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. 2017 Sep 1;10(9):1109-1115.
doi: 10.1242/dmm.030882.

NFκB Signaling in Alveolar Rhabdomyosarcoma

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Free PMC article

NFκB Signaling in Alveolar Rhabdomyosarcoma

Megan M Cleary et al. Dis Model Mech. .
Free PMC article

Abstract

Alveolar rhabdomyosarcoma (aRMS) is a pediatric soft tissue cancer commonly associated with a chromosomal translocation that leads to the expression of a Pax3:Foxo1 or Pax7:Foxo1 fusion protein, the developmental underpinnings of which may give clues to its therapeutic approaches. In aRMS, the NFκB-YY1-miR-29 regulatory circuit is dysregulated, resulting in repression of miR-29 and loss of the associated tumor suppressor activity. To further elucidate the role of NFκB in aRMS, we first tested 55 unique sarcoma cell lines and primary cell cultures in a large-scale chemical screen targeting diverse molecular pathways. We found that pharmacological inhibition of NFκB activity resulted in decreased cell proliferation of many of the aRMS tumor cultures. Surprisingly, mice that were orthotopically allografted with aRMS tumor cells exhibited no difference in tumor growth when administered an NFκB inhibitor, compared to control. Furthermore, inhibition of NFκB by genetically ablating its activating kinase inhibitor, IKKβ, by conditional deletion in a mouse model harboring the Pax3:Foxo1 chimeric oncogene failed to abrogate spontaneous tumor growth. Genetically engineered mice with conditionally deleted IKKβ exhibited a paradoxical decrease in tumor latency compared with those with active NFκB. However, using a synthetic-lethal approach, primary cell cultures derived from tumors with inactivated NFκB showed sensitivity to the BCL-2 inhibitor navitoclax. When used in combination with an NFκB inhibitor, navitoclax was synergistic in decreasing the growth of both human and IKKβ wild-type mouse aRMS cells, indicating that inactivation of NFκB alone may not be sufficient for reducing tumor growth, but, when combined with another targeted therapeutic, may be clinically beneficial.

Keywords: Cancer; IKKβ; NFκB; Rhabdomyosarcoma.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
In vivo efficacy of an NBD peptide on aRMS. (A) Tumor growth over time of SHO mice orthotopically allografted with aRMS tumor cells. Mice were treated with NBD peptide (black line; n=4; 10 mg/kg 3× per week by intraperitoneal injection) or vehicle (dashed line; n=4; 100 µl PBS 3× per week by intraperitoneal route), with endpoint measurement of tumor volume being 1.4cc. (B) qRT-PCR showing mRNA levels of Pax3:Foxo1 in lungs of mice treated with vehicle or NBD peptide. (C) Number of independent lung metastases counted during histological analysis.
Fig. 2.
Fig. 2.
IKKβ deletion in aRMS. (A) Kaplan–Meier survival curve of animals with p53 inactivation and Pax3:Foxo1 activation in the Myf6Cre lineage (IKKwt/wt) or IKKβ loss in combination with p53 inactivation and Pax3:Foxo1 activation (IKKnull/null). The addition of IKKβ deletion to Pax3:Foxo1, p53 mice significantly decreased tumor latency (paired t-test; P=0.0017). Conditional deletion of IKKβ protein was confirmed by western blot in all animals harboring the IKKβnull allele. (B) EMSA performed with IKKwt/wt or IKKβnull/null cell extracts. Arrowheads denote p65/DNA bound complexes. (C) Representative western blot of IKKβ protein expression in aRMS mice with IKKβnull allele. (D-F) Representative images of H&E (D), myogenin (E) or KI-67 (F) staining on tumors from IKKβnull/null mice (U65261) compared with those from IKKwt/wt control mice (U66564). (G,H) Anatomical site and tumor stage of tumors in IKKβwt/wt control mice compared to those with IKKβ deletion. U/G, urogenital; Unk, unknown.
Fig. 3.
Fig. 3.
Chemical screens for complementation of IKK genetic deletion. (A) Navitoclax (bold) was efficacious in abrogating tumor cell growth in aRMS tumors that expressed both active and inactive NFκB. Deeper red colors confer higher sensitivity. All concentrations listed are in nM. The complete results are listed in Table S2. (B) Combination indices reflecting mouse and human aRMS cell lines. A combination index 0<1 represents a synergistic combination, 1 represents a neutral combination, and 1>2.5 is antagonistic. Synergistic combinations were achieved at doses within the reported active NFκB inhibitory range.

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