within the cells. Moreover, the non-cancerous nucleus may respond differently to the retrograde mitochondrial signals than the nuclei within the cancer cells. This area of research requires additional investigation but has great potential to identify novel mechanisms of mitochondrial-nuclear interactions and new pathways that have not yet been identified.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.