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Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf–Mycl-driven mouse model of SCLC. RLF–MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF–MYCL genetically engineered mouse model displayed gene expression similarities with human RLF–MYCL SCLC. Together, our studies support RLF–MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC.Significance:The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF–MYCL gene fusion by developing a Rlf–Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis.This article is highlighted in the In This Issue feature, p. 2945

Off-target editing is one of the main safety concerns for the use of CRISPR-Cas9 genome editing in gene therapy. These unwanted modifications could lead to malignant transformation, which renders tumorigenicity assessment of gene therapy products indispensable. In this study, we established two in vitro transformation assays, the soft agar colony-forming assay (SACF) and the growth in low attachment assay (GILA) as alternative methods for tumorigenicity evaluation of genome-edited cells. Using a CRISPR-Cas9-based approach to transform immortalized MCF10A cells, we identified PTPN12, a known tumor suppressor, as a valid positive control in GILA and SACF. Next, we measured the limit of detection for both assays and proved that SACF is more sensitive than GILA (0.8% versus 3.1% transformed cells). We further validated SACF and GILA by identifying a set of positive and negative controls and by testing the suitability of another cell line (THLE-2). Moreover, in contrast to SACF and GILA, an in vivo tumorigenicity study failed to detect the known tumorigenic potential of PTPN12 deletion, demonstrating the relevance of GILA and SACF in tumorigenicity testing. In conclusion, SACF and GILA are both attractive and valuable additions to preclinical safety assessment of gene therapy products., Graphical abstract, Anchorage-independent growth is a feature of oncogenic transformed cells. Libertini and colleagues optimized and validated two in vitro transformation assays based on this criterion to be used on CRISPR-Cas9 genome-edited cells. Results demonstrate that these assays could be used for preclinical safety assessment of gene therapy products.

Murine tumor modeling is fundamental for the preclinical development of anti-cancer therapies. Use of immunocompetent mouse models is becoming increasingly relevant as we gain more knowledge of how cancer cells interact with the immune system in the tumor microenvironment and how we can harness the immune system to fight tumors. However, there are few intrinsically immunogenic preclinical tumor models, and the vast majority either do not respond to therapy or do not faithfully predict the responses of the therapy when applied in the clinic. Here, we discuss the limitations of commonly used murine tumor models in immuno-oncology and strategies to improve their immunogenicity and mutational burden to more accurately reflect the heterogeneity of patient tumors. © 2022 Wiley Periodicals LLC.

The utilization of suitable mouse models is a critical step in the drug discovery oncology workflow as their generation and use are important for target identification and validation as well as toxicity and efficacy assessments. Current murine models have been instrumental in furthering insights into the mode of action of drugs before transitioning into the clinic. Recent advancements in genome editing with the development of the CRISPR/Cas9 system and the possibility of applying such technology directly in vivo have expanded the toolkit of preclinical models available. In this review, a brief presentation of the current models used in drug discovery will be provided with a particular emphasis on the novel CRISPR/Cas9 models., Lay abstract Picking the right animal model is a critical step in the drug discovery oncology workflow. Current murine models have provided a key contribution to the advancement and refinement of drugs before transitioning into the clinic. The application of the CRISPR)/Cas system as a tool to genetically engineer preclinical models based on genomic information has revolutionized the field by reducing the cost and the time and while increasing versatility. In this review, the authors will provide a brief overview of preclinical models employed in drug discovery, with particular attention to the ones generated with the CRISPR/Cas9 technology.

Most experimental oncology therapies fail during clinical development despite years of preclinical testing rationalizing their use. This begs the question of whether the current preclinical models used for evaluating oncology therapies adequately capture patient heterogeneity and response to therapy. Most of the preclinical work is based on xenograft models where tumor mis-location and the lack of the immune system represent a major limitation for the translatability of many observations from preclinical models to patients. Genetically engineered mouse models (GEMMs) hold great potential to recapitulate more accurately disease models but their cost and complexity have stymied their widespread adoption in discovery, early or late drug screening programs. Recent advancements in genome editing technology made possible by the discovery and development of the CRISPR/Cas9 system has opened the opportunity of generating disease-relevant animal models by direct mutation of somatic cell genomes in an organ or tissue compartment of interest. The advent of CRISPR/Cas9 has not only aided in the production of conventional GEMMs but has also enabled the bypassing of the construction of these costly strains. In this review, we describe the Somatically Engineered Mouse Models (SEMMs) as a new category of models where a specific oncogenic signature is introduced in somatic cells of an intended organ in a post-natal animal. In addition, SEMMs represent a novel platform to perform in vivo functional genomics studies, here defined as DIVoS (Direct In Vivo Screening).

Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf–Mycl-driven mouse model of SCLC. RLF–MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF–MYCL genetically engineered mouse model displayed gene expression similarities with human RLF–MYCL SCLC. Together, our studies support RLF–MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC. Significance: The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF–MYCL gene fusion by developing a Rlf–Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis. This article is highlighted in the In This Issue feature, p. 2945

The ability to engineer specific mutations in mice has proven essential to advancing our understanding of the molecular basis of cancer. Chromosomal rearrangements, a common and clinically relevant class of cancer-causing mutations, have however remained difficult to faithfully recapitulate in vivo. The development of genetic tools for in vivo somatic genome editing has recently overcome this limitation and led to the generation of more sophisticated and accurate preclinical models of human cancers. Here, we review the potential applications of these new technologies to the study of tumor biology and discuss their advantages over more conventional strategies, their limitations, and the remaining challenges. Cancer Res; 76(17); 4918–23. ©2016 AACR.

Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions. A recently discovered example is a fusion between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK), generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC) and is clinically relevant because it confers sensitivity to ALK inhibitors. Despite their importance, modelling such genetic events in mice has proven challenging and requires complex manipulation of the germ line. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumours invariably harbour the Eml4-Alk inversion, express the Eml4-Alk fusion gene, display histopathological and molecular features typical of ALK(+) human NSCLCs, and respond to treatment with ALK inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms.

Progression to castration resistance is a major problem in the treatment of advanced prostate cancer and is likely to be driven by activation of several molecular pathways, including androgen receptor (AR) and cyclic AMP-dependent protein kinase A (PKA). In this study, we examined the therapeutic efficacy of a combined inhibition of the AR and the regulatory subunit type Iα (RIα) of protein kinase A with second generation antisense oligonucleotides (ODNs) in androgen-sensitive LNCaP and castration-resistant LNCaPabl tumors in vivo. We found that targeting the AR alone inhibited LNCaP, as well as LNCaPabl tumors. Combined inhibition resulted in an improved response over single targeting and even a complete tumor remission in LNCaPabl. Western blot analysis revealed that both ODNs were effective in reducing their target proteins when administered alone or in combination. In addition, treatment with the ODNs was associated with an induction of apoptosis. Our data suggest that dual targeting of the AR and PKARIα is more effective in inhibiting LNCaP and LNCaPabl tumor growth than single treatment and may give a treatment benefit, especially in castration-resistant prostate cancers.

To cite this article: Obiri DD, Flink N, Maier JV, Neeb A, Maddalo D, Thiele W, Menon A, Stassen M, Kulkarni RA, Garabedian MJ, Barrios AM, Cato ACB. PEST-domain-enriched tyrosine phosphatase and glucocorticoids as regulators of anaphylaxis in mice. Allergy 2012; 67: 175–182. Abstract Background: PEST-domain-enriched tyrosine phosphatase (PEP) is a protein tyrosine phosphatase exclusively expressed in hematopoietic cells. It is a potent negative regulator of T-cell receptor signalling that acts on receptor-coupled protein tyrosine kinases. PEST-domain-enriched tyrosine phosphatase is also expressed in mast cell and is positively regulated by glucocorticoids, but its function is unknown. In this communication, the function of PEP is analysed in mast cells. Methods: Signal transduction cascades following IgE receptor cross-linking were compared in bone marrow-derived mast cells (BMMC) from PEP−/− and PEP+/+ mice. Furthermore, antigen-induced passive systemic anaphylaxis (PSA) was analysed in PEP+/+ and PEP−/− mice. Results: Bone marrow-derived mast cells from PEP−/− mice showed impaired PLCγ1 phosphorylation and Ca2+ mobilization. Additionally, mice deficient in PEP showed impaired mast cell degranulation and were less susceptible to PSA. Treatment of wild-type BMMC or mice with an Au(I)–phosphine complex that selectively inhibits PEP activity produced defects in Ca2+ signalling pathway and reduced anaphylaxis similar to that caused by the deletion of the PEP gene. Glucocorticoid that negatively regulates a wide range of mast cell action increased PEP expression and only partially inhibited anaphylaxis. However, glucocorticoid potently inhibited anaphylaxis when combined with the PEP inhibitor. Conclusions: PEST-domain-enriched tyrosine phosphatase is an important positive regulator of anaphylaxis. Pharmacological inhibition of its activity together with glucocorticoid administration provide an effective rescue for PSA in mice.

Background MicroRNAs (miRNAs) are potential biomarkers in various malignancies. We aim to characterize miRNA expression in intrahepatic cholangiocarcinoma (ICC) and identify circulating plasma miRNAs with potential diagnostic and prognostic utility. Methods Using deep-sequencing techniques, miRNA expression between tumor samples and non-neoplastic liver parenchyma were compared. Overexpressed miRNAs were measured in plasma from an independent cohort of patients with cholangiocarcinoma using RT-qPCR and compared with that healthy volunteers. The discriminatory ability of the evaluated plasma miRNAs between patients and controls was evaluated with receiving operating characteristic (ROC) curves. Results Small RNAs from 12 ICC and 11 tumor-free liver samples were evaluated. Unsupervised hierarchical clustering using the miRNA expression data showed clear grouping of ICC vs. non-neoplastic liver parenchyma. We identified 134 down-regulated and 128 upregulated miRNAs. Based on overexpression and high fold-change, miR21, miR200b, miR221, and miR34c were measured in plasma from an independent cohort of patients with ICC (n = 25) and healthy controls (n = 7). Significant overexpression of miR-21 and miR-221 was found in plasma from ICC patients. Furthermore, circulating miR-21 demonstrated a high discriminatory ability between patients with ICC and healthy controls (AUC: 0.94). Conclusion Among the differentially expressed miRNAs in ICC, miR-21 and miR-221 are overexpressed and detectable in the circulation. Plasma expression levels of these miRNAs, particularly miR-21, accurately differentiates patients with ICC from healthy controls and could potentially serve as adjuncts in diagnosis. Prospective validation and comparison with other hepatobiliary malignancies is required to establish their potential role as diagnostic and prognostic biomarkers.

Background The Bag (Bcl-2 associated athanogene) family of proteins consists of 6 members sharing a common, single-copied Bag domain through which they interact with the molecular chaperone Hsp70. Bag5 represents an exception in the Bag family since it consists of 5 Bag domains covering the whole protein. Bag proteins like Bag1 and Bag3 have been implicated in tumor growth and survival but it is not known whether Bag5 also exhibits this function. Methods Bag5 mRNA and protein expression levels were investigated in prostate cancer patient samples using real-time PCR and immunoblot analyses. In addition immunohistological studies were carried out to determine the expression of Bag5 in tissue arrays. Analysis of Bag5 gene expression was carried out using one-way ANOVA and Bonferroni’s Multiple Comparison test. The mean values of the Bag5 stained cells in the tissue array was analyzed by Mann-Whitney test. Functional studies of the role of Bag5 in prostate cancer cell lines was performed using overexpression and RNA interference analyses. Results Our results show that Bag5 is overexpressed in malignant prostate tissue compared to benign samples. In addition we could show that Bag5 levels are increased following endoplasmic reticulum (ER)-stress induction, and Bag5 relocates from the cytoplasm to the ER during this process. We also demonstrate that Bag5 interacts with the ER-resident chaperone GRP78/BiP and enhances its ATPase activity. Bag5 overexpression in 22Rv.1 prostate cancer cells inhibited ER-stress induced apoptosis in the unfolded protein response by suppressing PERK-eIF2-ATF4 activity while enhancing the IRE1-Xbp1 axis of this pathway. Cells expressing high levels of Bag5 showed reduced sensitivity to apoptosis induced by different agents while Bag5 downregulation resulted in increased stress-induced cell death. Conclusions We have therefore shown that Bag5 is overexpressed in prostate cancer and plays a role in ER-stress induced apoptosis. Furthermore we have identified GRP78/BiP as a novel interaction partner of Bag5.

BACKGROUND AGR2 is a member of the endoplasmatic reticulum protein disulphide isomerase gene family implicated in tumor metastasis. Its expression pattern, function, and utility as a marker remains to be further investigated. METHODS Using real-time RT-PCR and immunohistochemistry, changes of expression in different tumor stages were explored in microdissected tumor samples. AGR2 transcript level in urine sediments was scrutinized for suitability as a tumor marker. AGR2 androgen regulation and function were analyzed in cellular prostate cancer models. RESULTS AGR2 is highly expressed in prostate cancer compared to benign tissue in particular also in low-grade tumors and PIN lesions. AGR2 transcripts were detected in urine sediments of patients undergoing prostate biopsy with significantly higher levels in tumor patients. The urine AGR2/PSA transcript ratio allowed much better discrimination between cancer and benign patients than serum total PSA or %freePSA. Prostate tumor cells express and secrete variable amounts of AGR2 protein, the highest level was found in PC3 cells. In androgen receptor-positive cell lines AGR2 is upregulated by androgens. Increased expression enhanced the migratory and invasive potential but decreased growth and proliferation in vitro and in vivo. CONCLUSION AGR2 enhances the invasion phenotype of prostate cancer cells while at the same time attenuating cell-cycle progression. This function, its expression pattern and the increased level of AGR transcripts in urine sediments of prostate cancer patients call for further exploration as a prostate cancer marker and a modulator of tumor growth and invasion. Prostate 71:575–587, 2011. © 2010 Wiley-Liss, Inc.

Introduction: Chromosomal rearrangements are often detected in several types of cancer and result in expression of aberrant genes with oncogenic functions. In spite of their importance in tumor formation, modeling such rearrangements has been proven challenging and requires heavy and long germline manipulation. Purpose of the study: in this study we propose to induce chromosomal rearrangements in vivo by delivering the CRISPR/Cas9 system making use of viral vectors. Experimental procedures: for in vitro studies we transfected NIH/3T3 cells with plasmids expressing the Cas9 protein and the specific sgRNA. For in vivo delivery of the CRISPR/Cas9 system to the lung of adult mice we made use of Adenoviral vectors containing the Cas9 and two sgRNAs expressed under the control of two independent U6 promoters. Imaging of the lungs was performed with μCT scan. Results: two sgRNAs to direct the endonuclease Cas9 to the intron 19 of Alk and the intron 14 of Eml4 to induce the Eml4-Alk inversion were designed to induce Eml4-Alk inversion, detected in a subset of patients with non-small cell lung cancer (NSCLC). Co-delivery of the sgRNAs and the Cas9 resulted in the expected rearrangements, indicating that two sgRNAs and the endonuclease are sufficient to induce chromosomal rearrangements in vitro. For in vivo delivery to the lung of adult mice, we generated an adenoviral system for the concomitant delivery of the Cas9 and the two sgRNAs (Ad-EA) or the Cas9 alone (Ad-Cas9). Within 8 weeks post-infection with Ad-EA, mice developed lung adenomas while mice infected with Ad-Cas9 were tumor free. All the tumors carried the genomic inversion Eml4-Alk and were negative for p63 and positive for TTF-1 resembling the histological feature of NSCLC. Finally we tested the sensitivity of the tumors to the ALK/MET inhibitor crizotinib, used in therapy for the treatment of ALK+ NSCLC. Radiological and histological analysis showed in all cases tumor regression, indicating that our CRISPR-induced mouse model closely resembles human NSCLC. Conclusion: In conclusion the strategy here presented expands significantly our possibility of modeling cancers driven by chromosomal rearrangements and represents an opportunity to test novel targeted therapies and thee mechanisms leading to drug resistance. Citation Format: Danilo Maddalo. In vivo chromosomal engineering with the CRISPR/Cas9 model: a new frontier for mouse modeling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-131. doi:10.1158/1538-7445.AM2015-LB-131

Nature 516, 423–427 (2014); doi:10.1038/nature13902 During the assembly of Fig. 1b of this Letter, the sequences of the targeted regions in Eml4 and Alk, shown in the schematic in the top panel, were inadvertently swapped. In addition, the legend for the top panel of Fig. 1b should have read: “b, A schematic of the loci before and after the inversion with the location of the primers used (top panel).

Purpose of the study: the purpose of this study is to investigate the role of the Bcl-2 associated athanogene 5 (Bag5) protein in stress tolerance and chemoresistance development in prostate cancer cells. Introduction: the Bcl-2 associated athanogene (Bag) family of proteins consists of six members sharing a common Bag domain that mediates the interaction with the molecular chaperone Hsp70. Bag family members like Bag1 and Bag3 have been already reported to be involved in the development of several cancers like breast prostate and lung cancer. However, the role of Bag5 in cancer has not been described yet. Considering the involvement of the Bag proteins in stress tolerance, we therefore investigated the role of Bag5 upon stress induction and its ability to induce tolerance to stress-inducing chemotherapeutic agents. Experimental procedures: for gene expression analysis real-time PCR assay was used. Immunofluorescence and cell fractionation were carried to analyze subcellular protein localization. Co-immunoprecipitation and GST-pull down assays were employed for protein-protein interaction analysis. GRP78 enzymatic activity was measured by in vitro ATPase assay and by intracellular refolding experiment. Clonogenic assay was used to determine cell growth upon chemotherapeutic agents treatment. Western blot experiment and Caspase-3 cleavage measurement were used to analyze stress induced cell death and activation of the unfolded protein response. Results: In order to determine if Bag5 plays a role in stress response, Bag5 gene and protein expression levels were analyzed upon treatment with the stress inducers thapsigargin and tunicamycin in 22Rv.1 and PC3 prostate cancer cell lines. In both cases we could appreciate a significant increase of Bag5 levels indicating its stress inducibility. Intriguingly we could observe that Bag5 accumulates into the endoplasmic reticulum once stress is induced. Moreover we could determine that Bag5 binds to the ER chaperone GRP78/BiP, enhancing its enzymatic activity. Since GRP78 plays a major role in the regulation of the unfolded protein response (UPR), we wanted to determine if Bag5 levels could influence UPR activation. Stable transfection of Bag5 in 22Rv.1 cells attenuated UPR activation as well as apoptosis compared to the control upon stress induction. Consistently with this observation, when Bag5 gene levels were reduced, cells became more sensitive to stress induction. Since UPR activation and stress tolerance represent the basic mechanism for chemoresistance development, we analyzed the effect of Bag5 overexpression when cells were treated with a range of chemotherapeutic agents including fenretinide, casodex, (−)epigallocatechingalle and taxol. Colony forming assay and Caspase-3 cleavage measurement showed in all the treatments an increased survival in Bag5 overexpressing cells compared to the vector control, indicating that Bag5 indeed plays a role in prostate cancer cell survival and might be responsible for chemoresistance development. Conclusions: In this work we showed for the first time that Bag5 can interact with the molecular chaperone GRP78/BiP and influence UPR activation. In addition, we provided the very first evidence that Bag5 could play a role in development of stress tolerance in prostate cancer cells.

Purpose of the study: The purpose of the study is to identify sequence(s) in Bag-1 proteins responsible for growth inhibition and to eventually derive a short peptide for cancer therapy. Introduction: Bag-1 family of proteins consists of four members (Bag- 1L, 1M, 1 and 1S) originating from different translation-initiation sites on the same mRNA. Clinical reports have shown a dual role of the Bag-1 proteins in tumor progression and in the inhibition of tumor growth. In this work we have identified a 19 amino acid sequence in the Bag-1 proteins that reduces prostate cancer cell growth ex vivo and in vivo. Experimental procedures: Inhibition of prostate cancer cell growth was analyzed in clonogenic assay and in athymic nude mouse xenograft models. Histological analysis for apoptosis was carried out on paraffin-embedded tumor samples. Structural analyses were carried out by NMR and circular dichroism. Interaction partners of the Bag-1 peptides were analyzed by GST-pull down and by co-immunoprecipitation assays. Significance of cellular levels of glucose-regulated proteins (GRP) 75 and 78 for the growth inhibitory action of the peptide was demonstrated by RNA interference experiments. Results: Overexpression of Bag-1 proteins in 22Rv.1 prostate tumor cells reduced the growth of these cells in a colony forming assay. A study making use of several deletion mutants identified a 69 amino acid Bag-1 sequence as responsible for the growth inhibition. NMR and circular dichroism studies showed this peptide to be unstructured but it nonetheless interacted with the substrate binding domain of GRP 75 and 78. The cellular levels of the GRPs were shown by RNA interference studies to be essential for the action of the peptide. Overexpression of the peptide in the 22Rv1 cells decreased growth in proliferation assay and in xenograft tumor models. The decreased growth in vivo was demonstrated to be due to apoptosis of the Bag-1 peptide expressing cells as showed by immuohistochemical analysis. The growth inhibitory activity could be narrowed down to a 7 amino acid core within a 19 amino acid Bag-1 peptide sequence. Amino acid exchanges in the core region impaired binding of the peptide to the GRPs and inhibition of tumor cell growth. Colony formation assay using a wide panel of prostate cells showed that the 19 amino acid peptide inhibits growth of only malignant but not benign cells and this correlates with the level of expression of the two GRP target proteins in the cells. Conclusion: Together these results demonstrate that a short peptide sequence derived from Bag-1 proteins that binds GRP75/78 specifically inhibits the growth of prostate cancer cells. 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 5403. doi:10.1158/1538-7445.AM2011-5403

Introduction and objectives: Bag1 (Bcl-2 associated athanogene-1) is a family of proteins conserved throughout evolution, promoting cell survival and inhibiting apoptosis under stressful conditions. They are found overexpressed in several types of tumors and associated with poor prognosis. Intriguingly, the nuclear isoform of the Bag-1 proteins, Bag-1L, also exhibits pro-apoptotic properties through an unknown mechanism. In extensive deletion mutagenesis studies we identified sequences in the Bag-1L protein that exert pro-apoptotic action. The objective of the study is to characterize the mechanism of action of these sequences and to explore their use as candidate peptides for prostate cancer therapy. Materials and Methods: In vitro inhibition of tumor cell growth was analyzed in clonogenic assay where prostate cells were transfected with plasmids expressing different Bag-1L peptides. In vivo growth inhibition was analysed by the injection of prostate cells stably expressing the Bag-1L peptides into the flank of athymic nude mice and analyzing their effect on tumor size in vivo. Histological analysis for apoptosis was carried out on the tumor samples. For analyzing the interaction partners of the Bag-1L peptides, the peptides were generated as fusion proteins with glutathione S transferase (GST), immobilized on glutathione agarose beads and allowed to interact with lysates from prostate cells. Specifically bound proteins were then eluted and analysed by mass spectrometry and by Western blotting. Results: Overexpression of a 68 amino acid sequence of Bag-1L reduced the growth of 22Rv.1 prostate cancer cells but not of benign prostate hyperplasia cells in a clonogenic assay. Stable overexpression of this peptide in the 22Rv.1 prostate cancer cells also reduced cell growth in vivo in athymic nude mice. Histological analysis revealed massive apoptosis in the tumor cells expressing the peptide. Further deletion analysis of the 68 amino acid peptide showed that the N-terminal 40 amino acids but not the C-terminal 29 amino acids are sufficient for the growth inhibitory action. Protein-protein interaction studies using GST-pull down assay demonstrated that the growth inhibitory function of the peptides correlated with their ability to bind glucose regulated proteins 75 and 78 (GRP75 and GRP78). GRP75 and GRP78 have been implicated in tumor formation through their action in the mitochondria and in the endoplasmic reticulum in the regulation of the unfolded protein response. Conclusions: We have therefore identified Bag1-L peptides which when expressed in prostate tumor cells reduce the growth in vitro as well as in a mouse model of tumor formation. The action of the peptides is to induce apoptosis and it is correlated with their ability to bind the molecular chaperones GRP75 and GRP78. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A216.

Peptide ligands that specifically target tumor cells represent useful tools for cancer therapy. They can be used for the transport and specific delivery of cell death-inducing sequences or chemotoxic drugs to tumor cells. Here we describe a peptide from a conserved region of Bag-1 (Bcl-2 associated athanogene-1) proteins that induces cell death without the need for a pro-drug conjugate or fusion with a death-inducing sequence. Stable expression of the Bag-1 peptide in 22Rv.1 prostate tumor cells interferes with tumor cell growth and survival in vivo and in vitro and elevates the level of apoptosis in tumor cells treated with the topoisomerase inhibitor II etoposide. Structural analysis using NMR and circular dichronism (CD) show that this peptide is completely unstructured. Nevertheless it interacts with specific sequences of the glucose regulated proteins GRP75 and GRP78. Domain mapping studies identified the ATPase domain of GRP78 and the substrate-binding domain of GRP75 as interaction sites of the Bag1-peptide. Both GRP75 and GRP78 belong to the heat shock protein family and act as protective chaperones in the unfolded protein response. In addition both proteins are over expressed in many human tumors and are found exclusively on the surface of tumor cells. In particular, the GRP78 protein that is present on the surface of tumor cells is glycosylated. Using deglycosylation and GST-pull down analysis, we can show that the Bag1-peptide also binds to the glycosylated form of GRP78 indicating its ability to target tumor cells by binding to the cell surface. Thus we have identified a candidate peptide for a broad-based tumor therapy that function by interfering with the action of GRP75 and GRP78. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B85.