Anscher Figure 5. Potential targets for intervention in the TGF- 1 pathway (partial list) [17, 74, 114]. The inactive form of TGF- 1 can be activated through the action of several factors (green ovals), including ROS, proteases, integrins, and throm- bospondin-1. The active form of TGF- 1 (yellow) can then signal through either Smad-dependent (light blue) or Smad- independent pathways (orange, pink, gray). Any point along these pathways might be targeted by potential inhibitors. Abbreviations: ERK, extracellular signal–related kinase; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3 kinase; ROS, reactive oxygen species; TGF, transforming growth factor. mechanisms, lead to parenchymal cell loss, affect DNA re- pair, suppress the immune system, or cause inflammation. The tumor itself may lead to architectural destruction, re- lease its own cytokines, affect the immune system, and alter vascular permeability. The patient may have underlying medical conditions predisposing to injury (e.g., diabetes, poor underlying pulmonary function, or certain collagen vascular diseases) or have an unknown genetic susceptibil- ity. All these factors may contribute to a varying degree in any given patient, and their relative importance cannot as yet be reliably determined. Nonetheless, it is becoming clear that abnormal microenvironmental conditions exist that are sustained long after the beam is turned off, the drugs are discontinued, and the tumor is eradicated, which appear to be responsible for the perpetuation of the tissue atrophy, loss of parenchymal cells, and excessive fibrosis character- istic of late normal tissue injury after cancer therapy. There is increasing evidence that chronic normal tissue hypoxia develops after exposure to radiation therapy, and that this phenomenon may be, in part, responsible for sus- taining the normal tissue injury phenotype [94 –96]. Hyp- oxia has been shown to promote the production of profibrotic cytokines and stimulate collagen deposition through the action of TGF- 1 [97, 98]. Hypoxia has been demonstrated after radiation in the lung, central nervous system, and kidney. It is thought that hypoxia develops as a result of decreased oxygen delivery resulting from vascular injury plus greater consumption by activated macrophages recruited to the site of injury in response to reactive oxygen www.TheOncologist.com 355 species–mediated tissue damage. In the lung, evidence of sustained oxidative stress and tissue hypoxia have been demonstrated 6 months after radiation in a rat model, and this finding was correlated with greater expression and ac- tivation of TGF- 1 and its signaling pathway [95]. West- bury et al. [99] evaluated a group of patients undergoing salvage mastectomy for recurrent breast cancer following radiation therapy. The patients received an injection of the hypoxic marker pimonidazole prior to surgery. Following surgery, the normal breast tissue in the specimens was eval- uated for the presence of pimonidazole staining, and for the presence of the hypoxic marker carbonic anhydrase IX (CA IX). The authors also looked for the presence of radiation damage histologically. In the only breast sample (of 12) that showed marked histologic changes consistent with a radia- tion effect, there was greater staining for pimonidazole, but not CA IX, suggesting an oxygen tension of 10 mmHg in this tissue. This is the first in vivo evidence demonstrating hypoxia in normal irradiated human tissue using this ap- proach. Given the relationship noted above between hyp- oxia and TGF- 1, these data further support the central role of TGF- 1 in the development of chronic radiation injury. THE IMPACT OF TGF- 1 BLOCKADE ON NORMAL TISSUE INJURY The TGF- 1 interactome is extremely complex in that many proteins interact with its transmembrane receptors and signaling proteins (Smads) within the cytoplasm and the nucleus, affecting signaling crosstalk and protein tran- scription [16]. In addition, there is increasing evidence that some forms of radiation injury may develop via Smad- independent TGF- 1 signaling [100, 101]. The dominant pathway at this time seems to involve the binding of TGF- 1 to its type II transmembrane receptor. This inter- action recruits the type I transmembrane receptor to form a complex with the type II receptor. This complex is phos- phorylated and activates the signaling proteins Smad2 and Smad3. These activated Smads bind with Smad4 and are translocated to the nucleus, where they bind to promoters and modulate transcription [74]. Given the vast number of potential targets in this pathway, most work has focused on interfering with the binding of TGF- 1 to its receptors, thus preventing initiation of the multitude of potential down- stream interactions. Several approaches have been taken to prevent the binding of TGF- 1 to its receptor, including the use of anti-TGF- 1 antibodies, small molecule receptor ty- rosine kinase inhibitors, and gene therapy approaches de- signed to induce production of a soluble TGF- 1 type II receptor. Anscher et al. [96] administered a single dose of anti-TGF- 1 antibody immediately after delivering the fi- nal fraction of right hemithorax irradiation (40 Gy in five