Tumors must adapt to the hypoxic environment in order to grow beyond a benign microscopic mass. In addition to transcriptional activation mediated by HIF-1a, hypoxia has also been reported to inhibit translation. The degree of translational inhibition is dependent on the duration as well as the severity of the hypoxic insult. Anoxia (<0.02% O 2 ) seems to have a more rapid and dramatic effect on translation as compared to hypoxia. We show here that prolonged hypoxia dramatically and reversibly inhibits translation in PC-3 cells. We also found that mTOR is inactivated and eIF-2a is phosphorylated during hypoxic treatment but only the eIF-2a phosphorylation correlates with the translational repression. We further used polysome analysis and microarray technology to analyze the impact of this translational repression on gene expression. We found that 33 mRNAs were refractory to this translational repression and that there was no correlation between mRNA induction and the ability to recruit ribosomes during hypoxia. We also found that ribosomal protein encoding mRNAs are more sensitive to this translational repression as compared to the majority of mRNAs. Although other reports have analyzed the effect of translation inhibition on gene expression under anoxic conditions, we believe that this is the first report in hypoxic cells. Our results show that the translational repression that occurs during hypoxia does impact gene expression in the highly transformed prostate cancer cell line, PC-3.

Prostate adenocarcinoma metastasizes to the skeleton more frequently than any other organ. An underlying cause of this phenomenon may be the ability of boneproduced factors to specifically select disseminated prostate cancer cells that are susceptible to their trophic effects. Platelet-derived growth factor (PDGF), a potent mitogen for both normal and tumor cells, is produced in several tissues including bone, where it is synthesized by both osteoblasts and osteoclasts. Here, we show that PDGF causes a significantly stronger activation of the Akt/PKB survival pathway in bone-metastatic prostate cancer cells compared to nonmetastatic cells. Normal prostate epithelial cells and DU-145 prostate cells, originally derived from a brain metastasis, are not responsive to PDGF. In contrast, epidermal growth factor stimulates Akt to the same extent in all prostate cells tested. This difference in PDGF responsiveness depends on the higher expression of a-PDGFR in bone-metastatic compared to nonmetastatic prostate cells and the lack of a-PDGFR expression in normal and metastatic prostate cells derived from tissues other than bone. Thus, a-PDGFR expression might identify prostate cancer cells with the highest propensity to metastasize to the skeleton.

In vitro and in vivo experimental studies have demonstrated the role of lysophosphatidic acid (LPA) signaling in tumor proliferation, invasiveness, and metastasis. Among LPA receptors, the overexpression of LPA receptor 3 (LPAR3) in transgenic mice has resulted in the highest rate of breast cancer metastasis. Our goal is to evaluate the LPA-producing enzyme autotaxin and LPAR3 as potential therapeutic targets in breast cancer patients. The expression of autotaxin and LPAR3 was examined by immunohistochemical analysis of 87 invasive human breast carcinomas. Carcinomas were more frequently positive for autotaxin and LPAR3 (24.4 and 43 %, respectively) compared to adjacent normal breast tissue (6.1 and 2.9 %, respectively). Increased stromal autotaxin expression was found in 16.3 % of the tumors. LPAR3 overexpression was associated with less differentiated tumors, human epidermal growth factor receptor 2 expression, and absence of progesterone receptors. The luminal type A carcinomas showed the lowest frequency of autotaxin and LPAR3 expression. Strong desmoplastic stromal reaction was more frequent among the carcinomas with autotaxin-positive tumor cells or autotaxin-positive stroma. Patients with carcinomas overexpressing LPAR3 in epithelial cells or autotaxin in stromal cells were more likely to have larger tumors, nodal involvement, and higher stage disease. Autotaxin overexpression in tumor cells also correlated with tumor size and clinical stage. Our data indicate that the increased expression of LPAR3 and autotaxin in human breast cancer is associated with tumor aggressiveness. They also suggest that LPA mediates tumor metastatic ability and peritumoral desmoplastic reaction through autocrine-paracrine mechanisms. A substantial portion of breast cancer patients might benefit from autotoxin/LPA receptor-targeted therapies.

Translation is often repressed in cell lines that are exposed to hypoxic conditions (0.5% -1.5% O 2 ) but this repression requires prolonged exposure (> 16 h). We report here that prolonged exposure to hypoxia results in the depletion of glucose from the media and that the loss of glucose correlates with the shut down in translation. Furthermore, we show that the addition of glucose or reoxygenation restores translation in hypoxic PC3 cells. This indicates that both glucose depletion and hypoxia are required for translational repression. We also show that eIF2a phosphorylation is reversed by glucose addition. Moreover, we present data that strongly indicate that eIF2a phosphorylation as well as the translational inhibition that occurs when cells are grown under conditions of glucose depletion and hypoxia is pancreatic eIF2a kinase (PERK) independent. We believe this is the first report to show that glucose depletion is required for translational repression under hypoxic conditions and that this explains why prolonged exposure to hypoxia is required for this inhibition. Since the physiological conditions that lead to tumor hypoxia would also likely lead to reduced glucose levels, understanding the interplay of glucose and hypoxia in regulating tumor metabolism will provide important information on the growth and development of solid tumors.

The insulin-like growth factor type 1 (IGF-I) plays an important role in neuronal physiology. Reduced IGF-I levels are observed during aging and this decrease may be important to age-related changes in the brain. We studied the effects of IGF-I on total protein oxidation in brain tissues and in cell cultures. Our results indicate that in frontal cortex the level of oxidized proteins is significantly reduced in transgenic mice designed to overproduce IGF-I compared with wild-type animals. The frontal cortex of IGF-I-overproducing mice exhibited high chymotrypsin-like activity of the 20S and 26S proteasomes. The proteasome can also be activated in response to IGF-I in cell cultures. Kinetic studies revealed peak activation of the proteasome within 15 min following IGF-I stimulation. The effects of IGF-I on proteasome were not observed in R -cells lacking the IGF-I receptor. Experiments using specific kinase inhibitors suggested that activation of proteasome by IGF-I involves phosphatidyl inositol 3-kinase and mammalian target of rapamycin signaling. IGF-I also attenuated the increase in protein carbonyl content induced by proteasome inhibition. Thus, appropriate levels of IGF-I may be important for the elimination of oxidized proteins in the brain in a process mediated by activation of the proteasome.