ATM inhibitor KU-55933 induces apoptosis and inhibits motility by blocking GLUT1-mediated glucose uptake in aggressive cancer cells with sustained activation of Akt.

Enhanced glucose uptake is coupled with elevated aerobic glycolysis (the Warburg effect) in cancer cells and is closely correlated with increased tumor aggressiveness and poor prognosis. We previously discovered that ATM, a protein kinase deficient in Ataxia-telangiectasia (A-T) disease, is an insulin-responsive protein that participates in insulin-mediated glucose uptake in muscle cells by stimulating glucose transporter 4 (GLUT4) translocation. However, the role of ATM in glucose uptake and tumorigenesis of cancer cells is unclear. In the present study, we found that aggressive breast and prostate cancer cell lines with overactivated Akt activity exhibit enhanced glucose uptake and GLUT1 translocation upon insulin treatment, and KU-55933, a specific inhibitor of ATM, inhibits insulin-mediated glucose uptake by blocking translocation of GLUT1 to the cell surface. KU-55933 also inhibits aerobic glycolysis and ATP production in these cells. Moreover, KU-55933 induces apoptosis and inhibits motility of cancer cells by inhibiting glucose uptake. Our results showed that while high concentration of glucose and insulin promote the expression of a mesenchymal biomarker (vimentin) in these cancer cells, KU-55933 strongly inhibits its expression as well as epithelial to mesenchymal transition. The roles of ATM in stimulating glucose uptake, glycolysis, motility, and proliferation of cancer cells were demonstrated by knocking-down ATM in these cells. KU-55933 treatment also inhibits tumor growth and metastasis in vivo in mouse mammary tumors through inhibition of GLUT1 translocation and vimentin expression. These results suggest that ATM acts as a promoter of tumorigenesis in cancer cells with overactivated Akt, and KU-55933 induces apoptosis and inhibits motility by blocking GLUT1-mediated glucose uptake and glycolysis in these cancer cells, which may lead to the use of KU-55933 and its analogs as new preventive or therapeutic agents against cancer.

Induction of the p53 Tumor Suppressor in Cancer Cells through Inhibition of Cap-Dependent Translation.

The p53 tumor suppressor plays a critical role in protecting normal cells from malignant transformation. Development of small molecules to reactivate p53 in cancer cells has been an area of intense research. We previously identified an internal ribosomal entry site (IRES) within the 5' untranslated region of p53 mRNA that mediates translation of the p53 mRNA independent of cap-dependent translation. Our results also show that in response to DNA damage, cells switch from cap-dependent translation to cap-independent translation of p53 mRNA. In the present study, we discovered a specific inhibitor of cap-dependent translation, 4EGI-1, that is capable of inducing the accumulation of p53 in cancer cells retaining wild-type p53. Our results show that 4EGI-1 causes an increase in p53 IRES activity, leading to increased translation of p53 mRNA. We also observed that 4EGI-1 induces cancer cell apoptosis in a p53-dependent manner. Furthermore, 4EGI-1 induces p53 in cancer cells without causing DNA double-strand breaks. In conclusion, we discovered a mechanistic link between inhibition of cap-dependent translation and enhanced p53 accumulation. This leads to apoptosis of cancer cells without causing collateral damage to normal cells, thus providing a novel and effective therapeutic strategy for cancer.

Targeting IRES-Mediated p53 Synthesis for Cancer Diagnosis and Therapeutics.

While translational regulation of p53 by the internal ribosome entry site (IRES) at its 5'-untranslated region following DNA damage has been widely accepted, the detailed mechanism underlying the translational control of p53 by its IRES sequence is still poorly understood. In this review, we will focus on the latest progress in identifying novel regulatory proteins of the p53 IRES and in uncovering the functional connection between defective IRES-mediated p53 translation and tumorigenesis. We will also discuss how these findings may lead to a better understanding of the process of oncogenesis and open up new avenues for cancer diagnosis and therapeutics.

Measuring relative utilization of aerobic glycolysis in breast cancer cells by positional isotopic discrimination.

The ability of cancer cells to produce lactate through aerobic glycolysis is a hallmark of cancer. In this study, we established a positional isotopic labeling and LC-MS-based method that can specifically measure the conversion of glucose to lactate in glycolysis. We show that the rate of aerobic glycolysis is closely correlated with glucose uptake and lactate production in breast cancer cells. We also found that the production of [3-(13) C]lactate is significantly elevated in metastatic breast cancer cells and in early stage metastatic mammary tumors in mice. Our findings may enable the development of a biomarker for the diagnosis of aggressive breast cancer.

Deregulation of Internal Ribosome Entry Site-Mediated p53 Translation in Cancer Cells with Defective p53 Response to DNA Damage.

Synthesis of the p53 tumor suppressor and its subsequent activation following DNA damage are critical for its protection against tumorigenesis. We previously discovered an internal ribosome entry site (IRES) at the 5' untranslated region of the p53 mRNA. However, the connection between IRES-mediated p53 translation and p53's tumor suppressive function is unknown. In this study, we identified two p53 IRES trans-acting factors, translational control protein 80 (TCP80), and RNA helicase A (RHA), which positively regulate p53 IRES activity. Overexpression of TCP80 and RHA also leads to increased expression and synthesis of p53. Furthermore, we discovered two breast cancer cell lines that retain wild-type p53 but exhibit defective p53 induction and synthesis following DNA damage. The levels of TCP80 and RHA are extremely low in both cell lines, and expression of both proteins is required to significantly increase the p53 IRES activity in these cells. Moreover, we found cancer cells transfected with a shRNA against TCP80 not only exhibit decreased expression of TCP80 and RHA but also display defective p53 induction and diminished ability to induce senescence following DNA damage. Therefore, our findings reveal a novel mechanism of p53 inactivation that links deregulation of IRES-mediated p53 translation with tumorigenesis.