Ror2 regulation of MMP2 expression in RCC cells is dependent upon an intact kinase domain.

<p>Quantitative RT-PCR for 786-0 cells shows a significant correlated decrease in <b>A</b>) Ror2 and <b>B</b>) MMP2 mRNA levels in both shRNA knockdown cell lines relative to control. <b>C</b>) Additionally, induction of wildtype Ror2 and Ror2-DM is evident following treatment with doxycycline (500 ng/ml). <b>D</b>) But a matching increase in MMP2 is only seen with overexpression of wildtype Ror2. For all quantitative RT-PCR assays, transcript values were normalized to β-actin RNA internal standard with fold change calculated in reference to either 786-0 control or unstimulated GFP expressing cells. Error bars represent SEM across triplicates of a representative duplicated experiment. Gelatin Zymography shows increased levels of both the precursor pro-MMP2 and its cleaved active form with overexpression of wildtype Ror2 relative to GFP vector control in both <b>E</b>) 786-0 and <b>F</b>) HEK293T cells. Quantification of the levels of active MMP2 normalized to the media only control (Con) is shown below each gel. <b>G</b>) Invasion of 786-0 cells seeded in Boyden matrigel coated chambers under normal culture conditions show a significant decrease upon treatment with MMP2 inhibitor (OA-Hy - 60 uM) in both independent shRNA knockdowns and Ror2 overexpression in comparison with vehicle control. The percentage of area covered was quantitated from random fields using ImageJ. Error bars represent stdev across duplicates of a representative duplicated experiment. <i>P</i>-values were calculated using an Anova one-way analysis (*<.05, **<.001).</p

Ror2 expression regulates cell invasion in RCC cells.

<p>Analysis of invasive potential using Boyden matrigel coated chamber assays under normal culture conditions with <b>A</b>) 786-0 cells show a significant reduction in invasion in both independent shRNA Ror2 knockdowns in comparison with parental 786-0 and vector control cells (representative images shown below). Additionally, <b>B</b>) 786-0 & <b>C</b>) HEK293T cells under normal culture conditions show a significant increase in cell invasion with overexpression of wild-type Ror2 that is reduced with expression of Ror2-DM (representative images shown below). The percentage of area covered was quantitated from random fields using ImageJ. <i>P</i>-values were calculated using an Anova one-way analysis with error bars representing stdev shown (*<.05, **<.001).</p

Summary of clinical characteristics of patients in the TCGA ccRCC dataset.

<p>Summary of clinical characteristics of patients in the TCGA ccRCC dataset.</p

Expression of Ror2 promotes <i>in vivo</i> tumor growth and invasion.

<p><b>A&C</b>) Tabulated rates of successful injection and growth of all 786-0 xenograft tumors are shown. <b>B</b>) Average tumor size (cm) with error bars showing stdev for resulting xenograft tumors following paired orthotropic injections of 786-0 cells containing either the control empty vector, pcDNA6 or hRor2 exhibited a trend to increased <i>in vivo</i> tumor growth with Ror2 overexpression. <b>D</b>) Average tumor size (mm) with error bars showing stdev for resulting xenograft tumors following subcutaneous injection of 786-0 cells containing either the control empty vector, GFP, wild-type Ror2, or Ror2-DM showed a significant increase in <i>in vivo</i> tumor growth with wild-type Ror2 overexpression. <i>P</i>-values were calculated using an Anova one-way analysis (*<.05 **<.01). <b>E</b>) Representative image of an orthotopic xenograft consisting of 786-0 vector control cells. <b>F</b>) Increased invasion of local tissues was evident with Ror2 expression, with arrows highlighting areas of continued invasion into the spleen.</p

High Ror2 expression correlates with increased tumor growth and stage in primary human RCC tumors.

<p>Tumor percentage breakdowns of TCGA ccRCC cases (468) stratified by Ror2-High and Ror2-Low expression show Ror2-High tumors exhibiting significant increases in <b>A</b>) tumor size, <b>B</b>) higher nuclear grade, <b>C</b>) clinical stage, and <b>D</b>) tumor stage. <i>P</i>-values were calculated using either an Anova one-way analysis or Fisher's exact test (*<.05, **<.001).</p