PDF file - 75K, Cell proliferation was evaluated by staining of Ki-67 and Ki-67 expression in 4TO7-Fra-1 cells (A) as well as 4TO7-sh1 and 4TO7-sh2 cells (B) was measured by FACS. The expression of Ki-67 was quantified by mean fluorescence intensity. Error bars represent mean � SD, #p=0.12, *p <0.05, versus control group

<p>PDF file - 2.1MB, Human breast cancer cells were treated with CTX (500uM) for 24hrs and the apoptosis were evaluated by Annexin V and PI double staining (A). Fra-1 expression in these breast cancer cells were detected by Western-blot (B)</p>

<p>PDF file - 159K, Cell proliferation was evaluated by cell cycle analysis. Cell cycle profiles of 4TO7 cells with Fra-1 extinction (A) and ectopic expression (B) were confirmed by FACS. Cells were fixed by 70% ethanol, incubated in PBS buffer with RNase A and then stained with propidium iodide (PI) overnight. Data were collected on the following day</p>

PDF file - 75K, Cell proliferation was evaluated by staining of Ki-67 and Ki-67 expression in 4TO7-Fra-1 cells (A) as well as 4TO7-sh1 and 4TO7-sh2 cells (B) was measured by FACS. The expression of Ki-67 was quantified by mean fluorescence intensity. Error bars represent mean � SD, #p=0.12, *p <0.05, versus control group

PDF file - 56K

PDF file - 4.2MB, Final tumor burden was depicted as size(A) and weight(B) on Day 23 after harvest, and the expression of Fra-1 in these tumors was further confirmed by western-blot(C)

PDF file - 2.1MB, Human breast cancer cells were treated with CTX (500uM) for 24hrs and the apoptosis were evaluated by Annexin V and PI double staining (A). Fra-1 expression in these breast cancer cells were detected by Western-blot (B)

Identification of specific oncogenic gene changes has enabled the modern generation of targeted cancer therapeutics. In high-grade serous ovarian cancer (OV), the bulk of genetic changes is not somatic point mutations, but rather somatic copy-number alterations (SCNAs). The impact of SCNAs on tumour biology remains poorly understood. Here we build haploinsufficiency network analyses to identify which SCNA patterns are most disruptive in OV. Of all KEGG pathways (N=187), autophagy is the most significantly disrupted by coincident gene deletions. Compared with 20 other cancer types, OV is most severely disrupted in autophagy and in compensatory proteostasis pathways. Network analysis prioritizes MAP1LC3B (LC3) and BECN1 as most impactful. Knockdown of LC3 and BECN1 expression confers sensitivity to cells undergoing autophagic stress independent of platinum resistance status. The results support the use of pathway network tools to evaluate how the copy-number landscape of a tumour may guide therapy.

Serous Ovarian Cancers (SOC) are frequently resistant to programmed cell death. However, here we describe that these programmed death-resistant cells are nonetheless sensitive to agents that modulate autophagy. Cytotoxicity is not dependent upon apoptosis, necroptosis, or autophagy resolution. A screen of NCBI yielded more than one dozen FDA-approved agents displaying perturbed autophagy in ovarian cancer. The effects were maximized via combinatorial use of the agents that impinged upon distinct points of autophagy regulation. Autophagosome formation correlated with efficacy in vitro and the most cytotoxic two agents gave similar effects to a pentadrug combination that impinged upon five distinct modulators of autophagy. However, in a complex in vivo SOC system, the pentadrug combination outperformed the best two, leaving trace or no disease and with no evidence of systemic toxicity. Targeting the autophagy pathway in a multi-modal fashion might therefore offer a clinical option for treating recalcitrant SOC.

Abstract The standard of care for ovarian cancer patients is carboplatin and paclitaxel. While many tumors initially respond to these drugs, patients often relapse with resistant disease. One of the mechanisms implicated in this development of resistance is evasion of apoptosis. Exploiting alternative death pathways, including necroptosis, is one strategy to treat such recurrent disease. Overexpressed in many cancers, inhibitor of apoptosis proteins (IAPs) represent one family of promising targets. Treatment of cells with an IAP antagonist can activate apoptosis or, if caspases are blocked, necroptosis. Among a panel of ovarian cancer cell lines screened for sensitivity to an IAP antagonist in combination with a pan-caspase inhibitor, only one (OVCAR3) was found to be sensitive, but this sensitivity was also exhibited by serous ovarian cancer cells isolated from patients. Combination treatment induces the formation of a p62-associated necrosome (RIPK1/RIPK3/FADD/caspase-8), and death can be rescued with inhibitors of RIPK1 and MLKL, two key proteins in the necroptosis pathway. All sensitive cell lines, but no resistant lines, express RIPK3 protein, and data from knockdown and ectopic expression studies demonstrate the dependency of cell death on kinase-active RIPK3, as well as caspase-8. Studies using a TNFα neutralizing antibody suggest that the combination elicits autocrine production of TNFα, which likely, in turn, activates the extrinsic death pathway in these cells. In conclusion, these findings illustrate that necroptosis can be induced in RIPK3-expressing ovarian cancer cells and indicate that in vivo studies are warranted. Citation Format: Katelyn E. McCabe, Karl Bacos, Dan Lu, Joe R. Delaney, Mitchell Vamos, Nicholas D. P. Cosford, Dwayne G. Stupack. Induction of necroptosis in ovarian cancer cells as a therapeutic strategy. [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 1340. doi:10.1158/1538-7445.AM2014-1340

Previous studies have implicated cancer stem cells in tumor recurrence and revealed that the stem cell gene SOX2 plays an important role in the tumor cell resistance to apoptosis. Nonetheless, the mechanism by which SOX2 regulates apoptosis signals remained undefined. Here, we demonstrated the surprising finding that silencing of the SOX2 gene effectively induces apoptosis via the activation of death receptor and mitochondrial signaling pathways in human non-small cell lung cancer cells. Unexpectedly, reverse transcription-PCR analysis suggested that downregulation of SOX2 leads to activation of MAP4K4, previously implicated in cell survival. Evaluation of the apoptotic pathways revealed an increased expression of key inducers of apoptosis, including tumor necrosis factor-α and p53, with concurrent attenuation of Survivin. Although p53 appeared dispensable for this pathway, the loss of Survivin in SOX2-deficient cells appeared critical for the observed MAP4K4 induced cell death. Rescue experiments revealed that SOX2-silencing-mediated killing was blocked by ectopic expression of Survivin, or by reduction of MAP4K4 expression. Clinically, expressions of Survivin and SOX2 were highly correlated with each other. The results reveal a key target of SOX2 expression and highlight the unexpected context-dependent role for MAP4K4, a pluripotent activator of several mitogen-activated protein kinase pathways, in regulating tumor cell survival.

Abstract Fra-1 is a member of the Fos transcription factor family that is highly expressed in multiple cancers, playing important roles in transformation, proliferation, and metastasis. In this study, we observed an inverse correlation between the expression of Fra-1 in human stage II breast cancer tissues and the corresponding level of clinical chemoresistance. Extending these findings in vitro, we found that knockdown of Fra-1 in breast tumor cells was sufficient to confer resistance to doxorubicin and cyclophosphamide, whereas enhanced Fra-1 expression could render these cells chemosensitive. The tumor cell side population, which is enriched for cancer stem cells, was found to be associated with chemoresistance. Increased side population fractions were detected among tumor cell lines subjected to Fra-1 knockdown. In contrast, enhanced expression of Fra-1 was correlated with a decreased side population fraction, and significantly, this finding was recapitulated in vivo, where tumors with enhanced expression of Fra-1 were found to have blunted growth. Tumor cells subjected to Fra-1 knockdown grew faster and were larger in size. Taken together, our findings suggest that Fra-1 may be an important prognostic marker for breast cancer therapy. Cancer Res; 72(14); 3451–6. ©2012 AACR.

Legumain is a member of the asparaginyl endopeptidase family that is over-expressed in response to hypoxic stress on mammary adenocarcinoma, colorectal cancer, proliferating endothelial cells, and tumor-associated macrophages (TAMs). Here, we demonstrate that elevated expression of legumain in ovarian cancer by a proteomic approach using isobaric tags for relative and absolute quantification (iTRAQ) followed by liquid chromatographymass spectrometry (LCMS/MS). To investigate the relationship between legumain expression and ovarian cancer development, we tested legumain expression in malignant human ovarian tumors (n?=?60), borderline ovarian tumors (n?=?20), benign ovarian tumors (n?=?20), and normal ovary samples (n?=?20) using immunohistochemical assay (IHC). A correlation between legumain expression, and clinocopathologic and biological variables was also established. Importantly, increased legumain expression was validated by real-time PCR and Western blots, correlated positively with an increased malignancy of ovarian tumors (P?<?0.01). In fact, patients with strong legumain expression had a worse prognosis (P?=?0.03). In addition, results of in vitro experiments revealed that over-expression of legumain correlates with increased cell migration and invasion of ovarian cancer cells. Although legumain's functional role and clinical utility remain to be established, our results indicated that a sensitive assay for early expression of legumain may serve as both a potential biomarker and a molecular target for treatment of ovarian cancer. J. Cell. Biochem. 113: 26792686, 2012. (c) 2012 Wiley Periodicals, Inc.

Most hepatocellular carcinoma (HCC) therapies fail to target cancer stem cells (CSCs) and monitor cancer progression or regression. The purpose of this study was to evaluate the possibility of cancer imaging and simultaneously monitoring targeted therapy in a single animal by anti-CD44 antibody-mediated liposomal nanoparticle. In this study, an in situ liver tumor model was applied for therapy by injecting 1.0 × 106 HepG2 cells carrying a reporter system encoding a double fusion (DF) reporter gene consisting of firefly luciferase (Fluc) and green fluorescent protein (GFP) into the liver of NOD/SCID mice. A strategy was developed which specifically targeted HCC via anti-CD44 antibody-mediated liposomal nanoparticle delivery, loaded of either doxorubicin (Dox) or a triple fusion (TF) gene containing the herpes simplex virus truncated thymidine kinase (HSV-ttk) and renilla luciferase (Rluc) and red fluorescent protein (RFP). The NOD/SCID mice were subsequently treated with ganciclovir (GCV) and the growth status of tumor was monitored by optical bioluminescence imaging (BLI) of Fluc and specific targeting of the liposomal nanoparticle was tracked by Rluc imaging. CD44 antibody-mediated liposomal nanoparticle, loaded of TF plasmids, were shown to be useful for monitoring and evaluating targeting efficacy and gene therapy by non-invasive molecular imaging. Here, we demonstrate the time intensive preclinical steps involved in molecular target identification, validation, and characterization by dual molecular imaging. This targeted and traceable therapeutic strategy has potential advantages to overcome the problems of conventional tumor therapy and may open a new application for the treatment of HCC by targeting CSCs.

Abstract Conventional therapies target rapidly proliferating non-tumorigenic cells and spare the relatively quiescent cancer stem cells (CSCs) and the CSCs will remain viable after therapy and re-establish the tumor. Thus developing therapeutic strategies to target CSCs to prevent tumor recurrence will be vital for cancer therapy. To date, it has been shown that CSCs in hepatocellular carcinoma (HCC) can be identified by several cell surface antigens CD133, CD90, CD44, OV6 and the epithelial cell adhesion molecule (EpCAM). Here, we developed a novel strategy which targeted CSCs by anti-CD44 antibody mediated nanoparticles delivery system loaded with a triple fusion gene containing the suicide gene-herpes simplex virus truncated thymidine kinase (TTK), renilla luciferase (RL) and RFP (TF, RL-RFP-TTK). The in situ liver cancer model was established by injection of 1.0×105 HepG2 cells, which carry a reporter system encoding the genes of firefly luciferase (FL) and GFP into the liver of NOD/SCID mice. The mice were subsequently treated with ganciclovir (GCV). Then the growth status of tumor was monitored by the optical bioluminescence imaging of firefly luciferase(FL) and the specific targeting of the nanoparticles was tracked by imaging of renilla luciferase(RL). Our results demonstrated that the anti-CD44 antibody mediated nanoparticles loaded with TTK could specifically target the CSCs of HCC, and thereafter were endocytosed by the plasma membrane to transport the triple fusion (TF, RL-RFP-TTK) into the cells, resulted in the apoptosis of the targeted cells. Taken together, our study demonstrated a novel therapeutic strategy by targeted CSCs, we also developed a useful multimodality imaging techniques to monitor HepG2 cells’ fate in vivo and assessed the targeting efficacy of the nanoparticles. This may open a new application for the treatment of the HCC by targeting CSCs. 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 2464. doi:10.1158/1538-7445.AM2011-2464

Fra-1 is a member of the Fos family of transcription factors, which is highly expressed in multiple tumors and plays important roles in the transformation of malignant cells. Prior studies clearly illustrated some of the mechanisms of Fra-1 involvement in tumor proliferation and metastasis. However, the function of Fra-1 in cancer stem cells (CSCs) still remains to be resolved. We either overexpressed or silenced Fra-1 in murine 4TO7 breast cancer cells, and investigated its effects on regulation of the cell cycle, maintenance of side populations (SP) and determination of sensitivity to chemotherapeutic drugs both in vitro and in vivo. Overexpression of Fra-1 in 4TO7 cells significantly decreased the population of G0/G1-phase cells by DNA content analysis of fixed cells with propidium iodide (PI), as well as the intracellular flow cytometry (FCM) analysis of Ki-67 expression in fixed and permeabilized 4TO7 cells. We also performed the Hoechst 33342 dye exclusion assay, and detected a remarkable decrease of SP cells, which was further confirmed by intracellular staining of cancer stem cell marker Sca-1. Importantly, overexpression of Fra-1 correlated with a significant increase in chemosensitivity to either Doxorubicin (Dox) or cyclophosphamide (CTX) treatment both in vitro and in vivo. In contrast, extinction of Fra-1 increased the proportion of both G0/G1-phase cells and SP cells. These events in turn increased resistance of 4TO7 cells to chemotherapy both in vitro and in vivo. Together, our findings indicate that up- or down- regulation of Fra-1 in murine 4TO7 breast cancer cells can drive CSCs out of or back into dormancy, which may subsequently render them sensitive or resistant to chemotherapeutic drugs. In conclusion, we describe here a novel function of transcription factor Fra-1 in breast CSCs, which may hold important implications for future diagnosis and treatment of breast cancer patients. 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 3342. doi:10.1158/1538-7445.AM2011-3342