Provocative finding
??This was a very provocative finding,?? Beachy said. ??It was clear that these bladder stem cells were the source of the intermediate cancers, or carcinomas in situ, that remain confined to the bladder lining. However, it was equally clear that sonic hedgehog expression must then be lost in order for those cancer cells to be able to invade surrounding tissue. We wondered whether the loss of this expression leads to increased tumor cell growth.??
In the current study, Beachy and Shin collaborated with Liao to obtain human surgical samples for study.
??With his help, we were able to obtain samples of invasive bladder cancer tissue from human patients that were not contaminated with benign cells,?? Beachy said. The researchers confirmed that, like the mice in the previous study, the human cancer tissue had lost the ability to express sonic hedgehog.
They then bred a strain of laboratory mice that were unable to produce a critical protein in the hedgehog signaling pathway called Smoothened. Mice without Smoothened developed invasive cancers much more quickly than their peers when a compound called N-butyl-N-4-hydroxybutyl nitrosamine, or BBN, was put in their drinking water. (Nitrosamines are carcinogens found in cigarette smoke; BBN is a type of nitrosamine that is specifically activated in the bladder.)
??We saw a striking effect when Smoothened was absent,?? said Beachy. ??While control mice developed invasive bladder cancer after about six months of receiving BBN, some of the mice lacking Smoothened developed fully invasive cancers as early as three months.??
Further investigation showed that sonic hedgehog signaling by cells in the bladder lining increased the levels of expression of a class of proteins called BMPs by the stromal tissue. These BMPs in turn stimulate the cells of the bladder lining to differentiate. But things go awry when sonic hedgehog expression is missing.
How cancer invades the bladder lining
??Wherever in the bladder lining that these cancer stem cells lose sonic hedgehog expression, there??s a corresponding drop in the levels of BMP signals in the stroma,?? said Beachy. ??That allows an invasion of cancer cells, which might usually be stopped in its tracks by the differentiation-inducing effects of BMP. With little or no BMP expression, that micro-invasion could persist, progress and form an invasive carcinoma that can kill you.??
The researchers wondered whether artificially activating the BMP pathway could mimic sonic hedgehog signaling and prevent the development of invasive cancers in their mouse model. They knew, from the work of co-author Spiekerkoetter that low doses of FK506 can activate the BMP signaling pathway. (Spiekerkoetter is currently testing whether FK506 treatment can help patients with pulmonary arterial hypertension, which is associated with a deficiency in BMP pathway activity.
The researchers gave laboratory mice BBN in their drinking water for four months ?? long enough to cause carcinomas in situ, but not invasive bladder cancers. They then split the mice into two groups, each of which continued to receive BBN for an additional month. One group also received a low dose of FK506 during that final month, while the other group received a placebo. They then assessed the health of the animals.
They found that none of the 10 mice that had received FK506 developed invasive bladder cancer after a total of five months exposure to BBN. In contrast, seven of nine animals that received the placebo did develop an invasive form of the disease. Beachy is now hoping to work with his collaborators to begin trials of FK506 in human patients with bladder carcinomas in situ.
??Bladder cancer has one of the highest recurrence rates of all cancers and poses many challenges in its clinical management,?? Liao said. ??These insights into the molecular mechanism of cancer progression in this clinically relevant animal model are truly exciting. As a urologist who cares for many bladder cancer patients, I feel the possibility of using an approved drug to curtail progression of early stage cancer to muscle-invasive disease, which typically requires complete removal of the bladder, could be a potential game changer.??
Other Stanford co-authors of the study are research associate Chen Zhao, former postdoctoral scholar Debashis Sahoo, PhD, and research associate Ying Pan.
The research was supported by the National Institutes of Health (grants CA160986, CA157877 and CA166232) and the Howard Hughes Medical Institute.
''Information about Stanford??s Department of Developmental Biology and Department of Urology, which also supported the work, is available at http://urology.stanford.edu andhttp://devbio.stanford.edu.
