Liane Fendt

Harald Niederstätter

Gabriela Huber

Bettina Zelger

Martina Dünser

Christof Seifarth

Alexander Röck

Georg Schäfer

Helmut Klocker

Walther Parson
email: walther.parson@i-med.ac.at

Accumulation of mutations over the entire mitochondrial genome of breast cancer cells obtained by tissue microdissection

Keywords:

Purpose:

The occurrence of heteroplasmy and mixtures is technically challenging for the analysis of mitochondrial DNA. More than that, observed mutations need to be carefully interpreted in the light of the phylogeny as mitochondrial DNA is a uniparental marker reflecting human evolution. Earlier attempts to explain the role of mtDNA in cancerous tissues led to substantial confusion in medical genetics mainly due to the presentation of low sequence data quality and misinterpretation of mutations representing a particular haplogroup background rather than being cancer specific. The focus of this study was to characterize the extent and level of mutations in breast cancer samples obtained by tissue microdissection by application of an evaluated full mtDNA genome sequencing protocol. Methods: We amplified and sequenced the complete mitochondrial genomes of microdissected breast cancer cells of 15 patients and compared the results to those obtained from paired non-cancerous breast tissue derived from the same patients. Results: We observed differences in the heteroplasmic states of substitutions between cancerous and normal cells, one of which was affecting a position that has been previously reported in lung cancer and another one that has been identified in 16 epithelial ovarian tumors, possibly indicating functional relevance. In the coding region we found full transitions in two cancerous mitochondrial genomes and 12 heteroplasmic substitutions as compared to the non-cancerous breast cells. Conclusions: We identified somatic mutations over the entire mtDNA of human breast cancer cells potentially impairing the mitochondrial OXPHOS system.

Background / Introduction

The involvement of mitochondria in carcinogenesis is based on the Warburg theory of an impaired energy metabolism in cancer cells triggered by mitochondrial dysfunction [START_REF] Warburg | On the origin of cancer cells[END_REF][START_REF] Wallace | A mitochondrial paradigm of metabolic and degenerative diseases aging and cancer: a dawn for evolutionary medicine[END_REF][START_REF] Brandon | Mitochondrial mutations in cancer[END_REF]. The role of mtDNA mutations in cancer has been discussed since the reporting of several somatic mutations in various types of cancer [START_REF] Bianchi | Mitochondrial genome instability in human cancers[END_REF]. Whereas the majority of the initial studies focused on the non-coding part of the mtDNA, namely the control region, the analysis of the coding region was targeted in detail more recently [START_REF] Santos | Mitochondrial DNA mutations in cancer: a review[END_REF]. Mitochondrial DNA sequence analysis is technically challenging due to complex phenomena such as heteroplasmy and mixtures of mtDNA types that overlay when characterized by direct sequence analysis, but also due to the fact that observed mutations -with respect to control or reference sequences -need to be carefully interpreted in the light of the phylogeny, particularly in the medical context. Hence, investigations on mtDNA mutations in cancer differed profoundly in data quality and their interpretation as discussed in [START_REF] Salas | A critical reassessment of the role of mitochondria in tumorigenesis[END_REF]. Still, the literature displays studies that need to be re-evaluated [START_REF] Chen | Study on the mutations in the D-loop region of mitochondrial DNA in cervical carcinoma[END_REF]. A recent study by He et al. [START_REF] Hey | Heteroplasmic mitochondrial DNA mutations in normal and tumour cells[END_REF] which is the first study comparing tumor to normal mtDNA with a second generation sequencing approach uncovers widespread heteroplasmy in normal cells as well as heteroplasmy and homoplasmy in cancer cells. Very surprisingly, the mtDNA of an individual that belongs to mitochondrial haplogroup J1c3a1 obviously lacks signature mutations on positions 15326, 2706, 4216, 3010 and 13934. By all means this is an unusual finding as these positions are yet considered to be stable in the phylogeny [START_REF] Van Oven | Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation[END_REF] and should be re-evaluated in that respect. Despite this fact, there is convincing evidence that a general feature of cancer cells. is the accumulation of somatic mutations scattered throughout the entire mitochondrial genome [START_REF] Wang | Somatic mutations of mitochondrial genome in early stage breast cancer[END_REF].

Theoretically, the high frequency of mtDNA variation in cancer may be explained by neutral random drift in clonally expanding cell populations [START_REF] Coller | High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection[END_REF]. However, the selective advantage of mtDNA changes in the development of tumors cannot be excluded either [START_REF] Maximo | Mitochondria and cancer[END_REF]. Recent findings by Beerenwinkel et al. [START_REF] Beerenwinkel | Genetic progression and the waiting time to cancer[END_REF] support the theory that tumors contain both neutral mutations (passengers) as well as a smaller fraction of positively-selected mutations (drivers) promoting tumor progression.

Assessment of tumor development and progression by sequence analysis gets technically challenging

when heteroplasmic mixtures contain a minor contribution of about 10% of the total signal [6;7;14] highlighting the paramount role of using appropriate quality of mtDNA raw data to prevent misinterpretation of the data. We have successfully designed a strategy for the amplification and Sanger sequencing of full mitochondrial genomes with reproducible high data quality [START_REF] Fendt | Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences[END_REF]. Only a systematic investigation of a representative number of tumor cells and corresponding normal cells allows for a meaningful evaluation of the role of mtDNA mutations for the development of cancer and the discussion of functional consequences. However, for the time being, there is insufficient reliable mtDNA data available addressing the comparison between neoplastic versus normal breast cancer cells.

In this study we performed a full mitochondrial genome analysis for somatic mutations in paired cancerous and non-cancerous tissues from 15 patients with diagnosed breast cancer using our evaluated protocols. The main aim of this study was to characterize the number and proportion in heteroplasmic mixtures of somatic mutations in primary cancerous tissues in comparison to noncancerous cells of the same individual.

Material and Methods

Clinical samples

Samples derived from patients with diagnosed invasive mamma carcinoma upon mastectomy or tumorectomy under informed and written consent. Criteria for exclusion were tumors which were smaller than 0.5 cm in diameter as well as non-invasive tumors. The study received declaration of no objection by the ethical committee of the Innsbruck Medical University.

Tissue specimens

Frozen and paraffin-embedded tissue samples were obtained directly from breast cancer patients who had undergone tumorectomy or mastectomy after fine needle biopsy diagnosis of breast cancer. The specimens were cooled in ice/water immediately and brought to the pathologist who performed a rapid section and isolated a frozen tissue slice that was embedded in Tissue-Tek OCT Compound (Sakura Finetek Germany GmbH, Staufen, Germany), snap frozen in liquid nitrogen and stored at -80°C until use. The rest of the tumorectomy was fixed and paraffin-embedded according to standard procedures.

Laser capture tissue microdissection

Frozen sections of the tumor specimens were stained with hematoxylin and eosin for pathological analysis and exact localization of the tumor regions. Parallel, unstained 8 µm frozen sections were used for laser capture microdissection (LCM). Slides were pretreated for 1 min in each of the following cooled solutions: 75% ethanol, DEPC-Water (nuclease free water) 100% ethanol (twice), xylene (twice) and then air dried. LCM was performed using a PixCell II LCM System (Arcturus Bioscience Inc., Mountain View, CA, USA) with 1,000 -3,000 laser impulses for each sample corresponding to about 10,000 to 30,000 captured cells.

DNA was extracted from single breast cancer and corresponding normal gland cells derived from laser capture microdissection of the frozen tissue blocks using EZ1 Robotic Workstation and the GenoPrep DNA from Tissue Kit (both Qiagen, Hilden Germany) according to the manufacturer's manual. DNA was resolved in water and quantified via real-time PCR [START_REF] Niederstätter | A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA[END_REF].

MtDNA amplification and sequencing

The entire human mitochondrial genome was amplified and sequenced as previously described [START_REF] Fendt | Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences[END_REF]. In brief, full mtDNA genomes were amplified as 2 overlapping fragments of 8.5 kb each in length using aliquots of 5,000 mitochondrial genomes for PCR. Subsequent chain termination sequencing was performed using 96 primers covering the coding region and 10 primers covering the control region [17]. Laser induced fluorescence capillary electrophoretic separation of purified sequencing products was performed on a 3100 DNA Analyzer (Applied Biosystems, Foster City, CA). Transformed cells were incubated at 37° C over night on LB agar plates containing 50μg/mL Kanamycin. Small amounts of bacterial colonies were picked using disposable pipette tips, transferred directly to 1.5 mL LB medium supplemented with 50 µg/mL Kanamycin in 96-well deep well plates, and incubated for 18 hours at 37 °C with constant shaking (200 rpm). Plasmid DNA (pDNA) minipreps from over-night suspension cultures were performed in the 96-well format with the montáge Plasmid Miniprep 96 Kit (Millipore, Billerica, MA, USA) according to the manufacturer's recommendations. 1 μL aliquots of pDNA minipreps were subjected to cycle sequencing (30 cycles comprising 95 °C for 15 s, 50 °C for 10 s and 60 °C for 4 min after an initial denaturation step of 2 min at 95 °C) using the primers M13 for (GTAAAACGACGGCCAGTGA) and M13 rev (GGAAACAGCTATGACCATG) and BigDye terminator chemistry (v1.1, Applied Biosystems).

Cloning and sequencing of selected samples

Electrophoresis of purified sequencing products was performed using a 3100 DNA Analyzer (Applied Biosystems).

Data analysis and quality assurance

Upon analysis of the raw data the sequences were aligned and the base-calls were reviewed independently by two scientists using the Sequencher software (v.4.8, Gene Codes Corporation, Ann Arbour, MI, USA). Consensus sequences were reported relative to the revised Cambridge Reference Sequence (rCRS) [18;19] following nomenclature guidelines for mtDNA typing suggested by [START_REF] Carracedo | DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing[END_REF][START_REF] Tully | Considerations by the European DNA profiling (EDNAP) group on the working practices nomenclature and interpretation of mitochondrial DNA profiles[END_REF][22].

Results

Somatic mutations and differences in the control region between breast tumor cells and corresponding normal breast tissue

In the 15 investigated sample pairs of cancerous and corresponding normal breast tissues we detected six differences in a total of five patients within the mtDNA control region (Table I). Four of the observed heteroplasmic mutations at positions 152, 215, 16304 and 16390 in the control region were also observed by Irwin et al. [START_REF] Irwin | Investigation of Heteroplasmy in the Human Mitochondrial DNA Control Region: A Synthesis of Observations from More Than 5000 Global Population Samples[END_REF] who investigated the occurrence of point-heteroplasmy in the control region of over 5,000 global population samples. Mutations at positions 152, 16304 and 16390 occur in multiple macro-haplogroup backgrounds (haplogroups L, M, N, and R) [START_REF] Malyarchuk | Molecular instability of the mitochondrial haplogroup T sequences at nucleotide positions 16292 and 16296[END_REF] whereas position 215 (8 observations of heteroplasmy in more than 5000 individuals) is not reported as evolutionary fast site [25;26] . The remaining two mutations were 16106G/ A and 16391G/ A . 16106A variant was described for haplotypes belonging to European haplogroup T [START_REF] Vernesi | Mitochondrial diversity in linguistic isolates of the Alps: a reappraisal[END_REF], and 16391A is common in clades I and A2g of haplogroup N [START_REF] Van Oven | Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation[END_REF]. Note that positions 152, 215 and 16391 harboured the heteroplasmic mixture in the normal tissue but were homoplasmic in the tumor.

Somatic mutations and differences in the coding region between breast tumor cells and corresponding normal breast tissue

In the mitochondrial coding region of the 15 analyzed patients a total of 14 mutations was detected between cancerous and corresponding non-cancerous reference tissue, five (35.7%) of which affected ND genes, two (14.3%) were located in the CO genes, four (28.6%) were found in mtRNA genes, and one hit (7.1%) was observed in the cytb gene (Table II). Two mutations in the cancerous cells (12875C, 12131C) were present in homoplasmic state. Eight positions harboured wild type and variant within the cancer cells but only wild type within the normal tissue (7379GA/ G , 5703GG/ A , 15341TT/ C , 2998TC/ T , 2145GA/ G , 12803GA/ G , 1632TC/ T , 1132TC/ T ). Four heteroplasmic substitutions observed within the normal mtDNA were not detected in the cancer cells (9966A/ G A, 5102A/ G A, 5390A/ G A, 1578A/ G A). It has to be noted that we also found (low level) heteroplasmy within four non-cancerous DNA samples (Figure 1, 4a-d).

For non-synonymous base substitutions (2145A/ G , 2998C/ T , 12803A/ G , 12875C, 12131C, 15341T/ C ) as well as for the mutations found in the rRNA and tRNA genes an evolutionary conservation analysis was performed. Positions 2998, 15341, 12803 and 12875 were conserved in an inter-vertebrate species comparison while position 12131 was not. (Figure 2).

The sensitivity of direct cycle sequencing to reliably display low level mixtures depends on numerous factors including the base composition of the adjacent nucleotide sequence and the uneven ddNTP incorporation rate. To clarify if the apparently fixed somatic mutations found in the tumor cells were actually present at a proportion below the detection limit of sequencing analysis in the normal tissue, clones of PCR products obtained from normal distant tissue samples were sequenced (Table III).

Discussion and Conclusions

In this study we sequenced the entire mitochondrial genomes of 15 primary breast cancer patients and observed in total 20 differences between the mtDNA of breast cancer cells and the corresponding distant normal tissue. Our results are based on an evaluated sequencing strategy for full mtDNA genome sequencing analysis [START_REF] Fendt | Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences[END_REF], yielding high quality sequencing electropherograms that are characterized by very low background noise levels, multiple sequence coverage, electronic data handling and hence unequivocal base calls. In addition, the data was checked for plausibility by constructing a phylogenetic tree of the detected lineages (Figure 3). All of the observed full mtDNA sequences could be assigned to known European and Asian mtDNA lineages. Haplotypes belonging to superhaplogroup H were found most commonly (53.3%) which is in agreement with the expected prevalence of this haplogroup in Europe [START_REF] Achilli | The molecular dissection of mtDNA haplogroup H confirms that the Franco-Cantabrian glacial refuge was a major source for the European gene pool[END_REF]. In addition, we observed two haplotypes from clusters HV and J (each 13.3%) and one representative of clusters K and R0a (6.7%). One sequence (6.7%) belonged to an Asian lineage (M35b). [START_REF] Rosson | Frequent mutations in the mitochondrial control region DNA in breast tissue[END_REF] observed various sets of somatic mutations in the mtDNA control region when comparing breast cancer cells and corresponding normal tissues. In a study by Tan et al. [START_REF] Tan | Comprehensive scanning of somatic mitochondrial DNA mutations in breast cancer[END_REF] fourteen of the 19 analyzed tumors displayed at least one somatic mtDNA mutation. Parella et al. [START_REF] Parrella | Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle aspirates[END_REF] found 12 somatic mutations in 18 primary breast tumors as compared to the corresponding mtDNAs. In contrast, Wang et al. [START_REF] Wang | Somatic mutations of mitochondrial genome in early stage breast cancer[END_REF] found only two heteroplasmic point substitutions within 10 sample pairs when analyzing the coding region.

The reasons for the different extent of mutations within tumor mtDNA remain unclear so far but it is conceivable that multiple parameters influence the induction of mtDNA mutations. Wang [START_REF] Wang | Somatic mutations of mitochondrial genome in early stage breast cancer[END_REF] compared mtDNA mutations in early stage breast cancer and suggested that the observed mutations resulted from somatic mutational events in primary cancerous or paracancerous tissues. Sigh et al.

[34] investigated the influence of mutations within nuclear genes on the mutation rate of mtDNA. They concluded that mutations in the polymerase domain of the POLG gene decrease mitochondrial activity and increase oxidative stress resulting in an increased leakage of ROS which in turn induce mutations and promote tumorigenesis.

The reporting of significantly different numbers of mutations in tumor cells might be also due to the misinterpretation of the resulting electropherograms based on ambiguous data [START_REF] Salas | A critical reassessment of the role of mitochondria in tumorigenesis[END_REF]. Also, the nature of observed mutations in our study differed from other reports. While we foundand confirmed by cloning 2 full transitions and 12 heteroplasmic mutations in the coding region (Table II), the majority of other studies observed largely apparent homoplasmic somatic mutations, for discussion see [START_REF] Santos | Mitochondrial DNA mutations in cancer: a review[END_REF]. Tzen et al. [35] as well as Kassauei et al. [29] observed that several mutations are present in heteroplasmy in healthy counterparts and became homoplasmic in cancer tissues. This is an interesting fact as the level of heteroplasmy is supposed to influence the phenotype in a way that if the pathogenic threshold is surpassed cellular dysfunction caused by defective mitochondria becomes apparent. This also explains the time-related and tissue-specific variability of clinical features displayed in mtDNA-related disorders. Hence, it is remarkable that Park et al. [START_REF] Park | A heteroplasmic not homoplasmic mitochondrial DNA mutation promotes tumorigenesis via alteration in reactive oxygen species generation and apoptosis[END_REF] found that a cell line carrying a heteroplasmic ND5 mtDNA mutation showed significantly enhanced tumor growth, while cells with the homoplasmic mutant variant inhibited tumor formation. In contrast, in a study of Petros et al. [START_REF] Petros | mtDNA mutations increase tumorigenicity in prostate cancer[END_REF], the introduction of the homoplasmic variant 8993G in a prostate cancer cell line led to a 7 times larger tumor growth in the resulting cybrid cell line as compared to the wild type-cybrids.

It is further worth mentioning that one cancer sample in our study harboured a mutation at the same position as observed by Aikhionbare et al. [START_REF] Aikhionbare | Mitochondrial DNA sequence variants in epithelial ovarian tumor subtypes and stages[END_REF] in 16 out of 102 epithelial ovarian tumors. In our case position 1632 (tRNA Val ) was affected by a heteroplasmic transition to C, while Aikhionbare et al. [START_REF] Aikhionbare | Mitochondrial DNA sequence variants in epithelial ovarian tumor subtypes and stages[END_REF] reported transversions to G in all of the observed tumor samples. A phylogenetic analysis of different species suggested that site 1632 is conserved (Figure 2) but might mean that the transversion has a more deleterious effect than the transition. Another interesting finding is the co-occurrence of mutational events at position 2998 (16s rRNA) in one cancer sample of this study and in a lung cancer sample published by Lorenc et al. [39]. While we found a heteroplasmic transition to C at that position, Lorenc et al. reported an insertion. Evolutionary comparisons suggested this position being a highly conserved site and therefore, an alteration of this position might have functional consequences.

The main yet unexplained question is: when do these mutations actually arise? Do the detected mutations induce cancer developmentthus being present at very low levels in pre-cancer states, or does cancer induce mutations? In a study of Wang et al. [START_REF] Wang | Somatic mutations of mitochondrial genome in early stage breast cancer[END_REF] one of the two detected cancer variants (8601G) also occurred in the paracancerous tissue, and the homoplasmic variant in the tumor arose either through random drift or selection, while the other detected cancer variant (2275C) arose only in the tumor, which was confirmed by subsequent cloning. In our study, tumor specificity of two apparently homoplasmic mutations in the ND4 and ND5 genes at positions 12875 and 12131 was confirmed by cloning and subsequent sequencing of the corresponding mtDNA segments from the normal tissue samples, for which only the wild type alleles were found. These findings support the model of induction of mutations within the tumor. Contrary, a study of Tzeng et al. [35] showed that mutations were found in 13 out of 53 analyzed ND4 genes of tumor cells but could also be identified in 11 samples of the corresponding normal tissues by DHPLC, indicating that most mtDNA mutations identified in tumors pre-existed already as minor components in heteroplasmic mixtures. These results suggested that mtDNA mutation occurred before tumorigenesis and became apparent in cancer cells.

MtDNA mutations in tumors obviously do not emerge randomly over the entire genome but rather affect phylogenetic informative positions as well as evolutionary fast sites, at least in the control region [START_REF] Vega | mtDNA mutations in tumors of the central nervous system reflect the neutral evolution of mtDNA in populations[END_REF]. This is also evident in our data, as transitions affecting positions 16390 and 16391 are found across the entire phylogeny and positions 152 as well as 16304 are known mutational hotspots [START_REF] Van Oven | Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation[END_REF]. Four of the observed mutations on positions 152, 215, 16304, 16390 were also determined as frequent hits at heteroplasmic sites in the control region of more than 5,000 global population samples [START_REF] Irwin | Investigation of Heteroplasmy in the Human Mitochondrial DNA Control Region: A Synthesis of Observations from More Than 5000 Global Population Samples[END_REF]. Only position 215 was not reported as evolutionary fast site [25;26] and therefore selection against it cannot be excluded, in particular as position 215 is situated close to the origin of heavy strand replication and a mutation there might influence mtDNA replication [5;23]. On the contrary, 215T can be found in sublineages of all 4 major macro-haplogroups M, N, R and L [START_REF] Van Oven | Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation[END_REF]. 16106A is a rare variant which has been observed in seven of 115 haplotypes of an alpine population [24;27]. It has been shown in a cell culture system that the rate of ROS induced lesions was 5 fold higher in the control region as compared to the coding region [START_REF] Rothfuss | Analysis of differential DNA damage in the mitochondrial genome employing a semi-long run real-time PCR approach[END_REF]. In the coding part of the mtDNA the literature displays the most affected regions to code for Complex I in the electron transport chain [5;42;43] which is consistent with our results (35.7 % of detected mutations were found in genes coding for Complex I). Functional consequences of these mutations are conceivable as they cause amino acid changes from serine to proline (12131C) and from isoleucine to threonine (12875C). It is possible that a small fraction of mutations act as driver mutations having functional consequences. This assumption goes with the findings of Zhidkov et al. [START_REF] Zhidkov | mtDNA mutation pattern in tumors and human evolution are shaped by similar selective constraints[END_REF] providing evidence for similar selective constraints for the patterns in mtDNA mutations in tumors and human evolution reflecting a response to positive selection pressures in cancer, rather than a random adaptive process.

On the contrary, mutations can become homoplasmic during the process of clonal expansion of tumor cells, representing a model of "rapid evolution". This theory is in agreement with the findings of Coller et al. [START_REF] Coller | High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection[END_REF] who explained the existence of homoplasmy in tumors solely as a consequence of tumor kinetics without any needs for positive selection, although they do not exclude the possibility of other mechanisms.

In conclusion, we identified somatic mutations over the entire mtDNA of human breast cancer cells potentially impairing the mitochondrial OXPHOS system. Two mutations affected positions that have already been reported by others. Our results suggest that mtDNA mutations may play an important role in the progression of breast cancer, but the mechanism by which mtDNA mutations contribute to cancer development remain unclear. Clearly, there is still more data needed from full mitochondrial genomes of cancer and corresponding non-cancerous tissues to get more insight in the processes involved. Sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) with accession numbers GU592019-GU592048.

For 4

 4 patients displaying point-heteroplasmy in the tumor tissue at positions 12875 (patient no. 1), 2998 (patient no. 8), 12131(patient no. 11), and 1632 (patient no. 13), the corresponding mtDNA segments from the non-cancerous normal tissue samples were amplified with the proofreading KOD hot start DNA polymerase (Novagen, Madison, WI, USA) and unlabelled primers: for 5'-TTGCTCATCAGTTGATGATACG-3' and rev 5'-AGCGGATGAGTAAGAAGATTCC-3' flanking position 12875, for 5'-CCCAACCTCCGAGCAGTACATG-3' and rev 5'-CGAACCTTTAATAGCGGCTGCACCAT-3' flanking position 2998, for 5'-AACCACGTTCTCCTGATCAAA-3' and rev 5'-GGTCGTAAGCCTCTGTTGTCAG-3' flanking position 12131 and for 5'-GTGGCAAGAAATGGGCTAC-3' and rev 5'-GCCAGGTTTCAATTTCTATCG-3' flanking position 1632. Ligation of the PCR products into the pCR 4-Blunt-TOPO vector and subsequent transformation into chemically competent One Shot TOP10 E. coli were performed using the Zero Blunt TOPO PCR cloning kit for sequencing (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol.

  Therefore we selected two patients where the corresponding cancerous samples harboured full transitions 12875T (patient 1) and 12131C (patient 11), one patient where the corresponding cancer sample displayed point heteroplasmy (1632C/ T , sample no. 13) and patient 8 whose corresponding cancerous sample harboured a partial transition at the evolutionarily well conserved position 2998T.None of the analyzed clones displayed any variants that would indicate low level contributionspotentially undetected by sequence analysis. The 95% confidence intervals further strengthened the assumption that there was no sequence variation in the corresponding reference tissue samples at positions 12875 (sample no. 1), 12131 (sample no. 11), 1632 (sample no.13) and 2998 (sample no. 8).

  When comparing our observations to previous studies with a similar setup, we noticed a variety of different findings, one being the number of observed mutations in the various tumor tissues. While Kassauei et al. [29] found 71 mutations in 15 complete mtDNA sequences of pancreatic ductal adenocarcinoma cells, Zhu et al. [30] found 45 somatic mutations in 15 complete mtDNA genomes of breast cancer tissues and Rosson et al.

Fig. 2 .

 2 Fig. 2. Evolutionary conservation analysis. Up to 7 vertebrate sequences comprising positions 1632, 2998, 12803, 15341, 12875, 2145 as well as 12131 were aligned. GenBank accession numbers are given in brackets.

Fig. 3 .

 3 Fig. 3. Depiction of the germ line and somatic mutations of the observed haplotypes in a

Table I . List of observed CR sequence differences between cancerous and corresponding normal breast tissues of 15 patients. patient No haplogroup normal tissue a cancer tissue a hits in the phylogeny b

 I

	5	H11a	16391G/ A	16391G	A2g, I
					B4f, C5c, D5a1, D5c1, E,
	9	H3	16390G	16390A/ G	F1a3, L0a2b, L0d2a, L1c4, L2, M27b, M48, N1b, N9b1b,
					R7a1b2, R8b, R9b1, U3a
	12	HV	215A/ G	215A	A2p, C1c1, J2a1, L0k2, M11
			16106G	16106G/ A	T
	13	M35b			H5, F3, L3d3, L3h2, M17c, M21d, M22, M25, M27c,
			16304C	16304T/ C	N9a2a, R0a2c, R5, R9, R31a,
					T2b, U5b3
	14	J1c	152C/ T	152T	common

a Minor contributions in point-heteroplasmic mixtures are indicated by reduced IUB base code letter size. Length variants and substitutions within the poly C stretches spanning positions 16183-16193 and 309-315 were disregarded. b A polymorphic site survey performed using databases EMPOP (www.empop.org), mtDB (http://www.genpad.uu.se/mtDB), mtDNA tree Build 08

[START_REF] Van Oven | Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation[END_REF]

(http://www.phylotree.org) mtDNA (http://ianlogan.co.uk/mtdna.htm) and MITOMAP (http://www.mitomap.org).

Table II . List of observed coding region sequence differences between cancerous and corresponding normal breast tissues of 15 patients patient

 II

	No	haplogroup	normal tissue a	cancer tissue a	gene	syn/non syn	hits in the literature b
	1	J1c2c	12875T	12875C	ND5	IleThr	-
	2	H	7379G	7379A/ G	CO I	syn	M34a (Chandrasekar 09)
	3	R0a1a	5703G	5703G/ A tRNA Asn		-
							G1c (Derenko 07), I1
							(Derenko 07, Finnilä 01),
	6	H5	9966A/ G	9966A	CO III	ValIle	L1c3a (Behar 08), M1a1c (Olivieri 06), M30c1a
							(Behar 08a), M31a2
							(Barik 08)
	7	H1	15341T 15341T/ C	cyt b	PheLeu	A5a (Tanaka 04), T1a1 (Coble 04)
			2998T	2998C/ T	16s rRNA		lung cancer (Lorenc 03)
	8	K1a1b1	2145G	2145A/ G	16s rRNA		L3e3b (Kivisild 06), H1b (Herrnstadt 02)
	11	H2a	12131T	12131C	ND 4	SerPro	-
	12	HV	12803G 12803A/ G	ND 5	SerAsp	-
			1632T	1632C/ T	tRNA Val		-
	13	M35b2	5102A/ G	5102A	ND 2	syn	R5 (Palanichamy 04), L3e2a (Behar 08)
			5390A/ G	5390A	ND 2	syn	U2e (Palanichamy 04, Achilli 05)
			1132T	1132C/ T	12s rRNA
	14	J1c					-
			1578A/ G	1578A	12sr RNA

a Minor contributions in point-heteroplasmic mixtures are indicated by reduced IUB base code letter size. Length variants and substitutions within the poly C stretches spanning positions 16183-16193 and 309-315 were disregarded. b A polymorphic site survey performed using databases EMPOP (www.empop.org), mtDB (http://www.genpad.uu.se/mtDB), mtDNA tree Build 08

[START_REF] Van Oven | Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation[END_REF]

(http://www.phylotree.org) mtDNA (http://ianlogan.co.uk/mtdna.htm) and MITOMAP (http://www.mitomap.org). References are available as supplementary file.

Table III . Frequency of variants found by cloning of PCR products obtained from normal distant breast tissues of patients no 1, 8, 11 and 13

 III

	Fig. 1
	Patient No	no. of clones/variants	relative frequency of variant (95% CI)
	1	53/0	0 (0.0000-0.0582)
	8	65/0	0 (0.000-0.048)
	11	48/0	0 (0.000-0.0639)
	13	80/0	0 (0.000-0.0393)

. Sequencing electropherograms showing different mutational conditions in the mitochondrial genomes of breast cancer cells as compared to the corresponding normal cells.

  Electropherograms of reverse strands are not shown. 1a/b: Full transitions on positions 12875 and 12131 from T in the normal to C in the corresponding cancerous mitochondrial genome from patients no 1 and 11. 2a-f: Heteroplasmic transitions in the tumor mt genome with the haplogroup -specific variant represented as the dominant type (position 7379: patient no. 2; 2145: patient no. 8; 2998: patient no. 8; 12803: patient no. 12; 1632: patient no. 13; 1132: patient no. 14). 3a/b: Heteroplasmic transitions in the tumor tissue where the haplogroup-specific variant constituted the minor contribution (position 5703: patient no. 3; 15341: patient no. 7). 4a-d: Heteroplasmic states in normal tissue that is not detected in mitochondrial tumor DNA (position 9966: patient no. 6; position 5390: patient no 13; 5102: patient no. 13; 1578: patient no. 14).

Acknowledgements

AR and WP were supported by the FWF Austrian Science Fund Translational Research Programme (L397). We are grateful to o.Univ.-Prof. Dr. Raimund Margreiter (Department of Operative Medicine, Innsbruck Medical University) for supporting the study. Abbreviations: mtDNA -mitochondrial DNA LCM -laser capture microdissection ND -NADH dehydrogenase COcytochrom oxidase cytbcytochrome b OXPHOSoxidative phosphorylation


Robert S Tolhurst

Ross S Thomas

Fiona J Kyle

Hetal Patel

Manikandan Periyasamy

Andrew Photiou

Paul T R Thiruchelvam

Chun-Fui Lai

Marwa Al-Sabbagh

Rosemary A Fisher

Marwa Al- Sabbagh

Sayka Barry

Tatjana Crnogorac-Jurcevic

Lesley-Ann Martin

Mitch Dowsett # ¶

R Charles Coombes

Tahereh Kamalati

Simak Ali
email: simak.ali@imperial.ac.uk

Laki Buluwela
email: l.buluwela@imperial.ac.uk

Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth

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Introduction

Two-thirds of breast cancers express estrogen receptor-α (ERα), and estrogen plays a critical role in the development and progression of these tumours. This understanding has led to the development of anti-estrogens, primarily tamoxifen, which compete with estrogen for binding to the ERα.

Treatment with tamoxifen for 5 years following surgery leads to 50% lower annual recurrence rate and a 28% decrease in annual rates of mortality in patients with early stage ER-positive breast cancer [START_REF] Osborne | Tamoxifen in the treatment of breast cancer[END_REF][START_REF] Ali | Endocrine-responsive breast cancer and strategies for combatting resistance[END_REF]. However, many patients who respond to tamoxifen, eventually relapse. Aromatase inhibitors act by preventing the conversion of androgens into estrogens by the aromatase enzyme, with new aromatase inhibitors displaying greater efficacy than tamoxifen. However, resistance to aromatase inhibitors also develops in many cases [START_REF] Johnston | Aromatase inhibitors for breast cancer: lessons from the laboratory[END_REF][START_REF] Chen | What do we know about the mechanisms of aromatase inhibitor resistance?[END_REF].

In a proportion of cases, patients who initially present with ERα-positive breast cancer, become ERα-negative [START_REF] Kuukasjarvi | Loss of estrogen receptor in recurrent breast cancer is associated with poor response to endocrine therapy[END_REF]. The mechanisms by which ERα expression is lost are unclear, although epigenetic silencing of the ERα gene may be involved [START_REF] Lapidus | The loss of estrogen and progesterone receptor gene expression in human breast cancer[END_REF]. In most cases, however, resistant tumours remain ERαpositive and show a response to a change of endocrine agent [START_REF] Ali | Endocrine-responsive breast cancer and strategies for combatting resistance[END_REF], indicating that ERα continues to be important in regulating tumour growth in these cases. For the latter, recent studies suggest that endocrine resistance could result from modulation of ERα activity by altered co-activator and co-repressor balance and/or crosstalk with growth factor signalling cascades, including phosphorylation of ERα at specific residues. In this context, elevated HER2 and EGFR expression have been observed in cell line models of tamoxifen resistance, whilst elevated ERK1/2 MAPK [START_REF] Gee | Phosphorylation of ERK1/2 mitogen-activated protein kinase is associated with poor response to anti-hormonal therapy and decreased patient survival in clinical breast cancer[END_REF] and high levels of phosphorylated AKT have been associated with poor response to tamoxifen and a worse patient prognosis [START_REF] Kirkegaard | AKT activation predicts outcome in breast cancer patients treated with tamoxifen[END_REF]. Further, phosphorylation of ERα at serine 118 (S118) is elevated in recurrence following tamoxifen treatment [START_REF] Sarwar | Phosphorylation of ERalpha at serine 118 in primary breast cancer and in tamoxifen-resistant tumours is indicative of a complex role for ERalpha phosphorylation in breast cancer progression[END_REF]. Finally, high-level expression of the coactivator AIB1 is associated with poor response to tamoxifen in ERα-positive breast cancer, with AIB1-and HER2-positive patients having the worst outcome following tamoxifen treatment [START_REF] Osborne | Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer[END_REF].

Ectopic expression of ERα in ERα-negative breast cancer cell lines and in immortalised non-tumourigenic breast cells inhibits their growth, despite showing estrogen-dependent stimulation of expression of estrogen-responsive genes [START_REF] Jiang | Growth regulation of estrogen receptor-negative breast cancer cells transfected with complementary DNAs for estrogen receptor[END_REF][START_REF] Zajchowski | Estrogen inhibits the growth of estrogen receptor-negative, but not estrogen receptor-positive, human mammary epithelial cells expressing a recombinant estrogen receptor[END_REF][START_REF] Lazennec | Expression of human estrogen receptor using an efficient adenoviral gene delivery system is able to restore hormonedependent features to estrogen receptor-negative breast carcinoma cells[END_REF]. By contrast, over-expression of ERα did not inhibit the growth of ERαpositive breast cancer cell lines [START_REF] Zajchowski | Estrogen inhibits the growth of estrogen receptor-negative, but not estrogen receptor-positive, human mammary epithelial cells expressing a recombinant estrogen receptor[END_REF], although conditional over-expression of ERα did lead to increased growth of MCF-7 cells in the absence of ligand [START_REF] Fowler | Increases in estrogen receptor-alpha concentration in breast cancer cells promote serine 118/104/106-independent AF-1 transactivation and growth in the absence of estrogen[END_REF],

suggesting that ERα over-expression in ERα-positive breast cancer cells may facilitate adaptation to estrogen deprivation.

To further investigate the consequences of ERα over-expression on the estrogen responses in ERα-positive breast cancer cells, we have transduced MCF-7 cells with an adenovirus encoding ERα. Prolonged culturing of the ERαtransduced cells in estrogen-free medium allowed the establishment of an estrogen-independent line, MLET5, in which ERα expression was maintained. MLET5 cells did not retain adenoviral sequences and microsatellite genotyping confirmed their lineage as MCF-7-derived. In further characterising MLET5 cells by gene expression microarray analysis, we found changes in apoptosis associated gene expression when compared to MCF-7 cells, so as to favour cell survival. As a result of these differences, we have gone on to show that the MLET5 line has altered cell survival characteristics, as indicated by a greatly reduced sensitivity to etoposide induced apoptosis. Together, these findings indicate that transient ERα over-expression in breast cancer cells could be sufficient to promote the development of endocrine resistance in breast cancer through altered expression of estrogen-regulated genes involved in cell survival.

Materials and Methods

Cell culture and cell lines MCF-7 cells were obtained from the ATCC (LGC Prochem, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich Ltd., UK) supplemented with 10% fetal calf serum (FCS) (First Link Ltd., UK). For culturing in estrogen-free conditions, MCF-7 cells were cultured in DMEM lacking phenol red (DMEM-PR) (Gibco-BRL, UK), supplemented with 10% dextrancoated charcoal-stripped FCS (DSS) (First Link Ltd., UK). MLET5 cells were routinely cultured in DMEM-PR, containing 10% DSS.

Recombinant adenovirus construction and infection of MCF-7 cells

The human ERα open reading frame was cloned into the adenoviral shuttle vector pAdTrack-CMV [START_REF] He | A simplified system for generating recombinant adenoviruses[END_REF], which encodes GFP, to generate pAdTrack-CMV-ERα. Recombinant adenoviral genome AdERα and control virus (AdGFP) were generated following recombination by co-transformation of E. coli BJ5183 cells with pAdEasy-1, as described and packaged in HEK293 cells, also as described [START_REF] He | A simplified system for generating recombinant adenoviruses[END_REF]. The viruses were purified by caesium chloride banding and viral particle concentration was determined by spectrophotometric analysis. MCF-7 cells (6x10 6 ) were seeded in 10-cm plates in DMEM-PR, containing 10% DSS and allowed to settle for 24 hours prior to infection. FACS analysis of single cell suspensions prepared 2 days following adenoviral transduction, was used to determine the percentage of cells transduced. Cell counts were performed using a haemocytometer with trypan blue exclusion for counting of viable cells.

Sulphorhodamine B (SRB) Growth assay

Sixteen hours following seeding of 3x10 3 cells in 96-well plates in DMEM-PR containing 10% DSS, the medium was replaced with fresh medium supplemented with 17ß-estradiol (E2), anti-estrogens, or an equivalent volume of the vehicle (ethanol). Medium was changed every three days. Cells were fixed using 40% (w/v) TCA, for one hour at 4°C, washed five times with distilled, deionised H 2 O, followed by incubation with 0.4% SRB in 1% acetic acid for one hour at room temperature. Excess dye was removed with five washes with 1% acetic acid and drying at room temperature. Absorbance at 480nm was determined following solubilisation of the dye by the addition of 100μl of 10mM Tris base to each well.

For measuring growth following addition of Etoposide (Sigma-Aldrich), 2x10 3 cells were seeded in each well. Medium supplemented with the inhibitors in a titration of two-fold dilutions, starting from 100µM, was added after 48 hours.

Cell growth was measured 48 and 72 hours after treatment, using the SRB assay, with the GI 50 being defined as the concentration of drug required to obtain 50% of the growth exhibited by untreated, control cells.

Cell cycle analysis

Cells were seeded in six-well plates (10 5 /well) in DMEM containing 10% FCS and allowed to adhere for 48 h, followed by the addition of 1nM-100 M etoposide (Sigma-Aldrich) or DMSO and incubation for 48 h. Cells were harvested, cell cycle Annexin V/propidium iodide staining and analysis was carried out as previously described [START_REF] Ali | The development of a selective cyclin-dependent kinase inhibitor that shows antitumor activity[END_REF].

RT-PCR analysis

RNA was prepared and RT-PCR performed as described previously [START_REF] Buluwela | Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-alpha and the transcriptional repressor PLZF[END_REF]. For quantitative RT-PCR (Q-RT-PCR) measurements, Taqman Gene Expression Assays were used with a 7900HT Fast Real-time PCR machine (Applied Biosystems). Primer details are given in supplementary information.

Immunoblotting MCF-7 and MLET cells (1x10 6 ) were seeded in 10-cm plates DMEM containing 10% FCS and lysed after 48 hours, as described [START_REF] Buluwela | Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-alpha and the transcriptional repressor PLZF[END_REF]. For experiments where ligands were to be added, the cells were incubated in DMEM-PR containing 10% DSS for three days prior to seeding. Ligands were added, as appropriate, with an equal volume of ethanol being added to the controls. Cell lysates were prepared 24 hours later. Immunoblotting was carried out as described previously [START_REF] Buluwela | Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-alpha and the transcriptional repressor PLZF[END_REF], using antibodies detailed in Supplementary Material.

Gene expression microarray analysis

For gene expression microarray analysis, MCF-7 and MLET5 cultures were seeded in estrogen-depleted medium as described above for 3 days, with three bioreplicate cultures used for each cell line and treatment. Following 16 hours treatment with 10nM E2, RNA was purified from the cultures (RNeasy, Quiagen) and used to probe bead arrays (Illumina, Human WG-6) through Cambridge Genomic Services (http://www.path.cam.ac.uk/cgs/). Hybridisation data were obtained using BeadStudio software (Illumina) and raw gene expression data analysed using GeneSpring GX 10 software (Agilent, Santa Clara USA). All samples were normalised by quantile normalisation to minimise variation between microarray chips. Data were filtered to include only those probes expressed in at least one sample (present and marginal flags). The three replicates for the no ligand and E2 treatments were compared by unpaired t tests, and differentially expressed genes were considered significant at a multiple testing corrected p value (Benjamini Hochberg FDR) of <0.05.

Results

Adenoviral transduction of ERα stimulates MCF-7 cell growth and expression of estrogen-regulated genes

MCF-7 cells were infected with an adenovirus encoding GFP for monitoring infection (AdGFP), into which the human ERα coding region was inserted (AdERα) (Supplementary Fig. 1). FACS analysis showed that 47%, 76% and 90% of the cells were GFP-positive using multiplicity of infection (MOI) of 400, 2,000 and 10,000 particles per cell, respectively. However, infection with AdGFP at MOI=10,000 resulted in cell death, so all subsequent infections were performed using MOI=2,000. Under these conditions, stimulation of growth was observed in the absence as well as in the presence of estrogen. ERα levels were 5-6-fold higher in the AdERα-infected cells, compared to the AdGFP-infected cells at the protein and RNA levels (Fig. 1A,B). However, in the presence of estrogen, expression of the estrogen-responsive PR and pS2 genes was similar in both cases (Fig. 1B-E), with inhibition of expression with the anti-estrogen ICI182,780 (ICI), but their expression was markedly elevated in the absence of ligand, suggesting that adenoviral over-expression of ERα leads to ligandindependent activation of E2-regulated genes, as well as growth in the absence of estrogen.

Survival of MCF-7 cells transduced with ERα is enhanced during long-term culturing in estrogen-depleted medium

Since transduction of ERα in MCF-7 cells conferred a growth advantage in estrogen-free culture conditions, we determined the effect of long-term culturing of AdERα-infected MCF-7 cells in E2-depleted growth medium (Fig. 1F). Under these conditions, cell numbers fell in all cultures over a period of 2-3 months, resulting in a complete loss in the mock and AdGFP-infected cultures by month 5. By contrast, cell numbers started to recover in the AdERα cultures. The loss of control AdGFP infected and mock infected MCF-7 cells, with the emergence of a small number of surviving cells following infection with AdERα over the course of 5-6 months, was observed in four additional, independent transduction experiments. In order to confirm these findings, the infections were repeated.

From 3 out of the 5 separate infections and selection over 8 months, the recovered cells were cultured, one of these giving rise to the MLET5 cell line.

Monitoring of the cultures by fluorescence microscopy showed no evidence of GFP expression 2-3 months following infection; nor was GFP expression evident in the MLET5 line (Supplementary Fig. 1E). Furthermore, there was no detectable expression of GFP by RT-PCR, nor was there any evidence for the presence of adenoviral sequences by PCR (data not shown). These observations are consistent with adenoviruses being non-integrative and, as a result of cell division, having transduced genomes that become diluted, resulting in loss of transgene expression. Finally, microsatellite genotyping analysis showed that MLET5 cells are indeed identical to MCF-7 cells for 10 markers examined (Table 1). All of the markers used are highly polymorphic with multiple alleles. Assuming a conservative figure of 0.5 for the frequency of each allele at the ten loci examined, the probability that the cell lines are not derived from MCF-7 is 0.5 16 (p<0.0001) (Supplementary Table 1).

MLET5 cells grow in an estrogen-independent manner, but show differential sensitivity to anti-estrogens

Estrogen is required for MCF-7 cell growth (Fig. 2A). By contrast, growth of the MLET5 cells was independent of E2, and was indistinguishable from growth in the absence of ligand (Fig. 2B). Moreover, in contrast to MCF-7 cells, the growth of MLET5 cells was only partially inhibited by anti-estrogens, (Fig. 2C-D). At the RNA level ER remained unchanged in MLET5 cells, although there was an apparent decrease in ER expression in the absence of E2 (Fig. 3A).

Overall however, ER protein levels were elevated in MLET5 cells compared with MCF-7 cells (Fig. 3H), which may be indicative of a post-transcriptional mechanism of ER protein stabilisation in MLET5 cells. Despite the elevated level of ER protein, expression of several well-characterised estrogen-regulated genes was reduced (PR, CTD, TFF1/pS2), at the mRNA and protein level (Fig. 3B-D and Fig. 3H), suggesting that the responsiveness of some, but not all E2regulated genes (GREB1, MYC, CCND1; Fig. 3F,G,H) is attenuated in MLET5 cells. This reduced stimulation in expression of some estrogen-regulated genes could be due to a reduction in ER phosphorylation in MLET5 cells. However, levels of Ser118 and Ser167 phosphorylation (Supplementary Fig. 2), previously associated with increased ER activity were found to be higher in MLET5 cells than in MCF-7 cells, with the higher levels of phosphorylation reflecting the elevated ER expression in MLET5 cells. Alternatively, altered ER transcriptional co-regulator levels could lead to attenuation of expression of regulated genes [START_REF] Lavinsky | Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes[END_REF][START_REF] Shang | Molecular determinants for the tissue specificity of SERMs[END_REF][START_REF] Keeton | Cell cycle progression stimulated by tamoxifenbound estrogen receptor-alpha and promoter-specific effects in breast cancer cells deficient in N-CoR and SMRT[END_REF]. Although expression of the p160 co-activators of ER SRC1 and AIB1

were not altered in MLET5 cells (Supplementary Fig. 3), there was a marked upregulation of the co-repressor SMRT in MLET5 cells, suggesting the possibility of its involvement in reduced expression of E2-responsive genes in MLET cells.

Gene expression microarray analysis of MLET5

In order to determine if reduced estrogen regulation was a general feature of ER activity in MLET5 cells, and to determine the gene expression changes that may have led to the development of the estrogen-independent outgrowth of MLET5 cells, gene expression microarray analysis was carried out. For this, RNA prepared from MCF-7 and MLET5 cells cultured in estrogen-depleted medium, followed by addition of E2 for 16 hours, was used to probe Illumina Human WG-6 bead chips. Of the 42,620 genes on the array, 4,353 genes showed significant differential expression in MCF-7 in the presence of E2, compared with the vehicle control, with similar numbers of genes showing up-and down-regulation. About half as many (2,134) genes showed significant differential expression in MLET5 cells, consistent with the reduced expression of estrogen-regulated genes observed using Q-RT-PCR (Fig. 3). Applying a fold change (FC) of 1.5 showed that 414 and 971 genes were up-, or down-regulated with estrogen in MLET5 and MCF-7 cells, respectively (Fig. 4A,B). Of the genes showing 1.5-fold upregulation in expression in E2-treated MCF-7 cells, 34% (171 of 501) genes were also up-regulated ≥1.5-fold in MLET5 cells (representing 79% of the genes upregulated by estrogen) and included known estrogen regulated targets such as GREB1, PDZK1, PGR, MYB, RET and IGFBP4 (Supplementary Table 2). It should also be noted that a further 140 genes with ≥1.5 fold induction by estrogen in MCF-7 cells showed a significant estrogen regulation in MLET5 cells per se.

Similarly, 115 (24%) of the estrogen down-regulated genes in MCF-7 cells were also down-regulated >1.5 fold in MLET5 and a further 140 genes showed significant down-regulation in MLET5 cells. These data are shown in a scatterplot analysis (Fig. 4C) and highlights the fact that most of the genes that show estrogen regulation in MCF-7 cells are also estrogen-regulated in MLET5 cells, indicating that despite their adaptation to growth in the absence of estrogen, MLET5 cells still feature a marked, but attenuated estrogen-regulated gene response.

In examining the MCF-7 and MLET5 gene expression profiles we also noted that a number of previously described estrogen up-regulated genes which showed moderate, but significant estrogen regulation in MCF-7 cells, were upregulated in MLET5 cells in the absence of ligand. For example, the expression of CXCL12 was 4.4 fold higher in MLET5 than in MCF-7. The same was also true for some estrogen down-regulated genes, such as INHBB, whose expression was 0.25 fold in vehicle treated MLET5 cells compared to MCF-7 cells. This suggests the possibility that in MLET5 cells ER regulates the expression of estrogen responsive genes in the absence of estrogen and that estrogen is able to further modulate the expression of these genes.

Estrogen regulation and apoptosis in MCF-7 and MLET5 cells

A total of 717 genes were differentially expressed (p<0.05, FC≥2.0) in MLET5 compared with MCF-7, whilst 886 genes showed differential expression in the presence of estrogen, with 563 of these genes being differentially regulated in both the vehicle and estrogen-treated samples (Fig 4D). Pathway analysis of the genes showing differential expression in MLET5 cells compared with MCF-7 cells (p<0.05, FC≥2.0) using gene set enrichment analysis (GSEA) identified cellular and molecular functional groupings (Supplementary Table 3), the most significant of which were functional groups encompassing apoptosis and included molecules involved in cell survival and the regulation of cell death. David (http://david.abcc.ncifcrf.gov/) also showed significant enrichment of cell death groups in MLET5 cells (Supplementary table 3).

With GSEA 5 of the top 6 annotated groups are associated with apoptosis and accounted for 22 genes showing differential expression in MLET5 v MCF-7 cells, including the key apoptotic regulators BCL2, BAD and BIK (Table 1; Supplementary Fig. 4). Q-RT-PCR showed that BCL2 expression was estrogen regulated in MCF-7 and MLET5 cells, but was dramatically elevated in MLET5 cells (Fig. 5A). Conversely, the BH3-only pro-apoptotic BIK (BCL2-interacting killer) gene was down-regulated by estrogen in MCF-7 and MLET5 cells and its expression was greatly reduced in MLET5 cells (Fig. 5B). The related gene BID, which showed estrogen-regulation, was also down-regulated in MLET5 cells (Fig. 5C), whereas expression of BAD, reduced upon estrogen treatment in MCF-7 cells, was increased in MLET5 cells (Fig. 5D). Western blotting confirmed Q-RT-PCR data, with reduced BID and BIK and elevated BCL2 and BAD (Fig. 5E).

These findings suggest that altered cell survival is a key feature in the progression of the estrogen-independent MLET5 cells. To test this, the response of MCF-7 and MLET5 cells to the pro-apoptotic drug, etoposide was compared. MLET5 cells were considerably less sensitive to etoposide, with a GI 50 = 19.3 µM, compared with a GI 50 = 4.6 µM for MCF-7 (t-test: p < 0.0001) (Fig. 6A,B).

Cell cycle analysis performed with etoposide at concentrations ranging from 1nM-100µM showed an accumulation of MCF-7 and MLET5 in G2/M (Fig. 6C). There was also an accumulation of MCF-7 cells in the sub-G1 phase, indicative of cells undergoing apoptosis, which was confirmed with direct determination of cells in apoptosis following staining for Annexin V, with 60% of the MCF-7 cells undergoing apoptosis in the presence of 100 µM etoposide (Fig. 6D). However, even at a concentration of 100µM, the proportion of MLET5 cells in apoptosis is similar to that seen for vehicle (DMSO) treated cells.

Discussion

Estrogen Receptor-α over-expression by adenoviral transduction results in ligand-independence in MCF-7 cells

In the normal breast, ERα expression is restricted to a small proportion of epithelial cells, with ERα expression being associated with a lack of estrogen regulated proliferation. In the majority of primary breast cancer patients, however, the cancer cells are distinguished by expression of ERα in proliferating cells, with levels of expression often being elevated. Although the presence of ERα in breast cancer is associated with the likelihood of response to endocrine therapies, the impact of ERα levels in breast cancer cells on prognosis and response to endocrine treatments remain unclear. Determination of ERα levels using a ligand binding assay showed that patients with high ERα levels had a shorter relapse-free period than patients with low ERα [START_REF] Hupperets | The prognostic significance of steroid receptor activity in tumor tissues of patients with primary breast cancer[END_REF]. Further, fluorescence in situ hybridization of ERα-positive breast tumors has shown that the ERα gene (ESR1) is amplified in about 20% of cases, the amplification being strongly associated with poor disease-free survival [START_REF] Nielsen | Amplification of ESR1 may predict resistance to adjuvant tamoxifen in postmenopausal patients with hormone receptor positive breast cancer[END_REF], whilst multiplex ligationdependent probe amplification-based copy number analysis shows a similar percentage of ESR1 amplification in breast cancer, an association with higher mitotic index and a trend towards higher grade [START_REF] Moelans | Molecular profiling of invasive breast cancer by multiplex ligation-dependent probe amplification-based copy number analysis of tumor suppressor and oncogenes[END_REF]. Other studies have shown similar levels of ERα gene amplification, but by contrast, ESR1 amplification was associated with better response to endocrine therapies [START_REF] Holst | Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer[END_REF][START_REF] Tomita | Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients[END_REF]. Further adding to the controversy are findings from other laboratories showing that the frequency of ESR1 amplification is considerably lower than 20% [START_REF] Albertson | Conflicting evidence on the frequency of ESR1 amplification in breast cancer[END_REF]. Additionally, there is no apparent relationship between ERα levels and response in patients with advanced disease; however, and response in the IMPACT trial which assessed tamoxifen, anastrozole or the combination in the neo-adjuvant setting, had significantly more responders in the patient group with higher ERα levels, but only in the combination arm [START_REF] Anderson | Predictors of response to aromatase inhibitors[END_REF]. Therefore, it remains unclear as to whether ERα levels are related to clinical response.

In order to better understand the consequences of high ERα expression in breast cancer cells, we over-expressed ERα in the MCF-7 ERα-positive breast cancer cell line. Interestingly, the transient over-expression of ERα led to the eventual outgrowth of cells that were estrogen-independent for growth. These cells were negative for GFP expression and PCR indicated that adenoviral sequences are absent from these lines. Further, in the generation of LTED cells, [START_REF] Chan | Molecular changes associated with the acquisition of oestrogen hypersensitivity in MCF-7 breast cancer cells on long-term oestrogen deprivation[END_REF] noted a transient over-expression of ERα levels in MCF-7 cells within three weeks of culturing in an estrogen deprived environment. This transient overexpression preceded susbsequent changes leading to estrogen independent growth, in which ERα over-expression was again seen. Taken together, these findings suggest that transient over-expression of ERα could play a role in the acquisition of an estrogen-independent phenotype by breast cancer cells. The mechanisms by which ERα over-expression may promote the development of resistant cells are unclear, but one possibility is that transient ERα overexpression provides a survival advantage that allows estrogen-independent cells to emerge. The minimum period of ERα over-expression that would be sufficient for establishment of the resistant lines is also unclear. However, at an MOI of 2,000 particles/cell, 90% of the cells were GFP-positive on day 3 following infection, falling to 48% on day 9 and no GFP expression was detectable after 12 weeks in culture (data not shown; and see Fig. 1F). Hence continued overexpression of transduced ERα was not required for the emergence of the MLET lines.

The MLET5 line was not responsive to estrogen for growth but was partially sensitive to anti-estrogens, indicating the continued importance of ERα in MLET5. Although ERα protein levels were significantly higher in MLET5 compared to MCF-7 cells, E2 induction of pS2 and CTD was markedly reduced.

Phosphorylation of ERα at S118 and S167 stimulates its activity [START_REF] Lannigan | Estrogen receptor phosphorylation[END_REF] and phosphorylation at these sites was seen to be increased in MLET5, suggesting that the reduced apparent ERα activity is not due to a reduction in ERα phosphorylation. However, levels of NCoR and particularly SMRT were elevated in MLET5 cells, compared to MCF-7 cells. This is in contrast to a previously described MCF-7 derived line, LCC1, generated through in vivo selection under conditions of low estrogen availability, and LCC9, derived from LCC1 following selection for growth in the presence of ICI [START_REF] Brunner | Acquisition of hormone-independent growth in MCF-7 cells is accompanied by increased expression of estrogen-regulated genes but without detectable DNA amplifications[END_REF]. LCC1 and LCC9 remain ERαpositive, but show reduced expression of the p160 co-activators (SRC1, TIF2 and AIB1) and the co-repressors NCoR and SMRT [START_REF] Naughton | Progressive loss of estrogen receptor alpha cofactor recruitment in endocrine resistance[END_REF]. The elevated levels of NCoR and SMRT may explain the reduced expression of pS2, CTD and PR in MLET5 cells. The reason for increased NCoR and SMRT proteins in the absence of increases in their mRNA is unclear. However, the ubiquitin ligase mSiah2 has been implicated in regulating proteasomal degradation of NCoR [START_REF] Zhang | Proteasomal regulation of nuclear receptor corepressormediated repression[END_REF]. Siah2 expression has been shown to be estrogen-regulated and to mediate the estrogen-stimulated down-regulation of NCoR, but not SMRT protein [START_REF] Frasor | Estrogen down-regulation of the corepressor N-CoR: mechanism and implications for estrogen derepression of N-CoR-regulated genes[END_REF],

although examination of the microarray data did not show a significant difference in Siah2 levels between MCF-7 and MLET5 cells, with 2.3 and 2.1 fold stimulation of Siah2 expression by estrogen, respectively.

High-level of AIB1 expression has been associated with a poor prognosis and non-responsiveness to tamoxifen in ERα-positive breast cancers, with AIB1positive patients also over-expressing HER1, 2 or 3, being most likely to relapse on tamoxifen [START_REF] Osborne | Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer[END_REF][START_REF] Shou | Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer[END_REF][START_REF] Kirkegaard | Amplified in breast cancer 1 in human epidermal growth factor receptor -positive tumors of tamoxifen-treated breast cancer patients[END_REF]. These findings indicate that crosstalk between ERα, AIB1 and growth factor receptor pathways are important in determining response and resistance to tamoxifen. HER2 expression and P-MAPK levels are elevated in the LTED cells [START_REF] Martin | Enhanced estrogen receptor (ER) alpha, ERBB2, and MAPK signal transduction pathways operate during the adaptation of MCF-7 cells to long term estrogen deprivation[END_REF], similar results being obtained in other LTED cells [START_REF] Santen | Long-term estradiol deprivation in breast cancer cells upregulates growth factor signaling and enhances estrogen sensitivity[END_REF].

However, there was no evidence for increased expression or activity of either EGFR or HER2 in MLET5 cells. Moreover, MAPK phosphorylation levels were comparable in MCF-7 and MLET5 cells, although there was an elevation in P-AKT levels and MLET5 cells were about 5-fold more sensitive to the PI3K inhibitor LY294002 than were MCF-7 cells (Supplementary Figure 2).

MLET5 cells are resistant to apoptosis

The gene expression microarray analysis showed that genes involved in cell survival and apoptosis are highly differentially regulated in MLET5 cells compared with MCF-7 cells. Examination of the proposed functions of these genes showed that the majority of the anti-apoptotic genes were up-regulated, whilst pro-apoptotic genes were down-regulated in MLET5 cells compared with MCF-7 cells. Of particular note were memebers of the Bcl2 gene family, particularly those genes whose expression is estrogen-regulated in MCF-7 cells.

Hence, expression of the estrogen-regulated, anti-apoptotic Bcl2 gene was dramatically elevated in MLET5 cells at the mRNA and protein levels, whereas expression of the pro-apoptotic Bcl2 antagonists Bik, Bid, but also BMF and BNIPL was repressed by estrogen and reduced in MLET5 cells. Although expression of the pro-apoptotic BAD gene did not follow this trend, its expression being elevated in MLET5 cells, levels of Bcl2 expression were increased to a considerably greater extent than those of Bad. It is interesting to note that analysis of the real-time RT-PCR data of Bcl2 and Bad expression shows that expression levels were similar in MCF-7 cells in the absence of estrogen, with Bcl2 levels in the presence of estrogen being 2-3 fold greater than levels of Bad. By contrast, in MLET5 cells, Bcl2 levels were 16 and 20-fold higher in MLET cells compared with MCF-7 cells in the absence and presence of estrogen, respectively. This, taken together with down-regulation of Bid, Bik, as well as BNIPL and BMF, is indicative of anti-apoptotic programming of MLET5 cells, a fact confirmed by the demonstration that MLET5 cells are significantly less sensitive to etoposide-induced apoptosis. Although Bcl2 over-expression has been noted in MCF-7 LTED cells [START_REF] Song | Down-regulation of Bcl-2 enhances estrogen apoptotic action in long-term estradiol-depleted ER(+) breast cancer cells[END_REF], it is not clear if this occurs in the context of other changes in gene expression indicative of overall cell survival, as we have seen in MLET5 cells. Given that Bcl2 positivity and high level Bad expression have been associated with better outcome in breast cancer patients [START_REF] Callagy | Bcl-2 is a prognostic marker in breast cancer independently of the Nottingham Prognostic Index[END_REF][START_REF] Cannings | Bad expression predicts outcome in patients treated with tamoxifen[END_REF], it is likely that the reduced expression of other pro-apoptotic proteins such as Bid and Bik are important for the resistance to apoptotic agents observed in MLET5 cells. Indeed, low Bik expression, as found in MLET5, has previously been associated with resistance to anti-estrogens in MCF-7 cells [START_REF] Coser | Antiestrogen-resistant subclones of MCF-7 human breast cancer cells are derived from a common monoclonal drug-resistant progenitor[END_REF]. Further confirmation of Bcl2 up-regulation was determined by analysing its expression in LTED cells (Supplementary Figure 5). In these cells there was an increase in Bcl2 expression as the cells progressed from the quiescent stage (weeks 2-12) to the estrogen hypersensitive stage (weeks 21-40) [START_REF] Chan | Molecular changes associated with the acquisition of oestrogen hypersensitivity in MCF-7 breast cancer cells on long-term oestrogen deprivation[END_REF]. As observed for MLET5 cells, Bik and Bid expression fell over this time course, levels of expression in the absence of estrogen being reduced to levels similar to, or lower than those seen in the presence of estrogen in MCF-7 cells. Interestingly, Bad expression was unaltered. These findings provide further confirm the association between the

  development of estrogen independence and altered expression of Bcl2, Bik and Bid in breast cancer cells. Taken together, our findings indicate that increased levels of ERα and/or transient over-expression of ERα in breast cancer cells may provide a mechanism for promoting estrogen-independence in breast cancer cells by facilitating cell survival under conditions of low estrogen availability, for example in patients treated with adjuvant aromatase inhibitor therapy. Our data, together with those of other groups who have over-expressed ERα in breast cancer cell lines, would indicate that breast cancer patients with high-level ERα expression would respond less well to endocrine treatment. Indeed, other studies have shown that ERα levels are elevated in LTED cells that emerge following longterm growth in estrogen-depleted media. In any case, our data indicate that even a transient over-expression of ERα would be sufficient for the development of resistance to aromatase inhibitors. In summary, the MLET5 line described here provides a useful new model for studying the mechanisms of endocrine response and resistance in breast cancer, and address the paucity of such lines that are currently available.

Figure 1 .

 1 Figure 1. Transduction of MCF-7 cells with an adenovirus encoding human ERα. (A) Immunoblots of total cell lysates prepared 48 hours following infection of MCF-7 cells with AdGFP or AdERα at MOI 400. (B) For preparing RNA, estrogen (E2; 10 nM) and/or ICI182, 780 (ICI; 100 nM) were added 24 hours prior to harvesting. RT-PCR was performed to analyse expression of the PR, pS2, ERα and GAPDH genes. The expression of each gene was normalised to GAPDH following densitometric analysis and relative expression calculated for each gene, relative to the no ligand control for AdGFP infected cells. The means for the relative expression levels of ERα (C), PR (D) and pS2 (E), as determined from densitometric analysis of RT-PCR carried out on three replicate RNA preparations are shown, the error bars represent the SEM. Significance for gene expression differences was calculated using the t-test. Statistically significant differences (p < 0.05) are shown for comparisons for any given treatment between the lines (*). (F) MCF-7 cells mock infected or infected with AdGFP or AdERα (MOI = 2,000), were grown over a period of 8 months, in estrogendepleted medium. The cell number was determined at monthly intervals. The graphs show the average cell numbers from five independent experiments, the error bars representing the SEM.

Figure 2 .

 2 Figure 2. The MLET lines grow in an estrogen-independent manner MCF-7 (A, C) and MLET5 (B, D) cells were grown in the absence of ligand (NL) or in the presence of increasing concentrations of 17ß-estradiol (E2) (A, B).Growth in the presence of anti-estrogens was carried out following the addition of E2 (10 nM), 4-hydroxytamoxifen (OHT; 100 nM), ICI 182, 780 (ICI; 100 nM) or Raloxifene (RAL; 100 nM) (C, D). Growth was determined using the SRB assay for three experiments. Error bars depict the standard errors of the mean.

Figure 3 .

 3 Figure 3. The expression of estrogen-regulated genes in MLET cells

Figure 4 .

 4 Figure 4. Gene expression microarray profiling of MCF-7 and MLET5 cells in the absence and presence of estrogen. (A, B) Venn Diagrams to show the number of genes whose expression was up-or down-regulated (FC≥1.5, p<0.05) following the addition of estrogen for 16 hours in MCF-7 and MLET5 cells. (C) A scatterplot is shown for genes whose expression is altered >2-fold with estrogen treatment in MCF-7 to show the fold change in expression with estrogen in MLET5 cells, where the estrogen regulation was deemed to be significant (p<0.05). Genes showing a 2-fold change in MLET5 treated with estrogen are similarly plotted to show genes in MCF-7 cells whose expression was significantly altered in MCF-7 cells following estrogen treatment. (D) The Venn diagram shows the number of differentially regulated genes in MCF-7 and MLET5 cells (p<0.05, FC≥2.0).

Figure 5 .

 5 Figure 5. Expression of genes associated with apoptosis is altered in MLET5 cells. (A-D) Q-RT-PCR for RNA prepared from estrogen-treated MCF-7 and the MLET5 cells. Gene expression is shown relative to the expression in MCF-7 cells in the absence of ligand. Error bars show the standard errors of the mean. Significance for gene expression differences was calculated using the t-

Figure 6 .

 6 Figure 6. MLET5 cells are resistant to apoptosis. MCF-7 and MLET cells were treated with etoposide at concentrations ranging from 1 nM -100 µM. (A-B) Cell growth was determined using the SRB assay. Shown are the results of three independent experiments. GI 50 values were determined as the concentration of etoposide that inhibited growth by 50%. SD = standard deviation. (C) MCF-7 cells were treated with etoposide at the concentrations shown, or with vehicle (DMSO) for 48 hours, prior to fixation, staining with propidium iodide (PI) and flow cytometric analysis. Shown are representative FACS profiles, with the black line representing the profile obtained for DMSO treated cells and the gray areas represent profiles for etoposide treated cells. (D) Cells treated for 48 hours with DMSO or etoposide were stained with an antibody for Annexin V and with PI. The percentage of cells that stained positive for Annexin V is shown for three independent experiments. Error bars represent the standard errors of the mean (SEM). Statistically significant differences between MCF-7 and MLET5 cells were reached at concentrations of etoposide of 50, 75 and 100 µM (t-test; p <0.05).

Acknowledgments

We would like to thank Georges Vassaux and Nick Lemoine for facilitating the generation of the adenoviruses used in this study. This work was made possible by grants from Cancer Research UK and the Breast Cancer Research Trust. We are grateful for support from the NIHR Biomedical Research Centre funding scheme. We also thank the CR-UK and the Dept of Health funded Imperial College Experimental Cancer Medicine Centre (ECMC).

Table 1. Genes associated with cell death showing differential expression in MLET5 v