AMF inhibition halts the development of the aggressive phenotype of breast cancer stem cells

    HPI/AMF inhibition halts the development of the aggressive phenotype of breast cancer stem cells Juan Carlos Gallardo-P´erez, Alhel´ı Ad´an-Ladr´on de Guevara, Alvaro Mar´ın-Hern´andez, Rafael Moreno-S´anchez, Sara Rodr´ıguez-Enr´ıquez PII: DOI: Reference:

S0167-4889(17)30167-2 doi:10.1016/j.bbamcr.2017.06.015 BBAMCR 18128

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

2 January 2017 13 June 2017 16 June 2017

Please cite this article as: Juan Carlos Gallardo-P´erez, Alhel´ı Ad´an-Ladr´ on de Guevara, Alvaro Mar´ın-Hern´ andez, Rafael Moreno-S´anchez, Sara Rodr´ıguez-Enr´ıquez, HPI/AMF inhibition halts the development of the aggressive phenotype of breast cancer stem cells, BBA - Molecular Cell Research (2017), doi:10.1016/j.bbamcr.2017.06.015

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HPI/AMF inhibition halts the development of the aggressive phenotype of breast cancer stem cells

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Juan Carlos Gallardo-Pérez*, Alhelí Adán-Ladrón de Guevara, Alvaro MarínHernández, Rafael Moreno-Sánchez and Sara Rodríguez-Enríquez*

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Departamento de Bioquímica, Instituto Nacional de Cardiología, Tlalpan, México, DF 14080, México.

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Authors for correspondence:

*Sara Rodríguez-Enríquez, Ph. D. and

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Juan Carlos Gallardo-Pérez, Ph. D. Instituto Nacional de Cardiología Departamento de Bioquímica Juan Badiano No. 1, Col. Sección XVI Tlalpan, México 14080 MEXICO [email protected]

Running Title: HPI/AMF inhibition blocks cancer stem cells phenotype Keywords: Breast cancer; stem cells; hexose-phosphate isomerase; metastatic phenotype Abbreviations: BCSC, breast cancer stem cell-like cells; CSC, cancer stem cells; HPI/AMF, hexose phosphate isomerase/autocrine motility factor; E4P, erythrose 4 phosphate; EMT, epithelial mesenchymal transition.

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Abstract

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Cancer stem cells are responsible for tumor recurrence and metastasis. A new highly reproducible procedure for human breast cancer MCF-7 stem

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cells (BCSC) isolation and selection was developed by using a combination

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of hypoxia/hypoglycemia plus taxol and adriamycin for 24 h. The BCSC enriched fraction (i) expressed (2-15 times) the typical stemness protein

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markers CD44+, ALDH1A3 and Oct 3/4; (ii) increased its clonogenicity index (20-times), invasiveness profile (>70%), migration capacity (100%) and

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ability to form mammospheres, compared to its non-metastatic MCF-7 counterpart. This isolation and selection protocol was successful to obtain stem cell enriched fractions from A549, SiHa and medulloblastoma cells.

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Since the secretion of HPI/AMF cytokine seems involved in metastasis, the

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effects of erytrose-4-phosphate (E4P) and 6-phosphogluconate (6PG),

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potent HPI inhibitors, on the acquisition of the breast stem cell-like phenotype were also evaluated. The presence of E4P during the BCSC selection deterred the development of the stemness phenotype, whereas both extracellular E4P (5-250 nM) and 6PG (1 µM) as well as siRNA HPI/AMF depressed the BCSC invasiveness ability (>90%), clonogenicity index (>90%) and contents (50-96%) of stemness (CD44, ALDH1A), pluripotency (p38 MAPK, Oct3/4, wnt/β-catenin) and EMT (SNAIL, MMP-1, vimentin) markers. The cytokine inhibitor repertaxin (10 nM) or the anti IL-8 or anti-TGF-β monoclonal antibodies (10 µg/mL) did not significantly affect the BCSC metastatic phenotype. E4P also diminished (75%) the formation and growth of MCF-7 stem cell mammospheres. These results suggested that E4P by directly interacting with extracellular HPI/AMF may be an effective strategy to deter BCSC growth and progression.

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1. Introduction

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Cancer stem cells (CSC; also named cancer initiating cells) are a marginal cell subpopulation (0.01-2.5% of total tumor mass) found in solid

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tumors which seems responsible for increasing tumorigenicity, drug and

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radiation resistance, tumor self-renewal, metastatic phenotype development

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and tumor recurrence [1-4].

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Mechanisms proposed to explain the maintenance of the CSC phenotype includes (i) activation by hypoxia (0.1-1%) of Oct-3/4 and Nanog

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factors involved in the CSC self-renewal which in turn promote progenitor

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stem cell maintenance, pluripotency and also self-renewal [reviewed by 5,

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6]; (ii) CSC interaction with fibroblasts, endothelial cells or immune cells [7] mediated by interleukins, cytokines and growth factors [8]; (iii) epigenetic modifications (chromatin remodeling, DNA methylation, histone modifications) and miRNAs overexpression (let-7, miR-200, miR-30, miR-16, miR181) contributing to the activation and maintenance of several pathways (wnt/β-catenin, hedgehog, BMP, TGF-β, Notch) involved in preserving the stem-cell phenotype [9-11]. Several strategies, based on the use of canonical anticancer drugs, have been assayed in experimental cell and animal models and patients attempting to target CSC within solid tumors [12-14], although unfortunately with poor outcomes [15] and severe side-effects [4, 15, 16].

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Other strategies based on the use of monoclonal antibodies

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(tocilizumab) or repertaxin to block several cytokines (IL-6, IL-8) actively secreted by metastatic phenotype-developing cells (breast and lung

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carcinomas) [17-21] have also been unsuccessful. Repertaxin also affects

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the functioning of the B-lymphocytes IL-8 dependent signaling [19, 22]

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compromising its clinical use. Further, repertaxin treatment may result ineffective against triple negative metastatic MDAMB-453 breast cancer cells

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because in these cells the IL-8 downstream factor PTEN is mutated inducing

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a constitutively AKT signaling activation which in turn promotes chemo-

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resistance [19].

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HPI/AMF (hexose phosphate isomerase/autocrine motility factor), also known as phosphoglucose isomerase (PGI) or glucose-6-phosphate isomerase (GPI), is the second glycolytic enzyme that catalyzes the interconversion of glucose-6-phosphate into fructose-6-phosphate. HPI/AMF may also act as a cytokine (or neurokine) and be actively expressed and secreted by malignant cells to trigger EMT and metastasis [23]. This cytokine initially isolated and purified from human malignant melanoma [24] mediates its biological effects through the interaction with its plasma membrane receptor (AMFR/gp78) [25]. HPI/AMF secretion to the extracellular milieu is significant in cancer cells [26], although non-cancer cells (neurons, articular chondrocytes, fibroblasts, normal breast cells) also

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secrete it at lower extents [26-28]. HPI/AMF inhibition by erytrose-4-

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phosphate (E4P) or glucose-6-phosphate (Glc6P) at 100-200 µM significantly decreased the migration rate of murine fibrosarcoma Gc-4PF

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cells [25]. Indeed, external E4P or 6-phosphogluconate (6PG) inhibit the

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HPI/AMF cytokine activity as revealed by the lower invasiveness ability and

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migrating protein levels of metastatic cells from human breast tumor

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microspheroids [26].

Therefore, HPI/AMF synthesis and secretion by breast cancer stem

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cells (BCSC) could be one of the primary mechanisms triggering EMT and

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tumor migration which in turn may be selectively inhibited by E4P. To

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assess this hypothesis, a reliable and reproducible protocol for isolating BCSC with high yields was developed which involved exposure to a variety of sequential stressful conditions [5, 29-31]. All of the metastasis and stemness markers assayed as well as several specific metastatic cell functions were substantially enhanced in the cell population isolated with the protocol here designed. Furthermore, these malignant properties were suppressed by therapeutically attractive doses of the HPI/AMF inhibitors E4P and 6PG.

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2. Material and methods

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2.1 Cell cultures

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Human breast non-metastatic MCF-7 (1 x 106 cells/mL), lung non-

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metastatic A549, desmoplastic cerebellar metastatic medulloblastoma DAOY, cervix metastatic SiHa cancer cell lines and mouse 3T3 fibroblasts

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(1 x 106 cells/mL) were grown in Dulbecco-MEM medium (DMEM)

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supplemented with 10% fetal bovine serum (GIBCO; Rockville, USA) plus 10,000 U penicillin/streptomycin (SIGMA; Steinheim, Germany). HUVEC,

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human umbilical vein endothelial cells (1x106 cells) were grown in 199

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medium (GIBCO; Rockville, USA) supplemented with 10% fetal bovine

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serum. Cancer and non-cancer cells were placed in a humidified atmosphere of 5% CO2/95% air at 37°C for 3-4 days until confluence of 8090% was reached. MCF-7 cells used in the present work proceeded from the original MCF-7 clone (American Type Culture Collection; Rockville, MD, USA). Genotyping of the four cancer cell lines used in the present work at the National Institute of Genomic Medicine, Mexico revealed that MCF-7 (5 out of 14), A549 (13 out of 16), DAOY (14 out of 14) and SiHa (11 out of 11) cells shared allelic markers with their original clones.

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2.2 Cancer stem cells (CSC) selection and isolation

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MCF-7 cells as well as medulloblastoma, lung and cervix carcinoma

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cells were exposed to three different stress conditions, individually and in

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combination. These were (i) hypoxia (0.1% or 1% O2); (ii) low glucose concentration (2.5 mM) and/or (iii) drug treatment. First, cells were cultured

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in a glucose-free DMEM medium containing 2.5 mM glucose plus taxol (100

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nM) for 12 h. Afterwards, old medium was replaced by complete DMEM containing 25 mM glucose plus doxorubicin (100 nM) and further exposed to

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severe hypoxia (0.1% O2) in an Oxygen Control Chamber (Coy Laboratory,

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Grass Lake, MI) for 12 h. To stabilize the stemness phenotype [32], the

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cells were finally cultured in serum-free DMEM containing 25 mM glucose and placed in an humidified atmosphere of 5% CO2/95% air at 37°C for 24 h until their use (Table S1 and Figs. S1A and S1B). CSC viability was assessed by trypan blue as previously described [33]. 2.3 Proliferation of breast cancer stem cells (BCSC) BCSC (5 x 105 cells/mL, viability >98 %) were grown in DMEM with penicillin/streptomycin and proliferation was evaluated in the absence or presence of E4P (24 nM) which was added daily from day 0 to day 7 of culture. As control, proliferation of (5 x 105 cells/mL) untreated MCF-7 cells was also performed for 7 days.

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2.4

siRNA plasmid construction and cell transfection

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siRNA for HPI/AMF was generated by sub-cloning specific sequences

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of 21 bp (5′-UGGUACCGCGAGCACCGCUTT-3′) into the pSIREN vector

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(Clontech, CA, USA) following manufacturer instructions [34]. One million BCSC were transfected with 1 μg of the siRNA construct using

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Lipofectamine 2000 (Invitrogen) for 6 h in serum-free DMEM and further 12–

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36 h incubation in DMEM with serum. Then, the cells were selected for 2 weeks with 450 mg G418/mL, cloned, and further expanded in the presence

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of G418 for 2-4 weeks. This relatively lengthy process was due to the slow

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proliferation rate of HPI/AMF siRNA cells. The siRNAs specificity was

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verified by Western blotting of the HPI/AMF protein content. Western blot assay and immunoprecipitation analyses BCSC, siRNA BCSC, MCF-7, A549, SiHa and medulloblastoma cells (5 x 106cells/mL) were dissolved in RIPA (PBS 1x pH 7.2, 1% IGEPAL NP40, SDS 0.1% and sodium deoxycholate 0.05%) lysis buffer plus 1 mM of PMSF (phenyl methanesulfonyl fluoride) and 1 tablet of complete protease inhibitors cocktail (Roche, Mannheimm, Germany). Protein samples (40 µg) were re-suspended in loading buffer plus 5% β-mercaptoethanol and loaded onto 10 or 12.5% polyacrilamide gel under denaturalizing conditions [26]. Electrophoretic transfer to PVDF membranes (BioRad; Hercules, CA, USA)

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was followed by overnight immunoblotting with 1:1000 dilution of CD133,

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CD44, ALDH1A3, Oct3/4, HPI/AMF, NFkappaB/p65, XIAP, caspase-3 and caspase-9, β- catenin, E-cadherin, N-cadherin, cytokeratin, fibronectin, IL-6,

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IL-8, TGF-β, SNAIL and vimentin; and 1:500 dilution of HPI/AMF receptor

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(gp78), p38 MAPK, WNT-1, MMP-1, Bcl2, c-IAP1 and α-tubulin antibodies

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(Santa Cruz; Santa Cruz, CA, USA) at 4°C. The hybridization bands were revealed with the corresponding secondary antibodies conjugated with

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horseradish peroxidase (Santa Cruz Biotechnology). The signal was

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detected by chemi-luminiscence using the ECL-Plus detection system

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(Amersham Bioscience; Little Chalfont, Buckinghamshire, UK).

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Densitometry analysis was performed using the Scion Image Software (Scion; Bethesda MD, USA) and normalized against its respective load control. Percentage of each band represents the mean ± SD of at least three independent experiments. To assess the gp78-HPI/AMF interaction, both proteins were immunoprecipitated with anti-gp78 or IgG1 (1 µg) for 1 h plus protein A (SigmaAldrich, St Louis, MO, USA) at 4°C. HPI/AMF and gp78 were detected with their respective specific antibodies (Santa Cruz) following manufacturer instructions.

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2.6 Colony formation assay

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BCSC and MCF7 cells were seeded at a density of 2 x 104 cells/mL in

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150x20 mm Petri dish in DMEM plus 10% FBS at 37°C for 96 h. Afterwards,

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cells were fixed for 20 min with cold ethanol (70% v/v), air-dried and stained with violet crystal. Petri dishes were scanned and colony numbers were

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2.7 3D mammosphere growth assay

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counted using the Clono-Counter software [35].

Mammospheres were formed by using the floating sphere-forming

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assay [36]. Briefly, 1 x105 BCSC and MCF7 cells were grown in Erlenmeyer

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flasks with serum-free DMEM and placed immediately under slow (20-50 rpm) orbital shaking for 10 days at 37ºC in 95% air/5% CO2. Fresh DMEM was added every 2 to 3 days to remove cellular debris and death cells. Mammosphere size was measured daily with a graduated reticule (1/10 mm; Zeiss, NY, USA) in an inverted phase contrast microscope (Zeiss, NY, USA). Tumor microspheroids were generated by using the liquid overlay modified technique [37]. BCSC and MCF-7 cells were incubated (1X105 cells) in DMEM in 2 % agarose-coated Petri dishes. After 5 days, DMEM was replaced with fresh medium and placed under slow (20-50 rpm) orbital shaking for additional 15 days at 37º C in 95% air/5% CO2. Fresh DMEM was added every 2 to 3 days to remove cellular debris and planktonic cells

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not-forming spheroids. Tumor microspheroid size was measured at different

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culture times with a graduated reticule in an inverted phase contrast microscope. The E4P IC50 value for stem cell microspheroids growth was

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determined in the presence of increasing E4P concentrations of 1, 10, 100

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and 1000 nM. E4P was added when spheroids reached 370 ± 30 μm of

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diameter (day 10 of culture). E4P inhibitory effect was registered measuring

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the spheroid diameter at day 20 of culture. 2.8 HPI/AMF activity

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The HPI activity was assayed in 50 mM MOPS buffer, pH 7.0,

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containing 1 mM NADP+, 2 U Glc6PDH (Roche, Manheim, Germany) and 4-

[38].

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5 x 103 permeabilized cells. The reaction was started by adding 2 mM Fru6P

2.9 Invasion and migration assays BCSC, siRNA BCSC, and MCF-7 cells (5 x 104 cells/mL) in serum-free DMEM were seeded on the upper compartment of Boyden chambers (Trevigen Inc., Helgerman, USA). The lower compartment was filled with DMEM without serum. After 24 h at 37°C, invasive cells in the lower compartment were loaded with 60 nM calcein AM (acetomethylester) for 60 min. Calcein fluorescence was detected at 485 nm excitation and 520 nm emission in a microplate reader (Nunclon TM, Roskilde, Denmark) [26]. For

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control assays, MDAMB-231 metastatic canonical breast cancer cells were

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used. The migration assay was performed as described elsewhere [26].

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Briefly, BCSC and MCF-7 cells were cultured in 6-multiwell plates at 80-90%

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confluence. Once cellular attachment was reached, each cell culture was

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wounded by using a plastic tip, washed with 37°C PBS buffer and incubated with serum-free DMEM. Cellular migration distance from the border to the

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center of the Petri dish was measured with a graduate reticule.

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2.10 Invasion assay in MCF-7 cells exposed to BCSC growing medium DMEM (15 mL) from 5-days BCSC cultures was removed and

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fractioned into sterile flasks until use. In parallel, medium of 70-80% confluent MCF-7 cell cultures was discarded and replaced by cell-free 5days growth BCSC culture medium and cells were further incubated for 24 h. Afterwards, invasion assays with Boyden chambers were performed [26]. 2.11 Detection of HPI/AMF protein in cytosol and extracellular milieu The presence of extracellular HPI/AMF protein was assayed in cellfree DMEM from 24 h BCSC, MCF7 and siRNA BCSC cell cultures. Cellfree DMEM was incubated with 10% trichloroacetic acid (TCA) at 4°C overnight [39]. Afterwards, the mixture was centrifuged once at 10,000 rpm for 30 min. The sediment was resuspended in loading buffer and loaded

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onto 12.5% SDS-PAGE gels. Electrophoretic transfer to PVDF membranes

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(BioRad; Hercules, CA, USA) was followed by overnight immunoblotting with 1:500 dilutions of HPI/AMF, IL-6, IL-8 and TGF-β antibodies (Santa Cruz, CA

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USA) at 4°C. The hybridization bands were revealed with the mouse

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secondary antibody conjugated with horseradish peroxidase (Santa Cruz

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Biotechnology). The signal was detected by chemi-luminiscence as described above. Due to the lack of a specific loading control for secreted

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proteins, the following protocol was used to standardize loading of

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extracellular proteins for Western blotting. Fifty μg of TCA-precipitated

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extracellular proteins from cell free-culture medium were loaded into 12%

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SDS-PAGE. After electrophoresis, in-gel proteins were electro-transferred onto a PVDF membrane, which was stained with Ponceau S Red Solution (0.1 %) for 10 min at room temperature. A band of approximately 50 KDa was used as loading control [40] in the blots where secreted HPI/AMF was assessed.

2.12 HPI/AMF and cytokine inhibition For secreted HPI/AMF blocking, HPI/AMF inhibitors E4P or 6PG (1, 10, 100 and 1000 nM) and anti HPI/AMF monoclonal antibody (10 µg/mL) were used. For cytokine IL-8 and TGF-β inhibition, monoclonal antibodies anti-IL8 and anti-TGFβ (both at 10 µg/mL) [41] were used; whereas for IL-6 inhibition, repertaxin (10 nM) was added. All inhibitors were directly added

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to the BCSC, MCF7, 3T3 and HUVEC cell cultures; monoclonal antibodies

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were directly added to BCSC and MCF-7 cells. After 24 h incubation, cellular viabilities, BCSC and MCF-7 cell invasion and contents of ALDH1A3,

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caspase-3, caspase-9, CD24, CD44, HPI/AMF, MMP-1, NFκB/p65, Oct3/4,

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SNAIL, Wnt-1, XIAP, β- catenin were performed. α-tubulin was employed as

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loading control.

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2.13 Statistical Analysis

Data are expressed as mean ± standard deviation of the indicated number of

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independent experiments. The experimental and control groups were

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statistically compared using an unpaired two-tailed Student’s t-test with P

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values < 0.01 or <0.05 as significance criterion.

3. Results

3.1 Characterization of MCF-7 breast cancer stem cells Three different protocols (hypoxia, hypoglycemia and drug treatment) were assayed alone [31, 42, 43] or in combination for MCF-7 breast cancer stem cells (BCSC) phenotype selection. Each protocol and their combinations were initially evaluated by assessing the canonical cancer stem cell biomarker CD44, as also described for other CSC isolation

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protocols [3, 44]. Cells exposed to hypoxia (1 or 0.1% O2) or hypoglycemia

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(2.5 mM glucose) or drugs (taxol or doxorubicin) for 24 h showed high viability (> 80%) and non-significant increase in CD44 marker (Table S1 and

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Fig. S1A), as also reported for hypoxic CSC ovarian cells [45]. Taxol plus

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doxorubicin treatment together with hypoxia or hypoglycemia did not induce

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significant CD44 increase either vs. parental MCF-7 cells (Fig. S1A). Longterm (24-72 h) exposures to nanomolar doses of taxol or doxorubicin lead to

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severe cell death [46, 47]. However, combination of hypoglycemia with taxol

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(100 nM) for only 12 h, followed by hypoxia (0.1 % O2) plus doxorubicin (100

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nM) by additional 12 h was the experimental protocol (named LGT/HD) that

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promoted an enhanced CD44 content (26-times vs. untreated MCF-7 cells), although viability of these last treated cells was also markedly low (23.5 ± 4%). Other less complex combinations did not induce significant CD44 increments (Table S1).

The same isolation and selection protocol was applied to lung A549, cervix SiHa and medulloblastoma DAOY cells for enrichment of their respective cancer stem cell fractions. Specific cancer stem cell markers were used for each cancer cell line [48-50]. The enrichment of the stem cell fractions was also remarkable (Table S2). These results indicated that stem cell enrichment by using a combination of hypoxia/hypoglycemia and drug

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treatment may be useful for different tumor cell lines, independently of their

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origin.

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3.2 Breast cancer stem cell phenotype

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BCSC showed a fibroblastoid, enlarged morphology (Fig. S1B) which

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was completely different to the parental MCF-7 cell morphology, but similar to that found in other breast (SUM-149 and SUM 159) cancer stem cells [3].

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The MCF-7 BCSC growth (Fig. 1A) revealed an increase in both number of cell generations (3.2 ± 0.8 vs. 0.9 ± 0.07 generations from day 3 to day 5 of

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culture) and final cellular densities (62%) attained after 7 days of culture vs. untreated MCF-7; similar observations have been previously reported for

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BCSC isolated by flow citometry [51, 52]. The contents (2-26 times) of CD44, ALDH1A3 and OCT3/4 significantly increased in BCSC vs. parental MCF-7 cells (Fig. 1B). BCSC also generated microspheroids of wider diameters (345 ± 45 m, n=60) compared to parental MCF-7 microspheroids (55 ± 10 m, n=60) after 10 days of culture in free-serum DMEM medium (Fig. S2A). Finally, BCSC showed a greater ability (20-times) to form colonies (from 5 ± 2 in MCF-7 cells to 103 ± 10 in BCSC; Fig. S2B), as previously reported for other BCSCenriched fractions [53].

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3.3 BCSC migration and invasiveness profiles

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Epithelial-mesenchymal transition (EMT), migration and invasiveness

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are typical characteristics of the CSC phenotype [reviewed by 9, 54, 55]. In

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order to assess whether our enriched BCSC fraction exhibits a metastatic profile, the contents of EMT and metastatic proteins as well as migration and

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invasiveness abilities were evaluated. Proteins involved in metastatic

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progression (wnt-1/β-catenin) and cellular migration (vimentin, cytokeratin, fibronectin, N-cadherin) increased by 3-85 times in MCF-7 BCSC vs.

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parental MCF-7 (Fig. 2A), except for MMP1. On the contrary, adherence

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proteins such as E-cadherin (EMT) were significantly (>90%) diminished

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indicating loss in the adherence capacity (Fig. 2A). Parental MCF-7 cells did not show migration (Fig. 2B) and invasiveness (Fig. 2C) abilities, even when the cells were pre-incubated for 24 h with high concentrations (3 μM) of the migration inducer HPI/AMF because the gp78 receptor was not expressed (Fig. 3A). In contrast, BCSC showed active migration (Fig. 2B) and invasiveness abilities (Fig. 2C), which was of a magnitude similar to that of the highly metastatic breast MDAMB231 cancer cells. The BCSC migration rate was slightly stimulated (10-14%) by the addition of exogenous HPI/AMF (3 μM, Fig. 2C) reaching the high migratory rate displayed by MDAMB-231 cells.

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3.4 HPI/AMF inhibition by external E4P blocks the development of the BCSC

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phenotype

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The addition of E4P (1 nM) to MCF-7 cells during the stem cell

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enrichment protocol significantly diminished the contents (56-98%) of the stemness markers CD44, ALDH1A3, Oct3/4, Wnt-1, β-catenin, SNAIL,

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MMP-1, as well as that of HPI/AMF (Fig. S2C). With 1 nM E4P, cells

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showed similar viability (20 ± 1 %) to that observed by using the LGT/HD protocol with no E4P (Table S1). At higher E4P doses (up to 10 nM),

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viability was compromised (3% or lower) and final cellular yield also severely

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decreased. Thus, E4P at 1 nM hindered the development of the stemness

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phenotype without inducing cellular death. It has been documented that several cytokines are secreted to the extracellular medium as metastatic inducers in breast and prostate cancer cells [56; reviewed by 57, 58]. Indeed, Western-blotting analysis revealed higher contents of HPI/AMF (27-times), TGF-β (6.3-times) and interleukins 6 and 8 (5-times) in the extracellular BCSC medium compared to that from parental MCF-7 cells (Fig. 3A). Moreover, the HPI/AMF receptor (gp-78; 20times) and the cytosolic HPI (3-times) increased in BCSC vs. MCF-7 cells (Fig. 3A) supporting the notion that some receptors involved in malignancy such as that for HPI/AMF are over-expressed after stemness activation.

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In order to establish which cytokine was involved in the metastatic

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progression of BCSC, inhibitors of HPI/AMF (E4P), IL-6 (repertaxin), IL-8 and TGF-β (antibodies against TGF-β or IL-8) were used. BCSC growth

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was suppressed by external E4P (24 nM) whereas MCF-7 growth was not

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affected (Fig. S1C). Incubation of BCSC with external E4P at 10 nM for 24 h

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(Figs. 3B, 3C and 5), a concentration lower than the concentration required to inhibit HPI/AMF cytokine function by 50% (IC50 proliferation value = 24 ±

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2.4 nM; n=5), as well as with lower (5 nM) or higher (250 nM) external E4P

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concentrations (Fig. S3) gradually inhibited the expression of several

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proteins associated with stemness (CD44, ALDH1A3), migration (wnt-1/β-

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catenin, MMP-1), EMT (SNAIL), and pluripotency (Oct-3/4) progression vs. BCSC with no external E4P (Fig. 3B). External E4P at 10 nM significantly diminished the content of intracellular HPI/AMF in BCSC (Fig. 3B), whereas intracellular HPI activity was not significantly affected (see next paragraph), indicating that the effect of external E4P occurs at the extracellular level. Perhaps external E4P by blocking HPI/AMF signaling induces repression of the HPI gene (and/or enhances HPI degradation). In fact, 10 nM external E4P for 24 h also blocked the HPI/AMF secretion by BCSC to the extracellular medium (data not shown). Expression of the stemness, migration and pluripotency proteins was more potently blocked by the HPI/AMF antibody (Fig. S3).

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On the other hand, the remaining intracellular HPI enzymatic activity,

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determined with a non-saturating Fru6P concentration of 0.2 mM, in 10 nM external E4P-treated and non-treated BCSC was 32-46% inhibited by 30 M

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added E4P, once the cells were permeabilized by repeated freezing/thawing

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cycles. The activities were 273 ± 62 (6) nmol / (min * 1x106 cells) and 324 ±

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58 (6) nmol / (min * 1x106 cells) in control and E4P-treated cells, respectively. In the presence of 30 μM E4P added to the reaction assay, the

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activities were 148 ± 51 (6) nmol / (min * 1x106 cells) and 219 ± 41 (6) nmol /

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(min * 1x106 cells) in control and E4P-treated cells, respectively. Although

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HPI activity was not significantly different in both E4P-treated and non-

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treated cells as it was observed for HPI protein content, similar behavior is also observed for other metabolic enzymes (GAPDH, GLUT1, LDHA, LDHB, 2-OGDH, IDH1) [59-62; Moreno-Sánchez et al., 2017, unpublished results]. Full inhibition of the HPI activity (with 0.2 mM Fru6P which corresponded to 2 times the KmFru6P value) was achieved by 1 mM E4P. The lowering in the content of all these metastasic proteins by external E4P or HPI/AMF antibody correlated with a diminished BCSC invasiveness ability (Fig. 3C). On the contrary, the IL-8 and TGF-β antibodies (10 µg/mL) and IL-6 inhibitor repertaxin (10 nM) were unable to significantly affect invasiveness (Fig. 3C) and stemness, EMT and pluripotency proteins levels (Fig. 3D) of BCSC. On the other hand, external E4P at 10 nM also blocked

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(65 ± 7.6 %; n=4) the invasiveness ability of MDAMB 231 cells (Fig. 3C),

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whereas repertaxin, anti-IL-8 or anti-TGF-β were innocuous (Fig. 3E).

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To establish whether external E4P impairs the HPI/AMF cytokine

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function by blocking the HPI/AMF-gp78 interaction, co-immuno-precipitation assays were carried out. The gp78 antibody indeed recognized the

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formation of the HPI/AMF-gp78 complex (Fig. 4), as previously described

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[63]. However, this complex was not apparent in the presence of 10 nM external E4P.

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It has been described for several cancer cell lines that the HPI/AMF cytokine pathway is linked to the activation of the MAPK signaling pathway

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[64], which in turn blocks the apoptotic onset through XIAP and NFkB/p65 up-regulation [65, 66]. HPI/AMF inhibition by 10 nM external E4P did not induce apoptosis in BCSC. However, external E4P at 24 nM promoted in BCSC a significant increment (2-3 times) in caspases 3 and 9 contents (Fig. S4A), correlating with apoptosis induction (Fig. S4B) and marked decreased contents (>90%) of proteins related to apoptosis inhibition (p38 MAPK, NFkB/p65 and XIAP). Inhibition of the BCSC invasiveness ability and decreased levels of EMT-related proteins were also induced by external 6PG, another HPI inhibitor (Fig. S5). In parental MCF-7 cells (Fig. 5), 3T3 mouse fibroblasts (Fig. S4C) and HUVEC human endothelial cells (Fig. S4D), external E4P or 6PG (1-50 μM) [26] did not affect cell viability after 24

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h incubation, suggesting that these two metabolites may be considered as

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drugs specifically targeting BCSC.

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3.5 HPI/AMF down-regulation restrains the development of the BCSC

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metastatic phenotype

It seems possible to down-regulate HPI expression in cancer cells

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despite its essential role for the glycolytic pathway [67]. Indeed, BCSC

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transfection with HPI/AMF siRNAs decreased HPI/AMF expression by 70%

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and fully blocked its secretion to the extracellular milieu, with no apparent

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effects on other glycolytic protein levels (Fig. 6A). HPI/AMF siRNA BCSC showed significantly lower metastatic protein levels (Fig. 6A) and migration capacity (Fig. 6B) vs. BCSC. However, the addition of exogenous HPI to HPI/AMF siRNA BCSC completely restored the metastatic phenotype (Figs. 6A and B).

3.6 Effect of external E4P on breast cancer stem cell mammospheres Cancer mammospheres is a tri-dimensional physiological model resembling the basic unit for cancer growth [68, 69] and also mimicking the CSC phenotype, i.e. cells derived from cancer mammospheres show high self-renewal activity, high differentiation potential and elevated EMT-markers

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such as vimentin, CD44, CD133 and ALDH1A3. Also, mammosphere-

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derived cells maintain high capacity to form new colonies [36, 70-72]. Therefore, external E4P was tested on BCSC mammospheres in order to

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evaluate changes in growth, contents of metastatic proteins, extracellular

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cytokine HPI/AMF levels and apoptosis induction.

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Maximal diameter (900 ± 90 µm, n=60 spheroids) of BCSC

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mammospheres was reached by day 20 of culture (Fig. 7A). The cell rearrangement from monolayers to tridimensional mammospheres promoted

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the stabilization and increase of some stem cell markers (CD44 and

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ALDH1A3 but not Oct3/4), metastatic proteins (vimentin, cytokeratin) and

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anti-apoptotic proteins (c-IAP and XIAP but not Bcl2) whereas HPI/AMF slightly decreased (Fig. 7B). The stem cell profile found in mammospheres also revealed the enhanced capacity to (i) express metastatic proteins; (ii) secrete HPI/AMF cytokine and (iii) avoid cellular death expressing antiapoptotic proteins.

Addition of external E4P at 80 nM, i.e. the concentration required to decrease mammospheres growth by 50%, significantly decreased the levels of stemness (20-76%), metastasis (50-90%), and anti-apoptosis markers (40-90%), and HPI/AMF secretion (>70%), indicating that extracellular HPI/AMF inhibition may revert the BCSC phenotype (Fig. 7C). Although long-term exposure to external E4P during mammospheres formation may

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bring about internalization of the inhibitor, intracellular HPI activity was not

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significantly inhibited (Table S3). In addition, tumor glycolysis was not altered either (data not shown; see also 26), indicating that external E4P

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affected exclusively HPI/AMF cytokine signal pathway in BCSC spheroids.

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In addition, E4P-induced down-regulation of Bcl-2, c-IAP and XIAP (Fig. 7C)

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suggested onset of apoptotic cell death [73] and in consequence cellular arrest in both mammospheres (Fig. 7D) and monolayer cell cultures (Fig.

4. Discussion

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S1C).

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4.1 Hypoxia, hypoglycemia and drug exposure as master regulators of the CSC phenotype

Low availability of oxygen and nutrients (i.e., glucose) are factors that stabilize and increase CSC proliferation inside solid tumors [5, 29-31]. For hypoxia, the molecular mechanisms associated to BCSC enrichment are mediated by HIF-1α stabilization which in turn, promotes over-expression of stemness proteins such as Oct3/4, Sox2 and Nanog [6]. For low glucose availability, mechanisms are not completely clear. However, it has been determined that the AMPK activation promotes CSC growth, self-renewal and differentiation [74] as well as autophagy onset for CSC survival under

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nutritional restriction [74, 75]. The molecular mechanism related to the

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enrichment of CSC under drug-exposure involves the over-expression of Pglycoprotein, a multidrug plasma membrane transporter/ATPase, which

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leads to decreased cell mortality and high drug-resistance [76]. This last

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characteristic is the principal factor to be considered in the therapy against

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CSC because it is linked to high recurrence, poor prognosis, poor clinical

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outcome, progression and death [77, 78].

By using the multi-stress cell selection protocol established in the

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present work, an enriched CSC fraction with a pronounced malignant

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phenotype was obtained. This protocol increased (a) the canonical CD44

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stem cell marker for breast tumor cells by 10-26-times vs. parental cells, value comparatively superior to those reported for stem SKBR3 breast cancer cells isolated from a single hypoxic exposure, in which the CD44 increment was 8-12 times vs. parental cells [5, 29-31, 79]. Similarly, this protocol also substantially increased the stemness markers CD44 in A549 cells, CD133 in DAOY cells and ALDH1 in SiHa cells in comparison to their parental cell lines (Table S2). Such marker enhancement was 7-12 times higher than those reported for A549, DAOY and SiHa cells in which CSC enrichment was performed by using only incubation with free-serum medium [80-82]; and (b) increased the CSC cellular yield (60 ± 3%, Fig. 1A) compared to other reported stem cells isolated from head, neck or

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squamous cell carcinoma biopsies (only 5%) by using a single drug

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treatment [83, 84].

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Exposure to a single stress (either hypoxia or hypoglycemia or drug

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treatment) induces a significant enrichment in stem cell markers, increased migration capacity and colony formation, and/or high resistance to

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chemotherapy [31, 42, 43]. However, the stem cell-like phenotype

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disappears once the stemness stimulus is suspended [43]. Therefore, the combination of several stresses is expected to potentiate and improve (i) the

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selection of cells with stem cell phenotype, as described in the present

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study, and (ii) its stability (cells from at least 5 culture passages, equivalent

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to 6.5 cell generations, exhibited maintenance of the stem-cell phenotype when cultured in the absence of the stemness stimuli; data not shown). 4.2 HPI/AMF inhibition by external E4P as therapeutic strategy against breast cancer stem cells

The low specificity of chemo- and radiotherapy eventually brings about cancer treatment failure and recurrence driven by the selection and survival of CSC. In this regard, multiple strategies have been developed for CSC abrogation including targeting specific surface markers (CD44, CD133), modulating signaling pathways (Notch, NFkB ), inhibiting ABC drug-efflux pumps (MDR inhibitors), and adjusting microenvironment signals (pH,

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substrate availability) [4, 85, 86]. Although some of them (in combination

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effects in patients are frequently observed [87-89].

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with traditional therapies) have been apparently successful, adverse side

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Several cytokines, chemokines and growth factors (CXCL12, TGF-, EGF, PDGF, FGF, IL6, IL8 and HPI/AMF) are released by fibroblasts, tumor-

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associated macrophages, neutrophils, stem cells and mast cells to the

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extracellular milieu to stimulate stem cells migration from tumors [8, 18, 20, 26, 90]. In consequence, blockage of these cytokines might in turn, inhibit

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migration and invasiveness of cancer and cancer stem cells [20, 26, 91]. All

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these cytokines including HPI/AMF are also secreted by non-cancer cells

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compromising the therapeutic use of cytokine inhibitors or antibodies [22, 92]. However, secreted HPI/AMF levels by non-cancer cells are substantially lower than those reached by cancer cells [93]. Thus, HPI/AMF has been proposed as a cancer biomarker [94, 95]. Furthermore, the use of repertaxin, a canonical cytokine inhibitor, and IL8 or TGF antibodies did not affect viability, migration and invasiveness of BCSC whereas external E4P showed high potency. In agreement with previous reports [26, 93, 96], both extracellular and intracellular HPI/AMF levels were markedly higher in cancer stem cells vs. non-stem cancer cells. In addition, HPI/AMF expression as well as the development of the stem cell phenotype were drastically and selectively

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depressed by external E4P. Thus, the E4P-HPI/AMF complex blocked the

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HPI/AMF interaction with its plasma membrane receptor gp78, suppressing the activation of the transduction pathways associated with the onset of

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EMT/migration/invasion and the development of a metastasis phenotype.

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This proposal was further supported by the similar results obtained with (i)

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6PG, which is another HPI/AMF ligand; (ii) HPI antibody, which sequestered extracellular HPI/AMF; and (iii) HPI down-regulation, which severely limited

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HPI/AMF secretion.

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External E4P also exerted deleterious effects on BCSC growth, which

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seemed associated with apoptosis induction as it has been determined for

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metastatic colon carcinoma exposed to E4P (500 mg/Kg weight for 15 days in BALB/C mice) [97]. As HPI affinity for E4P is much greater than for 6PG, E4P seems a more attractive new anti-metastatic drug to be considered for further studies and clinical trials [97]. It should be noted that E4P is a watersoluble small molecular weight inhibitor which does not have to be internalized by cancer cells to exert its anticancer effects. These E4P properties may facilitate its eventual clinical use.

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4.3 Multi-cellular tumor microspheroids of BCSC predicts behavior of stem

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cells inside solid tumors

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Tumor microspheroids resemble the initial stages of solid tumor

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development, in which hypoxic/hypoglycemic micro-regions gradually surge [98]. BCSC are able to form microspheroids [36, 71], although at slower

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rates than non-CSC, probably because cell-cell adherence proteins such as

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E-cadherin are scarce thus rather favoring migration and invasiveness processes [55]. The spheroid tridimensional rearrangement promoted

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stabilization and over-expression of EMT- and metastasis-related proteins.

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Remarkably, external E4P in the nanomolar range blocked BCSC

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microspheroid formation. This observation suggested that E4P may be a therapeutically viable new drug for deterring the development of metastatic phenotypes in both 2D and 3D tumor cell models. Author contributions

SRE and JCGP: experimental conception and design, analysis and interpretation of data; AAL, AMH and JCGP: acquisition and statistical analysis of data and development of methodology; SRE and RMS: writing of the manuscript; SRE and RMS: discussion on analysis and interpretation of data and manuscript revision. SRE, JCGP and RMS: obtained funding. All authors read and approved the final manuscript.

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Competing financial interests

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The authors declare no competing financial interests.

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Funding

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Authors thank Biol. Marco Antonio García-Amezcua for technical assistance. The present work was partially supported by grants from CONACyT-México

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to JCP (243249), SRE (107183), AMH (180322) and RMS (239930 and

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D

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281428).

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Figure 1. MCF-7 and breast cancer stem cell (BCSC) proliferation (A) and stemness markers (B). The culture medium was DMEM with 25 mM glucose

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Figure 3. External erythrose 4-phosphate (E4P) inhibits expression of stemness proteins and invasiveness in breast cancer stem cells. Contents

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of (A) extracellular cytokines and growth factors; and (B) stem cell markers, EMT, HPI/AMF and pluripotency related proteins. Where indicated, cells were incubated with 10 nM external E4P for 24 h prior to Western blot analyses. (C) Effect of external E4P, HPI/AMF antibody (10 g/mL), and IL6, IL8 and TGF-β inhibitors on cancer cell invasiveness. (D) Effect of IL-6, IL8 and TGF-β inhibitors on stemness, EMT and pluripotency protein levels in BCSC. (E) Effect of IL-6, IL8 and TGF-β inhibitors on MDAMB-231 invasiveness. n=4; *P<0.01 and **P<0.05 vs. MCF-7 (A) or BCSC untreated (B and C). For secreted proteins a band of  50 KDa of unknown identity but invariable presence was used as load control in Ponceau S stain transferred membranes. Abbreviations: C, cytosolic, R, receptor; RT, repertaxin; ab, antibody against IL-8 or TGF-β. Figure 4. HPI/AMF-gp78 interaction in BCSC. Representative coimmunoprecipitation blots of HPI/AMF and receptor gp78 of three

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independent BCSC batches. Protein A-agarose beads were overnight preincubated at 4°C with gp78 antibody or IgG. Then, the beads were further

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 Breast cancer stem cells secrete the hexosephosphate isomerase (HPI) cytokine  Secreted HPI/AMF induces EMT, invasiveness and metastatic phenotype  The external HPI inhibitor E4P halts the development of the stem cell-like phenotype  The external HPI inhibitors E4P and 6PG depressed the invasiveness and malignant phenotype  E4P interaction with HPI/AMF may be used as an alternative in anti-cancer therapy