Jeremy R. Graff

The Protein Kinase Cβ–Selective Inhibitor, Enzastaurin (LY317615.HCl), Suppresses Signaling through the AKT Pathway, Induces Apoptosis, and Suppresses Growth of Human Colon Cancer and Glioblastoma Xenografts

Activation of protein kinase Cbeta (PKCbeta) has been repeatedly implicated in tumor-induced angiogenesis. The PKCbeta-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses angiogenesis and was advanced for clinical development based upon this antiangiogenic activity. Activation of PKCbeta has now also been implicated in tumor cell proliferation, apoptosis, and tumor invasiveness. Herein, we show that Enzastaurin has a direct effect on human tumor cells, inducing apoptosis and suppressing the proliferation of cultured tumor cells. Enzastaurin treatment also suppresses the phosphorylation of GSK3betaser9, ribosomal protein S6(S240/244), and AKT(Thr308). Oral dosing with Enzastaurin to yield plasma concentrations similar to those achieved in clinical trials significantly suppresses the growth of human glioblastoma and colon carcinoma xenografts. As in cultured tumor cells, Enzastaurin treatment suppresses the phosphorylation of GSK3beta in these xenograft tumor tissues. Enzastaurin treatment also suppresses GSK3beta phosphorylation to a similar extent in peripheral blood mononuclear cells (PBMCs) from these treated mice. These data show that Enzastaurin has a direct antitumor effect and that Enzastaurin treatment suppresses GSK3beta phosphorylation in both tumor tissue and in PBMCs, suggesting that GSK3beta phosphorylation may serve as a reliable pharmacodynamic marker for Enzastaurin activity. With previously published reports, these data support the notion that Enzastaurin suppresses tumor growth through multiple mechanisms: direct suppression of tumor cell proliferation and the induction of tumor cell death coupled to the indirect effect of suppressing tumor-induced angiogenesis.

Increased AKT Activity Contributes to Prostate Cancer Progression by Dramatically Accelerating Prostate Tumor Growth and Diminishing p27Kip1 Expression

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancers, particularly in more advanced cancers, suggesting that the AKT/protein kinase B (PKB) kinase, which is negatively regulated by PTEN, may be involved in human prostate cancer progression. We now show that AKT activation and activity are markedly increased in androgen-independent, prostate-specific antigen-positive prostate cancer cells (LNAI cells) established from xenograft tumors of the androgen-dependent LNCaP cell line. These LNAI cells show increased expression of integrin-linked kinase, which is putatively responsible for AKT activation/Ser-473 phosphorylation, as well as for increased phosphorylation of the AKT target protein, BAD. Furthermore, expression of the p27Kip1 cell cycle regulator was diminished in LNAI cells, consistent with the notion that AKT directly inhibits AFX/Forkhead-mediated transcription of p27Kip1. To assess directly the impact of increased AKT activity on prostate cancer progression, an activated hAKT1 mutant was overexpressed in LNCaP cells, resulting in a 6-fold increase in xenograft tumor growth. Like LNAI cells, these transfectants showed dramatically reduced p27Kip1 expression. Together, these data implicate increased AKT activity in prostate tumor progression and androgen independence and suggest that diminished p27Kip1expression, which has been repeatedly associated with prostate cancer progression, may be a consequence of increased AKT activity.

Methylation Patterns of the E-cadherin 5′ CpG Island Are Unstable and Reflect the Dynamic, Heterogeneous Loss of E-cadherin Expression during Metastatic Progression

Metastatic progression of most common epithelial tumors involves a heterogeneous, transient loss of expression of the homotypic cell adhesion protein, E-cadherin, rather than the uniform loss of a functional protein resulting from coding region mutation. Indeed, whereas E-cadherin loss may promote invasion, reexpression may facilitate cell survival within metastatic deposits. The mechanisms underlying such plasticity are unclear. We now show that the heterogeneous loss of E-cadherin expression in primary human breast cancers reflects a heterogeneous pattern of promoter region methylation, which begins early prior to invasion. In cultured human tumor cells, such heterogeneous methylation is dynamic, varying from allele to allele and shifting in relation to the tumor microenvironment. Following invasion in vitro, which favors diminished E-cadherin expression, the density of promoter methylation markedly increased. When these cells were cultured as spheroids, which requires homotypic cell adhesion, promoter methylation decreased dramatically, and E-cadherin was reexpressed. These data show that the methylation associated with E-cadherin loss in human breast cancer is heterogeneous and unstable and suggest that such epigenetic plasticity may contribute to the dynamic, phenotypic heterogeneity that drives metastatic progression.

Mapping patterns of CpG island methylation in normal and neoplastic cells implicates both upstream and downstream regions in de novo methylation.

Promoter region CpG island methylation is associated with tumor suppressor gene silencing in neoplasia. GenBank sequence analyses revealed that a number of CpG islands are juxtaposed to multiple Alu repeats, which have been proposed as “de novo methylation centers.” These islands also contain multiple Sp1 elements located upstream and downstream of transcription start, which have been shown to protect CpG islands from methylation. We mapped the methylation patterns of the E-cadherin(E-cad) and von Hippel-Lindau (VHL) tumor suppressor gene CpG island regions in normal and neoplastic cells. Although unmethylated in normal tissue, these islands were embedded between densely methylated flanking regions containing multiple Alu repeats. These methylated flanks were segregated from the unmethylated, island CpG sites by Sp1-rich boundary regions. Finally, in human fibroblasts overexpressing DNA methyltransferase, de novomethylation of the E-cad CpG island initially involved sequences at both ends of the island and the adjacent, flanking regions and progressed with time to encompass the entire CpG island region. Together, these data suggest that boundaries exist at both ends of a CpG island to maintain the unmethylated state in normal tissue and that these boundaries may be progressively overridden, eliciting thede novo methylation associated with tumor suppressor gene silencing in neoplasia.

Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity

Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.

Hypomethylation of pericentromeric DNA in breast adenocarcinomas

Drug‐induced DNA demethylation in normal human cells and inherited localized hypomethylation in mitogen‐stimulated lymphocytes from patients with a rare recessive disease (ICF: i―mmunodeficiency, c―entromeric region instability, f―acial anomalies) are associated with karyotypic instability. This chromosomal recombination is targeted to heterochromatin in the vicinity of the centromere (pericentromeric region) of human chromosome 1. Pericentromeric rearrangements in this chromosome as well as overall genomic hypomethylation are frequently observed in many kinds of cancer, including breast adenocarcinoma. We found that almost half of 25 examined breast adenocarcinomas exhibited hypomethylation in satellite 2 DNA, which is located in the long region of heterochromatin adjacent to the centromere of chromosome 1 and is normally highly methylated. One of the 19 examined non‐malignant breast tissues displaying fibrocystic changes was similarly hypomethylated in this satellite DNA. We also looked at an opposing type of methylation alteration in these cancers, namely, hypermethylation in a tumor‐suppressor gene region that is frequently hypermethylated in breast cancers. We found that increased methylation in the E‐cadherin promoter region and decreased methylation in satellite 2 DNA were often present in the same breast cancers. While hypermethylation in certain tumor‐suppressor gene regions may favor tumorigenesis by repressing transcription, demethylation of other DNA sequences may predispose to cancer‐promoting chromosomal re‐arrangements. Int. J. Cancer 77:833–838, 1998.

Translational control and metastatic progression: enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs.

To form metastases, tumors must break from the primary tumor site, invade surrounding tissues, enter and survive within the circulation and ultimately colonize a distal tissue. Each of these steps requires the cooperative function of numerous proteins – proteins that facilitate angiogenesis (e.g., VEGF), cell survival (e.g., Bcl-2), invasion (e.g., MMPs), and autocrine growth stimulation (e.g., c-myc, cyclin D1). Although expression of these proteins is regulated at many levels by disparate stimuli, translation of these key malignancy-related proteins is regulated primarily by the activity of the mRNA cap-binding protein eIF-4E, the rate-limiting member of the eIF-4F translation initiation complex. By binding the cap structure at the 5′ terminus of cellular mRNAs, eIF-4E recruits mRNAs to the eIF-4F complex, which then scans from the 5′ cap through the untranslated region (5′UTR), unwinding secondary structure to reveal the translation initiation codon and to enable ribosome loading. Messenger RNAs with short unstructured 5′ UTRs are more easily translated than mRNAs harboring lengthy, highly structured 5′ UTRs, as these prohibit efficient scanning and start codon recognition. As such, the translation of these mRNAs, which typically encode proteins involved in angiogenesis (e.g., VEGF), tumor growth (cyclin D1) and survival (Bcl-2), is suppressed except when eIF-4E is engaged with the eIF-4F complex – a common event in many human and experimental cancers. This review focuses on the hypothesis that enhanced eIF-4E function contributes to metastatic progression by selectively upregulating the translation of key malignancy-related proteins that together conspire to drive the metastatic process.

Pak-1 Expression Increases with Progression of Colorectal Carcinomas to Metastasis

Purpose: The p21-activated kinase-1 (Pak-1) promotes cell motility and invasiveness. Pak-1 is activated by the Rac, Rho, and Cdc42 small GTPases in response to a variety of stimuli including ras and phosphatidylinositol 3′-kinase/AKT pathway activation. Because Pak-1 plays a central role in regulating cell motility and invasiveness, we sought to determine whether Pak-1 may be involved in the malignant progression of colorectal carcinoma. Experimental Design: Pak-1 expression was examined by immunohistochemistry in archived tissues from normal human colons, tubular and tubulovillous adenomas, invasive adenocarcinomas (stages I-III/IV), and lymph node metastases (184 total specimens from 38 patients). Specific cytoplasmic immunostaining was evaluated for overall intensity and uniformity to derive a combined histoscore (stain intensity × percentage of epithelium stained). Results: Pak-1 expression was increased significantly with colorectal cancer progression from normal tissue to lymph node metastases (P < 0.0001). Furthermore, Pak-1 expression was increased significantly in adenomas, invasive carcinomas, and lymph node metastases compared with normal colon (P < 0.0001). Strikingly, Pak-1 expression was significantly higher in lymph node metastases than in invasive cancers, adenomas, or normal colon (P < 0.0001). Moreover, in patients with multiple lesions representing different stages of disease, Pak-1 expression was increased specifically in the most advanced lesions. Conclusions: This study demonstrates that Pak-1 expression is increased significantly with malignant progression of human colorectal carcinoma. These data, along with numerous functional studies demonstrating a central role for Pak-1 activity in tumor invasiveness and motility, implicate Pak-1 as an exciting target for therapy of colorectal carcinoma.

eIF4E Activation Is Commonly Elevated in Advanced Human Prostate Cancers and Significantly Related to Reduced Patient Survival

Elevated eukaryotic translation initiation factor 4E (eIF4E) function induces malignancy in experimental models by selectively enhancing translation of key malignancy-related mRNAs (c-myc and BCL-2). eIF4E activation may reflect increased eIF4E expression or phosphorylation of its inhibitory binding proteins (4E-BP). By immunohistochemical analyses of 148 tissues from 89 prostate cancer patients, we now show that both eIF4E expression and 4E-BP1 phosphorylation (p4E-BP1) are increased significantly, particularly in advanced prostate cancer versus benign prostatic hyperplasia tissues. Further, increased eIF4E and p4E-BP1 levels are significantly related to reduced patient survival, whereas uniform 4E-BP1 expression is significantly related to better patient survival. Both immunohistochemistry and Western blotting reveal that elevated eIF4E and p4E-BP1 are evident in the same prostate cancer tissues. In two distinct prostate cancer cell models, the progression to androgen independence also involves increased eIF4E activation. In these prostate cancer cells, reducing eIF4E expression with an eIF4E-specific antisense oligonucleotide currently in phase I clinical trials robustly induces apoptosis, regardless of cell cycle phase, and reduces expression of the eIF4E-regulated proteins BCL-2 and c-myc. Collectively, these data implicate eIF4E activation in prostate cancer and suggest that targeting eIF4E may be attractive for prostate cancer therapy.

Therapeutic Inhibition of MAP Kinase Interacting Kinase Blocks Eukaryotic Initiation Factor 4E Phosphorylation and Suppresses Outgrowth of Experimental Lung Metastases

Activation of the translation initiation factor 4E (eIF4E) promotes malignant transformation and metastasis. Signaling through the AKT-mTOR pathway activates eIF4E by phosphorylating the inhibitory 4E binding proteins (4E-BP). This liberates eIF4E and allows binding to eIF4G. eIF4E can then be phosphorylated at serine 209 by the MAPK-interacting kinases (Mnk), which also interact with eIF4G. Although dispensable for normal development, Mnk function and eIF4E phosphorylation promote cellular proliferation and survival and are critical for malignant transformation. Accordingly, Mnk inhibition may serve as an attractive cancer therapy. We now report the identification of a potent, selective and orally bioavailable Mnk inhibitor that effectively blocks 4E phosphorylation both in vitro and in vivo. In cultured cancer cell lines, Mnk inhibitor treatment induces apoptosis and suppresses proliferation and soft agar colonization. Importantly, a single, orally administered dose of this Mnk inhibitor substantially suppresses eIF4E phosphorylation for at least 4 hours in human xenograft tumor tissue and mouse liver tissue. Moreover, oral dosing with the Mnk inhibitor significantly suppresses outgrowth of experimental B16 melanoma pulmonary metastases as well as growth of subcutaneous HCT116 colon carcinoma xenograft tumors, without affecting body weight. These findings offer the first description of a novel, orally bioavailable MNK inhibitor and the first preclinical proof-of-concept that MNK inhibition may provide a tractable cancer therapeutic approach.