Simultaneous Activation of Kras and Inactivation of p53 Induces Soft Tissue Sarcoma and Bladder Urothelial Hyperplasia

Xiaoping Yang,James Degregori,Elizabeth Kessler,Elizabeth Erin Genova,Yuan Li,Kendra Huber,Lih-Jen Su,Francisco G La Rosa,Jerome Schaack,Natalie J Serkova,Thomas W Flaig,Sumitra Deb

doi:10.1371/journal.pone.0074809

Abstract

The development of the Cre recombinase-controlled (Cre/LoxP) technique allows the manipulation of specific tumorigenic genes, temporarily and spatially. Our original intention of this study was to investigate the role of Kras and p53 in the development of urinary bladder cancer. First, to validate the effect of intravesical delivery on Cre recombination (Adeno-Cre), we examined activity and expression of β-galactosidase in the bladder of control ROSA transgenic mice. The results confirmed specific recombination as evidenced by β-galactosidase activity in the bladder urothelium of these mice. Then, we administered the same adenovirus into the bladder of double transgenic KrasLSLG12D/+. p53fl/fl mice. The virus solution was held in place by a distal urethral retention suture for 2 hours. To our surprise, there was a rapid development of a spindle-cell tumor with sarcoma characteristics near the suture site, within the pelvic area but outside the urinary track. Since we did not see any detectable β-galactosidase in the area outside of the bladder in the validating (control) experiment, we interpreted that this sarcoma formation was likely due to transduction by Adeno-Cre in the soft tissue of the suture site. To avoid the loss of skin integrity associated with the retention suture, we transitioned to an alternative technique without suture to retain the Adeno-Cre into the bladder cavity. Interestingly, although multiple Adeno-Cre treatments were applied, only urothelial hyperplasia but not carcinogenesis was observed in the subsequent experiments of up to 6 months. In conclusion, we observed that the simultaneous inactivation of p53 and activation of Kras induces quick formation of spindle-cell sarcoma in the soft tissues adjacent to the bladder but slow formation of urothelial hyperplasia inside the bladder. These results strongly suggest that the effect of oncogene regulation to produce either hyperplasia or carcinogenesis greatly depends on the tissue type.

Highlights

Kras, a well-known oncogene, and p53, a notable tumor suppressor gene, are two well-studied tumorigenic genes that have been associated with lung [1,2] [3], pancreas [4,5] [6] and colon cancer [7]
P53 is a nuclear phosphoprotein that plays a central role in controlling cell growth, and its mutations are commonly observed in high-grade urothelial carcinoma [8,9,10]
Adeno-Cre was instilled into the mouse urinary bladder through the urethra and X-gal staining was used to detect βgalactosidase activity, defining the Cre-recombination effect in the mouse urothelium

Summary

Introduction

A well-known oncogene, and p53, a notable tumor suppressor gene, are two well-studied tumorigenic genes that have been associated with lung [1,2] [3], pancreas [4,5] [6] and colon cancer [7]. The role of these two genes in the development of urothelial carcinoma is not well defined in invivo models. The loss of p53 in the context of simultaneously activated Hras is sufficient to promote urothelial tumorigenesis [13]. The role of Kras in urinary bladder cancer development has not been widely examined. Kras mutations may cooperate with β-catenin activation to induce urothelial cell carcinoma [18], consistent with our broad understanding of the Knudson “multiple-hit” hypothesis of tumorigenesis [19]. The mutant Kras induces neoplastic changes in a wide variety of tumor types [20], including squamous cell carcinoma of the oral cavity [21] and skin cancer [22]

Methods

Results

Conclusion