Ana Teresa Martins

Paula Monteiro

João Ramalho-Carvalho

Vera L Costa

Mário Dinis-Ribeiro

Conceição Leal

Rui Henrique

PhD Carmen Jerónimo
email: carmenjeronimo@ipoporto.min-saude.pt

High promoter methylation levels are predictive of poor prognosis in fine-needle aspirate washings of breast cancer lesions

Keywords: breast cancer, quantitative DNA methylation, epigenetic profile, fine needle aspirates, prognosis

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INTRODUCTION

Among all female cancers diagnosed worldwide in 2008, breast cancer accounted for 23% of cases, being the most common cancer in women, with an estimated 1.34 million new cases that year [START_REF] Ferlay | Estimates of cancer incidence and mortality in Europe in 2008[END_REF]. For 2009 in the United States, breast cancer is expected to account for 27% (192 370) of all new cancer cases in women, representing the second most deadly cancer, being responsible for 15% (40 170) of all cancer deaths in women in the same period [START_REF] Jemal | Cancer statistics[END_REF].

Thus, breast cancer constitutes a major health concern and continuous efforts are required to lower the mortality and morbidity rates associated with the disease.

Although ultrasound-guided breast biopsy is usually employed for breast cancer diagnosis, fine needle aspiration (FNA) biopsy of suspicious breast lesions provides a relatively simple, minimally invasive and rapid mean of triaging patients to more complex diagnostic procedures [START_REF] Jeronimo | Detection of gene promoter hypermethylation in fine needle washings from breast lesions[END_REF]. Because the accuracy of FNA largely depends on the cytopathologist proficiency both in performing the aspirate and the cytomorphological analysis [START_REF] Jeronimo | Detection of gene promoter hypermethylation in fine needle washings from breast lesions[END_REF][START_REF] Nemec | How should we screen for breast cancer? Mammography, ultrasonography, MRI[END_REF] and the identification of small pre-invasive lesions and well-differentiated tumors may be challenging [START_REF] Hamill | Improved breast cytology results with near patient FNA diagnosis[END_REF], analyses of tumor associated DNA alterations could augment the accuracy of the evaluation of cellular morphology [START_REF] Jeronimo | Detection of gene promoter hypermethylation in fine needle washings from breast lesions[END_REF].

Alterations in the methylation status of DNA are amongst the most frequent molecular changes associated with human cancers [START_REF] Esteller | Epigenetics in cancer[END_REF]. The search for genes in which promoter CpG islands are hypermethylated in cancer has been increasing, revealing unique profiles of hypermethylation that define each neoplasia. Because DNA methylation is linked to tissuespecific gene silencing [START_REF] Esteller | Epigenetics in cancer[END_REF], and methylation may, therefore, be used as a biomarker for cancer. For instance the GSTP1 gene is hypermethylated in 80 to 90% of patients with prostate cancer, but rarely in benign prostate tissues [START_REF] Esteller | Epigenetics in cancer[END_REF][START_REF] Esteller | Cancer epigenomics: DNA methylomes and histone-modification maps[END_REF].

The list of aberrantly methylated genes identified in breast cancer is long, covering most aspects of their biological functions: cell cycle regulation, apoptosis, DNA repair, hormone regulation, cell adhesion and invasion, angiogenesis, cellular growth-inhibitory signaling, among other [START_REF] Hinshelwood | Breast cancer epigenetics: normal human mammary epithelial cells as a model system[END_REF][START_REF] Liu | Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer[END_REF][START_REF] Lo | Epigenomics and breast cancer[END_REF]. In breast cancer, methylation patterns have been developed as biomarkers for early detection and subtype classification, as risk stratifiers, for monitoring prognosis, and as predictors of susceptibility or response to therapy [START_REF] Esteller | Epigenetics in cancer[END_REF][START_REF] Lo | Epigenomics and breast cancer[END_REF]. Recently, we reported that a defined methylated gene panel augments the accuracy of breast cancer detection in FNA washings [START_REF] Liu | Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer[END_REF]. Herein, we extended those observations and sought to determine whether quantitative promoter methylation of three gene loci included in the breast cancer detection panel might also carry prognostic information in addition to standard clinicopathologic parameters. These results could provide a supplementary ancillary tool to evaluate tumor aggressiveness and aid in therapeutic decision.

PATIENTS AND METHODS

Patients, sample collection, and DNA extraction

A total of 237 female patients with palpable suspicious breast lesions, consecutively submitted to FNA at the Portuguese Oncology Institute -Porto, Portugal, from 2002 to 2008, were enrolled in this study, following informed consent. Relevant clinical and pathological data was retrieved from the patient's clinical records. These studies were approved by the institutional review board (IRB) of Portuguese Oncology Institute -Porto.

FNA biopsy was performed pre-operatively using a 23-gauge needle attached to a 10-ml syringe and inserted into a syringe holder. The aspirates were smeared on microscope slides and routinely stained for cytopathological evaluation. Samples for methylation analysis were produced by washing the needle and syringe with 250 μl of phosphate buffer saline. The solution was spinned down, and the pellet was collected in a tube and stored at -80°C. DNA extraction was performed as previously described [START_REF] Jeronimo | Detection of gene promoter hypermethylation in fine needle washings from breast lesions[END_REF].

All the tumors were classified [START_REF] Page | Infiltrating carcinoma: major histological types[END_REF] and graded according to Bloom and Richardson's Modified system [START_REF] Elston | Assessment of histological grade[END_REF] by the same pathologist, and staged according to the AJCC system [START_REF] Greene | AJCC cancer staging manual[END_REF].

Bisulfite treatment and QMSP

This method allows for the assessment of the methylation status of individual CpG islands in genomic DNA. Sodium bisulfite conversion of unmethylated cytosine residues to uracil of genomic DNA obtained from FNA washings samples was performed as described previously [START_REF] Clark | High sensitivity mapping of methylated cytosines[END_REF]. The modified DNA was then used as a template for QMSP. Three promoters from genes involved in cell cycle regulation [Cyclin D2 (CCND2], apoptosis [Ras association domain family protein 1 (RASSF1A] and cell signaling [Adenomatous polyposis coli (APC)],

were assessed, using primers and probes described elsewhere [START_REF] Eads | MethyLight: a high-throughput assay to measure DNA methylation[END_REF][START_REF] Lehmann | Quantitative assessment of promoter hypermethylation during breast cancer development[END_REF].

To determine the relative levels of methylated promoter DNA in each sample (methylation level, ML), the values obtained for each target gene were compared with the values of the internal reference gene. The ratio was then multiplied by 1000 for easier tabulation ([ML = (target gene/ACTB) x 1000]. Fluorogenic QMSP assays were carried out in a reaction volume of 20 L in 96-well plates in an Applied Biosystems 7000 Sequence Detector (Perkin Elmer, Foster City, CA). PCR was performed in separate wells for each primer/probe set and each sample was run in triplicate. Additionally, multiple water blanks were used per plate, as previously detailed [START_REF] Jeronimo | Quantitative RARbeta2 hypermethylation: a promising prostate cancer marker[END_REF] and also a serial of dilutions of a positive control for constructing the corresponding calibration curve.

A given sample was considered positive when amplification was detected in at least two of the triplicates of the respective QMSP analysis. The QMSP threshold was determined adjusting the best fit of the slope and R2 based on the respective calibration curve.

Statistical analysis

The frequency of methylated and unmethylated cases, as well as the median and interquartile range of the methylation level for each gene in each group of tissue samples was determined. Methylation levels of the genes were expressed as continuous variables.

Values were analyzed using non-parametric tests, i.e., the chi-square test and the Mann-Whitney U test, to compare methylation frequencies and levels, respectively, between malignant and benign lesions. Receiver operator characteristic (ROC) curve analysis was used for each gene to determine the respective diagnostic performance, using the Area under the Curve [AUC, with 95% confidence interval (CI)]. This analysis was performed for all cases and also separately for premenopausal and postmenopausal cancer cases.

Histopathologic evaluation constituted the gold standard or reference test. Positivity for each methylated promoter was set as previously determined [START_REF] Liu | Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer[END_REF] and quantitative estimates of validity were determined.

The prognostic significance of clinical and pathological variables (age, tumor grade, pathological stage and hormone receptor status) was assessed by constructing overall, disease-specific and disease-free survival curves using the Kaplan-Meier method with log rank test (univariate test), and by a Cox-regression model comprising all variables (multivariate test). To test the prognostic significance of the methylation status for each gene, samples were categorized into two groups based on the methylation levels for that gene, using as threshold the value of the percentile 75 [START_REF] Henrique | High promoter methylation levels of APC predict poor prognosis in sextant biopsies from prostate cancer patients[END_REF]. Overall, disease-specific and diseasefree survival curves were then constructed based on each of the three genes (univariate analysis). A Cox-regression model comprising both clinical and epigenetic variables was then computed to assess the relative contribution of each variable to the assessment of follow-up status.

A P value smaller than 0.05 (two-sided) was considered to indicate statistical significance.

Statistical analyses were carried out using a computer-assisted program (SPSS, version 11.0, Chicago, IL).

RESULTS

Clinical and pathological data

We tested FNA washing samples from 237 suspicious breast lesions, 148 of which were cytopathologically diagnosed as malignant and 37 as benign. In the remaining 52 cases no definitive cytomorphological diagnosis was rendered (this category includes cases reported as "suspicious", "inconclusive", and "insufficient material").

Histopathological material for examination was available in 211 cases, comprising 178 malignant and 33 benign lesions. The relevant clinical and pathological characteristics of these patients are provided in Table 1. Of these 211 cases, 123 have been included in a previous study [START_REF] Liu | Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer[END_REF].

QMSP in breast cancer and non-cancerous breast tissues

QMSP was performed only in the 211 FNA washings in which a confirmatory histopathological diagnosis was available. The methylation frequencies and distribution of methylation levels are listed in Table 2. The frequency of promoter methylation was higher in malignant lesions for all genes, although a statistically significant difference was only observed for APC (P = 0.003). Breast cancers also displayed the highest methylation levels for all the analyzed genes (Table 2).

Performance of methylation markers in FNA washings

The diagnostic performance of the three genes was assessed using the cutoff values of methylation levels previously determined for each of these gene promoters (5.0 for APC, 2.0 for CCND2, and 50.0 for RASSF1A) [START_REF] Liu | Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer[END_REF]. ROC curve analysis allowed for the determination of the AUC (CI) for each gene: 0.74 (0.66-0.82) for APC, 0.76 (0.68-0.83) for CCND2, and 0.72 (0.63-0.81) for RASSF1A (Figure 1). Additionally, this analysis was performed independently for pre-and postmenopausal women (Figures 2A and2B, respectively). The AUC (CI) for each gene was: 0.73 (0.62-0.85) for APC, 0.69 (0.57-0.81) for CCND2, and 0.62 (0.49-0.76) for RASSF1A, for premenopausal women, and 0.82 (0.70-0.93) for APC, 0.86 (0.79-0.92) for CCND2, and 0.87 (0.81-0.94) for RASSF1A, for postmenopausal women.

Considering the above mentioned cutoff values for each gene, 123 (69%), 134 (75%), and 134 (75%) of malignant samples were positive for APC, CCND2, and RASSF1A, respectively, whereas 8 (24%), 14 (42%), and 13 (39%) of benign samples were positive for APC, CCND2, and RASSF1A, respectively. Validity and information estimates considering one, two or three positive markers are displayed in Table 3. The best balance between sensitivity and specificity was obtained with two positive markers (0.78 and 0.79, respectively).

Correlations between epigenetic data and clinicopathological parameters

Table 4 depicts the distribution of methylation levels for each gene according to standard clinicopathological parameters. No significant correlations were found between promoter methylation levels and patients' age, tumor grade or pathological stage. However, statistically significant differences were observed for RASSF1A and CCND2 methylation levels between estrogen receptor positive and estrogen receptor negative breast tumors (P = 0.003 and P < 0.001, respectively). Moreover, a statistically significant difference was observed for CCND2 methylation levels concerning progesterone receptor status (P = 0.011).

Survival analyses

The median follow-up of this series of breast cancer patients (n = 178) was 57.7 months (range: 0.5 to 90 months). A total of 19 patients (10.7 %) died from breast cancer during the follow-up period and 13 (7.3%) patients were lost to follow-up. For the purposes of survival analyses, all cases were coded based on gene methylation levels using as a threshold the value of percentile 75 for each gene. Moreover, grade 1 and 2 tumors were coupled in the same category, against grade 3 tumors. Among all clinical, pathological, and molecular variables analyzed, increased pathological stage, tumor grade, and high-methylation levels of RASSF1A were associated with worse overall survival in univariate analysis (P < 0.001, P = 0.018, and P = 0.040, respectively). Disease-specific survival curves using established clinical and pathological variables showed that advanced pathological stage and tumor grade were significantly associated with a worse outcome (P < 0.001 for both) (Figures 3 and4), whereas age, hormone receptor status and gene methylation levels did not show prognostic value within the available follow-up time.

Tumor recurrence was detected in 32 (18.0%) patients during the follow-up period. Advanced clinical stage, increased tumor grade, and high-methylation levels of RASSF1A (Figure 5)

were significantly associated with disease relapse in univariate analysis (P < 0.001, P < 0.001, and P = 0.004, respectively).

When clinical and epigenetic variables were introduced in a Cox-regression model for the prediction of relapse, pathological stage, tumor grade, and RASSF1A methylation levels were selected in the final step of the model as independent predictors (Table 5).

DISCUSSION

Cytological evaluation of suspicious breast lesions has been widely performed as an initial triaging procedure to identify malignant lesions and assist the clinician in setting the best strategy to obtain a definitive diagnosis and subsequent therapeutic decisions. However, cytomorphological assessment of breast FNA biopsy specimens meets with important limitations ranging from the cytopathologist's proficiency to the availability of representative material to render a definitive diagnosis. In previous studies, we demonstrated that FNA washings from suspicious breast lesions yield significant amounts of genomic DNA for methylation studies [START_REF] Jeronimo | Detection of gene promoter hypermethylation in fine needle washings from breast lesions[END_REF] and we confirmed the power of a small panel of methylation markers to identify malignant breast cells even in cases with low yield of cytological material, thus providing a valuable ancillary tool to routine cytomorphological observation [START_REF] Liu | Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer[END_REF]. In this study, we extended the spectrum of analysis of epigenetic markers in breast cancer, assessing the prognostic value of quantitative gene promoter methylation in a large series of breast cancer patients.

Overall, the population on which this study is based reflects the referral condition of a cancer institute. Indeed, benign lesions are less than 20% of all cases analyzed as most patients had been already triaged by the respective general physician based on clinical and imagiological information. Thus, most cases were highly suspicious of cancer and that condition was confirmed by FNA biopsy in the vast majority of cases. This finding highlights the usefulness of the FNA biopsy procedure, although in 22% (52 out of 237) of cases no definitive diagnosis was rendered based on cytomorphological evaluation.

The present series includes 123 of the cases previously reported by our research group [START_REF] Liu | Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer[END_REF] and it was extended with new consecutive cases, almost doubling the original series. This larger series of patients allowed us to perform a confirmatory test of the diagnostic performance of the small panel of methylation markers previously reported to augment the accuracy of FNA biopsy of breast lesions [START_REF] Liu | Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer[END_REF]. However, of the initial panel of four genes, only three loci were analyzed (APC, CCND2, and RASSF1A) owing to the scarcity of DNA available for each sample. Importantly, from our previous findings we concluded that two or three methylation markers would provide adequate ancillary information for breast cancer diagnosis in FNA biopsies [START_REF] Liu | Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer[END_REF]. In the present series, ROC curve analysis confirmed our previous findings concerning the diagnostic performance of individual methylation markers.

Interestingly, the validity estimates indicate that the best balance between sensitivity and specificity (around 80% for both) was obtained when two positive markers were used to identify a malignant lesion. It is noteworthy that we used the same cutoff values for gene methylation levels previously determined [START_REF] Liu | Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer[END_REF], a feature that provides additional validity to the present results.

The cancer specificity of our three gene panel is well demonstrated in the present study as the median levels of methylation at APC, CCND2, and RASSF1A promoters differed significantly between cancerous and non-cancerous samples, confirming our previous observations [START_REF] Liu | Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer[END_REF]. Importantly, these results are also in accordance with the findings of other researchers. Pu and co-workers reported on the ability of RARβ, RASSF1A, and CCND2 promoter methylation to identify malignancy in FNA samples with indeterminate cytological diagnosis [START_REF] Pu | Methylation profiling of benign and malignant breast lesions and its application to cytopathology[END_REF]. Moreover, aberrant methylation in at least one of a three gene panel which included RASSF1A, APC, and DAPK1 was positive in 76% of serum samples from breast cancer patients [START_REF] Dulaimi | Tumor suppressor gene promoter hypermethylation in serum of breast cancer patients[END_REF]. These studies confirmed the usefulness of epigenetic markers for early and accurate detection of breast cancer, in parallel with similar findings from our research group and others concerning prostate cancer [START_REF] Bardou | Progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases[END_REF].

The statistically significant age difference between patients carrying benign or malignant lesions might be one of the limitations of this study. However, there is a substantial overlap between the two groups (18 to 77 years vs. 29 to 92 years, respectively). Although associations between gene promoter methylation and age have been reported, they are not a universal finding and results are even conflicting concerning breast tissues. Whereas some researchers found age related variations for RASSF1A, with an odd pattern of increase from 32 to 55 years with a decline thereafter [START_REF] Euhus | DNA methylation in benign breast epithelium in relation to age and breast cancer risk[END_REF], other studies did not find such a correlation [START_REF] Van Der Auwera | Quantitative methylation profiling in tumor and matched morphologically normal tissues from breast cancer patients[END_REF].

Moreover, no correlation between age and APC or CCND2 promoter methylation has been reported thus far [START_REF] Euhus | DNA methylation in benign breast epithelium in relation to age and breast cancer risk[END_REF][START_REF] Jin | Adenomatous polyposis coli (APC) gene promoter hypermethylation in primary breast cancers[END_REF]. Finally, in the present study no association between age and methylation levels was apparent for any of the genes. Therefore, it is unlikely that age distribution is inducing significant bias in the performance of the epigenetic biomarkers.

The main novelty of this study, however, lies on the assessment of the prognostic value of methylation markers quantitatively determined in FNA washings from breast lesions. Indeed, to the best of our knowledge, this is the first study to demonstrate that high-methylation levels of the RASSF1A promoter (> p75) assessed in FNA washings is an independent predictor of poor outcome in breast cancer patients. The cutoff value (p75) was based in our previous studies in prostate cancer which demonstrated that high-methylation levels of APC were independent predictors of poor outcome [START_REF] Henrique | High promoter methylation levels of APC predict poor prognosis in sextant biopsies from prostate cancer patients[END_REF]. The biological rationale for the use of this cutoff is based on the association between promoter methylation (evaluated in the present study) and gene expression, as this depends on the density of CpG dinucleotide methylation. Thus, it is expectable that cases with the highest levels of methylation (e.g., > p75) will have the lowest levels of mRNA expression, and, thus, gene silencing will be more effective in this subgroup. Consequently, the loss of RASSF1A function will likely increment tumor aggressiveness and might justify the poorer prognosis. Remarkably, RASSF1A promoter methylation has been previously identified as a potential prognostic marker for breast cancer in different types of clinical samples. Indeed, Muller and co-workers reported that RASSF1A promoter methylation detected in sera or plasma from patients with primary or metastatic breast cancer was associated with poor outcome [START_REF] Muller | DNA methylation in serum of breast cancer patients: an independent prognostic marker[END_REF]. Following the same line of evidence, Hoque and co-workers found that RASSF1A promoter methylation was more frequent in advanced stage breast cancer patients [START_REF] Hoque | Detection of aberrant methylation of four genes in plasma DNA for the detection of breast cancer[END_REF]. Interestingly, RASSF1A promoter methylation seems to be one of the earliest epigenetic alterations in breast carcinogenesis as it has been found even in benign, yet atypical, breast lesions and carcinoma in situ [START_REF] Lehmann | Quantitative assessment of promoter hypermethylation during breast cancer development[END_REF]. Thus, it would be tempting to speculate whether those lesions with higher RASSF1A methylation levels would be more prone to progress to invasive cancer.

The only clinicopathological parameters that surfaced as independent predictors of outcome in the present series were pathological stage and tumor grade, whereas hormone receptor status did not. This was a somewhat unexpected result as the expression of estrogen and/or progesterone receptor is associated with favorable prognosis and it is highly predictive of response to endocrine treatment [START_REF] Bardou | Progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases[END_REF]. Because no selection bias was apparent in our series, this lack of prognostic value for hormone receptor status might be due to insufficient followup time. We also did not assess HER2 status in the present series as a significant number of cases were collected prior to the implementation of routine HER2 assessment in breast cancer at our institution and, thus, that information was not available in many cases.

Nonetheless, it is noteworthy that a molecular assay (quantitative RASSF1A promoter methylation) performed in an exiguous sample of cancer cells obtained by FNA was able not only to discriminate malignant from benign lesions, but also to convey relevant prognostic information when compared with standard parameters which require extensive tissue sampling and expert observation.

Further studies addressing the development of predictive models for pre-operative staging and therapy response based on epigenetic biomarkers might also provide valuable tools for breast cancer patient management.

Figure 1 -Figure 2 -

 12 Figure 1 -Receiver Operator Characteristic (ROC) curve for each individual gene (APC, RASSF1A and CCND2) in FNA washings from breast lesions

Figure 3 -

 3 Figure 3 -Disease-specific survival curve based on pathological stage in FNA washings from the 165 breast cancer patients.

Figure 4 -

 4 Figure 4 -Disease-specific survival curve based on tumor grade in FNA washings from the

Figure 5 -

 5 Figure 5 -Disease-free survival curve based on RASSF1A high-methylation levels in FNA washings from the 165 breast cancer patients. Abbreviations: P75, percentile 75 of promoter methylation level.

Table 1 -

 1 Clinical and pathological characteristics of patient's populations

	Malignant	Benign

Table 2 -

 2 Frequency of positive cases [n (%)] and distribution of methylation levels of cancerrelated genes [gene/ACTBx1000:median (IQR)]

			Benign	Malignant
	Gene	n (%)	Median (IQR)	n (%)	Median (IQR)	P value*
	APC	18 (55%)	0.12 (0-25.8)	144 (81%) 86.85 (1.41-723.78)	<0.001
	CCND2	22 (67%)	1.30 (0-11.0)	147 (83%) 86.77 (3.41-474.46)	<0.001
	RASSF1A 24 (73%) 14.49 (0-177.59) 153 (86%) 482.50 (52-2125.33)	<0.001
	IQR, interquartile range
	Table 3 -Validity estimates for increasing number of positive tested markers in the validation
	dataset of fine-needle aspirate washings from breast lesions (Se: sensitivity; Sp: specificity,
	LR+: positive likelihood ratio, PV+: positive predictive value, LR-: negative likelihood ratio,
	PV-: negative predictive value, AUC. Area under curve, α: estimated added information).
			Number of markers with positive result in each case
	Validity estimates	1	2	3

Table 4 -

 4 Distribution of gene promoter methylation levels and frequencies according to clinical and pathological parameters in breast cancers

APC CCND2 RASSF1A median (IQR) n (%) median (IQR) n (%) median (IQR) n (%)

				559.14)
		240.56	49	38.41 (0.72-	47	354.14 (14.19-	48
	3 (n = 57)	(3.81-	(86%)	501.99)	(82%)	2141.44)	(84%)
		892.83)
	Stage
		18.95 (0.01-	31	206.47	15	408.58 (108.28-	39
	I (n = 41)	403.29)	(76%)	(24.07-	(85%)	2341.60)	(95%)
				658.15)
		115.38	73	85.07 (1.19-	73	469.63 (24.44-	73
	II (n = 90)	(1.41-	(81%)	501.0)	(81%)	1839.15)	(81%)
		690.13)
		331.05	31	50.91 (1.34-	29	744.69 (183.31-	30
	III (n = 35)	(7.59-	(89%)	380.59)	(83%)	2695.73)	(86%)
		1089.66)
		778.89	3	120.45	3	1827.79	3
	IV (n = 4)	(0.75-	(75%)	(49.73-	(75%)	(538.34-	(75%)
		3045.21)		197.75)		3945.27)
	Estrogen receptor
	status
		140.28	118	191.28	122	622.98 (77.49-	128
	ER + (n = 142)	(1.55-	(83%)	(19.22-	(86%)	2566.22)***	(90%)
		875.94)		548.39)*
	ER -(n = 33)	8.08 (0-368.48)	24 (73%)	3.92 (0-67.07)*	24 (73%)	32.67 (0-1693.97)***	22 (67%)
	Progesterone
	receptor status
		110.12	91	180.36	97	479.49 (108.96-	99
	Age PgR + (n = 113)	(1.21-	(80%)	(18.26-	(86%)	2492.85)	(88%)
	< 62 yrs. PgR -(n = 62)	172.23 (7.01-817.12) 702.11) 64.0 (1.46-859.58)	75 (84%) 50 (82%)	138.32 (1.62-501.99) 522.07)** 33.94 (0.50-256.58)**	73 (82%) 48 (79%)	516.0 (52.45-1843.31) 624.29 (26.14-2141.44)	76 (85%) 50 (82%)
	702.11) 12.50 (0.09-IQR: interquartile range (p25-p75); Statistically significant differences: * P < 0.001; **P = 0.011; ***P = (77%) 440.20) (83%) 2618.30) (86%) ≥ 62 yrs. 69 50.11 (4.33-74 459.78 (51.92-77 0.003
	Grade
		41.33 (0-	33	167.66	28	462.52 (46.78-	25
	1 (n = 31)	465.0)	(74%)	(16.34-	(84%)	1814.36)	(81%)
				449.81)
	2 (n = 84)	54.74 (1.24-947.43)	68 (81%)	157.80 (11.50-	71 (84%)	634.0 (147.29-2599.72)	75 (89%)

Table 5 -

 5 Cox regression models assessing the potential of clinical and epigenetic variables in the prediction of overall survival, disease-specific or disease-free survival for 165 breast cancer patients

	Model Tested	Variables	Odds Ratio (OR) 95% CI* for OR	p

Acknowledgements:

V.L.C is supported by a grant from Fundação para a Ciência e a Tecnologia (SFRH/BD/23374/2005).

This study was funded by grants from Liga Portuguesa contra o Cancro -Núcleo Regional do Norte and the Comissão de Fomento da Investigação em Cuidados de Saúde -Ministério da Saúde (Project no. 21/2007).


Bruno M Simões

Marco Piva

Oihana Iriondo

M d.M Valentine Comaills

Jose A López-Ruiz

Iñaki Zabalza

Jon A Mieza

Olga Acinas

Maria D M Vivanco
email: mdmvivanco@cicbiogune.es

Vivanco

Estrogen affects the proportion of stem cells in the breast

Keywords: Estrogen, mammary gland, stem cells, breast cancer

There is increasing evidence that breast cancers contain tumor-initiating cells with stem cell properties. The importance of estrogen in the development of the mammary gland and in breast cancer is well known, but the influence of estrogen on the stem cell population has not been assessed. We show that estrogen reduces the proportion of stem cells in the normal human mammary gland and in breast cancer cells. The embryonic stem cell genes NANOG, OCT4 and SOX2 are expressed in normal breast stem cells and at higher levels in breast tumor cells and their expression decreases upon differentiation. Overexpression of each stem cell gene reduces estrogen receptor expression, and increases the number of stem cells and their capacity for invasion, properties associated with tumorigenesis and poor prognosis. These results indicate that estrogen reduces the size of the human breast stem cell pool and may provide an explanation for the better prognosis of estrogen receptorpositive tumors.

Introduction

The adult mammary epithelium consists of a bilayered structure with a central lumen, an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells. Estrogen is essential to normal mammary gland development, where it is responsible for epithelial cell proliferation and differentiation of healthy breast epithelium [START_REF] Bocchinfuso | Induction of mammary gland development in estrogen receptor-alpha knockout mice[END_REF]. In addition to its fundamental role in the normal physiology of the mammary gland, exposure to estrogen is an important determinant of risk for breast cancer. The estrogen receptor (ER) belongs to the family of steroid and nuclear hormone receptors, transcription factors that bind to DNA and regulate transcription of target genes in response to ligand binding. Tamoxifen, an antagonist of ER, is widely used as hormonal therapy in the treatment of ERpositive breast cancer, although very frequently tamoxifen resistance develops [START_REF] Ali | Endocrine-responsive breast cancer and strategies for combating resistance[END_REF]. Approximately three out of four cases of breast cancer express ER [START_REF] Ali | Endocrine-responsive breast cancer and strategies for combating resistance[END_REF], which is highly predictive of the clinical response to hormonal therapy. ER-positive tumors have a better prognosis than other types of breast tumors in terms of overall survival, while ER-negative tumors have a more aggressive phenotype [START_REF] Sotiriou | Breast cancer classification and prognosis based on gene expression profiles from a population-based study[END_REF][START_REF] Van De Vijver | A gene-expression signature as a predictor of survival in breast cancer[END_REF][START_REF] Sorlie | Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF].

The tissue expansion and remodeling that occurs in the mammary gland during the multiple cycles of pregnancy, lactation and involution throughout a woman's lifetime suggest that there are stem cells and early progenitor cells in the adult mammary gland [START_REF] Dontu | Breast cancer, stem/progenitor cells and the estrogen receptor[END_REF]. Adult stem cells are characterized by their self-renewal capacity, which allows the maintenance of the stem cell pool, and the ability to differentiate into the cell types of the tissue in which they reside, luminal and myoepithelial cells in the case of the mammary gland. Evidence has accumulated during the last several years supporting the existence of stem cells both in the mouse and human mammary gland [START_REF] Lamarca | Minireview: Hormones and mammary cell fate--what will i become when i grow up?[END_REF]. Different strategies have been used to identify and isolate human breast stem/progenitor cells including FACS sorting for the expression of cell surface antigens. For example, EMA + CALLA + and EMA -CALLA -cells can generate mixed colonies from a single cell [START_REF] Clayton | Growth and differentiation of progenitor/stem cells derived from the human mammary gland[END_REF]. In addition, an in vitro cell culture system has been described that allows the propagation of human mammary epithelial cells in an undifferentiated state based on their ability to proliferate in suspension as non-adherent mammospheres [START_REF] Dontu | In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells[END_REF].

The in vitro propagation of breast cancer-initiating cells as mammospheres from breast cancer lesions and cell lines has also been reported [START_REF] Ponti | Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties[END_REF][START_REF] Farnie | Novel cell culture technique for primary ductal carcinoma in situ: Role of notch and epidermal growth factor receptor signaling pathways[END_REF]. Compelling data supporting the existence of breast cancer stem cells showed that cells isolated as Lin-CD44 + CD24 -/low were capable of forming tumors in mice with higher efficiency than cells with alternate phenotypes [START_REF] Al-Hajj | Prospective identification of tumorigenic breast cancer cells[END_REF]. Interestingly, Shipitsin and collaborators showed that the gene expression profile of CD44 + cells resembles that of stem cells, and normal and tumor CD44 + cells are more similar to each other than to CD24 + cells from the same tissue [START_REF] Shipitsin | Molecular definition of breast tumor heterogeneity[END_REF]. Another fruitful approach identified aldehyde dehydrogenase activity (ALDH + ) as a shared common functional marker for both normal and malignant human mammary stem cells [START_REF] Ginestier | Aldh1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome[END_REF]. The identification of markers shared by both normal and malignant mammary stem cells lends support to the cancer stem cell hypothesis.

Recent studies suggest that cancer cells share molecular signatures that are similar to those of pluripotent embryonic stem (ES) cells [START_REF] Wong | Module map of stem cell genes guides creation of epithelial cancer stem cells[END_REF]. The regulatory networks controlling the function of ES cells, including key regulators of ES cell maintenance, namely Nanog, Oct4 and Sox2 [START_REF] Niwa | How is pluripotency determined and maintained?[END_REF][START_REF] Surani | Genetic and epigenetic regulators of pluripotency[END_REF], are also found in some adult stem cell populations [START_REF] Riekstina | Embryonic stem cell marker expression pattern in human mesenchymal stem cells derived from bone marrow, adipose tissue, heart and dermis[END_REF]19]. Moreover, genes associated with ES cell identity have been linked to tumor histology, supporting the possibility that these genes contribute to the stem cell-like phenotype of many tumors [START_REF] Ben-Porath | An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors[END_REF].

The proliferative role of estrogen in breast cancer cell lines and in ER-positive tumors is well established. Nevertheless, generally ER-positive tumors present a better prognosis. Presently, little is known about estrogen signaling in normal mammary epithelial cells and, particularly, in stem/progenitor cells [START_REF] Lamarca | Minireview: Hormones and mammary cell fate--what will i become when i grow up?[END_REF]. Therefore, we wished to investigate the effects of estrogen on the stem/progenitor cell population, both in normal breast and in breast cancer. To this end, we used the expression levels of embryonic stem cells transcription factors Nanog, Oct4 and Sox2 to monitor the differentiation status of breast stem cells in the presence of either estrogen or tamoxifen and then examined the outcome of changing the expression levels of these three factors on the stem cell phenotype. We found that estrogen induces the breast stem cells to differentiate, which may provide an explanation for the better prognosis of ER-positive breast tumors.

Materials and methods

Isolation of human breast epithelial cells

Normal breast tissue (n = 14) was obtained from women (average age 39) undergoing reduction mammoplasty with no previous history of breast cancer. Tumor samples were obtained from core biopsies (n = 8) or from women who underwent therapeutic surgery (n = 3). For histological information, please refer to Online Resource (Table 1). In all cases the samples were reviewed by a consultant breast pathologist. All patients provided written informed consent, and the procedures were approved by the local Hospital Research Ethics Committee and the 'Ethics Committee of Clinical Investigation of Euskadi'. The breast tissue was immediately processed as previously described [START_REF] Clayton | Growth and differentiation of progenitor/stem cells derived from the human mammary gland[END_REF].

For further details please see Online Resource.

Mammosphere culture

Breast epithelial cells were plated in 75 cm 2 ultralow attachment flasks (Corning) at a density of 10.000 cells/cm 2 in primary culture and 5.000 cells/cm 2 in passages. Human breast cancer cell lines MCF-7 and T47D were plated at a density of 2.500 cells/cm 2 in initial cultures and 1.000 cells/cm 2 during passage. All cells were grown and processed as described in [START_REF] Dontu | In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells[END_REF]. Experiments using primary epithelial cells treated with hormones were performed using samples from pre-menopausal women (n = 9). Please see Online Resources for further information (also for adherent cell culture).

Invasion assay

In vitro invasion and chemotaxis assays were performed in a 24-well BD Falcon TM HTS Multiwell Insert System containing an 8 m pore size PET (PolyEthylene Terepthalate) membrane. For invasion assays, the top of the upper wells was coated with 2 g of Matrigel Basement Membrane Matrix (BD) diluted in 50 l of DMEM:F-12 medium and allowed to air-dry overnight. The following day the Matrigel was re-hydrated and 100,000 cells, previously starved in serum-free medium for 24 h were added to the upper well. The lower well was filled with 300 l of chemoattractive medium containing 20% FBS and 300 l of MCF-7 cell-conditioned medium (0.45 m filtered). Medium alone was used as a negative control. After 72 h, cells on the upper surface of the membrane were removed by wiping with a cotton swab, and the cells remaining on the lower side of the membrane were fixed and stained with crystal violet. For chemotaxis assays the same procedure was followed but the upper wells were not coated with Matrigel. At least nine different fields from each well were counted to determine the number of invading cells. The percentage of invasion was calculated by dividing the mean number of cells that invaded through the Matrigel with those that migrated through the control.

Real-time polymerase chain reaction (PCR)

Total RNA was isolated using TRIzol (Invitrogen). When the cell number was lower than 0.5 x 10 6 cells the RNeasy Micro Kit (QIAGEN) was used. In all cases, RNA extraction was according to the instructions of the manufacturer. Real-time PCR was performed on a 7300 Real-Time PCR System (Applied Biosystems), using the iTaq™ SYBR ® Green Supermix with ROX (BioRad). cDNA was amplified using the following conditions: 95° C for 5 min, 40 cycles of amplification -95ºC for 15 sec, 55-65ºC for 1 min (annealing temperatures, AT, depending on the primers) -and a dissociation stage. 36B4 was used as a reference transcript for normalization. cDNA from the embryonal carcinoma cell line NTera2/D1, which expresses the stem cell genes NANOG, OCT4 and SOX2, was used as a positive control. The sequences of the primers used can be found as Online Resource.

Transcription assays and western blotting

The details for the transcription and western blotting assays, performed as in [START_REF] Gascoyne | Loss of mitotic spindle checkpoint activity predisposes to chromosomal instability at early stages of fibrosarcoma development[END_REF], can be found as Online Resource.

Lentivirus production

Lentivirus were produced by triple transfection of 293FT cells with the second generation packaging vector psPAX2 (Addgene plasmid 12260, deposited by Dr Didier Trono), the pMD.G vector (kind gift of Dr James Sutherland) encoding the envelope vesicular stomatitis virus glycoprotein (VSV-G), and either the pSin-EF2-Nanog-Pur, the pSin-EF2-Oct4-Pur or the pSin-EF2-Sox2-Pur vectors (Addgene plasmids 16578, 16579 and 16577, respectively, deposited by Dr James A Thomson). pSin-EF2-EGFP-Pur was used as control and obtained by replacing the Oct4 gene from the pSin-EF2-Oct4-Pur with an EGFP coding sequence. DNA plasmids were transfected and viral supernatant was collected every 24 hours for three days. The supernatant was filtered and concentrated 30-fold by ultracentrifugation. For lentiviral transductions, the concentrated supernatant containing polybrene (8 µg/ml final concentration, Sigma) was added to the cells at a 1:1 ratio. Cells were then incubated for 48 h prior to selection with 0.5-2 µg/ml of puromycin (Sigma).

Immunofluorescence

Cells grown on slides were fixed with 4% paraformaldehyde (Sigma), permeabilized with PBS 0.3% Triton-X-100 and blocked with 8% FBS in PBT (PBS containing 0.05% Tween-20). Cells were then incubated for 1 hour with one of the following primary antibodies: goat anti-NANOG (R&D Systems, AF1997), mouse anti-OCT3/4 (C-10, Santa Cruz, sc-5279) and goat anti-SOX2 (Y-17, Santa Cruz, sc-17320); and then with anti-goat alexa 568 (Molecular Probes, A11057) or anti-mouse alexa 647 (Molecular Probes, A31571) secondary antibodies. Finally, slides were mounted in Vectashield with DAPI (Vector) and visualized on the Leica confocal microscope.

Fluorescent activated cell sorting (FACS)

Human epithelial membrane antigen (EMA) and common acute lymphoblastic leukaemia antigen (CALLA) labeling was performed essentially as previously described [START_REF] Clayton | Growth and differentiation of progenitor/stem cells derived from the human mammary gland[END_REF]. For details please refer to Online Resource. The directly conjugated mouse PE anti-CD24 antibody (BD, clone ML5) and the directly conjugated mouse allophycocyanin (APC) anti-CD44 antibody (BD, G44-26) were used to label CD24 and CD44. Control samples were stained with isotype-matched control antibodies, PE conjugated non-specific mouse IgG2a,κ antibody (BD) and APC conjugated non-specific mouse IgG2b,κ antibody (BD) for CD24 and CD44, respectively. The viability dye 7-aminoactinomycin D (7AAD, BD) was added for dead cell exclusion.

Aldefluor assay

To measure cells with ALDH activity, the Aldefluor assay was carried out according to manufacturer's (Stemcell Technologies) guidelines. Briefly, dissociated single cells were suspended in Aldefluor assay buffer containing an ALDH substrate, bodipyaminoacetaldehyde (BAAA) at 1.5 mM, and incubated for 45 min at 37ºC. To distinguish between ALDH-positive and -negative cells, a fraction of cells was incubated under identical conditions in the presence of a 2-fold molar excess of the ALDH inhibitor, diethylaminobenzaldehyde (DEAB).

Results

Estrogen reduces the pool of stem/progenitor cells in the normal mammary gland

We wished to investigate whether the pluripotent factors Nanog, Oct4 and Sox2 were differentially expressed in the human mammary gland. To this end we assessed the expression of NANOG, OCT4 and SOX2 mRNAs in freshly isolated organoids from reduction mammoplasties, in breast epithelial cells derived from the organoids and grown as adherent cells in the presence of serum, and in mammospheres originating from single breast epithelial cells. We observed that, with respect to the expression in freshly isolated organoids, the expression of all three pluripotency genes was lower in the differentiated (adherent) cells and it was significantly higher in mammospheres (Fig. 1a).

Furthermore, when mammospheres were plated under differentiating conditions (with serum) there was a strong reduction in the expression of NANOG, OCT4 and SOX2 (Fig. 1b). In order to investigate the potential roles of estrogen and tamoxifen in cell-fate determination, we assessed the expression of NANOG, OCT4 and SOX2 in mammosphere cultures under different hormonal conditions. Progesterone receptor (PR, a known ER target) was induced in response to estrogen treatment of freshly isolated breast epithelial cells, indicating the presence of transcriptionally active ER (Fig. 1c). Naturally, the induction of PR by estrogen in the primary cells was more modest than that observed in ER-positive breast cancer cell lines since the expression of ER is lower [START_REF] Ricketts | Estrogen and progesterone receptors in the normal female breast[END_REF] and steroid hormone receptors in primary cells display lower transcriptional activity than in cancer cells [START_REF] Vivanco | A transition in transcriptional activation by the glucocorticoid and retinoic acid receptors at the tumor stage of dermal fibrosarcoma development[END_REF]. Nevertheless, estrogen treatment significantly reduced the expression of NANOG, OCT4 and SOX2 (Fig. 1d).

To confirm that culture of primary mammary epithelial cells as mammospheres enriches for stem/progenitor cells, the percentage of EMA + CALLA + was assessed in cells grown in suspension at very low density. As we previously reported, freshly isolated EMA + CALLA + (DP) cells from the mammary gland represent 0.5-1.4% of the total cell population [START_REF] Clayton | Growth and differentiation of progenitor/stem cells derived from the human mammary gland[END_REF]. This population of stem/progenitor cells was increased up to 5-fold in suspension cultures (Fig. 2a). Interestingly, estrogen treatment reduced the percentage of DP cells present in the mammospheres, while tamoxifen increased it with respect to the ethanol control (Fig. 2b). Furthermore, the expression levels of NANOG, OCT4 and SOX2 were strongly increased in the progenitor DP population when compared with the differentiated EMA + and CALLA + cell populations (Fig. 2c). These observations suggest that the stem cells proliferate more in the absence of estrogen or that estrogen induces differentiation of the progenitor cells and as a result depletes the pool of stem/progenitor cells.

Expression of NANOG, OCT4 and SOX2 in normal and tumor samples

The results above suggest that the pluripotency genes NANOG, OCT4 and SOX2 might be markers of stem/progenitor cells in the normal mammary gland. Next, we wished to assess whether this was also the case for breast tumors. To this end we compared NANOG and SOX2 expression in tumor samples and adjacent normal tissue. NANOG and SOX2 were more highly expressed in pathological samples (8/11 cases, Table 1 in Online Resource for Sox2 values) than in the normal counterpart samples (Fig. 3a). Furthermore, although material was too scarce to assess expression of the three markers, we could observe Sox2 protein expression in the tumor but not in the normal sample from the same patient (Fig. 3a, right). To monitor the effects of estrogen on the subpopulation of cancer stem/progenitor cells we examined the expression of NANOG and SOX2 in breast tumor samples from which individual cells had been isolated and grown in suspension for 6 days in the presence of ethanol, estrogen or tamoxifen. Quantification by real-time PCR showed that estrogen treatment strongly reduced the expression of NANOG and SOX2 in mammospheres, while tamoxifen increased their expression (Fig. 3b). We also determined the changes in NANOG, OCT4 and SOX2 expression in the CD44 + CD24 -/low population of breast cancer stem/progenitor cells. Interestingly, the expression of all three genes was significantly increased in the stem/progenitor CD44 + CD24 -/low subpopulation with respect to the expression in the rest of the tumor cell population (Fig. 3c). These results reinforce the value of these transcription factors as breast stem/progenitor cell markers both in normal and tumor samples.

Effects of estrogen and tamoxifen on mammosphere formation by MCF-7 breast cancer cells

To investigate further the potential role of Nanog, Oct4 and Sox2 in the regulation of breast stem cells, we used the ER-positive breast cancer cell line MCF-7. We plated MCF-7 cells on ultralow attachment plates at low density in the presence of estrogen, tamoxifen or ethanol. The number of mammospheres formed was highest in the presence of tamoxifen whereas estrogen treatment resulted in the formation of fewer, larger mammospheres (Fig. 4a). To ensure that the mammospheres formed were clonal in origin, tertiary mammospheres grown in the presence of hormones were dissociated and single cells were FACS sorted into ultralow attachment 96-well plates at a density of one cell per well and allowed to form mammospheres in the presence of the corresponding hormones. Estrogen reduced the frequency of mammosphere formation 6-fold compared with control cultures, while there was an increase induced by tamoxifen that was small but significant (Fig. 4b). Mammospheres formed in the presence of estrogen were larger than those with the other treatments, suggesting increased proliferation of the progeny of mammosphere-initiating cells, consistent with the known proliferative effect of estrogen on differentiated MCF-7 cells (Fig. 4c). In conclusion, estrogen inhibits the selfrenewal capacity of stem/progenitor cells within MCF-7 mammospheres, as measured by their capacity to be serially passaged.

NANOG, OCT4 and SOX2 expression reflects the differentiation state of MCF-7 cells

Next, we determined whether the expression of the pluripotency factors also mirrored the changes in the differentiation state of MCF-7 cells. We observed by quantitative PCR that the expression of NANOG and SOX2 was approximately 10-fold higher in suspension cultures than in adherent cultures, while the increase in OCT4 expression was small but significant (Fig. 5a). The high expression of these factors was maintained in suspension cultures serially passaged as many as 20 times (data not shown). Estrogen reduced the expression of NANOG, OCT4 and SOX2 in mammospheres, while tamoxifen increased it (Fig. 5b). Furthermore, when we plated tertiary mammospheres on normal tissue culture plates in the presence of serum (differentiation conditions), the expression of NANOG, OCT4 and SOX2 was strongly reduced (Fig. 5b). These observations suggest that the expression of NANOG, OCT4 and SOX2 is indicative of the self-renewal capacity of breast stem/progenitor cells and that estrogen induces these cells to mature and proliferate. The same results were observed using another breast cancer cell line that expresses ER, T47D (Online Resource 1). Therefore, these observations are not limited to MCF-7 cells, but rather a more general phenomenon in ER-positive breast cancer cell lines.

Overexpression of Nanog, Oct4 and Sox2 increases the stem/progenitor cell population

In order to investigate the relevance of Nanog, Oct4 and Sox2 in the maintenance of breast stem cell properties, we overexpressed each of these transcription factors in MCF-7 cells using lentivirus. The efficient expression of the three factors was confirmed by immunofluorescence (Fig. 6a). It has been shown that Nanog, Oct4 and Sox2 form autoregulatory and feedforward loops [START_REF] Boyer | Core transcriptional regulatory circuitry in human embryonic stem cells[END_REF] and, in agreement with this, we found that the overexpression of one of these factors increased the expression of each of the others (Fig. 6b). To monitor the effects of these factors on MCF-7 self-renewal, we tested the capacity of the cell lines to form mammospheres from cells plated at very low density. Nanog and Sox2 overexpressing cells formed mammospheres with higher efficiency than control cells and the mammospheres formed were smaller than those formed by control cells and had irregular shapes (Fig. 6c and6d), suggesting that their ability to self-renew was increased but their ability to proliferate and/or differentiate was compromised. This was not the case for Oct4, possibly because Oct4 protein expression was only detectable in approximately 20% of the cell population (Fig. 6a).

We, and others, have previously reported the lack or low expression of ER in breast stem/progenitor cells [START_REF] Clayton | Growth and differentiation of progenitor/stem cells derived from the human mammary gland[END_REF][START_REF] Kok | Mammosphere-derived gene set predicts outcome in patients with erpositive breast cancer[END_REF][START_REF] Lim | Aberrant luminal progenitors as the candidate target population for basal tumor development in brca1 mutation carriers[END_REF]. Here, we observed that MCF-7 cells overexpressing Nanog or Sox2 had reduced levels of ER expression, and this was even more evident when the cells were grown as mammospheres (Fig. 7a). Furthermore, expression of endogenous PR was also lower in cells overexpressing each of these factors (Fig. 7b). In addition, transient transfection assays showed that estrogen activation of ER-dependent transcription was reduced in cells expressing each of the pluripotency genes, most notably in Sox2-expressing cells (Fig. 7c), indicating that ER, although present at lower levels, is still transcriptionally active.

We next determined whether ectopic expression of Nanog, Oct4 and Sox2 affected the stem/progenitor cells as defined by different phenotypes. Increased expression of NANOG, OCT4 and SOX2 considerably increased the presence of the CD44 + CD24 -/low cell subpopulation (Fig. 8a), as well as the DP (EMA + CALLA + ) (Fig. 8b) and ALDH + (Fig. 8d) cell populations, most notably when cells were grown as mammospheres (Online Resource 2). In addition, treatment of the overexpressing cells with estrogen strongly reduced the percentage of stem/progenitor cells, while tamoxifen significantly increased it (Fig. 8c). Moreover, the overexpression of NANOG, OCT4 and SOX2 was associated with a remarkable increase in cellular invasion (Fig. 8e). These results indicate that Nanog, Oct4 and Sox2 are involved in the maintenance of the stem/progenitor cell population in breast cancer cells and that estrogen reduces the proportion of stem/progenitor cells.

Discussion

Here we demonstrate that stem cells isolated from both normal human breast and breast tumor cells display increased expression of the embryonic stem cell genes NANOG, OCT4 and SOX2. Moreover, we show that ectopic expression of any one of these factors, but in particular Nanog and Sox2, in breast cancer cells increases the pool of stem cells, the ability of the cells to form mammospheres and their capacity for invasion. The importance of estrogen and its receptor as negative regulators of the stem cell population is demonstrated by the fact that ER is expressed at low levels in stem cells and that estrogen inhibits expression of stem cell genes and mammosphere formation.

Our results suggest that ES, normal breast and breast cancer cells use the same genes to maintain a stem cell population. However, the expression of NANOG, OCT4 and SOX2 in tumors is higher than in normal cells. Several studies indicate that Nanog, Oct4 and Sox2 function as core transcription factors in the ES cell gene regulatory network [START_REF] Niwa | How is pluripotency determined and maintained?[END_REF][START_REF] Boyer | Core transcriptional regulatory circuitry in human embryonic stem cells[END_REF], and this is underlined by the essential role that these factors play in the induction of pluripotent stem cells from differentiated cells [START_REF] Okita | Generation of germline-competent induced pluripotent stem cells[END_REF][START_REF] Wernig | In vitro reprogramming of fibroblasts into a pluripotent es-cell-like state[END_REF][START_REF] Takahashi | Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[END_REF]. It has been proposed that the function of this network is to prevent the stabilization of differentiation signals that exist in the environment or that might be intrinsic to stem cells [START_REF] Kalmar | Regulated fluctuations in nanog expression mediate cell fate decisions in embryonic stem cells[END_REF]. We report here that one such signal comes from estrogen, which downregulates NANOG, OCT4 and SOX2 expression. Thus, by inhibiting the effects of estrogen, tamoxifen might prevent tumor stem cell differentiation leading to acquired resistance and the formation of more aggressive tumors in some cases. However, it is clear that ER-positive tumors do not represent a single entity and further molecular characterization would be helpful [START_REF] Badve | Oestrogen-receptor-positive breast cancer: Towards bridging histopathological and molecular classifications[END_REF].

Based on our results, it seems reasonable to hypothesize that ER-positive tumors will be found to express lower levels of NANOG, OCT4 and/or SOX2 and that this will be associated with a more favorable prognosis. We did not observe a clear association between the ER status and the expression levels of NANOG, OCT4 and SOX2, but this might be because of the small sample size and the unusual proportion of ER-positive and HER2-positive tumors in the sample analyzed. Analysis of a substantially larger number of samples is warranted to determine the potential of stem cell genes as markers of aggressiveness. In fact, high-grade ER-negative breast tumors have been reported to express an ES-like gene signature and activation of targets of Nanog, Oct4 and Sox2 is associated with aggressive tumor behavior [START_REF] Ben-Porath | An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors[END_REF]. Furthermore, there is precedence for expression of stem cell genes in tumors. Human gliomas, for example, express some embryonic stem cell-specific genes [START_REF] Clement | Hedgehoggli1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity[END_REF],

Sox2 is preferentially expressed in breast tumors with a basal-like phenotype [START_REF] Rodriguez-Pinilla | Sox2: A possible driver of the basal-like phenotype in sporadic breast cancer[END_REF] and a core embryonic transcriptional profile that includes OCT4 and NANOG was identified in stage 3 breast carcinomas [START_REF] Ezeh | Human embryonic stem cell genes oct4, nanog, stellar, and gdf3 are expressed in both seminoma and breast carcinoma[END_REF].

We observed that overexpression of any one of the stem cell factors (Nanog, Oct4 and Sox2) increased the capacity of MCF-7 cells to form mammospheres, as well as the size of the stem cell pool and invasiveness. These results are consistent with other reports on the roles of Nanog and Sox2 in breast tumorigenesis [START_REF] Jeter | Functional evidence that the self-renewal gene nanog regulates human tumor development[END_REF][START_REF] Chen | The molecular mechanism governing the oncogenic potential of sox2 in breast cancer[END_REF]. This suggests that high expression of NANOG, OCT4 and SOX2 is associated with a breast cancer stem cell phenotype and that targeting any one of these factors might provide an improved therapy for poorly differentiated tumors. Our results also suggest that mutations or epigenetic changes that lead to higher expression of either Nanog, Oct4 and/or Sox2 could contribute to the development of tumors with a cancer stem cell phenotype.

Previous studies demonstrated that breast cancer patients whose tumors displayed a CD44 + CD24 - /low phenotype have a poor prognosis, with shorter metastasis-free and overall survival [START_REF] Liu | The prognostic role of a gene signature from tumorigenic breast-cancer cells[END_REF][START_REF] Shipitsin | Molecular definition of breast tumor heterogeneity[END_REF]. These results suggest that the presence and frequency of CD44 + CD24 -/low tumorigenic breast cancer cell populations have prognostic relevance [START_REF] Shipitsin | The cancer stem cell hypothesis: In search of definitions, markers, and relevance[END_REF]. In addition, several groups have shown that the CD44 + CD24 -/low subpopulation of breast cancer cells expresses higher levels of pro-invasive genes and plays a role in the invasive step of metastasis [START_REF] Liu | The prognostic role of a gene signature from tumorigenic breast-cancer cells[END_REF][START_REF] Sheridan | Cd44+/cd24-breast cancer cells exhibit enhanced invasive properties: An early step necessary for metastasis[END_REF][START_REF] Abraham | Prevalence of cd44+/cd24-/low cells in breast cancer may not be associated with clinical outcome but may favor distant metastasis[END_REF]. Indeed, we observed higher expression of NANOG, OCT4 and SOX2 in the stem cell populations CD44 + CD24 -/low and EMA + CALLA + than in the rest of the sample population, and cells overexpressing these factors displayed an increase in the stem cell populations and were more invasive. The correlation between number/proportion of cells displaying stem-like features within the tumor and a worse prognosis has also been reported for ALDH1 expression in inflammatory breast cancer [START_REF] Ginestier | Wicha MS Aldehyde dehydrogenase 1-positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer[END_REF]. Furthermore, the intrinsic resistance of tumorigenic breast cancer cells to certain forms of therapy is reflected by the increase in the percentage of CD44 + CD24 -/low cells and in the formation of mammospheres from tumor samples evaluated after chemotherapy [START_REF] Li | Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy[END_REF]. Similarly, Pece and collaborators observed that poorly differentiated breast tumors contain more cancer stem cells than well-differentiated tumors [START_REF] Pece | Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content[END_REF].

Characterization of the pathways that regulate cancer stem cell differentiation might help to design better future therapies.

Our findings do not address the question of the origin of breast cancer stem cells [START_REF] Vivanco | Function follows form: Defining mammary stem cells[END_REF], but they suggest that certain types of tumors might arise through transformation of either ER-positive progenitors or ER-negative stem/early progenitors that may asymmetrically divide to self-renew and give rise to undifferentiated ER-positive progenitor cells. In response to estrogen, ER-positive progenitors might then secrete paracrine factors, such as amphiregulin [START_REF] Ciarloni | Amphiregulin is an essential mediator of estrogen receptor alpha function in mammary gland development[END_REF][START_REF] Lamarca | Estrogen regulation of mammary gland development and breast cancer: Amphiregulin takes center stage[END_REF], that influence the proliferation and/or differentiation of ER-negative cells.

The relevance of estrogen in the development of the mammary gland and in breast cancer has been known for many years, but its influence on the stem cell population is not fully understood (reviewed in [START_REF] Lamarca | Minireview: Hormones and mammary cell fate--what will i become when i grow up?[END_REF]). Our results suggest that estrogen reduces the pool of breast stem cells both in normal and breast cancer tissues. The number of mammospheres formed after serial passage at clonal density is believed to reflect stem cell self-renewal, whereas the size of the mammospheres formed reflects the rate of proliferation of more mature progenitor cells [START_REF] Dontu | In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells[END_REF]. We observed that estrogen treatment reduces the number of mammospheres but increases their size. This suggests that estrogen not only reduces the pool of self-renewing stem cells, presumably by promoting their differentiation, but also promotes the proliferation of more differentiated progenitors and tumor cells, as expected considering the known effects of estrogen on proliferation. In addition, tamoxifen antagonizes the mitogenic signals of estrogen in the tumor cells leading to smaller mammospheres. These results might seem paradoxical given that exposure to estrogen is considered to increase the risk of breast cancer (reviewed in [START_REF] Clemons | Estrogen and the risk of breast cancer[END_REF]). Although the complexity of estrogen signaling has already been highlighted [START_REF] Lewis-Wambi | Estrogen regulation of apoptosis: How can one hormone stimulate and inhibit?[END_REF].

Ironically, one of the strongest and most extensively documented protective factors for breast cancer is early age of first pregnancy [START_REF] Clemons | Estrogen and the risk of breast cancer[END_REF]. The protective effect of early childbirth has been attributed to mammary epithelial stem cell differentiation and a decrease in mammary stem cell numbers (reviewed in [START_REF] Britt | Pregnancy and the risk of breast cancer[END_REF][START_REF] Polyak | Pregnancy and breast cancer: The other side of the coin[END_REF]). Thus, given that estrogen is required for mammary gland differentiation during puberty [START_REF] Deroo | Profile of estrogen-responsive genes in an estrogen-specific mammary gland outgrowth model[END_REF], it is possible that estrogen can have the beneficial effect of reducing the size of the mammary stem cell pool. Furthermore, clinical benefit has been observed using estrogen to treat women with metastatic breast cancer resistant to classical hormone therapy [START_REF] Ellis | Lowerdose vs high-dose oral estradiol therapy of hormone receptor-positive, aromatase inhibitorresistant advanced breast cancer: A phase 2 randomized study[END_REF] and also in high doses to treat hormonally sensitive tumors [START_REF] Lewis-Wambi | Estrogen regulation of apoptosis: How can one hormone stimulate and inhibit?[END_REF], and higher circulating estrogen levels in postmenopausal breast cancer patients are associated with a less aggressive tumor phenotype [START_REF] Schneider | Circulating estradiol defines the tumor phenotype in menopausal breast cancer patients[END_REF].

The most striking evidence is that ER is a powerful predictive marker in breast cancer management and ER-positive tumors are more differentiated, and have a better outcome than basal or HER2positive tumors. Our results agree with all these well-reported clinical observations. In order to selfrenew, tumor cells should find ways to bypass the differentiation-promoting effect of estrogen, for example through loss of expression of ER. Consistent with this model, deletion of the breast cancer susceptibility gene BRCA1, which expands the stem cell population, reduces the expression of ER [START_REF] Liu | Brca1 regulates human mammary stem/progenitor cell fate[END_REF]. Moreover, women with ER-negative tumors are more likely to develop a second primary tumor and the second tumor is more likely also to be ER-negative [START_REF] Kurian | Second primary breast cancer occurrence according to hormone receptor status[END_REF]. In fact, it has been recently shown that poorly differentiated cancers contain more cancer stem cells than well-differentiated tumors [START_REF] Pece | Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content[END_REF],

and the presence of cancer stem cells was associated with high histological grade, ER-negativity and poor overall survival [START_REF] Zhou | The prognostic role of cancer stem cells in breast cancer: A meta-analysis of published literatures. Breast Cancer Res Treat[END_REF].

The protective effects of short-term stimulation with hormone, either by early pregnancy or with the hormones estrogen and progesterone, have also been extensively studied in animal models. For example, short-term hormone exposure can prevent mammary tumorigenesis in two genetically engineered mouse models [57]. Recently, it has been reported that estrogen and progesterone together increased stem cell numbers in mice, but no difference was observed using estrogen alone [START_REF] Asselin-Labat | Control of mammary stem cell function by steroid hormone signalling[END_REF][START_REF] Joshi | Khokha R Progesterone induces adult mammary stem cell expansion[END_REF]. These results are difficult to reconcile with the long-term protection against breast cancer offered by early pregnancy and with reports showing no difference in stem cell numbers between parous and virgin animals [START_REF] Britt | Pregnancy in the mature adult mouse does not alter the proportion of mammary epithelial stem/progenitor cells[END_REF] or a decrease in the number of stem cells after an early pregnancy [START_REF] Siwko | Evidence that an early pregnancy causes a persistent decrease in the number of functional mammary epithelial stem cells--implications for pregnancy-induced protection against breast cancer[END_REF], which was considered an explanation for pregnancy-induced protection against breast cancer. Clearly, this is still a controversial area and further studies will be needed to decipher fully the effects of exposure to steroid hormones and breast cancer risk.

To conclude, our results implicate Nanog, Oct4 and Sox2 in the maintenance of human mammary stem cells. Finally, this work provides an explanation for the better prognosis and less aggressive phenotype of ER-positive tumors.

Figure legends

Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8

 12345678 Fig. 1 Mammospheres derived from breast epithelial cells express high levels of NANOG, OCT4 and SOX2 that are reduced by estrogen. a Real-time PCR analysis of NANOG, OCT4 and SOX2 expression in the normal mammary gland (n = 12). RNA was isolated from freshly isolated organoids (org) and from normal mammary epithelial cells grown under different conditions: adherent (adh) or as secondary mammospheres (ms). Expression in organoids was set at 1. The median is indicated by the horizontal bar, the mean is indicated by a solid square and the outliers by open circles. ** p < 0.01 (Mann-Whitney U test). b Expression of NANOG, OCT4 and SOX2 in mammospheres grown under differentiating conditions (dif) compared to the expression in mammospheres (ms, set at 1). c Levels of progesterone receptor (PR) expression in mammospheres exposed to ethanol (OH, black bar, set at 1), estrogen (E2, dashed bar) or tamoxifen (Tam, gray bar). d Expression of NANOG, OCT4 and SOX2 in mammospheres in the presence of the indicated hormones

Acknowledgements

We thank the participating women for their willingness to cooperate with our study. We also thank Robert Kypta (CIC bioGUNE) for NTera2/D1 cells and critical reading of the manuscript, present and past members of the laboratory for help and discussions, and James Sutherland (CIC bioGUNE) for help and advice. This work was supported by grants from the 'Spanish Ministry of Education and Science', the 'Institute of Health Carlos III', the 'Department of Education of the Government of the Autonomous Community of the Basque Country', the 'Department of Industry, Tourism and Trade' (Etortek) and 'Department of Innovation Technology of the Government of the Autonomous Community of the Basque Country' to MV; 'Foundation for Science and Technology of the Portuguese Ministry of Science, Technology and Higher Education ' to BMS; 'Foundation La Caixa' to MV and MP; 'Department of Education of the Government of the Autonomous Community of the Basque Country' to OI.


MA Stephen Rd Johnston
email: stephen.johnston@rmh.nhs.uk

Commentary for BCRT 2010 "Are we missing the mTOR target in breast cancer?"

Abnormalities in the PI3K/Akt/mTOR intracellular signalling pathway have been implicated in several forms of human cancer. The central component of the pathway is the phosphatidylinositol 3-kinase (PI3K) heterodimer which comprises the p85 regulatory and p110 catalytic subunit, and mutations in the catalytic domain of PI3K have been identified in 20-25% of breast cancers [START_REF] Bachman | The PIK3CA gene is mutated with high frequency in human breast cancers[END_REF][START_REF] Saal | PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma[END_REF]. A further 15-35% of breast cancer patients demonstrate reduced expression of PTEN (phosphatase and tensin homolog deleted on chromosome ten), an endogenous inhibitor of the PI3K/AKT pathway [START_REF] Shoman | Reduced PTEN expression predicts relapse in patients with breast carcinoma treated by tamoxifen[END_REF]. Akt is the ultimate effector of the pathway and directs a multitude of intracellular effects related to growth, glucose metabolism, protein synthesis and cell survival. Activation of the PI3K/Akt pathway provides a cell with unrestricted growth and survival signals, and one important downstream consequence is the alleviation of the suppression by the tuberous sclerosis protein complex (TSC1/2) of the mammalian target of rapamycin (mTOR) [START_REF] Inoki | Dysregulation of the TSC-mTOR pathway in human disease[END_REF]. There are at least two groups of mTOR proteins, and the key protein involved in transmitting signals from PI3K-Akt is the mTORC1 complex. Within cells mTORC1 plays a critical role in the transduction of proliferative signals through phosphorylation of the translational regulator 4E-BP1 (eukaryotic initiation factor 4E-binding protein) and the ribosomal protein p70 s6k (the 70-kDa S6 kinase) that facilitate translation of mRNAs into proteins [START_REF] Hara | Regulation of eIF-4E BP1 phosphorylation by mTOR[END_REF][START_REF] Chung | PDGF-and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase[END_REF]. In recent years our understanding of this pathway in breast cancer has been exploited by the development of a number of targeted anti-cancer therapeutics, with mTOR inhibitors being the most clinically developed to date.

Rapamycin is the original inhibitor of mTOR that was found to be a potent fungicide with immunosuppressive properties, and therapeutics were developed for clinical use in transplantation to prevent graft rejection. Subsequently rapamycin was found to have anti-proliferative effects in a range of experimental tumours [START_REF] Eng | Activity of rapamycin (AY-22,989) against transplanted tumors[END_REF], and rapamycin analogues with a more favourable pharmacological profile were synthesised including CCI-779 (temsirolimus), RAD-001 (everolimus), and AP23573 (ridaforolimus). In various breast cancer cell lines with constitutive activation of the PI3K-Akt pathway due to either HER2 amplification, ER activation or PTEN deletions temsirolimus showed specific sensitivity [START_REF] Yu | mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer[END_REF], while everolimus (an oral hydroxyethyl ether of rapamycin) demonstrated impressive anti-proliferative activity against a wide variety of tumour models in vivo [START_REF] O'reilly | In vivo activity of RAD-001, an orally active rapamycin derivative, in experimental tumor models[END_REF]. In the clinic mTOR antagonists were found to be relatively well tolerated in early phase I/II trials [START_REF] Raymond | Safety and pharmacokinetics of escalated doses of weekly intravenous infusion of CCI-779, a novel mTOR inhibitor, in patients with cancer[END_REF][START_REF] O'donnell | Phase I pharmacokinetic and pharmacodynamic study of the oral mammalian target of rapamycin inhibitor everolimus in patients with advanced solid tumors[END_REF], and development programmes were established in various solid malignancies, in particular renal cell cancer and breast cancer.

But has the development of mTOR antagonists for breast cancer moved ahead too fast before it was really appreciated how best to utilise these drugs as targeted therapeutics? As a single agent temsirolimus showed only modest activity in the advanced breast cancer setting [START_REF] Chan | Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer[END_REF], and subsequently both temsirolimus and everolimus were developed in combination with endocrine therapy following encouraging evidence for additive or synergistic effects from several pre-clinical models of endocrine resistant breast cancer [START_REF] Boulay | Dual inhibition of mTOR and estrogen receptor signaling in vitro induces cell death in models of breast cancer[END_REF][START_REF] Degraffenried | Inhibition of mTOR activity restores tamoxifen response in breast cancer cells with aberrant Akt Activity[END_REF]. However, results from randomised clinical trials of mTOR antagonists in combination with endocrine therapy were mixed and failed to show substantial clinical benefit for the combination [START_REF] Baselga | Phase II, 3-arm study of CCI-779 in combination with letrozole in postmenopausal women with locally advanced or metastatic breast cancer: preliminary results[END_REF][START_REF] Baselga | Treatment of postmenopausal women with locally advanced or metastatic breast cancer with letrozole alone or in combinations with temsirolimus; a randomized 3arm, phase 2 study[END_REF][START_REF] Chow | Phase 3 study of temsirolimus with letrozole or letrozole alone in postmenopausal women with locally advanced or metastatic breast cancer[END_REF]. It became clear that as a targeted therapeutic, mTOR antagonists were being developed in unselected patients without understanding whether mTOR activation was indeed critical for all breast cancers, or indeed what the molecular consequence of mTOR inhibition would be. As such, it is now imperative that we refine the clinical development programme for mTOR antagonists by developing biomarkers that might select breast cancers most likely to respond to mTOR inhibition, and by understanding the evolving complexity of the PI3K/AkT/mTOR signalling pathway and applying that knowledge to design better therapeutic strategies.

In this month's edition of Breast Cancer Research and Treatment, three articles are published that contribute to a better understanding of mTOR as a target in breast cancer. In particular they relate to an evaluation of downstream mTOR targets and biomarkers for clinical response [START_REF] Macaskill | The mammalian target of rapamycin (mTOR) inhibitor everolimus (RAD001) in early breast cancer; results of a pre-operative study[END_REF][START_REF] Perez-Tenorio | Clinical potential of the mTOR targets S6K1 and S6K2 in breast cancer[END_REF], and review how best we might combine mTOR antagonists either with conventional cytotoxics, endocrine agents or other biological therapies for breast cancer [START_REF] Margariti | Overcoming drug resistance in breast cancer; could mTOR inhibitors be a myth or a real possibility for the future[END_REF]. Neo-adjuvant or short-term pre-operative studies can provide predictive biomarker data that may help select appropriate patients for future clinical trials. Macaskill et al evaluated the effects of just 14 days exposure to everolimus (5mg once daily) in 31 post-menopausal women with unselected early stage breast cancer [START_REF] Macaskill | The mammalian target of rapamycin (mTOR) inhibitor everolimus (RAD001) in early breast cancer; results of a pre-operative study[END_REF]. Paired biopsies taken at diagnosis and at surgery after drug exposure allowed an immuno-histochemical (IHC) assessment of changes in cell proliferation (Ki67), apoptosis (active caspase-3), ER and PgR, in addition to key activated components of the pathway (pAkt, p-S6 kinase, p-mTOR).

Increasingly, these type of studies are using Ki-67 as the primary endpoint and key biological readout for drug exposure in this setting [START_REF] Dowsett | Prognostic value of Ki67 expression after short-term presurgical endocrine therapy for primary breast cancer[END_REF]. Furthermore, short-term changes in Ki-67 can be correlated with both the tumour's molecular profile and changes in downstream signalling that in turn might identify predictive biomarkers for clinical benefit from the drug. The authors showed that reduction in proliferation was greatest in HER2+ tumours and/or those tumours with the highest pre-treatment Ki67 scores, and that this also correlated with inhibition of signalling demonstrated by associated reduction in p-Akt levels. In addition, a number of tumours showed no biological response to mTOR antagonism suggesting that some harbour innate denovo resistance. As such these data would suggest tumour profiles can be selected which may be more or less likely to benefit from mTOR inhibition, and in turn this could form key selection criteria for future clinical trials.

However, there are limitations to this short-term exposure in only a small group of patients in being able to correlate biological changes with clinical outcome, and to evaluating resistance mechanisms that might quickly develop and limit the drug's effectiveness. Larger neo-adjuvant studies that treat patients for 3-4 months prior to surgery can start to correlate biomarker data with traditional clinical response features. A recent randomised phase II study in 270 postmenopausal women with ER+ primary operable breast cancer evaluated the benefit of adding everolimus (10mg/day) or placebo to neo-adjuvant letrozole (2.5mg/day) for 16 weeks preoperatively [START_REF] Baselga | Phase II randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer[END_REF]. The primary endpoint of the study was clinical response, with a greater tumour shrinkage being observed for the combination (68.1% vs 59.1%). A significantly greater reduction in cell proliferation (Ki67) was seen in the letrozole/everolimus combination arm compared to letrozole. Associative correlative studies in pre-and post-treatment biopsies demonstrated expected down-regulation in progesterone receptor (PgR) and cyclin D1 in both treatment arms, with reductions in phosphorylated ribosomal protein S6 (mTOR downstream target) being observed only in the everolimus arm. Interestingly, in this study specific mutations in exon 9 of PIK3CA were found to be associated with a greater likelihood of an anti-proliferative response to the combination of letrozole plus everolimus compared to letrozole alone this was not observed in the study of Macaskill et al [START_REF] Macaskill | The mammalian target of rapamycin (mTOR) inhibitor everolimus (RAD001) in early breast cancer; results of a pre-operative study[END_REF], although they acknowledge that numbers in their study were too small. This particular PIK3CA mutation has been associated with a worse prognosis in breast cancer [START_REF] Barbareschi | Different prognostic roles of mutations in the helical and kinase domains of the PIK3CA gene in breast carcinomas[END_REF], and a greater likelihood of response to the combination implicates the PI3K/Akt/mTOR pathway in endocrine resistance. While these pre-operative studies have helped identify some tumour profiles that might predict for benefit from mTOR inhibition based on activation and/or dependence on the pathway (ie. high proliferation rate, HER2 amplification, ER+ve tumours with certain PI3KCA mutations), these biomarker profiles need to be validated prospectively in subsequent studies. What about the molecular downstream consequences of mTOR antagonism that may modify or nullify target inhibition?

Emerging evidence suggests an important negative feedback loop, whereby the mTOR activated kinase S6K1 phosphorylates and destabilizes the IRS1 and IRS2 proteins in insulin-like growth factor (IGF) responsive cells [START_REF] Sabatini | mTOR and cancer: insights into a complex relationship[END_REF]. In these cells, mTOR inhibition can lead to leads to a reduction in S6K1 activity, which in turn allows IRS1/2 expression to be increased with associated enhanced activation of IGFR1-dependent Akt activityas such, concern has been raised that loss of this negative feedback loop may overcome the anti-tumour effectiveness of mTOR blockade and limit their effectiveness [START_REF] O'reilly | mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt[END_REF][START_REF] Sun | Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition[END_REF]. This has supported by the clinical observation that phosphorylated Akt is upregulated in both tumour and skin biopsies of patients treated with everolimus [START_REF] Tabernero | Dose-and schedule-dependent inhibition of the mammalian target of rapamycin pathway with everolimus: a phase I tumor pharmacodynamic study in patients with advanced solid tumors[END_REF], although again Macaskill et all observed no change in pAkt in their short-term exposure study [START_REF] Macaskill | The mammalian target of rapamycin (mTOR) inhibitor everolimus (RAD001) in early breast cancer; results of a pre-operative study[END_REF]. While technical difference in detection of activated Akt can make such studies difficult, they have raised concern that loss of negative feedback may in time overcome the anti-tumour effectiveness of mTOR blockade alone, resulting in short term clinical remissions and perhaps rebound growth at the time of disease progression. As discussed below, this has driven the rationale either for better mTOR inhibitors, for drugs that provide dual blockade with PI3K, or indeed mTOR inhibition combined with blockade of upstream growth factors (HER2, IGFR) to block this compensatory feedback loop.

In addition to the role that the mTOR substrate S6 kinase plays in regulating IRS1 and IRS2, the S6K1 and S6K2 genes which code for these substrate proteins are amplified in breast cancer, which in theory could abrogate the effects of mTOR inhibition. Perez-Tenorio et al. in this edition of BCRT have studied S6K1 and S6K2 gene / protein expression in postmenopausal patients with early breast cancer followed up over many years within the previously reported Stockholm adjuvant trials of adjuvant tamoxifen in low-risk patients, and of radiotherapy versus chemotherapy in high risk patients [START_REF] Perez-Tenorio | Clinical potential of the mTOR targets S6K1 and S6K2 in breast cancer[END_REF]. They report that amplification / gain of these genes occurs in over 20% of breast cancer, and this is perhaps not surprising given that the loci sit within commonly amplified regions in 17q21-23 and 11q13, which may explain the association of S6K1 amplification with HER2 status. They report that S6K2 amplification correlates with ER status and Cyclin D1 amplification, and that in ER+ PgR-ve tumours nuclear expression of the S6K2 protein may be associated with a worse outcome from tamoxifen. While these data are clearly of interest, no studies to date have looked at amplification of these mTOR substrates in terms of responsiveness to mTOR inhibitionfor example, amplification of a downstream target could make blockade of mTOR ineffective if endogenous drive to protein translation is already occurring via S6Kinase activation. It will now be imperative to include this biomarker in subsequent studies of mTOR inhibitors.

So how can we improve upon the current mTOR antagonists that we have in the clinic, and should we be exploring a different strategy to utilise these drugs, namely combining them with other inhibitors to provide more effective blockade of the key components of intra-cellular signalling networks? In the third article in this edition of BCRT Margariti et al review the current progress that we have made with mTOR inhibition, in particular the attempts to reverse resistance to current clinical therapeutics including cytotoxics, endocrine therapy, and HER2 blockade with trastuzumab [START_REF] Margariti | Overcoming drug resistance in breast cancer; could mTOR inhibitors be a myth or a real possibility for the future[END_REF]. They conclude that while the pathway is a logical one to target and pre-clinical data have been especially encouraging, clinical progress with these drugs has been very modest to date. Heterogeneity of breast cancer and inability to target the right tumours may explain some of these results, although the most promising areas to develop further remain HER2+ tumours that may be resistant to trastuzumab, and endocrine resistant tumoursseveral key studied with existing mTOR antagonists in combination with trastuzumab and endocrine therapies are ongoing.

Ultimately we need to develop more complete mTOR antagonists, as well understand how best to use them in smarter targeted combinations. The current rapamycin analogues in development only block the mTORC1 complex, with no effect on the mTORC2 complex that independently activates Akt, thus potentially limiting their ability to totally block this pathway [START_REF] Jacinto | Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive[END_REF][START_REF] Sarbassov | Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex[END_REF]. Several novel targeted agents are in development against various components of the PI3K/Akt/mTOR pathway including mTOR inhibitors that target both mTORC1 and mTORC2, Akt inhibitors, PI3K inhibitors and dual mTOR/PI3K inhibitors (Table 1). For example the dual mTOR/PI3K inhibitor NVP-BEZ235 has previously demonstrated superior anti-proliferative activity to everolimus in cell lines [START_REF] Serra | NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations[END_REF], suggesting that greater blockade of the pathway can be achieved that just targeting mTOR alone. Likewise, a greater understanding of the pathway's molecular biology will allow partnering of key signal transduction inhibitors together to provide maximal "vertical" or "horizontal" blockade. For example dual vertical inhibition of IGF-1R signalling using either monoclonal antibodies or tyrosine kinase inhibitors could be used to overcome the rapamycin induced Akt activation loop. Preliminary evidence to support has come form some promising results in neuroendocrine tumours where octreotide (which targets IGF-IR signalling) was combined with everolimus [START_REF] Yao | Phase II study of RAD001 (everolimus) and depot octreotide in patients with advanced low grade neuro-endocrine carcinoma (LGNT)[END_REF]. More recently the mTOR inhibitor ridaforolimus has been combination with the IGF-1R antibody dalotozamab in patients with advanced solid tumours [START_REF] Cosimo | A phase I study of the oral mTOR inhibitor ridaforolimus in combination with the ICF-1R antibody in patients with advanced solid tumours[END_REF]. The most frequent adverse events were stomatitis, fatigue and hyperglycaemia which were mostly grade 1 -2.

Interestingly there were signs of activity in 5 heavily pre-treated breast cancer patients with 2 achieving partial responses, 1 stable disease and a further 2 demonstrating partial metabolic responses on PET scan. All 5 of these patients were ER+ and 4 had high expression of Ki67, consistent with data above relating to tumour profiles most likely to respond to mTOR inhibition.

So in conclusion, have we missed the mTOR target in breast cancer? Probably not completely is the answer, but we can certainly do betterperhaps the key lesson to remember is that development of a targeted agent in breast cancer requires a full understanding of the signalling pathway, and recognition that not all cancers will respond. Predictive biomarkers and understanding the consequences of target inhibition in-vivo allow identification of who best to treat, and how best to use the drug. Signalling networks are complex and sophisticated, and elucidating which ones are key oncogenic drivers and moreover what regulates them is central to getting the best out of modern targeted therapeutics. We should consider the current mTOR antagonists as first generation probes, and exploit the emerging science and clinical data to develop superior drugs against this pathway in smarter, better designed clinical trials.

Table 1 :

 1 Novel PI3K and mTOR inhibitors in development (adapted from ref 33) (33).

	Agent	Company	Molecular targets	Disease
	AZD8055	AstraZeneca	mTORC1 and mTORC2	Phase I -solid cancers
	SF1126	Semaphore Pharmaceuticals	PI3K, mTOR, DNA-PK	Phase I -solid tumours, myeloma
	XL-147	Exelixis	PI3K	Phase I -solid tumours, NSCLC
				Phase I -solid
	XL-765	Exelixis	PI3K / mTOR	tumours, NSCLC,
				glioma
	NVP-BEZ-235	Novartis	PI3K / mTOR	Phase I -solid tumours, Breast Ca
	NVP-BGT-226	Novartis	PI3K / mTOR	Phase I -solid tumours, Breast Ca
	GDC-0941	Genentech / Piramed Pharmaceuticals	PI3K / mTOR (weak)	Phase I -solid tumours
	GSK1059615	GlaxoSmithKline	PI3K	Phase I -solid tumours, Breast Ca