Paracrine regulation of matrix metalloproteinases contributes to cancer cell invasion by hepatocellular carcinoma-secreted 14-3-3σ

Abstract

14-3-3σ overexpression results in enhanced hepatocellular carcinoma (HCC) cell migration and HCC tumor vascular-invasion is significantly associated with 14-3-3σ expression. However, increased expression of 14-3-3σ paradoxically suppresses in vitro cell invasion of HCC. We hypothesize that surrounding tumor-associated stromal cells play a crucial role in 14-3-3σ-regulated HCC cell invasion. In this study, H68 fibroblasts, THP-1 and phorbol-12-myristate-13-acetate (PMA)-treated THP-1 (PMA-THP-1) cells were incubated with conditioned media of control (control-CM) and 14-3-3σ-overepxressing cells (14-3-3σ-CM), followed by co-culture with HCC cells. Invasiveness of HCC cells was examined by a Boyden chamber assay. HCC cells co-cultured with 14-3-3σ-CM treated cells significantly enhanced their invasive ability compared with control-CM treated cells. Moreover, incubation with 14-3-3σ-CM induced differential expression profiles of matrix metalloproteinases (MMPs) in fibroblasts (MMP-1, MMP-2, MMP-9, MMP-12 and MMP-14), THP-1 (MMP-1 and MMP-12) and PMA-THP-1 cells (MMP-2, MMP-12 and MMP-14). In contrast, silencing of 14-3-3σ by siRNA significantly abolished 14-3-3σ-CM induced MMPs. In addition, treatment with recombinant 14-3-3σ (r14-3-3σ) protein exhibits a similar expression profile of MMPs induced by 14-3-3σ-CM in fibroblasts, THP-1 and PMA-THP-1 cells. Finally, knockdown of aminopeptidase N (APN) significantly abrogated r14-3-3σ induced expression of MMPs in HS68 fibroblasts. These results suggest that HCC-secreted 14-3-3σ promotes expression of MMPs in cancerous surrounding cells via an APN dependent mechanism. 14-3-3σ has a paracrine effect in educating stromal cells in tumor-associated microenvironment.

Keywords: 14-3-3σ; hepatocellular carcinoma; invasion; matrix metalloproteinase; paracrine.

Figure 1. Co-culture of 14-3-3σ-CM treated stromal cells enhances cell invasion of Huh-7 cells

A. A flow chart of the experimental design. B. CM of control and 14-3-3σ overexpression cells was harvested. Subsequently, HS68, THP-1 and PMA-THP-1 cells (1 × 10⁴) were incubated with control-CM or 14-3-3σ-CM, followed by co-cultured parental Huh-7 cells (1 × 10⁵). Cell invasion was determined by a Boyden chamber assay. These results are from three independent experiments. Scale bars: mean ± SD. *, P<0.05.

Figure 2. 14-3-3σ-CM induces expression of MMPs in stromal cells

HS68, THP-1 and PMA-THP-1 cell were incubated with control-CM and 14-3-3σ-CM for 24 h and expression of MMPs was determined by Q-PCR. A. Expression of MMP-1, MMP-2, MMP-9, MMP-12 and MMP-14 in HS68 cells. B. Expression of MMP-1, MMP-2, MMP-9, MMP-12 and MMP-14 in THP-1 cells. C. Expression of MMP-1, MMP-2, MMP-9, MMP-12 and MMP-14 in PMA-THP-1 cells. D. Expression of MMP-1, MMP-2, MMP-9, MMP-12 and MMP-14 in 14-3-3σ overexpression compared to control Huh-7 cells. These results are from three independent experiments. Scale bars: mean ± SD. *, P<0.05, **, P<0.01.

Figure 3. 14-3-3σ silencing abolished 14-3-3σ-CM induced MMPs expression

A. Efficacy of 14-3-3σ silencing by siRNA was confirmed by Western blot analysis. B. A flow chart of the experimental design. C. HS68 fibroblasts were incubated with CM derived from 14-3-3σ silencing with siRNA or scramble siRNA in 14-3-3σ stable and control cells. Expression of MMPs was determined by Q-PCR. D. THP-1 cells were incubated with CM and expression of MMPs was determined by Q-PCR. E. PMA-THP-1 cells were incubated with CM and expression of MMPs was determined by Q-PCR. These results are from three independent experiments. Scale bars: mean ± SD. *, P<0.05.

Figure 4. Induced expression of MMPs in stromal cells by 14-3-3σ recombinant protein

A. Expression of 14-3-3σ in CM from cells of stable 14-3-3σ overexpression and transient transfection was determined by Western blot analysis. B. HS68 C. THP-1 and D. PMA-THP-1 cells were treated with different concentrations of recombinant 14-3-3σ (r14-3-3σ) protein for 24 h. Expression of MMPs was determined by Q-PCR. These results are from three independent experiments. Scale bars: mean ± SD. *, P<0.05, **, P<0.01.

Figure 5. The role of APN for uptake of 14-3-3σ in fibroblasts

A. Endogenous APN expression levels in Huh-7, HS68, THP-1 and PMA-THP-1 cells were determined by Q-PCR. B. HS68, THP-1 and PMA-THP-1 cells were treated with different concentrations (0-20 μg/ml) of r14-3-3σ for 24 h and the levels of 14-3-3σ were determined by Western blot analysis. Actin was used as a loading control. C. 14-3-3σ in subcellular fractions of membrane, nuclei and cytosol were determined by Western blot analysis. Lamin A/C was used as nuclear loading control. D. Silencing of APN by siRNA (vs. scramble siRNA) treated with control-CM and 14-3-3σ-CM or E. r14-3-3σ protein was determined by Q-PCR. F. Knockdown of APN with siRNA abolished the uptake of r14-3-3σ in HS68 cells was determined by Western blot analysis. Actin was used as a loading control. These results are from three independent experiments. Scale bars: mean ± SD. *, P<0.05, **, P<0.01.

Figure 6. Silencing of APN abrogated the effect of 14-3-3σ induced MMPs expression

A. HS68 cells were transfected with APN and scramble siRNAs for 24 h, followed by treatment with r14-3-3σ (10 μg/ml) for an additional 24 h. Expression of MMPs (MMP-1, MMP-2, MMP-9, MMP-12 and MMP-14) were determined by Q-PCR. B. and C. HS68 cells were transfected with scramble/APN siRNA or treated with 30 μM GM6001 followed by incubation with control-CM or 14-3-3σ-CM. These cells were subsequently co-cultured with Huh-7 cells and cell invasion of Huh-7 cells was determined by trans-well assay. These results are from three independent experiments. Scale bars: mean ± SD. *, P<0.05, **, P<0.01. D. An illustrated scheme for the role of HCC-secreted 14-3-3σ in regulating HCC tumor metastasis.