MicroRNAs (miRNAs) are short, noncoding RNAs that post-transcriptionally regulate gene expression. While hundreds of mammalian miRNA genes have been identified, little is known about the pathways that regulate the production of active miRNA species. Here we show that a large fraction of miRNA genes are regulated posttranscriptionally. During early mouse development, many miRNA primary transcripts, including the Let-7 family, are present at high levels but are not processed by the enzyme Drosha. An analysis of gene expression in primary tumors indicates that the widespread downregulation of miRNAs observed in cancer is due to a failure at the Drosha processing step. These data uncover a novel regulatory step in miRNA function and provide a mechanism for miRNA down-regulation in cancer.Supplemental material is available at http://www.genesdev.org.

Inappropriate kinase expression and subsequent promiscuous activity defines the transformation of many solid tumors including renal cell carcinoma (RCC). Thus, the expression of novel tumor-associated kinases has the potential to dramatically shape tumor cell behavior. Further, identifying tumor-associated kinases can lend insight into patterns of tumor growth and characteristics. Here, we report the identification of Ror2, a new tumor-associated kinase in RCC cell lines and primary tumors. Ror2 is an orphan receptor tyrosine kinase with physiological expression normally seen in the embryonic kidney. However, in RCC, Ror2 expression correlated with expression of genes involved at the extracellular matrix, including Twist and matrix metalloprotease-2 (MMP2). Expression of MMP2 in RCC cells was suppressed by Ror2 knockdown, placing Ror2 as a mediator of MMP2 regulation in RCC and a potential regulator of extracellular matrix remodeling. The suppression of Ror2 not only inhibited cell migration, but also inhibited anchorage independent growth in soft agar and growth in an orthotopic xenograft model. These findings suggest a novel pathway of tumor-promoting activity by Ror2 within a subset of renal carcinomas, with significant implications for unraveling the tumorigenesis of RCC.

SUMMARY 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.

␣-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPARs) mediate excitatory neurotransmission at neuronal synapses, and their regulated localization plays a role in synaptic plasticity. In Caenorhabditis elegans, the PDZ and PTB domain-containing protein LIN-10 is required both for the synaptic localization of the AMPAR subunit GLR-1 and for vulval fate induction in epithelia. Here, we examine the role that different LIN-10 domains play in GLR-1 localization. We find that an amino-terminal region of LIN-10 directs LIN-10 protein localization to the Golgi and to synaptic clusters. In addition, mutations in the carboxyl-terminal PDZ domains prevent LIN-10 from regulating GLR-1 localization in neurons but do not prevent LIN-10 from functioning in the vulval epithelia. A mutation in the amino terminus prevents the protein from functioning in the vulval epithelia but does not prevent it from functioning to regulate GLR-1 localization in neurons. Finally, we show that human Mint2 can substitute for LIN-10 to facilitate GLR-1 localization in neurons and that the Mint2 amino terminus is critical for this function. Together, our data suggest that LIN-10 uses distinct modular domains for its functions in neurons and epithelial cells and that during evolution its vertebrate ortholog Mint2 has retained the ability to direct AMPAR localization in neurons. INTRODUCTIONThe polarized trafficking and subcellular localization of specific synaptic proteins within neurons direct the flow of information within the nervous system. The localization of one synaptic component in particular, the ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type ionotropic glutamate receptor (AMPAR), to the postsynaptic density is a highly regulated process. AMPARs are gated by the neurotransmitter glutamate and mediate the bulk of excitatory transmission in the central nervous system. The mechanisms underlying synaptic plasticity stem, in part, from both the modulation of activity of these channels and the physical movement of these channels into and out of the synapse .One of the primary ways by which neurons regulate glutamate receptor activity is by regulating the amount of these receptors that reach the synaptic membrane surface (Bolton et al., 2000;Gomes et al., 2003). AMPAR subunits (up to four in mammals, referred to as GluR1-R4; two in Caenorhabditis elegans, referred to as GLR-1 and GLR-2) are multitransmembrane spanning proteins that assemble into tetrameric channels of differing subunit composition (Hollmann and Heinemann, 1994;Dingledine et al., 1999). Our current understanding of how AMPARs are mobilized within cells has focused on the proteins that interact with the cytosolically exposed carboxyl-terminal sequences of AMPARs. Different combinations of subunits present distinct arrays of carboxyl-terminal sequences, resulting in a specific pattern of localization for a given receptor subunit combination. For example, heteromeric channels of GluR1/GluR2 are brought to synapses in response ...

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 (17β-estradiol, E2) and 4-hydroxytamoxifen (4OHT) in breast cancer cells. The importance of AGR2 expression is highlighted here by the observation that (a) its knockdown inhibited the growth of both tamoxifen-sensitive and -resistant breast cancer cells, and (b) 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 highlights a mechanism where changes in FOXA1 activity obviate the need for ER in the regulation of this gene.

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.

Background: Expression of Ror2 leads to increased tumorigenicity in RCC. Results: Ror2 expression stabilizes an increased pool of soluble ␤-catenin, which enhances the response to Wnt stimulation. Conclusion: Ror2 promotes basal and canonical Wnt3a ligand-enhanced ␤-catenin signaling in RCC. Significance: This work demonstrates a novel Ror2-dependent tiered state of canonical Wnt signaling, promoting basal signals and priming RCC cells for a heightened response to the ligand.

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)-1␣ and -2␣ 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-1␣ or HIF-2␣. Subsequent evaluation of the ROR2 promoter suggests that HIF-2␣ 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.The tyrosine kinase Ror2 was initially identified as a homologue of the Trk neurotrophin receptors (1) and later as a member of the receptor tyrosine kinase superfamily (2). Ror2 is an orphan receptor with expression in the developing embryo identified in the embryonic limb buds, heart, primitive genitalia, developing somites, and mesenchymal cells in the developing lung, kidney, and cephalic regions (3-5). In the adult organism, Ror2 expression is present as a part of osteoblast differentiation, highly induced in the preosteoblast stage (6), and is suppressed as these cells terminally differentiate as osteocytes. This pattern of expression is inversely related to that of secreted frizzled related protein 1 (sFRP1), and can be transcriptionally suppressed by ectopic expression of sFRP1 in this cell type, but further insights into the major elements of Ror2 regulation are not known.Aside from developmental programs regulating bone morphogenesis and primitive organ development, Ror2 has only recently been recognized to play a role in the adult organism. We have identified Ror2 expression as a characteristic of many renal cell carcinoma (RCC) 2 cell lines and human tumors (7), where its expression is associated with increased cell migration and anchorage-independent growth. Ror2 also plays a prominent role in osteosarcoma (8) and has recently been identified in squamous cell carcinoma of the head and neck, where expression parallels the development of invasive features of these tumors (9). Furthermore, in a tumor genomic analysis of invasive gastric cancers, Ror2 was identified as a frequent target of mutagenesis (10). These findings place Ror2 as a frequently up-regulated feature of human cancers, ...