István Peták

Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy.

Adipose-derived mesenchymal stromal/stem cells (AD-MSC) may offer efficient tools for cell-based gene therapy approaches. In this study, we evaluated whether AD-MSC could deliver proapoptotic molecules for cancer treatment. Human AD-MSCs were isolated and transduced with a retroviral vector encoding full-length human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a proapoptotic ligand that induces apoptosis in a variety of human cancers but not normal tissues. Although several studies have documented the antitumor activity of recombinant human TRAIL, its use in vivo is limited by a short half-life in plasma due to a rapid clearance by the kidney. We found that these limitations can be overcome using stably transduced AD-MSC, which could serve as a constant source of TRAIL production. AD-MSC armed with TRAIL targeted a variety of tumor cell lines in vitro, including human cervical carcinoma, pancreatic cancer, colon cancer, and, in combination with bortezomib, TRAIL-resistant breast cancer cells. Killing activity was associated with activation of caspase-8 as expected. When injected i.v. or s.c. into mice, AD-MSC armed with TRAIL localized into tumors and mediated apoptosis without significant apparent toxicities to normal tissues. Collectively, our results provide preclinical support for a model of TRAIL-based cancer therapy relying on the use of adipose-derived mesenchymal progenitors as cellular vectors.

Staurosporine Induces Necroptotic Cell Death under Caspase-Compromised Conditions in U937 Cells

For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and mixed lineage kinase domain-like protein (MLKL), as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribose)polymerase (PARP) is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme.

Clinical significance of genetic alterations and expression of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas

The significance of epidermal growth factor receptor (EGFR) signaling is well studied in a number of different tumors, but limited data is available with regard to head and neck squamous cell carcinoma (HNSCC). Since anti-EGFR therapies are currently under investigation in these malignancies as well, comprehensive information about the alteration of EGFR in HNSCC is necessary to design the most appropriate therapeutic protocols. We examined retrospectively the gene copy number of EGFR by FISH and the protein expression by immunohistochemistry using different epitope-specific antibodies in paraffin-embedded primary tumors of five different regions, from 71 HNSCC patients who had not been treated with anti-EGFR therapy. In seven cases corresponding lymph node metastases were also available for comparative analyses. We also determined the mutational status of tyrosine kinase (TK) domain (exon 19 and 21) and the extracellular deletion mutation (vIII) of EGFR, the KRAS mutation at codon 12 and the presence of HPV infection. Eight of the 71 cases (11.3%) showed EGFR gene amplification (most of them localized into the hypopharyngeal region) and the increased gene copy number (amplification+polysomy) was 43.7%. Despite pronounced intratumoral heterogeneity of EGFR protein expression being found, the high EGFR expression correlated with poor prognosis. On the other hand, the phosphorylation of EGFR was associated with prolonged survival. No mutations in the TK domain of EGFR were found in any of the HNSCC patients and only two cases were KRAS mutant at codon 12. We detected vIII deletion mutation of EGFR in 21% of the samples, but there was no statistically significant correlation between the presence of vIII mutant form and patient survival. EGFR vIII mutation was, however, associated with increased gene copy number. Fourteen of 71 cases (19.7%) were HPV-positive and the incidence of infection showed a decreasing tendency from the oral cavity towards the larynx. Interestingly, in contrast to previous findings, we could not observe improved survival in HPV-positive patients compared to non-infected patients, most probably due to the fact that the majority of these HNSCC patients were smokers and alcohol consumers. In conclusion, we found that increased EGFR protein levels and gene copy numbers (not gene amplification alone) have prognostic significance in the investigated HNSCC patient population. However, the relatively high incidence of the EGFR-vIII mutant form warrants careful therapeutic decision-making when choosing between different anti-EGFR treatment options.

Epidermal Growth Factor Receptor (EGFR) High Gene Copy Number and Activating Mutations in Lung Adenocarcinomas Are Not Consistently Accompanied by Positivity for EGFR Protein by Standard Immunohistochemistry

The purpose of this study was to investigate whether detectable protein biomarker overexpression is a prerequisite for the presence of increased gene copy number or activating mutations and responsiveness to the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib in patients with lung adenocarcinomas. EGFR status was prospectively analyzed in tumor biopsy samples by three methods: protein expression (n = 117) by standardized immunohistochemistry (IHC), gene copy number (n = 97) by fluorescent in situ hybridization (FISH), and mutation analysis by sequencing (n = 126). Fifty-nine percent of the samples were positive by IHC, 40% were positive by FISH, and 13.5% contained activating kinase domain mutations. Thirty-four percent of the FISH-positive and 27% of the mutant samples were also IHC-negative. All EGFR mutant patients had major clinical responses (five complete response and five partial response) to gefitinib or erlotinib treatment, although three of these tumors were IHC-negative and four were FISH-negative. In a retrospective analysis of samples from nine patients with excellent therapeutic responses (three complete response, five partial response, one stable disease) to erlotinib or gefitinib, mutations were identified in eight cases, but IHC was negative in four of these tumors. These results indicate that molecular diagnostic methods appear to be most important for the identification of lung adenocarcinoma patients who may benefit from EGFR inhibitor treatments.

In vitro and in vivo antitumor effect of 2-methoxyestradiol on human melanoma.

2‐methoxyestradiol (2ME2) is an endogenous metabolite of estradiol with estrogen‐receptor‐independent antitumor and antiangiogenic activity. We examined the effects of 2ME2 on the cellular proliferation of 8 human melanoma cell lines. We show that 2ME2 inhibited cell proliferation by inducing apoptosis and an arrest in the G2/M phase, and the mechanism of action involved microtubules, mitochondrial damage and caspase activation. In male SCID mice, 2ME2 was effective in reducing primary tumor weight and the number of liver metastases after intrasplenic injection of human melanoma cells. In the metastases, we found a significantly higher rate of apoptotic cells after 2ME2 treatment. These findings on the antitumor effect of 2ME2 in cell culture as well as in an animal model may have implications for designing alternative treatment options for patients with advanced malignant melanoma.

Targeting death receptors in bladder, prostate and renal cancer

PURPOSE We describe key components of normal and aberrant death receptor pathways, the association of these abnormalities with tumorigenesis in bladder, prostate and renal cancer, and their potential application in novel therapeutic strategies targeted toward patients with cancer. MATERIALS AND METHODS A MEDLINE literature search of the key words death receptors, TRAIL (tumor necrosis factor related apoptosis inducing ligand), FAS, bladder, prostate, renal and cancer was done to obtain information for review. A brief overview of the TRAIL and FAS death receptor pathways, and their relationship to apoptosis is described. Mechanisms that lead to nonfunction of these pathways and how they may contribute to tumorigenesis are linked. Current efforts to target death receptor pathways as a therapeutic strategy are highlighted. RESULTS Activation of tumor cell expressing death receptors by cytotoxic immune cells is the main mechanism by which the immune system eliminates malignant cells. Death receptor triggering induces a caspase cascade, leading to tumor cell apoptosis. Receptor gene mutation or hypermethylation, decoy receptor or splice variant over expression, and downstream inhibitor interference are examples of the ways that normal pathway functioning is lost in cancers of the bladder and prostate. Targeting death receptors directly through synthetic ligand administration and blocking downstream inhibitor molecules with siRNA or antisense oligonucleotides represent novel therapeutic strategies under development. CONCLUSIONS Research into the death receptor pathways has demonstrated the key role that pathway aberrations have in the initiation and progression of malignancies of the bladder, prostate and kidney. This new understanding has resulted in exciting approaches to restore the functionality of these pathways as a novel therapeutic strategy.

Integrating molecular diagnostics into anticancer drug discovery

In the 1990s, the breast cancer drug trastuzumab (Herceptin; Genentech/Roche) — an antibody specific for human epidermal growth factor receptor 2 (HER2; also known as ERBB2) — was approved based on trials in which HER2 expression levels were used to select patients in clinical trials. This provided support for analogous efforts for drugs that target the epidermal growth factor receptor (EGFR). However, the development of these drugs, such as cetuximab (Erbitux; Bristol–Myers Squibb/Lilly) and gefitinib (Iressa; AstraZeneca), has revealed that EGFR expression is an insufficient and unreliable biomarker to select patients for EGFR-targeted therapies in both lung and colon cancer. Indeed, evidence on patient populations that are likely to respond to such therapies, on the basis of specific mutations in proteins of the targeted pathway, has only recently been clinically validated and incorporated into some of the drug labels. This article highlights lessons learned from the development of the first drugs targeting the EGFR family and discusses strategies to decrease the risk of failure in clinical development by more effectively integrating molecular diagnostics into anticancer drug discovery.

Major partial response to crizotinib, a dual MET/ALK inhibitor, in a squamous cell lung (SCC) carcinoma patient with de novo c-MET amplification in the absence of ALK rearrangement

The initial radiotherapy of a 73 years old Caucasian male patient with advanced squamous cell lung carcinoma was terminated due to severe pericarditis. Subsequently, the tumor sample was analyzed for possible targets with comprehensive molecular diagnostics. EGFR, KRAS and PIK3CA genes were wild type, ALK and ROS1 were negative for rearrangement, but c-MET was amplified by fluorescent in situ hybridization. The kinase inhibitor crizotinib is already in clinical use for the treatment of ALK positive non-small cell lung cancers, but it is also known to be a potent c-MET inhibitor. The patient was treated with the standard dose of twice a day 250 mg crizotinib as a monotherapy. Major partial response to therapy was confirmed by chest CT and PET/CT after 8 weeks on therapy. C-MET expression is associated with poor prognosis and resistance to EGFR inhibitors. This case may indicate that c-MET tyrosine kinase inhibitors can be an effective targeted treatment option for squamous cell carcinoma patients, and future clinical trials should be expanded for this patient group as well.

ROR1 expression is not a unique marker of CLL.

Recent studies have identified receptor tyrosine kinase‐like orphan receptor 1 (ROR1) on the surface of chronic lymphoid leukaemia (CLL) cells. In order to determine whether ROR1 expression is a suitable surrogate marker for the diagnosis of CLL we analysed the mRNA level of ROR1 in different types of non‐Hodgkin lymphomas (NHL), and detected elevated levels of ROR1 compared to control peripheral mononuclear cells in several entities (CLL ≥ mantle cell lymphoma (MCL) > marginal zone lymphoma (MZL) >> diffuse large B‐cell lymphoma > follicular lymphoma). ROR1 protein was expressed intensely on the cell surface of lymphoma cells with leukaemic blood count detected by three colour immunofluorescence. Our results indicate that ROR1 expression is not limited to CLL cases, but it is more prevalent in NHLs, mainly in MCL where it is expressed intensely and MZL where it is expressed moderately, suggesting a general role of ROR1 in lymphoma genesis and/or maintenance. Copyright

Proteasome inhibitors sensitize colon carcinoma cells to TRAIL-induced apoptosis via enhanced release of Smac/DIABLO from the mitochondria

The synergistic interaction between proteasome inhibitors and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising approach to induce cell death in tumor cells. However, the molecular and biochemical mechanisms of this synergism have been proven to be cell type specific. We therefore focused our investigation on TRAIL-resistant colon carcinoma cells in this study. DNA fragmentation, mitochondrial membrane depolarization and increased caspase-3-like enzyme activity was exclusively induced only by combined treatment with proteasome inhibitors (epoxomicin, MG132, bortezomib/PS-341) and TRAIL. The expression level of anti-apoptotic proteins (XIAP, survivin, Bcl-2, Bcl-Xl), regulated by NF-κB transcription factor, was not effected by any of these treatments. TRAIL alone induced only partial activation of caspase-3 (p20), while the combination of TRAIL and proteasome inhibition led to the full proteolytic activation of caspase-3 (p17). Only the combination treatment induced marked membrane depolarization and the release of cytochrome c, HtrA2/Omi and Smac/DIABLO. Apoptosis-inducing factor (AIF) was not released in any of these conditions. These results are consistent with a model where the full activation of caspase-3 by caspase-8 is dependent on the release of Smac/DIABLO in response to the combined treatment. This molecular mechanism, independent of the inhibition NF-kB activity, may provide rationale for the combination treatment of colon carcinomas with proteasome inhibitors and recombinant TRAIL or agonistic antibody of TRAIL receptors. (Pathology Oncology Research Vol 12, No 3, 133–142)