Metformin Restrains Pancreatic Duodenal Homeobox-1 (PDX-1) Function by Inhibiting ERK Signaling in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most potent and perilous diseases known, with a median survival rate of 3-5 months due to the combination of only advanced stage diagnosis and ineffective therapeutic options. Metformin (1,1-Dimethylbiguanide hydrochloride), the leading drug used for type 2 diabetes mellitus, emerges as a potential therapy for PDAC and other human cancers. Metformin exerts its anticancer action via a variety of adenosine monophosphate (AMP)-activated protein kinase (AMPK)- dependent and/or AMPK-independent mechanisms. We present data here showing that metformin downregulated pancreatic transcription factor pancreatic duodenal homeobox-1 (PDX-1), suggesting a potential novel mechanism by which metformin exerts its anticancer action. Metformin inhibited PDX-1 expression at both protein and mRNA levels and PDX-1 transactivity as well in PDAC cells. Extracellular signal-regulated kinase (ERK) was identified as a PDX-1-interacting protein by antibody array screening in GFP-PDX-1 stable HEK293 cells. Co-transfection of ERK1 with PDX-1 resulted in an enhanced PDX-1 expression in HEK293 cells in a dose-dependent manner. Immunoprecipitation/Western blotting analysis confirmed the ERK-PDX-1 interaction in PANC-1 cells stimulated by epidermal growth factor (EGF). EGF induced an enhanced PDX-1 expression in PANC-1 cells and this stimulation was inhibited by MEK inhibitor PD0325901. Metformin inhibited EGF-stimulated PDX-1 expression with an accompanied inhibition of ERK kinase activation in PANC- 1 cells. Taken together, our studies show that PDX-1 is a potential novel target for metformin in PDAC cells and that metformin may exert its anticancer action in PDAC by down-regulating PDX-1 via a mechanism involving inhibition of ERK signaling.

A phase III study of belagenpumatucel-L, an allogeneic tumour cell vaccine, as maintenance therapy for non-small cell lung cancer.

BACKGROUND: Treatment options after first-line chemotherapy are limited in non-small cell lung cancer (NSCLC). Belagenpumatucel-L is a therapeutic vaccine comprised of 4 transforming growth factor (TGF)-ß2-antisense gene-modified, irradiated, allogeneic NSCLC cell lines that may be useful for maintenance after initial treatment. METHODS: Stage III/IV NSCLC patients who did not progress after platinum-based chemotherapy were randomised 1:1 to receive maintenance belagenpumatucel-L or placebo. Patients were eligible for randomisation between one and four months from the end of induction chemotherapy. The primary endpoint was overall survival. RESULTS: This phase III trial enrolled 270 patients in the belagenpumatucel-L arm and 262 in the control arm. Belagenpumatucel-L was well tolerated with no serious safety concerns. There was no difference in survival between the arms (median survival 20.3 versus 17.8months with belagenpumatucel-L versus placebo, respectively; hazard ratio (HR) 0.94, p=0.594). There were also no differences in progression-free survival (4.3months versus 4.0 for belagenpumatucel-L vs placebo, respectively; HR 0.99, p=0.947). A prespecified Cox regression analysis demonstrated that the time elapsed between randomisation and the end of induction chemotherapy had a significant impact on survival (p=0.002) and that prior radiation was a positive prognostic factor (median survival 28.4months with belagenpumatucel-L versus 16.0months with placebo; HR 0.61, p=0.032). CONCLUSIONS: Although the overall trial did not meet its survival endpoint, improved survival for belagenpumatucel-L is suggested in patients who were randomised within 12weeks of completion of chemotherapy and in those who had received prior radiation. Further studies of belagenpumatucel-L in NSCLC are warranted.

Vertically integrated translational studies of PDX1 as a therapeutic target for pancreatic cancer via a novel bifunctional RNAi platform.

RNA interference (RNAi) represents a powerful, new tool for scientific investigation as well as a promising new form of targeted gene therapy, with applications currently in clinical trials. Bifunctional short hairpin RNA (shRNA) are synthetic RNAi molecules, engineered to utilize multiple endogenous RNAi pathways to specifically silence target genes. Pancreatic and duodenal homeobox 1 (PDX1) is a key regulator of pancreatic development, ß-cell differentiation, normal ß-cell function and pancreatic cancer. Our aim is to review the process of identifying PDX1 as a specific, potential RNAi target in pancreatic cancer, as well as the underlying mechanisms and various forms of RNAi, with subsequent testing and development of PDX1-targeted bifunctional shRNA therapy.

Assessment of intravenous pbi-shRNA PDX1 nanoparticle (OFHIRNA-PDX1) in yucatan swine.

PDX1 (pancreatic and duodenal homeobox 1) is overexpressed in pancreatic cancer, and its reduction results in tumor regression. Bi-functional pbi-shRNA PDX1 nanoparticle (OFHIRNA-PDX1) utilizes the endogenous micro-RNA biogenesis pathway to effect cleavage- and non-cleavage-dependent degradation of PDX1 mRNA. We have shown that OFHIRNA-PDX1 reduces pancreatic tumor volume in xenograft models. Thus, we are now exploring biorelevant large animal safety of OFHIRNA-PDX1. Mini pigs were chosen as the biorelevant species based on the similarity of human and pig PDX1 target sequence. In the initial study, animals developed fever, lethargy, hyporexia and cutaneous hyperemia following administration of OFHIRNA-PDX1. Twenty-one days later, the same animals demonstrated less toxicity with a second OFHIRNA-PDX1 infusion in conjunction with a prophylactic regimen involving dexamethasone, diphenhydramine, Indocin and ranitidine. In a new group of animals, PDX1 protein (31 kDa) expression in the pancreas was significantly repressed at 48 and 72 h (85%, P=0.018 and 88%, P=0.013; respectively) following a single infusion of OFHIRNA-PDX1 but recovered to normal state within 7 days. In conclusion, a single intravenous infusion of OFHIRNA-PDX1 in conjunction with premedication in pigs was well tolerated and demonstrated significant PDX1 knockdown.

A phase I trial of oral ridaforolimus (AP23573; MK-8669) in combination with bevacizumab for patients with advanced cancers.

AIMS: This phase I dose-escalation study was designed to evaluate the combination of the mammalian target of rapamycin inhibitor ridaforolimus with the vascular endothelial growth factor inhibitor bevacizumab. MATERIALS AND METHODS: Seventeen adult patients with refractory advanced solid tumours received oral ridaforolimus (30 or 40 mg) once daily for 5 days per week (QDx5/wk) combined with intravenous bevacizumab (10 mg/kg every 2 weeks [Q2wk] or 15 mg/kg every 3 weeks [Q3wk]). Patients were evaluated for dose-limiting toxicities, safety and anti-tumour activity. RESULTS: A 40 mg dose of ridaforolimus with either bevacizumab dosing schedule was the recommended phase II dose. No dose-limiting toxicities were reported; the most common drug-related adverse events were mucosal inflammation and anorexia. Seven patients, with clinical features that included primary tumour of the abdominal origin (colorectal, pancreatic or gynaecological cancers) and previous abdominal radiotherapy, reported serious adverse events related to bowel perforations. There were no objective responses, but 65% of patients had a best response of stable disease. CONCLUSION: Oral ridaforolimus (40 mg QDx5/wk) is feasible to combine with standard doses of bevacizumab, although careful patient selection would be needed to mitigate the risk of bowel perforation-related adverse events. Combination therapy produced prolonged stable disease in several heavily pretreated patients.

Immune response and survival of refractory cancer patients who received TGF-ß2 antisense/GM-CSF gene modified autologous tumor cell (TAG) vaccine.

TAG vaccine is a novel 'triad vaccine' that involves transfection of autologous tumor with a dual plasmid, TGFß2 antisense gene and GM-CSF gene. Patients with advanced cancer who failed standard therapy were treated. IFN-γ ELISPOT analysis (Enzyme-Linked Immunospot Assay for Interferon Gamma) using TAG autologous vaccine target cells was performed prior to vaccination and at week 12 after the third vaccination. The purpose of this assessment was to correlate the IFN-γ ELISPOT immune response with long-term survival of advanced cancer patients who received TAG vaccination. Twenty-three of 28 patients received ≥ 3 TAG vaccinations (two patients withdrew consent and three had disease progression prior to the third vaccination). Eleven patients demonstrated a positive ELISPOT response (>10 spots and ≥ 2 × baseline) at week 12 and 12 patients did not (P=0.002). Median survival from time of treatment between ELISPOT-positive and -negative groups was significantly different (550 vs 159 days, P=0.036), as was median survival from the time of procurement (627 vs 257 days, respectively, P=0.043). In conclusion, the IFN-γ ELISPOT assay may provide an effective measure of immune response following treatment with 'triad vaccines', but additional patient numbers and/or other immune modulatory parameters are necessary for future testing.

Clinical development directions in oncolytic viral therapy.

Oncolytic virotherapy is an emerging experimental treatment platform for cancer therapy. Oncolytic viruses are replicative-competent viruses that are engineered to replicate selectively in cancer cells with specified oncogenic phenotypes. Multiple DNA and RNA viruses have been clinically tested in a variety of tumors. This review will provide a brief description of these novel anticancer biologics and will summarize the results of clinical investigation. To date oncolytic virotherapy has shown to be safe, and has generated clinical responses in tumors that are resistant to chemotherapy or radiotherapy. The major challenge for researchers is to maximize the efficacy of these viral therapeutics, and to establish stable systemic delivery mechanisms.

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference.

RNA interference (RNAi) is a natural cellular regulatory process that inhibits gene expression by transcriptional, post-transcriptional and translational mechanisms. Synthetic approaches that emulate this process (small interfering RNA (siRNA), short hairpin RNA (shRNA)) have been shown to be similarly effective in this regard. We developed a novel 'bifunctional' RNAi strategy, which further optimizes target gene knockdown outcome. A bifunctional construct (bi-sh-STMN1) was generated against Stathmin1, a critical tubulin modulator that is overexpressed in human cancers. The bifunctional construct is postulated to concurrently repress the translation of the target mRNA (cleavage-independent, mRNA sequestration and degradation) and degrade (through RNase H-like cleavage) post-transcriptional mRNA through cleavage-dependent activities. Bi-sh-STMN1 showed enhanced potency and durability in parallel comparisons with conventional shRNA and siRNAs targeting the same sequence. Enhanced STMN1 protein knockdown by bi-sh-STMN1 was accompanied by target site cleavage at the mRNA level showed by the rapid amplification of complementary DNA ends (RACE) assay. Bi-sh-STMN1 also showed knockdown kinetics at the mRNA level consistent with its multieffector silencing mechanisms. The bifunctional shRNA is a highly effective and advantageous approach mediating RNAi at concentrations significantly lower than conventional shRNA or siRNA. These results support further evaluations.

Comparative assessment of siRNA and shRNA off target effects: what is slowing clinical development.

This review considers comparisons of the off-target effects of siRNA to shRNA and their potential impact on the efficacy and toxicity of RNAi based therapeutics.

Phase II trial of talabostat and docetaxel in advanced non-small cell lung cancer.

AIMS: Currently available therapies do improve survival in advanced stage non-small cell lung cancer (NSCLC), but only to a limited degree. Talabostat mesilate (PT-100) is an orally available amino boronic dipeptide that specifically inhibits dipeptidyl peptidases (including fibroblast activation protein) and enhances an immune response. The aim of this study was to determine the efficacy and safety of talabostat in NSCLC patients. MATERIALS AND METHODS: A phase II trial was conducted to evaluate talabostat in combination with docetaxel in patients with advanced NSCLC after failure of previous platinum-based chemotherapy. In total, 42 patients were enrolled. RESULTS: Talabostat was well tolerated. Two patients achieved a partial response and one achieved a complete response. CONCLUSION: There was no evidence that talabostat enhanced the clinical activity of docetaxel in patients with NSCLC.

Phase II trial of Belagenpumatucel-L, a TGF-beta2 antisense gene modified allogeneic tumor vaccine in advanced non small cell lung cancer (NSCLC) patients.

In a previous dose escalation trial we demonstrated dose related survival correlation to Belagenpumatucel-L. In order to further evaluate safety and response at the previously defined optimal dose and schedule and to gain preliminary evidence on a hypothesis that the level of circulating tumor cells (CTCs) in blood may correlate with the overall survival of patients with stage IV NSCLC, we initiated a phase II trial. Patients received intradermal immunization of 2.5 x 10(7) transfected allogeneic tumor cells (Belagenpumatucel-L, supplied by NovaRx) 1 x every month for a total of 16 months. Circulating tumor cells (Veridex, Raritan, NJ) were measured every 4 weeks. Twenty-one advanced NSCLC patients were enrolled on this study. No significant toxic effect was observed. Overall survival was 562 days. The median survival was 660 days in patients having less than 2 CTCs at baseline compared to 150 days in patients with 2 or more CTCs (P=0.025). Phase II results of safety and response are consistent with prior experience following treatment with Belagenpumatucel-L and there is a suggestion that the number of circulating tumor cells at baseline appears to correlate with overall survival. A larger clinical trial is warranted to further explore this observation.

MicroRNA profile analysis of human prostate cancers.

We examined the microRNA (miRNA) expression profile of 40 prostatectomy specimens from stage T2a/b, early relapse and non-relapse cancer patients, to better understand the relationship between miRNA dysregulation and prostate oncogenesis. Paired analysis was carried out with microdissected, malignant and non-involved areas of each specimen, using high-throughput liquid-phase hybridization (mirMASA) reactions and 114 miRNA probes. Five miRNAs (miR-23b, -100, -145, -221 and -222) were significantly downregulated in malignant tissues, according to significance analysis of microarrays and paired t-test with Bonferroni correction. Lowered expression of miR-23b, -145, -221 and -222 in malignant tissues was validated by quantitative reverse transcription (qRT)-PCR analyses. Ectopic expression of these miRNAs significantly reduced LNCaP cancer cell growth, suggesting growth modulatory roles for these miRNAs. Patient subset analysis showed that those with post-surgery elevation of prostate-specific antigen (chemical relapse) displayed a distinct expression profile of 16 miRNAs, as compared with patients with non-relapse disease. A trend of increased expression (>40%) of miR-135b and miR-194 was observed by qRT-PCR confirmatory analysis of 11 patients from each clinical subset. These findings indicate that an altered miRNA expression signature accompanied the prostate oncogenic process. Additional, aberrant miRNA expression features may reflect a tendency for early disease relapse. Growth inhibition through the reconstitution of miRNAs is potentially applicable for experimental therapy of prostate cancer, pending molecular validation of targeted genes.

Transcription factors: their potential as targets for an individualized therapeutic approach to cancer.

Pro-cancer signals are controlled by the expression and transcription of oncogenes. Transcription of DNA is dependent on the spatially and temporally coordinated interaction between transcriptional machinery (RNA polymerase II, transcription factors (TFs)) and transcriptional regulatory components (promoter elements, enhancers, silencers and locus control regions). Unique TFs have been identified in association with cancer. This review summarizes key oncogene-related TFs and organizes their pro-cancer activity according to the six hallmark functions (self sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of programmed cell death, limitless replicative potential, sustained angiogenesis and metastatic spread) proposed as constituting the infrastructure of the malignant process.

Modulation of miRNA activity in human cancer: a new paradigm for cancer gene therapy?

MicroRNAs (miRNAs) were discovered more than a decade ago as noncoding, single-stranded small RNAs (approximately 22 nucleotides) that control the timed gene expression pattern in Caenorhabditis elegans life cycle. A number of these evolutionarily conserved, endogenous miRNAs have been shown to regulate mammalian cell growth, differentiation and apoptosis. miRNAs are multispecific by nature. The individual miRNA is capable of modulating the expression of a network of mRNAs that it binds by imperfect sequence complementarity. Human cancers commonly exhibit an altered expression profile of miRNAs with oncogenic (miR-21, miR-106a and miR-155) or tumor-suppressive (let-7, miR-15a/16, miR-34a and miR-143/145) activity. As consistent with the natural function of miRNAs in specifying cellular phenotype, miRNA-based cancer gene therapy offers the theoretical appeal of targeting multiple gene networks that are controlled by a single, aberrantly expressed miRNA. Reconstitution of tumor-suppressive miRNA, or sequence-specific knockdown of oncogenic miRNAs by 'antagomirs,' has produced favorable antitumor outcomes in experimental models. We discuss pending issues that need to be resolved prior to the consideration of miRNA-based experimental cancer gene therapy. These include the need for definitive mRNA target validation, our incomplete understanding of rate-limiting cellular components that impact the efficiency of this posttranscriptional gene-silencing phenomenon, the possibility for nonspecific immune activation and the lack of a defined, optimal mode of delivery.

A phase I trial of intravenous infusion of ONYX-015 and enbrel in solid tumor patients.

ONYX-015 is an attenuated chimeric human group C adenovirus, which preferentially replicates in and lyses tumor cells that are p53 negative. The purpose of this phase I, dose-escalation study was to determine the safety and feasibility of intravenous infusion with ONYX-015 in combination with enbrel in patients with advanced carcinoma. Enbrel is a recombinant dimer of human tumor-necrosis factor (TNF)-alpha receptor, previously shown to reduce the level of functional TNF. Nine patients, three in each cohort received multiple cycles of ONYX-015 infusion (1 x 10(10), 1 x 10(11) and 1 x 10(12) vp weekly for 4 weeks/cycle) in addition to subcutaneous enbrel (only during cycle 1) injections per FDA-indicated dosing. Of the nine patients, four had stable disease. No significant adverse events were attributed to the experimental regimen, confirming that enbrel can be safely administered along with oncolytic virotherapy. Two of the three patients in cohort 3 had detectable viral DNA at days 3 and 8 post-ONYX-015 infusion. Their detectable circulating viral DNA was markedly higher during cycle 1 (with enbrel coadministration) as compared with cycle 2 (without enbrel) at the same time points. Area under the curve determinations indicate a marked higher level of TNF-alpha induction and accelerated clearance at cycle 2 in the absence of enbrel. Further assessment is recommended.

Proof concept for clinical justification of network mapping for personalized cancer therapeutics.

To identify signature targets associated with patient-specific cancer lesions based on tumor versus normal tissue differential protein and mRNA coexpression patterns for the purpose of synthesizing cancer-specific customized RNA interference knockdown therapeutics. Analysis of biopsied tissue involved two-dimensional difference in-gel electrophoresis (2D-DIGE) analysis coupled with MALDI-TOF/TOF mass spectrometry for proteomic assessment. Standard microarray techniques were utilized for mRNA analysis. Priority was assigned to overexpressed protein targets with co-overexpressed genes with a high likelihood of functional nodal centrality in the cancer network as defined by the interactive databases BIND, HPRD and ResNet. HPLC-grade small interfering RNA (siRNA) duplexes were utilized to assess knockdown of target proteins in expressive cell lines as measured by western blot. Seven patients with metastatic cancer underwent biopsy. One patient (RW001) had biopsies from two disease sites 10 months apart. Seven priority proteins were identified, one for each patient (RACK 1, Ras related nuclear protein, heat-shock 27 kDa protein 1, superoxide dismutase, enolase1, stathmin1 and cofilin1). Prioritized proteins in RW001 from the two disease sites over time were the same. We demonstrated >80% siRNA inhibition of RACK 1 and stathmin1 of inexpressive malignant cell lines with correlated cell kill. Identification of functionally relevant target gene fingerprints, unique to an individual's cancer, is feasible 'at the bedside' and can be utilized to synthesize siRNA knockdown therapeutics. Further animal safety testing followed by clinical study is recommended.

A review of vaccine clinical trials for non-small cell lung cancer.

Recent evidence suggests that vaccines which enhance tumour antigen recognition may provide clinical benefit to subsets of non-small cell lung cancer patients. In this review, a variety of peptide-, gene- and cell-based clinical vaccine approaches targeting non-small cell lung cancer patients are reviewed. Results consistently demonstrate lack of toxicity. Examples of prolonged stable disease, tumour shrinkage response and survival benefit in comparison with historical and low-dose control groups have been demonstrated. Specific vaccines fulfilling justification for Phase III evaluation based on these results include LBLP25, TGF-beta2 antisense gene vaccine and GVAX.

Herpes simplex virus 1 (HSV-1) for cancer treatment.

Cancer remains a serious threat to human health, causing over 500 000 deaths each year in US alone, exceeded only by heart diseases. Many new technologies are being developed to fight cancer, among which are gene therapies and oncolytic virotherapies. Herpes simplex virus type 1 (HSV-1) is a neurotropic DNA virus with many favorable properties both as a delivery vector for cancer therapeutic genes and as a backbone for oncolytic viruses. Herpes simplex virus type 1 is highly infectious, so HSV-1 vectors are efficient vehicles for the delivery of exogenous genetic materials to cells. The inherent cytotoxicity of this virus, if harnessed and made to be selective by genetic manipulations, makes this virus a good candidate for developing viral oncolytic approach. Furthermore, its large genome size, ability to infect cells with a high degree of efficiency, and the presence of an inherent replication controlling mechanism, the thymidine kinase gene, add to its potential capabilities. This review briefly summarizes the biology of HSV-1, examines various strategies that have been used to genetically modify the virus, and discusses preclinical as well as clinical results of the HSV-1-derived vectors in cancer treatment.

Phase 1/2 trial of autologous tumor mixed with an allogeneic GVAX vaccine in advanced-stage non-small-cell lung cancer.

Tumor vaccines composed of autologous tumor cells genetically modified to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) (GVAX) have demonstrated clinical activity in advanced-stage non-small-cell lung cancer (NSCLC). In an effort to remove the requirement for genetic transduction of individual tumors, we developed a 'bystander' GVAX platform composed of autologous tumor cells mixed with an allogeneic GM-CSF-secreting cell line. We conducted a phase I/II trial of this vaccine (3-12 biweekly vaccinations) in advanced-stage NSCLC. Tumors were harvested from 86 patients, tumor cell processing was successful in 76, and 49 proceeded to vaccination. The most common toxicity was local vaccine injection site reactions. Serum GM-CSF pharmacokinetics were consistent with secretion of GM-CSF from vaccine cells for up to 4 days with associated transient leukocytosis confirming the bioactivity of vaccine-secreted GM-CSF. Evidence of vaccine-induced immune activation was demonstrated; however, objective tumor responses were not seen. Compared with autologous GVAX vaccines prepared by transduction of individual tumors with an adenoviral GM-CSF vector, vaccine GM-CSF secretion was approximately 25-fold higher with the bystander GVAX vaccine used in this trial. However, the frequency of vaccine site reactions, tumor response, time to disease progression, and survival were all less favorable in the current study.