Inhibition of Cancer Relapse and Metastasis by BBI608.

Relapse after chemotherapy treatment is a common phenomenon (26). We hypothesized that stemness-high cancer cells are responsible for cancer relapse and that stemness inhibition can suppress cancer relapse. To effectively block cancer relapse it is, therefore, essential to target cancer stemness. Through a gene silencing-based approach, we identified Stat3 as a key driver for cancer stemness. We next set out to discover a druggable inhibitor of Stat3 to target cancer stemness, using in silico screening and computational biology to search for Stat3 binders, phenotype-driven testing to determine stemness inhibition, and Stat3-driven gene transcription to test for Stat3 inhibition. Through such an activity-oriented, quality-driven iterative research process, we identified BBI608.

To evaluate the phenomenon of tumor relapse, we used a pancreatic cancer xenograft model and the chemotherapy agent gemcitabine. Treatment with gemcitabine inhibited PaCa-2 xenograft tumor growth, with tumor growth inhibition of 47.5% on day 41 (Fig. 1A). However, after cessation of treatment on day 41, the tumors in the gemcitabine-treated animals soon relapsed and even outgrew the tumors in the vehicle control group (Fig. 1A). Treatment with BBI608 also significantly inhibited PaCa-2 xenograft tumor growth on day 41 (Fig. 1B). However, unlike the gemcitabine-treated animals, no tumor regrowth was observed in the animals administered BBI608 during the 22-d posttreatment observation period (Fig. 1B). Furthermore, no signs of toxicity as evidenced by body weight measurement were observed (Fig. S1 A and B). These data suggest that BBI608, unlike chemotherapy, can inhibit the cells within the tumor mass that are responsible for tumor relapse.

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Fig. 1.

Inhibition of cancer relapse by BBI608. (A) Immunosuppressed mice with established s.c. Paca-2 human pancreatic cancer were given gemcitabine (120 mg/kg) q3d, or vehicle control i.p. The animals received a total of 14 doses and were maintained 22 d for posttreatment observation. Tumor size was evaluated periodically during treatment and posttreatment observation. Each point represents the mean ± SEM of seven tumors. (B) Immunosuppressed mice with established s.c. PaCa-2 human pancreatic cancer were given BBI608 (20 mg/kg) daily or vehicle control i.p. and monitored as in A. (C) Immunosuppressed mice bearing intrasplenically inoculated HT29 human colon cancer cells were given daily BBI608 (20 mg/kg) or vehicle control i.p. Primary tumors at spleen and spontaneous liver metastases were examined macroscopically. A representative photograph is shown.

As another model for cancer relapse, we used the intrasplenic-nude mouse model system (ISMS) model to evaluate cancer relapse in a metastatic setting. This model involves the injection of colon cancer cells (HT29) into the spleen capsule of nude mice, and these colon cancer cells can form liver metastases spontaneously in a few weeks. To test the potential therapeutic role of BBI608 against metastasis, we used the ISMS model. BBI608 was found to effectively block spleen and liver metastases in the ISMS model (Fig. 1C). These data demonstrate that BBI608 is effective at blocking metastasis in vivo.

Depletion of Stemness-High Cancer Cells by BBI608.

We hypothesized that the antirelapse activity of BBI608 may be attributed to its effect on stemness-high cancer cells. To test this hypothesis, we used two human xenograft tumor models. In the pancreatic tumor (PaCa2) model animals were treated with vehicle, gemcitabine, or BBI608. Tumors were collected after 7 d of treatment, single-cell suspensions obtained from the tumors, and frequency of stemness-high cancer cells was determined by their self-renewal capacity as measured by their ability to grow as spheres when cultured under serum-free, attachment-free stem cell culture conditions. Compared with control-treated animals, BBI608 treatment decreased the stemness-high subpopulation by fivefold (Fig. 2A). By contrast, gemcitabine treatment caused a threefold increase in stemness-high cell population. Similar data were also observed in a head and neck tumor (FaDu) model treated with vehicle, carboplatin, or BBI608 (Fig. 2B). These data demonstrate that BBI608 is effective at targeting the stemness-high cancer cells in vivo, whereas standard chemotherapy caused an enrichment of the stemness-high cancer cell subpopulation.

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Fig. 2.

Depletion of stemness-high cancer cells by BBI608 in vivo. Mice were administered either vehicle, (A) gemcitabine (120 mg/kg [PaCa2]), (B) carboplatin (30 mg/kg [FaDu]), or 20 mg/kg of BBI608 i.p. After killing, tumors were collected after 7 or 14 d of treatment, for Paca-2 and FaDu cells, respectively. Single-cell suspensions were obtained after animal killing and sterile removal of tumors. Live cells were then counted and used to measure their ability to form spheres when cultured in cancer stem cell media.

BBI608 Can Block Survival and Self-Renewal of Stemness-High Cancer Cells.

To determine the effect of BBI608 on stemness-high cancer cells, we examined self-renewal of stemness-high cancer cells isolated or enriched by stem-cell culture selection (Fig. 3A), side population-based enrichment (Fig. 3B), or cancer stem cell surface markers (Fig. 3C). As shown in Fig. 3A, stemness-high colon cancer cells grown under stem cell culture conditions form spheres, and treatment with BBI608 blocked spherogenesis, suggesting that BBI608 inhibits self-renewal of stemness-high cancer cells

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Fig. 3.

Inhibition of self-renewal of stemness-high cancer cells by BBI608. (A) Spheres derived from DLD1 and HCT116 cell lines were dissociated to single cells and allowed to form spheres in suspension with cancer stem cell medium for 48 h before treatment with BBI608 (2 μM). After 24 h, the drugs were removed and the cells were cultured in fresh cancer stem cell medium for another 24 h. Representative images are shown. (B) SW480 colon cancer cells were isolated by FACS based on Hoechst dye exclusion and were cultured for 72 h in cancer stem cell conditions before the addition of the indicated concentrations of therapeutic agents. Sphere growth was scored by counting the number of spheres possessing >50 cells. SP, side population; NSP, nonside population. (C) CD44^high FaDu cells were isolated by FACS and were cultured for 72 h in cancer stem cell conditions before the addition of the indicated therapeutic agents (400 nM BBI608, 2 μM imatinib, 10 μM sunitinib, 10 μM erlotinib, 100 nM doxorubicin). Sphere growth was scored by counting the number of spheres possessing >50 cells. Representative images are shown. (D) CD34⁺ bone marrow mononuclear cells were treated with either DMSO or BBI608 for 6 h at 37 °C. Cells were then washed and plated in complete Methocult H4434 medium. Colonies of both erythroid and myeloid lineages containing >50 cells per colony were counted. Each treatment was performed in triplicate.

We determined whether the stemness-high population was resistant to chemotherapeutics and targeted kinase inhibitors. Stemness-high cancer cells were isolated by sorting for exclusion of Hoechst dye, followed by culture in the serum-free stem cell media. Sorting based on Hoechst dye exclusion provides two cell populations: a stemness-high side population and a stemness-low nonside population. Both the stemness-high side population and nonside population were killed by BBI608, with the side population being more sensitive to BBI608 (Fig. 3B). By contrast, whereas treatment of the nonside population with doxorubicin resulted in inhibition of viability, treatment of the side population with doxorubicin had little effect on viability (Fig. 3B). Stemness-high cancer cells were also isolated by sorting for high CD44 expression followed by culture in the serum-free stem cell media. Treatment of these cells with BBI608 resulted in inhibition of spherogenesis (Fig. 3C). By contrast, treatment with imatinib, sunitinib, erlotinib, or doxorubicin had little effect on the spherogenesis (Fig. 3C).

We next determined whether BBI608 affects normal stem cells. To address this issue we obtained CD34⁺ hematopoietic stem cells, treated them with BBI608, and then assessed their ability to form both erythroid and myeloid colonies. No inhibition of colony formation of CD34⁺ hematopoietic stem cells was observed by BBI608 treatment at up to 30 μM (the highest concentration tested) (Fig. 3D).

We next compared the potency of BBI608 and clinically used targeted therapeutics against stemness-high cancer cells and heterogeneous cancer cells under the same conditions. As shown in Table 1, stemness-high cancer cells displayed between threefold and 10-fold resistance to all targeted therapeutics tested, whereas the IC₅₀ for BBI608 was lower in the stemness-high cancer cell population than in the bulk cancer cells. We next determined the IC₅₀ of the inhibitory activity of BBI608 in stemness-high cancer cells isolated from a variety of human cancer cell lines, including head and neck, lung, brain, colon, liver, ovarian, pancreatic, and kidney cancer cell lines. As shown in Table 2, BBI608 was highly effective at targeting stemness-high cancer cells. These data suggest that stemness-high cancer cells are resistant to conventional chemotherapeutic and targeted agents tested but are sensitive to cancer stemness inhibitor BBI608.

BBI608 Down-Regulates Stemness Gene Expression.

Multiple pathways regulating the self-renewal of stem cells have been identified (16). Stat3, the target of BBI608, regulates many of the genes implicated in cancer stem cell self-renewal, including c-Myc and β-catenin (27⇓⇓⇓⇓–32). We investigated inhibition of these signaling pathways after treatment with BBI608. We found that BBI608 treatment resulted in a dose-dependent decrease in Nanog, Axl, Sox-2, Klf4, survivin, c-Myc, Bmi-1, and β-catenin protein levels (Fig. 4A). Levels of some proteins, such as c-Myc and Axl, were decreased as early as 3 h after treatment, whereas most were lower at 6 h, with the majority of proteins still reduced after 24 h (Fig. 4A). To expand on this observation, we analyzed the changes in gene expression after treatment with BBI608 using a cancer stem cell PCR array. Numerous key molecular markers and genes responsible for cancer stem cell proliferation and self-renewal were found to be down-regulated by BBI608 treatment (Fig. 4B), among them Nanog, Smo, Axl, Atm, and Bmi-1. Given that BBI608 treatment resulted in inhibition of multiple self-renewal pathways, we next compared the effect of BBI608 with chemo- and targeted therapeutics on stemness gene expression. Treatment of stemness-high cancer cells with BBI608 resulted in decreased expression of the self-renewal genes β-catenin, Nanog, Smo, and Sox2 (Fig. 5). By contrast, treatment of stemness-high cancer cells with the chemotherapeutic agents gemcitabine or carboplatin either had no effect or resulted in increased cancer stem cell gene expression (Fig. 5). Likewise, treatment with the kinase inhibitor sunitinib also resulted in increased cancer stem cell gene expression (Fig. 5). Taken together, these results demonstrate that treatment with BBI608 resulted in decreased expression of multiple cancer stem cell genes, whereas treatment with chemo- or targeted therapeutics resulted in increased cancer stem cell gene expression.

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Fig. 4.

BBI608 inhibits stemness gene expression. (A) FaDu cancer stem cells were treated for 3, 6, or 24 h with BBI608 at either 1 or 2 μM or with DMSO (0). Cell lysates from these treated cells were then analyzed by Western blotting. BBI608 down-regulates a number of stemness related proteins involved in the growth and maintenance of cancer stem cells. Actin is shown as a loading control. (B) FaDu sphere cultures were treated for 6 h with DMSO (control) or BBI608 at 2 μM. RNA was isolated and reverse transcribed and then the resulting cDNA analyzed using a quantitative PCR cancer stem cell array. GAPDH was used as a housekeeping gene to which the data were normalized. Data shows the top genes down-regulated after treatment with BBI608 normalized to the control treated sample.

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Fig. 5.

Comparison between BBI608 and current cancer therapeutics. FaDu cancer stem cells were treated for 24 h with DMSO (control), BBI608 (2 μM), sunitinib (20 μM), gemcitabine (2 μM), or carboplatin (32 μM). RNA was isolated, reverse transcribed, and then analyzed by quantitative PCR for stem cell genes: β-catenin, Nanog, SMO, and SOX2. GAPDH was used as a housekeeping gene to which the data were normalized.