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Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma (NB), but resistance to ALK inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing NB; inhibition of miR-1304-5p decreased, while mimics increased the sensitivity of NB cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in NB, inducing apoptosis both in vitro and in vivo. In particular, on combined treatment of NB patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth was observed although tumours rapidly regrew on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk NB although prolonged therapy is likely required to prevent relapse, rendering high-risk NB a chronic rather than a lethal disease.

Resistance to anaplastic lymphoma kinase (ALK)-targeted therapy in ALK-positive non-small cell lung cancer has been reported, with the majority of acquired resistance mechanisms relying on bypass signaling. To proactively identify resistance mechanisms in ALK-positive neuroblastoma (NB), we herein employ genome-wide CRISPR activation screens of NB cell lines treated with brigatinib or ceritinib, identifying PIM1 as a putative resistance gene, whose high expression is associated with high-risk disease and poor survival. Knockdown of PIM1 sensitizes cells of differing MYCN status to ALK inhibitors, and in patient-derived xenografts of high-risk NB harboring ALK mutations, the combination of the ALK inhibitor ceritinib and PIM1 inhibitor AZD1208 shows significantly enhanced anti-tumor efficacy relative to single agents. These data confirm that PIM1 overexpression decreases sensitivity to ALK inhibitors in NB, and suggests that combined front-line inhibition of ALK and PIM1 is a viable strategy for the treatment of ALK-positive NB independent of MYCN status., Anaplastic lymphoma kinase (ALK) inhibitors are currently being considered in neuroblastoma (NB), but its acquired resistance is reported in non-small cell lung cancers. Here, the authors have found PIM1 overexpression decreases sensitivity to ALK inhibitors in NB and combined ALK and PIM1 inhibition enhances anti-tumour efficacy in vitro and in PDX models.

Anaplastic lymphoma kinase (ALK)-targeted therapy is a promising strategy for the treatment of ALK-positive neuroblastoma (NB). Several ALK tyrosine kinase inhibitors (ALK TKIs) have been approved for the treatment of Non-Small Cell Lung Carcinoma (NSCLC) and are currently in clinical trials for NB therapy. Recent evidence reported that ALK TKI resistance can occur in cancer patients, making the long-term efficacy of these therapies challenging. With the use of high-throughput and genome-wide CRISPR-Cas9 knockout screens, we identified loss of genes associated with decreased sensitivity to ALK TKIs in ALK-positive NB cells. Specifically, we discovered that CRISPR-Cas9 mediated knockout of miR-1304-5p desensitises ALK-positive NB to the ALK TKIs brigatinib and ceritinib. Inhibition of miR-1304-5p decreased, while mimics increased the sensitivity of NB cells to these ALK TKIs. Furthermore, we showed that miR-1304-5p targets the RAS/MAPK pathway, thereby decreasing cell viability via induction of apoptosis. Among the targets of miR-1304-5p, NRAS expression levels are of prognostic significance to NB patients suggesting it may drive an aggressive disease phenotype. Indeed, a combination treatment applying ALK TKIs and miR-1304-5p mimics increased treatment response. A similar effect was achieved using RAS pharmacological inhibition, which resulted in synergistic activity in MYCN Wild Type (WT) but had less effect on MYCN-amplified cells, suggesting that this pathway could be differentially regulated according to MYCN status. miR-1304-5p and NRAS expression, in patient-derived xenografts (PDXs) of high-risk NB harbouring ALK mutations, confirmed the relevance of this regulatory axis in NB and suggested that the reduced ALK TKI response in MYCN-positive PDXs could be due to inhibition of miR-1304-5p, and the combination of ALK and NRAS inhibition showed extraordinary anti-tumor efficacy compared to single agents. These findings suggest that the miR-1304-5p/NRAS axis alters the sensitivity of NB to ALK inhibitors and suggest that the modulation of this pathway in combination with ALK inhibition is a promising approach to improve NB treatment response and ultimately patient survival. Citation Format: Perla Pucci, Liam Lee, Suzanne Turner, Leila Jahangiri, Jamie Matthews, Amos Burke, Patrick Reynolds, Lukas Kenner, Olaf Merkel, Fikret Rifatbegovic, Ji Luo, Peter Ambros, MiaoJun Han. High-throughput CRISPR-Cas9 knockout screens identify loss of miRNA1304-5p targeting the RAS/MAPK pathway as a modulator of resistance to ALK inhibitors in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5344.

INTRODUCTION Primary liver tumors and liver metastasis currently represent the leading cause of cancer-related deaths. The liver intimately cross-talks with the gut and performs many essential functions related to digestion, metabolism of nutrients, and clearance of bacterial metabolites. Diseased livers are often associated with altered gut bacterial composition, or dysbiosis, and it has been suggested that gut bacterial products contribute to malignant transformation of hepatocytes. The liver is exposed to the gut microbiome through the portal vein and is an immunological organ that is heavily populated by immune cells. Emerging studies have shown that gut commensal bacteria are important regulators of antitumor immunity. Although it has been established that the gut microbiome influences the efficacy of cancer immunotherapy, the role of gut bacteria in antitumor surveillance in the liver is poorly understood. RATIONALE The liver is exposed to gut bacterial metabolites and products by way of blood from the intestine, which comprises 70% of the whole liver blood supply. Changes in the gut microbiome may affect immune cell function in the liver, and commensal bacteria can mediate the metabolism of primary into secondary bile acids, which recirculate back into the liver through the enterohepatic circulation. Given that bile acids are known to be involved in liver cancer development, we focused on the role of bile acids in immunosurveillance of tumors growing in the liver. We altered gut bacteria and examined changes of hepatic immune cells and antitumor immunity directed against liver tumors. Uncovering how the gut microbiome uses bile acids to shape immunity to liver cancer may have future therapeutic applications. RESULTS Using one primary liver model and three liver metastasis models, we found that altering commensal gut bacteria induced a liver-selective antitumor effect. A selective increase of hepatic CXCR6 + natural killer T (NKT) cells was observed, independent of mouse strain, gender, or presence of liver tumors. The accumulated hepatic NKT cells showed an activated phenotype and produced more interferon-γ upon antigen stimulation. In vivo studies using both antibody-mediated cell depletion and NKT-deficient mice confirmed that NKT cells mediated the inhibition of tumor growth in the liver. Further investigation showed that NKT cell accumulation was regulated by the expression of CXCL16, the solo ligand for CXCR6, on liver sinusoidal endothelial cells, which form the lining of liver capillaries and the first barrier for the blood coming from the gut entering the liver. Primary bile acids increased CXCL16 expression, whereas secondary bile acids showed the opposite effect. Removing gram-positive bacteria by antibiotic treatment with vancomycin, which contains the bacteria mediating primary-to-secondary bile acid conversion, was sufficient to induce hepatic NKT cell accumulation and decrease liver tumor growth. Feeding secondary bile acids or colonization of bile acid–metabolizing bacteria, reversed both NKT cell accumulation and inhibition of liver tumor growth in mice with altered gut commensal bacteria. In nontumor liver tissue from human patients with primary liver cancer, primary bile acid chenodeoxycholic acid (CDCA) levels correlated with CXCL16 expression, whereas an inverse correlation was observed with secondary bile acid glycolithocholate (GLCA), suggesting that the finding may apply to humans. CONCLUSION We describe a mechanism by which the gut microbiome uses bile acids as messengers to control a chemokine-dependent accumulation of hepatic NKT cells and antitumor immunity in the liver, against both primary and metastatic liver tumors. These findings not only have possible implications for future cancer therapeutic studies but also provide a link between the gut microbiome, its metabolites, and immune responses in the liver.

Myeloid-derived suppressor cells are key components of tumor-induced immune suppression. They are composed of a heterogeneous population of immature myeloid cells that abrogates innate and adaptive immune responses. Myeloid-derived suppressor cells accumulate not only in peripheral blood, secondary lymphoid organs and tumors, but also in the liver in preclinical tumor models and in hepatocellular carcinoma patients. The liver, continuously exposed to food and microbial antigens from the intestine, avoids autoimmune damage through the use of specialized mechanisms of immune tolerance. In the context of cancer, myeloid-derived suppressor cells profit the intrinsic tolerogenic properties of the liver to accumulate and exert various immune-suppressive and tumor-promoting mechanisms which go from inducing immune cell dysfunction to supporting the generation of liver metastases. In this review, we seek to describe the phenotype, function, accumulation and therapeutic targeting of hepatic myeloid-derived suppressor cells both in preclinical settings and in the context of human hepatocellular carcinoma.

Cross-priming of CD8 + T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8 + T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8 + T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8 + T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells.

PDZ domains represent one group of the major structural units that mediate protein interactions in intercellular contact, signal transduction and assembly of biological machineries. Tax-interacting protein (TIP)-1 protein is composed of a single PDZ domain that distinguishes TIP-1 from other PDZ domain proteins that more often contain multiple protein domains and function as scaffolds for protein complex assembly. However, the biological functions of TIP-1, especially in cell transformation and tumor progression, are still controversial as observed in a variety of cell types. In this study, we have identified ARHGEF7, a guanine nucleotide exchange factor for Rho GTPases, as one novel TIP-1-interacting protein in human glioblastoma cells. We found that the presence of TIP-1 protein is essential to the intracellular redistribution of ARHGEF7 and rhotekin, one Rho effector and the spatiotemporally coordinated activation of Rho GTPases (RhoA, Cdc42 and Rac1) in migrating glioblastoma cells. TIP-1 knockdown resulted in both aberrant localization of ARHGEF7 and rhotekin, as well as abnormal activation of Rho GTPases that was accompanied with impaired motility of glioblastoma cells. Furthermore, TIP-1 knockdown suppressed tumor cell dispersal in orthotopic glioblastoma murine models. We also observed high levels of TIP-1 expression in human glioblastoma specimens, and the elevated TIP-1 levels are associated with advanced staging and poor prognosis in glioma patients. Although more studies are needed to further dissect the mechanism(s) by which TIP-1 modulates the intracellular redistribution and activation of Rho GTPases, this study suggests that TIP-1 holds potential as both a prognostic biomarker and a therapeutic target of malignant gliomas.

Tax-interacting protein 1 (TIP-1, also known as Tax1bp3) inhibited proliferation of colon cancer cells through antagonizing the transcriptional activity of beta-catenin. However, in this study, elevated TIP-1 expression levels were detected in human invasive breast cancers. Studies with two human invasive breast cancer cell lines indicated that RNAi-mediated TIP-1 knockdown suppressed the cell adhesion, proliferation, migration and invasion in vitro, and inhibited tumor growth in mammary fat pads and pulmonary metastasis in athymic mice. Biochemical studies showed that TIP-1 knockdown had moderate and differential effects on the beta-catenin-regulated gene expression, but remarkably down regulated the genes for cell adhesion and motility in breast cancer cells. The decreased expression of integrins and paxillin was accompanied with reduced cell adhesion and focal adhesion formation on fibronectin-coated surface. In conclusion, this study revealed a novel oncogenic function of TIP-1 suggesting that TIP-1 holds potential as a prognostic biomarker and a therapeutic target in the treatment of human invasive breast cancers.

Aim: The gut microbiome can modify tumor immunity and has been suggested to be involved in the development and growth of liver cancer as well as metastasis in the liver. However, it remains unknown how the gut microbiome controls hepatic immune responses. This study was designed to exam the effect of the gut microbiome on liver antitumor immunity, and to study potential mechanism. Experimental Procedure: An antibiotic cocktail containing 0.5g/L vancomycin, 0.5 g/L neomycin and 0.6 g/L primaxin in drinking water was given to reduce mouse gut microbiota. Control mice were kept on regular water. EL4 thymoma cells were injected s.c. to induces spontaneous liver metastasis. B16 melanoma and CT26 colon cancer cells were injected intrasplenically to form liver metastasis. Lung metastasis was induced by tail injection of tumor cells. Spontaneous hepatocellular carcinomas were studied in TRE-MYC mice. Gut bacteria and metabolic studies were performed. Results: Antibiotic cocktail efficiently depleted gut bacteria. Removing gut commensal bacteria did not affect the growth of primary s.c. EL4 tumors, but impaired formation of liver metastasis in different models. The inhibitory effect on liver metastasis by removing gut microbiome was found after intrasplenic injection of tumor cells to form liver metastasis using both B16 melanoma and CT26 colon cancer tumor cells as well as in TRE-MYC mice. Interestingly, formation of lung metastasis caused by tail vein injection of B16 cells was not impaired by antibiotics treatment, suggesting a liver specific effect. The inhibition of liver metastasis by antibiotic treatment was absent in Rag1 knockout mice, suggesting that the observed mechanism is mediated by the adaptive immune system. A detailed mechanism how the gut microbiome causes metabolic liver changes and thereby growth of liver tumors will be presented. Conclusion: Our results suggest that the gut microbiome affects the liver immune microenvironment and modulates antitumor immunity Citation Format: Chi Ma, Miaojun Han, Bernd Heinrich, Qiong Fu, Qianfei Zhang, Xin W. Wang, Giorgio Trinchieri, Tim Greten. Gut microbiome controls growth of liver tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A02.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death. Non-alcoholic fatty liver disease (NAFLD) affects a large proportion of the US population and is considered to be a metabolic predisposition to liver cancer. However, the role of adaptive immune responses in NAFLD-promoted HCC is largely unknown. Here we show, in mouse models and human samples, that dysregulation of lipid metabolism in NAFLD causes a selective loss of intrahepatic CD4(+) but not CD8(+) T lymphocytes, leading to accelerated hepatocarcinogenesis. We also demonstrate that CD4(+) T lymphocytes have greater mitochondrial mass than CD8(+) T lymphocytes and generate higher levels of mitochondrially derived reactive oxygen species (ROS). Disruption of mitochondrial function by linoleic acid, a fatty acid accumulated in NAFLD, causes more oxidative damage than other free fatty acids such as palmitic acid, and mediates selective loss of intrahepatic CD4(+) T lymphocytes. In vivo blockade of ROS reversed NAFLD-induced hepatic CD4(+) T lymphocyte decrease and delayed NAFLD-promoted HCC. Our results provide an unexpected link between lipid dysregulation and impaired anti-tumour surveillance.

Background Upon co-stimulation with CD3/CD28 antibodies, activated CD4 + T cells were found to lose their susceptibility to HIV-1 infection, exhibiting an induced resistant phenotype. This rather unexpected phenomenon has been repeatedly confirmed but the underlying cell and molecular mechanisms are still unknown. Methods We first replicated the reported system using the specified Dynal beads with PHA/IL-2-stimulated and un-stimulated cells as controls. Genome-wide expression and analysis were then performed by using Agilent whole genome microarrays and established bioinformatics tools. Results We showed that following CD3/CD28 co-stimulation, a homogeneous population emerged with uniform expression of activation markers CD25 and CD69 as well as a memory marker CD45RO at high levels. These cells differentially expressed 7,824 genes when compared with the controls on microarrays. Series-Cluster analysis identified 6 distinct expression profiles containing 1,345 genes as the representative signatures in the permissive and resistant cells. Of them, 245 (101 potentially permissive and 144 potentially resistant) were significant in gene ontology categories related to immune response, cell adhesion and metabolism. Co-expression networks analysis identified 137 “key regulatory” genes (84 potentially permissive and 53 potentially resistant), holding hub positions in the gene interactions. By mapping these genes on KEGG pathways, the predominance of actin cytoskeleton functions, proteasomes, and cell cycle arrest in induced resistance emerged. We also revealed an entire set of previously unreported novel genes for further mining and functional validation. Conclusions This initial microarray study will stimulate renewed interest in exploring this system and open new avenues for research into HIV-1 susceptibility and its reversal in target cells, serving as a foundation for the development of novel therapeutic and clinical treatments.

Glioblastoma is the most common and fatal type of primary brain tumors featured with hyperplastic blood vessels. Here, we performed meta-analyses of published data and established a correlation between high TIP-1 expression levels and the poor prognosis of glioblastoma patients. Next, we explored the biological relevance of TIP-1 expression in the pathogenesis of glioblastoma. By using orthotopic and heterotopic mouse models of human glioblastomas, this study has characterized TIP-1 as one contributing factor to the tumor-driven angiogenesis. In vitro and in vivo functional assays, along with biochemical analyses with microarrays and antibody arrays, have demonstrated that TIP-1 utilizes multiple pathways including modulating fibronectin gene expression and uPA protein secretion, to establish or maintain a pro-angiogenic microenvironment within human glioblastoma. In conclusion, this work supports one hypothesis that TIP-1 represents a novel prognostic biomarker and a therapeutic target of human glioblastoma.

Recent studies have resulted in a variety of therapeutic options for cancer. However, tumor patients, even a same patient at different disease stages, respond(s) to a treatment protocol with different efficacy. A concept of personalized medicine has been developed to deliver individually tailored treatment upon the unique responsiveness of each patient. Currently, it is still challenging to predict treatment outcomes due to the genetic complexity and heterogeneity of cancers, which underlies the varied responses to treatment. To efficiently design treatment strategies and monitor the outcomes of therapies for individual patient, tumor responsiveness to a specific treatment regimen needs to be assesses in a timeand cost-efficient manner. Non-invasive imaging technologies have demonstrated great potentials in diagnosis and treatment management by monitoring individual patient’s disease condition and progression. Currently, anatomic and functional imaging modalities have been generally applied to detect, stage, and monitor tumors. Compared to the anatomic imaging that measures tumor size, functional or molecular imaging provides more information on tumor metabolism, biomarker expression, cell death or proliferation and thus is more relevant to the imaging assessment of tumor responsiveness to a treatment regimen, especially when the treatment affects tumors through blocking the tumor progression instead of shrinking the tumor size. Discovery of novel probes that specifically binds to tumor-limited targets with sound biological relevance is a limiting factor to develop such functional imaging modality. Compared to antibody (~150 kD), peptide is in a much smaller size (1-2 kD) that enables an improved tissue penetration, faster clearance from circulation system, and less immunogenic property that are expected for a imaging probe, especially in the repeated assessment of treatment responsiveness in solid tumors. Advances in phage display-related technologies have facilitated the discovery and development of peptide derivatives as imaging probes for a variety of tumors. By using one example of HVGGSSV peptide that has

Non-alcoholic steatohepatitis (NASH), a common condition in obese patients, is an important risk factor for HCC. However, only little information exists about the immunological mechanisms of how NASH promotes hepatocarcinogenesis. Methionine choline deficient (MCD) diet is widely used to induce NASH. In this study we fed Lap-tTA-myc mice, which develop HCC, with MCD diet. MCD diet accelerated tumor development in MYC on mice. A robust decrease of hepatic CD4 but not CD8 T cells was observed in NASH mice, which was exacerbated by MYC transgene expression. As expected higher level of cell death was detected in these hepatic CD4 T cells. Phenotypic analysis demonstrated that these CD4 T cells had a CD44highCD62Llow effector memory phenotype and produced more IFNγ. Selective CD4 T cell depletion further accelerated HCC development suggesting an important role of these cells for tumor development. Next, we studied the cause of CD4 T cells. In co-culture experiments we demonstrated that lipid-laden hepatocytes from mice on MCD diet induced selective CD4 but not CD8 T cells death through a contact-independent mechanism. FFAs were found from hepatocyte culture medium, and linoleic acid recapitulates the selective toxicity to CD4 T cells. Blocking ROS abolished linoleic acid-induced CD4 T cell death. Our results suggest the critical role of CD4 T cell in the disease progression of NASH to HCC, and provide a new link between lipid metabolism dysfunction with impaired anti-tumor surveillance. Citation Format: Chi Ma, Miaojun Han, Masaki Terabe, Jay Berzofsky, Dean Felsher, Tim Greten. The role of CD4 T cells in murine model of NASH-promoted HCC. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B44.

Meeting abstracts Tumor cell vaccines are widely used as a potential therapeutic. Different approaches are currently being applied to kill tumor cell vaccines prior to injection to prevent outgrowth of metastasis at the site of injection. We and others have previously shown that the mode by which

Background Malignant gliomas represent one group of tumors that poorly respond to ionizing radiation (IR) alone or combined with chemotherapeutic agents because of the intrinsic or acquired resistance. In this study, TIP-1 was identified as one novel protein that confers resistance of glioma cells to IR. Methodology/Principal Findings Meta-analysis indicated that high TIP-1 expression levels correlate with the poor prognosis of human malignant gliomas after radiotherapy. Studies with established human glioma cell lines demonstrated that TIP-1 depletion with specific shRNAs sensitized the cells to IR, whereas an ectopic expression of TIP-1 protected the glioma cells from the IR-induced DNA damage and cell death. Biochemical studies indicated that TIP-1 protein promoted p53 ubiquitination and resulted in a reduced p53 protein level. Furthermore, p53 and its ubiquitination are required for the TIP-1 regulated cellular response to IR. A yeast two-hybrid screening identified that TIP-1, through its single PDZ domain, binds to the carboxyl terminus of LZAP that has been studied as one tumor suppressor functioning through ARF binding and p53 activation. It was revealed that the presence of TIP-1 enhances the protein association between LZAP and ARF and modulates the functionality of ARF/HDM2 toward multi-ubiquitination of p53, while depleting TIP-1 rescued p53 from polyubiquitination and degradation in the irradiated glioma cells. Studies with a mouse xenograft model indicated that depleting TIP-1 within D54 cells improved the tumor growth control with IR. Conclusions/Significance This study provided the first evidence showing that TIP-1 modulates p53 protein stability and is involved in the radioresistance of malignant gliomas, suggesting that antagonizing TIP-1 might be one novel approach to sensitize malignant gliomas to radiotherapy.

Therapy resistance and invasion are two common features of malignant glioma. Our previous studies showed that TIP-1 regulates sensitivity of glioma cells to ionizing radiation. It was also found that TIP-1 protein level was elevated in human malignant glioma compared to the normal brain tissue counterparts, and correlated with the malignancy of gliomas. This observation was supported with a survival study within animal models, in which TIP-1 knockdown within glioma cell lines significantly retarded progression of the intracranially implanted tumors and extended survival time of the mice. In vitro and in vivo studies indicated that it is the reduced migration and invasion capability, but not the cell proliferation rate, that contributed to the retarded tumor progression of gliomas with TIP-1 knockdown. The glioma cells with TIP-1 knockdown also showed morphological changes with reorganized cytoskeleton and reduced efficiency of reorientation in directional migration. TIP-1 was reported to associate to beta-catenin and Rhotekin, two proteins that are involved in the cell skeleton reorganization and cell migration. Their interactions are through the single PDZ domain within TIP-1 and the classic Type I PDZ-binding motifs located at the carboxyl terminus of beta-catenin and Rhotekin respectively. It was found that TIP-1 knockdown altered intracellular location of Rhotekin. Mislocalization of Rhotekin at the cell migration leading edge resulted in irregular activation of RhoA, which generally regulates cell rear body retraction. Further, we identified that beta-PIX shares a similar PDZ-binding motif and associates to TIP-1 in vitro and in vivo. Beta-PIX is recruited to the leading edge by Scribble for Rac1 or Cdc42 activation and thus regulates cell front protrusion. Knockdown of TIP-1 within the invasive glioma cells disrupted the association of beta-PIX and Scribble, resulted in mislocalization of beta-PIX and reduced Rac1 activation. Taken together, these data illustrated that TIP-1 is one critical modulator in migration of the invasive glioma through regulating two small GTPases (RhoA and Rac1), and targeting TIP-1 might represent one novel approach to treat the highly invasive and mortal glioma 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 1410. doi:10.1158/1538-7445.AM2011-1410

Malignant gliomas represent one group of tumors that poorly respond to chemo- and radio-therapies because of the intrinsic or acquired resistance. Identification of novel molecules that are involved in the therapeutics resistance is critical to develop new therapeutic modalities. Our previous studies show that TIP-1 involves in tumor cells response to ionizing radiation (IR). In this study, we found that TIP-1 expression level correlates to the clinical staging and overall patient survival time after diagnosis of malignant gliomas. We hypothesized that TIP-1 is one novel predictive biomarker of glioma cell response to treatment, and targeting TIP-1 represents one novel strategy to combat the disease of high mortality. Studies on cell-based models indicated that TIP-1 deficiency resulted in the enhanced sensitivity of D54 and GL261 glioma cell lines to IR and chemotherapeutic agents, whereas ectopic expression of TIP-1 conferred resistance to the therapeutics in the cells. The data also showed that sensitivity of the glioma cell lines to IR correlates to stability and activation of p53 protein after IR treatment. Yeast two-hybrid screening identified C53/LZAP as one TIP-1 interacting protein within glioma cells. PDZ domain within TIP-1 binds to one classic type I PDZ-binding motif at the carboxyl terminus of C53/LZAP protein. C53/LZAP has been studied as one tumor suppressor and modulator of the cancer cell susceptibility to genotoxic stress including IR and several chemotherapeutic agents. C53/LZAP interacts with ARF and protects p53 from Mdm2-mediated protein degradation. Our data demonstrated that, after irradiation, presence of TIP-1 enhanced C53/LZAP interaction with ARF, thus promoted Mdm2-mediated p53 ubiquitination and resulted in less stable p53 protein. On the other hand, with absence of TIP-1 protein, IR treatment resulted in more stabilized and activated p53 protein that drives IR-induced apoptosis of the glioma cells. Taken together, our data suggests that TIP-1 is one novel modulator of p53 tumor suppressor and contributes to chemo- and radio-resistance of glioma cells. Therefore, targeting TIP-1 represents one novel approach for the improved therapy of malignant glioma. 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 1642. doi:10.1158/1538-7445.AM2011-1642