Triple-negative breast cancers (TNBC), which include the basal-like and claudin-low disease subtypes, are aggressive malignancies for which effective therapeutic targets are lacking. NF-κB activation has an established role in breast malignancy, and it is higher in TNBC than other breast cancer subtypes. On this basis, we hypothesized that proteins derived from NF-κB target genes might be molecular targets for TNBC therapy. In this study, we conducted a microarray-based screen for novel NF-κB–inducible proteins as candidate therapeutic targets, identifying tropomodulin 1 (TMOD1) as a lead candidate. TMOD1 expression was regulated directly by NF-κB and was significantly higher in TNBC than other breast cancer subtypes. TMOD1 elevation is associated with enhanced tumor growth in a mouse tumor xenograft model and in a 3D type I collagen culture. TMOD1-dependent tumor growth was correlated with MMP13 induction, which was mediated by TMOD1-dependent accumulation of β-catenin. Overall, our study highlighted a novel TMOD1-mediated link between NF-κB activation and MMP13 induction, which accounts in part for the NF-κB–dependent malignant phenotype of TNBC. Cancer Res; 75(1); 1–11. ©2014 AACR.

Pediatric tumors harbor very low numbers of somatic mutations and therefore offer few targets to improve therapeutic management with targeted drugs. In particular, outcomes remain dismal for patients with metastatic alveolar rhabdomyosarcoma (aRMS), where the chimeric transcription factor PAX3/7-FOXO1 has been implicated but problematic to target. In this report, we addressed this challenge by developing a two-armed screen for druggable upstream regulatory kinases in the PAX3/7-FOXO1 pathway. Screening libraries of kinome siRNA and small molecules, we defined PLK1 as an upstream-acting regulator. Mechanistically, PLK1 interacted with and phosphorylated PAX3-FOXO1 at the novel site S503, leading to protein stabilization. Notably, PLK1 inhibition led to elevated ubiquitination and rapid proteasomal degradation of the PAX3-FOXO1 chimeric oncoprotein. On this basis, we embarked on a preclinical validation of PLK1 as a target in a xenograft mouse model of aRMS, where the PLK1 inhibitor BI 2536 reduced PAX3-FOXO1–mediated gene expression and elicited tumor regression. Clinically, analysis of human aRMS tumor biopsies documented high PLK1 expression to offer prognostic significance for both event-free survival and overall survival. Taken together, these preclinical studies validate the PLK1–PAX3-FOXO1 axis as a rational target to treat aRMS. Cancer Res; 75(1); 1–13. ©2014 AACR.

Pediatric tumors harbor very low numbers of somatic mutations and therefore offer few targets to improve therapeutic management with targeted drugs. In particular, outcomes remain dismal for patients with metastatic alveolar rhabdomyosarcoma (aRMS), where the chimeric transcription factor PAX3/7-FOXO1 has been implicated but problematic to target. In this report, we addressed this challenge by developing a two-armed screen for druggable upstream regulatory kinases in the PAX3/7-FOXO1 pathway. Screening libraries of kinome siRNA and small molecules, we defined PLK1 as an upstream-acting regulator. Mechanistically, PLK1 interacted with and phosphorylated PAX3-FOXO1 at the novel site S503 leading to protein stabilization. Notably, PLK1 inhibition led to elevated ubiquitination and rapid proteasomal degradation of the PAX3-FOXO1 chimeric oncoprotein. On this basis, we embarked on a preclinical validation of PLK1 as target in a xenograft mouse model of aRMS, where the PLK1 inhibitor BI 2536 reduced PAX3-FOXO1-mediated gene expression and elicited tumor regression. Clinically, analysis of human aRMS tumor biopsies documented high PLK1 expression to offer prognostic significance for both event-free and overall survival. Taken together, these preclinical studies validate the PLK1 – PAX3-FOXO1 axis as a rational target to treat alveolar rhabdomyosarcoma.

Osteopontin (OPN) has been implicated widely in tumor growth and metastasis, but the range of its contributions is not yet fully understood. In this study, we show that genetic ablation of Opn in mice sensitizes them to diethylnitrosamine (DEN)-induced hepatocarcinogenesis. Opn-deficient mice (Opn−/− mice) exhibited enhanced production of proinflammatory cytokines and compensatory proliferation. Administering OPN antibody or recombinant OPN protein to wild-type or Opn−/− mice-derived macrophages, respectively, had little effect on cytokine production. In contrast, overexpression of intracellular OPN (iOPN) in Opn-deficient macrophages strongly suppressed production of proinflammatory cytokines. In addition, we found that iOPN was able to interact with the pivotal Toll-like receptor (TLR) signaling protein MyD88 in macrophages after stimulation with cellular debris, thereby disrupting TLR signaling in macrophages. Our results indicated that iOPN was capable of functioning as an endogenous negative regulator of TLR-mediated immune responses, acting to ameliorate production of proinflammatory cytokines and curtail DEN-induced hepatocarcinogenesis. Together, our results expand the important role of OPN in inflammation-associated cancers and deepen its relevance for novel treatment strategies in liver cancer. Cancer Res; 75(1); 1–12. ©2014 AACR.

Osteopontin (OPN) has been implicated widely in tumor growth and metastasis but the range of its contributions are not yet fully understood. In this study, we show that genetic ablation of OPN in the mouse sensitizes them to diethylnitrosamine (DEN)-induced hepatocarcinogenesis. Opn deficient mice (Opn-/- mice) exhibited enhanced production of proinflammatory cytokines and compensatory proliferation. Administering OPN antibody or recombinant OPN protein to wild type (WT) or Opn-/- mice-derived macrophages, respectively, had little effect on cytokine production. In contrast, overexpression of intracellular Opn (iOpn) in Opn-deficient macrophages strongly suppressed production of proinflammatory cytokines. In addition, we found iOPN was able to interact with the pivotal Toll-like receptor (TLR) signaling protein MyD88 in macrophages after stimulation with cellular debris, thereby disrupting TLR signaling in macrophages. Our results indicated that iOPN was capable of functioning as an endogenous negative regulator of TLR-mediated immune responses, acting to ameliorate production of proinflammatory cytokines and curtail DEN-induced hepatocarcinogenesis. Together, our results expand the important role of OPN in inflammation-associated cancers and deepen its relevance for novel treatment strategies in liver cancer.