PAX3-FKHR induces morphological change and enhances cellular proliferation and invasion in rhabdomyosarcoma

Abstract

Alveolar rhabdomyosarcoma (ARMS) is consistently associated with the characteristic translocations t(2;13)(q35;q14) and t(1;13)(p36;q14), which encode for the PAX3-FKHR and PAX7-FKHR fusion oncoproteins respectively. We have investigated the relationship between PAX3-FKHR expression and ARMS histogenesis in primary tumors and cell culture systems. In a blinded histological review of discrepant primary tumors in which there was PAX3-FKHR expression but embryonal histology, we found small areas of alveolar histology in 6 of 11 cases. This suggests that histology alone may under-represent the association between PAX3-FKHR and ARMS, and we investigated this link by examining the effect of ectopic PAX3-FKHR expression on RMS cells. Two cell lines, RD and HX170C, were stably transfected with a PAX3-FKHR expression construct. In cloned transfectants derived from both lines, PAX3-FKHR expression resulted in increased proliferative rate in vitro and promoted cell growth in the absence of added growth factors. Tumors that formed as xenografts in immunodeficient mice were faster growing, more locally invasive, and had a denser, more pleomorphic architecture than untransfected or empty vector transfected tumors. The characteristic clefts and alveolar spaces of ARMS, however, were not seen. In contrast, tumors grown as xenografts from individual clones derived from ARMS cell lines showed all of the classical morphological features of ARMS suggesting divergence in vivo from precursor cells propagated in culture.

Figure 1.

Schema of blinded histological review.

Figure 2.

Tumors grown from individual clones derived from cell lines. A: Tumor from a clone of cell line RH30 showing giant cells, clefts, and alveolar histology. B: Desmin staining of a clone derived from RD showing focal desmin positivity and staining of giant cells.

Figure 3.

Representative Western blot showing expression of the HA epitope on tagged PAX3-FKHR in different clones of RD cells transfected with pCMV-P3F. a: Coomassie blue-stained gel made in parallel with blotted gel to demonstrate equivalence of loading. b: Band at about 120 kd corresponding to the expected size of PAX3-FKHR. C5 through C15 are different clones whereas “mixed” represents a mixed population of RD cells transfected with pCMV-P3F. L is protein standard ladder, WT is untransfected RD, and POS is a positive control cell line that highly expresses an HA tagged protein.

Figure 4.

a-e: Comparative proliferation assays. 100,000 cells of RD or 500,000 cells of HX170C were plated on day 1 and viable cell number determined after differing incubation times by trypan blue exclusion. Vector equals empty vector transfected cells, M and CMV refer to PAX3-FKHR expression construct with MyoD and CMV immediate early promoters respectively, and c refers to individual colony numbers.

Figure 5.

Mean tumor weight following subcutaneous injection in NOD-SCID mice of RD or HX170C rhabdomyosarcoma cells transfected with empty vector (vec) or pCMV-P3F. RD-P3f-13 and RD-P3f-14 refer to the same pCMV-P3F clones as used in the in vitro proliferation assays. Tumors were grown for four weeks. There were four mice per group in RD experiments and one mouse per group in HX170c experiments.

Figure 6.

Characteristic histological appearances of tumors grown as xenografts from clones of cell stably transfected with either empty vector or PAX3-FKHR. Cell lines and magnification on left. Cells were transfected with vector (a, c, e, g) or PAX3-FKHR (b, d, f, h). In b, there is evidence of increased local invasion. Individual muscle fibers are separated by infiltrating tumor cells. In contrast, in a the tumor has a well circumscribed edge and there is no evidence of significant splitting of individual fibers. d shows the increase in cellularity, nuclear size, and cellular pleomorphism in the PAX3-FKHR transfected tumor and f confirms the increased proliferation rate. g and h show a similar increase in cellularity and the grouping of cells into fascicular bundles that result from the expression of PAX3-FKHR in HX170C cells.