HDAC6 Deacetylase Activity Is Required for Hypoxia-Induced Invadopodia Formation and Cell Invasion

<div><p>Despite significant progress in the cancer field, tumor cell invasion and metastasis remain a major clinical challenge. Cell invasion across tissue boundaries depends largely on extracellular matrix degradation, which can be initiated by formation of actin-rich cell structures specialized in matrix degradation called invadopodia. Although the hypoxic microenvironment within solid tumors has been increasingly recognized as an important driver of local invasion and metastasis, little is known about how hypoxia influences invadopodia biogenesis. Here, we show that histone deacetylase 6 (HDAC6), a cytoplasmic member of the histone deacetylase family, is a novel modulator of hypoxia-induced invadopodia formation. Hypoxia was found to enhance HDAC6 tubulin deacetylase activity through activation of the EGFR pathway. Activated HDAC6, in turn, triggered Smad3 phosphorylation resulting in nuclear accumulation. Inhibition of HDAC6 activity or knockdown of the protein inhibited both hypoxia-induced Smad3 activation and invadopodia formation. Our data provide evidence that hypoxia influences invadopodia formation in a biphasic manner, which involves the activation of HDAC6 deacetylase activity by EGFR, resulting in enhanced Smad phosphorylation and nuclear accumulation. The identification of HDAC6 as a key participant of hypoxia-induced cell invasion may have important therapeutic implications for the treatment of metastasis in cancer patients.</p> </div

Hypoxia promotes invadopodia formation.

<p>HT-1080 cells were cultured on FITC-gelatin-coated slides in normoxia or hypoxia for 4 h (<b>B</b>) or 10 h (<b>A, C</b>). (<b>A</b>) Percentage of cells forming invadopodia. (<b>B</b>) Micrographs of actin (green), cortactin (red) nucleus (blue) and merged images are shown. The associated graph shows the number of F-actin-positive and cortactin-positive invadopodia per cell as described under Materials and Methods. (<b>C</b>) Quantification of ECM degradation (area/cell). The associated micrographs show representative ECM degradation area for a single cell. Columns correspond to the mean ± SEM; ** p < 0.01, *** p < 0.001; scale bars correspond to 5 µm.</p

The Smad3-dependent signaling pathway is involved in hypoxia-induced invadopodia formation.

<p>(<b>A, B</b>) HT-1080 cells were seeded on gelatin-coated slides and incubated in the presence or absence of TGFβ or in hypoxia for 3, 6 or 16 h. Cells were labeled for p-Smad3 and nucleus (DAPI) and analyzed by confocal microscopy. (<b>A</b>) Micrographs of p-Smad3 (red), nucleus (blue) and merged images (binary mask-overlay). (<b>B</b>) Graph showing the percentage of colocalization of p-Smad3 with nucleus measured as described under Materials and Methods. (<b>C</b>) Cells were seeded on FITC-gelatin-coated slides, preincubated in the presence or absence of SIS3 or Ly364947 for 30 min and incubated in normoxia in the presence or absence of TGFβ or hypoxia for 10 h. The graph shows the percentage of cells forming invadopodia. Columns correspond to the mean ± SEM; ** p = 0.01, *** p < 0.001; Scale bars correspond to 5 µm.</p

Proposed mechanisms involved in invadopodia formation under hypoxia.

<p>The hypoxic microenvironment in solid tumor is proposed to influence invadopodia formation through a biphasic mechanism involving the regulation of HDAC6, followed by activation of the TGFβ/Smad3-dependent signaling pathway. First, hypoxia leads to the activation of the EGF/EGFR pathway, which enhances HDAC6 tubulin deacetylase activity. In parallel, hypoxia increases the expression of the proprotein convertase furin, resulting in enhanced TGFβ processing and bioactivation. HDAC6 tubulin deacetylation may allow the desequestration of Smad3 from the MT network leading to its activation and nuclear translocation. Smad3 translocation leads to enhanced transcription of TGFβ inducible genes and invadopodia production. In this model, HDAC6 might also translocate to the cell periphery where it was shown to be involved in cortactin deacetylation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055529#pone.0055529-Kaluza1" target="_blank">[31]</a> an event needed for the formation of actin-rich invadopodia structures.</p

Endogenous TGFβ is involved in hypoxia-induced invadopodia formation.

<p>(<b>A</b>, <b>B</b>) HT-1080 cells were cultured on FITC-gelatin-coated slides in normoxia or hypoxia for 10 h in the presence or absence of TGFβ added at the indicated concentrations. (<b>A</b>) Percentage of cells forming invadopodia. (<b>B</b>) Quantification of ECM degradation (area/cell). (<b>C</b>, <b>D</b>) HT-1080, HT-1080-hFur and HT-1080-PDX cells were cultured on FITC-gelatin-coated slides in normoxia or hypoxia for 10 h. (<b>C</b>) Percentage of cells forming invadopodia. (<b>D</b>) Quantification of ECM degradation (area/cell). (<b>E</b>) Total TGFβ was quantitated in supernatants of cells cultured in normoxia or hypoxia for 24 h. Columns correspond to the mean ± SEM; ** p < 0.01, *** p < 0.005.</p

Hypoxia induces HDAC6 deacetylase activity.

<p>(<b>A</b>) HT-1080 cells were seeded on gelatin-coated slides and incubated in normoxia or hypoxia for 2 h or 4 h and cell lysates immunoblotted for HDAC6, acetylated-tubulin, and tubulin. (B, C) Cells seeded on gelatin-coated slides were incubated in normoxia for 4 h in the presence or absence of (<b>B</b>) TGFβ or (<b>C</b>) EGF at the indicated concentrations. Immunoblots of HDAC6, acetylated-tubulin, and tubulin.</p

EGF enhances TGFβ signaling and Smad3 nuclear accumulation.

<p>HT-1080 cells were seeded on gelatin coated-slides and incubated in normoxia or hypoxia for 6 h in the presence or absence of TGFβ EGF, Tyrphostin AG1478 (2 µM) and Ly364947 (500 nM). Percentage of colocalization of p-Smad3 with nucleus measured as described under Materials and Methods.</p

The EGFR signaling pathway is involved in hypoxia-induced HDAC6 activation.

<p>(<b>A</b>) HT-1080 cells were seeded on gelatin-coated slides and incubated in normoxia or hypoxia for the indicated times. Cell lysates were immunoprecipitated (IP) with anti-EGFR antibodies and immunoblotted with anti-phosphotyrosine and anti-EGFR antibodies. (<b>B</b>) HT-1080 cells were seeded on gelatin-coated slides and incubated in normoxia or hypoxia for 2 h and 4 h in the presence or absence of Tyrphostin AG1478 or Ly364947 at the indicated concentration. Cell lysates were immunoblotted with acetylated-tubulin and tubulin antibodies.</p

HDAC6 is involved in hypoxia-induced invadopodia formation.

<p>(<b>A)</b> HT-1080 (CTL), HT-1080 transfected with control shRNA (sh-CTL) or shRNA targeting HDAC6 (shHDAC6) or (<b>B</b>) untransfected HT-1080 cells incubated in the presence or absence of Tubacin or TGFβ were cultured on gelatin-coated slides in normoxia or hypoxia for 6 h. (<b>A, B</b>) Graph showing the percentage of colocalization of p-Smad3 with nucleus (DAPI) measured as described under Materials and Methods. (<b>A</b>) Immunoblot showing inhibition of HDAC6 expression by HDAC6 shRNA. (<b>C</b>) HT-1080 cells were cultured on FITC-gelatin-coated slides in normoxia or hypoxia for 10 h and treated with DMSO (vehicle CTL), Tubacin or Nitubacin (Negative CTL). The graph shows the percentage of cells forming invadopodia. (<b>D-F</b>) HT-1080 (CTL), HT-1080 transfected with shCTL (sh-CTL) and HT-1080 transfected with shHDAC6 (shHDAC6) were cultured on FITC-gelatin-coated slides (D) or allowed to invade collagen gels (E, F) in normoxia or hypoxia. (<b>D</b>) Percentage of cells forming invadopodia for cells incubated for 10 h. (<b>E, F</b>) Cells were allowed to invade collagen gels for 24 h and stained as described under Materials and Methods. (<b>E)</b> Relative fluorescence intensity of the cells according to the depth of invasion. The arbitrary index of invasion was calculated as described under Materials and Methods. (<b>F</b>) Maximal depth of invasion. Columns correspond to the mean ± SEM; * p < 0.04; ** p < 0.002, *** p < 0.001.</p

The MEK/ERK Pathway Promotes NOTCH Signalling in Pancreatic Cancer Cells

<div><p>Activation of the NOTCH receptors relies on their intracellular proteolysis by the gamma-secretase complex. This cleavage liberates the NOTCH intracellular domain (NIC) thereby allowing the translocation of NIC towards the nucleus to assemble into a transcriptional platform. Little information is available regarding the regulatory steps operating on NIC following its release from the transmembrane receptor up to its association with transcriptional partners. Interfering with these regulatory steps might potentially influences the nuclear outcome of NOTCH signalling. Herein, we exploited a reliable model to study the molecular events occurring subsequent to NOTCH1 cleavage. In pancreatic cancer cells, pulse of NOTCH1 activation led to increased expression of NOTCH target genes namely HES1 and c-MYC. We uncovered that, upon its release, the NOTCH1 intracellular domain, NIC1, undergoes a series of post-translational modifications that include phosphorylation. Most interestingly, we found that activation of the MEK/ERK pathway promotes HES1 expression. Inhibition of the gamma-secretase complex prevented the MEK/ERK-induced HES1 expression suggesting a NOTCH-dependent mechanism. Finally, higher levels of NIC1 were found associated with its transcriptional partners [CBF1, Su(H) and LAG-1] (CSL) and MASTERMIND-LIKE 1 (MAML1) upon MEK/ERK activation providing a potential mechanism whereby the MEK/ERK pathway promotes expression of NOTCH target genes. For the first time, our data exposed a signalling pathway, namely the MEK/ERK pathway that positively impacts on NOTCH nuclear outcome. </p> </div