Effects of salinomycin and niclosamide on small cell lung cancer and small cell lung cancer circulating tumor cell lines

The high mortality rate of cancer patients is due to the refractoriness of metastatic cells to current treatments. Tumors seem to contain a small subpopulation of cancer stem cells (CSCs) which sustain primary tumors and metastases [1‐4]. CSCs are chemoresistant due to efficient DNA damage repair, the activation of survival pathways, decreased apoptosis, immune evasion and the adaptation to a hostile microenvironment [5]. Therefore, conventional chemotherapy eliminates the bulk of the tumor cells but CSCs survive and reconstitute the tumor as well as metastases [6]. Tumor dissemination is accomplished by circulating tumor cells (CTCs) which may show CSCs characteristics provided that these cells play a decisive role in cancer metastasis [7]. Thus, targeting CSCs in combination with standard chemotherapy may improve outcomes of cancer patients and procure long-lasting responses. However, attempts to target and eliminate CSC by suitable drugs were clinically not successful so far [8]. CSCs have been identified in most solid tumors and this subpopulation has been found to be enriched after chemotherapy [4, 9‐12]. For most solid tumors, expression of CD133, CD44, CD24, CD166, epithelial cell adhesion molecule (EpCAM), aldehyde dehydrogenases (ALDH) and ATP-binding cassette subfamily G member 2 (ABCG2) have been proposed as selective markers [9, 10, 13‐15]. Furthermore, altered developmental pathways such as Notch, Wnt and Hedgehog drive CSC growth, progression and drug resistance [16, 17]. Additionally, CSCs express pluripotency transcription factors OCT4, Nanog and Sox2 and show enhanced tumor-sphere formation [18‐20]. Increased oxidative phosphorylation OXPHOS is a hallmark of resistance to chemotherapy and, therefore, the OXPHOS inhibitor salinomycin was shown to selectively kill CSCs [21‐24]. CD133⁺ colorectal CSC-like cells were sensitive to salinomycin treatment, but not to the conventional anticancer drug oxaliplatin [25]. A few clinical case reports have documented effectiveness of salinomycin in therapy-resistant cancer patients, e.g., a patient with metastatic invasive ductal breast cancer treated with salinomycin showed induction of clinical tumor regression [26, 27]. In particular, salinomycin-induced ER Ca²⁺ depletion inhibits Wnt signaling by down-regulating β-catenin [28, 29]. Additionally, other drugs such as niclosamide, effectively inhibit activation of the Wnt/β-catenin signaling and reduce the formation of primary and secondary tumorspheres [30]. The anthelminthic niclosamide showed similar effects as salinomycin on cancer stemness as well as uncoupling of mitochondrial phosphorylation [31].

Metastasis-initiating capabilities of CTCs based on stemness properties are difficult to detect since CTCs show extensive heterogeneity and only an extremely small fraction of these cells is able to establish secondary lesions [32, 33]. In vitro expansion of relevant CTCs has been reported for only a limited number of tumors and cell lines so far [34]. We have obtained five permanent CTC cell lines from blood samples of patients bearing extended disease small cell lung cancer (ED-SCLC) [35]. Small cell lung cancer (SCLC) comprises approximately 15% of all lung cancers and is found disseminated in the great majority of patients at first presentation [36]. Patients respond well to first-line platinum-based combination therapy with response rates on the order of 70%–90% in limited disease and 50%– 60% in extended disease [36, 37]. However, nearly all patients with SCLC eventually relapse with chemo- and radioresistant tumors which are difficult to treat and have a dismal prognosis [38]. These characteristics of SCLC suggest that it may be enriched in CSCs and the general resistance is effected by a CSC subpopulation [39, 40]. In the present study, the SCLC CTC and tumor cell lines were used to study chemosensitivity to the CSC-targeting drugs salinomycin and niclosamide and to characterize their expression of selected CSC markers and pluripotent transcription factors.

In the present study, the cytotoxic activities of the CSC-selective drugs salinomycin and niclosamide were tested against SCLC and SCLC CTC cell lines. The five permanent CTC cell lines, namely BHGc7, 10, 16, 26 and UHGc5, express the typical SCLC markers, such as CD56/NCAM, enolase-2, chromogranin, synaptophysin and EpCAM (except UHGc5), as well as unique mutations of p53 [35, 43]. All CTC lines grow as loosely attached single cells and spontaneously form large TOS in suspension in regular tissue culture [43]. Spheroids have been demonstrated to enrich the CSC fraction and the CTC lines express components of the noncanonical WNT pathway and interact with macrophages, a typical feature of CSCs [45‐47].

With the single exception of the SCLC NCI-H69 cell line, salinomycin has more pronounced cytotoxic effects against the SCLC CTC cell lines including BHGc16, BHGc16 TOS, BHGc26 and UHGc5 TOS. TOS show higher resistance to chemotherapeutics such as cisplatin, topotecan and epirubicin compared to the corresponding single cell suspensions [35]. However, in response to salinomycin, BHGc10 TOS and BHGc26 TOS were more resistant, whereas BHGc7 TOS and UHGc5 TOS were more sensitive than the related single cell suspensions. BHGc16 TOS showed no alteration in chemosensitivity to this drug compared to single cells. In general, SCLC CTC spheroids are not sensitized to salinomycin and the difference in chemosensitivity between single cells and aggregates is far lower than the 100fold disparity observed in breast cancer mammospheres and tumor cells exhibiting a CD44⁺/CD24⁻ CSC phenotype [29]. Cell death mechanisms induced by the ionophore salinomycin seem to be dependent on the impairment of mitochondrial function, generation of reactive oxygen species (ROS) and induction of apoptosis [29, 48‐50]. The in vitro IC₅₀ values of salinomycin vary between 0.3 and 10 μM, depending on the cell type used and treatment period [51, 52]. However, salinomycin is not suitable for clinical application and the pleiotropic effects may be linked to its direct effects on DNA through intercalation [53].

Niclosamide showed high cytotoxic activity against most SCLC lines with exception of GLC14 and GLC16. Among the SCLC CTC lines, BHGc7 TOS and UHGc5 TOS were more resistant to niclosamide than the corresponding single cell fractions in contrast to BHGc10 TOS, BHGc16 TOS and BHGc26 TOS which exhibited the reversed effect. Again, this CSC-selective drug exhibits heterogenous activity against the SCLC CTC spheroids. Niclosamide has antitumor activity via inhibition of NF-κB, an increase in ROS and blocking of the Wnt/β-catenin, STAT3 and NOTCH signaling pathways but also targets mitochondria in cancer cells [54, 55]. However, the multitude of activities of niclosamide may be explained by its function as nonselective DNA intercalator [56]. Niclosamide induces mitochondrial uncoupling, which renders mutant p53 cells, such as SCLC cells, susceptible to mitochondrial-dependent apoptosis [57, 58]. In conclusion, salinomycin and niclosamide lack consistent cytotoxic activity against the multicellular CTC TOS in contrast to CSC-enriched spheres of other tumor types.

SCLC is sensitive to the first-line therapy in most patients but invariably recurs as chemo- and radioresistant tumor within approximately one year [36]. This natural history of SCLC would fit a CSC model with elimination of the bulk tumor with initial cycles of chemotherapy and regrowth of the small population of resistant CSCs. Furthermore, the CSCs are hypothesized to be part of the large number of CTCs in SCLC and to constitute the true metastasis-inducing cells, as so-called circulating CSCs [7]. However, the present findings with the CSC-selective drugs salinomycin and niclosamide are not compatible with a consistent CSC phenotype of the SCLC cells and spheroids. Possible CSC markers described in SCLC cell line studies comprise CD44, CD90, CD133, CD87, Oct-4, SOX2, ALDH1, uPAR/CD87, CD166/ALCAM, BMI-1, PODXL-1, and PTCH [59]. CD44 plays an important role in tumor initiation and metastasis and seems to be involved in sphere formation in primary SCLC lines [13, 60]. Spheroid-forming cells were mainly found within the CD44⁺ CD90⁺ subpopulation expressing increased levels of Nanog and Oct-4. CD133 (Prominin 1) has been used to identify CSCs in lung cancer and was linked to high levels of ABCG2 and chemoresistance [61]. Several SCLC cell lines were found to contain a consistent side population (SP) fraction that comprises <1% of the bulk population [62, 63]. The SCLC cell line NCI-H446 exhibits a high degree of stemness, tumorigenicity and plasticity correlated to expression of CD133, Sall4, Oct-4, nestin, neural cell adhesion molecule (NCAM), S100β, vimentin, CD44 and CD105 [64]. CD133 positivity was described in H345 and NCI-H69 SCLC cell lines and the putative CSC markers urokinase plasminogen activator/receptor (uPA/uPAR CD87) are involved in cell migration, invasion and inhibition of apoptosis in SCLC CSCs [65, 66]. However, in a panel of six SCLC cell lines CD133⁻/CD87⁻ cells showed the highest tumorigenic potential [67]. Significant correlation between SOX2 mRNA expression levels and the number of distant metastatic sites was established in SCLC patients [68]. In conclusion, the correlation of the CSC phenotype in SCLC cells with markers previously established in other tumors is questionable and seems not to be of general validity.

In respect to CTCs, CD44 has been identified as CSC marker of gastric cancer and most of the CTC-positive patients showed CD44-positive CTCs [69]. In breast cancer patients, bone marrow metastases are enriched in CD44^high/CD24^low cells, which have also been found in pleural metastases [70]. Recent studies have demonstrated that stem cell markers are frequently overexpressed in CTCs of breast cancer [40]. Early dissemination in SCLC is most likely related to the extremely high numbers of circulating tumor cells (CTCs) reaching counts of over several hundred CTCs per 7.5 ml blood compared to a 3–5 CTCs/7.5 ml as prognostic threshold for other solid tumors [71]. Our results demonstrate that SCLC tumor and CTC cells express CD133, but with exception of the BHGc10 CTC line obtained from a patient with primary resistance to cisplatin and GLC16, this antigen was mainly found in DMS53, GLC14 and NCI-H69 cell lines derived from patients prior to therapy [41, 42]. CD24 was co-expressed with CD133 in NCI-H69, DMS53, and GLC14 tumor cells and detectable in all CTC SCLC cell lines. In ovarian cancer, CD24⁺ CSCs were reported to be less proliferative and more resistant to chemotherapy compared to CD24⁻ cells [72]. With exception of NCI-H69, CD44 was low or absent from all CD133⁺ SCLC cells and, in particular, from all SCLC CTC lines. No expression of the chemoresistance mediator ABCG2 could be detected in the SCLC lines studied here (data not shown). In conclusion, CD133 is not expressed in response to prior chemotherapy and a CD24⁺/CD44⁻/ABCG2⁻ phenotype does not fit to the panel of markers generally expressed by typical CSCs of solid tumors, with the possible exception of BHGc10. Furthermore, SOX2 seems to represent a general marker of the neuroendocrine SCLC tumors which is not restricted to CSCs. Expression of Oct-3/4 and Nanog is limited to BHGc16 und BHGc26 which lack significant expression of CD133 and GLC14/16.

The numerous studies reporting CSC traits in SCLC tumor cells point to a possible role of this phenotype in resident SCLC primaries and secondaries but our results preclude a role of CSC characteristics among SCLC CTCs [73, 74]. It cannot be excluded that CSC plasticity allows for a switch to a non-CSC cell type in the circulation [6]. SCLC dissemination seems to be related to metastasis-initiating CTCs which exhibit a CD44⁻/CD24⁺/ABCG2⁻ salinomycin-sensitive phenotype which lack CD133 expression. With exception of SOX2, pluripotent stem cell transcription factors Oct-3/4 and Nanog are expressed to a minor extent in the CTC cell lines studied. The SCLC CTC cell lines are chemosensitive to topotecan and epirubicin as single suspensions in good agreement with the lack of ABCG2 expression [35]. However, TOS are chemoresistent and likewise radioresistant due to large size and hypoxic conditions in the core region [75, 76]. CSCs were reported to reside in hypoxic niches but SCLC CTCs recapitulate this strategy by the existence of quiescent and hypoxic cells in the TOS.