OncKRAS and oncNRAS increase tumorigenicity of RUCH-2 cells

The transplantation of RUCH-2 cell lines were performed with 8 Nu/Nu mice for each cohort.

In contrast to TE617.T cells, 9 × 10⁶ RUCH-2 cells in matrigel were implanted. All mice were sacrificed when a tumor reached a diameter of 1.5 cm or latest after 21 days.

6.2.2.1 OncKRAS and oncNRAS increase tumor growth and weight of RUCH-2 xenografts

The transplantation of RUCH-2 KRAS (Fig. 38B) and RUCH-2 NRAS (Fig. 38C) cells resulted in a strongly enhanced tumor growth in comparison to simultaneously transplanted RUCH-2 pMSCV cells. In contrast, RUCH-2 HRAS cells did not grow and the tumor volume started to regress early after transplantation (Fig. 38A). However, since BrdU incorporation in cultured RUCH-2 HRAS cells was significant (see section 6.1.5) and since RUCH-2 KRAS and RUCH-2 NRAS xenotransplants grew very fast, it is likely that the RUCH-2 HRAS cells have died prior or during the transplantation procedure. Nevertheless, this study should be repeated to see whether this assumption is true or not.

Figure 38: OncKRAS and oncNRAS increase tumor growth and weight of RUCH-2 xenografts

9 × 10⁶ RUCH-2 pMSCV, RUCH-2 HRAS (A), RUCH-2 KRAS (B) or RUCH-2 NRAS (C) cells in 200 µl PBS were transplanted into the left or right flanks, respectively, of Nu/Nu mice (n=8 for each cohort). Tumor size was measured with a caliper every or every second day after transplantation and the approximate tumor volume was calculated. The results are shown in the tumor growth curves in the left panels. At the end of the study tumors were isolated, weighed and measured to determine the exact tumor volume. The results are shown in the respective middle and right panels. For statistical analyses multiple tests (growth curve) and non-parametric t-tests (Mann Whitney; for tumor weight and exact tumor volume) were performed. * indicate significance compared to characteristics of RUCH-2 pMSCV xenografts. *p<0.05, **p<0.01, ***p<0.001, ****p< 0.0001

The approximate tumor volume of RUCH-2 xenografts with oncKRAS or oncNRAS mutations in living mice, and their weight and exact tumor volume after tumor isolation were significantly increased, when compared to RUCH-2 pMSCV xenografts (Fig. 38). For oncKRAS this result fits to cell culture experiments, in which oncKRAS also increased the proliferation rate. However, the in vivo results for the oncNRAS-expressing RUCH-2 cell line are different from the in vitro situation, in which oncNRAS did not increase the proliferation rate, even not after 72 h of incubation with BrdU (compare section 6.1.5).

6.2.2.2 OncRAS may downregulate GLI1 expression in tumor cells and upregulate Gli2 expression in stromal cells of RUCH-2 xenografts

In vitro experiments using oncRAS-expressing RUCH-2 cell lines showed a decrease in GLI1 and GLI2 mRNA level and a simultaneous increase in SHH mRNA level in comparison to the control (compare section 6.1.2). The expression of these genes was also analyzed in the

xenografts. In addition and in order to see, whether RUCH-2 cell with oncRAS secrete SHH in vivo, the expression of Gli1 and Gli2 was measured in the tumor stroma.

In xenografts derived from oncKRAS-expressing RUCH-2 cells the expression of hGLI1 and hSHH was somewhat lower in comparison to RUCH-2 pMSCV xenografts. However, these results were not significant. In addition, the level of hGLI2 remained unchanged (Fig. 39A).

When the expression level of mGli1 and mGli2 were measured, an increase in xenografts derived from oncKRAS-expressing RUCH-2 was detected, which was not significant for Gli1 but significant for Gli2. This indicates a potential impact of oncKRAS-expressing tumor cells on HH signaling in surrounding stromal cells (Fig. 39B). However, since SHH was rather downregulated in these xenografts, the increase in mGli1 or mGli2 is rather not mediated by secretion of the SHH ligand by tumor cells.

Figure 39: OncKRAS significantly induces mGli2 expression in the stroma of RUCH-2 xenografts

Xenograft tumors were subjected to RNA isolation, cDNA synthesis and subsequent qRT-PCR analyses of the HH signaling pathway genes hGLI1/mGli1, hGLI2/mGli2, hSHH using species specific primers for human (A) and murine genes (B). The data were normalized to hHPRT for human gene expression (A) and to mHprt for murine gene expression (B) and are shown as fold expression to RUCH-2 pMSCV xenografts that were set to 1. Bars represent the mean ± SEM of 8 isolated tumors of each cohort. For statistical analyses non-parametric t-tests (Mann Whitney) were performed. **p<0.01 compared to expression level of RUCH-2 pMSCV xenografts.

Gene expression analyses of xenografts derived from oncNRAS-expressing RUCH-2 cells revealed that, as seen in cell culture, oncNRAS decreased hGLI1 and hGLI2 in the tumor cells. Whereas the decrease in GLI1 was significant, the decrease in hGLI2 was not. In contrast to cell culture, oncNRAS also decreased hSHH in comparison to RUCH-2 pMSCV xenografts (Fig. 40A). In the murine stroma, no significant changes in mRNA level of mGli1 and mGli2 were measured (Fig. 40B).

Figure 40: OncNRAS significantly decreases hGLI1 expression in tumor cells of RUCH-2 xenografts Xenograft tumors were subjected to RNA isolation, cDNA synthesis and subsequent qRT-PCR analyses of the HH signaling pathway genes hGLI1/mGli1, hGLI2/mGli2, hSHH using species specific primers for human (A) and murine genes (B). The data were normalized to hHPRT for human gene expression (A) and to mHprt for murine gene expression (B) and are shown as fold expression to RUCH-2 pMSCV xenografts that were set to 1. Bars represent the mean ± SEM of 8 isolated tumors of each cohort. For statistical analyses non-parametric t-tests (Mann Whitney) were performed. ***p<0.001 compared to expression level of RUCH-2 pMSCV xenografts.

Taken together, the results show that transplanted RUCH-2 cells with oncKRAS or oncNRAS behave somewhat different when compared to cell culture (compare section 6.1.2). Thus, hGLI1 is only moderately decreased in RUCH-2 oncKRAS xenografts (it however is significantly decreased in RUCH-2 oncNRAS xenografts) and an upregulation of hSHH is observed in none of the analyzed xenografts. Nevertheless, the expression of mGli2 in the tumor stroma is significantly increased, at least in oncKRAS xenografts.

6.2.3 Chapter summary

In chapter 6.2, the impact of oncRAS on growth and HH signaling activity in ERMS xenotransplants is described. As in cell culture, the tumor growth is significantly stimulated by oncRAS mutations in ERMS cell lines. In addition, oncRAS tend to decrease tumorintrinsic GLI1 expression, although the respective results are only significant for oncNRAS-expressing RUCH-2 xenografts. In contrast, the transplantation of oncRAS cells rather results in a decrease in SHH expression (which was significant for oncH-/NRAS expressing TE617.T cells), whereas SHH expression of cultured oncRAS RUCH-2 or oncRAS TE617.T cells is elevated or decreased, respectively. Finally, tumor-extrinsic Gli2 expression in oncKRAS-expressing RUCH-2 xenografts is increased, which may indicate that the transplanted tumor cells indeed secrete HH ligands and thus may affect HH signaling activity in the tumor microenvironment.