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

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.


INTRODUCTION
The tumor microenvironment is comprised of multiple cell types including cancer cells, carcinoma-associated fibroblasts, invading inflammatory cells, and endothelial cells [1][2][3][4]. Cancer cells interact and associate with surrounding stromal cells and subsequently "educate" stromal cells to induce tumor growth, invasion as well as metastasis [5][6][7][8][9]. Thus, interplay and synergism of cancer cells with tumor-associated stromal cells are crucial in promoting tumor progression.

DISCUSSION
We have shown the paracrine role of HCC-secreted 14-3-3σ in modulating cancer cell invasion via stimulating MMP expression in carcinoma associated stromal cells. We have provided evidence that HCC-secreted 14-3-3σ educates stromal cells in the tumor microenvironment. Tumor associated stromal cells play critical role for tumor progression [5][6][7][8][9]. In HCC, an earlier study has demonstrated that depleting tumor-associated macrophages significantly enhances the therapeutic effect   of sorafenib by suppressing metastasis and angiogenesis in an in vivo xenograft nude mice model [41]. This result affirms the role of tumor associate stromal cells in promoting tumor metastasis and growth. In this work, we found that incubation of HS68, THP-1 and PMA-THP-1 cells with 14-3-3σ-CM or r14-3-3σ induced expression of different MMP expression. Since the expression levels of 14-3-3σ in HS68, THP-1 and PMA-THP-1 are barely detectable (Supplementary Figure S6), it is reasonable to assume that these cells uptake 14-3-3σ secreted from HCC ( Figure 6D). In this study, we found that increased HCC-secreted 14-3-3σ results in inducing expression of MMPs in stromal cells via an APN-dependent mechanism. However, the molecular mechanism of differential expression of 14-3-3σ in HCC and associated stromal cells needs further investigation.
Expression of 14-3-3σ is silenced in about 80% of breast tumors but highly expressed in correlation with malignant phenotype of basal-like breast cancer [42]. Expression of 14-3-3σ is associated with poor prognostic outcomes in basal-like subtypes of breast cancer. This indicates that 14-3-3σ contributes to a more aggressive tumor progression [42]. 14-3-3σ is overexpressed in 77.1% of HCC tumors [15]. Although 14-3-3σ does not play as an independent prognostic factor [15], it was suggested that 14-3-3σ combined with EZH2 significantly correlates with worse clinical HCC outcomes [16]. Here, we demonstrate that an increase of HCC-secreted 14-3-3σ results in synergizing stromal cells and contributes to tumor invasion. Taken together, it is likely that 14-3-3σ overexpression associate with more aggressive or highly invasive cancer cells of HCC by paracrine regulation.
Taken together, in this study, we provided evidence that carcinoma associated stromal cells play important roles in 14-3-3σ-promoting tumor progression. 14-3-3σ may synergize with other signals from its microenvironment milieu and associate with tumor-associated stromal cells in regulating HCC tumor progression.
To prepare 14-3-3σ-CM and control conditioned media (control-CM), the supernatants of 14-3-3σ overexpression and control stable cells were harvested and centrifuged at 300 g for 10min. The supernatants were transfer to Amicon Ultra-15 filter devices (Millipore, Billerica, MA, USA) followed by centrifugation at 3,000 g for 15min.

Invasion assay
The cell invasion assay was performed by a modified Boyden chamber system with Bio-coat FluoroBlock cell culture insert (BD Biosciences, San Jose, CA, USA) coated with 0.1% matrigel containing 0.1% bovine serum albumin (BSA)-DMEM medium in the upper chambers. DMEM medium with 100 μg/mL fibronectin (BD Biosciences), epidermal growth factor (80 ng/mL), and 10% BSA were added to the bottom chambers. Parental Huh-7 cells were trypsinized and re-suspended, followed by incubation with pre-warmed CellTracker dye (CellTracker™, Invitrogen Life Technologies, Grand Island, NY, USA) for 45 minutes. 1 × 10 5 of CellTracker-stained Huh-7 cells combined with 1 × 10 4 of CM-incubated HS68, THP-1 or PMA-THP-1 cells were co-cultured in the upper chambers for 16 h. For MMP inhibitor experiment, HS68 cells were treated 30 μM GM6001 (Millipore, Billerica, MA, USA) for 1hr. The invasive cells were fixed with 2% formaldehyde for 20 minutes and efficiency of invasiveness was determined by fluorescence microscope with emission wavelength at 490 nm.

Quantitative real-time PCR (Q-PCR)
Total RNA was extracted by use of the RNAspin Mini Kit (QIAGEN, Valencia, CA, USA) and cDNA was synthesized from 2 to 5 μg RNA by use of the random primers and SuperScript™ III Reverse Transcriptase cDNA Synthesis Kit (Invitrogen™ Life Technology). Quantitative real-time PCR using SYBR Green (Kapabiosystem, Wilmington, MA, USA) with specific oligonucleotide primers of MMP-1, MMP-2, MMP-9, MMP-12, MMP-14 and aminopeptidase N (APN) (Supplementary Table S1) were detected by the AB 7900HT system (Applied Biosystems, Foster city, CA, USA). Applied Biosystems Relative Quantification Manager Software version 1.2 was used to analyze the relative gene expression in each sample by the comparative cycle threshold (Ct) method. Gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase.

Transfection of siRNAs
Silencing of 14-3-3σ and APN with siRNA (small interfering RNA) was purchased from Invitrogen™ (Supplementary Table S2), including scramble siRNA (Cat.No.12935-112). Transfections of siRNAs were performed by using Lipofectamine™ RNAiMAX (Invitrogen™ Life Technology) and harvested at the indicated time for further analysis.

Subcellular fractionation
HS68 cells were treated with 5 or 10 μg/ml r14-3-3σ for 1hr. Cells were harvested and the subcellular fractions of membrane, nuclei and cytosol were prepared by ProteoExtract subcellular proteome extraction kit (Calbiochem, San Diego, CA, USA).

Statistical analysis
A Student's t-test was used to analyze differences between the two experimental groups. A P value <0.05 was considered statistically significant.