David J. Bearss

The nuclease hypersensitivity element III(1) upstream of the P1 promoter of c-MYC controls 85-90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand of the DNA in this region can form two different intramolecular G-quadruplex structures, only one of which seems to be biologically relevant. This biologically relevant structure is the kinetically favored chair-form G-quadruplex, which is destabilized when mutated with a single G --> A transition, resulting in a 3-fold increase in basal transcriptional activity of the c-MYC promoter. The cationic porphyrin TMPyP4, which has been shown to stabilize this G-quadruplex structure, is able to suppress further c-MYC transcriptional activation. These results provide compelling evidence that a specific G-quadruplex structure formed in the c-MYC promoter region functions as a transcriptional repressor element. Furthermore, we establish the principle that c-MYC transcription can be controlled by ligand-mediated G-quadruplex stabilization.

PURPOSE: Epithelial-mesenchymal transition (EMT) has been associated with metastatic spread and EGF receptor (EGFR) inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using non-small cell lung carcinoma (NSCLC) cell lines and patients treated in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) study. EXPERIMENTAL DESIGN: We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from patients with NSCLC. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE study, and potential therapeutic targets associated with EMT were identified. RESULTS: Compared with epithelial cells, mesenchymal cells showed significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend toward greater sensitivity to the Axl inhibitor SGI-7079, whereas the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In patients with NSCLC, the EMT signature predicted 8-week disease control in patients receiving erlotinib but not other therapies. CONCLUSION: We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype.

YY1 is a mammalian zinc-finger transcription factor with unusual structural and functional features. It has been implicated as a positive and a negative regulatory factor that binds to the CCATNTT consensus DNA element located in promoters of many cellular and viral genes. A mammalian cDNA that encodes a YY1-binding protein and possesses sequence homology with the yeast transcriptional factor RPD3 has been identified. A Gal4 DNA binding domain-mammalian RPD3 fusion protein strongly represses transcription from a promoter containing Gal4 binding sites. Association between YY1 and mammalian RPD3 requires a glycine-rich region on YY1. Mutations in this region abolish the interaction with mammalian RPD3 and eliminate transcriptional repression by YY1. These data suggest that YY1 negatively regulates transcription by tethering RPD3 to DNA as a cofactor and that this transcriptional mechanism is highly conserved from yeast to human.

To identify new diagnostic markers and drug targets for pancreatic cancer, we compared the gene expression patterns of pancreatic cancer cell lines growing in tissue culture with those of normal pancreas using cDNA microarray analysis. Fluorescently (cyanine 5) labeled cDNA probes, made individually from mRNA samples of nine pancreatic cell lines, were each combined with fluorescently (cyanine 3) labeled universal reference mRNA. The mixed probes of each sample were then hybridized with 5760 cDNA arrays (5289 unique cDNA sequences) printed on individual microscope slides. Fluorescently (cyanine 5) labeled normal pancreas mRNA was also compared with the same universal reference mRNA reference pool. The expression ratios of neoplastic versus normal pancreas cells were then calculated by multiplying the ratio of cancer versus the universal reference mRNA and the ratio of the universal reference mRNA cell versus normal pancreas. For 5289 different genes interrogated by the arrays, 30 of them showed an expression ratio 2 SD from the mean in at least three of the nine pancreatic cell lines studied. To confirm the expression profiles of these genes, quantitative reverse transcription-PCR and Northern blot were carried out for 25 of the overexpressed genes. To verify the overexpression in patient samples, two of the overexpressed genes, c-Myc and Rad51, were selected to undergo analysis by reverse transcription-PCR in frozen tumor tissues and by immunostaining in paraffin-embedded tissue section microarrays. The results of these experiments are in agreement with the microarray data. Potential up-regulated targets of note from this study include urokinase-type plasminogen activator receptor, serine/threonine kinase 15, thioredoxin reductase, and CDC28 protein kinase 2, as well as several others.

Cationic porphyrins are being studied as possible anticancer agents because of their ability to bind to and stabilize DNA guanine quadruplexes (G-quadruplexes). We have shown previously that the cationic porphyrin TMPyP4 is able to bind to and stabilize G-quadruplexes in human telomere sequences, resulting in inhibition of telomerase activity. To better understand the mechanism of action behind telomerase inhibition by TMPyP4, we performed a cDNA microarray analysis on cells treated with TMPyP4 and TMPyP2, a positional isomer of TMPyP4 that has low affinity for G-quadruplexes. Analysis of time course data from the microarray experiments revealed that TMPyP4 and TMPyP2 treatment altered the expression of several gene clusters. We found that c-MYC, an oncogene nearly ubiquitous in human tumors that bears the potential in its promoter to form a G-quadruplex, was among the genes specifically down-regulated by TMPyP4, but not by TMPyP2. The hTERT gene, which encodes the catalytic subunit of telomerase, is transcriptionally regulated by c-MYC, and we have found that TMPyP4 also causes a decrease in human telomerase reverse transcriptase transcripts, suggesting two possible mechanisms for the effect of TMPyP4 on telomerase activity. We also show that TMPyP4, but not TMPyP2, is able to prolong survival and decrease tumor growth rates in two xenograft tumor models. We believe that, because of the actions of TMPyP4 in decreasing both c-MYC protein levels and telomerase activity, as well as its anticancer effects in vivo, it is a worthwhile agent to pursue and develop further.