DNA damage response and repair data with pharmacological modulators of Tousled.

Human Tousled kinase 1 (TLK1) plays an important role in chromatin remodeling, replication, and DNA damage response and repair. TLK1 activity is immediately, but transiently, downregulated after genotoxic insult, and its recovery is important for exit from checkpoint arrest and cell survival after radiation. The data in this article compliments research presented in the paper titled, "Tousled kinase activator, gallic acid, promotes DNA repair and suppresses radiation cytotoxicity in salivary gland cells" [1]. The identification of small molecule activators and inhibitors of TLK1 provided an opportunity to pharmacologically alter the protein׳s activity to elucidate its role in DNA damage response pathways. TLK1 effectors, gallic acid (GA) and thioridazine (THD) activate and inhibit the kinase, respectively, and the data report on the impact of these compounds and the significance of TLK1 to DNA break repair and the survival of human salivary acinar cells.

Transcriptional and translational dual-regulated oncolytic herpes simplex virus type 1 for targeting prostate tumors.

The aim of this project was to demonstrate that an oncolytic herpes simplex virus type 1 (HSV-1) can replicate in a tissue- and tumor-specific fashion through both transcriptional (prostate-specific promoter, ARR(2)PB) and translational (5'-untranslated regions (5'UTRs) of rFGF-2) regulation of an essential viral gene, ICP27. We generated two recombinant viruses, ARR(2)PB-ICP27 (A27) and ARR(2)PB-5'UTR-ICP27 (AU27) and tested their efficacy and toxicity both in vitro and in vivo. The ARR(2)PB promoter caused overexpression of ICP27 gene in the presence of activated androgen receptors (ARs) and increased viral replication in prostate cells. However, this transcriptional upregulation was effectively constrained by the 5'UTR-mediated translational regulation. Mice bearing human prostate LNCaP tumors, treated with a single intravenous injection of 5 x 10(7) plaque-forming units (pfu) of AU27 virus exhibited a >85% reduction in tumor size at day 28 after viral injection. Although active viral replication was readily evident in the tumors, no viral DNA was detectable in normal organs as measured by real-time PCR analyses. In conclusion, a transcriptional and translational dual-regulated (TTDR) viral essential gene expression can increase both viral lytic activity and tumor specificity, and this provides a basis for the development of a novel tumor-specific oncolytic virus for systemic treatment of locally advanced and metastatic prostate cancers.

eIF4E-targeted suicide gene therapy in a minimal residual mouse model for metastatic soft-tissue head and neck squamous cell carcinoma improves disease-free survival.

BACKGROUND: Translation initiation factor eIF4E unwinds long 5'-untranslated regions of certain tightly regulated mRNAs and, thereby, facilitates their translation into proteins. eIF4E has been shown to be overexpressed in a majority of solid tumors, including head and neck cancers. To exploit this dysregulation, a long 5'-untranslated region was spliced upstream of a thymidine kinase (Tk) gene to enhance translation of this "suicide" gene within cells overexpressing eIF4E. We investigated the efficacy of therapy with an adenovirus incorporating this novel suicide gene (Ad-HSV-UTk) following cytoreductive tumor surgery in improving disease-free and overall survival in a mouse soft-tissue metastasis model for head and neck squamous cell carcinoma. MATERIALS AND METHODS: SCC-7 (orally-derived mouse SCCa) cells were treated with Ad-HSV-Tk, Ad-HSV-UTk, Ad-null, or saline and characterized for eIF4E and Tk levels by Western blot analysis. Cytotoxicities for cells treated with Ad-HSV-Tk, Ad-HSV-UTk, or Ad-null were quantified by MTS assay. Mice bearing SCC-7-induced tumors received cytoreduction followed by Ad-HSV-UTk + ganciclovir (GCV) or control treatment and were followed for disease-free and overall survival. RESULTS: SCC-7 cells showed uniformly high levels of eIF4E but elevated Tk for Ad-HSV-Tk- and Ad-HSV-UTk-treated cells over Ad-null-treated cells. Cytotoxicities for Ad-HSV-Tk- and Ad-HSV-UTk-treated cells were, correspondingly, observed to be 100-fold more sensitive than Ad-null-treated cells to GCV treatment. Cytoreduced mice receiving Ad-HSV-UTk + GCV treatment showed significantly longer disease-free survival (P = 0.0045) than control arm mice. CONCLUSIONS: Ad-HSV-UTk suicide gene therapy prolonged disease-free survival in a mouse minimal residual soft-tissue head and neck squamous cell carcinoma metastasis model.

Cancer-specific targeting of a conditionally replicative adenovirus using mRNA translational control.

BACKGROUND: In view of the limited success of available treatment modalities for a wide array of cancer, alternative and complementary therapeutic strategies need to be developed. Virotherapy employing conditionally replicative adenoviruses (CRAds) represents a promising targeted intervention relevant to a wide array of neoplastic diseases. Critical to the realization of an acceptable therapeutic index using virotherapy in clinical trials is the achievement of oncolytic replication in tumor cells, while avoiding non-specific replication in normal tissues. In this report, we exploited cancer-specific control of mRNA translation initiation in order to achieve enhanced replicative specificity of CRAd virotherapy agents. Heretofore, the achievement of replicative specificity of CRAd agents has been accomplished either by viral genome deletions or incorporation of tumor selective promoters. In contrast, control of mRNA translation has not been exploited for the design of tumor specific replicating viruses to date. We show herein, the utility of a novel approach that combines both transcriptional and translational regulation strategies for the key goal of replicative specificity. METHODS: We describe the construction of a CRAd with cancer specific gene transcriptional control using the CXCR4 gene promoter (TSP) and cancer specific mRNA translational control using a 5'-untranslated region (5'-UTR) element from the FGF-2 (Fibroblast Growth Factor-2) mRNA. RESULTS: Both in vitro and in vivo studies demonstrated that our CRAd agent retains anti-tumor potency. Importantly, assessment of replicative specificity using stringent tumor and non-tumor tissue slice systems demonstrated significant improvement in tumor selectivity. CONCLUSIONS: Our study addresses a conceptually new paradigm: dual targeting of transgene expression to cancer cells using both transcriptional and mRNA translational control. Our novel approach addresses the key issue of replicative specificity and can potentially be generalized to a wide array of tumor types, whereby tumor selective patterns of gene expression and mRNA translational control can be exploited.

Rat adenocarcinoma cell line infected with an adenovirus carrying a novel herpes-simplex virus-thymidine kinase suicide gene construct dies by apoptosis upon treatment with ganciclovir.

BACKGROUND: Eukaryotic initiation factor 4E (eIF4E) facilitates the translation of mRNAs with long 5' untranslated regions and thus regulates protein synthesis. This protein has been found in elevated quantities in breast, colon, and head and neck cancers. To exploit this dysfunction, the 619 base pair 5' untranslated regions of fibroblast growth factor-2 was spliced upstream of the herpes simplex virus thymidine kinase gene in an adenovirus vector (Ad-HSV-UTK), with the expectation that TK will be expressed in cells that overexpress eIF4E and, thus, render these cells susceptible to ganciclovir. In this study, we investigated the in vitro activity of this suicide gene therapy against the rat Mat BIII breast adenocarcinoma cell line, and assessed whether apoptosis was the responsible mechanism of cell killing. METHODS: Mat BIII cells were infected with Ad-HSV-UTK, and optimal multiplicity of infection was determined using green fluorescent protein tagged adenovirus. Western blot analysis was used to detect eIF4E and TK expression. Cell viability was assessed by the MTT assay. Induction of apoptosis was determined using annexin V-FITC and propidium iodine detection kit and a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling in situ cell death detection kit. RESULTS: Western Blot analysis confirmed successful infection of the cell line. Marked cytotoxicity was noted by the MTT assay in the infected group with a 100-fold less concentration of ganciclovir compared with the control groups. Annexin V-FITC/propidium iodide revealed apoptosis in infected cells following treatment with ganciclovir. CONCLUSION: Suicide gene therapy targeting the overexpression of eIF4E induces apoptosis and cell death in rat Mat BIII mammary adenocarcinoma cells.

Correlation of TLK1B in elevation and recurrence in doxorubicin-treated breast cancer patients with high eIF4E overexpression.

BACKGROUND: Tousled-like kinase 1B (TLK1B), a mammalian threonine kinase, facilitates the repair of DNA breaks. Eukaryotic initiation factor 4E (eIF4E) overexpression leads to the upregulation of TLK1B. Doxorubicin, commonly used in the adjuvant setting for breast cancer, causes DNA breaks. We hypothesized that the degree of TLK1B elevation is correlated with eIF4E overexpression and translates clinically to an increased risk for recurrence in breast cancer patients treated with doxorubicin-based adjuvant chemotherapy. STUDY DESIGN: We prospectively accrued 152 patients with stage I to III breast cancer treated with a doxorubicin-based chemotherapy in an adjuvant setting. Standardized treatment and surveillance protocols were used. eIF4E and TLK1B protein levels were quantified using Western blots, and patients were divided into tertiles based on previously reported stratification of eIF4E and TLK1B levels. Primary end points were cancer recurrence and death. Statistical analysis included Spearman's correlation, Kaplan-Meier survival analysis, log rank test, and the Cox proportional hazard model. RESULTS: The degree of TLK1B overexpression was highly correlated with the degree of eIF4E elevation (r=0.25, p=0.0025, Spearman rank correlation). Patients whose tumors were in the highest tertile for eIF4E overexpression had a higher risk for cancer recurrence and cancer death (p=0.015 and 0.049, respectively, log rank test). After adjusting for T-stage, nodal status, age, and estrogen receptor and progesterone receptor status, patients with tumors in the highest tertile of TLK1B overexpression treated with doxorubicin were 1.7-fold more likely to suffer recurrence than those in the low TLK1B group treated similarly (p=0.0078, CI, 1.17 to 2.75, Cox model). CONCLUSIONS: TLK1B overexpression was highly correlated with the level of eIF4E elevation. High TLK1B in cancer specimens was associated with a higher risk for cancer recurrence in patients treated with doxorubicin-based adjuvant chemotherapy.

Cancer-specific targeting of an adenovirus-delivered herpes simplex virus thymidine kinase suicide gene using translational control.

Two technical hurdles, gene delivery and target specificity, have hindered the development of effective cancer gene therapies. In order to circumvent the problem of tumor specificity, the suicide gene, HSV-1 thymidine kinase (HSV-Tk), was modified with a complex 5' upstream-untranslated region (5'-UTR) that limits efficient translation to cells expressing high levels of the translation initiation factor, eIF4E. Since previous studies have shown that most tumor cells express elevated levels of eIF4E, tumor-specific gene delivery was optimized by incorporation of the 5'-UTR-modified suicide gene (HSV-UTk) into an adenovirus vector (Ad-CMV-UTk). The efficacy of this novel approach of targeting suicide gene expression and limiting cytotoxicity by means of translational restriction was tested in vitro with the use of the human breast cancer cell lines (MCF-7, MDA-MB435, and ZR-75-1). As controls, normal MCF10A, HMEC, and HMSC cell lines that express relatively low levels of eIF4E were used. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantify HSV-Tk mRNA for cells infected with Ad-CMV-UTk as well as with Ad-CMV-Tk (a control adenovirus in which HSV-Tk is not regulated at the level of translation). Translation of HSV-Tk in the Ad-infected cells was measured by Western blot analysis. In addition, cytotoxicity was determined following treatment with the pro-drug ganciclovir (GCV) using an MTT viability assay. Finally, microPET imaging was used to assess cancer cell-specific expression of HSV-Tk and expression in normal tissues in vivo after intraperitoneal injection of Ad-CMV-Tk or Ad-CMV-UTk. These data collectively showed enhanced cancer cell-specific gene expression and reduced normal tissue gene expression for the Ad-HSV-UTk compared to the Ad-CMV-Tk, leading to increased cancer cell-enhanced GCV cytotoxicity. These results indicate that translational targeting of suicide gene expression in tumor cells in vitro and in vivo is effective and may provide a platform for enhanced cancer gene therapy specificity.

High eIF4E, VEGF, and microvessel density in stage I to III breast cancer.

OBJECTIVE: In a prospective trial, to determine if eIF4E overexpression in breast cancer specimens is correlated with VEGF elevation, increased tumor microvessel density (MVD) counts, and a worse clinical outcome irrespective of nodal status. SUMMARY AND BACKGROUND DATA: In vitro, the overexpression of eukaryotic initiation factor 4E (eIF4E) up-regulates the translation of mRNAs with long 5'-untranslated regions (5'-UTRs). One such gene product is the vascular endothelial growth factor (VEGF). METHODS: A total of 114 stage I to III breast cancer patients were prospectively accrued and followed with a standardized clinical surveillance protocol. Cancer specimens were quantified for eIF4E, VEGF, and MVD. Outcome endpoints were cancer recurrence and cancer-related death. RESULTS: eIF4E overexpression was found in all cancer specimens (mean +/- SD, 12.5 +/- 7.6-fold). Increasing eIF4E overexpression correlated with increasing VEGF elevation (r = 0.24, P = 0.01, Spearman's coefficient), and increasing MVD counts (r = 0.35, P < 0.0002). Patients whose tumor had high eIF4E overexpression had shorter disease-free survival (P = 0.004, log-rank test) and higher cancer-related deaths (P = 0.002) than patients whose tumors had low eIF4E overexpression. Patients with high eIF4E had a hazard ratio for cancer recurrence and cancer-related death of 1.8 and 2.1 times that of patients with low eIF4E (respectively, P = 0.009 and P = 0.002, Cox proportional hazard model). CONCLUSIONS: In breast cancer patients, increasing eIF4E overexpression in the cancer specimens correlates with higher VEGF levels and MVD counts. Patients whose tumors had high eIF4E overexpression had a worse clinical outcome, independent of nodal status. Thus, eIF4E overexpression in breast cancer appears to predict increased tumor vascularity and perhaps cancer dissemination by hematogenous means.

A prospective trial on initiation factor 4E (eIF4E) overexpression and cancer recurrence in node-positive breast cancer.

OBJECTIVE: A previous study of patients with stage I to III breast cancer showed that those patients whose tumors were in the highest tertile of eIF4E overexpression experienced a higher risk for recurrence. This study was designed to determine whether high eIF4E overexpression predicts cancer recurrence independent of nodal status by specifically targeting patients with node-positive disease. METHODS: The prospective trial was designed to accrue 168 patients with node-positive breast cancer to detect a 2.5-fold increase in risk for recurrence. eIF4E level was quantified by Western blots as x-fold elevated compared with breast tissues from noncancer patients. End points measured were disease recurrence and cancer-related death. Statistical analyses performed include survival analysis by the Kaplan-Meier method, log-rank test, and Cox proportional hazard model. RESULTS: One hundred seventy-four patients with node-positive breast cancer were accrued. All patients fulfilled study inclusion and exclusion criteria, treatment protocol, and surveillance requirements, with a compliance rate >95%. The mean eIF4E elevation was 11.0 +/- 7.0-fold (range, 1.4-34.3-fold). Based on previously published data, tertile distribution was as follow: 1) lowest tertile (<7.5-fold) = 67 patients, 2) intermediate tertile (7.5-14-fold) = 54 patients, and 3) highest tertile (>14-fold) = 53 patients. At a median follow up of 32 months, patients with the highest tertile had a statistically significant higher cancer recurrence rate (log-rank test, P = 0.002) and cancer-related death rate (P = 0.036) than the lowest group. Relative risk calculations demonstrated that high eIF4E patients had a 2.4-fold increase in relative risk increase for cancer recurrence (95% confidence interval, 1.2-4.1; P = 0.01). CONCLUSIONS: In this prospective study designed to specifically address risk for recurrence in patients with node-positive breast cancer, the patients whose tumors were in the highest tertile of eIF4E overexpression had a 2.4-fold increase in relative risk for cancer recurrence. Therefore, eIF4E overexpression appears to be an independent predictor of a worse outcome in patients with breast cancer independent of nodal status.

TLK1B is elevated with eIF4E overexpression in breast cancer.

INTRODUCTION: The overexpression of eukaryotic initiation factor 4E (eIF4E), a critical component of the "RNA helicase" necessary for the initiation of protein synthesis of mRNAs with long 5' prime untranslated regions (5'UTRs), can result in malignant transformation. In a prospective study on breast cancer outcome of women with stage I to III disease, eIF4E overexpression was an independent predictor of cancer recurrence (RR = 7.3, CI = 1.58-33.9). Dysregulation of Tousled-like kinase 1B (TLK1B), a threonine kinase with a highly conserved gene sequence, has been linked to defects in cell division and DNA replication. In cell lines, TLK1B overexpression has been recently associated with resistance to radiation. The 5'UTR of TLK1B is long (1088 nt) and the structure is complex. Our hypothesis is that TLK1B elevation is correlated with the overexpression of eIF4E in human breast carcinoma. MATERIAL AND METHODS: Eighty-seven patients with invasive breast cancer and 11 patients with benign breast disease were accrued prospectively. Clinical data collected include age, race, stage, grade of tumor, ER, and PR status. TLK1B and eIF4E levels were quantified by Western blot analysis. Statistical analysis was performed using Spearman correlation, paired and unpaired t test, and multivariate analysis. RESULTS: In the 87 cancer specimens from patients with breast carcinoma, eIF4E level was elevated by a mean of 9.5-fold (range = 1.8-48.4), and TLK1B was elevated by a mean of 9.4-fold (range = 1.0-58.0) when compared to the 11 specimens from noncancer patients. Multivariate analysis performed demonstrates the degree of eIF4E overexpression is independent of age, race, tumor grade, and ER or PR status of the tumor. Similarly, the degree of TLK1B elevation is independent of age, tumor grade, and ER or PR status of the tumor. Using the Spearman correlation, the degree of TLK1B elevation was strongly correlated with the degree of eIF4E overexpression (r = 0.39, P = 0.001). CONCLUSIONS: Both eIF4E and TLK1B are elevated in breast cancer specimens but not in benign breast specimens from noncancer patients. The degree of TLK1B elevation is correlated with the degree of IF4E overexpression. Both eIF4E and TLK1B overexpression are independent of tumor grade, tumor stage, and ER and PR status.

Prospective study of eukaryotic initiation factor 4E protein elevation and breast cancer outcome.

OBJECTIVE: To validate the authors' initial hypothesis-generating observation that eukaryotic initiation factor 4E (eIF4E) protein elevation predicts a higher cancer recurrence rate in patients with stage 1 to 3 breast cancer. SUMMARY BACKGROUND DATA: Tumor size and nodal status continue to be the two most important independent prognostic markers in breast cancer, despite well-documented limitations. In a previous smaller retrospective study, eIF4E, important in the regulation of protein synthesis of mRNAs with long or complex 5' untranslated regions, appeared promising as an independent predictor of breast cancer recurrence. METHODS: Specimens and clinical data from 191 patients with stage 1 to 3 breast cancer were accrued prospectively. Data collected include stage of disease, tumor grade, age at diagnosis, and menopausal status. Endpoints measured were disease recurrence and cancer-related death. eIF4E protein level was quantified using Western blot analysis. Immunohistochemical staining was used to determine estrogen receptor, progesterone receptor, and HER-2/neu receptor status. Statistical analysis include Cox proportional hazards model, log-rank test, Kaplan-Meier survival curve, Fisher exact test, and t test. RESULTS: Patients were divided into three groups based on tertile distribution of eIF4E: low, defined as less than 7.5-fold elevation (n = 64); intermediate, defined as 7.5- to 14-fold elevation (n = 61); and high, defined as more than 14-fold elevation (n = 66). The relative risk for cancer recurrence with intermediate elevation was 4.1 times that of patients with low elevation. For patients with high elevation, the relative risk for recurrence was higher, at 7.2 times that of the low group. The relative risk for cancer-related death for high elevation was 7.3 times that of patients with low eIF4E. Using multivariate analysis, high eIF4E remained an independent predictor of cancer recurrence after adjusting for tumor size, tumor grade, nodal disease, estrogen receptor status, progesterone receptor status, and menopausal status. CONCLUSIONS: High eIF4E is an independent predictor of cancer recurrence in patients with stage 1 to 3 breast cancer. The relative risk for cancer recurrence increases with eIF4E protein elevation. High eIF4E elevation is also associated with an increased relative risk for cancer-related death.