A canonical Wnt signal maintains adult mammary ductal stem cell activity, and this signal requires the Wnt signaling reception, LRP5. However, previous data from our laboratory have shown that LRP5 and LRP6 are co-expressed in mammary basal cells and that LRP6 is active, leading us to question why LRP6 is insufficient to mediate canonical signaling in the absence of LRP5. Here, we show that at endogenous levels of LRP5 and LRP6 both receptors are required to signal in response to some Wnt ligands both in vitro (in mouse embryonic fibroblasts and mammary epithelial cells) and in vivo (in mammary outgrowths). This subgroup of canonical ligands includes Wnt1, Wnt9b, and Wnt10b; the latter two are expressed in mammary gland. In contrast, the ligand commonly used experimentally, Wnt3a, prefers LRP6 and requires just one receptor regardless of cellular context. When either LRP5 or LRP6 is overexpressed, signaling remains ligand-dependent, but the requirement for both receptors is abrogated (regardless of ligand type). We have documented an LRP5-6 heteromer using immiscible filtration assisted by surface tension (IFAST) immunoprecipitation. Together, our data imply that under physiological conditions some Wnt ligands require both receptors to be present to generate a canonical signal. We have designed a model to explain our results based on the resistance of LRP5-6 heteromers to a selective inhibitor of E1/2-binding Wnt-LRP6 interaction. These data have implications for stem cell biology and for the analysis of the oncogenicity of LRP receptors that are often overexpressed in breast tumors.

The mouse mammary gland is an outstanding developmental model that exemplifies the activities of many of the effector pathways known to organize mammalian morphogenesis; furthermore, there are well-characterized methods for the specific genetic manipulation of various mammary epithelial cell components. Among these signaling pathways, Wnt signaling has been shown to generate plasticity of fate determination, expanding the genetic programs available to cells in the mammary lineage. It is responsible first for the appearance of the mammary fate in embryonic ectoderm and then for maintaining bi-potential basal stem cells in adult mammary ductal trees. Recent technical developments have led to the separate analysis of various mammary epithelial cell subpopulations, spurring the investigation of Wnt-dependent interactions. Although Wnt signaling was shown to be oncogenic for mouse mammary epithelium even before being identified as the principle oncogenic driver for gut epithelium, conclusive data implicating this pathway as a tumor driver for breast cancer lag behind, and we examine potential reasons.

CRD-BP/IGF2BP1 has been characterized as an "oncofetal" RNA binding protein typically highly expressed in embryonic tissues, suppressed in normal adult tissues, but induced in many tumor types. In this study, we show that adult breast tissues express ubiquitous but low levels of CRD-BP protein and mRNA. Although CRD-BP mRNA expression is induced in breast tumor cells, levels remain ∼1000-fold lower than in embryonic tissues. Despite low expression levels, CRD-BP is required for clonogenic growth of breast cancer cells. We reveal that because the most common protein isoform in normal adult breast and breast tumors has an N-terminal deletion (lacking two RNA recognition motif (RRM) domains) and is therefore missing antibody epitopes, CRD-BP expression has been under-reported by previous studies. We show that a CRD-BP mutant mouse strain retains expression of the shorter transcript (ΔN-CRD-BP), which originates in intron 2, suggesting that the impact of complete ablation of this gene in mice is not yet known. Either the full-length CRD-BP or the N-terminally truncated version can rescue the clonogenicity of CRD-BP knockdown breast cancer cells, suggesting that clonogenic function is served by either CRD-BP isoform. In summary, although CRD-BP expression levels are low in breast cancer cells, this protein is necessary for clonogenic activity. Background: The RNA binding protein, coding region determinant-binding protein (CRD-BP), is expressed by tumor cells and protects key mRNAs. Results: This study identifies a novel variant of CRD-BP and finds that CRD-BP is required for breast tumor cell clonogenicity. Conclusion: CRD-BP has tumorigenic activity and is ubiquitously expressed in breast epithelium. Significance: Under-reporting of CRD-BP isoforms suggests that published studies may be incomplete.

Although antiestrogen therapies are successful in many patients with estrogen receptor alpha-positive (ERα+) breast cancer, 25% to 40% fail to respond. Although multiple mechanisms underlie evasion of these treatments, including tumor heterogeneity and drug-resistant cancer stem cells (CSC), further investigations have been limited by the paucity of preclinical ERα+ tumor models. Here, we examined a mouse model of prolactin-induced aggressive ERα+ breast cancer, which mimics the epidemiologic link between prolactin exposure and increased risk for metastatic ERα+ tumors. Like a subset of ERα+ patient cancers, the prolactin-induced adenocarcinomas contained two major tumor subpopulations that expressed markers of normal luminal and basal epithelial cells. CSC activity was distributed equally across these two tumor subpopulations. Treatment with the selective estrogen receptor downregulator (SERD), ICI 182,780 (ICI), did not slow tumor growth, but induced adaptive responses in CSC activity, increased markers of plasticity including target gene reporters of Wnt/Notch signaling and epithelial–mesenchymal transition, and increased double-positive (K8/K5) cells. In primary tumorsphere cultures, ICI stimulated CSC self-renewal and was able to overcome the dependence of self-renewal upon Wnt or Notch signaling individually, but not together. Our findings demonstrate that treatment of aggressive mixed lineage ERα+ breast cancers with a SERD does not inhibit growth, but rather evokes tumor cell plasticity and regenerative CSC activity, predicting likely negative impacts on patient tumors with these characteristics. Significance: This study suggests that treatment of a subset of ERα+ breast cancers with antiestrogen therapies may not only fail to slow growth but also promote aggressive behavior by evoking tumor cell plasticity and regenerative CSC activity. Cancer Res; 78(7); 1672–84. ©2018 AACR.

Lrp5 is typically described as a Wnt signaling receptor, albeit a less effective Wnt signaling receptor than the better studied sister isoform, Lrp6. Here we show that Lrp5 is only a minor player in the response to Wnt3a-type ligands in mammary epithelial cells; instead Lrp5 is required for glucose uptake, and glucose uptake regulates the growth rate of mammary epithelial cells in culture. Thus, loss of Lrp5 leads to profound growth suppression, whether growth is induced by serum or by specific growth factors, and this inhibition is not due to loss of Wnt signaling. Depletion of Lrp5 decreases glucose uptake, lactate secretion and oxygen consumption rates; inhibition of glucose consumption phenocopies loss of Lrp5 function. Both Lrp5 knockdown and low external glucose induce mitochondrial stress, revealed as the accumulation of reactive oxygen species (ROS) and the activation of the ROS-sensitive checkpoint, p38α. In contrast, loss of function for Lrp6 reduces Wnt responsiveness but has little impact on growth. This highlights the distinct functions for these two Lrp receptors, and an important Wnt-ligand independent role of Lrp5 in glucose uptake in mammary epithelial cells.