Search

Purpose: To compare napabucasin (generator of reactive oxygen species) plus paclitaxel with paclitaxel only in patients with second-line advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma. Patients and Methods: In the double-blind, phase III BRIGHTER study (NCT02178956), patients were randomized (1:1) to napabucasin (480 mg orally twice daily) plus paclitaxel (80 mg/m2 i.v. weekly for 3 of 4 weeks) or placebo plus paclitaxel. The primary endpoint was overall survival (OS). Secondary endpoints included progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), and safety. Results: Overall, 714 patients were randomized (napabucasin plus paclitaxel, n = 357; placebo plus paclitaxel, n = 357). 72.1% were male, 74.6% had gastric adenocarcinoma, and 46.2% had peritoneal metastases. The study was unblinded following an interim analysis at 380 deaths. The final efficacy analysis was performed on 565 deaths (median follow-up, 6.8 months). No significant differences were observed between napabucasin plus paclitaxel and placebo plus paclitaxel for OS (6.93 vs. 7.36 months), PFS (3.55 vs. 3.68 months), ORR (16% vs. 18%), or DCR (55% vs. 58%). Grade ≥3 adverse events occurred in 69.5% and 59.7% of patients administered napabucasin plus paclitaxel and placebo plus paclitaxel, respectively, with grade ≥3 diarrhea reported in 16.2% and 1.4%, respectively. Conclusions: Adding napabucasin to paclitaxel did not improve survival in patients with pretreated advanced gastric or GEJ adenocarcinoma. Consistent with previous reports, the safety profile of napabucasin was driven by manageable gastrointestinal events; grade ≥3 diarrhea occurred at a higher frequency with napabucasin plus paclitaxel versus placebo plus paclitaxel.

Supplementary Figure from Randomized, Double-Blind, Placebo-Controlled Phase III Study of Paclitaxel ± Napabucasin in Pretreated Advanced Gastric or Gastroesophageal Junction Adenocarcinoma

Supplementary Data from ARQ 197, a Novel and Selective Inhibitor of the Human c-Met Receptor Tyrosine Kinase with Antitumor Activity

Supplementary Table from Randomized, Double-Blind, Placebo-Controlled Phase III Study of Paclitaxel ± Napabucasin in Pretreated Advanced Gastric or Gastroesophageal Junction Adenocarcinoma

The met proto-oncogene is functionally linked with tumorigenesis and metastatic progression. Validation of the receptor tyrosine kinase c-Met as a selective anticancer target has awaited the emergence of selective c-Met inhibitors. Herein, we report ARQ 197 as the first non-ATP–competitive small molecule that selectively targets the c-Met receptor tyrosine kinase. Exposure to ARQ 197 resulted in the inhibition of proliferation of c-Met–expressing cancer cell lines as well as the induction of caspase-dependent apoptosis in cell lines with constitutive c-Met activity. These cellular responses to ARQ 197 were phenocopied by RNAi-mediated c-Met depletion and further demonstrated by the growth inhibition of human tumors following oral administration of ARQ 197 in multiple mouse xenograft efficacy studies. Cumulatively, these data suggest that ARQ 197, currently in phase II clinical trials, is a promising agent for targeting cancers in which c-Met-driven signaling is important for their survival and proliferation. Mol Cancer Ther; 9(6); 1544–53. ©2010 AACR.

Supplementary Data from Randomized, Double-Blind, Placebo-Controlled Phase III Study of Paclitaxel ± Napabucasin in Pretreated Advanced Gastric or Gastroesophageal Junction Adenocarcinoma

Purpose:To compare napabucasin (generator of reactive oxygen species) plus paclitaxel with paclitaxel only in patients with second-line advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma.Patients and Methods:In the double-blind, phase III BRIGHTER study (NCT02178956), patients were randomized (1:1) to napabucasin (480 mg orally twice daily) plus paclitaxel (80 mg/m2 i.v. weekly for 3 of 4 weeks) or placebo plus paclitaxel. The primary endpoint was overall survival (OS). Secondary endpoints included progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), and safety.Results:Overall, 714 patients were randomized (napabucasin plus paclitaxel, n = 357; placebo plus paclitaxel, n = 357). 72.1% were male, 74.6% had gastric adenocarcinoma, and 46.2% had peritoneal metastases. The study was unblinded following an interim analysis at 380 deaths. The final efficacy analysis was performed on 565 deaths (median follow-up, 6.8 months). No significant differences were observed between napabucasin plus paclitaxel and placebo plus paclitaxel for OS (6.93 vs. 7.36 months), PFS (3.55 vs. 3.68 months), ORR (16% vs. 18%), or DCR (55% vs. 58%). Grade ≥3 adverse events occurred in 69.5% and 59.7% of patients administered napabucasin plus paclitaxel and placebo plus paclitaxel, respectively, with grade ≥3 diarrhea reported in 16.2% and 1.4%, respectively.Conclusions:Adding napabucasin to paclitaxel did not improve survival in patients with pretreated advanced gastric or GEJ adenocarcinoma. Consistent with previous reports, the safety profile of napabucasin was driven by manageable gastrointestinal events; grade ≥3 diarrhea occurred at a higher frequency with napabucasin plus paclitaxel versus placebo plus paclitaxel.

To compare napabucasin (generator of reactive oxygen species) plus paclitaxel with paclitaxel only in patients with second-line advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma.In the double-blind, phase III BRIGHTER study (NCT02178956), patients were randomized (1:1) to napabucasin (480 mg orally twice daily) plus paclitaxel (80 mg/m2 intravenously weekly for 3 of 4 weeks) or placebo plus paclitaxel. The primary endpoint was overall survival (OS). Secondary endpoints included progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), and safety.Overall, 714 patients were randomized (napabucasin plus paclitaxel, n = 357; placebo plus paclitaxel, n = 357). 72.1% were male, 74.6% had gastric adenocarcinoma, and 46.2% had peritoneal metastases. The study was unblinded following an interim analysis at 380 deaths. The final efficacy analysis was performed on 565 deaths (median follow-up, 6.8 months). No significant differences were observed between napabucasin plus paclitaxel and placebo plus paclitaxel for OS (6.93 vs. 7.36 months), PFS (3.55 vs. 3.68 months), ORR (16% vs. 18%), or DCR (55% vs. 58%). Grade {greater than or equal to}3 adverse events occurred in 69.5% and 59.7% of patients administered napabucasin plus paclitaxel and placebo plus paclitaxel, respectively, with grade {greater than or equal to}3 diarrhea reported in 16.2% and 1.4%, respectively.Adding napabucasin to paclitaxel did not improve survival in patients with pretreated advanced gastric or GEJ adenocarcinoma. Consistent with previous reports, the safety profile of napabucasin was driven by manageable gastrointestinal events; grade {greater than or equal to}3 diarrhea occurred at a higher frequency with napabucasin plus paclitaxel versus placebo plus paclitaxel.

Spontaneous tumor regression following bacterial infection has been observed for hundreds of years. These observations along with anecdotal medical findings in 1890s led to the development of Coley's "toxins," consisting of killed Streptococcus pyogenes and Serratia marcescens bacteria, as the first cancer immunotherapy. The use of this approach, however, was not widely accepted at the time especially after the introduction of radiation therapy as a treatment for cancer in the early 1900s. Over the last 30-40 years there has been renewed interest in the use of bacteria to treat human solid tumors. This is based on the observation that various nonpathogenic anaerobic bacteria can infiltrate and replicate within solid tumors when given intravenously. Bacteria tested as potential anticancer agents include the Gram-positive obligate anaerobes Bifidobacterium and Clostridium, as well as the gram-negative facultative anaerobe Salmonella. Recent advances in synthetic biology and clinical success in cancer immunotherapy provide renewed momentum for developing bacteria-based cancer immunotherapy for cancer treatment and should allow greater potential for the development of novel therapeutic approaches for this devastating disease.

Spontaneous tumor regression following bacterial infection has been observed for hundreds of years. These observations along with anecdotal medical findings in 1890s led to the development of Coley's "toxins," consisting of killed Streptococcus pyogenes and Serratia marcescens bacteria, as the first cancer immunotherapy. The use of this approach, however, was not widely accepted at the time especially after the introduction of radiation therapy as a treatment for cancer in the early 1900s. Over the last 30-40 years there has been renewed interest in the use of bacteria to treat human solid tumors. This is based on the observation that various nonpathogenic anaerobic bacteria can infiltrate and replicate within solid tumors when given intravenously. Bacteria tested as potential anticancer agents include the Gram-positive obligate anaerobes Bifidobacterium and Clostridium, as well as the gram-negative facultative anaerobe Salmonella. Recent advances in synthetic biology and clinical success in cancer immunotherapy provide renewed momentum for developing bacteria-based cancer immunotherapy for cancer treatment and should allow greater potential for the development of novel therapeutic approaches for this devastating disease.

Summary Background Napabucasin is a first-in-class cancer stemness inhibitor that targets STAT3, which is a poor prognostic factor in colorectal cancer. This study aimed to test napabucasin in advanced colorectal cancer. Methods This study was a double-blind randomised phase 3 trial done at 68 centres in Canada, Australia, New Zealand, and Japan. Patients with advanced colorectal cancer with a good Eastern Cooperative Oncology Group (ECOG) performance status (0–1) for whom all available standard therapies had failed were eligible for the study. Patients were randomly assigned (1:1) to receive placebo or napabucasin through a web-based system with a permuted block method, after stratification by ECOG performance status, KRAS status, previous VEGF inhibitor treatment, and time from diagnosis of metastatic disease. Napabucasin 480 mg or matching placebo was taken orally every 12 h. All patients received best supportive care. The primary endpoint was overall survival assessed in an intention-to-treat analysis. This is the final analysis of this trial, which is registered at ClinicalTrials.gov, number NCT01830621. Findings Accrual began on April 15, 2013, and was stopped for futility on May 23, 2014, at which point 282 patients had undergone randomisation (138 assigned to the napabucasin group and 144 to the placebo group). Overall survival did not differ significantly between groups: median overall survival was 4·4 months (95% CI 3·7–4·9) in the napabucasin group and 4·8 months (4·0–5·3) in the placebo group (adjusted hazard ratio [HR] 1·13, 95% CI 0·88–1·46, p=0·34). The safety population included 136 patients in the napabucasin group and 144 patients in the placebo group. More patients who received napabucasin had any grade of treatment-related diarrhoea (108 [79%] of 136 patients), nausea (69 [51%]), and anorexia (52 [38%]) than did patients who received placebo (28 [19%] of 144 patients, 35 [24%], and 23 [16%], respectively). The most common severe (grade 3 or worse) treatment-related adverse events were abdominal pain (five [4%] patients receiving napabucasin vs five [3%] receiving placebo), diarrhoea (21 [15%] vs one [1%]), fatigue (14 [10%] vs eight [6%]), and dehydration (six [4%] vs one [1%]). 251 (89%) patients had data on pSTAT3 expression, of whom 55 (22%) had pSTAT3-positive tumours (29 in the napabucasin group, 26 in the placebo group). In a prespecified biomarker analysis of pSTAT3-positive patients, overall survival was longer in the napabucasin group than in the placebo group (median 5·1 months [95% CI 4·0–7·5] vs 3·0 months [1·7–4·1]; HR 0·41, 0·23–0·73, p=0·0025). Interpretation Although there was no difference in overall survival between groups in the overall unselected population, STAT3 might be an important target for the treatment of colorectal cancer with elevated pSTAT3 expression. Nevertheless, these results require validation. Funding Canadian Cancer Society Research Institute and Boston Biomedical.

Partial or even complete cancer regression can be achieved in some patients with current cancer treatments. However, such initial responses are almost always followed by relapse, with the recurrent cancer being resistant to further treatments. The discovery of therapeutic approaches that counteract relapse is, therefore, essential for advancing cancer medicine. Cancer cells are extremely heterogeneous, even in each individual patient, in terms of their malignant potential, drug sensitivity, and their potential to metastasize and cause relapse. Indeed, hypermalignant cancer cells, termed cancer stem cells or stemness-high cancer cells, that are highly tumorigenic and metastatic have been isolated from cancer patients with a variety of tumor types. Moreover, such stemness-high cancer cells are resistant to conventional chemotherapy and radiation. Here we show that BBI608, a small molecule identified by its ability to inhibit gene transcription driven by Stat3 and cancer stemness properties, can inhibit stemness gene expression and block spherogenesis of or kill stemness-high cancer cells isolated from a variety of cancer types. Moreover, cancer relapse and metastasis were effectively blocked by BBI608 in mice. These data demonstrate targeting cancer stemness as a novel approach to develop the next generation of cancer therapeutics to suppress cancer relapse and metastasis.

The gene-silencing activity of a small interfering RNA (siRNA) is determined by various factors. Considering that RNA interference (RNAi) is an unparalleled technology in both basic research and therapeutic applications, thorough understanding of the factors determining RNAi activity is critical. This report presents observations that siRNAs targeting KRT7 show cell-line-dependent activity, which correlates with the expression level of KRT7 mRNA. By modulating the target mRNA level, it was confirmed that highly expressed genes are more susceptible to siRNA-mediated gene silencing. Finally, several genes that show different expression levels in a cell-line dependent manner were tested, which verified the expression-level-dependent siRNA activities. These results strongly suggest that the abundance of target mRNA is a critical factor that determines the efficiency of the siRNA-mediated gene silencing in a given cellular context. This report should provide practical guidelines for designing RNAi experiments and for selecting targetable genes in RNAi therapeutics studies.

The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.

In most applications, small interfering RNAs are designed to execute specific gene silencing via RNA interference (RNAi) without triggering nonspecific responses such as immunostimulation. However, in anticancer therapeutics, immunostimulation combined with specific oncogene silencing could be beneficial, resulting in the synergistic inhibition of cancer cell growth. In this study, we report an immunostimulatory long double-stranded RNA (dsRNA) structure with the ability to trigger RNAi-mediated specific target gene silencing, termed as long interfering dsRNA (liRNA). liRNA targeting Survivin mRNA not only efficiently and specifically triggered target gene silencing via RNAi, but also stimulated the protein kinase R pathway to induce the expression of interferon β. As a result, the ability of Survivin-targeting liRNA to inhibit cancer cell growth was superior over conventional small interfering RNA or nontargeting dsRNA structures. Our results thus provide a simple yet efficient dual function immunostimulatory RNAi-triggering structure, which is potentially applicable for the development of anticancer therapeutics.

Since the discovery of double-stranded (ds) RNA-mediated RNA interference (RNAi) phenomenon in Caenorhabditis elegans, specific gene silencing based upon RNAi mechanism has become a novel biomedical tool that has extended our understanding of cell biology and opened the door to an innovative class of therapeutic agents. To silence genes in mammalian cells, short dsRNA referred to as small interfering RNA (siRNA) is used as an RNAi trigger to avoid nonspecific interferon responses induced by long dsRNAs. An early structure-activity relationship study performed in Drosophila melanogaster embryonic extract suggested the existence of strict siRNA structural design rules to achieve optimal gene silencing. These rules include the presence of a 3' overhang, a fixed duplex length, and structural symmetry, which defined the structure of a classical siRNA. However, several recent studies performed in mammalian cells have hinted that the gene silencing siRNA structure could be much more flexible than that originally proposed. Moreover, many of the nonclassical siRNA structural variants reported improved features over the classical siRNAs, including increased potency, reduced nonspecific responses, and enhanced cellular delivery. In this review, we summarize the recent progress in the development of gene silencing siRNA structural variants and discuss these in light of the flexibility of the RNAi machinery in mammalian cells.

RNA interference (RNAi) has been established as an important research tool that carries great potential for gene therapy. However, targeted induction of RNAi in vivo has met with significant challenges. In this study, a novel pSLS plasmid capable of expressing short hairpin RNAs (shRNAs) was transformed into attenuated Salmonella enterica serovar typhimurium strain 7207 (SL). In vitro infection studies with the transformed S. enterica containing pSLS (SL-pSLS-CAT) demonstrated that expression of shRNA targeting the CTNNB1 gene induced potent and specific silencing of CTNNB1 expression in cultured SW480 cells. CTNNB1 knockdown in SW480 cells was associated with markedly reduced proliferation and cell death compared with that of control infected cells. In addition, SL-pSLS-CAT-mediated CTNNB1 knockdown markedly reduced tumor growth in SW480 xenograft mice. These tumors exhibited reduced levels of CTNNB1, as well as c-Myc and cyclin D1. Finally, SL-pSLS-CAT treatment also resulted in reduced expression levels of these genes in polyps, mucosal tissues and in small intestines of APC(Min) mice. Taken together, these data suggest that attenuated shRNA-expressing Salmonella may be a powerful new tool for in vitro gene silencing, functional genomics, and the development of RNAi-based anticancer or human immunodeficiency virus therapeutics.

The met proto-oncogene is functionally linked with tumorigenesis and metastatic progression. Validation of the receptor tyrosine kinase c-Met as a selective anticancer target has awaited the emergence of selective c-Met inhibitors. Herein, we report ARQ 197 as the first non-ATP–competitive small molecule that selectively targets the c-Met receptor tyrosine kinase. Exposure to ARQ 197 resulted in the inhibition of proliferation of c-Met–expressing cancer cell lines as well as the induction of caspase-dependent apoptosis in cell lines with constitutive c-Met activity. These cellular responses to ARQ 197 were phenocopied by RNAi-mediated c-Met depletion and further demonstrated by the growth inhibition of human tumors following oral administration of ARQ 197 in multiple mouse xenograft efficacy studies. Cumulatively, these data suggest that ARQ 197, currently in phase II clinical trials, is a promising agent for targeting cancers in which c-Met-driven signaling is important for their survival and proliferation. Mol Cancer Ther; 9(6); 1544–53. ©2010 AACR.

RNA interference (RNAi) has become an indispensable technology for biomedical research and has demonstrated the potential to become a new class of therapeutic. Current RNAi technology in mammalian cells relies on short interfering RNA (siRNA) consisting of symmetrical duplexes of 19-21 base pairs (bp) with 3' overhangs. Here we report that asymmetric RNA duplexes with 3' and 5' antisense overhangs silence mammalian genes effectively. An asymmetric interfering RNA (aiRNA) of 15 bp was incorporated into the RNA-induced silencing complex (RISC) and mediated sequence-specific cleavage of the target mRNA between base 10 and 11 relative to the 5' end of the antisense strand. The gene silencing mediated by aiRNA was efficacious, durable and correlated with reduced off-target silencing by the sense strand. These results establish aiRNA as a scaffold structure for designing RNA duplexes to induce RNAi in mammalian cells.

Activation of DNA damage checkpoint pathways, including Chk2, serves as an anticancer barrier in precancerous lesions. In an effort to identify small-molecule activators of Chk2, the authors developed a quantitative cell-based assay using a high-content analysis (HCA) platform. Induction of phosphorylated Chk2 was evaluated using several different parameters, including fold induction, Kolmogorov-Smirnov score, and percentage of positively stained cells. These measurements were highly correlated and provided an accurate method for compound ranking/binning, structure-activity relationship studies, and lead identification. Screening for Chk2 activators was undertaken with a target-focused library and a diversified library from ArQule chemical space. Several compounds exhibited submicromolar EC( 50) values for phosphorylated Chk2 induction. These compounds were further analyzed for Chk2-dependent cytotoxicity, as assessed through a high-content cell death assay in combination with siRNA silencing of Chk2 expression. Several compounds were identified and showed specific inhibition or lethality in a target-dependent manner. Therefore, identification of DNA damage checkpoint pathway activators by HCA is an attractive approach for discovering the next generation of targeted cancer therapeutics.

Since its discovery in 1998 RNA interference (RNAi), a potent and highly selective gene silencing mechanism, has revolutionized the field of biological science. The ability of RNAi to specifically down-regulate the expression of any cellular protein has had a profound impact on the study of gene function in vitro. This property of RNAi also holds great promise for in vivo functional genomics and interventions against a wide spectrum of diseases, especially those with "undruggable" therapeutic targets. Despite the enormous potential of RNAi for medicine, development of in vivo applications has met with significant problems, particularly in terms of delivery. For effective gene silencing to occur, silencing RNA must reach the cytoplasm of the target cell. Consequently, various strategies using chemically modified siRNA, liposomes, nanoparticles and viral vectors are being developed to deliver silencing RNA. These approaches, however, can be expensive and in many cases they lack target cell specificity or clinical compatibility. Recently, we have shown that RNAi can be activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture and deliver silencing shRNA to target cells. This new approach, termed TransKingdom RNAi (tkRNAi), has several key advantages. First, tkRNAi may provide a viable means to accomplish therapeutic RNAi since non-pathogenic bacteria have a proven safety record in clinical applications. Second, tkRNAi eliminates the cost of siRNA manufacture since silencing shRNA are produced inside bacteria. Moreover, the intracellular mechanism of shRNA release inherent to tkRNAi may circumvent, or mitigate, the activation of host immune responses. Finally, tkRNAi may facilitate high-throughput in vivo functional genomics screening since bacteria-based RNAi libraries can be easily constructed, stored, reproduced and amplified, thereby allowing for the creation of a stable gene silencing system.

Cequent Pharmaceuticals, Inc. is a recently established biopharmaceutical company that aims to develop clinically compatible therapies based on RNAi, a potent gene-silencing mechanism discovered in 1998. The company’s proprietary technology, transkingdom RNAi (tkRNAi), uses nonpathogenic bacteria to produce and deliver shRNA into target cells to induce RNAi. Our initial focus is on the development of a tkRNAi-based therapy for familial adenatomous polyposis, an inherited form of colon cancer. Cequent’s first tkRNAi-based drug for familial adenatomous polyposis, CEQ501, is currently in advanced preclinical testing. As part of its ongoing activities, Cequent plans to develop additional tkRNAi-based products for indications within and outside the GI tract. Our overall goal is to establish tkRNAi as a platform for developing a wide range of RNAi-based therapies.

Most efforts thus far have been devoted to develop apoptosis inducers for cancer treatment. However, apoptotic pathway deficiencies are a hallmark of cancer cells. We propose that one way to bypass defective apoptotic pathways in cancer cells is to induce necrotic cell death. Here we show that selective induction of necrotic cell death can be achieved by activation of the DNA damage response pathways. While beta-lapachone induces apoptosis through E2F1 checkpoint pathways, necrotic cell death can be selectively induced by beta-lapachone in a variety of cancer cells. We found that beta-lapachone, unlike DNA damaging chemotherapeutic agents, transiently activates PARP1, a main regulator of the DNA damage response pathway, both in vitro and in vivo. This occurs within minutes of exposure to beta-lapachone, resulting in selective necrotic cell death. Inhibition of PAR blocked beta-lapachone-induced necrosis. Furthermore, necrotic cell death induced by beta-lapachone was significantly reduced in PARP1 knockout cell lines. Our data suggest that selective necrotic cell death can be induced through activation of DNA damage response pathways, supporting the idea of selective necrotic cell death as a therapeutic strategy to eliminate cancer cells.

The etiology and significance of genomic instability (GIN), a hallmark of human cancers, remains controversial. The paradigm that inactivation of tumor suppressors [e.g. p53 or adenomatous polyposis coli (APC) genes] leads to GIN is largely based on experiments in vitro and in animal models. It remains unclear whether GIN is a cause or a result of cancer, particularly in patients. Precancerous Barrett's esophagus (BE) provides a clinical model to investigate GIN in cancer progression. We analyzed specimens from endoscopic biopsies or esophagectomies in patients with BE (ten cases, including five cases with multilayered epithelium (ME)), BE-associated esophageal adenocarcinoma (ten cases), or with normal gastro-esophageal junction (five cases). Chromosomal enumeration probe Cep 7, 11, 12, 17 and 18 were detected by fluorescence in situ hybridization (FISH). Expression of p53 and APC were determined by immunohistochemistry. Increased p53 expression, a measurement of p53 mutations, was observed in BE with high grade dysplasia (HGD) and in BE-associated esophageal cancer (EC). The expression of wild type APC was decreased in BE with HGD and in advanced EC. Chromosomal abnormalities were found in all EC samples. Numeric changes of chromosome 7, 11 and 12 were observed in BE in 14%, 64% and 43% of cases, respectively. Aneusomy of chromosome 11 and 12 were found in ME and in BE without dysplasia, in the presence of normal expression pattern of p53 and APC. Our results suggest that GIN is an early event that occurs at precancerous stages prior to changes in tumor suppressor genes (p53 and APC) in BE-associated tumorigenesis in patients, suggesting that GIN may serve as a causative link between chronic inflammation and cancer.

We propose that the lethality of commonly used anticancer drugs, e.g., methotrexate and cis-platinum are due, at least in part, to an increase of the E2F-1–mediated apoptotic cascade. The drugs directly or indirectly decrease deoxynucleoside triphosphates. The E2F family acts to provide control of S phase by transcribing genes required for deoxynucleoside triphosphate and DNA synthesis. Thus, a mechanism for control of E2F-1 is essential, a signal safeguarding against aberrant or uncontrolled cell proliferation. We have proposed a feedback control by NTPs that down-regulates E2F-1. Here, we provide evidence in support of this hypothesis.

The DNA synthetic S phase of the unperturbed cell cycle is a closed system, as compared to regulation of G(1) by external growth factors. The E2F family provides internal control in S phase by transcribing genes required for deoxynucleotide triphosphate (dNTP) and DNA synthesis. Furthermore, over expression of E2F-1 activates programmed cell death (apoptosis), a safeguarding signal of aberrant growth of cells that have become carcinogenic. Mechanisms for control of E2F-1 are thus essential. The hypothesis is proposed that deoxythymidine triphosphate (dTTP) allosterically feedback controls E2F-1 to regulate both DNA synthesis and apoptosis. This may act either upon production of E2F-1 or its degradation.

s-Lapachone is an ortho naphthoquinone, originally isolated from a tree whose extract has been used medicinally for centuries. Recent investigations suggest its potential application against numerous diseases. Its lethality at micromolar (μm) concentrations against a variety of cancer cells in culture indicates its potential against tumor growth. A few experiments with positive results have been performed that apply the compound to tumors growing in animals. Particularly promising is the remarkably powerful synergistic lethality between s-lapachone and taxol against several tumor cell lines implanted into mice; the mice did not appear to be adversely affected. Enhanced lethality of X-rays and alkylating agents to tumor cells in culture was reported when s-lapachone was applied during the recovery period, because of inhibition of DNA lesion repair. Clinical trials are still to be initiated. The detailed mechanism of cell death induced by s-lapachone remains for investigation. DNA topoisomerase I was the first biochemical target of s-lapachone to be discovered, although its role in cell death is not clear. A proposed mechanism of cell death is via activation of a futile cycling of the drug by the cytoplasmic two-electron reductase NAD(P) H: quinone oxidoreductase, also known as NQO1, DTdiaphorase and Xip3. Death of NQO1 expressing cells is prevented by the NQO1 inhibitor dicoumarol, and cells with low NQO1 are resistant. At higher drug concentrations the production of reactive oxygen species (ROS) appears to be responsible. Furthermore, this process is p53- and caspase- independent. Either apoptotic or necrotic cell death can result, as reported in various studies performed under differing conditions. s-Lapachone is one of a few novel anticancer drugs currently under active investigation, and it shows promise for chemotherapy alone and especially in combinations.

RNAi (RNA interference) technology has the potential to target any genes causing disease, including conventionally “undruggable” targets in cancer. We previously discovered aiRNA (asymmetric interfering RNA), a next generation of gene-silencing technology with improved gene silencing efficiency and reduced off-target effects in comparison with siRNA. We have recently developed a nanoscale formulation that encapsulates therapeutic aiRNAs targeting CTNNB1 and PD-L1, named BBI-801. Here we investigate the in vivo delivery and antitumor activity of BBI-801 encapsulating aiRNAs targeting CTNNB1 and PD-L1. CTNNB1 encodes undruggable β-catenin which is a cancer stemness gene that is broadly implicated in multiple cancer types PD-L1 gene encodes a key immune checkpoint factor that mediates cancer immune evasion. In our in vivo studies, we have achieved prolong silencing of β-Catenin/PD-L1 mRNA and protein in a dose-dependent manner in a wide variety of murine tumor models, including subcutaneous human tumor xenografts, orthotopic human liver and lung tumors, as well as syngeneic mouse colorectal, breast and lung tumors. Our biodistribution analysis of fluorescence-labeled aiRNA demonstrated that the delivery of BBI-801 to xenograft tumors happens within 5 minutes of aiRNA administration and lasts at least 8 hours. In all the models we examined, significant tumor growth inhibition by BBI-801 was achieved not only in β-Catenin over-expressed colorectal tumor models, SW480 and APCmin, but also in the rest of β-Catenin normal-expressed tumor models. Finally, BBI-801 is well tolerated and no signs of toxicity were observed after repeated dosing. These exciting data support further investigation of the anti-tumor potential of BBI-801 as an anticancer therapeutic in variety of tumor indications. Citation Format: Youzhi Li, Yuan Gao, Yuxin Wang, Jie Su, Eric Hsu, Ewa Wybieralska, Janet Huang, Keyur Gada, Jun Oishi, Xiaoshu Dai, Erina Koga, Wei Li, Xiangao Sun, Emily Brooks, Chiang J. Li. In vivo delivery of asymmetric gene-silencing RNAs targeting CTNNB1 and PD-L1 show a broad spectrum of potent antitumor activities in preclinical cancer models [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 LB-069. doi:10.1158/1538-7445.AM2017-LB-069

In recent years evidence has accumulated in support of the cancer stem cell (CSC) model in cancer chemotherapy resistance, highlighting the urgency and necessity of identifying CSC targets for developing novel cancer therapeutics. A number of studies have suggested that Nanog is a crucial factor that can confer stemness properties to a portion of the heterogeneous cancer cell population. Although latent in normal somatic cells, aberrant expression of Nanog has been reported in many types of human cancers. Importantly, the expression levels of Nanog are often positively correlated with treatment resistance and poor survival of cancer patients. Various studies have shown that upregulation of Nanog expression enhances the tumorigenicity both in vivo and in vitro whereas repression or ablation of Nanog inhibits tumor initiation. Thus, expression of the stemness factor Nanog is linked to tumor progression, therapeutic resistance, relapse and metastasis. However, Nanog is considered a non-druggable target. Here we provide data to support a role for STK33 (Serine Threonine Kinase 33) as a novel regulator of Nanog and as a potential therapeutic target. Ectopic STK33 expression promotes stemness phenotypes of cancer cells and increases expression levels of CSC drivers including Nanog, KLF4, and SOX2. On the other hand, knockdown of STK33 inhibits expression of Nanog and results in a reduction of the stemness phenotype. STK33 directly interacts with Nanog and appears to promote its stabilization through phosphorylation, resulting in increased Nanog transcriptional activity. Moreover, BBI-503 (Amcasertib), a first-in-class cancer stemness inhibitor, potently inhibits STK33, which led to inhibition of phosphorylation of Nanog. Collectively, our data demonstrate STK33 is a critical element in the signaling network that governs the stemness of cancer cells, and as a promising therapeutic target for cancer. Citation Format: Susan L. Tran, Yudai Furuta, Chen Zhu, Ao Yang, Xiangao Sun, Harry A. Rogoff, Chiang J. Li. Identification of STK33 as a cancer stemness kinase and regulator of Nanog function [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 LB-142. doi:10.1158/1538-7445.AM2017-LB-142

Cancer stem cells (CSCs) are considered to play an important role in cancer therapy resistance, metastasis, and recurrence. Wnt/β-catenin is one of key signaling pathways that confer cancer stemness properties to a portion of the heterogeneous cancer cell population. Dysregulation of the Wnt/β-catenin pathway may lead to accumulation of β-catenin and aberrant activation of its target gene transcription. However, it has been proven difficult to design a therapeutic targeting β-catenin. Identification of a drugguble target for the β-catenin pathway is highly desirable given the importance of β-catenin in promoting cancer stemness. Here, we demonstrate that Serine Threonine Kinase 17A (STK17A), a novel member of the death-associated protein family of serine/threonine kinases, promotes cancer stemness phenotypes via β-catenin. Over-expression of STK17A resulted in increased anchorage-independent spherogenesis and invasive phenotype of cancer cells, whereas STK17A gene silencing reduced these malignant phenotypes. Overexpression of STK17A led to increased transcriptional activity of β-catenin and upregulation of its target genes. These changes were accompanied by increased phosphorylation of β-catenin at ser675. Cell-free in vitro kinase assay revealed that STK17A directly phosphorylates β-catenin at ser675 in an ATP-dependent manner. Moreover, BBI-503 (Amcasertib), a first-in-class cancer stemness kinase inhibitor, was found to potently inhibit STK17A, leading to inhibition of β-catenin phosphorylation at ser675. These results suggest that inhibition of STK17A is a promising approach for targeting cancer stemness. Citation Format: Taiki Kida, Harry A. Rogoff, Chiang J. Li. STK17A, a novel serine threonine kinase, promotes cancer stemness phenotypes by phosphorylating β-catenin [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 LB-023. doi:10.1158/1538-7445.AM2017-LB-023

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor constitutively activated in many cancer types, and has been identified as a major mediator of cancer stemness (Li et.al., PNAS 2015). The Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2) is the reactive oxygen species (ROS) sensor and a master regulator of antioxidant gene expression. NRF2 has recently been implicated in contributing to cancer stem cell phenotypes, chemoresistance, and is associated with poor clinical prognosis. Hypoxia is considered a major feature of the tumor microenvironment as it can promote tumor progression, increased stemness characteristic, and resistance to conventional therapeutic intervention. Hypoxia can regulate a series of stress-sensor transcription factors, notably, the reactive oxygen species (ROS) sensor and response modulator NRF2. We studied NRF2 transcript and protein levels in breast cancer cell lines MCF7 and AU565 cells under both hypoxic and normoxic conditions. Our data shows that in response to STAT3 pathway activators, NRF2 is upregulated at both mRNA and protein levels under hypoxia and this upregulation is STAT3 dependent. NRF2 ChIP-Seq experiments demonstrate that both hypoxia and STAT3 stimulation can modulate NRF2 binding in MCF-7 breast cancer cells. Surprisingly, under hypoxic conditions, STAT3 activation dramatically enhances (10 fold) NRF2 binding sites across the genome. Downstream analysis of NRF2 target genes revealed a significant change from stress response to activation of gene signaling pathways involved in stemness-high cancer cells and metastatic cells under both hypoxic and STAT3 stimulation. Finally, we investigated whether the stemness inhibitor BBI-608 (Napabucasin) could reduce chemotherapy-induced NRF2 upregulation. Our results indicate that STAT3 modulates NRF2 levels and transcriptional activity, and that stemness inhibitor BBI-608 can prevent this upregulation. Our data revealed STAT3-NRF2-Hypoxia as a novel reinforcing regulatory mechanism for promoting cancer stemness and chemoresistance. Citation Format: Luz Elisa Tavera, Karen Simon, Juying Li, Katherine Geromini, Zhuo Zhang, Sarah Keates, Harry A. Rogoff, Chiang J. Li. Identification of STAT3-NRF2-hypoxia as a novel reinforcing mechanism for promoting cancer stemness [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 LB-143. doi:10.1158/1538-7445.AM2017-LB-143

Cancer stem cells (CSCs), a highly malignant tumor cell subpopulation capable of self-renewal, are considered to be fundamentally responsible for malignant growth and tumor recurrence. Emerging evidences indicate that CSCs and CSC pathways, such as STAT3, beta-Catenin, CD44 and Nanog pathway, are involved in the immune evasion in cancers. With the exception of a small percentage of colorectal cancers (CRC) patients that display microsatellite instability (MSI), the vast majority of CRC patients have been found to be resistant to immune checkpoint therapies. BBI-608 (napabucasin) is an orally-administered first-in-class cancer stemness inhibitor that works by targeting STAT3, which lead to inhibition of multiple cancer stemness pathways, including Stat3 and β-catenin pathways. In this study, we investigate the effect of cancer stemness inhibition on sensitizing CRC to immune checkpoint inhibitors in preclinical models. In the syngeneic tumor model, anti-PD-1 antibody monotherapy produced low level and temporary antitumor activity with rapid development of complete resistance to anti-PD1 in the MSS CT26 CRC model. The anti-PD-1 antibody treated CT26 tumors exhibited increased p-STAT3 activation and overexpression of a variety of stemness factors, including Nanog, CD44 and CD133, as well as enrichment of sphere-forming stemness-high cancer cells. BBI-608 was able to reduce basal as well as anti-PD1-induced STAT3 activation and other CSC features within CT26 tumors. Combination of stemness inhibitor BBI-608 with anti-PD-1 antibody to treat CT26 tumors led to tumor complete response (CR) virtually in all treated CT26 tumors with 40% of the mice remain tumor-free for 30 days following treatment termination. This combination also had a synergistic effect on the influx of tumor infiltrating CD8+ T cells, which likely contributed to the rapid tumor regression. Finally, mice CR-induced by BBI-608 and anti-PD-1 antibody were able to reject CT26 tumors upon rechallenge, but not the unrelated breast cancer 4T1 tumors. Our data suggests cancer stemness pathways contribute to immunotherapy resistance in MSS CRC and inhibition of cancer stemness by BBI-608 sensitizes colorectal cancer to immune checkpoint inhibition. This study provides compelling preclinical evidence to support the investigation of the combination of BBI608 with immune checkpoint inhibitors in CRC. Citation Format: Yuan Gao, Youzhi Li, Eric Hsu, Yuxin Wang, Janet Huang, Emily Brooks, Chiang J. Li. Inhibition of cancer stemness sensitizes colorectal cancer to immune checkpoint inhibitors [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 LB-140. doi:10.1158/1538-7445.AM2017-LB-140

Systemic administration of small molecule toll-like receptor (TLR)-7 agonists leads to potent activation of innate immunity and to the generation of anti-tumor immune responses. However, activation of TLRs with small molecule agonists may lead to the induction of TLR tolerance, defined as a state of hyporesponsiveness to subsequent agonism, which may limit immune activation, the generation of anti-tumor responses and clinical response. Our data reveal that dose scheduling impacts on the efficacy of systemic therapy with the selective TLR7 agonist, 6-amino-2-(butylamino)-9-((6-(2-(dimethylamino)ethoxy)pyridin-3-yl)methyl)-7,9-dihydro-8H-purin-8-one (DSR-6434). In a preclinical model of renal cell cancer, systemic administration of DSR-6434 dosed once weekly resulted in a significant anti-tumor response. However, twice weekly dosing of DSR-6434 led to the induction of TLR tolerance, and no anti-tumor response was observed. We show that TLR7 tolerance was independent of type I interferon (IFN) negative feedback because induction of TLR7 tolerance was also observed in IFN-α/β receptor knockout mice treated with DSR-6434. Moreover, our data demonstrate that treatment of bone marrow-derived plasmacytoid dendritic cells (BM-pDC) with DSR-6434 led to downregulation of TLR7 expression. From our data, dose scheduling of systemically administered TLR7 agonists can impact on anti-tumor activity through the induction of TLR tolerance. Furthermore, TLR7 expression on pDC may be a useful biomarker of TLR7 tolerance and aid in the optimization of dosing schedules involving systemically administered TLR7 agonists.

BACKGROUND: Human multiple myeloma (MM) remains an incurable hematological malignancy. We have reported that beta-lapachone, a pure compound derived from a plant, can induce cell death in a variety of human carcinoma cells, including ovary, colon, lung, prostate, pancreas, and breast, suggesting a wide spectrum of anticancer activity. MATERIALS AND METHODS: We first studied antisurvival effects of beta-lapachone in human MM cells by colony formation assay. To determine whether the differential inhibition of colony formation occurs through antiproliferative activity, we performed MTT assays. The cytotoxicity of beta-lapachone on human peripheral blood mononuclear cells was also measured by MTT assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the propidium iodide staining procedure to determine the sub-GI fraction, Annexin-V staining for externalization of phosphatidylserine, and fragmentation of cellular genomic DNA subjected to gel electrophoresis. To investigate the mechanism of anti-MM activity, we examined Bcl-2 expression, cytochrome C release, and poly (ADP ribose) polymerase cleavage by Western blot assay. RESULTS: We found that beta-lapachone (less than 4 microM) inhibits cell survival and proliferation by triggering cell death with characteristics of apoptosis in ARH-77, HS Sultan, and MM.1S cell lines, in freshly derived patient MM cells (MM.As), MM cell lines resistant to dexamethasone (MM.1R), doxorubicin (DOX.40), mitoxantrone (MR.20), and mephalan (LR5). Importantly, after treatment with beta-lapachone, we observed no apoptosis in peripheral blood mononuclear cells in either quiescent or proliferative states, freshly isolated from healthy donors. In beta-lapachone treated ARH-77, cytochrome C was released from mitochondria to cytosol, and poly (ADP ribose) polymerase was cleaved, signature events of apoptosis. Finally, the apoptosis induced by beta-lapachone in MM cells was not blocked by either interleukin-6 or Bcl-2, which confer multidrug resistance in MM. CONCLUSIONS: Our results suggest potential therapeutic application of beta-lapachone against MM, particularly to overcome drug resistance in relapsed patients.

To date much attention has been focused on regulation of apoptosis in proliferating cells. However, recent evidence shows that regulation of apoptosis in quiescent tissue plays an important role in homeostasis of the organism. This review examines the implications of apoptosis of quiescent cells for both tumourigenesis and viral infection such as HIV. In this article we propose a dual role for cellular activation in the homeostasis regulation. In this model cellular mitogens not only activate quiescent cells into the active cell cycle, but under certain conditions, loss of quiescence may result in apoptosis. The loss of quiescence-associated apoptosis may play a significant role in tumourigenesis and viral infections.

HIV-1 infection causes B cell hyperactivation. Tat protein, a potent virus-encoded transactivator, has the potential to activate B cells based on its pleiotropic biological properties: (1) Tat regulates cellular gene expression; (2) Tat modulates growth of various cell types; and (3) Tat is released from infected T cells and acts on bystander uninfected cells in a paracrine fashion. To test a possible activating effect of Tat on B cells, we examined the effect of purified Tat on the expression of Fas, an activation marker, in B cells in primary culture. Flow cytometric analysis demonstrated that treatment of peripheral blood mononuclear cells with Tat, at concentrations in the range of extracellular Tat as determined in vivo, up-regulated Fas expression in B cells. Reverse transcriptase-PCR further demonstrated that Tat induced Fas expression in B cells at the mRNA level. These results indicate that exogenous Tat alone can activate B cells, suggesting that Tat may contribute to B cell hyperactivation during the early stage of HIV-1 infection and activation-induced B cell death mediated by Fas during the late stage of HIV-1 infection.

There were errors in both the Funding and Competing interests. The correct versions of both are: Funding: This study was supported by the BIDMC Department of Medicine Foundation and Global Research Laboratory Award (Ministry of Education Science and Technology, Korea). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have read the journal's policy and have the following conflicts: CJL has a management position at Boston Biomedical, Inc, a company that develops cancer stemness inhibitors. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Background: Wilms' tumor gene 1 (WT1), broadly expressed in hematological malignancies and solid tumors, is a promising candidate for development as a cancer vaccine. WT4869, one of our WT1 vaccine candidates demonstrated early signs of clinical activity in Azacitidine (AZA)-resistant higher-risk MDS patients with a median survival (OS) of 13.0 months in a phase 1/2 study (Suzuki T, Ueda Y, Ogura M, et al. A Phase 1/2 Study of WT1 Peptide Cancer Vaccine WT4869 in Patients with Myelodysplastic Syndromes (MDS). Blood. 2015;126:2868). In this study, we report the characterization of DSP-7888 and the potential synergic effect with anti-PD-1 antibody. DSP-7888, which is a novel cocktail peptide vaccine designed to induce cytotoxic T lymphocytes (CTLs) that recognize WT1 antigen in an HLA-A*02:01, HLA-A*02:06 or HLA-A*24:02 restricted manner, has entered early clinical trials in patients with MDS and pediatric brain cancer in Japan and solid tumors in the U.S. Methods: WT1-specific T cells were measured by WT1 tetramer staining following stimulation of human peripheral blood mononuclear cells (PBMCs) with DSP-7888. In vivo CTL inducing activity of DSP-7888 was evaluated by ELISPOT assay and 51Cr-release assay after immunization of HLA-A2.1/DR1 transgenic (Tg) mice or HLA-A24.2 Tg mice. The response of induced CTLs against the WT1 epitope was measured by IFN-ɣ ELISA assay following co-culture with irradiated cancer cells. The frequency of PD-1 positive CTLs in spleen and tumor was analyzed by flow cytometry. The anti-tumor effect of DSP-7888 with or without anti-PD-1 antibody was evaluated using HLA-A24.2 Tg mice bearing mouse EL4 cells expressing both HLA-A24.2 and WT1. Results: HLA-A*02:01- or HLA-A*24:02-restricted and WT1-specific CTLs were induced in HLA-A2.1/DR1 Tg mice, HLA-A24.2 Tg mice and human PBMCs. Two groups of DSP-7888-induced CTLs recognized WT1 in an HLA-A*02:01-restricted manner and one in an HLA-A*24:02-restricted manner. Compared to its component vaccine without helper peptide, DSP-7888 induced a large number of WT1-specific CTLs in HLA-A2.1/DR1 Tg mice. The activity of DSP-7888-induced CTLs was relatively maintained in the environment including cancer cells. In addition, it was further activated by treatment with anti-PD-1 antibody. On the other hand, the activity of the component vaccine-induced CTLs was very low even when treated with anti-PD-1 antibody. In DSP-7888-administered tumor-bearing mice, half of WT1-specific CTLs in spleen expressed PD-1; however, in tumor more than 90% of the CTLs expressed this protein. Combination therapy of DSP-7888 and anti-PD-1 antibody showed a higher anti-tumor effect than each monotherapy alone in this model. Conclusions: In this study, DSP-7888 induced CTLs that recognize multiple WT1 epitopes. The helper peptide included in DSP-7888 enhanced the response of the CTL induction, and contributed to the lasting cytotoxic activities even in the tumor immunosuppressive environment, suggesting potential of DSP-7888 as a cancer vaccine. In addition, the in vitro treatment with anti-PD-1 antibody furthermore enhanced the activity of DSP-7888-induced CTLs. The combination of DSP-7888 and anti-PD-1 antibody may induce multiple WT1 epitope-specific CTLs and maintain the intratumoral cytotoxic activity. Further evaluations of DSP-7888 are warranted. Disclosures Goto: Sumitomo Dainippon Pharma Co.,Ltd: Employment. Nakamura:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Suginobe:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Takasu:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Takanashi:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Ban:Sumitomo Dainippon Pharma Co.,Ltd.: Employment. Li:Sumitomo Dainippon Pharma Co.,Ltd.: Employment.

Recent studies indicate that cancer stem cells (CSCs) exist in most hematological and solid tumors. CSCs are characterized by their ability to self-renew and their capacity to differentiate into the multitude of cells that comprise the tumor mass. Moreover, these cells have been shown to be intrinsically resistant to conventional anticancer therapies. Despite their fundamental role in cancer pathogenesis, the cellular origin of CSCs remains highly controversial. The aim of this study was to examine whether heterogeneous cancer cells can acquire stem cell-like properties in response to chemotherapy. We demonstrate that carboplatin can induce the self-renewal (spherogenesis) and pluripotency (Sox2 and Oct3/4 expression) of hepatocellular carcinoma (HCC) cells grown under stem cell culture conditions. Moreover, we show that non-CSC cells, obtained by side population flow cytometric sorting using Hoechst 33342, can acquire stem-like properties after exposure to carboplatin. Finally, we show that knockdown of Sox2 and Oct3/4 gene expression in HCC cells can reduce carboplatin-mediated increases in sphere formation and increase cellular sensitivity to chemotherapy. Taken together, our data indicate that bulk cancer cells may be an important source of CSCs during tumor development, and that targeting Sox2 and/or Oct3/4 may be a promising approach for targeting CSCs in clinical cancer treatment.

The development of gene interfering RNA (iRNA) molecules such as small interfering RNAs (siRNAs) and antagomirs provides promising therapeutic modalities for targeting specific mRNAs and microRNAs (miRNAs) involved in disease mechanisms. Therapeutic iRNA strategy against cancer or hypermutable viruses prefers targeting multiple genes simultaneously to achieve synergistic inhibition and to prevent resistance.In the present study, we report chemically synthesized, multi-target gene interfering RNA structures based upon branched, tripodal interfering RNAs (termed T-tiRNAs).The T-tiRNAs could simultaneously inhibit up to three different mRNAs or miRNAs by harboring three siRNA or antagomir units. Moreover, when complexed with cationic delivery vehicles, T-tiRNAs showed enhanced gene interfering activity over conventional siRNAs or antagomirs as a result of increased intracellular delivery.The data obtained in the present study provide an example of synthetic multi-functional RNA structures that enable multiple gene interference in mammalian cells, which could become powerful tools for an efficient combinatorial iRNA strategy.

No curative drug against HIV has yet been found, despite enormous efforts aimed at reverse transcriptase and a variety of other targets. The long terminal repeat (LTR) of HIV-1 has recently become a promising site for antiviral action. This article briefly summarizes information on the nature of this target and potential anti-LTR expression drugs.

Transcription of type 1 human immunodeficiency virus (HIV-1) provirus is governed by the viral long terminal repeat (LTR). Drugs can block HIV-1 replication by inhibiting activity of its LTR. We report that topotecan, beta-lapachone, and curcumin are potent and selective inhibitors of HIV-1 LTR-directed gene expression, at concentrations that have minor effects on cells. At these concentrations, each drug inhibited p24 antigen production in cells either acutely or chronically infected with HIV-1. Their target is transcriptional function of the LTR.

WOS: 000275794300014, PubMed ID: 20198329, The purpose of the study was to investigate whether carvedilol has an antiproliferative effect alone and whether carvedilol provides an additive, synergistic or antagonistic effect on imatinib mesylate-induced cytotoxicity in both C6 glioma monolayer and spheroid culture. The C6 rat glioma chemoresistant experimental brain tumour cell line, that is notoriously difficult to treat with combination chemotherapy, was used both in monolayer and spheroid Cultures. We treated C6 glioma cells with carvedilol alone and a combination of carvedilol and imatinib mesylate at a concentration of 10 mu M. Following treatment, we evaluated cell proliferation index. bromodeoxyuridine labelling index (BrDU-LI), cell cycle distributions, apoptotic cell percentages, cAMP levels and three dimensional cell morphology at monolayer cultures. In addition BrDU-LI, volume and morphology of spheroids were also assessed. Carvedilol and imatinib mesylate alone reduced cell number. BrDU-LI cAMP levels and spheroid volume. Carvedilol and imatinib mesylate arrested cells at G0/G1 phase in a time-dependent manner and time-independent manner, respectively. Carvedilol increased apoptosis rate only at the 24th h but imatinib mesylate did for all time intervals. Interestingly carvedilol, drug with well-known protective effect on mitochondria, induced severe mitochondria damage, and imatinib mesylate induced autophagy confirmed only by transmission electron microscopy. These results suggest that carvedilol showed antitumour activity against rat C6 glioma cells and a combination of carvedilol with imatinib mesylate resulted in enhanced in vitro antitumour activity., Istanbul UniversityIstanbul University [484/05052006]; Novartis AG, Basel, Switzerland; Novartis AG, Istanbul, Turkey, The present work was supported by the Research Fund of Istanbul University, project number 484/05052006. Imatinib was provided by Novartis AG, Basel, Switzerland and Novartis AG, Istanbul, Turkey. The authors thank Fusun Oncu and Ebru Karabulut for their technical assistance. Some parts of this manuscript was presented at the 12th World Congress on Advances in Oncology and and 10th International Symposium On Molecular Medicine, October, 2007, Hersonissos, Crete, Greece.

RNA interference (RNAi) is a potent and specific mechanism for eliminating the mRNA of specific genes. This gene silencing mechanism occurs naturally and is highly conserved from plants to human cells, holding promise for functional genomics and for revolutionizing medicine due to its unlimited potential to treat genetic, epigenetic, and infectious disease. However, efforts to unleash the enormous potential of RNAi have met with significant challenges. Delivery is problematic because short interfering RNAs (siRNA) are negatively charged polymers that inefficiently enter cells and undergo rapid enzymatic degradation in vivo. In addition, the synthesis of siRNAs is expensive for long-term research and therapeutic applications. Recently, we have shown that nonpathogenic bacteria can be engineered to activate RNAi in mammalian cells (TransKingdom RNA interference; tkRNAi). This new approach offers several advantages and has significant implications. First, this method allows the establishment of a long-term stable gene silencing system in the laboratory against genes of interests in vitro and in vivo, and enables high-throughput functional genomics screening in mammalian systems. RNAi libraries can be constructed, stored, reproduced, amplified, and used with the help of E. coli as currently done with gene cloning. Second, this technology provides a clinically compatible way to achieve RNAi for therapeutic applications due to the proven clinical safety ofnonpathogenic bacteria as a gene carrier, tkRNAi also eliminates the siRNA manufacture issue, and may circumvent or mitigate host interferon-like responses since siRNA is produced intracellularly.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that mediate efficient gene silencing in a sequence-specific manner by utilizing the endogenous RNA interference (RNAi) pathway. The current standard synthetic siRNA structure harbors a 19–base-pair duplex region with 3′ overhangs of 2 nucleotides (the so-called 19+2 form). However, the synthetic 19+2 siRNA structure exhibits several sequence-independent, nonspecific effects, which has posed challenges to the development of RNAi therapeutics and specific silencing of genes in research. In this study, we report on the identification of truncated siRNA backbone structures with duplex regions shorter than 19 bp (referred to as asymmetric shorter-duplex siRNAs or asiRNAs) that can efficiently trigger gene silencing in human cell lines. Importantly, this asiRNA structure significantly reduces nonspecific effects triggered by conventional 19+2 siRNA scaffold, such as sense-strand–mediated off-target gene silencing and saturation of the cellular RNAi machinery. Our results suggest that this asiRNA structure is an important alternative to conventional siRNAs for both functional genomics studies and therapeutic applications.

RNA interference (RNAi) is a potent and specific mechanism for eliminating the mRNA of specific genes. This gene silencing mechanism occurs naturally and is highly conserved from plants to human cells, holding promise for functional genomics and for revolutionizing medicine due to its unlimited potential to treat genetic, epigenetic, and infectious disease. However, efforts to unleash the enormous potential of RNAi have met with significant challenges. Delivery is problematic because short interfering RNAs (siRNA) are negatively charged polymers that inefficiently enter cells and undergo rapid enzymatic degradation in vivo. In addition, the synthesis of siRNAs is expensive for long-term research and therapeutic applications. Recently, we have shown that nonpathogenic bacteria can be engineered to activate RNAi in mammalian cells (TransKingdom RNA interference; tkRNAi). This new approach offers several advantages and has significant implications. First, this method allows the establishment of a long-term stable gene silencing system in the laboratory against genes of interests in vitro and in vivo, and enables high-throughput functional genomics screening in mammalian systems. RNAi libraries can be constructed, stored, reproduced, amplified, and used with the help of E. coli as currently done with gene cloning. Second, this technology provides a clinically compatible way to achieve RNAi for therapeutic applications due to the proven clinical safety ofnonpathogenic bacteria as a gene carrier, tkRNAi also eliminates the siRNA manufacture issue, and may circumvent or mitigate host interferon-like responses since siRNA is produced intracellularly.

K-Ras, the first oncogene identified in human cancer, is mutated in about 30% of human solid tumors. K-Ras protein, a small membrane-bound GTP-binding protein, acts as a molecular switch to transduce cell proliferation signals. Activating mutations of K-Ras lock the Ras protein into the hyper-active GTP-bound state, resulting in the activation of numerous signaling pathways that control cell survival and proliferation. Ras is also an important oncoprotein in many cancers where it is not mutated since Ras can be functionally activated through aberrant activation of other signal transduction elements. Activated K-Ras proteins are, therefore, found in a large proportion of all human cancers, and occupy a central position of interest. Hypermalignant cancer cells, termed cancer stem cells (CSCs), that are highly tumorigenic and metastatic have been isolated from cancer patients with a variety of tumor types and found to have high stemness properties. These stemness-high cancer cells are hypothesized to be fundamentally responsible for cancer metastasis and relapse. Furthermore, a number of stemness genes, such as beta-catenin, nanog, Sox2, Oct3/4 have been implicated in cancer cell stemness. The role of oncogenes such as K-Ras in cancer cell stemness, however, is not clear. To elucidate the role of K-Ras in the maintenance of cancer cell stemness, we employed asymmetric silencing RNA technology (aiRNA) which is able to silence target genes with high potency and precision. Moreover, aiRNA technology can be readily applied to CSCs. Here we report, to our surprise, that CSCs are not simply addicted to activating mutations of K-Ras, or activation of downstream regulators of the Ras pathway. However, CSCs with amplified mutant K-Ras are highly sensitive to K-Ras silencing. Moreover, the DNA copy number of the mutant K-Ras directly predicts the sensitivity of CSCs to K-Ras silencing. Our studies suggest that amplified mutated K-Ras is required for the maintenance of malignancy and cancer cell stemness, which may have significant implications for understanding the connections between oncogenes and cancer cell stemness, and for developing cancer stem cell inhibitors. Citation Format: Jun Oishi, Hiroki Umehara, Nithya Jesuraj, Jelena Barbulovic, Xiangao Sun, Chiang J. Li. Specific and potent silencing of K-Ras by asymmetric silencing RNA (aiRNA) reveals addiction of cancer stem cells to mutant K-Ras amplification. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-141. doi:10.1158/1538-7445.AM2015-LB-141

Cancer cells are extremely heterogeneous, even in each individual patient, in terms of their malignant potential, drug-senstivity, and their potential to metastasize and cause relapse. Subpopulations of cancer cells with extremely high tumorigenic potential have been isolated from cancer patients with a variety of tumor types and found to have high stemness properties termed cancer stem cells. These stemness-high cancer cells are extremely tumorigenic and are resistant to conventional therapeutics due to activation of pro-survival and anti-apoptotic pathways, overexpression of drug efflux pumps, and increased DNA repair capacity. Moreover, chemotherapy and radiation have been found to induce stemness genes in cancer cells, converting stemness-low cancer cells to stemness-high cancer cells. Such highly tumorigenic and drug-resistant stemness-high cancer stem cells are, therefore, likely to be “left-over” following chemotherapy or radiotherapy and ultimately responsible for relapse. We hypothesized that cancer stemness inhibition is sufficient to suppress metastasis and relapse. Stemness, initially defined by the expression of stem cells genes, is a property shared by embryonic stem cells and adult stem cells. It has been demonstrated that the gene expression profiles of cancer stem cells more closely resemble embryonic stem cells than adult stem cells, suggesting the feasibility to identify molecular targets that are required for cancer stemness, but not (or less so) by normal adult stem cells. Through gene-silencing approaches, we have identified Stat3 as critically important for maintaining cancer stemness, yet largely dispensable for adult stem cells. Here we show that BBI608, a small molecule identified by its ability to inhibit gene-transcription driven by Stat3 and cancer cell stemness properties, displays anticancer properties that are highly different from chemotherapeutics agents. Stemness-high cancer cells enriched by multiple techniques are resistance to chemotherapeutics, yet highly sensitive to the stemness inhibitor BBI608. Blockade of spherogenesis and reduction of stemness gene expression by BBI608 were observed in stemness-high cancer cells isolated from a variety of cancer types. While treatment of xenografted tumor models with chemotherapeutics enriched stemness-high cancer cells, BBI608 induced significant depletion of stemness-high populations in vivo. Moreover, the inhibition of stemness by BBI608 is sufficient to suppress cancer relapse and metastasis in xenografted human cancers in mice. These data demonstrate targeting cancer stemness as an effective way to suppress cancer relapse and metastasis. Citation Format: Youzhi Li, Harry A. Rogoff, Sarah Keates, Yuan Gao, Sylaja Murikipudi, Keith Mikule, David Leggett, Wei Li, Arthur Pardee, Chiang J. Li. Inhibition of stemness by BBI608 is sufficient to suppress cancer relapse and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-253. doi:10.1158/1538-7445.AM2015-LB-253