Lung cancer is the leading cause of cancer and cancer deaths worldwide, with non-small-cell lung carcinomas (NSCLC) representing 85% of all diagnosed lung cancers. Platinum-combination chemotherapy is the current standard treatment for NSCLC, however, associated toxicities and resistance limit its efficacy. Our laboratory previously identified activating transcription factor 3 (ATF3), a stress-inducible gene whose elevated and sustained expression can trigger apoptosis to a wide variety of stressors, as a key regulator of cisplatin cytotoxicity as well. Thus, enhanced and sustained induction of ATF3 by combining platins with other ATF3 inducers potentially represents an effective therapeutic strategy. A chemical library screen identified vorinostat and topotecan as ATF3 inducers that also enhance cisplatin cytotoxicity. ATF3 plays a significant role in cisplatin, vorinostat and topotecan and their combinations cytotoxicity. Importantly, vorinostat and topotecan induced synergistic cytotoxicity with cisplatin in NSCLC cell lines and their cisplatin resistance sub-lines with enhanced ATF3 expression observed. Our study suggests a potential novel therapeutic approach where ATF3 inducing agents in combination with platins represents a rational combination based therapeutic strategy.

Epidermal growth factor receptor (EGFR) is highly expressed in head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) and is a key regulator of tumor cell growth and survival. Erlotinib, also known as tarceva, (a first-generation) and afatinib, also known as giotrif, (a second-generation) are tyrosine kinase inhibitors (TKIs) of EGFR. These TKIs are recognized therapeutic agents in these tumor types, as they inhibit EGFR signaling but show limited activity as single agents. Novel strategies will likely require EGFR-TKIs combination with an agent(s) that will enhance their therapeutic efficacy. Recently, we have demonstrated that combining statins, inhibitors of the mevalonate pathway, with erlotinib enhanced EGFR inhibition and induced synergistic cytotoxicity through the activation of cellular integrated stress response pathway (ISR) regulated by the induction of activating transcription factor 3 (ATF3). In our Phase I clinical trial, combining rosuvastatin with erlotinib, while demonstrating clinical activity, this treatment also showed statin-induced myopathies likely the result of diminished ubiquinone levels, which limited their utilization. Therefore, alternative strategies are warranted. Targeting geranylgeranyl diphosphate (GGPP) synthesis or its incorporation, a downstream mevalonate metabolite, represents such an approach with the potential to circumvent statin-associated toxicities but retain the efficacy in combination with EGFR inhibitors. In this project, we evaluated the effect of the combination of geranylgeranyl transferase-I inhibitor (GGTI-298) with the EGFR inhibitor, tarceva, (aim 1) and a GGPP synthase inhibitor, digeranyl bisphosphonate (DGBP), with the EGFR inhibitor, afatinib, (aim 2). For aim 1, we demonstrated that GGTI-298 treatment induced ATF3 expression in SCC9 and SCC25 cells and in a cohort of ex-vivo tumor tissues. Furthermore, GGTI-298 and tarceva induced synergistic cytotoxicity in SCC cells that was dependent on ATF3 expression, as ATF3 deficient murine embryonic fibroblasts (ATF3-/- MEFs) displayed attenuated cytotoxicity in response to GGTI-298 alone and in combination with tarceva. Similarly, SCC9 sub-lines that were selected as resistant to GGTI-298 through prolonged exposure to this agent also failed to demonstrate synergy with treatment of GGTI-298 in combination with tarceva. For aim 2, we demonstrated that the specific GGPP synthase inhibitor, DGBP, induced cytotoxicity in SCC cells. We further demonstrated this specificity as specific shRNA targeting of GGPP synthase as well as the inhibitor DGBP significantly enhanced the cytotoxic activity of the EGFR-TKI afatinib in SCC cells. DGBP as well as afatinib treatments induced ATF3 expression in SCC cells in vitro and in a cohort of ex-vivo tumor tissues. Co-administration of the downstream metabolite GGPP inhibits the induction of ATF3 and the cytotoxic and apoptotic effects associated with DGBP treatment. Furthermore, the synergistic cytotoxicity induced by the combination of DGBP and afatinib in SCC cells was also dependent on the expression of ATF3 through the induction of cellular stress response pathways. Taken together, these results suggest the potential clinical utility of combining downstream mevalonate inhibitors (GGTI-298 or DGBP) with EGFR inhibitors in HNSCC patients as a novel and more refined combination therapeutic approach.

No description available.

Current research suggests resveratrol, a phytoalexin found predominately in grapes, may function as a chemopreventive and/or chemotherapeutic agent for various cancers, including colorectal cancer. However, the underlying mechanism(s) involved in these activities remain elusive. Thus, the objective of the studies discussed here sought to investigate the effect of resveratrol treatment on gene modulation in human colorectal cancer cells in order to identify and characterize novel molecular targets that contribute to the observed anticancer activities of resveratrol. Here, we identify activating transcription factor 3 (ATF3) and early growth response-1 (Egr-1) as novel targets of resveratrol and provide data to elucidate the mechanism(s) of regulation and how each target contributes to the anticancer effect of resveratrol in colorectal cancer cells. We demonstrate the involvement of resveratrol in ATF3 transcriptional regulation, which is facilitated by Egr-1 and Krüppel-like factor 4 interactions, and show that ATF3 contributes, at least partially, to resveratrol-induced apoptosis (Chapter 3). Moreover, we suggest that increased Egr-1 transcriptional activity by resveratrol requires posttranslational acetylation of Egr-1 in a SIRT1-independent manner. This acetylation by resveratrol may contribute to Egr-1-mediated expression of the pro-apoptotic protein nonsteroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) induced by the phytoalexin (Chapter 4). Taken together, the work presented here provide (1) novel mechanisms by which resveratrol induces ATF3 and Egr-1 expression and (2) represent additional explanations for the anti-tumorigenic/anti-carcinogenic effects of resveratrol in human colorectal cancer cells.

No description available.

No description available.

No description available.

Die Zellen des Hodgkin-Lymphoms sind, neben einer permanenten Aktivierung des NF-kB Signalweges, durch eine konstitutive AP-1 Aktivität gekennzeichnet. Die vorliegende Arbeit beschäftigte sich mit der Analyse der aberranten AP-1 Aktivität in Zellen des Hodgkin-Lymphoms. Von besonderem Interesse ist in diesem Zusammenhang der JUN Promotor, da c-Jun unter anderem die Fähigkeit besitzt seinen eigenen Promotor positiv zu regulieren (Autoregulation). Im Rahmen dieser Arbeit wurden Faktoren, die mit dem JUN Promotor in Zellen des Hodgkin-Lymphoms assoziiert sind, über verschiedene chromatographische Reinigungsschritte angereichert, mittels Massenspektrometrie identifiziert und hinsichtlich ihres Einflusses auf die c-Jun Expression analysiert. Dabei wurden die zwei Faktoren ATF-3, aus der Familie der AP-1 Proteine, und p52, aus der NF-kB Familie, hinsichtlich ihres Einflusses auf die Überexpression von c-Jun in Hodgkinzellen genauer untersucht. Die hier aufgeführten Ergebnisse tragen zu einem besseren Verständnis der Regulation der konstitutiven AP-1 Aktivität in den Hodgkinzellen bei. Im Rahmen dieser Arbeit konnten zwei Faktoren identifiziert werden, die maßgeblich an der Regulation der Expression von c-Jun in Hodgkinzellen beteiligt sind. Eine besondere Rolle kommt dabei dem Transkriptionsfaktor p52 zu, da dieser unter anderem auch die Expression anderer Mitglieder der AP-1 Familie reguliert. Ein weiterer Befund dieser Arbeit, dass p50 und p52 die zentralen Komponenten der konstitutiven NF-kB Aktivität sind, rückt p52 in das Zentrum zukünftiger Forschung. Die Befunde dieser Arbeit belegen, dass die aberrante Aktivierung von AP-1 im Hodgkin-Lymphom nicht auf ein einzelnes Ereignis in der Zelle zurückzuführen ist, sondern das Ergebnis eines komplexen Zusammenspiels vieler Faktoren ist. / The cells of Hodgkin''s lymphoma are characterized by a permanent activation of the NF-kB signaling pathway and a constitutive AP-1 activation. The present study focused on the analysis of aberrant AP-1 activity in cells of Hodgkin''s lymphoma. Of particular interest in this context is the JUN promoter, since c-Jun has the ability to regulate its own promoter (autoregulation). In this work different JUN promoter associated factors were enriched through various chromatographic purification steps, identified by Mass spectrometry and analyzed in terms of its influence on the expression of c-Jun. The focus was on specific factors in cells of Hodgkin''s lymphoma. The two factors ATF-3 and p52, were analyzed in terms of their influence on the overexpression of c-Jun in cells of Hodgkin''s lymphoma. Another interesting finding of this study is, p50 and p52 are central components of the constitutive NF-kB activity. This puts p52 in the center of future research. The results presented here contribute to a better understanding of the regulation of the constitutive AP-1 activity in the Hodgkin cells. In this work two Factors (ATF3 and p52) could be identified, which are are involved in the regulation of the expression of c-Jun in Hodgkin cells. A special role is played by the transcription factor p52, which also regulates the expression of other members of the AP-1 family. The findings of this study also show that the aberrant activation of AP-1 in Hodgkin''s lymphoma is not due to a single event in the cell. It is rather a result of a complex interplay of many factors.

DNA-damaging chemotherapeutics are a consistently employed for the treatment of cancer, and are regularly part of first-line combination therapeutic regimens. However, these regimens often display limited activity in cancers including of the lung and breast. Novel therapeutic strategies are urgently required. Understanding the molecular mechanisms regulating DNA-damaging chemotherapeutics activity will lead to such novel strategies. Activating transcription factor 3 (ATF3) is a stress inducible gene that plays a significant role in regulating cellular stress including DNA damage. This thesis investigates the role of ATF3 in mediating the cytotoxic effects of two commonly employed DNA-damaging chemotherapeutics, cisplatin and doxorubicin. This study also identifies other independent ATF3 inducing agents in potential novel combination therapeutic strategies. In this study, cell line models of cisplatin-resistance were generated independently from two non-small cell lung cancer (NSCLC) cell lines, Calu6 and H23. Full transcriptome RNA-sequencing analysis identified ATF3 as the most highly dysregulated apoptosis regulatory gene in the cisplatin-resistant compared to their respective parental cell lines following treatment with cisplatin. Further characterization identified cisplatin induced activation of JNK as the key regulator of ATF3 induction in these cell lines. Restoring JNK activity resulted in induced ATF3 expression and re-sensitization of the resistant cell lines to cisplatin treatment. FDA-approved 1200 compound library screens were employed to identify agents that can enhance cisplatin cytotoxicity as well as whose cytotoxicity was dependent on ATF3 expression. Vorinostat, an HDAC inhibitor, was identified in both screens and importantly displayed synergistic cytotoxicity in combination with cisplatin. In addition, ATF3 was also induced with treatments of the DNA damaging agent doxorubicin in these NSCLC cell lines including in the cisplatin resistant models. This work suggested a different mechanism of ATF3 induction by doxorubicin and the potential role of ATF3 in mediating doxorubicin cytotoxicity was further investigated in breast cancer cells. Doxorubicin robustly increased ATF3 expression in human breast cancer cell lines and tumor tissue ex-vivo. Loss of ATF3 in MEFs resulted in reduced sensitivity to doxorubicin treatment compared to wild-type MEFs. Employing the same library screen as above, compounds with ATF3 dependent mechanisms that could enhance doxorubicin cytotoxicity were identified. Vorinostat, as well as, the nucleoside analogues trifluridine and 6-mercaptopurine induced ATF3 expression and enhanced doxorubicin cytotoxicity. This study demonstrated that ATF3 plays an important role in mediating the cytotoxic effect of the DNA-damaging chemotherapeutics cisplatin and doxorubicin. It also provides rationale for ATF3 as a potential therapeutic target, and for the incorporation of ATF3 inducing agents in novel combination therapeutic strategies.

No description available.