Inhibition of nuclear factor kappa-B signaling reduces growth in medulloblastoma in vivo

Abstract

Background: Medulloblastoma is a highly malignant pediatric brain tumor that requires surgery, whole brain and spine irradiation, and intense chemotherapy for treatment. A more sophisticated understanding of the pathophysiology of medulloblastoma is needed to successfully reduce the intensity of treatment and improve outcomes. Nuclear factor kappa-B (NFκB) is a signaling pathway that controls transcriptional activation of genes important for tight regulation of many cellular processes and is aberrantly expressed in many types of cancer.

Methods: To test the importance of NFκB to medulloblastoma cell growth, the effects of multiple drugs that inhibit NFκB, pyrrolidine dithiocarbamate, diethyldithiocarbamate, sulfasalazine, curcumin and bortezomib, were studied in medulloblastoma cell lines compared to a malignant glioma cell line and normal neurons. Expression of endogenous NFκB was investigated in cultured cells, xenograft flank tumors, and primary human tumor samples. A dominant negative construct for the endogenous inhibitor of NFκB, IκB, was prepared from medulloblastoma cell lines and flank tumors were established to allow specific pathway inhibition.

Results: We report high constitutive activity of the canonical NFκB pathway, as seen by Western analysis of the NFκB subunit p65, in medulloblastoma tumors compared to normal brain. The p65 subunit of NFκB is extremely highly expressed in xenograft tumors from human medulloblastoma cell lines; though, conversely, the same cells in culture have minimal expression without specific stimulation. We demonstrate that pharmacological inhibition of NFκB in cell lines halts proliferation and leads to apoptosis. We show by immunohistochemical stain that phosphorylated p65 is found in the majority of primary tumor cells examined. Finally, expression of a dominant negative form of the endogenous inhibitor of NFκB, dnIκB, resulted in poor xenograft tumor growth, with average tumor volumes 40% smaller than controls.

Conclusions: These data collectively demonstrate that NFκB signaling is important for medulloblastoma tumor growth, and that inhibition can reduce tumor size and viability in vivo. We discuss the implications of NFκB signaling on the approach to managing patients with medulloblastoma in order to improve clinical outcomes.

Figure 1

Pharmacological inhibition of NFκB in medulloblastoma cell lines. A: Dose Dependent Survival: Dose response of medulloblastoma cell lines (cell number normalized to control) to NFκB inhibitors: pyrrolidine dithiocarbamate = PDTC, diethyldithiocarbamate = DDTC, sulfasalazine = SAS, curcumin = CURC, bortezomib = BTZ. (n = 3) Drug applied on day 0, cell count day 3 B: Time dependent survival: Time course of Daoy and D425 cells in response to IC90 values of PDTC (cell number normalized to control). Daoy 325 nM PDTC; D425 350 nM PDTC. (n = 3) Drug was applied only once on Day 0 C: Apoptosis: Annexin V staining for apoptosis in cell lines after 3 days in culture with designated concentrations of PDTC (n = 3 or more)

Figure 2

Pharmacological inhibition of NFκB in other cells. A: DDTC, PDTC in glioma: Dose response of human U-87MG glioma cells to PDTC and DDTC after 4 days in culture (cell number normalized to control). n = 3 B: PDTC in immature neurons: Dose response of mouse neurosphere stem cells to PDTC (mM): Western analysis probing with anti-cleaved caspase 3(Casp 3) (band ~19kD).

Figure 3

NFκB expression in medulloblastoma in vitro and in vivo. A: Activation of NFκB: Timecourse of NFκB activation by 15 ng/mL TNFα in Daoy cells by probing for nuclear p65, nuclear phos(S276) p65, & cytoplasmic IκB. nuc = nuclear fraction, cyto = cytoplasmic fraction. p65 band ~70kD; phos p65 band ~97kD; IκB band ~37kD B: Tissue Expression: expression of NFκB p65 subunit in D425 cells grown in culture (CC) compared to D425 cells grown in a flank tumor model (FT). Nuclear (nuc) and cytoplasmic (cyto) fractions were separated by differential centrifugation following lysis by nitrogen cavitation as described. C: p65 expression by Western in cultured cells vs flank tumors for 2 additional cell lines. CC = cells in culture, FT = flank tumor. p65 band~60kD. Extracts were obtained using the NE-PER kit as described.

Figure 4

Primary tumor expression of NFκB. A-C: Phosphorylated p65 in primary medulloblastoma: immunohistochemical staining of three different primary medulloblastoma tumors with phos(S276)p65 antibody. Magnification: A = 10X; B = 20X; C = 10X. C shows endothelial cell positivity in addition to tumor cells. D: Expression of p65 and phos (S536) p65 in normal brain and tumor in humans [CX = cerebral cortex, CB1 and CB2 = cerebella from 2 different autopsy specimens; mb4, mb2, mb8 = surgical samples of 3 different patient medulloblastoma tumors] along with normal C57Bl/6 mouse cerebellum (CB) compared to Smo/Smo mouse (Smo) cerebellar tumor.

Figure 5

Dominant negative inhibitor. A. Induction of NFκB in 4H10 cells with expression of dnIκB (-DOX) or without dnIκB (+DOX) by 15 ng/mL TNFα for 30 min. nuc = nuclear fraction, cyto = cytoplasmic fraction. B. Mean tumor growth with and without dnIκB: Tumor volumes measured in athymic mice beginning at 21 days after xenograft 4H10 cell implantation and ending at day 37. C: Xenograft tumors were removed from animals and measured in 3 dimensions. Volumes were calculated using 4/3 π ABC, the volume of an ellipsoid, where A = length, B = width, C = breadth. No DOX represents animals not given doxycycline, therefore expression of dnIκB was permitted, blocking NFκB activation. D. Western of total tissue lysates from 4H10 (dnIκB) tumors removed from xenograft animals. Three tumors from animals with a normal diet (No DOX) are compared to three tumors from animals receiving DOX, which shuts off expression of dnIκB.