The Lineage Determining Factor GRHL2 Collaborates with FOXA1 to Establish a Targetable Pathway in Endocrine Therapy-Resistant Breast Cancer.

Notwithstanding the positive clinical impact of endocrine therapies in estrogen receptor-alpha (ERα)-positive breast cancer, de novo and acquired resistance limits the therapeutic lifespan of existing drugs. Taking the position that resistance is nearly inevitable, we undertook a study to identify and exploit targetable vulnerabilities that were manifest in endocrine therapy-resistant disease. Using cellular and mouse models of endocrine therapy-sensitive and endocrine therapy-resistant breast cancer, together with contemporary discovery platforms, we identified a targetable pathway that is composed of the transcription factors FOXA1 and GRHL2, a coregulated target gene, the membrane receptor LYPD3, and the LYPD3 ligand, AGR2. Inhibition of the activity of this pathway using blocking antibodies directed against LYPD3 or AGR2 inhibits the growth of endocrine therapy-resistant tumors in mice, providing the rationale for near-term clinical development of humanized antibodies directed against these proteins.

Expression of Ror2 mediates invasive phenotypes in renal cell carcinoma.

Ror2 is a Wnt ligand receptor that is overexpressed in a variety of tumors including clear cell renal cell carcinoma (ccRCC). Here we demonstrate that expression of wild type Ror2 results in increased tumorigenic properties in in vitro cell culture and in vivo xenograft models. In addition, Ror2 expression produced positive changes in both cell migration and invasion, which were dependent on matrix metalloprotease 2 (MMP2) activity. Mutations in key regions of the kinase domain of Ror2 resulted in the abrogation of increased tumor growth, cell migration, and cell invasion observed with expression of wild-type Ror2. Finally, we examined Ror2 expression as a prognostic biomarker for ccRCC utilizing the TCGA ccRCC dataset. High expression of Ror2 showed a significant correlation with higher clinical stage, nuclear grade, and tumor stage. Furthermore, high expression of Ror2 in ccRCC patients correlated with significant lower overall survival, cancer specific survival, and recurrence free survival. Together, these findings suggest that Ror2 plays a central role in influencing the ccRCC phenotype, and can be considered as a negative prognostic biomarker and potential therapeutic target in this cancer.

Delineation of a FOXA1/ERα/AGR2 regulatory loop that is dysregulated in endocrine therapy-resistant breast cancer.

UNLABELLED: Tamoxifen, a selective estrogen receptor (ER) modulator (SERM), remains a frontline clinical therapy for patients with ERα-positive breast cancer. However, the relatively rapid development of resistance to this drug in the metastatic setting remains an impediment to a durable response. Although drug resistance likely arises by many different mechanisms, the consensus is that most of the implicated pathways facilitate the outgrowth of a subpopulation of cancer cells that can either recognize tamoxifen as an agonist or bypass the regulatory control of ERα. Notable in this regard is the observation here and in other studies that expression of anterior gradient homology 2 (AGR2), a known proto-oncogene and disulfide isomerase, was induced by both estrogen (17ß-estradiol, E2) and 4-hydroxytamoxifen (4OHT) in breast cancer cells. The importance of AGR2 expression is highlighted here by the observation that (i) its knockdown inhibited the growth of both tamoxifen-sensitive and -resistant breast cancer cells and (ii) its increased expression enhanced the growth of ERα-positive tumors in vivo and increased the migratory capacity of breast cancer cells in vitro. Interestingly, as with most ERα target genes, the expression of AGR2 in all breast cancer cells examined requires the transcription factor FOXA1. However, in tamoxifen-resistant cells, the expression of AGR2 occurs in a constitutive manner, requiring FOXA1, but loses its dependence on ER. Taken together, these data define the importance of AGR2 in breast cancer cell growth and highlight a mechanism where changes in FOXA1 activity obviate the need for ER in the regulation of this gene. IMPLICATIONS: These findings reveal the transcriptional interplay between FOXA1 and ERα in controlling AGR2 during the transition from therapy-sensitive to -resistant breast cancer and implicate AGR2 as a relevant therapeutic target.

Receptor tyrosine kinase-like orphan receptor 2 (Ror2) expression creates a poised state of Wnt signaling in renal cancer.

Expression of the receptor tyrosine kinase-like orphan receptor 2 (Ror2) has been identified in an increasing array of tumor types and is known to play a role as an important mediator of Wnt signaling cascades. In this study, we aimed to clarify Ror2 interactions with the Wnt pathways within the context of renal cell carcinoma (RCC). An examination of Ror2 expression in primary human RCC tumors showed a significant correlation with several Wnt signaling genes, including the classical feedback target gene Axin2. We provide evidence that Ror2 expression results in a partially activated state for canonical Wnt signaling through an increased signaling pool of ß-catenin, leading to an enhancement of downstream target genes following Wnt3a stimulation in both renal and renal carcinoma-derived cells. Additionally, inhibition of low-density lipoprotein receptor-related protein 6 (LRP6) with either siRNA or dickkopf decreased the response to Wnt3a stimulation, but no change was seen in the increased ß-catenin pool associated with Ror2 expression, suggesting that LRP6 cofactor recruitment is necessary for a Wnt3a-induced signal but that it does not participate in the Ror2 effect on ß-catenin signaling. These results highlight a new role for Ror2 in conveying a tonic signal to stabilize soluble ß-catenin and create a poised state of enhanced responsiveness to Wnt3a exogenous signals in RCC.

Identification of Ror2 as a hypoxia-inducible factor target in von Hippel-Lindau-associated renal cell carcinoma.

Ror2 is an orphan receptor tyrosine kinase with expression normally restricted to early stages of development. However, emerging evidence has placed aberrantly expressed Ror2, leading to an invasive phenotype, in several cancers including renal cell carcinoma (RCC). Although Ror2 is currently identified as up-regulated in an assortment of cancers, neither the regulatory role or mechanism of action have been delineated. We sought to place Ror2 in the most commonly mutated pathway of RCC, the loss of the tumor suppressor von Hippel-Lindau (VHL), which causes hypoxia-inducible factor (HIF)-1alpha and -2alpha stabilization and the transcriptional activation of a broad repertoire of response genes. We found that Ror2 was indeed associated with the pVHL loss in RCC as well as with VHL somatic mutations tightly coordinated with the induction of RCC. Additionally, knockdown and rescue analysis of HIF expression suggests that Ror2 is dependent on pathologic stabilization of either HIF-1alpha or HIF-2alpha. Subsequent evaluation of the ROR2 promoter suggests that HIF-2alpha and its dimerization partner, aryl hydrocarbon nuclear transferase localize to the ROR2 promoter via a cryptic transcriptional element. This data substantiates a unique regulation pattern for Ror2 in the VHL-HIF axis that has the potential to be applied to other cancer etiologies.

Molecularly targeted therapy in renal cell carcinoma.

Recent developments in the molecular biology of renal cell carcinoma have identified multiple pathways associated with the development of this cancer. Multiple strategies have been investigated targeting these pathways, with significant clinical benefits shown in early studies. This review aims to overview the findings of recent clinical trials and clarify the development of these compounds for use in renal cell carcinoma. The authors also aim to clarify the molecular pathways implicated in renal cell carcinoma and the clinical results in metastatic renal cell carcinoma with agents targeting these pathways. The relevant literature was reviewed concerning pathways implicated in the pathophysiology of renal cell carcinoma including pathways activated secondary to von Hippel-Lindau gene inactivation and PI-3 kinase/Akt/mammalian target of rapamycin pathway activation. Therapeutic targeting based upon underlying molecular biology in renal cell carcinoma has strong rationale. Substantial clinical activity has been reported with various agents targeting these pathways, most notably with vascular endothelial growth factor-targeted therapy. However, investigation is needed to optimally utilize these agents at the appropriate stage of disease and in the best combinations for maximal clinical benefit.