Jing Hu - US grants

DESCRIPTION (provided by applicant): Histone deacetylase (HDAC) inhibitor trichostatin A (TSA) exerts potent anti-tumor effect. Our findings suggest that Nuclear Factor (B (NF-(B)is a primary transcription factor target of TSA. NF-kappaB is important in many aspects of tumor development and treatment. Our long-term goal is to elucidate the molecular mechanisms by which HDAC inhibitor TSA down-regulates NF-kappaB and NF-kappaB controlled target gene expression. The specific hypothesis of this proposal is that TSA-induced p100 processing and p52 acetylation plays a causal role in the regulation of NF-kappaB activity and NF-kappaB controlled cyclin D1 gene transcription. We base that hypothesis on the observations that 1) TSA promotes NF-kappaB2/p100 processing, 2) TSA enhances p52 level and acetylation, 3) Accumulation of acetylated p52 coincides with disruption of NF-kappaB DNA binding and cyclin D1 downregulation. Based on these observations, our specific aims are to: 1. Determine the role of p100 acetylation in HDAC inhibitor TSA induced NF-kappaB2/p100 processing. We will use p100 acetylation mutants to test the hypothesis that p100 processing into p52 requires acetylation at certain sites. 2. Identify the acetylation sites on p52 and ascertain their causal role in regulating NF-kappaB DNA binding upon TSA treatment. The Electrophoretic-Mobility Shift Assay (EMSA) of nuclear protein from cells transfected with p52 acetylation mutants or wild type p52 should reveal which acetylation sites are required for TSA disruption of NF-kappaB DNA binding. 3. Determine the possible causal role of p52 acetylation in TSA down regulated cyclin D1 gene expression. If p52 acetylation at certain sites contributes to the down regulation of cyclin D1 expression by TSA, then transfection of p52 mutated at those sites will fail to down regulate cyclin D1 RNA and protein expression. In addition, we will determine whether p52 acetylation affects the binding of transcription factors Sp-1, AP-1, CREB to cyclin D1 promoter using EMSA and chromatin immunoprecipitation (ChIP).

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Dietary isothiocynates (ITC), including phenethylisothiocynate (PEITC), are major bioactive components of cruciferous vegetables. ITCs may greatly account for the cancer protective effect of cruciferous vegetables on various cancers such as colon cancer. We recently demonstrated that PEITC inhibits cap-dependent translation and inhibition of protein translation is critical for PEITC-induced apoptosis. Our long-term goal is to define the molecular mechanism of ITCs' chemoprotective actions. Our specific hypothesis is that the modulation of translational regulation contributes to PEITC-mediated chemoprotective effects. In this proposed study, we will focus on determining the potential upstream signaling and downstream target of PEITC-mediated translation inhibition. We also propose to determine whether the mechanistic information obtained from in vitro studies can be extended to in vivo. More specifically, we propose to: Aim 1. Examine the signaling pathways involved in the regulation of eIF4E availability for translation initiation. We will: (A) identify the signaling events responsible for PEITC-mediated inhibition of 4E-BP1 phosphorylation; and (B) determine whether PEITC inhibits eIF4E availability through targeting Mnk1/eIF4E phosphorylation pathway. Aim 2. Analyze PEITC-mediated changes in polyribosome profile. Polysome profile will be used to: A) Evaluate overall translation rate; B) Identify potential translational target mRNAs. Aim 3. Validate the role of eIF4E availability in PEITC-caused inhibition of tumor growth. To establish in vivo relevance, we will examine whether overexpression of eIF4E overcomes PEITC-mediated inhibition of xenograft growth. Tumors will be examined to determine the extent to which PEITC-induced inhibition of translation observed in cells (Aim 1 and 2) correlates with its effect in vivo. PUBLIC HEALTH RELEVANCE The proposed studies will provide in-depth mechanistic information regarding whether and how translational regulation plays a critical role in PEITC's chemoprotective effects. A better understanding of the linkage between translational regulation and PEITC's effects on tumor growth may ultimately result in discovery of effective biomarkers to assess dietary agent cancer protective effect. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Histone deacetylase inhibitors (HDACis) are a promising new class of anticancer drugs. Two HDACis are licensed by the United States FDA for the treatment of advanced cutaneous T-cell lymphoma. Although clinical trials with HDACis as monotherapy in solid tumors have not been successful, HDACi shows promise in combination therapy for patients with recurrent metastatic lung cancer, suggesting that the full therapeutic potential of current HDACis will probably be best realized through a combination with other anticancer agents. However, currently there is little information to direct the clinical use of HDACi in combination with other agents. As colorectal cancer (CRC) remains an essentially incurable disease, it is critical to identify mechanism of HDACi resistance that can lead to combination strategies that increase HDACi therapeutic potential in CRC. The overall goal of this project is to uncover the HDAC mechanism responsible for the limited efficacy of HDACi treatment in colorectal cancer. There are 18 HDACs in humans and these different HDACs are not redundant in their function. In CRC, one of the most cancer relevant HDACs is HDAC2. We have found that in CRC cells, HDAC2 knockdown enhances the anti-tumor effect of SAHA, whereas overexpression of HDAC2 confers resistance to SAHA treatment. These findings strongly suggest that HDAC2 influences HDACi sensitivity and contributes to refractoriness to HDACi therapy in CRC cells. But the underlying mechanism by which HDAC2 works remains undefined. Clinical evidence suggests that HDACi refractoriness may involve mechanisms independent of deacetylase inhibition. Intriguingly, we have discovered that HDAC2 possesses a deacetylase-independent sumoylation-promoting activity and promotes sumoylation of Gsk3¿ (a key component of Wnt signaling). Based on our preliminary results, we hypothesize that HDAC2 sumoylation-promoting activity contributes to HDACi refractoriness in CRC cells by upregulating the Wnt/¿-catenin pathway through enhancing Gsk3¿ sumoylation. In this proposal, we will determine the role of the Wnt/¿-catenin pathway in HDAC2-mediated refractoriness in HDACi therapy. We will then validate the role of HDAC2 sumoylation-promoting activity in HDACi sensitivity of CRC cells. Finally, we will dissect how HDAC2 upregulates the Wnt/¿-catenin pathway through enhancing Gsk3¿ sumoylation. The successful completion of this proposed study could reveal a possible cause of insensitivity to HDACi treatment in CRC.

DESCRIPTION (provided by applicant): About 85% of human colorectal cancers (CRCs) are associated with inactivation mutations of the tumor suppressor adenomatous polyposis coli (APC) gene. Loss-of-function mutations are generally not drug-able as it is difficult to restore a function that is lost, but understanding the molecular consequences of APC mutation and how they mechanistically link to CRC development could suggest new intervention approaches for this disease. Mice study indicates that one such consequence is the induction of HDAC2 (histone deacetylase 2) expression. HDAC2 is also elevated in human colon cancers, suggesting that APC mutation-mediated HDAC2 induction is a common feature seen in both APCmin/+ mice and human CRC. Genetic knockout of the endogenous HDAC2 diminishes adenoma formation in APCmin/+ mice, indicating the critical role of HDAC2 in intestinal tumorigenesis and strongly suggesting that targeting HDAC2 could be a promising approach for CRC treatment. Targeted cancer therapy relies on a thorough characterization and validation of the cancer target, but how HDAC2 acts to promote intestinal tumorigenesis remains unclear. The best known feature of HDACs is their deacetylase activity. Until now, the role of HDAC2 in cancer is solely attributed to its deacetylase activity. However, we have recently revealed that HDAC2 possesses a deacetylase-independent sumoylation-promoting activity. Based on our published evidence and preliminary results that HDAC2 promotes sumoylation of substrates with pivotal roles in human cancer, we hypothesize that HDAC2 sumoylation-promoting activity contributes to intestinal tumorigenesis. In this application, we will first conduct detailed characterization of the motifs required for HDAC2 sumoylation-promoting activity; we will then validate the functional role of HDAC2 sumoylation- promoting activity in both cell culture and in mice. Completion of the study will reveal information essential for the design and development of inhibitors that target HDAC2 sumoylation activity. The results of the proposed experiments could add new insights into intestinal tumorigenesis, with implication for alternative approach to treating APC-mutated CRC.