Targeted deletion of RasGRP1 impairs skin tumorigenesis.

Ras is frequently activated in cutaneous squamous cell carcinoma, a prevalent form of skin cancer. However, the pathways that contribute to Ras-induced transformation have not been entirely elucidated. We have previously demonstrated that in transgenic mice, overexpression of the Ras activator RasGRP1 promotes the formation of spontaneous skin tumors and enhances malignant progression in the multistage carcinogenesis skin model that relies on the oncogenic activation of H-Ras. Utilizing a RasGRP1 knockout mouse model (RasGRP1 KO), we now show that lack of RasGRP1 reduced the susceptibility to skin tumorigenesis. The dependency on RasGRP1 was associated with a diminished response to the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Specifically, we found impairment of epidermal hyperplasia induced by TPA through keratinocyte proliferation. Using a keratinocyte cell line that carries a ras oncogenic mutation, we also demonstrated that RasGRP1 could further activate Ras in response to TPA. Thus, we propose that RasGRP1 upregulates signaling from Ras and contributes to epidermal tumorigenesis by increasing the total dosage of active Ras.

RasGRP1 is essential for ras activation by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in epidermal keratinocytes.

RasGRP1 is a guanine nucleotide exchange factor for Ras that binds with high affinity to diacylglycerol analogs like the phorbol esters. Recently, we demonstrated a role for RasGRP1 in skin carcinogenesis and suggested its participation in the action of tumor-promoting phorbol esters like 12-O-tetradecanoylphorbol-13-acetate (TPA) on Ras pathways in epidermal cells. Given the importance of Ras in carcinogenesis, we sought to discern whether RasGRP1 was a critical pathway in Ras activation, using a RasGRP1 knockout (KO) mouse model to examine the response of keratinocytes to TPA. In contrast to the effect seen in wild type keratinocytes, Ras(GTP) levels were barely detected in RasGRP1 KO cells even after 60 min of exposure to phorbol esters. The lack of response was rescued by enforced expression of RasGRP1. Furthermore, small hairpin RNA-induced silencing of RasGRP1 abrogated the effect of TPA on Ras. Analysis of Ras isoforms showed that both H-Ras and N-Ras depended on RasGRP1 for activation by TPA, whereas activation of K-Ras could not be detected. Although RasGRP1 was dispensable for ERK activation in response to TPA, JNK activation was reduced in the KO keratinocytes. Notably, TPA-induced phosphorylation of JNK2, but not JNK1, was reduced by RasGRP1 depletion. These data identify RasGRP1 as a critical molecule in the activation of Ras by TPA in primary mouse keratinocytes and suggest JNK2 as one of the relevant downstream targets. Given the role of TPA as a skin tumor promoter, our findings provide additional support for a role for RasGRP1 in skin carcinogenesis.

RasGRP1 transgenic mice develop cutaneous squamous cell carcinomas in response to skin wounding: potential role of granulocyte colony-stimulating factor.

Models of epidermal carcinogenesis have demonstrated that Ras is a critical molecule involved in tumor initiation and progression. Previously, we have shown that RasGRP1 increases the susceptibility of mice to skin tumorigenesis when overexpressed in the epidermis by a transgenic approach, related to its ability to activate Ras. Moreover, RasGRP1 transgenic mice develop spontaneous papillomas and cutaneous squamous cell carcinomas, some of which appear to originate in sites of injury, suggesting that RasGRP1 may be responding to signals generated during the wound-healing process. In this study, we examined the response of the RasGRP1 transgenic animals to full-thickness incision wounding of the skin, and demonstrated that they respond by developing tumors along the wounded site. The tumors did not present mutations in the H-ras gene, but Rasgrp1 transgene dosage correlated with tumor susceptibility and size. Analysis of serum cytokines showed increased levels of granulocyte colony-stimulating factor in transgenic animals after wounding. Furthermore, in vitro experiments with primary keratinocytes showed that granulocyte colony-stimulating factor stimulated Ras activation, although RasGRP1 was dispensable for this effect. Since granulocyte colony-stimulating factor has been recently associated with proliferation of skin cancer cells, our results may help in the elucidation of pathways that activate Ras in the epidermis during tumorigenesis in the absence of oncogenic ras mutations.

A 2-substituted prodiginine, 2-(p-hydroxybenzyl)prodigiosin, from Pseudoalteromonas rubra.

In the course of work aimed at the discovery of new pharmaceutical lead compounds from marine bacteria, a lipophilic extract of the bacterium Pseudoalteromonas rubra displayed significant cytotoxicity against SKOV-3, a human ovarian adenocarcinoma cell line. Bioassay-directed fractionation of this extract resulted in the isolation of a series of known and new prodiginine-type azafulvenes. The structure of the major metabolite was elucidated by interpretation of spectroscopic data as a 2-substituted prodigiosin, which we named 2-(p-hydroxybenzyl)prodigiosin (HBPG).

RasGRP1 overexpression in the epidermis of transgenic mice contributes to tumor progression during multistage skin carcinogenesis.

RasGRP1 is a guanine nucleotide exchange factor for Ras, activated in response to the second messenger diacylglycerol and its ultrapotent analogues, the phorbol esters. We have previously shown that RasGRP1 is expressed in mouse epidermal keratinocytes and that transgenic mice overexpressing RasGRP1 in the epidermis under the keratin 5 promoter (K5.RasGRP1) are prone to developing spontaneous papillomas and squamous cell carcinomas, suggesting a role for RasGRP1 in skin tumorigenesis. Here, we examined the response of the K5.RasGRP1 mice to multistage skin carcinogenesis, using 7,12-dimethylbenz(a)anthracene as carcinogen and 12-O-tetradecanoylphorbol-13-acetate (TPA) as tumor promoter. We found that whereas tumor multiplicity did not differ between transgenic and wild-type groups, the transgenic tumors were significantly larger than those observed in the wild-type mice (wild-type, 4.58 +/- 0.25 mm; transgenic, 9.83 +/- 1.05 mm). Histologic analysis further revealed that squamous cell carcinomas generated in the transgenic mice were less differentiated and more invasive than the wild-type tumors. Additionally, 30% of the transgenic mice developed tumors in the absence of initiation, suggesting that RasGRP1 overexpression could partially substitute for the initiation step induced by dimethylbenz(a)anthracene. In primary keratinocytes isolated from K5.RasGRP1 mice, TPA stimulation induced higher levels of Ras activation compared with the levels measured in the wild-type cells, indicating that constitutive overexpression of RasGRP1 in epidermal cells leads to elevated biochemical activation of endogenous Ras in response to TPA. The present data suggests that RasGRP1 participates in skin carcinogenesis via biochemical activation of endogenous wild-type Ras and predisposes to malignant progression in cooperation with Ras oncogenic signals.

Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin.

RasGRP1 is a guanine nucleotide exchange factor for Ras and a receptor of the second messenger diacylglycerol and its ultrapotent analogues, the phorbol esters. We have recently shown expression of RasGRP1 in the epidermal keratinocytes where it can mediate Ras activation in response to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, a well-known mouse skin tumor promoter. To explore the participation of RasGRP1 in skin carcinogenesis, we targeted the overexpression of RasGRP1 to basal epidermal keratinocytes using the keratin 5 promoter. These transgenic mice were viable and indistinguishable from their littermates, with normal differentiation and skin architecture. However, a percentage of the adult transgenic population developed spontaneous skin tumors, mainly squamous cell papillomas. The transgene was detected in the tumors as well as in primary keratinocytes isolated from transgenic mice. The transgenic keratinocytes also displayed elevated levels of active, GTP-loaded Ras compared with the levels observed in keratinocytes derived from wild-type littermates. We noticed a correlation between tumor incidence and wounding, which suggests that RasGRP1 overexpression may confer sensitivity to promotional stimuli, like wound repair mechanisms. Interestingly, we also found elevated levels of granulocyte colony-stimulating factor in conditioned media derived from transgenic keratinocytes subjected to in vitro wounding. Taken together, these data are the first to provide evidence of a novel role for RasGRP1 in skin carcinogenesis and suggest that RasGRP1 may participate in tumorigenesis through modulation of Ras and autocrine pathways.

The exchange factor and diacylglycerol receptor RasGRP3 interacts with dynein light chain 1 through its C-terminal domain.

RasGRP3 is an exchange factor for Ras-like small GTPases that is activated in response to the second messenger diacylglycerol. As with other diacylglycerol receptors, RasGRP3 is redistributed upon diacylglycerol or phorbol ester binding. Several factors are important in determining the pattern of translocation, including the potency of the diacylglycerol analog, the affinity of the receptor for phospholipids, and in some cases, protein-protein interactions. However, little is known about the mechanisms that play a role in RasGRP3 redistribution aside from the nature of the ligand. To discover potential protein binding partners for RasGRP3, we screened a human brain cDNA library using a yeast two-hybrid approach. We identified dynein light chain 1 as a novel RasGRP3-interacting protein. The interaction was confirmed both in vitro and in vivo and required the C-terminal domain encompassing the last 127 amino acids of RasGRP3. A truncated mutant form of RasGRP3 that lacked this C-terminal domain was unable to interact with dynein light chain 1 and displayed a dramatically altered subcellular localization, with a strong reticular distribution and perinuclear and nuclear localization. These findings suggest that dynein light chain 1 represents a novel anchoring protein for RasGRP3 that may regulate subcellular localization of the exchange factor and, as such, may participate in the signaling mediated by diacylglycerol through RasGRP3.

Differential effects of bryostatin 1 and 12-O-tetradecanoylphorbol-13-acetate on the regulation and activation of RasGRP1 in mouse epidermal keratinocytes.

The antitumor agent bryostatin 1 and the tumor-promoting phorbol esters function as structural mimetics of the second lipid messenger diacylglycerol (DAG) by binding to the C1 domain of DAG receptors. However, bryostatin 1 and the phorbol esters often differ in their cellular actions. In mouse skin, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent tumor promoter, whereas bryostatin 1 lacks this activity and antagonizes the tumor-promoting effects of TPA. Although protein kinase C mediates many of the effects of DAG on skin, the exact mechanisms responsible for the biology of bryostatin 1 and TPA in the epidermis have not been elucidated. We recently reported that the novel DAG receptor RasGRP1 is expressed in mouse keratinocytes and mediates TPA-induced Ras activation. This finding prompted us to examine the regulation of RasGRP1 by bryostatin 1. We found that whereas TPA induced translocation of RasGRP1 to both the plasma and internal membranes of the keratinocytes, bryostatin 1 recruited RasGRP1 only to internal membranes and the nuclear envelope. In addition, TPA led to a concentration-dependent down-regulation of RasGRP1, whereas bryostatin 1 failed to induce full RasGRP1 down-regulation. Interestingly, bryostatin 1 was less effective than TPA at activating Ras. The results presented here suggest the possibility that a differential modulation of RasGRP1 by bryostatin 1 compared with TPA could participate in the disparate responses of the epidermal cells to both DAG analogues. This result may have implications in the understanding of the antitumor effects of bryostatin 1 in the skin.

RasGRP1 represents a novel non-protein kinase C phorbol ester signaling pathway in mouse epidermal keratinocytes.

The mouse skin model of carcinogenesis has been instrumental in our appreciation of the multistage nature of carcinogenesis. In this system, tumor promotion is a critical step in the generation of tumors and is usually achieved by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Although it is generally assumed that protein kinase C (PKC) is the sole receptor for TPA in this system, we sought to evaluate whether non-PKC pathways could also contribute to the effects of phorbol esters in skin. We documented expression of the high affinity non-PKC phorbol ester receptor and Ras activator RasGRP1 in mouse primary keratinocytes. Overexpression of RasGRP1 in keratinocytes increased the level of active GTP-loaded Ras. TPA treatment further elevated this Ras activation in a PKC-independent manner and induced the translocation and down-regulation of RasGRP1. Overexpression of RasGRP1 in keratinocytes also caused apoptosis. Finally, induction of keratinocyte differentiation by elevation of extracellular calcium suppressed expression of endogenous RasGRP1, whereas overexpression of RasGRP1 inhibited expression of the differentiation markers keratins 1 and 10 induced by high calcium in the medium. Taken together, our results demonstrate that RasGRP1 is an additional diacylglycerol/phorbol ester receptor in epidermal keratinocytes and suggest that activation of this novel receptor may contribute to some of the phorbol ester- and Ras-mediated effects in mouse epidermis.

Modulating protein kinase C (PKC) to increase the efficacy of chemotherapy: stepping into darkness.

The identification of molecules that promote chemotherapeutic resistance would allow rationally designed approaches to abrogate this resistance, thereby possibly improving clinical outcomes for patients with cancer. In this regard, the PKC family is attractive for targeting, because it is comprised of a family of isoforms that play key roles in multiple cellular processes and can contribute to cellular transformation. Encouraging in vitro data originally showed that approaches to modulate PKC activity through small-molecule inhibitors or genetic manipulation could affect tumor cell survival. Recently, some of these approaches have begun clinical testing. Early-stage clinical trials revealed scattered clinical responses to these agents, but the most recent clinical trials have shown that combining modulators of PKC with standard chemotherapy does not improve outcome over chemotherapy alone. In this review, we will trace the development of these approaches, and discuss possible explanations for the recent negative results. Importantly, we will suggest guidelines for the clinical evaluation of PKC modulators.

Structural basis of RasGRP binding to high-affinity PKC ligands.

The Ras guanyl releasing protein RasGRP belongs to the CDC25 class of guanyl nucleotide exchange factors that regulate Ras-related GTPases. These GTPases serve as switches for the propagation and divergence of signaling pathways. One interesting feature of RasGRP is the presence of a C-terminal C1 domain, which has high homology to the PKC C1 domain and binds to diacylglycerol (DAG) and phorbol esters. RasGRP thus represents a novel, non-kinase phorbol ester receptor. In this paper, we investigate the binding of indolactam(V) (ILV), 7-(n-octyl)-ILV, 8-(1-decynyl)benzolactam(V) (benzolactam), and 7-methoxy-8-(1-decynyl)benzolactam(V) (methoxylated benzolactam) to RasGRP through both experimental binding assays and molecular modeling studies. The binding affinities of these lactams to RasGRP are within the nanomolar range. Homology modeling was used to model the structure of the RasGRP C1 domain (C1-RasGRP), which was subsequently used to model the structures of C1-RasGRP in complex with these ligands and phorbol 13-acetate using a computational docking method. The structural model of C1-RasGRP exhibits a folding pattern that is nearly identical to that of C1b-PKCdelta and is comprised of three antiparallel-strand beta-sheets capped against a C-terminal alpha-helix. Two loops A and B comprising residues 8-12 and 21-27 form a binding pocket that has some positive charge character. The ligands phorbol 13-acetate, benzolactam, and ILV are recognized by C1-RasGRP through a number of hydrogen bonds with loops A and B. In the models of C1-RasGRP in complex with phorbol 13-acetate, benzolactam, and ILV, common hydrogen bonds are formed with two residues Thr12 and Leu21, whereas other hydrogen bond interactions are unique for each ligand. Furthermore, our modeling results suggest that the shallower insertion of ligands into the binding pocket of C1-RasGRP compared to C1b-PKCdelta may be due to the presence of Phe rather than Leu at position 20 in C1-RasGRP. Taken together, our experimental and modeling studies provide us with a better understanding of the structural basis of the binding of PKC ligands to the novel phorbol ester receptor RasGRP.