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.

The eukaryotic translation initiation factor 4E (eIF4E) is frequently overexpressed in human cancers in relation to disease progression and drives cellular transformation, tumorigenesis, and metastatic progression in experimental models. Enhanced eIF4E function results from eIF4E overexpression and/or activation of the ras and phosphatidylinositol 3-kinase/AKT pathways and selectively increases the translation of key mRNAs involved in tumor growth, angiogenesis, and cell survival. Consequently, by simultaneously and selectively reducing the expression of numerous potent growth and survival factors critical for malignancy, targeting eIF4E for inhibition may provide an attractive therapy for many different tumor types. Recent work has now shown the plausibility of therapeutically targeting eIF4E and has resulted in the advance of the first eIF4E-specific therapy to clinical trials. These studies illustrate the increased susceptibility of tumor tissues to eIF4E inhibition and support the notion that the enhanced eIF4E function common to many tumor types may represent an Achilles' heel for cancer. [Cancer Res 2008;68(3):631-4] eIF4E Function and mRNA Discrimination Eukaryotic translation initiation factor 4E (eIF4E) binds the 5 ¶,7-methylguanosine cap structure of mRNAs, delivering these mRNAs to the eIF4F translation initiation complex, which is composed of eIF4E, the scaffolding protein eIF4G, and the ATPdependent RNA helicase eIF4A. The eIF4F complex then scans through the 5 ¶ untranslated region (UTR), unwinding mRNA secondary structure to expose the translation initiation codon and enable translation (1). eIF4F complex assembly is rate limiting for translation initiation and is largely dependent on eIF4E availability. Under normal cellular conditions, eIF4F complex assembly is limited as eIF4E is sequestered from eIF4G by binding to 4E binding proteins (4EBP). Stimulation of the phosphatidylinositol 3-kinase/AKT/mTOR pathway leads to hierarchical 4EBP phosphorylation, dislodging 4EBP from eIF4E and enabling assembly of the eIF4F complex (1).Enhanced eIF4F complex formation increases the translation of all cap-dependent mRNAs and thereby increases global protein synthesis rates. However, mRNAs vary widely in their inherent ''translatability, '' largely as a function of differences in the length and structure of their 5 ¶ UTRs. Cellular mRNAs most sensitive to alterations in eIF4E availability and eIF4F complex formation (i.e., weak mRNAs) have lengthy, G+C rich, highly structured 5 ¶ UTRs that encumber efficient RNA unwinding by eIF4F and subsequently prevent ribosome loading. Consequently, these mRNAs are most sensitive to eIF4E availability and are poorly translated under normal conditions when eIF4F complex formation is limiting. In contrast, most cellular mRNAs have relatively short, unstructured 5 ¶ UTRs (e.g., h-actin) that enable efficient scanning, initiation codon recognition, and ribosome loading and translation even when eIF4F complex levels are limiting. Whereas strong mRNAs (such as h-act...

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. [Cancer Res 2009; 69(9):3866-73]

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.

Purpose: The transforming growth factor-h (TGF-h) signaling pathway has been frequently implicated in breast cancer. An intronic variant (Int7G24A) of TGF-h receptor type I (TGFBR1) is associated with kidney and bladder cancers in our recent study.We hypothesize that this germline variant may be involved in development and progression of breast cancer. Experimental Design: Case-control studies were designed from archived paraffin-embedded tissue specimens from the same geographic area with a homogenous ethnic population.We analyzed 223 patients (25 with preinvasive tumors and 198 with invasive and metastatic breast cancers) and 153 noncancer controls.The Int7G24A was identified by PCR-RFLP. Another germline deletion (TGFBR1*6A) and somatic mutations in theTGFBR1 were also analyzed by PCR and single-strand conformational polymorphism. Results: The Int7G24A allele was evident in 32% of patients with preinvasive neoplasms and 48% of patients with invasive breast cancers compared with 26% controls (P = 0.00008). In addition, 11 (5.6%) homozygous Int7G24A carriers were found in patients with invasive breast cancers, whereas only 3 (2 %) homozygous carriers were found in the control group. The TGFBR1*6A allele was not significantly associated with breast cancer patients and only one somatic mutation was found in 71breast cancers. Conclusion: These data suggest that the germline Int7G24A variant may represent a risk factor for invasive breast cancer and a marker for breast cancer progression. A separate study with alarger sample size is warranted to validate the association of the Int7G24A with human breast cancer.

TGF-␤ signaling is frequently perturbed in many human cancers, including renal cell carcinomas (RCCs) and transitional cell carcinomas (TCCs) of the bladder. Genetic alterations of the TGF-␤ type 1 receptor (TGFBR1) may contribute to these perturbations. We therefore examined variations in the TGFBR1 gene by PCR, SSCP and RFLP in carcinomas of the urinary system and in tissues from noncancer, age-matched controls. A G3 A variant 24 bp downstream of the exon/intron 7 boundary of the TGFBR1 gene (Int7G24A) was evident in patients with RCC (46.5%, n ‫؍‬ 86) and bladder and upper urinary tract TCC (49.2%, n ‫؍‬ 65) significantly more frequently than in age-matched controls (28.3%, n ‫؍‬ 113, p < 0.002 by 2 test). Moreover, 8 homozygous variant carriers were found in the cancer groups, whereas not a single homozygous variant carrier was found in the control group. The Int7G24A allele (both heterozygous G/A and homozygous A/A carriers) was associated with increased RCC incidence (OR ‫؍‬ 2.20, 95% CI 1.22-3.96) and TCC incidence (OR ‫؍‬ 2.45, 95% CI 1.89 -3.16). One somatic mutation of serine to phenylalanine at codon 57 of the TG-FBR1 gene was confirmed in an upper urinary tract TCC. In conclusion, the Int7G24A variant in the TGFBR1 gene is significantly more frequent in patients with RCC and TCC than normal age-matched controls, suggesting that it may represent a risk factor for the development of kidney and bladder carcinomas. © 2004 Wiley-Liss, Inc. Key words: TGF-␤ type 1 receptor; renal cell carcinoma; bladder transitional cell carcinoma; upper urinary tract transitional cell carcinoma; genetic variant; SSCPCancer of the urinary system is among the 5 most frequent malignancies in the United States based on recent cancer statistics. 1 More than 7% of all cancers in 2004 will be cancers of the kidney or urinary bladder. RCCs, which kill nearly 35% of afflicted patients, account for the majority of kidney neoplasms. TCC of the bladder and upper urinary tract is the fourth most frequent malignancy in men. 1 Advances in the genetic understanding of renal neoplasms have been made through successful cloning and mutational screening of the VHL, TSC and MET proto-oncogenes. However, many sporadic RCCs lack evidence of involvement of these 3 genes. Thus, other genes involved in cell growth regulation may be associated with sporadic RCC. Acquisition of TGF-␤ resistance has been suggested to be an important progression factor in human RCC and bladder TCC. 2,3 Disruption of the TGF-␤ signaling pathway has been hypothesized as the predominant mechanism through which many types of tumor cells gain a selective growth advantage. 4 We focused on the TGFBR1 gene, an important component of the TGF-␤ signaling pathway, for genetic analysis in cancers of the kidney and urinary bladder.The TGF-␤ signal is transduced by 2 transmembrane serinethreonine kinase receptors. TGF-␤ binds first to TGFBR2 homodimers, which then form heterotetramers with 2 TGFBR1 molecules. As a consequence, the TGFBR2 kinase phosphorylates and activates TGFBR1. ...

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomicsbased systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification.

Background:Melanoma is a disease that primarily arises in the skin but is a derivative of the neural crest. Eukaryotic translation initiation factor 4E (eIF4E) regulates translation of multiple malignancy-associated mRNAs and is overexpressed in many epithelial tumours. However, expression in human tumours derived from the neural crest is unknown. Here, we determined the association of eIF4E and phospho-eIF4E expression in melanocytic lesions with malignant conversion, metastatic potential and patient survival.Methods:Archived formalin-fixed, paraffin-embedded surgical specimens from 114 patients with melanocytic lesions were stained immunohistochemically for eIF4E and phospho-eIF4E and evaluated semiquantitatively. The relationship between cytoplasmic and nuclear eIF4E and phospho-eIF4E protein expression, melanocytic lesion subtype and tumour progression was determined. Kaplan–Meier survival analyses and Cox proportional hazard regression were performed.Results:Increased eIF4E and phospho-eIF4E expression was highly associated with malignancy (P<0.0001). High nuclear phospho-eIF4E was associated with synchronous or future metastasis (P=0.0059). Kaplan–Meier analyses demonstrated highly significant associations between high histoscores for cytoplasmic and nuclear phospho-eIF4E and reduced survival in all patients (P=0.0003 and 0.0009, respectively).Conclusions:Increased melanoma expression of eIF4E and phospho-eIF4E is associated with metastatic potential, reduced survival and increased risk of death.