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Notch1-WISP-1 axis determines the regulatory role of mesenchymal stem cell-derived stromal fibroblasts in melanoma metastasis - PubMed

  • ️Fri Jan 01 2016

Notch1-WISP-1 axis determines the regulatory role of mesenchymal stem cell-derived stromal fibroblasts in melanoma metastasis

Hongwei Shao et al. Oncotarget. 2016.

Abstract

Mesenchymal stem cells-derived fibroblasts (MSC-DF) constitute a significant portion of stromal fibroblasts in the tumor microenvironment (TME) and are key modulators of tumor progression. However, the molecular mechanisms that determine their tumor-regulatory function are poorly understood. Here, we uncover the Notch1 pathway as a molecular determinant that selectively controls the regulatory role of MSC-DF in melanoma metastasis. We demonstrate that the Notch1 pathway's activity is inversely correlated with the metastasis-regulating function of fibroblasts and can determine the metastasis-promoting or -suppressing phenotype of MSC-DF. When co-grafted with melanoma cells, MSC-DFNotch1-/- selectively promote, while MSC-DFN1IC+/+ preferentially suppress melanoma metastasis, but not growth, in mouse models. Consistently, conditioned media (CM) from MSC-DFNotch1-/- and MSC-DFN1IC+/+ oppositely, yet selectively regulates migration, but not growth of melanoma cells in vitro. Additionally, when co-cultured with metastatic melanoma cells in vitro, MSC-DFNotch1-/- support, while MSC-DFN1IC+/+ inhibit melanoma cells in the formation of spheroids. These findings expand the repertoire of Notch1 signaling as a molecular switch in determining the tumor metastasis-regulating function of MSC-DF. We also identified Wnt-induced secreted protein-1 (WISP-1) as a key downstream secretory mediator of Notch1 signaling to execute the influential role of MSC-DF on melanoma metastasis. These findings reveal the Notch1-WISP-1 axis as a crucial molecular determinant in governing stromal regulation of melanoma metastasis; thus, establishing this axis as a potential therapeutic target for melanoma metastasis.

Keywords: Notch1; WISP-1/CCN4; cancer-associate fibroblasts; melanoma; mesenchymal stem cells.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors have declared that no conflicts of interest exists.

Figures

Figure 1
Figure 1. Generation and characterization of BM-derived MSC-DF

(A) MSC-DF are αSMA+/vimentin+/FSP1+ by IF. (B) Effect of Notch1 activation on MSC-DF cell growth and migration. top: expression of mutant N1IC and elevated levels of Hes1 and Hey1 protein in Cre/Lenti-transduced MSC-DFN1IC+/+. Decreased cell growth (middle) and migration (bottom) rates of MSC-DFN1IC+/+ vs. MSC-DFLSL-N1IC (non-transduced (−) or GFP/Lenti-transduced). (C) Effect of Notch1 deletion on MSC-DF cell growth and migration. top: Notch1 deletion at protein levels; middle: comparable growth rate of MSC-DFNotch1−/− vs. MSC-DFNotch1+/+; bottom: MSC-DFNotch1−/− migrate faster than MSC-DFNotch1+/+ (LPF: low power field).

Figure 2
Figure 2. Differential effects of Notch1 activation and inactivation in MSC-DF on melanoma cell behavior in vitro

(A) Cell growth of three melanoma cells is not affected by CM of MSC-DFN1IC+/+ or MSC-DFNotch1−/−. NS: no significance. White bars represent non-transduced cells and grey bars are GFP/lenti-transduced cells, while black bars are Cre/lenti-transduced cells. (B) Effect of CM from MSC-DFN1IC+/+ and MSC-DFNotch1−/− on C8161 migration. CM of MSC-DFN1IC+/+ (top) inhibit while CM of MSC-DFNotch1−/− (bottom) promote melanoma cell migration. *P < 0.01. (C) MSC-DFN1IC+/+ (GFP+) mitigate, yet MSC-DFNotch1−/− (GFP+) increase spheroid formation of C8161 (DsRed+) in co-culture. C8161 alone don't form typical spheroid. Quantification of spheroids formed with MSC-DFN1IC+/+ or MSC-DFNotch1−/− is showed in the left panel. Data are analyzed by Student's t-test and presented as mean ± SD based on three independent experiments. *P < 0.01.

Figure 3
Figure 3. MSC-DFNotch1−/− selectively promote melanoma invasion and metastasis in vivo

(A) Melanoma growth in mouse skin. top: Tumor weight; bottom: three representative images of resected tumors/group. Solo-grafted melanoma grew slowly compared with co-grated melanoma (a). Growth rate of 3× solo-grafted melanoma was comparable to co-grafted melanoma cells (b). No significant difference for tumor growth with MSC-DFNotch1−/− (e) vs. MSC-DFNotch1+/+ (non-transduced (c) or GFP/lenti-transduced (d)). (B) Tumor lung metastasis was robustly increased when co-grafted with MSC-DFNotch1−/− vs. MSC-DFNotch1+/+. Five representative IVIS images of lungs/group are shown. Metastatic foci pointed by arrows in lung were detected by H&E staining. % of lung metastasis and tumor burden in lung are exhibited. (C) MSC-DFNotch1−/− significantly promote melanoma Invasion. Five representative H&E images of tumor sections/groups are shown. Dash lines highlight tumor boundaries. Arrowheads point to melanoma cell invasion. % of local invasion detected by H&E staining is shown. In panel A, B, C: b: (3×) C8161; c: C8161 + MSC-DFNotch1+/+ (non-transduced); d: C8161 + MSC-DFNotch1+/+ (GFP/lenti-transduced); e: C8161 + MSC-DFNotch1−/− (Cre/lenti-transduced).

Figure 4
Figure 4. MSC-DFN1IC+/+ selectively inhibit melanoma invasion and metastasis in vivo

(A) Growth of co-grafted melanoma in mice. Top: images and weights of tumors resected from each group (n = 6/group). No significant difference in growth of tumor co-grafted with MSC-DFN1IC+/+ vs. MSC-DFLSL-N1IC. (B) MSC-DFN1IC+/+ significantly diminished lung metastasis, both tumor loading in lung and % of metastasis. Metastatic foci pointed by arrowheads in lung sections were confirmed by H&E staining. (C) MSC-DFN1IC+/+ significantly inhibit tumor invasion. Six representative images of H&E staining of tumor tissues/groups are shown. Dash lines highlight tumor boundaries. Arrowheads point to invading tumor cells. Rate of local invasion in different groups is summarized.

Figure 5
Figure 5. Expression of WISP1 depends upon Notch1

(A) Heatmap of differentially expressed gene profiles from duplicates of MSC-DFNotch1−/− vs. MSC-DFNotch1+/+ by microarray analysis. (B) Heatmap of differentially expressed CCN family genes (top). Relative levels of CCN1-5 genes in MSC-DFNotch1−/− vs. MSC-DFNotch1+/+ (bottom), based on unpaired Student's t-test between the two conditions. (C) Deletion of Notch1 in MSC-DFNotch1−/− (top) or expression of N1IC in MSC-DFN1IC+/+ (bottom) and downregulation of WISP1 in MSC-DFNotch1−/− are validated by immunoblotting.

Figure 6
Figure 6. WISP-1 mediates Notch1-determined metastasis-regulating function of MSC-DF

(A) top: Immunoblot shows reconstituted overexpression of WISP-1 in MSC-DFNotch1−/− ; bottom: CM of WISP-1hi/MSC-DFNotch1−/− and WISP-1lo/MSC-DFNotch1−/− don't affect C8161 cell proliferation. (B) CM of WISP-1hi/MSC-DFNotch1−/− inhibit DsRed+-C8161 migration. (C) Effect of γhWISP-1 on DsRed+ C8161 migration. Quantitative data in A, B, C are analyzed by Student's t-test and presented as mean ± SD of three independent experiments. (D) WISP-1hi/MSC-DFNotch1−/− could largely reverse WISP-1lo/MSC-DFNotch1−/−-induced metastasis-promoting effects (see Figure 3B-e). Five representative IVIS images of lungs are shown. Metastatic foci in lung are confirmed by H&E staining. (E) WISP-1hi/MSC-DFNotch1−/− significantly mitigate WISP-1lo/MSC-DFNotch1−/−-induced melanoma invasion (see Figure 3C-e). Five representative H&E images are shown. Arrows points to invading melanoma cells in skin tissues. Rate of local invasion is summarized.

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