• Consequences of Oncogene and Tumor Suppressor Gene Targeting for Human Cancer Development and Therapeutic Efficacy

      Weiss, Michele; Weber, David J., Ph.D. (2009)
      Finding genes mutated at high frequencies in multiple human cancers uncovers the importance of these genes in the advancement of the disease. Understanding the effects of such mutations on tumor biology is an important and clinically relevant task. p53 and PTEN are the most mutated tumor suppressors and PI3K is one of the most mutated oncogenes in human malignancy. Mismatch repair proteins are also positioned as key regulators of neoplastic transformation such that, when lost, lead to tumorigenesis for 15% of all malignancies. All of these genes are implicitly involved in cell processes such as cell cycle arrest, apoptosis, and the maintenance of genome stability. However, each protein has a unique action in preventing the tumorigenic process, and when mutated, may cooperate or may lie in divergent paths towards malignancy. An examination of the phenotypic effects of alterations in p53, PI3K, and PTEN in cancer progression and therapeutic efficacy was achieved through the deletion of p53, the constitutive activation of PI3K, or the deletion of PTEN via somatic cell gene targeting in the non-tumorigenic cell line, MCF-10A. Deletion of p53 eliminated canonical p53 functions as well as produced p53-/- clones with varying phenotypes due to chromosomal instability and with treatment-schedule dependent responses to DNA damage. The knockin of two "hotspot" PIK3CA mutations allowed for growth factor-independent cell proliferation partly mediated by GSK3β. Moreover, GSK3β inhibitors Lithium Chloride or SB216763 were selectively toxic for PIK3CA mutant clones. The loss of PTEN in MCF-10A cells allowed for increased resistance to anoikis and increased sensitivity to DNA damage. Alternatively, the role of mismatch repair deficiency brought about by loss of MLH1 was examined by the targeted correction of MLH1 in HCT116 cancer cells, recapitulating cancer gene therapy. However, the heterogeneous nature of tumor cell pools or cancer cell compensatory mechanisms allowed for mismatch repair deficiency to be maintained in one corrected clone. In conclusion, the model systems reviewed herein will allow for a better understanding of specific downstream effects of these alterations as well as the consequences of potential tumor-specific responses cancer treatment options such as chemotherapy or cancer gene therapy.
    • Hyper-radiosensitization Induced by the Histone Deacetylase Inhibitor Vorinostat, in glioblastoma

      Diss, Eric; Carrier, France (2010)
      Glioblastoma is an aggressive form of brain cancer that limits patients to an average survival of 12 months after diagnosis [9]. Long term survival is limited by an inability to completely eradicate glioblastomas even with high dose radiation. Glioblastoma's aggressiveness allows it to regenerate rapidly if even trace amounts of the cancerous cells are alive [9]. Even when combined with drugs such as temozolomide current standards of care call for partial brain radiation of 60 Gy [10]. Such high doses have detrimental effects on patients to include extreme nausea, skin damage, hair loss, general malaise, and links have been found to reduction in life expectancy [9]. In order to reduce these adverse effects, drugs that induce radiosensitization such as Vorinostat (SAHA), are key to furthering cancer research. By inducing a conformational change to a more open form in chromatin structure, HDAC inhibitors could sensitize cancer cells to radiation treatments that are harmful to the brain.
    • Immunohistochemistry Analysis of Ki-67 and P53 Proteins in Oral Lichen Planus Compared to Other Oral Epithelial Lesions

      Akbar, Aqdar Abdullah; Meiller, Timothy F. (2014)
      Aim: The goal of this project was to determine if selected biomarkers of oral cancer are expressed in lichen planus and to identify the relationship, if any, of these biomarkers to malignant transformation. Methods: Six each of archived specimens with confirmed histopathological diagnosis of oral benign fibroma, oral reticular lichen planus, oral erosive lichen planus, oral mild to moderate dysplasia, oral severe dysplasia, and oral squamous cell carcinoma were selected. A total of 35 acceptable specimens were stained using an immunohistochemistry method for Ki67 and P53 proteins. Specimens were scanned with Aperio Scanscope CS and slides were analyzed visually, for expression patterns, and also using Aperio software. Results: The results of the present study demonstrated that the expression of P53 in oral lichen planus was 40.3 %, compared to 31.6% in oral benign fibroma, 46.3% in oral erosive lichen planus, 43.9% in oral mild to moderate dysplasia, 46.4% in oral severe dysplasia, and 47.2% in oral squamous cell carcinoma. On the other hand, Ki67 expression for oral lichen planus was 32.3% compared to 19.1% in oral benign fibroma, 40.3% in oral erosive lichen planus, 36% in oral mild to moderate dysplasia, 44.3% in oral severe dysplasia, and 48.9% in oral squamous cell carcinoma. Conclusion: Expression of the P53 and Ki67 biomarkers do not consistently correlate with malignant transformation of oral lichen planus appear to be associated with premalignant lesions of dysplasia. P53 and Ki67 are not a definitive diagnostic tool and further investigation to other biomarkers is needed.
    • Inactivation of the p53 tumor suppressor gene in human esophageal carcinoma

      Huang, Ying; Meltzer, Stephen J.; Schulze, Dan H. (Dan Howard) (1994)
      The p53 tumor suppressor gene is thought to play a critical role in the control of cell growth and tumor development. The p53 is the most commonly mutated gene found in human tumors. To study the role of p53 in esophageal tumorigenesis and the possible mechanism(s) which lead to p53 inactivation, we examined esophageal cancers for p53 alterations. By a combination of Southern blot and PCR analyses, we found that 58% of esophageal cancers contained p53 allelic deletions, and most of these tumors revealed mutations in the remaining allele. p53 gene mutations were examined in fifteen tumor samples by single strand conformation polymorphism and DNA sequence analyses. Four nonsense mutations were found which resulted from either base substitution mutations or deletion-generated frameshift mutations. Seven missense mutations substituted a single amino acid in different exons of the p53 hot spot region (exons 5-8). One single base pair deletion resulted in a reading frameshift, while the remaining mutation was silent. p53 mRNA expression was examined in eight esophageal cancers and their accompanying normal tissues by Northern analysis. Three tumors expressed aberrant-sized p53 transcripts and one revealed total lack of p53 mRNA expression. No abnormal p53 mRNA was observed in any accompanying normal tissues. Aberrant-sized mRNA was found in one adenocarcinoma and its adjacent Barrett's dysplasia, suggesting that genetic alteration of p53 may have occurred before flank carcinoma, early in neoplastic progression. In addition, abnormal p53 mRNA expression in two tumors correlated with p53 intragenic deletion and/or allelic deletion. p53 intragenic deletions flanked by short direct repeats were found in two esophageal cancers. One was forty-five bp deletion, involving all RNA splice consensus sequences at the 3{dollar}\sp\prime{dollar} end of intron 7; Northern blot analysis revealed a total lack of p53 mRNA expression in this tumor. The other deletion removed 5 nucleotides within exon 8, resulting in a reading frameshift. Short direct repeats were found at the deletion breakpoints of the p53 gene in both tumors, with both deletions removing one repeat and the sequence between the two repeats. These results imply that a "slipped mispairing" mechanism occurring during DNA replication may have been involved in the generation of deletions in these esophageal cancers. We conclude that multiple mechanisms lead to p53 inactivation in human esophageal cancers, and our results strongly suggest that inactivation of the p53 gene is involved in esophageal cancer formation.
    • Neuronal Intrinsic Apoptosis Mechanisms and Their Modulation by Sp1 Inhibition

      Makarevich, Oleg; Stoica, Bogdan A.; 0000-0003-2061-2285 (2021)
      Drug-induced DNA damage, reactive oxygen species (ROS), inflammatory mediators and central neurotoxicity have all been shown to play a role in cognitive impairments after chemotherapy, after CNS injury or in neurodegenerative diseases. Intrinsic apoptosis is a regulated cell death pathway implicated in many of these conditions. This pathway proceeds sequentially through DNA damage responses, including phosphorylation of ATM, H2AX and Tumor Protein 53 (p53), transcriptional activation of pro-apoptotic BH3-only proteins, and mitochondrial outer membrane permeabilization (MOMP), activating caspase-dependent and caspase-independent apoptosis. Previous work has indicated that Sp1 may regulate p53’s transcriptional profile and ability to promote apoptosis after DNA damage. To examine the role of Sp1 in DNA-damage-induced apoptosis, my work has utilized Mithramycin, a drug that binds G-C rich DNA to compete with Sp1 chromatin binding. Our hypothesis is that Mithramycin competes with Sp1 chromatin binding and thus protects neurons from DNA damage-induced, p53-dependent intrinsic apoptosis. However, it is vital to recognize that neurons increase their resistance to cytotoxic stimuli as they mature, leading to decreased levels of apoptosis. Thus, we additionally characterized the changes in the intrinsic apoptosis pathway upon neuronal maturation. Despite significant attenuation of the intrinsic apoptosis pathway in mature neurons, we found evidence that Mithramycin can still attenuate DNA-damage dependent intrinsic apoptosis in this paradigm to the extent that it is induced by neurons. Additional experiments showed that mature animal cortices or hippocampi do not demonstrate evidence of end-stage apoptosis (caspase activity) in response to DNA damage, unlike those in immature animals. However, in vivo administration of Mithramycin was able to attenuate cortical expression of p53-dependent genes. Thus, our studies provide evidence that: 1) Mithramycin attenuates activation of the DNA damage-dependent intrinsic apoptosis pathway via indirect inhibition of p53-dependent transcription and 2) Mature neurons restrict intrinsic apoptosis pathway activation in response to DNA damage. Therefore, Sp1 is likely to play a significant role in DNA damage-dependent neuronal intrinsic apoptosis, and Mithramycin treatment is protective when this pathway is activated. However, mature neurons significantly downregulate this pathway, thus limiting the potential therapeutic effects of Mithramycin in adult animals.
    • S100B regulation of p53 phosphorylation by PKC(alpha) and structural characterization of zinc(2+)-binding to calcium(2+)-S100B

      Wilder, Paul T.; Weber, David J., Ph.D. (2004)
      The S100B protein is a small acidic metal binding protein that has been found to be associated with neurological disorders and traumas, diabetes, and cancer. This work examines Zn2+ regulation of S100B Ca 2+-binding affinity and target protein interaction and also explores S100B interaction and regulation of an important target protein, tumor suppressor p53. It was found that S100B binds p53 preventing PKC phosphorylation of p53 (1), and this interaction connects the molecular and cellular functions of S100B with clinical data that associates S100B expression to several forms of cancer. In order to further characterize this S100B inhibition of PKC activities, peptides based on the PKC phosphorylation domains of p53 (residues 367--388), neuromodulin (residues 37--53), and the regulatory domain of PKC (residues 19--31) were synthesized and shown to be substrates for PKC and the catalytic domain of PKC, PKM, which lacks the Ca2+ and lipid regulatory region of PKC. It was necessary to use PKM in order to separate the influence of Ca2+ on the activation S100B from its effects on PKC allowing kinetic parameters of the PKC-dependent phosphorylation and its Ca2+-dependent inhibition by S100B to be determined for the peptides. While Ca2+-binding appears to be necessary for target protein binding by S100B, Zn2+-binding potentiates both S100B Ca2+-binding affinity and its interaction with certain target proteins. In order to determine how structural changes in S100B upon Zn2+ binding regulate its function, the high resolution structures of the Zn2+-Ca2+-S100B was completed using multi-dimensional heteronuclear NMR experiments. Studies were done to determine the amino acid ligands used for coordinating Zn2+ in S100B revealing what appears to be a conserved Zn2+/Cu2+-binding site at the dimer interface found in several S100 proteins. The structure shows that Zn2+-binding induces rearrangement of the Ca 2+-binding EF-hands in S100B that may contribute to the change in Ca2+-binding affinity found in S100B. Furthermore, there is an extension of the final helix IV and orienting of amino acids involved in target protein interaction upon Zn2+-binding by Ca2+ S100B that may influence target protein affinity.
    • Structure, Function, and Inhibition of S100B

      Charpentier, Thomas H.; Weber, David J., Ph.D. (2009)
      Aberrant levels of the small dimeric protein S100B have been found in malignant melanoma, renal cell cancer, and astrocytomas. S100B may aid in cancer progression via its interaction with and down regulation of the tumor suppressor p53, in a Ca²⁺ and possibly Zn²⁺ dependent manner. S100B bound to Ca²⁺ undergoes a conformational change exposing a hydrophobic cleft for the p53-S100B interaction. S100B binds to the C-terminus and tetramerization domains (319-393) of p53. Experiments reducing S100B expression via siRNA restores p53 levels in primary malignant melanoma cells. Thus, several small molecules have been identified that bind S100B and inhibit the Ca²⁺-S100B-p53 interaction. One of these small molecules is pentamidine, an FDA approved drug. We have characterized the interaction between Ca²⁺-S100B and pentamidine via nuclear magnetic resonance (NMR) and X-ray crystallography. We obtained crystal structures of pentamidine bound to Zn²⁺-Ca²⁺-S100B. The previously solved NMR structure of Zn²⁺-Ca²⁺-S100B was compared to the X-ray crystal structure solved here. We characterized the Zn²⁺ ligands in each structure to determine if Zn²⁺ binding changed the pentamidine interaction with S100B. A goal of the Weber lab has been to identify small molecules to inhibit the p53-S100B interaction and we have been moderately successful. We have identified three additional small molecules found through screens performed by our lab and the high throughput screening core (UMAB). SBi132, SBi279, and SBi523 (S100B inhibitor ###) were shown to interact with S100B through NMR and X-ray crystallography. Other small molecules derived from pentamidine or SBi132 interact with the "hinge" region of S100B, while other screening molecules were found to covalently bind to cysteine 84 on helix 4 of S100B. To characterize the S100B-p53 protein-protein interaction further, TRTK-12 a peptide derived from the CapZ protein, was used to study the effects of peptide bound to S100B. Surprisingly, the Ca²⁺ coordination for both EF-hands of S100B were not affected by TRTK-12. The X-ray structure of TRTK-12 peptide bound to S100B did show differences in temperature factor. These differences in peptide binding can aid us in identifying inhibitors of the S100B-p53 complex and restore p53 levels in malignant melanoma.