Natalia Issaeva

Reactivation of Mutant p53 and Induction of Apoptosis in Human Tumor Cells by Maleimide Analogs

Reactivation of mutant p53 is likely to provide important benefits for treatment of chemotherapy- and radiotherapy-resistant tumors. We demonstrate here that the maleimide-derived molecule MIRA-1 can reactivate DNA binding and preserve the active conformation of mutant p53 protein in vitro and restore transcriptional transactivation to mutant p53 in living cells. MIRA-1 induced mutant p53-dependent cell death in different human tumor cells carrying tetracycline-regulated mutant p53. The structural analog MIRA-3 showed antitumor activity in vivo against human mutant p53-carrying tumor xenografts in SCID mice. The MIRA scaffold is a novel lead for the development of anticancer drugs specifically targeting mutant p53.

Poly(ADP-Ribose) Polymerase Is Hyperactivated in Homologous Recombination–Defective Cells

Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gammaH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers.

Rescue of mutants of the tumor suppressor p53 in cancer cells by a designed peptide

We designed a series of nine-residue peptides that bound to a defined site on the tumor suppressor p53 and stabilized it against denaturation. To test whether the peptides could act as chaperones and rescue the tumor-suppressing function of oncogenic mutants of p53 in living cells, we treated human tumor cells with the fluorescein-labeled peptide Fl-CDB3 (fluorescent derivative of CDB3). Before treatment, the mutant p53 in the cell was predominantly denatured. Fl-CDB3 was taken up into the cytoplasm and nucleus and induced a substantial up-regulation of wild-type p53 protein and representative mutants. The mutants, His-273 and His-175 p53, adopted the active conformation, with a dramatic decrease in the fraction of denatured protein. In all cases, there was p53-dependent induction of expression of the p53 target genes mdm2, gadd45, and p21, accompanied by p53-dependent partial restoration of apoptosis. Fl-CDB3 sensitized cancer cells that carried wild-type p53 to p53-dependent γ-radiation-induced apoptosis. Although Fl-CDB3 did not elicit a full biological response, it did bind to and rescue p53 in cells and so can serve as a lead for the development of novel drugs for anticancer therapy.

Characterization of HPV and host genome interactions in primary head and neck cancers

Significance A significant proportion of head and neck cancer is driven by human papillomavirus (HPV) infection, and the expression of viral oncogenes is involved in the development of these tumors. However, the role of HPV integration in primary tumors beyond increasing the expression of viral oncoproteins is not understood. Here, we describe how HPV integration impacts the host genome by amplification of oncogenes and disruption of tumor suppressors as well as driving inter- and intrachromosomal rearrangements. Tumors that do and do not have HPV integrants display distinct gene expression profiles and DNA methylation patterns, which further support the view that the mechanisms by which tumors with integrated and nonintegrated HPV arise are distinct. Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twenty-five cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter- and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis.

6-Thioguanine Selectively Kills BRCA2-Defective Tumors and Overcomes PARP Inhibitor Resistance

Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.

PRIMA-1Met/APR-246 induces wild-type p53-dependent suppression of malignant melanoma tumor growth in 3D culture and in vivo

Disseminating malignant melanoma is a lethal disease highly resistant to radio- and chemotherapy. Therefore, the development of new treatment strategies is strongly needed. Tumor suppressor p53-mediated apoptosis is essential for the response to radio- and chemotherapy. Although p53 is not frequently mutated in melanoma, it is inactivated by integrin αv-mediated signaling, as we previously demonstrated 1, which may account, at least partially, for increased apoptosis resistance of malignant melanoma. In this study we addressed the question whether functional restoration of p53 by APR-246 (PRIMA-1Met), which can reactivate mutant p53 and induce massive apoptosis in cancer cells, is able to restore the function of inactive p53 in melanoma. Using a three-dimensional collagen gel (3D-collagen) to culture melanoma cells carrying wild-type p53, we found that APR-246 treatment resulted in activation of p53, leading to increased expression of p53 pro-apoptotic targets Apaf1 and PUMA and activation of caspase- 9 and -3. Moreover, APR-246 triggered melanoma cell apoptosis that was mediated by p53 and caspase 9. Importantly, APR-246 treatment also suppressed human melanoma xenograft tumors in vivo in a p53-dependent manner. Thus, wild-type p53 reactivation may provide a novel approach for malignant melanoma treatment, with APR-246 as a candidate drug for such a development.

TrkC signaling is activated in adenoid cystic carcinoma and requires NT-3 to stimulate invasive behavior

Treatment options for adenoid cystic carcinoma (ACC) of the salivary gland, a slowly growing tumor with propensity for neuroinvasion and late recurrence, are limited to surgery and radiotherapy. Based on expression analysis performed on clinical specimens of salivary cancers, we identified in ACC expression of the neurotrophin-3 receptor TrkC/NTRK3, neural crest marker SOX10, and other neurologic genes. Here, we characterize TrkC as a novel ACC marker, which was highly expressed in 17 out of 18 ACC primary-tumor specimens, but not in mucoepidermoid salivary carcinomas or head and neck squamous cell carcinoma. Expression of the TrkC ligand NT-3 and Tyr-phosphorylation of TrkC detected in our study suggested the existence of an autocrine signaling loop in ACC with potential therapeutic significance. NT-3 stimulation of U2OS cells with ectopic TrkC expression triggered TrkC phosphorylation and resulted in Ras, Erk 1/2 and Akt activation, as well as VEGFR1 phosphorylation. Without NT-3, TrkC remained unphosphorylated, stimulated accumulation of phospho-p53 and had opposite effects on p-Akt and p-Erk 1/2. NT-3 promoted motility, migration, invasion, soft-agar colony growth and cytoskeleton restructuring in TrkC-expressing U2OS cells. Immunohistochemical analysis demonstrated that TrkC-positive ACC specimens also show high expression of Bcl2, a Trk target regulated via Erk 1/2, in agreement with activation of the TrkC pathway in real tumors. In normal salivary gland tissue, both TrkC and Bcl2 were expressed in myoepithelial cells, suggesting a principal role for this cell lineage in the ACC origin and progression. Sub-micromolar concentrations of a novel potent Trk inhibitor AZD7451 completely blocked TrkC activation and associated tumorigenic behaviors. Pre-clinical studies on ACC tumors engrafted in mice showed efficacy and low toxicity of AZD7451, validating our in vitro data and stimulating more research into its clinical application. In summary, we describe in ACC a previously unrecognized pro-survival neurotrophin signaling pathway and link it with cancer progression.

Functional p53 chimeras containing the Epstein–Barr virus Gly-Ala repeat are protected from Mdm2- and HPV-E6-induced proteolysis

Functional inactivation of the tumor suppressor protein p53 by accelerated ubiquitin/proteasome-dependent proteolysis is a common event in tumor progression. Proteasomal degradation is inhibited by the Gly-Ala repeat (GAr) of the Epstein–Barr virus nuclear antigen-1, which acts as a transferable element on a variety of proteasomal substrates. We demonstrate that p53 chimeras containing GAr domains of different lengths and positions within the protein are protected from proteolysis induced by the ubiquitin ligases murine double minute 2 and E6-associated protein but are still ubiquitinated and retain the capacity to interact with the S5a ubiquitin-binding subunit of the proteasome. The GAr chimeras transactivate p53 target genes, induce cell cycle arrest and apoptosis, and exhibit improved growth inhibitory activity in tumor cells with impaired endogenous p53 activity.

Reverse Phase Protein Array profiling of oropharyngeal cancer and significance of PIK3CA mutations in HPV-associated head and neck cancer

PURPOSE Human papilloma virus (HPV)-associated (HPV+) oropharyngeal squamous cell carcinomas (OPSCC) have different molecular and biologic characteristics and clinical behavior compared with HPV-negative (HPV-) OPSCC. PIK3CA mutations are more common in HPV(+) OPSCC. To define molecular differences and tumor subsets, protein expression and phosphorylation were compared between HPV(+) and HPV(-) OPSCC and between tumors with and without PIK3CA mutations. EXPERIMENTAL DESIGN Expression of 137 total and phosphorylated proteins was evaluated by reverse-phase protein array in 29 HPV(+) and 13 HPV(-) prospectively collected OPSCCs. Forty-seven OPSCCs were tested for hotspot-activating mutations in PIK3CA and AKT. Activation of PIK3CA downstream targets and sensitivity to pathway inhibitors were determined in HPV(+) head and neck cancer cells overexpressing wild-type or mutant PIK3CA. RESULTS Analyses revealed 41 differentially expressed proteins between HPV(+) and HPV(-) OPSCC categorized into functional groups: DNA repair, cell cycle, apoptosis, phosphoinositide 3-kinase (PI3K)/AKT/mTOR, and receptor kinase pathways. All queried DNA repair proteins were significantly upregulated in HPV(+) samples. A total of 8 of 33 HPV(+) and 0 of 14 HPV(-) tumors contained activating PIK3CA mutations. Despite all activating PIK3CA mutations occurring in HPV(+) samples, HPV(+) tumors had lower mean levels of activated AKT and downstream AKT target phosphorylation. Ectopic expression of mutant PIK3CA in HPV(+) cells increased mTOR, but not AKT activity. HPV E6/E7 overexpression inhibited AKT phosphorylation in HPV-negative cells. Mutant PIK3CA overexpressing cells were more sensitive to a dual PI3K/mTOR inhibitor compared with an AKT inhibitor. CONCLUSIONS Protein expression analyses suggest that HPV(+) and HPV(-) OPSCC differentially activate DNA repair, cell cycle, apoptosis, PI3K/AKT/mTOR, and receptor kinase pathways. PIK3CA mutations are more common in HPV(+) OPSCC and are associated with activation of mTOR, but not AKT. These data suggest that inhibitors for mTOR may have activity against HPV(+) PIK3CA mutant oropharyngeal cancers.

Protoporphyrin IX Interacts with Wild-type p53 Protein in Vitro and Induces Cell Death of Human Colon Cancer Cells in a p53-dependent and -independent Manner

Photodynamic therapy (PDT) of cancer is an alternative treatment for tumors resistant to chemo- and radiotherapy. It induces cancer cell death mainly through generation of reactive oxygen species by a laser light-activated photosensitizer. It has been suggested that the p53 tumor suppressor protein sensitizes some human cancer cells to PDT. However, there is still no direct evidence for this. We have demonstrated here for the first time that the photosensitizer protoporphyrin IX (PpIX) binds to p53 and disrupts the interaction between p53 tumor suppressor protein and its negative regulator HDM2 in vitro and in cells. Moreover, HCT116 colon cancer cells exhibited a p53-dependent sensitivity to PpIX in a dose-dependent manner, as was demonstrated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and fluorescence-activated cell sorter (FACS) analysis of cell cycle profiles. We have also observed induction of p53 target pro-apoptotic genes, e.g. puma (p53-up-regulated modulator of apoptosis), and bak in PpIX-treated cells. In addition, p53-independent growth suppression by PpIX was detected in p53-negative cells. PDT treatment (2 J/cm2) of HCT116 cells induced p53-dependent activation of pro-apoptotic gene expression followed by growth suppression and induction of apoptosis.