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. 2017 Apr;108(4):696-703.
doi: 10.1111/cas.13190. Epub 2017 Apr 12.

Podoplanin promotes progression of malignant pleural mesothelioma by regulating motility and focus formation

Shinji Takeuchi  1 , Koji Fukuda  1 , Tadaaki Yamada  1 , Sachiko Arai  1 , Satoshi Takagi  2 , Genichiro Ishii  3 , Atsushi Ochiai  3 , Shotaro Iwakiri  4 , Kazumi Itoi  4 , Hisanori Uehara  5 , Hiroshi Nishihara  6 , Naoya Fujita  2 , Seiji Yano  1
Affiliations

Podoplanin promotes progression of malignant pleural mesothelioma by regulating motility and focus formation

Shinji Takeuchi et al. Cancer Sci. 2017 Apr.

Abstract

Malignant pleural mesothelioma (MPM) is characterized by dissemination and aggressive growth in the thoracic cavity. Podoplanin (PDPN) is an established diagnostic marker for MPM, but the function of PDPN in MPM is not fully understood. The purpose of this study was to determine the pathogenetic function of PDPN in MPM. Forty-seven of 52 tumors (90%) from Japanese patients with MPM and 3/6 (50%) MPM cell lines tested positive for PDPN. Knocking down PDPN in PDPN-high expressing MPM cells resulted in decreased cell motility. In contrast, overexpression of PDPN in PDPN-low expressing MPM cells enhanced cell motility. PDPN stimulated motility was mediated by activation of the RhoA/ROCK pathway. Moreover, knocking down PDPN with short hairpin (sh) RNA in PDPN-high expressing MPM cells resulted in decreased development of a thoracic tumor in mice with severe combined immune deficiency (SCID). In sharp contrast, transfection of PDPN in PDPN-low expressing MPM cells resulted in an increase in the number of Ki-67-positive proliferating tumor cells and it promoted progression of a thoracic tumor in SCID mice. Interestingly, PDPN promoted focus formation in vitro, and a low level of E-cadherin expression and YAP1 activation was observed in PDPN-high MPM tumors. These findings indicate that PDPN is a diagnostic marker as well as a pathogenetic regulator that promotes MPM progression by increasing cell motility and inducing focus formation. Therefore, PDPN might be a pathogenetic determinant of MPM dissemination and aggressive growth and may thus be an ideal therapeutic target.

Keywords: YAP1; focus formation; mesothelioma; motility; podoplanin.

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Figures

Figure 1
Figure 1
Knocking down podoplanin (PDPN) inhibited the motility of human mesothelioma cells. (a) Expression of PDPN in human mesothelioma cell lines was determined using Western blotting. (b) H226 cells that expressed high levels of PDPN were treated with scrambled siRNA or PDPN‐specific siRNA and expression of PDPN was determined using Western blotting. (c) The viability of the resulting cells was determined using an MTT assay. (d) The motility of the resulting cells was assessed with a wound healing assay. (e) The motility of H226 cells treated with PDPN‐shRNA or Luc‐shRNA (a negative control) was assessed using a wound healing assay. The data shown are representative of at least three independent experiments with similar results.
Figure 2
Figure 2
Overexpression of podoplanin (PDPN) enhanced motility of human mesothelioma cells. (a) MSTO‐211H cells were transfected with the PDPN gene (MSTO‐211H/PDPN) or a vector control gene. Expression of PDPN was determined using Western blotting. (b) The motility of the resulting cells was determined using a wound healing assay. (c) The motility of the resulting cells was also determined with a migration assay using a transwell system. (d) The motility determined in (c) was quantified. The data shown are representative of at least three independent experiments with similar results. *P < 0.05.
Figure 3
Figure 3
Podoplanin (PDPN) enhanced the motility via RhoA activation in mesothelioma cells. RhoA‐GTP binding of MSTO‐211H cells transfected with the control gene or PDPN (a) and of H226 cells treated with control shLuc or PDPN‐shRNA (b) was determined as described in the Materials and Methods. (c) The effect of a ROCK inhibitor (Y‐27632) on the viability of MSTO‐211H/PDPN was determined with an MTT assay. (d) The effect of Y27632 was determined using a wound healing assay. Data shown are the representative of at least three independent experiments with similar results. *P < 0.05.
Figure 4
Figure 4
Podoplanin (PDPN) promoted the progression of mesothelioma cells that were orthotopically implanted in SCID mice. (A) H226/ShLuc or H226/ShPDPN cells (1 × 106) were orthotopically implanted in the thoracic cavity of SCID mice. Seventy days after tumor cell implantation, the mice were euthanized and tumor development was evaluated. (b) H. MSTO‐211H/Vector or MSTO‐211H/PDPN cells (1 × 106) were orthotopically implanted in the thoracic cavity of SCID mice. Twenty‐one days after tumor cell implantation, the mice were sacrificed and tumor development was evaluated. n = 5/group. (c) H. MSTO‐211H/Vector or MSTO‐211H/PDPN cells (1 × 106) were orthotopically implanted in the thoracic cavity of SCID mice. The survival of the mice was evaluated. (n = 20/group). (d) Ki‐67‐positive tumor cells were determined based on the immunohistochemistry of thoracic tumors. *P < 0.05.
Figure 5
Figure 5
Podoplanin (PDPN) promoted focus formation in human mesothelioma cells. Confluent cultures of human mesothelioma cells, H. MSTO‐211H/Vector or MSTO‐211H/PDPN cells (a) and H226/ShLuc or H226/ShPDPN cells (b) in 35‐mm dishes were incubated for additional 2 weeks and stained with crystal violet; the number of foci was counted under a microscope. Data are representative of three independent experiments with similar results. *P < 0.001.
Figure 6
Figure 6
Podoplanin (PDPN) resulted in increased nuclear localization of YAP1 and decreased expression of E‐cadherin in thoracic tumors produced by mesothelioma cells. Thoracic tumors produced by H226/ShLuc or H226/ShPDPN cells were harvested 70 days after inoculation, and which produced by H. MSTO‐211H/Vector or MSTO‐211H/PDPN cells were harvested 21 days after inoculation. YAP1‐positive tumor cells in nucleus were determined by immunohistochemistry (a) (b), and expression of EMT‐related proteins was determined using western blotting (c) (d) in these thoracic tumors. *P < 0.001.
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References

    1. Imai M, Hino O. Environmental carcinogenesis. Cancer Sci 2015; 106: 1483–5. - PMC - PubMed
    1. Kato Y, Fujita N, Kunita A et al Molecular identification of Aggrus/T1alpha as a platelet aggregation‐inducing factor expressed in colorectal tumors. J Biol Chem 2003; 278: 51599–605. - PubMed
    1. Suzuki‐Inoue K, Kato Y, Inoue O et al Involvement of the snake toxin receptor CLEC‐2, in podoplanin –mediated platelet activation, by cancer cells. J Biol Chem 2007; 282: 25993–6001. - PubMed
    1. Fujita N, Takagi S. The impact of Aggrus/podoplanin on platelet aggregation and tumour metastasis. J Biochem 2012; 152: 407–13. - PubMed
    1. Martin‐Villar E, Megias D, Castel S, Yurrita MM, Vilaro S, Quintanilla M. Podoplanin binds ERM proteins to activate RhoA and promote epithelial–mesenchymal transition. J Cell Sci 2006; 119: 4541–53. - PubMed

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