Stress resistant human embryonic stem cells as a potential source for the identification of novel cancer stem cell markers

Stress resistant human embryonic stem cells as a potential source for the identification of novel cancer stem cell markers

Cancer Letters 289 (2010) 208–216 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet Stress ...

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Cancer Letters 289 (2010) 208–216

Contents lists available at ScienceDirect

Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Stress resistant human embryonic stem cells as a potential source for the identification of novel cancer stem cell markers Shaker A. Mousa a, Thangirala Sudha a, Evgeny Dyskin a, Usawadee Dier a, Christine Gallati a, Christine Hanko a, Sridar V. Chittur b, Abdelhadi Rebbaa c,* a

The Pharmaceutical Research Institute at Albany, Albany College of Pharmacy and Health Sciences, Albany, NY, United States Center for Functional Genomics, University at Albany, State University of New York, Albany, NY, United States c Department of Pathology, University of Pittsburgh, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, PA, United States b

a r t i c l e

i n f o

Article history: Received 16 April 2009 Received in revised form 11 August 2009 Accepted 12 August 2009

Keywords: Cancer stem cells Drug resistance Markers Trichostatin A cDNA array

a b s t r a c t Cancer stem cells are known for their inherent resistance to therapy. Here we investigated whether normal stem cells with acquired resistance to stress can be used to identify novel markers of cancer stem cells. For this, we generated a human embryonic stem cell line resistant to Trichostatin A and analyzed changes in its gene expression. The resistant cells over-expressed various genes associated with tumor aggressiveness, many of which are also expressed in the CD133+ glioma cancer stem cells. These findings suggest that stress-resistant stem cells generated in vitro may be useful for the discovery of novel markers of cancer stem cells. Ó 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The development of resistance to chemotherapy represents an adaptive biological response by tumor cells that leads to treatment failure and patient relapse. In recent years, it has become obvious that cancer cells can develop resistance not only to classical cytotoxic drugs, but also to the newly discovered targeted therapies [1,2]. Important progress has been made in identifying putative mechanisms responsible for this phenomenon [3] such as alterations in drug transport and drug or target modifying enzymes [4–6]. Subsequently, a number of drug resistance reversing agents were discovered and, although they were very effective in vitro, most did not perform as well in vivo [7]. In view of the significance of new drug discovery in this area, research interest was re-directed toward the study of

* Corresponding author. Address: Children’s Hospital of Pittsburgh of UPMC, Department of Pathology, Room B269, 45th Street & Penn Avenue, Pittsburgh, PA 15201, United States. Tel.: +1 412 692 7043. E-mail address: [email protected] (A. Rebbaa). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.08.018

alternative drug resistance mechanisms such as those mediated by deficiencies in apoptosis or senescence pathways [8,9]. The effectiveness of these strategies in the control of cancer progression, particularly in vivo, is still under investigation. In addition to these mechanisms, the concept of cancer stem cells (CSCs) responsible for tumor origin, maintenance, and resistance to treatment has gained prominence in the last few years [10–12]. This prompted interest in identifying and targeting cancer stem cell populations within tumors, and generated high expectations that this approach may become an important treatment modality to prevent disease recurrence. However, in order to target CSCs, specific molecular markers must be identified. Until now, only a very limited number of such markers have been discovered, mainly due to the fact that their identification necessitates the challenging task of isolating CSCs from existing tumors. Most current approaches to fractionate this cell population from diverse cancers take advantage of common cell surface markers such as CD133 and CD44 [11], however, since none of these markers is

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exclusively expressed by solid tumor CSCs, it is imperative to delineate more specific and universal markers. In the search for alternative approaches to accelerate the discovery of novel cancer stem cell markers, we adopted a strategy that takes into account their inherent resistance to therapy. Considering the possibility that normal stem cells may acquire a resistance phenotype even before becoming tumorigenic (Fig. 1), we sought to determine whether stress-resistant stem cells generated in vitro can be used as a source to identify novel markers of CSCs. For this, we selected the human embryonic stem cell line (BG01V) for resistance to the histone deacetylase inhibitor Trichostatin A. The resulting cells were characterized for their response to stress, expression of markers of self renewal and pluripotency, and for the expression of putative markers of cancer stem cells. This resulted in the identification of various candidate genes, some of which are already known as markers of cancer stem cells. Validation of this approach was carried out by comparing genes expression profiles between the TSA-resistant stem cells and the previously described glioma cancer stem cells isolated based on over-expression of the CD133 antigen [13]. We show that almost half of the genes that characterize this cancer stem cell line, including the CD133, were also differentially expressed in the TSA-resistant stem cells. Our findings suggest that stress-resistant embryonic stem/progenitor cells generated in vitro can be used as a source to identify putative markers of cancer stem cells, without the need of isolating the latter cells from existing tumors. 2. Material and methods 2.1. Cell culture and drug selection The human embryonic stem cells BG01V were obtained from ATCC (Manassas, VA) and maintained on mitomycin C-treated MEF (CF-1) MITC (ATCC, Manassas, VA) feeder

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layer. Undifferentiated hESC colonies were dissected using a glass pipette and re-plated onto a fresh feeder layer every 7 days. DMEM/F12 (Invitrogen, Carlsbad, CA), supplemented with 20% knockout serum replacement (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM b-mercaptoethanol (Sigma), 0.4 ng/ml bFGF (Invitrogen), 50 U/ml penicillin (Invitrogen) and 50 lg/ml streptomycin (Invitrogen), is used as hESC culture medium, and the cells were incubated at 37 °C in 95% air and 5% carbon dioxide (CO2). In some experiments, human embryonic stem cells were propagated using the Feeder-free mTeSRTM1 medium from StemCell Technologies according to the manufacturer’s protocol. Selection experiments were carried out by cultivating human embryonic stem cells in stepwise-increasing concentrations of the histone deacetylase inhibitor, Trichostatin A (TSA) according to the previously described procedure [14]. The cells were allowed to adapt to a given stress level before transferring them to the next level. After several adaptation cycles, the generated TSA-resistant stem cells were characterized at the cellular and molecular levels. To compare population doubling between parental and the TSA-resistant cells, cells were seeded at 1  104 cells per cm2 of growth area and sub-cultured every 5 days. A Coulter Counter was used to determine cell number, and cumulative population doubling level at each sub-cultivation was calculated from the cell count and added to the previous population doubling level (PDL) as described previously [15]. Cellular response to stress was determined by cultivation for 48 h in the absence or the presence of increasing concentrations of TSA for up to 500 nM. Cell viability was determined using the MTT assay as described previously [16]. Briefly, the cells cultured in a 24 well plate under feeder-free conditions (using the mTeSRTM1 medium) were treated with increasing concentrations of TSA for 48 h. Fifty microliter of MTT solution (5 mg/ml) was added to each well (containing 500 ll of medium) and incubated

Stress

Normal stem cell

Stress resistance genes

Oncogenes

Cancer stem cell

Stress resistance genes and oncogenes

Aggressive cancer Fig. 1. Schematic representing hypothetical processes leading to the formation of cancer stem cells and aggressive tumors. The accumulation of damage caused by stress to normal stem cells may either cause induction of oncogenes, then stress resistance genes (red arrows), or stress resistance genes then oncogenes (black arrows). Cancer stem cells are characterized by the expression of both oncogenes and stress resistance genes.

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for 4 h at 37 °C. The cells were then solubilized by addition of 500 ll of 10% SDS/0.01 M HCl and optical density was measured in100 ll of this solution using an ELISA plate reader with an activation wavelength of 570 nm and reference wavelength of 650 nm. 2.2. Immunostainings Colonies grown in Lab-TekTM Chamber Slides (Nunc, Rochester, NY) were fixed in 4% paraformaldehyde for 5 min and after washing, they were incubated at 4 °C for 15 h with antibodies against the following stem cells markers: Rabbit anti-human Oct3/4 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human SSEA-4, rabbit anti mouse SSEA-1 antibodies (Millipore, Billerica, MA), mouse anti-human TRA-1-81 and mouse anti-human TRA-160 (Abcam, Cambridge, MA). After wash with PBS, the cells were incubated for 1 h at 4 °C with fluorescence labeled secondary antibodies and after washing three times with PBS, expression of stem cell markers was detected using confocal microscopy.

SYBR Green PCR buffer, 3 ll of 25 mM MgCl2, 2 ll of dNTP mix (2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, and 5 mM dUTP), 0.125 ll of AmpliTag Gold DNA polymerase (5 units/ll AmpliTag Gold DNA polymerase), and 10.125 ll of H2O. The reaction was amplified with iCycler iQ Multicolor Real Time PCR Detector (Bio-Rad) for 37 cycles with melting at 94 °C for 30 s, an annealing at 58 °C for 30 s, and extension at 72 °C for 1 min in iCycler iQ PCR 96-well plates (Bio-Rad).

2.5. Statistical analysis Data are expressed as means ± SE. Differences in measured variables between the experimental and control groups were assessed by Student’s t test. Statistical calculations were performed using the Statview statistical

A

BG01V

BG01V-TSAR

2.3. Microarray analysis of differentially expressed genes mRNA was extracted using the RNAeasy kit (Qiagen). RNA labeling, hybridization on GE Healthcare CodeLink Gene Expression Bioarrays (Human Whole Genome) and data analysis were performed by GenUs Biosystems. Two biological replicates for each sample were analyzed. Intensity values after hybridization were normalized to the median intensity of the chip and ratios between samples were calculated for each gene in either stress-resistant or parental stem cell lines. Relative changes in expression levels for each gene in both cell lines were calculated (heatmap). Validation of microarray data by quantitative RT-PCR was performed as described above for selected genes.

Adh.

EBs

B

12 BG01V BG01V-TSAR

10

Total RNA was isolated using Qiagen Rneasy mini kit (Qiagen Inc., Valencia, CA) as recommended by the supplier. Total RNA was quantified by OD at 260 nm. Using equal amount of total RNA (200 ng), stimulated under various conditions, mRNA was primed with random hexamers, and complementary DNA (cDNA) was synthesized from mRNA by TaqMan reverse transcription with MultiScribe reverse transcriptase (PE Applied Biosystems, Foster, CT) according to the manufacturer’s description. The final cDNA product was used for subsequent cDNA amplification by polymerase chain reaction. cDNA was amplified and quantitated by using SYBR Green PCR reagents from PE Applied Biosystems according to the manufacturer’s instructions. The cDNA for GAPDH was amplified by using a specific forward primer (50 -GAA GGT GAA GGT CGG AGT C-30 ) and a specific reverse primer (50 -GAA GAT GGT GAT GGG ATT TC-30 ). The list of primers used to detect known and putative stem cell markers is presented in the Supplemental data S3. The PCR reaction mixture (final volume 25 ll) contained 5 ll of cDNA, 1 ll of 10 lM forward primer, 1 ll of 10 lM reverse primer, 2.5 ll of PCR 10

Cumulative PD

2.4. Gene expression analysis by quantitative PCR 8 6 4 2 0

5

10

15 20 25 Days of cultivation

30

35

Fig. 2. Morphological and proliferative characteristics of parental (BG01V) and the TSA-resistant (BG01V-TSAR) stem cells. Panel A. The two cell lines were cultured either on mouse fibroblasts under normal culture conditions, or in medium with fibroblasts and bFGF (in order to form embryoid bodies, EBs). Adherent (Adh) colonies and EBs formation were monitored under light microscopy and photographs taken under 20 magnification. Panel B. Proliferation rate of the two cell lines was followed over a 30 days period. Cell numbers were determined at the end of every passage and cumulative population doublings (PD) were calculated in relation to cell numbers at the first passage. Data represents mean ± SE of three determinations.

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package (Abacus Concepts, Berkeley, CA). Ps < 0.05 were considered as statistically significant. 3. Results The generation of stress-resistant stem cells was achieved after serial cultivation of the human embryonic stem cells BG01 V in the presence of sub-toxic amounts of the histone deacetylase inhibitor, Trichostatin A (TSA), used here as the stressor. Morphologically, the resistant cells (BG01-TSAR) grow as colonies of undifferentiated cells resembling their parental ones (Fig. 2A). The proliferative behavior of resistant stem cells was also similar to their parental counterparts, and both cell lines were able to form embryoid bodies (EBs) when cultivated in the absence of serum (Fig. 2A). The two cell lines also seemed to grow at comparative rates (Fig. 2B) suggesting that the resistant stem cells maintained key characteristics pertaining to morphology and proliferative behavior. 3.1. Comparison of resistance to stress Although both parental and the TSA-resistant stem cells displayed similar phenotypes when cultivated in the absence of stress, their survival abilities in the presence of TSA were quite different. As shown in Fig. 3A, parental colonies become disintegrated as a result of accelerated death of individual cells when exposed to 5  108 M of the drug, however the resistant cells continued to proliferate under such conditions. The difference in stress response between the two cell lines can be better appreciated by comparing surviving fractions after 4 days in culture using the MTT assay (Fig. 3B), as even 108 M of TSA was toxic for the parental cells. Interestingly the resistant cells were less responsive not only to TSA, but also to the DNA damaging drug doxorubicin and the DNA de-methylating agent 5 Aza-C (Fig. 3B and C). This multi-drug resistance characteristic

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mirrors those of cancer cells after being continuously cultivated in the presence of drugs [14], suggesting that common pathways may account for resistance to stress in both cancer and stem cells. Moreover, since TSA is known to induce histone hyperacetylation, our findings (Fig. 3) suggest that alteration in the epigenetic makeup of stem cells may be sufficient for causing stress resistance.

3.2. Molecular characterization of the TSA-resistant stem cells Immunohistochemistry analyses were carried out to compare expression of self renewal and pluripotency markers between parental and the TSA-resistant cell lines. As shown in Fig. 4 (Panels A and B), there was an overall decrease in expression particularly of SSEA-4, TRA-1-60 and TRA1-81 in the BG01-TSAR compared to parental cells. SSEA-1 which is a marker for differentiation was not detected in any of the two cell lines (data not shown). Western blot analysis (Fig. 4C) confirmed that the expression levels of the stem cell markers OCT4 and Sox2 did not change, however, there was a slight increase in expression of the histone deacetylase Sirt1. The later event may represent a cellular adaptation to the reduced activity of other HDACs caused by TSA. As changes in certain stem cell markers (TRA-1-81 and TRA-1-61) did not appear to affect the proliferative behavior of the TSA-resistant stem cells when compared to their parental counterparts (Fig. 2), it is suggested that alternate proliferative pathways to those controlled by the stemness maintenance genes described above may be activated in the resistant cells.

3.3. Analysis of global gene expression profiles A comprehensive analysis of gene expression profiles between parental and the TSA-resistant stem cells was carried out using the Affymatrix cDNA microarray. The scatter plot (Fig. 5A) indicates that expression of

Fig. 3. Comparison of cellular response to stress. Panel A. Parental and TSA-resistant stem cells were incubated in the absence or the presence of TSA at the indicated concentration for 48 h. Photographs were taken to illustrate colony disintegration due to individual cell death. Panels B and C. Parental (BG01V) and the TSA-resistant stem cells (BG01V-TSAR) were incubated for 72 h in the absence or in the presence of doxorubicin (Dox.), TSA, or 5 azacytidine (5-AzaC) at the indicated concentrations. The media was then replaced by DMEM free of drugs and number of colonies determined after additional 72 h incubation. The data represent means of three determinations ± SE.

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A

BG01V

BG01V-TSAR

Oct-4

SSEA-4

TRA-1-81

TRA-1-60

Fluorescence intensity (A. U.)

B

BG01V

400

C

BG01V-TSAR

WT

TSAR

Oct3/4

350 300

Sox2

250 200 150

Sirt1

100 50

β-actin

0 Oct-4

SSEA-4

TRA-1-81

TRA-1-61

Fig. 4. Analysis of cancer stem cell marker expression. Panel A. Parental (BG01V) and the TSA-resistant (BG01V-TSAR) were allowed to form colonies on coverslips and then fixed and stained with antibodies against markers of cancer stem cells. The resulting reactions were visualized under fluorescence microscope. Panel B. Leica Confocal Software (LCS) quantification of fluorescence intensity of the images shown in panel A. Panel C. Western blot analysis of expression of the stem cells markers Oct3/4 and Sox2, compared to those of Sirt1 and b-actin in wild type (WT) BG01V and the their (TSA) resistant TSAR counterparts BG01V-TSAR.

most genes was similar between the two cell lines, however, about 1800 genes were differentially expressed, with more than half of these genes up-regulated in the resistant cells. Interestingly, there were no dramatic changes in the expression profiles of major stem cell markers between the two cell lines (Supplemental data S1) as the relative amount were less than two times in most cases, providing further explanation for the observed retention of stem cell characteristics by the TSA-resistant cell line (Fig. 2).

In contrast to this, expression of genes with relevance to stress resistance and metastasis (two major characteristics of cancer stem cells) were enhanced in the resistant stem cells (Supplemental data S2). Among these, known markers of cancer stem cells including SNAI2 [17] and Abcg2 [18], were differentially expressed. It has been shown for instance that in human breast cancer, the expression of SNAI1 and/or the homologous SNAI2 (Slug) were associated with local or distant metastasis, tumor recurrence, and poor prognosis in different tumor series [19].

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A

B

0.0016 WT TSA-R

Normalized Mean Expression

0.0014 0.0012 0.0010 0.0008 0.0006 0.0004 0.0002 0.0000 ATF-3

NUAK-2

EGR-1

ID2

CYR61

uPA

Claudin-4

Fig. 5. Differentially expressed genes between parental and the TSA-resistant stem cells. Panel A. A Scatter plot indicating that expression of most genes was not changed between the two cell lines, however a significant number (about 1800) were differentially expressed. The proportion of up-regulated genes in the resistant cells (TSAR) appears to be higher than the down-regulated fraction (induced in the wild type cells, WT). Panel B. Expression of selected genes was validated by quantitative PCR. The data represent mean of three determinations ± SE.

SNAI2 has been also shown to be implicated in diseases of melanocyte development and cancer in humans [19]. In addition, this gene controls key aspects of stem cell function in mouse and human [17], suggesting that it may represent a useful tool for the identification and targeting of cancer stem cells. Genes implicated in cellular response to stress in general (i.e. bZip transcription factor, ATF3), response to chemotherapy (i.e. Abcc2), or metastasis (i.e. Claudin-4, Cyr61 and PLAU) were also induced in the TSA-resistant stem cells (Supplemental data S2). ATF3 was reported to directly regulates the proliferating cell nuclear antigen (PCNA)-associated factor KIAA0101/p15(PAF). Furthermore, expression of ATF3 and p15 (PAF) stimulated the DNA repair machinery in response to stress and their deficiency impaired the repair mechanisms [20]. Over-expression of ATF3 was also found to result in the development of squamous metaplastic lesions in mice, suggesting that this transcription factor acts as a mammary

oncogene [21]. As this gene is also highly expressed in human breast tumors, it may have potential use as a biomarker or a therapeutic target for these diseases. Abcg2 is a well described drug resistance gene and a marker of cancer stem cells [18]. Enhanced expression in the TSA-resistant stem cells (Supplemental data S2) of this gene as well as that of Abca1, a member of the same family, provided further support the concept that these cells are indeed multi-drug resistant and mimic the behavior of cancer stem cells. It also strengthen the rational for potential use of these cells as a source for the identification of novel markers to identify and target cancer stem cells. Claudin-4 is frequently over-expressed in several neoplasias, including ovarian, breast, pancreatic, and prostate cancers [22]. Although the exact role of this protein in tumorigenesis is not well defined, recent studies indicated that increase in its expression was associated with poor prognosis and high tumor grade in breast cancer [23], and also that this

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Table 1 Common genes expressed in the TSAR and in the CD133+ glioma cancer stem cells. Affymatrix ID

Gene symbol

NCBI ID

Genes down regulated 8131844 8040742 8118564 8018966 7929072 7913694 7979473 7973377 8045499 8115147 8090433 7978932 8125530 8178193 8018937 8015835 8125436 8042207 8125556 7918359 8088803 8005586 8076690 8062880 7934615 7966259 8178826

GPNMB MAPRE3 HLA-DQA2 TIMP2 IFIT5 FUCA1 DHRS7 BCL2L2 HNMT CD74 MGLL SOS2 HLA-DMB HLA-DRA USP36 DUSP3 HLA-DRB5 COMMD1 HLA-DPA1 AMIGO1 EIF4E3 ZNF179 C22orf9 YWHAB DLG5 GLTP HLA-DQB1

NM_001005340 NM_012326 NM_020056 NM_003255 NM_012420 NM_000147 NM_016029 NM_004050 NM_006895 NM_001025159 NM_007283 NM_006939 NM_002118 NM_019111 NM_025090 NM_004090 NM_002125 NM_152516 NM_033554 NM_020703 NM_173359 NM_007148 NM_001009880 NM_003404 NM_004747 NM_016433 M24364

Genes upregulated 7962455 8137252 NM_130759 8161919 0.23079 8021826 7991246 8138941 8146921 8059279 7940028 8099476 8162940 8059249 7946446 8122045 8093518

NELL2 GIMAP1 NEDD4L TLE1 SOCS6 SALL3 RLBP1 NT5C3 RDH10 EPHA4 SERPING1 PROM1 (CD133) ABCA1 OBSL1 NRIP3 AKAP7 FGFR3

NM_006159 NM_130759 NM_015277 NM_005077 NM_004232 NM_171999 NM_000326 NM_001002009 NM_172037 NM_004438 NM_000062 NM_006017 NM_005502 NM_015311 NM_020645 NM_01637 NM_000142

molecule can be used for imaging and targeted therapy for pancreatic cancer [24]. In light of this, claudin-4 and perhaps other members of its family may represent promising targets for cancer detection, diagnosis, and therapy. The potential role of claudin-4 as a marker for cancer stem cells in these tumors is however not yet reported. The cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, thought to be implicated in cellular growth and differentiation. Recent evidence indicated that this gene was also associated with increased migration and expression of matrix metalloproteinase (MMP)-13 in human chondrosarcoma cells [25]. Cyr61 and the urokinase plasminogen activator (uPA) were found to act as mediators of sphingosine 1phosphate (S1P)-induced cancer cell invasion [26]. The uPa related pathways also play a role in cancer stem cell biology as described by a recent study showing that uPAR-positive cells in all the SCLC lines tested displayed multi-drug resistance, high clonogenic activity, and co-expression of the cancer stem cell markers CD44 and MDR1 [27]. These observations suggest that a putative relationship may exist between Cyr61 and cancer stem cells. The differential expression for some of the genes described above and others shown in Supplemental data S2 was validated by quantitative PCR (Fig. 5B) and five out the seven genes tested were found to be increased in

the TSA-resistant cells. Taken together, these findings suggest that the development of stress resistance in normal stem cells cause them to acquire certain characteristics of cancer stem cells, particularly those related to the over-expression of genes responsible for tumor aggressiveness. 3.4. Comparison of genes that characterize the TSA-resistant stem cells and the CD133+ glioma cancer stem cells To further evaluate the authenticity of differentially expressed genes between parental and the TSA-resistant stem cells as potential markers of cancer stem cells, we compared them to those that characterize the previously described brain tumor cancer stem cell line, the CD133 positive glioma [13]. The data presented in Table 1 indicates that the CD133 antigen, based on which glioma cancer stem cells were isolated, was also enhanced in the TSA-resistant cell line. Moreover, 17 out of the 39 genes induced in the CD133+ glioma cancer stem cells [13] were also induced in the TSA-resistant stem cell line, and 27 out of the 68 genes decreased in the CD133+ glioma cancer stem cells were also decreased (Table 1). Notably, FGF receptor which is known to play a key role in cancer cell proliferation was induced in both the TSA-resistant stem cells and the glioma cancer stem cells. The oncogenic potential of this receptor was demonstrated using the murine bone marrow (BM) cells transduced with retroviral vectors containing either wild-type FGFR3 or an activated mutant form of the receptor, FGFR3-TD. Mice transplanted with FGFR3-TDexpressing BM developed a marked leukocytosis and lethal hematopoietic cell infiltration of multiple tissues within 6 weeks of transplantation [28]. Another potential marker of cancer stem cells identified by this approach is the Ephrin A4 receptor, known to participate in tumor aggression [29]. Its close family member EphB2 was found to be over-expressed in the CD44-positive colorectal adenoma cells [30], suggesting that it may represent a potential marker for this cell population. Other genes such as NELL2, SALL1, SOCS6, ZFP516, and the CD74 antigen may also be associated with cancer stem cell identity as they were expressed in both the CD133+ cells and the TSA-resistant hESCs. Further studies will be needed to confirm or rule out the possibility that these genes may be considered as markers of cancer stem cells.

4. Discussion Stress-induced accumulation of damage in normal cells is considered a major causative factor of cancer and other chronic diseases. Based on this well known concept, continuous exposure stem cells to stress will likely cause them to become senescent [31–33], to die [34,35], or survive and feed neoplastic formation [36–38]. Cancer stem cells are a rare population of tumor cells, characterized by enhanced expression of both oncogenes and stress resistance genes. Since in the process of their biogenesis, they may acquire the resistance phenotype even before becoming cancerous (Fig. 1), stress-resistant stem cells generated in vitro could share similarities with cancer stem cells, particularly with regards to the expression of genes associated with tumor aggressiveness. Based on this, the present study was designed to test the hypothesis that stress-resistant stem/ progenitor cells generated in vitro, can be used as a source for discovery of novel markers of tumor aggressiveness that characterize cancer stem cells. The findings presented here provided evidence supporting the validity of this concept and shed light on a new avenue that may lead to the discovery of a new generation of therapeutics. The TSA-resistant cells generated in vitro by continuous exposure to stepwise increased concentrations of the drug, maintained major stem cell characteristics including their ability to proliferate, to form embryoid bodies, and to express self renewal and pluripotency markers (Figs. 2 and 4). However, a noticeable phenotypic difference between the two cell lines was that the resistant cells had superior

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ability to survive in the presence of relatively high concentrations of TSA, 5-AzaC and doxorubicin (Fig. 3). This multidrug resistance characteristic was strikingly similar to that observed in cancer cells selected with TSA [14], suggesting that the underlying molecular mechanisms of resistance to stress may be similar between stem and cancer cells. In this regard, therapy-resistant stem cells generated in vitro may also represent a suitable model to study the pathways that control stress resistance in general. By comparing global gene expression profiles between the TSA-resistant and parental stem cells, various genes implicated not only in stress resistance, but also in metastasis were identified (Fig. 5 and Supplemental data S2). This suggests the existence of a positive relationship between resistance to therapy and metastatic potential, investigation of which may establish which one is a causative factor of the other. Regardless of this, our findings suggest that the acquisition of both resistance to stress and metastatic potential may occur prior to the onset oncogenic transformation and the biogenesis of cancer stem cells. Further studies to determine the tumorigenic potential of resistant stem cells in animal models would be required to establish this concept. It is also noteworthy that, although the comparison of gene expression profiles between parental and the TSAresistant cells provided information on possible mechanisms of resistance to stress in these cells, it does not indicate whether these genes are expressed in actual cancer stem cells. To address this, we compared gene expression profiles between the TSA-resistant stem cells and those of the previously reported CD133+ glioma cells [13], the most described model of brain tumor cancer stem cells. Interestingly, many genes, including the CD133 antigen itself, were found to be commonly expressed between the two cell lines (Table 1). As some of these genes have a demonstrated role in cancer cell survival and aggressiveness, their potential role as cancer stem cell markers is greater. Overall, findings from this study suggest that putative markers of cancer stem cells can be identified without having to isolate these cells from existing tumors. This can be achieved by using stem cells trained in vitro to become stress-resistant. This approach is expected to open new opportunities for accelerated discovery and targeting of cancer stem cells in situ and the eradication of aggressive tumors. The discovered markers may also represent valuable tools for the isolation of novel cancer stem cell lines to be used not only for research purposes to understand the molecular mechanisms leading to their biogenesis, but also as drug testing tools to facilitate the design of novel therapeutic modalities. Conflict of interest The authors declare that there is no conflict of interest associated with this work. Acknowledgement This work was supported in part by the Pharmaceutical Research Institute in Albany, NY.

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