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ecular mechanism underlying ALK mutationsmediated tumorigenesis, we selected H694R and E1384K ALK mutants for further studies because they demonstrated the highest ability to promote growth on the xenograft tumors. To confirm the results of H694R Epoxomicin and E1384K mutants obtained in H1299 cells , we repeated the studies by overexpressing H694R and E1384K in NIH3T3 cells, that is an additional cell line generally utilized to assess oncogenic property of ALK alterations in non–lung cancer genetic background . Consistent using the results on the H1299 cell model, overexpression of H694R or E1384K mutant in NIH3T3 cells considerably enhanced the kinase activity along with the downstream signaling of ALK as compared with wild type counterpart . The enhanced tyrosine kinase activity of H694R and of E1384K was further validated by in vitro kinase assay .
Moreover, we also examined the effects of H694R and E1384K mutations on protein stability and subcellular localization of ALK protein. Our results showed that wild Epoxomicin type, H694R, or E1384K mutant ALK proteins shared a PP1 half life of approximately 3. 5 hours soon after cycloheximide therapy and uniform cytoplasmic localization . Next, we examined the oncogenic effects of H694R and E1384K mutations in H1299 and NIH3T3 stable cells. In comparison with mock control, overexpression of wild type ALK only slightly enhanced proliferative activity soon after 7 days and showed a substantial boost in cell migration assay and anchorage independent growth in soft agar. In contrast, the expression of H694R or E1384K mutant ALK exhibited considerably improved oncogenic properties in all three assays compared using the wild type counterpart .
To validate the oncogenic property of H694R and E1384K mutants in vivo, H1299 cells were injected into nude mice, along with the growth curve on the xenografted tumors was measured. Once more, cells stably expressing wild type Erythropoietin ALK had slightly improved tumor PP1 volume 5 weeks soon after injection. In contrast, the tumors expressing H694R or E1384K showed a substantial upshift within the growth curve as early as 2 weeks soon after injection, along with the difference continued to expand throughout the assay period . No substantial difference within the growth curve was noted amongst the tumors with ALK mutants. To correlate the tumorigenic ability of ALK mutations with their kinase Epoxomicin activities, we performed IHC staining on sections from xenografted tumors working with antibodies against phospho Y1604 ALK, phospho STAT3, and phospho AKT.
Our results consistently showed that the ALK activity, as measured by the phosphorylated proteins of ALK, STAT3, and AKT, only marginally improved PP1 in tumors expressing wild type ALK but was considerably upregulated in H694R and E1384K mutant expressing xenografted tumors . Taken with each other, our findings illustrated that H694R and E1384K mutations led to constitutive activation of ALK activity and its downstream effectors STAT3, AKT, and ERK, which, in turn, promoted tumorigenesis with no altering ALK protein stability or subcellular localization.
H694R and E1384K Mutation Bearing Tumors Sensitive to Treatment of ALK Epoxomicin Inhibitors To investigate whether or not tiny molecule ALK inhibitor could suppress ALK mutation mediated tumorigenic properties, cells or xenografted tumors expressing wild type, H694R, or E1384K mutant ALKs were treated with WHI P154, which could repress kinase activity of ALK . The results demonstrated that WHI P154 therapy showed a dose dependent inhibition of growth in cells expressing wild type or mutant ALKs . Analytically, the half maximal cell growth inhibitory concentration of H694R and E1384K mutations were 2. 28 to 2. 86 folds reduce than that of wild type. It was concluded that cells expressing H694R or E1384K mutant ALKwere much more sensitive to inhibitory effect of WHI P154 than cells expressing wild type ALK . The effects of WHI P154 on cell migration and AIG were also examined in H1299 stable cells.
Consistently, PP1 WHI P154 treatments resulted in a profound inhibition of cell migration and AIG in H1299 expressing either wild type or mutant ALKs compared with DMSO control . Offered the stronger effects of mutant ALK than wild type ALK on the cell migration and AIG, it was no surprise that WHI P154 inhibited the mutant ALK far more than the wild type. Notably, the oncogenic effects of mutant ALK became comparable to the wild type ALK in both assays soon after WHI P154 therapy, indicating the ALK inhibitor reversed the property of mutant ALK back to the basal level. As shown in Figure 4B, WHI P154 therapy repressed phosphorylation of ALK Y1604 in a dose dependent manner, suggesting that WHI P154 inhibited the aforementioned oncogenic effects of ALK by suppressing its kinase activity. Because the WHI P154 was lately reported to be an inhibitor of JAK3/STAT3 also, to further validate the therapeutic efficacy of ALK inhibitor in mutations induced oncogenesis, a far more distinct ALK inhibitor NVP TAE684 was integrated . Similarly, TAE684 therapy efficiently inhibited the