No abstract

Spatial control of mRNA translation can generate cellular asymmetries and functional specialization of polarized cells like neurons. A requirement for the translational repressor Nanos (Nos) in the Drosophila larval peripheral nervous system (PNS) implicates translational control in dendrite morphogenesis [1]. Nos was first identified by its requirement in the posterior of the early embryo for abdomen formation [2]. Nos synthesis is targeted to the posterior pole of the oocyte and early embryo through translational repression of unlocalized nos mRNA coupled with translational activation of nos mRNA localized at the posterior pole [3, 4]. Abolishment of nos localization prevents abdominal development, whereas translational derepression of unlocalized nos mRNA suppresses head/thorax development, emphasizing the importance of spatial regulation of nos mRNA [3, 5]. Loss and overexpression of Nos affect dendrite branching complexity in class IV dendritic arborization (da) neurons, suggesting that nos also might be regulated in these larval sensory neurons [1]. Here, we show that localization and translational control of nos mRNA are essential for da neuron morphogenesis. RNA-protein interactions that regulate nos translation in the oocyte and early embryo also regulate nos in the PNS. Live imaging of nos mRNA shows that the cis-acting signal responsible for posterior localization in the oocyte/embryo mediates localization to the processes of class IV da neurons but suggests a different transport mechanism. Targeting of nos mRNA to the processes of da neurons may reflect a local requirement for Nos protein in dendritic translational control.

With the advent of molecularly targeted agents, treatment of metastatic renal cell carcinoma (mRCC) has improved significantly. Agents targeting the vascular endothelial growth factor receptor (VEGFR) and the mammalian target of rapamycin complex 1 (mTORC1) are more effective and less toxic than previous standards of care involving cytotoxic and cytokine therapies. Unfortunately, many patients relapse following treatment with VEGFR and mTORC1 inhibitors as a result of acquired resistance mechanisms, which are thought to lead to the reestablishment of tumor vasculature. Specifically, the loss of negative feedback loops caused by inhibition of mTORC1 leads to upregulation of downstream effectors of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway and subsequent activation of hypoxia-inducible factor, an activator of angiogenesis. De novo resistance involving activated PI3K signaling has also been observed. These observations have led to the development of novel agents targeting PI3K, mTORC1/2 and PI3K/mTORC1/2, which have demonstrated antitumor activity in preclinical models of RCC. Several agents-BKM120, BEZ235 and GDC-0980-are being investigated in clinical trials in patients with metastatic/ advanced RCC, and similar agents are being tested in patients with solid tumors. The future success of mRCC treatment will likely involve a combination of agents targeting the multiple pathways involved in angiogenesis, including VEGFR, PI3K and mTORC1/2.Renal cell carcinoma (RCC) accounts for more than 2% of all human cancers, and its incidence is steadily rising by approximately 2% per year.

Intracellular mRNA localization is a conserved mechanism for spatially regulating protein production in polarized cells, such as neurons. The mRNA encoding the translational repressor Nanos (Nos) forms ribonucleoprotein (RNP) particles that are dendritically localized in Drosophila larval class IV dendritic arborization (da) neurons. In nos mutants, class IV da neurons exhibit reduced dendritic branching complexity, which is rescued by transgenic expression of wild-type nos mRNA but not by a localization-compromised nos derivative. While localization is essential for nos function in dendrite morphogenesis, the mechanism underlying the transport of nos RNP particles was unknown. We investigated the mechanism of dendritic nos mRNA localization by analyzing requirements for nos RNP particle motility in class IV da neuron dendrites through live imaging of fluorescently labeled nos mRNA. We show that dynein motor machinery components mediate transport of nos mRNA in proximal dendrites. Two factors, the RNA-binding protein Rumpelstiltskin and the germ plasm protein Oskar, which are required for diffusion/entrapment-mediated localization of nos during oogenesis, also function in da neurons for formation and transport of nos RNP particles. Additionally, we show that nos regulates neuronal function, most likely independent of its dendritic localization and function in morphogenesis. Our results reveal adaptability of localization factors for regulation of a target transcript in different cellular contexts.

Developmental control of translation is frequently mediated by regulatory elements that reside within 3' untranslated regions (3' UTRs). Two stem-loops within the nanos 3' UTR translational control element (TCE) act independently to direct translational repression of maternal nanos mRNA in the ovary or embryo. We have previously shown that the nanos TCE can also function in select somatic sites. Using an ectopic expression screen, we now identify a new site of TCE function, the dorsal pouch epithelium. Analysis of TCE mutants reveals that TCE activity in the dorsal pouch does not depend on either of the stem-loops required for maternal TCE function, but instead requires a third feature-a sequence that closely matches the Bearded box, a regulatory motif found in the 3' UTRs of several Notch pathway genes. In addition, we identify pleiohomeotic mRNA as an endogenous candidate for regulation by Bearded box-like motifs in the dorsal pouch. Together, these results suggest that the TCE has appropriated a conserved regulatory motif to expand its function to somatic tissues.

The advent of molecular targeted therapies offers the hope of therapeutic advance in the fight against cancer. However, this hope is tempered by recent findings that certain targeted therapies may have unique side effects. The hedgehog (HH) pathway is a potential target for treatment of several cancers, including basal cell carcinoma (BCC) and a subset of medulloblastoma (MB). Recent clinical trials in adults have shown responses to HH pathway inhibition in both BCC and MB. However, concerns have been raised about the use of HH pathway inhibitors in children because of the role the HH pathway plays in development. Indeed, young mice treated with the HH pathway inhibitor, HhAntag, developed severe bone defects including premature differentiation of chondrocytes, thinning of cortical bone and fusion of the growth plate. In an effort to lessen the severity of bone defects caused by HhAntag, we treated young mice simultaneously with HhAntag and parathyroid hormone related protein (PTHrP), which functions downstream of Indian Hedgehog (IHH) to maintain chondrocytes in a proliferative state. The results show that while treatment with PTHrP causes a significant increase in trabecular bone, it does not prevent fusion of the growth plate induced by HhAntag.

Current treatment for the most common pediatric brain tumor, medulloblastoma (MB) compromises neurological and endocrine development, particularly in younger patients. Mutations in Patched (PTCH1), the receptor for Hedgehog (Hh) proteins, cause MB in approximately 30% of patients. Loss of PTCH1 leads to constitutive activation of Smoothened (SMO) and increased expression of downstream target genes such as GLI1. Previously, we investigated the use of the SMO inhibitor, HhAntag, in a mouse model of MB. Treatment of Ptch1+/−p53−/− mice with HhAntag completely eliminates spontaneous and transplanted tumors. However, treatment of young mice with HhAntag results in permanent defects in bone structure. Histological analyses of long bones from these mice showed a significant reduction in proliferating chondrocytes and an increase in the number of hypertrophic chondrocytes. In our current study, we have further characterized the bone toxicities caused by HhAntag treatment and are testing potential molecules that might lessen the severity of these toxicities. We treated P10-old mice for 4 or 6 days and analyzed their limbs at 0, 2, or 4 days after HhAntag treatment. We found that the severity of bone defects increases with the length of time the mice are treated with HhAntag. MicroCT and MRI analysis showed that exposure to HhAntag for as little as 4 days caused premature growth plate fusion and an increase in bone mineral density. In an effort to protect proliferating chondrocytes from the toxicities caused by HhAntag, we have identified several candidate molecules that have been shown to alter developing bone. One candidate, Parathyroid Hormone-Related Protein (PTHrP), is a downstream effector of the Hh pathway. Hh signaling activates the expression of PTHrP, which then signals to proliferating chondrocytes, preventing them from differentiating. Previous studies show that overexpression of PTHrP in developing bones delays chondrocyte hypertrophy. Therefore, exogenous PTHrP may offset the premature differentiation evident in HhAntag treated animals. We have identified a physiologically relevant dose of PTHrP that is sufficient to mediate a change in bone morphology. We are currently treating mice simultaneously with PTHrP and HhAntag, in an effort to reduce the HhAntag associated bone toxicities. Another candidate is the proteasome inhibitor, bortezomib. Bortezomib has been shown to alter Hh pathway activity and therefore may also reduce the toxicities associated with HhAntag treatment. We are currently establishing the proper dosing strategy appropriate for bortezomib treatment of young mice. We have shown that treatment of young mice with HhAntag leads to severe bone defects and are currently employing a multi-drug approach to lessen the severity of these defects. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C110.