Plasma Membrane Of Tumor Cells Research Articles

Inhibition of the ecto-beta subunit of F1F0-ATPase inhibits proliferation and induces apoptosis in acute myeloid leukemia cell lines

BackgroundLeukemia, a heterogeneous clonal disorder of hematopoietic progenitor cells, presents a world-wide health problem, especially in childhood. F1F0 ATPase, an inner mitochondrial enzyme, is expressed on the plasma membrane of tumor cells, and its inhibition induces both anti-angiogenic and anti-tumorigenic activity.MethodsMonoclonal Antibody (McAb) against ATPase was produced by polyethylene glycol-mediated fusions and screened by ELISA. Proliferation, cell cycle and apoptosis of cells were analyzed when the surface ATPase of cells was blockaded with McAb.ResultsWe detected cell-membrane expression of the F1F0 ATPase β subunit on 0.1% to 56% of the 11 cell lines derived from leukemia, including acute myeloid leukemia (AML). We produced a monoclonal antibody, McAb7E10, which recognizes both the native and recombinant ATPase β subunit, with a dissociation constant (KD) of 3.26E–10. We demonstrate that McAb7E10 binds to ATPase at the cell surface, where it is able to inhibit ATP synthesis. McAb7E10 significantly inhibited proliferation of AML cell lines in vitro: the relative inhibitory rates of 50 μg/mL McAb7E10 treated MV4-11and HL-60 cells were 69.6% and 81.9% respectively. Cell cycle analysis indicated that McAb7E10 significantly induced apoptosis in MV4-11 and HL-60 cells: the relative rates of apoptosis in 5, 10 and 50ug/mL McAb7E10 treated MV4-11 cells was 3.6 ± 0.83%, 8.4 ± 1.69% and 17.3 ± 2.56% compared to 1.5% ± 0.85% in mouse IgG treated cells (p < 0.01). The relative rate of apoptosis in 5, 10 and 50ug/mL McAb7E10 treated HL-60 cells was 5.5 ± 2.37%, 11.3 ± 3.62% and 19.9 ± 3.31% compared to 1.56% ± 0.97% in mouse IgG treated cells (p < 0.01). Annexin V staining demonstrated that the relative apoptotic rates in 50 μg/mL McAb7E10 treated MV4-11 and HL-60 cells were 50.5% ± 7.04% and 32.9% ± 4.52%, respectively, significantly higher than IgG control antibody treated cells were 21.9% ± 3.11% and 15.3% ± 3.95%, p < 0.01.ConclusionsThese findings indicate that ectopic expression of ATPase β subunit may be a tumor-associated antigen in hematological malignancies. The F1F0 ATPase β subunit provides a potential target for immunotherapy in AML and hematological malignancies.

The Odyssey of Hsp60 from Tumor Cells to Other Destinations Includes Plasma Membrane-Associated Stages and Golgi and Exosomal Protein-Trafficking Modalities

BackgroundIn a previous work we showed for the first time that human tumor cells secrete Hsp60 via exosomes, which are considered immunologically active microvesicles involved in tumor progression. This finding raised questions concerning the route followed by Hsp60 to reach the exosomes, its location in them, and whether Hsp60 can be secreted also via other mechanisms, e.g., by the Golgi. We addressed these issues in the work presented here.Principal FindingsWe found that Hsp60 localizes in the tumor cell plasma membrane, is associated with lipid rafts, and ends up in the exosomal membrane. We also found evidence that Hsp60 localizes in the Golgi apparatus and its secretion is prevented by an inhibitor of this organelle.Conclusions/SignificanceWe propose a multistage process for the translocation of Hsp60 from the inside to the outside of the cell that includes a combination of protein traffic pathways and, ultimately, presence of the chaperonin in the circulating blood. The new information presented should help in designing future strategies for research and for developing diagnostic-monitoring means useful in clinical oncology.

Abstract 3805: Identification of a distinctive pattern of lipid composition in Irvalec® resistant tumor cells

Abstract Irvalec® is a marine derived synthetic depsipeptide showing cytotoxic activity against a wide diversity of tumor cells both in vitro and in vivo. The compound is currently undergoing Phase II clinical studies in patients with solid tumors. The existing data on the mechanism of action of Irvalec® indicate that the compound inserts and self-organize in the plasma membrane of tumor cells, rapidly inducing loss of membrane integrity and cell permeabilization, giving rise to a fast and dramatic necrotic death. By continuous exposure of human colon carcinoma HCT-116 cells to stepwise increasing concentratrions of Irvalec®, a resistant subclone of the cell line was established, named HCT-116-Irv. In dose-response cytotoxicity assays, HCT-116-Irv cells showed a GI50 value &amp;gt;50 μM, representing a specific resistance index to the compound greater than 10.8 as compared to their wt counterpart. The resistant cells did not show any of the morphological and physiological responses to Irvalec® observed in parental cells and no overexpression of Pgp efflux pump or significant cross-resistance to other antitumoral drugs was detected. Due to the presumably direct effect of Irvalec® on the cell membrane, we searched for possible changes in the lipid composition of the resistant cells, acquired during the process of resistance development. After fractionation of the lipid extracts using thin layer cromatography (TLC), two differential fractions, that were completely absent in the resistant cells as compared to wt cells, were identified and purified. Interestingly, one of these fractions, named F4, was found to be able to interact with a biotinilated derivative of Irvalec®, using a standard overlay assay. These results suggested that the lipid composition could be directly related to the sensitivity of tumor cells to Irvalec®. Efforts are now focused on the chemical characterization of the selected lipid fraction and its putative biological role mediating the cytotoxic response to Irvalec®. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3805. doi:1538-7445.AM2012-3805

Loss of EBP50 stimulates EGFR activity to induce EMT phenotypic features in biliary cancer cells

Scaffold proteins form multiprotein complexes that are central to the regulation of intracellular signaling. The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is highly expressed at the plasma membrane of normal biliary epithelial cells and binds epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with oncogenic properties. This study investigated EBP50-EGFR interplay in biliary cancer. We report that in a collection of 106 cholangiocarcinomas, EBP50 was delocalized to the cytoplasm of tumor cells in 66% of the cases. Ectopic expression of EBP50 was correlated with the presence of satellite nodules and with the expression of EGFR, which was at the plasma membrane, implying a loss of interaction with EBP50 in these cases. In vitro, loss of interaction between EBP50 and EGFR was mimicked by EBP50 depletion using a small interfering RNA approach in human biliary carcinoma cells co-expressing the two proteins at their plasma membrane, and in which interaction between EBP50 and EGFR was validated. EBP50 depletion caused an increase in EGFR expression at their surface, and a sustained activation of the receptor and of its downstream effectors (extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3) in both basal and EGF-stimulated conditions. Cells lacking EBP50 showed epithelial-to-mesenchymal transition-associated features, including reduction in E-cadherin and cytokeratin-19 expression, induction of S100A4 and of the E-cadherin transcriptional repressor, Slug, and loss of cell polarity. Accordingly, depletion of EBP50 induced the disruption of adherens junctional complexes, the development of lamellipodia structures and the subsequent acquisition of motility properties. All these phenotypic changes were prevented upon inhibition of EGFR tyrosine kinase by gefitinib. These findings indicate that loss of EBP50 at the plasma membrane in tumor cells may contribute to biliary carcinogenesis through EGFR activation.

The Human Splice Variant Δ16HER2 Induces Rapid Tumor Onset in a Reporter Transgenic Mouse

Several transgenic mice models solidly support the hypothesis that HER2 (ERBB2) overexpression or mutation promotes tumorigenesis. Recently, a HER2 splice variant lacking exon-16 (Δ16HER2) has been detected in human breast carcinomas. This alternative protein, a normal byproduct of HER2, has an increased transforming potency compared to wild-type (wt) HER2 receptors. To examine the ability of Δ16HER2 to transform mammary epithelium in vivo and to monitor Δ16HER2-driven tumorigenesis in live mice, we generated and characterized a mouse line that transgenically expresses both human Δ16HER2 and firefly luciferase under the transcriptional control of the MMTV promoter. All the transgenic females developed multifocal mammary tumors with a rapid onset and an average latency of 15.11 weeks. Immunohistochemical analysis revealed the concurrent expression of luciferase and the human Δ16HER2 oncogene only in the mammary gland and in strict correlation with tumor development. Transgenic Δ16HER2 expressed on the tumor cell plasma membrane from spontaneous mammary adenocarcinomas formed constitutively active homodimers able to activate the oncogenic signal transduction pathway mediated through Src kinase. These new transgenic animals demonstrate the ability of the human Δ16HER2 isoform to transform “per se” mammary epithelium in vivo. The high tumor incidence as well as the short latency strongly suggests that the Δ16HER2 splice variant represents the transforming form of the HER2 oncoprotein.

Irvalec Inserts into the Plasma Membrane Causing Rapid Loss of Integrity and Necrotic Cell Death in Tumor Cells

Irvalec is a marine-derived antitumor agent currently undergoing phase II clinical trials. In vitro, Irvalec induces a rapid loss of membrane integrity in tumor cells, accompanied of a significant Ca2+ influx, perturbations of membrane conductivity, severe swelling and the formation of giant membranous vesicles. All these effects are not observed in Irvalec-resistant cells, or are significantly delayed by pretreating the cells with Zn2+. Using fluorescent derivatives of Irvalec it was demonstrated that the compound rapidly interacts with the plasma membrane of tumor cells promoting lipid bilayer restructuration. Also, FRET experiments demonstrated that Irvalec molecules localize in the cell membrane close enough to each other as to suggest that the compound could self-organize, forming supramolecular structures that likely trigger cell death by necrosis through the disruption of membrane integrity.

Abstract 2301: E2-induced T47D:A18/PKCα tumor regression is accompanied by translocation of ERα to extranuclear sites in association with caveolin-1

Abstract Background: Acquired resistance to tamoxifen (TAM) is a serious clinical problem in the treatment of breast cancer patients. Our laboratory developed and characterized a TAM-resistant and 17 beta-estradiol (E2) independent preclinical model where PKCα was stably transfected into T47D:A18 cells compared with the E2-dependent T47D:A18/neo cells (Chisamore et al, 2001). We and others have shown that PKCα overexpression is associated with TAM resistance in cell lines and in the clinic (Tonetti et al, 2003, Assender et al, 2007). Additionally the T47D:A18/PKCα model exhibits E2-induced tumor regression in vivo or when grown in matrigel (3D matrix) but not on 2D plastic. Recently we have demonstrated that tumor regression involves participation of estrogen receptor alpha (ERα), Fas/FasL and the extracellular matrix (Zhang et al, 2009). Interestingly the membrane impermeable E2-BSA conjugate mediates growth inhibition of T47D:A18/PKCα colonies in matrigel similar to E2 suggesting a potential role of extranuclear ERα. On the basis of our preliminary data we investigated whether extranuclear ERα signaling pathway might play a critical role in E2-induced tumor regression. Materials and methods: Four micrometer thick sections were prepared from paraffin embedded T47D:A18/PKCα tumor tissues from untreated (control) and E2-treated mice and T47D:A18/neo tumors. Fluorescence tagged secondary antibodies were used for immunofluorescence staining. Co-immunoprecipitation (coIP) was done with control and E2 treatment group tumor extracts. Results: Immunofluorescence images of T47D:A18/PKCα tumor sections from untreated control mice showed that the ERα was mainly localized in the nucleus. Interestingly, treatment with E2 resulted in significant translocation of ERα from the nucleus to the cytoplasm and the plasma membrane of tumor cells. However in T47D:A18/neo tumors the majority of ERα was found in the nucleus suggesting the involvement of ERE mediated classical pathway in tumor growth. Confocal microscopy clearly indicates the colocalization of ERα with caveolin-1 in E2-treated T47D:18/PKCα tumor sections. Furthermore coIP performed with T47D:A18/PKCα tumor extracts revealed greater association of ERα with caveolin-1 in E2-treated tumors compared with control tumors. Discussion: We present evidence that E2-induced regression of our clinically relevant PKCα-overexpressing tumor model is accompanied by translocation of ERα to extranuclear sites in association with caveolin. This result suggests that E2-induced tumor regression is likely to be mediated by a membrane-associated signaling pathway. Therapies that stimulate the extranuclear ERα signaling pathway in PKCα overexpressing tumors may be an attractive approach to treat endocrine resistant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2301. doi:10.1158/1538-7445.AM2011-2301

Mechanism of Beclin1 as a favorable molecules marker is that Beclin1 inhibits tumor proliferation and cell cycle arrest in stage IIIB colon cancers

Objective Beclin1 is a multi-function protein，bridging autophagy，apoptosis and differentiation. Our previous study found that Beclin1 was a favorable prognostic biomarker in locally advanced colon carcinomas. This study investigated the expression of Beclin1 in human colon cancers and its mechanism as a favorable molecules marker. Methods A total of 104 cases of colon cancer specimens and clinical data were obtained from Chinese stage ⅢB colon cancer patients with intact follow-up data who subjected to radical resection between Jan，1999 and July，2003 in the cancer center of Sun Yat-Sen University. The specimens were constructed from formalin-fixed，paraffin-embedded tissues. The expression of Beclin1 in primary tumors matched with adjacent noncancerous tissue were examined with immunohistochemistry technique. Additionally，immunofluorescence double staining technique was used to investigate the molecular mechanism by analyzing coexpression of the protein PCNA and Beclin1. Results The expression of Beclin1 was observed in plasma-membrane，cytoplasm and nucleus in tumor cells. In the tumor tissues analyzed，immunopositivity was observed for Beclin1 in 87(83.7% ) of the 104 tumor tissues. In contrast，adjacent non-cancerous tissues and normal tissues showed no or very weak expression of Beclin1. The 5-year overall survival rate for Beclin1 immunopositivity cases was 69.8%. Beclin1 immunonegativity was 47.1%. Both univariate analysis and multivariate analysis showed that Beclin1 and T stage were independent prognostic factors. The immunofluorescence double staining showed the expression of Beclin1 negatively correlated with the expression of PCNA，which indicates Beclin inhibits tumor proliferation and cell cycle arrest. Conclusions Beclin1 plays important roles in the regulation of the life span of human colon cells by inhibiting tumor proliferation and cell cycle arrest in stage ⅢB colon cancers. Beclin1 is a favorable prognostic biomarker in locally advanced colon carcinomas and provides the information regarding the likely targeted therapy intervention. In future，with the progress of autophagy analysis technique，it is critical to determine the autophagy pathway responsible for colon cancer in cell lines and animal models to define targets to fight the locally advanced colon cancer.

IGF2 is Highly Expressed in Pediatric Undifferentiated Sarcomas and Reveals Two Distinct Cytoplasmic Trafficking Patterns

Pediatric undifferentiated soft tissue sarcomas (USTSs) are tumors composed of primitive mesenchymal cells. As such, they form an attractive model for studying the early events in sarcoma development. In an effort to better understand the critical molecular aberrations leading to sarcoma development, gene expression array analysis and post-array validation techniques were applied to several USTSs; the results were consistent with upregulation of the excitatory components of the insulin-like growth factor (IGF) pathway. Particularly high expression of the insulin-like growth factor 2 (IGF2) ligand was seen and confirmed by real-time reverse transcriptase-polymerase chain reaction. Immunohistochemistry performed using antibodies against IGF2 revealed overexpression of the IGF2 protein in 19 of 21 (90%) USTSs and revealed 2 distinct staining patterns, 1 in which there was diffuse cytoplasmic staining (16/19) and 1 in which there was punctate perinuclear positivity (3/19). Using ultrastructural immunogold localization of IGF2, it was determined that IGF2 was primarily localized to Golgi-derived vesicles and multivesicular bodies in tumor cells showing the punctate pattern, and to both the cytoplasm and plasma membrane of tumor cells showing the diffuse pattern. The results suggest that upregulation of the IGF pathway in pediatric USTSs is a critical early event in the development of sarcomas. Furthermore, findings from the immunocytochemical and immunogold analyses confirm the presence of 2 different cytoplasmic trafficking patterns and storage motifs of IGF2 within this type of tumor. Given that in one subcellular pattern the IGF2 protein does not appear to reach the membrane, these findings may have functional significance.

Metastasis-inducing S100A4 and RANTES cooperate in promoting tumor progression in mice.

BackgroundThe tumor microenvironment has been described as a critical milieu determining tumor growth and metastases. A pivotal role of metastasis-inducing S100A4 in the development of tumor stroma has been proven in animal models and verified in human breast cancer biopsies. Expression and release of S100A4 has been shown in various types of stroma composing cells, including fibroblasts and immune cells. However, the events implicated in upstream and downstream pathways regulating the activity of the extracellular S100A4 protein in the tumor milieu remain unsolved.Methodology/Principal FindingsWe studied the interplay between the tumor cell-derived cytokine regulated-upon-activation, normal T-cell expressed and secreted (RANTES; CCL5) and S100A4 which were shown to be critical factors in tumor progression. We found that RANTES stimulates the externalization of S100A4 via microparticle shedding from the plasma membrane of tumor and stroma cells. Conversely, the released S100A4 protein induces the upregulation of fibronectin (FN) in fibroblasts and a number of cytokines, including RANTES in tumor cells as well as stimulates cell motility in a wound healing assay. Importantly, using wild type and S100A4-deficient mouse models, we demonstrated a substantial influence of tumor cell-derived RANTES on S100A4 release into blood circulation which ultimately increases the metastatic burden in mice.Conclusions/SignificanceAltogether, the data presented strongly validate the pro-metastatic function of S100A4 in the tumor microenvironment and define how the tumor cell-derived cytokine RANTES acts as a critical regulator of S100A4-dependent tumor cell dissemination. Additionally, for the first time we demonstrated the mechanism of S100A4 release associated with plasma membrane microparticle shedding from various cells types.

Abstract 5527: Short-chain glucosylceramide improves doxorubicin delivery in spontaneous mouse breast carcinomas

Abstract Background: Insufficient delivery of chemotherapeutic agents to their intended intracellular targets within the tumor remains a major obstacle in clinical oncology. Most of these drugs are amphiphilic and their diffusion through the plasma membrane is, therefore, slow and a major limitation for sufficient uptake within the tumor cell. We have found that certain short-chain sphingolipids enhance the intratumoral drug accumulation by modulation of inward translocation of cytostatics over the plasma membrane. Here, we examine this novel approach in an in vivo mouse model of aggressive invasive lobular breast carcinoma (ILC), designed to closely mimic its human counterpart. Results: In a panel of cell lines, short-chain sphingolipids, such as C8-glucosylceramide (GC) increase the intracellular accumulation of doxorubicin up to 4-fold. A clinically approved drug carrier (100 nm liposomes) was employed for co-delivery of both lipid and drug to the tumor tissue. The addition of 10 mol% GC markedly enhanced the cellular doxorubicin content in vivo, as was demonstrated with intravital fluorescence microscopy. In the metastatic and aggressive mouse ILC tumors, the efficacy of GC doxorubicin liposomes was significantly improved compared to the currently applied conventional doxorubicin liposomes. The enrichment with the GC membrane modulator resulted in a significant tumor growth delay (p = 0.0119) and an overall survival benefit (p = 0.0143) for mice treated with the GC enriched liposomal doxorubicin as compared to conventional liposomes. Conclusion: Short-chain sphingolipids improve the translocation of various cytostatics, including doxorubicin, over the plasma membrane of tumor cells. This membrane modulating strategy can be successfully applied in vivo, e.g. as a co-formulation with doxorubicin liposomes, thereby enhancing their anti-tumor efficacy. Furthermore, our data suggest that GC-enriched doxorubicin liposomes hold promise as anti-neoplastic therapy in human breast cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5527.

CX3CR1/CX3CL1 axis negatively controls glioma cell invasion and is modulated by transforming growth factor-beta1

The chemokine CX3CL1 is constitutively expressed in the central nervous system by neurons and astrocytes controlling neuronal survival and neurotransmission. In this work, we analyzed the expression and function of the chemokine CX3CL1 and its receptor, CX3CR1, by human glioma cells. We show that both molecules are expressed on the tumor cell plasma membrane and that soluble CX3CL1 accumulates in the culture supernatants, indicating that the chemokine is constitutively released. We found that CX3CR1 is functional, as all the cell lines adhered to immobilized recombinant CX3CL1 and migrated in response to the soluble form of this chemokine. In addition, the blockade of endogenous CX3CL1 function by means of a neutralizing monoclonal antibody markedly delayed tumor cell aggregation and increased their invasiveness. We also show that CX3CL1 expression is potently modulated by the transforming growth factor-beta1 (TGF-beta1), a key regulator of glioma cell invasiveness. Indeed, both the treatment of glioma cells with recombinant TGF-beta1 and the inhibition of its endogenous expression by siRNA showed that TGF-beta1 decreases CX3CL1 mRNA and protein expression. Overall, our results indicate that endogenously expressed CX3CL1 negatively regulates glioma invasion likely by promoting tumor cell aggregation, and that TGF-beta1 inhibition of CX3CL1 expression might contribute to glioma cell invasive properties.

HERG1 Channels In Cancer Cells: Physical and Functional Interaction With Integrin Receptors

The expression and activity of different channel types mark and regulate specific stages of cancer progression, from cell proliferation and apoptosis, to invasiveness, angiogenesis and metastatic spread. As is being increasingly recognized, some of these roles can be attributed to signaling mechanisms independent of ion flow. Evidence is particularly extensive for K+ channels. For example, intracellular signaling cascades can be triggered when ion channels form protein complexes with other membrane proteins such as integrins or growth factor receptors.Work in our lab has established that hERG1 K+ channels are often aberrantly expressed in primary human cancers and exert pleiotropic effects in cancer cells, in turn regulating cell proliferation, cell motility and invasiveness or stimulating the process of neo-angiogenesis. hERG1 can induce such diverse effects since it triggers and modulates intracellular signaling cascades. This role depends on the formation, on the plasma membrane of tumor cells, of macromolecular complexes with integrin receptors, in particular with the β1 subunit. Recent FRET experiments have clearly shown that hERG1 and β1 directly interact, the intermolecular distance between the two proteins being around 4 nm. Moreover, the hERG1 protein inside the complex could function differently from its classical role in excitable cells, i.e. independently of ion flux, but through a conformational coupling with the partner protein(s). On the whole, data gathered so far allow us to propose a novel antineoplastic therapeutical approach, based on the targeting and unlocking of the β1 /hERG1 complex, in order to impair the hERG1-mediated signaling in cancer cells.

Estradiol Induced Regression of T47D:A18/PKCα Tumor Involves Translocation of Estrogen Receptor alpha from the Nucleus to the Cytoplasm.

Abstract Background: Resistance to tamoxifen (TAM) is one of the most challenging problems in the treatment of breast cancer. In our laboratory we have developed a PKCα overexpressing T47D:A18 cell line which is TAMresistant and 17β estradiol (E2) independent compared to the E2 dependent T47D:A18/neo cell line (Chisamore et al, 2001). PKCα overexpression is known to be associated with TAM treatment failure in breast cancer (Tonetti et al, 2003). This T47D:A18/PKCα model exhibits E2-induced tumor regression in in vivo or when grown in matrigel (3D matrix) but not on 2D plastic. Recently we have demonstrated that tumor regression involves participation of estrogen receptor alpha (ERα), Fas/FasL and the extracellular matrix (Zhang et al, 2009). Interestingly membrane impermeable E2-BSA conjugate mediates growth inhibition of T47D:A18/PKCα colonies in matrigel similar to E2 suggesting a potential role of extranuclear ERα. On the basis of our preliminary data we investigated whether extranuclear ERα may play a critical role in E2-induced tumor regression.Materials and methods: T47D:A18/PKCα cells were injected into mammary fat pads of 20 ovariectomized athymic mice. Mice were left untreated and tumors were allowed to grow until the mean tumor size reached 0.5 cm2, then 10 mice were implanted with a 1.0 cm E2 capsule and the other 10 continued with no treatment (control). T47D:A18/neo tumors were also established in 10 athymic mice in the presence of E2. Four micrometer thick sections were prepared from the paraffin embedded T47D:A18/PKCα tumor tissues of control and E2-treated mice and T47D:A18/neo tumors. For immunofluorescence staining, alexa fluor 488 and Cy3 fluorescence tagged secondary antibodies were used against ERα and β-actin primary antibodies respectively.Results: Immunofluorescence images of T47D:A18/PKCα tumor sections from untreated control mice showed that the ERα was mainly localized in the nucleus. Interestingly, treatment with E2 resulted in significant translocation of ERα from the nucleus to the cytoplasm and the plasma membrane of tumor cells. Immunofluorescence analysis of tumor sections obtained from E2 treated mice indicated that approximately 5-10% of cells contained ERα localized to the plasma membrane whereas the majority of ERα was localized to the cytosol. However in T47D:A18/neo tumors the majority of ERα was found in the nucleus suggesting the involvement of the ERE mediated classical pathway in tumor growth.Discussion: This is the first time we are reporting that E2-induced T47D:A18/PKCα tumor regression is accompanied by translocation of ERα to extranuclear sites. E2-induced tumor regression observed in our pre-clinical model suggests E2 or an E2-like compound may be a potential treatment option for patients harboring PKCα overexpressing tumors. Furthermore the extranuclear ERα signaling pathway may be an attractive therapeutic target to treat PKCα overexpressing tumors that are refractory to current endocrine therapies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4140.

Abstract C51: Anticancer peptides PNC-27/-28: Cancer cell killers by binding to HDM2 and pore formation in cancer cell membranes

Abstract Studies in vitro and in vivo have clearly demonstrated the efficiency of the novel anti-cancer peptides PNC-27 and PNC-28 in inducing the death of a variety of cancer cells by necrosis without any effect on normal cells. The peptides were derived from the HDM2-binding domain of p53 (PNC-27: AA12-26; PNC-28: AA17-26) that were linked to membrane resident peptide (MRP) AA sequence from the homeodomain protein antennapedia of the fruit fly. Recently we have shown that in contrast to the necrotic cell death of cancer cells by external application of PNC-28, the intracellular over-expression of AAs17-26 results in the cancer cells' apoptosis while normal cells showed no response. We now provide evidence for the critical role for the expression of HDM2 in the plasma membrane of cancer cells and its absence in the plasma membrane of normal cells as a target molecule for PNC-27. In direct competition experiments anti-HDM2 antibodies (Ab) blocked PNC-27 induced cell killing by &amp;gt;80% while control incubations with non-specific Ab at identical protein concentrations did not hinder PNC-27 from efficiently killing cancer cells. Immunofluorescence staining of HDM2 showed the presence of HDM2 on the plasma membrane of cancer cells (MIA-PaCa-2, BMRPA1.TUC3, A2058) which was absent in normal cell plasma membrane. Furthermore, co-localization of HDM2 and PNC-27 was observed on the plasma membrane of the cancer cells, suggesting their neighborliness. The findings were further confirmed by HDM2-positive immunoblots (IB) of purified plasma membrane from several cancer cells while plasma membrane isolated from healthy normal cells only delivered negative IB results. Real-time imaging using spinning disc confocal microscopy show that exposure to PNC-27 of MIA PaCa-2 cells preloaded with mitotracker dye in presence of propidium iodide (PI) results in the expansion within &amp;lt;3min of the cells' plasma membrane followed &amp;lt;6 min by rounding and disruption of the mitochondria and the disappearance of the dye which was paralleled by the rapid appearance of PI in the nuclei. Interestingly, the cells' lysosomes pre-loaded with lysosensor dye remained intact throughout this process strongly suggesting that the cells were not killed due to lysosomal death. We further confirmed the colocalization of PNC-27 and HDM2 in cancer cell plasma membrane by TEM analysis, where cells were stained with gold-conjugated antibodies to HDM2 (6nm) and MRP (15nm) which stained around a ring-like structures in the plasma membrane of tumor cells, but not normal cells. Immuno-gold SEM demonstrated that PNC-27 and HDM2 were stained on the plasma membrane of MIAPaCa-2 cells with multiple 15nm or 6nm gold-conjugated antibodies organized around pore-like structures with diameter of 30–50nm. We have also recently found PNC-27-mediated cytotoxicity in cancer cells to be highly temperature-sensitive and that PNC-27 plasma membrane insertion is temperature-independent whereas pore formation and cytotoxicity is temperature-dependent. These findings suggest that amphipatic PNC-27 is inserted into the plasma membrane of cancer cells where, through random movement and interaction with HDM2, the HDM2-PNC-27 complexes assemble into oligomeric membrane pores. Influx of free PNC-27 molecules through the newly formed membrane pores into the cells' cytoplasm leads to the assembly of pores in mitochondrial membranes and rapid cell death. Absence of HDM2 in normal untransformed cells prevents the formation of membrane pores and, thus, cell death. Citation Information: Cancer Res 2009;69(23 Suppl):C51.

Prognostic significance of epithelial–mesenchymal transition in malignant pleural mesothelioma☆☆☆

Epithelial-to-mesenchymal transition (EMT) is a morphologic transdifferentiation of carcinomas, conferring increased tumour invasiveness, but may also be applied to the epithelioid versus sarcomatoid histotype of malignant pleural mesothelioma (MPM). Herein, we correlated proteins of a putative MPM-EMT axis, including periostin, epidermal growth factor receptor (EGFR), integrin beta1, phosphatase and tensin homologue (PTEN), integrin-linked kinase (ILK), p21 and p27, with clinico-pathologic parameters, in particular overall survival (OS). A retrospective cohort of 352 mostly untreated patients with MPM was investigated by immunohistochemistry of a tissue microarray. Protein expression intensities were semi-quantitatively scored from 0 to 3 in their respective compartments, including peritumoural stroma as well as tumour cell plasma membrane, cytoplasm or nucleus. Data were correlated with histotype and survival outcome. A total of 32% of the tumours were diagnosed as epithelioid, 13% as sarcomatoid and 55% as biphasic histotype. High expression of membranous EGFR and integrin beta1 as well as nuclear p27 correlated with the epithelioid and high expression of cytoplasmic tumoural and stromal periostin with the sarcomatoid histotype. The median survival time of the 128 patients with complete follow-up data was 11.7 months. Univariate survival analysis revealed age, epithelioid histotype and any therapy as prognosticators for better OS. High expression of cytoplasmic PTEN or ILK as well as high expression of nuclear p21 or p27 correlated with increased, whereas high expression of cytoplasmic periostin with decreased OS (all p values <0.05). Multivariate Cox regression revealed any treatment, low cytoplasmic periostin and high cytoplasmic PTEN as independent prognosticators for better OS. Activation of periostin-triggered EMT is associated with the sarcomatoid histotype and has an impact on shorter survival of MPM patients. Finally, only the high expression of PTEN and the low expression of cytosolic periostin could be shown to be independent prognostic factors for longer OS.

LAT1 expression in non-small-cell lung carcinomas: Analyses by semiquantitative reverse transcription-PCR (237 cases) and immunohistochemistry (295 cases)

Objective System l-amino acid transport mediates the uptake of aromatic neutral amino acids and nutritionally essential amino acids from extracellular fluids. Little is known about the role of l-amino acid transporter 1 (LAT1), a member of the system l-amino acid transporter family, in non-small-cell lung carcinomas (NSCLCs). Patients and methods We examined (i) LAT1 mRNA levels in 40 normal lung tissues (NLTs) and 237 NSCLCs using semiquantitative RT-PCR, (ii) LAT1 protein expression in 295 NSCLCs using immunohistochemistry, and (iii) whether LAT1 mRNA and protein expressions were related to clinicopathologic findings and outcome. Results The LAT1 mRNA level was significantly higher in all NSCLCs (6.81 ± 1.13) than in NLT (1.00 ± 0.18). The LAT1 mRNA level showed no association with clinicopathologic findings or outcome. LAT1 protein was detected with a diffuse or granular appearance within the cytoplasm and/or on the plasma membrane of tumor cells. When tumors were graded as positive if staining indicating a plasma membrane expression of LAT1 protein made up more than 10% of the tumor, the frequency of this membrane expression was found to be associated with tumor histology, differentiation grade, pathologic stage, T classification, pleural invasion, lymph-vessel invasion, and overall survival rate. Conclusion Detection of a plasma membrane expression of LAT1 protein would appear to be of value in informing the prognosis in NSCLC cases.

Highly Efficient In Vivo Agonist-Induced Internalization of sst2 Receptors in Somatostatin Target Tissues

The successful peptide receptor imaging of tumors, as exemplified for somatostatin receptors, is based on the overexpression of peptide receptors in selected tumors and the high-affinity binding to these tumors of agonist radioligands that are subsequently internalized into the tumor cells in which they accumulate. Although in vitro studies have shown ample evidence that the ligand-receptor complex is internalized, in vivo evidence of agonist-induced internalization of peptide receptors, such as somatostatin receptors, is missing. Rats subcutaneously transplanted with the somatostatin receptor subtype 2 (sst(2))-expressing AR42J tumor cells were treated with intravenous injections of various doses of the sst(2) agonist [Tyr(3), Thr(8)]-octreotide (TATE) or of the sst(2) antagonist 1,4,7,10-tetraazacyclododecane-N,N',N'',N''',-tetraacetic acid (DOTA)-Bass and were sacrificed at various times ranging from 2.5 min to 24 h after injection. The tumors and pancreas were then removed from each animal. All tissue samples were processed for sst(2) immunohistochemistry using sst(2)-specific antibodies. Compared with the sst(2) receptors in untreated animals, which localized at the plasma membrane in pancreatic and AR42J tumor cells, the sst(2) receptors in treated animals are detected intracellularly after an intravenous injection of the agonist TATE. Internalization is fast, as the receptors are already internalizing 2.5 min after TATE injection. The process is extremely efficient, as most of the cell surface receptors internalize into the cell and are found in endosomelike structures after TATE injection. The internalization is most likely reversible, because 24 h after injection the receptors are again found at the cell surface. The process is also agonist-dependent, because internalization is seen with high-affinity sst(2) agonists but not with high-affinity sst(2) antagonists. The same internalization properties are seen in pancreatic and AR42J tumor cells. They can further be confirmed in vitro in human embryonic kidney-sst(2) cells, with an immunofluorescence microscopy-based sst(2) internalization assay. These animal data strongly indicate that the process of in vivo sst(2) internalization after agonist stimulation is fast, extremely efficient, and fully functional under in vivo conditions in neoplastic and physiologic sst(2) target tissues. This molecular process is, therefore, likely to be responsible for the high and long-lasting uptake of sst(2) radioligands seen in vivo in sst(2)-expressing tumors.

N-cadherin serves as diagnostic biomarker in intrahepatic and perihilar cholangiocarcinomas

As a definite immunoprofile of this tumor is missing, the histopathologic diagnosis of intrahepatic cholangiocarcinoma is difficult. The aim of this study was to explore E- and N-cadherin expressions in intrahepatic bile duct tumors, and to determine their potential interest in differential diagnosis. Normal liver tissue, 5 cirrhosis with ductular reaction, 5 focal nodular hyperplasia, 5 bile duct hamartomas, 5 bile duct adenomas, and 45 intrahepatic cholangiocarcinomas from Causasian patients were studied. Tissue-microarrays including 20 esophageal, 86 gastric, 8 small bowel, 64 colonic, 18 pancreatic, 6 gallbladder, and 7 extrahepatic biliary tract adenocarcinomas, 22 hepatocellular carcinomas, and normal tissues were constructed. Immunohistochemistry was performed using E-cadherin, N-cadherin, NCAM, Hep Par1, and cytokeratins 7, 19 and 20. Immunoblot analysis of frozen liver tissues was performed to control the specificity of E- and N-cadherin antibodies used. In normal liver, epithelial cells of intrahepatic bile ducts, whatever their caliber, as well as hepatocytes, coexpressed E- and N-cadherins at their plasma membranes. In cirrhosis, ductular reactions completely expressed E- and N-cadherins. All the benign lesions and 30 of the 45 intrahepatic cholangiocarcinomas (23/29 peripheral and 7/16 hilar) also expressed N-cadherin. E-cadherin was detected in all the lesions. The expression of N-cadherin at the plasma membrane of tumor cells was significantly more frequent in peripheral than in hilar intrahepatic cholangiocarcinomas (P=0.003). Among noncholangiocarcinomas, only 1% gastric and 66% gallbladder adenocarcinomas and all the hepatocellular carcinomas expressed N-cadherin at the membrane of tumor cells. Finally, for the diagnosis of intrahepatic cholangiocarcinomas, the specificity value of membranous expression of N-cadherin was 88%, whereas that of the combination cytokeratin 7/membranous N-cadherin was 98%. In the gastrointestinal and liver tract, membranous N-cadherin is restricted to the hepatocytes and intrahepatic biliary cells. In combination with cytokeratin 7 and Hep Par1, N-cadherin is a reliable tool for the histopathological diagnosis of primary hepatic tumors.

Autologous Large Multivalent Immunogen Vaccine in Patients With Metastatic Melanoma and Renal Cell Carcinoma

To evaluate the safety and activity of large multivalent immunogen (LMI), prepared by immobilizing autologous tumor cell plasma membrane on 5-microm diameter silica beads, in patients with melanoma and renal cell carcinoma (RCC). Thirty patients with stage IV metastatic melanoma and 31 patients with stage IV RCC were randomly assigned to 1 of 3 trial arms and received monthly treatment with (1) LMI alone, (2) cyclophosphamide followed 8 days later with LMI, or (3) the same treatment as in arm 2 with IL-2 given for 5 days beginning 1 week after LMI administration. No grade 4 toxicities were observed. For patients with melanoma, median overall survival time for all 30 patients was 20.4 months [95% confidence interval (CI): 8.0-not assessable], and median progression-free survival was 2.8 months (95% CI: 1.9-6.3). For patients with RCC, median overall survival exceeded 46.2 months (95% CI: 30.3-not assessable), and median progression-free survival was 12.2 months (95% CI: 4.6-not assessable). Two patients had a partial response to LMI treatment. Based on our results that demonstrate the safety and tolerability of LMI vaccine, further development of this therapy is warranted to evaluate its clinical efficacy.