Search

The globo-series glycosphingolipids (SSEA3, SSEA4, and Globo H) were shown to express in many cancers selectively, and a combination of anti-SSEA4 and anti-Globo H antibodies was able to suppress tumor growth in mice inoculated with breast cancer cell lines. To further understand the effect, we focused on the combined effect of the two antibodies in target binding and antibody-dependent cellular cytotoxicity (ADCC) in vitro. Here, we report that the binding of anti-Globo H antibody (VK9) to MDA-MB231 breast cancer cells was influenced by anti-SSEA4 antibody (MC813-70), and a combination of both antibodies induced a similar effect as did anti-SSEA4 antibodies alone in a reporter-based ADCC assay, indicating that SSEA4 is a major target in breast cancer due to its higher expression than Globo H. Furthermore, we showed that a homogeneous anti-SSEA4 antibody (chMC813-70-SCT) designed to maximize the ADCC activity can be used to isolate a subpopulation of natural killer (NK) cells that exhibit an ∼23% increase in killing the target cells as compared to the unseparated NK cells. These findings can be used to predict a therapy outcome based on the expression levels of antigens and evaluate therapeutic antibody development.

Epidermal growth factor receptor (EGFR) remains the sole druggable molecular target other than the PD1/PD-L1 pathway with meaningful clinical benefit in squamous cell carcinoma of head and neck (SCCHN). Human epidermal growth factor receptor 3 (HER3) confers the resistance to EGFR-targeted treatment in SCCHN. Thus, it is essential to determine the distribution and regulatory mechanisms of HER3 in SCCHN. We explored the prevalence of HER3 expression and its distribution within SCCHN by immunohistochemical staining and clinicopathological correlations were analyzed. The regulatory mechanism of HER3 expression was then dissected in vitro, using RT-PCR, Western blotting, and immunoprecipitation in a set of SCCHN cell lines. Subsequent in vivo validation in the murine model was also performed. We found that concomitant high expression of HER3 and its ligand NRG1 in SCCHN is associated with the increased presence of regional lymphatic metastasis and the majority of HER3 is located on the differentiated tumor cells. Further investigation revealed that HER3 is under positive control of NOTCH1 through transcriptional activation and inhibition of protein degradation through the polyubiquitination machinery via AKT pathway and USP8 deubiquitinating enzyme. In addition, loss of function of NOTCH1 suppresses HER3 expression through increased phosphorylation of serine 473 of AKT in SCCHN cells, and promotes the aggressiveness of the tumor cells. These data indicated that the level of HER3 is regulated by NOTCH1 in SCCHN both transcriptionally and post-translationally, and NOTCH1 is in a higher hierarchy in the regulatory system of the AKT pathway. Since NOTCH1 is inactivated in approximately 10% of SCCHN cases and this aberration strongly impacts the AKT pathway and diminishes HER3, exclusion of patients with NOTCH1-inactivated SCCHN may be beneficial for future clinical trials of HER3-targeting antibodies.

Ovarian cancer has a high recurrence rate after platinum‐based chemotherapy. To improve the treatment of ovarian cancer and identify ovarian cancer‐specific antibodies, we immunized mice with the human ovarian carcinoma cell line, SKOV‐3, and generated hybridoma clones. Several rounds of screening yielded 30 monoclonal antibodies (mAbs) with no cross‐reactivity to normal cells. Among these mAbs, OV‐Ab 30‐7 was found to target integrin α3 and upregulate p53 and p21, while stimulating the apoptosis of cancer cells. We further found that binding of integrin α3 by OV‐Ab 30‐7 impaired laminin‐induced focal adhesion kinase phosphorylation. The mAb alone or in combination with carboplatin and paclitaxel inhibited tumor progression and prolonged survival of tumor‐bearing mice. Moreover, immunohistochemical staining of ovarian patient specimens revealed higher levels of integrin α3 in cancer cells compared with normal cells. By querying online clinical databases, we found that elevated ITGA3 expression in ovarian cancer is associated with poor prognosis. Taken together, our data suggest that the novel mAb, OV‐Ab 30‐7, may be considered as a potential therapeutic for ovarian cancer., The novel mAb, OV‐Ab 30‐7, can induce ovarian cancer cell apoptosis, and blockage integrin‐laminin signaling, and may be considered as a potential therapeutic for ovarian cancer.

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in tumor‐associated endothelial cells, where it modulates tumor‐promoting angiogenesis, and it is also found on the surface of tumor cells. Currently, there are no Ab therapeutics targeting VEGFR2 approved for the treatment of prostate cancer or leukemia. Therefore, development of novel efficacious anti‐VEGFR2 Abs will benefit cancer patients. We used the Institute of Cellular and Organismic Biology human Ab library and affinity maturation to develop a fully human Ab, anti‐VEGFR2‐AF, which shows excellent VEGFR2 binding activity. Anti‐VEGFR2‐AF bound Ig‐like domain 3 of VEGFR2 extracellular region to disrupt the interaction between VEGF‐A and VEGFR2, neutralizing downstream signaling of the receptor. Moreover, anti‐VEGFR2‐AF inhibited capillary structure formation and exerted Ab‐dependent cell‐mediated cytotoxicity and complement‐dependent cytotoxicity in vitro. We found that VEGFR2 is expressed in PC‐3 human prostate cancer cell line and associated with malignancy and metastasis of human prostate cancer. In a PC‐3 xenograft mouse model, treatment with anti‐VEGFR2‐AF repressed tumor growth and angiogenesis as effectively and safely as US FDA‐approved anti‐VEGFR2 therapeutic, ramucirumab. We also report for the first time that addition of anti‐VEGFR2 Ab can enhance the efficacy of docetaxel in the treatment of a prostate cancer mouse model. In HL‐60 human leukemia‐xenografted mice, anti‐VEGFR2‐AF showed better efficacy than ramucirumab with prolonged survival and reduced metastasis of leukemia cells to ovaries and lymph nodes. Our findings suggest that anti‐VEGFR2‐AF has strong potential as a cancer therapy that could directly target VEGFR2‐expressing tumor cells in addition to its anti‐angiogenic action., Affinity‐maturated anti‐vascular endothelial growth factor receptor 2 (VEGFR2) therapeutic Ab has strong clinical potential for cancer treatment due to its simultaneous inhibition of tumor angiogenesis in vascular endothelial cells and tumorigenesis in VEGFR2‐expressing tumor cells.

Cancer cell migration plays a crucial role during the metastatic process. Reversible tyrosine phosphorylation by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) have been implicated in the regulation of cancer cell migration and invasion. However, the underlying mechanisms have not been fully elucidated. Here, we show that depletion of the FERM and PDZ domain-containing protein tyrosine phosphatase PTPN3 enhances lung cancer cell migration/invasion and metastasis by promoting actin filament assembly and focal adhesion dynamics. We further identified Src and DAAM1 (dishevelled associated activator of morphogenesis 1) as interactors of PTPN3. DAAM1 is a formin-like protein involved in the regulation of actin cytoskeletal remodeling. PTPN3 inhibits Src activity and Src-mediated phosphorylation of Tyr652 on DAAM1. The tyrosine phosphorylation of DAAM1 is essential for DAAM1 homodimer formation and actin polymerization. Ectopic expression of a DAAM1 phosphodeficient mutant inhibited F-actin assembly and suppressed lung cancer cell migration and invasion. Our findings reveal a novel mechanism by which reversible tyrosine phosphorylation of DAAM1 by Src and PTPN3 regulates actin dynamics and lung cancer invasiveness.

Although epithelial cell adhesion molecule (EpCAM) has previously been shown to promote tumor progression, the underlying mechanisms remain largely unknown. Here, we report that the EGF-like domain I within the extracellular domain of EpCAM (EpEX) binds EGFR, activating both AKT and MAPK signaling to inhibit forkhead transcription factor O3a (FOXO3a) function and stabilize PD-L1 protein, respectively. Treatment with the EpCAM neutralizing antibody, EpAb2-6, inhibited AKT and FOXO3a phosphorylation, increased FOXO3a nuclear translocation, and upregulated high temperature requirement A2 (HtrA2) expression to promote apoptosis while decreasing PD-L1 protein levels to enhance the cytotoxic activity of CD8+ T cells. In vivo, EpAb2-6 markedly extended survival in mouse metastasis and orthotopic models of human colorectal cancer. The combination of EpAb2-6 with atezolizumab, an anti-PD-L1 antibody, almost completely eliminated tumors. Moreover, the number of CD8+ T cells in combination-treated tumors was increased compared with atezolizumab alone. Our findings suggest a new combination strategy for cancer immunotherapy in patients with EpCAM-expressing tumors. Significance: This study shows that treatment with an EpCAM neutralizing antibody promotes apoptosis while decreasing PD-L1 protein to enhance cytotoxic activity of CD8+ T cells.

Epithelial cell adhesion molecule (EpCAM) is highly expressed in colon cancers, but its role in cancer progression remains to be elucidated. In this work, we found that the extracellular domain of EpCAM (EpEX) activated EGFR and downstream ERK1/2 signaling to promote colon cancer cell migration and proliferation, as well as tumor growth. Mechanistically, we discovered that EpEX-EGFR-ERK1/2 signaling positively regulated intramembrane proteolysis (RIP) of EpCAM and shedding of the intracellular domain (EpICD). Treatment with an EGFR inhibitor ablated the EpEX-induced phosphorylation of ERK1/2 and AKT. Additionally, treatment with inhibitors of either EGFR or MEK decreased EpEX-induced EpICD shedding and further revealed that EpICD is necessary for nuclear accumulation of β-catenin and the induction of HIF1α target gene expression in vitro and in vivo. Moreover, an anti-EpCAM neutralizing monoclonal antibody, EpAb2-6, inhibited the nuclear translocation of EpICD and β-catenin and induced apoptosis in colon cancer cells. Importantly, analysis of colorectal cancer tissues showed that nuclear accumulation of EpICD was highly correlated with metastasis and poor prognosis, suggesting that it may play an important functional role in cancer progression. Thus, we provide novel insights into the mechanisms and functions of EpEX-mediated signaling, which may be considered as a promising target for the treatment of colon cancer.

Mutation of KRAS in non-small-cell lung cancer (NSCLC) shows a poor response to epidermal growth factor receptor (EGFR) inhibitors and chemotherapy. Currently, there are no direct anti-KRAS therapies available. Thus, new strategies have emerged for targeting KRAS downstream signaling. Panobinostat is a clinically available histone deacetylase inhibitor for treating myelomas and also shows potentiality in NSCLC. However, the therapeutic efficacy of panobinostat against gefitinib-resistant NSCLC is unclear. In this study, we demonstrated that panobinostat overcame resistance to gefitinib in KRAS-mutant/EGFR-wild-type NSCLC. Combined panobinostat and gefitinib synergistically reduced tumor growth in vitro and in vivo. Mechanistically, we identified that panobinostat-but not gefitinib-inhibited TAZ transcription, and the combination of panobinostat and gefitinib synergistically downregulated TAZ and TAZ downstream targets, including EGFR and EGFR ligand. Inhibition of TAZ by panobinostat or short hairpin RNA sensitized KRAS-mutant/EGFR-wild-type NSCLC to gefitinib through abrogating AKT/mammalian target of rapamycin (mTOR) signaling. Clinically, TAZ was positively correlated with EGFR signaling, and coexpression of TAZ/EGFR conferred a poorer prognosis in lung cancer patients. Our findings identify that targeting TAZ-mediated compensatory mechanism is a novel therapeutic approach to overcome gefitinib resistance in KRAS-mutant/EGFR-wild-type NSCLC.

Lung cancer is the leading cause of cancer-related death worldwide. Most targeted drugs approved for lung cancer treatment are tyrosine kinase inhibitors (TKIs) directed against EGFR or ALK, and are used mainly for adenocarcinoma. At present, there is no effective or tailored targeting agent for large cell carcinoma (LCC) or small cell lung cancer (SCLC). Therefore, we aimed to identify targeting peptides with diagnostic and therapeutic utility that possess broad subtype specificity for SCLC and non-small cell lung cancer (NSCLC). We performed phage display biopanning of H460 LCC cells to select broad-spectrum lung cancer-binding peptides, since LCC has recently been categorized as an undifferentiated tumor type within other histological subcategories of lung cancer. Three targeting phages (HPC1, HPC2, and HPC4) and their respective displayed peptides (HSP1, HSP2, and HSP4) were able to bind to both SCLC and NSCLC cell lines, as well as clinical specimens, but not to normal pneumonic tissues. In vivo optical imaging of phage homing and magnetic resonance imaging (MRI) of peptide-SPIONs revealed that HSP1 was the most favorable probe for multimodal molecular imaging. Using HSP1-SPION, the T2-weighted MR signal of H460 xenografts was decreased up to 42%. In contrast to the tight binding of HSP1 to cancer cell surfaces, HSP4 was preferentially endocytosed and intracellular drug delivery was thereby effected, significantly improving the therapeutic index of liposomal drug in vivo. Liposomal doxorubicin (LD) conjugated to HSP1, HSP2, or HSP4 had significantly greater therapeutic efficacy than non-targeting liposomal drugs in NSCLC (H460 and H1993) animal models. Combined therapy with an HSP4-conjugated stable formulation of liposomal vinorelbine (sLV) further improved median overall survival (131 vs. 84 days; P = 0.0248), even in aggressive A549 orthotopic models. Overall, these peptides have the potential to guide a wide variety of tailored theranostic agents for targeting therapeutics, non-invasive imaging, or clinical detection of SCLC and NSCLC.

Background and objectives Squamous cell carcinoma of head and neck (SCCHN) is the fifth most prevalent cancer worldwide. Because the anatomical complexity of this region, complete surgical resection is often not achievable and conventional chemotherapy would aid locoregional control and mitigate distant metastasis. Nonetheless, the nonspecific cytotoxicity and short in vivo half-life of conventional chemotherapeutic drugs limit their effects. Given the high frequency of overexpression of wild type epidermal growth factor receptor (EGFR), we exploit EGFR as a homing beacon for drug delivery system with cytotoxic payloads. Materials and methods We generated fully human anti-EGFR single chain variable fragment (scFv)-conjugated immunoliposomes (IL) containing doxorubicin and vinorelbine and tested their anti-neoplastic efficacy in vitro and in vivo. Result Our IL enhanced endocytosis and significantly reduced the half maximal inhibitory concentrations of the therapeutic payloads when compared to non-targeting liposomal counterparts in various cell lines of SCCHN. Furthermore, median survival time was significantly prolonged in subcutaneous and orthotopic SCCHN xenograft murine models treated with our IL formulations than those treated with non-targeting counterparts (94 days versus 60 days and 72 days versus 56 days, respectively) without evident increased systemic toxicity. Conclusion The therapeutic index of the chemotherapeutic payloads was augmented by our EFGR-targeting IL formulation and they are warranted for further development and preclinical trial.

While there has been extensive development of anti-cancer drugs for treatment of prostate cancer, the therapeutic efficacy of such drugs remains inadequate in many cases. Here, we performed in vitro biopanning of the PC3 human prostate carcinoma cell line to select prostate cancer-specific peptides by phage display. We successfully identified specific peptides targeting prostate cancer cells, and their specificity was confirmed by cellular ELISA and flow cytometry. Moreover, we found that the phage clones also recognize other prostate cancer cell lines and surgical specimens from prostate cancer patients. The tumor targeting ability of these phages was validated in a xenograft model, in which high accumulation of targeting phage was observed. To investigate whether selected peptides are able to target tumors and enhance drug delivery into cancer cells, we synthesized peptide-PEGylated lipids and post-inserted them into preformed liposomal doxorubicin and vinorelbine. The results of our cellular uptake and MTT assays indicate that peptide-conjugated liposomes exhibit enhanced drug intracellular delivery and cytotoxicity. The conjugation of targeting peptide to imaging agents, such as quantum dots (QDs) and superparamagnetic iron oxide nanoparticles (SPIONs), results in more precise delivery of these agents to tumor sites. Furthermore, administration of liposomal doxorubicin and vinorelbine conjugated with targeting peptides was found to markedly increase the inhibition of human prostate tumor growth in mouse xenograft and orthotopic models. These results indicate that targeting peptide, SP204, has significant potential for targeted therapy and molecular imaging in prostate cancer.

The objective of this study was to evaluate radiolabeled DOTA-SP90 as a radiotracer for breast cancer. The in vitro competition assay showed that radiolabeled DOTA-SP90 had significant binding affinity to BT-483 cancer cells. Biodistribution, nanoSPECT/CT and nanoPET/CT imaging results indicated that radiolabeled DOTA-SP90 can accumulate in tumors. In addition, radiolabeled DOTA-SP90 peptides can also detect metastatic tumors. Therefore, radiolabeled SP90 peptide may provide the potential capability as diagnostic agent for breast cancer patients.

Paclitaxel (PTX) exhibits potent antineoplastic activity against various human malignancies; however, clinical application must overcome the inherent hydrophobicity of this molecule. The commercialized Taxol formulation utilizes Cremophor EL (CrEL)/ethanol as a solvent to stabilize and dispense PTX in an aqueous solution. However, adverse CrEL-induced hypersensitivity reactions have been reported in ~30% of recipients, and 40% of patients receiving premedication may also experience this adverse effect. Therefore, the development of a CrEL-free delivery system is crucial, in order to fully exploit the therapeutic efficacy of PTX. In the present study, a novel liposomal PTX (lipo-PTX) formulation was optimized with regards to encapsulation rate and long-term stability, arriving at a molar constituent ratio of soybean phosp hatidylcholine:cholesterol:N-(carbonyl-methoxy-poly-ethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt:PTX at 95:2:1:2. Comparable doses of lipo-PTX and Taxol were bioequivalent in terms of therapeutic efficacy in xenograft tumor models. However, the systemic side effects, including hematopoietic toxicity, acute hypersensitivity reactions and cardiac irregularities, were significantly reduced in lipo-PTX-treated mice compared with those infused with reference formulations of PTX. In conclusion, the present study reported that lipo-PTX exhibited a higher therapeutic index than clinical PTX formulations.

Epithelial cell adhesion molecule (EpCAM) is known to be overexpressed in epithelial cancers associated with enhanced malignant potential, particularly colorectal carcinoma (CRC) and head and neck squamous cell carcinoma (HNSCC). However, it is unknown whether progression of malignance can be directly inhibited by targeting EpCAM. Here, we have generated five novel monoclonal antibodies (mAbs) against EpCAM. One of these anti-EpCAM mAbs, EpAb2-6, was found to induce cancer cell apoptosis in vitro, inhibit tumor growth, and prolong the overall survival of both a pancreatic cancer metastatic mouse model and mice with human colon carcinoma xenografts. EpAb2-6 also increases the therapeutic efficacy of irinotecan, fluorouracil, and leucovorin (IFL) therapy in a colon cancer animal model and gemcitabine therapy in a pancreatic cancer animal model. Furthermore, EpAb2-6, which binds to positions Y95 and D96 of the EGF-II/TY domain of EpCAM, inhibits production of EpICD, thereby decreasing its translocation and subsequent signal activation. Collectively, our results indicate that the novel anti-EpCAM mAb can potentially be used for cancer-targeted therapy.

Nanocarriers, such as liposomes, have the potential to increase the payload of chemotherapeutic drugs while decreasing toxicity to non-target tissues; such advantageous properties can be further enhanced through surface conjugation of nanocarriers with targeting moieties. We previously reported that SP94 peptides, identified by phage display, exhibited higher binding affinity to human hepatocellular carcinoma (HCC) than to hepatocytes and other normal cells. Here, we confirm the tumor-targeting properties of SP94 peptide by near-infrared fluorescence imaging. Non-targeted PEGylated liposomal doxorubicin (LD) and SP94‑conjugated PEGylated liposomal doxorubicin (SP94‑LD) were compared by assessing pharmacokinetics, tissue distribution, and antitumor efficacy in xenograft-bearing mice, in order to investigate the effectiveness of SP94‑mediated targeting for cancer therapy. SP94‑LD demonstrated a significant increase in drug accumulation in tumors, while its plasma residence time was the same as its non-targeted equivalent. Consistent with this result, conjugation of targeting peptide SP94 enhances the therapeutic efficacy of liposomal doxorubicin in mouse models with hepatocellular carcinoma xenografts. Furthermore, combination targeted therapy exhibited a significant enhancement against orthotopic tumor growth, and markedly extended the survival of mice compared with all other treatments. Our study shows that SP94‑mediated targeting enhances antitumor efficacy by improving tumor pharmacokinetics and tissue distribution, allowing large amounts of antitumor drugs to accumulate in tumors.

Glutathione S-transferase mu 3 (GSTM3) is an enzyme involving in the detoxification of electrophilic compounds by conjugation with glutathione. Higher GSTM3 mRNA levels were reported in patients with ERα-positive breast cancer who received only tamoxifen therapy after surgery. Thus, this study aimed to clarify the oncogenic characteristics of GSTM3 in breast cancer and the mechanism of tamoxifen resistance. GSTM3 expression in human breast tumour tissues (n = 227) was analysed by RT-PCR and quantitative PCR. Western blot, promoter activity assays, and chromatin immunoprecipitation (ChIP) assays were used to investigate the mechanism of GSTM3 gene regulation. Hydrogen peroxide (H2O2)-induced cytotoxicity in breast cancer cells was detected by MTT assays and flow cytometry. The oncogenic characteristics of GSTM3 in MCF-7 cells were examined by siRNA knockdown in soft agar assays and a xenograft animal model. GSTM3 mRNA was highly expressed in ER- and HER2-positive breast cancers. Moreover, patients who received adjuvant Herceptin had increased GSTM3 mRNA levels in tumour tissue. Oestrogen-activated GSTM3 gene expression through ERα-mediated recruitment of SP1, EP300, and AP-1 complexes. GSTM3-silenced MCF-7 cells were more sensitive to H2O2, with significantly inhibited proliferation and colony formation abilities. Tamoxifen-resistant (Tam-R) cells lacking GSTM3 showed enhanced sensitivity to H2O2, but this result was contrary to that obtained after short-term tamoxifen exposure. The animal model suggested that GSTM3 silencing might suppress the tumourigenic ability of MCF-7 cells and increase tumour cell apoptosis. ROS production is one mechanism by which cancer drugs kill tumour cells, and according to our evidence, GSTM3 may play an important role in preventing breast cancer treatment-induced cellular cytotoxicity.

Thyroid hormone status has long been implicated in cancer development. Here we investigated the role of thyroxine (T4) in colorectal cancer cell lines HCT 116 (APC wild type) and HT-29 (APC mutant), as well as the primary cultures of cancer cells derived from patients. Cell proliferation was evaluated with standard assay and proliferation marker expression. β-Catenin activation was examined according to nuclear β-catenin accumulation and β-catenin target gene expression. The results showed that T4 increased colorectal cancer cell proliferation while cell number and viability were elevated by T4 in both established cell lines and primary cells. Moreover, the transcriptions of proliferative genes PCNA, CCND1, and c-Myc were enhanced by T4 in the primary cells. T4 induced nuclear β-catenin accumulation, as well as high cyclin D1 and c-Myc levels compared to the untreated cells. In addition, the β-catenin-directed transactivation of CCND1 and c-Myc promoters was also upregulated by T4. CTNNB1 transcription was raised by T4 in HCT 116, but not in HT-29, while the boosted β-catenin levels were observed in both. Lastly, the T4-mediated gene expression could be averted by the knockdown of β-catenin. These results suggested that T4 promotes β-catenin activation and cell proliferation in colorectal cancer, indicating that an applicable therapeutic strategy should be considered.

The anti-cancer efficacy of TNF-related apoptosis-inducing ligand (TRAIL)-based therapy is limited due to systemic toxicity, poor bioavailability, and the development of TRAIL-resistance. We developed a tumor-targeted LCPP (Lipid/Calcium/Phosphate/Protamine) nanoparticle (NP) to deliver TRAIL plasmid DNA (pDNA) into hepatocellular carcinoma (HCC) cells. TRAIL pDNA was encapsulated in a pH stimuli-responsive calcium phosphate (CaP) core, and protamine was added to facilitate nuclear delivery of pDNA. In addition, the intracellular release of Ca2+ from the CaP core overcame TRAIL resistance via calcium influx-dependent DR5 upregulation. TRAIL expression also attenuated fibrosis in the liver tissues surrounding HCCs by reverting activated hepatic stellate cells (HSCs) to a quiescent state or by directly inducing apoptosis in activated HSCs. Conclusion: TRAIL pDNA delivered by HCC-targeted LCPP NPs in combination with conventional sorafenib treatment attenuated HCC progression as well as liver fibrosis. Overall, our study presents an effective TRAIL-based cancer therapy that could be developed for clinical applications. This article is protected by copyright. All rights reserved.

Metastasis-mediated death remains a major challenge in cancer treatment due to the lack of identifiable biomarkers for early diagnosis. Identifying tumor-specific biomarkers is critical for the development of diagnostic and therapeutic tools. In the present study, we found that podocalyxin-like 1 (PODXL), a cell surface glycoprotein, was overexpressed in cancer tissues and was upregulated in lymph node metastatic tumor cells. The expression of PODXL was associated with the migratory ability of human oral squamous cell carcinoma (OSCC). Knockdown of PODXL by small hairpin RNA in the SAS OSCC cell line reduced tumor migration and invasion, and inhibited cell proliferation and colony formation. Suppression of PODXL resulted in downregulation of focal adhesion kinase (FAK) and paxillin phosphorylation. PODXL silencing inhibited filopodia formation, and suppressed F-actin and cortactin colocalization. In addition, PODXL expression was associated with the DNA methylation status, and treatment with the DNA methyltransferase inhibitor 5-aza-deoxycytidine increased the PODXL transcriptional level. Moreover, DNA microarray analysis data revealed that suppression of PODXL significantly affected subsets of genes associated with extracellular matrix organization, the epithelial-mesenchymal transition, and the expression of metastasis-related cytokines. Collectively, these data showed that the overexpression of PODXL may be associated with tumor aggressiveness and that PODXL could be a diagnostic biomarker for metastatic OSCC.

Background Even though new molecular targeted agents are now being developed, they are still premature in application of clinical use for triple negative breast cancers (TNBCs). Therefore, it is highly demanded to explore the effective antitumor agents against TNBCs. Novel non-camptothecin topoisomerase I inhibitors are discovered and evaluated in this study. Methods Human MDA-MB-231 (BCRC-60425), BT-549 (ATCC® HTB-122™) and MCF-7 (BCRC-60436) were used in this study. Top1 assay was determined by gel mobility assay to validate the Top1-mediated DNA cleavages at different concentrations of non-camptothecin compounds. Cell viability analysis (MTT assay), comet assay and flow cytometry analysis were used to evaluate their growth inhibitory activity, DNA damage, and induction of cell arrest. Mechanistic pathways were studied and validated through western blot analysis, flow cytometry and confocal microscopy analysis. Binding interactions between top1 and non-camptothecin compound were analyzed by computational analysis (Discovery Studio). Results The IC50 values (growth inhibitory activity) of these non-camptothecin compounds are in the micromolar to nanomolar range (1.8 μm to 190 nM) against MDA-MB-231, BT-549 and MCF-7 cell lines. These compounds significantly inhibited the process in which supercoiled DNA strand transforms into its relaxed state and showed antitumor spectra similar to camptothecin rather than doxorubicin. Comet tails were observed to increase significantly with various doses of compounds in a dose-dependent manner. In addition, their mode of actions were shown to involve G2/M arrest of the cell cycle along with a dose-dependent increase in protein levels of cleaved caspase-3 and cleavage of cPARP. Except apoptosis pathway, non-camptothecin compounds also induce necrosis, and autophagy. Favorable ADMET characteristics of non-camptothecin compounds were observed using ADMET Descriptors. Conclusions Our results have provided evidence for therapeutic intervention in the treatment of TNBC using new top1 inhibitors, non-camptothecin compounds. Legal entity responsible for the study Wen-Shan Li. Funding Ministry of Science and Technology, Taiwan and Academia Sinica, Taiwan. Disclosure All authors have declared no conflicts of interest.

Background EpCAM is a type I transmembrane glycoprotein with an extracellular domain (EpEX) and an intracellular domain (EpICD), which have 265 and 26 amino acid residues, respectively. EpCAM is highly expressed in advanced epithelial cancers and tumor-initiating cells, but its role in cancer progression remains to be elucidated. Methods To identify cell signaling pathways that are stimulated by EpEX, we used a Human Phospho-RTK Array Kit to screen for phosphorylation of RTKs. Inhibition of EpEX-induced EGFR-PI3K-AKT signaling was analyzed by small molecule inhibitors or shRNA knockdown. Results Here, we found that EpEX activated AKT signaling, thereby inducing the phosphorylation and nuclear exclusion of FOXO3a. The EGFR inhibitor, AG1478, and MEK inhibitor, U0126, both decreased the production of EpICD, which was found to be necessary for nuclear accumulation of β-catenin protein and expression of HIF1α target genes in vitro and in mouse xenograft models. We also demonstrated that treatment with an anti-EpCAM neutralizing antibody, EpAb2-6, decreased the ADAM17 and γ-secretase activity, the EpCAM-downstream gene expression, and tumor colony and sphere formation. We also found that EpAb2-6 inhibited EpEX-activated EGFR-PI3K-AKT signaling and induced apoptotic signaling through FOXO3a activation of HTRA2 gene expression. Importantly, we also showed that EpAb2-6 inhibited the nuclear translocation of EpICD and oncogenic signaling through β-catenin. Finally, in both metastatic and orthotopic animal models of colorectal cancer, EpAb2-6 therapy exhibited an antitumor effect and markedly extended the survival time of mice. Conclusions Our results demonstrate that EpEX contributes to malignancy by functioning as a growth factor, which activates EpICD-mediated signaling, thereby enhancing colon cancer cell survival. To the best of our knowledge, hEpAb2-6 is the first humanized anti-EpCAM antibody to directly trigger apoptosis in cancer cells. Thus, we provide novel insight into EpEX-EGFR signaling, which can be considered as a promising target for treatment of colon cancer. Legal entity responsible for the study The authors. Funding Academia Sinica. Disclosure All authors have declared no conflicts of interest.

Epithelial cell adhesion molecule (EpCAM) is highly expressed in advanced epithelial cancers and tumor-initiating cells, but its role in cancer progression remains to be elucidated. Here, we show that the extracellular domain of EpCAM (EpEX) acts through its EGF-like domain I to bind EGFR and activates downstream ERK1/2 and AKT signaling. Inhibition or shRNA knockdown of EGFR ablated EpEX-induced ERK1/2 and AKT phosphorylation in colon cancer cells. EGFR signaling, stimulated by either EpEX or EGF, then induces regulated intramembrane proteolysis of EpCAM, releasing both EpEX and EpCAM intracellular domain (EpICD). MEK inhibitor, U0126, prevented EpEX-induced EpCAM proteolysis by ADAM17 and presenilin 2 proteases. Furthermore, EGFR inhibitor, AG1478, and MEK inhibitor, U0126, both decreased the production of EpICD, which was found to be necessary for nuclear accumulation of β-catenin protein and expression of HIF1α target genes in vitro and in mouse xenograft models. In clinical samples from colorectal carcinoma patients, high levels of nuclear EpICD predicted metastasis and poor prognosis. Importantly, we also showed that EpAb2-6, an anti-EpCAM neutralizing monoclonal antibody, inhibited EpEX-activated EGFR-PI3K-AKT signaling and induced apoptotic signaling through FOXO3a activation of HTRA2 gene expression. This antibody also inhibited the nuclear translocation of EpICD and oncogenic signaling through β-catenin. Finally, in both metastatic and orthotopic animal models of colorectal cancer, EpAb2-6 therapy exhibited an antitumor effect and markedly extended the survival time of mice. Taken together, the results demonstrate that EpEX contributes to malignancy by functioning as a growth factor, which activates EpICD-mediated signaling, thereby enhancing colon cancer cell survival. Furthermore, the data indicate that EpEX can be considered as a promising target for treatment of colon cancer. Citation Format: Kang-Hao Liang, Hsien-Cheng Tso, Shao-Hsi Hung, Mei-Ying Liao, Han-Chung Wu. The molecular mechanisms of EpCAM in regulating tumor progression in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4670.

The efficacy of systemic cytotoxic chemotherapy has been widely assessed in patients with advanced hepatocellular carcinoma (HCC). For example, doxorubicin is the most commonly studied chemotherapeutic agent for HCC. However, it has been shown to have a response rate of only 10-20% in clinical trial. In addition, its potential benefit has been reduced by the related adverse effect. So far, the multikinase inhibitor, sorafenib, is considered to provide survival benefit over supportive care. However, the long term prognosis of those cancer patients still remain poor. Therefore, in the present experiment, we proposed to use the so-called peptide targeting chemotherapy to overcome the adverse event in the conventional targeted chemotherapy. In order to perform this experiment, we have construct some specific peptides which can bind specifically to the cancer cells and cancer vascular endothelia by using a phage displayed 12-mer random peptide library. We have obtained 3 different peptides and one control peptide. Each contains 12 amino acids: a. L-peptide: RLLDTNRPLLPY (anti-different cancer cell membrane); b. control peptide: RLLDTNRGGGGG; c. SP-94-peptide: SFSHHTPILP (anti-NPC tumor cell and hepatoma cell membranes) and d. PC5-52-peptide: SVSVGMKPSPRP (anti-tumor endothelia). Those L-peptide (L-P), SP-peptide (SP-P), PC5-52-peptide and a control peptide (C-P) were linked to liposomal iron oxide nanoparticles; and also used those peptides to link liposomal doxorubicin (L-D). Using peptide linked liposomal iron oxide, we can localize the peptide targeted tumor cells and tumor endothelia, and then we used those peptides linked liposomal doxorubicin to treat SCID mice bearing different cancer xenografts. Our results showed that when L-P-L-D containing 2mg/kg of SCID mouse body weight was used to treat xenografts bearing SCID mice, the tumor could be well controlled, and no specific adverse event was seen. However, when the control peptide was used to replace the specific peptide, the xenograft size was also shown decrease, but the visceral organs revealed marked apoptotic change. In conclusion, the specific peptides linked liposomal doxorubicin nanoparticles can be used for treatment of SCID mice bearing cancer xenografts with minimal adverse event, especially in the SCID miceγspecies (NGS), which show a remarkable tumor suppression. Note: This abstract was not presented at the meeting. Citation Format: Chin-Tarng Lin, Cheng-Der Wu, Jen-Chien Lee, Han-Chung Wu, Chung-Wei Lee, Ming-Chieh Hsu. Preclinical verification of the efficacy of targeting peptides linked liposomal nanoparticles for therapy of hepatocellular and nasopharyngeal carcinomas and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2184.

Pancreatic adenocarcinoma is an aggressive disease with a high mortality rate. In this study, we have newly generated a monoclonal antibody (mAb), Pa65-2, which specifically binds to pancreatic cancer cells and tumor blood vessels. The target protein of Pa65-2 is identified as human clathrin heavy chain (CHC). In vitro and In vivo study showed that suppression of CHC either by shRNA or by Pa65-2 inhibited tumor growth and angiogenesis. One of the key functions of CHC was to bind with the hypoxia-inducing factor (HIF)-1α protein, increasing the stability of this protein and facilitating its nuclear translocation, thereby regulating the expression of VEGF. Taken together, our findings indicate that CHC plays a role in the processes of tumorigenesis and angiogenesis. Pa65-2 antibody or CHC shRNA can potentially be used for pancreatic cancer therapy.

Due to the development of drug resistance, the outcome for the majority of patients with acute myeloid leukemia (acute myelogenous leukemia; AML) remains poor. To prevent drug resistance and increase the therapeutic efficacy of treating AML, the development of new combinatory drug therapies is necessary. Sonic hedgehog (Shh) is expressed in AML biopsies and is essential for the drug resistance of cancer stem cells of AML. AML patients are frequently infected by bacteria and exposed to lipopolysaccharide (LPS). LPS itself, its derivatives, and its downstream effectors, such as tumor necrosis factor-α (TNF-α) and interferons (IFNs), have been shown to provoke anti-tumor effects. The application of a Shh inhibitor against AML cells in the presence of LPS/TNF-α/IFNs has not been investigated. We found that the Shh inhibitor cyclopamine in combination with LPS treatment synergistically induced massive cell apoptosis in THP-1 and U937 cells. The cytotoxic effects of this combined drug treatment were confirmed in 5 additional AML cell lines, in primary AML cells, and in an AML mouse model. Replacing cyclopamine with another Shh inhibitor, Sant-1, had the same effect. LPS could be substituted by TNF-α or IFNs to induce AML cell death in combination with cyclopamine. Our results suggest a potential strategy for the development of new therapies employing Shh antagonists in the presence of LPS/TNF-α/IFNs for the treatment of AML patients.

Building on the initial successful optimization of a novel series of tetraindoles, a second generation of the compounds with changes in the core phenyl ring was synthesized to improve anticancer properties. 17 new compounds with different rigidity, planarity, symmetry and degree of conjugation of their core structures to 5-hydroxyindole units were synthesized. All the compounds were fully characterized and tested against breast cancer cell line (MDA-MB-231). The results revealed that the core structure is required for activity and it should be aromatic, rigid, planar, symmetrical and conjugated for optimal activity. Compound 29, which has strong anticancer activity against various tumor-derived cell lines, including Mahlavu (hepatocellular), SK-HEP-1 (hepatic), HCT116 (colon), MIA PaCa-2 (pancreatic), H441 (lung papillary), A549 (lung), H460 (non-small cell lung) and CL1-5 (lung carcinoma) with IC50 values ranging from 0.19 to 3.50μM, was generated after series of successive optimizations. It was found to induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in the non-obese diabetic-severe combined immunodeficiency (NOD/SCID) mice bearing xenografted MIA PaCa-2 human pancreatic cancer.

The derivation of induced pluripotent stem cells (iPSCs) requires not only efficient reprogramming methods, but also reliable markers for identification and purification of iPSCs. Here, we demonstrate that surface markers, epithelial cells adhesion molecule (EpCAM) and epithelial cadherin (E-cadherin) can be used for efficient identification and/or isolation of reprogrammed mouse iPSCs. By viral transduction of Oct4, Sox2, Klf4 and n- or c-Myc into mouse embryonic fibroblasts, we observed that the conventional mouse embryonic stem cell (mESC) markers, alkaline phosphatase (AP) and stage-specific embryonic antigen 1 (SSEA1), were expressed in incompletely reprogrammed cells that did not express all the exogenous reprogramming factors or failed to acquire pluripotent status even though exogenous reprogramming factors were expressed. EpCAM and E-cadherin, however, remained inactivated in these cells. Expression of EpCAM and E-cadherin correlated with the activation of Nanog and endogenous Oct4, and was only seen in the successfully reprogrammed iPSCs. Furthermore, purification of EpCAM-expressing cells at late reprogramming stage by FACS enriched the Nanog-expressing cell population suggesting the feasibility of selecting successful reprogrammed mouse iPSCs by EpCAM expression. We have thus identified new surface markers that can efficiently identify successfully reprogrammed iPSCs and provide an effective means for iPSC isolation.

The purpose of this abstract was to assess the efficacy of peptide targeted chemotherapy for pancreatic carcinoma. Previously we have constructed four peptides that bind specifically to cancer cell lines, which were derived from three different carcinoma cell lines and their vascular endothelia: L-peptide: (L-P, anti-cancer cell membrane), RLLDTNRPLLPY; SP-94-P (anti-hepato-pancreatic cancer cell membrane), SFSHHTPILP; PC5-52-peptide (anti-tumor endothelia), SVSVGMKPSPRP; and control peptide, RLLDTNRGGGGG. These peptides were linked to pegylated liposomal iron oxide nanoparticles to identify the targeted tumor cells and vascular endothelia using MRI analysis, and were also linked to dextran coated liposomal doxorubicin (L-D) for treatment of non-obese diabetic, severe combined immunodeficiency (NOD-SCID) mice bearing pancreatic cancer cell (PANC-1) xenografts. Our results demonstrated that the tumor intensity of MRI is clearly decreased after SP-94-P-iron oxide was applied, and that in combination of application of L-P linked L-D (L-P-L-D) plus SP-94-P linked L-D (SP-94-P-L-D) and PC5-52-P linked L-D (PC5-52-P-L-D), they could inhibit pancreatic tumor growth with very mild adverse events. The use of the control peptide linked L-D also led to a decrease of the xenograft size, but also induced marked apoptotic change of their visceral organs. It is concluded that the combination of L-P-L-D, SP-94-P-L-D and PC5-52-P-L-D to treat pancreatic cancer xenograft in NOD SCID mice can clearly inhibit pancreatic cancer growth with minimal adverse events. Citation Format: Chin-Tarng Lin, Chen-Der Wu, Jen-Chieh Lee, Han-Chung Wu, Chung-Wei Lee, Chin-Feng Lin, Ming-Chieh Hsu. Peptide-targeted chemotherapy for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3880.

Hepatocellular carcinoma is the fourth leading cause of cancer death worldwide. Novel treatment strategies derived from increased knowledge of molecular oncology are constantly being developed to cure this disease. Here, we used phage display to identify a novel peptide (SP94), which binds specifically to hepatocellular carcinoma cells. In vitro, the phage clone PC94 was shown to bind to hepatocellular carcinoma cell lines by ELISA and flow cytometry analysis. In vivo, PC94 homed specifically to tumor tissues but not to normal visceral organs in severe combined immunodeficient mice bearing human hepatocellular carcinoma xenografts. This homing ability could be competitively inhibited by synthetic peptide, SP94. Immunohistochemical staining confirmed that PC94 localized to tumor tissues and that it could not be detected in SP94-competed tumor tissues. In addition, PC94 recognized the tumor tissue but not nontumor tissue in surgical specimens from hepatocellular carcinoma patients, with a positive rate of 61.3% (19 of 31). With the conjugation of SP94 and liposomal doxorubicin, the targeted drug delivery system enhanced the therapeutic efficacy against hepatocellular carcinoma xenografts through enhanced tumor apoptosis and decreased tumor angiogenesis. Our results indicate that SP94 has the potential to improve the systemic treatment of patients with advanced hepatocellular carcinoma. [Mol Cancer Ther 2008;7(3):579–89]

To understand whether the p53-regulated mdm2 gene expression was altered by the Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC), the NPC-TW01 cell line was infected by EBV through IgA receptor-mediated endocytosis. The mdm2 gene was expressed only in a small fraction of the NPC cell population and could be enhanced in the EBV-infected (EBV+) cells. In the animals bearing EBV+ and EBV- NPC xenografts, the MDM2+ cells only appeared in clusters in both EBV+ and EBV- tumors with stronger expression in EBV+ cells. Cotransfection of pmdm2-Luc plus pSV40-p53 plus pCMV-LMP1 in the NPC-TW06 line that had p53 heterozygous point mutation showed stronger mdm2 promoter activity than cells cotransfected with pmdm2-Luc plus pSV40-p53, but no mdm2 promoter activity was seen in cells cotransfected with pmdm2-Luc plus pCMV-LMP1. Only the EBV-LMP1 but not the EBV-LMP2A gene could enhance p53 to upregulated mdm2 expression. Tumor cells in NPC biopsy specimens revealed similar mdm2 expression as in the animal model. It is concluded that although EBV can indirectly enhance mdm2 gene expression in tumor cells that express this gene, it cannot turn on or directly regulate mdm2 expression in cells that do not express this gene. In other words, EBV plays a role as an enhancer in NPC tumorigenesis.

Epithelial cell adhesion molecule (EpCAM) is highly expressed in advanced epithelial cancers and tumor-initiated cells (TICs), but its roles in cancer progression remain to be elucidated. Here, we showed that the extracellular domain of EpCAM (EpEX) could bind to EGFR through EGF-like domain I, and subsequently activated its downstream molecules, ERK1/2 and Akt. EGFR inhibitor and knockdown of EGFR by shRNA ablated EpEX-induced ERK1/2 phosphorylation. Regulated intramembrane proteolysis (RIP) of EpCAM was induced similarly by EpEX and EGF through EGFR-dependent activation of ERK pathway. MEK inhibitor, U0126, could abolish ADAM17 and PS2 phosphorylation induced by EpEX. EpAb2-6, an anti-EpEX neutralizing monoclonal antibody, inhibits EpEX-activated EGFR-PI3K-AKT pathway in detached colon cancer cells. Moreover, intracellular domain of EpCAM (EpICD), the product of RIP-induced cleavage of EpCAM, is necessary for nuclear accumulation of β-catenin, and their target gene expressions in vitro and in mouse xenograft models. We also found that an increase of nuclear EpICD observed in CRCs predicted metastasis and poor prognosis in CRC patients. Finally, in animal model studies, EpAb2-6 therapy exhibited an enhanced antitumor effect and markedly extended the survival time of mice with human colorectal cancer in metastatic and orthotopic models. These results demonstrate that EpEX works as a growth factor in activating EGFR-mediated signaling, and as a potential target for treatment of colon cancer. This research was supported by grants from Academia Sinica and Ministry of Science and Technology [MOST 104-0210-01-09-02,MOST 105-0210-01-13-01], and the National Science Council (NSC103-2321-B-001-064), Taiwan (to H-C Wu). Citation Format: Kang-Hao Liang, Jun-Kai Lai, I-I Kuan, Hsien-Cheng Tso, Han-Chung Wu. EpCAM/EpEX regulate tumor progression through EGFR signaling in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 327. doi:10.1158/1538-7445.AM2017-327

Backgrounds: Being a crucial hormone regulating growth, metabolism and various physiological processes, the status of thyroid hormone has long been implicated in cancer risks and tumor developments. In the present study, we aimed to look at the role of thyroid hormone, thyroxine (T4), in colorectal cancer cell proliferation and β-catenin activation, which is highly involved in both normal and oncogenic developments of the gut. Materials and Methods: The effects of T4 in colorectal cancer cell lines HCT 116 (APC wild type) and HT-29 (APC mutant) as well as the primary cells derived from colorectal cancer patients were studied. Cell proliferation was evaluated according to cell counting, MTT assay and qRT-PCR. The activation of β-catenin was examined using Western blotting, qRT-PCR and immunoprecipitation. Results: The results showed that T4 increased the cell number of both HCT 116 and HT-29 cells compared to the untreated cells. In both cell lines, T4 induced nuclear β-catenin accumulation and the protein levels of WNT/β-catenin targets Cyclin D1 and c-Myc. The mRNA expression of CTNNB1 was elevated by T4 in HCT 116 cells, but not in HT-29 cells. In APC wild type HCT 116 cells, T4 increased the WNT4 mRNA expression and decreased the association between β-catenin and the WNT-regulated β-catenin destruction complex. Moreover, the cell numbers of T4-treated primary cells were higher compared to the untreated cells while the mRNA expressions of proliferative genes PCNA, CCND1 and c-Myc were elevated by T4. In the primary cells, T4-induced nuclear β-catenin accumulation, protein levels of Cyclin D1 and c-Myc, and mRNA expressions of CTNNB1 and WNT4 were also observed. Conclusions: T4 promoted cell proliferation and β-catenin activation in colorectal cancer. In the cells with different APC mutation status, the activation of β-catenin was regulated by different mechanisms. Citation Format: Yee-Shin Lee, Meng-Ti Hsieh, Yu-Tang Chin, Aleck Hercbergs, Paul J. Davis, Han-Chung Wu, Hung-Yun Lin. Thyroid hormone, thyroxine, promotes cell proliferation and β-catenin activation in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3625. doi:10.1158/1538-7445.AM2017-3625

We have generated a novel monoclonal antibody (mAb), OCAb9-1, which specifically binds to various types of cancer cell lines, but not to normal cells. According to the results of immunoaffinity chromatography, LC-MS/MS analysis, co-immunoprecipitation, and RNA interference studies, the target protein of OCAb9-1 is the epithelial cell adhesion molecule (EpCAM). EpCAM is a type I transmembrane glycoprotein which is highly expressed in epithelial-transformed neoplasia and tumor-initiating cells (TICs). However, regulation of EpCAM gene expression in tumors and its role in tumorigenesis are not fully understood. In the present study, we show that EpCAM expression is elevated in several cancer cell lines and tumor tissues. Loss-of-function experiments were performed to demonstrate that EpCAM negatively regulates expression of p53 and p21, and promotes tumor cell growth, colony formation, migration and invasion. The median overall survival of tumor-bearing mice treated with OCAb9-1 was significantly higher than that of PBS-treated mice. Moreover, we report that the interplay between SUZ12 and JMJD3 results in dynamic regulation of lysine 27 trimethylation of histone 3 (H3K27me3). Taken together, our findings suggest that the anti-EpCAM mAb may be suitable for use in cancer diagnosis, prognosis, imaging and therapy. Furthermore, EpCAM overexpression in cancer cells is strongly associated with tumor progression, and may be regulated by epigenetic mechanisms.

Currently, epithelial ovarian cancer is viewed as a heterogeneous disease with five major histological subtypes. Clear cell carcinoma represents a specific histological subtype of epithelial ovarian cancer that demonstrates more aggressive clinical behavior and drug resistance compared with other subtypes. Nevertheless, clear cell carcinoma is treated in the same manner as the other subtypes without any particular consideration to its unique clinical characteristics. To improve the therapeutic efficacy of the current liposomal doxorubicin approach for the treatment of clear cell carcinoma, we aimed to develop a novel peptide-conjugated liposomal doxorubicin to actively target this subtype. Two phage clones (OC-6 and OC-26) that specifically bound to clear cell carcinoma were isolated from a phage peptide display library after biopanning procedures. The peptide sequences were translated and aligned (OCSP-6 for OC-6, and OCSP-26 for OC-26, respectively). Peptide-conjugated nanoparticles demonstrated better tumor endocytosis and time-dependent gradual increase of intracellular drug uptake than non-targeting liposomal nanoparticles. Furthermore, peptide-conjugated liposomal doxorubicin better controlled tumors than did non-targeting liposomal doxorubicin. The current work may pave a new way for the development of drugs that target each subtype of epithelial ovarian cancer in the future.

Hepatoma-derived growth factor (HDGF) overexpression is involved in liver fibrosis and carcinogenesis. However, the receptor(s) and signaling for HDGF remain unclear. By using affinity chromatography and proteomic techniques, nucleolin (NCL) was identified and validated as a HDGF-interacting membrane protein in hepatoma cells. Exogenous HDGF elicited the membrane NCL accumulation within 0.5 hour by protein stabilization and transcriptional NCL upregulation within 24 hours. Blockade of surface NCL by antibodies neutralization potently suppressed HDGF uptake and HDGF-stimulated phosphatidylinositol 3-kinase (PI3K)/Akt signaling in hepatoma cells. By using rescectd hepatocellular carcinoma (HCC) tissues, immunohistochemical analysis revealed NCL overexpression was correlated with tumour grades, vascular invasion, serum alpha-fetoprotein levels and the poor survival in HCC patients. Multivariate analysis showed NCL was an independent prognostic factor for survival outcome of HCC patients after surgery. To delineate the role of NCL in liver carcinogenesis, ectopic NCL overexpression promoted the oncogenic behaviours and induced PI3K/Akt activation in hepatoma cells. Conversely, NCL knockdown by RNA interference attenuated the oncogenic behaviours and PI3K/Akt signaling, which could be partially rescued by exogenous HDGF supply. In summary, this study provides the first evidence that surface NCL transmits the oncogenic signaling of HDGF and facilitates a novel diagnostic and therapeutic target for HCC.

Metastatic disease is the leading cause of cancer mortality. Identifying biomarkers and regulatory mechanisms is important toward developing diagnostic and therapeutic tools against metastatic cancer. In this study, we demonstrated that podocalyxin-like 1 (PODXL) is overexpressed in breast tumor cells and increased in lymph node metastatic cancer. Mechanistically, we found that the expression of PODXL was associated with cell motility and invasiveness. Suppression of PODXL in MDA-MB-231 cells reduced lamellipodia formation and focal adhesion kinase (FAK) and paxillin phosphorylation. PODXL knockdown reduced the formation of invadopodia, such as inhibiting the colocalization of F-actin with cortactin and suppressing phosphorylation of cortactin and neural Wiskott-Aldrich syndrome protein. Conversely, overexpression of PODXL in MCF7 cells induced F-actin/cortactin colocalization and enhanced invadopodia formation and activation. Invadopodia activity and tumor invasion in PODXL-knockdown cells are similar to that in cortactin-knockdown cells. We further found that the DTHL motif in PODXL is crucial for regulating cortactin phosphorylation and Rac1/Cdc42 activation. Inhibition of Rac1/Cdc42 impeded PODXL-mediated cortactin activation and FAK and paxillin phosphorylation. Moreover, inhibition of PODXL in MDA-MB-231 cells significantly suppressed tumor colonization in the lungs and distant metastases, similar to those in cortactin-knockdown cells. These findings show that overexpression of PODXL enhanced invadopodia formation and tumor metastasis by inducing Rac1/Cdc42/cortactin signaling network.

Epithelial cell adhesion molecule (EpCAM) is highly expressed in epithelial-transformed neoplasia and tumor-initiated cells (TICs), but the role that EpCAM plays in the stemness properties of TICs is still unclear. Here we show that EpCAM and reprogramming factors (c-Myc, Oct4, Nanog, and Sox2) were concomitantly elevated in TICs, which were shown to have superior self-renewal, invasiveness, and tumor-initiating abilities. Elevation of EpCAM enhanced tumorsphere formation and tumor initiation. Knockdown of EpCAM inhibited the expressions of reprogramming factors and epithelial-mesenchymal transition genes, thereby suppressing tumor initiation, self-renewal, and invasiveness. In addition, EpCAM, especially intracellular domain of EpCAM (EpICD), bound to and activated the promoter of reprogramming factors. Treatment with the inhibitor of γ-secretase (DAPT) led to the blockage of the expressions of reprogramming factors and epithelial-mesenchymal transition genes, which was accompanied by the reduction of tumor self-renewal and invasion. Furthermore, the increased release of EpEX enhanced production of EpICD and regulated the expression of reprogramming factors. Together, these findings suggest that EpCAM plays an important role in regulating cancer-initiating abilities in TICs of colon cancer. This discovery can be used in the development of new strategies for cancer therapy.

Nucleolin (C23, NCL) mRNA was up-regulated in nasopharyngeal carcinoma (NPC) cells compared to that of normal nasomucosal (NNM) cells using a cDNA microarray approach. The level of nucleolin protein was also up-regulated in 13 NPC cell lines, 30 biopsy specimens and nine other cancer cell lines compared to five NNM cells or normal stromal cells, which were analyzed using immunoblotting or immunohistochemistry. We transfected nucleolin antisense oligodeoxynucleotides (phosphorothioate-modified oligodeoxynucleotides; S-ODNs) into NPC-TW01 cells to knockdown nucleolin expression to evaluate the function of nucleolin in cancer cells. Nucleolin knockdown induced NPC cells but not NNM cells to undergo apoptosis. Furthermore, treatment of NPC-TW01 xenograft tumors with nucleolin antisense oligodeoxynucleotides suppressed the growth of xenograft tumors without obvious side effects. Therefore, we suggest that nucleolin may be a potential cancer therapeutic target and that nucleolin antisense oligodeoxynucleotides may be used as a potential drug for therapy in NPC.

The identification of molecules that are down-regulated in malignant phenotype is important for understanding tumor biology and their role in tumor suppression. We compared the expression profile of four normal nasal mucosal (NNM) epithelia and a series of nasopharyngeal cancinoma (NPC) cell lines using cDNA microarray and confirmed the actual expression of the selected genes, and found osteoprotegerin (OPG) to be ubiquitously deficient in NPC cells. We also found OPG to be down-regulated in various cancer cell lines, including oral, cervical, ovarian, lung, breast, pancreas, colon, renal, prostate cancer, and hepatoma. Administration of recombinant OPG (rOPG) brought about a reduction in cancer cell growth through apoptotic mechanism. We generated eleven monoclonal antibodies (MAbs) against OPG to study OPG's expression and biological functions in cancer cells. OPG was detected in the tumor stromal regions, but not in the cancer cell per se in surgical specimens of liver cancer. Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) revealed that OPG was down-regulated in NPC tissues compared with normal nasal polyp (NNP) tissues. In addition, we showed OPG silencing to be associated with promoter methylation as well as histone modifications. In OPG-silenced cancer cell lines, the OPG gene promoter CpG dinucleotides were highly methylated. Compared to normal cells, silenced OPG gene in cancer cells were found to have reduced histone 3 lysine 4 tri-methylation (H3K4me3) and increased histone 3 lysine 27 tri-methylation (H3K27me3). Taken together, these results suggest that OPG silencing in carcinoma cancer cells occurs through epigenetic repression.

Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in South China, Singapore, and Taiwan. At present, its etiological factors are not well defined. To identify which genetic alterations might be involved in NPC pathogenesis, we identified genes that were differentially expressed in NPC cell lines and normal nasomucosal cells using subtractive hybridization and microarray analysis. Most NPC cell lines and biopsy specimens were found to have higher expression levels of the gene encoding nucleolar and coiled-body phosphoprotein 1 (NOLC1) as compared with normal cells. Severe combined immunodeficiency mice bearing NPC xenografts derived from NOLC1-short hairpin-RNA-transfected animals were found to have 82% lower levels of tumor growth than control mice as well as marked tumor cell apoptosis. Measuring the expression levels of genes related to cell growth, apoptosis, and angiogenesis, we found that the MDM2 gene was down-regulated in the transfectants. Both co-transfection and chromatin immunoprecipitation experiments showed that tumor protein 53-regulated expression of the MDM2 gene requires co-activation of NOLC1. These findings suggest that NOLC1 plays a role in the regulation of tumorigenesis of NPC and demonstrate that both NOLC1 and tumor protein 53 work together synergistically to activate the MDM2 promoter in NPC cells.

Nasopharyngeal carcinoma (NPC) is prevalent in south-eastern Asia, and its tumourigenesis is rather complex. The purpose of this research was to identify the pivotal genes that may be altered during the early stage of NPC progression. Eleven genes were selected by comparative microarray analysis of NPC versus normal nasomucosal cells. The expression of SPARC (secreted protein, acidic, cysteine-rich) was statistically significantly down-regulated in NPC cells. In exploring the mechanism underlying the decreased transcription of SPARC in NPC cells, we found that the transcription factor SRY (sex-determining region Y)-box 5 (SOX-5) is up-regulated in NPC cells. RNA interference of SOX-5 by short hairpin RNA (shRNA) in NPC cells caused a dramatic increase in SPARC and chromosome immunoprecipitation assay showed that SOX-5 can bind directly to the SPARC promoter, suggesting that SOX-5 acts as a key transcriptional repressor of SPARC. We further demonstrated that shRNA knockdown of SOX-5 suppressed the proliferation of NPC cells, as well as their migratory ability, which was also observed when SPARC was over-expressed in NPC cells. Alternatively, blocking SPARC with an antagonistic antibody reversed the effects of SOX-5 knockdown. In 66 NPC patients, over-expression of SOX-5 in tumour cells correlated clinically with poor survival. Our study suggests that SOX-5 transcriptionally down-regulates SPARC expression and plays an important role in the regulation of NPC progression. SOX-5 is a potential tumour marker for poor NPC prognosis.

Antiangiogenesis therapies for the treatment of cancers hold the promise of high efficacy and low toxicity. In vivo phage display was used to identify peptides specifically targeting tumor blood vessels. The peptide SP5-52 recognized tumor neovasculature but not normal blood vessels in severe combined immunodeficiency mice bearing human tumors. Synthetic peptide was shown to inhibit the binding of PC5-52 phage particles to the tumor mass in the competitive inhibition assay. Several selected phage clones displayed the consensus motif, proline-serine-proline, and this motif was crucial for peptide binding to the tumor neovasculature. SP5-52 peptides also bound vascular endothelial growth factor–stimulated human umbilical vein endothelial cells and blood vessels of human lung cancer surgical specimens. Furthermore, this targeting phage was shown to home to tumor tissues from eight different types of human tumor xenografts following in vivo phage display experiments. An SP5-52 peptide-linked liposome carrying doxorubicin enhanced the therapeutic efficacy of the drug, markedly decreased tumor blood vessels, and resulted in higher survival rates of human lung and oral cancer–bearing xenograft mice. The current study indicates that ligand-targeted therapy offers improved therapeutic effects over conventional anticancer drug therapy, and that the peptide SP5-52 specifically targets tumor neovasculature and is a good candidate for targeted drug delivery to solid tumors. [Cancer Res 2007;67(22):10958–65]

It was proposed that Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC); however, the molecular mechanisms involved in the effect of EBV on NPC host genes have not yet been well defined. For this study, two sets of microarray experiments, NPC (EBV-free) vs normal epithelial cells and EBV(+) vs EBV(-) NPC arrays, were analyzed and the datasets were cross-compared to identify any correlation between gene clusters involved in EBV targeting and the NPC host gene expression profiles. Statistical analysis revealed that EBV seems to have a preference for targeting more genes from the differentially expressed group in NPC cells than those from the ubiquitously expressed group. Furthermore, this trend is also reflected in log ratios where the EBV target genes of the differentially expressed group origin showed greater log ratios than genes with an origin from the ubiquitously expressed NPC group. Taken together, the genome-wide comparative scanning of EBV and NPC transcriptomes has successfully demonstrated that EBV infection has an intensifying effect on the signals involved in NPC gene expression both in breadth (the majority of the genes) and in depth (greater log ratios).

Nasopharyngeal carcinoma (NPC) is a common cancer among Chinese living in southern China, Taiwan, and Singapore. The 5-year survival rate in the early stage of NPC has been reported as high as 90 to 95% with the use of radiotherapy, but in the advanced cases, even with the use of both chemotherapy and radiotherapy, the survival rate is still

A membrane invasion culture system was used to study the ability of EBV to enhance invasion and migration of nasopharyngeal carcinoma (NPC) cells. Semi-reverse transcriptase-PCR analysis of matrix proteinases and angiogenic factors from EBV-infected, or EBV-positive (EBV+), cells demonstrated different degrees of elevated gene expression. In our animal model, EBV+ tumors grew faster and larger than EBV-free, or EBV-negative (EBV-), tumors and also had clonal EBV terminal repeat sequences. Double-localization of EBV and certain host proteins in EBV+ tumors and biopsy specimens demonstrated that EBV up-regulates host genes only in cells that express those genes but not in cells that do not express them. Double-localization of EBV and host genes in NPC biopsy specimens all showed EBV- tumor cells expressing those host genes. Our data strongly suggest that EBV infection enhances progression of NPC tumor growth. They do not rule out a role for EBV infection in the induction and early promotion of NPC development. Unidentified factors may also enhance NPC tumor growth independent of the effects of EBV.

Epithelial cell adhesion molecule (EpCAM) is known to be overexpressed in epithelial cancers associated with enhanced malignant potential, particularly colorectal carcinoma (CRC) and head and neck squamous cell carcinoma (HNSCC). However, it is yet to be determined whether targeting EpCAM can directly inhibit the progression of malignance. Here, we have generated six novel monoclonal antibodies (mAbs) against EpCAM; these antibodies specifically bind to CRC and HNSCC cells, but not normal cells. One EpCAM mAb, EpAb2-6, was found to induce cancer cell apoptosis in vitro, inhibit tumor growth, and prolong the overall survival of mice with human colon or oral carcinoma xenografts. EpAb2-6 also increases the therapeutic efficacy of irinotecan, fluorouracil, and leucovorin (IFL) therapy in tumor-bearing mice. Subsequent analysis suggested that EpAb2-6 can potentially inhibit the growth and proliferation of tumorspheres. Investigation of the mechanism by which EpAb2-6 inhibits tumor growth revealed that EpAb2-6 inhibits tumor-necrosis-factor α converting enzyme (TACE/ADAM17) and γ-secretase activation and blocks EpCAM intracellular domain (EpICD) cleavage, thus preventing the nuclear localization of EpICD. Furthermore, EpAb2-6, which binds to positions in the EGF-II/TY domain of EpCAM, inhibits production of EpICD, thereby decreasing translocation of β-catenin into the nucleus and activation of p53 and p21 expression. Collectively, our results indicate that novel EpCAM mAbs can potentially be used for cancer-targeted therapy. Citation Format: Han-Chung Wu, Yi-Hsuan Chi, Mei-Ying Liao. An effective anti-EpCAM antibody EpAb2-6 for the treatment of colon cancer and its underlying mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2906. doi:10.1158/1538-7445.AM2014-2906

Krüppel-like factor 4 (KLF4) is a C2H2 zinc-finger transcription factor known to regulate multiple cellular processes including cell proliferation, differentiation and stemness. Cell scattering is characterized by disruption of E-cadherin-mediated intercellular adhesion and acquisition of fibroblast-like morphology with increased motility and invasiveness, a cellular process reminiscent of epithelial-to-mesenchymal transition (EMT). We have previously demonstrated the involvement of KLF4 in hepatocyte growth factor (HGF)-induced cell scattering (Lai et al., J. Cell Sci., in press). Still, the question as to whether KLF4 is generally involved in cell-scattered response induced by other stimuli such as 12-O-Tetradecanoylphorbol-13-Acetate (TPA) has never been formerly addressed and thus the main focus of this study. Using human hepatocellular carcinoma cell line HepG2 as the cellular model, we herein presented evidence that KLF4 was clearly up-regulated following TPA stimulation. RT-PCR analysis and luciferase reporter assay revealed that both the mRNA and promoter activity of KLF4 was markedly increased by TPA, indicating that TPA up-regulated KLF4 at the level of transcription. Of note, knockdown of KLF4 severely impaired the level of TPA-induced HepG2 cell scattering, highlighting the requirement of KLF4 in TPA-induced scattered response. Additionally, we found that the promoting effect of TPA on KLF4 expression was clearly blocked when cells were pre-treated with the pan-PKC inhibitor Gö6983 as well as the PKCα-specific inhibitor Gö6976. Likewise, TPA-induced cell scattering and KLF4 up-regulation were ablated when ERK activity was inhibited by U0126. Taken together, our results defined the involvement of KLF4 in TPA-induced cell scattered response. Particularly, TPA engages the PKCα-ERK signaling axis to induce transcriptional up-regulation of KLF4, leading to the initiation of cell scattering. Our findings further implicate the possible involvement of KLF4 in other TPA-induced cellular responses such as cell migration and invasion. Citation Format: Jun-Kai Lai, Han-Chung Wu, Chia-Che Chang. 12-O-tetradecanoylphorbol-13-acetate (TPA) induces cell scattering by upregulating krüppel-like factor 4 (KLF4) through the PKCα-ERK signaling axis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 296. doi:10.1158/1538-7445.AM2013-296

Pancreatic adenocarcinoma is an aggressive disease with a high mortality rate. Currently, treatment options are limited. In an effort to improve the efficacy of treatments for pancreatic adenocarcinoma, we have newly generated a monoclonal antibody (mAb), Pa65-2, which specifically binds to various cancer cells and tumor blood vessels but not to normal cells. According to immunoaffinity chromatography, LC-MS/MS analysis, co-immunoprecipitation and RNA interference studies, the target protein of Pa65-2 is human clathrin heavy chain (CHC). Knocking down CHC with shRNA reduced tumor cell growth, colony formation and invasion in vitro, and inhibited tumor angiogenesis and growth. One of the key functions of CHC was to bind with the transcription factor subunit HIF-1α, increasing the stability of this protein and facilitating its nuclear translocation, thereby regulating the expression of VEGF. In animal studies using NOD/SCID mice bearing pancreatic cancer xenografts, Pa65-2 and CHC shRNA could each inhibit tumor growth and angiogenesis by regulating HIF-1α and VEGF. Furthermore, the expression of CHC, HIF-1α and VEGF was significantly correlated with tumor stage, suggesting that CHC might be a useful prognostic indicator for cancer. Considered together, our results indicate that CHC interacts with and stabilizes HIF-1α, and plays a role in HIF-1α nuclear localization and HRE promoter binding, leading to an increased production of VEGF. These results suggest that the CHC mAb, Pa65-2, can potentially be used to inhibit tumor angiogenesis and growth. 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 2520. doi:1538-7445.AM2012-2520

MiR-1 (microRNA-1) can be used as a positive control and for optimizing transfection conditions in microRNA experiments (e.g., Ambion Pre-miR miRNA Starter Kit, QIAGEN Syn-hsa-miR-1 miScript miRNA mimic positive control). We noticed that transfection of nasopharyngeal carcinoma cells with miR-1 reveals a typical apoptotic process when observed by time-lapse microscopy. Annexin V and TUNEL staining and caspase assay confirm that miR-1 induces nasopharyngeal carcinoma cell apoptosis. MiR-1 transfection of HeLa, Cal-27, KYSE30 and NPC-TW06 cell lines which express low levels of endogenous miR-1 also induces apoptosis. However, miR-1 transfection of cell lines such as SW620, HepG2, HEK-293T, SAS and PC-13 which express high levels of endogenous miR-1 does not induce apoptosis. To determine miR-1 target genes that are involved in apoptosis, we analyzed microRNA and pathway databases, and cDNA expression microarrays from miR-1 transfected cells. Using qRT-PCR analysis and luciferase reporter vector assays, we found that miR-1 directly targets PTMA (prothymosin alpha). We used PTMA siRNA to down regulate PTMA mRNA levels and observed whether PTMA siRNA could induce apoptosis. Knocking down PTMA expression alone did not induce apoptosis but could accelerate apoptotic progression in cells treated with apoptosis inducers. We also found that transfection of miR-1 could up regulated some pro-apoptotic proteins (HTRA, SMAC, p53, TNF-R1, TNF-R2, TRAILR-1, TNF-alpha, TNF-beta). They are not miR-1 directly target genes but involve in miR-1 inducing apoptosis. We conclude that miR-1 can induced apoptosis in low levels of endogenous miR-1 cell lines. This is a novel model of microRNA-induced cell apoptosis. The apoptotic inducers including actinomycin D, camptothecin and etoposide are also the chemotherapeutic drugs in clinical cancer therapy and PTMA siRNA can accelerate apoptotic progression in cells treated with those apoptosis inducers. Therefore PTMA siRNA may have potential applications as an adjuvant in cancer chemotherapy. 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 194. doi:1538-7445.AM2012-194

We have recently developed practical software programs for optimization of cancer targeting peptides, including combinatorial construction of 3D peptide library (Buildpep), binding pocket analyzer (PscanMS) and high accuracy protein-ligand scoring program (HotLig). Based on these programs, a strategy of structure-based optimization of cancer targeting peptides in silico is developed to target human GRP78 which consists of 654 amino acids. The homology modeling of human GRP78 was constructed through aligning amino acid sequence and 3D structure with its homologs. This protein contains two major structural domains, the peptide-binding domain and the ATPase. Based on the results from the molecular modeling, new peptide analogs were designed and examined for optimized binding to the targeted GRP78 through in silico analysis. We had thus delineated the pharmacophore description of binding motif targeting peptide-binding domain of GRP78 as Pro-X1-Leu-X2. Peptides possessing this motif can be applied in cancer-targeted drug delivery system. We have thus designed several new tumor-targeted peptides [P-12, P-13, and P-6]. Their ability to bind target protein was confirmed in subsequent in vitro binding through Biacore analysis. We next examined their capacity to target cancer cells in the in vitro binding with various cancer cells, and in vivo tumor imaging and targeted chemotherapeutic studies. The results indicate that these peptides exhibit promising therapeutic potential by their selective binding to cancer cells, but not normal cells. Notably, these peptide analogs can bind to breast cancer stem cell (CSC) population in xenografts of primary breast cancer suggesting that they can target breast CSC enriched subpopulation. The microSPECT/CT molecular imaging of a primary human breast tumors, BC0244, xenograft with 188Re-liposome-PEG-P12 or P13 peptide showed significantly more uptake of radioactivity as compared to 188Re- liposome-PEG-L-peptide in the BC0244 breast tumors in NOD/SCID mice at 6, 24 and 48 h after intravenous injection, and even greater difference was found as compared to that of 188Re-liposome alone. To further compare P12 labeled liposome with L-peptide labeled liposome for therapeutic efficacy, mice bearing BC0244 xenografts were distributed into 4 groups for treatment with P12-Lipo-Dox, L-peptide-Lipo-Dox, Lipo-Dox, and PBS. The tumor growth of P12-lipo-Dox group was 1.57-fold slower than that of the L-peptide group; both of them showed significant increase in the therapeutic efficiency than Lipo-Dox control. These optimized cancer targeting peptides may be useful for imaging and targeted therapy of cancers. 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 1366. doi:10.1158/1538-7445.AM2011-1366