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ENTPD5 Expression Correlates with AKT Activation in Human Cancer Cell Lines and Primary Tumor Samples PTEN mutation and AKT activation are common features for human cancer. To check whether what was observed in PTEN null MEFs is also true for human cancer cells, we screened a panel of human cancer cell lines for the expression of PTEN, activated AKT, and ENTPD5. As shown in Figure 7A, AKT activation was seen in human prostate cancer lines C42 and LNCaP cells, and in these two cell lines, elevated ENTPD5 expression was also observed. We also examined ENTPD5 expression and AKT activation in primary human tumor samples by staining two adjacent sections from a formalin-fixed, paraffin-embedded human primary prostate cancer sample with rabbit monoclonal antibodies against human ENTPD5 and phosphoAKT, respectively. The specificity of this anti-ENTPD5 antibody was verified by western blotting analysis using LNCaP cell lines with or without their ENTPD5 knocked down (Figure S6A). The staining intensity for ENTPD5 in tumor was significantly greater compared with adjacent normal tissue and was correlated with pAKT staining (Figure 7B). Out of 10 samples from patients between age 57 and 76, only one tumor sample from a 57-year-old patient and another sample collected from a patient who had just gone through therapy did not show strongENTPD5staining, and the same tumors were also negative for pAKT (Figure S6B2 and Figure S6B10). The remaining eight samples all showed greater tumor staining of pAKT and ENTPD5 (Figure 7B and Figures S6B3¨CS6B9). Because microarray data of many tumors are publicly available, we also analyzed a group of recently publicized microarray data from human prostate cancer samples (Bermudo et al., 2008). We found that ENTPD5 is highly expressed in all 20 tumor samples compared to normal prostate epithelium cells (Figure S7). In addition, after clustering all gene expression profiles from prostate tumor microarray data using SOM (self-organization method), we identified dozens of genes associated with AKT activation, including Her-3, PI3KCB, Ras, S6 kinase, CD36, IL8, EGF, osteropontin, and FoxO1, which are significantly coregulated with ENTPD5 (Figure S7). ENTPD5 Is Important for Cancer Cell Growth To verify the functional significance of ENTPD5 expression in human cancer cells, we generated a cell line from the original LNCaP cells in which an shRNA against human ENTPD5 could be induced by Dox. In these cells, knockdown of ENTPD5 by adding Dox to the culture media also lowered N-glycosylation (Figure 7C, comparing lanes 7 and 8, 9 and 10, and 11 and 12) and induced BiP expression (Figure 7C, lanes 8, 10, and 12). These phenotypes were rescued by the expression of a wildtype shRNA-resistant ENTPD5 transgene, but not by the active site mutant ENTPD5 (Figure 7C, lanes 13¨C24). Several growth factor receptors were also checked in these cell lines after Dox treatment. As shown in Figure 7D, EGFR and Her2/ErbB-2 were significantly down, and IGFRb was slightly down (Figure 7D, lanes 1¨C4). They were restored to the normal level by the expression of shRNA-resistant ENTPD5 transgene (Figure 7D, lanes 5 and 6), but not the active site mutant (Figure 7D, lanes 7 and 8). Consistently, when the cell number was measured after 4 day knockdown of ENTPD5, only about half of LNCaP cells were there, compared to a control knockdown cell line, and the defect was rescued by expression of wild-type ENTPD5 transgene, but not active site mutant (Figure 7E). To test whether knocking down ENTPD5 in LNCaP cells also has an effect on their growth in vivo, we implanted the LNCaP cells bearing a Dox-inducible shRNA targeting ENTPD5 in matrigel in nude mice. As a control, LNCaP cells with a Dox-inducible shRNA targeting GFP were also implanted. After the xenograft tumors reached the size of 500 mm3, a cohort of seven mice were fed with Dox-containing water. The level of ENTPD5 in these tumors was measured after 6 weeks. Compared with mice fed with normal water, the ENTPD5 levels in ENTPD5-targeting shRNA containing tumors from mice fed with Dox-containing water were significantly lower except in one mouse (Figure 7F). Whereas ENTPD5-targeting shRNA containing tumors in mice fed with normal water continued to grow, the tumors in mice fed with Dox-containing water shrank (Figure 7G). Amazingly, when these tumor samples were analyzed under a microscope after fixing and staining with hematoxylin and eosin, there were very few tumor cells left in the matrigel in tumors grown in Dox-fed mice, whereas in mice fed with normal water, the matrigel was filled with tumor cells (Figure 7H). The GFP shRNA-containing tumors did not respond to Dox treatment and continued to grow during the period of experiment. |
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