Wnt signaling pathway is aberrantly activated in a variety of cancers, especially in colorectal cancer (CRC), because of mutations in the genes encoding adenomatous polyposis coli (APC), β-catenin and Axin. Small-molecule antagonists of Wnt/β-catenin signaling are attractive candidates for developing effective therapeutics for CRC. In this study, we have identified a novel Wnt signaling inhibitor, isopropyl 9-ethyl-1- (naphthalen-1-yl)-9H-pyrido[3,4-b]indole-3- carboxylate (Z86). Z86 inhibited Wnt reporter activities and the expression of endogenous Wnt signaling target genes in mammalian cells and antagonized the second axis formation of Xenopus embryos induced by Wnt8. We showed that Z86 treatment inhibits GSK3β (Ser9) phosphorylation, leading to its overactivation and promoting the phosphorylation and degradation of β-catenin. In vitro, Z86 selectively inhibited the growth of CRC cells with constitutive Wnt signaling and caused obvious G1-phase arrest of the cell cycle. Notably, in a nude mouse model, Z86 inhibited dramatically the xenografted tumor growth of CRC. Daily intraperitoneal injection of Z86 at 5 mg/kg resulted in >70% reduction in the tumor weight of HCT116 cell origin that was associated with decreased GSK3β (Ser9) phosphorylation and increased β-catenin phosphorylation. Taken together, our findings provide a novel promising chemotype for CRC therapeutics development targeting the canonical Wnt signaling.

Background: HER receptor tyrosine kinases (EGFR/HER1; HER2; HER3; HER4) and their soluble ligands represent a robust system involved in the regulation of a diverse array of cellular processes. Deregulation of HER receptors, notably HER2, has been linked to the initiation and progression of breast cancer and other solid tumors. Relatively less is known about the role of HER receptor soluble ligands in tumorigenesis and responsiveness to HER targeted therapies. Here we will discuss the impact of the HER3 ligand heregulin (HRG) on the sensitivity of breast cancers to HER tyrosine kinase inhibitors (TKIs), and identify TKI strategies to treat HRG-driven breast cancers. Methods: The effects of exogenous HRG (50ng/ml) on the antitumor activity of a panel of TKIs with different enzymatic properties e.g. a reversible, selective inhibitor (lapatinib) and irreversible, pan-HER inhibitors (neratinib; CI-1033) were assessed in HER2+ breast cancer (BC) cell lines. The concentration of TKIs used was in the 1–2.5 uM range, and cells were treated over a 72 hr course. Vehicle treatment alone served as controls. The impact of the above mentioned treatments on total HER receptor and specific EGFR, HER2, and HER3 phosphotyrosine sites, in addition to the phosphorylation state of components of downstream MAPK and PI3K signaling pathways was analyzed through western blot. In addition to studying the effects of exogenous HRG, the impact of TKIs on a model of triple negative BC (TNBC) known to produce HRG in an autocrine manner was also evaluated. QRT-PCR was used to assess the effects of TKIs on HER receptor mRNA levels. Results: Lapatinib inhibited proliferation and phosphorylation of EGFR, HER2, HER3, and downstream MAPK and PI3K signaling pathways in HER2+ SKBR3 and BT474 cell lines. Pre-treatment with HRG abrogated the antitumor effects of a therapeutic concentration of lapatinib (1 uM), and reversed the inhibitory effects of lapatinib on the phosphorylation of HER receptors and components of their downstream signaling pathways e.g. Erk1/2 and Akt. Neratinib also blocked proliferation and phosphorylation in HER2+ BC cells. In contrast to lapatinib, the antitumor effects of neratinib were not reversed by exogenous HRG. Interestingly, treatment with neratinib and similar pan-HER irreversible TKIs, but not reversible TKIs, resulted in loss of HER2 and EGFR protein expression. QRT-PCR was used to evaluate if this effect was at the level of transcription. Furthermore, neratinib inhibited cell proliferation and blocked EGFR signaling in a TNBC model driven by autocrine produced HRG. Conclusions: Our findings suggest that HRG is a mediator of therapeutic resistance to lapatinib in HER2+ and TNBC. Neratinib demonstrates profound effects on HER signaling by markedly reducing HER2 and EGFR protein expression and blocking the pro-tumorigenic effects of autocrine or paracrine expression of HRG. In contrast to HRG, we previously showed that EGF, an EGFR specific ligand did not reverse the antitumor effects of lapatinib in breast cancer cells. Thus, the selection of HER targeted therapies in a given tumor should take into account not only the HER receptor expression profile, but also the presence of autocrine or paracrine derived ligands activating HER receptors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-03.

The regulation of gene expression through histone posttranslational modifications plays a crucial role in breast cancer progression. However, the molecular mechanisms underlying the contribution of histone modification to tumor initiation remain unclear. To gain a deeper understanding of the role of the histone modifier Enhancer of Zeste homology 2 (Ezh2) in the early stages of mammary tumor progression, we employed an inducible mammary organoid system bearing conditional Ezh2 alleles that faithfully recapitulates key events of luminal B breast cancer initiation. We showed that the loss of Ezh2 severely impairs oncogene-induced organoid growth, with Ezh2-deficient organoids maintaining a polarized epithelial phenotype. Transcriptomic profiling showed that Ezh2-deficient mammary epithelial cells up-regulated the expression of negative regulators of Wnt signaling and down-regulated genes involved in mTORC1 (mechanistic target of rapamycin complex 1) signaling. We identified Sfrp1 , a Wnt signaling suppressor, as an Ezh2 target gene that is derepressed and expressed in Ezh2-deficient epithelium. Furthermore, an analysis of breast cancer data revealed that Sfrp1 expression was associated with favorable clinical outcomes in luminal B breast cancer patients. Finally, we confirmed that targeting Ezh2 impairs mTORC1 activity through an indirect mechanism that up-regulates the expression of the tumor suppressor Pten. These findings indicate that Ezh2 integrates the mTORC1 and Wnt signaling pathways during early mammary tumor progression, arguing that inhibiting Ezh2 or therapeutically targeting Ezh2-dependent programs could be beneficial for the treatment of early-stage luminal B breast cancer.