Anwesha Dey

Double-edged swords as cancer therapeutics: simultaneously targeting p53 and NF-|[kappa]|B pathways

The p53 and nuclear factor-κB (NF-κB) pathways play crucial roles in human cancer, in which inactivation of p53 and hyperactivation of NF-κB is a common occurrence. Activation of p53 and inhibition of NF-κB promotes apoptosis. Although drugs are being designed to selectively activate p53 or inhibit NF-κB, there is no concerted effort yet to deliberately make drugs that can simultaneously do both. Recent results suggest that a surprising selection of small molecules have this desirable dual activity. In this Review we describe the principles behind such dual activities, describe the current candidate molecules and suggest mechanisms and approaches to their further development.

Updates on p53: modulation of p53 degradation as a therapeutic approach

The p53 pathway is aberrant in most human tumours with over 50% expressing mutant p53 proteins. The pathway is critically controlled by protein degradation. Here, we discuss the latest developments in the search for small molecules that can modulate p53 pathway protein stability and restore p53 activity for cancer therapy.

Nutlin-3 inhibits the NFκB Pathway in a p53 Dependent Manner: Implications in Lung Cancer Therapy

Nutlins were identified as the first potent and specific small molecule Mdm2 antagonists that inhibit the p53- Mdm2 interaction. We show in this study that Nutlin-3 can downregulate TNFα induced activation of the NF-κB reporter in lung cancer cells. Activation of p53 dependent transcription is not compromised when Nutlin-3 is combined with TNFα. Instead, this combination treatment decreases cell viability in a p53 dependent manner. We show that Nutlin-3 strikingly inhibits the protein expression of NF-κB target genes ICAM-1 and MCP-1 while other targets like Bcl-xL and FLIP are not affected, thereby suggesting that the inhibition is promoter specific. This inhibition of ICAM-1 and MCP-1 by Nutlin-3 is again dependent on the p53 status in cells. Furthermore, we show that Nutlin-3 strongly inhibits protein expression of ICAM-1 and MCP-1 induced by IL1, another NF-κB activating stimuli. Nutlin-3 does not inhibit Akt phosphorylation, IκBα phosphorylation, IκBα degradation, p65 modification or p65 DNA binding in the cell lines tested. This study suggests the potential of Nutlin-3 as a bitargeted anti-cancer drug by simultaneously causing p53 activation and NF-κB suppression. It also suggests that Nutlin-3 could be evaluated for treatment of lung cancer as a single agent or in combination therapy by targeting its effect on ICAM-1 and MCP-1 which are known to be critical for cancer cell invasion, thereby downregulating tumor formation and metastasis. This study also suggests biomarkers of response for evaluation of Nutlin-3 in the clinic.

Cyclin-dependent kinase inhibitors sensitize tumor cells to nutlin-induced apoptosis: a potent drug combination.

Current chemotherapy focuses on the use of genotoxic drugs that may induce general DNA damage in cancer cells but also high levels of toxicity in normal tissues. Nongenotoxic activation of p53 by targeting specific molecular pathways therefore provides an attractive therapeutic strategy in cancers with wild-type p53. Here, we explored the antitumor potential of cyclin-dependent kinase (CDK) inhibitors in combination with a small molecule inhibitor of p53-murine double minute 2 (MDM2) interaction. We show that low doses of CDK inhibitors roscovitine and DRB synergize with the MDM2 antagonist nutlin-3a in the induction of p53 activity and promote p53-dependent apoptosis in a dose- and time-dependent manner. Statistical measurement of the combination effects shows that the drug combination is additive on the reduction of cell viability and synergistic on inducing apoptosis, a critical end point of cytotoxic drugs. The degree of apoptosis observed 24 to 48 h after drug treatment correlated with the accumulation of p53 protein and concomitant induction of proapoptotic proteins Puma and PIG3. The antiproliferative and cytotoxic effects of this drug combination are validated in a range of tumor-derived cells including melanoma, colon carcinoma, breast adenocarcinoma, and hepatocarcinoma cells. Furthermore, this drug combination does not induce phosphorylation of Ser15 on p53 and does not induce genotoxic stress in the cell. Given that many cytotoxic drugs rely on their ability to induce apoptosis via DNA damage–mediated activation of p53, the data presented here may provide a new therapeutic approach for the use of CDK inhibitors and MDM2 antagonists in combinatorial drug therapy. (Mol Cancer Res 2007;5(11):1133–45)

Hexamethylene Bisacetamide (HMBA) simultaneously targets AKT and MAPK pathway and represses NF-κB activity: Implications for cancer therapy

Hexamethylene Bisacetamide (HMBA) is a hybrid polar compound originally developed as a differentiation inducing agent. We show in this study that HMBA can inhibit activation of several NF-κB target genes in both lung and breast cancer cell lines. Furthermore, consistent with its ability to inhibit NF-κB function, HMBA can also sensitize cells to apoptosis. We show that HMBA mediates inhibition of the Akt and ERK/MAPK cascade, both of which are critical for cell survival and proliferation and are well known regulators of NF-κB activation. We also show that PTEN negative breast cancer cells which have hyper activation of the PI3K/Akt pathway show increased sensitivity to growth inhibitory effects of combination of HMBA and TNFα. Furthermore, HMBA can decrease the kinase activity of the IKK complex leading to defective phosphorylation of IκBα and Ser536 of p65. This study gives mechanistic insight into the mechanism of action of HMBA, provides the rationale for the potential use of HMBA in combination with various existing kinase inhibitors in combination therapy and also suggests useful biomarkers for monitoring tumor response to HMBA.

HEXIM1 and the Control of Transcription Elongation: From Cancer and Inflammation to AIDS and Cardiac Hypertrophy

Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) is an inhibitor of positive transcription elongation factor b (P-TEFb) that has recently been shown to be involved in cancers, AIDS, cardiac hypertrophy, and inflammation. It was first cloned from vascular smooth muscle cells (VSMCs) treated with hexamethylene bis-acetamide (HMBA), a compound that suppresses the proliferation of VSMCs. Little was known about the biological function of HEXIM1 till the discovery of its association with P-TEFb. P-TEFb, a protein complex composed of cyclin-dependent kinase 9 and a cyclin partner, plays a key role in regulation of RNA polymerase II elongation. When associated with 7SK small nuclear RNA, HEXIM1 binds to P-TEFb and inhibits the kinase activity of P-TEFb. This finding provides the molecular basis for the inhibitory function of HEXIM1 in P-TEFb-dependent transcription, such as human immunodeficiency virus Tat transactivation and NF-κB-mediated transcription. Recent evidences suggest an essential role of HEXIM1 in several diseases through transcriptional regulation.

Nucleophosmin Interacts with HEXIM1 and Regulates RNA Polymerase II Transcription

Hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) was identified earlier as an inhibitor of positive transcription elongation factor b (P-TEFb), which is a key transcriptional regulator of RNA polymerase II (Pol II). Studies show that more than half of P-TEFb in cells is associated with HEXIM1, which results in the inactivation of P-TEFb. Here, we identify a nucleolar protein, nucleophosmin (NPM), as a HEXIM1-binding protein. NPM binds to HEXIM1 in vitro and in vivo, and functions as a negative regulator of HEXIM1. Over-expression of NPM leads to proteasome-mediated degradation of HEXIM1, resulting in activation of P-TEFb-dependent transcription. In contrast, an increase in HEXIM1 protein levels and a decrease in transcription are detected when NPM is knocked down. We show that a cytoplasmic mutant of NPM, NPMc+, associates with and sequesters HEXIM1 in the cytoplasm resulting in higher RNA Pol II transcription. Correspondingly, cytoplasmic localization of endogenous HEXIM1 is detected in an acute myeloid leukemia (AML) cell line containing the NPMc+ mutation, suggesting the physiological importance of HEXIM1-NPMc+ interaction. Over-expression of NPM has been detected in tumors of various histological origins and our results may provide a possible molecular mechanism for the proto-oncogenic function of NPM. Furthermore, considering that 35% of AML patients are diagnosed with NPMc+ mutation, our findings suggest that in some cases of AML, RNA Pol II transcription may be disregulated by the malfunction of NPM and the mislocation of HEXIM1.

Modulating the p53 pathway.

p53 is a transcription factor that protects cells against stress, by modulating genes that induce growth arrest, repair, apoptosis, senescence or altered metabolism. Activation of p53 can potentially be used to modulate disease states. We describe here recent developments that attempt to modulate the function of p53 and outline strategies that are being investigated for pharmacological intervention in the p53 pathway. These include interruption of the interactions between p53 and its negative regulators, restabilization of mutant/misfolded p53, activation of p53 dependant transcription and modulation of p53 function systemically to affect the therapeutic profile of existing drugs. In addition the development of new animal models to investigate the above developments, including mice and zebrafish will be highlighted.

Ubiquitination of HEXIM1 by HDM2

Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) is an inhibitor of the positive transcription elongation factor b (P-TEFb), which controls RNA polymerase II transcription and human immunodeficiency virus Tat transactivation. In cells, more than half of P-TEFb is associated with HEXIM1 resulting in the inactivation of P-TEFb. Recently, we found that nucleophosmin (NPM), a key factor involved in p53 signaling pathway, interacts with HEXIM1 and activates P-TEFb-dependent transcription. Here we report that human double minute-2 protein (HDM2), a p53-specific E3 ubiquitin ligase, specifically ubiquitinates HEXIM1 through the lysine residues located within the basic region of HEXIM1. However, the HDM2-induced HEXIM1 ubiquitination does not lead to proteasome-mediated protein degradation. Fusion of ubiquitin to HEXIM1 demonstrates stronger inhibition on P-TEFb-dependent transcription. Our results demonstrate that HDM2 functions as a specific E3 ubiquitin ligase for HEXIM1, suggesting a possible role for HEXIM1 ubiquitination in the regulation of P-TEFb activity.