Side population and cancer stem cells: Therapeutic implications

Cancer Letters 288 (2010) 1–9

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Mini-review

Side population and cancer stem cells: Therapeutic implications Lidia Moserle a,1, Margherita Ghisi a,1, Alberto Amadori a,b, Stefano Indraccolo b,* a b

Department of Oncology and Surgical Sciences, University of Padova, Italy Istituto Oncologico Veneto, IRCCS, Padova, Italy

a r t i c l e

i n f o

Article history: Received 17 March 2009 Received in revised form 15 May 2009 Accepted 18 May 2009

Keywords: Side population Cancer stem cells Chemotherapy Interferon

a b s t r a c t New studies indicate that the side population (SP) and cancer stem cells (CSC) drive and maintain many types of human malignancies. SP and CSC appear to be highly resistant to chemo- and radio-therapy and this knowledge is now reshaping our therapeutic approach to cancer. Several studies have pioneered the possibility of specifically targeting CSC and SP cells by exploiting pathways involved in drug resistance, or forcing these cells to proliferate and differentiate thus converting them into a target of conventional therapies. Moreover, certain cytokines – such as IFN-a – appear to modulate SP and stem cell functions, and this associates with remarkable therapeutic activity in animal models. These recent findings underscore the need of a more comprehensive view of the interactions between cytokines and key regulatory pathways in SP and CSC. Ó 2009 Published by Elsevier Ireland Ltd.

1. Introduction More than two thirds of cancer patients currently receive either chemotherapy or radiotherapy, which in many cases have beneficial effects and can improve quality of life and survival. In spite of the broad use of these therapies, however, many unresolved issues still remain regarding the mechanisms that determine remission or the subsequent relapse. During the last years, new studies have described the rather heterogeneous tumorigenic potential of cancer cells and led to the development of the cancer stem cell (CSC) hypothesis. This hypothesis postulates that only a fraction of cells within a tumor is endowed with stem cell-like features, including unlimited proliferative potential and asymmetric cell division, giving rise to all the other components of the neoplasm. This cellular subset, whose size may vary in different tumors, can initiate and sustain tumor growth and is believed to drive relapse.

* Corresponding author. Istituto Oncologico Veneto, IRCCS, via Gattamelata, 64, 35128 Padova, Italy. Tel.: +39 0498215875; fax: +39 0498072854. E-mail address: [email protected] (S. Indraccolo). 1 The first two authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.canlet.2009.05.020

The prospective identification of CSC in tumors has proven to be challenging. Two different approaches have been proposed: the first one tracks specific surface markers that are selectively expressed on CSC but not on the bulk of tumor cells. The second approach exploits some functional characteristics, such as a unique pattern of staining with certain dyes, to identify stem-like cells. This latter method is used for the detection by dual-wavelength flow cytometry of the so-called side population (SP) on the basis of the ability of these cells to efflux the fluorescent DNA-binding dye Hoechst 33342. SP cells express high levels of ATPbinding cassette (ABC) transporter family members, such as MDR1 and ABCG2, responsible for the extrusion of both Hoechst 33342 and some drugs [1,2]. In 1996, SP cells were first identified in mouse bone marrow as a distinct, minor, cell population highly enriched for hematopoietic stem cell markers and endowed with long-term repopulating capacity [3]. Since their discovery, SP cells have been identified in many tissues with high regenerative capacities, including skin [4,5], lung [6], skeletal muscle [7], brain [8], heart [7], liver [9], kidneys [10] and mammary gland [11]. Moreover, recent work has led to the detection of the SP in a variety of tumor types, including leukemia, glioma, medulloblastoma, hepatocarcinoma, breast, prostate, thyroid, colorectal and ovarian carcinoma [12–18].

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The association between stem cells and SP is sound in normal tissues and hematopoietic malignancies. In contrast, in normal epithelial tissues, such as the mammary gland [19], and certain solid tumors, such as gastrointestinal and thyroid cancer [14,20], SP cells do not appear to be highly enriched in stem cells. In any case, in several experimental systems, malignant SP cells have been shown to be endowed with higher clonogenic and tumorigenic potential than non-SP cells. Due to these features, SP cells can be responsible for the aggressive behavior of certain tumors as well as for the development of drug resistance; thus, strategies targeting this subset may have therapeutic implications. 2. Chemo-resistance Experimental studies suggest that SP and CSC have higher resistance to chemotherapy than the bulk of cancerous cells. Hirshmann-Jax and colleagues first demonstrated that neuroblastoma SP cells were less sensitive to mitoxantrone [16]. Similar results were obtained by Szotek’s group, who showed that the SP fraction of ovarian cancer could not be inhibited by doxorubicin, unlike non-SP cells [12]. Moreover, the SP of human malignant glioma cell lines and primary glioblastoma was shown to increase following treatment with temolozomide, suggesting that this drug preferentially kills non-SP cells [21]. Furthermore, studies focused on CD133+ brain tumor stem cells disclosed a survival advantage compared to CD133 cells to temolozomide, carboplatin, paclitaxel and etoposide [22]. Resistance could depend, among others, on certain features that both CSC and SP share with normal stem cells. One key property is resistance to apoptosis. Stem cells are programmed to be long-lived, in order to maintain the progenitor pool from which differentiated cells derive. For this purpose, stem cells activate some protective mechanisms that shield them from senescence and cellular stress. These mechanisms include: (I) activation of some self-renewal pathways, such as TGF-b, Sonic Hedgehog (SHH), Wnt/b-cat or BMI-1; (II) expression of anti-apoptotic proteins like BCL-2; (III) enhanced capability to repair DNA damage after genotoxic stress; (IV) generation of autocrine loops through the production of growth factors like epidermal growth factor (EGF); and (V) over-expression of drug-effluxing pumps and metabolic mediators, that allow the cell to rapidly eliminate or degrade toxic compounds and radical oxygen species. Importantly, it appears that resistance to apoptosis, which can be limited to CSC initially, is often rapidly acquired also by the bulk of tumor cells at relapse, perhaps due to the genetic instability which distinguishes tumor from normal cells. As a consequence of this, chemotherapy invariably causes bone marrow toxicity due to its effects on trans-amplifying, progenitor and even more differentiated cells, whereas tumors may initially regress but subsequently become completely resistant to chemotherapy. A second key property is expression of certain pumps, including ABCC1, ABCG2 and MDR1, which are the principal mediators of multidrug-resistance identified so far. They are promiscuous transporters of both hydrophobic

and hydrophilic compounds and can help the cell extruding several drugs including vinblastine, paclitaxel, mitoxantrone, doxorubicin, topotecan, methotrexate and imatinib mesylate [23]. The exact physiological role of these pumps is not yet fully understood, but it is known that they are involved in cellular protection against exogenous products and in resistance to hypoxic stress, mediated by an increased ability to consume hydrogen peroxide [24] and a reduced accumulation of toxic heme metabolites [25]. In normal stem cells they seem to have a role in the repression of cellular differentiation and are normally turned off in committed progenitors and mature cells [1]. Their enhanced expression in SP may explain their particular drug resistance and their tolerance to hypoxic conditions and make these proteins ideal targets for cancer therapy. The last property concerns cell proliferation. Eventually, many chemotherapies rely on cycling cells in order to cause lethal cellular damage. Normal stem cells – especially hematopoietic stem cells [26] – are quiescent in the absence of specific stimulation from the microenvironment; if this property is indeed shared by CSC – an issue which is not yet firmly established – then lack of therapeutic efficacy of cytotoxic drugs does not come unexpected. Concerning SP, at present there are somewhat conflicting observations on their cell cycle kinetics. In fact, whereas many groups described SP cells as slow proliferating cells [1,12,27], others found that SP are characterized by a high proliferation rate [28–30]. This controversial behavior could depend on the stage of differentiation of the cells giving rise to the SP fraction. In some tumors, SP could derive from the transformation of immature stem cells and be slowly cycling; in others, instead, SP cells could originate from the transformation of a more differentiated cell and hence proliferate more. Moreover, the SP population can be heterogeneous and comprise both slow-cycling cells as well as more differentiated and highly proliferative progenitors similar to transit amplifying cells [14]. Alternatively, the proliferative status of SP cells might vary based on their tissue of origin and its growth and renewal kinetics, or be determined – in the case of SP cells in tumors – from the individual repertoire of mutations affecting cell cycle properties. The effects of chemotherapy on SP cells are therefore not easy to predict and could be heterogeneous. 3. Radio-resistance Radiation therapy is typically applied in multiple doses over several weeks to minimize damage to normal tissues and achieve optimal therapeutic effects, but this fractionated schedule is sometimes associated to accelerated repopulation, suggesting that a subset of radiation-resistant cells undergo proliferation. Experimental data from breast or brain cancer models indicate that CSC are endowed with resistance to radiation and suggest that CSC numbers in a tumor can affect relapse after radiotherapy [23,31,32]. In a glioblastoma model it was demonstrated that the CD133+ fraction, enriched in CSC, is more resistant to radiation-induced apoptosis than the CD133 counter-

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part. Moreover, the percentage of CD133+ cells following irradiation was increased both in vitro and in tumors [33]. Other groups showed in breast cancer cell lines a similar enrichment for the progenitor cell-containing SP after irradiation [34]. As mentioned before, experimental evidence indicate that CSC, like normal stem cells, are provided with very efficient mechanisms of DNA damage repair and can activate DNA damage check points more readily than the rest of the cancerous cells. For example, in glioblastoma cell lines, the CD133+ subpopulation displays a basal activation of some proteins involved in the DNA damage check point, such as the checkpoint kinases Chk1/2. Inhibition of these kinases with a small molecule inhibitor disabled radioresistance of CSC-enriched cells [33]. However, efficient DNA damage repair probably is just one of the mechanisms that account for the particular resistance of CSC to radiation. As an alternative mechanism, albeit not mutually exclusive, Phillips et al. argued that reduced levels of reactive oxygen species (ROS), indicating higher levels of radical scavengers, and the activation of the Jagged1–Notch1 axis could contribute to the decreased radio-sensitivity of the CSC-enriched mammospheres compared to adherent cells from the same line [35]. This hypothesis has recently been confirmed by the Clarke’s group, who found a striking association of ROS levels and radioresistance in breast cancer stem cells. Importantly, pharmacologic depletion of ROS scavengers in CSC markedly impaired their clonogenicity and resulted in radiosensitization [36]. Among other mechanisms of adaptation to radiotherapy, it has been observed that activation of the Wnt/bcatenin pathway may be enhanced by DNA damage and can promote genomic instability. Thus, b-catenin activation might both favor the conversion of non-tumorigenic stem cells into CSC or SP and allow tumor cells to survive after irradiation [34,37]. Finally, the tumor microenvironment might also influence distribution of CSC within the tumor mass and their resistance to radiation. For example, there are some clues that hypoxia can favor stem cells expansion and maintenance in tumors [27,38]. Moreover, preclinical and clinical studies show that local tumor control after radiotherapy inversely correlates with tumor hypoxia [39]. Thus, in order to improve the efficacy of radiation therapy, it will be important to validate methods to determine SP and CSC content and their spatial distribution in tumors. Modern radiotherapy techniques allow the delivery of an inhomogeneous dose distribution with a high precision within the tumor; so, if we could identify CSC-rich niches within the tumor bulk, we might have a tool to boost the radiation dose specifically in those areas without increasing toxicity to normal tissues [40].

4. Overcoming CSC and SP resistance to cancer therapeutics Standard chemo- and radiotherapy clinical endpoints are usually based on radiologic documentation of tumor shrinkage. However, if the CSC hypothesis is correct and CSC are more resistant to therapy than non-CSC, changes

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in tumor volume after therapy, which are governed by modification of the total burden of cancer cells, may not correlate with tumor eradication. For permanent control of at least certain tumor types it appears fundamental to eliminate the rare CSC subpopulation. Thus, it is of primary importance to optimize cancer therapies in order to target CSC. There are at least two possible strategies of targeting CSC and SP within a tumor: (I) the introduction of treatments able to target molecular pathways specifically over-activated in malignant stem cells; and (II) the sensitization of CSC to standard therapies by blocking those mechanisms which provide a survival advantage to these cells and may account for their resistance to cancer therapeutics. 4.1. Pathway-directed interventions Adult stem cells share with their malignant counterparts the activation of a number of distinct signalling pathways implicated in the control of proliferation, differentiation and self-renewal, including the SHH/ Smoothened (SMO), Wnt/b-catenin, Notch and BMI-1 pathways. Components of these pathways provide new targets for the development of therapeutic strategies able to hit CSC, although some concern remains about the possibility that treatments targeting pathways shared for survival and maintenance by normal stem cells and CSC could cause toxicity also to normal tissues. Over-activation of the Wnt/b-catenin axis is associated to malignant transformation and progression of colorectal cancer, and high levels of nuclear b-catenin are thought to trigger epithelial to mesenchimal transition and promote dissemination of cancer cells [41]. SP cells from different cancer cell lines show increased expression of genes involved in the WNT/b-catenin signalling pathway when compared to non-SP cells [17]; moreover, mouse mammary epithelial SP undergo expansion following treatment with Wnt effectors [42]. The use of selective anti-Wnt antibodies, Wnt protein inhibitors or repressors disrupting nuclear LEF/TCF/b-catenin complexes may counteract the nuclear accumulation of b-catenin, thereby inhibiting proliferation and maintenance of CSC and SP [43,44]. The use of these drugs is now being evaluated as a possible therapeutic approach for Wnt-dependent tumors such as colon cancer, Barrett’s esophagus or hepatocellular carcinoma [45]. Aberrant Notch activity is involved in the pathogenesis of many human malignancies, including T-ALL, lymphoma, medulloblastoma, colorectal, pancreatic, mammary, ovarian and lung carcinomas. Notch-ligand interaction is a highly conserved mechanism that regulates specific cell fate decisions during development. In addition to its functions in developmental and cell maturation processes, studies indicate that Notch activation plays a role in the regulation of self-renewal and proliferation of adult stem cells as well as of CSC [46]. Recently, c-secretase inhibitors (GSI), as well as various pharmaceutical or genetic inhibitors of Notch have been suggested as potential novel cancer therapeutics [47]. In medulloblastoma, Fan et al. demonstrated that GSI treatment results in specific deple-

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tion of both the CD133+ brain CSC population and of the SP, associated to an increase in the apoptotic rates and a loss of tumorigenic capacity [48]. These findings imply that targeting Notch could be a possible strategy to hit CSC. Notch genes are preferentially expressed by glioblastoma and breast SP cells [13]; thus, inhibition of this pathway could also affect SP levels. SHH signalling is important in development. Damage to components of this pathway can results into severe birth defects. Mutations in SHH receptor patched (PTCH), or in the signalling element SMO are present in different aggressive forms of cancer, such as basal cell carcinoma (BCC), medulloblastoma, rhabdomyosarcoma. Moreover, the aberrant activation of the SHH cascade is often detected in glioma, gastrointestinal, pancreas, prostate, breast and small-cell lung cancers. The inhibition of SHH cascade by SMO-signalling inhibitors, cyclopamine alkaloid or the anti-SHH antibody results, both in vitro and in vivo, in the growth arrest and apoptosis of metastatic cancer cell lines, but not of normal cells [49,50]. Moreover, cyclopamine has shown a high treatment efficacy in animal models of medulloblastoma, pancreatic and prostatic carcinoma [46]. In addition, in gliomas the SP fraction, marked by Hoechst dye excretion, was significantly reduced or eliminated by cyclopamine [45]. The results obtained so far suggest that blocking the SHH cascade may hit the SP subset in selected malignancies [50]. Further evidence that triggering specific signal transduction pathways could affect cancer stem cells has been provided by the elegant study by Piccirillo et al., showing inhibition of the tumorigenic potential of human brain tumor-initiating cells by bone morphogenetic proteins, especially BMP-4, by a mechanism involving Smad signalling, reduction of cell proliferation and increased neural differentiation [51]. 4.2. Sensitization strategies: can we convert SP and CSC into a target of conventional therapies? In several tumor types, cancer stem cells, or at least a substantial fraction of them, express ABC transporters. Many efforts have been dedicated to develop specific inhibitors of ABC transporters and to verify their efficacy, generally in combination with a range of chemotherapy regimens. However, clinical trials failed, probably because of the presence of additional transporters, not targeted by inhibitors, or the pharmacokinetic interaction between the chemotherapeutic agent and ABC inhibitors [52]. Importantly, indirect modulation of ABC levels appears to represent a feasible alternative. Several studies have indeed demonstrated that the activity of the serine/threonine kinase Akt may influence the intracellular localization of ABCG2, thus modifying the capability of cells to extrude drugs [53,54]. Akt acts as a downstream mediator of the PI3K/mTOR pathway (PhoshatidylInositol 3-Kinase/mammalian Target of Rapamycin) and it is implicated in the control of cellular growth, survival and metabolism [55,56]. The use of compounds that directly inhibit Akt or other components of this pathway could impair the resistance properties of CSC and SP cells. Treatment of cells with the PI3K inhibitors LY294002 or wortmannin led to a decrease of SP fraction by inducing the transloca-

tion from membrane to intracellular sites of ABCG2 [53,54]. Moreover, inhibition of Akt signalling thought PI3K inhibitors attenuated the efflux of the doxorubicin that is actively extruded by ABCG2, thus increasing its therapeutic efficacy. The PI3K/mTOR pathway could also be modulated by targeting mTOR activity; indeed, recent studies have demonstrated that the mTOR inhibitor rapamycin reduces SP fraction in tumor cell lines [57,58]. Rapamycin has recently been used in clinical trials in leukemia treatment combined with conventional therapy, and may target leukemia stem cells [59–61]. Akt activity could also be directly down-regulated by imatinib mesylate [62]. Imatinib mesylate is a specific small molecule inhibitor which binds to the bcr–abl fusion product involved in the pathogenesis of chronic myeloid leukemia (CML). The blockade of bcr–abl activity allows the cells to complete differentiation and die in a few days like normal mature granulocytes [63]. Intriguingly, imatinib stimulates ABCG2-specific ATPase activity and it is both a substrate and an inhibitor of ABCG2 [20,64,65]. Bcr–abl enhances ABCG2 expression, by activation of the PI3K/Akt pathway, and imatinib-mediated bcr–abl inhibition decreases Akt phosphorylation and reduces expression of ABCG2. In an experimental model where bcr–abl+ cells were enforced to express high levels of ABCG2, however, the activity of the pump was clearly demonstrated to protect the cells from imatinib cytotoxicity [62]. Conversely, Chu et al. demonstrated that imatinib decreases the SP subset of head-and-neck squamous carcinoma cells, and allows greater doxorubicin retention presumably via inhibition of Akt which eventually regulates ABCG2 function [66]. The PI3K/mTOR/Akt axis is involved in insulin-like growth factor-mediated signalling and this pathway could be modulated by EGF and EGFR inhibitors. Over-expression of EGFR, Her2 and/or their ligands has been associated to many forms of cancer (skin, lung, colon, prostate, pancreas, breast, brain and ovarian cancer). A few years ago, selective inhibition of EGFR signalling has become clinical practice for treatment of lung, breast and colon cancer [50,67,68]. EGF exerts a stimulatory effect on the phosphorylation state of Akt and it increases the relative expression of ABCG2 at the cell surface [54,69]. EGFR activation consequently results in an increase in SP; on the contrary, treatment of cells with EGFR inhibitors, such as gefitinib or CI1033, decreases the SP subset and the ability of cells to extrude doxorubicin [69,70]. Among the EGFR-targeting drugs so far available, there are the anti-Her2 antibody trastuzumab, anti-EGFR antibodies and EGFR-specific tyrosine kinase inhibitors [50]. In breast cancer the addition of trastuzumab to adjuvant chemotherapy reduces the recurrence rate by almost 50%; moreover, trastuzumab reduced the stem cell-like population in certain breast cancer cell lines [71]. Importantly, these findings have recently been strengthened by the observation that also lapatinib, a small molecule inhibitor of HER2, is capable to target this population in patients with HER2+ tumors [72]. These effects could be exploited to develop new therapeutic strategies; in a recent study hyaluronan oligomers (o-HA) were demonstrated to antagonize the malignant properties of glioma cells by inhibiting activation of EGFR and Akt, decreasing

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ABCG2 expression and increasing methotrexate cytotoxicity [73]. In addition to the above mentioned strategies, in the future new approaches for targeting CSC can be envisioned. In this regard, immunotherapy will likely be re-directed to target tumor-initiating cells. Although this mission will demand time, in order to identify appropriate CSC-specific antigens to be used as targets for antibodies or cytotoxic T lymphocytes, the idea is promising, as indicated by the recent finding that loading dendritic cells with antigens obtained from glioma-initiating cells promotes better immune control of tumor growth compared to vaccination with DC loaded with antigens derived from the bulk of tumor cells [74]. 5. Exploiting cytokines to tackle SP and CSC cells In some tissues, such as the hematopoietic system [26], stem cells are quiescent, which may contribute to shield them from conventional therapies. Although it is likely that the overall turnover kinetics of different tissues could in part modulate stem cell dormancy, this concept has led to the suggestion that their sensitivity to conventional therapies could be increased by inducing the entrance of the cell into the cell cycle [75]. The use of this approach against SP cells, however, should take into account the controversial findings on SP proliferative capacity, as discussed above. Although the biology of CSC is as yet largely unexplored, it seems that certain cytokines, including granulocyte–macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and IL-3 could induce proliferation of specific cancer or normal stem cells, such as leukemic progenitors or bone marrow stem cells [76]. Cytokines were originally discovered as key regulators of immune and inflammatory responses; however, elevated expression of some cytokines has been detected in multiple epithelial tumors and it has been speculated that certain cytokines may contribute to both positive and negative regulation of CSC behavior [77]. Importantly, a recent study has reinforced this hypothesis, by showing that the multifunctional cytokine IL-6 could be a potential regulator of normal and tumor stem cell self-renewal, through MAPK-dependent up-regulation of the transmembrane receptor Notch-3 and its ligand Jagged-1 in tumor mammospheres [78]. These pioneer studies represent conceptual advances in our understanding of the role of cytokines in cancer. 6. The intriguing case of interferon-a (IFN-a) IFNs are pleiotropic cytokines involved in host defence against viral infections and in the regulation of cell proliferation and differentiation. IFNs are divided into two major types: type I IFNs include IFN-a and IFN-b, sharing the same receptor; type II IFN is represented by IFN-c [79]. Antitumor activities of IFNs have been attributed mainly to stimulation of the immune system, inhibition of tumor cell proliferation or anti-angiogenic effects [80–82]. IFN-a has been widely used to treat cancer patients with some clinical benefit at cost of high toxicity. Occasionally, major

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clinical responses have been reported in tumors of different origins, such as ovarian cancer [83–85], renal cell carcinoma [86], melanoma [87] and myeloproliferative neoplasms [88,89]. The molecular mechanisms which determine the sensitivity or resistance of tumors to IFNs have still to be fully elucidated. In our experience with IFN-a administrated by gene therapy, we showed marked anti-tumor effects in orthotropic models of ovarian cancer [90]. Therapeutic efficacy depended on both the timing of administration of the IFN-a gene and tumor cell-related factors. Surprisingly, we found that ovarian cancer xenografts containing large numbers of SP cells were extremely IFN-a-sensitive; we showed a marked anti-proliferative and pro-apoptotic effects of IFN-a on this subset [29]. In agreement with these findings, Kayo et al. measured a reduction in SP cells after IFN-a treatment of adult T-cell leukemia/lymphoma (ATLL) cells [91]; in this study, however, IFN-a did not decrease viability of ATLL cells, as observed in epithelial ovarian cancer (EOC) models, whereas it increased expression of CD25, a marker of maturation. These effects were accompanied by sustained phosphorylation of Stat1 and Stat5 in ATLL cells, although the authors unfortunately did not investigate whether levels of Stat activation could differ between SP and non-SP cells. Interestingly, IFN-a has been demonstrated to induce differentiation of CML progenitors [92,93] as well as of hepatic progenitors from which hepatocellular carcinoma could arise [22]. Together, these findings suggest that the screening of tumor samples for their SP content could form a basis for rationale-based administration of IFN-a to cancer patients. This could be promising especially after standard chemotherapy, when drug-resistant SP cells could rapidly repopulate the tumor [52,94] (Fig. 1). Do IFNs act as inhibitors of pumps responsible of SP phenotype or do they directly hit SP cells? Although this question is still substantially unanswered, microarray analysis of IFN-a treated SP cells in ovarian cancer samples did not disclose changes in ABCG2 and MDR1 levels, but it revealed strong up-regulation of transcripts, including TRAIL and GBP-1, involved in pro-apoptotic and anti-proliferative signals. Moreover, we also found increased expression of other transcripts, such as IFI16, USP18, IFIH1 (MAD5) and PLSCR-1, associated to terminal differentiation of tumor cells [29]. Overall, the transcriptional signature of SP and non-SP cells was qualitatively similar, but the intensity of the measured changes differed between these two subsets, SP cells being more reactive than non-SP cells to IFN-a. Understanding the mechanism(s) involved in the modulation of the transcriptional response to this cytokine represents a goal of future studies in our laboratory. Finally, it is important to point out that treatment of mesenchimal progenitor cells from aggressive fibromatosis with IFN-b had opposite effects on SP cells [95] (Fig. 1). The observation that type I IFN increases the proportion of cells that exclude Hoechst dye and sort to the SP subset raises the possibility that type I IFN signalling positively regulates the number of precursor cells that drive tumor formation and maintenance in aggressive fibromatosis. These results – which have been strengthened also by elegant studies in mice predisposed to developing aggressive fibro-

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Fig. 1. Effects of IFN-a on SP cells. In many tumor types, IFN-a treatment is associated with a reduction in SP cells by inducing a specific set of genes (among others: TRAIL, IFI16, IFIH1, USP18 and PLSCR-1) followed by apoptosis and differentiation [29,91]. Whether this has therapeutic efficacy or not, however, depends on SP levels in tumors. IFN-a-therapy may lead to regression of tumors with high-SP levels (A), whereas minimally affecting the growth of tumors with low SP levels (B). In selected malignancies, however, such as aggressive fibromatosis, type I IFN increases SP cell number [95], thus driving the development of aggressive tumors (C). Orange: SP cells and Green: non-SP cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

matosis (Apc/Apc1638N) crossed with mice deficient for the type I IFN receptor [95] – imply that IFN treatment is a less than optimal therapy for this tumor type and that one should be cautious in generalizing the results obtained in EOC [29] to other tumor types. Intriguingly, a recent study has shown that whereas chronic activation of the IFN-a pathway in hematopoietic stem cells impairs their function, acute IFN-a treatment might be capable of stimulating proliferation of dormant hematopoietic stem cells [96]. These new findings provide a valuable example of the complexity of IFN-a effects on stem cell biology and have far reaching therapeutic implications. Altogether, these studies highlight the importance to dig into the interactions of cytokines with SP and CSC and raise the question of whether it might be worth to develop novel therapies aimed at modulating levels of specific cytokines, such as IL-6 or IFN-a, involved in the regulation of stem cell behavior. In either case, given the importance of cytokines in the homeostatic control of the immune system, targeted interventions into the tumor microenvironment, also by smart gene delivery protocols [97], will be key to limit toxicity and overcome current limitations of cytokine therapy.

7. Conclusions Cancer could arise from a subset of cells that shares some features with stem cells, identified using either stem cell markers or SP phenotype. CSC and SP cells appear to be highly resistant to chemo- and radiotherapy and this knowledge begins to change our therapeutic approach to cancer. Several studies have pioneered the possibility of specifically targeting CSC and SP cells by exploiting pathways involved in drug resistance. Alternatively, these bona-fide quiescent cells might be forced to proliferate and differentiate, thus becoming a target of conventional therapies. The open question is whether it might be possible to impair tumor-initiating cells without damaging their normal counterparts. In this regard, the safety of interfering with stem cell-specific pathways, a certainly attractive possibility, remains to be established. Further studies are warranted to define more stringent cancer stem cell phenotypes and reliable procedures to identify these cells and investigate the genetic and functional differences between cancer stem cells and their normal counterparts. Finally, certain cytokines, provisionally including G-CSF, GM-CSF, IL-3, IL-6 and IFN-a seem to modulate CSC functions, and this is certainly a notion which could have

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therapeutic implications. In our opinion, a more comprehensive view of the interactions between cytokines and key regulatory pathways in CSC will represent a future mainstay in this field. Conflict of interest None declared. Acknowledgments We are grateful to Ms. Colette Case for help in the preparation of the manuscript. This work was supported in part by Grants from Associazione Italiana per la Ricerca sul Cancro (AIRC) and Fondazione Italiana per la Ricerca sul Cancro (FIRC); Ministry of University and Research, 60% and PRIN; Ministry of Health, Oncology Program 2006 and Ricerca Finalizzata 2007; Banca Popolare di Verona. References [1] A. Hadnagy, L. Gaboury, R. Beaulieu, D. Balicki, SP analysis may be used to identify cancer stem cell populations, Exp. Cell Res. 312 (2006) 3701–3710. [2] C. Wu, B.A. Alman, Side population cells in human cancers, Cancer Lett. 268 (2008) 1–9. [3] M.A. Goodell, K. Brose, G. Paradis, A.S. Conner, R.C. Mulligan, Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo, J. Exp. Med. 183 (1996) 1797–1806. [4] S. Yano, Y. Ito, M. Fujimoto, T.S. Hamazaki, K. Tamaki, H. Okochi, Characterization and localization of side population cells in mouse skin, Stem Cells 23 (2005) 834–841. [5] G. Larderet, N.O. Fortunel, P. Vaigot, M. Cegalerba, P. Maltere, O. Zobiri, X. Gidrol, G. Waksman, M.T. Martin, Human side population keratinocytes exhibit long-term proliferative potential and a specific gene expression profile and can form a pluristratified epidermis, Stem Cells 24 (2006) 965–974. [6] S.M. Majka, M.A. Beutz, M. Hagen, A.A. Izzo, N. Voelkel, K.M. Helm, Identification of novel resident pulmonary stem cells: form and function of the lung side population, Stem Cells 23 (2005) 1073– 1081. [7] C.M. Martin, A.P. Meeson, S.M. Robertson, T.J. Hawke, J.A. Richardson, S. Bates, S.C. Goetsch, T.D. Gallardo, D.J. Garry, Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart, Dev. Biol. 265 (2004) 262– 275. [8] M. Kim, C.M. Morshead, Distinct populations of forebrain neural stem and progenitor cells can be isolated using side-population analysis, J. Neurosci. 23 (2003) 10703–10709. [9] K. Shimano, M. Satake, A. Okaya, J. Kitanaka, N. Kitanaka, M. Takemura, M. Sakagami, N. Terada, T. Tsujimura, Hepatic oval cells have the side population phenotype defined by expression of ATPbinding cassette transporter ABCG2/BCRP1, Am. J. Pathol. 163 (2003) 3–9. [10] A.L. Allan, S.A. Vantyghem, A.B. Tuck, A.F. Chambers, Tumor dormancy and cancer stem cells: implications for the biology and treatment of breast cancer metastasis, Breast Dis. 26 (2006) 87–98. [11] A.J. Alvi, H. Clayton, C. Joshi, T. Enver, A. Ashworth, M.M. Vivanco, T.C. Dale, M.J. Smalley, Functional and molecular characterisation of mammary side population cells, Breast Cancer Res. 5 (2003) 1R–8R. [12] P.P. Szotek, R. Pieretti-Vanmarcke, P.T. Masiakos, D.M. Dinulescu, D. Connolly, R. Foster, D. Dombkowski, F. Preffer, D.T. Maclaughlin, P.K. Donahoe, Ovarian cancer side population defines cells with stem cell-like characteristics and mullerian inhibiting substance responsiveness, Proc. Natl. Acad. Sci. USA 103 (2006) 11154–11159. [13] L. Patrawala, T. Calhoun, R. Schneider-Broussard, J. Zhou, K. Claypool, D.G. Tang, Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2 cancer cells are similarly tumorigenic, Cancer Res. 65 (2005) 6207–6219. [14] N. Mitsutake, A. Iwao, K. Nagai, H. Namba, A. Ohtsuru, V. Saenko, S. Yamashita, Characterization of side population in thyroid cancer cell lines: cancer stem-like cells are enriched partly but not exclusively, Endocrinology 148 (2007) 1797–1803.

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