Although there is consensus that nitric oxide (NO) modulates tumor development, it is unsettled whether it inhibits or stimulates angiogenesis and metastatic spread. The cytostatic effect of NO lends support to the belief that it has antitumor action. However, conflicting data from non-comparable experimental systems leave unsettled the effect of NO on tumor progression. Hence, a comprehensive investigation of the effects of NO on tumor progression is needed. It requires an adequate model system that takes into account two coordinate events that cooperate to produce NO: (i) The activation of NOS2 gene expression, and (ii) The transport of adequate amounts of the NO substrate L-arginine. A highly useful tumor model system employs the polyoma middle T oncogenic transgene (PyV-mT). It elicits rapid, reproducible, multifocal metastatic mammary adenocarcinoma in 100 percent of females. Sustained and copious NO production occurs via the transcriptionally controlled inducible nitric oxide synthase (NOS2) enzyme. Transport of the sole substrate for NO production, L-arginine is mediated by specific transporters (CATs). When PyV-mT is expressed in either Nos2-/- and Cat2-/- mice, the models serve to define their individual roles in breast tumor progression. We postulate that the CAT2 arginine transporter plays a central role in the regulated production of NO via NOS2. The consequences of NO produced from NOS2 and arginine transport via CAT2, will be assessed by measuring the occurrence, growth, angiogenesis and metastasis of mammary adenocarcinoma using """"""""designer"""""""" bigenic and trigenic mice. The PyV-mT transgene is a surrogate for receptor tyrosine kinase (specifically cErbB2) and is an extremely oncogenic transgene. However, the cErbB2 transgene is a more indolent oncogene exhibiting slower tumor kinetics. Transplantation studies will determine whether the stimulatory effect of NO on mammary tumor formation is epithelial cell autonomous. Alternatively, transplant experiments will reveal if circulating and/or stromal tissues are responsible for the differences in tumor progression observed between Nos2-/- and Nos2+/+ mice. An analysis of the microvascular density will be used to measure angiogenesis and the extent of apoptosis will be assessed. The role of CAT2 mediated L-arginine transport on NO production will be assessed in tumor cell lines and in appropriate tumor bearing mice. The proposed experiments directly test the role of Nos2 and Cat2 genes on angiogenesis, tumor growth, and metastasis in this model. The mammary tumor progression exhibited by these model systems have important morphological and etiological similarities with human breast cancer development. Hence, the knowledge gained will be worthwhile since both the transport of arginine and the production of NO can be modulated by diet and/or arginine analogues that modify NOS2 activity or substrate flux.
