Carlos Cordon-Cardo

Molecular pathways of urothelial development and bladder tumorigenesis

Bladder cancer is the fifth most common human malignancy and the second most frequently diagnosed genitourinary tumor after prostate cancer. The majority of malignant tumors arising in the urinary bladder are urothelial carcinomas. Clinically, superficial bladder tumors (stages Ta and Tis) account for 75% to 85% of neoplasms, while the remaining 15% to 25% are invasive (T1, T2-T4) or metastatic lesions at the time of initial presentation. Several studies have revealed that distinct genotypic and phenotypic patterns are associated with early vs. late stages of bladder cancer. Early superficial disease appears to segregate into 2 main pathways: (1) superficial papillary bladder tumors, which are characterized by gain-of-function mutations affecting oncogenes such as H-RAS, FGFR3, and PI3K, and deletions of the long arm of chromosome 9 (9q); (2) Carcinoma in situ, a flat high grade lesion considered to be a precursor of invasive cancer, is characterized by loss-of-function mutations affecting tumor suppressor genes, such as p53, RB, and PTEN. Based on these data, a model for bladder tumor progression has been proposed in which 2 separate genetic pathways characterize the evolution of early bladder neoplasms. Several molecular markers have been correlated with tumor stage, but the rationale for these 2 well-defined genetic pathways still remains unclear. Normal urothelium is a pseudo-stratified epithelium that coats the bladder, composed of 3 cell types: basal, intermediate, and superficial (umbrella) cells. We have identified a series of markers that are differently expressed in these distinct cells types, and postulated a novel model for urothelium development and configuration. Briefly, it is our working hypothesis that 2 distinct progenitor cells are responsible for basal/intermediate cells and umbrella cells, respectively. Basal and intermediate cells are characterized by a p63 positive phenotype, as well as expression of high molecular weight cytokeratins (CKs), such as CK5, CK10, and CK14. On the contrary, umbrella cells display a p63 negative phenotype and are characterized by expression of 2 specific low molecular weight CKs: CK18 and CK20. Neither urothelial stem cells nor bladder cancer stem cells have been identified to date. In this review, we will further expand on the issues discussed above.

Intravesical Delivery of Rapamycin Suppresses Tumorigenesis in a Mouse Model of Progressive Bladder Cancer

Early-stage bladder cancer occurs as two distinct forms: namely, low-grade superficial disease and high-grade carcinoma in situ (CIS), which is the major precursor of muscle-invasive bladder cancer. Although the low-grade form is readily treatable, few, if any, effective treatments are currently available for preventing progression of nonmuscle-invasive CIS to invasive bladder cancer. Based on our previous findings that the mammalian target of Rapamycin (mTOR) signaling pathway is activated in muscle-invasive bladder cancer, but not superficial disease, we reasoned that suppression of this pathway might block cancer progression. To test this idea, we performed in vivo preclinical studies using a genetically engineered mouse model that we now show recapitulates progression from nonmuscle-invasive CIS to muscle-invasive bladder tumors. We find that delivery of Rapamycin, an mTOR inhibitor, subsequent to the occurrence of CIS effectively prevents progression to invasive bladder cancer. Furthermore, we show that intravesical delivery of Rapamycin directly into the bladder lumen is highly effective for suppressing bladder tumorigenesis. Thus, our findings show the potential therapeutic benefit of inhibiting mTOR signaling for treatment of patients at high risk of developing invasive bladder cancer. More broadly, our findings support a more widespread use of intravesical delivery of therapeutic agents for treatment of high-risk bladder cancer patients, and provide a mouse model for effective preclinical testing of potential novel agents.

A comparison of the outcomes of neoadjuvant and adjuvant chemotherapy for clinical T2-T4aN0-N2M0 bladder cancer.

Despite evidence supporting perioperative chemotherapy, few randomized studies compare neoadjuvant and adjuvant chemotherapy for bladder cancer. Consequently, the standard of care regarding the timing of chemotherapy for locally advanced bladder cancer remains controversial. We compared patient outcomes following neoadjuvant or adjuvant systemic chemotherapy for cT2‐T4aN0‐N2M0 bladder cancer.

A BAC-based transgenic mouse specifically expresses an inducible Cre in the urothelium.

Cre-loxp mediated conditional knockout strategy has played critical roles for revealing functions of many genes essential for development, as well as the causal relationships between gene mutations and diseases in the postnatal adult mice. One key factor of this strategy is the availability of mice with tissue- or cell type-specific Cre expression. However, the success of the traditional molecular cloning approach to generate mice with tissue specific Cre expression often depends on luck. Here we provide a better alternative by using bacterial artificial chromosome (BAC)-based recombineering to insert iCreERT2 cDNA at the ATG start of the Upk2 gene. The BAC-based transgenic mice express the inducible Cre specifically in the urothelium as demonstrated by mRNA expression and staining for LacZ expression after crossing with a Rosa26 reporter mouse. Taking into consideration the size of the gene of interest and neighboring genes included in a BAC, this method should be widely applicable for generation of mice with tissue specific gene expression or deletions in a more specific manner than previously reported.

Mouse models of human bladder cancer as a tool for drug discovery.

Muscle‐invasive bladder cancer is a deadly condition in dire need of effective new treatments. This unit contains a description of mouse models suitable for the evaluation of potential new therapies. Included is a genetically engineered mouse model of bladder cancer generated by the delivery of an adenovirus expressing Cre recombinase into the bladder lumen. Also described is an orthotopic mouse model created by the instillation of human bladder tumor cells into the bladder lumen of immune deficient mice. Protocols are also provided on the use of these models for the preclinical evaluation of new chemical entities, with mTOR inhibitors shown as an example. Curr. Protoc. Pharmacol. 49:14.14.1‐14.14.18.