Characterization of Factors Involved in Localized Translation Near Mitochondria by Ribosome-Proximity Labeling.

Mitochondria exert their many functions through a repertoire of hundreds of proteins. The vast majority of these proteins are encoded in the nuclear genome, translated in the cytosol and imported into the mitochondria. Current models, derived mainly from work in yeast, suggest that the translation of many of these proteins can occur in close vicinity to the mitochondria outer membrane by localized ribosomes. Here, we applied ribosome-proximity biotin labeling to address this possibility. A clear biotinylation of ribosomes by mitochondrial Tom20-BirA fusion protein was observed in a human cell line. Isolation of these ribosomes revealed their preferred association with mRNAs encoding mitochondrial proteins. Furthermore, knock down of the mitochondrial protein receptor Tom70 resulted in a decrease in ribosomes translating mRNAs encoding proteins predicted to be recognized by Tom70. Intriguingly, levels of ribosomes translating mRNAs encoding targets of Tom20 were increased. We also knocked down the RNA binding protein CLUH that is implicated in regulation of mRNA encoding mitochondrial proteins, and found an increase in association of CLUH targets with mitochondria-proximal ribosomes. This is consistent with a role for CLUH in maintaining mRNAs encoding mitochondrial proteins in the cytosol. Overall, these data shed light on factors that contribute to association of translating ribosomes with human mitochondria and may suggest a co-translational mode of protein import into this organelle.

Nanoparticles target early-stage breast cancer metastasis in vivo.

Despite advances in cancer therapy, treating cancer after it has metastasized remains an unmet clinical challenge. In this study we demonstrate that 100 nm liposomes target triple-negative murine breast-cancer metastases post intravenous administration. Metastatic breast cancer was induced in BALB/c mice either experimentally, by a tail vein injection of 4T1 cells, or spontaneously, after implanting a primary tumor xenograft. To track their biodistribution in vivo the liposomes were labeled with multi-modal diagnostic agents, including indocyanine green and rhodamine for whole-animal fluorescent imaging, gadolinium for magnetic resonance imaging (MRI), and europium for a quantitative biodistribution analysis. The accumulation of liposomes in the metastases peaked at 24 h post the intravenous administration, similar to the time they peaked in the primary tumor. The efficiency of liposomal targeting to the metastatic tissue exceeded that of a non-liposomal agent by 4.5-fold. Liposomes were detected at very early stages in the metastatic progression, including metastatic lesions smaller than 2 mm in diameter. Surprisingly, while nanoparticles target breast cancer metastasis, they may also be found in elevated levels in the pre-metastatic niche, several days before metastases are visualized by MRI or histologically in the tissue. This study highlights the promise of diagnostic and therapeutic nanoparticles for treating metastatic cancer, possibly even for preventing the onset of the metastatic dissemination by targeting the pre-metastatic niche.

Forchlorfenuron disrupts SEPT9_i1 filaments and inhibits HIF-1.

Forchlorfenuron (FCF) is a synthetic plant cytokinin that has been shown to alter yeast and mammalian septin organization. Septins are a highly conserved family of GTP-binding cytoskeletal proteins. Mammalian septins are involved in diverse cellular processes including tumorigenesis. We have been studying the interaction between septin 9 isoform 1 (SEPT9_i1) and hypoxia inducible factor-1α (HIF-1α), the oxygen regulated subunit of HIF-1. HIF-1 is a key transcription factor in the hypoxic responses pathway, and its activation has been observed in carcinogenesis and numerous cancers. SEPT9_i1/HIF-1α interaction plays an important role in upregulation of HIF-1 transcriptional activity by preventing HIF-1α's ubiquitination and degradation leading to increased tumor growth and angiogenesis. We tested the hypothesis whether FCF affects SEPT9_i1 filamentous structures and consequently HIF-1 pathway in cancer cells. We showed that FCF suppresses tumorigenic properties, including proliferation, migration and transformation, in prostate cancer cells. FCF did not alter SEPT9_i1 steady state protein expression levels but it affected its filamentous structures and subcellular localization. FCF induced degradation of HIF-1α protein in a dose- and time-dependent manner. This inhibition was also shown in other common cancer types tested. Rapid degradation of HIF-1α protein levels was accompanied by respective inhibition in HIF-1α transcriptional activity. Moreover, HIF-1α protein half-life was markedly decreased in the presence of FCF compared with that in the absence of FCF. The FCF-induced degradation of HIF-1α was mediated in a significant part via the proteasome. To the best of our knowledge, this is the first demonstration of specific manipulation of septin filaments by pharmacological means having downstream inhibitory effects on the HIF-1 pathway.