Anchorage-independent Growth Potential Research Articles

Auger Emitter Conjugated PARP Inhibitor for Therapy in Triple Negative Breast Cancers: A Comparative In-Vitro Study.

Simple SummaryTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer, with a high recurrence rate. Since treatment of BRCAmut TNBC patients with PARP inhibitor (PARPi), targeting the nuclear protein PARP1, shows varied responses, its therapeutic efficacy is currently evaluated in combination with chemotherapy. Auger emitters (AEs) are radionuclides that can cause DNA damage when delivered close to the DNA. Due to the nuclear location of PARP1, radiolabelling of PARPi with AEs provide an efficient nuclear delivery mechanism. This study shows the radiosynthesis of an AE radiolabelled PARPi ([125I]-PARPi-01) and its therapeutic effect as monotherapy or in combination with chemotherapeutics in a panel of TNBC cell lines. We found that [125I]-PARPi-01 efficiently induces DNA damage with therapeutic effect irrespective of BRCA mutation. All responsive cell lines have homologous recombination deficiency. Short pretreatment with doxorubicin significantly reduces clonogenic survival of both responsive and resistant cell lines.PARP1 inhibitors (PARPi) are currently approved for BRCAmut metastatic breast cancer, but they have shown limited response in triple negative breast cancer (TNBC) patients. Combination of an Auger emitter with PARPis enables PARP inhibition and DNA strand break induction simultaneously. This will enhance cytotoxicity and additionally allow a theranostic approach. This study presents the radiosynthesis of the Auger emitter [125I] coupled olaparib derivative: [125I]-PARPi-01, and its therapeutic evaluation in a panel of TNBC cell lines. Specificity was tested by a blocking assay. DNA strand break induction was analysed by γH2AX immunofluorescence staining. Cell cycle analysis and apoptosis assays were studied using flow cytometry in TNBC cell lines (BRCAwt/mut). Anchorage independent growth potential was evaluated using soft agar assay. [125I]-PARPi-01 showed PARP1-specificity and higher cytotoxicity than olaparib in TNBC cell lines irrespective of BRCA their status. Cell lines harbouring DNA repair deficiency showed response to [125I]-PARPi-01 monotherapy. Combined treatment with Dox-NP further enhanced therapeutic efficiency in metastatic resistant BRCAwt cell lines. The clonogenic survival was significantly reduced after treatment with [125I]-PARPi-01 in all TNBC lines investigated. Therapeutic efficacy was further enhanced after combined treatment with chemotherapeutics. [125I]-PARPi-01 is a promising radiotherapeutic agent for low radiation dosages, and mono/combined therapies of TNBC.

PTCH1 regulates anchorage-independent growth and bone invasion of non-small cell lung cancer cells.

Acquisition of metastatic potential by cancer cells is related to cancer stemness and anchorage-independent growth. The onset and progression of cancer are known to involve Hedgehog (HH) signaling that is activated by the binding of HH to the Patched 1 (PTCH1) receptor. However, the functions and mechanisms of action of PTCH1 in the context of bone metastasis remain to be elucidated. In this study, lentivirally-delivered shRNA was used to deplete PTCH1 levels, which resulted in the inhibition of spherical colony formation by the human non-small cell lung cancer (NSCLC) cell line; this suggested that PTCH1 promotes anchorage-independent growth. Concordantly, knockdown of PTCH1 resulted in significantly reduced migration and invasion of NSCLC cells; this was accompanied by the downregulation of MMP7 and SOX2. PTCH1 knockdown resulted in decreased bone destruction and osteoclastogenesis in a mouse bone metastasis model. These results indicate that PTCH1 may be an important regulator of bone invasion, and strongly suggest that knockdown of PTCH1 may decrease the anchorage-independent growth and metastatic potential of NSCLC.

MTH1 is involved in the toxic and carcinogenic long-term effects induced by zinc oxide and cobalt nanoparticles.

The nanoparticles (NPs) exposure-related oxidative stress is considered among the main causes of the toxic effects induced by these materials. However, the importance of this mechanism has been mostly explored at short term. Previous experience with cells chronically exposed to ZnO and Co NPs hinted to the existence of an adaptative mechanism contributing to the development of oncogenic features. MTH1 is a well-described enzyme expressed exclusively in cancer cells and required to avoid the detrimental consequences of its high prooxidant microenvironment. In the present work, a significantly marked overexpression was found when MTH1 levels were monitored in long-term ZnO and Co NP-exposed cells, a fact that correlates with acquired 2.5-fold and 3.75-fold resistance to the ZnO and Co NPs treatment, respectively. The forced stable inhibition of Mth1 expression by shRNA, followed by 6 additional weeks of exposure, significantly reduced this acquired resistance and sensitized cells to the oxidizing agents H2O2 and KBrO3. When the oncogenic phenotype of Mth1 knock-down cells was evaluated, we found a decrease in several oncogenic markers, including proliferation, anchorage-independent cell growth, and migration and invasion potential. Thus, MTH1 elicits here as a relevant player in the NPs-induced toxicity and carcinogenicity. This study is the first to give a mechanistic explanation for long-term NPs exposure-derived effects. We propose MTH1 as a candidate biomarker to unravel NPs potential genotoxic and carcinogenic effects, as its expression is expected to be elevated only under exposure conditions able to induce DNA damage and the acquisition of an oncogenic phenotype.

Three Prime Repair Exonuclease 1 (TREX1) expression correlates with cervical cancer cells growth in vitro and disease progression in vivo

Alterations in specific DNA damage repair mechanisms in the presence of human papillomavirus (HPV) infection have been described in different experimental models. However, the global effect of HPV on the expression of genes involved in these pathways has not been analyzed in detail. In the present study, we compared the expression profile of 135 genes involved in DNA damage repair among primary human keratinocytes (PHK), HPV-positive (SiHa and HeLa) and HPV-negative (C33A) cervical cancer derived cell lines. We identified 9 genes which expression pattern distinguishes HPV-positive tumor cell lines from C33A. Moreover, we observed that Three Prime Repair Exonuclease 1 (TREX1) expression is upregulated exclusively in HPV-transformed cell lines and PHK expressing HPV16 E6 and E7 oncogenes. We demonstrated that TREX1 silencing greatly affects tumor cells clonogenic and anchorage independent growth potential. We showed that this effect is associated with p53 upregulation, accumulation of subG1 cells, and requires the expression of E7 from high-risk HPV types. Finally, we observed an increase in TREX1 levels in precancerous lesions, squamous carcinomas and adenocarcinomas clinical samples. Altogether, our results indicate that TREX1 upregulation is important for cervical tumor cells growth and may contribute with tumor establishment and progression.

Abstract 2080: Role of differentially expressed micro RNAs in non-WNT, non-SHH medulloblastoma

Abstract Medulloblastoma is a common malignant brain tumor in children. It has four distinct molecular subgroups namely WNT, SHH, Group 3 and Group 4. The molecular subgroups differ in their clinical characteristics including age, incidence of metastasis and survival. Group 3 and Group 4 medulloblastomas have overlapping gene expression profile but are distinct in their clinical behavior. Group 3 medulloblastomas have the worst overall survival rates whereas Group 4 medulloblastomas have intermediate survival. Group 3 and Group 4 tumors are not associated with any known signaling pathway and due to their overlapping expression profiles it is often difficult to accurately identify the subgroup, which is very crucial for the appropriate treatment design. MiR-592, located in the intronic region of GRM8 gene, is over expressed in Group 4 medulloblastoma along with its host gene, thus acting as a good marker for Group 4 tumors. MiR-204, on the other hand is expressed from the 6th intron of TRPM3 gene and expressed in some Group 3 and majority of Group 4 medulloblastomas. In order to delineate the role of miR-592 and miR-204 in medulloblastoma biology, effect of expression of these microRNAs on medulloblastoma cell growth and behavior were investigated. The micro RNAs were expressed in a lentiviral mediated inducible manner in Group 3 medulloblastoma cell lines D283, D425 and D341 and their effects on cell proliferation, anchorage-independent growth and invasion potential were studied. MiR-592 expression had marginal effects on the anchorage independent growth potential and the tumorigenic potential of the Group 3 cell line where as miR-204 expression led to significant decrease in the anchorage independent growth, tumorigenic potential, as well as invasion potential of the medulloblastoma cell lines. Further in order to decipher the molecular mechanisms underlying these micro RNA mediated effects, RNA-sequencing analysis of miR-592 and miR-204 expressing medulloblastoma cells were performed to identify genes that are differentially expressed upon micro RNA expression. The results helped us to identify the molecular pathways that are being affected by the micro RNA expression and how they affect the malignant behavior of medulloblastoma cells. Citation Format: Raikamal Paul, Rakesh Jalali, Epari Sridhar, Aliasagar Moiyadi, Neelam Shirsat. Role of differentially expressed micro RNAs in non-WNT, non-SHH medulloblastoma [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 2080.

Abstract 5524: Tumor suppressor Numb represses monocarboxylate transporters to reduce the glycolysis in oral carcinoma

Abstract Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide. Our previous studies have shown that Numb pluripotent gene drives suppressor activity against OSCC, and it is co-targeted by multiple oncogenic miRNAs. In this study, we further identified that Numb mRNA expression was down-regulated in a large fraction of OSCC tumors. We established the Numb-knockout (KO) OSCC cell subclones using CRISPR-Cas9 system. We also generated lentiviruses for exogenous expression of Numb 1 and Numb 4 isoforms. The anchorage independent growth potential was found increased in Numb-KO cell subclones. However, such eligibility was repressed after the rescue of the Numb expression by lentiviral infection. In addition, the level of lactate production and the ratio of ECAR/OCR in the Numb-KO cell subclones were higher than parental cells. The knockout of Numb was able to up-regulate the expression of monocarboxylate transporter (MCT) family members MCT1 and MCT4, but not other metabolism genes, to increase the lactate production. Knockdown of both MCT1 and MCT4 significantly decreased the anchorage independent growth and the lactate production of OSCC cells. This study provides new evidences denoting that Numb represses MCTs to attenuate the transformative capability and the glycolysis in oral carcinoma. Citation Format: Chung-Hsien Chou, Chun-Yu Fan Chaing, Shu-Chun Lin, Kuo-Wei Chang, Hsi-Feng Tu. Tumor suppressor Numb represses monocarboxylate transporters to reduce the glycolysis in oral carcinoma [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 5524.

Inhibition of cancer progression by a novel trans-stilbene derivative through disruption of microtubule dynamics, driving G2/M arrest, and p53-dependent apoptosis

Resveratrol, a trans-stilbene polyphenolic compound and its synthetic analogs are widely used bioactive molecules due to their remarkable chemo-preventive potential. Here, we have identified a novel synthetic trans-stilbene compound, Z-DAN-11 ((Z)-3-(3, 4-dimethoxyphenyl)-2-(3, 4, 5-trimethoxyphenyl) acrylonitrile) which shows remarkable efficacy in blocking tumor growth and progression both in vitro and in vivo. Z-DAN-11 inhibits proliferation of cancer cells in vitro through microtubule depolymerization that induced G2/M arrest and consequently leads to apoptotic cell death. More importantly, Z-DAN-11 shows limited cytotoxicity to normal cells as compared to cancer cells. Quite interestingly, we have found that Z-DAN-11-mediated ROS production helps in dramatic alteration in the mitochondrial redox status which critically contributes to the apoptosis. Mechanistic studies reveal that Z-DAN-11 induces the expression of pro-apoptotic proteins and decreases anti-apoptotic protein expression that decisively helps in the activation of caspase 8, caspase 9, and caspase 3, leading to cleavage of PARP1 and cell death via intrinsic and extrinsic pathways of apoptosis. Moreover, Z-DAN-11-mediated apoptosis of cancer cells is through a partial p53-dependent pathway, since both HCT116 p53−/− cells as well as p53-silenced cells (siRNA) were able to block apoptosis partially but significantly. Importantly, Z-DAN-11 also imparts its anti-tumorigenic effect by inhibiting clonogenic property and anchorage-independent growth potential of cancer cells at concentrations at least 10 times lower than that required for inducing apoptosis. Finally, in vivo study with immuno-competent syngeneic mice shows Z-DAN-11 to be able to impede tumor progression without any adverse side-effects. Hence, we identified a novel, synthetic trans-stilbene derivative with anti-tumorigenic potential which might tremendously help in devising potential therapeutic strategy against cancer.

Gamma-Klotho exhibits multiple roles in tumor growth of human bladder cancer

Alpha-Klotho (KLα) and beta-Klotho (KLβ) have recently been reported to correlate with cancer prognosis in some malignancies and we previously reported the association between KLα, KLβ, and urothelial carcinoma of the bladder (UCB), indicating that KLβ acts as a tumor promoter. However, the association between gamma-Klotho (KLγ) and cancer prognosis remains unclear. In the present study, we evaluated the association between KLγ and UCB. To evaluate the effect of KLγ on human bladder cancer cell lines in vitro assays were performed. Exogenous KLγ increased the ability of human bladder cancer cells to proliferate, migrate, invade, form colonies, and provide anchorage-independent growth potential. In in vivo assays, eighteen mice bearing xenografts inoculated using UM-UC-3, were randomly divided into three groups and treated with a small interfering RNA (siRNA) by intratumoral administration once a week for four weeks. Knockdown of KLγ with siRNA led to a dramatic change in tumor growth and suggested that KLγ had effects on tumor growth, including promotion of cell proliferation, inhibition of apoptosis, and enhancement of the epithelial-mesenchymal transition. To confirm the study, human tissue samples were used and patients were divided into two groups according to KLγ expression level. High expression of KLγ was significantly associated with higher stage and grade cancer and the presence of lymphovascular invasion compared to patients with lower expression of KLγ. Our results suggest that KLγ plays an important role in tumor invasion and progression and these results may lead to the development of new therapies and diagnostic methods for UCB.

Runx3 in Immunity, Inflammation and Cancer.

In this chapter we summarize the pros and cons of the notion that Runx3 is a major tumor suppressor gene (TSG). Inactivation of TSGs in normal cells provides a viability/growth advantage that contributes cell-autonomously to cancer. More than a decade ago it was suggested that RUNX3 is involved in gastric cancer development, a postulate extended later to other epithelial cancers portraying RUNX3 as a major TSG. However, evidence that Runx3 is not expressed in normal gastric and other epithelia has challenged the RUNX3-TSG paradigm. In contrast, RUNX3 is overexpressed in a significant fraction of tumor cells in various human epithelial cancers and its overexpression in pancreatic cancer cells promotes their migration, anchorage-independent growth and metastatic potential. Moreover, recent high-throughput quantitative genome-wide studies on thousands of human samples of various tumors and new investigations of the role of Runx3 in mouse cancer models have unequivocally demonstrated that RUNX3 is not a bona fide cell-autonomous TSG. Importantly, accumulating data demonstrated that RUNX3 functions in control of immunity and inflammation, thereby indirectly influencing epithelial tumor development.

Abstract 2128: Influence of estrogen receptor status on acquisition of doxorubicin resistance in breast cancer cells

Abstract Breast cancer is the most commonly diagnosed invasive cancer in women. Doxorubicin is being the mainstay treatment for solid cancers including breast cancer. However, chemotherapy resistance, either innate or acquired, is a major limitation in breast cancer treatment. The mechanism of resistance as well as the role of estrogen receptor (ER) status in doxorubicin resistance in breast cancer is not clear. Therefore, objective of this study was to determine whether ER status influence the doxorubicin resistance, and to further identify the molecular mechanism in this process. To address this question, ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cell lines were given continuous treatment of clinically relevant concentration of doxorubicin and the pattern of resistance was monitored. Resistance was evaluated using various parameters such as cell count, cell growth and cytotoxicity by MTT assay, cell cycle analysis using flow cytometer. Expression of genes and protein related to cell cycle and cell survival, drug transport, DNA damage repair, metastasis/invasion and epigenetic regulatory complex were measured by qRT- PCR and western blot respectively. Soft agar assay and wound healing assay were performed to determine the anchorage-independent growth and migration potential of resistance cells. Results of MTT assay, and cell cycle revealed that ER-positive MCF-7 cells developed relatively earlier and high level of resistance when compared to MDA-MB-231 cells. This was further confirmed by additional features such as, cancer stem cell markers, epithelial to mesenchymal transition, and increased tumorigenicity in MCF-7 cells than in MDA-MB-231 cells during resistance development. These changes were associated with alteration in drug transporters, cell cycle genes and epigenetic regulatory proteins such as HDAC1 and DNMT1. Pretreatment with demethylating agent 5-aza-deoxycytidine and HDAC inhibitor Trichostatin A significantly resensitized resistance MCF-7 and MDA-MB-231 cells to doxorubicin in comparable to sensitive parental cells. However, this resenstitivty was higher in MCF-7 cells when compared to MDA-MB-231 cells. In summary, result of this study suggests that acquisition of doxorubicin resistance depends on status of estrogen receptor in breast cancer cell lines. Additionally, this differential resistance pattern could be due to differences in epigenetic changes in these two types of breast cancers, thus the acquired resistance could be potentially resensitized using epigenetic therapy Citation Format: Logeswari Ponnusamy, Prathap Kumar Salakatte Mahalingaiah, Kamaleshwar P. Singh. Influence of estrogen receptor status on acquisition of doxorubicin resistance in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2128.

Overexpression of PGC-1α enhances cell proliferation and tumorigenesis of HEK293 cells through the upregulation of Sp1 and Acyl-CoA binding protein

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC‑1α), a coactivator interacting with multiple transcription factors, regulates several metabolic processes. Although recent studies have focused on the role of PGC‑1α in cancer, the underlying molecular mechanism has not been clarified. Therefore, we evaluated the role of PGC‑1α in cell proliferation and tumorigenesis using human embryonic kidney (HEK)293 cells and colorectal cancer cells. We established stable HEK293 cell lines expressing PGC‑1α and examined cell proliferation, anchorage-independent growth, and oncogenic potential compared to parental HEK293 cells. To identify the molecular PGC‑1α targets for increased cell proliferation and tumorigenesis, the GeneFishing™ DEG (differentially expressed genes) screening system was used. Western blot analysis and immunofluorescence staining were performed for a regulated gene product to confirm the results. Forced expression of PGC‑1α in HEK293 cells promoted cell proliferation and anchorage-independent growth in soft agar. In addition, HEK293 cells that highly expressed PGC‑1α showed enhanced tumor formation when subcutaneously injected into the bilateral flanks of immunodeficient mice. The results of the GeneFishing DEG screening system identified one upregulated gene (Acyl-CoA binding protein; ACBP). Real-time RT-PCR, western blot analysis, and immunofluorescence staining showed that ACBP was markedly increased in HEK293 cells stably overexpressing PGC‑1α (PGC‑1α-HEK293 cells) compared to those expressing an empty vector. In PGC‑1α, ACBP, and specificity protein 1 (Sp1) siRNA knockdown experiments in PGC‑1α-HEK293 and SNU-C4 cells, we also observed inhibition of cell proliferation, reduced expression of antioxidant enzymes, and increased H2O2-induced reactive oxygen species production and apoptosis. These findings suggest that PGC‑1α may promote cell proliferation and tumorigenesis through upregulation of ACBP. We provide evidence that increased Sp1 expression might contribute to enhanced ACBP expression by PGC‑1α. The current results also suggest that PGC‑1α, whose expression is related to enhanced cell proliferation and tumorigenesis, may be a good candidate molecular target for cancer therapy.

Focus formation: a cell-based assay to determine the oncogenic potential of a gene.

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals(1). In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)(2). When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci(1) that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

Abstract B02: Protein Kinase C iota is required for the maintenance of a tumor-initiating cell phenotype in lung squamous cell carcinoma.

Abstract Background: Protein kinase C iota (PKCι) is an oncogene that is frequently targeted for tumor-specific gene amplification in human cancers harboring the 3q26 amplicon including Non-Small Cell Lung Carcinoma (NSCLC). PKCι is required for the maintenance of the transformed phenotype of NSCLC cells in vitro and in vivo. In addition, PKCι is critical for lung tumorigenesis in the LSL-K-rasG12D mouse model of NSCLC. Recently, we demonstrated that genetic disruption of Prkcι/λ; in LSL-K-rasG12D mice blocks K-ras-mediated expansion and morphological transformation of bronchioalveolar stem cells (BASCs), the first identifiable step in K-ras-mediated transformation in this model. These results demonstrate that PKCι plays a requisite role in tumor initiation, and suggest that PKCι may serve a similar role in the tumor-initiating activity of lung cancer stem cells. Our current studies aim to identify and characterize the molecular signaling pathway(s) by which PKCι regulates the biology of human lung squamous cell carcinoma (LSCC) tumor initiating cells (TICs) isolated from LSCC cell lines and primary tumors. Methods: Cultures enriched in TICs were established from LSCC lines or primary human LSCC tumors that harbor PRKCI amplification. PKCι expression in TIC cultures was inhibited by lentiviral shRNA, and the effect of PKCι knockdown on their stem-like properties was assessed. Whole genome deep sequencing analysis was performed on mRNA from parental, control TIC and PKCι knockdown TIC cultures in order to identify PKCι-regulated targets and signaling pathways in TICs. Results: LSCC TIC cultures express putative stem cell markers, and exhibit clonal expansion, self-renewal, enhanced anchorage-independent growth and tumorigenic potential in vivo. RNAi-mediated knockdown of PKCι inhibited TIC growth, clonal expansion, transformed growth and tumor initiation. mRNA sequencing revealed that PKCι regulates the expression and function of critical molecules involved in the stem-like phenotype of LSCC TICs. Details of PKCι-mediated signaling in LSCC TICs will be discussed. Conclusions: PKCι is required for maintenance of a highly tumorigenic stem-like population of cells in human LSCC. Given the prevalence of 3q26 amplification in human cancer, PKCι may regulate the biology of TICs in tumor types other than LSCC. Our results provide a rationale for therapeutic targeting of PKCι in LSCC TICs. Citation Format: Verline Justilien, Michael P. Walsh, Syed A. Ali, E. A. Thompson, Alan P. Fields. Protein Kinase C iota is required for the maintenance of a tumor-initiating cell phenotype in lung squamous cell carcinoma. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B02.

Abstract P4-09-06: miR-187 is an independent prognostic factor in lymph node-positive breast cancer patients.

Abstract Purpose: MicroRNAs (miRNAs) involved in cancer progression have now become the focus of much attention as they represent a new class of biomarkers and potential drug targets. Here, we describe an integrated bioinformatics, functional analysis and translational pathology approach for the identification of novel miRNAs involved in breast cancer progression. Experimental Design Differential gene expression can, in part, be attributed to the activity of specific miRNAs. Given a database of miRNA binding site motifs and gene expression levels determined by transcriptomic profiling, correspondence analysis, between group analysis and co-inertia analysis can be combined to produce a ranked list of miRNAs associated with a specific gene signature and phenotype. Here, using two independent breast cancer cohorts, this approach was employed to produce a ranked list of miRNAs associated with disease progression. Functional studies were subsequently carried out in MCF7 cells assessing for alterations in growth, tumorigenicity and agressiveness and miRNA expression was evaluated in two cohorts of breast cancer patients by locked nucleic acid in situ hybridisation on tissue microarrays. Results: CIA identified miR-187 as a key miRNA associated with poor outcome in breast cancer. Ectopic expression of miR-187 in MCF7 cells resulted in a more aggressive phenotype (evidenced by increased anchorage-independent growth, migratory and invasive potential). In a test cohort (n = 117) breast cancer patients, high expression of miR-187 was associated with a trend towards reduced breast cancer-specific survival (BCSS) (p = 0.058), and a significant association with reduced BCSS in lymph node-positive patients (p = 0.036). In a validation cohort (n = 470), high miR-187 was significantly associated with reduced BCSS in the entire cohort (p = 0.021) and, again, in lymph node-positive patients (p = 0.012). Multivariate cox regression analysis revealed that miR-187 is an independent prognostic factor in both TMA cohorts (Cohort 1 HR-7.369 (95% CI 2.048–26.509, p = 0.002); Cohort 2 HR-2.798 (95% CI 1.518–5.157, p = 0.001). Conclusions: miR-187 expression in breast cancer leads to the formation of a more aggressive, invasive phenotype and acts as an independent predictor of outcome. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-09-06.

Smac mimetic Birinapant induces apoptosis and enhances TRAIL potency in inflammatory breast cancer cells in an IAP-dependent and TNF-α-independent mechanism

X-linked inhibitor of apoptosis protein (XIAP), the most potent mammalian caspase inhibitor, has been associated with acquired therapeutic resistance in inflammatory breast cancer (IBC), an aggressive subset of breast cancer with an extremely poor survival rate. The second mitochondria-derived activator of caspases (Smac) protein is a potent antagonist of IAP proteins and the basis for the development of Smac mimetic drugs. Here, we report for the first time that bivalent Smac mimetic Birinapant induces cell death as a single agent in TRAIL-insensitive SUM190 (ErbB2-overexpressing) cells and significantly increases potency of TRAIL-induced apoptosis in TRAIL-sensitive SUM149 (triple-negative, EGFR-activated) cells, two patient tumor-derived IBC models. Birinapant has high binding affinity (nM range) for cIAP1/2 and XIAP. Using isogenic SUM149- and SUM190-derived cells with differential XIAP expression (SUM149 wtXIAP, SUM190 shXIAP) and another bivalent Smac mimetic (GT13402) with high cIAP1/2 but low XIAP binding affinity (K (d) > 1 μM), we show that XIAP inhibition is necessary for increasing TRAIL potency. In contrast, single agent efficacy of Birinapant is due to pan-IAP antagonism. Birinapant caused rapid cIAP1 degradation, caspase activation, PARP cleavage, and NF-κB activation. A modest increase in TNF-α production was seen in SUM190 cells following Birinapant treatment, but no increase occurred in SUM149 cells. Exogenous TNF-α addition did not increase Birinapant efficacy. Neutralizing antibodies against TNF-α or TNFR1 knockdown did not reverse cell death. However, pan-caspase inhibitor Q-VD-OPh reversed Birinapant-mediated cell death. In addition, Birinapant in combination or as a single agent decreased colony formation and anchorage-independent growth potential of IBC cells. By demonstrating that Birinapant primes cancer cells for death in an IAP-dependent manner, these findings support the development of Smac mimetics for IBC treatment.

Ethanol promotes cytotoxic effects of tumor necrosis factor-related apoptosis-inducing ligand through induction of reactive oxygen species in prostate cancer cells

Effective treatment of prostate cancer (PCa) remains a major challenge due to chemoresistance to drugs including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Ethanol and ethanol extracts are known apoptosis inducers. However, cytotoxic effects of ethanol on PCa cells are unclear. In this study we utilized PC3 and LNCaP cell culture models. We used immunohistochemical analysis, western blot analysis, reactive oxygen species (ROS) measurement, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) Cell Proliferation Assay, Annexin-V staining and flow cytometry for quantification of apoptosis. In vitro soft agar colony formation and Boyden chamber invasion assays were used. Tumorigenicity was measured in a xenotransplantation mouse model. Here, we demonstrate that ethanol enhances the apoptosis-inducing potential of TRAIL in androgen-resistant PC3 cells and sensitizes TRAIL-resistant, androgen sensitive LNCaP cells to apoptosis through caspase activation, and a complete cleavage of poly (ADP)-ribose polymerase, which was in association with increased production of ROS. The cytotoxicity of ethanol was suppressed by an antioxidant N-acetyl cystein pretreatment. Furthermore, ethanol in combination with TRAIL increased the expression of cyclin-dependent kinase inhibitor p21 and decreased the levels of Bcl-2 and phosphorylated-AKT. These molecular changes were accompanied by decreased proliferation, anchorage-independent growth and invasive potential of PC3 and LNCaP cells. In vivo studies using a xenotransplantation mouse model with PC3 cells demonstrated significantly increased apoptosis in tumors treated with ethanol and TRAIL in combination. Taken together, use of ethanol in combination with TRAIL may be an effective strategy to augment sensitivity to TRAIL-induced apoptosis in PCa cells.

Fas Ligand Enhances Malignant Behavior of Tumor Cells through Interaction with Met, Hepatocyte Growth Factor Receptor, in Lipid Rafts

Many late-stage cancer cells express Fas ligand (FasL) and show high malignancy with metastatic potential. We report here a novel signaling mechanism for FasL that hijacks the Met signal pathway to promote tumor metastasis. FasL-expressing human tumor cells express a significant amount of phosphorylated Met. The down-regulation of FasL in these cells led to decreased Met activity and reduced cell motility. Ectopic expression of human FasL in NIH3T3 cells significantly stimulated their migration and invasion. The inhibition of Met and Stat3 activities reverted the FasL-associated phenotype. Notably, FasL variants activated the Met pathway, even though most of their intracellular domain or Fas binding sites were deleted. FasL interacted with Met through the FasL(105-130) extracellular region in lipid rafts, which consequently led to Met activation. Knocking down Met gene expression by RNAi technology reverted the FasL-associated motility to basal levels. Furthermore, treatment with synthetic peptides corresponding to FasL(117-126) significantly reduced the FasL/Met interaction, Met phosphorylation, and cell motility of FasL(+) transfectants and tumor cells. Finally, the transfectants of truncated FasL showed strong anchorage-independent growth and lung metastasis potential in null mice. Collectively, our results establish the FasL-Met-Stat3 signaling pathway and explains the metastatic phenotype of FasL-expressing tumors.

Abstract 1143: EZH2 promotes oral squamous cell carcinoma initiation and progression through epigenetic silencing of FHL1

Abstract Oral squamous cell carcinoma (OSCC) is widely represented as a heterogeneous tumor with more aggressive phenotypes, but the basis for these characteristics remains unknown. In the current study, we show that up-regulation of the Polycomb complex histone methytransferase EZH2 is critical to maintain the malignant phenotypes in OSCC and associated significantly with oral squamous cell carcinoma development in patients with oral leukoplakia (OLP), which represents oral precancerous lesion as well as the worse prognosis of patients with locally advanced OSCC. Patients with OLP (n=76) and OSCC (n=124) from two independent cohort were included in this study. Immunohistochemical analyses on EZH2 in the two cohort revealed that the higher EZH2 levels were strongly associated with dysplasia (P < 0.001) and OSCC development (P < 0.0001) in patients with OLP, as well as worse overall survival (P = 0.001) and disease free survival (P = 0.002) in patients with locally advanced OSCC. Furthermore, upon EZH2 silencing by small interfering RNA (siRNA)-mediated knockdown, the proliferation, anchorage-independent growth and invasion potential of OLP cell line and OSCC cell lines were remarkably suppressed. Available publicly H3K27 modifications based on CpG array and three public microarray datasets (GEO) analyses identified human four-and-half LIM protein 1(FHL1) as a novel candidate target of EZH2 in OSCC. Further depletion of EZH2 by RNA interference (RNAi), the expression of FHL1 was notably restored in OLP and OSCC cell lines. In addition, ablation of FHL1 by RNA interference promoted significantly proliferation and invasion potential of OSCC cells. Together, our findings indicate that aberrantly elevated EZH2 promotes aggressive phenotypes of OLP and OSCC cells and plays a critical role in initiation and progression of OSCC. meanwhile, FHL1 may be a novel target of EZH2 for transcriptional repression in OSCC initiation and progression. ACKNOWLEDGMENTS: This study was supported by the National Natural Science Foundation of China (Grant No. 30973343), Projects of the Shanghai Science and Technology Committee (Grant No. 08JC1414400 and 10XD1402500) and the Shanghai Leading Academic Discipline Project (S30206). 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 1143. doi:1538-7445.AM2012-1143

Augmented autocrine bone morphogenic protein (BMP) 7 signaling increases the metastatic potential of mouse breast cancer cells

As malignant breast cancers progress, they acquire the ability to spread to other regions of the body, including bone and lung, but the molecular mechanism underlying the increase in metastatic potential is not fully understood. Here we studied murine 4T1E/M3 highly bone marrow metastatic breast cancer cells, which we established previously. These cells show upregulated expression of bone morphogenetic protein (BMP) 7 and BMP receptors, as well as augmented phosphorylation of Smad1/5/8. Both anchorage-independent cell growth measured in colony forming assays and cell migration measured in wound healing assays were suppressed in 4T1E/M3 cells following treatment with a neutralizing anti-BMP7 antibody or knockdown of BMP7 gene expression. In addition, metastasis of 4T1E/M3 cells to the spine and lung and intracellular levels of phosphorylated Smad1/5/8 were suppressed by knocking down BMP7. Conversely, overexpression of BMP7 in the weakly metastatic parental 4T1E cells augmented their anchorage-independent growth, migration and metastasis to spine and lung. Taken together, our results strongly suggest that augmented autocrine BMP7 signaling leads to increases in the anchorage-independent cell growth, migration and metastatic potential in our bone marrow metastatic breast cancer model.