Antitumorigenesis of antioxidants in a transgenic Rac1 model of Kaposi's sarcoma

Abstract

Kaposi's sarcoma (KS) is the major AIDS-associated malignancy. It is characterized by the proliferation of spindle cells, inflammatory infiltrate, and aberrant angiogenesis caused by Kaposi's sarcoma herpesvirus (KSHV) infection. Small GTPase Rac1, an inflammatory signaling mediator triggering reactive oxygen species (ROS) production by NADPH-oxidases, is implicated in carcinogenesis and tumor angiogenesis. Here, we show that expression of a constitutively active Rac1 (RacCA) driven by the alpha-smooth muscle actin promoter in transgenic mice is sufficient to cause KS-like tumors through mechanisms involving ROS-driven proliferation, up-regulation of AKT signaling, and hypoxia-inducible factor 1-alpha-related angiogenesis. RacCA-induced tumors expressed KS phenotypic markers; displayed remarkable transcriptome overlap with KS lesions; and were, like KS, associated with male gender. The ROS scavenging agent N-acetyl-cysteine inhibited angiogenesis and completely abrogated transgenic RacCA tumor formation, indicating a causal role of ROS in tumorigenesis. Consistent with a pathogenic role in KS, immunohistochemical analysis revealed that Rac1 is overexpressed in KSHV(+) spindle cells of AIDS-KS biopsies. Our results demonstrate the direct oncogenicity of Rac1 and ROS and their contribution to a KS-like malignant phenotype, further underscoring the carcinogenic potential of oxidative stress in the context of chronic infection and inflammation. They define the RacCA transgenic mouse as a model suitable for studying the role of oxidative stress in the pathogenesis and therapy of KS, with relevance to other inflammation-related malignancies. Our findings suggest host and viral genes triggering Rac1 or ROS production as key determinants of KS onset and potential KS chemopreventive or therapeutic targets.

Fig. 1.

KS-like tumorigenesis in RacCA transgenic mice. (A) Macroscopic image of a representative tail tumor in a male RacCA^+/+ mouse from the FVB/N strain. (B) Tail KS-like tumor in a male RacCA^+/+ mouse from the C57BL/6 strain. (C) KS-like tumor on the nose of a RacCA^+/+ mouse (FVB/N). H&E staining of tumor tissues from an early-stage RacCA tumor (D) and a late-stage RacCA tumor (E). (F) Tumor-free survival of homozygous male RacCA mice.

Fig. 2.

Immunophenotype of the RacCA tumor tissue. Immunofluorescence double staining showing colocalization of Rac1– and α-SMC-actin–expressing cells with KS markers CD31, CD34, and vWF.

Fig. 3.

Global gene expression of RacCA tumors resembles KS and KSHV-induced Kaposi-like mouse tumors. (A) Heat map representation of 450 significantly up- or down-regulated genes (P < 0.05) from the 723 human KS signature genes that were present on the Affymetrix mouse 430 2.0 chip. A total of 408 genes (91%) in this 408-gene signature were equally up- or down-regulated between RacCA-mKS and KS sample groups. Control mouse and human SCC samples possess less than 10% of the KS signatures. Selected up- (red) or down-regulated (blue) genes are listed on the right. The full list of genes is available in Table S2. (B) Distribution of KS signature. Dark gray, genes shared by KS, RacCA-mKS, and KSHV-mKS; orange, genes shared by KS and RacCA-mKS; green, genes shared by KS and KSHV-mKS; yellow, genes in KS only. Gene list is available in Table S3. (C) Up-regulation of angiogenic gene expression in RacCA tumors compared with surrounding uninvolved tissue. Bars represent mean fold increase (triplicates ± SEM) in mRNA levels quantified by real-time quantitative RT-PCR.

Fig. 4.

Rac1 is overexpressed in AIDS-KS, mECK36 (KSHV-mKS), and RacCA-mKS tumors. Immunofluorescence detection of Rac1 and KSHV LANA expression in AIDS-KS tumor biopsies (A), KSHV-mKS (mECK36 tumors) (C), and RacCA tumors (D) compared with normal human skin tissue (B) and mouse skin tissue (D).

Fig. 5.

Causal role of ROS in RacCA tumorigenesis. (A) Up-regulation of AKT isoform gene expression in RacCA tumors compared with uninvolved adjacent tissues. Bars represent mean fold increase (triplicates ± SEM) in mRNA levels quantified by real-time quantitative RT-PCR. (B) AKT phosphorylation levels (as percentage of AKT phosphorylated/total AKT signal) in RacCA tumor compared with smooth muscle and skin tissue, as determined by MSD (duplicate ± SEM; see Materials and Methods). (C) Activities of immunoprecipitated PTEN from various tissues measured by release of free phosphate (see Materials and Methods) (triplicates ± SEM). (D) Lucigenin luminescence determination of (·O₂⁻) production in the tumor tissue and adjacent skin tissue compared with the tail skin tissue from WT mice skin tissue (triplicates ± SEM). (E) Effects on cell proliferation of NAC (10 mM) or H₂O₂ (1 μM and 100 μM). Bars show percentage increase in cell number after 24 h of incubation (triplicates ± SEM). (F) Dose-dependent inhibition of AKT phosphorylation in cultured RacCA tumor cells by NAC (as percentage of AKT phosphorylated/total AKT signal) determined by MSD (duplicate ± SEM). (G) Effect of NAC (20 mM) on colony-forming ability of cultured RacCA tumor cells. (H) Dose-dependent reduction of VEGF secretion by 24 h of NAC treatment in cultured RacCA tumor cells. (I) Oral administration of NAC (10 mg/mL in drinking water) prevents tumor formation in homozygous RacCA mice. (J) Western blot showing increased HIF1-α level in RacCA tumor tissue (T1, T2). (K) NAC treatment (10 mM) reduced HIF1-α level in cultured RacCA tumor cells after 24 h. (L) Three months of oral administration of NAC after tumor onset (14 months) stabilized tumor progression. Significance was established by t test: *P < 0.05; ^†P < 0.01; ^‡P < 0.001; ^§total AKT detection did not surpass background levels.