Potential Key Target for Deadly Brain Cancer Identified

Potential Key Target for Deadly Brain Cancer Identified

Inder Verma, PhD

Building on previous work showing increased NF-kB activity in lung cancers, a team at the laboratory of genetics, The Salk Institute for Biological Studies, La Jolla in California, has recently published a study showing this activity is also enhanced in glioblastoma, one of the most lethal forms of human cancers.

Through the development and experimental manipulation of anin vivomouse model, andin vitrostudies using mouse and human glioma cell lines, researchers have concluded that possible therapeutic avenues to pursue for glioblastoma multiforme include inhibition of NF-kB activity or targeting genes induced by NF-kB activity.^1,2

“This is a disease for which there has been practically no improvement in treatment outcome for years,” said Inder Verma, PhD, professor in the Salk Institute’s Laboratory of Genetics and senior author of the paper. “It is clear that even if a surgeon removes 99.99% of a glioblastoma multiforme tumor, what is left behind will come back and grow into more tumor.”³

Methods

First, the team created a mouse model of the mesenchymal subtype of glioblastoma, by injecting Cre-inducible oncogenic lentiviral vectors that contained either shNF1-shp53 or HRasV12-shp53 in the cortex of glial fibrillary acidic protein (GFAP) Cre mice.

Tumors were evoked in each case, and analysis revealed high expression of NF-kB target genes versus normal brain tissue. Further analyses included using 005 cells (tumor-derived mouse cell line with properties of a brain-tumor—initiating stem cell), and a RNA-Seq analysis of 005 tumors that showed a high expression of NF-kB target genes confirming that their lentivirus-induced MES glioma tumors had constitutive NF-kB activity.¹

Possible Therapeutic Approaches

The experiments then turned to blocking activity in the NF-kB pathway by inhibiting a key molecule, IKK2 an important kinase in the NF-kB activation cascade. By injecting 005 cells with an effective vector they were able to knockdown IKK2 and confirmed it using Western blot analysis. Subsequent quantitative real-time polymerase chain reaction (RT-PCR) analysis showed the expected decreased expression of NF-kB target genes, and also found a reduction in proliferation versus parental 005 cells.

When the knockdown IKK2 cells and control 005 cells were transplanted into mice, both developed glioma tumors. However, the knock down tumors did not develop as quickly, and Kaplan Meier analysis showed that control mice survived for about 40 days versus about 60 days for the IKK2 knockdown transplanted mice, respectively. Similar results were obtained when mice were given orthotopic transplants of cells with either Cre-inducible deletion of IKK2 or transduced with a nondegradable mutant form of IkBa (the IkBaM super repressor).¹

The team next sought to inhibit another key step in the activation cascade and utilized a peptide previously shown to enter the CNS and inhibit NF-kB in vivo.⁴This peptide, NF-kappaB essential modifier-binding domain (NBD), selectively inhibits NF-kB by blocking the interaction between NF-kB essential modifier (NEMO), a regulatory protein, and the IKK complex.

Experiments with glioma cell lines confirmed that NBD wild type (NBDwt) decreased the expression of NF-kB target genes. Mice were treated with NBDwt or NBD-mutant (NBDmut) peptide, intraperitoneally (daily dose 10 mg/kg), for 20 days following transplantation with 005 cells. A third group of mice were untreated. At 20 days, mice treated with the NBDwt peptide developed tumors with a longer latency versus mice treated or untreated with NBDmut peptide. Comparing the expression of NF-kB genes showed NBDwt peptide was inhibiting the activity of NF-kB and lowered the expression of Ki67. The researchers concluded that this indicates the NF-kB cascade is primarily pro-proliferative in their model of glioma.

Using a human glioma cell line with constitutive activity of NF-kB, they confirmed that treatment with NBDwt, but not NBDmut peptide decreased the expression of NF-kB target genes. When U87 glioma cells (expressing luciferase) were injected into the brains of nonobese, diabetic-severe immunodeficient (NOD — SCID) mice, treatment with NBDwt resulted in almost doubling of median survival time from less than 30 days (controls) to more than 50 days. “We could increase survival time from one month without treatment to 3 months with treatment,” said Verma. “That’s a profound increase in life expectancy, especially considering a mouse only lives for two years.”³Mice in the control group had succumbed to the disease after 30 days, but it was decided to continue treating the mice receiving NBDwt. Unfortunately, at 40 days they showed signs of toxicity, confirmed by analysis to be liver toxicity with elevated serum levels of L-alanine-2-oxoglutarate aminotransferase (ALT). Thus, NBDwt peptide can slow tumor growth in human and mouse models and can prolong survival.

Silencing a NF-kBTarget — Gene, Timp 1

“The ultimate goal is to block NF-kB, but because it turns on many genes—at least 100—our aim became finding the handful of genes that directly affect tumor growth,” said Verma.“Then we can be more selective in treatment.”³

It has recently been shown that Timp 1 is one of the mediators of NF-kB—induced lung cancer growth,⁶and the research team found that it is highly expressed in their model glioma tumors, and significantly upregulated versus pooled normal controls. It was found that Timp 1 expression in 005 cells decreased when an inhibitor of NF-kB known as 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide) or TPCA-1, was added. Similar results were obtained when NF-kB was inhibited by either deletion of IKK2 or expression of the super repressor in AR53Cre-ErT2 cells. Reduced levels of Timp 1 also resulted when 005 tumors were treated with NBDwt peptide versus controls.¹

Linking their experimental data to real patients, the team consulted the REpository for Molecular BRain Neoplaisia DaTa (REMBRANT) data set, and found when Timp 1 expression is down regulated, glioma patients have a more favorable prognosis than patients with an upregulation of Timp 1 expression.¹

Future Therapeutic Investigations

The authors concluded that their experiments confirm approaches inhibiting NF-kB or downstream target genes may be worthwhile avenues to pursue for the treatment of glioblastoma. Discussing the options, they noted that blocking the NF-kB cascade can lead to toxicities because of the pleiotropic actions of NF-kB. Although treatment with NBDwt peptide looked as though it would be promising, treatment had to be halted in the mouse model because of toxicity. The authors suggested that this may be overcome by targeting the peptide specifically to the liver, possibly using tumor-homing nanoparticles. Alternatively, on the basis of the team’s work on the glioblastoma mouse model and patient-derived cells, inhibiting a gene such as Timp 1 may be another opportunity to reduce tumor cell proliferation and prolong survival.¹

The authors also concluded that there are other opportunities to pursue to improve outcomes in glioblastoma, and these approaches administered locally following surgery may be expected to block cancer growth and recurrence, with reduced toxicity.¹

References

1. Friedmann-Morovinski D, Narasimamurthy R, Xia Y, Myskiw C, Soda Y, Verma IM. Targeting NF-kB in glioblastoma: a therapeutic approach.Science Advances. 08 Jan 2016:Vol. 2, no. 1, e1501292DOI: 10.1126/sciadv.1501292.
2. Xia Y, Shen S, Verma IM. NF-kB, an active player in human cancers.Cancer Immunol Res. 2014;2(9):823-830.
3. Salk. Scientists find key driver for treatment of deadly brain cancer. http://www.salk.edu/news-release/scientists-find-key-driver-for-treatment-of-deadly-brain-cancer/. Accessed January 15, 2016.
4. Ghosh A, Roy A, Liu X, et al. Selective inhibition of NF-kappaB activation prevents dopaminergic neuronal loss in a mouse model of Parkinson's disease.Proc Natl Acad Sci U S A. 2007;104(47):18754-18759.
5. Hayden MS, Ghosh S. Signaling to NF-kappaB.Genes Dev. 2004 ;18(18):2195-2224.
6. Xia Y, Yeddula N, Leblanc M, et al. Reduced cell proliferation by IKK2 depletion in a mouse lung-cancer model.Nat Cell Biol. 2012;14(3):257-65.