Ji Hyeon Kim,Soeun Park,Eunsun Jung,Jinwoo Shin,Yoon-Jae Kim,Ji Young Kim,Jonathan L. Sessler,Jae Hong Seo,Jong Seung Kim
A dual-action niclosamide-based prodrug that targets cancer stem cells and inhibits TNBC metastasis
cancer stem cells,triple-negative breast cancer,niclosamide,Acetazolamide
Significance Cancer stem cells (CSCs) are a key driver of tumor relapse and metastasis, leading to poor clinical outcomes. A key current challenge is finding ways to eradicate them and suppress their characteristics. Niclosamide is an approved anthelmintic drug that has also been shown to suppress CSCs via signal transducer and transcriptional activator 3 (STAT3) inactivation. Conjugation of a niclosamide core to a CAIX inhibitor gives the prodrug Nic-A, a conjugate that provides for a reduction of CSC-like characteristics, including suppression of tumor growth and metastasis in mouse models. Nic-A exhibits good bioavailability and acceptable solubility. As such, it constitutes a promising new strategy for anti-CSC therapeutic discovery.
A dual-action niclosamide-based prodrug that targets cancer stem cells and inhibits TNBC metastasis

Cancer stem cells (CSCs) are a key driver of tumor relapse and metastasis, leading to poor clinical outcomes. A key current challenge is finding ways to eradicate them and suppress their characteristics. Niclosamide is an approved anthelmintic drug that has also been shown to suppress CSCs via signal transducer and transcriptional activator 3 (STAT3) inactivation. Conjugation of a niclosamide core to a CAIX inhibitor gives the prodrug Nic-A, a conjugate that provides for a reduction of CSC-like characteristics, including suppression of tumor growth and metastasis in mouse models. Nic-A exhibits good bioavailability and acceptable solubility. As such, it constitutes a promising new strategy for anti-CSC therapeutic discovery.

Triple-negative breast cancer (TNBC) accounts for approximately 10 to 15% of all breast cancer subtypes and constitutes a highly heterogeneous and aggressive phenotype (1, 2). The lack of established molecular targets, such as the estrogen receptor, progesterone receptor, or the human epidermal growth factor receptor 2, severely restricts therapeutic options for TNBC (3). TNBC tumors typically harbor a high cancer stem-like population and exhibit a high propensity for drug resistance; in fact, the majority of TNBC patients who receive chemotherapy eventually experience metastasis (4). Tumor metastasis is associated with 90% of cancer-related deaths, highlighting what is an unmet clinical need (5). There is evidence that tumor initiation, invasion, and metastasis are driven by a small subset of cells known as cancer stem cells (CSCs) (6–8). Therefore, the elimination of CSCs from the primary tumor is expected to improve cancer prognoses (9). Nevertheless, the development of de novo drugs targeting CSCs represents a challenge due to the complex and heterogeneous nature of these unique cell populations (10, 11). Here we report a niclosamide prodrug (Nic-A) that selectively targets the signal transducer and transcriptional activator 3 (STAT3) signaling pathway and effectively eliminates CSCs. STAT3 signaling is a key biological pathway for the regulation of tumor cell survival, migration, angiogenesis, immune activation, and cancer stemness (12). STAT3 is a transcription factor that is predominantly activated via the phosphorylation of a tyrosine residue (Tyr705) and mediates cellular response to various growth factors and cytokines. Activated phospho-STAT3 translocates to the nucleus and upregulates the expression of key genes, including cyclin D1, bcl-2, survivin, and Sox2, that play a pivotal role in proliferation, differentiation, epithelial-to-mesenchymal transition (EMT), and apoptosis (13–15). Notably, the aberrant activation of STAT3 preserves stem-like characteristics and promotes self-renewal capacity in many cancers (16–18). Several preclinical studies support the suggestion that STAT3 is constitutively activated in CSC-enriched populations of TNBC, whereas blocking or reducing STAT3 signaling suppresses the CSC-load, EMT, tumor growth, and metastasis (19, 20). Niclosamide is an U.S. Food and Drug Administration (FDA)-approved anthelmintic drug for the treatment of tapeworm infections in humans and animals. Preclinical studies have led to the suggestion that niclosamide could be repurposed as a cancer therapy due to its ability to inhibit STAT3 (21, 22). Several studies have shown that niclosamide attenuates features associated with stemness in breast cancer, as well as suppression of 3 dimension (3D) spheroid formation and induction of CSC apoptosis (23). However, to date, there are few reports demonstrating the eradication of cancer stem-like features through selective niclosamide delivery. Moreover, delivery strategies for niclosamide have been limited to encapsulation in nanoparticles (24, 25) and poor solubility and nonspecific delivery remain obstacles to its therapeutic development (26). Niclosamide is known to bind to STAT3 and inhibit its phosphorylation (27). It contains multiple functional groups that can bind tightly to the Src homology 2 domain of STAT3, and it appears that its interaction with STAT3 is stabilized by two hydrogen bonds, one of which is formed by the niclosamide phenolic moiety (28). The signature nature of this interaction led us to suggest that new prodrugs based on niclosamide could be developed by protecting the phenol group of niclosamide to interfere with STAT3 binding. Such considerations prompted us to prepare the prodrug Nic-A. Nic-A also contains acetazolamide, a carbonic anhydrase IX (CAIX) inhibitor (Fig. 1A) expected to improve selective uptake. CAIX is predominantly overexpressed in TNBC and participates in multiple aspects of tumorigenesis in EMT acquisition, abnormal vascularization, and dissemination in hypoxic tumor microenvironments (29, 30). Acetazolamide acts as a CAIX inhibitor as the result of interactions between its sulfonamide unit and the active zinc ion in CAIX (31). Prodrug Nic-A was thus designed to function as a dual-action drug that targets both CAIX and the STAT3 protein as shown in Fig. 1B. Nic-A possesses a glutathione (GSH)-responsive disulfide linker, which contributes to its enhanced selectivity for CSCs over normal cells. CSCs are characterized by a high level of GSH, presumably as protection against reactive oxygen species (ROS)-mediated DNA damage from perturbed redox homeostasis (32). As with other disulfide-linked prodrugs (33), it was thus expected that Nic-A would be selectively activated in the reductive environment of CSCs to release free niclosamide and acetazolamide (34). As detailed below, Nic-A was found to exhibit superior anti-CSC and anti-metastatic effects relative to a CAIX nontargeted reference molecule (R1) and a noncleavable control (R2) (Fig. 1C). These findings highlight the promise of Nic-A as a potential therapeutic that may have a role to play in addressing the current limitations of metastatic TNBC therapy.

The potential dual action capability of Nic-A against TNBC is predicated on the tethering of a CAIX-targeting acetazolamide derivative to a niclosamide moiety via a GSH-responsive disulfide linker. In terms of specifics, we appreciated that to be effective under physiological conditions Nic-A would need to possess reasonable aqueous solubility. Therefore, a solubilizing tetraethylene glycol linker was included within Nic-A. Niclosamide itself shows poor absorption in the body. While this allows it to be used safely as an anthelminthic drug (35), it hampers its repurposing as an anticancer drug. This limitation prompted us to develop a niclosamide-based prodrug. As inferred from a molecular docking study between STAT3 and niclosamide (Fig. 1A), we hypothesized that blocking the phenolic group with a removable moiety would render niclosamide incapable of inhibiting STAT3 unless it is reduced by GSH, thus converting it into a potential prodrug. SI Appendix, Scheme S1 illustrates the synthetic route used to access Nic-A. Briefly, the N-methylamine group in compound 1 was reacted with phosgene and triethylamine (TEA) to yield the carbamoyl chloride intermediate 1-1. Compound 2 was formed when a solution of niclosamide and TEA in dichloromethane were added to 1-1 in the presence of a catalytic amount of dimethylaminopyridine. To prepare the asymmetric self-immolative disulfide bond in R1, a solution of sodium methoxide was added slowly to a mixture of compound 2 and 2-propyne-S-thioacetate at 0 °C to generate the corresponding propargyl thiol in situ. This latter intermediate was substituted directly with pyridyl thiol to yield R1. Nic-A was then prepared via a Cu(II)-mediated “click” reaction between R1 and an azide-labeled acetazolamide (Az-N3). Reference compound R2, a niclosamide-containing analogue of Nic-A lacking the cleavable disulfide linker, was synthesized by first reacting niclosamide with propargyl bromide in DMF, followed by a click reaction with Az-N3 (SI Appendix, Scheme S2). Additionally, a resorufin fluorophore containing probe, Res-A, was synthesized using similar synthetic procedures and used to study the release chemistry underlying the proposed Nic-A activation (SI Appendix, Scheme S3). All new compounds were analyzed by 1H and 13C NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) and yielded data consistent with their proposed structures (SI Appendix, Figs. S1–S26). Gratifyingly, Nic-A displayed better water-solubility than the niclosamide, an attribute considered advantageous in the context of drug administration (SI Appendix, Fig. S27).

Fig. 2A shows the proposed GSH-induced activation and payload release pathway for Nic-A. As noted above, the disulfide linker is GSH-sensitive and is known to undergo scission in the presence of high GSH concentrations (33). In the case of Nic-A, the resulting free thiol is expected to undergo intramolecular thiol attack on the tertiary carbamate to liberate niclosamide in free form. To test this assumption, Nic-A was treated with 1 mM GSH in phosphate-buffered saline (PBS) (pH = 7.4) at 37 °C. Free niclosamide has a characteristic absorbance in the 300 to 450 nm spectral region. This allowed the putative cleavage reaction to be followed spectroscopically. As expected, an increase in the intensity for the spectral features ascribable to niclosamide were seen as a function of time (Fig. 2B and SI Appendix, Fig. S28). In contrast, no appreciable spectral changes were seen in the absence of GSH, a finding taken as support for the notion that the tertiary carbamate bond will prove stable under normal physiological conditions where GSH levels are relatively low (Fig. 2C). As can be seen from an inspection of Fig. 2D, Nic-A cleavage is essentially complete within 3 h of exposure to 1 mM GSH. The resulting products were further analyzed using high-performance liquid chromatography (HPLC). Under our conditions of HPLC analysis, Nic-A has a retention peak at 13.2 min. However, after incubation with GSH (1.0 mM) for 4 h, this peak disappeared and other peaks with retention times of 20 min (active niclosamide) and 2.5 min (presumed by-product) were seen (Fig. 2E). ESI-MS spectrometric analysis provided confirmation that the peak at 20 min was niclosamide (SI Appendix, Fig. S29). Repeat studies using different GSH concentrations (0 to 4 mM) also triggered an increase in the absorbance at 376 nm (Fig. 2 F and G and SI Appendix, Fig. S30) provided the GSH concentrations were ≥0.5 mM. These results highlight the potential of Nic-A as a safe and selective drug delivery system that should allow release of niclosamide through reduction of the disulfide linker mediated by GSH overexpressed in the tumor.

Immunocytochemical analyses were carried out and provided confirmation of the expectation that TNBC (BT549 and MDA-MB-231) cells express CAIX at higher levels than normal mammary epithelial MCF10A cells (Fig. 3A). This observation led us to infer that Nic-A would accumulate preferentially in TNBC cells as a result of the incorporated acetazolamide CAIX-targeting group. We also note that MDA-MB-231 (460.8 nmol/mL), Hs578T (468.9 nmol/mL), and BT549 cells (628.6 nmol/mL) are characterized by nearly 20-fold higher GSH levels than nonmalignant MCF10A cells (26.1 nmol/mL) (Fig. 3B). Thus, not only selective targeting, but also effective cleavage and release of niclosamide was expected. The above assumptions were tested using Res-A, an analog of Nic-A, designed to release a resorufin fluorophore upon GSH-based cleavage. As illustrated in Fig. 3C, the proposed activation mechanism is expected to be identical to that of Nic-A with the exception of the release of resorufin rather than niclosamide. Internal charge transfer is inhibited when resorufin is conjugated by a carbamate bond with the consequence that, as prepared, Res-A, is essentially nonemissive. On the other hand, once GSH-mediated disulfide cleavage occurs, resorufin is released, giving rise to a bright fluorescent emission.51 First, we measured fluorescence emission of Res-A in PBS (pH 7.4, 37 °C). No appreciable time-dependent increase in fluorescence was seen, leading us to infer that Res-A is inherently stable in PBS (Fig. 3D). In contrast, an increase in the fluorescence intensity is seen upon incubation of Res-A with GSH (Fig. 3E). Res-A was then studied in BT549 and MCF10A cells (Fig. 3F). In the BT549 cells the red fluorescence of free resorufin was observed after exposure to Res-A (10 μM) as monitored at 3 and 24 h. In contrast, minimal fluorescence was seen in the case of the MCF10A under otherwise identical study conditions. A xenograft mouse model was used to confirm the tumor selectivity of Res-A (Fig. 3 G–I). Specifically, in vivo fluorescence imaging revealed that after treatment with Res-A (20 mg/kg BW via tail vein injection), resorufin-based fluorescence is seen primarily in the tumor, as opposed to organs such as the heart, liver, lung, kidney, and intestine.

In TNBC, STAT3 is overexpressed and constitutively active, playing a crucial role in many signaling pathways that promote cell proliferation, anti-apoptosis, migration, and invasion (36). The publicly available cancer genome atlas (TCGA) dataset provides support for the expectation that outcomes are significantly worse in patients with high STAT3 mRNA expression (Fig. 4A). As part of the present study, we first identified the expression levels of STAT3 and phosphorylated STAT3 (p-STAT3) in a panel of breast cancer cell lines (Fig. 4B). Although expression of STAT3 was seen in various cell lines, considerably higher phosphorylation of STAT3 (Y705) was seen in the TNBC cell lines. To explore the anti-proliferative effect of Nic-A in TNBC cells, BT549 and MDA-MB-231 cells were treated with Nic-A, R1, R2 or niclosamide at various concentrations for 48 h. Following either Nic-A or R1 treatment, the viability of TNBC cells was drastically reduced. In contrast, R2, an inactive analog of Nic-A lacking the disulfide linkage (vide supra), had a negligible effect on cell proliferation. MCF10A cells proved less sensitive to Nic-A treatment than the TNBC cell lines, displaying IC50 values that were 6 ~10-fold higher; in contrast, niclosamide engendered substantial cytotoxicity even in nonmalignant cells (Fig. 4C). These results lead us to suggest that Nic-A can selectively inhibit the proliferation of TNBC cells while giving rise to minimal cytotoxicity in normal cells that are characterized by relatively low GSH and CAIX levels. In BT549 and MDA-MB-231 cells, Nic-A (10 μM, 48 h) induces significant increases in the proportion of early and late apoptotic cells (Fig. 4D), accompanied by caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage (Fig. 4E and SI Appendix, Fig. S31). Similar to Nic-A, niclosamide treatment was found to promote apoptosis in TNBC cells. The anti-proliferative effect of selective STAT3 inhibitors has been ascribed to dysregulation of the STAT3 downstream target genes that govern cell survival (36, 37). Therefore, we further investigated whether STAT3 signaling could be disrupted by Nic-A. Treatment with Nic-A (10 μM, 48 h) resulted in a marked decrease in the expression of STAT3 and phosphorylation (Y705) of STAT3, as well as downregulation of its downstream factors cyclin D1 and survivin (Fig. 4F and SI Appendix, Fig. S32). After treatment with Nic-A (10 μM, 24 h), a marked decrease in STAT3 phosphorylation was observed in both the cytoplasm and nucleus of BT549 cells (Fig. 4G and SI Appendix, Fig. S33).

Oncogenic STAT3 is preferentially activated in the CD44high/CD24low and ALDH1+ stem-like subpopulations responsible for tumor propagation to distant organs (19, 37). Using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data cohort, we analyzed the correlation between STAT3 and CSC markers, CD44 and ALDH1A1. A substantial positive correlation between STAT3 and either CD44 (Fig. 5A) or ALDH1A1 (Fig. 5B) was seen. In the analysis of the publicly available TCGA dataset, particularly high CD44 or ALDH1A1 expression levels were observed in breast cancer patients, with high STAT3 mRNA expression correlating with relatively poor overall survival (Fig. 5 C and D). These data highlight the clinical significance of STAT3 in TNBC, a highly heterogeneous phenotype with elevated expression of either CD44 or aldehydedehydrogenase 1 (ALDH1). CAIX promotes the survival and invasion of tumor cells as well as cancer stemness in hypoxic microenvironments, while the forced knockdown of CAIX expression or its pharmacologic inhibition impairs CSC-like traits, mammary tumor growth, and distant metastasis in vivo (30, 38). According to the Gene Expression database of Normal and Tumor tissues 2 dataset, breast cancer patients with high levels of CAIX mRNA expression had substantially lower overall survivals (SI Appendix, Fig. S34A). In breast cancer, analysis of the METABRIC gene data revealed a proportionately significant correlation between CAIX and either CD44 or ALDH1A1 (Fig. 5 E and F). A Kaplan–Meier analysis confirmed that high CAIX levels were associated with considerably poorer overall survival in patients with high expression levels of CD44 or ALDH1A1 (log-rank, P < 0.0001, Fig. 5 G and H and SI Appendix, Fig. S34B). In light of the above genomic data analyses, we compared ALDH1-positive and -negative 4T1 cells to determine whether STAT3 or CAIX is elevated in the CSC population. We found that the CD44, ALDH1, and phospho-STAT3 protein levels were upregulated during CSC-enriched mammosphere formation. CAIX was also found to be elevated in the mammospheres (Fig. 5I), mimicking a hypoxic environment (39). In accord with our prior studies, fluorescence-activated cell sorting analysis revealed that revealed that CD44 and phospho-STAT3 (Y705) were elevated in the ALDH1-positive 4T1 cell populations (Fig. 5 J–L) (39, 40). It is noteworthy that CAIX was expressed at a higher level in ALDH1-positive cells than in their negative counterparts, leading us to suggest that CAIX may be a crucial factor for determining the fate of CSCs (Fig. 5M). We thus evaluated CSC-like features, including the CD44high/CD24low phenotype, ALDH1 activity, and mammosphere-forming ability after exposure to Nic-A and the reference compounds R1 and R2, as well as niclosamide. Nic-A (10 μM, 48 h) induced the greatest reduction in the CD44high/CD24low population in MDA-MB-231 cells (Fig. 5N). ALDH1 is a detoxifying enzyme that scavenges ROS produced by chemotherapy or radiation therapy and can protect CSCs from DNA damage (41). Elevated ALDH1 levels in breast cancer are correlated with development of resistance to chemotherapies, including cyclophosphamide, doxorubicin, and paclitaxel, which are first-line options for TNBC (41). Nic-A (10 μM, 48 h) administration led to a sharp decrease in ALDH1 activity in BT549 cells and provided a greater effect than niclosamide (Fig. 5O). Mammosphere formation was impaired upon exposure to Nic-A, as evidenced by a statistically significant reduction in the number and volume of mammospheres (89.1% and 86.3% reduction compared to control groups, respectively) derived from BT549 cells (Fig. 5P). This finding is thought to reflect decreases in ALDH1 activity and CD44high/CD24low subpopulations, as well as dysregulation of STAT3.

To test whether the above in vitro observations would be recapitulated in vivo, we studied the impact of Nic-A and R1 on tumor growth in a TNBC xenograft model. To create this model, MDA-MB-231 cells were injected into the mammary gland fat pads of female athymic BALB/c nude mice. When the tumor volumes reached approximately 100 mm3, either Nic-A (5 mg/kg BW/day), R1 (2.5 mg/kg BW), or a control medium, was injected intravenously twice weekly for 8 wk. Nic-A treatment inhibited tumor growth by 89.4% compared to controls, and to a larger extent than an analogous treatment with R1 (Fig. 6A). No histopathological abnormalities were observed in the lungs, liver, and kidneys after Nic-A or R1 administration (SI Appendix, Fig. S35A), and no statistically meaningful changes were observed in the body weights of animals in the two groups (Fig. 6B). Blood biochemical analyses, as effected using alanine aminotransferase (ALT), aspartate aminotransferase (AST) and blood urea nitrogen (BUN) assays, revealed that Nic-A and R1 did not engender any adverse effects on either liver or kidney function (SI Appendix, Fig. S35B). The marked reductions in tumor size following Nic-A treatment were verified using in vivo bioluminescence imaging (BLI) and ex vivo resection of the whole tumor burden (Fig. 6 C and D and SI Appendix, Figs. S36 and S37, respectively). Tumor cell proliferation Ki-67 positive cells were significantly reduced by 64.2% in the presence of Nic-A, when compared to the control group (SI Appendix, Fig. S38A). Apoptotic cell death determined by expression of cleaved caspase-3 was also dramatically increased in the Nic-A-treated groups (SI Appendix, Fig. S38B). Immunohistochemical analyses for phosphor-STAT3, CD44, and ALDH1 were carried out using xenograft tumor samples. Nic-A significantly decreased the proportion of phospho-STAT3–positive cells compared to both the control and R1-treated groups (Fig. 6E). The marked decrease in phospho-STAT3 expression in mice treated with Nic-A rather than R1 is attributed to the effective delivery of niclosamide resulting from the tumor-homing ability of the CAIX-targeting ligand, as well as CAIX inhibition. Nic-A treatment also resulted in a marked reduction in the expression of ALDH1 (Fig. 6F) and CD44, in agreement with the in vitro observations (SI Appendix, Fig. S39). Angiogenesis is a crucial process for tumor progression and metastasis, since it results in the production of new endothelial blood vessels that deliver nutrients and oxygen to the tumor mass (42). To explore the effect of Nic-A on tumor angiogenesis, microvessel density levels in malignant tissue were determined using the endothelial cell adhesion molecule-1 (CD31) (43). The number of CD31-positive blood vessels was decreased in the peritumoral and intratumoral areas in the Nic-A-treated groups, and a statistically significant difference was also seen between the Nic-A and R1 treatment groups (Fig. 6 G and H and SI Appendix, Fig. S40). Recent reports have noted that niclosamide exerts anti-angiogenic properties, including impairment of proliferation and migration in human umbilical vein endothelial cells as well as disruption of endothelial cord-like structure formation. An animal study has provided evidence that vascular endothelial growth factor (VEGF)-induced angiogenesis is markedly attenuated in vivo upon niclosamide treatment (44). The hypoxia-inducible enzyme CAIX is involved in the regulation of intracellular pH and acidification/alkalinization in the tumor microenvironment, thereby contributing to enhanced tumor cell dissemination (45). Recent studies have shown that combination treatments involving CAIX activity blockade in conjunction with angiogenesis inhibitors suppresses TNBC tumor growth and distant metastasis (46). Nic-A was designed to embody both features in a single molecular construct. The anti-angiogenic property was associated with a marked downregulation of CAIX (SI Appendix, Fig. S41) in xenograft tumors. The combination of anti-angiogenic properties and anti-hypoxic effector function embodied in Nic-A could provide substantial advantages in reducing metastases and preventing advanced cancer progression. To evaluate further the impact of Nic-A on metastasis, we generated a metastatic allograft TNBC model derived from a CSC-enriched population (Fig. 6I). After treatment with Nic-A or R1 for 5 wk, lung metastatic lesions were assessed by means of BLI (Fig. 6J). The luminescence intensity of the lung metastasis areas was suppressed in the Nic-A-treated mice (Fig. 6J). Primary tumor growth was also repressed by Nic-A administration (SI Appendix, Fig. S42), concomitant with a marked reduction in the expression of the murine CSC markers CD49f and ALDH1 (SI Appendix, Fig. S43 A and B). Since the chemical structure of Nic-A comprises a CAIX targeting ligand added to R1, we suggest that the amount of niclosamide released in tumors is higher for Nic-A than in the case of R1. In other words, the tumor-homing ability of CAIX-targeting ligand in Nic-A is expected to contribute to a reduction in distant metastasis (47). There were no statistically significant differences in the ALT, AST, or BUN levels in the animals (SI Appendix, Fig. S44), leading us to propose that Nic-A does not adversely affect hepatic or renal function. The EMT marker vimentin regulates cellular dynamics and preserves the integrity and flexibility of cells, and may promote tumor cell motility and metastatic spread (48). Animals administered Nic-A exhibited a marked decrease in vimentin expression in both allografts and xenograft tumors (SI Appendix, Figs. S43C and S45). A kinetic investigation of cell migration was performed on MDA-MB-231 and BT549 cells to explore further the effect of Nic-A on the migratory capability of cells in vitro. Nic-A was found to inhibit TNBC cell migration in a dose-dependent manner in vitro (SI Appendix, Fig. S46). This finding leads us to suggest that the disruption of EMT dynamics via downregulation of vimentin may contribute to the inhibition of migratory ability and thus the prevention of TNBC metastasis.

The newly synthesized prodrug detailed in this report, Nic-A, is a stable conjugate containing an acetazolamide subunit linked to a niclosamide core via a tertiary carbamate tether and a disulfide linker that is expected to be cleaved by GSH overexpressed in TNBC. Nic-A displays high selectivity toward CSCs associated with TNBC and induces TNBC apoptosis concomitant with caspase-3 activation and suppression of STAT3 signaling. Nic-A also targets hallmarks of CSCs, such as ALDH1 activity, CD44high/CD24low subpopulations, and the ability to generate mammospheres. Studies of ALDH1-positive TNBC cells revealed a substantial correlation between CSC-like characteristics and the expression levels of STAT3 and CAIX. Nic-A treatment also reduced angiogenesis and tumor growth in TNBC xenograft tumors, as well as engendering significant reductions in Ki-67 expression and increased apoptosis. Notably, Nic-A inhibited distant metastasis in TNBC allografts produced from a CSC-enriched population, presumably as the result of STAT3 inactivation and CAIX downregulation. Compared with single niclosamide, Nic-A with a disulfide release trigger and CAIX targeting displays improved selectively and an enhanced anti-metastatic effect; however, premature drug release in the bloodstream or in normal tissue cannot be ruled out based on the results obtained to date. Preclinical studies are currently planned to evaluate the pharmacokinetics, pharmacodynamics, and toxicity of Nic-A and address these potential concerns.

All reagents and assay kits used in this study are commercially available and used directly without purification. Experimental protocols including cell culture, confocal imaging, living animals, as well as the details of experimental instruments are described in SI Appendix.

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