Qing Ren

Malignant Transformation of Immortalized HaCaT Keratinocytes through Deregulated Nuclear Factor κB Signaling

Previous studies addressing functional aspects of nuclear factor kappaB (NF-kappaB) activation in normal and transformed keratinocytes revealed complex and seemingly contradictory roles of this transcription factor in this cell type. In normal skin, NF-kappaB signaling seems to inhibit squamous cell carcinoma development whereas, in squamous cell carcinoma themselves, deregulated NF-kappaB expression and/or signaling is frequently observed. To further investigate this paradox, we focused on NF-kappaB activation as it relates to the transformed phenotype of immortalized but nontumorigenic human keratinocytes (HaCaT cells). We observed that NF-kappaB activity contributed to survival and growth of cultured HaCaT keratinocytes as shown by use of pharmacologic NF-kappaB inhibitors, RNA interference, and inducible overexpression of a dominant interfering IkappaB construct. NF-kappaB activation was largely provided through interaction with extracellular matrix components because preventing cell attachment by forced suspension culture markedly reduced NFkappaB signaling associated with cell death (anoikis); conversely, anoikis was partially reversed by NF-kappaB activation induced either by tumor necrosis factor-alpha treatment or by overexpressing the NF-kappaB p65 subunit in HaCaT cells. Furthermore, overexpression of NF-kappaBp65 in HaCaT cells induced colony formation in soft agar and tumorigenicity in nude mice. In summary, as opposed to normal keratinocytes, immortalized HaCaT keratinocytes provide a cellular context in which deregulated NF-kappaB signaling supports multiple malignant traits in vitro and in vivo.

Inhibition of p73 function by Pifithrin-α as revealed by studies in zebrafish embryos

The p53 family of proteins contains two members that have been implicated in sensitization of cells and organisms to genotoxic stress, i.e., p53 itself and p73. In vitro, lack of either p53 or p73 can protect certain cell types in the adult organism against death upon exposure to DNA damaging agents. The present study was designed to assess the relative contribution of p53 to radiation resistance of an emerging vertebrate model organism, i.e., zebrafish embryos. Consistent with previous reports, suppressing p53 protein expression using antisense morpholino oligonucleotides (MOs) increased survival and reduced gross morphological alterations in zebrafish embryos exposed to ionizing radiation. By contrast, a pharmacological inhibitor of p53 function (Pifithrin-α (PFT-α)) caused developmental abnormalities affecting the head, brain, eyes and kidney function and did not protect against lethal effects of ionizing radiation when administered at 3 hours post fertilization (hpf). The phenotypic abnormalities associated with PFT-α treatment were similar to those caused by antisense MO knock down (kd) used to reduce p73 expression. PFT-α also inhibited p73-dependent transcription of a reporter gene construct containing canonical p53-responsive promoter sequences. Notably, when administered at later stages of development (23 hpf), PFT-α did not cause overt developmental defects but exerted radioprotective effects in zebrafish embryos. In summary, this study highlights off-target effects of the pharmacological p53 inhibitor PFT-α related to inhibition of p73 function and essential roles of p73 at early but not later stages of zebrafish development.

Nuclear factor kappaB inhibitors alleviate and the proteasome inhibitor PS-341 exacerbates radiation toxicity in zebrafish embryos.

Inflammatory changes are a major component of the normal tissue response to ionizing radiation, and increased nuclear factor κB (NF-κB) activity is an important mediator of inflammatory responses. Here, we used zebrafish embryos to assess the capacity of two different classes of pharmacologic agents known to target NF-κB to modify radiation toxicity in the vertebrate organism. These were proteasome inhibitors, including lactacystin, MG132, and PS-341 (Bortezomib/VELCADE), and direct inhibitors of NF-κB activity, including ethyl pyruvate (EP) and the synthetic triterpenoid CDDO-TFEA (RTA401), among others. The proteasome inhibitors either did not significantly affect radiation sensitivity of zebrafish embryos (MG132, lactacystin) or rendered zebrafish embryos more sensitive to the lethal effects of ionizing radiation (PS-341). Radiosensitization by PS-341 was reduced in fish with impaired p53 expression or function but not associated with enhanced expression of select p53 target genes. In contrast, the direct NF-κB inhibitors EP and CDDO-TFEA significantly improved overall survival of lethally irradiated zebrafish embryos. In addition, direct NF-κB inhibition reduced radiation-induced apoptosis in the central nervous system, abrogated aberrations in body axis development, restored metabolization and secretion of a reporter lipid through the gastrointestinal system, and improved renal clearance compromised by radiation. In contrast to amifostine, EP and CDDO-TFEA not only protected against but also mitigated radiation toxicity when given 1 to 2 hours postexposure. Finally, four additional IκB kinase inhibitors with distinct mechanisms of action similarly improved overall survival of lethally irradiated zebrafish embryos. In conclusion, inhibitors of canonical pathways to NF-κB activation may be useful in alleviating radiation toxicity in patients. [Mol Cancer Ther 2009;8(9):2625–34]

Differential radiation sensitization of human cervical cancer cell lines by the proteasome inhibitor velcade (bortezomib, PS-341)

Purpose/objective(s)Cervical cancer is one of the deadliest cancers in women with a death toll of 230,000 worldwide each year, nearly 80% in developing countries. Radiotherapy (RT) is a major treatment modality for advanced cervical cancer but the local relapse rate is 30–44% in patients treated with RT alone and 19–25% in patients treated with concurrent chemoradiotherapy. Previous studies have shown that the transcription factor NF-κB is constitutively expressed in human cervical squamous cell carcinomas. NF-κB activation also contributes to the resistance of cervical cancer cells to apoptosis induced by chemotherapeutic agents and radiation. Therefore, inhibition of NF-κB in tumor cells may render them more sensitive to chemo/radiation therapies. The objective of this study is to investigate the potential of radiosensitization of NF-κB inhibition by Velcade in human cervical cancer cell lines.Materials and methods We used the human cervical cancer cell lines HeLa and SiHa. Both are highly radioresistant and chemoresistant as compared to other cervical cancer cell lines. These cells had been treated with Velcade before they were irradiated with different doses of ionizing radiation. MTT metabolic assays and clonogenic cell survival assays were performed to evaluate the effects of Velcade on radiation resistance.ResultsInhibition of NF-κB by Velcade alone decreased metabolic potential (MTT) and clonogenic survival in SiHa, but not in HeLa cells. Furthermore, pre-treatment of SiHa, but not HeLa cells with Velcade enhanced radiation sensitivity.ConclusionsInhibition of NF-κB by the proteasome inhibitor Velcade enhances radiosensitivity of certain human cervical carcinoma cancer cells in vitro. These results raise the possibility that inhibition of NF-κB will result in radiosensitization only in those tumor cells which are more dependent on NF-κB for their metabolism and survival, however, the radiosensitivity of “NF-κB independent” cells are not likely influenced by it.

Gene expression signatures modulated by epidermal growth factor receptor activation and their relationship to cetuximab resistance in head and neck squamous cell carcinoma

BackgroundAberrant activation of signaling pathways downstream of epidermal growth factor receptor (EGFR) has been hypothesized to be one of the mechanisms of cetuximab (a monoclonal antibody against EGFR) resistance in head and neck squamous cell carcinoma (HNSCC). To infer relevant and specific pathway activation downstream of EGFR from gene expression in HNSCC, we generated gene expression signatures using immortalized keratinocytes (HaCaT) subjected to ligand stimulation and transfected with EGFR, RELA/p65, or HRASVal12D.ResultsThe gene expression patterns that distinguished the HaCaT variants and conditions were inferred using the Markov chain Monte Carlo (MCMC) matrix factorization algorithm Coordinated Gene Activity in Pattern Sets (CoGAPS). This approach inferred gene expression signatures with greater relevance to cell signaling pathway activation than the expression signatures inferred with standard linear models. Furthermore, the pathway signature generated using HaCaT-HRASVal12D further associated with the cetuximab treatment response in isogenic cetuximab-sensitive (UMSCC1) and -resistant (1CC8) cell lines.ConclusionsOur data suggest that the CoGAPS algorithm can generate gene expression signatures that are pertinent to downstream effects of receptor signaling pathway activation and potentially be useful in modeling resistance mechanisms to targeted therapies.

Differential regulation of p53 function by the N-terminal ΔNp53 and Δ113p53 isoforms in zebrafish embryos

BackgroundThe p53 protein family coordinates stress responses of cells and organisms. Alternative promoter usage and/or splicing of p53 mRNA gives rise to at least nine mammalian p53 proteins with distinct N- and C-termini which are differentially expressed in normal and malignant cells. The human N-terminal p53 variants contain either the full-length (FL), or a truncated (ΔN/Δ40) or no transactivation domain (Δ133) altogether. The functional consequences of coexpression of the different p53 isoforms are poorly defined. Here we investigated functional aspects of the zebrafish ΔNp53 ortholog in the context of FLp53 and the zebrafish Δ133p53 ortholog (Δ113p53) coexpressed in the developing embryo.ResultsWe cloned the zebrafish ΔNp53 isoform and determined that ionizing radiation increased expression of steady-state ΔNp53 and Δ113p53 mRNA levels in zebrafish embryos. Ectopic ΔNp53 expression by mRNA injection caused hypoplasia and malformation of the head, eyes and somites, yet partially counteracted lethal effects caused by concomitant expression of FLp53. FLp53 expression was required for developmental aberrations caused by ΔNp53 and for ΔNp53-dependent expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A, p21, Cip1, WAF1). Knockdown of p21 expression markedly reduced the severity of developmental malformations associated with ΔNp53 overexpression. By contrast, forced Δ113p53 expression had little effect on ΔNp53-dependent embryonal phenotypes. These functional attributes were shared between zebrafish and human ΔNp53 orthologs ectopically expressed in zebrafish embryos. All 3 zebrafish isoforms could be coimmunoprecipitated with each other after transfection into Saos2 cells.ConclusionsBoth alternative N-terminal p53 isoforms were expressed in developing zebrafish in response to cell stress and antagonized lethal effects of FLp53 to different degrees. However, in contrast to Δ113p53, forced ΔNp53 expression itself led to developmental defects which depended, in part, on p21 transactivation. In contrast to FLp53, the developmental abnormalities caused by ΔNp53 were not counteracted by concomitant expression of Δ113p53.

CHK1 affects cell sensitivity to microtubule-targeted drugs.

Microtubules are the target of many anticancer drugs. Understanding the mechanism by which cells respond to different microtubule‐targeted drugs is important to resolve the drug resistance and to gain better tumor control. We report here for the first time that CHK1, an essential protein in mammalian cells, affects cell sensitivity to microtubule‐targeted drugs. By using a pair of transformed rat fibroblast cell lines, we show that compared with their counterpart B4 cells, A1‐5 cells with higher CHK1 expression are more resistant to taxotere, a microtubule stabilizer, but are more sensitive to nocodazole, a microtubule destabilizer. We also show that the altered sensitivities of A1‐5 cells to either taxotere or to nocodazole are related to the lesser microtubule‐formation in the cells. In addition, we show that the altered drug sensitivities and less microtubules‐formation shown in A1‐5 cells could be efficiently reversed by Chk1 siRNA. Taken together, these results indicate that CHK1 is one of the factors affecting cell resistance to taxotere and sensitiveness to nocodazole, suggesting that CHK1 is involved in affecting microtubule dynamics and could be inhibited for taxotere sensitization in CHK1 highly expressed tumor cells.

PET imaging of EGFR expression in nude mice bearing MDA-MB-468, a human breast adenocarcinoma.

Background and objectiveCetuximab is a monoclonal antibody that binds to and inhibits the epidermal growth factor receptor (EGFR). EGFR overexpression has been observed in a subset of breast cancers. The purpose of this study was to evaluate 64Cu-labeled cetuximab as an imaging agent using MDA-MB-468 breast cancer cells. MethodsCetuximab was coupled with an N-sulfosuccinimide ester of DOTA, purified, and labeled with the positron-emitting nuclide, 64Cu. Receptor-binding specificity and affinity of 64Cu-DOTA-cetuximab were studied using human MDA-MB-468 breast cancer cells, which express high levels of EGFR. Micropositron emission tomography and biodistribution studies were performed in athymic nude mice bearing MDA-MB-468 cell xenografts. Blocking studies with cold cetuximab were also performed to determine the specific binding of cetuximab. ResultsThe radiochemical yield was 97.1±1.1%. The specific activity was 1.5 Ci/&mgr;m cetuximab and the affinity to EGFR-positive MDA-MB-468 cells was high (KD=0.4 nmol/l). Both biodistribution and micropositron emission tomographic imaging studies with 64Cu-DOTA-cetuximab showed higher tumor uptake at 24 h (20.91±2.49% ID/g, standardized uptake values of 9.6) than at 4 h (11.65±3.89% ID/g, standardized uptake values of 4.9). Tumor uptake was significantly reduced from 20.91±2.49% ID/g at 24 h to 14.42±0.85% ID/g in a 1-h blocking study (P=0.00). ConclusionCetuximab can be labeled with 64Cu without compromising its biological activity. The tumor uptake was excellent with high tumor/muscle (7.97±1.78 at 4 h, 15.91±6.04 at 24 h) and reasonable tumor/blood (0.5±0.18 at 4 h, 2.12±0.86 at 24 h) ratios. Blocking studies showed the specific binding of the labeled antibody to tumor tissue.

Nuclear Factor κB Inhibitors Alleviate and the Proteasome Inhibitor PS-341 Exacerbates Radiation Toxicity in Zebrafish Embryos

Inflammatory changes are a major component of the normal tissue response to ionizing radiation, and increased nuclear factor κB (NF-κB) activity is an important mediator of inflammatory responses. Here, we used zebrafish embryos to assess the capacity of two different classes of pharmacologic agents known to target NF-κB to modify radiation toxicity in the vertebrate organism. These were proteasome inhibitors, including lactacystin, MG132, and PS-341 (Bortezomib/VELCADE), and direct inhibitors of NF-κB activity, including ethyl pyruvate (EP) and the synthetic triterpenoid CDDO-TFEA (RTA401), among others. The proteasome inhibitors either did not significantly affect radiation sensitivity of zebrafish embryos (MG132, lactacystin) or rendered zebrafish embryos more sensitive to the lethal effects of ionizing radiation (PS-341). Radiosensitization by PS-341 was reduced in fish with impaired p53 expression or function but not associated with enhanced expression of select p53 target genes. In contrast, the direct NF-κB inhibitors EP and CDDO-TFEA significantly improved overall survival of lethally irradiated zebrafish embryos. In addition, direct NF-κB inhibition reduced radiation-induced apoptosis in the central nervous system, abrogated aberrations in body axis development, restored metabolization and secretion of a reporter lipid through the gastrointestinal system, and improved renal clearance compromised by radiation. In contrast to amifostine, EP and CDDO-TFEA not only protected against but also mitigated radiation toxicity when given 1 to 2 hours postexposure. Finally, four additional IκB kinase inhibitors with distinct mechanisms of action similarly improved overall survival of lethally irradiated zebrafish embryos. In conclusion, inhibitors of canonical pathways to NF-κB activation may be useful in alleviating radiation toxicity in patients. [Mol Cancer Ther 2009;8(9):2625-34] Introduction Normal tissue damage limits the dose of ionizing radiation that can be safely administered to treat neoplastic disease. A well-known example of this problem is inflammation of the oral mucosa and of the lining of the gastrointestinal tract in tumor patients receiving chemotherapy or radiation.1 Depending on the area of the body treated with radiation, other organ sites including the lungs and the pericardium also manifest radiation-induced inflammation. A pervasive feature of ionizing radiation–associated inflammation is the increased presence of proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6, both locally and in the circulation.2 In contrast to intracellular regulators of the DNA damage response, these and other inflammatory mediators act in a paracrine fashion affecting diverse cell types in the tissue microenvironment or even at a distance.3 This circumstance highlights the necessity to use animal models to investigate the relative contribution of inflammatory changes to the overall response to radiation-induced cell and tissue injury in a multicellular organism. In recognition of this need, we recently established zebrafish embryos as a facile vertebrate in vivo system to monitor the effects of radiation protectors on normal tissues during development.4 The nuclear factor κB (NF-κB) family of transcription factors represents a diverse and shared signaling mechanism activated during cell stress responses.5 In addition, deregulated NF-κB signaling has been implicated in the malignant phenotype and treatment resistance of select tumor forms.6-10 The canonical pathway to NF-κB activation leads to IκB kinase β (IKKβ)-dependent phosphorylation and subsequent proteasomal degradation of the NF-κB inhibitor IκB, increased nuclear presence of NF-κB dimers, and enhanced NF-κB– dependent transcriptional activity.5 Whole-body radioprotection through antiinflammatory agents has very recently been shown in animal models. Specifically, certain triterpenoids (CDDO and derivatives thereof) have been shown to selectively protect normal mouse tissues against the deleterious effects of ionizing radiation.11 Furthermore, ethyl pyruvate (EP), a derivative of the end product of glycolysis, similarly protects normal cells against the deleterious effects of radiation both in vitro and in mice.12 Among other molecular targets, both drugs inhibit activation of NF-κB. EP inhibits NF-κB signaling through direct molecular interaction with a reactive cysteine of the p65 subunit of NF-κB13 whereas CDDOTFEA binds to a reactive cysteine (Cys179) of IKKα, thus inhibiting its kinase activity.14 However, these drugs also target other signaling molecules and pathways of potential relevance to the radiation response, including signal transducers and activators of transcription 3 and Jaks.15, 16 In addition to these agents proteasome inhibitors have been shown to inhibit NF-κB–dependent transcription, and one of these (PS-341; Bortezomib; VELCADE) Studies Borbala Daroczi,1 Gabor Kari,1 Qing Ren,1 Adam P. Dicker,1,3 and Ulrich Rodeck2,3 1Departments of Radiation Oncology, and 2Dermatology and Cutaneous Biology, and 3Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania Reprinted with permission from the American Association of Cancer Research, “Nuclear factor κB inhibitors alleviate and the proteasome inhibitor PS-341 exacerbates radiation toxicity in zebrafish embryos”, Moleculaer Cancer Therapy, 2009;8(9), pages 2625-2634.