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The peroxisome proliferator-activated receptor (PPAR) is one member of the nuclear receptor superfamily that contains in excess of 80 described receptors. PPAR activators are a diverse group of agents that range from endogenous fatty acids or derivatives (linolenic, linoleic, and 15-deoxy-^12,14-prostaglandin J₂) to Food and Drug Administration-approved thiazolidinedione drugs [pioglitazone (Actos) and rosiglitazone (Avandia)] for the treatment of diabetes. Once activated, PPAR will preferentially bind with retinoid X receptor and signal antiproliferative, antiangiogenic, and prodifferentiation pathways in several tissue types, thus making it a highly useful target for down-regulation of carcinogenesis. Although PPAR- activators show many anticancer effects on cell lines, their advancement into human advanced cancer clinical trials has met with limited success. This article will review translational findings in PPAR activation and targeting in carcinogenesis prevention as they relate to the potential use of PPAR activators clinically as cancer chemoprevention strategies.

Heat shock protein 90 (HSP90) is a ubiquitously expressed chaperone that is involved in the posttranslational folding and stability of proteins. Inhibition at the NH₂-terminal ATP-binding site leads to the degradation of client proteins by the ubiquitin proteasome pathway. Inhibition of HSP90 leads to the degradation of known oncogenes, such as ERB-B2, BRAF, and BCR-ABL, leading to the combinatorial blockade of multiple signal transduction pathways, such as the RAS-RAF-mitogen-activated protein/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. Multiple structurally diverse HSP90 inhibitors are undergoing early clinical evaluation. The clinical focus of these drugs should be solid tumors, such as breast, prostate, and lung cancers, along with malignant melanoma, in addition to hematologic malignancies, such as chronic myeloid leukemia and multiple myeloma. HSP90 inhibitors can be used as single agents or in combination with other targeted treatments or conventional forms of treatment such as chemotherapy and radiotherapy. Clinical trials evaluating efficacy of these agents should include innovative designs to capture cytostasis evidenced by clinical nonprogression and enrichment of patient populations by molecular characterization. The results of clinical trials evaluating the efficacy of drugs targeting this exciting target are awaited.

In October 2006 the Food and Drug Administration recommended an update in the tamoxifen label to reflect the increased risk of recurrence in breast cancer patients who are cytochrome P450 2D6 (CYP2D6) poor metabolizers. This recommendation was based on only a few studies at that time. More clinical studies addressing the relation between the CYP2D6 genotype and tamoxifen efficacy have been published since, mostly describing Caucasian populations in the adjuvant treatment setting. An updated analysis of the literature is presented. Furthermore, the possibility to implement CYP2D6 genotyping in clinical practice is evaluated by analyzing the results of six studies on mainly Caucasian patients using adjuvant tamoxifen. Three studies were consistent with the FDA advice, but the three other studies showed contradictory results. Although some of the published criticism on the negative studies is justified, this does not imply that these results should be discarded. The reviewed literature is put in perspective acknowledging the limiting effect of Mendelian randomization on confounding and the limitations of the various study designs. The current accumulation of data showing worse clinical outcome in patients with decreased CYP2D6 metabolism in other types of populations still indicates that the CYP2D6 genotype may well become a clinically relevant predictive marker. The CYP2D6 genotype might be one of the first predictors of therapeutic response in cancer care based on germline DNA creating the possibility to analyze blood instead of tumor.

Purpose: S100 proteins are implicated in metastasis development in several cancers. In this study, we analyzed the prognostic role of mRNA levels of all S100 proteins in early stage non–small cell lung cancer (NSCLC) patients as well as the pathogenetic of S100A2 in the development of metastasis in NSCLC.

Experimental Design: Microarray data from a large NSCLC patient cohort was analyzed for the prognostic role of S100 proteins for survival in surgically resected NSCLC. Metastatic potential of the S100A2 gene was analyzed in vitro and in a lung cancer mouse model in vivo. Overexpression and RNAi approaches were used for analysis of the biological functions of S100A2.

Results: High mRNA expression levels of several S100 proteins and especially S100A2 were associated with poor survival in surgically resected NSCLC patients. Upon stable transfection into NSCLC cell lines, S100A2 did not alter proliferation. However, S100A2 enhanced transwell migration as well as transendothelial migration in vitro. NOD/SCID mice injected s.c. with NSCLC cells overexpressing S100A2 developed significantly more distant metastasis (64%) than mice with control vector transfected tumor cells (17%; P < 0.05). When mice with S100A2 expressing tumors were treated i.v. with shRNA against S100A2, these mice developed significantly fewer lung metastasis than mice treated with control shRNA (P = 0.021).

Conclusions: These findings identify S100A2 as a strong metastasis inducer in vivo. S100A2 might be a potential biomarker as well as a novel therapeutic target in NSCLC metastasis.

Purpose: Peripheral T-cell lymphoma, unspecified (PTCL-U) comprises histopathologically and clinically heterogeneous groups. The purpose of this study was to identify subgroups with distinct genetic, histopathologic, and prognostic features.

Experimental Design: We used array comparative genomic hybridization (CGH) for high-resolution analysis of 51 PTCL-U patients and the array data for examining possible correlations of histopathologic and clinical features. Moreover, we compared the genetic, histopathologic, and prognostic features of the PTCL-U cases with those of 59 cases of lymphoma-type adult T-cell leukemia/lymphoma (ATLL).

Results: We identified 32 regions with frequent genomic imbalance, 1 region with high copy number gain at 14q32.2, and 1 region with homozygous loss at 9p21.3. Gains of 7p and 7q and loss of 9p21.3 showed a significant association with poor prognosis. PTCL-U cases with genomic imbalance showed distinct histopathologic and prognostic features compared with such cases without alteration and a marked genetic, histopathologic, and prognostic resemblance to lymphoma-type ATLL.

Conclusions: The array CGH enabled us to identify the frequently altered genomic regions with strong prognostic power among PTCL-U cases. A correlative analysis using the array CGH data disclosed a subgroup in PTCL-U with genomic alterations and with histopathologic and clinical relevance. In addition to histopathologic similarity, the strong genetic and prognostic resemblance between PTCL-U cases with genomic imbalance detected by array CGH and lymphoma-type ATLL seems to support the notion that the former may constitute a distinct PTCL-U subgroup.

Purpose: Progression to the castration-resistant state is the incurable and lethal end stage of prostate cancer, and there is strong evidence that androgen receptor (AR) still plays a central role in this process. We hypothesize that knocking down AR will have a major effect on inhibiting growth of castration-resistant tumors.

Experimental Design: Castration-resistant C4-2 human prostate cancer cells stably expressing a tetracycline-inducible AR-targeted short hairpin RNA (shRNA) were generated to directly test the effects of AR knockdown in C4-2 human prostate cancer cells and tumors.

Results:In vitro expression of AR shRNA resulted in decreased levels of AR mRNA and protein, decreased expression of prostate-specific antigen (PSA), reduced activation of the PSA-luciferase reporter, and growth inhibition of C4-2 cells. Gene microarray analyses revealed that AR knockdown under hormone-deprived conditions resulted in activation of genes involved in apoptosis, cell cycle regulation, protein synthesis, and tumorigenesis. To ensure that tumors were truly castration-resistant in vivo, inducible AR shRNA expressing C4-2 tumors were grown in castrated mice to an average volume of 450 mm³. In all of the animals, serum PSA decreased, and in 50% of them, there was complete tumor regression and disappearance of serum PSA.

Conclusions: Whereas castration is ineffective in castration-resistant prostate tumors, knockdown of AR can decrease serum PSA, inhibit tumor growth, and frequently cause tumor regression. This study is the first direct evidence that knockdown of AR is a viable therapeutic strategy for treatment of prostate tumors that have already progressed to the castration-resistant state.

Purpose: Secretory clusterin (sCLU)/apolipoprotein J is an extracellular chaperone that has been functionally implicated in DNA repair, cell cycle regulation, apoptotic cell death, and tumorigenesis. It exerts a prosurvival function against most therapeutic treatments for cancer and is currently an antisense target in clinical trials for tumor therapy. However, the molecular mechanisms underlying its function remained largely unknown.

Experimental Design: The molecular effects of small interfering RNA-mediated sCLU depletion in nonstressed human cancer cells were examined by focusing entirely on the endogenously expressed sCLU protein molecules and combining molecular, biochemical, and microscopic approaches.

Results: We report here that sCLU depletion in nonstressed human cancer cells signals stress that induces p53-dependent growth retardation and high rates of endogenous apoptosis. We discovered that increased apoptosis in sCLU-depleted cells correlates to altered ratios of proapoptotic to antiapoptotic Bcl-2 protein family members, is amplified by p53, and is executed by mitochondrial dysfunction. sCLU depletion-related stress signals originate from several sites, because sCLU is an integral component of not only the secretory pathway but also the nucleocytosolic continuum and mitochondria. In the cytoplasm, sCLU depletion disrupts the Ku70-Bax complex and triggers Bax activation and relocation to mitochondria. We show that sCLU binds and thereby stabilizes the Ku70-Bax protein complex serving as a cytosol retention factor for Bax.

Conclusions: We suggest that elevated sCLU levels may enhance tumorigenesis by interfering with Bax proapoptotic activities and contribute to one of the major characteristics of cancer cells, that is, resistance to apoptosis.

Purpose: This study evaluates the proapoptotic function of integrin β₃ in human hepatocellular carcinoma (HCC).

Experimental Design: The expression of integrin β₃ in 67 HCC specimens paired with corresponding neighboring nontumorous tissue was studied by quantitative real-time PCR and Western blot. The proapoptotic function of integrin β₃ in SMMC-7721 human hepatoma cells overexpressing ITGB3 (gene coding integrin β₃) was determined through colony formation, serum starvation, and anoikis assay.

Results: Compared with neighboring pathologically normal liver tissue, ~60% of the HCC specimens showed a significant down-regulated level of integrin β₃ expression. Transient expression of integrin β₃ in SMMC-7721 resulted in an enhanced level of apoptosis and suppression of colony formation. Cell growth inhibition on serum/ligand deprivation and incidences of anoikis were remarkably increased in SMMC-7721 with stable expression of integrin β₃ in comparison with vector control transfectants. In addition, expression of fibrinogen and vitronectin, two native ligands for integrin _vβ₃ in liver, was inhibited, which was correlated with the decreased integrin β₃ expression. Replenishing these ligands to the starved SMMC-7721 stable transfectants effectively restored the proapoptotic function of integrin β₃.

Conclusions: Down-regulation of integrin β₃ and its ligands in liver is related to the aggressive growth of HCC. Thus, reconstitution of integrin β₃ in HCC may be a potential therapeutic approach to inhibit aggressive growth of liver cancer.

Purpose: In previous studies, we have used human embryonic stem cells (hESC) to generate a tissue microenvironment in immunocompromised mice as an experimental approach for studying human tumorigenesis. We now examine the attributes of such a cellular microenvironment in supporting the growth of human cancer cells freshly harvested from malignant ovarian ascites and to determine whether there are differences among subsets of ascites-derived cancer cells in terms of tumorigenic capacity in the conventional murine xenograft model and in the hESC-derived microenvironment.

Experimental Design: Freshly harvested malignant ovarian ascites-derived cancer cells and six derivative ovarian cancer cell subpopulations (CCSP) were characterized for ovarian cancer–associated biomarker expression both in vitro and in vivo and for their capacity to generate tumors in the two models.

Results: Ovarian cancer–associated biomarkers were detected in the ascites-derived cancer cells and in the six newly established CCSPs. Nevertheless, certain CCSPs that did not develop into tumors in a conventional murine xenograft model did generate tumors in the hESC-derived cellular microenvironment, indicating variable niche dependency for the tumorigenic capacity of the different CCSPs. The hESC-derived microenvironment provided an improved niche for supporting growth of certain tumor cell subpopulations.

Conclusions: The results highlight the experimental utility of the hESC-derived cellular microenvironment to enable functional distinction of CCSPs, including the identification of cells that do not grow into a tumor in the conventional direct tumor xenograft platform, thereby rendering such cells accessible to characterization and testing of anticancer therapies.

Purpose: p27 localization and expression has prognostic and predictive value in cancer. Little is known regarding expression patterns of p27 in renal cell carcinoma (RCC) or how p27 participates in disease progression or response to therapy.

Experimental Design: RCC-derived cell lines, primary tumors, and normal renal epithelial cells were analyzed for p27 expression, phosphorylation (T157 of the NLS), and subcellular localization. RCC-derived cell lines were treated with phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors and effects on p27 localization were assessed. The potential contribution of cytoplasmic p27 to resistance to apoptosis was also evaluated.

Results: p27 was elevated in tumors compared with matched controls, and cytoplasmic mislocalization of p27 was associated with increasing tumor grade. Cytoplasmic localization of p27 correlated with phosphorylation at T157, an AKT phosphorylation site in the p27 NLS. In RCC cell lines, activated PI3K/AKT signaling was accompanied by mislocalization of p27. AKT activation and phosphorylation of p27 was associated with resistance to apoptosis, and small interfering RNA knockdown of p27 or relocalization to the nucleus increased apoptosis in RCC cells. Treatment with the PI3K inhibitors LY294002 or wortmannin resulted in nuclear relocalization of p27, whereas mTOR inhibition by rapamycin did not.

Conclusions: In RCC, p27 is phosphorylated at T157 of the NLS, with increasing tumor grade associated with cytoplasmic p27. PI3K inhibition (which reduces AKT activity) reduces T157 phosphorylation and induces nuclear relocalization of p27, whereas mTOR inhibition does not. Clinical testing of these findings may provide a rational approach for use of mTOR and PI3K/AKT pathway inhibitors in patients with RCC.

Purpose: The effects of pan–histone deacetylase (HDAC) inhibitors on cancer cells have shown that HDACs are involved in fundamental tumor biological processes such as cell cycle control, differentiation, and apoptosis. However, because of the unselective nature of these compounds, little is known about the contribution of individual HDAC family members to tumorigenesis and progression. The purpose of this study was to evaluate the role of individual HDACs in neuroblastoma tumorigenesis.

Experimental Design: We have investigated the mRNA expression of all HDAC1-11 family members in a large cohort of primary neuroblastoma samples covering the full spectrum of the disease. HDACs associated with disease stage and survival were subsequently functionally evaluated in cell culture models.

Results: Only HDAC8 expression was significantly correlated with advanced disease and metastasis and down-regulated in stage 4S neuroblastoma associated with spontaneous regression. High HDAC8 expression was associated with poor prognostic markers and poor overall and event-free survival. The knockdown of HDAC8 resulted in the inhibition of proliferation, reduced clonogenic growth, cell cycle arrest, and differentiation in cultured neuroblastoma cells. The treatment of neuroblastoma cell lines as well as short-term-culture neuroblastoma cells with an HDAC8-selective small-molecule inhibitor inhibited cell proliferation and clone formation, induced differentiation, and thus reproduced the HDAC8 knockdown phenotype. Global histone 4 acetylation was not affected by HDAC8 knockdown or by selective inhibitor treatment.

Conclusions: Our data point toward an important role of HDAC8 in neuroblastoma pathogenesis and identify this HDAC family member as a specific drug target for the differentiation therapy of neuroblastoma.

Purpose: During cancer progression, the oncoprotein MUC1 binds β-catenin while simultaneously inhibiting the degradation of the epidermal growth factor receptor (EGFR), resulting in enhanced transformation and metastasis. The purpose of this study was to design a peptide-based therapy that would block these intracellular protein-protein interactions as a treatment for metastatic breast cancer.

Experimental Design: The amino acid residues responsible for these interactions lie in tandem in the cytoplasmic domain of MUC1, and we have targeted this sequence to produce a MUC1 peptide that blocks the protumorigenic functions of MUC1. We designed the MUC1 inhibitory peptide (MIP) to block the intracellular interactions between MUC1/β-catenin and MUC1/EGFR. To allow for cellular uptake we synthesized MIP adjacent to the protein transduction domain, PTD4 (PMIP).

Results: We have found that PMIP acts in a dominant-negative fashion, blocking both MUC1/β-catenin and MUC1/EGFR interactions. In addition, PMIP induces ligand-dependent reduction of EGFR levels. These effects correspond to a significant reduction in proliferation, migration, and invasion of metastatic breast cancer cells in vitro, and inhibition of tumor growth and recurrence in an established MDA-MB-231 immunocompromised (SCID) mouse model. Importantly, PMIP also inhibits genetically driven breast cancer progression, as injection of tumor-bearing MMTV-pyV mT transgenic mice with PMIP results in tumor regression and a significant inhibition of tumor growth rate.

Conclusions: These data show that intracellular MUC1 peptides possess significant antitumor activity and have important clinical applications in the treatment of cancer.

Purpose: Angiogenesis plays a critical role in breast cancer development and progression. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that regulates endothelial cell proliferation and survival. We investigated the effects of motesanib, a novel, oral inhibitor of VEGF receptors 1, 2, and 3; platelet-derived growth factor receptor; and Kit receptor, on the growth of xenografts representing various human breast cancer subtypes.

Experimental Design: Athymic nude mice were implanted with MCF-7 (luminal) or MDA-MB-231 (mesenchymal) tumor fragments or Cal-51 (mixed/progenitor) tumor cells. Once tumors were established, animals were randomized to receive increasing doses of motesanib alone or motesanib plus cytotoxic chemotherapy (docetaxel, doxorubicin, or tamoxifen).

Results: Across all three xenograft models, motesanib treatment resulted in significant dose-dependent reductions in tumor growth, compared with vehicle-treated controls, and in marked reductions in viable tumor fraction and blood vessel density. No significant effect on body weight was observed with compound treatment compared with control-treated animals. Motesanib did not affect the proliferation of tumor cells in vitro. There was a significantly greater reduction in xenograft tumor growth when motesanib was combined with docetaxel (MDA-MB-231 tumors) or with the estrogen receptor modulator tamoxifen (MCF-7 tumors), compared with either treatment alone, but not when combined with doxorubicin (Cal-51 tumors).

Conclusions: Treatment with motesanib alone or in combination with chemotherapy inhibits tumor growth in vivo in various models of human breast cancer. These data suggest that motesanib may have broad utility in the treatment of human breast cancer.

Purpose:N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE; tesmilifene) is thought to potentiate the antineoplastic effect of cytotoxic drugs. In a phase III randomized trial for metastatic breast cancer using doxorubicin with or without DPPE, addition of the latter resulted in a significant improvement in overall survival and a trend toward a difference in progression-free survival but, paradoxically, no difference in objective tumor response. Here we tested the hypothesis that DPPE targets breast tumor-initiating cells (TICs).

Experimental Design: Human breast TICs from pleural effusions were identified as CD44⁺:CD24^–/low cells by flow cytometry and functionally by their ability to form nonadherent spheres in culture. Mouse mammary TICs from two different models of breast cancer were identified as cells capable of initiating spheres in culture and secondary tumors following transplantation into the mammary gland of syngeneic mice.

Results: We show that at physiologically attainable concentrations, treatment with DPPE alone reduced tumorsphere formation and viability of CD44⁺:CD24^–/low breast cancer cells. The kinetics of killing varied for the different breast tumor cells and required continuous exposure to the drug. Whereas doxorubicin killed CD44⁺:CD24^–/low and CD44^–:CD24⁺ cells equally well, DPPE induced apoptosis preferentially in CD44⁺:CD24^–/low cells. Treatment of Her2/Neu⁺ mammary tumor cells with DPPE in vitro efficiently killed TICs, as determined by flow cytometry and transplantation assays; DPPE further cooperated with doxorubicin to completely eradicate tumorigenic cells.

Conclusions: Our results show that continuous treatment with DPPE alone directly targets breast TICs, and provide rationale to test for cooperation between DPPE and known drugs with efficacy toward breast cancer subtypes.

Purpose: β-Lapachone (ARQ 501, a formulation of β-lapachone complexed with hydroxypropyl-β-cyclodextrin) is a novel anticancer agent with selectivity against prostate cancer cells overexpressing the NAD(P)H:quinone oxidoreductase-1 enzyme. Lack of solubility and an efficient drug delivery strategy limits this compound in clinical applications. In this study, we aimed to develop β-lapachone–containing polymer implants (millirods) for direct implantation into prostate tumors to test the hypothesis that the combination of a tumor-specific anticancer agent with site-specific release of the agent will lead to significant antitumor efficacy.

Experimental Design: Survival assays in vitro were used to test the killing effect of β-lapachone in different prostate cancer cells. β-Lapachone release kinetics from millirods was determined in vitro and in vivo. PC-3 prostate tumor xenografts in athymic nude mice were used for antitumor efficacy studies in vivo.

Results: β-Lapachone killed three different prostate cancer cell lines in an NAD(P)H:quinone oxidoreductase-1–dependent manner. Upon incorporation of solid-state inclusion complexes of β-lapachone with hydroxypropyl-β-cyclodextrin into poly(d,l-lactide-co-glycolide) millirods, β-lapachone release kinetics in vivo showed a burst release of ~0.5 mg within 12 hours and a subsequently sustained release of the drug (~0.4 mg/kg/d) comparable with that observed in vitro. Antitumor efficacy studies showed significant tumor growth inhibition by β-lapachone millirods compared with controls (P < 0.0001; n = 10 per group). Kaplan-Meier survival curves showed that tumor-bearing mice treated with β-lapachone millirods survived nearly 2-fold longer than controls, without observable systemic toxicity.

Conclusions: Intratumoral delivery of β-lapachone using polymer millirods showed the promising therapeutic potential for human prostate tumors.

Purpose: To characterize the functional role of c-Jun NH₂-terminal kinase (JNK) and other apoptotic pathways in grape seed extract (GSE)-induced apoptosis in human leukemia cells by using pharmacologic and genetic approaches.

Experimental Design: Jurkat cells were treated with various concentrations of GSE for 12 and 24 h or with 50 µg/mL GSE for various time intervals, after which apoptosis, caspase activation, and cell signaling pathways were evaluated. Parallel studies were done in U937 and HL-60 human leukemia cells.

Results: Exposure of Jurkat cells to GSE resulted in dose- and time-dependent increase in apoptosis and caspase activation, events associated with the pronounced increase in Cip1/p21 protein level. Furthermore, treatment of Jurkat cells with GSE resulted in marked increase in levels of phospho-JNK. Conversely, interruption of the JNK pathway by pharmacologic inhibitor (e.g., SP600125) or genetic (e.g., small interfering RNA) approaches displayed significant protection against GSE-mediated lethality in Jurkat cells.

Conclusions: The result of the present study showed that GSE induces apoptosis in Jurkat cells through a process that involves sustained JNK activation and Cip1/p21 up-regulation, culminating in caspase activation.

Purpose: Prosurvival Bcl-2 proteins inhibit the mitochondrial and death receptor-mediated apoptotic pathways. Obatoclax is a small-molecule antagonist of the BH3-binding groove of Bcl-2 proteins that may enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity and efficacy.

Experimental Design: Human pancreatic cancer cell lines were incubated with obatoclax and/or TRAIL and cell viability, Annexin V labeling, caspase cleavage, and cytochrome c release were measured. In drug-treated cell lines, protein-protein interactions were studied by immunoprecipitation. Bax/Bak activation was analyzed using conformation-specific antibodies. Lentiviral short hairpin RNA was used to knockdown Bim, Bid, and apoptosis-inducing factor (AIF) expression.

Results: Obatoclax reduced the viability of PANC-1 and BxPC-3 cell lines and synergistically enhanced TRAIL-mediated cytotoxicity. Obatoclax enhanced TRAIL-mediated apoptosis, as shown by Annexin V labeling, which was accompanied by caspase activation (caspase-8, -9, and -3) and cleavage of Bid. Obatoclax potentiated TRAIL-mediated Bax/Bak activation and the release of mitochondrial cytochrome c, Smac, and AIF. Mechanisms underlying the apoptotic effect of obatoclax include displacement of Bak from its sequestration by Bcl-x_L or Mcl-1 and release of Bim from Bcl-2 or Mcl-1. Bid knockdown by short hairpin RNA attenuated caspase cleavage and cytotoxicity of obatoclax plus TRAIL. Bim knockdown failed to inhibit the cytotoxic effect of obatoclax alone or combined with TRAIL yet attenuated TRAIL-mediated cytotoxicity. AIF knockdown attenuated cytotoxicity of the drug combination.

Conclusions: Obatoclax potentiates TRAIL-mediated apoptosis by unsequestering Bak and Bim from Bcl-2/Bcl-x_L or Mcl-1 proteins. This drug combination enhances Bid-mediated cross-talk between the mitochondrial and death receptor-mediated apoptotic pathways and may represent a novel therapeutic strategy against pancreatic cancer.

Purpose: Hypoxia is a cause for resistance to cancer therapies. Molecularly targeted recombinant cytotoxins have shown clinical efficacy in the treatment of patients with primary brain tumors, glioblastoma multiforme, but it is not known whether hypoxia influences their antitumor effect.

Experimental Design: We have exposed glioblastoma multiforme cells, such as U-251 MG, U-373 MG, SNB-19, and A-172 MG, to either anoxia or hypoxia and then reoxygenated them while treating with an interleukin (IL)-13-based diphtheria toxin (DT)-containing cytotoxin, DT-IL13QM. We measured the levels of immunoreactive IL-13R2, a receptor that mediates IL-13-cytotoxin cell killing, and the levels of active form of furin, a protease that activates the bacterial toxin portion in a cytotoxin.

Results: We found that anoxia/hypoxia significantly alters the responsiveness of glioblastoma multiforme cells to DT-IL13QM. Interestingly, bringing these cells back to normoxia caused them to become even more susceptible to the cytotoxin than the cells maintained under normoxia. Anoxia/hypoxia caused a highly prominent decrease in the immunoreactive levels of both IL-13R and active forms of furin, and reoxygenation not only restored their levels but also became higher than that in normoxic glioblastoma multiforme cells.

Conclusions: Our results show that a recombinant cytotoxin directed against glioblastoma multiforme cells kills these cells much less efficiently under anoxic/hypoxic conditions. The reoxygenation brings unexpected additional benefit of making glioblastoma multiforme cells even more responsive to the killing effect of a cytotoxin.

Purpose: Carcinoembryonic antigen (CEA) is a tumor-associated protein expressed on a variety of adenocarcinomas. To develop an immunotherapy for patients with cancers that overexpress CEA, we isolated and genetically modified a T-cell receptors (TCRs) that specifically bound a CEA peptide on human cancer cells.

Experimental Design: HLA-A2.1 transgenic mice were immunized with CEA:691-699. A CEA-reactive TCR was isolated from splenocytes of these mice and was genetically introduced into human peripheral blood lymphocytes via RNA electroporation or retroviral transduction. Amino acid substitutions were introduced throughout the complementarity determining regions (CDR1, CDR2, and CDR3) of both TCR and β chains to improve recognition of CEA.

Results: Murine lymphocytes bearing the CEA-reactive TCR specifically recognized peptide-loaded T2 cells and HLA-A2.1⁺ CEA⁺ human colon cancer cells. Both CD8⁺ and CD4⁺ human lymphocytes expressing the murine TCR specifically recognized peptide-loaded T2 cells. However, only gene-modified CD8⁺ lymphocytes specifically recognized HLA-A2.1⁺ CEA⁺ colon cancer cell lines, and tumor cell recognition was weak and variable. We identified two substitutions in the CDR3 of the chain that significantly influenced tumor cell recognition by human peripheral blood lymphocytes. One substitution, T for S at position 112 (S112T), enhanced tumor cell recognition by CD8⁺ lymphocytes, and a second dually substituted receptor (S112T L110F) enhanced tumor cell recognition by CD4⁺ T cells.

Conclusions: The modified CEA-reactive TCRs are good candidates for future gene therapy clinical trials and show the power of selected amino acid substitutions in the antigen-binding regions of the TCR to enhance desired reactivities.