Small-molecule antagonists of the oncogenic Tcf/beta-catenin protein complex

Key molecular lesions in colorectal and other cancers cause beta-catenin-dependent transactivation of T cell factor (Tcf)-dependent genes. Disruption of this signal represents an opportunity for rational cancer therapy. To identify compounds that inhibit association between Tcf4 and beta-catenin, we screened libraries of natural compounds in a high-throughput assay for immunoenzymatic detection of the protein-protein interaction. Selected compounds disrupt Tcf/beta-catenin complexes in several independent in vitro assays and potently antagonize cellular effects of beta-catenin-dependent activities, including reporter gene activation, c-myc or cyclin D1 expression, cell proliferation, and duplication of the Xenopus embryonic dorsal axis. These compounds thus meet predicted criteria for disrupting Tcf/beta-catenin complexes and define a general standard to establish mechanism-based activity of small molecule inhibitors of this pathogenic protein-protein interaction.     

肿瘤的节拍化疗

节拍化疗指以肿瘤内增殖的血管内皮细胞为靶点,通过持续应用低毒性剂量的药物而抑制肿瘤血管生成的一种化疗模式。节拍化疗在头颈部肿瘤、乳腺癌、消化道肿瘤、卵巢癌和前列腺癌中的成功应用显示了其潜在价值。节拍化疗的作用机制除抗肿瘤血管生成外,还包括刺激免疫和直接抗肿瘤效应,其与其他治疗模式的联合应用尚待深入探索。     

Targeting stroma to treat cancers

All cancers depend on stroma for support of growth. Leukemias, solid tumors, cancer cells causing effusions, metastases as well as micro-disseminated cancer cells release factors that stimulate stromal cells, which in turn produce ligands that stimulate cancer cells. Therefore, elimination of stromal support by destroying the stromal cells or by inhibiting feedback stimulation of cancer growth is in the focus of many evolving therapies. A stringent evaluation of the efficacy of stromal targeting requires testing in animal models. Most current studies emphasize the successes of stromal targeting rather than deciphering its limitations. Here we show that many of the stromal targeting approaches, while often reducing tumor growth rates, are rarely curative. Therefore, we will also discuss conditions where stromal targeting can eradicate large established tumors. Finally, we will examine still unanswered questions of this promising and exciting area of cancer research.     

Evaluation of a reductively activated duocarmycin prodrug against murine and human solid cancers

In treating cancer with clinically approved chemotherapies, the high systemic toxicity and lack of selectivity for malignant cells often result in an overall poor response rate. One pharmacological approach to improve patient response is to design targeted therapies that exploit the cancer milieu by reductively activating prodrugs, which results in the selective release of the free drug in the tumor tissue. Previously, we characterized prodrugs of seco-CBI-indole₂ (CBI-indole₂) designed to be activated in hypoxic tumor microenvironments, wherein the tumor maintains higher concentrations of “reducing” nucleophiles capable of preferentially releasing the free drug by nucleophilic attack on a weak N-O bond. Of these prodrugs, BocNHO-CBI-indole₂ (BocNHO) surpassed the efficacy of the free drug, CBI-indole₂, when examined in vivo in the murine L1210 leukemia model and demonstrated reduced toxicity suggesting a targeted or sustained release in vivo. Herein, we further examine the biological activity of the BocNHO prodrug in murine breast cancer, as well as human prostate and lung cancer cell lines, in vitro. Notably, BocNHO manifests potent antiproliferative and cytotoxic activity in all three tumor cell lines. However, in comparison to the activity observed in the murine cancer cell line, the human cancer cell lines were less sensitive, especially at early timepoints for cytotoxicity. Based on these findings, BocNHO was tested in a more clinically relevant orthotopic lung tumor model, revealing significant efficacy and reduced toxicity compared with the free drug. The data suggests that this pharmacological approach to designing targeted therapies is amenable to human solid tumors.     

Targeting chemotherapy to solid tumors with long-circulating thermosensitive liposomes and local hyperthermia.

The effectiveness of the combination of long-circulating, thermosensitive liposomes and hyperthermia is described. Small-sized, thermosensitive liposomes that encapsulate doxorubicin (DXR-PEG-TSL (SUV)) have a prolonged circulation time and are extravasated to targeted solid tumors in vivo, where they preferentially release the agent in an anatomical site subjected to local hyperthermia. Liposomes were prepared by the incorporation of amphipathic polyethyleneglycol (PEG) to prolong their circulation time. DXR-PEG-TSL (SUV) was retained longest and was accumulated most efficiently in solid tumors in Balb/c mice. The combination of DXR-PEG-TSL (SUV) and hyperthermia at the tumor sites 3 h after injection, gave high concentrations of doxorubicin in tumor tissue and resulted in more effective tumor retardation and increased survival time. A large amount of DXR-PEG-TSL (SUV) was extravasated into the tumors during circulation for 3 h after injection, suggesting that the encapsulated drug was released into the interstitial spaces of the lesions by local hyperthermia. This system is expected to be clinically valuable for the delivery of a wide range of chemotherapeutic agents in the treatment of solid tumors.     

Targeting the tumor microenvironment to enhance antitumor immune responses

The identification of tumor-specific antigens and the immune responses directed against them has instigated the development of therapies to enhance antitumor immune responses. Most of these cancer immunotherapies are administered systemically rather than directly to tumors. Nonetheless, numerous studies have demonstrated that intratumoral therapy is an attractive approach, both for immunization and immunomodulation purposes. Injection, recruitment and/or activation of antigen-presenting cells in the tumor nest have been extensively studied as strategies to cross-prime immune responses. Moreover, delivery of stimulatory cytokines, blockade of inhibitory cytokines and immune checkpoint blockade have been explored to restore immunological fitness at the tumor site. These tumor-targeted therapies have the potential to induce systemic immunity without the toxicity that is often associated with systemic treatments. We review the most promising intratumoral immunotherapies, how these affect systemic antitumor immunity such that disseminated tumor cells are eliminated, and which approaches have been proven successful in animal models and patients.     

PIN1 overexpression and beta-catenin gene mutations are distinct oncogenic events in human hepatocellular carcinoma

The peptidyl-proplyl-isomerase, PIN1, upregulates beta-catenin by inhibiting its interaction with APC. beta-catenin accumulation occurs in about 70% of hepatocellular carcinoma (HCC), of which only 20% are due to beta-catenin mutations. The role of PIN1 in beta-catenin upregulation in HCC was investigated. PIN1 was shown to be overexpressed in more than 50% of HCC. All cases with PIN1 overexpression also showed beta-catenin accumulation, with 68% of cases showing concomitant beta-catenin and cyclin D1 accumulation. PIN1 was shown to contribute to beta-catenin and cyclin D1 overexpression directly by in vitro cell-line transfection experiments. Finally, we showed that PIN1 overexpression and beta-catenin gene mutations appeared to be mutually exclusive events, leading to beta-catenin accumulation in HCC. These results showed that PIN1 overexpression leading to beta-catenin accumulation might be a critical event in hepatocarcinogenesis, and that PIN1 is a potential target for therapeutic intervention in HCC.     

Targeting Bcl-2 stability to sensitize cells harboring oncogenic ras

The pro-survival factor Bcl-2 and its family members are critical determinants of the threshold of the susceptibility of cells to apoptosis. Studies are shown that cells harboring an oncogenic ras were extremely sensitive to the inhibition of protein kinase C (PKC) and Bcl-2 could antagonize this apoptotic process. However, it remains unrevealed how Bcl-2 is being regulated in this apoptotic process. In this study, we investigate the role of Bcl-2 stability in sensitizing the cells harboring oncogenic K-ras to apoptosis triggered by PKC inhibitor GO6976. We demonstrated that Bcl-2 in Swiss3T3 cells ectopically expressing or murine lung cancer LKR cells harboring K-ras rapidly underwent ubiquitin-dependent proteasome pathway after the treatment of GO6976, accompanied with induction of apoptosis. In this process, Bcl-2 formed the complex with Keap-1 and Cul3. The mutation of serine-17 and deletion of BH-2 or 4 was required for Bcl-2 ubiquitination and degradation, which elevate the signal threshold for the induction of apoptosis in the cells following PKC inhibition. Thus, Bcl-2 appears an attractive target for the induction of apoptosis by PKC inhibition in cancer cells expressing oncogenic K-ras.     

Prenylation of oncogenic human PTP protein tyrosine phosphatases

Many isoprenylated proteins are known to participate in signal transduction, but not all have been identified. Using an in vitro prenylation screen, two human cDNAs (PTP and PTP ) homologous to the rat PRL-1 and human OV-1 protein tyrosine phosphatase genes were identified. PTP and PTP were farnesylated in vitro by mammalian farnesyl:protein transferase, and epitope-tagged PTP was prenylated in epithelial cells. Overexpression of PTP and PTP in epithelial cells caused a transformed phenotype in culture and tumor growth in nude mice. Thus, PTP and PTP represent a novel class of isoprenylated, oncogenic protein tyrosine phosphatases.     

Vaccination against human cancers (review)

Classical and molecular immunological means of active tumor-specific immunization against human cancers yielded whole cell or tumor cell lysate vaccines of preventive value (reduced relapse rates) and dendritic cell-peptide or genetically engineered vaccines that may induce remissions even in metastatic disease. Active tumor-specific immunization was often successful in the past 50 years against experimental tumors maintained in the laboratory. During the epochs of classical and molecular immunology several vaccines were generated and used for the reduction of relapse rates of human cancer after surgical removal of the primary or metastatic tumors. Whole cell vaccines consist of X-irradiated autologous or allogeneic tumor cells coadministered with immunostimulants (BCG, Detox). Tumor cells haptenized biologically (as in viral oncolysates) or chemically were also used. Dendritic cell vaccines are prepared by transfection or transduction with tumor antigen-encoding DNA or by pulsing the cells with antigenic peptides in vitro; or collecting dendritic cells that engulfed apoptotic tumor cell DNA and/or peptide antigens in vivo for reinjection into the patient. Genetically engineered tumor cells are prepared in vitro to express MHC and peptides, costimulatory molecules (B7.1) and cyto- or lymphokines (interferons, interleukins, hematopoietic growth factors) for vaccination of patients. Antibody- and immune T cell-mediated immune reactions to autologous tumor cells are newly generated and/or quantitatively increased in immunized patients but do not always correlate with clinical response. Most vaccines are claimed to have reduced relapse rates presumably by inducing effective host immunity against micrometastases. Dendritic cell-peptide vaccines could induce partial or occasionally complete remissions in metastatic disease. The wrong antigenic presentation may result in tolerance induction toward the tumor; occasionally tumor enhancement may occur. Human tumor antigens when presented appropriately (with costimulatory molecules and with IL-2, IL-12) break the host's natural tolerance toward its tumor and induce rejection strength immune reactions even in patients with metastatic disease. Immune T cells thus generated could be collected for adoptive immunotherapy. For successful active specific immunization against human cancers the understanding of the immunoevasive maneuvers of the tumor cell (through FasL --> Fas; TRAIL; CD40L --> CD40; TGFbeta etc. systems) is essential.     

Targeting dendritic cells to enhance DNA vaccine potency

DNA vaccination that can induce both cellular and humoral immune responses has become an attractive immunization strategy against cancer and infection. Dendritic cells (DCs) play a critical role in the induction of immune responses by DNA vaccination. However, a major problem of DNA vaccination is its limited potency, because only a very limited fraction of injected DNA molecules are taken up by DCS: In this study, we describe a novel DNA vaccination strategy to enhance uptake and presentation of antigens by DCS: Specifically, we developed a DNA vaccine based upon expression of a model hepatitis B virus (HBV) e antigen fused to an IgG Fc fragment. After vaccination, the DNA are taken up by cells that produce and secrete the antigen-Fc fusion proteins. The secreted fusion proteins, in addition to inducing B cells, are efficiently captured and processed by DCs via receptor-mediated endocytosis and then presented to the MHC class II and as -I (cross-priming). The results of this study demonstrate that broad enhancement of antigen-specific CD4+ helper, CD8+ cytotoxic T-cell, and B-cell responses can be achieved by this DNA vaccination strategy. Thus, the strategy capable of inducing all arms of the adaptive immunity may provide a novel, generic design for the development of therapeutic and preventive DNA vaccines.     

Mutational analysis of oncogenic AKT E17K mutation in common solid cancers and acute leukaemias

Mounting evidence indicates that alterations of AKT signalling play important roles in cancer development. An earlier study discovered an oncogenic AKT1 gene mutation (AKT1 E17K) in breast, colorectal and ovarian cancers. The aim of this study was to see whether the AKT1 E17K mutation is common in breast, colorectal, lung, gastric and hepatocellular carcinomas and acute leukaemias. We analysed the presence of the AKT1 E17K mutation in 731 cancer tissues by a single-strand conformation polymorphism assay. In addition, we analysed the corresponding sequences of AKT1 E17K in AKT2 and AKT3 genes. Overall, we detected the four AKT1 E17K mutations in the breast cancers (4/93; 4.3%), but none in other cancers. There was no AKT2 or AKT3 mutation in the cancers. This study demonstrated that the AKT1 E17K mutation occurs in breast cancers at a low frequency, and that it is rare in other common cancers, including colorectal, lung, gastric and hepatocellular carcinomas and acute leukaemias. Despite the confirmed oncogenic function of the AKT1 E17K, the rare incidences of the mutation suggest that it may not play a crucial role in the development of the most common types of human cancers.     

Modulation of the oncogenic potential of beta-catenin by the subcellular distribution of plakoglobin

Plakoglobin (Pg) and beta-catenin are homologous proteins that function in cell-cell adhesion and signaling. The cadherin-associated form of these proteins mediates adhesion, whereas the cytosolic/nuclear form has a signaling role. Despite their interactions with common cellular partners, beta-catenin has a well-documented oncogenic potential while Pg has a less characterized tumor suppressor activity. We showed previously that Pg overexpression in Pg-deficient SCC9 cells (SCC9-Pg-WT) induced Bcl-2 expression and inhibited apoptosis. To assess the exact role of Pg in Bcl-2 expression, we generated and characterized SCC9 transfectants expressing Pg with a restricted cytoplasmic (Pg-NES) or nuclear (Pg-NLS) distribution. We show that Bcl-2 was expressed regardless of Pg localization, although its level was substantially lower in SCC9-Pg-NLS cells. Bcl-2 expression coincided with increased nuclear beta-catenin levels (Pg-NES) or a decrease in the level of total and nuclear beta-catenin associated with N-cadherin and alpha-catenin (Pg-WT and -NLS) cells. Bcl-2 expression also was induced in SCC9 cells overexpressing beta-catenin. In contrast, SCC9 cells expressing mutant Pg proteins, unable to interact with N-cadherin and alpha-catenin, had noticeably lower Bcl-2 levels. Our data suggest that Bcl-2 expression is induced by beta-catenin and modulated by Pg. We show that the inhibition of beta-catenin-dependent TCF transactivation had no effect on Bcl-2 levels, suggesting that induction of Bcl-2 expression by beta-catenin and its modulation by Pg may involve factors other than, or in addition, to, TCF. These results provide a possible mechanism for the tumor suppressor activity of Pg via its role as a regulator of the oncogenic potential beta-catenin.     

Targeting oncogenes to improve breast cancer chemotherapy

Despite recent advances in treatment, breast cancer remains a serious health threat for women. Traditional chemotherapies are limited by a lack of specificity for tumor cells and the cell cycle dependence of many chemotherapeutic agents. Here we report a novel strategy to help overcome these limitations. Using triplex-forming oligonucleotides (TFOs) to direct DNA damage site-specifically to oncogenes overexpressed in human breast cancer cells, we show that the effectiveness of the anticancer nucleoside analogue gemcitabine can be improved significantly. TFOs targeted to the promoter region of c-myc directly inhibited gene expression by approximately 40%. When used in combination, specific TFOs increased the incorporation of gemcitabine at the targeted site approximately 4-fold, presumably due to induction of replication-independent DNA synthesis. Cells treated with TFOs and gemcitabine in combination showed a reduction in both cell survival and capacity for anchorage-independent growth (approximately 19% of untreated cells). This combination affected the tumorigenic potential of these cancer cells to a significantly greater extent than either treatment alone. This novel strategy may be used to increase the range of effectiveness of antitumor nucleosides in any tumor which overexpresses a targetable oncogene. Multifaceted chemotherapeutic approaches such as this, coupled with triplex-directed gene targeting, may lead to more than incremental improvements in nonsurgical treatment of breast tumors.