Probes for INS

Abstract

PURPOSE:

Four consensus molecular subtypes (CMS1-4) of colorectal cancer (CRC) were identified in primary tumors and found to be associated with distinctive biological features and clinical outcomes. Given that distant metastasis largely accounts for CRC-related mortality, we examined the molecular and clinical attributes of CMS in metastatic CRC (mCRC).

EXPERIMENTAL DESIGN:

We developed a CRC-focused Nanostring based CMS classifier that is ideally suited to interrogate archival tissues. We successfully employ this panel in the CMS classification of FFPE tissues from mCRC cohorts, one of which is comprised of paired primary tumors and metastases. Finally, we developed novel mouse implantation models to enable modelling of CRC in vivo at relevant sites.

RESULTS:

Using our classifier we find that the biological hallmarks of mCRC, including CMS, are in general highly similar to those observed in non-metastatic early stage disease. Importantly, our data demonstrate that CMS1 has the worst outcome in relapsed disease, compared to other CMS. Assigning CMS to primary tumors and their matched metastases revealed mostly concordant subtypes between primary and metastasis. Molecular analysis of matched discordant pairs revealed differences in stromal composition at each site. The development of two novel in vivo orthotopic implantation models further reinforces the notion that extrinsic factors may impact on CMS identification in matched primary and metastatic CRC.

CONCLUSION:

We describe the utility of a Nanostring panel for CMS classification of FFPE clinical samples. Our work reveals the impact of intrinsic and extrinsic factors on CRC heterogeneity during disease progression.

Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson's disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson's disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3'UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson's disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3'UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct spatial expression of GDNF. Furthermore, our results suggest that 3'UTR targeting may constitute a useful tool in analyzing gene function.

A subset of midbrain dopamine (DA) neurons express vesicular glutamate transporter 2 (VgluT2), which facilitates synaptic vesicle loading of glutamate. Recent studies indicate that such expression can modulate DA-dependent reward behaviors, but little is known about functional consequences of DA neuron VgluT2 expression in neurodegenerative diseases like Parkinson's disease (PD). Here, we report that selective deletion of VgluT2 in DA neurons in conditional VgluT2-KO (VgluT2-cKO) mice abolished glutamate release from DA neurons, reduced their expression of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB), and exacerbated the pathological effects of exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, viral rescue of VgluT2 expression in DA neurons of VglutT2-cKO mice restored BDNF/TrkB expression and attenuated MPTP-induced DA neuron loss and locomotor impairment. Together, these findings indicate that VgluT2 expression in DA neurons is neuroprotective. Genetic or environmental factors causing reduced expression or function of VgluT2 in DA neurons may place some individuals at increased risk for DA neuron degeneration. Therefore, maintaining physiological expression and function of VgluT2 in DA neurons may represent a valid molecular target for the development of preventive therapeutic interventions for PD.