J. Giannios’s research while affiliated with St. Lucas Andreas Hospital and other places

IntroductionAdenosquamous Ca is an aggressive and highly metastatic variant of adenocarcinoma with both glandular and squamous differentiation. Usually, it occurs in chemoradiated patients.Methods Tumour cells were obtained from a resected pancreatic adenosquamous Ca which already had metastasised to regional lymph nodes. Methylation-specific PCR (MSP) detected methylated DNA template of p16. The methylated CpG islands in a promoter of p16 inhibited transcription by preventing RNA polymerase and the RNA transcription machinery from producing messenger RNA leading to gene inactivation. SSCP analysis has detected mutated K-Ras. We constructed an adenovirus p16 expression vector and we inserted p16cDNA into a cassette cosmid containing an adenovirus type 5 genome. Subsequently, we produced a recombinant adenovirus termed as SVN-22/3 by cotransfection of expression cosmid and adenovirus DNA-terminal protein complex into cells by calcium phosphate precipitation.ResultsAfter 1 h treatment with SVN-22/3, pancreatic Ca cells expressed high levels of p16 gene mRNA according to NB hybridisation analysis. The adenoviral mediated gene transfer of wt p16-INK4A formed a heterodimer with cyclin-dept kinase 4 and 6 preventing their interaction with cyclin D. PCR analysis has showed downregulation of cyclin D1 and K-Ras. Subsequent treatment with docetaxel has exerted a synergistic antimitotic effect exhibited by BrdU and Ki-67. Flow-cytometry has reported diploid DNA. A biochemical assay showed activation of caspase-3/CPP32 pathway which led to electron cytological signs of D2 apoptotic stage forming apoptotic bodies which were phagocytosed by adjacent tumour cells leading to a bystander killing effect.Conclusion Concluding, by restoring wild-type p16 protein in chemoresistant adenosquamous carcinoma cells, we have achieved to block cyclin D1 which led to inactivation of K-Ras allowing induction of apoptosis after the antimitotic action of docetaxel.

IntroductionWe investigate if venom proteome of vipera ammodytes can induce apoptosis in CRC.Methods We developed a large scale expression system with CRC cell line which we modified genetically by cloning. We expressed cDNA encoding multimodular disintegrin (rMDVA) comprising 3 domains from the venom of vipera ammodytes. The cDNA of rMDVA was cloned into the plasmid vector pcDNAIII, which was transferred into CRC. The multimodular peptide rMDVA was encapsulated in the hydrophilic phase of liposomes termed as LIP-rMDVA. The in vivo studies with human metastatic CRC were performed in an orthotopic xenograft-model.ResultsThe venom proteome contained 140 proteins. We isolated the multimodular disintegrin displaying cysteine rich disulfide bond 2RGD and C-terminal motif (ammodystatin), the dimeric MLD/VGD motif (VADD), and metalloprotease-domain (ammodylysin). Ammodystatin with the 2 (ARG-GLY-ASP) RGD motifs interacts with integrins avb5, a5b1 inhibiting invasion and angiogenesis/vasculogenesis. It also blocked the bFGF-induced avb3 integrin and avb5 downregulating VEGF, and Ras. It also disrupts the actin cytoskeleton inducing G1 DNA replicative phase arrest, and blocks endothelial cell (EC) secretion of MMP-2, MMP-9 and urokinase-type plasminogen activator with its receptor inhibiting proteolysis and dissolution of ECM blocking invasion. Ammodystatin and ammodylysin lead to type I PCD/apoptosis of tumour cells in masses up to 2 mm and EC. Beyond that size, ammodystatin inhibits the development of the required vascular network according to HUVEC, MVD and IHC. VADD causes burring and blocked adhesion of a4b1 integrin to VCAM1. LDH, BrdU and MTT exhibited inhibition of tumour and EC proliferation. TEM exhibited apoptotic signs of irreversible D2 stage in tumour and ECs.Conclusion Novel liposomal formulation LIP-rMDVA is an effective angiostatic and cytostatic agent against metastatic CRC inducing apoptosis with minimum systemic toxicity.

IntroductionMetastatic pancreatic Ca leads to fatalities due to resistance in conventional anticancer therapies. By activation of immunological mechanisms we aim to circumvent the chemoresistant mechanisms.Methods Animal models characterized by metastatic pancreatic Ca refractory to conventional treatment were developed and treated with IV administration of the Gene-Modified Cellular Vaccine(GMCV) termed as SV/AS (under patent),which is composed of Autologous Adipose-derived Mesenchymal Stem Cells (AADMSCs), which were transfected with lipid-cation imunodominant molecule Hsp70.ResultsPost-treatment, we observed molecular remission in all tumour/metastatic sites, and activation of CD4+ T-cells by antigen presenting cells (APC), enhancement of MHC class I expression, generation of tumor-specific cytotoxicity with CTLs induced by the antigenic fingerprint/repertoire, activation of natural-killer (NK) cells, generation of peptide-specific tumour immunity induced by CD91 and C19 overexpression on dendritic cells (DCs),CD40 on macrophages, and LOX-1,CD14 and TLR2–4 on monocytes. Furthermore, hsp70 induced Th1-type immune response inducing secondary necrosis, which is the most potent immunogenic mode of cell death, and phagocytosis of tumour cells by activated macrophages leading to a lethal bystander effect. Finally, we observed repair of damaged tissue and organs by renewal of injured cells.Conclusion The Gene Modified Cellular Vaccine (GMCV) consisting of autologous adipose mesenchymal stem cells expressing Hsp70 activated the innate and adaptive immunity leading to eradication of metastatic pancreatic Ca cells, while there was stem cell renewal of injured cells.

IntroductionThe unmet medical need for metastatic gastric Ca of the antrum is very high because of potent chemoresistance. We aim to circumvent these resistant factors.Methods We obtained surgically tumour cells from patients with stage IV gastric Ca overexpressing MUC1, sGC heterodimer a1b1, and bcl-2. We synthesised 6 mer homopyrimidine triplex ((PNA)2/RNA)) hybridised to the 5' end (Leader), and 10 mer purine/pyrimidine duplex (PNA/RNA) hybridised to the 3'-end (Trailer) of the AUG start codon region on the mRNA of sGCa1/b1. ClampPNA anti-sGCa1/b1 was incorporated in the polar phase, and docetaxel was entrapped in the lipid phase, which was surrounded by the stealth polymer layer, and biological recognition layer with linked chimeric MAbs against MUC1 of a nanoparticle formulation (SV-IV), with which we treated xenograft animal models developed from gastric Ca cells obtained from the stage IV patients.ResultsPost-treatment, we observed that downregulated MUC1 blocked binding of TKIs, such as cetuximab and trastuzumab, by inhibiting direct steric hindrance onto HER2 and EGFR via the MUC1 cytoplasmic tail. MUC1 phosphorylation was inhibited, blocking Ras/Raf/(Mek)Erk1/2/MAPK, PI3K/AKT, VEGF and MMP-2. The clamp PNA hybridised to the leader, and trailer region of the AUG start codon region on mRNA sGC forming Watson-crick double helices, which inhibited sGCa1/b1 and nitric oxide inducing p53-indept apoptosis or type I PCD. Inhibition of B-Raf, downregulated VEGFR-1-2-3, PDGFR-b, FIR-3, FGFR-1 and upregulated p21CIP1, p16INK4a, and p27KIP1. Downregulation of ERK1/2 blocked angiogenesis and lymphangiogenesis. RIN, TSC2, PTEN were upregulated, and PI3K/AKT/mTOR pathway was blocked. Inhibition of eIF4E led to strong autocrine/paracrine downregulation, inducing apoptosis. Docetaxel phosphorylated bcl-2 releasing beclin-1, which with upregulated BIM induced caspase-indept type II PCD or autophagy.Conclusion Administration of immunochemogene formulation SV-IV eradicated metastatic gastric Ca of the antrum after circumvention of chemoresistance.

IntroductionCurrent anticancer therapies succeed at eradicating bulky disease but miss a tumour reservoir consisting of stem/progenitor cells (SCs) that lead to disease recurrence and metastasis. Tumour stem cells are characterised by upregulation of hTERT which is the limiting factor for telomerase activity. DNA methylation leads to hTERT gene expression.Methods We obtain specimens of surgically resected oesophageal Ca from distant lymph node metastatic sites from patients chemoresistant to vinca alkaloids like vinorelbine-tartrate, and isolate the mutated tumour stem cells. We treat them with DARPins targeting DNMT1 conjugated to docetaxel.ResultsPost-treatment, we observed downregulation of DNMT1 blocking methylation of hTERT promoter inhibiting its transcription and subsequent telomerase activity. Stem cell markers p63, CD44, CD117, CD90, CD133, BCRP1, b-catenin and methylation marker SCNN1B were downregulated. After DNA demethylation, there was upregulation of thymosin b10, p16/Rb, RASSF2, p53 and PTEN lipid/protein phosphatase. Subsequently, there was inhibition of chemoresistant factors, such as choline-kinase, PI3K-AKT/PKB-mTOR, Ras/Raf/Erk/TGFa/EGFR, COX-2, PGE2, PDGF, VEGF, cyclin D1, HIF-1a, survivin and aurora – A/STK15/BTAK. There was enhancement in adheren junction formation. Docetaxel polymerised microtubules blocking cell cycle at G2/M while it phosphorylated antiapoptotic bcl-2 upregulating tumour suppressor gene beclin1 which induced autophagy. Activation of procaspase 10 and procaspase 8 formed DISC which induced type I, II and III PCD and aponecrosis of mutated tumour stem cells.Conclusion Combination of molecularly targeted therapies such as DARPins and conventional cytostatic agents such as docetaxel could provide a potent strategy to eradicate metastatic mutated ESCC stem cells resistant to conventional chemotherapy with vinca alkaloids such as vinorelbine.

IntroductionColorectal carcinoma (CRC) cells develop resistance to cetuximab through insertion mutations at exon 20, ubiquitination and c-Src which activates EGFR in the absence of ligand despite treatment with cetuximab. We aim to circumvent this acquired resistance.MethodsCRC cells were obtained from metastatic patients resistant to cetuximab due to insertion mutations at exon 20, ubiquitination and overexpressed c-Src. Orthotopic mouse CRC models generated from our patients' tumour cells were injected with multitargeted siRNA against HSP90, UbcH5 and c-Src.ResultsMulti-targeted siRNA inhibited expression of the E2 enzyme UbcH5, blocking the covalent attachment of ubiquitin to target protein EGFR, and neutralizing the multi-enzyme cascade. E1 deactivated ubiquitin, blocking transfer to the cysteine residue of E2 ubiquitin conjugating enzyme (UbcH5). This inhibited the E2 ligation of ubiquitin via its carboxy terminus to lysine residues of the protein substrate EGFR. Multi-targeted siRNA inhibited expression of HSP90 resulting in degradation of EGFR with kinase domain deletion type mutations in exon 19, substitutions in exon 21, and resistant insertion mutations at exon 20. Inhibition of c-Src circumvented transactivation and inhibited EGFR signalling, inhibiting tumour proliferation, and metastasis to the liver, and peritoneum. Addiction to EGFR was circumvented. Inhibition of EGFR blocked the activation of downstream mediators including STAT3, AKT, Erk/MAPK and PI3K, while IRF-1 was upregulated. There was enhanced cell to cell adhesion and membrane localisation of b-catenin, while MMP-9 invasive activity was blocked. HIF-1a/Met pathway was blocked downregulating CAIX. VEGFR-2 and VEGFR-3 were blocked inhibiting vascularisation and lymphangiogenesis, respectively. We observed type I, II and III PCD in tumour cells.Conclusion These results indicate that systemic treatment of multitargeted siRNA against UbcH5, c-Src and HSP90 circumvented resistance to cetuximab suppressing tumour growth and metastasis in orthotopic mouse mCRC model.

IntroductionMetastatic oesophageal squamous cell Ca (ESCC) is incurable due to chemoresistance caused by cancer stem cells due to overexpression of oncomirs which upregulate oncogenes and hypermethylation in CpG islands which inactivates tumour suppressor genes.Methods We obtained metastatic ESCC and CSCs from patients and we injected them in xenograft animal models which were treated with LNA oligonucleotides targeting DICER where the 2'-oxygen is bridged to the 4' position via a methylene linker leading to formation of a rigid bicycle locked into a C3 endo (RNA) sugar conformation encapsulated in PEG colloidal nanoparticles with linked Abs targeting CD44. Microarray, RT-PCR, IHC, flow cytometry, MTT, BrdU, TUNEL and TEM were used.ResultsThere was inhibition of Dicer RNAIII endonuclease which blocked exportin5 cleavage blocking formation of mature oncogenic miRNA segments. This inhibition of oncomirs led to silencing of oncogenes such as transcription factors, apoptotic inhibitors, chromatin modifiers, growth factors (tyrosine kinases-integral membrane proteins), signal transducers (cytoplasmic regulators, membrane associated G-proteins, GTPase exchange factors and serine/threonine kinases). Dicer silencing led to inhibition of angiogenesis, invasion, metastasis, ESCC and CSC proliferation by inhibiting stem cell pathways Bmi-1, Notch, SHH and Wnt. There was inhibition of hypermethylation of CpG islands reactivating apoptotic tumour suppressor genes inducing irreversible D2 stage of type I PCD/apoptosis which led to a bystander killing effect. BrdU and MTT exhibited inhibition of DNA synthesis and metabolic activity of ESCC and CSCs.Conclusion Silencing of DICER exerted a synergistic apoptotic effect by activation of tumour suppressor genes after demethylation and inhibition of oncomirs and linked oncogenes leading to eradication of chemoresistant CSCs and ESCCs.

IntroductionVinorelbine, which is an anticancer cytostatic agent, may shrink tumours but it stimulates production of more cancer stem cells, which then metastasise as a way to survive the cytostatic action of this drug.Methods Colorectal Ca obtained from patients were treated with vinorelbine. Post-treatment, the tumours were analysed for cancer stem cells using multi-colour flow cytometry methods for detecting markers and receptors on the surface of tumour cells.ResultsPost-treatment, we observed remission of the tumour cells and relapse of cancer stem cells characterised by a high proliferation index. There was overexpression of cancer stem cells (CSC) markers, Nanog and BMI1 which renew the CSCs. Other CSC markers that were overexpressed include CD44, CD133, CD44 and DR5 exhibiting cancer cell positive resilience, and chemoresistance. Vinorelbine activated the Notch signalling pathway, and phosphorylated the prosurvival mTOR pathway resulting in mitochondrial polarisation, and enhanced tumour cell proliferation. A common factor overexpressed in all cancer stem cells was Oct4, POU transcription factor protein that converts adult stem cells in cancer stem cells. This makes the square to sickle shift making super cancer cells, which form new incurable tumours despite extensive treatment with vinorelbine. Overexpression of these cancer stem cell markers is associated with resistance to vinorelbine leading to poor outcome for patients with colorectal Ca. This creates radioresistance, and chemoresistance for all conventional chemotherapeutic agents that might be administered after vinorelbine treatment. Thus, although vinorelbine reduces tumour size, it doesn't result in long term cures due to induction of metastasis and recurrence. Between 25%>40% of tumour cells consisted of cancer stem cells capable of propagating, reproducing and building metastatic and chemoresistant tumours after vinorelbine treatment.Conclusion To circumvent the distant metastasis of CRC induced by vinorelbine treatment due to stimulation of chemoresistant cancer stem cells, we will need new biological therapies which will eradicate with induction of apoptosis the CSCs through targeting of specific proteins on their plasma membrane.

IntroductionFailure of tumour cells to undergo PCD cause resistance to chemoradiological therapies due to overexpression of oncogenes and transcriptionally repressed apoptotic TSGs due to methylation.Methods We obtained surgically 19 pancreatic Ca specimens from 12 males and 7 females consisting of 9 ductal adeno Ca, 4 Ca of the papilla of Vateri, 1 acinus Ca, 1 neuroendocrine Ca, 1 intrapancreatic distal bile duct and 3 periampullary Ca. Genomic DNA of tumours was analysed for CpG island hypermethylation by using MS-PCR. All of the tumours showed hypermethylation of TSGs: DUSP6/MKP-3 88%, RASSF1A 80%, RARb2 70%, BRCA1 50%. IHC, WB, SB and RT-PCR exhibited overexpression of DNMT1, CaSm, mcl-1 and bcl-2. We treated the samples with the isolated tumour cells with anti-CaSm scFv attached onto high energy-radioisotopes, docetaxel and 21-nucleotide double-stranded siRNA segment generated against DNMT1ResultsPost-treatment, we detected re-expression of DUSP6/MKP-3, RASSF1A, RARb2, BRCA1 after inhibition of DNMT1mRNA. There was downregulation of oncogene CaSm due to targeted scFv and inactivation of bcl-2 and mcl-1 due to phosphorylation by docetaxel. We detected upregulation of p21waf1, p27kip, Bid and Bak. The radioisotopes induced DNA double strand breaks in tumour cells arresting synergistically with docetaxel their growth at G2/M. We detected externalisation of PS, depolarisation of ΔΨΜ, activation of caspases 3, 7, 8, 9, bax, cleavage of PARP and DNA fragmentation. TEM exhibited apoptotic bodies which were phagocytosed by adjacent tumour cells leading to a bystander killing effect.Conclusion We have achieved to eradicate pancreatic Ca cells with combined chemoradioimmuno-therapy after circumvention of chemo- and radioresistant mechanisms such as hypermethylation of TSGs RASSF1A, RARb2, DUSP6/ MKP-3, BRCA1 and overexpression of oncogenes bcl-2, CaSm and mcl-1.

IntroductionVinorelbine in advanced gastro-intestinal stromal tumour (GIST) cells induces tumour relapse with enhanced angiogenesis and metastasis by inducing an innate cancer cellular stress response, which enhances the expression of GRP78 that blocks cell death or apoptosis increasing growth, and spread of GIST due to chemoresistance. We aim to circumvent this with the use of induced pluripotent stem cells encoded with antisense GRP78 shRNA.Methods We take induced pluripotent stem cells(iPSCs), which we infected them with a DNA vector that encoded an RNA molecule of 67 nucleotides. The sequence of this small hairpin RNA (shRNA) is designed to suppress the GRP78 gene. GIST cells were obtained from patients, and they were implanted in animal models, which were treated with vinorelbine. After tumour relapse, there was induction of enhanced angiogenesis, and metastasis. These chemoresistant tumour cells were treated with the induced pluripotent stem cells, which were encoded with shRNA against GRP78.ResultsPost-treatment, stem cells encoded with anti-GRP78 shRNA converted dicer into a siRNA molecule generating a long lasting RNAi silencing effect of GRP78, which spreads to adjacent tumour cells inducing a gene silencing bystander effect (GSBE). Capillary growth into the tumours were blocked, while VEGF and bFGF were downregulated. PKG was upregulated inhibiting b-catenin. Integration of endothelial precursor cells and tumour cells was blocked inhibiting growth of mosaic blood vessels. This leads to inhibition of tumour spread or metastasis, while the existing tumours die from lack of nutrients/oxygen, and a waste disposal pathway. TEM exhibited induction of type I PCD or apoptosis in tumour cells leading to a bystander killing effect. Thus, anti-GRP78 induced pluripotent stem cells (iPSCs) circumvented vinorelbine induced angiogenesis, and metastasis eradicating chemoresistant GIST cells.Conclusion Vinorelbine induced angiogenesis, and metastatic spread in GIST are circumvented with induced pluripotent stem cells (iPSCs) encoded with anti-GRP78 shRNA, which induces apoptosis after a gene silencing bystander effect (GSBE).