OR09-1 Estrogen Drives Atypical Cell-autonomous WNT4 Signaling In Lobular Carcinoma

Abstract

Invasive lobular carcinoma (ILC) is an understudied breast cancer subtype. Clinical features of ILC suggest exquisite dependence on estrogen and the estrogen receptor (ER), and that ILC patients should have good outcomes with anti-estrogen treatment. However, relative to other breast cancers, patients with ILC have poorer long-term outcomes and poorer anti-estrogen response. Advances in ILC treatment are hindered by our poor understanding of the distinct biology of ILC, especially regarding ER signaling. We previously identified the Wnt ligand WNT4 as an ILC-specific ER transcriptional target critical for estrogen-induced growth and anti-estrogen resistance in ILC cells. We hypothesize that characterizing the WNT4 signaling network in ILC will identify novel biomarkers and therapeutic targets for ILC patients. Wnt signaling typically requires the enzyme PORCN, leading us to test whether PORCN inhibitors blocked growth of WNT4-dependent ILC cells. However, PORCN inhibition did not phenocopy WNT4 knockdown, suggesting an atypical role for PORCN in WNT4 signaling. WNT4 was over-expressed in a diverse cell line panel including ILC. Conditioned medium was assessed for Wnt secretion and activity, and dependence on Wnt secretion proteins PORCN and WLS. WLS was universally required for Wnt secretion and paracrine signaling. In contrast, WNT4 secretion was PORCN-independent in all models. Surprisingly, secreted WNT4 did not present paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we identified cell autonomous non-canonical Wnt signaling activation, independent of secretion, by WNT4. To define downstream cell-autonomous WNT4 signaling in ILC cells, we utilized reverse phase protein arrays (RPPA) and identified ER-mediated protein changes blocked by concurrent WNT4 knockdown. These experiments identified putative WNT4 signaling targets including Akt/mTOR signaling; estrogen treatment activated mTOR signaling, but not Akt, in ILC cells in a WNT4-dependent manner. Consistent with this, ILC cell lines were sensitive to mTOR-targeting inhibitors (eg everolimus) but not PI3K- or Akt-targeting inhibitors. Activation of mTOR by WNT4 was associated with atypical intracellular activation of DVL proteins (Wnt second messengers), as we identified that WNT4 can directly activate DVL independent of membrane receptors. Estrogen regulates ILC cell proliferation and survival by upregulating WNT4, subsequently initiating a novel cell-autonomous WNT4 signaling pathway that leads to mTOR activation. Further understanding of this pathway will develop strategies to target WNT4 signaling therapeutically, as currently available drugs (eg targeting PORCN) are ineffective. PORCN-independent cell-autonomous Wnt signaling may be critical in contexts otherwise considered to have dysfunctional Wnt signaling.