Adaptation of Poe : From The Domain of Arnheim to Konziki-no-shi and Panorama-to-kitan

特集 北アメリカの文学柴田元幸教授退官記念号論

Inhibition of cancer-type amino acid transporter LAT1 suppresses B16-F10 melanoma metastasis in mouse models

Abstract Metastasis is the leading cause of mortality in cancer patients. L-type amino acid transporter 1 (LAT1, SLC7A5) is a Na+-independent neutral amino acid transporter highly expressed in various cancers to support their growth. Although high LAT1 expression is closely associated with cancer metastasis, its role in this process remains unclear. This study aimed to investigate the effect of LAT1 inhibition on cancer metastasis using B16-F10 melanoma mouse models. Our results demonstrated that nanvuranlat (JPH203), a high-affinity LAT1-selective inhibitor, suppressed B16-F10 cell proliferation, migration, and invasion. Similarly, LAT1 knockdown reduced cell proliferation, migration, and invasion. LAT1 inhibitors and LAT1 knockdown diminished B16-F10 lung metastasis in a lung metastasis model. Furthermore, nanvuranlat and LAT1 knockdown suppressed lung, spleen, and lymph node metastasis in an orthotopic metastasis model. We discovered that the LAT1 inhibitor reduced the cell surface expression of integrin αvβ3. Our findings revealed that the downregulation of the mTOR signaling pathway, induced by LAT1 inhibitors, decreased the expression of integrin αvβ3, contributing to the suppression of metastasis. These results highlight the critical role of LAT1 in cancer metastasis and suggest that LAT1 inhibition may serve as a potential target for anti-metastasis cancer therapy

NRFL-1, the C. elegans NHERF orthologue, interacts with amino acid transporter 6 (AAT-6) for age-dependent maintenance of AAT-6 on the membrane.

The NHERF (Na(+)/H(+) exchanger regulatory factor) family has been proposed to play a key role in regulating transmembrane protein localization and retention at the plasma membrane. Due to the high homology between the family members, potential functional compensations have been a concern in sorting out the function of individual NHERF numbers. Here, we studied C. elegans NRFL-1 (C01F6.6) (nherf-like protein 1), the sole C. elegans orthologue of the NHERF family, which makes worm a model with low genetic redundancy of NHERF homologues. Integrating bioinformatic knowledge of C. elegans proteins into yeast two-hybrid scheme, we identified NRFL-1 as an interactor of AAT-6, a member of the C. elegans AAT (amino acid transporter) family. A combination of GST pull-down assay, localization study, and co-immunoprecipitation confirmed the binding and characterized the PDZ interaction. AAT-6 localizes to the luminal membrane even in the absence of NRFL-1 when the worm is up to four-day old. A fluorescence recovery after photobleaching (FRAP) analysis suggested that NRFL-1 immobilizes AAT-6 at the luminal membrane. When the nrfl-1 deficient worm is six-day or older, in contrast, the membranous localization of AAT-6 is not observed, whereas AAT-6 tightly localizes to the membrane in worms with NRFL-1. Sorting out the in vivo functions of the C. elegans NHERF protein, we found that NRFL-1, a PDZ-interactor of AAT-6, is responsible for the immobilization and the age-dependent maintenance of AAT-6 on the intestinal luminal membrane

Immobilization of AAT-6 on the intestinal apical membrane by NRFL-1.

<p><b><i>A</i></b>, FRAP analysis of the AAT-6 dynamics was performed in 4-day old worms. Confocal images of AAT-6 (AAT-6<i>¹⁻⁵¹⁷</i>::GFP::AAT-6<i>^518–523</i>) was compared between <i>aat-6</i> and <i>nrfl-1</i>;<i>aat-6</i> genetic backgrounds. Top pictures indicate representative images prior to photobleaching (<i>pre-bleach</i>), immediately after photobleaching (<i>post-bleach</i>), and 300 sec after photobleaching (<i>recovery</i>). <b><i>B</i></b>, Graph depicts the time course of recovery of AAT-6 fluorescence for <i>nrfl-1</i>;<i>aat-6</i> (•) and <i>aat-6</i> (□). Recovery was measured with the pre-bleach fluorescence intensity being 100% and the post-bleach intensity being 0%. The recovery curves were generated from 5 separate experiments and the values were expressed as mean ± S.E. (n = 5). Scale bars: 2 µm.</p

Expression and protein profile of NRFL-1 (C01F6.6) in <i>C. elegans</i>. <i>A</i>

<p><i>–</i><b><i>D</i></b>, Expression of NRFL-1 in worm. <i>gfp::nrfl-1</i> expression was detected in excretory canal (<i>arrow</i> in <b><i>A</i></b> and <b><i>B</i></b>) and luminal membrane of intestinal epithelial cells (<i>single-arrowhead</i> ) in the anterior (<b><i>B</i></b>), middle (<b><i>C</i></b>) and posterior (<b><i>D</i></b>) intestine. <i>gfp::nrfl-1</i> was not detected on the basal side (<i>double-arrowhead</i> in <b><i>B</i></b>, <b><i>C</i></b> and <b><i>D</i></b>). In <b><i>A</i></b>, a worm with <i>gfp::nrfl-1</i> expression restricted to the excretory system was imaged for clarity. Such worms occasionally occurred in the transgenic population. In <b><i>C</i></b> and <b><i>D</i></b>, cytosolic dispersion of NRFL-1 was seen. Scale bars, 25 µm. Ten worms examined for each. <b><i>E</i></b>, Luminal enrichment of NRFL-1. Endogenous NRFL-1 was detected by anti-NRFL-1 antibody along the luminal side of the intestine. Non-specific signal on the body-wall is arrowed (<i>left</i>). IFB-2 was immune-labeled in a similar pattern (<i>middle</i>). Merged image shows that NRFL-1 is distributed apical to IFB-2 (<i>right</i>). Scale bar, 25 µm. Confocal images of a representative intestinal section (whole-worm) from seven independent experiments are shown. <b><i>F</i></b>, Immunoblot of endogenous and recombinant NRFL-1. Left (<i>recombinant</i>), middle (<i>wild type</i>) and right (<i>nrfl-1</i>) lanes are for recombinant C01F6.6a and C01F6.6b proteins (untagged), lysate from wild-type, and lysate from <i>nrfl-1</i>(<i>tm3501</i>), respectively. Note that the band at ∼72 kDa in the middle lane was not detected in the right and the band did not match either C01F6.6a (∼62 kDa) or C01F6.6b (∼78 kDa). Sixty microgram of protein was loaded for the lysates. A representative blot from three separate experiments is shown. <b><i>G</i></b>, Dephosphorylation of endogenous NRFL-1. The wild-type lysate was incubated with (+) and without (−) phosphatase inhibitors for the indicated period. Over incubation, the bands for inhibitor-free lysate migrated towards a position corresponding to the recombinant C01F6.6a. Ninety microgram of protein was loaded for the lysate. The results are confirmed in duplicate experiments.</p

Interaction of AAT-6 with NRFL-1 <i>in vivo</i>. <i>A</i>

<p>, Expression of AAT-6 in worms. AAT-6<i>¹⁻⁵¹⁷</i>::GFP::AAT-6<i>^518–523</i> was localized to the luminal surface (<i>single arrowhead</i>) but not to the basal side (<i>double arrowhead</i>) of the intestinal epithelia. Scale bar, 25 µm. Non-specific fluorescence on gut granules was seen. More than twenty worms were analyzed. <b><i>B</i></b>, Co-localization of AAT-6 and NRFL-1. GFP fluorescence from AAT-6<i>¹⁻⁵¹⁷</i>::GFP::AAT-6<i>^518–523</i> (<i>top</i>) was co-localized with immunostaining of NRFL-1 by anti-NRFL-1 antibody visualized by Cy3-labeled secondary antibody (<i>middle</i>). Bottom image is merged from top and middle images. Confocal images of a representative intestine section (whole worm) are shown. Scale bar: 25 µm. More than five worms were analyzed. <b><i>C</i></b>, Intensity profile along the line A–B in the merged image shows an overlapping of the peaks of the NRFL-1and the AAT-6 signal. <b><i>D</i></b>, Immunoprecipitation of NRFL-1/AAT-6 complex from worm lysate. The worm lysate was immunoprecipitated with anti-GFP monoclonal antibody (mouse) and immunoblotted using anti-NRFL-1 antibody. <i>Top</i>: input (2.5%). <i>Middle</i> and <i>bottom</i>: immunoprecipitant was immunoblotted using anti-NRFL-1 antibody and anti-GFP antibody (chicken), respectively. In the middle blot, two major bands of NRFL-1 detected (<i>arrowheads</i>), probably reflecting partial dephosphorylation during immunoprecipitation process. A representative blot of two separate experiments is shown.</p

NRFL-1, the worm orthologue of NHERF family.

<p><b><i>A</i></b>, Schematic illustration of <i>nrfl-1</i> transcript (modified from WormBase). Black boxes, exons; grey box, spliced leader 1 (SL1); CC, two bases of cytosine constituting the 5′UTR; ATG, the start codon; TAA, the stop codon; AATAAA, a putative polyadenylation signal; poly-A, poly adenosine tail. The alleles <i>tm3501</i> and <i>ok2292</i> lack 953-basepair and 1117-basepair of the genomic DNA, respectively, indicated by the horizontal lines. <b><i>B</i></b>, NRFL-1 protein. The <i>nrfl-1</i>gene products contain either a single or double PDZ domains with PDZ II shared by all. The variants C01F6.6a, c, and e have PDZ I and PDZ II (double-domain group). PDZ I is structurally related to PDZ II with 45% identity and 59% similarity. <b><i>C</i></b>, Pairwise domain comparison between NRFL-1 and two human NHERF proteins: NHERF 1 and NHERF 2. PDZ domains were specified by ExPASy Prosite <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043050#pone.0043050-Gasteiger1" target="_blank">[67]</a> and compared by BLAST <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043050#pone.0043050-Altschul1" target="_blank">[26]</a>. For each pair, identity/similarity (%/%) is assigned. The red lines indicate the most remarkable pair(s) for each of NRFL-1 PDZ domains with respect to sequence identity.</p

Maintenance of AAT-6 on the intestinal luminal membrane by NRFL-1. <i>A</i>

<p>, The localization of AAT-6<i>¹⁻⁵¹⁷</i>::GFP::AAT-6<i>^518–523</i> was compared between <i>aat-6</i> and <i>nrfl-1</i>(<i>tm3501</i>);<i>aat-6</i> genetic backgrounds. Epifluorescence images of the distribution of AAT-6 in the intestine are shown for worms two, four and six days after hatching. In six-day old worm, the membranous localization of AAT-6 decayed in <i>nrfl-1</i>(<i>tm3501</i>);<i>aat-6</i>, whereas AAT-6 was retained on the luminal membrane in six days in the presence of NRFL-1 (<i>aat-6</i>). Scale bars: 100 µm. Representative pictures from more than ten worms analyzed for each are shown. <b><i>B</i></b>, Fluorescence intensity was measured to quantify the age-dependent regulation. The intensity of the intestinal luminal surface was peaked at day four with significantly stronger signal in <i>aat-6</i> (gray column) compared with <i>nrfl-1</i>(<i>tm3501</i>);<i>aat-6</i> (white column). The intensity at day six did not differ significantly between the strains (<i>luminal membrane</i>). <b><i>C</i></b>, The fluorescence intensity of the whole intestine showed a similar pattern (<i>whole intestine</i>). The localization index, luminal intensity divided by whole intestine intensity, quantifies the membranous localization. The six-day old <i>nrfl-1</i>(<i>tm3501</i>);<i>aat-6</i> worm had a significantly lower score, showing age-dependent decay in luminal localization (<i>localization index</i>). Gray column, <i>aat-6</i>. White column, <i>nrfl-1</i>(<i>tm3501</i>);<i>aat-6</i>. Values are presented with mean ± S.E. (n = 5). <b><i>D</i></b>, Immunoblot and densitometric analysis of AAT-6<i>¹⁻⁵¹⁷</i>::GFP::AAT-6<i>^518–523</i> in six-day old worm. Densitometric analysis followed by anti-GFP antibody immunobloting exhibited no significant difference in band intensity between the genetic backgrounds. A representative blot was presented with actin as a loading control. The bar graph indicates the relative band intensities of the respective sample. Values are presented with mean ± S.E. (n = 4).</p