Targeting Tumor Invasion

by Stephen Western
Astrocytoma Options.com

While most conventional chemotherapeutics are designed to kill or slow the proliferation of tumor cells, few of these agents are effective in preventing the migration, invasion into healthy tissue, and metastasis of tumor cells. Inhibiting tumor cell migration and invasion with currently available agents is a critically important but underexplored area of research.

Fluvoxamine

In March of 2016 a Japanese group published research showing that the cheap, off-patent anti-depressant fluvoxamine inhibits GBM migration and invasion both in vitro, and in vivo in an orthotopic mouse model using human glioma-initiating cells [23].

In this study, 18 antidepressants and antipsychotics were screened in vitro for their ability to inhibit actin polymerization in GBM cell lines, a process essential for cell migration and invasion. Of these, the most potent inhibitor was found to be fluvoxamine. Fluvoxamine treatment of three GBM cell lines inhibited cell migration in a dose-dependent manner. In contrast, fluvoxamine had no effect on cell proliferation in the same three cell lines.

In follow-up studies, the brains of nude mice were injected with human glioma-initiating cells. One week later, fluvoxamine treatment of the mice was initiated by daily intraperitoneal injection. The dosage of fluvoxamine used in the mice had no effect on mouse body weight. At four weeks, tumors were isolated and histologically examined. Untreated tumors showed a disorderly invasion into healthy tissue, while tumors from fluvoxamine-treated mice were significantly smaller with invading cells forming island-like shapes that clumped together at the tumor border. CD133 positive cells (a marker of glioma stem-like cells) were found localized to the small tumor area in the fluvoxamine-treated mice, while CD133+ cells had spread out from the main tumor in untreated control mice. Vascular endothelial cells and proliferative Ki-67-positive cells were also greatly reduced in the fluvoxamine-treated tumors. Survival of mice in the fluvoxamine-treated group was extended.

Curcumin (preclinical evidence)

The plant polyphenol curcumin, derived from turmeric (discussed in the Supplements section) is also reported to have JAK/STAT3 pathway inhibiting activity (5). In a preclinical study of murine gliomas (5), curcumin was proven to be a STAT3 inhibitor by downregulating the activating kinase JAK1 and 2. Even more significantly, a 10 micromole per litre concentration of curcumin (likely achievable in brain tissue) led to significant reductions in transcripts of MMP-9 and Snail, which are both relevant to glioma invasion. An even smaller concentration of curcumin, 5 micromoles per litre (certainly achievable in brain tissue), led to a significant reduction in the number of migrating cells in a cell migration assay. When these cells were transfected with a persistently activated form of STAT3, curcumin's invasion-reducing ability was blunted, showing the importance of STAT3 inhibition in curcumin's effects. When mice implanted with glioma tumours were fed curcumin in their diet (0.05% by weight), there was a significant decrease in migration of glioma cells to the contra-lateral side of the brain.

Minocycline

In a 2013 study (20), SKOV-3 ovarian cancer cells were exposed to 10 micromolar (uM) concentration of minocycline for 18 hours. Cell migration, cell invasion, and cell adhesion were significantly reduced by this treatment. Mice bearing ovarian tumor xenografts were treated with intraperitoneal minocycline at a dose of 30 mg/kg mouse body weight. Plasma interleukin-6 (IL-6) levels were significantly decreased at 4 and 24 hours after minocycline treatment. IL-6 is a cytokine which leads to STAT3 activation. In the ovarian cancer tumors, minocycline treatment led to significant reductions in interleukin-6, and phosphorylated (activated) STAT3 at both the 4 and 24 hour time points.

Inhibiting NF-KB

The primary disulfiram metabolite, diethyldithiocarbamate, inhibits single cell migration of cholangiocarcinoma cells at concentrations as low as 0.1 - 1 micromolar (uM). This effect is dependent on NF-KB inhibition by disulfiram and metabolites (21).

Inhibiting GRP78

Glucose-regulated protein, 78 kDa (GRP78) is a stress-related protein that co-ordinates the unfolded protein response and relieves endoplasmic reticulum (ER) stress. It is associated with chemotherapy resistance and is especially active in glioblastoma samples. Knockdown of GRP78 using siRNA (small interfering RNA) technology leads to increased sensitivity to radiation, decreased tumorsphere formation, and decreased migration and invasion in glioblastoma cells in vitro (22).

Pterostilbene

A Taiwanese group published a study (22) in early 2015 showing that the naturally-occuring nutraceutical pterostilbene at a 1 µM concentration significantly inhibited the migration and invasion of GBM cells in vitro. Pterostilbene's mechanism of action was found to be the induction of microRNA-205, leading to decreased expression of GRP78 and other tumour-associated proteins such as c-Myc. Via the same mechanism, pterostilbene also suppressed glioma stem cell properties and increased radiosensitivity in glioblastoma cells (see the Radiation page).

Inhibiting glycogen synthase kinase 3 beta (GSK3B)

An abstract from the 2015 annual meeting of SNO describes preclinical work in Japan with GSK3B inhibitors and their effect on glioma cell invasion. Four drugs, cimetidine, lithium carbonate, olanzapine, and valproic acid were shown to inhibit migration and invasion of GBM cells (U87, T98G, U251) in a dose-dependent manner. Then, these same drugs were given orally to mice with GBM xenografts once daily for two weeks. According to the abstract, "all drugs reduced satellite lesions and nestin-positive cells in vivo." The abstract does not tell us drug doses, or whether the drugs were effective individually, or only when combined in vivo. We hope to see the full study published soon.

Another study with cimetidine also showed anti-migratory effects on GBM cells in vitro, as described on the Repurposed Drugs page.

Inhibiting Matrix Metalloproteinase-2 and -9 (MMP2 and MMP9)

MMP-2 and MMP-9 are proteases which degrade extracellular matrix and are therefore critically important for cell invasion.

Minocycline

Minocycline hydrochloride is a tetracycline-derived antibiotic used to treat bacterial infections, including E. coli. Several preclinical studies have suggested a potential use for minocycline in glioma, leading to a phase I/II trial of repeat irradiation, Avastin, and minocycline for recurrent glioblastoma. The main target of minocycline in the context of glioma is the invasion-promoting capacity of glioma-associated microglia (discussed on the Targeting Tumour-Associated Macrophages and Microglia page). One study (17) showed that minocycline, fed in the drinking water to immunocompetent mice with GL-261 gliomas, reduced tumour volume by 46 to 78% compared to untreated controls, depending on the timing of minocycline initiation (starting on day 7 versus day 1). The mechanism involved was the inhibition by minocycline of microglial expression of membrane type 1 matrix metalloprotease (MT1-MMP), a necessary co-factor to convert inactive pro-MMP-2 to active MMP-2.

A more recent study by the same group (18) published in April 2014 in the International Journal of Cancer, found that MMP-9 (the other major matrix metalloprotease associated with glioma invasion) is mainly secreted by glioma-associated microglia, rather than by glioma cells. When microglia were cultured in glioma-conditioned medium, microglial MMP-9 mRNA was increased 17-fold after 24 hours, showing that glioma cells influence microglia to produce invasion-promoting substances, such as MMP-9.

In vitro, a 50 micromolar concentration of minocycline inhibited microglial MMP-9 production, and 25 micromolar minocycline reduced the microglial expression of toll-like receptor 2 (TLR2), the main cell-surface receptor for glioma-microglia signalling. Next, GL-261 brain-tumour bearing immunocompetent mice were given minocycline at a dose of 30 mg per kg body weight (comparable to the human dose of 200 mg total per day) via their drinking water, starting on the day of tumour cell implantation. Minocycline treatment led to a small, but statistically significant increase in median survival. It must be remembered that unlike humans, experimental mice do not usually undergo surgery as part of their glioma "treatment" and would be killed by the rapidly growing bulk tumour, rather than by the invasive component. Median survival compared to untreated controls is therefore probably not the best in vivo indicator of efficacy for agents which primarily target glioma invasion. Regardless, in these experiments minocycline was shown to be moderately to substantially effective as a single agent when considering either survival or tumour volume reduction versus untreated controls.

Chlorotoxin

Chlorotoxin, a scorpion venom derived from the species Leiurus quinquestriatus, is being studied as an anti-invasive glioma therapy. Chlorotoxin selectively binds to the surface of glioma cells, but not to normal brain. A preclinical study showed that the chlorotoxin receptor on glioma cells is MMP-2, which it selectively binds, modulating its surface expression and inhibiting its invasion-promoting role (12). Chlorotoxin also inhibits chloride ion channels, which are likewise important for glioma invasion (13, 14). A phase II trial was carried out utilizing a synthetic version of chlorotoxin prepared to carry a dose of radioactive Iodine-131 to glioma cells. Due to the glioma-specific binding activity of chlorotoxin, the synthetic form is also used as an aid in glioma imaging.

Inhibiting Id-1

Id-1 (inhibitor of DNA-binding protein 1) represses the function of basic helix-loop-helix transcription factors. Several studies have shown Id-1 to be implicated in various cancer promoting functions such as glioma stem cell maintenance and increased microvessel density. At least two studies have shown that Id-1 promotes glioma migration and invasion (15, 16). One of these studies showed that Id-1 is positively regulated by the COX-2 enzyme via its product prostaglandin E2 (PGE2). Therefore COX-2 inhibitors such as Celebrex could be one way to block Id-1 expression. The other study (16) demonstrated that Id-1 regulates the invasiveness of U251 glioblastoma cells. In this study, cannabidiol (the second most prevalent cannabinoid in the cannabis plant) was used to inhibit Id-1 gene expression. When injected intraperitoneally into mice, cannabidiol downregulated Id-1 in the implanted U251 tumours, and decreased the tumour area by 95% compared to controls. Cannabidiol is also discussed on the Supplements page.

Inhibiting Lactic Acid

As discussed on the Diet page, cancer cells preferably derive their energy from aerobic glycolysis outside the mitochondria, rather than through oxidative respiration in the mitochondria. The main byproduct of this glycolytic metabolism is lactic acid, which accumulates outside the cell and acidifies the extracellular environment. A recent preclinical study (10) showed that lactic acid facilitates tumour cell migration by increasing thrombospondin-1 (THBS-1) production, which in turn activates transforming growth factor beta-2 (TGFb2), a well-known immunosuppressive and pro-invasive cytokine. Perhaps this aspect of migratory cell biology could be targeted with dichloroacetate (see discussion in Repurposed Drugs), which causes pyruvate to enter the mitochondria, preventing its glycolytic conversion to lactic acid. The primary clinical use of DCA has in fact been to treat mitochondrial disorders involving lactic acidosis. As discussed in Targeting Tumour Metabolism, DCA may be most effectively used in glioblastomas, which are known to have highly hypoxic regions and extracellular lactate accumulation. This may not be the case for lower grade gliomas, especially IDH1-mutant gliomas, which have silenced lactate dehydrogenase A genes, and reduced expression of glycolytic genes (via reduced HIF1 activity).

Summary

The ideas in this section are exploratory. The inhibition of cell proliferation, which is the target of most conventional chemotherapy, is a far more developed practice than the inhibition of cell migration and invasion, though both are of equal importance. As migrating cells have increased resistance to apoptosis, it is these cells along with the likewise therapy-resistant glioma stem cells which will require the most effort in terms of strategic targeting.

References

1. STAT3 silencing inhibits glioma single cell infiltration and tumor growth. Priester et al. 2013.
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2. Reciprocal activation of transcription factors underlies the dichotomy between proliferation and invasion of glioma cells. Dhruv et al. 2013.
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3. STATs in cancer inflammation and immunity: a leading role for STAT3. Yu. 2009.
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4. Persistently activated STAT3 maintains constitutive NF-kappaB activity in tumors. Lee et al. 2009.
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5. Dietary curcumin attenuates glioma growth in a syngeneic mouse model by inhibition of the JAK1,2/STAT3 signaling pathway. Weissenberger et al. 2010.
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6. A conceptually new treatment approach for relapsed glioblastoma: Coordinated undermining of survival paths with nine repurposed drugs (CUSP9) by the International Initiative for Accelerated Improvement of Glioblastoma Care. Kast et al. 2013.
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7. European Medicines Agency recommends suspension of marketing authorisations for oral ketoconazole. Press release. July 2013.
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8. Matrix metalloproteinase-2 and -9 in glioblastoma: a trio of old drugs - captopril, disulfiram and nelfinavir - are inhibitors with potential as adjunctive treatments in glioblastoma. Kast et al. 2012.
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9. Modulating antiangiogenic resistance by inhibiting the signal transducer and activator of transcription 3 pathway in glioblastoma. de Groot et al. 2012.
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10. Lactate-modulated induction of THBS-1 activates transforming growth factor (TGF)-beta2 and migration of glioma cells in vitro. Seliger et al. 2013.
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11. Targeting Src family kinases inhibits bevacizumab-induced glioma cell invasion. Huveldt et al. 2013.
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12. Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2. Deshane et al. 2003.
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13. Use of chlorotoxin for targeting of primary brain tumors. Soroceanu et al. 1998.
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14. A role for ion channels in glioma cell invasion. Mcferrin et al. 2006.
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15. COX-2 overexpression increases malignant potential of human glioma cells through Id1. Xu et al. 2013.
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16. Id-1 is a key transcriptional regulator of glioblastoma aggressiveness and a novel therapeutic target. Soroceanu et al. 2013.
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17. Minocycline reduces glioma expansion and invasion by attenuating microglial MT1-MMP expression. Markovic et al. 2011.
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18. Glioma associated microglial MMP9 expression is up regulated by TLR2 signalling and sensitive to minocycline. Hu et al. 2014.
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19. Minomycin (minocycline hydrochloride) Product Information.
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20. Minocycline Suppresses Interleukine-6, Its Receptor System and Signaling Pathways and Impairs Migration, Invasion and Adhesion Capacity of Ovarian Cancer Cells: In Vitro and In Vivo Studies. Ataie-Kachoie et al. 2013.
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21. Diethyldithiocarbamate suppresses an NF-kappaB dependent metastatic pathway in cholangiocarcinoma cells. Srikoon et al. 2013.
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22. Pterostilbene suppressed irradiation-resistant glioma stem cells by modulating GRP78/miR-205 axis. Huynh et al. 2015.
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23. Fluvoxamine, an anti-depressant, inhibits human glioblastoma invasion by disrupting actin polymerization. Hayashi et al. 2016.
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This page was created on 12/28/2013 and last updated on 04/21/2019