Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway - PubMed (original) (raw)

Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway

Igor Matushansky et al. J Clin Invest. 2007 Nov.

Abstract

Malignant fibrous histiocytoma (MFH), now termed high-grade undifferentiated pleomorphic sarcoma, is a commonly diagnosed mesenchymal tumor, yet both the underlying molecular mechanisms of tumorigenesis and cell of origin remain unidentified. We present evidence demonstrating that human mesenchymal stem cells (hMSCs) are the progenitors of MFH. DKK1, a Wnt inhibitor and mediator of hMSC proliferation, is overexpressed in MFH. Using recombinant proteins, antibody depletion, and siRNA knockdown strategies of specific Wnt elements, we show that DKK1 inhibits hMSC commitment to differentiation via Wnt2/beta-catenin canonical signaling and that Wnt5a/JNK noncanonical signaling regulates a viability checkpoint independent of Dkk1. Finally, we illustrate that hMSCs can be transformed via inhibition of Wnt signaling to form MFH-like tumors in nude mice, and conversely, MFH cells in which Wnt signaling is appropriately reestablished can differentiate along mature connective tissue lineages. Our results provide mechanistic insights regarding the cell of origin of MFH, establish what we believe is a novel tumor suppressor role for Wnt signaling, and identify a potential therapeutic differentiation strategy for sarcomas.

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Figures

Figure 1

Figure 1. MFH associated with hMSCs.

(A) Unsupervised hierarchical clustering of a panel of STS (described in detail in ref. 9) using a stem cell gene list (Supplemental Table 1). (B) Immunohistochemical staining of differentiation-associated proteins on the indicated tumor specimens from a tissue microarray. A single stain representative of 10 tissue samples each of MFH, LS, and leiomyosarcomas (LMS), and of 6 tissue samples each of fibrosarcomas (FS) are shown. Original magnification, ×200.

Figure 2

Figure 2. DKK1 and β-catenin patterns in hMSCs and MFH.

(A) ANOVA of gene expression analysis of the indicated sarcomas listed in order of genes overexpressed in MFH. (B) DKK1 levels by RT-PCR in the indicated sarcomas. (C) Dkk1 expression by IHC staining in the indicated sarcomas and percentage of samples expressing intense staining. Original magnification, ×200. (D) Cell density of hMSCs proliferating in vitro in relation to β-catenin activity. Immunoblot analysis of Dkk1 and β-catenin in total cellular extracts (E), nuclear extracts, (F) and cytoplasmic extracts (G) in the indicated samples. Immunoblot analysis of LEF1, c-myc, and cyclin D1 tubulin (H) in the indicated samples.

Figure 3

Figure 3. Wnt signaling in hMSCs.

(A) Expression of Wnt2 and Wnt5a by RT-PCR in the indicated sarcomas. (B) Immunoblot analysis of Wnt2 and Wnt5a in the indicated samples. (C) ELISA of β-catenin in the indicated samples following specified treatments.

Figure 4

Figure 4. Wnt5a regulates a commitment viability checkpoint.

(A) Expression of EMofMD by RT-PCR in hMSCs grown in either continuous culture medium (CCM), ADM, or ODM, as indicated. (B) Cell cycle analysis of hMSCs after 7 days of culture under the specified treatments.

Figure 5

Figure 5. Wnt5a signals through Jnk.

(A) Relative activity of Wnt noncanonical signaling and its inhibitors: Wnt/Ca pathway (Ca and hispidin), Wnt/Rho pathway (Rho and Y-276632), and Wnt/Jnk (JNK and SP600125) in specified samples under the indicated conditions. (B) Relative activity of Wnt/Jnk in specified samples under the indicated conditions. All measurements (A and B) indicated are the average of 2 independent time series.

Figure 6

Figure 6. Differentiation and transformation via Wnt signaling.

MFH cells cultured in ADM (A); ODM (B); ADM, rWnt2, and rWnt5a (C); and ODM, rWnt2, and rWnt5a (D). hMSCs cultured in the absence (E) or presence of hrDkk1 (G); SV40-hMSCLTag cultured in the absence (F) or presence of hrDkk1 (H); tumor volumes at the indicated days of subcutaneously injected SV40-hMSCLTag tumorigenic clones (upper line) and SV40-hMSCLTag cells (lower line) as controls into the flanks of Balb/c nude mice. Clones from H&E staining of tumors formed following subcutaneous injection of in vitro–transformed cells at low (I) and high (J) power. Original magnification, ×200; ×400 (inset).

Figure 7

Figure 7. Schematic diagram illustrating a working hypothesis regarding tumorigenesis.

Enhanced recruitment of adult stem cells and/or tissue precursors under conditions that result in tissue damage may promote development of the first oncogenic event that predisposes that cell to accumulate further genetic damage, ultimately resulting in tumor initiation and progression. See text for details.

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