Rho kinase proteins display aberrant upregulation in vascular tumors and contribute to vascular tumor growth

Abstract

Background: The serine/threonine protein kinases ROCK1 and 2 are key RhoA-mediated regulators of cell shape and cytoskeletal dynamics. These proteins perform multiple functions in vascular endothelial cell physiology and are attractive targets for cancer therapy based on their roles as oncogenes and metastatic promoters. Given their critical functions in both of these processes, we hypothesized that molecular targeting of ROCK proteins would be exceedingly effective against vascular tumors such as hemangiomas and angiosarcomas, which are neoplasms composed of aberrant endothelial cells.

Methods: In this study, we compared ROCK1 and 2 protein expression in a large panel of benign and malignant vascular tumors to that of normal vasculature. We then utilized shRNA technology to knockdown the expression of ROCK1 and 2 in SVR tumor-forming vascular cells, and evaluated tumor size and proliferation rate in a xenograft model. Finally, we employed proteomics and metabolomics to assess how knockdown of the ROCK paralogs induced alterations in protein expression/phosphorylation and metabolite concentrations in the xenograft tumors.

Results: Our findings revealed that ROCK1 was overexpressed in malignant vascular tumors such as hemangioendotheliomas and angiosarcomas, and ROCK2 was overexpressed in both benign and malignant vascular tumors including hemangiomas, hemangioendotheliomas, hemangiopericytomas, and angiosarcomas. shRNA-mediated knockdown of ROCK2, but not ROCK1, in xenograft vascular tumors significantly reduced tumor size and proliferative index compared to control tumors. Proteomics and metabolomics analysis of the xenograft tumors revealed both overlapping as well as unique roles for the ROCK paralogs in regulating signal transduction and metabolite concentrations.

Conclusions: Collectively, these data indicate that ROCK proteins are overexpressed in diverse vascular tumors and suggest that specific targeting of ROCK2 proteins may show efficacy against malignant vascular tumors.

Keywords: Angiosarcoma; Hemangioendothelioma; Hemangioma; Hemangiopericytoma; Rho kinase; Rock; Vascular sarcoma; shRNA.

Fig. 1

ROCK1 and ROCK2 are overexpressed in vascular tumors. a Box and whisker plots indicating the mean immunohistochemical staining score for ROCK1 in a panel of human tissues obtained from normal endothelium and benign, borderline, and malignant vascular tumors. The number of tumor samples tested (N) for each tissue type is indicated to the right of the plot. For statistical analysis, the Mann-Whitney rank sum test was used. Statistical significance was determined if the two-sided P value of the test was <0.05. b Representative 600× images of immunohistochemical staining for ROCK1 in a panel of human tissues obtained from normal endothelium and benign, borderline, and malignant vascular tumors (a = negative control, b = positive control, c = normal endothelium, d = capillary hemangioma, e = cavernous hemangioma, f = granulomatous hemangioma, g = hemangiopericytoma, h = angiosarcoma). Negative controls lacking the primary antibody and positive controls from the kidney were used to ensure immunopositivity was reliable. Brown staining indicates immunopositivity. c Box and whisker plots indicating the mean immunohistochemical staining score for ROCK2 in a panel of human tissues obtained from normal endothelium and benign, borderline, and malignant vascular tumors. The number of tumor samples tested (N) for each tissue type is indicated to the right of the plot. For statistical analysis, the Mann-Whitney rank sum test was used. Statistical significance was determined if the two-sided P value of the test was <0.05. d Representative 600× images of immunohistochemical staining for ROCK2 in a panel of human tissues obtained from normal endothelium and benign, borderline, and malignant vascular tumors (a = negative control, b = positive control, c = normal endothelium, d = capillary hemangioma, e = cavernous hemangioma, f = granulomatous hemangioma, g = hemangiopericytoma, h = angiosarcoma). Negative controls lacking the primary antibody and positive controls from the kidney were used to ensure immunopositivity was reliable. Brown staining indicates immunopositivity. Please see Additional file 3 for high resolution image

Fig. 2

shRNA-mediated knockdown of ROCK1 & 2 in SVR cells. a qPCR quantification of the levels of ROCK1 and 2 mRNA in SVR cells harboring shRNA vectors for scrambled control, ROCK1, or ROCK2. GAPDH mRNA was used as a normalization control for RQ calculations. For statistical analysis, the Student’s t-test was used. Statistical significance was determined if the two-sided P value of the test was <0.05. b Images were collected at 400× total magnification for SVR cells harboring shRNA vectors for scrambled control, ROCK1, or ROCK2 or treated with the pharmacological inhibitor Y-27632 (24 h; 10 μM). Please see Additional file 4 for high resolution image

Fig. 3

ROCK1 & 2 knockdowns in a xenograft vascular tumor model. a qPCR quantification of the levels of ROCK1 and ROCK2 mRNA in the SVR xenograft tumors relative to the scrambled control. GAPDH mRNA was used as a normalization control for RQ calculations. For statistical analysis, the Student’s t-test was used. Statistical significance was determined if the two-sided P value of the test was <0.05. b Representative images of mice harboring subcutaneous scrambled control, ROCK1 shRNA, and ROCK2 shRNA vascular xenograft tumors. c Box and whisker plot showing the distribution of tumor weights in scrambled control, ROCK1 shRNA and ROCK2 shRNA vascular xenograft tumors. Asterisks indicate p value <0.0005. (d) Low magnification H&E staining of whole tumor sections showing relative tumor sizes following removal from the host. Please see Additional file 5 for high resolution image

Fig. 4

ROCK2 knockdown results in reduced proliferation in a xenograft vascular tumor model. a Histogram depicting immunopositivity for Ki67 in scrambled control and ROCK2 knockdown xenograft vascular tumors. Data presented is the mean plus or minus the standard deviation. Asterisks indicate p value <0.005. b Representative images of scrambled control (a) and ROCK2 knockdown (b) angiosarcoma tumors stained via immunohistochemistry for the proliferative marker Ki67 (immunopositivity is brown). Positive control (c) is Ki67 staining of a breast carcinoma and negative control (d) is the xenograft vascular tumor minus primary antibody incubation. Please see Additional file 6 for high resolution image

Fig. 5

Omics characterization of ROCK1 and 2 shRNA knockdown SVR cells. a Lysates from SVR tumors harboring shRNA vectors for scrambled control, ROCK1, or ROCK2 were subjected to Full Moon Phospho Explorer Antibody arrays. Two-fold or more protein expression/modification changes between the knockdown and the control tumors are depicted via heatmap analysis. Each lane is the mean of three independent biological replicates. (red = upregulated; green = downregulated) b Western blot analysis detecting the expression of key cell cycle/survival regulators in protein extracts collected from scrambled control and ROCK2 knockdown xenograft vascular tumors. c Lysates from SVR tumors harboring shRNA vectors for scrambled control, ROCK1, or ROCK2 were subjected to ¹H NMR analysis metabolomics analysis. Two-fold or more metabolite concentration changes between the knockdown and the control cells are depicted via heatmap analysis. Each column represents the triplicate mean of an individual biological replicate (red = upregulated; green = downregulated). Please see Additional file 7 for high resolution image