Dazhi Yang

Wealth of opportunity - the C1 domain as a target for drug development.

The diacylglycerol-responsive C1 domains of protein kinase C and of the related classes of signaling proteins represent highly attractive targets for drug development. The signaling functions that are regulated by C1 domains are central to cellular control, thereby impacting many pathological conditions. Our understanding of the diacylglycerol signaling pathways provides great confidence in the utility of intervention in these pathways for treatment of cancer and other conditions. Multiple compounds directed at these signaling proteins, including compounds directed at the C1 domains, are currently in clinical trials, providing strong validation for these targets. Extensive understanding of the structure and function of C1 domains, coupled with detailed insights into the molecular details of ligand - C1 domain interactions, provides a solid basis for rational and semi-rational drug design. Finally, the complexity of the factors contributing to ligand - C1 domain interactions affords abundant opportunities for manipulation of selectivity; indeed, substantially selective compounds have already been identified.

RasGRP3 Contributes to Formation and Maintenance of the Prostate Cancer Phenotype

RasGRP3 mediates the activation of the Ras signaling pathway that is present in many human cancers. Here, we explored the involvement of RasGRP3 in the formation and maintenance of the prostate cancer phenotype. RasGRP3 expression was elevated in multiple human prostate tumor tissue samples and in the human androgen-independent prostate cancer cell lines PC-3 and DU 145 compared with the androgen-dependent prostate cancer cell line LNCaP. Downregulation of endogenous RasGRP3 in PC-3 and DU 145 cells reduced Ras-GTP formation, inhibited cell proliferation, impeded cell migration, and induced apoptosis. Anchorage-independent growth of the PC-3 cells and tumor formation in mouse xenografts of both cell lines were likewise inhibited. Inhibition of RasGRP3 expression reduced AKT and extracellular signal-regulated kinase 1/2 phosphorylation and sensitized the cells to killing by carboplatin. Conversely, exogenous RasGRP3 elevated Ras-GTP, stimulated proliferation, and provided resistance to phorbol 12-myristate 13-acetate-induced apoptosis in LNCaP cells. RasGRP3-overexpressing LNCaP cells displayed a markedly enhanced rate of xenograft tumor formation in both male and female mice compared with the parental line. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses, caused the cells to resume the LNCaP morphology, and suppressed growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. We conclude that RasGRP3 contributes to the malignant phenotype of the prostate cancer cells and may constitute a novel therapeutic target for human prostate cancer.

RasGRP3, a Ras activator, contributes to signaling and the tumorigenic phenotype in human melanoma

RasGRP3, an activator for H-Ras, R-Ras and Ras-associated protein-1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from hepatocyte growth factor (HGF) or epidermal growth factor (EGF) stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target.

The synthetic bryostatin analog Merle 23 dissects distinct mechanisms of bryostatin activity in the LNCaP human prostate cancer cell line

Bryostatin 1 has attracted considerable attention both as a cancer chemotherapeutic agent and for its unique activity. Although it functions, like phorbol esters, as a potent protein kinase C (PKC) activator, it paradoxically antagonizes many phorbol ester responses in cells. Because of its complex structure, little is known of its structure-function relations. Merle 23 is a synthetic derivative, differing from bryostatin 1 at only four positions. However, in U-937 human leukemia cells, Merle 23 behaves like a phorbol ester and not like bryostatin 1. Here, we characterize the behavior of Merle 23 in the human prostate cancer cell line LNCaP. In this system, bryostatin 1 and phorbol ester have contrasting activities, with the phorbol ester but not bryostatin 1 blocking cell proliferation or tumor necrosis factor alpha secretion, among other responses. We show that Merle 23 displays a highly complex pattern of activity in this system. Depending on the specific biological response or mechanistic change, it was bryostatin-like, phorbol ester-like, intermediate in its behavior, or more effective than either. The pattern of response, moreover, varied depending on the conditions. We conclude that the newly emerging bryostatin derivatives such as Merle 23 provide powerful tools to dissect subsets of bryostatin mechanism and response.

Phorbol Esters and Diacylglycerol: The PKC Activators

Protein kinase C (PKC) represents the most prominent of the families of signaling proteins integrating response to the ubiquitous lipophilic second messenger sn-1,2-diacylglycerol and to its ultrapotent analogs, the tumor-promoting phorbol esters. Response is mediated through twin conserved zinc finger structures, the C1 domains. The C1 domains function as hydrophobic switches, for which ligand binding completes a hydrophobic surface on the face of the C1 domain, driving membrane association of PKC and enzymatic activation. Since the lipid bilayer provides critical contacts for ligand binding, along with the C1 domain, membrane heterogeneity provides an important mechanism for diversity, as do the differential functions of the twin C1 domains. Consistent with such mechanistic diversity, PKC ligands can differ dramatically in biological consequences. Thus, whereas PKC ligands have provided the paradigm for tumor promoters, some PKC ligands in fact function as inhibitors of tumor promotion. Reflecting the central role of PKC in cellular signaling, PKC has emerged as a promising therapeutic target for cancer with several PKC ligands currently in clinical trials.

Abstract 4058: Diverse patterns of biological response to phorbol esters and related protein kinaseC activators in LNCaP human prostate cancer cells

All protein kinase C activators are not equivalent. Whereas phorbol 12-myristate 13-acetate (PMA) is the paradigmatic tumor promoter, bryostatin 1 or ingenol 3-angelate are in clinical trials as cancer chemotherapeutic agents. To better understand the structural features contributing to different biological outcomes, we tested a series of phorbol esters differing widely in hydrophobicity in two systems in which bryostatin 1 and PMA give very different responses. With U937 human leukemia cells, PMA inhibits cell growth and induces cell attachment, unlike bryostatin 1. We found that all the phorbol derivatives, like PMA, inhibited growth in a dose dependent manner. They likewise all induced attachment, a measure of differentiation, but several (sapintoxin D, phorbol 12, 13-dibenzoate, phorbol 12, 13-dihexanoate) differed in displaying a biphasic curve for this latter response. In the LNCaP cells, PMA inhibits growth and induces tumor necrosis factor alpha (TNF-alpha) secretion, whereas bryostatin 1 does not. We found that inhibition of growth in response to the phorbol esters ranged from full to partial (31-100 % of the PMA response) and the dose response curves ranged from monophasic to steeply biphasic. Similar divergent behavior was observed for induction of TNF-alpha secretion. For example, the secretion induced by phorbol 12, 13-dibenzoate and phorbol 12, 13-didecanoate was only 22% and 40% of the maximal response, respectively, and the response induced by sapintoxin D was very biphasic: 10 and 30 nM drug induced 56-59%, and 3000 and 10,000 nM induced only 8% and 5% of the maximal response. While none of the compounds induced as little response as did bryostatin 1, the results suggest that the difference was more quantitative in this system than qualitative. We conclude, moreover, that the hydrophobicity of the compounds was not the critical determinant of activity. Unlike the biological response, the translocation pattern of GFP-PKC delta did correlate with hydrophobicity, as the more lipophilic compounds PMA, octylindolactam V and phorbol 12, 13-didecanoate induced translocation to the plasma membrane, followed by translocation to internal membranes, while the more hydrophilic compounds translocated GFP-PKC delta mostly to the internal membranes. We thus conclude that the overall pattern of PKC delta translocation by itself cannot predict the different responses observed. Finally, Nano-pro technology, which can fingerprint the phosphorylation pattern of a protein, is revealing that the different phorbol esters induce divergent phosphorylation patterns of PKC delta, the isoform largely responsible for the phorbol ester induced apoptosis and TNF-alpha secretion in the LNCaP cells, and may provide a powerful tool for viewing the integrated outcome of the numerous elements impinging on this kinase following ligand interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4058. doi:10.1158/1538-7445.AM2011-4058

Abstract 5029: RasGRP3- a new therapeutic target in human melanoma

RasGRP3, a RasGEF (Ras guanine nucleotide exchange factor), is an activator of H-Ras, R-ras and Rap1, Here, we report that its expression level was increased in multiple human melanoma cell lines, reaching a level of protein expression approaching that of Ramos cells in the case of the SK-MEL-5 melanoma line. Likewise, RasGRP3 expression was variably elevated in human melanoma tissue samples. The importance of RasGRP3 expression for the melanoma cell lines was demonstrated by down regulating its expression, which inhibited cell proliferation and blocked both colony formation in soft agar and xenograft tumor formation in immunodeficient mice in the case of both the M14 and SK-Mel-5 cell lines. Finally, RasGRP3 was involved in downstream signaling in these cells. Suppression of RasGRP3 expression reduced both basal and HGF induced AKT phosphorylation in the M14 and SK-Mel-5 cells. Consistent with these results, we found that overexpression of RasGRP3 in human primary melanocytes changed their morphology, enhanced cell proliferation, and caused xenograft tumor formation. Suppression of the RasGRP3 overexpression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. We conclude that RasGRP3 represents a potential therapeutic target in melanoma. Even more generally, the high level of expression and functional role of RasGRP3 in melanoma emphasizes that this RasGEF may play a more general role in regulation of Ras family members than had been initially appreciated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5029.