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Epigenetic upregulation of urokinase plasminogen activator promotes the tropism of mesenchymal stem cells for tumor cells - PubMed

Epigenetic upregulation of urokinase plasminogen activator promotes the tropism of mesenchymal stem cells for tumor cells

Sai Murali Krishna Pulukuri et al. Mol Cancer Res. 2010 Aug.

Abstract

A major obstacle for the effective treatment of cancer is the invasive capacity of the tumor cells. Previous studies have shown the capability of mesenchymal stem cells (MSC) to target these disseminated tumor cells and to serve as therapeutic delivery vehicles. However, the molecular mechanisms that would enhance the migration of MSCs toward tumor areas are not well understood. In particular, very little is known about the role that epigenetic mechanisms play in cell migration and tropism of MSCs. In this study, we investigated whether histone deacetylation was involved in the repression of urokinase plasminogen activator (uPA) expression in MSCs derived from umbilical cord blood (CB) and bone marrow (BM). Induction of uPA expression by histone deacetylase inhibitors trichostatin A and sodium butyrate was observed in CB- and BM-derived MSCs examined. In vitro migration assays showed that induction of uPA expression by histone deacetylase inhibitors in CB- and BM-derived MSCs significantly enhanced tumor tropism of these cells. Furthermore, overexpression of uPA in CB-MSCs induced migration capacity toward human cancer cells in vitro. In addition, our results showed that uPA-uPAR knockdown in PC3 prostate cancer cells significantly inhibited tumor-specific migration of uPA-overexpressing MSCs. These results have significant implications for the development of MSC-mediated, tumor-selective gene therapies.

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Figures

Figure 1
Figure 1. TSA induces accumulation of acetylated histones H3 and H4 in chromatin associated with the uPA gene

(A) Nuclear extracts were isolated from control and TSA-treated CB- and BM-MSCs, and immunoblot analysis was performed using anti-acetyl histone H3, anti-acetyl histone H4, and histone H3 antibodies. Histone H3 was utilized as a loading control. (B) Densitometric analysis of immunoblots. Data are normalized to H3, averaged, and expressed as percentage of control (CTL = 1). (C) Schematic representation of the uPA promoter region and the location of primers used for PCR amplification in the ChIP assay. Bent arrow, transcriptional start site. (D) Chromatin fragments from CB- and BM-MSCs cultured with (+) or without (−) TSA for 16 hrs were immunoprecipitated with antibody to acetylated (Ac) histones H3 and H4 or control normal rabbit serum (NRS).

Figure 2
Figure 2. Effects of trichostatin A (TSA) on uPA expression and cellular migration in MSCs

(A) uPA mRNA expression (top) and activity levels (bottom) in control and TSA-treated CB- and BM-MSCs were analyzed by RT-PCR and fibrin zymography, respectively. (B) uPA mRNA expression (top) and activity levels (bottom) in control and SB-treated CB- and BM-MSCs were analyzed by RT-PCR and fibrin zymography, respectively. (C) The migration capacity of the control and TSA-treated CB-MSCs toward human cancer cells PC3 and MDA231 were assessed in vitro by transwell migration assay. Columns, percentage of the DMEM control (*p <0.05); bars, SD. (D) The migration capacity of the control and TSA-treated CB-MSCs toward non-tumor cells RWPE1 and HEK293 were assessed as described in (C).

Figure 3
Figure 3. Overexpression of uPA enhances the tumor-specific migration ability of MSCs

(A) RT-PCR (top) and immunoblot (bottom) analyses of CB-MSCs stably transduced with mock, uPA expression vector or empty vector. GAPDH was used as loading control for RNA and protein analysis. (B) The migration capacity of the uPA-overexpressing MSCs toward PC3 cells was assessed in vitro by transwell migration assay. Columns, percentage of the vector control (*p <0.05); bars, SD. (C) The migration capacity of the uPA-overexpressing MSCs toward MDA-231 and U251 was assessed as described in (B). (D) The migration capacity of the uPA-overexpressing MSCs toward RWPE1 and HEK293 was assessed as described in (B).

Figure 4
Figure 4. Activation of ERK by uPA mediates the tumor-specific migration capacity of MSCs

(A) Immunoblot analysis shows increased levels of ERK1/2 phosphorylation in uPA-expressing MSCs (uPA-10). GAPDH was used as a loading control. (B) Pretreatment of ERK inhibitor significantly decreased uPA-expressing MSC migration toward PC3 and MDA231 cells compared with cells treated with nonspecific isotype IgG. Columns, percentage of the vector control (*p <0.05); bars, SD.

Figure 5
Figure 5. Expression levels of the uPA and uPAR in human tumor and non-tumor cell lines

(A) uPA and uPAR mRNA expression in RWPE1, HEK293, U251, MDA231 and PC3 cells were analyzed by quantitative RT-PCR. (B) Immunoblot analysis of uPA and uPAR proteins in HEK293, U251, MDA231 and PC3 cells. GAPDH was used as a loading control.

Figure 6
Figure 6. Effects of tumor cells expressing uPA and uPAR on the migration ability of MSC/uPA

The migration capacity of the uPA-overexpressing MSCs (uPA-10) toward shRNA-transfected PC3 cells was assessed in vitro by transwell migration assay. Columns, percentage of the vector control (*p <0.05); bars, SD.

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References

    1. Rao JS. Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer. 2003;3:489–501. - PubMed
    1. Buzanska L, Machaj EK, Zablocka B, Pojda Z, Domanska-Janik K. Human cord blood-derived cells attain neuronal and glial features in vitro. J Cell Sci. 2002;115:2131–8. - PubMed
    1. Chen N, Hudson JE, Walczak P, et al. Human umbilical cord blood progenitors: the potential of these hematopoietic cells to become neural. Stem Cells. 2005;23:1560–70. - PMC - PubMed
    1. Kramm CM, Sena-Esteves M, Barnett FH, et al. Gene therapy for brain tumors. Brain Pathol. 1995;5:345–81. - PubMed
    1. Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res. 2005;65:3307–18. - PubMed

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